WO2001070984A2 - Anti-tissue factor antibodies with enhanced anticoagulant potency - Google Patents

Abstract

The invention concerns anti-tissue factor (anti-TF) antibodies with enhanced anticoagulant potency, and methods and means for identifying, producing and using such antibodies. The anti-TF antibodies of the present invention are designed to comprise a region binding to an epitope in the C-terminal macromolecular substrate binding region of TF.

Description

ANTI-TISSUE FACTOR ANTIBODIES WITH ENHANCED ANTICOAGULANT POTENCY

Background of the Invention Field of the Invention

This invention concerns methods for engineering anti-tissue factor (anti-TF) antibodies, especially those havmg enhanced anticoagulant potency The invention further concerns anti-TF antibodies, methods and means for producing them, compositions compπsing the antibodies and their use in the diagnosis, management, prevention and treatment of vaπous diseases and disorders Descπption of the Related Art A Tissue factor Tissue factor (TF) is the receptor for coagulation factor Vila (FVIIa) and the zymogen precursor factor

VII (FVII) Native human TF (hTF) is a 263 ammo 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, Morπssey et al , [1987] Cell 50 129-135) The TF extracellular domain is composed of two immunoglobulin-like fϊbronectin 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 β-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 cerevmae (Waxman et al , [1992] Biochemistry 31 3998-4003, Ruf et al , [1991] J Bio Chem

266 2158-2166 and Shigematsu et al , [1992] J Biol Chem 267 21329-21337) The crystal structures of the 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 Phel40 make a large contribution (1 kcal/mol) to the free energy (ΔG) 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 (ΔΔG = 0 3 - 1 0 kcal mol ') Mutations of TF residues Thrl7, Argl31, Leu 133 and Val207, each of which contact FVIIa in the crystal structure, have no effect on affinity for FVIIa Lysl 5Ala and Tyrl 85Ala mutations result in small increases in affinity (ΔΔG = -0 4 kcal mol ') (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 m the area of amino acids 157-168 contnbute 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 FVH/FVIIa binding It has been shown that lysme 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) Lysme residues 165 and 166 are located on the C-terminal fϊbronectin 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

Lysl65Ala-Lysl66Ala variant (hTFAA) composing 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 vaπant partially blocks thrombus formation without increasing bleeding tendency (Kelley et al , (1997) Blood 89, 3219-3227) However, high doses of the vaπant 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 γ-carboxyglutamic acid- containing (Gla) domain with phosphohpid

TF is expressed constitutively on cells separated from plasma by the vascular endothehum (Carson, S D and J P Brozna, [1993] Blood Coag Fibπnol 4 281-292) Its expression on endothehal cells and monocytes is induced by exposure to inflammatory cytokines or bacterial hpopolysacchaπde (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 seπne 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 Fibπnol 4 281-292, Davie, E W et al , [1991] Biochemistry 30 10363-10370, Rapaport, S I and L V M Rao, [1992] Arteπoscler Thromb 12 11 11-1121) The complex initiates the extπnsic 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 pro thrombin to thrombm, which carries out many biological functions (Badimon, L et al , [1991] Trends Cardiovasc Med I 261-267) Among the most important functions of thrombin is the conversion of fibπnogen 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 vaπety of clinical manifestations including arterial and venous thrombosis, septic shock, adult respiratory distress syndrome (ARDS), disseminated mtravascular coagulation (DIC) and various other disease states (HaskeL E J et al , [1991] Circulation 84 821-827, Hoist, J et al , [1993] Haemostasis 23 (suppl 1) 112-117, Creasey. A A et al , [1993] J Clin Invest 9 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-tissue factor antibodies

Monoclonal antibodies in humanized or chimaeπc forms are successfully used to treat a variety of diseases (Vaswani and Hamilton, [1998] Ann Allergy Asthma Immunol 81 105-119, Vaughan et al , [1998]

Nature Biotechnology 16 535-539)

Antibodies reactive with hTF have been described (Tanaka et al , [1985] Throm Res 40 745-756, Tanaka et al , [1986] Chem Abstracts, 104 366 4921 lz, Momssey et al , [1988] Throm Res 52 247-260, U S Patent No 5,223,427, Ruf et al , P992] J Crystal Growth 122 253-264, Huang et al , [1998] 275 873-894) Anti-TF monoclonal antibodies have been shown to inhibit tissue factor activity in vaπous primate and non- pπmate species (Momssey et al , [1988] supra, Huang et al [1998] supra) Neutralizing anti-TF monoclonal antibodies have been shown to prevent death in a baboon model of sepsis (Taylor et al , [1991] Circ Shock 33 127), and attenuate endotoxin-induced DIC in rabbits (Warr et al , [1990], Blood 75 1481)

Inhibition of TF initiated blood coagulation by antibodies reactive with tissue factor has been proposed as a therapeutic modality (European Patent No 0 266 993 Bl), and the use of antibodies that specifically recognize TF at the site of thrombogenesis is currently viewed as a promising strategy for treating vaπous thrombotic disorders In fact, in vivo studies with anti-TF monoclonal antibodies demonstrated efficient anticoagulant activities (Levi et al , [1 94] J Chn Invest 93, 1 14-120, Taylor et al , [1991] Circulatory Shock 33, 127-134, Himber et al , [1997] Thromb Haemostasis 78, 1142-1149, Pawashe et al , [1994] Circ Res 74, 56-63, Ragni et al , [1996] Circulation 93, 1913-1918, Jang et al , [1992] Artenoscl Thromb 2, 948-954,

Thomas et al , [ 1993] Stroke 24, 847-854, Gohno et al , [ 1996] Nature Med 2, 35-40) The use of a CDR- grafted anti-hTF antibody has been descπbed for the attentuation or prevention of tissue factor mediated coagulation (International Publication No WO 96/40921)

However, the precise TF binding sites of the antibodies used in the foregoing in vivo studies, with the exception of the antibody used by Levi et al supra, are not known The location of the antibody binding epitope may represent a critical factor in determining the inhibitory potencies of antibodies, because the cofactor function of TF involves several defined regions of the TF molecule As a cofactor for factor Vila (FVIIa), the cell surface exposed TF immobilizes FVII/FVIla to the cell membrane thereby stabilizing the overall conformation of FVIIa (Waxman et al , [1993] Biochemistry 32, 3005-3012) The binding to TF also leads to the correct spatial orientation of the catalytic domain and the positioning of the active site in respect to the phosphohpid membrane (McCallum et al , [1997] J Biol Chem 272, 30160-30166, Banner et al , [1996] Nature 380, 41-46) Most of the TF-FVIIa contact surface area is provided by the FVIIa light chain interaction with TF A smaller, yet critical contact surface lies between the N-terminal TF domain and the FVIIa catalytic domain This contact is thought to play a main role in the enhancement of catalysis towards small synthetic as well as to macromolecular substrates (Dickmson et al , [1996] Proc Natl Acad Sci USA 93, 14379-14384,

Dickinson and Ruf, [1997] J Biol Chem 272, 19875-19879) In addition, TF participates in direct interaction with substrates (Huang et al , [1996] J Biol Chem 271, 21752-21757) via residues K165 and K166 (Huang et al , supra, Ruf et al , [1992] J Biol Chem 267, 6375-6381, Roy et al , [1991] J Biol Chem 266, 22063- 22066, Kelley et al , [1995] Biochemistry 34, 10383-10392), and neighboring residues (Ruf et al , [1992] J_ Biol Chem 267 22206-22210) in the C-termmal domain of TF To add to this complex cofactor-enzyme- substrate interplay, recent observations suggested that the γ-carboxyglutamic acid-rich (Gla) domain of FVIIa contributes to substrate interaction (Huang et al , [1996] supra, Ruf et al , [1991] J Biol Chem 266, 15719- 15725, Martin et al , [1993] Biochemistry 32, 13949-13955, Ruf et al , [1999] Biochemistry 38, 1957-1966) Thus, anti-TF antibodies by virtue of their epitope location may interfere with one or several of these TF- mediated processes, which could translate into differences in their anticoagulant effectiveness Such antibody epitope-dependent differences in potencies could be exacerbated under non-equilibrium conditions, which most likely prevail under therapeutic conditions In this setting, antibody and the substrates circulating in blood would simultaneously interact with exposed TF In view of the limited characterization of most anti-TF antibodies known in the art, and the complexity of the mechanism by which TF exerts its thrombotic activity, it has so far been impossible to reliably engineer anti-TF antibodies with enhanced anticoagulant potency

It is an objective of the present invention to determine which characteristics of anti-TF antibodies have the most profound effect on their anticoagulant properties It is another objective, to design anti-TF antibodies with enhanced anticoagulant potency

Summary of the Invention

The present invention is based in part on the expeπmental finding that potency differences between various anti-TF antibodies can be explained by the location of the TF epitopes to which the antibodies bind and consequently, by the particular mode of inhibition Anti-TF antibodies which bind to an epitope overlappmg with the C-terminal macromolecular substrate-bmding region of TF, and thus interfere with the TF-substrate interaction, are the most potent anticoagulant agents This finding permits, for the first time, the purposeful design of anti - TF antibodies with high potency to treat or inhibit thrombosis

Accordingly, one aspect of invention concerns a method for identifying anti-tissue factor (anti-TF) antibodies with enhanced anticoagulant potency, comprising (a) subjecting a plurality of anti-TF antibodies to epitope mapping, and (b) selecting antibodies binding to an epitope comprising at least part of the C-terminal macromolecular substrate-binding region of tissue factor (TF) The tissue factor is preferably human, and the macromolecular substrate preferably is Factor X (FX) or Factor IX (FIX) In a particularly preferred embodiment, the antibody selected recognizes an epitope which includes a TF region directly interacting with substrate factor FX or FIX, preferably by bindmg to a site which prevents or blocks association of TF with a Gla domain of the substrate factor In another preferred embodiment, the antibody selected binds an epitope compπsing residues K165, K166 and K201 of hTF In yet another preferred embodiment, the epitope further comprises residues N199, R200 and 1152 of hTF In a further preferred embodiment, the epitope additionally comprises residue Y156 of hTF In a particular embodiment, the method is used to identify antibodies that bind essentially to the same hTF epitope as any of antibodies D3, 5G6 and TF8-5G9 In some instances, it might be advantageous to select antibodies that have the binding properties specified above, and do not interfere with the association of hTF and Factor Vila (FVIIa) All antibodies identified in accordance with the present invention may be poly- or monoclonal antibodies (as hereinafter defined), and may be rodent, (e g murine), humanized or human antibodies The invention also covers compositions compnsing the antibodies identified in accordance with the present invention, and methods of using such antibodies to block a TF-FVIIa mediated or associated process or event, or to prevent or treat a TF-FVIIa related disease or disorder, including but not limited to, thrombotic and coagulopathic disorders In another aspect, the invention concerns a method for producing an antibody having enhanced anticoagulant potency, comprising raising antibodies against an antigen comprising at least part of the C- terminal macromolecular substrate binding region of tissue factor (TF) Again, the antibodies may be poly- or monoclonal antibodies (as hereinafter defined), including rodent, e g murine, humanized and human antibodies In a preferred embodiment, the antibodies are raised against an antigen compnsing the entire C-terminal macromolecular substrate-binding region of TF, preferably human TF (hTF) Preferably, the antigen used to raise the antibodies compπses residues K165, K166 and K201 , and optionally residues N199, R200 and 1152 of hTF The antigen may additionally contain residue Y156 of hTF

In yet another aspect, the invention concerns an anti-tissue factor (anti-TF) antibody heavy chain vaπable domain comprising the amino acid sequence of SEQ ID NO 1 (VH SEQUENCE OF MURINE D3, Figure 8) or SEQ ID NO 2 (VH SEQUENCE OF HUMANIZED D3H44, Figure 8)

In a further aspect, the invention concerns an anti-tissue factor (anti-TF) light chain vaπable domain comprising the ammo acid sequence of SEQ ID NO 3 (VL SEQUENCE OF MURINE D3, Figure 9) or SEQ ID NO 4 (VL SEQUENCE OF HUMANIZED D3H44, Figure 9)

In a further aspect, the invention concerns an anti-tissue factor (anti-TF) heavy chain variable domain compnsing the ammo acid sequence of SEQ ID NO 5 (VH SEQUENCE OF MURINE 5G6 - Figure 15)

In a different aspect, the invention concerns an anti-tissue factor (anti-TF) light chain variable domain comprising the amino acid sequence of SEQ ID NO 6 (VL SEQUENCE OF MURINE 5G6 - Figure 15)

In another aspect, the invention concerns isolated nucleic acid compnsing a sequence encoding an anti- tissue factor (anti-TF) antibody heavy chain variable domain of SEQ ID NO 1 , 2 or 5 In yet another aspect, the invention concerns isolated nucleic acid compnsing a sequence encoding an anti-tissue factor (anti-TF) antibody light chain variable domain of SEQ ID NO 3, 4 or 6

In a further aspect, the invention concerns a vector comprising, and capable of expressing, a nucleic acid as hereinabove defined, a recombinant host cell transformed with such vector, a cell culture comprising such recombinant host cell, and a method for expressing said nucleic acid to produce the encoded polypeptide The invention also concerns a humanized anti-tissue factor (anti-TF) antibody comprising a heavy and a light chain variable domain, wherein the heavy chain vanable domain comprises hypervaπable regions CDR- Hl having the sequence of GFNIKEYYMH (SEQ ID NO 7), CDR-H2 having the sequence of LIDPEQGNTIYDPKFQD (SEQ ID NO 8) and CDR-H3 having the sequence of DTAAYFDY (SEQ ID NO 9) In a particular embodiment, the humanized anti-TF antibody of the present invention has a light chain variable domain comprising hypervaπable regions CDR-L1 having the sequence of RASRDIKSYLN (SEQ ID NO 10),

CDR-L2 having the sequence of YATSLAE (SEQ ID NO 1 1) and CDR-L3 having the sequence of LQHGESPWT (SEQ ID NO 12) Preferably, both the heavy and light chain hypervanable regions are provided in a human framework region Particular antibodies that are within the scope of the present invention include, without limitation (a) murine antibody D3 (D3Mur), (b) humanized antibody D3H44, (c) murine antibody 5G6, and (d) antibodies specifically binding essentially the same epitope as any one of antibodies (a) - (c)

In another aspect, the invention concerns isolated nucleic acid comprising a sequence encoding a humanized anti-TF antibody heavy or light chain variable domain as hereinabove defined, a vector comprising and capable of expressing such nucleic acid, a recombinant host cell transformed with such vector, a cell culture comprising such recombinant host cell, and a method for expressing said nucleic acid to produce the encoded polypeptide

In another aspect, the invention concerns a composition comprising an anti-tissue factor (anti-TF) antibody identifiable by the method of claim 1, in admixture with a pharmaceutically acceptable earner The antibody preferably is an anti-hTF antibody, and is preferably humanized or human The composition may, for example, comprise an antibody selected from the group consisting of (a) murine antibody D3 (D3Mur), (b) humanized antibody D3H44, (c) murine antibody 5G6, and (d) an antibody specifically binding essentially the same epitope as any one of antibodies (a)-(c), in admixture with a pharmaceutically acceptable carrier

The invention further concerns a method for the prevention or treatment of a TF-FVIIa related disease or disorder, such as thrombotic or coagulopathic disorder, comprising administering to a subject an effective amount of an anti-tissue factor (anti-TF) antibody of the present invention

The invention also concerns diagnostic methods, diagnostic kits and articles of manufacture comprising one or more antibodies of the present invention, optionally in combination with one or more further active ingredients useful in the desired diagnostic or therapeutic application Brief Descnption of the Drawings

Fig 1 Inhibitory characteπstics of anti-tissue factor antibodies (a) Amidolytic activity of sTF FVIIa towards the small synthetic substrate Chromozym t-PA

Antibodies were incubated together with sTF (lOnM) and FVIIa (lOnM) in HBS buffer, 5mM CaCl2 for 15mιn Chromozym t-PA (0 5mM) was added and the rates of substrate cleavage were measured The results were expressed in percent of control rates (average of 3 expeπments ± SD) (b)

