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Thromb Haemost 2001; 85(03): 379-389
DOI: 10.1055/s-0037-1615593
Rapid Communications
Schattauer GmbH

Generation of a Humanized, High Affinity Anti-tissue Factor Antibody for Use as a Novel Antithrombotic Therapeutic

Leonard Presta
,
Paul Sims
2   BioAnalytical Technology
,
Y. Gloria Meng
2   BioAnalytical Technology
,
Paul Moran
3   Cardiovascular Research
,
Sherron Bullens
3   Cardiovascular Research
,
Stuart Bunting
3   Cardiovascular Research
,
Jill Schoenfeld
3   Cardiovascular Research
,
David Lowe
3   Cardiovascular Research
,
Jadine Lai
,
Patricia Rancatore
4   Recovery Sciences
,
Mark Iverson
4   Recovery Sciences
,
Amy Lim
4   Recovery Sciences
,
Vanessa Chisholm
5   Molecular Biology
,
Robert F. Kelley
6   Protein Engineering, Genentech Inc., South San Francisco, CA, USA
,
Markus Riederer
7   Preclinical Research Department, F. Hoffmann-La Roche Ltd., Basel, Switzerland
,
Daniel Kirchhofer
3   Cardiovascular Research
› Author Affiliations
Further Information

Publication History

Received 21 June 2000

Accepted after revision 14 September 2000

Publication Date:
08 December 2017 (online)

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Summary

Blocking the cofactor function of human tissue factor may be beneficial in various coagulation-mediated diseases. The murine antibody D3 binds to the membrane proximal substrate interaction region of human tissue factor and blocks tissue factor function even in the presence of bound factor VIIa. The cloned murine D3 antibody was humanized and affinity matured by exchanging amino acids in the complementarity determining regions as well as in the antibody framework. The humanized antibody, D3H44, bound to tissue factor with a 100-fold increased affinity (KD 0.1 nM) as compared to the original murine and chimeric versions. Depending on the particular disease, different pharmacokinetic properties of the antibody may be required and, therefore, several antibody variants – F(ab), F(ab’)2, IgG2, IgG4 and IgG4b – were generated. In vitro, the humanized D3 antibodies displayed potent inhibition of plasma clotting and tissue factor: factor VIIa-mediated activation of factors IX and X (e. g. D3H44-F(ab’)2, IC50 (F.X) 47 pM). In addition, D3H44-F(ab’)2 completely prevented fibrin deposition in a human ex vivo thrombosis model under venous blood flow conditions (IC50 37 nM). The humanized D3 antibodies may be utilized for treatment of cardiovascular diseases which involve tissue factor activity, e. g. acute coronary syndrome and venous thrombosis.

Keywords

Tissue factor - coagulation factor VII - humanization - anticoagulants - thrombosis
 

