WO2011150470A1

WO2011150470A1 - Vitronectin:keratinocyte growth factor chimeras

Info

Publication number: WO2011150470A1
Application number: PCT/AU2011/000700
Authority: WO
Inventors: Zee Upton
Original assignee: Queensland University Of Technology
Priority date: 2010-06-03
Filing date: 2011-06-03
Publication date: 2011-12-08
Also published as: US20100303884A1; US8871709B2; US20130243843A1; BR112012030821A2; US9562086B2; CN103038351A; RU2012156861A; KR20130115994A; DK2576790T3; CN106243228A; HK1183908A1; EP2576790B1; EP2576790A4; JP2013530161A; AU2011261177A1; JP5843175B2; AU2011261177B2; NZ603942A; CA2801240A1; EP2576790A1

Abstract

Isolated protein complexes are provided comprising keratinocyte growth factor and vitronectin, or at least domains thereof that enable binding to and activation of both a keratinocyte growth factor receptor and an integrin receptor for vitronectin. These protein complexes include synthetic proteins where the keratinocyte growth factor and vitronectin sequences are joined by a linker sequence. In particular forms, vitronectin sequences do not include a C-terminal heparin binding domain. Also provided are uses of these protein complexes for stimulating or inducing cell migration and/or proliferation in wound healing, tissue engineering, cosmetic and therapeutic treatments such as skin replacement, skin replenishment and treatment of burns where epithelial cell migration is required. In other embodiments, the invention provides inhibition of cancer cell metastasis, particularly in relation to breast cancer.

Description

U2011/000700

TITLE

VITRONECTIN:KERATINOCYTE GROWTH FACTOR CHIMERAS

FIELD OF THE INVENTION THIS INVENTION relates to protein complexes having respective domains that enable binding to and activation of both a keratinocyte growth factor (KGF) receptor and an integrin receptor for vitronectin (VN). In particular embodiments, this invention relates to chimeric proteins comprising keratinocyte growth factor receptor-binding domains and an integrin receptor-binding domain of vitronectin. More particularly, this invention relates to protein complexes and chimeric proteins that stimulate cell migration and to compositions and methods that promote or induce cell migration and/or proliferation. These compositions and . methods have use in wound healing, tissue engineering, cosmetic and therapeutic treatments such as skin replacement, and skin replenishment and treatment of bums where epithelial cell migration and/or proliferation is required. In other embodiments, the invention provides treatment provided by the present invention related to prevention or inhibition of cancer cell metastasis, particularly in relation to breast cancer.

BACKGROUND OF THE INVENTION

Keratinocyte growth factor is a mitogenic peptide growth factor involved in a broad range of cellular processes including hyperplasia, DNA synthesis, differentiation, cell cycle progression, and inhibition of apoptosis (Marchese et al,

1990, J. Cell Physiol. 144:326-32). These effects are mediated through binding to its tyrosine-kinase linked cell surface receptor, the KGF receptor.

Vitronectin is a glycoprotein that is highly abundant in the blood and in the extra cellular matrix (ECM). Primarily synthesized in the liver, but expressed by many other cell types, VN circulates in the blood in a closed conformation and is deposited in the ECM in an open, or extended, conformation (Schvartz et al, 1999,

Int. J. Biochem. Cell Biol. 31 :531 -44). Both conformations are believed to bind IGF-.

II (Uptor. et al, 1999, Endocrinology 140:2928-3 1 ; International Publication WO

02/24219; McMurty et al, 1996, Endocrinology 150: 149-60) and also bind multiple other ligands including collagen (Morris et al, 1994, J. Bio. Chem. 269:23845-52), glycosaminoglycans (Francois et al, 1999, J. Bio. Chem. 274:3761 1 - 19), many other ECM proteins, and a wide variety of integrins, particularly the a_v integrins. Indeed, the primary role of vitronectin is as an ECM organization molecule that provides adhesive links to these cell surface integrin receptors via an RGD binding motif. The VN receptors (a_v integrins) have been shown to regulate the actin cytoskeleton rearrangement required for growth and invasion, hence, VN binding coordinates cell adhesion and movement (DePasquale, 1998, Histochemistry and Cell Biology 1 10:485-94; Huang, 2000, Oncogene 19: 1915-23).

However, the relative contributions of KGF and VN, and their respective domains, present in protein complexes, in terms of stimulating biological responses such as cell migration and/or proliferation, have remained elusive.