Prolongation of human plasma clotting by anti-TF antibodies The antibodies were incubated with TF reagent (Innovm) for 15mιn, then added to human citrated plasma The increase in clotting times is reported as the ratio of clotting times m the presence of antibody and baseline values The results are the average of two independent experiments (filled circle) D3, (open circle) D3 Fab, (x) 5G6, (filled square) 7G11, (open square) 6B4, (filled triangle) HTF1, (open triangle) lsotype-matched control Ab ig 2 Inhibition of fibπnopeptide A (FPA) generation by anti-TF antibodies in a human ex-vivo blood flow system The FPA concentrations in plasma are expressed in percent of control values Each value is the average of 3-8 experiments except for the highest concentrations of D3 Fab and 5G6 (n=l and n=2, resp ) and for two 6B4 concentrations (n=2 for 1 5μg/ml and 15μg/ml) The average of all control values (n=36) was 1348 6 ± 46 1 ng/ml (±SEM) filled circle, D3, open circle, D3 Fab, x. 5G6, open square, 6B4, filled triangle, HTF1 ig 3 Effects of anti-tissue factor antibodies on TF-dependent FX activation in human plasma The inhibited lates of FXa generation during the initial phase of 45sec were calculated and expressed as fractional activity (vi/vo) (filled circle) D3, (x) 5G6, (filled square) 7G11, (open square) 6B4, (filled triangle) HTF1, (open tπangle) lsotype-matched control Ab Prolongation of prothrombin time (PT) by anti-tissue factor antibodies Prolongation of clotting times are reported as the ratio of clotting times in the presence of antibody and baseline values The results are the average of two independent expenments (filled circle) D3, (x) 5G6, (filled square) 7G11 , (open square) 6B4, (filled tπangle) HTF1 Effects of sTF mutations on antibody binding The changes in binding affinities are expressed as the

Kpj ratios of sTF mutants and sTF wildtype (Kj3(mut)/ Kj) (wt)) The Kj values were calculated from surface plasmon resonance measurements with immobilized antibodies Localization of the antibody epitopes on the crystal structure of the sTF FVIIa complex FVIIa is colored with the light chain in orange and the heavy chain in green The active site inhibitor (D-Phe-L- Phe-Arg chloromethyl ketone) is in red and the calcium atoms in yellow Tissue factor (grey) is in a solvent accessible representation and the antibody epitope residues are shown in red color The figures were produced using Insight II (MSI, San Diego) rystal structure of muπne D3 F(ab) Ribbon diagram of VH (dark grey) and VL (light grey) backbones is shown Side chains of residues changed or investigated during the humamzation are shown and labeled, side chain nitrogens and oxygens are dark grey Spheres represent two internal water molecules equence alignment of VH domains of murine D3 (D3Mur), consensus human subgroup III (HumVHIII), and humanized D3H44 CDRs are underlined and differences between sequences are noted by * CDR's are defined according to Kabat et al , Sequences of Proteins of Immunological Interest, 5^th Ed Public Health Service, National Institute of Health, Bethesda, MD (1991) except for CDR-H1 which was defined using a combination of CDR-H1 definitions from Kabat et al (supra) and Chothia et al , Nature 342 877-833 (1989), l e , CDR-H 1 was defined as extending from residues H26- H35 in the heavy chain equence alignment of VL domains of murine D3 (D3Mur), consensus human kappa subgroup I

(Hum l), and humanized D3H44 CDRs are underlined and differences between sequences are noted by * Residue numbering is according to Kabat et al (1991 ), supra nhibition of the rate of FX activation by antibody F(ab) using membrane TF(mTF) FVIIa complex The antibodies were mcubated with mTF and FVIIa for 20 mm before FX was added Ahquots were taken at different time points and quenched in 20 mM EDTA In the second stage of the assay, a chromogenic substrate S-2765 was added and the amidolytic activity measured at 405 nm on a kinetic microplate reader The initial rates are calculated and the inhibition expressed as fractional rates (vi/vo) of FXa generation hibition of the rate of F IX activation by antibody F(ab) using membrane TF(mTF) FVIIa complex

The antibodies were incubated with mTF and FVIIa for 20 mm before F IX was added Ahquots were taken at different time points and quenched in 30 mM EDTA-60% ethyleneglycol In the second stage of the assay, a chromogenic substrate #299 was added and the amidolytic activity measured at 405 nm on a kinetic microplate reader The initial rates are calculated and the inhibition expressed as fractional rates (vi/vo) of FIXa generation Fig 12 Effects of antibody F(ab) and F(ab')2 on prothrombin time (PT) in human plasma E coli expressed F(ab) of D3C2 (chimeπc F(ab)), D3H18, D3H31 , D3H44 and F(ab') of D3H44 were incubated m human plasma for 5 mm Clotting was initiated by addition of human tissue factor reagent (Innovm)

Clotting times were measured on an ACL 300 instrument The prolongation of the clotting time is expressed as the ratio of inhibited clotting (with antibody) and uninhibited clotting time (buffer control) The indicated antibody concentrations are the concentrations m plasma Fig 13 Ammo acid sequence of human tissue factor (hTF) (SEQ ID NO 13) Fig 14 Ribbon representation of the structure of the extracellular portion of human tissue factor

Fig 15 Heavy chain variable domain sequence of murine anti-TF antibody 5G6 (SEQ ID NO 5) Light chain vaπable domain sequence of murine anti-TF antibody 5G6 (SEQ ID NO 6) Fig 16 Binding of anti-tissue factor antibodies to tissue factor IgGl , IgG2, IgG4 and IgG4b Fig 17 Prolongation of human plasma clotting time (PT) for the full length versions and Fab and F(ab'), versions of D3H44

Detailed Description of the Prefeπed Embodiments Definitions

Abbreviations used throughout the description include FIXa for Factor IXa, FXIa for Factor XIa, FXa for Factor Xa, TF for tissue factor, FVII for zymogen factor VII, FVIIa for Factor Vila, TF-FVIIa for tissue factor-Factor Vila complex, FVII/FVIIa for FVII and/or FVIIa, sTF for soluble tissue factor composed of the extracellular domain residues 1-219 in the hTF sequence of Figure 13 (SEQ ID NO 13), hTFAA, the sTF variant containing Lys to Ala substitutions at positions 165 and 166 of the native hTF sequence, TF7I-C for the Kunitz type TF-FVIIa inhibitor of the same name in Dennis et al , (l 994) J Biol Chem 269(35) 22129-22136, K_D for equilibrium dissociation constant, PT for prothrombin time, APTT for activated partial thromboplastin time The term "anticoagulant potency" is used to refer to the ability of a substance, e g an antibody herein, to prevent, inhibit or prolong blood coagulation m an in vitro or in vivo assay of blood coagulation Blood coagulation assays are known in the art and include, for example, prothrombin time assays such as those described in Example 1 herein, the human ex vivo thrombosis model described by Kirchhofer et al , Arterioscler Thromb Vase Biol 15, 1098-1106 (1995), and Kirchhofer et al , J Clin Invest 93, 2073-2083 (1994), and in the examples of the present application, and assays based on the measurement of Factor X activation m human plasma, as described m the examples of the present application

The anticoagulant potency of an antibody of the present invention is "enhanced", if its ability to prevent, inhibit or prolong blood coagulation surpasses the ability of an anti-TF antibody that binds to a TF epitope other than an epitope comprising at least part of the C-termmal macromolecular substrate-binding region of TF, as determined in a standard in vivo or in vitw assay of blood coagulation, such as the assays referred to above Preferably, the anti-TF with enhanced anticoagulant potency achieves the same effect (prevention, inhibition or prolongation) at a lower dose and/or in a shorter time than a reference antibody binding to a different TF epitope Preferably, the difference between the potency of an antibody withm the scope of the present invention and a reference antibody is at least about 1 5 fold, more preferably at least about 2-fold, even more preferably at least about 3-fold, most preferably at least about 5-fold, as determined by side-by-side comparison in a selected standard blood coagulation assay

The "C-termmal macromolecular substrate-bindmg region of TF" is defined as the C-termmal region within the three-dimensional structure of TF that is responsible for the interaction of TF with its macromolecular substrate Factor X (FX) of Factor IX (FIX) In hTF, the FX interaction region is located within the second FNIII module of the extracellular domain of hTF as defined by Muller et al , J Mol Biol 256, 144-159 (1996), including the β-strands β8_A to β 16₀ shown in Figure 3 of Muller et al , supra, and in Figure 14 herein The mam portion of the macromolecular substrate binding region of hTF includes residues Lys 165, Lys 166 (Roy et al , (1991) supra, Ruf et al , (1992) J Biol Chem 267 6375-6381, Huang et al , (1996) J Biol Chem 271 21752- 21757), Tyr 157, Lys 159, Ser 163, Gly 164, Tyr 185 (Kirchhofer et al , (1999) Thromb Haemost Suppl 300, abstract, Kirchhofer et al , (2000) Biochemistry, 39 7380-7387) There are additional hTF residues which contribute to F X interaction such as Tyr 156, Tip 158, Lys 169, Asn 173, Glu 174, Asn 199, Arg 200, Lys 201 and Asp 204 The substrate F IX interacts with about the same hTF region, the main interaction region (Lys 165, Lys 166, Tyr 157, Lys 159, Ser 163, Gly 164, Tyr 185) being identical to that for F X The only difference observed concerned the hTF residues Tip 158 and Asp 204 both of which may be less important for F IX interaction than for F X interaction

The term "epitope" is used to refer to binding sites for (monoclonal or polyclonal) antibodies on protein antigens

Antibodies which bind to the C-terminal macromolecular substrate-binding region of TF are identified by "epitope mapping " There are many methods known in the art for mapping and characteπzmg the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999 Competition assays are discussed below According to the gene fragment expression assays, the open reading frame encoding the protein is fragmented either randomly or by specific genetic constructions and the reactivity of the expressed fragments of the protein with the antibody to be tested is determined The gene fragments may, for example, be produced by PCR and then transcribed and translated into protein in vitro, m the presence of radioactive ammo acids The binding of the antibody to the radioactively labeled protein fragments is then determined by immunoprecipitation and gel electrophoresis Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries) Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody m simple binding assays The latter approach is suitable to define linear epitopes of about 5 to 15 ammo acids

An antibody binds "essentially the same epitope" as a reference antibody, when the two antibodies recognize identical or steπcally overlapping epitopes The most widely used and rapid methods for determining whether two epitopes bind to identical or steπcally overlapping epitopes are competition assays, which can be configured in all number of different formats, using either labeled antigen or labeled antibody Usually, the antigen is immobilized on a 96-well plate, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive or enzyme labels The term amino acid or amino acid residue, as used herein, refers to naturally occurring L amino acids or to D amino acids as described further below with respect to variants. The commonly used one- and three- letter abbreviations for amino acids are used herein (Bruce Alberts et al., Molecular Biology of the Cell, Garland Publishing, Inc., New York (3d ed. 1994)). A "TF-FVIIa mediated or associated process or event", or equivalently, an "activity associated with plasma FVIIa", according to the present invention is any event which requires the presence of TF-FVIIa. The general mechanism of blood clot formation is reviewed by Ganong, in Review of Medical Physiology, 13th ed., Lange, Los Altos CA, pp411-414 (1987) and Bach (1988) CRC Crit. Rev. Biochem. 23(4):359-368. Coagulation requires the confluence of two processes, the production of thrombin which induces platelet aggregation and the formation of fibrin which renders the platelet plug stable. The process comprises several stages each requiring the presence of discrete proenzymes and procofactors. The process ends in fibrin crosslinking and thrombus formation. Fibrinogen is converted to fibrin by the action of thrombin. Thrombin, in turn, is formed by the proteolytic cleavage of prothrombin. This proteolysis is effected by FXa which binds to the surface of activated platelets and in the presence of FVa and calcium, cleaves prothrombin. TF-FVIIa is required for the proteolytic activation of FX by the extrinsic pathway of coagulation. Therefore, a process mediated by or associated with TF-FVIIa, or an activity associated with FVIIa includes any step in the coagulation cascade from the formation of the TF-FVII complex to the formation of a fibrin platelet clot and which initially requires the presence TF-FVIIa. For example, the TF-FVIIa complex initiates the extrinsic pathway by activation of FX to FXa, FIX to FIXa, and additional FVII to FVIIa. TF-FVIIa mediated or associated process, or FVIIa activity, can be conveniently measured employing standard assays such as those described in Roy, S., (1991) J. Biol. Chem. 266:4665-4668, and O'Brien, D., et al., (1988) J. Clin. Invest. 82:206-212 for the conversion of Factor X to Factor Xa in the presence of Factor VII and other necessary reagents.

A "TF-FVIIa related disease or disorder" is meant to include chronic thromboembolic diseases or disorders associated with fibrin formation including vascular disorders such as deep venous thrombosis, arterial thrombosis, stroke, tumor metastasis, thrombolysis, arteriosclerosis and restenosis following angioplasty, acute and chronic indications such as inflammation, septic shock, septicemia, hypotension, adult respiratory distress syndrome (ARDS), disseminated intravascular coagulopathy (DIC) and other diseases. The TF-FVIIa related disorder is not limited to in vivo coagulopathic disorders such as those named above but includes ex vivo TF- FVIIa related processes such as coagulation that may result from the extracorporeal circulation of blood, including blood removed in-line from a patient in such processes as dialysis procedures, blood filtration, or blood bypass during surgery.

"Bleeding disorders" are characterized by a tendency toward hemorrhage, both inherited and acquired. Examples of such bleeding disorders are deficiencies of factors VIII, IX, or XI. Examples of acquired disorders include acquired inhibitors to blood coagulation factors e.g., factor VIII, von Willebrand factor, factors IX, V,

XI, XII and XIII, hemostatic disorders as a consequence of liver disease which included decreased synthesis of coagulation factors, bleeding tendency associated with acute and chronic renal disease and hemostasis after trauma or surgery. The terms "tissue factor protein" and "mammalian tissue factor protein" are used to refer to a polypeptide having an amino acid sequence corresponding to a naturally occumng mammalian tissue factor or a recombinant tissue factor as described below Naturally occurring TF includes human species as well as other animal species such as rabbit, rat, porcine, non human primate, equine, muπne, and ovine tissue factor (see, for example, Hartzell et al , (1989) Mol Cell Biol , 9 2567-2573, Andrews et al , (1991) Gene, 98 265-269, and

Takayenik et al , (1991) Biochem Biophys Res Comm , 181 1145-1150) The amino acid sequence of human tissue factor is shown in Figure 13 (SEQ ID NO 13) The amino acid sequence of the other mammalian tissue factor proteins are generally known or obtainable through conventional techniques

As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (I e , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment "Treatment" is an intervention performed with the intention of preventing the development or alteπng the pathology of a disorder Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented Accordingly, "treatment" in the context of the present invention is an intervention performed with the intention of preventing a TF-FVIIa mediated or associated process or event, or a TF-FVIIa related disease or disorder, or a bleeding disorder, as hereinabove defined

"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc Preferably, the mammal is human

"Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the same structural characteπstics While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-hke molecules that lack antigen specificity Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas

"Native antibodies" and "native immunoglobulins" are usually heterotetrameπc glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages vanes among the heavy chains of different immunoglobulin lsotypes Each heavy and light chain also has regularly spaced lntracham disulfide bridges Each heavy chain has at one end a variable domain (V_H) followed by a number of constant domains Each light chain has a variable domain at one end (V_L) and a constant domain at its other end, the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light- chain vanable domain is aligned with the variable domain of the heavy chain Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains

