WO1998039436A2 - Compositions de muteine du facteur de croissance des fibroblastes, et procedes d'utilisation de ces compositions - Google Patents

Compositions de muteine du facteur de croissance des fibroblastes, et procedes d'utilisation de ces compositions Download PDF

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WO1998039436A2
WO1998039436A2 PCT/JP1998/000878 JP9800878W WO9839436A2 WO 1998039436 A2 WO1998039436 A2 WO 1998039436A2 JP 9800878 W JP9800878 W JP 9800878W WO 9839436 A2 WO9839436 A2 WO 9839436A2
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fgf
replacement
amino acid
modified
heparin
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PCT/JP1998/000878
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WO1998039436A3 (fr
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Hengyi Zhu
Ramnarayan Kalyanaraman
Takatoshi Kawai
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Eisai Co., Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C07K14/501—Fibroblast growth factor [FGF] acidic FGF [aFGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • FGF FGF genes and peptides.
  • DNA encoding modified FGF peptides and compositions containing the modified FGF peptides are provided.
  • the FGF peptides can be used in methods of treating heparin-associated disorders. BACKGROUND OF THE INVENTION During the last thirty years, a great deal of attention has been directed towards the identification and characterization of factors that stimulate the growth, proliferation and differentiation of specific cell types. Numerous growth factors and families of growth factors that share structural and functional features have been identified. Many of these factors have multifunctional activities and affect a wide spectrum of cell types.
  • FGF fibroblast growth factor
  • Acidic FGF has an acidic isoelectric point with a pi of about 5.4.
  • the other members of the FGF family have subsequently been identified on the basis of amino acid sequence homoiogies with aFGF and bFGF and common physical and biological properties. These proteins are widely distributed in tissues, such as the central and peripheral nervous system, retina, kidney and myocardium.
  • FGFs have extremely high affinities for heparin, which is a highly sulfated, negatively charged polysaccharide containing repeating disaccharide structure of varying lengths as found in other glycosaminoglycans, and many of the key amino acid residues required for heparin binding have been identified (Presta (1 992) Biochem. Biophys. Res. Commun. 1 85: 1098-1 1 07; Thompson et al. ( 1 994) Biochemistry 33:3831 -3840; Li et al. ( 1 994) Biochemistry 33: 10999-1 1007).
  • aFGF and bFGF possess two potential binding domains for heparin, one being located near the amino-terminal region, while the other is near the carboxy-terminal region (residues 1 8-22 and 107 to 1 1 0 for bFGF and 9-1 2 and 1 00-102 for aFGF; e.g., see Gospodarowicz et al. ( 1 987) Endocrin. Rev. 8:95-1 1 4; Baird et al. ( 1 988) Proc. Natl. Acad. Sci. U.S.A. 85:2324-2328) .
  • heparin binding is not absolutely required for the binding of an FGF to its receptor, heparin has been reported to modulate one or more activity of FGFs including increasing receptor affinity, conferring protection from heat and acid inactivation and proteolytic degradation, and is also essential for the mitogenic activity of bFGF stimulated cells (e.g. , see Shi et al. (1 993) Mol. Cell. Biol. 1 3:3907-391 8; Roghani et al. ( 1 994) J. Biol. Chem. 269:3976-3984; Gospodarowicz et al. (1 986) J. Cell Biol. 1 28:475-484: Yanyon et al. (1 991 ) Cell 64:841 -848) .
  • FGFs exhibit a mitogenic effect on a wide variety of mesenchymal, endocrine and neural cells. They are also important in differentiation and development. Of particular interest is their stimulatory effect on collateral vascularization and angiogenesis. Such effects have stimulated considerable interest in FGFs as therapeutic agents, for example, as pharmaceutical for wound healing, neovascularization, nerve regeneration and cartilage repair. In addition to potentially useful proliferative effects, FGF-induced mitogenic stimulation may, in some instances, be detrimental. For example, cell proliferation and angiogenesis are an integral aspect of tumor growth.
  • FGF FGF-like growth factor receptor
  • bFGF bFGF
  • aFGF or other FGFs should have a therapeutic use in treatment of tumorigenic conditions, restenosis, and other such conditions in which an FGF peptide plays a pathogenic role.
  • FGFs are mediated by high affinity receptor tyrosine kinases on the cell surface membranes or FGF-responsive cells [e.g., see Lee et al. , ( 1 989) Science 245, 57-60; Imamura et a/. , B.B.R.C. 1 55, 583-590 ( 1 989); Huang and Huang, ( 1 986) J. Biol. Chem. 261 , 9568-9571 ; Moscatelli, ( 1 987) J. Cell. Phvsiol. 1 31 , 1 23-1 30; Verdier et al. ( 1 997) Genomics 40, 1 51 -1 54; U.S. Patent No. 5,288,855].
  • FGF-responsive cells e.g., see Lee et al. , ( 1 989) Science 245, 57-60; Imamura et a/. , B.B.R.C. 1 55, 583-590 ( 1 989); Huang and Huang, ( 1
  • Lower affinity receptors also play a role in mediating FGF activities.
  • the high affinity receptor proteins constitute a family of structurally related FGF receptors.
  • Four FGF receptor genes have been identified and at least two of these genes generate multiple mRNA transcripts via alternative splicing of the primary transcript [e.g., see U.S. Patent No. 5,288,855; Kiefer et aL, (1 991 ) Growth Factors 5: 1 1 5-1 271. This splicing potentially creates a large number of different molecular forms that can interact with FGF family members, thereby permitting cells to respond to different FGF family members.
  • alternative splicing of a single gene results in the receptor FGFR2, which has high affinity for acidic and basic FGFs but no detectable affinity for KGF, and the KGF receptor, which has high affinity for KGF but reduced affinity for basic FGF.
  • alternative splicing of FGFR1 produces variants that have about a 50-fold decreased the affinity for basic FGF, but unchanged acidic FGF binding.
  • Receptor expression is altered by physical, chemical, and hormonal injury as well as in certain pathological conditions such as restenosis, tumors and selected proliferative diseases.
  • Receptor messenger RNA and protein is expressed in melanoma cells (see, e.g., Becker et aL . ( 1 992) Oncogene ⁇ V.
  • the receptor message is not normally expressed in the palmar fascia, but is present in the proliferative hand disease Dupuytren's Contracture, (see, e.g., Gonzales et aL ( 1 992) Amer. J. Pathol. 141 : 61 -671 ).
  • Quiescent smooth muscle cells (SMCs) do not respond to bFGF, but proliferating SMCs, in a model of restenosis after balloon angioplasty, strongly respond to exogenous bFGF (see, e.g. , Casscells et aL ( 1 992) Proc. Natl. Acad. Sci. U.S.A. 89:71 59-71 63).
  • SMCs smooth muscle cells
  • Coronary artery thrombosis plays a pivotal role in the pathogenesis of acute coronary syndromes including, but not limited to: unstable angina, non Q-wave myocardial infarction and sudden death.
  • Thrombotic occlusion of the artery is thought to be responsible for most of the acute manifestations of coronary artery diseases.
  • antithrombolytic therapy is a mainstay in the early management and treatment of patients suffering from acute coronary syndromes ⁇ e.g. , see van den Bos et al. ( 1 993) Circulation 88:2058-2066; Bombardini et al. (1 997) An ⁇ iology 48:969-976; Walenga et al. ( 1 997) Curr. Qpin. Pulm. Med. 3:291 -302).
  • Heparin is the most widely used antithromoblytic agent for acute management of thrombosis and is the treatment of choice for preventing and treating venous thromboembolism.
  • the anticoagulant effect of heparin is not linked to a cellular target but is presumed to be exerted in conjunction with antithrombin III to inhibit the activity of soluble circulatory enzymes involved in the fibrinolytic blood clotting cascade, particularly Factor Xa and Factor Ma.
  • heparin is widely used as the injectable anticoagulant of choice, it has several potential short comings. For example, the systemic administration of high levels of heparin used to impede local thrombus deposition also can results in the global reduction in Factor Xa and/or Factor Ha activity.
  • a complication of systemic heparin therapy is severe bleeding in patients because of the reduced capability of blood to coagulate (e.g. , Visentin et al. (1 995) Curr. Qpin. Hematol. 2:351 -357). Severe bleeding is a serious thromboembolic complication of heparin therapy and can result in crippling disabilities and/or death (e.g. , see Sodian et al. (1 997) ASAIO J. 43:M430- M433).
  • Heparin-induced thrombocytopenia is an immunoglobulin-mediated adverse drug reaction associated with a high risk of thrombotic complications.
