WO1998039436A2

WO1998039436A2 - Fibroblast growth factor mutein compositions and methods of use therefor

Info

Publication number: WO1998039436A2
Application number: PCT/JP1998/000878
Authority: WO
Inventors: Hengyi Zhu; Ramnarayan Kalyanaraman; Takatoshi Kawai
Original assignee: Eisai Co Ltd
Current assignee: Eisai Co Ltd
Priority date: 1997-03-03
Filing date: 1998-03-03
Publication date: 1998-09-11
Anticipated expiration: 1999-09-03
Also published as: WO1998039436A3

Abstract

Isolated DNA molecules that encode fibroblast growth factor (FGF) mutein polypeptides and the encoded FGF muteins are provided. The FGF muteins exhibit reduced receptor binding activity, but retain the ability to bind to heparin. Methods for treating heparin-related disorders by administering a therapeutically effective amount of an FGF mutein are also provided.

Description

DESCRIPTION

FIBROBLAST GROWTH FACTOR MUTEIN COMPOSITIONS AND METHODS OF

USE THEREFOR

RELATED APPLICATIONS

Benefit of priority is claimed to U.S. Provisional application Serial No. 60/040,785, entitled "BASIC FGF MUTEIN AND ASSAY FOR MODULATORS OF FGF ACTIVITY", filed March 3, 1 997. Where permitted, the subject matter of the above-noted U.S. provisional application is incorporated herein by reference in its entirety. FIELD OF THE INVENTION The present invention relates to mutagenized fibroblast growth factor

(FGF) genes and peptides. In particular, 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.

Fibroblast growth factors and fibroblast growth factor receptors

One family of growth factors that has a broad spectrum of activities is the fibroblast growth factor (FGF) family [e.g., see Johnson et al. , Advan. Cancer Res. 60: 1 -41 ( 1 993)] This family of proteins includes FGFs designated FGF-1 through FGF-1 2 (or acidic FGF (aFGF), basic FGF (bFGF), int-2, hst/K- FGF, FGF-5, FGF-6, keratinocyte growth factor (KGF), FGF-8, FGF-9, FGF-10, FGF-1 1 and FGF-1 2, respectively) . Acidic and basic FGF, which were the first members of the FGF family that were characterized, are about 55 % identical at the amino acid level and are highly conserved among species. Basic FGF has a molecular weight of approximately 1 6 kD, is basic and temperature sensitive and has a high isoelectric point [pi = 9.6; e.g., see in The Cytokine FactsBook, Callard and Gearing, eds., p.1 21 , Academic Press, Inc., London]. 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.

In addition, 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). For example, 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) .

Although 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. Members of the FGF family, including bFGF, are thought to play a pathophysiological role, for example, in tumor development, rheumatoid arthritis, proliferative diabetic retinopathies and other complications of diabetes. Because FGFs are associated with many disease states, they are therapeutic targets. For example, antagonists of bFGF activity and/or 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.

The effects of 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]. 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. For example, 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. Similarly, 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. 2303- 231 3). 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). Heparin-induced thrombosis and thrombocytopenia

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. As a result, 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) Anαiology 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. Although 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).

The most notorious complication of systemic heparin therapy is heparin-induced thrombocytopenia. Heparin-induced thrombocytopenia (HIT) is an immunoglobulin-mediated adverse drug reaction associated with a high risk of thrombotic complications. The pathogenic antibody, usually immunoglobulin (lg)G (HIT-lgG), recognizes a multimolecular complex of heparin and platelet factor 4, a heparin-binding protein normally contained in platelet alpha granules, resulting in platelet activation via platelet Fc receptors. There are an array of disorders or side-effects of heparin treatment that require treatment. Thus, there is a need to develop pharmacological products that modulate the activity of heparin. Therefore it is an object herein to provide mutagenized FGF peptides and compositions containing these FGF mutein peptides that modulate, particularly inhibit, the activity of heparin. It also an object herein to provide methods for ameliorating heparin-induced or heparin- related conditions, such as coagulants for heparin-associated bleeding, antagonists of heparin-induced angiogenesis in ophthalmic disorders, and for treating heparin-induced thrombocytopenia and thrombosis. SUMMARY OF THE INVENTION Isolated DNA encoding 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. In particular, 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. In addition, the muteins will optionally include replacement of the glu96 residue. In preferred embodiments, 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. Presently preferred FGF 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) .

In other embodiments, 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.

Pharmaceutical 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. In particular, 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.

Articles of manufacture containing packaging material, an FGF mutein polypeptide provided herein, which is effective for ameliorating the symptoms of a heparin-related disorder, antagonizing the effects of heparin or heparin binding to an endogenous FGF polypeptide, within the packaging material, and a label that indicates that the compound or salt thereof is used for antagonizing the effects of heparin, treating a heparin-related disorder, or inhibiting the binding of heparin to an FGF peptide are provided.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference herein.

The 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. The nucleotides, which occur in the various DNA fragments, are designated with the standard single-letter designations used routinely in the art.

As used herein, 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. For example, bFGF (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. As used herein, 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. Preferably, 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.

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.

Other 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 Molecυlar Biology of the Gene, 4th Edition, 1 987, The Bejacmin/Cummings Pub. co., p.224).

Such substitutions are preferably made in accordance with those set forth in TABLE 1 as follows:

TABLE 1

Original residue Conservative substitution

Ala (A) Gly; Ser

Arg (R) Lys

Cys (C) Ser Gin (Q) Asn

Glu (E) Asp Gly (G) Ala; Pro His (H) Asn; Gin

He (I) Leu; Val

Leu (L) lie; Val

Lys (K) Arg; Gin; Glu Met (M) Leu; Tyr; lie iginal residue Conservative substitution

Phe (F) Met; Leu; Tyr

Ser (S) Thr

Thr (T) Ser

Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) lie; Leu

Other substitutions are also permissible and may be determined empirically or in accord with known conservative substitutions. As used herein, 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. 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. 4,868.1 1 3, published International Application WO 90/08771 (and the corresponding U.S. patent, upon its issuance), which is based on U.S. Application Serial No. 07/304,281 , filed January 31 , 1 989, and Miyamoto et aL ( 1 993) Mol. Cell. Biol. 1 3:4251 -4259) , and any DNA fragment that may be produced from any of the preceding DNA fragments by substitution of degenerate codons. It is understood that once the complete amino acid sequence of a peptide, such as an FGF peptide, and one DNA fragment encoding such peptide are available to those of skill in this art, it is routine to substitute degenerate codons and produce any of the possible DNA fragments that encode such peptide. It is also generally possible to synthesize DNA encoding such peptide based on the amino acid sequence. As used herein, 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. As used herein, 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. Thus, 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.

As used herein, operative linkage or operative association of heterologous DNA to regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences, refers to the functional relationship between such DNA and such sequences of nucleotides. For example, 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. As used herein, 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. In addition, 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. As used herein, 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).

As used herein, heparin is the heterogenous, sulfated anionic polysacchaπde 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. As used herein, heparin also refers to low molecular weight heparin analogs (i.e. , LMWH; commercially available as "FRAGMIN").

As used herein, hepaπn-like substances are molecules that have oligosacchaπde structures related to heparin and exhibit an anti-coagulant activity of substantially similar to heparin.

As used herein, a heparin-induced or heparin-related disorder is a disorder in which the administration of heparin or hepaπn-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.

As used herein, 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.

As used 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.

As used herein, 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. As used herein, systemic administration refers to adminstration, such as intravenously or intramuscularly, whereby the administered composition enters the bloodstream.

As used herein, 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.

As used herein, 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. As used 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. As used herein, 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. For example, hydroxy groups can be esterified or etherified. As used herein, 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. 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.

As used herein, adequately pure or "pure" per se means sufficiently pure for the intended use of the adequately pure compound.

As used herein, 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. To produce a prodrug, 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. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, once a pharmaceutically active compound is identified, those of skill in the pharmaceutical art generally can design prodrugs of the compound [see, e.g., Nogrady (1 985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392].

As used herein, the IC₅₀ refers to an amount, concentration or dosage of a particular compound that achieves a 50% inhibition of a maximal response. As used herein, EC₅₀ 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. As used herein, 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. The effectiveness of a potential heparin antagonist can be assessed using methods known to tnose of skill in the art For example, 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.

As used herein, the abbreviations for any group or other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see,

( 1 972) Biochem. 1 1 :942-944).