SUMMARY OF THE INVENTIO

The present inventors have discovered that protein complexes comprising KGF and VN stimulate cell migration and/or proliferation by binding and synergistically co-activating keratinocyte growth factor receptors and VN-binding integrin receptors.

Therefore, the invention is broadly directed to isolated protein complexes that comprise a receptor-binding domain of keratinocyte growth factor and a domain of vitronectin that is capable of binding an integrin receptor, wherein the isolated protein complex can co-activate the keratinocyte growth factor receptor and integrin receptor to thereby elicit a biological response.

In a first aspect, the invention provides an isolated protein complex in the form of a synthetic chimeric protein comprising an amino acid:sequence of:

(i) keratinocyte growth factor, or at least a domain of keratinocyte growth factor which is capable of binding a keratinocyte growth factor receptor; and

(ii) one- or more domains of vitronectin (VN) including at least an integrin-binding domain of VN.

Preferably, the integrin receptor is an a_v integrin.

More preferably, the integrin receptor is an _νβί integrin or an a._vfi₅ integrin. This aspect of the invention also includes within its scope amino acid deletions, additions, substitutions and/or mutations of amino acid sequences corresponding to (i) and (ii) above. 2011/000700

In a second aspect, the invention provides an isolated nucleic acid encoding the isolated protein complex of the first aspect.

In a third aspect, the invention provides a genetic construct comprising the isolated nucleic acid of the second aspect operably linked to one or more regulatory sequences in an expression vector.

Preferably, the genetic construct is an expression construct.

In a fourth aspect, the invention provides a host cell comprising the genetic construct of the third aspect.

In a fifth aspect, the invention provides a pharmaceutical composition comprising the isolated protein complex of the first aspect and a pharmaceutically- acceptable carrier, diluent or excipient.

This aspect of the invention also contemplates a pharmaceutical composition comprising the host cell of the fourth aspect, which cell expresses said synthetic protein(s).

In a sixth aspect, the invention provides an antibody specific for the synthetic protein of the first aspect.

In a seventh aspect, the invention provides a method of promoting cell migration including the step of using a synthetic protein to bind both a keratinocyte growth factor receptor and an integrin receptor.

Preferably, the integrin receptor is an a_v integrin.

More preferably, the integrin receptor is an _νβ3 integrin or an _v s integrin. In a preferred embodiment, this aspect of the invention relates to promotion or induction of epithelial/keratinocyte/fibroblast cell migration and/or proliferation to facilitate wound healing in mammals, preferably humans.

Preferably, said synthetic protein is as according to the first aspect of the invention.

In an eighth aspect, the invention provides a method of preventing cell migration and/or proliferation, including the step of preventing, inhibiting or otherwise reducing binding of both a keratinocyte growth factor receptor and an integrin receptor by a complex comprising keratinocyte growth factor and vitronectin.

Preferably, the integrin receptor is an a_v integrin. More preferably, the integrin receptor is an α_νβ3 integrin or an _vPs integrin.

In a preferred embodiment, this aspect of the invention relates to prevention or inhibition of metastatic cancer cell migration and/or proliferation in mammals, preferably humans.

A particular example contemplated by this aspect of the invention is prevention or inhibition of breast cancer metastasis.

It will also be appreciated that the methods of the seventh and eighth aspects may encompass prophylactic and therapeutic methods of treatment.

In a ninth aspect, the invention provides use of the isolated protein complex of the first aspect to produce a molecule that:

(i) is an agonist of protein complexes comprising keratinocyte growth factor and vitronectin; or

(ii) is an antagonist of protein complexes comprising keratinocyte growth factor and vitronectin.

In one embodiment, the invention provides use of the synthetic protein of the first aspect to produce a molecule that:

(i) is an agonist of GF:VN protein complexes; or

(ii) is an antagonist of KGF:VN protein complexes.

Agonists and/or antagonists produced according to this aspect of the invention may have particular efficacy in promoting wound healing, tissue engineering, skin regeneration and/or prevention of cancer cell metastasis or hyperproliferative disorders of the skin, such as scarring and psoriasis.

In a tenth aspect, the invention provides a biomaterial that comprises the isolated protein complex of the first aspect.