  • the pathogenic antibody usually immunoglobulin (lg)G (HIT-lgG)
  • HIT-lgG immunoglobulin-binding protein normally contained in platelet alpha granules, resulting in platelet activation via platelet Fc receptors.
  • FGF mutein polypeptides and compositions containing the mutein FGF polypeptides are provided.
  • the FGF muteins are useful in methods of modulating the activity of heparin, and can be used for treating heparin-induced and heparin-related disorders. Since the muteins provide information regarding requisites for high affinity binding, the muteins are also useful for rational drug design of FGF, particularly FGF-2, antagonists.
  • DNA encoding FGF muteins that bind to heparin but have little or substantially reduced FGF receptor binding activity compared to wild type are provided.
  • DNA encoding FGF muteins having amino acid substitutions, preferably alanine or a conservative amino acid substitution therefor, corresponding to positions 95, 101 or 1 04 of FGF-2 (basic FGF) are provided.
  • the muteins will optionally include replacement of the glu96 residue.
  • the DNA encodes an FGF-1 thru FGF- 1 0 set forth in SEQ ID NOs. 1 -1 0, respectively, that contains an amino acid replacement corresponding (by alignment of conserved residues) to position 95, 1 01 or 1 04 of FGF-2 or combinations thereof and optionally additionally replacement of Glu96, which is highly conserved among FGF peptides.
  • FGF-2 muteins are FGF-2 (bFGF) muteins in which the replacement amino acid is glycine, serine, alanine, methionine, leucine or tyrosine such that the resulting mutein retains heparin binding ability but has reduced, substantially reduced (preferably at least about 1 0-fold, more preferably at least about 1 00-fold or more) binding affinity for FGF receptors, particularly FGFR 1 (for FGF-2) .
  • the FGF muteins further include replacement of one or more cys residues, particularly those that contribute to aggregation and decrease solubility. These residues correspond to Cys78 and Cys96 in FGF-2.
  • Compositions containing a FGF mutein peptide with amino acid replacements corresponding to positions one or more of 95, 1 01 and 104 and optionally 96 of FGF-2 are provided. Such compositions when formulated for pharmaceutical use can be used as coagulants for heparin-associated bleeding, antagonists of heparin-induced angiogenesis, and for treating heparin-induced thrombocytopenia and thrombosis.
  • Particularly preferred are FGF-2 mutein peptides in which the Glu96 and Ala104 are replaced with glycine, serine or alanine, more preferably alanine.
  • compositions containing a therapeutically effective amount of one of these FGF mutein for treating heparin-related disorders are also provided.
  • the compositions may be formulated for oral, intravenous or parenteral administration.
  • the compositions may be formulated for administration sublingually, as aerosols, as suppositories, and for ophthalmic application.
  • Methods of treating heparin-related disorders by administering a therapeutically effective amount of an FGF mutein that binds heparin but does not bind to its cognate receptor are also provided.
  • methods of treating heparin-related disorders such as excessive bleeding resulting from the anticoagulant activity of the systemic administration of heparin, heparin-induced and heparin-associated thrombocytopenia and thrombosis or the undesired stimulation of angiogenesis mediated by the interaction of heparin with an FGF, e.g., FGF-2, are provided.
  • amino acids which occur in the various amino acid sequences appearing herein, are identified according to their well-known, three-letter or one-letter abbreviations.
  • nucleotides which occur in the various DNA fragments, are designated with the standard single-letter designations used routinely in the art.
  • FGF refers to polypeptides having amino acid sequences of native FGF proteins. Such polypeptides include, but are not limited to, FGF-1 - FGF-1 2.
  • FGF-2 should be generally understood to refer to polypeptides having substantially the same amino acid sequences and receptor- targeting activity as that of bovine bFGF or human bFGF. It is understood that differences in amino acid sequences can occur among FGFs of different species as well as among FGFs from individual organisms or species.
  • Reference to FGFs is also intended to encompass proteins isolated from natural sources as well as those made synthetically, as by recombinant means or possibly by chemical synthesis.
  • an FGF mutein is modified a member of the FGF family of peptides that contains at least one ammo acid residue that differs from naturally-occurring FGF peptides.
  • the FGF muteins have replacements in ammo acid residues corresponding to positions 95, 1 01 , 1 04 of bFGF.
  • Preferred replacement ammo acids are alanine, phenylalanine, serine, glycine, methionme, leucine and tyrosine, more preferably alanine, serine and glycine.
  • positions in FGF-2 For purposes herein, reference is made the positions in FGF-2. Corresponding positions in other FGF polypeptides may be determined by sequence comparison in which homologous regions are aligned. With respect to the FGF family, such alignment is well known to those of skill in the art Identification of corresponding residues is exemplified herein.
  • oositions may also be replaced with conservative ammo acid substitutions that do not substantially alter active.
  • Suitable conservative substitutions of ammo acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule Those of skill in this art recognize that, in general, single ammo acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et aL Molec ⁇ lar Biology of the Gene, 4th Edition, 1 987, The Bejacmin/Cummings Pub. co., p.224).
  • DNA encoding an FGF peptide or polypeptide reactive with an FGF receptor refers to any of the DNA fragments set forth herein as coding such peptides, to any such DNA fragments known to those of skill in the art, any DNA fragment that encodes an FGF and any FGF that may be isolated from a human cell library using any of the preceding DNA fragments as a probe to isolate any DNA fragment that encodes any of the FGF peptides set forth in SEQ ID NOs. 1 -1 0 (such DNA sequences are available in publicly accessible databases, such as DNA " (July, 1 993 release from DNASTAR, Inc.
  • vector or plasmid refers to discrete elements that are used to introduce heterologous DNA into cells for either expression of the heterologous DNA or for replication of the cloned heterologous DNA. Selection and use of such vectors and plasmids are well within the level of skill of the art.
  • expression vector includes vectors capable of expressing DNA fragments that are in operative linkage with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments.
  • an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA.
  • Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or may integrate into the host cell genome.
  • operative linkage or operative association of heterologous DNA to regulatory and effector sequences of nucleotides refers to the functional relationship between such DNA and such sequences of nucleotides.
  • operative linkage of heterologous DNA to a promoter refers to the physical and functional relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA in reading frame.
  • a promoter region refers to the portion of DNA of a gene that controls transcription of DNA to which it is operatively linked.
  • a portion of the promoter region includes specific sequences of DNA that are sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter region is referred to as the promoter.
  • the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of the RNA polymerase. These sequences may be cis acting or may be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. For use herein, inducible promoters are preferred.
  • the promoters are recognized by an RNA polymerase that is expressed by the host. The RNA polymerase may be endogenous to the host or may be introduced by genetic engineering into the host, either as part of the host chromosome or on an episomal element.
  • transfection refers to the taking up of DNA or RNA by a host cell. Transformation refers to this process performed in a manner such that the DNA is rephcable, either as an extrachromosomal element or as part of the chromosomal DNA of the host.
  • Methods and means for effecting transfection and transformation are well known to those of skill in this art (see, e.g., Wigler et aL ( 1 979) Proc. Natl. Acad. Sci. USA 76: 1 373-1 376: Cohen et aL ( 1 972) Proc. Natl. Acad. Sci. USA 69:21 1 0).
  • heparin is the heterogenous, sulfated anionic polysaccha ⁇ de composed of D-glucuronic acid and D-glucosamine, bound to a protein core as the "proteoglycan” or in a free form that has potent anticoagulant activity.
  • heparin also refers to low molecular weight heparin analogs (i.e. , LMWH; commercially available as "FRAGMIN").
  • hepa ⁇ n-like substances are molecules that have oligosaccha ⁇ de structures related to heparin and exhibit an anti-coagulant activity of substantially similar to heparin.
  • a heparin-induced or heparin-related disorder is a disorder in which the administration of heparin or hepa ⁇ n-like substances causes or contributes to the pathology or adverse effects thereof.
  • Such disorders include, but are not limited to: proliferative disorders arising from heparin-induced angiogenesis, heparin-induced and heparin-associated thrombocytopenia and thrombosis and excessive bleeding caused by or associated with the anticoagulant activity of heparin.
  • treatment means any manner in which the symptoms or pathology of a condition, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein.
  • amelioration of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition.
  • local application or administration refers to administration of an FGF mutein or FGF mutein composition to the site, such as into the lens area of the eye following cataract surgery, to prevent the undesired proliferation of endothelial cells resulting from systemic heparin administration.
  • systemic administration refers to adminstration, such as intravenously or intramuscularly, whereby the administered composition enters the bloodstream.
  • topical application refers to application to the surface of the body, such as to the skin, eyes, mucosa and lips, which can be in or on any part of the body, including but not limited to the epidermis, any other dermis, or any other body tissue.
