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. 2000 Aug 1;349(Pt 3):805–812. doi: 10.1042/bj3490805

Fusion of the tissue factor extracellular domain to a tumour stroma specific single-chain fragment variable antibody results in an antigen-specific coagulation-promoting molecule.

J F Rippmann 1, K Pfizenmaier 1, R Mattes 1, W J Rettig 1, D Moosmayer 1
PMCID: PMC1221208  PMID: 10903142

Abstract

Solid tumours growing beyond a size of 1-2 mm in diameter induce supporting connective tissue structures, the tumour stroma, comprising activated fibroblasts and newly formed blood vessels, embedded in an extracellular matrix. The selective destruction of this tissue or the inhibition of its function (e.g. tumour neoangiogenesis) may result in the destruction of tumour nodules, thus providing novel opportunities for tumour therapy. Our approach aims at an antibody-mediated induction of coagulation in tumour nodules to cut off their blood supply. As a target structure the fibroblast activation protein (FAP) is used, which is specifically and abundantly expressed on the activated fibroblasts of the tumour stroma. We constructed a fusion protein comprising a single-chain module of a FAP-specific humanized antibody [single-chain fragment variable (scFv) OS4] and the extracellular domain of human tissue factor. The fusion protein, designated TFOS4, was produced in the Proteus mirabilis protoplast expression system with a yield of 15 microg/ml. Biochemical characterization of TFOS4 revealed high-affinity binding to cellular FAP. Further, TFOS4 bound to factor VIIa and also exerted allosteric activation of factor VIIa. A complex of TFOS4 and factor VIIa bound to FAP-expressing cells efficiently generated activated factor X. Finally, cell-bound TFOS4 selectively induced plasma coagulation, implying its activity under physiological conditions, notably with relevant concentrations of coagulation factors and their natural inhibitors. These findings suggest that TFOS4 has the potential to increase the procoagulant state in a cell-type-specific fashion. No systemic coagulation or side effects were observed when TFOS4 was injected intravenously into normal mice, indicating the biosafety and specificity of the recombinant protein.

