EP0668875A1 - Auf blutgerinnsel gerichtetes antikoagulanz, verfahren zur herstellung desselben und zu seiner verwendung - Google Patents

Auf blutgerinnsel gerichtetes antikoagulanz, verfahren zur herstellung desselben und zu seiner verwendung

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Publication number
EP0668875A1
EP0668875A1 EP93922464A EP93922464A EP0668875A1 EP 0668875 A1 EP0668875 A1 EP 0668875A1 EP 93922464 A EP93922464 A EP 93922464A EP 93922464 A EP93922464 A EP 93922464A EP 0668875 A1 EP0668875 A1 EP 0668875A1
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EP
European Patent Office
Prior art keywords
clot
targeting
anticoagulant
inhibitor
molecule
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93922464A
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English (en)
French (fr)
Other versions
EP0668875A4 (de
Inventor
Paul Eisenberg
Dennis Brian Rylatt
Carmel Judith Hillyard
Peter Gregory Bundesen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Agen Ltd
Original Assignee
Agen Ltd
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Publication date
Application filed by Agen Ltd filed Critical Agen Ltd
Publication of EP0668875A1 publication Critical patent/EP0668875A1/de
Publication of EP0668875A4 publication Critical patent/EP0668875A4/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a clot-targeting, anticoagulant molecule, a clot- targeting, anticoagulant binding molecule, compositions for treating a clot in a mammal, methods of treating a clot in a mammal, and kits for treating a clot in a mammal.
  • Blood coagulation is the result of a complex series of events ultimately leading to the proteolytic conversion of circulating soluble fibrinogen to insoluble fibrin. Fibrin formation is a normal physiological response to vascular tissue injury of various aetiology. If the thrombotic process is overwhelming or persistent, partial or total vascular occlusion may result.
  • the clinical consequences of thrombosis include: coronary artery disease (e.g. myocardial ischaemia), cerebral ischaemia (e.g. transient cerebral ischaemic attacks), or peripheral vascular disease (e.g. deep venous thrombosis, peripheral artery occlusion). Atherosclerosis and its complications are the prime cause of death in western society.
  • the atherosclerotic process is associated with extracellular lipid uptake and accumulation within fibroblasts and smooth muscle cells of lipid, resulting in the pathological lesion referred to as an atherosclerotic plaque or atheroma .
  • the factors that promote the development of atherosclerotic plaques are unknown; however, plasma hyperlipaemia, diabetes, hypertension, and cigarette smoking are known risk factors.
  • the natural history of thrombosis is dependent on a balance between procoagulant activity at the site of the thrombus and fibrinolytic activity, which induces dissolution of the thrombus.
  • Clot-associated procoagulant activity is supported by the recruitment of circulating coagulation factors and their activation on the surface of the clot. There is a need for an agent which can prevent clots and which is effective in reducing mortality associated with thrombotic disease, and which is not also associated with untoward bleeding side effects.
  • An object of this invention is to provide a clot-targeting, anticoagulant molecule.
  • compositions for treating a clot in a mammal methods of treating a
  • the inventors realised that it is factor Xa associated with or entrapped within the clot which induces prothrombin activation, thereby increasing local thrombin concentrations, which in turn provides the focus for extension of the thrombus particularly when this enzyme is subsequently exposed by the process of clot dissolution it could easily initiate clot reformation depending on the local balance of fibrinolytic activators and inhibitors.
  • whole-blood clots particularly when platelet rich, induce activation of the coagulation system by at least two phenomena: one is ascribable to the activity of thrombin bound to fibrin, and the other to clot-associated Xa activity.
  • Both clot-associated thrombin and Factor Xa appear to be relatively protected from inhibition by anti-thrombin III or heparin-anti-thrombin III.
  • Factor Xa activity associated with whole-blood clots induces thrombin formation de novo, even when clot-bound thrombin is inhibited.
  • the aim of the present invention is to provide a high concentration of an anticoagulant agent at the surface of a clot.
  • Clot-targeted anticoagulants provide a novel approach for preventing the local progression of clotting while not inhibiting circulating clotting activity.
  • This novel approach has particular applicability to the treatment of coronary thrombosis after angioplasty or thrombolysis, conditions in which the high doses of conventional anticoagulants required to prevent reformation of the coronary clot result in a high incidence of bleeding complications.
  • a clot-targeting, anticoagulant molecule comprising a clot-targeting binding molecule coupled to an anticoagulant.