  • Topical administration or application means the direct contact of the FGF mutein composition with tissue, such as skin or membrane, particularly the cornea, or oral, vaginal or buccal mucosa.
  • Topical administration also includes application to hardened tissue such as teeth and appendages of the skin such as nails and hair.
  • a composition formulated for topical administration is generally liquid or semi-liquid carriers such a gel, lotion, emulsion, cream, plaster, or ointment, a spray or aerosol, or a "finite" carrier, i.e., a non-spreading substance that retains its form, such as a patch, bioadhesive, dressing and bandage. It may be aqueous or non-aqueous; it may be formulated as a solution, emulsion or a suspension.
  • biological activity refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures. Biological activity may be detected by in vitro assays, such as those described herein.
  • an effective amount of a compound for treating a disorder is an amount that is sufficient to ameliorate, or in some manner reduce a symptom or stop or reverse progression of a condition. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective.
  • pharmaceutically acceptable salts, esters or other derivatives of the compounds include any salts, esters or derivatives that may be readily prepared by those of skill in this art using known methods for such derivatization and that produce compounds that may be administered to animals or humans without substantial toxic effects and that either are pharmaceutically active or are prodrugs.
  • hydroxy groups can be esterified or etherified.
  • substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography [TLC], gel electrophoresis and high performance liquid chromatography [HPLC], used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance.
  • TLC thin layer chromatography
  • HPLC high performance liquid chromatography
  • Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art.
  • a substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.
  • adequately pure or “pure” per se means sufficiently pure for the intended use of the adequately pure compound.
  • a prodrug is a compound that, upon jn vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound.
  • the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes.
  • the prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug.
  • the IC 50 refers to an amount, concentration or dosage of a particular compound that achieves a 50% inhibition of a maximal response.
  • EC 50 refers to a dosage, concentration or amount of a particular test compound that elicits a dose-dependent response at 50% of maximal expression of a particular response that is induced, provoked or potentiated by the particular test compound.
  • an heparin antagonist is a compound, such as an FGF mutein described herein, that inhibits heparin-induced physiological responses. The antagonist may act by interfering with the interaction of heparin by for example, binding to and sequestering free heparin present in blood.
  • a potential heparin antagonist can be assessed using methods known to tnose of skill in the art
  • the properties of a potential FGF mutein antagonist may be assessed as a function of its ability to bind to heparin and reduced ability to bind one or more FGF receptor using a purified FGF receptor binding assay or a heparin binding assay.
  • DNA encoding an FGF polypeptide for mutagenesis reactions may be isolated, synthesized or obtained from commercial sources (the ammo acid sequences of FGF-1 to FGF-1 0 are set forth in SEQ ID NOs. 1 -10; DNA sequences may be based on these ammo acid sequences or may be those that are known to those of skill in this art (see, e.g., DNA* (July, 1 993 release from DNASTAR, Inc. Madison, Wl); see, also U.S. Patent No. 4,956,455, U.S. Patent No. 5, 1 26,323, U.S. Patent No. 5, 1 55,21 7, U.S. Patent No.
  • DNA is introduced into a plasmid for expression in a desired host.
  • the host is a bacterial host
  • the sequences of nucleotides in the plasmids that are regulatory regions, such as promoters and operators, are operationally associated with one another for transcription of the sequence of nucleotides that encode an FGF mutein.
  • the sequence of nucleotides encoding the FGF mutein may also include DNA encoding a secretion signal, whereby the resulting peptide is a precursor of the FGF mutein.
  • the DNA plasmids also include a transcription terminator sequence.
  • the promoter regions and transcription terminators are each independently selected from the same or different genes.
  • T7 phage promoter and other T7-l ⁇ ke phage promoters include, but are not limited to, the T3, T5 and SP6 promoters, the trp, Ipp, and lac promoters, such as the
  • Particularly preferred plasmids for transformation of J . coli cells include the pET expression vectors (see, U.S patent 4,952,496; available from NOVAGEN, Madison, Wl) .
  • the plasmid pET1 1 d is a prokaryotic expression vector that contains a multiple cloning site for inserting heterologous DNA templates downstream of a bacte ⁇ ophage T7 promoter. Transformation into a bacterial host that expresses T7 RNA polymerase, e.g., E ⁇ coli strain BL2KDE3), results in high level, recombinant expression of the heterologous protein.
  • pET1 1 d was used for the site-directed mutagenesis and mtracellular expression of bFGF and bFGF muteins.
  • a synthetic DNA encoding human bFGF e.g., see SEQ ID N0:2; R & D Systems, Minneapolis, MN] was digested with the restriction endonucleases Nco ⁇ and Bam - ⁇ and placed in operable association with the T7 promoter by gating into the ⁇ /col and Bam ⁇ of pET1 1 d.
  • the resulting plasmid was transformed in a competent bacteria host for recombinant expression of the encoded polypeptide.
  • DNA expression vectors encoding other FGF polypeptides may be constructed using similar methods to those described herein or by using other methods and commercially available vectors known to those of skill in the art [see, e.g. , Sambrook et aL. (1 989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]. 3. DNA mutagenesis
  • the introduction of a mutation into the coding region of an FGF polypeptide may be effected using any method known to those of skill in the art, including site-specific or site-directed mutagenesis of DNA encoding the protein.
  • site-directed mutagenesis may be performed as described herein or using mutagenesis kits available from a variety of commercial sources [e.g., see Clontech, Transformer Site-directed Mutagenesis Kit, Item No. PT1 1 30-1 ].
  • Site-specific mutagenesis is typically effected using mesophilic or thermophihc PCR-based mutagenesis or using a phage vector that has single- and double-stranded forms, such as M 1 3 phage vectors, which are well-known and commercially available.
  • a phage vector that has single- and double-stranded forms such as M 1 3 phage vectors, which are well-known and commercially available.
  • Other suitable phagemid vectors that contain a single-stranded phage origin of replication may be used (see, e.g., Veira et al. ( 1 987) Meth. Enzymol. 15:3) .
  • site-directed mutagenesis is performed by preparing a single-stranded vector that encodes the protein of interest (i.e., a member of the FGF family).
  • oligonucleotide primer that contains the desired mutation within a region of homology to the DNA in the single-stranded vector is annealed to the template followed by addition of a DNA polymerase, such as E coli polymerase I Klenow fragment, which uses the double stranded region as a primer to produce a heteroduplex in which one strand encodes the altered sequence and the other the original sequence.
  • a DNA polymerase such as E coli polymerase I Klenow fragment
  • the heteroduplex is introduced into appropriate bacterial cells and clones that include the desired mutation are selected.
  • the encoded FGF mutein may be expressed recombinantly in appropriate host organisms to produce the modified protein.
  • site-directed mutagenesis was performed to introduce amino acid substitutions in the solvent-accessible residues that neighbor Glu96 of FGF-2 within a 7.5 angstrom radius based on the crytsal structure of bFGF (Erickson et al. ( 1 991 ) Proc. Natl. Acad. Sci. U.S.A. 88:3441 - 3445; Zhang et al. ( 1 991 ) Proc. Natl. Acad. Sci. U.S.A. 88:3446-3450; Zhu et al. ( 1 991 ) Science 251 :90-93) .
  • crytsal structure of bFGF revealed that residues Leu55, Val63, Ile65, Phe94, Phe95, Arg97, Leu98, Asn 1 04, Thr 1 05, Tyr1 06 and Arg 1 07 are within a 7.5 angstrom radius.
  • the resulting muteins were then expressed and purified to near homogeneity employing a heparin-Sepharose column followed by a CM- Sepharose column.
  • the binding affinities of these muteins to soluble FGFR1 ?- tissue plasminogen activator (TPA) fusion protein were determined and compared with wild-type bFGF.
  • TPA tissue plasminogen activator
  • Host organisms for recombinant production of FGF muteins include those organisms in which recombinant production of heterologous proteins have been carried out, such as, but not limited to, bacteria (for example, E. coli), yeast (for example, Saccharomvces cerevisiae and Pichia pastoris), mammalian cells, insect cells.
  • bacteria for example, E. coli
  • yeast for example, Saccharomvces cerevisiae and Pichia pastoris
  • mammalian cells insect cells.
  • Presently preferred host organisms are strains of bacteria.
  • Most preferred host organisms are strains of J . coli. Expression of a recombinant bFGF protein in yeast and J . coli is described in Barr et a/. , J. Biol. Chem.
  • the DNA encoding an FGF mutein is introduced into a plasmid in operative linkage to an appropriate promoter for expression of polypeptides in a selected host organism.
  • the DNA fragment encoding the FGF mutein may also include a protein secretion signal that functions in the selected host to direct the mature polypeptide into the periplasm or culture medium.