The term "vaπable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen However, the variability is not evenly distributed throughout the variable domains of antibodies It is concentrated in three segments called hypervaπable regions both in the light chain and the heavy chain vaπable doma s The more highly conserved portions of vaπable domains are called the framework region (FR) The vaπable domains of native heavy and light chains each compπse four FRs (FR1 , FR2, FR3 and FR4, respectively), largely adopting a β-sheet configuration, connected by three hypervaπable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure The hypervaπable regions in each chain are held together in close proximity by the FRs and, with the hypervaπable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al , Sequences of Proteins of Immunological Interest, 5th Ed Public Health Service, National Institutes of Health, Bethesda, MD (1991), pages 647-669) The constant domains are not involved directly in binding an antibody to an antigen, but exhibit vaπous effector functions, such as participation of the antibody in antibody- dependent cellular toxicity

The term "hypervaπable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding The hypervaπable region comprises amino acid residues from a "complementarity determining region" or "CDR" (i e residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain vanable domain and 31-35 (HI), 50-65 (H2) and 95-102 (H3) m the heavy chain variable domain,

Kabat et al , Sequences of Proteins of Immunological Interest, 5th Ed Public Health Service, National Institutes of Health, Bethesda, MD (1991)) and/or those residues from a "hypervaπable loop" (i e residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain vaπable domain and 26-32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain, Chofhia and Lesk J Mol Biol 196 901-917 (1987)) "Framework" or "FR" residues are those vaπable domain residues other than the hypervaπable region residues as herein defined

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a smgle antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily Pepsin treatment yields an F(ab'), fragment that has two antigen-combining sites and is still capable of cross-linking antigen "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non- covalent association It is in this configuration that the three hypervaπable regions of each variable domain interact to define an antigen-bmding site on the surface of the V_H-V_L dimer Collectively, the six hypervaπable regions confer antigen-binding specificity to the antibody However, even a single variable domain (or half of an Fv compnsing only three hypervaπable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain including one or more cysteιne(s) from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteine resιdue(s) of the constant domams bear a free thiol group F(ab'), antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them Other chemical couplings of antibody fragments are also known The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (λ), based on the amino acid sequences of their constant domams

Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes There are five major classes of immunoglobulins IgA, IgD, IgE, IgG, and

IgM, and several of these may be further divided into subclasses (isotypes), e g , IgGl , IgG2, IgG3, IgG4, IgAl, and IgA2 The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies

(including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e g , bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity

"Antibody fragments" comprise a portion of a full length antibody, generally the antigen binding or variable domain thereof Examples of antibody fragments include Fab, Fab', F(ab , and Fv fragments, diabodies, linear antibodies, single-cham antibody molecules, and multispecific antibodies formed from antibody fragments

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i e , the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts Monoclonal antibodies are highly specific, being directed against a single antigenic site Furthermore, in contrast to conventional

(polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen The modifier "monoclonal" indicates the character of the antibody as bemg obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybπdoma method first described by Kohler et al Natui e 256 495 (1975), or may be made by recombinant DNA methods (see, e g , U S Patent No 4,816,567) The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al , Natuie 352 624-628 (1991) and Marks e α/ , J Mol Biol 222 581-597 (1991), for example The monoclonal antibodies herein specifically include "chimeπc" antibodies (immunoglobulins) m which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the cham(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U S Patent No 4,816,567, and Morrison et al , Pioc Natl

Acad Sci USA 81 6851-6855 (1984))

"Humanized" forms of non-human (e g , murine) antibodies are chimeπc antibodies which contain minimal sequence denved from non-human lmmunoglobuhn For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervaπable region residues of the recipient are replaced by hypervaπable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity In some instances, framework region (FR) residues of the human immunoglobulm are replaced by corresponding non-human residues Furthermore, humanized antibodies may compnse residues which are not found in the recipient antibody or in the donor antibody These modifications are made to further refine antibody performance In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domams, in which all or substantially all of the hypervanable regions correspond to those of a non-human immunoglobulm and all or substantially all of the FRs are those of a human immunoglobulm sequence The humanized antibody optionally also will comprise at least a portion of an immunoglobulm constant region (Fc), typically that of a human immunoglobulm For further details, see Jones et al , Nature 321 522-525 (1986), Reichmann et al , Nature

332 323-329 (1988), and Presta, Curr Op Struct Biol 2 593-596 (1992)

"Single-chain Fv" or "sFv" antibody fragments compnse the V_H and V_L domains of antibody, wherein these domains are present in a single polypeptide chain Generally, the Fv polypeptide further compπses a polypeptide linker between the V_H and V_L domains which enables the sFv to form the desired structure for antigen binding For a review of sFv see Pluckthun m The Pharmacology of Monoclonal Antibodies, vol 1 13,

Rosenburg and Moore eds Springer- Verlag, New York, pp 269-315 (1994)

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain vanable domain (V_H) connected to a light chain variable domain (V_L) in the same polypeptide chain (V_H - V_L) By using a linker that is too short to allow painng between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites Diabodies are descπbed more fully in, for example, EP 404,097, WO 93/11161, and Holhnger et al , Proc Nat! Acad Sci USA 90 6444-6448 (1993)

The expression "linear antibodies" when used throughout this application refers to the antibodies descnbed in Zapata et al Protein Eng 8(10) 1057-1062 (1995) Briefly, these antibodies comprise a pair of tandem Fd segments (V_H-C_H1-V_H-C_H1) which form a pair of antigen binding regions Linear antibodies can be bispecific or monospecific

Methods for carrying out the invention A Antibody preparation

Methods for humanizing nonhuman TF antibodies and generating variants of anti-TF antibodies are descnbed in the examples below In order to humanize an anti-TF antibody, the nonhuman antibody starting mateπal is prepared Where a variant is to be generated, the parent antibody is prepared Exemplary techniques for generating such nonhuman antibody starting material and parent antibodies will be described in the following sections

(i) Polyclonal antibodies Methods of preparing polyclonal antibodies are known in the art Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or lntrapeπtoneal injections It may be useful to conjugate the immunizing agent to a protein known to be immunogemc in the mammal bemg immunized, such as serum albumin, or soybean trypsin inhibitor Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM (u) Monoclonal antibodies

Monoclonal antibodies may be made usmg the hybπdoma method first described by Kohler et al , Nature, 256 495 (1975), or may be made by recombinant DNA methods (U S Patent No 4,816,567)

In the hybπdoma method, a mouse or other appropriate host animal, such as a hamster or macaque monkey, is immunized as heremabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization Alternatively, lymphocytes may be immunized in vitro Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybπdoma cell (Goding, Monoclonal Antibodies Principles and Practice, pp 59-103, [Academic Press, 1986])

The hybπdoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoπbosyl transferase (HGPRT or HPRT), the culture medium for the hybπdomas typically will include hypoxanthine, ammopteπn, and thymidme (HAT medium), which substances prevent the growth of HGPRT-deficient cells

Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOP-21 and M C -11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland USA Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J Immunol , 133 3001 (1984), Brodeur et al , Monoclonal Antibody Production Techniques and Applications, pp 51-63, Marcel Dekker, Inc , New York, [1987]) Culture medium in which hybπdoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen Preferably, the binding specificity of monoclonal antibodies produced by hybπdoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA)

The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al , Anal Biochem , 107 220 (1980)

After hybπdoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the cells may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies Principles and Practice, pp 59-103 (Academic Press, 1986)) Suitable culture media for this purpose include, for example, DMEM or RPMI-1640 medium In addition, the hybπdoma cells may be grown in vivo as ascites tumors in an animal

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulm purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography DNA encoding the monoclonal antibodies is readily isolated and sequenced usmg conventional procedures (e g , by using ohgonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies) The hybπdoma cells serve as a preferred source of such DNA Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulm protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells Recombinant production of antibodies will be described in more detail below (in) Humanized antibodies

Example 2 below descπbes procedures for humamzation of an anti-TF antibody Generally, a humanized antibody has one or more amino acid residues introduced into it from a nonhuman source These non-human ammo acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain Humamzation can be essentially performed following the method of Winter and co-workers [Jones et al , Nature, 321 522-525 (1986), Riechmann et al , Nature, 332 323- 327 (1988), Verhoeyen et al , Science, 239 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody

(iv) Amino acid sequence variants of antibodies

Example 2 also describes methodologies for generating ammo acid sequence vanants of an anti-TF antibody with enhanced affinity relative to the parent antibody

Amino acid sequence vanants of the anti-TF antibody are prepared by introducing appropriate nucleotide changes into the anti-TF antibody DNA, or by peptide synthesis Such vanants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the anti-TF antibodies of the examples herein Any combination of deletion, insertion, and substitution is made to arπve at the final construct, provided that the final construct possesses the desired characteristics The ammo acid changes also may alter post-translational processes of the humanized or vanant anti-TF antibody, such as changing the number or position of glycosylation sites

A useful method for identification of certain residues or regions of the anti-TF antibody that are prefeπed locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells Science, 244 1081-1085 (1989) Here, a residue or group of target residues are identified {e g , charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalamne) to affect the interaction of the amino acids with TF antigen Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed anti-TF antibody variants are screened for the desired activity

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging m length from one residue to polypeptides containing a hundred or more residues, as well as mtrasequence insertions of single or multiple amino acid residues Examples of terminal insertions include an anti-TF antibody with an N- terminal methionyl residue or the antibody fused to an epitope tag Other msertional variants of the anti-TF antibody molecule mclude the fusion to the N- or C-terminus of the anti-TF antibody of an enzyme or a polypeptide which increases the serum half-life of the antibody (see below)

Another type of variant is an amino acid substitution variant These variants have at least one amino acid residue in the anti-TF antibody molecule removed and a different residue inserted in its place The sites of greatest interest for substitutional mutagenesis include the hypervaπable regions, but FR alterations are also contemplated Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions" If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 1 , or as further described below in reference to amino acid classes, may be introduced and the products screened

Table 1

Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain Naturally occurπng residues are divided into groups based on common side-chain properties

(1) hydrophobic norleucine, met, ala, val, leu, lie,

(2) neutral hydrophihc cys, ser, thr,

(3) acidic asp, glu, (4) basic asn, gin, his, lys, arg,

(5) residues that influence chain orientation gly, pro, and

(6) aromatic trp, tyr, phe

Non-conservative substitutions will entail exchangmg a member of one of these classes for another class Any cysteine residue not involved in maintaining the proper conformation of the humanized or variant anti-TF antibody also may be substituted, generally with seπne, to improve the oxidative stability of the molecule and prevent aberrant crosslinking Conversely, cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment)

A particularly preferred type of substitutional variant involves substituting one or more hypervaπable region residues of a parent antibody (e g a humanized or human antibody) Generally, the resulting vaπant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated A convenient way for generating such substitutional variants is affinity maturation using phage display Bπefly, several hypervaπable region sites (e g 6-7 sites) are mutated to generate all possible amino substitutions at each site The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle The phage-displayed variants are then screened for their biological activity (e g binding affinity) as herein disclosed In order to identify candidate hypervaπable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervaπable region residues contributing significantly to antigen binding Alternatively, or m addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and human TF Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development Another type of amino acid vaπant of the antibody alters the original glycosylation pattern of the antibody By alteπng is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody

Glycosylation of antibodies is typically either N-hnked or O-hnked N-hnked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue The tπpeptide sequences asparagme-X- senne and asparagine-X-threonme, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain Thus, the presence of either of these tπpeptide sequences in a polypeptide creates a potential glycosylation site O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly seπne or threonine, although 5-hydroxyprolιne or 5-hydroxylysιne may also be used

Addition of glycosylation sites to the antibody is conveniently accomplished by altenng the amino acid sequence such that it contains one or more of the above-described tnpeptide sequences (for N-hnked glycosylation sites) The alteration may also be made by the addition of, or substitution by, one or more seπne or threonine residues to the sequence of the oπgmal antibody (for O-hnked glycosylation sites) Nucleic acid molecules encoding amino acid sequence vanants of the anti-TF antibody are prepared by a variety of methods known in the art These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurπng amino acid sequence variants) or preparation by ohgonucleotide- mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared vaπant or a non- vaπant version of the anti-TF antibody (v) Human antibodies

Human antibodies can be produced using vaπous techniques known in the art, including phage display libraries [Hoogenboom and Winter, J Mol Biol , 227 381 (1991), Marks et al , J Mol Biol , 222 581 (1991)] The techniques of Cole et al and Boerner et al are also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p 77 (1 85) and Boerner et al , J Immunol , 147(1) 86-95 (1991)] Similarly, human antibodies can be made by introducing of human immunoglobulm loci into transgenic animals, e g , mice m which the endogenous immunoglobulm genes have been partially or completely inactivated Upon challenge, human antibody production is observed, which closely resembles that seen in humans m all respects, including gene rearrangement, assembly, and antibody repertoire This approach is described, for example, in U S Patent Nos 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, and in the following scientific publications Marks et al , Bio/Technology 10,

779-783 (1992), Lonberg et al , Nature 368 856-859 (1994), Momson, Nature 368, 812-13 (1994), Fishwild et al , Nature Biotechnology 14, 845-51 (1996), Neuberger, Nature Biotechnology 14, 826 (1996), Lonberg and Huszar, Intern Rev Immunol L3 65-93 (1995) (vi) Antibody fragments In certain embodiments, the humanized or variant anti-TF antibody is an antibody fragment Various techniques have been developed for the production of antibody fragments Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e g Moπmoto et al , J Biochem Biophvs Methods 24 107- 117 ( 1992) and Brennan et al , Science 229 81 ( 1985)) However, these fragments can now be produced directly by recombinant host cells For example, Fab'-SH fragments can be directly recovered from E coli and chemically coupled to form F(ab')₂ fragments (Carter et al , Bio/Technology 10 163-167 (1992)) In another embodiment, the F(ab')₂ is formed using the leucine zipper GCN4 to promote assembly of the F(ab'), molecule. According to another approach, Fv, Fab or F(ab')_: fragments can be isolated directly from recombinant host cell culture Other techniques for the production of antibody fragments will be apparent to the skilled practitioner

(vn) Multispecific antibodies

In some embodiments, it may be desirable to generate multispecific (e g bispecific) humanized or variant anti-TF antibodies having binding specificities for at least two different epitopes Exemplary bispecific antibodies may bind to two different epitopes of the TF protein Alternatively, an anti-TF arm may be combined with an arm which binds to a tπggenng molecule on a leukocyte such as a T-cell receptor molecule

(e g , CD2 or CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRIl (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the TF-expressmg cell Bispecific antibodies may also be used to localize cytotoxic agents to cells which express TF These antibodies possess a TF-bmdmg arm and an arm which binds the cytotoxic agent (e g , sapoπn, anti-interferon-α, vmca alkaloid, πcin A chain, methotrexate or radioactive isotope hapten) Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e g , F(ab'), bispecific antibodies)

According to another approach for making bispecific antibodies, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture The preferred interface comprises at least a part of the C_H3 domain of an antibody constant domain. In this method, one or more small ammo acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e g , tyrosme or tryptophan) Compensatory "cavities" of identical or similar size to the large side chaιn(s) are created on the interface of the second antibody molecule by replacing large ammo acid side chains with smaller ones (<? g , alanine or threonine) This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. See WO96/27011 published September 6, 1996

Bispecific antibodies include cross-lmked or "heteroconjugate" antibodies For example, one of the antibodies m the heteroconjugate can be coupled to avidin, the other to biotm Heteroconjugate antibodies may be made using any convenient cross-linking methods Suitable cross-linking agents are well known in the art, and are disclosed in US Patent No 4,676,980, along with a number of cross-linking techniques Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature For example, bispecific antibodies can be prepared using chemical linkage Brennan et al , Science 229 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab fragments These fragments are reduced m the presence of the dithiol complexmg agent sodium arsemte to stabilize vicinal dithiols and prevent intermolecular disulfide formation The Fab' fragments generated are then converted to thiomtrobenzoate (TNB) derivatives One of the Fab'-TNB derivatives is then reconverted to the

Fab'-thtol by reduction with mercaptoethylamme and is mixed with an equimolar amount of the other Fab'- TNB derivative to form the bispecific antibody The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes In yet a further embodiment, Fab'-SH fragments directly recovered from E coli can be chemically coupled in viti o to form bispecific antibodies, e g Shalaby et al J Exp Med 175 217-225 (1992)