A. Preparation of DNA encoding FGF muteins 1 . DNA encoding FGF polypeptides

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. 4,868.1 1 3, published International Applications WO 95/24414 and WO 90/08771 (and the corresponding U.S. patent, upon its issuance), which is based on U.S. Application Serial No. 07/304,281 , filed January 31 , 1 989, and Miyamoto et aL (1 993) Mol. Cell. Biol. 1 3:4251 -4259). Specific reference to ammo acid sequence positions of bFGF is relative to the 146 ammo acid isoform of bFGF, which is generated from N-terminal truncation of the 1 55 ammo acid isoform set forth in SEQ ID NO: 2 [e.g., see International application Publication No. WO 86/07595]. 2. DNA constructs for recombinant production of FGF muteins

DNA is introduced into a plasmid for expression in a desired host. In preferred embodiments, 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.

In preterred embodiments 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.

A wide variety of multipurpose expression vectors suitable for the site- directed mutagenesis of heterologous proteins are known to those of skill in the art and are commercially available. Expression vectors containing mducible promoters or constitutive promoters that are linked to regulatory regions are preferred. Such promoters include, but are not limited to, the T7 phage promoter and other T7-lιke phage promoters, such as the T3, T5 and SP6 promoters, the trp, Ipp, and lac promoters, such as the |acUV5, from J . coli; the P1 0 or polyhedron gene promoter of baculovirus/msect cell expression systems and mducible promoters from other eukaryotic expression systems. 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) . For example, the plasmid pET1 1 d is a prokaryotic expression vector that contains a multiple cloning site for inserting heterologous DNA templates downstream of a bacteπophage 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. As exemplified below, pET1 1 d was used for the site-directed mutagenesis and mtracellular expression of bFGF and bFGF muteins. For instance, 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 Λ/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 [e.g., SEQ ID NOs: 1 and 3-1 0] 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. For example, 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. 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) . In general, 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). An 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. 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.

As exemplified below, 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) . The analysis of the 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.

Of these residues Leu55, Val63, Phe95, Arg97, Leu98, Asn1 04 and Arg 107 of FGF-2 are solvent accessible. Each of these residues was changed to an alanine following the site-directed mutagenesis procedure set forth below in Example 1 A.2 and the receptor binding activity was determined as in Example 2A.

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 (see EXAMPLE 2A) were determined and compared with wild-type bFGF. Upon identification of each crucial residue, the corresponding neighboring residues within a 7.5 A radius were further examined. Those which are solvent accessible were subjected to a second-round metagenesis and testing in the receptor binding assay.

Site-directed mutagenesis was performed to introduce amino acid substitutions in the solvent-accessible residues that are within a 7.5 angstrom radius of amino acid Asn104 of bFGF. Analysis of the crystal structure of bFGF revealed that Leu23, Glu96, Arg97, Leu98, Glu99, Asn1 01 , Asn1 02, Tyr103, Thr1 05, Tyr106, Ile1 37, Phe 1 39, Leu 140 and Pro1 41 are within a 7.5 angstrom radius. Of these residues, residues Glu96, Arg97, Leu98, Glu99, Asn1 01 , Asn102, Tyr103, Leu 1 40 and Pro141 are water-accessible. Each of these residues was changed to an alanine following the site-directed mutagenesis procedure set forth below in Example 1 A.2 and the receptor binding activity was determined as in Example 2A. B. Preparation of FGF mutein polypeptides

1 . Host organisms for recombinant production of FGF muteins Host organisms 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. 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. , 263: 1 6471 -1 6478 ( 1 988) and in International PCT Application Serial No. PCT/US93/05702. Expression of recombinant FGF proteins may be performed as described herein; and DNA encoding FGF proteins may be used as the starting materials for the methods herein.

2. Methods for recombinant production of FGF muteins

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.

Methods of transforming 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.

Once the FGF mutein-encoding DNA fragment has been introduced into the host cell, 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. In a preferred embodiment, 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 lacUVδ promoter (see, e.g. , Muller-Hill et a _. ( 1 968) Proc. Natl. Acad. Sci. USA 59: 1 259-1 2649) . Addition of IPTG induces expression of the T7 RNA polymerase and the T7 promoter, which is recognized by the T7 RNA polymerase. In more preferred embodiments, 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). In addition, a variety of chromatographic methods, such as ion-exchange chromatography or immunoaffinity chromatography using antibodies raised against an FGF polypeptide, may also be used.

As exemplified below, bFGF muteins in which amino acid residues Phe⁹⁵, Asn¹⁰¹ and Asn¹⁰⁴ 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. C. FGF muteins

Muteins of 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. In particular, 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-3 that has been modified by replacement of the valine residue at position 1 1 8;

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; and FGF-10 that has been modified by replacement of the lysine residue at position 86.

Other muteins include:

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; and

FGF-1 0 has been modified by replacement of the asparagine residue at position 91 .

In preferred embodiments, the FGF has been mutagenized to introduce an amino acid substitution at positions corresponding to residues Phe⁹⁵, Asn¹⁰¹ or Asn¹⁰⁴ of bFGF, such that the resulting peptide has reduced binding to the cognate FGF receptor, but retains heparin binding activity. Preferably, the substituting ammo acid residue is phenylalanine, glycine, serine or alanine and more preferably alanine.

Also provided are muteins in which in addition to the above-noted modifications, also have the Glu at the position corresponding the Glu96 in FGF- 2 replaced, preferably with alanine, phenylalanine, serine or glycine Table 2 indicates the positions of the residues of FGF-1 and FGF-3 through FGF-1 0 that correspond to the above-identified residues of bFGF as determined by the alignment of homologous regions of the sequence of ammo acids set forth in SEQ ID NOs: 1 and 3-1 0

TABLE 2

In certain preferred embodiments, 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. In more preferred embodiments, the substituting ammo acid residue is alanine.

Muteins in which two or three of the above residues are modified are also provided herein. In addition, FGF muteins in which positions corresponding to one or more of the Cys⁷⁸ and Cys⁹⁶ of FGF-2 have been replaced with serine residues are contemplated herein. For example, 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-3 by replacement of the cysteine residue at position 50; FGF-

4 by replacement of the cysteine residue at 88; 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 serine; FGF-9 by replacement of the cysteine residue at position 68. Most preferred is the FGF-2 mutein in which Asn 1 04 and the Glu96 residues are replaced with alanine, phenylalanine, serine or glycine. D. Evaluation of the bioactivity of FGF muteins

1 . FGF receptor binding assays

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. For example, 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. 1 31 : 1 23-1 30]; mitogenic assays [Gospardarowicz et al. ( 1 984) Proc. Natl. Acad. Sci. U.S.A. 81 :6963- 6967; Thomas et al. (1 984) Proc. Natl. Acad. Sci. U.S.A. 81 :357]; stimulation of angiogenesis in vitro [see, e.g., European Patent Application No. EP 645 451 ]; cell proliferation assays [see, e.g.. International Application Publication No. WO 92/1 2245]; assays measuring the release of cellular proteases [Mostacelli et al. (1 986) Proc. Natl. Acad. Sci. U.S.A. 83:2091 -2095: Phadke ( 1 987) Biochem. Biophvs. Res. Comm. 1 42:448-453]; and, assays for the promotion of FGF-mediated neurite outgrowth and neuron survival [Togari et al. ( 1 983) Biochem. Biophvs. Res. Comm. 1 1 4: 1 1 89-1 1 93; Wagner et al. ( 1 986) J. Cell Biol. 1 03: 1 363-1 3671.

In addition, 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].

2. Heparin binding assays

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. For example, 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. WO 92/1 2245); affinity chromatography by immobilizing the FGF mutein measuring the binding of labeled or unlabeled heparin or by calculating a thermodynamic dissociation constant for heparin affinity for each FGF mutein (e.g., see European patent application Publication No. EP 0 645 451 ).

Using such assays, 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. ( 1 982) Exp. Pathol. 21 :62-67; International Patent Application Publication No WO 92/08473]. FGF muteins that exhibit the in vitro activities that correlate with the in vivo effectiveness will then be formulated in suitable pharmaceutical compositions and used as therapeutics. E. Formulation of pharmaceutical compositions 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. Typically the FGF muteins described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art.

About 1 0 to 500 mg of 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. The amount of active substance in those compositions or preparations is such that a suitable dosage in the range indicated is obtained.

To prepare compositions, one or more FGF mutein is mixed with a suitable pharmaceutically acceptable carrier. Upon mixing or addition of the

FGF mutein(s), 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.

Pharmaceutical carriers or vehicles suitable for administration of the FGF muteins provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. In addition, 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 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. Typically, 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. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

If oral administration is desired, the FGF mutein should be provided in a composition that protects it from the acidic environment ot the stomach. For example, 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. For the purpose of oral therapeutic administration, 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. 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. In addition, 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. 5,292,362, 5,282,851 , 5,273,056, 5,229, 1 27, 4, 51 7,295 and 4,328,803), VISCOAT (fluorine-containing (meth)acrylates, such as, 1 H, 1 H,2H,2H-hepta- decafluorodecylmethacrylate; see, e.g., U.S. Patent Nos. 5,278, 1 26, 5,273,751 and 5,214,080; commercially available from Alcon Surgical, Inc.), ORCOLON (see, e.g., U.S. Patent Nos. 5,273,056; commercially available from Optical Radiation Corporation), methylcellulose, methyl hyaluronate, polyacrylamide and polymethacrylamide (see, e.g., U.S. Patent No. 5,273,751 ). 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. or 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. For the indications herein, 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.