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Selected References

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  1. Banner D. W., D'Arcy A., Chène C., Winkler F. K., Guha A., Konigsberg W. H., Nemerson Y., Kirchhofer D. The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor. Nature. 1996 Mar 7;380(6569):41–46. doi: 10.1038/380041a0. [DOI] [PubMed] [Google Scholar]
  2. Baugh R. J., Broze G. J., Jr, Krishnaswamy S. Regulation of extrinsic pathway factor Xa formation by tissue factor pathway inhibitor. J Biol Chem. 1998 Feb 20;273(8):4378–4386. doi: 10.1074/jbc.273.8.4378. [DOI] [PubMed] [Google Scholar]
  3. Bromberg M. E., Konigsberg W. H., Madison J. F., Pawashe A., Garen A. Tissue factor promotes melanoma metastasis by a pathway independent of blood coagulation. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8205–8209. doi: 10.1073/pnas.92.18.8205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brooks P. C., Strömblad S., Klemke R., Visscher D., Sarkar F. H., Cheresh D. A. Antiintegrin alpha v beta 3 blocks human breast cancer growth and angiogenesis in human skin. J Clin Invest. 1995 Oct;96(4):1815–1822. doi: 10.1172/JCI118227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Davie E. W., Fujikawa K., Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry. 1991 Oct 29;30(43):10363–10370. doi: 10.1021/bi00107a001. [DOI] [PubMed] [Google Scholar]
  6. Dittmar S., Ruf W., Edgington T. S. Influence of mutations in tissue factor on the fine specificity of macromolecular substrate activation. Biochem J. 1997 Feb 1;321(Pt 3):787–793. doi: 10.1042/bj3210787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Drake T. A., Ruf W., Morrissey J. H., Edgington T. S. Functional tissue factor is entirely cell surface expressed on lipopolysaccharide-stimulated human blood monocytes and a constitutively tissue factor-producing neoplastic cell line. J Cell Biol. 1989 Jul;109(1):389–395. doi: 10.1083/jcb.109.1.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dübel S., Breitling F., Fuchs P., Braunagel M., Klewinghaus I., Little M. A family of vectors for surface display and production of antibodies. Gene. 1993 Jun 15;128(1):97–101. doi: 10.1016/0378-1119(93)90159-z. [DOI] [PubMed] [Google Scholar]
  9. Folkman J. Antiangiogenic gene therapy. Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9064–9066. doi: 10.1073/pnas.95.16.9064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garin-Chesa P., Old L. J., Rettig W. J. Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7235–7239. doi: 10.1073/pnas.87.18.7235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Huang X., Molema G., King S., Watkins L., Edgington T. S., Thorpe P. E. Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor vasculature. Science. 1997 Jan 24;275(5299):547–550. doi: 10.1126/science.275.5299.547. [DOI] [PubMed] [Google Scholar]
  12. Kao J., Fan Y. G., Haehnel I., Clauss M., Stern D. Endothelial-monocyte activating polypeptides (EMAPs): tumor derived mediators which activate the host inflammatory response. Behring Inst Mitt. 1993 Aug;(92):92–106. [PubMed] [Google Scholar]
  13. Kujau M. J., Hoischen C., Riesenberg D., Gumpert J. Expression and secretion of functional miniantibodies McPC603scFvDhlx in cell-wall-less L-form strains of Proteus mirabilis and Escherichia coli: a comparison of the synthesis capacities of L-form strains with an E. coli producer strain. Appl Microbiol Biotechnol. 1998 Jan;49(1):51–58. doi: 10.1007/s002530051136. [DOI] [PubMed] [Google Scholar]
  14. Martin D. M., Boys C. W., Ruf W. Tissue factor: molecular recognition and cofactor function. FASEB J. 1995 Jul;9(10):852–859. doi: 10.1096/fasebj.9.10.7615155. [DOI] [PubMed] [Google Scholar]
  15. McCallum C. D., Su B., Neuenschwander P. F., Morrissey J. H., Johnson A. E. Tissue factor positions and maintains the factor VIIa active site far above the membrane surface even in the absence of the factor VIIa Gla domain. A fluorescence resonance energy transfer study. J Biol Chem. 1997 Nov 28;272(48):30160–30166. doi: 10.1074/jbc.272.48.30160. [DOI] [PubMed] [Google Scholar]
  16. Moosmayer D., Dübel S., Brocks B., Watzka H., Hampp C., Scheurich P., Little M., Pfizenmaier K. A single-chain TNF receptor antagonist is an effective inhibitor of TNF mediated cytotoxicity. Ther Immunol. 1995 Feb;2(1):31–40. [PubMed] [Google Scholar]