  • References

  • 1 Rapaport SI, Rao LVM. The tissue factor pathway: how it has become a ‘Prima Ballerina‘. Thromb Haemost 1995; 74: 7-17.
    PubMedGoogle Scholar
  • 2 Mann KG. Biochemistry and physiology of blood coagulation. Thromb Haemost 1999; 82: 165-74.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 3 Davie EW. Biochemical and molecular aspects of the coagulation cascade. Thromb Haemost 1995; 74: 1-6.
    PubMedGoogle Scholar
  • 4 Wilcox JN, Smith KM, Schwartz SM, Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci USA 1989; 86: 2839-43.
    CrossrefPubMedGoogle Scholar
  • 5 Annex BH, Denning SM, Channon KM, Sketch Jr MH, Stack RS, Morrissey JH, Peters KG. Differential expression of tissue factor protein in directional atherectomy specimens from patients with stable and unstable coronary syndromes. Circulation 1995; 91: 619-22.
    CrossrefPubMedGoogle Scholar
  • 6 Moreno PR, Bernardi VH, Lopez-Cuellar J, Murcia AM, Palacios IF, Gold HK, Mehran R, Sharma SK, Nemerson Y, Fuster V, Fallon JT. Macrophages, smooth muscle cells, and tissue factor in unstable angina. Circulation 1996; 94: 3090-7.
    CrossrefPubMedGoogle Scholar
  • 7 Marmur JD, Thiruvikraman SV, Fyfe BS, Guha A, Sharma SK, Ambrose JA, Fallon JT, Nemerson Y, Taubmann MB. Identification of active tissue factor in human coronary atheroma. Circulation 1996; 94: 1226-32.
    CrossrefPubMedGoogle Scholar
  • 8 Kaikita K, Ogawa H, Yasue H, Takeya M, Takahashi K, Saito T, Hayasaki K, Horiuchi K, Takizawa A, Kamikubo Y, Nakamura S. Tissue factor expression on macrophages in coronary plaques in patients with unstable an-gina. Arterioscler Thromb Vasc Biol 1997; 17: 2232-7.
    CrossrefPubMedGoogle Scholar
  • 9 Mallat Z, Hugel B, Ohan J, Leseche G, Freyssinet J-M, Tedgui A. Shed membrane microparticles with procoagulant potential in human athero-sclerotic plaques. Circulation 1999; 99: 348-53.
    CrossrefPubMedGoogle Scholar
  • 10 Toschi V, Gallo R, Lettino M, Fallon JT, Gertz SD, Fernandez-Ortiz A, Chesebro JH, Badimon L, Nemerson Y, Fuster V, Badimon JJ. Tissue factor modulates the thrombogenicity of human atherosclerotic plaques. Circulation 1997; 95: 594-9.
    CrossrefPubMedGoogle Scholar
  • 11 Badimon JJ, Lettino M, Toschi V, Fuster V, Berrozpe M, Chesebro JH, Badimon L. Local inhibition of tissue factor reduces the thrombogenicity of disrupted human atherosclerotic plaques. Circulation 1999; 99: 1780-7.
    CrossrefPubMedGoogle Scholar
  • 12 Weinberg JB, Pippen AMM, Greenberg CS. Extravascular fibrin formation and dissolution in synovial tissue of patients with osteoarthritis and rheumatoid arthritis. Arthritis Rheumat 1991; 34: 996-1005.
    CrossrefPubMedGoogle Scholar
  • 13 Zacharski LR, Brown FE, Memoli VA, Kisiel W, Kudryk BJ, Rousseau SM, Hunt JA, Dunwiddie C, Nutt EM. Pathways of coagulation activation in situ in rheumatoid synovial tissue. Clin Immunol Immunopathol 1992; 63: 155-62.
    CrossrefPubMedGoogle Scholar
  • 14 Miller CL, Graziano C, Lim RC, Chin M. Generation of tissue factor by patient monocytes: correlation to thromboembolic complications. Thromb Haemost 1981; 46: 489-95.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 15 Blakowski SA, Zacharski LR, Beck JR. Postoperative elevation of human peripheral blood monocyte tissue factor coagulant activity. J Lab Clin Med 1986; 108: 117-20.
    PubMedGoogle Scholar
  • 16 Pawashe AB, Golino P, Ambrosio G, Migliaccio F, Ragni M, Pascucci I, Chiariello M, Bach R, Garen A, Konigsberg WK, Ezekowitz MD. A monoclonal antibody against rabbit tissue factor inhibits thrombus formation in stenotic injured rabbit carotid arteries. Circ Res 1994; 74: 56-63.
    CrossrefPubMedGoogle Scholar
  • 17 Ragni M, Cirillo P, Pascucci I, Scognamiglio A, D’Andrea D, Eramo N, Ezekowitz MD, Pawashe AB, Chiariello M, Golino P. Monoclonal antibody against tissue factor shortens tissue plasminogen activator lysis time and prevents reocclusion in a rabbit model of carotid artery thrombosis. Circulation 1996; 93: 1913-8.