  • the clot-targeting, anticoagulant molecule may further comprise at least one thrombolytic coupled to the clot binding molecule or may further comprise a thrombolytic coupled to the anticoagulant.
  • the clot-targeting binding molecule may be one capable of coupling to another coagulant molecule in vivo.
  • a clot-targeting binding molecule which is capable of coupling to a coagulant molecule in vivo as well as a composition comprising such a molecule together with an acceptable carrier, diluent, excipient and/or adjuvant.
  • the clot-targeting, anticoagulant molecule of the first embodiment may comprise one or more clot-targeting binding molecules coupled to one or more anticoagulants.
  • composition for treatment of a clot in a mammal comprising a clot-targeting, anticoagulant molecule of the first embodiment together with an acceptable carrier, diluent and/or excipient.
  • treatment of a clot includes treatment to maintain the size of a clot in mammal, treatment to reduce a clot in mammal and a treatment to prevent reformation of a clot in mammal.
  • a method of treating a clot in a mammal comprising administering to a mammal requiring such treatment an effective amount of a clot-targeting, anticoagulant molecule of the first embodiment or a composition of the second embodiment.
  • the method of the third embodiment may further include administering to the mammal an anticoagulant molecule.
  • a method of treating a clot in a mammal comprising administering to a mammal requiring such treatment an effective clot treating amount of a clot-targeting binding molecule and an anticoagulant capable of being bound by the clot- targeting, binding molecule in vivo.
  • the clot-targeting binding molecule may be administered simultaneously or before or after the administration of the anticoagulant capable of being bound by the clot-targeting, anticoagulant molecule.
  • the idea for this embodiment is that the clot-targeting molecule targets and binds to a clot in the mammal and then binds to the anticoagulant capable of being bound to it which prior to now is circulating in the system of the mammal after having been administered to the mammal.
  • the clot-targeting molecule binds to the anticoagulant capable of being bound to it and then targets and binds to a clot in the mammal.
  • a kit for treating a mammal requiring treatment selected from the group consisting of a treatment to treat a clot therein, a treatment to reduce a clot therein and a treatment to prevent reformation of a clot therein, comprising (a) a clot- targeting, binding molecule; and (b) an anticoagulant capable of being bound by the clot-targeting, binding molecule in vitro and/or in vivo.
  • the kit of the fifth embodiment may further comprise an acceptable carrier, diluent and/or excipient.
  • composition of the second embodiment may be administered parenterally, e.g. by injection and by intra-arterial
  • compositions can also be delivered locally by drug delivery catheter devices, in order to increase the dose. It is also possible to administer the compositions orally provided the compositions contain materials such as liposomes which substantially prevent the anticoagulant molecule from being decomposed in the digestive tract.
  • the anticoagulant molecule may be coupled to a carrier which enables it to pass through to the blood stream without substantially decomposing in the digestive tract.
  • the mammal is a bovine, human, ovine, equine, caprine, Leporine, feline or canine vertebrate.
  • the mammal is a human
  • the composition is a pharmaceutical composition
  • the carrier, diluent, excipient and/or adjuvant are pharmaceutically acceptable.
  • composition is typically a veterinary composition and the carrier, diluent, excipient and/or adjuvant are veterinarily acceptable.
  • the clot-targeting, anticoagulant molecule or its salt may be prepared in sterile aqueous or oleaginous solution or suspension.
  • Aqueous solutions or suspensions may further comprise one or more buffering agents
  • Su i table buffering agents include sodium acetate, sodium citrate, sodium borate or sodium tartrate, for example.
  • the dosage form of the clot-targeting, anticoagulant molecule will comp ⁇ se from 0.01% to 99% by weight of the active substance.
  • dosage forms according to the invention will comprise from 0.1% to about 20%, more typically 0.5% to 10% by weight of the active substance.
  • compositions of the second embodiment may be prepared by means known in the art for the preparation of pharmaceutical compositions including blend i ng, grinding, homogenising, suspending, dissolving, emulsifying d i spersing and mixing of the clot-targeting, anticoagulant molecule together with the selected excipient(s), carrier(s), adjuvant(s) and/or diluent(s).
  • a molecule of the first embodiment or a composition of the second embodiment will usually be administered by i n j ect i on.
  • a suitable treatment may comprise the administration of a single dose
  • An alternat i ve suitable treatment may comprise the administration of a single dose or mult i ple doses of the clot-targeting, anticoagulant binding molecule of the first embodiment or a composition of the second embodiment, followed by or preceded by or simultaneously administrating at least one anticoagulant.
