EP1263960A2 - Anti-tissue factor antibodies with enhanced anticoagulant potency - Google Patents

Anti-tissue factor antibodies with enhanced anticoagulant potency

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Publication number
EP1263960A2
EP1263960A2 EP01924131A EP01924131A EP1263960A2 EP 1263960 A2 EP1263960 A2 EP 1263960A2 EP 01924131 A EP01924131 A EP 01924131A EP 01924131 A EP01924131 A EP 01924131A EP 1263960 A2 EP1263960 A2 EP 1263960A2
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EP
European Patent Office
Prior art keywords
antibody
antibodies
seq
sequence
fviia
Prior art date
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EP01924131A
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German (de)
English (en)
French (fr)
Inventor
Daniel K. Kirchhofer
David G. Lowe
Leonard G. Presta
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Genentech Inc
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Genentech Inc
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Publication of EP1263960A2 publication Critical patent/EP1263960A2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'

Definitions

  • 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 comp ⁇ sing the antibodies and their use in the diagnosis, management, prevention and treatment of va ⁇ ous diseases and disorders Desc ⁇ ption of the Related Art
  • a Tissue factor Tissue factor (TF) is the receptor for coagulation factor Vila (FVIIa) and the zymogen precursor factor
  • hTF Native human TF
  • the TF extracellular domain is composed of two immunoglobulin-like f ⁇ bronectin 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 ⁇ -sheets with Ig superfamily type C2 homology
  • 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 ( ⁇ G) of binding to FVIIa (Kelley et al , (1995) supra)
  • Substitution of Lys20 and Asp58 with alanine residues leads to 78- and 30- fold reductions in FVIIa affinity respectively
  • Lysl65Ala-Lysl66Ala variant 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
  • sTF Lysl65Ala-Lysl66Ala variant
  • the va ⁇ ant partially blocks thrombus formation without increasing bleeding tendency (Kelley et al , (1997) Blood 89, 3219-3227)
  • high doses of the va ⁇ ant 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 ⁇ -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 Fib ⁇ nol 4 281-292) Its expression on endothehal cells and monocytes is induced by exposure to inflammatory cytokines or bacterial hpopolysaccha ⁇ de (Drake et al ,
  • FVII can be activated by peptide bond cleavage to yield se ⁇ ne 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 Fib ⁇ nol 4 281-292, Davie, E W et al , [1991] Biochemistry 30 10363-10370, Rapaport, S I and L V M Rao, [1992] Arte ⁇ oscler Thromb 12 11 11-1121)
  • the complex initiates the ext ⁇ nsic pathway by activation of FX to Factor Xa (FXa), FIX to Factor IXa (FIXa), and additional FVII to FVIIa
  • FXa Factor Xa
  • FIXa FIX to Factor IXa
  • FVIIa Factor IXa
  • the action of TF-FVIIa leads ultimately to the conversion of pro thrombin to thrombm, which carries out many biological functions (Badimon,
  • 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 va ⁇ ous primate and non- p ⁇ mate 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 endo
  • 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
  • FVIIa factor Vila
  • the cell surface exposed TF immobilizes FVII/FVIla to the cell membrane thereby stabilizing the overall conformation of FVIIa
  • 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 F
  • the present invention is based in part on the expe ⁇ mental 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
  • 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)
  • tissue factor is preferably human
  • the macromolecular substrate preferably is Factor X (FX) or Factor IX (FIX)
  • 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
  • the antibody selected binds an epitope comp ⁇ sing residues K165, K166 and K201 of hTF
  • the epitope further comprises residues N199, R200 and 1152 of hTF
  • the epitope further comprises residues N199, R200 and
  • the invention concerns an anti-tissue factor (anti-TF) antibody heavy chain va ⁇ able 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)
  • the invention concerns an anti-tissue factor (anti-TF) light chain va ⁇ able 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)
  • 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)
  • 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)
  • 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
  • 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
  • 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
  • anti-TF humanized anti-tissue factor
  • 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 hyperva ⁇ able 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)
  • the humanized anti-TF antibody of the present invention has a light chain variable domain comprising hyperva ⁇ able regions CDR-L1 having the sequence of RASRDIKSYLN (
  • 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)
  • 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
  • the invention concerns a composition
  • 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 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
  • 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 t ⁇ angle) lsotype-matched control
  • PT prothrombin time
  • 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 t ⁇ angle) 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 mu ⁇ ne 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
  • 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 va ⁇ able 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
  • 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 thrombo
  • 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
  • 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
  • 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
  • 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)
  • FX macromolecular substrate Factor X
  • FIX Factor IX
  • 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 ⁇ -strands ⁇ 8 A to ⁇ 16 0 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
  • epitopope 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"
  • epitope mapping There are many methods known in the art for mapping and characte ⁇ zmg 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
  • 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 label
  • an antibody binds "essentially the same epitope" as a reference antibody, when the two antibodies recognize identical or ste ⁇ cally overlapping epitopes.