  • the resulting FGF mutein can be purified by methods routinely used in the art for wild type FGF, including, methods described hereinafter in the Examples.
  • suitable host cells preferably bacterial cells, and more preferably EL. coli cells, as well as methods applicable for culturing said cells containing a gene encoding a heterologous protein, are generally known in the art. See, for example, Sambrook et aL . ( 1 989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • the desired FGF mutein is produced by subjecting the host cell to conditions under which the promoter is induced, whereby the operatively linked DNA is transcribed.
  • the promoter is the T7 RNA polymerase promoter and the I . coli host strain BL21 (DE3) includes DNA encoding T7 RNA polymerase operably linked to the ac operator and a promoter, preferably the lacUV ⁇ promoter (see, e.g. , Muller-Hill et a . ( 1 968) Proc. Natl. Acad. Sci. USA 59: 1 259-1 2649) .
  • the DNA construct includes a transcription terminator that is recognized by T7 RNA polymerase.
  • the DNA construct includes a transcription terminator that is recognized by T7 RNA polymerase. 3.
  • Purification of FGF muteins Generally, recombinantly expressed human FGF muteins may be purified according to standard methods used for the purification of the corresponding wild type FGFs (e.g., see Zhu et al. J. Biol. Chem. 270:21 869-21 871 ( 1 995); U.S. Patent No. 5, 1 20,71 5).
  • chromatographic methods such as ion-exchange chromatography or immunoaffinity chromatography using antibodies raised against an FGF polypeptide, may also be used.
  • bFGF muteins in which amino acid residues Phe 95 , Asn 101 and Asn 104 have been replaced were prepared following the methods and teachings described herein.
  • the DNA encoding each of these human bFGF muteins was inserted in pET1 1 d in operable association with the T7 promoter and the resulting plasmids were transformed into competent BL2 KDE3).
  • the expression of the FGF mutein was induced and each FGF mutein was purified using ion-exchange chromatography. The bioactivity of each bFGF mutein was determined using one or more assay described herein.
  • FGF family members including FGF-1 , FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9 and FGF-10 are provided.
  • muteins include: FGF-1 that has been modified by replacement of the asparagine residue at position 1 1 0 with another amino acid;
  • FGF-2 that has been modified by replacement of the asparagine residue at position 104 with another amino acid
  • FGF-3 that has been modified by replacement of the asparagine residue at position 1 27 with another amino acid
  • FGF-4 that has been modified by replacement of the asparagine residue at position 1 67 with another amino acid
  • FGF-5 that has been modified by replacement of the asparagine residue at position 1 72 with another amino acid
  • FGF-6 that has been modified by replacement of the asparagine residue at position 1 59 with another amino acid
  • FGF-7 that has been modified by replacement of the asparagine residue at position 149 with another amino acid
  • FGF-8 that has been modified by replacement of the asparagine residue at position 1 39 with another amino acid
  • FGF-9 that has been modified by replacement of the asparagine residue at position 146 with another amino acid
  • FGF-10 that has been modified by replacement of the valine residue at position 95 with another amino acid.
  • the position numbers are determined by reference to SEQ ID NO. 1 -10 for FGF-1 to FGF-10, respectively; and the replacement amino acid is selected such that the resulting mutein has substantially reduced binding affinity for FGF receptor-1 (FGFR1 ) compared to wild type.
  • Other muteins include:
  • FGF-1 that has been modified by replacement of the leucine residue at position 101 ;
  • FGF-2 that has been modified by replacement of the phenylalanine residue at position 95;
  • FGF-4 that has been modified by replacement of the lysine residue at position 1 58
  • FGF-5 that has been modified by replacement of the arginine residue at position 1 63;
  • FGF-6 that has been modified by replacement of the arginine residue at position 1 50;
  • FGF-7 that has been modified by replacement of the lysine residue at position 140;
  • FGF-8 that has been modified by replacement of the threonine residue at position 1 30;
  • FGF-9 that has been modified by replacement of the arginine residue at position 1 37
  • FGF-10 that has been modified by replacement of the lysine residue at position 86.
  • FGF-1 has been modified by replacement of the phenylalanine residue at position 100 with another amino acid
  • FGF-3 has been modified by replacement of the phenylalanine residue at position 1 1 7;
  • FGF-4 has been modified by replacement of the phenylalanine residue at position 1 57;
  • FGF-5 has been modified by replacement of the phenylalanine residue at position 1 62;
  • FGF-6 has been modified by replacement of the phenylalanine residue at position 149
  • FGF-7 has been modified by replacement of the phenylalanine residue at position 1 39;
  • FGF-8 has been modified by replacement of the phenylalanine residue at position 1 29;
  • FGF-9 has been modified by replacement of the phenylalanine residue at position 1 36;
  • FGF-1 0 has been modified by replacement of the phenylalanine residue at position 85;
  • FGF-1 has been modified by replacement of the asparagine residue at position 1 07 with another amino acid
  • FGF-2 has been modified by replacement of the asparagine residue at position 1 01 ;
  • FGF-3 has been modified by replacement of the leucine residue at position 1 24;
  • FGF-4 has been modified by replacement of the asparagine residue at position 1 64;
  • FGF-5 has been modified by replacement of the asparagine residue at position 1 69;
  • FGF-6 has been modified by replacement of the asparagine residue at position 1 56;
  • FGF-7 has been modified by replacement of the asparagine residue at position 146;
  • FGF-8 has been modified by replacement of the asparagine residue at position 1 36
  • FGF-9 has been modified by replacement of the asparagine residue at position 143
  • FGF-1 0 has been modified by replacement of the asparagine residue at position 91 .
  • the FGF has been mutagenized to introduce an amino acid substitution at positions corresponding to residues Phe 95 , Asn 101 or Asn 104 of bFGF, such that the resulting peptide has reduced binding to the cognate FGF receptor, but retains heparin binding activity.
  • the substituting ammo acid residue is phenylalanine, glycine, serine or alanine and more preferably alanine.
  • the FGF is FGF-2, is encoded by the
  • DNA bFGF as set forth in SEQ ID N0:2 and the replacement ammo acid residue is glycine, serine or alanine.
  • the substituting ammo acid residue is alanine.
  • FGF muteins in which two or three of the above residues are modified are also provided herein.
  • FGF muteins in which positions corresponding to one or more of the Cys 78 and Cys 96 of FGF-2 have been replaced with serine residues are contemplated herein.
  • FGF- 1 can be further modified by replacement of the cysteine residues at positions 31 or 1 32 or positions 31 and 1 32;
  • FGF-5 has been by replacement of the cysteine residues at position 1 9, 93, or 202, or at least two of positions 1 9, 93, or 202, or at all of positions 1 9, 93, and 202;
  • FGF-6 by replacement of the cysteine at position 80;
  • FGF-7 by replacement of the cysteine residues at position 1 8, 23, 32, 46, 71 or 1 33, or at least two of positions 1 8, 23, 32, 46, 71 or 1 33, or at least three of positions 1 8, 23, 32, 46, 71 or 1 33, or at least four of positions 1 8, 23, 32, 46, 71 or 1 33, or at least five of positions 1 8, 23, 32, 46, 71 or 1 33, or at positions 1 8, 23, 32, 46, 71 or 1 33;
  • FGF-8 by replacement of the cysteine residues at position 1 0, 1 9, 1 09 or 1 27, or at least two of positions 1 0, 1 9, 1 09 or 1 27, or at least three of positions 1 0, 1 9, 109 and 1 27 with se
  • Standard physiological, pharmacological and biochemical procedures are available for testing the FGF muteins to identify those that lack or have greatly reduced FGF receptor binding activity.
  • Numerous assays are known to those of skill in the art for evaluating the ability of FGF muteins to modulate the activity of one or more FGF peptide.
  • the properties of a potential antagonist may be assessed as a function of its ability to inhibit FGF activity including the ability in vitro to compete for binding to FGF receptors present on the surface of tissues or recombinant cell lines, cell-based competitive assays fsee, e.g., Mostacelli et al. ( 1 987) J. Cell. Phvsiol.
  • FGF muteins lacking FGF receptor binding activity may be identified by the inability of a sub-type specific FGF mutein to interfere with one or more FGF peptide binding to different tissues or cells expressing different FGF receptor subtypes, or to interfere with the biological effects of an FGF peptide [see, e.g., International Patent Application Publication No. WO 95/2441 4].
  • the heparin binding activity of the FGF muteins can be measured using the methods described herein or other methods known to those of skill in the art.