If administered intravenously, 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. Such 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].

Finally, 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. F. Methods of treating heparin-related disorders

Methods using 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. In particular, 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. In certain embodiments, 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 Glu⁹⁶ and Leu¹⁴⁰ 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

As noted above, heparin is a widely used antithromoblytic agent for acute management of thrombosis and is a treatment of choice for preventing and treating venous thromboembolism. Although heparin is widely used as the injectable anticoagulant of choice, it has several potential shortcomings 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 lla activity Thus, 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). A notorious complication of systemic heparin therapy is heparin-induced thrombocytopenia. 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. Preferably, the medicament containing the FGF mutein is administered intravenously (IV), although treatment by localized administration of the composition may be tolerated in some instances. Generally, 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. Alternatively, 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.

Ophthalmic Disorders

Pharmaceutical 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)]. In particular, 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.

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. EXAMPLE 1

Preparation of FGF muteins A. 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) . [¹²⁵l]bFGF was obtained from NEN Research Products. Anti-bFGF monoclonal antibody was purchased from Upstate Biotechnology. Alkaline phosphatase-conjugated anti-mouse l_BG 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 crystal structure of bFGF was obtained from the protein data bank (Koetzle et al. (1 977) J. Mol. Biol 1 1 2:535-542; Abola et al. ( 1 987)ln Allen, F.H., Bergerhoff, G. and Slevers, R. (eds), Crystallographic Databases - Information Content, Software Systems, Scientific Applications, Data Commission of the International Union of Crystallography, Cambridge, pp. 1 07- 1 32) (code 3FGF) and analyzed computationally as described previously (seem Zhu et a/.( 1 995) J. Biol. Chem.270:21 869-21 874) . Solvent-accessible residues ( > 10 A²) within a 7.5 A radius of Glu96 and Asn 104 were chosen for site- directed mutagenesis. B. Mutagenesis, protein expression and purification

The construction of the human bFGF gene into the pET1 1 d vector, mutagenesis and expression and purification are described below. Briefly, after site-directed mutagenesis, the expression vector was transformed into the BL2I(DE3) Escherichia coli strain. Cultures were grown to an A₆₀₀ 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-/?-σ-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 Λ/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 Phe⁹⁵, Asn¹⁰¹ or Asn¹⁰⁴. 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

BL2 KDE3) [Novagen, Madison, Wl], which contains a copy of the T7 RNA polymerase gene under the control of the iacUVδ operon promoter

Transformants were selected for resistance to ampicillm and the cells from single colony transformants were grown at 37°C to mid-log phase (A₆₀₀ = 0.8) in LB medium [Sambrook et a/. , 1 989] supplemented with 40 μg/ml ampicillm.

Recombinant expression of FGF muteins was induced by the addition of the of 0.4 mM ιsopropyl- ?-D-thιogalactopyranosιde (IPTG) and expression was allowed to proceed for an additional 4 hours at 37°C

Cells were pelleted by centrifugation, lysed and the cellular debris was removed by centrifugation. The cytoplasmic fraction containing the soluble FGF muteins was loaded onto a carboxymethyl-Sepharose (CM-Sepharose) column (e.g., Pharmacia) and the bound bFGF muteins were eluted from the column using a high salt gradient (e.g. , NaCl or NH⁴OAc) . 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).

Samples of the purified protein fractions were subject to electrophoresis on 1 2% SDS-polyacrylamide gels and resolved proteins were visualized by staining with Coomassie Blue 250. The concentration and purity of the various FGF muteins were determined using a scanning laser densitometry and bovine serum albumin as a standard or by using a commercially available kit based on the method of Bradford [e.g. , Bio-Rad].

EXAMPLE 2 Assays for measuring the binding FGF muteins to an FGF receptor

A. Soluble FGF receptor assay

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 ¹²⁵l-bFGF for binding to one or more FGF receptor or FGF-binding fragment thereof . In one embodiment, 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.

1 . Isolation of DNA encoding the shorter form of human fibroblast growth factor receptor 1 (FGFR1 )

The nucleotide sequence of the DNA encoding the shorter form of human basic fibroblast growth factor receptor 1 (FGFR 1 ) has been determined [e.g. , N. Itoh et al. , (1 990) Biochem. Biophvs. Res. Comm 1 69:680-685], This shorter form of FGFR1 is a 731 amino acid polypeptide that has a signal peptide, two extracellular immunoglobulin-like domains, a transmembrane domain and an intracellular tyrosine kinase domain. Based on the reported sequence, two oligonucleotides complementary to sequences flanking the FGFR1 coding region were synthesized and used as primers in polymerase chain reactions (PCR) to isolate a DNA encoding a full- length human FGFR1 from a human aorta cDNA library (Quickclone, Clontech, Palo Alto, CA). PCR amplification was performed using a commercially available PCR kit according to manufacturer's instructions (Perkin Elmer Cetus, Norwalk, CT). An oligonucleotide corresponding to nt -20 to + 5, relative to the A of the ATG initiation codon of FGFR1 , [e.g., N. Itoh et al., ( 1 990) Biochem. Biophvs. Res. Comm. 1 69:680-6851 and an oligonucleotide complementary to nt 221 8- 2243 were used as primers to amplify a 2,243 bp PCR product encoding the entire FGRF1 coding region.

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. Simultaneously, 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. 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.

Thus, 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.

2. isolation of DNA encoding human tissue plasminogen activator

The nucleotide sequence of the DNA encoding human tissue plasminogen activator (tPA) has been determined [e.g., see Pennica et al. ( 1 983) Nature 301 :214-221 ]. Human tPA is a 562 amino acid polypeptide which is processed during secretion to its mature form by cleavage of a 35 amino acid signal peptide. Several regions of the primary structure of mature tPA have a high degree of homology to known structural domains of other proteins, such as homology to the finger and growth factor domains, the Kringle 1 and Kringle 2 domains of plasminogen and prothrombin and the C-terminal serine protease domain [e.g., see Ny et al. Proc. Natl. Acad. Sci. U.S.A. 81:5355-5359].

Based on the reported sequence, 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. (1 983) Nature 301 :214- 221 ] and an oligonucleotide complementary to nt 1 558 to nt 1 584 were used to amplify a 1 591 bp DNA encoding the entire human tPA prepro polypeptide.

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. Concurrently, 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).

The Sail site and the amino acid substitution 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). The BamHl site at nt 652 to nt 657 and translational stop codon at amino acid codon 1 80 (nt 642-644) 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 amplified PCR fragment was incubated with Sajl and BamHl and subjected to agarose gel electrophoresis according to the standard procedures [Sambrook et al. , (1 989) Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, New York]. The 585 bp DNA was isolated from gel by electroelution and recovered by precipitation with ethanol. 3. Construction of a vector for expressing human FGFR1 -tPA fusion protein

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

BamHl, subjected to agarose gel electrophoresis and the 1 ,426 bp DNA fragment was excised from the gel and isolated as described above. 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

1 ,434 bp fragment (nt 2-1 435 of SEQ ID No: 1 1 ) was isolated and ligated into the 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.

4. Expression of FGFR1 -tPA chimeric protein in mammalian cells Baby hamster kidney cells (BHK cells; Waechter, D.E., et al. ( 1 982) Proc.

Natl. Acad. Sci., USA:79: 1 1 06) were transfected with 5 μg of the FGFR1 -tPA- containing expression plasmid using the CellPhect calcium phosphate method according to manufacturer's instructions (Pharmacia, Sweden). Transfectants were selected for the presence of the DHFR gene by selecting resistance to methotrexate and maintained in Dulbecco's Eagle medium containing 10% fetal bovine serum and 250 nM methotrexate. Upon expression, the recombinant FGFR 1 -tPA fusion protein is secreted into the surrounding culture medium. Recombinant FGFR1 -tPA fusion protein expression in BHK cells was monitored by sandwich enzyme-linked immunosorbent assays (sandwich ELISAs). A mouse IgG monoclonal antibody specific for human tPA, designated 1 4-6, was used as the capture antibody and a polyclonal, rabbit anti-lgG antibody conjugated to horseradish peroxidase was used as the secondary-labelled antibody.