  • the treatment will consist X administering from one to five doses da i ly of the clot-targeting, anticoagulant molecule for a period of from one day to several days.
  • a further treatment ⁇ may be given during the lifetime of the patient.
  • the treatment will consist of the administrat i on of the clot-targeting, anticoagulant molecule of the first embodiment or a compos i t i on of the second embodiment for a period of from one day to several days.
  • the administered dosage of the clot-targeting, anticoagulant molecule of the first embodiment or a composition of the second embodiment can vary and depends on several factors, such as the condition of the patient. Dosages w i ll range from O.OOOlmg to 200 mg per kg. Usually, the dose of the act i ve substance will be from O.OOlmg to lOmg per kg of body weight, more typ i cally 0.1 mg - 10 mg per kg of body weight. In the case of a bolus infusion the dose of the active substance is typically 0. lmg to 50mg per kg of body weight.
  • epitope is used herein to refer to a binding site on or in a target clot which is specifically recognized by a clot binding molecule, and is not limited to binding sites recognized by antibodies.
  • Other fibrin targeting and binding molecules include D-dimer monoclonal antibody DD 3B6/22, fibrin specific antibodies such as fibrin antifibrin antibody MA 59D8, bifunctional antibodies which bind to fibrin or platelets or other clot components.
  • Examples of molecules with affinity for platelets and which can bind to platelets include platelet binding antibodies, or platelet binding molecules, bifunctional antibodies which bind to activated platelets, activated platelet binding antibodies (e.g. MAb 7E3, MAb 50H.19, MAb PADGEM and MAb P256, see S. F. Rosebrough et al. 1990; 31: 1048 the contents of which are incorporated herein by cross reference), activated platelet binding molecules and an antigen-binding fragment of a clot-targeting antibody, (e.g. MAb P256, F(ab') 2 , and Fab' fragments of MAb P256, see for example A.W.J. Stuttle, J.M. Ritter, A.M.
  • platelet binding antibodies e.g. MAb 7E3, MAb 50H.19, MAb PADGEM and MAb P256, see S. F. Rosebrough et al. 1990; 31: 1048 the contents of which are incorporated herein by cross reference
  • the clot targeting and binding molecule may be any substance having a preferential affinity for a clot component including monoclonal or polyclonal antibodies, enzymes, or other binding proteins or substances (or binding fragments thereof).
  • the CBM is usually an antibody, molecular recognition unit, or an antigen-binding fragment of an antibody, such as a F(ab') 2 , Fab', Fv or VH fragment.
  • Antibodies which recognize at least one of the constituents of clots may be prepared by conventional techniques using clots, or the purified constituents thereof, as immunogens. These antibodies may be monoclonal or polyclonal in nature. Either the intact antibody, or specific
  • the antibody or antibody fragment may be polyvalent, divalent or univalent.
  • anticoagulants including thrombin inhibitors can be included into the molecules of the invention, such as hirudin and hirudin analogues, C- terminal hirudin peptides and analogues, tick anticoagulant peptide (TAP), heparin, coumarin, short peptide inhibitors of thrombin such as small peptide- like inhibitors such as * PPACK (d-phe-pro-arg-chloromethylketone), DuP 714 (ac-d-phe-pro-boroarginine), agratropin (2R, 4R)-4- methyl -1- [N2 -(3-methyl- 1,2,3,4,-tetrahydro -8-quinolinesulphonyl)-L- arginyl]-2- piperidinecarboxylic acid monohydrate, tick anticoagulant peptide or extrinsic pathway inhibitor or tissue pathway inhibitor.
  • TRIP tick anticoagulant peptide
  • heparin heparin
  • coumarin short
  • Fibrinogen receptor antagonists which are suitable anticoagulants for use in the present invention include cyclo(S,S)-N ⁇ -acetyl-Cys-(N ⁇ -methyl)Arg-Gly-Asp-Pen NH 2 Ali et al., EP 0341 915, cyclo(S,S)-(2-mercapto) benzoyl-(N ⁇ -methyl)Arg-Gly- Asp-(2-mercapto), EP Application No.
  • Patent 4,952,562 Ali et al., PCT US 90/06514; Alig. et al., EP 0 381 033; and Alig et al., EP 0 384 362; the contents of all of which are incorporated herein by cross reference as well as and the cyclic peptides.