  • competition assays which can be configured in all number of different formats, using either labeled antigen or labeled antibody
  • 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.
  • amino acid or amino acid residue refers to naturally occurring L amino acids or to D amino acids as described further below with respect to variants.
  • 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 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.
  • 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.
  • Blooding 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,
  • 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, mu ⁇ ne, 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
  • treatment is an approach for obtaining beneficial or desired clinical results
  • 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 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
  • 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 are glycoproteins having the same structural characte ⁇ stics 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 heterotetrame ⁇ c 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
  • va ⁇ able 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
  • the variability is not evenly distributed throughout the variable domains of antibodies It is concentrated in three segments called hyperva ⁇ able regions both in the light chain and the heavy chain va ⁇ able doma s
  • the more highly conserved portions of va ⁇ able domains are called the framework region (FR)
  • the va ⁇ able domains of native heavy and light chains each comp ⁇ se four FRs (FR1 , FR2, FR3 and FR4, respectively), largely adopting a ⁇ -sheet configuration, connected by three hyperva ⁇ able regions, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure
  • the hyperva ⁇ able regions in each chain are held together in close proximity by the FRs and, with the hyperva ⁇ able regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al , Sequences of Proteins
  • hyperva ⁇ able region when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding
  • the hyperva ⁇ able 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,
  • CDR complementarity determining region
  • 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 hyperva ⁇ able regions of each variable domain interact to define an antigen-bmding site on the surface of the V H -V L dimer
  • the six hyperva ⁇ able regions confer antigen-binding specificity to the antibody
  • 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 cyste ⁇ ne(s) from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteine res ⁇ due(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 ( ⁇ ), based on the amino acid sequences of their constant domams
  • immunoglobulins can be assigned to different classes There are five major classes of immunoglobulins IgA, IgD, IgE, IgG, and
  • IgM immunoglobulin
  • subclasses e g , IgGl , IgG2, IgG3, IgG4, IgAl, and IgA2
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , and ⁇ , respectively
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known
  • antibody herein is used in the broadest sense and specifically covers monoclonal antibodies
  • Antibody fragments comprise a portion of a full length antibody, generally the antigen binding or variable domain thereof
  • antibody fragments include Fab, Fab', F(ab , and Fv fragments, diabodies, linear antibodies, single-cham antibody molecules, and multispecific antibodies formed from antibody fragments
  • monoclonal antibody 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
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hyb ⁇ doma 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 ⁇ / , J Mol Biol 222 581-597 (1991), for example
  • 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 ⁇ / , J Mol Biol 222 581-597 (1991), for example
  • the monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al
  • humanized forms of non-human (e g , murine) antibodies are chime ⁇ c antibodies which contain minimal sequence denved from non-human lmmunoglobuhn
  • humanized antibodies are human immunoglobulins (recipient antibody) in which hyperva ⁇ able region residues of the recipient are replaced by hyperva ⁇ able region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity
  • framework region (FR) residues of the human immunoglobulm are replaced by corresponding non-human residues
  • humanized antibodies may compnse residues which are not found in the recipient antibody or in the donor antibody
  • 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 comp ⁇ ses a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding
  • sFv see Pluckthun m The Pharmacology of Monoclonal Antibodies, vol 1 13,
  • 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 )
  • V H heavy chain vanable domain
  • V L light chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites
  • Diabodies are desc ⁇ bed more fully in, for example, EP 404,097, WO 93/11161, and Holhnger et al , Proc Nat! Acad Sci USA 90 6444-6448 (1993)
  • 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 H 1-V H -C H 1) which form a pair of antigen binding regions
  • Linear antibodies can be bispecific or monospecific
  • 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 lntrape ⁇ toneal 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 hyb ⁇ doma 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)
  • 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 hyb ⁇ doma cell (Goding, Monoclonal Antibodies Principles and Practice, pp 59-103, [Academic Press, 1986])
  • the hyb ⁇ doma 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
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells
  • the culture medium for the hyb ⁇ domas typically will include hypoxanthine, ammopte ⁇ n, 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
  • 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 hyb ⁇ doma cells are growing is assayed for production of monoclonal antibodies directed against the
  • 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)
  • hyb ⁇ doma cells After hyb ⁇ doma 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.