5. Purification of FGFR1 -tPA chimeric protein

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]. The column was then washed with 10 column volumes of column buffer and bound fusion protein was eluted from the column by the addition of 0.2 M glycine-HCI, pH 2.5. Fractions (0.5 ml) were collected into a tube containing 0.5 ml of 1 M Tris-HCl, pH 8.0 to neutralize the acidic eluate. Eluted fractions were monitored for the presence of FGFR1 -tPA protein by measuring the absorbance of each fraction at 280 nm. The FGFR1 - tPA-containing fractions were dialyzed against PBS and concentrated to a final concentration of 1 .5-2.0 mg/ml using Centriprep filters (AMICON).

6. Analysis of bFGF-FGFR1 interaction The soluble, recombinant FGFR1 -tPA fusion protein was immobilized to a solid support by attachment to the surface of the wells of an enzyme-linked immunosorborbent assay plate (High binding plates, COSTAR) . A 0.1 ml aliquot of a 10 μg/ml solution of rFGFR1 -tPA in PBS was added and the plate was incubated for approximately 1 6 hr at 4°C. Unbound fusion protein was removed by washing three times with an equal volume of cold PBS.

To each well, a 0.1 ml aliquot of blocking buffer (25 mM HEPES, pH 7.5, 100 mM NaCl and 0.5% gelatin) was added, and the samples incubated for 1 hr at ambient temperature to prevent non-specific binding of reagents. The wells were washed three times with binding buffer (25 mM HEPES, pH 7.5, 100 mM NaCl and 0.3 % gelatin) followed by addition of 0.1 ml of binding buffer supplemented with 2 μg/ml heparan sulfate and a range of 1 -20ng/ml of labelled ¹²⁵l-bFGF (800-1 200Ci/mmol; Amersham, Arlington Heights, IL) and incubated in the absence or presence of 2.5 μg/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 amount of bound radiolabelled bFGF in each well was calculated and the specificity of bFGF binding was analyzed according to Scatchard [Scatchard ( 1 949) Ann. N.Y. Acad. Sci. 51 :6601. From this analysis, a 280 pM dissociation constant (K_D) for the binding of bFGF to the recombinant FGFR1 - tPA fusion protein of was calculated. This value correlates well with 1 30 pM K_D value reported for bFGF binding to native FGFR1 receptors expressed in smooth muscle cells [Saltis et al. ( 1 995) Arteriosclerosis 1 1 8:77-871. B. Membrane-bound FGF receptor assays

1 . Competitive inhibition of FGF binding

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 ¹²⁵l-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 '²⁵l-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.

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. Following removal of the low affinity sample, 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. Competitive inhibition of EGF binding

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. Rb-1 cells were grown as described above and incubated in binding buffer containing 2 ng/ml of ¹²⁵I-EGF

( > 750Ci/mmol; Amersham) under similar conditions. Non-specific binding of radiolabelled EGF was estimated in parallel reactions performed in an excess of unlabeled EGF.

After washing the cells twice with cold PBS, specifically bound EGF was dissociated from the cells by addition of a solution of 0.1 % Triton-X-1 00 and 5 min incubation at ambient temperature. The amount of radioactivity in each supernatant was measured using a gamma counter. C. Inhibition of ³H-thymidine incorporation

The incorporation of radiolabelled nucleotides into newly synthesized cellular DNA may be used as an indicator of cell proliferation. SMCs, such as rat aortic Rb-1 cells, incorporate tritiated thymidine into DNA upon stimulation with bFGF, PDGF or EGF. 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 0⁴ 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 μg/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.

After washing twice in serum-free DMEM medium, the follow was added to each well: 400 μl of serum-free DMEM, 50 μl of 3 ng/ml of unlabelled bFGF in DMEM and 50 μl of known concentration test compound in DMEM 1 0% DMSO for 23 hr at 37°C in a 5 % C0₂ atmosphere. To each well, 10μl of tritiated thymidine (³H-thymidine, 50 μCi/ml) was added and cells were incubated for 1 hour at 37°C. The medium was removed and the cells were washed twice with cold PBS. An 500 μl aliquot of a cold 1 0% TCA solution was added to each well and the cells incubated at 4°C overnight. After washing three times in cold PBS, the cells were incubated in 500 μl of 0.5 N NaOH for 30 min and the pH of the sample was neutralized by the addition of an equal volume of 0.5 N HCl. The amount of radioactivity present the supernatant of each well was determined using a liquid scintillation counter.

EXAMPLE 3 Analysis of the bioactivity of FGF muteins

Analysis of the crystal structure of bFGF indicates that Leu55, Val63, Ile65, Phe94, Phe95, Arg97, Leu98, Asn 104, Thr1 05, Tyr1 06 and Arg 1 07 are within a 7.5 A radius of Glu96. Of these residues, Leu55, Val63, Phe95,

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. To evaluate whether neighboring residues of Glu96 are involved in high-affinity receptor binding, 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. Substitution of Leu55, Val63, Arg97, Leu98 and Arg 1 07 by alanine gave muteins exhibiting receptor binding affinities similar to wild-type bFGF. By contrast, replacement of residues Phe95 and Asn104 with alanine gave -6.28- and 434-fold reductions of receptor binding affinities, respectively. A. FGF receptor binding

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 ¹²⁵l-bFGF for binding in the Soluble Receptor assay described in EXAMPLE 2A and the values compared to wild type bFGF. The IC₅₀ and the IC₅₀ mutein/IC₅₀ wildtype ratio for the bFGF muteins are shown in the following tables.

Site-directed metagenesis of residues within a 7.5 A radius of Asn104

To explore further potentially critical residues around Asn104, residues within a 7.5 A radius of Asn 104 were examined. The crystal structure of bFGF indicates that Leu23, Glu96, Arg97, Leu98, Glu99, Asn101 , Asn102, Tyr103, Thr1 05, Tyr1 06, Ile 1 37, Phe 1 39, Leu 1 40, and Pro141 are such neighbors of Asn104. 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 A²) (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:

Substitution of residues Leu55, Val63, Arg97, Leu98 and Arg 1 07 by alanine gave muteins with nearly unchanged receptor binding affinity compared with the wild-type. By contrast, replacement of residues Phe95 and Asn104 reduced the receptor binding affinity -6.3- and 434-fold, respectively, compared with the wild-type Since residue Asn 1 04 is conserved among nine members of the FGF family, this result suggests that the conserved asparagine residue in other members of FGF family may also play an important role in high-affinity receptor binding. The F95A mutation has a moderate effect on receptor binding affinity, suggesting that Phe95 is either close to the receptor binding site or is a part of the structural epitope.

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. Since 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 dimeπzation Based on the discovery of this Asn 1 04 residue as a novel receptor binding epitope encouraged us to extend further our affinity epitope, 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 . Most of the resulting muteins were similar to wild-type bFGF in their binding affinity for FGFR1 -TPA, but three, 1 01 A, Y103A and L1 40A, showed reductions of 7.7-, 402- and 245-fold.

As shown in the Tables, the replacement of ammo acid residues Leu55, Val63, Arg97, Leu98, Asn 102 and Asn1 07 with alanine resulted in FGF muteins have receptor binding affinities comparable to wildtype bFGF. In contract, the replacement of Phe95, Asn1 01 Tyr103, Asn104 and Leu 1 40 with an alanine residue resulted in a 6.3-, a 7.7-, 402-, a 434-, and a 245-fold reduction in bFGF mutein receptor binding affinity, respectively. Each FGF mutein should retained the ability to bind to heparin. For example, 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) . Several qualitative principles regarding protein-protein interactions have been deduced from the structure of numerous crystal structures of protein- protein complexes. First, hydrogen bonding and other electrostatic interactions are critical in determining specificity in the interactions. Second, predominantly buried hydrophobic surfaces are an important element in tight binding. Finally, sufficient conformational rigidity in unbound ligand and acceptor is required to obviate and unacceptable entropy loss upon binding. Thus, the residues on bFGF identified for high-affinity receptor binding here and elsewhere elucidate the highly specific interaction between bFGF and FGFR1 ?.

The site-directed mutagenesis experiments on bFGF show that Lys1 1 0, Tyr1 1 1 and Trp1 1 4 in the interstrand 9/10 domain are not required for the receptor binding affinity, but are important in stimulating the proliferation of 3T3 fibroblasts. Furthermore, replacement of this loop in bFGF with the corresponding loop structure from FGF1 altered the receptor binding specificity of FGF2, indicating that this loop is a receptor binding specificity determinant of fibroblast growth factors. On the other hand, site-directed mutagenesis studies on FGFR show that the active core of the receptor (the loop II, the inter-loop ll/lll sequence, the N-terminus of loop III and glycosaminoglycan) can bind to acidic fibroblast growth factor (aFGF), bFGF and KFGF. The specificity of an FGF ligand bound to the active core is determined by how the ligand interacts with the highly conserved invariant III domain on the front face of loop III. Based on this reports, it appears that the highly conserved residues Tyr24, Glu96, Tyr103, Asn104, Leu 140 among nine members of the FGF family bind to this active core of the receptor, whereas the distinct FGF type-specific interstrand 9/10 domain may interact with receptor domains on the front of loop III. Glu96 and Asn104 of bFGF are crucial polar residues that are required for high-affinity receptor binding and may function as the primary recognition site for this process. The identification of the binding epitope on FGF-2 will be useful in identifying FGFR 1 antagonists by de novo design and by computational substructure screening. Furthermore, double muteins lacking these residues, but retaining the ability to bind to heparin can be used to treat heparin-mediated disorders.