  • the anticoagulant included in the molecules of the invention, acts as an antithrombin agent and/or inhibitor of earlier steps in the coagulation cascade such as factor Xa (for example TAP), factor Va, factor X, factor IXa, factor Villa, factor Vila, the prothrombinase complex, vitamin-K-dependent
  • anticoagulants include factor Xa inhibitor including TAP, factor Va inhibitor, factor Villa inhibitor, factor Vila inhibitor, factor IXa inhibitor, prothrombinase complex inhibitor, vitamin-K-dependent gamma carboxylation of prothrombin inhibitor, factor X inhibitor, factor VII inhibitor, factor V inhibitor, factor Vffl inhibitor, and factor IX inhibitor.
  • Thrombolytic agents such as urokinase, scuPA streptokinase or tPA may also be coupled to the clot binding molecule of the invention to prevent arterial wall intimal proliferation.
  • Anti growth factors e.g. anti-PDGF and other targeted growth factors e.g. FGF's to induce reendothelialization
  • the clot-targeting, anticoagulant molecule of the invention may be a single molecule with clot binding and anticoagulant moieties, or a complex of two or more molecules.
  • the term "molecule” will be used to cover both moieties and molecules, and "conjugate” to cover both a single hybrid molecule with clot binding and anticoagulant moieties and two conjugated molecules one of which has clot binding behaviour and the other of which has anticoagulant behaviour.
  • the conjugate is obtained by coupling a CBM to an anticoagulant (ACM).
  • ACM anticoagulant
  • TMB thrombolytic
  • TBM thrombolytic binding molecule
  • the CBM, the ACM and optionally the TMB and/or TBM may be coupled together directly or indirectly (e.g. via a linker molecule), and by covalent or non- covalent means (or a combination thereof).
  • the clot-targeting, anticoagulant binding molecule of the invention may be a single molecule with clot binding and anticoagulant binding moieties, or a complex of two or more molecules.
  • the conjugate is obtained by coupling a CBM to an anticoagulant binding molecule (ACBM).
  • ACBM anticoagulant binding molecule
  • the conjugate is obtained by coupling a CBM to an ACBM and a TMB and/or TBM.
  • the CBM, the ACBM and optionally the TMB and/or TBM may be coupled together directly or indirectly (e.g. via a linker molecule), and by covalent or non-covalent means (or a combination thereof).
  • anticoagulant molecule such as [anticoagulant-antibody to clot], [anticoagulant-antibody to clot-thrombolytic agent], [anticoagulant-thrombolytic- antibody to clot] and [thrombolytic-anticoagulant-antibody to clot]
  • antibody is understood to encompass clot targeting and binding monoclonal or polyclonal antibodies and clot targeting and binding fragments thereof in particular, F(ab') , Fab', Fv or VH fragments, especially D-dimer including MAb DD-3B6/22, F(ab') 2 fragments of MAb DD-3B6/22, Fab' fragments of MAb DD-3B6/22, all the fibrin targeting monoclonal antibodies disclosed in U.S.
  • Patent No. 4,758,524 (the contents of all of which is incorporated herein by cross reference), monoclonal antibody DD-1D2/48, F(ab') 2 fragments of MAb DD-1D2/48, Fab' fragments of MAb DD-1D2/48, monoclonal antibody DD-1C3/108, F(ab') 2 fragments of MAb DD-1C3/108, Fab' fragments of MAb DD-1C3/108, and all the other fibrin targeting and binding antibodies disclosed elsewhere in this specification, and any other monoclonal either human or mouse or other mammal which has specificity for fibrin or D dimer but not fibrinogen or fragments thereof, fibrin specific antibodies such as fibrin antifibrin antibody MA 59D8, monoclonal antibody DD-1C3/108, bifunctional antibodies which target and bind to fibrin or target and bind to platelets, particularly activated platelets or other clot components or a clot antigen-binding fragment of an antibody, such as a F(a
  • the antibody or antibody fragment may be polyvalent, divalent or univalent.
  • MAb DD-3B6/22 is commercially available from Agen Biomedical Limited, 11 Durbell Street, Acacia Ridge, Qld 4110 and Agen Inc., 20 Waterview Boulevard, Parsippany, New Jersey, U.S.A. 07054.
  • MAb DD-1D2/48 and MAb DD1C3/108 are also available from these sources.