    CrossrefPubMedGoogle Scholar
  • 18 Himber J, Kirchhofer D, Riederer M, Tschopp TB, Steiner B, Roux SP. Dissociation of antithrombotic effect and bleeding time prolongation in rabbits by inhibiting tissue factor function. Thromb Haemost 1997; 78: 1142-9.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 19 Thomas WS, Mori E, Copeland BR, Yu J-Q, Morrissey JH, del Zoppo GJ. Tissue factor contributes to microvascular defects after focal cerebral ischemia. Stroke 1993; 24: 847-54.
    CrossrefPubMedGoogle Scholar
  • 20 Golino P, Ragni M, Cirillo P, Avvedimento VE, Feliciello A, Esposito N, Scognamiglio A, Trimarco B, Iaccarino G, Condorelli M, Chiariello M, Ambrosio G. Effects of tissue factor induced by oxygen free radicals on coronary flow during reperfusion. Nature Med 1996; 2: 35-40.
    CrossrefPubMedGoogle Scholar
  • 21 Kappelmayer J, Bernabei A, Edmunds Jr H, Edgington TS, Colman RW. Tissue factor is expressed on monocytes during simulated extracorporeal circulation. Circulation Res 1993; 72: 1075-81.
    CrossrefPubMedGoogle Scholar
  • 22 Jang Y, Guzman LA, Lincoff AM, Gottsauner-Wolf M, Forudi F, Hart CE, Courtman DW, Ezban M, Ellis SG, Topol EJ. Influence of blockade at specific levels of the coagulation cascade on restenosis in a rabbit athero-sclerotic femoral artery injury model. Circulation 1995; 92: 3041-50.
    CrossrefPubMedGoogle Scholar
  • 23 Courtman DW, Schwartz SM, Hart CE. Sequential injury of the rabbit abdominal aorta induces intramural coagulation and luminal narrowing independent of intimal mass. Circ Res 1998; 82: 996-1006.
    CrossrefPubMedGoogle Scholar
  • 24 Taylor Jr. FB, Chang A, Ruf W, Morrissey JH, Hinshaw L, Catlett R, Blick K, Edgington TS. Lethal E. coli septic shock is prevented by blocking tissue factor with monoclonal antibody. Circulatory Shock 1991; 33: 127-34.
    PubMedGoogle Scholar
  • 25 Levi M, ten Cate H, Bauer KA, van der Poll T, Edgington TS, Büller HR, van Deventer SJH, Hack CE, ten Cate JW, Rosenberg RD. Inhibition of endotoxin-induced activation of coagulation and fibrinolysis by pentoxifyl-line or by a monoclonal anti-tissue factor antibody in chimpanzees. J Clin Invest 1994; 93: 114-20.
    CrossrefPubMedGoogle Scholar
  • 26 Kirchhofer D, Moran P, Chiang N, Kim J, Riederer MA, Eigenbrot C, Kelley RF. Epitope location on tissue factor determines the anticoagulant potency of monoclonal anti-tissue factor antibodies. Thromb Haemost 2000; 84: 1072-81.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 27 Ruf W, Edgington TS. An anti-tissue factor monoclonal antibody which inhibits TF·VIIa complex is a potent anticoagulant in plasma. Thromb Haemost 1991; 66: 529-33.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 28 Fiore MM, Neuenschwander PF, Morrissey JH. An unusual antibody that blocks tissue factor/factor VIIa function by inhibiting cleavage only of macromolecular substrates. Blood 1992; 80: 3127-34.
    PubMedGoogle Scholar
  • 29 Huang M, Syed R, Stura EA, Stone MJ, Stefanko RS, Ruf W, Edgington TS, Wilson IA. The mechanism of an inhibitory antibody on TF-initiated blood coagulation revealed by the crystal structures of human tissue factor, Fab 5G9 and TF·5G9 complex. J Mol Biol 1998; 275: 873-94.
    CrossrefPubMedGoogle Scholar
  • 30 Huang Q, Neuenschwander PF, Rezaie AR, Morrissey JH. Substrate recognition by tissue factor-factor VIIa. Evidence for interaction of residues Lys165 and Lys166 of tissue factor with the 4-carboxyglutamate-rich domain of factor X. J Biol Chem 1996; 271: 21752-7.
    CrossrefPubMedGoogle Scholar
  • 31 Dittmar S, Ruf W, Edgington TS. Influence of mutations in tissue factor on the fine specificity of macromolecular substrate activation. Biochem J 1997; 321: 787-93.
    CrossrefPubMedGoogle Scholar