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT:

(A) NAME: Eisai , Ltd.

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(C) CITY: Bunkyo-ku Tokyo

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( i ) INVENTOR :

(A) NAME : Kalyanaraman Ramnarayan

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(C) CITY: San Diego

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( i ) INVENTOR :

(A) NAME: Takatoshi Kawai

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(ii) TITLE OF THE INVENTION: FGF MUTEIN COMPOSITIONS AND METHODS OF EREFOR

(iii) NUMBER OF SEQUENCES: 12

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Brovm, Martin, Haller & McClain

(B) STREET: 1660 Union Street

(C) CITY: San Diego

(D) STATE: CA

(E) COUNTRY: USA

(F) ZIP: 92101-2926

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ Version 1.5

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: Herewith

(C) CLASSIFICATION: (vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 60/040,785

(B) FILING DATE: 03-MAR-1997

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Seidman, Stephanie L

(B) REGISTRATION NUMBER: 33,779

(C) REFERENCE/DOCKET NUMBER: 6790-1208PC

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 619-238-0999

(B) TELEFAX: 619-238-0062

(C) TELEX:

2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 155 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

Met Ala Glu Gly Glu lie Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe 1 5 10 15

Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser 20 25 30

Asn Gly Gly His Phe Leu Arg lie Leu Pro Asp Gly Thr Val Asp Gly 35 40 45

Thr Arg Asp Arg Ser Asp Gin His lie Gin Leu Gin Leu Ser Ala Glu 50 55 60

Ser Val Gly Glu Val Tyr lie Lys Ser Thr Glu Thr Gly Gin Tyr Leu 65 70 75 80

Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gin Thr Pro Asn Glu 85 90 95

Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105 110 lie Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys 115 120 125

Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gin Lys Ala 130 135 140 lie Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 468 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..468

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 1..468

(D) OTHER INFORMATION: /product= "bFGF"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

ATG GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC TTG CCC GAG GAT GGC 48 Met Ala Ala Gly Ser lie Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly -9 -5 1 5

GGC AGC GGC GCC TTC CCG CCC GGC CAC TTC AAG GAC CCC JAG CGG CTG 96 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 10 15 20

TAC TGC AAA AAC GGG GGC TTC TTC CTG CGC ATC CAC CCC GAC GGC CGA 144 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg lie His Pro Asp Gly Arg 25 30 35

GTT GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTT CAA CTT 192 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His lie Lys Leu Gin Leu 40 45 50 55

CAA GCA GAA GAG AGA GGA GTT GTG TCT ATC AAA GGA GTG TGT GCT AAC 240 Gin Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Ala Asn 60 65 70

CGT TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT 288 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 75 80 85

GTT ACG GAT GAG TGT TTC TTT TTT GAA CGA TTG GAA TCT AAT AAC TAC 336 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 90 95 100

AAT ACT TAC CGG TCA AGG AAA TAC ACC AGT TGG TAT GTG GCA TTG AAA 384 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 105 110 115

CGA ACT GGG CAG TAT AAA CTT GGA TCC AAA ACA GGA CCT GGG CAG AAA 432 Arg Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gin Lys 120 125 130 135

GCT ATA CTT TTT CTT CCA ATG TCT GCT AAG AGC TGA 468

Ala He Leu Phe Leu Pro Met Ser Ala Lys Ser *

140 145

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 239 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

Met Gly Leu He Trp Leu Leu Leu Leu Ser Leu Leu Glu Pro Gly Trp 1 5 10 15

Pro Ala Ala Gly Pro Gly Ala Arg Leu Arg Arg Asp Ala Gly Gly Arg 20 25 30

Gly Gly Val Tyr Glu His Leu Gly Gly Ala Pro Arg Arg Arg Lys Leu 35 40 45

Tyr Cys Ala Thr Lys Tyr His Leu Gin Leu His Pro Ser Gly Arg Val 50 55 60

Asn Gly Ser Leu Glu Asn Ser Ala Tyr Ser He Leu Glu He Thr Ala 65 70 75 80

Val Glu Val Gly He Val Ala He Arg Gly Leu Phe Ser Gly Arg Tyr 85 90 95

Leu Ala Met Asn Lys Arg Gly Arg Leu Tyr Ala Ser Glu His Tyr Ser 100 105 110

Ala Glu Cys Glu Phe Val Glu Arg He His Glu Leu Gly Tyr Asn Thr 115 120 125

Tyr Ala Ser Arg Leu Tyr Arg Thr Val Ser Ser Thr Pro Gly Ala Arg 130 135 140

Arg Gin Pro Ser Ala Glu Arg Leu Trp Tyr Val Ser Val Asn Gly Lys 145 150 155 160

Gly Arg Pro Arg Arg Gly Phe Lys Thr Arg Arg Thr Gin Lys Ser Ser 165 170 175

Leu Phe Leu Pro Arg Val Leu Asp His Arg Asp His Glu Met Val Arg 180 185 190

Gin Leu Gin Ser Gly Leu Pro Arg Pro Pro Gly Lys Gly Val Gin Pro 195 200 205

Arg Arg Arg Arg Gin Lys Gin Ser Pro Asp Asn Leu Glu Pro Ser His 210 215 220

Val Gin Ala Ser Arg Leu Gly Ser Gin Leu Glu Ala Ser Ala His 225 230 235

(2) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 206 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

Met Ser Gly Pro Gly Thr Ala Ala Val Ala Leu Leu Pro Ala Val Leu 1 5 10 15

Leu Ala Leu Leu Ala Pro Trp Ala Gly Arg Gly Gly Ala Ala Ala Pro 20 25 30

Thr Ala Pro Asn Gly Thr Leu Glu Ala Glu Leu Glu Arg Arg Trp Glu 35 40 45

Ser Leu Val Ala Leu Ser Leu Ala Arg Leu Pro Val Ala Ala Gin Pro 50 55 60

Lys Glu Ala Ala Val Gin Ser Gly Ala Gly Asp Tyr Leu Leu Gly He 65 70 75 80

Lys Arg Leu Arg Arg Leu Tyr Cys Asn Val Gly He Gly Phe His Leu 85 90 95

Gin Ala Leu Pro Asp Gly Arg He Gly Gly Ala His Ala Asp Thr Arg 100 105 110

Asp Ser Leu Leu Glu Leu Ser Pro Val Glu Arg Gly Val Val Ser He 115 120 125

Phe Gly Val Ala Ser Arg Phe Phe Val Ala Met Ser Ser Lys Gly Lys 130 135 140

Leu Tyr Gly Ser Pro Phe Phe Thr Asp Glu Cys Thr Phe Lys Glu He 145 150 155 160

Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly 165 170 175

Met Phe He Ala Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn Arg 180 185 190

Val Ser Pro Thr Met Lys Val Thr His Phe Leu Pro Arg Leu 195 200 205

(2) INFORMATION FOR SEQ ID NO : 5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 268 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :

Met Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe Ser His Leu He Leu 1 5 10 15

Ser Ala Trp Ala His Gly Glu Lys Arg Leu Ala Pro Lys Gly Gin Pro 20 25 30 Gly Pro Ala Ala Thr Asp Arg Asn Pro He Gly Ser Ser Ser Arg Gin 35 40 45

Ser Ser Ser Ser Ala Met Ser Ser Ser Ser Ala Ser Ser Ser Pro Ala 50 55 60

Ala Ser Leu Gly Ser Gin Gly Ser Gly Leu Glu Gin Ser Ser Phe Gin 65 70 75 80

Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly 85 90 95

He Gly Phe His Leu Gin He Tyr Pro Asp Gly Lys Val Asn Gly Ser 100 105 110

His Glu Ala Asn Met Leu Ser Val Leu Glu He Phe Ala Val Ser Gin 115 120 125

Gly He Val Gly He Arg Gly Val Phe Ser Asn Lys Phe Leu Ala Met 130 135 140

Ser Lys Lys Gly Lys Leu His Ala Ser Ala Lys Phe Thr Asp Asp Cys 145 150 155 160

Lys Phe Arg Glu Arg Phe Gin Glu Asn Ser Tyr Asn Thr Tyr Ala Ser 165 170 175

Ala He His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu 180 185 190

Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro 195 200 205

Gin His He Ser Thr His Phe Leu Pro Arg Phe Lys Gin Ser Glu Gin 210 215 220

Pro Glu Leu Ser Phe Thr Val Thr Val Pro Glu Lys Lys Asn Pro Pro 225 230 235 240

Ser Pro He Lys Ser Lys He Pro Leu Ser Ala Pro Arg Lys Asn Thr 245 250 255

Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 260 265

(2) INFORMATION FOR SEQ ID NO : 6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 198 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :

Met Ser Arg Gly Ala Gly Arg Leu Gin Gly Thr Leu Trp Ala Leu Val 1 5 10 15 Phe Leu Gly He Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Thr 20 25 30

Arg Ala Asn Asn Thr Leu Leu Asp Ser Arg Gly Trp Gly Thr Leu Leu 35 40 45

Ser Arg Ser Arg Ala Gly Leu Ala Gly Glu He Ala Gly Val Asn Trp 50 55 60

Glu Ser Gly Tyr Leu Val Gly He Lys Arg Gin Arg Arg Leu Tyr Cys 65 70 75 80

Asn Val Gly He Gly Phe His Leu Gin Val Leu Pro Asp Gly Arg He 85 90 95

Ser Gly Thr His Glu Glu Asn Pro Tyr Ser Leu Leu Glu He Ser Thr 100 105 110

Val Glu Arg Gly Val Val Ser Leu Phe Gly Val Arg Ser Ala Leu Phe 115 120 125

Val Ala Met Asn Ser Lys Gly Arg Leu Tyr Ala Thr Pro Ser Phe Gin 130 135 140

Glu Glu Cys Lys Phe Arg Glu Thr Leu Leu Pro Asn Asn Tyr Asn Ala 145 150 155 160

Tyr Glu Ser Asp Leu Tyr Gin Gly Thr Tyr He Ala Leu Ser Lys Tyr 165 170 175

Gly Arg Val Lys Arg Gly Ser Lys Val Ser Pro He Met Thr Val Thr 180 185 190

His Phe Leu Pro Arg He 195

(2) INFORMATION FOR SEQ ID NO : 7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 194 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :

Met His Lys Trp He Leu Thr Trp He Leu Pro Thr Leu Leu Tyr Arg 1 5 10 15

Ser Cys Phe His He He Cys Leu Val Gly Thr He Ser Leu Ala Cys 20 25 30

Asn Asp Met Thr Pro Glu Gin Met Ala Thr Asn Val Asn Cys Ser Ser 35 40 45

Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp He 50 55 60 Arg Val Arg Arg Leu Phe Cys Arg Thr Gin Trp Tyr Leu Arg He Asp 65 70 75 80

Lys Arg Gly Lys Val Lys Gly Thr Gin Glu Met Lys Asn Asn Tyr Asn 85 90 95

He Met Glu He Arg Thr Val Ala Val Gly He Val Ala He Lys Gly 100 105 110

Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr 115 120 125

Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu He Leu 130 135 140

Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly 145 150 155 160

Gly Glu Met Phe Val Ala Leu Asn Gin Lys Gly He Pro Val Arg Gly 165 170 175

Lys Lys Thr Lys Lys Glu Gin Lys Thr Ala His Phe Leu Pro Met Ala 180 185 190

He Thr

( 2 ) INFORMATION FOR SEQ ID NO : 8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 215 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :

Met Gly Ser Pro Arg Ser Ala Leu Ser Cys Leu Leu Leu His Leu Leu 1 5 10 15

Val Leu Cys Leu Gin Ala Gin Val Thr Val Gin Ser Ser Pro Asn Phe 20 25 30

Thr Gin His Val Arg Glu Gin Ser Leu Val Thr Asp Gin Leu Ser Arg 35 40 45

Arg Leu He Arg Thr Tyr Gin Leu Tyr Ser Arg Thr Ser Gly Lys His 50 55 60

Val Gin Val Leu Ala Asn Lys Arg He Asn Ala Met Ala Glu Asp Gly 65 70 75 80

Asp Pro Phe Ala Lys Leu He Val Glu Thr Asp Thr Phe Gly Ser Arg 85 90 95

Val Arg Val Arg Gly Ala Glu Thr Gly Leu Tyr He Cys Met Asn Lys 100 105 110

Lys Gly Lys Leu He Ala Lys Ser Asn Gly Lys Gly Lys Asp Cys Val 115 120 125 Phe Thr Glu He Val Leu Glu Asn Asn Tyr Asn Ala Leu Gin Asn Ala 130 135 140

Lys Tyr Glu Gly Trp Tyr Met Ala Phe Thr Arg Lys Gly Arg Pro Arg 145 150 155 160

Lys Gly Ser Lys Thr Arg Gin His Gin Arg Glu Val His Phe Met Lys 165 170 175

Arg Leu Pro Arg Gly His His Thr Thr Glu Gin Ser Leu Arg Phe Glu 180 185 190

Phe Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu Arg Gly Ser Gin Arg 195 200 205

Thr Trp Ala Pro Glu Pro Arg 210 215

(2) INFORMATION FOR SEQ ID NO : 9 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 208 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :

Met Ala Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gin Asp Ala 1 5 10 15

Val Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp Ser Pro Val Leu 20 25 30

Leu Ser Asp His Leu Gly Gin Ser Glu Ala Gly Gly Leu Pro Arg Gly

35 40 45

Pro Ala Val Thr Asp Leu Asp His Leu Lys Gly He Leu Arg Arg Arg 50 55 60

Gin Leu Tyr Cys Arg Thr Gly Phe His Leu Glu He Phe Pro Asn Gly 65 70 75 80

Thr He Gin Gly Thr Arg Lys Asp His Ser Arg Phe Gly He Leu Glu 85 90 95

Phe He Ser He Ala Val Gly Leu Val Ser He Arg Gly Val Asp Ser 100 105 110

Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu 115 120 125

Lys Leu Thr Gin Glu Cys Val Phe Arg Glu Gin Phe Glu Glu Asn Trp 130 135 140

Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr Lys His Val Asp Thr Gly Arg 145 150 155 160

Arg Tyr Tyr Val Ala Leu Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr 165 170 175 Arg Thr Lys Arg His Gin Lys Phe Thr His Phe Leu Pro Arg Pro Val

180 185 190

Asp Pro Asp Lys Val Pro Glu Leu Tyr Lys Asp He Leu Ser Gin Ser 195 200 205

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 181 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTISENSE: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Met Glu Ser Lys Glu Pro Gin Leu Lys Gly He Val Thr Arg Leu Phe

1 5 10 15

Ser Gin Gin Gly Tyr Phe Leu Gin Met His Pro Asp Gly Thr He Asp

20 25 30

Gly Thr Lys Asp Glu Asn Ser Asp Tyr Thr Leu Phe Asn Leu He Pro

35 40 45

Val Gly Leu Arg Val Val Ala He Gin Gly Val Lys Ala Ser Leu Tyr

50 55 60

Val Ala Met Asn Gly Glu Gly Tyr Leu Tyr Ser Ser Asp Val Phe Thr 65 70 75 80

Pro Glu Cys Lys Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val He

85 90 95

Tyr Ser Ser Thr Leu Tyr Arg Gin Gin Glu Ser Gly Arg Ala Trp Phe

100 105 110

Leu Gly Leu Asn Lys Glu Gly Gin He Met Lys Gly Asn Arg Val Lys

115 120 125

Lys Thr Lys Pro Ser Ser His Phe Val Pro Lys Pro He Glu Val Cys

130 135 140

Met Tyr Arg Glu Pro Ser Leu His Glu He Gly Glu Lys Gin Gly Arg 145 150 155 160

Ser Arg Lys Ser Ser Gly Thr Pro Thr Met Asn Gly Gly Lys Val Val

165 170 175

Asn Gin Asp Ser Thr 180

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1440 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (ix) FEATURE:

(A) NAME/KEY: Coding Sequence

(B) LOCATION: 9...1427

(D) OTHER INFORMATION: FGFRl-tPA fusion protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

AAGCTTGG ATG TGG AGC TGG AAG TGC CTC CTC TTC TGG GCT GTG CTG GTC 50 Met Trp Ser Trp Lys Cys Leu Leu Phe Trp Ala Val Leu Val 1 5 10

ACA GCA ACA CTC TGC ACC GCT AGG CCG TCC CCG ACC TTG CCT GAA CAA 98 Thr Ala Thr Leu Cys Thr Ala Arg Pro Ser Pro Thr Leu Pro Glu Gin 15 20 25 30

GAT GCT CTC CCC TCC TCG GAG GAT GAT GAT GAT GAT GAT GAC TCC TCT 146 Asp Ala Leu Pro Ser Ser Glu Asp Asp Asp Asp Asp Asp Asp Ser Ser 35 40 45