  • SPDP N-Succinimidyl-3,2-(pyridyldithio) propionate
  • MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester
  • cross linking agents include cross-linking agents include 4,4'- dithiobisphenylazide, dithiobis-(succinimidylpropionate), 2- iminothiolane, dimethyl-3,3'-dithiobispropionimidate.2HCl,
  • the coupling may also be noncovalent, for example, b y (a) attach i ng biotin to one and avidin (or streptavidin) to the other), (b) attaching an ant i antibod y to one, which then binds the other, (c) attaching Protein A to one, which then binds the Fc portion of the other, or (d) attaching a sugar to one and a corresponding lectin to the other.
  • b y (a) attach i ng biotin to one and avidin (or streptavidin) to the other), (b) attaching an ant i antibod y to one, which then binds the other, (c) attaching Protein A to one, which then binds the Fc portion of the other, or (d) attaching a sugar to one and a corresponding lectin to the other.
  • the binding characteristics of the CBM and TBM the anticoagulant properties of the ACM and the thrombolytic propert i es of the TMB should be changed as little as possible. It may be advantageous to provide a spacer moiety between the CBM and ACM and optionally TMB and TBM to reduce steric hindrance.
  • the CBM may be coupled directly to an ACM
  • the CBM may be coupled directly to an TMB and/or TBM or indirectly via coupling to an ACM coupled to the CBM.
  • the binding characteristics of the CBM, ACBM and TBM and the thrombolytic properties of the TMB should be changed as little as possible. It may be advantageous to provide a spacer moiety between the CBM and ACBM and optionally TMB and TBM to reduce steric hindrance.
  • the CBM may be coupled to directly to an ACBM or indirectly via an TMB and/or TBM.
  • the CBM may be coupled directly to an TMB and/or TBM or indirectly via coupling to an ACBM coupled to the CBM.
  • a CBM/ACM conjugate may be an antibody coupled to an anticoagulant.
  • One method of constructing such a conjugate is the following: (a) preparing F(ab')' 2 fragments of a selected antibody to a clot component by pepsin digestion; (b) reducing and treating the fragments with Ellman's reagent to produce Fab' fragments of the selected antibody; and (c) coupling the Ellman's reagent-treated Fab' fragment to a selected anticoagulant and optionally a TMB and/or TBM, to produce a clot-targeting, anticoagulant molecule.
  • the CBM/ACBM conjugate may be a hybrid antibody.
  • One method of constructing such a conjugate is the following: (a) preparing F(ab')' 2 fragments of a selected antibody to a clot component by pepsin digestion; (b) reducing and treating the fragments with Ellman's reagent to produce Fab' fragments of the selected antibody; (c) thiolysing a selected anticoagulant-specific antibody or a selected anti-coagulant antibody; and (d) coupling the thiolated Fab' fragment to the Ellman's reagent-treated Fab' fragment to produce a hybrid anti-clot antibody anticoagulant specific antibody conjugate.
  • the CBM/ACBM/TBM conjugate may also be a hybrid antibody.
  • One method of constructing such a conjugate is the following: (a) preparing F(ab')' fragments of a selected antibody to a clot component by pepsin digestion; (b) reducing and treating the fragments with Ellman's reagent to produce Fab' fragments of the selected antibody; (c) thiolysing a selected anticoagulant- specific antibody or a selected anti-coagulant antibody; (d) thiolysing a selected thrombolytic-specific antibody or a selected thrombolytic antibody; and (e) coupling the thioylated Fab' fragments of (c) and (d) to the Ellman's reagent- treated Fab' fragment to produce a hybrid anti-clot antibody anticoagulant specific antibody and thrombolytic-specific antibody conjugate.
  • the hybrid or chimeric antibody of the present invention thus comprises two "half molecules," one with specificity for clot component(s) (the CBM) and the other with specificity for an anticoagulant (the ACBM) .
  • the antibody's own disulfide bonds couple the ACBM to the CBM to form an appropriate conjugate.
  • Such a hybrid antibody, or F(ab') 2 fragment of such a molecule has advantages which include ease of preparation, the preservation of the correct stoichiometry and stereochemistry of both antibodies and the retention of the binding affinity of each fragment.
  • the use of a peptide to bind to the clot has the further advantage that the entire conjugate may be prepared without any need for a bifunctional coupling agent.
  • a DNA sequence encoding the clot-binding peptide and the anticoagulant-binding peptide as a single transcriptional unit is provided, and the desired conjugate is expressed as a fusion protein, with the two moieties joined by a simple peptide bond, or with a peptide spacer of desired length.