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described For example, bispecific antibodies have been produced using leucme zippers Kostelny et al , J Immunol 148(5) 1547-1553 (1992) The leucme zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion The antibody homodimers were reduced at the hmge region to form monomers and then re-oxidized to form the antibody heterodimers This method can also be utilized for the production of antibody homodimers The "diabody" technology described by Hollinger et al , Proc Natl Acad Sci USA 90 6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments The fragments comprise a heavy-chain variable domain

(V_H) connected to a light-chain variable domain (V_L) by a linker which is too short to allow pairing between the two domains on the same chain Accordingly, the V_H and V_L domains of one fragment are forced to pair with the complementary V_L and V_H domains of another fragment, thereby forming two antigen-bmding sites Another strategy for making bispecific antibody fragments by the use of single-cham Fv (sFv) dimers has also been reported See Gruber et al J Immunol 152 5368 (1994) Alternatively, the bispecific antibody may be a "linear antibody" produced as described m Zapata et al Protein Eng 8( 10) 1057-1062 (1995)

Antibodies with more than two valencies are contemplated For example, tπspecific antibodies can be prepared Tutt et al , J Immunol 147 60 ( 1991 ) (viu) Other modifications Other modifications of the humanized or variant anti-TF antibody are contemplated It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance the effectiveness of the antibody for instance in treating cancer For example, cysteine resιdue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation m this region The homodimeπc antibody thus generated may have improved intemahzation capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) See Caron et al , J Exp Med 176 1 191-1195 (1992) and

Shopes, B J Immunol 148 2918-2922 (1992) Homodimeπc antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al Cancer Research 53 2560-2565 (1993) Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities See Stevenson et al , Anti-Cancei Drug Design 3 219-230 (1989)

The invention also pertains to immunoconjugates comprising the antibody described herein conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i e a radioconjugate)

Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above Enzymatically active toxms and fragments thereof which can be used include diphtheria A chain, nonbindmg active fragments of diphtheria toxin, exotoxm A chain (from Pseudomonas aei uginosά), πcm A chain abπn A chain, modeccm A chain, α-sarcm, Aleurites foi du proteins, dianthm proteins, Phytolaca amei icana proteins (PAPI, PAPII, and PAP-S) momordica charantia inhibitor, curc , crotm, sapaonaπa officmahs inhibitor, gelomn, mitogelhn. restπctocm, phenomycin, enomycm and the tπcothecenes A variety of radionuchdes are available for the production of radioconjugated anti-TF antibodies Examples include ^2PBι, ^{1 I}I, ^lIn, ⁹⁰Y and 1⁸⁶Re

Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succmιmιdyl-3-(2-pyπdyldιthιol) propionate (SPDP), lminothiolane (IT), bifunctional derivatives of lmidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccimmidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazomum derivatives (such as bιs-(p-dιazonιumbenzoyl)-ethylenedιamιne), diisocyanates (such as tolyene 2,6- dusocyanate), and bis-active fluoπne compounds (such as l,5-dιfluoro-2,4-dmιtrobenzene) For example, a πcin immunotoxin can be prepared as described in Vitetta et al , Science 238 1098 (1987) Carbon- 14-labeled l-ιsothιocyanatobenzyl-3-methyldιethylene tπaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radtonucleotide to the antibody See W094/11026

In another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "hgand" (e g , avidin) which is conjugated to a cytotoxic agent (e g , a radionuchde)

The anti-TF antibodies disclosed herein may also be formulated as lmmunoliposomes Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al , Proc Natl Acad Sci USA 82 3688 (1985), Hwang et al , Proc Natl Acad Sci USA 77 4030 (1980), and U S Pat Nos 4,485,045 and 4,544,545 Liposomes with enhanced circulation time are disclosed in U S Patent No 5,013,556

Particularly useful liposomes can be generated by the reverse phase evaporation method with a hpid composition comprising phosphatidylchohne, cholesterol and PEG-deπvatized phosphatidylethanolamine (PEG-PE) Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al , J Bio! Chem 257 286-288 (1982) via a disulfide interchange reaction A chemotherapeutic agent (such as Doxorubtcin) is optionally contained withm the l posome See Gabizon et al , J National Cancer Inst 81(19) 1484 (1989)

The antibody of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e g , a peptidyl chemotherapeutic agent, see WO81/01 145) to an active anti-cancer drug See, for example, WO 88/07378 and U S Patent No 4,975,278

The enzyme component of the mimunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form

Enzymes that are useful m the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs, arylsulfatase useful for converting sulfate-contammg prodrugs into free drugs, cytosme deaminase useful for converting non-toxic 5- fluorocytosme into the anti-cancer drug, 5-fluorouracιl, proteases, such as serratia protease, thermolysm, subtilism, carboxypeptidases and cathepsms (such as cathepsms B and L), that are useful for converting peptide-contaimng prodrugs into free drugs, D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents. carbohydrate-cleaving enzymes such as β-galactosidase and neuramimdase useful for converting glycosylated prodrugs into free drugs, β-lactamase useful for converting drugs deπvatized with β-lactams into free drugs, and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs deπvatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes", can be used to convert the prodrugs of the invention into free active drugs (see, e g , Massey,

Nature 328 457-458 (1987)) Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population

The enzymes of this invention can be covalently bound to the anti-TF antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above Alternatively, fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known m the art (see, e g , Neuberger et al , Nature 312 604-608 [ 1984])

In certain embodiments of the invention, it may be desirable to use an antibody fragment, rather than an intact antibody, to increase tumor penetration, for example In this case, it may be desirable to modify the antibody fragment in order to increase its serum half life This may be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment (e g , by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, e g , by DNA or peptide synthesis) See W096/32478 published October 17, 1996 The salvage receptor binding epitope generally constitutes a region wherein any one or more amino acid residues from one or two loops of a Fc domain are transferred to an analogous position of the antibody fragment Even more preferably, three or more residues from one or two loops of the Fc domain are transferred Still more preferred, the epitope is taken from the CH2 domain of the Fc region (e g , of an IgG) and transferred to the CHI, CH3, or V_H region, or more than one such region, of the antibody Alternatively, the epitope is taken from the CH2 domain of the Fc region and transfeπed to the C_L region or V_L region, or both, of the antibody fragment

In one most prefeπed embodiment, the salvage receptor binding epitope comprises the sequence PKNSSMISNTP (SEQ ID NO 14), and optionally further comprises a sequence selected from the group consisting of HQSLGTQ (SEQ ID NO 15), HQNLSDGK (SEQ ID NO 16), HQNISDGK (SEQ ID NO 17), or VISSHLGQ (SEQ ID NO 18), particularly where the antibody fragment is a Fab or F(ab')₁ In another most preferred embodiment, the salvage receptor binding epitope is a polypeptide containing the sequence(s) HQNLSDGK (SEQ ID NO 16), HQNISDGK (SEQ ID NO 17), or VISSHLGQ (SEQ ID NO 18) and the sequence PKNSSMISNTP (SEQ ID NO 14)

Covalent modifications of the humanized or variant anti-TF antibody are also included withm the scope of this invention They may be made by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable Other types of covalent modifications of the antibody are introduced into the molecule by reacting targeted ammo acid residues of the antibody with an organic deπvatizmg agent that is capable of reacting with selected side chains or the N- or C-termmal residues Exemplary covalent modifications of polypeptides are described in US Patent 5,534,615, specifically incorporated herein by reference A preferred type of covalent modification of the antibody compπses linking the antibody to one of a variety of nonprotemaceous polymers, e g , polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth m U S Patent Nos 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 or 4,179,337 B Vectors, Host Cells and Recombinant Methods The invention also provides isolated nucleic acid encoding the humanized or vaπant anti-TF antibody, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibody

For recombinant production of the antibody, the nucleic acid encoding it may be isolated and inserted into a rephcable vector for further cloning (amplification of the DNA) or for expression In another embodiment, the antibody may be produced by homologous recombination, e g as described in US Patent 5,204,244, specifically incorporated herein by reference DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e g , by using ohgonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) Many vectors are available The vector components generally include, but are not limited to, one or more of the following a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, e g , as described in US Patent 5,534,615 issued July 9, 1996 and specifically incorporated herein by reference

Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above Suitable prokaryotes for this purpose include eubacteπa, such as Gram-negative or Gram-positive organisms, for example, Enterobacteπaceae such as Escherichia, e g , E coli,

Enterobactei , Erwinia, Klebsiella, Proteus, Salmonella, e g , Salmonella typhimurium, Serratia, e g , Serratia marcescans, and Shigella, as well as Bacilli such as B subtihs and B licheniformis (e g , B licheniformis 41 P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P aeruginosa, and Streptomyces One preferred E coli cloning host is E coli 294 (ATCC 31 ,446), although other strains such as E coli B, E coh X1776 (ATCC 31,537), and E coli W3110 (ATCC 27,325) are suitable These examples are illustrative rather than limiting

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-TF antibody-encoding vectors Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms However, a number of other genera, species, and strains are commonly available and useful herem, such as Schizosaccharomyces pombe,

Kluyveromyces hosts such as, e g , K lactis, K fragihs (ATCC 12,424), K bulgaucus (ATCC 16,045) K wickeramu (ATCC 24,178), K waltu (ATCC 56,500), K drosophilarum (ATCC 36,906), K thermotolerans, dLΑ& K marxianus, yarrowia (EP 402,226), Pichia pastoi is (EP 183,070), Candida Trichoderma reesia (EP 244,234), Neurospora ciassa, Schwanmomvces such as Schwannwmvces occidentals, and filamentous fungi such as, e g , Neurospoi a Pemcillium, Tolypocladium, and Aspeigillus hosts such as A nidulans and A nigei

Suitable host cells for the expression of glycosylated anti-TF antibody are derived from multicellular organisms Examples of invertebrate cells include plant and insect cells Numerous baculoviral strains and variants and coπesponding permissive insect host cells from hosts such as Spodoptei a frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombvx mon have been identified A vaπety of viral strains for transfection are publicly available, e g , the L-l variant of Autographa cahfomica NPV and the Bm-5 strain of Bombvx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of ^' Spodoptera frugiperda cells Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells m culture

(tissue culture) has become a routine procedure Examples of useful mammalian host cell lines are monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651) human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al J Gen Vuol 36 59 [1977]), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al , Ptoc Natl Acad Sci USA 77 4216 [1980]), mouse sertoli cells (TM4, Mather, Biol Repi od 23 243-251 [1980]), monkey kidney cells (CVl ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HeLa, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al Annals N Y Acad Sci 383 44-68 [1982]), MRC 5 cells, and FS4 cells

Host cells are transformed with the above-described expression or cloning vectors for anti-TF antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences

The host cells used to produce the anti-TF antibody of this invention may be cultured in a variety of media Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM)

(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturmg the host cells In addition, any of the media described m Ham et al Meth Enz 58 44 (1979), Barnes et al . Anal Biochem 102 255 (1980), U S Pat Nos 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90/03430, WO 87/00195, or U S Patent Re 30,985 may be used as culture media for the host cells Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferπn, or epidermal growth factor), salts (such as sodium chloride calcium magnesium, and phosphate), buffers (such as HEPES), nu leotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled m the art

The culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan

When using recombinant techniques, the antibody can be produced intracellularly, in the peπplasmic space, or directly secreted into the medium If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centπfugation or ultrafiltration Carter et al Bio/Technologv 10 163 167 (1992) describe a procedure for isolating antibodies which are secreted to the peπplasmic space of E coh Briefly cell paste is thawed in the presence of sodium acetate (pH 3 5) EDTA and phenylmethylsulfonylfluoπde (PMSF) over about 30 mm Cell debris can be removed by centπfugation Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Milhpore Pelhcon ultrafiltration unit A protease inhibitor such as PMSF may be included m any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique The suitability of protein A as an affinity hgand depends on the species and lsotype of any immunoglobulm Fc domain that is present in the antibody Protein A can be used to purify antibodies that are based on human γl , γ2, or γ4 heavy chains (Lindmark et al , J Immunol Meth 62 1- 13 (1983)) Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al , EMBOJ

5 15671575 (1986)) The matπx to which the affinity hgand is attached is most often agarose, but other matrices are available Mechanically stable matrices such as controlled pore glass or poly(styrenedιvιnyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose Where the antibody comprises a C_H3 domain, the Bakerbond ABX™ resin (J T Baker, Phillipsburg, NJ) is useful for purification Other techniques for protein punfication such as fractionation on an ion- exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on hepaπn SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2 5-4 5, preferably performed at low salt concentrations (e g ,from about 0-0 25M salt) C Pharmaceutical Formulations

Therapeutic formulations of the antibody are prepared for storage by mixing the antibody having the desired degree of punty with optional physiologically acceptable carriers, excipients or stabilizers (Remington's

Pharmaceutical Sciences 16th edition, Osol, A Ed (1980)), in the form of lyophilized formulations or aqueous solutions Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methtonine, preservatives (such as octadecyldime hylbenzyl ammonium chloπde, hexamethomum chloride, benzalkomum chloride, benzethonium chloπde, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophihc polymers such as polyvinylpyrrohdone, amino acids such as glycine, glutamine, asparagine, histidine, argimne, or lysine, monosacchaπdes, disacchaπdes, and other carbohydrates including glucose, mannose, or dextnns, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt-formmg counter-ions such as sodium, metal complexes (e g Zn-protein complexes), and or non- lomc surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG)

The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other (see Section F below) Such molecules are suitably present in combination in amounts that are effective for the purpose intended

The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatm-microcapsule and poly-(mefhylmefhacylate) microcapsule, respectively, m colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or m macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A Ed (1980)

The formulations to be used for in vivo administration must be sterile This is readily accomplished by filtration through sterile filtration membranes Sustained-release preparations may be prepared Suitable examples of sustained-release preparations include sem permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are m the form of shaped articles, e g , films, or microcapsule Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vιnylalcohol)), polylactides (U S Pat No 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene- vmyl acetate, degradable lactic acid-glycohc acid copolymers such as the Lupron Depot™ (injectable microspheres composed of lactic acid-glycohc acid copolymer and leuprohde acetate), and poly-D-(-)-3- hydroxybutyπc acid While polymers such as ethylene-vinyl acetate and lactic acid-glycohc acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in lmmunogemcity

Rational strategies can be devised for stabilization depending on the mechanism involved For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophihzing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matnx compositions

D Non-therapeutic Uses for the Antibody

The antibodies of the invention may be used as affinity purification agents In this process, the antibodies are immobilized on a solid phase such as Sephadex resin or filter paper, using methods well known in the art The immobilized antibody is contacted with a sample containing the TF protein (or fragment thereof) to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the TF protein, which is bound to the immobilized antibody Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5 0, that will release the TF protein from the antibody

Anti-TF antibodies may also be useful in diagnostic assays for TF protein, e g detecting its expression m specific cells, tissues, or serum Such diagnostic methods may be useful in the diagnosis of various disorders associated with the aberrant expression, e g over- or underexpression of TF For example, overexpression and/or aberrant utilization of TF has been linked to the pathophysiology of both thrombosis and sepsis, and TF has been implicated in tumor metastasis Accordingly, anti-TF antibodies may be useful m the diagnosis of these diseases For diagnostic applications, the antibody typically will be labeled with a detectable moiety Numerous labels are available which can be generally grouped into the following categories

(a) Radioisotopes, such as ³⁵S, ¹ C, ^l25I, Η, and ^13II The antibody can be labeled with the radioisotope using the techniques descnbed in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al , Ed Wiley-Interscience, New York, New York, Pubs (1991) for example and radioactivity can be measured using scintillation counting

(b) Fluorescent labels such as rare earth chelates (europium chelates) or fluorescem and its derivatives, rhodamme and its derivatives, dansyl, Lissamine, phycoerythπn and Texas Red are available The fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra, for example Fluorescence can be quantified using a fluoπmeter