  • the hyb ⁇ doma 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 hyb ⁇ doma cells serve as a preferred source of such DNA
  • 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 Recomb
  • 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
  • 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 ar ⁇ ve 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 prefe ⁇ ed locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells Science, 244 1081-1085 (1989)
  • 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
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined
  • ala scanning or random mutagenesis is conducted at the target codon or region and the expressed anti-TF antibody variants are screened for the
  • 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
  • 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)
  • 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 hyperva ⁇ able 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
  • 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 occur ⁇ ng residues are divided into groups based on common side-chain properties
  • 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 se ⁇ ne, 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 hyperva ⁇ able region residues of a parent antibody (e g a humanized or human antibody)
  • a parent antibody e g a humanized or human antibody
  • the resulting va ⁇ ant(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 B ⁇ efly, several hyperva ⁇ able 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
  • alanine scanning mutagenesis can be performed to identify hyperva ⁇ able region residues contributing significantly to antigen binding Alternatively, or m addition, it may be beneficial to
  • N-hnked or O-hnked N-hnked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue
  • the t ⁇ peptide 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
  • O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly se ⁇ ne or threonine, although 5-hydroxyprol ⁇ ne or 5-hydroxylys ⁇ ne may also be used
  • 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 occur ⁇ ng amino acid sequence variants) or preparation by ohgonucleotide- mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared va ⁇ ant or a non- va ⁇ ant version of the anti-TF antibody (v) Human antibodies
  • Human antibodies can be produced using va ⁇ ous 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
  • 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 Mo ⁇ moto 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
  • bispecific antibodies may bind to two different epitopes of the TF protein
  • an anti-TF arm may be combined with an arm which binds to a t ⁇ ggenng molecule on a leukocyte such as a T-cell receptor molecule
  • 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 , sapo ⁇ n, anti-interferon- ⁇ , vmca alkaloid, ⁇ cin 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)
  • 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 H 3 domain of an antibody constant domain.
  • 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 cha ⁇ n(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
  • 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'
  • 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
  • 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)
  • 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
  • sFv single-cham Fv
  • the bispecific antibody may be a "linear antibody" produced as described m Zapata et al Protein Eng 8( 10) 1057-1062 (1995)
  • t ⁇ specific 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 res ⁇ due(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation m this region The homodime ⁇ c 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
  • Homodime ⁇ c 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)
  • 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)
  • 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)
  • 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 uginos ⁇ ), ⁇ cm A chain ab ⁇ n A chain, modeccm A chain, ⁇ -sarcm, Aleurites foi du proteins, dianthm proteins, Phytolaca amei icana proteins (PAPI, PAPII, and PAP-S) momordica charantia inhibitor, curc , crotm, sapaona ⁇ a officmahs inhibitor, gelomn, mitogelhn.
