KR20230111872A - Novel FGF-1 variants and uses thereof - Google Patents

Abstract

It relates to novel FGF-1 variants and uses thereof. According to the novel FGF-1 variants and uses thereof according to one aspect, the novel FGF-1 variants and uses thereof have increased proteolytic stability and therapeutic effects compared to wild-type FGF1, so that they can be usefully used in wound healing, tissue regeneration, inhibition of cancer growth, and inhibition of angiogenesis.

Description

Novel FGF-1 variants and uses thereof {Novel FGF-1 variants and uses thereof}

It relates to novel FGF-1 variants and uses thereof.

Human growth factors play a pivotal role in orchestrating many complex processes such as wound healing, tissue regeneration, angiogenesis and tumorigenesis. Accordingly, there is tremendous interest in using growth factors as protein therapeutics to accelerate wound healing and regeneration processes or to inhibit cancer growth and angiogenesis in a variety of diseases and conditions. However, although numerous recombinant growth factors have been developed as therapeutics, only a few candidates have been effective enough to receive clinical approval. This is mostly due to the short effective half-life of growth factors in vivo, which is usually due to low stability and rapid blood clearance. Therapeutic growth factors must remain active at the wound site for a long period of time to be effective. However, growth factors can be denatured or degraded upon exposure to physiological temperature and proteases. Since proteases such as plasmin and metalloproteinases are particularly active in tissue remodeling, resistance to protease-mediated degradation may be of particular importance.

Various growth factors have previously been modified to enhance thermal and proteolytic stability, which has been shown to enhance their biological activity in both in vitro functional assays and in vivo experiments. For example, growth hormone releasing hormone (GHRH) designed to be more resistant to the serine protease, oxidation and dipeptidyl peptidase IV has been shown to be more potent than wild type GHRH in inducing weight gain in pigs. However, most of these constructs were rationally designed based on protein-specific hypotheses and tested in a low-throughput fashion. Thus, generating new growth factor variants with enhanced stability can be a difficult and slow process. In the case of engineered proteins with increased proteolytic stability, single mutations often occur directly adjacent to the predicted cleavage site, based on primary sequence specificity or mass spectrometry after proteolysis. However, since proteolytic stability for a particular protease is multifactorial, this method is unreliable. First, the activity of a protease in cleaving a specific amino acid sequence is greatly influenced by the number of amino acids in the vicinity of the cleavage site. Although the amino acids directly upstream of the predicted cleavage site (P1) match the primary specificity of the protease, up to 8 amino acids can determine whether cleavage occurs at a particular site and the rate at which cleavage occurs. In the publication of Gosalia et al., they confirmed that even changing the two amino acids upstream of the correct P1 site (P2, P3) in combination dramatically affects the rate of proteolytic cleavage of the peptide substrate. Second, proteolytic cleavage of protein substrates is also determined by the steric accessibility of the cleavage site by the protease's enzymatic binding pocket. Although computational methods have improved to predict how mutations affect protein structure, predicting how mutations might change the accessibility of potential cleavage sites by proteases remains computationally expensive and technically challenging. Therefore, there is a serious limitation in predicting mutations that will increase proteolytic stability through rational design. The lack of readily adaptable methods for manipulating proteolytic stability may partly explain the limited development of proteolytic stability growth factors to date.

The present invention meets this need by providing methods for engineering proteolytically stable growth factors. The present invention also provides proteolytically stable FGF peptide variants produced by the methods described, as well as such FGF peptide variants.

One aspect is to provide a variant of human fibroblast growth factor 1 (FGF1) that exhibits increased proteolytic stability compared to wild-type FGF1 of SEQ ID NO: 1.

Another aspect is to provide a mutant in which any one amino acid selected from the group consisting of Q40P, S47I, H93G, and K112N in the amino acid sequence of SEQ ID NO: 1 is substituted.

Another aspect is to provide a nucleic acid encoding the FGF1 variant polypeptide.

Another aspect is to provide a composition for wound healing and skin regeneration comprising the FGF1 variant as an active ingredient.