TCA GAG GAG AAA GAA ACA GAT AAC ACC AAA CCA AAC CCC GTA GCT CCA 194 Ser Glu Glu Lys Glu Thr Asp Asn Thr Lys Pro Asn Pro Val Ala Pro 50 55 60

TAT TGG ACA TCC CCA GAA AAG ATG GAA AAG AAA TTG CAT GCA GTG CCG 242 Tyr Trp Thr Ser Pro Glu Lys Met Glu Lys Lys Leu His Ala Val Pro 65 70 75

GCT GCC AAG ACA GTG AAG TTC AAA TGC CCT TCC AGT GGG ACC CCA AAC 290 Ala Ala Lys Thr Val Lys Phe Lys Cys Pro Ser Ser Gly Thr Pro Asn 80 85 90

CCC ACA CTG CGC TGG TTG AAA AAT GGC AAA GAA TTC AAA CCT GAC CAC 338 Pro Thr Leu Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Pro Asp His 95 100 105 110

AGA ATT GGA GGC TAC AAG GTC CGT TAT GCC ACC TGG AGC ATC ATA ATG 386 Arg He Gly Gly Tyr Lys Val Arg Tyr Ala Thr Trp Ser He He Met 115 120 125

GAC TCT GTG GTG CCC TCT GAC AAG GGC AAC TAC ACC TGC ATT GTG GAG 434 Asp Ser Val Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys He Val Glu 130 135 140

AAT GAG TAC GGC AGC ATC AAC CAC ACA TAC CAG CTG GAT GTC GTG GAG 482 Asn Glu Tyr Gly Ser He Asn His Thr Tyr Gin Leu Asp Val Val Glu 145 150 155

CGG TCC CCT CAC CGG CCC ATC CTG CAA GCA GGG TTG CCC GCC AAC AAA 530 Arg Ser Pro His Arg Pro He Leu Gin Ala Gly Leu Pro Ala Asn Lys 160 165 170

ACA GTG GCC CTG GGT AGC AAC GTG GAG TTC ATG TGT AAG GTG TAC AGT 578 Thr Val Ala Leu Gly Ser Asn Val Glu Phe Met Cys Lys Val Tyr Ser 175 180 185 190

GAC CCG CAG CCG CAC ATC CAG TGG CTA AAG CAC ATC GAG GTG AAT GGG 626 Asp Pro Gin Pro His He Gin Trp Leu Lys His He Glu Val Asn Gly 195 200 205 AGC AAG ATT GGC CCA GAC AAC CTG CCT TAT GTC CAG ATC TTG AAG ACT 674 Ser Lys He Gly Pro Asp Asn Leu Pro Tyr Val Gin He Leu Lys Thr 210 215 220

GCT GGA GTT AAT ACC ACC GAC AAA GAG ATG GAC GTG CTT CAC TTA AGA 722 Ala Gly Val Asn Thr Thr Asp Lys Glu Met Asp Val Leu His Leu Arg 225 230 235

AAT GTC TCC TTT GAG GAC GCA GGG GAG TAT ACG TGC TTG GCG GGT AAC 770 Asn Val Ser Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly Asn 240 245 250

TCT ATC GGA CTC TCC CAT CAC TCT GCA TGG TTG ACC GTT CTG GAA GCC 818 Ser He Gly Leu Ser His His Ser Ala Trp Leu Thr Val Leu Glu Ala 255 260 265 270

CTG GAA GAG AGG CCG GCA GTG ATG ACC TCG CCC CTG TAC GTC GAC GCC 866 Leu Glu Glu Arg Pro Ala Val Met Thr Ser Pro Leu Tyr Val Asp Ala 275 280 285

CGA TTC CCA AGA GGA GCC AGA TCT TAC CAA GTG ATC TGC AGA GAT GAA 914 Arg Phe Pro Arg Gly Ala Arg Ser Tyr Gin Val He Cys Arg Asp Glu 290 295 300

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

CTC AGA AGC AAC CGG GTG GAA TAT TGC TGG TGC AAC AGT GGC AGG GCA 1010 Leu Arg Ser Asn Arg Val Glu Tyr Cys Trp Cys Asn Ser Gly Arg Ala 320 325 330

CAG TGC CAC TCA GTG CCT GTC AAA AGT TGC AGC GAG CCA AGG TGT TTC 1058 Gin Cys His Ser Val Pro Val Lys Ser Cys Ser Glu Pro Arg Cys Phe 335 340 345 350

AAC GGG GGC ACC TGC CAG CAG GCC CTG TAC TTC TCA GAT TTC GTG TGC 1106 Asn Gly Gly Thr Cys Gin Gin Ala Leu Tyr Phe Ser Asp Phe Val Cys 355 360 365

CAG TGC CCC GAA GGA TTT GCT GGG AAG TGC TGT GAA ATA GAT ACC AGG 1154 Gin Cys Pro Glu Gly Phe Ala Gly Lys Cys Cys Glu He Asp Thr Arg 370 375 380

GCC ACG TGC TAC GAG GAC CAG GGC ATC AGC TAC AGG GGC ACG TGG AGC 1202 Ala Thr Cys Tyr Glu Asp Gin Gly He Ser Tyr Arg Gly Thr Trp Ser 385 390 395

ACA GCG GAG AGT GGC GCC GAG TGC ACC AAC TGG AAC AGC AGC GCG TTG 1250 Thr Ala Glu Ser Gly Ala Glu Cys Thr Asn Trp Asn Ser Ser Ala Leu 400 405 410

GCC CAG AAG CCC TAC AGC GGG CGG AGG CCA GAC GCC ATC AGG CTG GGC 1298 Ala Gin Lys Pro Tyr Ser Gly Arg Arg Pro Asp Ala He Arg Leu Gly 415 420 425 430

CTG GGG AAC CAC AAC TAC TGC AGA AAC CCA GAT CGA GAC TCA AAG CCC 1346 Leu Gly Asn His Asn Tyr Cys Arg Asn Pro Asp Arg Asp Ser Lys Pro 435 440 445

TGG TGC TAC GTC TTT AAG GCG GGG AAG TAC AGC TCA GAG TTC TGC AGC 1394 Trp Cys Tyr Val Phe Lys Ala Gly Lys Tyr Ser Ser Glu Phe Cys Ser 450 455 460

ACC CCT GCC TGC TCT GAG GGA AAC AGT GAC TGA TACTTTGGGA TCC 1440

Thr Pro Ala Cys Ser Glu Gly Asn Ser Asp * 465 470

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 472 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 12 :