  • the ability of FGF muteins to bind to heparin can be determined by methods including, but not limited to, heparin or heparin sulfate chromatography (Zhang et al. ( 1 991 ) Proc. Natl. Acad. Sci. U.S.A. 88:3441 - 3445; International patent application No.
  • the relative affinities of the FGF muteins for FGF receptors and heparin have been and can be assessed. Those that possess the desired in vitro properties, such as significantly reduced FGF receptor binding affinity for one or more FGF receptor and normal heparin binding activity, are selected.
  • the selected FGF muteins that exhibit desirable activities, e.g., specifically bind to heparin but do not bind to their cognate receptor, may be therapeutically useful in the methods described herein and are tested for such uses employing the above-described assays from which the in vivo effectiveness may be evaluated [Gospodarowicz et al. ( 1 987) Endocrin. Rev. 8:95-1 14; Buntrock et al.
  • compositions are provided for use in the methods herein that contain therapeutically effective amounts of an FGF mutein or peptide-encoding fragment thereof.
  • the FGF mutein are preferably formulated into suitable pharmaceutical preparations such as tablets, capsules or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparation.
  • suitable pharmaceutical preparations such as tablets, capsules or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparation.
  • the FGF muteins described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art.
  • an FGF mutein or mixture of FGF muteins or a physiologically acceptable salt is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form as called for by accepted pharmaceutical practice.
  • a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc. is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form as called for by accepted pharmaceutical practice.
  • the amount of active substance in those compositions or preparations is such that a suitable dosage in the range indicated is obtained.
  • one or more FGF mutein is mixed with a suitable pharmaceutically acceptable carrier. Upon mixing or addition of the
  • the resulting mixture may be a solution, suspension, emulsion or the like.
  • Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the FGF mutein in the selected carrier or vehicle.
  • the effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined.
  • compositions suitable for administration of the FGF muteins include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • the active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action or have other action.
  • the FGF muteins may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients. In instances in which the FGF muteins exhibit insufficient solubility, methods for solubilizing compounds may be used.
  • Such methods include, but are not limited to, using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such as tween, or dissolution in aqueous sodium bicarbonate.
  • cosolvents such as dimethylsulfoxide (DMSO)
  • surfactants such as tween
  • dissolution in aqueous sodium bicarbonate Such methods are known to those of skill in this art, and include, but are not limited to, using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such as tween, or dissolution in aqueous sodium bicarbonate.
  • Derivatives of the compounds such as salts of the compounds or prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions.
  • the concentrations or FGF muteins are effective for delivery of an amount, upon administration, that ameliorates the symptoms of the disorder for which the FGF muteins are administered.
  • the compositions are formulated for single dosage administration.
  • the FGF muteins may be prepared with carriers that protect them against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems,
  • the FGF mutein is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.
  • the therapeutically effective concentration may be determined empirically by testing the activity of the FGF muteins in known in vitro and in vivo model systems for the treated disorder.
  • the compositions can be enclosed in ampules, disposable syringes or multiple or single dose vials made of glass, plastic or other suitable material. Such enclosed compositions can be provided in kits.
  • the concentration of FGF mutein in the drug composition will depend on absorption, inactivation and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • the composition may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of , the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated.
  • the FGF mutein should be provided in a composition that protects it from the acidic environment ot the stomach.
  • the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine.
  • the composition may also be formulated in combination with an antacid or other such ingredient.
  • Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules.
  • the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules or troches.
  • Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder, such as, but not limited to, gum tragacanth, acacia, corn starch or gelatin; an excipient such as microcrystalline cellulose, starch and lactose, a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a glidant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, and fruit flavoring.
  • a binder such as, but not limited to, gum tragacanth, acacia, corn starch or gelatin
  • an excipient such as microcrystalline cellulose, starch and lactose, a disintegrating agent such as, but not limited to, alginic acid and corn
  • the dosage unit form When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil.
  • dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents.
  • the compounds can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like.
  • a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
  • the FGF muteins or peptides thereof can also be mixed with other active materials, that do not impair the desired action, or with materials that supplement the desired action, including viscoelastic materials, such as hyaluronic acid, which is sold under the trademark HEALON (solution of a high molecular weight (MW of about 3 millions) fraction of sodium hyaluronate; manufactured by Pharmacia, Inc. see, e.g., U.S. Patent Nos.
  • HEALON solution of a high molecular weight (MW of about 3 millions) fraction of sodium hyaluronate
  • the viscoelastic materials are present generally in amounts ranging from about 0.5 to 5.0%, preferably 1 to 3 % by weight of the conjugate material and serve to coat and protect the treated tissues.
  • the compositions may also include a dye, such as methylene blue or other inert dye, so that the composition can be seen when injected into the eye or contacted with the surgical site during surgery.
  • Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil like sesame oil, coconut oil, peanut oil, cottonseed oil, etc.
  • a sterile diluent such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil like sesame oil, coconut oil, peanut oil, cottonseed oil, etc.
  • a synthetic fatty vehicle like ethyl oleate or the like, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • Parental preparations can be enclosed in ampules, disposable syringes or multiple dose vials made of glass, plastic or other suitable material. Buffers, preservatives, antioxidants and the like can be incorporated as required.
  • the ophthalmologic indications herein are typically treated locally either by the application of drops to the affected tissue(s), contacting with a biocompatible sponge that has absorbed a solution of the FGF muteins or by injection of a composition.
  • the composition will be applied during or immediately after surgery in order to prevent closure of the trabeculectomy, prevent a proliferation of keratocytes following excimer laser surgery, prevent the proliferation of lens epithelial cells following cataract surgery or to prevent a recurrence of pterygii.
  • the composition may also be injected into the affected tissue following surgery and applied in drops following surgery until healing is completed. For example, to administer the formulations to the eye, it can be slowly injected into the bulbar conjunctiva of the eye.
  • suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • PBS physiological saline or phosphate buffered saline
  • suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • Liposomal suspensions, including tissue-targeted liposomes may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. For example, liposome formulations may be prepared as described in U.S. Patent No. 4,522,81 1 .
  • the active compounds may be prepared with carriers that protect the compound against rapid elimination from the body, such as time release formulations or coatings.
  • carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others. Methods for preparation of such formulations are known to those skilled in the art.
  • the compounds may be formulated for local or topical application, such - as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application.
  • Such solutions may be formulated as 0.01 % - 1 00% (weight to volume) isotonic solutions, pH about 5-7, with appropriate salts.
  • the compounds may be formulated as aeorsols for topical application, such as by inhalation [see, e.g., U.S. Patent Nos. 4,044, 1 26, 4,41 4,209, and 4,364,923].
  • the FGF mutein may be packaged as articles of manufacture containing packaging material, an acceptable composition containing an FGF mutein provided herein, which is effective for treating the particular disorder, and a label that indicates that the FGF mutein or salt thereof is used for treating FGF-mediated disorders or one or more FGF peptide from binding to its receptor.
  • FGF mutein and FGF mutein peptide compositions containing therapeutically effective concentrations of the FGF mutein or FGF mutein peptide for treating disorders, particularly disorders associated with the systemic administration of heparin, in which heparin causes or contributes to the pathology are provided herein.
  • FGF muteins that specifically bind to heparin but have reduced FGF receptor binding affinity may be used to prevent excessive bleeding resulting from the anti-coagulant activity of heparin, heparin-induced thrombosis and thrombocytopenia and to prevent the potentiation of undesired growth and proliferation of FGF-sensitive cells occurring in angiogenesis and ophthalmic disorders, are provided herein.
  • any FGF mutein that specifically binds to heparin that has significantly reduced FGF receptor binding activity may be used in the methods of treating heparin-related disorders provided herein.
  • the methods of treating heparin-related disorders use the FGF mutein compositions and pharmaceutical compositions provided herein whereas in other embodiments the methods use previously described FGF muteins that fail to recognize their cognate receptor but retain a high affinity for heparin (e.g., ammo acid substitutions corresponding to residues Glu 96 and Leu 140 of bFGF, Springer et al ( 1 994) J. Biol. Chem. 269: 26879-26884- Zhu et al ( 1 995) J Biol. Chem. 270: 10222-1 0230. Heparin-induced thrombosis and thrombocytopenia
  • heparin is a widely used antithromoblytic agent for acute management of thrombosis and is a treatment of choice for preventing and treating venous thromboembolism.
  • heparin is widely used as the injectable anticoagulant of choice, it has several potential shortcomings
  • the systemic administration of high levels of heparin used to impede local thrombus deposition also can results in the global reduction in Factor Xa and/or Factor lla activity
  • a complication of systemic heparin therapy is severe bleeding in patients because of the reduced capability of blood to coagulate (e.g., Visentin et al. ( 1 995) Curr. Qpin Hematol. 2:351 -357) .