Another aspect is to provide a composition for inhibiting cancer growth and angiogenesis comprising the FGF1 variant.

One aspect provides a variant of human fibroblast growth factor 1 (FGF1) that exhibits increased proteolytic stability compared to wild-type FGF1 of SEQ ID NO: 1.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature and laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization used herein are those well known and commonly used in the art. Standard techniques are used for nucleic acid and peptide synthesis. Techniques and procedures are generally performed according to conventional methods in the art and in accordance with various general references provided throughout this document (see generally Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference). The nomenclature used herein and the laboratory procedures of analytical and synthetic organic chemistry described below are well known and commonly used in the art. Standard techniques, or variations thereof, are used for chemical synthesis and chemical analysis.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single-stranded or double-stranded form. Unless specifically limited, the term includes nucleic acids comprising known analogues of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with a mixed-base and/or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Pro bes 8:91-98 (1994)). The term nucleic acid is used interchangeably with genes, cDNAs, and mRNAs encoded by genes. Moreover, as used herein, a nucleic acid encoding a polypeptide variant of the invention is defined as comprising a nucleic acid sequence complementary to such nucleic acid sequence.

The term "gene" refers to a segment of DNA involved in producing a polypeptide chain. This may include intervening sequences (introns) between individual coding segments (exons), as well as regions preceding and following coding regions (leaders and trailers).

The term "isolated" when applied to a nucleic acid or protein means that the nucleic acid or protein is essentially free of other cellular components with which it is associated in its natural state. It can be dry or aqueous, but is preferably in a homogeneous state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. Proteins, which are the dominant species present in the formulation, are substantially purified. Specifically, an isolated gene is separated in an open reading frame that flanks the gene and encodes a protein other than the gene of interest. The term “purified” means that the nucleic acid or protein produces essentially one band on an electrophoretic gel. In particular, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. An isolated nucleic acid may be a component of an expression vector.

Typically, an isolated polypeptide of the invention has a level of purity, preferably expressed as a range. The lower end of the purity range for a polypeptide is about 60%, about 70% or about 80% and the upper end of the purity range is about 70%, about 80%, about 90%, about 95% or greater than about 95%. When a polypeptide is greater than about 90% pure, that purity is also preferably expressed as a range. The lower end of the purity range is about 90%, about 92%, about 94%, about 96% or about 98%. The upper end of the purity range is about 92%, about 94%, about 96%, about 98% or about 100% pure.

Purity is determined by any art-recognized analytical method (eg, band intensity on a silver stained gel, polyacrylamide gel electrophoresis, HPLC, mass spectrometry, or similar means).

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code as well as subsequently modified amino acids such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, i.e., hydrogen-bonded α carbons, carboxyl groups, amino groups, and R groups, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogues have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimetic" refers to a chemical compound that differs from the general chemical structure of an amino acid, but has a structure that functions in a manner similar to naturally occurring amino acids.

Amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, eg, hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account one or more of the foregoing characteristics are well known to those skilled in the art and include (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg) , (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu) (original residues: exemplary substitutions). Accordingly, embodiments of the present disclosure contemplate functional or bioequivalents of polypeptides or proteins as set forth above. Specifically, embodiments of the present invention provide variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to a parent polypeptide. In various embodiments, the invention provides variants having this level of identity to a portion of a wild-type growth factor comprising a parent polypeptide sequence, eg, wild-type FGF1 (SEQ ID NO: 1). In various embodiments, the variant has at least about 95%, 96%, 97%, 98% or 99% sequence identity to a wild-type growth factor comprising a parent polypeptide or a portion of a parent polypeptide, e.g., wild-type FGF1 (SEQ ID NO: 1) as defined herein.

"Conservatively modified variants" apply to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, “conservatively modified variant” refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, or to essentially the same sequence if the nucleic acid does not encode an amino acid sequence. Due to the degeneracy of the genetic code, multiple functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon may be changed to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variances are "silent variances", which are one species of conservatively modified variances. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One skilled in the art will recognize that each codon of a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to yield functionally identical molecules. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

The term "parent polypeptide" refers to a wild-type polypeptide and the amino acid sequence or nucleotide sequence of the wild-type polypeptide is part of a publicly accessible protein database (eg, EMBL Nucleotide Sequence Database, NCBI Entrez, ExPasy, Protein Data Bank, etc.).