  • One particularly preferred example is a DNA sequence encoding a clot-binding antibody fragment (which could be just the binding site, not a complete Fab) and anticoagulant antibody (which could be just the binding site, not a complete
  • the desired conjugate is expressed as a fusion protein, with the two moieties joined by a simple peptide bond, or with a peptide spacer of desired length.
  • the divalent peptide may be prepared by direct chemical synthesis.
  • the anticoagulant and thrombolytic may be molecules which may be activated in vivo on attachment to the clot by light irradiation (particularly far
  • anticoagulants including thrombin inhibitors are targeted to a clot by combining them with clot binding molecules to form a hybrid molecule one part of which can bind to a clot component such as fibrin or platelets with high affinity and the other part being able to bind and inactivate thrombin or other procoagulant molecules located at or within the clot.
  • hybrid molecules can be constructed which comprise one half monoclonal antibody with high affinity for a clot component and one half a specific thrombin inhibitor such as a hirudin.
  • the anticoagulant inactivates thrombin and/or other procoagulant molecules located either bound to the clot surface or activated in the vicinity of the clot.
  • This approach has two major advantages over the current antithrombotic agents. Firstly, it targets the clot and thus brings anticoagulant to the clot where thrombolysis causes a local high concentration of procoagulant thrombin. Thus, it enhances the specificity of anticoagulants for ongoing thrombotic processes and increases the local concentration of anticoagulants. Second, the half-life of the desired anticoagulant effect is increased permitting shorter administration of the anticoagulant. These effects result in a decrease in the amount of drug that must be administered, an increase in efficacy and an increase in safety.
  • a clot targeted anticoagulant molecule is made by coupling the fibrin specific monoclonal antibody DD-3B6/22 with an anticoagulant peptide (for example PPACK, hirulog or hirudin) or with an anticoagulant (for example heparin, or coumarin) and a thrombolytic agent (example tPA or urokinase).
  • an anticoagulant peptide for example PPACK, hirulog or hirudin
  • an anticoagulant for example heparin, or coumarin
  • a thrombolytic agent example tPA or urokinase
  • a pharmaceutical composition for treatment selected from the group consisting of a treatment to treat a clot therein, a treatment to reduce a clot therein and a treatment to prevent reformation of a clot therein is then made by mixing the clot targeted anticoagulant molecule or the clot targeted anticoagulant thrombolytic molecule together with a pharmaceutically acceptable carrier, diluent or excipient.
  • a human may be treated to reduce the clot therein by administering to the human requiring treatment such as treatment to treat a clot therein, treatment to reduce a clot therein and treatment to prevent reformation of a clot therein, an effective clot treating amount of the pharmaceutical composition.
  • Use of monoclonal antibody DD-3B6/22 is especially advantageous to target a clot as there is no cross reaction of the monoclonal antibody with fibrinogen, fibrinogen fragment D, fibrinogen fragment E or non
  • dosage forms in accordance with the invention are as follows:
  • Fab-SH fragment of DD-3B6/22 was prepared by the method described by John eLfi! Throm. Res. 58 273-281 1990. Briefly purified antibody was digested with pepsin and F(ab')2 fragments purified. Fab-SH is prepared from
  • the anti-thrombin peptide derivative PPACK was reacted with SPDP (Pharmacia) under the conditions recommended by the manufacturer. Purification of the N-terminally modified derivative was carried out by reverse phase chromatography on a C-18 column using a gradient of 0-60% acetonitrile containing 0.1% trifluoracetic acid. The SPDP-labelled peptide was recovered after freeze drying. (c) Formation of Fab-PPACK derivative.
  • the pH of the Fab-SH solution was adjusted to pH 7.0 by the addition of 0.2M Sodium phosphate buffer pH 8.0. 10ml of 2mg ml fragment was reacted with
  • This assay measures the ability of the reagent to inhibit the action of thrombin on the thrombin specific chromogenic substrate S-2238. Inhibitors of this
  • Targeted anticoagulant reagent inhibits the activity of thrombin on the chromogenic substrate in a dose dependant manner.
  • the Fab fragment of the antibody or the antibody fragment alone has no such inhibitory activity (not shown). 3. Thrombin clotting time inhibition assay
  • This assay measures the ability of the reagent to inhibit the activity of thrombin to form clots in plasma.
  • Thrombin clotting time was measured as described in "Practical Haematology", Dacie JV and Lewis SM eds, Churchill Livingstone Publishers Edinburgh UK 1 84 pp 218-219.
  • SUBSTITUTE SHEET This assay measures the ability of clot bound targeted anticoagulant reagent to inhibit clot extension.