Met Trp Ser Trp Lys Cys Leu Leu Phe Trp Ala Val Leu Val Thr Ala

1 5 10 15

Thr Leu Cys Thr Ala Arg Pro Ser Pro Thr Leu Pro Glu Gin Asp Ala

20 25 30

Leu Pro Ser Ser Glu Asp Asp Asp Asp Asp Asp Asp Ser Ser Ser Glu

35 40 45

Glu Lys Glu Thr Asp Asn Thr Lys Pro Asn Pro Val Ala Pro Tyr Trp

50 55 60

Thr Ser Pro Glu Lys Met Glu Lys Lys Leu His Ala Val Pro Ala Ala 65 70 75 80

Lys Thr Val Lys Phe Lys Cys Pro Ser Ser Gly Thr Pro Asn Pro Thr

85 90 95

Leu Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Pro Asp His Arg He

100 105 110

Gly Gly Tyr Lys Val Arg Tyr Ala Thr Trp Ser He He Met Asp Ser

115 120 125

Val Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys He Val Glu Asn Glu

130 135 140

Tyr Gly Ser He Asn His Thr Tyr Gin Leu Asp Val Val Glu Arg Ser 145 150 155 160

Pro His Arg Pro He Leu Gin Ala Gly Leu Pro Ala Asn Lys Thr Val

165 170 175

Ala Leu Gly Ser Asn Val Glu Phe Met Cys Lys Val Tyr Ser Asp Pro

180 185 190

Gin Pro His He Gin Trp Leu Lys His He Glu Val Asn Gly Ser Lys

195 200 205

He Gly Pro Asp Asn Leu Pro Tyr Val Gin He Leu Lys Thr Ala Gly

210 215 220

Val Asn Thr Thr Asp Lys Glu Met Asp Val Leu His Leu Arg Asn Val 225 230 235 240

Ser Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly Asn Ser He

245 250 255

Gly Leu Ser His His Ser Ala Trp Leu Thr Val Leu Glu Ala Leu Glu

260 265 270

Glu Arg Pro Ala Val Met Thr Ser Pro Leu Tyr Val Asp Ala Arg Phe

275 280 285

Pro Arg Gly Ala Arg Ser Tyr Gin Val He Cys Arg Asp Glu Lys Thr 290 295 300

Gin Met He Tyr Gin Gin His Gin Ser Trp Leu Arg Pro Val Leu Arg 305 310 315 320

Ser Asn Arg Val Glu Tyr Cys Trp Cys Asn Ser Gly Arg Ala Gin Cys

325 330 335

His Ser Val Pro Val Lys Ser Cys Ser Glu Pro Arg Cys Phe Asn Gly

340 345 350

Gly Thr Cys Gin Gin Ala Leu Tyr Phe Ser Asp Phe Val Cys Gin Cys

355 360 365

Pro Glu Gly Phe Ala Gly Lys Cys Cys Glu He Asp Thr Arg Ala Thr

370 375 380

Cys Tyr Glu Asp Gin Gly He Ser Tyr Arg Gly Thr Trp Ser Thr Ala 385 390 395 400

Glu Ser Gly Ala Glu Cys Thr Asn Trp Asn Ser Ser Ala Leu Ala Gin

405 410 415

Lys Pro Tyr Ser Gly Arg Arg Pro Asp Ala He Arg Leu Gly Leu Gly

420 425 430

Asn His Asn Tyr Cys Arg Asn Pro Asp Arg Asp Ser Lys Pro Trp Cys

435 440 445

Tyr Val Phe Lys Ala Gly Lys Tyr Ser Ser Glu Phe Cys Ser Thr Pro

450 455 460

Ala Cys Ser Glu Gly Asn Ser Asp 465 470

Claims

1 . An isolated DNA molecule, comprising a sequence of nucleotides that encodes a fibroblast growth factor (FGF) mutein selected from the group _ consisting of FGF-1 , FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9 and FGF-1 0, wherein: the FGF-1 has been modified by replacement of the asparagine residue at position 1 1 0 with another amino acid; the FGF-2 has been modified by replacement of the asparagine residue at position 1 04 with another amino acid; the FGF-3 has been modified by replacement of the asparagine residue at position 1 27 with another amino acid; the FGF-4 has been modified by replacement of the asparagine residue at position 1 67 with another amino acid; the FGF-5 has been modified by replacement of the asparagine residue at position 1 72 with another amino acid; the FGF-6 has been modified by replacement of the asparagine residue at position 1 59 with another amino acid; the FGF-7 has been modified by replacement of the asparagine residue at position 149 with another amino acid; the FGF-8 has been modified by replacement of the asparagine residue at position 1 39 with another amino acid; the FGF-9 has been modified by replacement of the asparagine residue at position 146 with another amino acid; the FGF-10 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.

2. An isolated DNA molecule, comprising a sequence of nucleotides that encodes a fibroblast growth factor (FGF) mutein selected from among FGF- 1 , FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9 and FGF-1 0, wherein: the FGF-1 has been modified by replacement of the asparagine residue at position 1 07 with another amino acid; the FGF-2 has been modified by replacement of the asparagine residue at position 101 with another amino acid; the FGF-3 has been modified by replacement of the leucine residue at position 1 24 with another amino acid; the FGF-4 has been modified by replacement of the asparagine residue at position 1 64 with another amino acid; the FGF-5 has been modified by replacement of the asparagine residue at position 1 69 with another amino acid; the FGF-6 has been modified by replacement of the asparagine residue at position 1 56 with another amino acid; the FGF-7 has been modified by replacement of the asparagine residue at position 146 with another amino acid; the FGF-8 has been modified by replacement of the asparagine residue at position 1 36 with another amino acid; the FGF-9 has been modified by replacement of the asparagine residue at position 143 with another amino acid; the FGF-10 has been modified by replacement of the asparagine residue at position 91 with another amino acid; the position numbers are determined by reference to SEQ ID NO. 1 -1 0 for FGF-1 to FGF-1 0, respectively; and the replacement amino acid is selected such that the resulting peptides has substantially reduced binding activity for FGFR1 compared to the wild type FGF.

3. An isolated DNA molecule, comprising a sequence of nucleotides that encodes a fibroblast growth factor (FGF) mutein selected from the group consisting of FGF-1 , FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9 and FGF-10, wherein: the FGF-1 has been modified by replacement of the phenylalanine residue at position 100 with another amino acid; the FGF-2 has been modified by replacement of the phenylalanine residue at position 95 with another amino acid; the FGF-3 has been modified by replacement of the phenylalanine residue at position 1 1 7 with another amino acid; the FGF-4 has been modified by replacement of the phenylalanine residue at position 1 57 with another amino acid; the FGF-5 has been modified by replacement of the phenylalanine residue at position 1 62 with another amino acid; the FGF-6 has been modified by replacement of the phenylalanine residue at position 149 with another amino acid; the FGF-7 has been modified by replacement of the phenylalanine residue at position 1 39 with another amino acid; the FGF-8 has been modified by replacement of the phenylalanine residue at position 1 29 with another amino acid; the FGF-9 has been modified by replacement of the phenylalanine residue at position 1 36 with another amino acid; the FGF-1 0 has been modified by replacement of the phenylalanine residue at position 85 with another amino acid; the position numbers are determined by reference to SEQ ID NO. 1 -1 0 for FGF-1 to FGF-10, respectively; and the replacement amino acid is selected such that the resulting peptides has substantially reduced binding activity for FGFR1 compared to the wild type FGF.

4. The DNA molecule of any of claims 1 -3, wherein the replacement amino acid is alanine, phenylalanine, glycine or serine, methionine, or tyrosine.

5. The DNA molecule of any of claims 1 -3, wherein the replacement amino acid is alanine, glycine or serine.

6. The DNA molecule of any of claims 1 -3, wherein the replacement amino acid is alanine.

7. The DNA molecule of any of claims 1 -6, wherein: the FGF mutein is further modified by replacement of the Glu positions 1 02, 96, 1 1 9, 1 59, 1 64, 1 51 , 141 , 1 31 , 1 37 and 87 in FGF-1 - FGF-10, respectively with an amino acid that results in an FGF mutein that does not bind to FGFR 1 ; and the position numbers are determined by reference to SEQ ID NO. 1 -1 0 for

FGF-1 to FGF-10, respectively; and the replacement amino acid is selected such that the resulting mutein.

8. The DNA molecule of any of claims 1 -7, wherein the replacement amino acid is alanine, phenylalanine, serine, glycine, methionine, leucine or tyrosine.

9. The DNA molecule of any of claims 1 -8, wherein the FGF mutein is further modified by replacement of one or more cysteine residues, whereby aggregation of the resulting peptide is reduced compared to the wild type peptide.

1 0. The DNA molecule of any of claims 7-9, wherein the replacement amino acid is alanine, glycine or serine.

1 1 . The DNA molecule of claim 1 that encodes an FGF-2 mutein, wherein the sequence of nucleotides that encodes the FGF-2 mutein encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, except that the asparagine residue at position 104 is replaced with alanine, serine or glycine.

1 2. A fibroblast growth factor mutein polypeptide encoded by the DNA molecule of any of claims 1 -1 2.

1 3. A pharmaceutical composition, comprising a therapeutically effective amount of an FGF mutein of claim 1 2 in a vehicle suitable for topical, local or systemic administration, wherein the amount is effective for ameliorating adverse effects of heparin.

1 4. Use of an FGF an FGF mutein that binds to heparin but has substantially reduced FGF receptor binding activity compared to wild-type for treating a heparin-related disorder, wherein the therapeutically effective amount of the FGF mutein ameliorates at least one symptom of the heparin-related disorder.

1 5. Use of an FGF mutein that binds to heparin but has substantially reduced FGF receptor binding activity compared to wild-type for formulation of a medicament for treating a heparin-related disorder, wherein the therapeutically , effective amount of the FGF mutein ameliorates at least one symptom of the heparin-related disorder.

1 6. Use of an FGF mutein of claim 1 2 for formulation of a medicament for treatment of a heparin-related disorder.

1 7. Use of the pharmaceutical composition of claim 1 3 for treating a heparin-related disorder, wherein the FGF mutein ameliorates at least one symptom of the heparin-related disorder.

1 8. The use of any of claims 14-1 7, wherein the heparin-related disorder is selected from the group consisting of excessive bleeding induced by heparin, ophthalmic disorders and heparin-associated thrombocytopenia and thrombosis.

1 9. A method of treating a heparin-related disorder, comprising administering a therapeutically effective amount of an FGF mutein that binds to heparin but has substantially reduced FGF receptor binding activity compared to wild-type, whereby the therapeutically effective amount of the FGF mutein ameliorates at least one symptom of the heparin-related disorder.

20. The method of claim 1 9, wherein the heparin-related disorder is selected from the group consisting of excessive bleeding induced by heparin, ophthalmic disorders and heparin-associated thrombocytopenia and thrombosis.