The term "mutant polypeptide" or "polypeptide variant" or "mutein" or "mutant polypeptide" refers to a form of a polypeptide wherein the amino acid sequence differs from the amino acid sequence of its corresponding wild-type (parent) form, naturally occurring form, or any other parent form. A mutant polypeptide may contain one or more mutations, eg, substitutions, insertions, deletions, etc., that result in a mutant polypeptide.

The term "corresponding to a parent polypeptide" (or a grammatical variant of the term) is used to describe a polypeptide of the invention, wherein the amino acid sequence of the polypeptide differs from the amino acid sequence of the corresponding parent polypeptide only by the presence of at least an amino acid variance. Typically, the amino acid sequences of the variant polypeptide and the parent polypeptide exhibit a high percentage of identity. In one example, “corresponding to a parent polypeptide” means that the amino acid sequence of the variant polypeptide has at least about 50% identity, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 98% identity to the amino acid sequence of the parent polypeptide. In another example, a nucleic acid sequence encoding a variant polypeptide has at least about 50% identity, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 98% identity to a nucleic acid sequence encoding a parent polypeptide. In some embodiments, the parent polypeptide corresponds to FGF1 of SEQ ID NO: 1.

In some embodiments, the variant is a proteolytically stable variant compared to wild-type growth factor. In an exemplary embodiment, the variant exhibits increased proteolytic stability compared to wild type. In some embodiments, the variant is any variant of a wild-type growth factor. In some embodiments, the variant is an antagonist to the growth factor receptor to which wild-type growth factor binds.

In some embodiments, the variant is a variant of FGF1. In some embodiments, variants of human fibroblast growth factor 1 (FGF1) are provided comprising at least one member selected from amino acid substitutions, amino acid deletions, amino acid additions, and combinations thereof. In some embodiments, a variant of human fibroblast growth factor 1 (FGF1) is provided, wherein the resulting FGF1 variant comprises at least one member selected from amino acid substitutions, amino acid deletions, amino acid additions, and combinations thereof, wherein the resulting FGF1 variant exhibits increased proteolytic stability compared to wild-type FGF1 of SEQ ID NO: 1. In some embodiments, the FGF1 variant comprises amino acid substitutions, amino acid deletions, amino acid additions in the β-loop or near the C-terminus, and combinations thereof. In some embodiments, the FGF1 variant is a fibroblast growth factor receptor (FGFR) antagonist. The present invention provides a FGF1 polypeptide comprising at least one amino acid at at least one position not found in the parent FGF1 polypeptide (wild type, SEQ ID NO: 1).

The variant of FGF1 may be a variant in which any one amino acid selected from the group consisting of Q40P, S47I, H93G, and K112N in the amino acid sequence of SEQ ID NO: 1 is substituted.

In some embodiments, the variant is an isolated variant. In some embodiments, the variant exhibits at least one desirable characteristic not present in the present polypeptide. Exemplary characteristics include, but are not limited to, increased proteolytic stability, increased thermal stability, increased or decreased conformational flexibility, and increased antagonistic activity. As will be appreciated by those skilled in the art, a variant may exhibit any combination of two or more of these improved characteristics.

In some embodiments, the variant FGF1 is an antagonist to the FGFR receptor. In some embodiments, the FGF1 variant has a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.

In some embodiments, the growth factor variant has at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 96%, 97%, 98% or 99% sequence identity to the parent polypeptide. In some embodiments, a growth factor variant of the invention has at least about 99.2%, at least about 99.4%, at least about 99.6% or at least about 99.8% sequence identity to the parent polypeptide.

In some embodiments, the FGF1 variant has at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 96%, 97%, 98% or 99% sequence identity to the parent polypeptide. In some embodiments, an FGF1 variant of the invention has at least about 99.2%, at least about 99.4%, at least about 99.6% or at least about 99.8% sequence identity to the parent polypeptide.