  • Preparation of plasma and whole blood clots and measurement of thrombin-dependant clot associated procoagulant activity was carried out essentially as described by Eisenberg et al J. Clin. Invest. 91 1877- 1883 1993 (incorporated herein by cross reference). Briefly plasma clots were developed in barium-adsorbed plasma repeated with CaCl 2 to a final concentration of 25mM. The clot formed was washed extensively and then incubated in a solution of citrated plasma containing variable concentrations of inhibitor or buffer as a control.
  • the Fab-SH fragment of DD-3B6/22 was prepared by the method described by John eL_a] Throm. Res. 58 273-281 1990 except that the Fab-TNB derivative was prepared instead of the Fab-SH derivative. Briefly purified antibody was digested with pepsin and F(ab') 2 fragments purified. Fab-SH is prepared from F(ab') 2 by mild reduction with beta mercaptoethylamine and reaction with lO M Ellman's reagent (5,5' Dithio-bis[-2-nitrobenzoic acid]). The derivative was purified by gel filtration chromatography in a 0.2M phosphate buffer at pH 7.4.
  • Peptides were prepared by the Merrifield procedure (Hodges and Merrifield Anal.Biochem. 65 7241 195) essentially as described by Kemp et al Science 241 1352- 1354 1988.
  • the peptides prepared were based on the hirulog structure (Manganore et al Biochemistry 29 7095-7101 1990), but with addition of a cysteine residue to enable conjugation of the peptide to the hinge region of the
  • Fab-TNB (-2mg/ml) was a incubated with the cysteine containing hirulog derivatives at a 1:20 molar ratio. The reaction mixture turned yellow in colour after 2min. The reaction was then stopped with the addition of 50 ⁇ l of 300mM iodoacetamide. This was wrapped in foil and incubated for 30min at room temperature. Unreacted hirulog derivative was removed from conjugate by gel filtration chromatography on a column of Ultragel Ac44 in PBS.
  • the thrombin clotting assay was used to establish that the conjugate contained anticoa ulant activit .
  • RNA messenger RNA
  • single and double stranded complementary DNA may be synthesised.
  • the ds-cDNA may be cloned into lambda-gtlO arms and packaged into a phage library.
  • the heavy chain clone gamma-M/l.T (Tyler et al., Proc. Natl. Acad. of Sci., 1982; 79: 2008-2012) and the light chain clone pH76-kap ⁇ a-10 (Adams et al., Biochem., 1980; 19: 2711-2719) were used to source ds-DNA inserts for the screening of the gtlO library.
  • N-terminal sequence of the 3B6/22 light chain N- terminal residues from the intact 3B6/22 Ig may be removed sequentially by Ed an degradation in an Applied Biosystems sequencer. The N-terminal sequence of the light chain can be thus deduced from the mixed sequence by comparison with the sequence of the cloned heavy chain.
  • the variable region of the kappa light chain not present in gtlO library clones may be amplified by PCR from ss-cDNA.
  • the redundant, forward (sense) primer N960 may be designed from the deduced amino terminal kappa sequence.
  • a typical reaction 100 ⁇ l volume
  • a typical reaction 100 ⁇ l volume
  • a typical reaction 100 ⁇ l volume
  • a mixed A,C,G and T deoxynucleotide dNTP solution
  • each primer 10 pMolar each
  • 1 ⁇ l of internal primers 0.05-0.1 pM
  • Mg + + to a final concentration of 1-5 mM a reaction buffer appropriate for the particular polymerase chosen (supplied by manufacturer), and water to 100 ⁇ l.
  • the reactants were mixed and
  • paraffin oil Sigma biochemical
  • a thermal cycler Corbett Research, Australia.
  • the general strategy for each of the examples consisted of a denaturation step at 93°C (usually 1 minute), an annealing step between 50 and 65°C for 1 minute and an extension step at 72°C for 2 minutes. Annealing temperatures were adjusted as required to give final product.
  • Vb variable domain
  • the product was digested with Thai and Eco Rl, and cloned into the Msc 1/Eco Rl-digested expression vectors pPOW (Power et al, Gene, 1992; 113: 95-99) and transformed into E coli strains TG-1 (Gibson TJ, 1984, "Studies on the Epstein-Barr virus genome", PhD thesis, Cambridge University, England) and 14 ⁇ l respectively. 3.