  • PAPI Phytolaca amei icana proteins
  • radionuchdes are available for the production of radioconjugated anti-TF antibodies Examples include 2P B ⁇ , 1 I I, l In, 90 Y and 1 86 Re
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succm ⁇ m ⁇ dyl-3-(2-py ⁇ dyld ⁇ th ⁇ ol) 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 b ⁇ s-(p-d ⁇ azon ⁇ umbenzoyl)-ethylened ⁇ am ⁇ ne), diisocyanates (such as tolyene 2,6- dusocyanate), and bis-active fluo ⁇ ne compounds (such as l,5-d ⁇ fluoro-2,4-dm ⁇ trobenzene)
  • 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)
  • a "receptor” such as streptavidin
  • 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-de ⁇ vatized 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!
  • chemotherapeutic agent such as Doxorubtcin
  • a chemotherapeutic agent 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
  • a prodrug e g , a peptidyl chemotherapeutic agent, see WO81/01 1405
  • 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-fluorourac ⁇ l, 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.
  • alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs
  • arylsulfatase useful
  • carbohydrate-cleaving enzymes such as ⁇ -galactosidase and neuramimdase useful for converting glycosylated prodrugs into free drugs, ⁇ -lactamase useful for converting drugs de ⁇ vatized with ⁇ -lactams into free drugs, and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs de ⁇ vatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs
  • 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,
  • 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])
  • an antibody fragment rather than an intact antibody, to increase tumor penetration, for example
  • 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
  • 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
  • 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 de ⁇ vatizmg 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 comp ⁇ ses 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
  • the nucleic acid encoding it may be isolated and inserted into a rephcable vector for further cloning (amplification of the DNA) or for expression
  • 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 eubacte ⁇ a, such as Gram-negative or Gram-positive organisms, for example, Enterobacte ⁇ aceae such as Escherichia, e g , E coli,
  • 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
  • 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
  • Saccharomyces cerevisiae or common baker's yeast
  • Schizosaccharomyces pombe 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, dL ⁇ & 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
  • invertebrate cells include plant and insect cells
  • Numerous baculoviral strains and variants and co ⁇ esponding 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 va ⁇ ety 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
  • 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)
  • 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, transfer ⁇ n, 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 GENTAMYCINTM 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
  • hormones and/or other growth factors such as insulin, transfer ⁇ n, or epidermal growth factor
  • salts such as sodium chloride calcium magnesium, and
  • 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
  • the antibody can be produced intracellularly, in the pe ⁇ plasmic 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 cent ⁇ fugation or ultrafiltration Carter et al Bio/Technologv 10 163 167 (1992) describe a procedure for isolating antibodies which are secreted to the pe ⁇ plasmic space of E coh Briefly cell paste is thawed in the presence of sodium acetate (pH 3 5) EDTA and phenylmethylsulfonylfluo ⁇ de (PMSF) over about 30 mm Cell debris can be removed by cent ⁇ fugation 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
  • the mat ⁇ x 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(styrened ⁇ v ⁇ nyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose
  • the Bakerbond ABXTM resin J T Baker, Phillipsburg, NJ
  • Other techniques for protein punfication such as fractionation on an ion- exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on hepa ⁇ n SEPHAROSETM 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 subjecte
  • 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).