The invention provides conjugates of the variants of the invention with one or more conjugation partners. Exemplary conjugation partners include polymers, targeting agents, therapeutic agents, cytotoxic agents, chelating agents, and detectable agents. One skilled in the art will recognize that there is overlap between these non-limiting categories of agents.

A conjugation partner or “modifier” can be any conjugateable moiety. Exemplary modifiers are discussed below. A modifying group can be selected for its ability to modify a property (eg, biological or physiochemical property) of a given polypeptide. Exemplary polypeptide properties that can be altered by use of modifying groups include, but are not limited to, pharmacokinetics, pharmacodynamics, metabolic stability, biodistribution, water solubility, lipophilicity, tissue targeting ability, and therapeutic activity profile. Modifiers are useful for modification of polypeptides used in diagnostic applications or in vitro biological assay systems.

In some embodiments, growth factor variants including FGF1 variants, eg, as described herein, are combined with an Fc moiety. The Fc-moiety may be derived from a human or animal immunoglobulin (Ig), preferably an IgG. IgG can be IgG1, IgG2, IgG3 or IgG4 (see eg FIG. 34 ). It is also preferred that the Fc-moiety is derived from the heavy chain of an immunoglobulin, preferably an IgG. More preferably, the Fc-moiety comprises a portion, eg a domain, of an immunoglobulin heavy chain constant region. Such Ig constant regions preferably include at least one Ig constant domain selected from any of the hinge, CH2, CH3 domains, or any combination thereof. In some embodiments, the Fc-moiety includes at least CH2 and CH3 domains. More preferably, the Fc-moiety comprises an IgG hinge region, CH2 and CH3 domains.

A number of water soluble polymers are known to those skilled in the art and are useful in the practice of the present invention. The term water soluble polymer includes sugars (eg, dextran, amylose, hyaluronic acid, poly(sialic acid), heparan, heparin, etc.); poly(amino acids) such as poly(aspartic acid) and poly(glutamic acid); nucleic acids; synthetic polymers (eg, poly(acrylic acid), poly(ether), such as poly(ethylene glycol)); Includes species such as peptides, proteins, etc. The present invention can be practiced with any water soluble polymer, the only limitation being that the polymer must contain a point to which the remainder of the conjugate can be attached. See, eg, Harris, Macronol. Chem. Phys. C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987); Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992); Zalipsky, Bioconjugate Chem. 6: 150-165 (1995); and Bhadra, et al., Pharmazie, 57:5-29 (2002).

In another embodiment similar to that discussed above, the modified sugar comprises a water insoluble polymer rather than a water soluble polymer. The conjugates of the present invention may also include one or more water insoluble polymers. This embodiment of the invention is illustrated by the use of conjugates as vehicles to deliver therapeutic polypeptides in a controlled manner. Polymeric drug delivery systems are known in the art. See, eg, Dunn et al., Eds. Polymeric Drugs And Drug Delivery Systems, ACS Symposium Series Vol. 469, American Chemical Society, Washington, D.C. 1991]. One skilled in the art will recognize that virtually any known drug delivery system can be applied to the conjugates of the present invention.

In various embodiments, the variant is conjugated to a component of a matrix for tissue regeneration. Exemplary matrices are known in the art, and it is within the ability of those skilled in the art to select and modify appropriate matrices of growth factor variants of the invention, including, for example, FGF1 variants. Growth factor variants of the present invention, including, for example, FGF1 variants, are generally used in regenerative medicine applications, including, for example, regeneration of eye, liver, muscle, nerve and heart tissue.