(c) Various enzyme-substrate labels are available and U S Patent No 4,275,149 provides a review of some of these The enzyme generally catalyzes a chemical alteration of the chromogenic substrate which can be measured using various techniques For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometπcally Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate Techniques for quantifying a change in fluorescence are described above

The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor Examples of enzymatic labels include luciferases (e g , firefly luciferase and bacterial luciferase, U S Patent No 4,737,456), lucifeπn, 2,3-dιhydrophthalazιnedιones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sacchande oxidases (e g , glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocychc oxidases (such as uncase and xanthme oxidase), lactoperoxidase, microperoxidase, and the like Techniques for conjugating enzymes to antibodies are descnbed in O'Sulhvan et al , Methods for the Preparation of Enzyme- Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzvm (ed J Langone & H Van Vunakis), Academic press, New York, 73 147-166 (1981)

Examples of enzyme-substrate combinations include, for example

(l) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e g , orthophenylene diamine (OPD) or 3,3',5,5'-tetramethyl benzidine hydrochloπde (TMB)), (n) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate, and

(m) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e g , p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate 4-methylumbellιferyl-β-D-galactosιdase

Numerous other enzyme-substrate combinations are available to those skilled in the art For a general review of these, see U S Patent Nos 4,275,149 and 4,318,980 Sometimes, the label is indirectly conjugated with the antibody The skilled artisan will be aware of various techniques for achieving this For example, the antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa Biotm binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten (e g , digoxm) and one of the different types of labels mentioned above is conjugated with an anti- hapten antibody (e g , anti-digoxm antibody) Thus, indirect conjugation of the label with the antibody can be achieved

In another embodiment of the invention, the anti-TF antibody need not be labeled, and the presence thereof can be detected using a labeled antibody which binds to the TF antibody

The antibodies of the present invention may be employed any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays Zola, Monoclonal Antibodies A Manual of Techniques, pp 147-158 (CRC Press, Inc 1987)

Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody The amount of TF protein in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies To facilitate determining the amount of standard that becomes bound, the antibodies generally are msolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogemc portion, or epitope, of the protein to be detected In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex See, e g , US Pat No 4,376,110 The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti- immunoglobulm antibody that is labeled with a detectable moiety (indirect sandwich assay) For example, one type of sandwich assay is an ELISA assay, m which case the detectable moiety is an enzyme

For lmmunohistochemistry, the tumor sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example

The antibodies may also be used for in vivo diagnostic assays Generally, the antibody is labeled with a radio nuchde (such as '"In, "Tc, ^l C, ^π,I, ^P51, ³H, ³²P or ³⁵S) so that the tumor can be localized using lmmunoscintiography

E Diagnostic Kits

As a matter of convenience, the antibody of the present invention can be provided in a kit, i e , & packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay Where the antibody is labeled with an enzyme, the kit will include substrates and cofactors required by the enzyme (e g , a substrate precursor which provides the detectable chromophore or fluorophore) In addition, other additives may be included such as stabilizers, buffers (e g , a block buffer or lysis buffer) and the like The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropnate concentration

F Therapeutic Uses for the Antibody

For therapeutic applications, the anti-TF antibodies of the invention are administered to a mammal, preferably a human, m a pharmaceutically acceptable dosage form such as those discussed above, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, lntrapeπtoneal, intra-cerebrospinal, subcutaneous, lntra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes The antibodies also are suitably administered by intra tumoral, peπtumoral, lntralesional, or penlesional routes, to exert local as well as systemic therapeutic effects The intrapeπtoneal route is expected to be particularly useful, for example, in the treatment of ovarian tumors

For the prevention or treatment of disease, the appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician The antibody is suitably administered to the patient at one time or over a series of treatments.

The anti-TF antibodies are useful in the treatment of various neoplastic and non-neoplastic diseases and disorders, such as TF-FVIIa related diseases or disorders Such diseases or disorders include, for example, chronic thromboembolic diseases or disorders associated with fibπn formation including vascular disorders such as deep venous thrombosis, arterial thrombosis, stroke, tumor metastasis, thrombolysis, arteriosclerosis and restenosis following angioplasty, acute and chronic indications such as inflammation, septic shock, septicemia, hypotension, adult respiratory distress syndrome (ARDS), disseminated lntravascular coagulopathy (DIC) The TF-FVIIa related disorder is not limited to in vivo coagulopathic disorders such as those named above but includes ex vivo TF-FVIIa related processes such as coagulation that may result from the extracorporeal circulation of blood, including blood removed in-line from a patient in such processes as dialysis procedures, blood filtration, or blood bypass duπng surgery

Dependmg on the type and severity of the disease, about 1 μg/kg to about 50 mg/kg (eg , 0.1- 20mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily or weekly dosage might range from about 1 μg/kg to about 20 mg/kg or more, depending on the factors mentioned above For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs However, other dosage regimens may be useful The progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic tumor imaging

According to another embodiment of the invention, the effectiveness of the antibody in preventing or treating disease may be improved by administering the antibody serially or in combination with another agent that is effective for those purposes, such as commercially available forms of hepaπn, low molecular weight hepaπn and or inhibitors of platelet glycoprotein Ilbllla, and or coumaπn and or other anticoagulant or antiplatelet agents or one or more conventional therapeutic agents such as, for example, alkylating agents, fohc acid antagonists, anti-metabolites of nucleic acid metabolism, antibiotics, pyπmidme analogs, 5-fluorouracιl, cisplatm, puπne nucleosides, amines, ammo acids, tπazol nucleosides, or corticosteroids Such other agents may be present in the composition being admmistered or may be administered separately Also, the antibody is suitably administered serially or in combination with radiological treatments, whether involving irradiation or administration of radioactive substances. G Articles of Manufacture

In another embodiment of the invention, an article of manufacture containing mateπals useful for the treatment of the disorders described above is provided The article of manufacture comprises a container and a label Suitable containers include, for example, bottles, vials, syringes, and test tubes The containers may be formed from a vanety of materials such as glass or plastic The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) The active agent in the composition is the anti-TF antibody The label on, or associated with, the container indicates that the composition is used for treating the condition of choice The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline,

Ringer's solution and dextrose solution It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, synnges, and package inserts with instructions for use

The following examples are offered by way of illustration and not by way of limitation The disclosures of all citations in the specification are expressly incorporated herein by reference

EXAMPLES

EXAMPLE 1 This example describes the determination of the bindmg epitopes of 5 neutralizing anti-TF antibodies and establishes the respective roles of binding affinity and epitope location on the anticoagulant potencies in different systems Interestingly, the results demonstrate that the anticoagulant potencies have no correlation with antibody binding affinities Rather, potency is pnmaπly determined by the precise location of the antibody- binding site on the TF molecules MATERIALS AND METHODS Materials Fatty acid-free BSA was from Calbiochem (La Jolla, CA) Human recombinant FVIIa was a gift from

Mark O'Connell (Genentech, Inc ) FX was from Haematologic Technologies Inc (Essex Junction, VT) Thrombin inhibitor napsagatran was a gift from Dr Kurt Hilpert (Roche, Switzerland) Chromozym t-PA was from Boehnnger Mannheim (Indianapolis, IN) Truncated transmembrane tissue factor comprising residues 1 - 243 (TFj-243) was generated and rehpidated as described (47,48) FX chromogenic substrate S2765 was from Diapharma Group Inc (Columbus, OH)

Preparation of murine D3 Fab fragments

Fab fragments were prepared from the D3 antibody by digestion with papain in the presence of cysteine A concentrated solution of the D3 Mab was prepared for digestion by dialysis versus 0 1 M sodium acetate pH 5 5, 1 mM EDTA To this solution (1 1 6 mg/mL antibody) was added solid cysteine to a final concentration of 50 mM Sufficient papain (Worthmgton Biochemical Corp , Lakewood, NJ) was added to give a 1 100 weight ratio to antibody and the solution was incubated at 37 °C After 8 hours the digestion was quenched by addition of 100 mM lodoacetamide to inactivate the papam Residual intact antibody and Fc fragments were removed by passing the solution over a Protein A-Sepharose column The Fab fragments in the flow-through fraction were further purified by affinity chromatography on a column of immobilized soluble TF 1-219 (sTF) The affinity column was prepared by using a 1 x 5 mL NHS-activated HiTrap column (Pharmacia Biotech, Piscataway, NJ) following the instructions supplied by the manufacturer The final coupling density achieved was 25 mg of sTF per mL of resin D3 Fab were eluted from this column by washing with a solution of 0 1 M acetate pH 3, 0 2 M NaCl and the Fab containing fractions were pooled and neutralized

Clotting assays

For pre-incubation assays, 20 μl of antibody was added to 180 μl re pidated human tissue factor (Innovin, Dade Behπng Inc , Newark, DE) and incubated at 37 C for 15 mm 100 μl of normal citrated human plasma (Peninsula Blood Bank, Burhngame, CA) was added and clotting times were measured using an MLA Electra 800 (Medical Laboratory Automation Inc , Pleasant, NY)

Prothrombin time assays

Prothrombin time (PT) assays, antibody was added to citrated human plasma After 5mιn incubation, clotting was started by adding human tissue factor reagent Innovin Clotting times were measured on an ACL300 using the PT mode (Coulter Corp , Miami, FL) For both assays, the antibody concentrations are reported as final concentrations in the reaction mixture (including the tissue factor reagent)

Site-directed mutagenesis expression and put ification ofsTF mutants

Expression of sTF mutants (TF 1-219) E coli and subsequent purification on a D3 antibody affinity column was carried out as described earlier (Kelley et al , [1995] Biochemistry 34 10383-10392) For sTF mutants which did not bind to the D3 column (N199A R200A and K201 A D204A), a 7G1 1 antibody column was used This column was prepared by coupling the 7G11 antibody to CNBr-activated Sepharose 4B

(Pharmacia, Piscataway, NJ) according to the manufacturer's instructions Cell pellets were stored at -20 C for at least 1 hr The osmotic shock supernatants were applied to the antibody affinity column which was equilibrated with 50 mM Tπs-HCl, pH 8 0, 500 mM NaCl (buffer A) To remove nonspecifically bound proteins, the column was washed with 6 column volumes of buffer A and 50 mM Tπs-HCl pH 8 0, 1 0 M NaCl, 0 5M tetramethylammomum chloride sTF mutants were eluted with 0 1 M sodium acetate, pH 3 0, 0 2 M NaCl

Fractions were neutralized and peak fractions concentrated using a Centπprep 10 ( Amicon, Beverly, MA) Protein concentrations were determined by absorbance measurements using an λ280 of 29 4 mM cm calculated from quantitative amino acid analysis data An λ280 of24 mM cm was used for the Trp to Phe mutants of sTF Determination ofanti-TF antibody 's TF binding affinity and antibody epitope mapping

The binding affinity of sTF for immobilized antibody was determined by surface plasmon resonance (SPR) measurements on a Pharmacia BIAcore 2000 instrument (Pharmacia Biosensor) Each antibody was coupled to the sensor chip surface at a level of 2000-3000 resonance units using amme coupling chemistry (Pharmacia Biosensor) In a typical experiment, 4 different antibodies were immobilized on each of the 4 flow cells of the sensor chip so that sensorgrams could be recorded simultaneously for all 4 antibodies Sensorgrams were recorded for sTF binding at concentrations ranging from 15 6 nM to 500 nM m 2 -fold increments The kinetic constants were determined by non linear regression analysis according to a 1 1 binding model using software supplied by the manufacturer Dissociation constants were calculated from the kinetic constants In experiments to determine competition between the antibody and FVIIa for binding to sTF, the same sTF concentration series was prepared m the presence of 5 μM human, recombinant FVIIa These solutions were incubated at ambient temperature for 30 minutes pnor to injection onto the sensor chip The epitopes on sTF for binding the monoclonal antibodies were determined by measuring the effect of amino acid substitutions in sTF on the affinity for immobilized antibody Affinities were determined by SPR measurements as described above for the wild-type protem

Monoclonal antibodies

Monoclonal antibody 7G1 1 was generated by immunizing female BALB/c mice subcutaneously 3 times, lntrapeπtoneally 3 times with 20μg sTF in MPL/TDM adjuvant (Ribi Immunochem Research, Hamilton, MT), at 2 week intervals These mice were further boosted 8 times into footpads with lOμg sTF m lOOul

MPL/TDM Adjuvant every week 5G6 was generated by immunizing female BALB/c mice via footpad with lOμg sTF in lOOul MPL/TDM adjuvant, 13 times every week Four days after the last boost, popliteal lymph nodes were removed and fused with mouse myeloma cells P3X63Ag8U 1 (Yelton et al , [1978] Cuπ Top Microbiol Immunol 81 1-7) using 35% polyefhyleneglycol as described (Chuntharapai and Kim, [1997] Methods Enzymol 288 15-27) Hybπdoma cell lines secreting antibody specific for sTF, as determined by

ELISA, were cloned twice by limiting dilution and further characterized Ascites were produced in BALB/c mice and monoclonal antibodies were punfied using protein G conjugated Sepharose 4B The generation of D3 antibody was described previously (Paborsky et al , [1990] Prot Engineering 3 547-553) and the antibody 6B4 came from a separate immunization protocol The HTF1 antibody was described by Carson et al (Carson et al , [1987] supra)

FX activation in human plasma

The antibodies were diluted in human citrated plasma from a donor plasma pool (Peninsula Blood Bank, Burhngame, CA) for lOmin at room temperature At the end of the incubation period the thrombin inhibitor napsagatran (Hilpert et al , [1994] J Med Chem 37 3889-3901 , Gast and Tschopp [1995] Blood Coag Fibπnolysis 6 533-560) was added FX activation was started with rehpidated TFj -243 in 20mM hepes, pH 7 5, 0 5% BSA (HBS buffer) containing 15mM CaCl2 The reaction mixture contained 33% plasma and the concentrations of rehpidated TFι _243, napsagatran and CaCl2 were 0 4nM, 0 5μM and 5mM, respectively 50μl ahquots taken at 15sec intervals were quenched in 150μl of 20mM EDTA In the second stage, 50μl of 1 5mM FXa chromogenic substrate S2765 was added and increase in absorbance at 405nm monitored on a kinetic microplate reader (Molecular Devices, Menlo Park, CA) The initial rates were calculated by the linear fit of the values over a 45 sec period The values of ahquots taken at later time points indicated that the linear phase of the reaction was limited to this short time period Control experiments in which rehpidated TFj-243 was omitted showed that there was no increase in chromogenic activity, indicating the absence of any FXa generation without TF Also, napsagatran had no effect on FXa amidolytic activity towards S2765 under the employed conditions, which is consistent with the reported high selectivity towards thrombin (Hilpert et al , [1994] supra)

From standard curves with FXa incubated with plasma and all other components used m the assay, it was calculated that under non-inhibited conditions an average of 8 6 nM ± 0 9 FXa/min (±S D ) was generated

The amidolytic activities of several coagulation factors, such as factors Ha, Vila, IXa and XIa were tested under identical assay conditions to test whether other coagulation factors generated during the reaction might have contributed to the amidolytic activity measured in the second stage of the assay Only factor XIa displayed significant amidolytic activity towards S2765, which was about 25% of the FXa activity To assess a possible contribution of factor XIa in our assay system, the inhibitory activity of the D3 antibody was examined m factor Xl-deficient plasma (George King Bio-Medical, Overland Park, KS) The IC50 value of 5 2±0 lμg/ml (±SD, n=4) was similar to the value determined in normal human plasma In addition, expeπments carried out in factor II-, and factor Vlll-deficient plasmas (Ameπcan Diagnostica) gave similar results (6 4±0 9μg/ml and 7 6±1 5μg/ml respectively) Together, these results strongly suggested that rates of S2765 cleavage accurately reflected the concentration of FXa generated by rehpidated TFj_243 FVIIa complex in plasma Amidolytic activity of soluble TF FVIIa complex The antibodies were incubated with sTF and FVIIa in HBS buffer containing 5mM CaCl2 for 20mιn prior to addition of Chromozym t-PA The final concentration of the reactants was as follows lOnM sTF, lOnM FVIIa, 0 5mM Chromozym t-PA The rates of amidolytic activity were measured at 405nm on a kinetic microplate reader (Molecular Devices) The background activity was defined as the amidolytic activity of FVIIa in the absence of sTF and was subtracted from the obtained values Human ex-vivo thrombosis model