  • 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 chlo ⁇ de, hexamethomum chloride, benzalkomum chloride, benzethonium chlo ⁇ de, 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 immunoglobulin
  • 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)
  • 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
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(v ⁇ nylalcohol)), polylactides (U S Pat No 3,773,919), copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene- vmyl acetate, degradable lactic acid-glycohc acid copolymers such as the Lupron DepotTM (injectable microspheres composed of lactic acid-glycohc acid copolymer and leu
  • 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
  • the antibodies of the invention may be used as affinity purification agents
  • 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
  • Radioisotopes such as 35 S, 1 C, l25 I, ⁇ , and 13I I
  • 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
  • Fluorescent labels such as rare earth chelates (europium chelates) or fluorescem and its derivatives, rhodamme and its derivatives, dansyl, Lissamine, phycoeryth ⁇ n 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 fluo ⁇ meter
  • the enzyme generally catalyzes a chemical alteration of the chromogenic substrate which can be measured using various techniques
  • the enzyme may catalyze a color change in a substrate, which can be measured spectrophotomet ⁇ cally
  • 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
  • enzymatic labels include luciferases (e g , firefly luciferase and bacterial luciferase, U S Patent No 4,737,456), lucife ⁇ n, 2,3-d ⁇ hydrophthalaz ⁇ ned ⁇ ones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, sacchande oxidases (e g , glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocychc oxidases (such as uncase and xanthme oxida
  • enzyme-substrate combinations include, for example
  • the label is indirectly conjugated with the antibody
  • 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
  • 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)
  • an anti- hapten antibody e g , anti-digoxm antibody
  • 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)
  • 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
  • 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
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich sandwich
  • 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, ⁇ , I, P5 1, 3 H, 32 P or 35 S) so that the tumor can be localized using lmmunoscintiography
  • a radio nuchde such as '"In, "Tc, l C, ⁇ , I, P5 1, 3 H, 32 P or 35 S
  • 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
  • the kit will include substrates and cofactors required by the enzyme (e g , a substrate precursor which provides the detectable chromophore or fluorophore)
  • substrates and cofactors required by the enzyme e g , a substrate precursor which provides the detectable chromophore or fluorophore
  • 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
  • the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropnate concentration
  • 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, lntrape ⁇ toneal, intra-cerebrospinal, subcutaneous, lntra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes
  • the antibodies also are suitably administered by intra tumoral, pe ⁇ tumoral, lntralesional, or penlesional routes, to exert local as well as systemic therapeutic effects
  • the intrape ⁇ toneal route is expected to be particularly useful, for example, in the treatment of ovarian tumors
  • 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
  • diseases or disorders include, for example, chronic thromboembolic diseases or disorders associated with fib ⁇ n 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 du ⁇
  • about 1 ⁇ g/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 ⁇ g/kg to about 20 mg/kg or more, depending on the factors mentioned above
  • the treatment is repeated until a desired suppression of disease symptoms occurs
  • 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
  • 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 hepa ⁇ n, low molecular weight hepa ⁇ n and or inhibitors of platelet glycoprotein Ilbllla, and or couma ⁇ n 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, py ⁇ midme analogs, 5-fluorourac ⁇ l, cisplatm, pu ⁇ ne nucleosides, amines, ammo acids, t ⁇ azol nucleosides, or corticosteroids
  • the antibody is suitably administered serially or in combination with radiological treatments, whether involving irradiation or administration of radioactive substances.
  • an article of manufacture containing mate ⁇ als useful for the treatment of the disorders described above 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 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
  • 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 pnma ⁇ ly 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
  • 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)
  • 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 m
  • Prothrombin time (PT) assays antibody was added to citrated human plasma After 5m ⁇ n 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)
  • 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)
  • a 7G1 1 antibody column was used This column was prepared by coupling the 7G11 antibody to CNBr-activated Sepharose 4B
  • 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)
  • SPR surface plasmon resonance
  • 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
  • the same sTF concentration series was prepared m the presence of 5 ⁇ M human, recombinant FVIIa These solutions were incubated at ambient temperature for 30 minutes pnor to injection
  • Monoclonal antibody 7G1 1 was generated by immunizing female BALB/c mice subcutaneously 3 times, lntrape ⁇ toneally 3 times with 20 ⁇ g sTF in MPL/TDM adjuvant (Ribi Immunochem Research, Hamilton, MT), at 2 week intervals These mice were further boosted 8 times into footpads with lO ⁇ g sTF m lOOul
  • MPL/TDM Adjuvant every week 5G6 was generated by immunizing female BALB/c mice via footpad with lO ⁇ g 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] Cu ⁇ Top Microbiol Immunol 81 1-7) using 35% polyefhyleneglycol as described (Chuntharapai and Kim, [1997] Methods Enzymol 288 15-27) Hyb ⁇ doma cell lines secreting antibody specific for sTF, as determined by
  • the antibodies were diluted in human citrated plasma from a donor plasma pool (Peninsula Blood Bank, Burhngame, CA) for lOmin at room temperature
  • the thrombin inhibitor napsagatran (Hilpert et al , [1994] J Med Chem 37 3889-3901 , Gast and Tschopp [1995] Blood Coag Fib ⁇ nolysis 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 TF ⁇ _243, napsagatran and CaCl2 were 0 4nM, 0 5 ⁇ M and 5mM, respectively 50 ⁇ l ahquots taken at 15sec intervals were quenched in 150 ⁇ l of 20mM EDTA In the second stage, 50 ⁇ l of 1 5mM
  • Tissue factor-expressing human J82 cells epidermal carcinoma, ATCC HTB1
  • epidermal carcinoma ATCC HTB1
  • 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
  • DMEM-1% (w/v) BSA The details of the experimental system were described recently (Kirchhofer et al , [1995] supra,
  • 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.