In some embodiments, the present invention provides conjugates that are selectively localized to specific tissues due to the presence of a targeting agent as a component of the conjugate. In an exemplary embodiment, the targeting agent is a protein. Exemplary proteins include transferrin (brain, blood pool), HS-glycoprotein (bone, brain, blood pool), antibodies (brain, tissue with antibody-specific antigen, blood pool), clotting factors V to XII (damaged tissue, thrombosis, cancer, blood pool), serum proteins such as α-acid glycoprotein, fetuin, α-fetal protein (brain, blood pool), β2-glycoprotein (liver, atherosclerotic plaque, brain, blood pool), G-CS F, GM-CSF, M-CSF, and EPO (immune stimulation, cancer, blood pool, red blood cell overproduction, neuroprotection), albumin (increase half-life), IL-2, and IFN-a.

In another embodiment, the present invention provides a conjugate between a growth factor variant of the present invention, including, for example, a FGF1 variant, and a therapeutic moiety. Therapeutic moieties useful in the practice of this invention include drugs from a broad class of drugs having a variety of pharmacological activities. Methods for conjugating therapeutic and diagnostic agents to a variety of different species are well known to those skilled in the art. See, eg, Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996; and Dunn et al., Eds. Polymeric Drugs And Drug Delivery Systems, ACS Symposium Series Vol. 469, American Chemical Society, Washington, D.C. 1991].

Another aspect is to provide a nucleic acid encoding the FGF1 variant polypeptide.

In some embodiments, the invention provides an isolated nucleic acid encoding a growth factor variant according to any of the embodiments set forth above, including, for example, a FGF1 variant. In some embodiments, the invention provides nucleic acids complementary to such nucleic acids.

In some embodiments, the invention provides an expression vector comprising a nucleic acid encoding a polypeptide variant according to any of the embodiments set forth above, operably linked to a promoter.

Another aspect is to provide a composition for wound healing and skin regeneration comprising the FGF1 variant as an active ingredient.

Another aspect is to provide a composition for inhibiting cancer growth and angiogenesis comprising the FGF1 variant.

Growth factor variants, including FGF1 variants, and their conjugates of the present invention have a wide range of pharmaceutical applications.

Accordingly, in another aspect, the invention provides a pharmaceutical composition comprising at least one polypeptide or polypeptide conjugate of the invention and a pharmaceutically acceptable diluent, carrier, vehicle, excipient or combination thereof. The pharmaceutical composition of the present invention is suitable for use in a variety of drug delivery systems. Formulations suitable for use in the present invention are described in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th ed. (1985)]. For a brief review of drug delivery methods, see Langer, Science 249:1527-1533 (1990).

The pharmaceutical composition may be formulated for any suitable mode of administration including, for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration such as subcutaneous injection, the carrier preferably includes water, saline, alcohol, fat, wax or buffer. For oral administration, any of the above carriers or solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate can be used. Biodegradable matrices such as microspheres (eg polylactate polyglycolate) can also be used as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are disclosed, for example, in US Pat. Nos. 4,897,268 and 5,075,109.

Generally, the pharmaceutical composition is administered subcutaneously or parenterally, eg intravenously. Accordingly, the present invention provides a composition for parenteral administration, which comprises a compound dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, such as water, buffered water, saline, PBS, or the like. The composition may also include detergents such as Tween 20 and Tween 80; stabilizers such as mannitol, sorbitol, sucrose, and trehalose; and preservatives such as EDTA and meta-cresol. The composition may contain pharmaceutically acceptable auxiliary substances as necessary to mimic physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like.

These compositions can be sterilized by conventional sterilization techniques or can be sterile filtered. The resulting aqueous solution can be packaged for use as is, or can be lyophilized, and the lyophilized preparation is combined with a sterile aqueous carrier prior to administration. The pH of the formulation will typically be between 3 and 11, more preferably between 5 and 9, and most preferably between 7 and 8.

In some embodiments, glycopeptides of the invention may be incorporated into liposomes formed from standard vesicle-forming lipids. See, eg, Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980)], and US Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. A variety of methods are available for preparing liposomes. Targeting of liposomes with various targeting agents (eg, sialyl galactosides of the present invention) is well known in the art (see, eg, US Pat. Nos. 4,957,773 and 4,603,044).