In particular embodiments the biomaterial may be a surgical implant, prosthesis, scaffold, wound or burn dressing, or the like suitably impregnated, coated or otherwise comprising an isolated protein Complex of the invention. 11 000700

5

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 . The amino acid sequence of vitronectin (SEQ ID NO: I ), including residue references for the various domains within vitronectin, as well as residue modification sites, ligand binding sites and protease recognition sites.

Figure 2. The structural relationship of (a) full-length VN (75 kDa) and (b) yolk VN (54 kDa) showing ligand binding sites. Both mammalian and avian serum VIM have the same domain structure, however, there are differences in the amino acid sequence. Yolk VN (5_.4 kDa) is a truncated form of these proteins. The abbreviations used are: Som B, Somatomedin B; Connecting, Connecting domain; Hemopexin, Hemopexin-like repeat; HBD, Heparin binding domain; PAI- I , plasminogen activator inhibitor- 1 ; uPAR, urokinase plasminogen activator receptor; TAT, thrombin-antithrombin II I complex; uPA, urokinase plasminogen activator; -, polyanionic region (basic region); + + +, polycationic region (acidic region).

Figure 3. Amino acid sequence of (A) mature vitronectin protein (SF.Q ID NO:2), (B) mature KGF (SEQ ID NO:3), (C) preferred linker sequences (SEQ ID

NOs:4-9), and (D) to (H) embodiments of KGF and VN-containing chimeric proteins (SEQ ID NOs: 10- 14). ·

Figure 4. (A) A VN:KGF chimeric protein stimulates primary keratinocyte cell migration. Migration of isolated skin keratinocyte cells seeded within the inner ■ chamber of a seeding insert which was removed to allow outward migration, in response to the culture well being coated with a VN:KGF chimera and controls. Each bar represents the average area (+^■/- SEM) of cell coverage after 24 hours incubation and are obtained from at least three replicate experiments in which treatments were analysed in triplicate wells. (B) A VN:KGF chimeric protein stimulates primary fibroblast ceil migration. Migration of isolated skin fibroblast cells seeded within the inner chamber of a seeding insert which was removed to allow outward migration, in response to the culture well being coated with a VN:KGF chimera and controls. Each bar represents the average area (+/- SEM) of cell coverage after 24 hours incubation and are obtained from at least three replicate experiments in which treatments were analysed in triplicate wells. (C) A VN: KGF chimeric protein stimulates primary keratinocyte cell proliferation. Proliferation of isolated skin keratinocyte cells in response to the culture well being coated with a VN:KGF chimera and controls. Each bar represents the average absorbance (+/- SEM) of DNA-binding GR dye (representative of cell number) after 72 hours incubation and are obtained from at least three replicate experiments in which treatments were analysed in triplicate wells. (D) A VN:KGF chimeric protein stimulates primary fibroblast cell proliferation. Proliferation of isolated skin fibroblast cells ^'in response to the culture well being coated with a VN:KGF chimera and controls. Each bar represents the average absorbance (+/- SEM) of DNA-binding GR dye (representative of cell number) after 24 hours incubation and are obtained from at least three replicate experiments in which treatments were analysed in triplicate wells.

Figure 5. Primary keratinocyte and fibroblast signaling responses to VN.vKGF chimeric proteins and controls. VN:KGF chimeric proteins promote similar levels of realtive activation of ERK l /2 and AKT signaling pathways as respective controls: (A) keratinocyte ERK l /2; (B) keratinocyte AKT; (C) fibroblast ERK l /2; (D) fibroblast AKT.

DETAILED DESCRIPTION OF THE INVENTION

The present invention has arisen from the discovery that protein complexes comprising KGF and VN bind and exert their biological effect on cell migration through the KGF receptor and the VN-binding integrin receptor expressed by responsive cells. More particularly, this dual binding event synergistically stimulates cell migration and/or proliferation.

Although not wishing to be bound by any particular theory, it is thought that a domain of VN which interacts with or binds KGF is the polyanionic region of VN corresponding to amino acids 53-64 of mature VN (SEQ ID NO:2).

This discovery has led the present inventors to provide an isolated protein complex that comprises at least the minimal domain or region of KGF capable of binding the KGF receptor in combination with the integrin-binding domain of VN.

Even more particularly, a single, contiguous protein may be produced which comprises these domains.

Such protein complexes, in the form o a single synthetic protein, coordinately bind or co-ligate the KGF receptor and the VN-binding integrin receptor and are therefore useful agents for the promotion of ceil migration and/or proliferation and wound healing. Analogously, prevention of the GF receptor and the VN-binding integrin receptor co-ligation can be used to prevent cancer cell metastasis.

