Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-03-08
2004-03-09
Gambel, Phillip (Department: 1644)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C530S387100, C530S387300, C530S388200, C530S388250, C435S069600, C435S252300, C435S320100, C435S455000, C435S326000
Reexamination Certificate
active
06703494
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention concerns methods for engineering anti-tissue factor (anti-TF) antibodies, especially those having enhanced anticoagulant potency. The invention further concerns anti-TF antibodies, methods and means for producing them, compositions comprising the antibodies and their use in the diagnosis, management, prevention and treatment of various diseases and disorders.
2. Description of the Related Art
A. Tissue Factor
Tissue factor (TF) is the receptor for coagulation factor VIIa (FVIIa) and the zymogen precursor factor VII (FVII). Native human TF (hTF) is a 263 amino acid residue glycoprotein composed of an extracellular domain (residues 1 to 219), a single transmembrane domain (residues 220-242), and a short cytoplasmic domain (residues 243 to 263) (Fisher et al., [1987
] Thromb. Res.
48:89-99, Morrissey et al., [1987
] Cell
50:129-135). The TF extracellular domain is composed of two immunoglobulin-like fibronectin type III domains of about 105 amino acids each (Huang et al., [1998
] J. Mol. Biol.
275:873-894). Each domain is formed by two anti-parallel &bgr;-sheets with Ig superfamily type C2 homology.
The protein interaction of FVIIa with TF is mediated entirely by the TF extracellular domain (Muller et al., [1994
] Biochem.
33:10864-10870; Gibbs et al., [1994
] Biochem.
33:14003-14010; Ruf et al. [1994
] Biochem.
33:1565-1572) which has been expressed in
E. coli,
cultured Chinese Hamster Ovary (CHO) cells and
Saccharomyces cerevisiae
(Waxman et al., [1992
] Biochemistry
31:3998-4003; Ruf et al., [1991
] J. Bio. Chem.
25 266:2158-2166 and Shigematsu et al., [1992
] J. Biol. Chem.
267:21329-21337). The crystal structures of hTF extracellular domain and its complex with active site inhibited FVIIa have recently been determined by x-ray crystallography (Harlos et al., [1994
] Nature
370:662-666; Muller et al., [1994
] Biochemistry
33:10864; Muller et al., [1996
] J. Mol. Biol.
256:144-159; Banner et al., [1996
] Nature
380:41-46).
The hTF extracellular domain has also been extensively characterized by alanine scanning mutagenesis (Kelley et al., [1995
] Biochemistry,
34:10383-10392; Gibbs et al., [1994] supra; Ruf et al., [1994] supra). Residues in the area of amino acids 16-26 and 129-147 contribute to the binding of FVIIa as well as the coagulant function of the molecule. Residues Lys20, Trp45, Asp58, Tyr94, and Phe140 make a large contribution (1 kcal/mol) to the free energy (&Dgr;G) of binding to FVIIa (Kelley et al., (1995) supra). Substitution of Lys20 and Asp58 with alanine residues leads to 78- and 30-fold reductions in FVIIa affinity respectively (Kelley et al., [1995] supra). A set of 17 single-site mutants at other nearby sites that are in contact with FVIIa result in modest decreases in affinity (&Dgr;&Dgr;G=0.3-1.0 kcal mol
−1
). Mutations of TF residues Thr17, Arg131, Leu133 and Val207, each of which contact FVIIa in the crystal structure, have no effect on affinity for FVIIa. Lys15Ala and Tyr185Ala mutations result in small increases in affinity (&Dgr;&Dgr;G=−0.4 kcal mol
−1
) (Kelley et al., [1995] supra). The 78-fold decrease in affinity imposed by the alanine substitution of Lys20 in hTF can be reversed by substituting a tryptophan for Asp58 (Lee and Kelley, [1998
] J. Biol. Chem.
273:4149-4154).
Residues in the area of amino acids 157-168 contribute to the procoagulant function of TF-FVIIa (Kelley et al., [1995] supra; Ruf et al., [1992
] J. Biol. Chem.
267:22206-22210) but are not important for FVII/FVIIa binding. It has been shown that lysine residues 165 and 166 are important to TF cofactor function but do not participate in FVIIa complex formation (Roy et al., [1991
] J. Biol. Chem.
266:22063; Ruf et al., [1992
] J. Biol. Chem.