Standard methods for coupling targeting agents to liposomes can be used. Such methods generally involve the incorporation of a targeting agent or a lipid component, such as phosphatidylethanolamine, that can be activated for attachment of a derivatized lipophilic compound, such as a lipid-derivatized glycopeptide of the present invention, into the liposome.

Targeting mechanisms generally require that the targeting moiety be positioned on the surface of the liposome in a manner available for interaction with a target, eg, a cell surface receptor. Carbohydrates of the present invention may be attached to lipid molecules before liposomes are formed using methods known to those skilled in the art (eg, alkylation or acylation of hydroxyl groups present in carbohydrates using long-chain alkyl halides or fatty acids, respectively).

Alternatively, liposomes can be constructed in such a way that the connector portion is incorporated into the membrane first when forming the membrane. The connector portion should have a lipophilic portion that is firmly embedded and anchored in the membrane. It must also have reactive moieties that are chemically available on the aqueous surface of the liposome. The reactive moiety is selected so that it is chemically compatible to form a stable chemical bond with a targeting agent or carbohydrate that is subsequently added. In some embodiments, it is possible to attach the targeting agent directly to the connector molecule, but in most cases it is more appropriate to use a third molecule that acts as a chemical bridge, thus linking the connector molecule in the membrane with the targeting agent or carbohydrate extending three-dimensionally at the vesicle surface.

For example, growth factor variants produced by the methods of the present invention, including FGF1 variants, can be used as diagnostic reagents. For example, labeled compounds can be used to locate areas of inflammation or tumor metastasis in a patient suspected of having inflammation. For this use, compounds may be labeled with 125 I, 14 C, or tritium.

In various embodiments, the present invention provides methods for preventing, ameliorating or treating a disease state. In these embodiments, the invention provides methods comprising administering to a subject in need thereof an amount of a polypeptide variant of the invention sufficient to prevent, ameliorate, or treat a disease state. An exemplary disease state is cancer. The disclosed agonist variants may be useful in promoting cell growth, particularly for angiogenesis, and in the treatment of cardiovascular, hepatic, musculoskeletal and neurological diseases.

For example, certain polypeptide variants of the invention are useful for the prevention or treatment of hyperproliferative diseases or disorders, such as various forms of cancer.

In an exemplary embodiment, the present invention provides a method of treating cancer in a subject in need thereof. The method comprises administering to a subject a therapeutically effective amount of a polypeptide variant of the invention.

It is contemplated that the polypeptide variants of the present invention can be used in the treatment of various FGF responsive disorders, including, for example, FGF responsive tumor cells in various eye disorders, lung cancer, breast cancer, colon cancer, prostate cancer, ovarian cancer, head and neck cancer, ovarian cancer, multiple myeloma, liver cancer, gastric cancer, esophageal cancer, kidney cancer, nasopharyngeal cancer, pancreatic cancer, mesothelioma, melanoma and glioblastoma.

In an exemplary embodiment, the cancer is carcinoma, eg of the colorectal, squamous cell, hepatocyte, kidney, breast or lung.

Polypeptide variants can be used to inhibit or reduce the proliferation of tumor cells. In this approach, tumor cells are exposed to a therapeutically effective amount of a polypeptide variant to inhibit or reduce proliferation of the tumor cells. In certain embodiments, the polypeptide variant inhibits tumor cell proliferation by at least 50%, 60%, 70%, 80%, 90%, 95% or 100%.

In certain embodiments, the polypeptide variant is used to inhibit or reduce the proliferation of tumor cells, wherein the variant reduces the ability of FGF1 to bind FGFR. In certain embodiments, FGF1 polypeptide variants are used to inhibit or promote wound healing.

Additionally, polypeptide variants can be used to inhibit or slow tumor growth or development in mammals. In such methods, an effective amount of a polypeptide variant is administered to a mammal to inhibit or slow tumor growth in the mammal. Thus, polypeptide variants can be used, for example, to treat tumors in mammals. The method comprises administering to a mammal a therapeutically effective amount of a polypeptide variant. Polypeptide variants can be administered alone or in combination with another pharmaceutically active molecule to treat tumors.