  • Heparin-induced thrombocytopenia HIT is an immunoglobulin-mediated adverse drug reaction associated with a high risk of thrombotic complications.
  • Methods of treating heparin-induced and heparin-related disorders such excessive bleeding in patients that arise from the anticoagulant activity of heparin and methods of treating thrombocytopenia and thrombosis by administering a therapeutically effective amount of FGF mutein that binds to heparin but has significantly reduced receptor binding activity are provided.
  • the medicament containing the FGF mutein is administered intravenously (IV), although treatment by localized administration of the composition may be tolerated in some instances.
  • the medicament containing the FGF mutein is injected into the circulatory system of a subject in order to deliver a dose to bind the desired amount of heparin.
  • the FGF mutein can be formulated for topical or local administration and applied at the desired location (i.e., at a wound) . Dosages may be determined empirically, but will typically be in the range of about 0.01 mg to about 1 00 mg of the compound per kilogram of body weight are expected to be employed as a daily dosage.
  • compositions provided herein may be used in methods of treating ophthalmic disorders resulting from heparin potentiation of FGF- mediated hyper-proliferation of lens epithelial cells, fibroblasts or keratinocytes [e.g., see Dell Drug Discov. Today 1:221 -222 ( 1 996)].
  • ophthalmic disorders that may be treated using the methods and compositions provided herein include, but are not limited to, diabetic retinopathy, corneal clouding following excimer laser surgery, closure of trabeculectomies, hyperproliferation of lens epithelial cells following cataract surgery and the recurrence of pterygii.
  • the FGF mutein compositions for treating ophthalmic disorders may be formulated for local or topical application and administered by topical application of an effective concentration to the skin and mucous membranes, such as in the eye.
  • the compositions may also include a dye, such as methylene blue or other inert dye, so that the composition can be seen when injected into the eye or contacted with the surgical site during surgery.
  • the effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined.
  • FGF muteins Materials and Methods Materials
  • a human synthetic bFGF gene was purchased from R and D Systems (Minneapolis, MN).
  • Expression vector pET1 1 d and bacterial strain BL2 KDE3) were obtained from Novagen (Madison, Wl).
  • Baculovirus transfection vector PVL1 393 was obtained from PharMingen (San Diego, CA).
  • a Magic Mini preparation kit was obtained from Promega (Madison, Wl).
  • Heparin-Sepharose was obtained from Pharmacia-LKB Biotechnology (Uppsala, Sweden) . Heparin was purchased from Sigma (St Louis, MO) .
  • FGFR1 J-TPA fusion protein was a gift from Eisai (Tsukuba, Japan) .
  • [ 125 l]bFGF was obtained from NEN Research Products.
  • Anti-bFGF monoclonal antibody was purchased from Upstate Biotechnology.
  • Alkaline phosphatase-conjugated anti-mouse l B G antibodies were purchased from Bio-Rad. Prestained protein molecular weight standards were purchased from GIBCO/BRL. All other chemicals were of reagent grade, purchased from Sigma. Identification of residues for mutagenesis
  • the expression vector was transformed into the BL2I(DE3) Escherichia coli strain. Cultures were grown to an A 600 of 0.8 in LB medium containing 40 yg/ml ampicillin at 37°C. Expression of bFGF and muteins was induced by adding 0.4 mM isopropyl-/?- ⁇ -thiogalactopyranoside and the cultures were further grown for 3 h. The bFGF was purified using a CM Sepharose column, followed by a heparin-Sepharose column. The concentration of wild-type bFGF and its mutants was then determined. C. Preparation of mutagenized FGF peptides by site-directed mutagenesis
  • Site-directed mutagenesis was and can be performed using a commercially available site-directed mutagenesis kit [Clontech, Palo Alto, CA] - according to the instructions provided by the manufacturer. Plasmid isolation, production of competent cells and transformation were carried out according to published procedures (Sambrook et aL . ( 1 989) Molecular Cloning, a Laboratory Manual Cold, Spring Harbor Laboratory Press, Cold Spring Harbor, NY) . Purification of DNA fragments was achieved using the Magic mini-prep kit, purchased from Promega, (Madison, Wl) . 1 . Mutagenesis of bFGF
  • a synthetic DNA encoding human bFGF [SEQ ID NO:2; commercially available from R & D Systems, Minneapolis, MN] was digested with the restriction endonucleases ⁇ /col and BamYW and ligated into the Nco ⁇ and Bam ⁇ sites of pET 1 1 d.
  • the bFGF-pET1 1 d DNA template was denatured in an excess of two complementary primers: a bFGF-specific primer containing the desired substitutions in the bFGF coding region; and a BamYW selection primer provided by the manufacturer.
  • the BamYW specific primer introduces a mutation into the resulting plasmid that inactivates the BamYW site in the multiple cloning site thereby allowing for enrichment of mutagenized plasmids during transformation using BamYW.
  • Oligonucleotide primers used for site-directed mutagenesis of human bFGF were synthesized based on the reported bFGF sequence (SEQ ID NO:2) except for a substitution in the sequence of nucleotides encoding amino acid positions Phe 95 , Asn 101 or Asn 104 .
  • the two primers were annealed to the denatured template by slow cooling, followed by in vitro second strand synthesis and ligation.
  • Unmutagenized vector DNA was digested with BamYW and a portion of the partially digested ligation mixture was used to transform competent E ⁇ coli mutS strain BMH 71 -1 8, which was provided by the manufacturer.
  • Plasmid DNA was purified from the resulting Amp R transformants using a Magic mini-prep kit [Promega, Madison, Wl] and plasmid DNA isolated from single colony transformants was sequenced to verify the presence of each bFGF mutation. 2. Recombinant expression and purification of mutagenized bFGF
  • Plasmids encoding bFGF muteins were transformed into the E. coli strain
  • CM-Sepharose carboxymethyl-Sepharose
  • Pharmacia carboxymethyl-Sepharose
  • the bound bFGF muteins were eluted from the column using a high salt gradient (e.g. , NaCl or NH 4 OAc) .
  • the bFGF mutein-containing fractions were pooled, dialyzed against buffer A [25 mM Tris-HCl, pH 7.5; 0.6 M NaCl] and loaded onto a heparin-Sepharose column (Pharmacia) equilibrated in buffer A The column was washed extensively with buffer B (buffer A supplemented to 1 .0 M NaCl), and bound FGF muteins were eluted from the column by the addition of buffer C (buffer A supplemented to 2.0 M NaCl).
  • the binding activity of the FGF muteins for one or more FGF receptor was and can be determined by testing the ability of an FGF mutein to compete with 125 l-bFGF for binding to one or more FGF receptor or FGF-binding fragment thereof .
  • a recombinant FGF receptor fusion protein was used in which the extracellular domain of a human FGF receptor, FGFR 1 , was fused to the amino terminal fragment of tissue plasminogen activator (tPA) protein. This fusion protein retains the ability to bind FGF, such as bFGF [Zhu et al. ( 1 995) J. Biol. Chem. 270:21 869-21 8741.
  • FGFR 1 human basic fibroblast growth factor receptor 1
  • PCR polymerase chain reactions
  • the full-length FGFR1 -encoding DNA was used as a template for a subsequent PCR reaction, performed as described above, to amplify a 869 bp DNA fragment encoding only the FGFR1 extracellular domain.
  • a Hindi 11 restriction endonuclease site was introduced upstream of the FGFR1 initiation codon and a Sail site was introduced downstream of the second immunoglobulin-like extracellular domain (Igll) to facilitate cloning of the amplified product.
  • Igll immunoglobulin-like extracellular domain
  • the Hindlll site was introduced at nt -8 to -3 during the PCR reaction by synthesizing an oligonucleotide primer corresponding to nt -1 2 to + 22 that introduced nucleotide changes at three positions in the FGFR 1 sequence: nt -3 (G to T), nt -6 (A to G) and nt -8 (G to A).
  • the Sail site was introduced at nt 849 to nt 854 by synthesizing an oligonucleotide primer complementary to nt 823 to 857 containing nucleotide substitutions at three positions in the FGFR 1 sequence: nt 849 (C to G), nt 851 (G to C) and nt 854 (G to C).
  • the 857 bp PCR fragment was incubated with Hindlll and Sail and purified by agarose gel electrophoresis according to the standard procedures [Sambrook et a/. , ( 1 989) Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, New York] .
  • the DNA was isolated from gel by electroelution and recovered by precipitation with ethanol.
  • the resulting Hindlll to Sail DNA fragment consists of nt -7 to nt 849 of the FGFR1 cDNA described by Itoh et al. and encodes amino acid residues 1 to 284 of the shorter form of the bFGF receptor.