  • 32 Roy S, Hass PE, Bourell JH, Henzel WJ, Vehar GA. Lysine residues 165 and 166 are essential for the cofactor function of tissue factor. J Biol Chem 1991; 266: 22063-6.
    PubMedGoogle Scholar
  • 33 Ruf W, Miles DJ, Rehemtulla A, Edgington TS. Cofactor residues lysine 165 and 166 are critical for protein substrate recognition by the tissue factor-factor VIIa protease complex. J Biol Chem 1992; 267: 6375-81.
    PubMedGoogle Scholar
  • 34 Ruf W, Miles DJ, Rehemtulla A, Edgington TS. Tissue factor residues 157-167 are required for efficient proteolytic activation of factor X and factor VII. J Biol Chem 1992; 267: 22206-10.
    PubMedGoogle Scholar
  • 35 Kirchhofer D, Lipari MT, Moran P, Eigenbrot C, Kelley RF. The tissue factor region that interacts with substrates factor IX and factor X. Biochemistry 2000; 39: 7380-7.
    CrossrefPubMedGoogle Scholar
  • 36 King DJ, Adair JR. Recombinant antibodies for the diagnosis and therapy of human disease. Curr Opin Drug Discovery Develop 1999; 2: 110-7.
    PubMedGoogle Scholar
  • 37 Farah RA, Clinchy B, Herrera L, Vitetta ES. The development of monoclonal antibodies for the therapy of cancer. Crit Rev Eukar Gene Expression 1998; 8: 321-56.
    PubMedGoogle Scholar
  • 38 Coller BS. GPIIb/IIIa antagonists: pathophysiologic and therapeutic insights from studies of c7E3 Fab. Thromb Haemost 1997; 78: 730-5.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 39 Vaughan TJ, Osbourn JK, Tempest PR. Human antibodies by design. Nature Biotechnology 1998; 16: 535-9.
    CrossrefPubMedGoogle Scholar
  • 40 Carter P, Presta L, Gorman CM, Ridgway JBB, Henner D, Wong WLT, Rowland AM, Kotts C, Carver ME, Shepard HM. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci USA 1992; 89: 4285-9.
    CrossrefPubMedGoogle Scholar
  • 41 Paborsky LR, Caras IW, Fisher KL, Gorman CM. Lipid association, but not the transmembrane domain, is required for tissue factor activity. J Biol Chem 1991; 266: 21911-6.
    PubMedGoogle Scholar
  • 42 Mimms LT, Zampighi G, Nozaki Y, Tanford C, Reynolds JA. Phospholipid vesicle formation and transmembrane protein incorporation using octyl glucoside. Biochemistry 1981; 20: 833-40.
    CrossrefPubMedGoogle Scholar
  • 43 Kelley RF, Costas KE, O’Connell MP, Lazarus RA. Analysis of the factor VIIa binding site on human tissue factor: effects of tissue factor mutations on the kinetics and thermodynamics of binding. Biochemistry 1995; 34: 10383-92.
    CrossrefPubMedGoogle Scholar
  • 44 Paborksy LR, Fendly BM, Fisher KL, Lawn RM, Marks BJ, McCray G, Tate KM, Vehar GA, Gorman CM. Mammalian cell transient expression of tissue factor for the production of antigen. Prot Engineering 1990; 3: 547-53.
    CrossrefPubMedGoogle Scholar
  • 45 Presta LG, Chen H, O’Connor SJ, Chisholm V, Meng YG, Krummen L, Winkler M, Ferrara N. Humanization of an anti-vascular endothelial growth factor monoclonal antibody for therapy of solid tumors and other disorders. Cancer Res 1997; 57: 4593-9.
    PubMedGoogle Scholar
  • 46 Eigenbrot C, Randal M, Presta L, Kossiakoff AA. X-ray structures of the antigen-binding domains from three variants of humanized anti-p185HER2 antibody 4D5 and comparison with molecular modeling. J Mol Biol 1993; 229: 969-95.
    CrossrefPubMedGoogle Scholar
  • 47 Werther WA, Gonzalez TN, O’Connor SJ, McCabe S, Chan B, Hotaling T, Champe M, Fox JA, Jardieu PM, Berman PW, Presta LG. Humanization of an anti-lymphocyte function-associated antigen (LFA-1) monoclonal antibody and reengineering of the humanized antibody for binding to rhesus LFA-1. J Immunol 1996; 157: 4986-95.
    PubMedGoogle Scholar
  • 48 Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 1985; 82: 488-92.
    CrossrefPubMedGoogle Scholar
  • 49 Kabat EA, Wu TT, Perry HM, Gottesmann KS, Foeller C. Sequences of proteins of immunological interest. Public Health Service, Natl Inst Health, Bethesda, MD: 1991
    PubMedGoogle Scholar