  • 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
  • the 7G11 binding site is formed by a clearly defined patch of surface exposed residues with a calculated solvent accessible
  • 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
  • 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
  • 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
  • VL and VH domains 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 mu ⁇ ne 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])
  • the plasmid contains a DNA fragment encoding a consensus human K subgroup I light chain (VL ⁇ I-CL), a consensus human subgroup III heavy chain (VHIII- CH1) and an alkaline phosphatase promoter
  • VL ⁇ I-CL consensus human K subgroup I light chain
  • VHIII- CH1 consensus human subgroup III heavy chain
  • alkaline phosphatase promoter The use of the consensus sequences for VL and VH has been described previously (Carter et al , supra)
  • F(ab)-1 therefore consisted of a complete human framework (VLU K subgroup I and VH subgroup III) with the six complete mu ⁇ ne CDR sequences
  • Plasmids for all other F(ab) variants were constructed from the plasmid template of F(ab)-1 Plasmids were transformed lnto ⁇ 1 coli strain XL-1 Blue (Stratagene, San Diego, CA) for preparation of double- and smgle-stranded DNA
  • 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 ⁇ g/ml carbenicilhn, and a single colony selected for protein expression
  • Sealkeen filter Cells were harvested by cent ⁇ fugation 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 T ⁇ s-1 mM EDTA- 20%sucrose, pH 8 0 250 ⁇ l 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 cent ⁇ fuged 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 T ⁇ s-1 mM EDTA, pH 7 5 The column was washed with 10 ml of 10 mM T ⁇ s-1 mM EDTA, pH 7 5, and eluted with 3 ml 0 3 M gly
  • 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 (h ⁇ s)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 cent ⁇ fugation (4300xg, 30m ⁇ n) to remove cellular debris The supernatant was filtered (0 2 ⁇ m) and loaded on to SP Sepharose FF (Amersham Pharmacia
  • 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
  • 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 chime ⁇ c 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
  • F IX was from Haematologic Technologies Inc , (Essex Jet , VT) and F X was from Enzyme Research Laboratories (South Bend, IN) Dioleoyl l ,2-d ⁇ acyl-sn-glycero-3-(phospho-L-se ⁇ ne) (PS) and oleoyl 1,2- d ⁇ acyl-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
  • Malhnckrodt Inc Pans, KY
  • Fatty acid-free BSA was from Calbiochem (Calbiochem-Novabiochem Corp , La Joll
  • Membrane TF 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
  • 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 ⁇ g/ml mTF (membrane protein concentration), 2 nM FVIIa and 400 nM F IX in HBSA 100 ⁇ l ahquots of the reaction mixture were taken at 30 s intervals and quenched in 96- well plates (Costar, Corning Inc , Corning, NY) containing 125 ⁇ l of 30 mM EDTA-buffer-60% (v/v) efhyleneglycol
  • 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
  • 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-mu ⁇ ne change in its light chain framework, Tyr L71 , as well as one change which is neither
  • 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
  • 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
  • F IXa activity was measured by addmg chromogenic substrate #299 and momtonng absorb

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US6904707B2 (en) 2003-07-01 2005-06-14 Softspikes, Llc Indexable shoe cleat with improved traction

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JP2003527861A (ja) 2003-09-24
CA2402596A1 (en) 2001-09-27
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AU5081401A (en) 2001-10-03
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