  • tPA tissue plasminogen activator
  • oligonucleotides complementary to sequences flanking the tPA coding region were synthesized and used as primers in PCR reactions to isolate a full-length cDNA encoding human tPA from a human placenta cDNA library (Clontech, Palo Alto, CA).
  • An oligonucleotide corresponding to nt -6 to + 21 , relative to the A of the initiation codon of the of human tPA prepro polypeptide [e.g., see Pennica et al.
  • the full-length DNA was used as a template for a subsequent PCR reaction to amplify a 599 bp DNA encoding the a portion of the signal peptide- finger-growth factor-first Kringle domains of tPA, and which also to introduce an in-frame amber stop codon [i.e. , UGA] at amino acid codon 1 80 of mature tPA sequence.
  • a S_a]l restriction endonuclease site and a mutation substituting a Pro for an Arg at position -6 were introduced upstream of the first Ser codon of mature tPA and a BarnHI site was introduced downstream of newly introduced translational stop codon to allow for convenient subcloning of the amplified product.
  • the substitution of Pro for Arg at amino acid residue position -6 introduces a proteolytic cleavage site for thrombin in the linker sequence (i.e., Phe-Pro-Arg-Gly at positions -7 to -4).
  • nt 76 to 81 and 91 and 92 were introduced at nt 76 to 81 and 91 and 92 (nt -30 to -25 and -1 5 and -1 4, respectively, relative to the first nucleotide of mature tPA) during the PCR reaction by synthesizing an oligonucleotide primer corresponding to nt 72 to nt 1 1 1 containing nucleotide substitutions at six positions in the tPA sequence: nt 76 (A to G), nt 79 (C to G), nt 81 (T to C), nt 91 (A to C) and nt 92 (G to C).
  • nt 652 to nt 657 and translational stop codon at amino acid codon 1 80 were introduced by synthesizing an oligonucleotide primer complementary to nt 623 to 661 containing nucleotide substitutions at three positions in the tPA sequence: nt 644 (C to A), nt 655 (A to T) and nt 657 (G to C).
  • the isolated Sajl to BamHl fragment encoding the portion of human tPA was ligated into the Sail and BamHl sites of pUC 1 8 to generate plasmid HTPA3/4-pUC 1 8.
  • HTPA3/4-pUC1 8 was then digested with Hindlll and Sail into which the isolated Hindlll to Sajl FGFR1 -encoding fragment was inserted.
  • the plasmid carrying the FGFR1 -tPA chimeric DNA was digested with Hindlll and
  • the resulting DNA encodes a 472 amino acid peptide comprised of amino acids 1 - 284 of human FGFR 1 , a 1 o amino acid linker sequence VDARFPRGAR, derived from the human tPA signal peptide, and amino acids 1 -1 78 from human tPA.
  • the resulting DNA encoding the FGFR1 -tPA fusion protein is shown in SEQ ID No: 1 1 and the encoded amino acids are set forth in SEQ ID No: 1 2.
  • the DNA of SEQ ID No. 1 1 was digested with Hindlll to BamHl and the
  • pK4K a mammalian expression vector pK4K for recombinant expression of the FGFR1 -tPA fusion protein (Niidome, T. et al. ( 1 994) Biochem. Biophvs. Res. Commun. 203, 1 821 -1 827).
  • the plasmid pK4K is a pBR322-based vector that has unique Hindlll and BamHl sites for directional cloning of heterologous DNAs whose expression is under the control of the SV40 early promoter. This plasmid also contains the Mactamase and DHFR genes for use as selectable markers in prokaryotes and eukaryotic organisms, respectively.
  • Recombinant FGFR1 -tPA fusion protein expression in BHK cells was monitored by sandwich enzyme-linked immunosorbent assays (sandwich ELISAs).
  • sandwich enzyme-linked immunosorbent assays sandwich enzyme-linked immunosorbent assays (sandwich ELISAs).
  • the recombinant FGFR 1 -tPA fusion protein was purified from condition medium of BHK-expressing cells by affinity chromatography. Transfected cells were grown as described above and the condition medium was harvested. The osmolarity of the conditioned medium was adjusted to a final concentration of 0.5 M NaCl by the addition of solid NaCl. The sample was applied onto a column of Cellulofine (Seikagaku Kogyo, Tokyo, Japan) conjugated with anti- tPA 14-6 monoclonal antibody previously equilibrated in column buffer [50 mM Tris-HCl, pH 7.5, and 0.5 M NaCl].
  • blocking buffer 25 mM HEPES, pH 7.5, 100 mM NaCl and 0.5% gelatin
  • binding buffer 25 mM HEPES, pH 7.5, 100 mM NaCl and 0.3 % gelatin
  • binding buffer 25 mM HEPES, pH 7.5, 100 mM NaCl and 0.3 % gelatin
  • binding buffer 25 mM HEPES, pH 7.5, 100 mM NaCl and 0.3 % gelatin
  • 0.1 ml of binding buffer supplemented with 2 ⁇ g/ml heparan sulfate and a range of 1 -20ng/ml of labelled 125 l-bFGF (800-1 200Ci/mmol; Amersham, Arlington Heights, IL) and incubated in the absence or presence of 2.5 ⁇ g/ml unlabelled bFGF or varying concentrations of an FGF mutein for 3 hr at ambient temperature.
  • the buffer was removed by aspiration and the wells were washed twice each with PBS and a solution of 25 mM HEPES, pH 7.5, containing 2 M NaCl.
  • Bound bFGF was dissociated from the immobilized fusion protein by the addition of two aliquots of a solution of 25 mM sodium acttate, pH 4.0, containing 2 M NaCl. The two sodium acetate washes were combined and the amount of radioactivity present was determined using a gamma counter.
  • the rat aortic smooth muscle cell line, Rb-1 expresses high and low affinity FGF receptors [e.g., see Nachtigal et al. ( 1 989) In Vitro Cell. & Develop. Biol. 25:892-897].
  • the binding activity of the FGF muteins was and can also be determined by the ability of an FGF mutein to compete with 125 l-bFGF for binding to the FGF receptors expressed on cell surface of such cells [e.g., see, Mostacelli et al. ( 1 987) J. Cell. Phvsiol. 1 31 : 1 23-1 301.
  • Rb-1 cells were grown in 24-well plates to near-confluence in Dulbecco's modified Eagle's medium (DMEM; GIBCO BRL) supplemented with 10% fetal bovine serum, penicillin ( 100 unit/ml) and streptomycin (100 ug/ml). The culture medium was removed by aspiration and the cells were incubated in binding buffer [serum-free DMEM supplemented with 20 mM HEPES (pH 7.5) and 0.1 % BSA] containing 2 ng/ml recombinant human ' 25 l-bFGF (800-1 200 Ci/mmol; Amersham, Arlington Heights, IL) and varying concentrations of test- compound, for 2 hr at ambient temperature. The nonspecific binding of iodinated bFGF to Rb-1 cells was estimated in parallel reactions performed in the presence of an excess of unlabeled bFGF.
  • binding buffer [serum-free DMEM supplemented with 20 mM HEPES
  • the cells were washed twice with cold phosphate-buffered saline (PBS) and the bFGF bound to low affinity heparin sulfate proteoglycan (HSPG) receptors was dissociated by the addition to each well of a 1 ml solution of 25 mM HEPES (pH 7.5) containing 2 M NaCl.
  • the bFGF bound to high affini ty FGF receptors was dissociated by the addition to each well of a 1 ml solution of 25 mM sodium acetate (pH 4.0) containing 2 M NaCl.
  • a 1 ml aliquot from each well was transferred to a polypolyene tube and the amount of radioactivity present in the low and affinity samples was determined using a gamma counter. 2.
  • the specificity of the FGF muteins was and can be examined by measuring the ability of compounds to inhibit the binding of epidermal growth factor (EGF) to the surface of Rb-1 cells.
  • EGF epidermal growth factor
  • Rb-1 cells were grown as described above and incubated in binding buffer containing 2 ng/ml of 125 I-EGF
  • SMCs such as rat aortic Rb-1 cells
  • FGF muteins The activity of FGF muteins can be assessed by measuring tritiated thymidine incorporation into the DNA of cultured SMCs incubated in the presence of bFGF, PDGF or EGF.
  • An inoculum of approximately 2 X 1 0 4 Rb-1 cells was added to a plurality of wells and the cells cultured for three days as described in EXAMPLE 1 B(i) .
  • the cells were washed twice with serum-free medium [DMEM supplemented with 0.1 % BSA, 5 ⁇ g/ml transferrin, I mM sodium pyruvate, penicillin ( 1 00 unit/ml) and streptomycin ( 1 00 ug/ml)] and cultured for an additional three days in serum-free DMEM medium.