Tissue factor-expressing human J82 cells (epithelial carcinoma, ATCC HTB1) were grown on Thermanox plastic covershps as described (Kirchhofer et al , [1995] Arterioscler Thromb Vase Biol 15 1098-1106) The covershps with the cell monolayer were then positioned in parallel plate perfusion chambers and the entire system including tubings, mixing devices and parallel plate chambers was filled with DMEM-1% (w/v) BSA The details of the experimental system were described recently (Kirchhofer et al , [1995] supra,

Kirchhofer et al , [1994] J C n Invest 93 2073-2083) Blood was then drawn from the antecubital vein of a healthy donor at a rate of 1 mL/min Immediately before enteπng the mixing chambers the flowing blood was infused with the antibodies at a rate of 50 μL/min by use of an infusion pump (Infu 362, Datex AG, Switzerland) The homogenous blood-antibody mixture then entered three parallel plate perfusion devices containing the J82 cell monolayers The blood flow of 1 mL/min resulted in a shear rate of 65 s on the covershps which corresponded to venous blood flow conditions After a 3 5 minute perfusion period the cell layer was washed and covershps removed for visual inspection of deposited fibrin Fibπnopeptide A (FPA) levels were measured in the blood leaving the perfusion device as described previously (Kirchhofer et al , [1994] and [1995] supra) RESULTS

Functionally different anti-TF antibodies

As seen in Fig la, the antibodies 7G11, 6B4 and HTF1 completely inhibited sTF FVIIa-dependent activity towards Chromozym t-PA, indicating interference with the proper formation of the sTF FVIIa complex In contrast. 5G6 antibody did not inhibit at all, whereas D3 reduced activity by about 20%, reaching a plateau at higher concentration The inhibition by D3 was also seen when the smaller antibody Fab was used (Fig la) The inhibitory effect of D3 was dependent on low sTF concentrations, since no inhibition occurred at high sTF concentrations (120-200nM) m the presence of molar excess antibody (data not shown) In agreement, D3 did not affect the amidolytic activity when rehpidated TF 1.243 was used, which binds FVIIa with much higher affinity than sTF (data not shown) However, both D3 and 5G6 inhibited TF FVIIa-mediated activation of macromolecular substrate as well as the other antibodies This was shown by results obtained from clotting assays in which antibodies were pre-incubated with TF reagent (Fig lb) Anticoagulant potencies ofanti-TF antibodies

The results of the amidolytic assays indicated two fundamentally different types of anti-TF antibodies Two antibodies of each group (D3 and 5G6 vs 6B4 and HTFl) were selected and their anticoagulant potencies in a human ex-vivo blood flow system determined (Kirchhofer et al , [1994] and [1995] supra) In this system the antibodies were infused to flowing non-anticoagulated human blood, which then entered parallel plate devices containing a monolayer of TF-expressing J82 cells The shear rate at the cell layer was 65 s simulating venous blood flow conditions In controls this resulted in the generation of fibπnopeptide A (FPA) and the deposition of polymerized fibπn onto the cell monolayer The average FPA levels of 36 control samples was

1348 ± 46 1 ng/ml plasma (±SEM), which was similar to earlier reported FPA concentrations using the same system (1192 ± 69 ng/ml, (Kirchhofer et al , [1995] supra)) Infusion of D3 and 5G6 potently inhibited FPA generation with IC50 values of 16μg/ml and 50μg/ml, respectively (Fig 2) Compared to full length D3, the inhibition by the D3 Fab was weaker (IC50 36μg/ml), most likely due to reduced avidity for surface TF as compared to the bivalent full length D3 antibody Surprisingly, HTFl antibody did not inhibit at the highest tested concentration of 50μg/ml, while 6B4 showed rather weak inhibitory activity with about 40% inhibition at 150μg/ml (Fig 2) Consistent with the observed reduction of FPA levels by D3, D3 Fab and 5G6, a visual inspection of the cell layers showed that only little if any fibrin was deposited, while HTFl and 6B4-treated samples were indistinguishable from controls Next, the measurement of FX activation in human plasma was used as another way to quantify anticoagulant potencies of the antibodies Similar to the blood flow system, where antibody is not pre- equihbrated with TF but infused to the flowing blood, the antibodies were added to plasma and coagulation was triggered with rehpidated TF 1-243 We found that the tested antibodies inhibited the initial rates of FX activation in a concentration-dependent manner (Fig 3) The antibodies D3 and 5G6 were more potent than HTFl, 6B4 and 7G11 The concentrations which inhibited the rates by 50% were as follows 7 2±1 Oμg/ml (±SD, n=5) for D3, 15 5±1 3μg/ml (±SD, n=5) for 5G6, 43 4±6 8μg/ml (±SD, n=4) for 6B4, 147 8±8 6μg/ml (±SD, n=5) for 7G1 1 and 150 0±31 l μg/ml (±SD, n=4) for HTFl

Furthermore, similar potency differences between the antibodies were found when clotting times were measured in PT assays using the same incubation protocol as for FX activation rate assays (Fig 4) The antibody concentrations which prolonged the clotting time by 1 5-fold were lOμg/ml for D3, 27μg/ml for 5G6, 133μg/ml for 6B4 and 500μg/ml for 7G11 The highest tested concentration of HTFl (40μg/ml) had no effect (Fig 4) Determination of kinetic constants of anti-TF antibodies

Because the inhibitory potencies of the examined antibodies could merely be a reflection of their binding affinities to sTF, we determined the kinetic constants of each antibody A comparison of the calculated KD values (Table 2) and the inhibitory activities of each antibody showed that there is no correlation between affinities and anticoagulant potencies In fact, D3 was consistently the most potent anticoagulant, yet it displayed the weakest affinity for TF, while HTFl and 7G1 1 were the strongest binders, but had the weakest anticoagulant activities This lack of correlation was also seen when on-rates were compared which, with the exception of HTFl, were in a similar range (2 3x10 - 6 0x10 M sec ) Table 2

In addition, competition experiments with FVIIa and sTF showed that in the presence of molar excess of FVIIa (> 1 OOx), the antibodies 7G 11 , 6B4 and HTF 1 did not bind to TF, whereas the affinity of D3 and 5G6 was only reduced by 4-5-fold This was consistent with the results from amidolytic assays (fig la), indicating that D3 and 5G6 had a fundamentally different inhibitory mechanism than the other antibodies Determination of antibody epitopes

The results so far indicated that the antibodies' anticoagulant potencies could be related to their specific inhibitory mechanism and, thus, to the precise binding site on TF To determine the antibody epitopes, a large number of sTF mutants were generated by expression in E coh and subsequent affinity purification on a D3 column (Kelley et al , [1995] supra) The bindmg of the antibodies to each sTF mutant was measured on a BIAcore instrument The results, summarized in Fig 5, show the affinity loss expressed as the ratio of Krj values of sTF mutant and wild-type sTF Residues that increased the ratio by more than 3-fold were considered important for antibody binding The two double mutants sTF N199A R200A and sTF K201 A D204A did not bind to the D3 affinity column and were purified on a 7G 1 1 antibody affinity column As expected, D3 had the largest loss m affinity to these two mutants (Kpj (mut)/ K j (wt) >5000) Since sTF D204A alone had the same affinity to the antibodies as wildtype sTF, we concluded that K201 m the K201 A D204A double mutant was the critical residue for antibody binding Other residues found to be important for D3 binding were 1152, Y156 and K165 K166 With the exception of Y156L, the same sTF mutants also showed decreased binding to the 5G6 antibody

In contrast to D3 and 5G6 which bound to the C-termmal TF domain, the three antibodies 7G 1 1 , 6B4 and HTFl bound to residues located in the N-termmal TF domain The TF mutants which had the greatest loss in affinity to 7G11 were K46A (5000x) and Y51 A (32x) Additional mutants with significant affinity losses were S47A, K48A, F50A and T52A The sTF mutants that affected binding of the 6B4 antibody were Y10A,

F76A, Y94A, E99A and L104A El 05 A Three of these mutants, F76A, Y94A and E99A, also reduced binding affinity of the HTFl antibody (Fig 5) Location of antibody epitopes on the crystal structure of the TF FVIIa complex

As seen in the crystal structure of the TF FVIIa complex (Banner et al , [1996] supra), the 7G11 binding site is formed by a clearly defined patch of surface exposed residues with a calculated solvent accessible

2 area of 397A (Fig 6) This is relatively small compared to buried surface areas of typical antigens (Huang et al , [1998] supra) and thus, the antibody binding region may not have been identified in its entirety This region is important for FVIIa binding and the F50 residue makes a critical hydrophobic contact to the second epidermal growth factor domain of FVIIa (Banner et al , [1996] supra)

The TF residues that were important for 6B4 binding defined a large surface area located on the 'back side' of TF as compared to the 7G11 epitope (Fig 6) With the premise that the epitope is within the perimeter

2 defined by the identified TF residues, the area of the hypothetical epitope was calculated to be 594 A The epitope extended into a TF region which contacts the catalytic domain of FVIIa and included the residues F76 and Y94 For the HTFl antibody only a relatively small number of TF mutants were examined and the epitope is somewhat less well defined (Fig 6) Nevertheless, the HTFl and 6B4 epitopes were largely the same, since three identified binding residues including F76 and Y94 were shared by both antibodies The epitopes of the D3 and 5G6 antibodies were very similar, being located outside of the FVIIa-TF contact region As shown in Figure 6, the epitope runs from the bottom to the top of the C-terminal TF domain and is approximately opposite to the mam TF-FVIIa contact region Accordingly, antibody binding may not interfere with TF FVIIa complex formation DISCUSSION By using a large panel of sTF mutants the binding epitopes of 5 anti-TF antibodies was determined and a clear picture of how they exert their anticoagulant effect gained They bound to three distinct regions of TF and either interfered with FVIIa-TF association (7G11, 6B4, HTFl) or with TF-macromolecular substrate mteraction (D3, 5G6) The anticoagulant potencies were determined in whole blood and plasma-based systems in which the antibodies and coagulation factors were allowed to simultaneously interact with TF First, in the human ex -vivo blood flow system D3 and 5G6 potently inhibited the generation of FPA and the deposition of fibrin onto the J82 cell layer In contrast, the antibodies 6B4 and HTFl were at least one order of magnitude less potent and, in the case of HTFl, virtually inactive Secondly, since coagulation in the blood flow system was shown to proceed via direct TF FVIIa mediated activation of FX (Kirchhofer et al , [1995] supra), it was reasoned that the differential effects on FPA generation should have a correlate in the inhibition of FX activation rates and prothrombin times in human plasma Indeed, it was found that D3 and 5G6 were by far the most potent antibodies tested

The apparent differences in anticoagulant activities could not be explained by differences m the binding affinities of the antibodies In fact, D3 was the weakest binder (KQ 7nM) and 7G 11 the strongest (Krj 0 2nM), yet D3 was about 20-fold more potent in inhibiting FX activation Because the TF-imtiated coagulation is a rapid process it seemed possible that the potency differences reflected differences in on-rates However, with the exception of HTFl, the experimentally determined on-rates were withm a narrow range and showed no correlation with the observed anticoagulant potencies These findings suggested that the epitope locations rather than on-rate constants or binding affinities were the main determinants of anticoagulant potencies of the anti-TF antibodies Inspection of the epitopes on the crystal structure of the TF FVIIa complex revealed that all antibodies bound to functionally important regions of TF, but impacted different aspects of TF function The binding epitope of D3 and 5G6 overlapped with a TF region that does not contact FVIIa, but interacts with macromolecular substrate binding This explained why the antibodies had little or no effect on amidolytic activity towards a small synthetic substrate The epitope residues K165 and K166 were previously found to be critical for TF FVIIa mediated activation of FX (Ruf et al , [1992] supra, Roy et al , [1991] supra, Kelley et al , [1995] supra) and FIX (Huang et al , [1996] supra) These two residues and the additional epitope residues Y156 and K201 are all part of a distinct surface-exposed TF region which directly interacts with substrates FX and FIX (Kirchhofer et al , [1999] Thromb Haemost Suppl [abstract], 300, Kirchhofer et al , (2000) Biochemistry 39 7380-7387) This region may extend into the FVIIa Gla domain (Martin et al , [1993] supra, Ruf et al ,

[1999] supra) and most likely contacts the Gla domains of substrates (Huang et al , [1996] supra, Martin et al , [1993] supra) Thus, D3 and 5G6 by binding to this region will stencally prevent the association of the substrate Gla domains with TF and thus interfere with proper substrate orientation to form a productive ternary TF-FVIIa- substrate complex This further provided a basis of explaining the excellent anticoagulant potencies of D3 and 5G6 First, since the epitope is not within the TF-FVIIa contact region, these antibodies were able to bind to TF during and after the rapid formation of TF FVII and TF FVIIa complexes in plasma Secondly, they competed with a rather low affinity TF-substrate interaction event Moreover, the epitope residues K165 and K166 were shown to be important for the FVIIa and FXa-dependent activation of TF FVII (Dittmar et al , [1997] Biochem J 321 787-793) Therefore, both antibodies could have interfered with the activation of TF-bound zymogen FVII, a reaction which in all likelihood is the first activation step for coagulation in plasma (Rapaport and Rao

[1995] Thromb Haemost 74 7-17), as well as in the employed blood flow system with J82 cells (Sakai et al , [1989] J Biol Chem 264 9980-9988) In fact, D3H44-F(ab')₂ was found to strongly inhibit the FXa-mediated activation of zymogen FVII (Kirchhofer et al , (2001) Biochemistry 40 675-682) In agreement, the closely related antibody TF8-5G9 (Momssey et al , [1988] supra, Ruf and Edgmgton [1991] supra, Huang et al , [1998] supra, Fiore et al , [1992] supra) which bmds to the same TF region (Huang et al , [1998] supra), is a potent anticoagulant in plasma clotting assays (Ruf and Edgmgton [1991] supra) and inhibits TF-dependent FVII conversion to FVIIa (Fiore et al , [1992] supra) A comparison with TF8-5G9 revealed that all of the identified D3 and 5G6 epitope residues were also found as contact residues in the crystal structure of TF8-5G9 Fab bound to soluble TF (Huang et al , [1998] supra) Yet, despite having an apparently identical epitope the D3 and TF8- 5G9 (Fiore et al , [1992] supra) antibodies differed from 5G6 in their ability to weakly inhibit the amidolytic activity (Chromozym t-PA) of the sTF FVIIa complex at low sTF concentrations These results indicated that there existed subtle differences in antibody binding to an apparently identical epitope

The weaker anticoagulant potencies of 7G1 1, HTFl and 6B4 were surpπsmg, since they were as potent as D3 and 5G6 when allowed to pre-bind to TF in plasma clotting assays The identification of the antibody binding sites on TF provided a basis for explaining these results 7G11 bound to a TF region proximal to the light chain of FVIIa One of the binding residues, F50, makes an important contact to the second epidermal growth factor domain of FVIIa (Banner et al , [1996] supra, Zhang et al , [1999] J Mol Biol 285 2089-2104) suggesting that the antibody interfered with the formation of the TF FVIIa complex Both 6B4 and HTFl interfered with a shared FVIIa contact site, which was distinct from the 7G 11 epitope In agreement, 6B4 did not prevent binding of 7G11 to TF in cross-blocking expenments (data not shown) Epitope residues F76 and Y94 make direct contacts to the FVIIa catalytic domain residue Met306 Mutating this Met306 or TF residue Y94 strongly impaired macromolecular substrate activation (Dickinson et al , [1996] supra, Kelley et al , [1995] supra, Ruf et al , [1995] Biochemistry 34 6310-6315), and binding of 6B4 or HTFl should consequently have deleterious effects as well This contact site is also important for the TF-dependent enhancement of FVIIa activity towards small synthetic substrates (Dickinson et al , [1996] supra), thus explaining the observed inhibitory effects of 6B4 and HTFl in the amidolytic assay The results are also consistent with a previous report demonstrating that HTFl interfered with the binding of TF to FVIIa (Carson et al , [1987] supra) There is a possibility that 6B4 had additional effects on the macromolecular substrate-TF interaction 6B4 did not bind to the mam substrate interaction region around K165 and K166 (fig 6), but the epitope residues L104 E105 were proximal to residues N199 R200 which are part of the FX recognition region (Kirchhofer et al , [1999] supra) Thus, 6B4 binding could have resulted in additional steπc effects on substrate-TF interaction