  • 50 Chothia C, Lesk AM, Tramontano A, Levitt M, Smith-Gill SJ, Air G, Sheriff S, Padlan EA, Davies D, Tulip WR, Colman PM, Spinelli S, Alzari PM, Poljak RJ. Conformations of immunoglobulin hypervariable regions. Nature 1989; 342: 877-83.
    CrossrefPubMedGoogle Scholar
  • 51 Landschulz WH, Johnson PF, McKnight SL. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 1988; 240: 1759-64.
    CrossrefPubMedGoogle Scholar
  • 52 Eaton DL, Woods WI, Eaton D, Hass PE, Hollingshead P, Wion K, Mather J, Lawn RM, Vehar GA, Gorman C. Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule. Biochemistry 1986; 25: 8343-7.
    CrossrefPubMedGoogle Scholar
  • 53 Angal S, King DJ, Bodmer MW, Turner A, Lawson AD, Roberts G, Pedley B, Adair JR. A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody. Molec Immunol 1993; 30: 105-8.
    CrossrefPubMedGoogle Scholar
  • 54 Bloom JW, Madanat MS, Marriot D, Wong T, Chan S-Y.. Intrachain disulfide bond in the core hinge region of human IgG4. Prot Sci 1997; 6: 407-15.
    PubMedGoogle Scholar
  • 55 Kirchhofer D, Tschopp TB, Baumgartner HR. Active site-blocked factors VIIa and IXa differentially inhibit fibrin formation in a human ex vivo thrombosis model. Arterioscler Thromb Vasc Biol 1995; 15: 1098-106.
    CrossrefPubMedGoogle Scholar
  • 56 Kirchhofer D, Tschopp TB, Hadvary P, Baumgartner HR. Endothelial cells stimulated with tumor necrosis factor- express varying amounts of tissue factor resulting in inhomogenous fibrin deposition in a native blood flow system. J Clin Invest 1994; 93: 2073-83.
    CrossrefPubMedGoogle Scholar
  • 57 Orvim U, Barstad M, Stormorken H, Brosstad F, Sakariassen KS. Immuno-logic quantification of fibrin in thrombi formed in flowing native human blood. Br J Haematol 1996; 95: 389-98.
    CrossrefPubMedGoogle Scholar
  • 58 Paborsky LR, Tate KM, Harris RJ, Yansura DG, Band L, McCray G, Gorman CM, O’Brien DP, Chang JY, Swartz JR, Fung VP, Thomas JN, Vehar GA. Purification of recombinant human tissue factor. Biochemistry 1989; 28: 8072-7.
    CrossrefPubMedGoogle Scholar
  • 59 Refino CJ, Himber J, Burcklen L, Moran P, Peek M, Suggett S, Devaux B, Kirchhofer D. A human antibody that binds to the γ-carboxyglutamic acid domain of factor IX is a potent antithrombotic in vivo. Thromb Haemost 1999; 82: 1188-95.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 60 Xiang J, Sha Y, Jia Z, Prasad L, Delbaere LT. Framework residues 71 and 93 of the chimeric B72.3 antibody are major determinants of the conformation of heavy-chain hypervariable loops. J Mol Biol 1995; 253: 385-90.
    CrossrefPubMedGoogle Scholar
  • 61 Tramontano A, Chothia C, Lesk AM. Framework residue 71 is a major determinant of the position and conformation of the second hypervariable region in the VH domains of immunoglobulins. J Mol Biol 1990; 215: 175-82.
    CrossrefPubMedGoogle Scholar
  • 62 Cacia J, Keck R, Presta LG, Frenz J. Isomerization of an aspartic acid residue in the complementarity-determining regions of a recombinant antibody to human IgE: identification and effect on binding affinity. Biochemistry 1996; 35: 1897-903.
    CrossrefPubMedGoogle Scholar
  • 63 Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE. The protein data bank. Nucleic Acid Res 2000; 28: 235-42.
    CrossrefPubMedGoogle Scholar
  • 64 Ruf W, Stura EA, LaPolla RJ, Syed R, Edgington TS, Wilson IA. Purification, sequence and crystallisation of an anti-tissue factor Fab and its use for the crystallization of tissue factor. J Crystal Growth 1992; 122: 253-64.
    CrossrefPubMedGoogle Scholar
  • 65 Roitt IM. Essential Immunity. 6th ed. Blackwell Scientific Publications; Oxford: 1988: 44.
    Google Scholar
  • 66 Colcher D, Goel A, Pavlinkova G, Beresford G, Booth B, Batra SK. Effects of genetic engineering on the pharmacokinetics of antibodies. Quarterly J. Nuclear Med 1999; 43: 132-9.
    PubMedGoogle Scholar