Generally, a therapeutically effective amount of a polypeptide variant will range from about 0.1 mg/kg to about 100 mg/kg, optionally from about 1 mg/kg to about 100 mg/kg, optionally from about 1 mg/kg to 10 mg/kg. The amount administered will depend on variables such as the type and severity of the disease or indication being treated, the general state of health of the particular patient, the relative biological potency of the polypeptide variant to be delivered, the formulation of the polypeptide variant, the presence and type of excipients in the formulation, and the route of administration. The initial dosage administered may be increased beyond the upper limit in order to rapidly achieve the desired blood concentration or tissue level, or the initial dosage may be less than the optimal dosage and the daily dosage may be gradually increased during the course of treatment depending on the particular circumstances. Human dosages can be optimized in conventional Phase I dose escalation therapy studies designed to run, for example, from 0.5 mg/kg to 20 mg/kg. The frequency of dosing may vary depending on factors such as the route of administration, dosage and the disease state being treated. Exemplary dosing frequencies are once daily, once weekly and once every two weeks. A preferred route of administration is parenteral, eg intravenous infusion. Formulation of protein-based drugs is within the ordinary skill in the art. In some embodiments of the invention, polypeptide variants, eg, protein-based polypeptide variants, are lyophilized and reconstituted in buffered saline upon administration.

Polypeptide variants can be administered alone or in combination with other pharmaceutically active ingredients. Other active ingredients, such as immunomodulatory agents, may be administered with the polypeptide variant, or may be administered before or after the polypeptide variant.

Formulations comprising a polypeptide variant for therapeutic use typically include the polypeptide variant in combination with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" means buffers, carriers, and excipients suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with sound medical judgment and commensurate with a reasonable benefit/risk ratio. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the recipient. In this regard, pharmaceutically acceptable carriers are intended to include any and all buffers, solvents, dispersion media, coatings, tonicity and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.

The formulations may conveniently be presented in dosage unit form and may be prepared by any suitable method, including any method well known in the art of pharmacy. See Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990)].

In an exemplary embodiment, the polypeptide variant is used for diagnostic purposes either in vitro or in vivo, and the polypeptide variant is typically directly or indirectly labeled with a detectable moiety. A detectable moiety can be any moiety capable of directly or indirectly generating a detectable signal. For example, the detectable moiety may be a radioactive isotope such as 3H, 14C, 32P, 35S, or 125I; Fluorescent or chemiluminescent compounds such as Cy5.5, fluorescein isothiocyanate (GE Healthcare), Alexa Fluro® dye (Invitrogen), IRDye® infrared dye (LI-COR® Biosciences), rhodamine, or luciferin; enzymes such as alkaline phosphatase, beta-galactosidase, or horseradish peroxidase; spin probes such as spin labels; or colored particles, such as latex or gold particles. Polypeptide variants are described, for example, in Hunter et al. (1962) Nature 144: 945; David et al. (1974) Biochemistry 13: 1014; Pain et al. (1981) J. Immunol Meth 40: 219; and Nygren (1982) J. Histochem and Cytochem. 30: 407] can be conjugated to the detectable moiety using a number of approaches known in the art. Labels can be detected, for example, visually or with the aid of a spectrophotometer or other detector or other suitable imaging system.

Polypeptide variants can be used in a wide range of immunoassay techniques available in the art. Exemplary immunoassays include, for example, sandwich immunoassays, competitive immunoassays, immunohistochemical procedures.

According to the novel FGF-1 variant and its use according to one aspect, it has increased proteolytic stability and therapeutic effect compared to wild-type FGF1, so that it can be usefully used for related applications, such as wound healing, tissue regeneration, inhibition of cancer growth, and inhibition of angiogenesis.

1 is a graph showing the thermal stability of FGF-1 variants according to one embodiment.
Figure 2 is a graph showing the thermal stability of FGF-1 variants according to another embodiment.
Figure 3 is a graph showing the binding ability of FGF-1 variants according to one embodiment.
Figure 4 is a graph showing the proteolytic stability of FGF-1 variants according to one embodiment.