A distinguishing characteristic of antibodies 7G11 , 6B4 and HTFl is their competition with local FVIIa contact sites within the context of an overall large contact surface (Banner et al , [1996] supra) and high affinity FVII-TF interaction Whereas this inhibitory mechanism provided potent inhibition in pre-incubation experiments (Fig lb), it did not do so under the non-equihbπum conditions of our experimental systems One likely explanation is that once TF FVIIa complexes were formed, the antibodies would have little inhibitory effect since inhibition would mainly be determined by the FVIIa/TF off-rate Similar conclusions were made by Ruf and Edgmgton (1991, supra) and Fiore et al (1992, supra) using different in- vitro systems to evaluate two antibodies which interfered with TF-FVIIa association Nevertheless, at appropπate doses such types of antibodies demonstrated inhibition of the coagulation system in animal experiments (Taylor et al , [1991] Circulatory Shock 33 127-134, Himber et al , [1997] Thromb Haemostasis 78 1 142-1149, Pawashe et al , [1994] Circ Res 74 56-63, Ragm et al , [1996] 93 1913-1918, Thomas et al , [1993] Stroke 24 847-854, Gohno et al , [1996] Nature Med 2 35-40) A caveat to this comparison is that the antibodies used for the animal experiments have not been characteπzed in much detail and no epitope map information is available

Furthermore, the expenments would predict that an antibody like D3 or 5G6 should be efficacious at significantly lower doses Consistent with this view the closely related TF8-5G9 antibody appeared extremely potent in inhibiting coagulation m a chimpanzee study (Levi et al , [1994] J Clm Invest 93 1 14-120) and was also very effective in a tumor metastasis model (Mueller et al , [1992] supra) However, a direct comparison of two well-defined, different type-antibodies has yet to be done

The findings suggested that the anticoagulant potencies of anti-TF antibodies is not pπmaπly determined by the binding affinity, but rather by the epitope location and consequently by the particular mode of inhibition Even though the translation of the results obtained from blood/plasma based ln-vitro systems into an in- vivo setting has obvious limitations, the findings may nevertheless have some implications in regard to the use of anti-TF antibodies in anticoagulant therapy As suggested by this study, the choice of an anti-TF antibody may be important in terms of the expected efficacy, since the epitope location will strongly influence the antibody's potency to inhibit thrombosis EXAMPLE 2

Humamzation of a munne anti-human tissue factor monoclonal antibody D3 MATERIALS AND METHODS Cloning of Murine D3 The muπne anti-human TF mAb D3 was generated and cloned at Genentech (Paborsky et al , [1990]

Prot Engineering 3 547-553) Protein sequence analysis of the purified antibody provided an N-terminal sequence for the heavy chain, EVQLQQSGAELVRPGALVKLSCKASGFNIKD (SEQ ID NO 19), and for the light chain, DIKMTQSPSSMSASLGESVTITCKASRDIK (SEQ ID NO 20) Total RNA was purified from D3 hybπdoma cell line (1D4(14)_D3) using the standard RNA ST AT protocol (Tel-Test-Inc , Fnendswood, TX) cDNA was made using Ohgo dT and Superscπpt II RNase H-Reverse Transcπptase according to the manufacturers instructions (Gibco BRL, Gai hersburg, MA) PCR amplification was performed in a 50 μl reaction using 3 units of UlTma DNA Polymerase (Perkm Elmer, Foster City, CA) with lx buffer, 4 mM MgCl, 40 μM dNTPs, and 0 7 - 1 0 μM forward and reverse primers Specific pπmers used were heavy chain forward, 5'-TACAAACGCGTACGCTGARGTNCARYTNCARCARWSNGGNGC-3' (SEQ ID NO 21), heavy chain reverse, 5 '-C AGTGG ATAG AC AGATGGGC CCGTCGTTTTGGC-3 ' (SEQ ID NO 22), light chain forward,

5 '-GCATACGCTGAYATHAARATG ACNC AR WSNCC-3 ' (SEQ ID NO 23), light chain reverse, 5'- TGGTGCAGCCACGGTCCGTTTKAKYTCCARYTTKGT-3' (SEQ ID NO 24) Separate reactions were set up for heavy chain and light chain and cycled with the following conditions 95° for 2 mm, 30 cycles of (95° 20 sec, 60° 30 sec, 72° 1 mm), 4° hold m a Perkin Elmer 9600 After purification on Qiaquick columns (Qiagen), 1/10 of the PCR reaction was cloned into pCR-Blunt (Invitrogen) Sequence analysis of the clones revealed that amino acid 7 of both the heavy and light chains were Arg instead of the expected Ser, the codon WSN used in the primers can result in Arg or Ser The PCR was repeated on individual clones in pCR-Blunt to change the Arg to Ser The same reverse primers were used and new heavy chain forward primer, 5'- AGGTACAAACGCGTACGCTGAAGTGCAACTCCAGCAAAGTGG-3' (SEQ ID NO 25) and light chain forward primer, 5'-GCATACGCTGAT ATAAAAATGACGCAGTCGCCATCC-3 ' (SEQ ID NO 26) PCR used UlTma and the same conditions as above The heavy chain PCR fragment was digested with BsiWI and Apal while the light chain PCR fragment was digested with EcoRV and RsrII Each resulting digested fragment was cloned into a previously described F(ab) chimeπc expression plasmid (Presta et al , Cancer Res 57 4593- 4599 [1997]) DNA sequence of heavy chain fragment BsiWI to Apal (SEQ ID NO 27)

5'-

GTACGCTGAAGTGCAACTCCAGCAAAGTGGCGCTGAGCTTGTGAGGCCAGGGCCTTAGTCAAGTTG TCCTGCAAAGCTTCTGGCTTCAACATTAAAGACTACTATATGCACTGGGTGAAGCAGAGGCCTGAA CAGGGCCTGGAGTTGATTGGATGGATTGATCCTGAGAATGGTAATACTATTTATGACCCGAAGTTC CAGGACAAGGCCAGTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCT

GACATCTGAGGACACTGCCGTCTATTACTGTGCTAGAGATACTGCGGCATACTTTGACTACTGGGG CCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACGGGCCC-3'

DNA sequence of light chain fragment EcoRV to RsrII (SEQ ID NO 28) 5'-

GATATCAAAATGACGCAGTCGCCATCCTCCATGTCTGCATCGCTGGGAGAGAGTGTCACTATCACT TGCAAGGCGAGTCGGGACATTAAAAGCTATTTAAGCTGGTACCAGCAGAAACCATGGAAATCTCCT AAGACCCTGATCTATTATGCCACAAGCTTGGCGGATGGGGTCCCATCAAGATTCAGTGGCAGTGGA TCTGGGCAAGATTATTCTCTAACCATCAGCAGCCTGGAGTCTGACGATACAGCAACTTATTACTGT

CTACAGCATGGTGAGAGCCCATTCACGTTCGGCTCGGGGACAAAGTTGGAACTCAAACGGACCG-3' Computer Graphics Models of Murine and Humanized F(ab)s

Sequences of the VL and VH domains (SEQ ID NOS 3 and 1 , respectively) were used to construct a computer graphics model of the murine D3 VL-VH domains This model was used to determine which framework residues should be incorporated into the humanized antibody A model of the humanized F(ab) was also constructed to verify correct selection of muπne framework residues Construction of models was performed as described previously (Carter et al , Proc Natl Acad Sci USA 89 4285-4289 [1992], Eigenbrot et al , J Mol Biol 229 969-995 [1993])

Construction of Humanized F(ab)s The plasmid pEMXl used for mutagenesis and expression of F(ab)s in E coli has been described previously

(Werther et al , J Immunol 157 4986-4995 [1996]) Bπefly, the plasmid contains a DNA fragment encoding a consensus human K subgroup I light chain (VLκI-CL), a consensus human subgroup III heavy chain (VHIII- CH1) and an alkaline phosphatase promoter The use of the consensus sequences for VL and VH has been described previously (Carter et al , supra) To construct the first F(ab) vaπant of humanized D3, F(ab)-1, site-directed mutagenesis (Kunkel, Proc

Natl Acad Sci USA 82 488-492 [1985]) was performed on a deoxyuπdine-containing template of pEMXl The six CDRs were changed to the muπne D3 sequence, the residues included in each CDR were from the sequence-based CDR definitions (Kabat et al , Sequences of proteins of immunological interest, Ed 5, Public Health Service, National Institutes of Health, Bethesda, MD [1991] except for CDR-H1 which was defined using a combination of CDR-H1 definitions from Kabat et al (supra) and Chothia et al , Nature 342 877-833

(1989), l e , CDR-H1 was defined as extending from residues H26-H35 in the heavy chain) F(ab)-1 therefore consisted of a complete human framework (VLU K subgroup I and VH subgroup III) with the six complete muπne CDR sequences Plasmids for all other F(ab) variants were constructed from the plasmid template of F(ab)-1 Plasmids were transformed lnto ϋ¹ coli strain XL-1 Blue (Stratagene, San Diego, CA) for preparation of double- and smgle-stranded DNA For each variant, DNA coding for light and heavy chains was completely sequenced using the dideoxynucleotide method (Sequenase, U S Biochemical Corp , Cleveland, OH) Plasmids were transformed into E coli strain 16C9, a derivative of MM294, plated onto Luna broth plates containing 50 μg/ml carbenicilhn, and a single colony selected for protein expression The single colony was grown in 5 ml Luna broth- 100 μg/ml carbenicilhn for 5-8 h at 37°C The 5 ml culture was added to 500 ml AP5-50 μg/ml carbenicilhn and allowed to grow for 20 h in a 4 L baffled shake flask at 30°C AP5 media consists of 1 5 g glucose, 1 1 0 g Hycase SF, 0 6 g yeast extract (certified), 0 19 g MgS04 (anhydrous), 1 07 g NH4C1, 3 73 g

KC1, 1 2 g NaCl, 120 ml 1 M tnethanolamine, pH 7 4, to 1 L water and then sterile filtered through 0 1 μm

Sealkeen filter Cells were harvested by centπfugation in a 1 L centrifuge bottle at 3000xg and the supernatant removed After freezing for 1 h, the pellet was resuspended in 25 ml cold 10 mM Tπs-1 mM EDTA- 20%sucrose, pH 8 0 250 μl of 0 1 M benzamidme (Sigma, St Louis, MO) was added to inhibit proteolysis After gentle stirring on ice for 3 h, the sample was centπfuged at 40,000xg for 15 mm The supernatent was then applied to a protein G-Sepharose CL-4B (Pharmacia, Uppsala, Sweden) column (0 5 ml bed volume) equilibrated with 10 mM Tπs-1 mM EDTA, pH 7 5 The column was washed with 10 ml of 10 mM Tπs-1 mM EDTA, pH 7 5, and eluted with 3 ml 0 3 M glycine, pH 3 0, into 1 25 ml 1 M Tπs, pH 8 0 The F(ab) was then buffer exchanged into PBS using a Centπcon-30 (Amicon, Beverly, MA) and concentrated to a final volume of 0 5 ml SDS-PAGE gels of all F(ab)s were run to ascertain purity and the concentration of each variant was determined by amino acid analysis F(ab)s were quantified by measuring OD280 ^an^ amino acid analysis, concentrations used in assays were from the amino acid analysis A chimeπc F(ab) was used as the standard in the binding assays This chimeπc F(ab) consisted of the entire murine D3 VH domain fused to a human CHI domain at amino acid SerHl 13 and the entire murine D3 VL domain fused to a human CL domain at amino acid LysL107 Expression and purification of the chimeπc F(ab) were identical to that of the humanized F(ab)s

Construction and purification of D3H44-F(ab')2 D3H44-F(ab')2 was generated by the addition of the heavy chain hinge (CPPCPAPELLGG) to the C- terminus of the D3H44-F(ab), followed by the GCN4 leucme zipper (51 ) and a (hιs)6 tag for purification D3H44-F(ab')2 was expressed in E coli and the cell paste was diluted 1 5 (w/v) in 20 mM sodium phosphate pH 7 4, 50 mM NaCl, then lysed using an M 1 10Y microfluidizer (Micro fluidics Corp , Newton, MA) Polyethylene inline (BASF Corp , Rensselaer, NY) was added to a final concentration of 0 2%, followed by centπfugation (4300xg, 30mιn) to remove cellular debris The supernatant was filtered (0 2 μm) and loaded on to SP Sepharose FF (Amersham Pharmacia Biotech, Uppsala, Sweden) under conditions in which F(ab') 2 flowed through The SP Sepharose FF flow through fraction was applied to Chelatmg Sepharose FF (Amersham Pharmacia Biotech, Uppsala, Sweden), charged with Cu2+ and equilibrated in 2 mM lmidazole, pH 7 0, 250 mM NaCl D3H44-F(ab')2 was eluted using 200 mM lmidazole pH 7 0 The Chelatmg Sepharose FF elution pool was adjusted to pH 4 0, and the leucine zιpper/(hιs)6 tag was cleaved using pepsin Following pepsin cleavage, D3H44-F(ab')2 was applied to SP Sepharose High Performance (Amersham Pharmacia Biotech, Uppsala, Sweden) and eluted using a linear gradient from 0 to 0 12 M sodium acetate in 25 mM MES pH 5 6 SP Sepharose High Performance gradient fractions were analyzed by SDS-PAGE and pooled Finally, D3H44- F(ab')2 was formulated by ultrafiltration using a 10 kDa regenerated cellulose membrane (Milhpore Corp , Bedford, MA), followed by diafiltration into 20 mM sodium acetate pH 5 5, 0 14 M NaCl Formulated D3H44-

F(ab')2 purity was >99 9% by an E coli protein impurity assay The endotoxin level in the formulated D3H44-F(ab')2 was <0 01 EU/mg

Construction ofChimei ic and Humanized IgG

For generation of human IgG2 and IgG4 variants of humanized D3, the humanized VL and VH domams from (F(ab)-D3H44) were subcloned separately into previously described pRK vectors (Eaton et al ,

Biochemistry 25 8343-8347 [1986]) containing the constant domams of human IgG2 or IgG4 The IgG4b variant includes a Ser H241 Pro change that improves formation of the inter-heavy chain disulfides m the hmge, resulting in a more homogeneous production of IgG4 antibody (Angal S, King DJ, Bodmer MW, Turner A, Lawson AD, Roberts G, Pedley b, Adair JR A single ammo acid substitution abolishes the heterogeneity of chimeπc mouse/human (IgG4) antibody Molec Immunol 1993,30 105-108, Bloom JW, Madanat MS, Mamot D, Wong T, Chan S-Y Intrachain disulfide bond in the core hmge region of human IgG4 Prot Sci 1997,6 407- 415) The DNA coding for the entire light and the entire heavy chain of each variant was verified by dideoxynucleotide sequencing The IgG variants were punfied using Protein A-Sepharose Construction and Punfication of IgG 1

Tissue Factor Binding Assay

Maxisorp plates (Nunc, Roskilde, Denmark) were coated overnight at 4 C with 100 μl/well of 10 μg/ml human soluble tissue factor in coat buffer (50 mM carbonate buffer, pH 9 6) The plates were blocked with 150 μl/well blocking buffer (PBS, 0 5% BSA, pH 7 2) for 1 h at room temperature The standard and samples were diluted in assay buffer (PBS, 0 5% BSA, 0 05% Tween20, pH 7 2) and incubated on the plates for