267:6375). Lysine residues 165 and 166 are located on the C-terminal fibronectin type III domain of TF on the opposite surface of the molecule from residues found to be important for FVIIa binding on the basis of mutagenesis results (Kelley et al., (1995) supra). Alanine substitution of these lysine residues results in a decreased rate of FX activation catalyzed by the TF-FVIIa complex (Ruf et al., (1992) supra). The Lys165Ala-Lys166Ala variant (hTFAA) comprising residues 1-219 of hTF (sTF) inhibits the extrinsic pathway of blood coagulation in vitro through competition with membrane TF for binding to FVIIa. In a rabbit model of arterial thrombosis the variant partially blocks thrombus formation without increasing bleeding tendency (Kelley et al., (1997) Blood 89, 3219-3227). However, high doses of the variant are required for the antithrombotic effect, in part because FVIIa binds to cell surface TF approximately 1000-fold more tightly than to sTF (Kelley et al. (1997) supra). The greater apparent affinity is due to interaction of the FVIIa &ggr;-carboxyglutamic acid-containing (Gla) domain with phospholipid.
TF is expressed constitutively on cells separated from plasma by the vascular endothelium (Carson, S. D. and J. P. Brozna, [1993
] Blood Coag. Fibrinol.
4:281-292). Its expression on endothelial cells and monocytes is induced by exposure to inflammatory cytokines or bacterial lipopolysaccharide (Drake et al., [1989
] J. Cell Biol.
109:389). Upon tissue injury, the exposed extracellular domain of TF forms a high affinity, calcium dependent complex with FVII. Once bound to TF, FVII can be activated by peptide bond cleavage to yield serine protease FVIIa. The enzyme that catalyzes this step in vivo has not been elucidated, but in vitro FXa, thrombin, TF-FVIIa and FIXa can catalyze this cleavage (Davie, et al., [1991
] Biochemistry
30:10363-10370). FVIIa has only weak activity upon its physiological substrates FX and FIX whereas the TF-FVIIa complex rapidly activates FX and FIX.
The TF-FVIIa complex constitutes the primary initiator of the extrinsic pathway of blood coagulation (Carson, S. D. and Brozna, J. P., (1993)
Blood Coag. Fibrinol.
4:281-292; Davie, E. W. et al., [1991
] Biochemistry
30:10363-10370; Rapaport, S. I. and L. V. M. Rao, [1992
] Arterioscler. Thromb.
12:1111-1121). The complex initiates the extrinsic pathway by activation of FX to Factor Xa (FXa), FIX to Factor IXa (FIXa), and additional FVII to FVIIa. The action of TF-FVIIa leads ultimately to the conversion of prothrombin to thrombin, which carries out many biological functions (Badimon, L. et al., [1991
] Trends Cardiovasc. Med.
1:261-267). Among the most important functions of thrombin is the conversion of fibrinogen to fibrin, which polymerizes to form a clot.
The involvement of this plasma protease system has been suggested to play a significant role in a variety of clinical manifestations including arterial and venous thrombosis, septic shock, adult respiratory distress syndrome (ARDS), disseminated intravascular coagulation (DIC) and various other disease states (Haskel, E. J. et al., [1991
] Circulation
84:821-827; Holst, J. et al., [1993
] Haemostasis
23 (suppl. 1):112-117; Creasey, A. A. et al., [1993
] J. Clin. Invest.
91:2850-2860; see also, Colman R. W. [1989
] N. Engl. J. Med
320:1207-1209; Bone, R. C. [1992
] Arch. Intern. Med.
152:1381-1389). Overexpression and/or aberrant utilization of TF has been linked to the pathophysiology of both thrombosis and sepsis (Taylor et al., [1991
] Circ. Shock
33:127; Warr et al., [1990
] Blood
75:1481; Pawashe et al., [1994
] Circ. Res.
74:56). TF is expressed on cells found in the atherosclerotic plaque (Wilcox et al., [1989
] Proc. Natl. Acad. Sci. U.S.A.
86:2839). Additionally, TF has been implicated in tumor metastasis (Bromberg et al., [1995
] Proc. Natl. Acad. Sci. USA,
92:8205).
B. Anti-T
Kirchhofer Daniel K.
Lowe David G.
Presta Leonard G.
Gambel Phillip
Genentech Inc.
Shin Elinor K.
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