  17. Nagy J. A., Brown L. F., Senger D. R., Lanir N., Van de Water L., Dvorak A. M., Dvorak H. F. Pathogenesis of tumor stroma generation: a critical role for leaky blood vessels and fibrin deposition. Biochim Biophys Acta. 1989 Feb;948(3):305–326. doi: 10.1016/0304-419x(89)90004-8. [DOI] [PubMed] [Google Scholar]
  18. Neuenschwander P. F., Morrissey J. H. Roles of the membrane-interactive regions of factor VIIa and tissue factor. The factor VIIa Gla domain is dispensable for binding to tissue factor but important for activation of factor X. J Biol Chem. 1994 Mar 18;269(11):8007–8013. [PubMed] [Google Scholar]
  19. Pai L. H., Pastan I. Clinical trials with Pseudomonas exotoxin immunotoxins. Curr Top Microbiol Immunol. 1998;234:83–96. doi: 10.1007/978-3-642-72153-3_6. [DOI] [PubMed] [Google Scholar]
  20. Ran S., Gao B., Duffy S., Watkins L., Rote N., Thorpe P. E. Infarction of solid Hodgkin's tumors in mice by antibody-directed targeting of tissue factor to tumor vasculature. Cancer Res. 1998 Oct 15;58(20):4646–4653. [PubMed] [Google Scholar]
  21. Rippmann J. F., Klein M., Hoischen C., Brocks B., Rettig W. J., Gumpert J., Pfizenmaier K., Mattes R., Moosmayer D. Procaryotic expression of single-chain variable-fragment (scFv) antibodies: secretion in L-form cells of Proteus mirabilis leads to active product and overcomes the limitations of periplasmic expression in Escherichia coli. Appl Environ Microbiol. 1998 Dec;64(12):4862–4869. doi: 10.1128/aem.64.12.4862-4869.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ruf W., Kalnik M. W., Lund-Hansen T., Edgington T. S. Characterization of factor VII association with tissue factor in solution. High and low affinity calcium binding sites in factor VII contribute to functionally distinct interactions. J Biol Chem. 1991 Aug 25;266(24):15719–15725. [PubMed] [Google Scholar]
  23. Ruf W., Rehemtulla A., Morrissey J. H., Edgington T. S. Phospholipid-independent and -dependent interactions required for tissue factor receptor and cofactor function. J Biol Chem. 1991 Feb 5;266(4):2158–2166. [PubMed] [Google Scholar]
  24. Schullek J. R., Ruf W., Edgington T. S. Key ligand interface residues in tissue factor contribute independently to factor VIIa binding. J Biol Chem. 1994 Jul 29;269(30):19399–19403. [PubMed] [Google Scholar]
  25. Shigematsu Y., Miyata T., Higashi S., Miki T., Sadler J. E., Iwanaga S. Expression of human soluble tissue factor in yeast and enzymatic properties of its complex with factor VIIa. J Biol Chem. 1992 Oct 25;267(30):21329–21337. [PubMed] [Google Scholar]
  26. Shoji M., Hancock W. W., Abe K., Micko C., Casper K. A., Baine R. M., Wilcox J. N., Danave I., Dillehay D. L., Matthews E. Activation of coagulation and angiogenesis in cancer: immunohistochemical localization in situ of clotting proteins and vascular endothelial growth factor in human cancer. Am J Pathol. 1998 Feb;152(2):399–411. [PMC free article] [PubMed] [Google Scholar]
  27. Sipkins D. A., Cheresh D. A., Kazemi M. R., Nevin L. M., Bednarski M. D., Li K. C. Detection of tumor angiogenesis in vivo by alphaVbeta3-targeted magnetic resonance imaging. Nat Med. 1998 May;4(5):623–626. doi: 10.1038/nm0598-623. [DOI] [PubMed] [Google Scholar]
  28. Weiner L. M., Clark J. I., Davey M., Li W. S., Garcia de Palazzo I., Ring D. B., Alpaugh R. K. Phase I trial of 2B1, a bispecific monoclonal antibody targeting c-erbB-2 and Fc gamma RIII. Cancer Res. 1995 Oct 15;55(20):4586–4593. [PubMed] [Google Scholar]
  29. Welt S., Divgi C. R., Scott A. M., Garin-Chesa P., Finn R. D., Graham M., Carswell E. A., Cohen A., Larson S. M., Old L. J. Antibody targeting in metastatic colon cancer: a phase I study of monoclonal antibody F19 against a cell-surface protein of reactive tumor stromal fibroblasts. J Clin Oncol. 1994 Jun;12(6):1193–1203. doi: 10.1200/JCO.1994.12.6.1193. [DOI] [PubMed] [Google Scholar]
  30. Willems G. M., Janssen M. P., Salemink I., Wun T. C., Lindhout T. Transient high affinity binding of tissue factor pathway inhibitor-factor Xa complexes to negatively charged phospholipid membranes. Biochemistry. 1998 Mar 10;37(10):3321–3328. doi: 10.1021/bi972194+. [DOI] [PubMed] [Google Scholar]
  31. Zacharski L. R., Memoli V. A., Ornstein D. L., Rousseau S. M., Kisiel W., Kudryk B. J. Tumor cell procoagulant and urokinase expression in carcinoma of the ovary. J Natl Cancer Inst. 1993 Aug 4;85(15):1225–1230. doi: 10.1093/jnci/85.15.1225. [DOI] [PubMed] [Google Scholar]
  32. Zetter B. R. On target with tumor blood vessel markers. Nat Biotechnol. 1997 Nov;15(12):1243–1244. doi: 10.1038/nbt1197-1243. [DOI] [PubMed] [Google Scholar]

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