  • serum-free medium [DMEM supplemented with 0.1 % BSA, 5 ⁇ g/ml transferrin, I mM sodium pyruvate, penicillin ( 1 00 unit/ml) and streptomycin ( 1 00 ug/ml)] and cultured for an additional three days in serum-free DMEM medium.
  • Arg97, Leu98, Asn 104 and Arg 1 07 are solvent accessible and Thr105, Ile65, Phe94 and Try1 06 are either fully buried or hardly solvent accessible.
  • each solvent-accessible residue > 10 A was replaced with alanine and receptor binding affinities of the resulting muteins using the radiolabled receptor competition assay were determined. All of the expressed recombinant proteins were partially soluble and could be purified to homogeneity using a CM-Sepharose column, followed by a heparin-Sepharose column.
  • the relative receptor binding affinities of the solvent-accessible bFGF alanine muteins for one or more FGF receptor was determined by testing the ability of an FGF mutein to compete with 125 l-bFGF for binding in the Soluble Receptor assay described in EXAMPLE 2A and the values compared to wild type bFGF.
  • the IC 50 and the IC 50 mutein/IC 50 wildtype ratio for the bFGF muteins are shown in the following tables.
  • Residues Leu23, Thr1 05, Tyr106, Ile1 37 and Phe 1 39 are either fully buried or hardly solvent accessible, whereas Tyr103, Leu 140, Pro141 , Glu96, Arg97, Leu98, Glu99, Asn 101 and Asn102 are solvent accessible ( > 10 A 2 ) (Table 4). Replacement of these solvent-exposed residues with alanine and expression of the resulting muteins yielded partially soluble proteins muteins. These were purified and their receptor binding affinities were determined by a radiolabeled receptor competitive assay.
  • Table 4 shows that substitution of Asn1 01 , Tyr103, and Leu140 with alanine reduces the receptor binding affinities -7.72-, 402- and 245-fold, respectively, compared with the wild-type protein, confirming the importance of these three residues for high-affinity receptor binding:
  • the mutein N 1 04A exhibits a reduction in binding by a factor of 400, but like wild-type bFGF, N 104A binds tightly to heparin-Sepharose and can be eluted from a heparin-Sepharose column with 2 M NaCl buffer, suggesting that replacement of Asn 104 by alanine does not affect heparin affinity and there is no global conformational change in the N 104A mutein.
  • N 1 04A binds strongly to heparin, the effect of the mutation on receptor binding is unlikely to be due to an effect on ligand dime ⁇ zation
  • mapping studies to the neighbors of Asn104 were performed. On this site-directed mutagenesis was effected to replace following neighboring solvent-accessible residues by alanine: Arg97, Leu98, Glu99, Asn101 , Asn1 02, Tyr1 03, Leu 1 40 and Pro141 .
  • the FGF Asn104 mutein still binds tightly to heparin-Sepharose and therefore, these FGF muteins, particularly Asn1 04, and double muteins Glu have the desired properties for treating heparin-related disorders.
  • the present data, together with identification of Glu96 as a crucial residue indicate that the primary high-affinity FGFR binding site involves at least four hydrophobic residues (Tyr24, Tyr1 03, Leu 1 40 and Met142) and two polar residues (Glu96 and Asn 104) .
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE: (ix) FEATURE:
  • AAA ACG CAG ATG ATA TAC CAG CAA CAT CAG TCA TGG CTG CGC CCT GTG 962 Lys Thr Gin Met He Tyr Gin Gin His Gin Ser Trp Leu Arg Pro Val 305 310 315
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE

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Abstract

L'invention décrit des mollécules d'ADN isolées qui codent des polypeptides de mutéine du facteur de croissance de fibroblaste (FCF) ainsi que les mutéines FCF codées. Les mutéines FCF présentent une activité de liaison de récepteur réduite mais gardent leur aptitude à se fixer à l'héparine. Des procédés de traitement de troubles liés à l'héparine consistent à administrer une quantité thérapeutiquement efficace d'une mutéine FCF.
PCT/JP1998/000878 1997-03-03 1998-03-03 Compositions de muteine du facteur de croissance des fibroblastes, et procedes d'utilisation de ces compositions WO1998039436A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0960117A1 (fr) * 1996-08-30 1999-12-01 The Johns Hopkins University School Of Medicine Facteurs homologues du facteur de croissance des fibroblastes (fhf) et ses modes d'utilisation
WO2002014471A2 (fr) * 2000-08-15 2002-02-21 Phage Biotechnology Corporation Procede de production du facteur de croissance de fibroblastes humain acide biologiquement actif et son utilisation pour favoriser l'angiogenese
US7252818B2 (en) 1998-07-24 2007-08-07 Cardiovascular Biotherapeutics, Inc. Method of producing biologically active human acidic fibroblast growth factor and its use in promoting angiogenesis
US7294706B2 (en) * 2001-08-01 2007-11-13 New York University Identification of receptor and heparin binding sites in FGF4 by structure-based mutagenesis

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996022369A1 (fr) * 1994-10-13 1996-07-25 Amgen Inc. Analogues de facteur de croissance du fibroblaste acide a stabilite et activite biologique renforcees
WO1996038167A1 (fr) * 1995-05-31 1996-12-05 Osteosa, Inc. Utilisation de facteurs de croissance des fibroblastes pour stimuler la croissance osseuse

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1996022369A1 (fr) * 1994-10-13 1996-07-25 Amgen Inc. Analogues de facteur de croissance du fibroblaste acide a stabilite et activite biologique renforcees
WO1996038167A1 (fr) * 1995-05-31 1996-12-05 Osteosa, Inc. Utilisation de facteurs de croissance des fibroblastes pour stimuler la croissance osseuse

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Title
BURGESS, W.H., ET AL.: "possible dissociation of the heparin-binding and mitogenic activities of heparin-binding (acidic Fibroblast) growth factor-1 from its receptor-binding activities by site-directed mutagenesis of a single lysine residue" THE JOURNAL OF CELL BIOLOGY, vol. 111, November 1990, pages 2129-2138, XP002074068 *
SPRINGER, B.A., ET AL.: "identification and concerted function of two receptor binding surfaces on basic fibroblast growth factor required for mitogenesis" THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 43, October 1994, pages 26879-26884, XP002073992 cited in the application *
WANG W -P ET AL: "CLONING OF THE GENE CODING FOR HUMAN CLASS 1 HEPARIN-BINDING GROWTH FACTOR AND ITS EXPRESSION IN FETAL TISSUES" MOLECULAR AND CELLULAR BIOLOGY, vol. 9, no. 6, June 1989, pages 2387-2395, XP000039606 *
ZHU, H., ET AL.: "Glu-96 of basic fibroblast growth factor is essential for high affinity receptor binding" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 270, no. 37, September 1995, pages 21869-21874, XP002073993 cited in the application *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0960117A1 (fr) * 1996-08-30 1999-12-01 The Johns Hopkins University School Of Medicine Facteurs homologues du facteur de croissance des fibroblastes (fhf) et ses modes d'utilisation
EP0960117A4 (fr) * 1996-08-30 2000-06-14 Univ Johns Hopkins Med Facteurs homologues du facteur de croissance des fibroblastes (fhf) et ses modes d'utilisation
US6635744B1 (en) 1996-08-30 2003-10-21 The Johns Hopkins University School Of Medicine Fibroblast growth factor homologous factor-4
US7252818B2 (en) 1998-07-24 2007-08-07 Cardiovascular Biotherapeutics, Inc. Method of producing biologically active human acidic fibroblast growth factor and its use in promoting angiogenesis
WO2002014471A2 (fr) * 2000-08-15 2002-02-21 Phage Biotechnology Corporation Procede de production du facteur de croissance de fibroblastes humain acide biologiquement actif et son utilisation pour favoriser l'angiogenese
WO2002014471A3 (fr) * 2000-08-15 2003-03-13 Phage Biotechnology Corp Procede de production du facteur de croissance de fibroblastes humain acide biologiquement actif et son utilisation pour favoriser l'angiogenese
US6642026B2 (en) 2000-08-15 2003-11-04 Phage Biotechnology Corporation Method of producing biologically active human acidic fibroblast growth factor and its use in promoting angiogenesis
AU2001288256B2 (en) * 2000-08-15 2006-03-30 New Technologies Holdings Pte Ltd A method of producing biologically active human acidic fibroblast growth factor and its use in promoting angiogenesis
US7294706B2 (en) * 2001-08-01 2007-11-13 New York University Identification of receptor and heparin binding sites in FGF4 by structure-based mutagenesis

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