2 h at room temperature 100 μl of 1 10,000 goat anti-human F(ab)-HRP (Cappel, Costa Mesa, CA) was added and the plates were incubated for 1 h at room temperature 100 μl of the substrate 3,3',5,5'-tetramefhyl benzidine (TMB) (Kirkegaard & Perry, Gaithersburg, MD) was added After 5 mm, 100 μl of 1 M H3PO4 was added to stop the reaction The plate was washed with wash buffer (PBS, 0 05% Tween 20, pH 7 2) between each step The absorbance was read at 450 nm on a Titerek stacker reader (ICN, Costa Mesa, CA) The standard and samples were fit by Kaleidagraph 3 0 8 (Synergy Software, Reading, PA) using a four parameter fit regression The OD450 at the IC50 of the standard was determined The concentration of sample needed to obtain this OD was determined and the ratio of this value versus the IC50 of the standard was calculated BIAcore™ Biosensor Assays TF binding of the humanized and chimeπc F(ab)s were compared using a BIAcore™ biosensor

(Karlsson et al , 1994) Concentrations of F(ab)s were determined by quantitative ammo acid analysis TF was coupled to a CM-5 biosensor chip through primary amine groups according to manufacturer's instructions (Pharmacia) Off-rate kinetics were measured by saturating the chip with F(ab) (35 ml of 2 μM F(ab) at a flow rate of 20 μl min) and then switching to buffer (PBS-0 05% polysorbate 20) Data points from 0 - 4500 sec were used for off-rate kinetic analysis The dissociation rate constant (koff) was obtained from the slope of the plot of ln(R0/R) versus time, where R0 is the signal at t=0 and R is the signal at each time point

On-rate kinetics were measured using two-fold serial dilutions of F(ab) (0 0625 - 2 μM) The slope, K_s, was obtained from the plot of ln(-dR/dt) versus time for each F(ab) concentration using the BIAcore™ kinetics evaluation software as described in the Pharmacia Biosensor manual R is the signal at time t Data between 80 and 168, 148, 128, 114, 102, and 92 sec were used for 0 0625, 0 125, 0 25, 0 5, 1, and 2 μM F(ab), respectively The association rate constant (ko_n) ^was obtained from the slope of the plot of K_s versus F(ab) concentration At the end of each cycle, bound F(ab) was removed by injecting 5 μl of 50 mM HC1 at a flow rate of 20 μl/mm to regenerate the chip Bioassays Reagents

F IX was from Haematologic Technologies Inc , (Essex Jet , VT) and F X was from Enzyme Research Laboratories (South Bend, IN) Dioleoyl l ,2-dιacyl-sn-glycero-3-(phospho-L-seπne) (PS) and oleoyl 1,2- dιacyl-sn-glycero-3-phosphocholme (PC) from Avanti Polar Lipids Inc (Alabaster, AL) F IXa chromogenic substrate #299 was from American Diagnostica (Greenwich, CT) and FXa chromogenix substrate S-2765 was from Diapharma Group Inc (Columbus, Ohio) Innovin was obtained from Dade International Inc (Miami, FL) E hyleneglycol (analytical grade) was from Malhnckrodt Inc (Pans, KY) Fatty acid-free BSA was from Calbiochem (Calbiochem-Novabiochem Corp , La Jolla, CA) TF (1-234) lacking the cytoplasmic domain was produced as described (Paborsky et al , (1989) Biochemistry 28 8072, Paborsky et al , (1991) J Biol Chem

266 1911) and rehpidated with PC/PC (7 3 molar ratio) according to Mimms et al , (1981) Biochemistry 20 833- 840)

Activation ofFLXby membrane tissue factor (mTF) FVIIa complex

Membrane TF (mTF) was prepared from a human embryonic kidney cell line (293) expressing full length TF (1-263) (Kelley et al , Blood 89 3219-3227 [1997]) The cells were washed m PBS, detached with 10

7 mM EDTA and centπfuged twice (2500 rpm for 10 mm) The cell pellet (4-5x10 cells/ml) was resuspended in

Tπs, pH 7 5, and homogenized in PBS using a pestle homogemzer, followed by centnfugation (2500 rpm on a

Beckman GSA) for 40 mm at 4 C The protein concentration of the cell membrane fraction was determined and o the membranes stored in ahquots at -80 C until use Prior to the addition of F IX, the antibodies were incubated in microtiter tubes (8 8x45 mm, OPS,

Petaluma, CA) together with mTF and FVIIa in HBSA buffer (20 mM Hepes, pH 7 5, 150 mM NaCl, 5 mM CaCl2, 0 5 mg/ml BSA) for 20 mm at room temperature The final concentration in the reaction mixture for the reactants were as follows 150 μg/ml mTF (membrane protein concentration), 2 nM FVIIa and 400 nM F IX in HBSA 100 μl ahquots of the reaction mixture were taken at 30 s intervals and quenched in 96- well plates (Costar, Corning Inc , Corning, NY) containing 125 μl of 30 mM EDTA-buffer-60% (v/v) efhyleneglycol

After adding 25 μl of 5 mM F IX substrate #299, F IXa amidolytic activity was measured at 405 nm on a kinetic microplate reader (Molecular Devices, Menlo Park, CA) Inhibition by the tested antibodies was expressed as fractional rates (vi vo) of F IXa generation

Activation ofFXby mTF FVIIa complex The experiments were carried out m a similar fashion as described for F IX activation The concentration in the reaction mixture of the reactants were as follows 200 nM FX, 150 μg/ml mTF, 30 pM FVIIa in HBSA At 30 s intervals, 50 μl ahquots were quenched in 150 μl 20 mM EDTA and the FXa amidolytic activity measured by adding 50 μl of 1 5 mM S-2765 RESULTS Transplanting the murine D3 CDRs onto the human framework (VLK subgroup I, VH subgroup III)

(Carter et al , Proc Natl Acad Sci USA 89 4285-4289 [1992], Presta et al , Cancer Res 57 4593-4599 [1997]) resulted in a F(ab) which lacked binding to human TF Based on the computer graphic model of murine D3 F(ab) (Fig 7), several amino acid residues in the CDRs as well as framework region of light and heavy chains were altered using site-directed mutagenesis in order to optimize antigen binding The engineered antibody thus evolved, D3H44 F(ab), exhibited acceptable binding and efficacy in all of the biological assays, including the prothrombin tune assays Figs 10-12 D3H44 has four human-to-murme changes in its heavy chain framework Gly H49, Ala H67, Ala H71 , and Ala H78 (Fig 8) D3H44 also has one human-to-muπne change in its light chain framework, Tyr L71 , as well as one change which is neither human nor murine, Val L46 In the CDRs, D3H44 has seven differences from the murine D3 parent Glu H31 (CDR-Hl), Leu H50 and Gin H54 (CDR-H2), Arg L24 and Asn L34 (CDR-L1), Glu L56 (CDR-L2), and Trp L96 (CDR-L3) (Figs 8, 9)

Since a crystal structure of huTF-TF8-5G9 (Huang et al , J Mol Biol 275 873-894 [1998]) was available in the public Protein Data Bank crystal structure database (coordmates PDB1AHW), the effect of alteπng some of the sequence of the chimenc D3 F(ab) to that of TF8-5G9 was investigated First, three residues in CDR-H3 were altered D3Ch Thr96-Ala97-Ala98 to TF8 Asn96-Ser97-Tyr98 This resulted in a 20- fold reduction m binding (20 3±0 69, n=2) Given that these CDR-H3 residues interact with huTF in the crystal structure, the severe reduction in binding was unexpected Second, in CDR-H2 D3Ch Asp H65 was changed to TF8 Gly, bmdmg was reduced by 14-fold (14 2±2 7, n=3) Inspection of the huTF-TF8 crystal structure shows that residue H65 is not in contact with huTF and the change to Gly should not have affected bindmg Taken together, these data suggest that the D3 antibody does not bind to huTF in the same manner as TF8-5G9

Binding of anti-tissue factor antibodies (IgGl, IgG2, IgG4 and IgG4b) to tissue factor is shown in Figure 16 Each of E coli produced IgGl and CHO produced IgG2, IgG4 and IgG4b bound immobilized TF

Inhibition of the rates of F X and F IX activation by full length versions and a F(ab')2 version of the D3H44 antibody are shown in Table 3

Table 3

Antibodies were incubated with rehp TF (1-234) (0 04 nM) and F Vila (0 04 nM) for 20 mm and the reaction started by adding F X (200 nM) Ahquots were taken at different time points and quenched in EDTA In the second stage of the assay, F Xa activity was measured by adding chromogenic substrate S2765 and monitoring absorbance at 405 nM on a kinetic microplate reader IC50 values were calculated by non-linear curve fitting using fractional activities (vi/vo) of initial substrate activation rates vs antibody concentration For F IX assays the concentration of reactants was 1 nM rehp TF( 1-234), 1 nM FVIIa, 400 nM F IX Reaction ahquots were quenched in EDTA-60% (v/v) efhyleneglycol In the second stage of the assay, F IXa activity was measured by addmg chromogenic substrate #299 and momtonng absorbance at 405 nM on a kinetic microplate reader IC50 values were calculated as described above for F X

Prolongation of clotting time for the full length versions and Fab and F(ab')2 versions of D3H44 are shown in Figure 17

All references cited throughout the specification, including the examples, and all references cited therein are hereby expressly incorporated by reference

Claims

WHAT IS CLAIMED IS

1 A method for identifying anti-tissue factor (anti-TF) antibodies with enhanced anticoagulant potency, compnsing (a) subjecting a plurality of anti-TF antibodies to epitope mapping, and (b) selecting from said plurality, antibodies binding to an epitope comprising at least part of the C-termmal macromolecular substrate-binding region of tissue factor (TF)

2 The method of claim 1 wherein said TF is human (hTF)

3 The method of claim 2 wherem said macromolecular substrate is Factor X (F X) or Factor IX (F IX)

4 The method of claim 3 wherem said epitope includes an hTF region directly interacting with F X or F IX

5 The method of claim 4 wherein said region includes residues interacting with a Gla domain of

6 The method of claim 2 wherein said epitope comprises residues K165, K166 and K201 of hTF 7 The method of claim 6 wherein said epitope additionally compnses residues N199, R200 and

1152 of hTF

8 The method of claim 6 wherein said epitope additionally comprises residue Y 156 of hTF

9 The method of claim 2 wherein at least one antibody selected binds essentially to the same hTF epitope as an antibody selected from the group consisting of D3, 5G6, and TF8-5G9

10 The method of claim 2 wherein at least one antibody selected binds essentially to the same hTF epitope as antibody D3

11 The method of claim 2 wherein at least one antibody selected binds essentially to the same hTF epitope as antibody 5G6 12 The method of claim 2 wherein at least one antibody selected does not interfere with the hTF-

Factor Vila (FVIIa) association

13 The method of claim 2 wherein said antibodies are humanized

14 The method of claim 2 wherein said antibodies are human

15 A composition compnsing an antibody identified according to the method of claim 2, in admixture with a pharmaceutically acceptable earner

16 A method for blocking a TF-FVIIa associated process or event, comprising administering an antibody identified according to the method of claim 2

17 A method of treating a TF-VIIa related disease or disorder, comprising administering to an individual an effective amount of an anti-TF antibody identified according to the method of claim 2 18 The method of claim 17 wherein said disease or disorder is a thrombotic or coagulopathic disorder

19 An anti-tissue factor (anti-TF) antibody heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 1 or SEQ ID NO 2 20 An anti-tissue factor (anti-TF) light chain vaπable domain compnsing the amino acid sequence of SEQ ID NO 3 or SEQ ID NO 4

21 An anti-tissue factor (anti-TF) heavy chain variable domain comprising the ammo acid sequence of SEQ ID NO 5 22 An anti-tissue factor (anti-TF) light chain variable domain compnsing the amino acid sequence of SEQ ID NO 6

23 An isolated nucleic acid comprising a sequence encoding an anti-TF antibody heavy chain variable domain of SEQ ID 1 , 2, or 5

24 An isolated nucleic acid compnsing a sequence encoding an anti-TF antibody light chain vaπable domain of SEQ ID NO 3, 4, or 6

25 A humanized anti-tissue factor (anti-TF) antibody comprising a heavy and a light chain vanable domain, wherem said heavy chain variable domain compπses hypervanable regions CDR-Hl having the sequence of GFNIKEYYMH (SEQ ID NO 7), CDR-H2 having the sequence of LIDPEQGNTIYDPKFQD (SEQ ID NO 8) and CDR-H3 having the sequence of DTAAYFDY (SEQ ID NO 9) 26 The humanized anti-TF antibody of claim 25 wherein said hypervanable regions are provided in a human framework region

27 The humanized anti-TF antibody of claim 26 comprising a heavy chain variable domain of SEQ ID NO 2

28 The humanized anti-TF antibody of claim 25 wherein said light chain variable domain comprises hypervaπable regions CDR-L1 havmg the sequence of RASRDIKSYLN (SEQ ID NO 10), CDR-L2 having the sequence of YATSLAE (SEQ ID NO 11) and CDR-L3 having the sequence of LQHGESPWT (SEQ ID NO 12)

29 The humanized anti-TF antibody of claim 28 wherein said hypervaπable regions are provided in a human framework region 30 The humanized anti-TF antibody of claim 29 comprising a light chain variable domain of SEQ

ID NO 4

31 An antibody selected from the group consisting of (a) murine antibody D3 (D3Mur), (b) humanized antibody D3H44, (c) muπne antibody 5G6, and (d) an antibody comprises essentially the same hypervanable regions as any one of antibodies of (a) - (c) 32 An isolated nucleic acid molecule comprising a sequence encoding a humanized antibody heavy chain variable domain compnsing hypervaπable regions CDR-Hl having the sequence of GFNIKEYYMH (SEQ ID NO 7), CDR-H2 having the sequence of LIDPEQGNTIYDPKFQD (SEQ ID NO 8) and CDR-H3 having the sequence of DTAAYFDY (SEQ ID NO 9), or humanized antibody light chain variable domain compnsing hypervaπable regions CDR-L1 having the sequence of RASRDIKSYLN (SEQ ID NO 10), CDR-L2 having the sequence of YATSLAE (SEQ ID NO 11) and CDR-L3 having the sequence of

LQHGESPWT (SEQ ID NO 12)

33 A vector comprising and capable of expressing the nucleic acid of claim 32

34 A recombmant host cell transformed with a vector of claim 33

35. A method of producing a humanized antibody heavy or light chain comprising expressing, in a recombinant host cell, a nucleic acid encoding an antibody heavy or light chain and comprising the hypervariable regions of claim 32.

36. The method of claim 35 wherein nucleic acid molecules encoding an antibody heavy and light chains are coexpressed in said recombinant host cell.

37. A composition comprising an anti-tissue factor (anti-TF) antibody identifiable by the method of claim 1, in admixture with a pharmaceutically acceptable carrier.

38. The composition of claim 37 wherein said antibody is an anti-human TF (anti-hTF) antibody.

39. The composition of claim 38 wherein said antibody is humanized. 40. A composition comprising an antibody selected from the group consisting of (a) murine antibody D3 (D3Mur), (b) humanized antibody D3H44, (c) murine antibody 5G6, and (d) an antibody comprising essentially the same hypervariable regions as any one of antibodies (a)-(c), in admixture with a pharmaceutically acceptable carrier.

41. A method for the prevention or treatment of a TF-FVIIa related disease or disorder, comprising administering to a subject an effective amount of an anti-tissue factor (anti-TF) antibody of claim 25.

42. The method of claim 41 wherein said disorder is a thrombotic or coagulopathic disorder.

43. The method of claim 42 wherein said disorder is selected from the group consisting of: deep venous thrombosis, arterial thrombosis, stroke, tumor metastasis, thrombolysis, arteriosclerosis and restenosis following angioplasty, acute and chronic indications such as inflammation, septic shock, septicemia, hypotension, adult respiratory distress syndrome (ARDS), and disseminated intravascular coagulopathy (DIC).