Hereinafter, an example will be described in detail for a more detailed explanation. However, these examples do not limit the present invention.

Example 1. Preparation of FGF-1 variants

To increase the stability of FGF-1, we designed two new point mutations, Q40P and S47I. In addition, five stabilizing single substitutions (H21Y, L44F, H93G and F108Y) were included. All mutant proteins were expressed in yields of 20–60 mg of pure protein per liter of bacterial culture. All substitutions and all recombinant proteins that alter the heparin-binding affinity showed very similar elution profiles during purification on heparin-cellose columns. Confirmed by circular dichroism (CD) and fluorescence of basic structural variants. There were no significant changes in the fluorescence or far and near UV CD spectra of the mutants, suggesting that secondary and tertiary structures were preserved. The CD spectrum showed a minimum and a positive peak at about 206 nm, which is typical of b-sheet-rich proteins of the b-II type. Between 220 nm and 240 nm, the center was near 228 nm.

Example 2. Thermal stability of FGF-1 variants

Thermal stability of the FGF- variants was confirmed, and the results are shown in Table 1, FIGS. 1 and 2 below.

Example 3. Binding ability of FGF-1 variants

The binding ability of the novel FGF-1 variants was confirmed, and the results are shown in FIG. 3 .

As shown in Figure 3, it was found that all mutants could bind. There was no difference in the ability to compete with 125I-labeled wild-type FGF-1 with a similar affinity to the wild-type protein for cell surface receptors present in NIH3T3 cells.

Example 4. Proteolytic stability of FGF-1

The proteolytic stability of the novel FGF-1 variants was confirmed, and the results are shown in FIG. 4 .

Since wild-type FGF-1 has a T den value close to 40 °C, biological activity was performed at 37 °C. The advantage of stable mutants may be due to their resistance to proteolysis. It is well known that the unfolded portion of mutants that are stable at physiological temperatures is much lower than that of the wild type, and the unfolded protein is much more susceptible to protease attack. Selected stable mutants were trypsinized to investigate the dependence between proteolysis susceptibility, thermodynamic stability and biological activity. As shown in Figure 4, wild-type FGF-1 was almost completely digested after 1 hour incubation with protease at 37 °C. In contrast, the triple mutant Q40P/S47I/H93G showed the longest half-life and the strongest mitotic properties, no obvious fragmentation was observed even after 1 h of trypsin digestion, and the intensity of the band corresponding to the intact form of FGF-1 did not change.

<110> SP2 Therapeutics Inc. <120> Novel FGF-1 variants and uses thereof <130> PN211204 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 140 <212> PRT <213> Homo sapiens <400> 1 Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140

Claims (10)

In the wild-type FGF-1 amino acid sequence of SEQ ID NO: 1, any one amino acid selected from the group consisting of Q40P, S47I, H21Y, L44F, H93G, F108Y and K112N is substituted, FGF-1 variant. The variant according to claim 1, wherein the substitutions are Q40P, S47I, and H93G. The variant according to claim 1, wherein the FGF-1 variant is a fibroblast growth factor receptor (FGFR) antagonist. The variant of claim 1 , wherein the FGF-1 variant is conjugated to a member selected from a detectable moiety, a water soluble polymer, a water insoluble polymer, a therapeutic moiety, a targeting moiety, and combinations thereof. The variant according to claim 1, wherein the FGF-1 variant is conjugated to a detectable moiety selected from radioisotopes, paramagnetic substances, fluorophores, and combinations thereof. A composition for wound treatment or skin regeneration comprising the FGF-1 variant of claim 1 as an active ingredient. A pharmaceutical composition for preventing or treating cancer or inhibiting angiogenesis, comprising the FGF-1 variant of claim 1 as an active ingredient. The pharmaceutical composition of claim 7, wherein the cancer is selected from colon, oral cavity, hepatocytes, kidney, breast, lung, ovary, stomach, brain, prostate, and combinations thereof. A nucleic acid encoding the FGF-1 variant polypeptide of claim 1. An isolated cell in which the nucleic acid of claim 9 is expressed.