  • Transformed E coli were screened for the presence of plasmids carrying the Vh gene fragment and selected clones (hereafter referred to as pP3B6Vh pr [G3B6Vh) sequenced to check the integrity of the cloning procedure.
  • ( ii ) Amplification and cloning of the light chain variable domain and construction of a composite single-chain antibody domain (scFv ) .
  • Oligonucleotide primers were synthesised to simultaneously amplify (as in i(l) above) and add to the cloned light chain gene, in a PCR amplif i cation reaction, a Bst E2 site and a sequence coding for a linker (amino acid sequence - (GGGGS)3-) at the 5' end and a peptide epitope-Eco Rl sequence at the 3' end.
  • the product was digested with Bst E2 and Eco Rl and cloned into the Bst E2,/Eco Rl digested plasmids described as pP3B6Vh pr pG3B6Vh above and transformed into the E coli strains described in i above.
  • Oligonucleotide NSfil5 was used to add a Sfi 1 restriction site to the 5' end of the scFv gene construct in P P3B6scFv, and (NVKFORNOT) was used to add a Not 1 site to the 3' end of the scFv domain.
  • the product was digested with these restriction enzymes and cloned into the likewise restricted vector pHFA, a derivative of the vector pHEN, and the construct transferred into the E coli strain HB2151, a non-supE phenotypic strain.
  • Clones, referred to as pHFA3B6, were identified by hybridisation and were tested for expression of a
  • Recombinant E coli were grown in lOmls of 2X-YT medium (10 gm yeast extract, 19 gm tryptone, 5 gm NaCl per litre overnight. Overnight cultures were diluted to an ODgrjO 0.5 into 100 ml of fresh medium and grown to mid- log phase (OD600 0.5-0.9). Cultures of pG3B6scFv and pHFA 3B6 were induced upon the addition of isopropyl-B-D-thiogalactopyranoside; (IPTG; Sigma 15502) to a concentration of ImM, and growth continued at 30-37°C as required for a further 4 hours. pHFA 3B6/22 cultures were maintained at 30°C for up to 24 hours post-induction.
  • 2X-YT medium 10 gm yeast extract, 19 gm tryptone, 5 gm NaCl per litre overnight. Overnight cultures were diluted to an ODgrjO 0.5 into 100 ml of fresh medium and grown to mid

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EP93922464A 1992-10-12 1993-10-12 Auf blutgerinnsel gerichtetes antikoagulanz, verfahren zur herstellung desselben und zu seiner verwendung. Withdrawn EP0668875A4 (de)

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US5840733A (en) * 1996-07-01 1998-11-24 Redcell, Canada, Inc. Methods and compositions for producing novel conjugates of thrombin inhibitors and endogenous carriers resulting in anti-thrombins with extended lifetimes
US7754208B2 (en) 2001-01-17 2010-07-13 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
CA2450828C (en) * 2001-06-26 2013-11-26 Agen Biomedical Limited Humanized antibodies derived from dd-3b6/22, specific for the d-dimer fragment of fibrin
US20050261241A1 (en) 2004-05-19 2005-11-24 Celsus Biopharmaceuticals, Inc. Use of dermatan sulfates and/or desulfated heparins to treat or prevent heparinoid-induced autoimmune responses
US20080181892A1 (en) * 2004-08-11 2008-07-31 Trubion Pharmaceuticals Binding Domain Fusion Protein
HUE026303T2 (hu) 2005-07-25 2016-06-28 Emergent Product Dev Seattle B-sejt csökkentés CD37-specifikus és CD20-specifikus kapcsoló molekulák alkalmazásával
US8815804B2 (en) * 2006-02-06 2014-08-26 Sanford-Burnham Medical Research Institute Methods and compositions related to targeting tumors and wounds
EP2418223A3 (de) 2006-06-12 2013-01-16 Emergent Product Development Seattle, LLC Einkettige, multivalente Bindungsproteine mit Effektorfunktion
DK2132228T3 (da) 2008-04-11 2011-10-10 Emergent Product Dev Seattle CD37-immunterapeutisk middel og kombination med bifunktionelt kemoterapeutisk middel deraf
CA2724630A1 (en) 2008-05-16 2009-11-19 Bayer Healthcare Llc Targeted coagulation factors and method of using the same
CN108367004B (zh) 2015-09-21 2022-09-13 阿帕特夫研究和发展有限公司 Cd3结合多肽
PE20190966A1 (es) 2016-11-16 2019-07-08 Bayer Healthcare Llc Factor viii direccionado a los globulos rojos y metodo para su uso

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