Throughout ^"this specification, unless otherwise indicated, "comprise",

"comprises" and "comprising" are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers.

In the particular context of keratinocyte growth factor receptor-binding domains and i-ntegrin-binding domains, such a domain will comprise an amino acid sequence of the domain, together with other, additional aminQ acids as desired.

It will be understood also that such a domain may "consist essentially of the amino acid sequence of the domain, together with n more than ten, preferably no more than five or even more preferably no more than four, three, two, or one additional amino acids.

It will be understood also that such a domain may "consist of the amino acid sequence of the domain, in the absence of any additional amino acids.

For the purposes of this invention, by "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form.

As used herein, by "synthetic" is meant not naturally occurring but made through human technical intervention. In the context of synthetic proteins and nucleic acids, this encompasses molecules produced by recombinant, chemical synthetic or combinatorial techniques as are well understood in the art.

By "protein" is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids, D- or L- amino acids as are well understood in the art. The term "protein" also includes and encompasses such terms as "glycoprotein", "lipoprotein" and the like, as are commonly used in the art. A "peptide" is a protein having less than fifty (50) amino acids.

A "polypeptide" is a protein having fifty (50) or more amino acids.

As hereinbefore described, the present invention provides,, in one particular aspect, an isolated protein complex in the form of a synthetic chimeric protein comprising an amino acid sequence of:

(i) a keratinocyte growth factor or at least a domain of a keratinocyte growth factor which is capable of binding a keratinocyte growth factor receptor; and

(ii) vitronectin, or at least an integrin-binding domain of vitronectin.

As used herein, a "chimeric protein" comprises a contiguous sequence of amino acids derived from an integrin receptor-binding domain of vitronectin and a keratinocyte growth factor or at least a receptor-binding domain of a keratinocyte growth factor.

As used herein, "keratinocyte growth factor" is a biologically active protein that is capable of regulating cell growth, differentiation, survival and/or migration in vitro and/or in vivo (Marchese el at , 1990, J. Cell Physiol. 144:326-32; UniProtKB/Swiss-Prot: #P21781 , mature protein comprises amino acid residues 32- 194 of the complete sequence).

isolated protein complexes in the form of synthetic chimeric proteins of the invention comprise a keratinocyte growth factor or at least a domain of a keratinocyte growth factor which is capable of binding a keratinocyte growth factor receptor.

In this context, by "domain" is meant at least that portion of region of a keratinocyte growth factor that is capable of binding a keratinocyte growth factor receptor. Typically, although not exclusively, the keratinocyte growth factor receptor is expressed by a cell and binding or ligation of the keratinocyte growth factor receptor by the at least a domain of a keratinocyte growth factor elicits a cellular response such as cell growth, differentiation, survival and/or migration.

It will also be understood that another component of isolated protein complexes in the form of synthetic chimeric proteins of the invention is at least an. integrin-binding domain of vitronectin.

Preferably, the integrin receptor is an a_v integrin.

More preferably, the integrin receptor is an α_νβ₃ integrin or an s integrin. Although not wishing to be bound by any particular theory, it is proposed that synthetic chimeric proteins are able to co-ligate and co-activate a receptor for keratinocyte growth factor and an integrin receptor for VN to thereby stimulate, induce, augment or otherwise promote cell growth, differentiation, survival and/or cell migration.

An advantage of chimeric proteins according to the invention is that they are readily produced by chemical synthetic or recombinant means 'and are expected to be more stable in vivo, as they do not rely on maintaining the protein-protein interactions that are required in non-covalent oiigo-protein complexes.

The present invention contemplates embodiments of synthetic chimeric proteins that do not include the C-terminal heparin binding domain (HBD) and/or the polyanionic region of VN. By "C-terminal HBD" is meant residues 347-459 of the mature VN sequence (SEQ ID NO:2). Xu el al. (2001 , Proteins 44:3 12-20) have argued that VN contains a second HBD in its central region. The present invention does not contemplate this alleged HBD.

With regard to VN proteins and amino acid sequences thereof that do not include the C-terminal HBD and/or the polyanionic region, these may be naturally occurring proteins such as the 54kD chicken yolk VN" (lacking a C-terminal HBD) or may be engineered by deletion, mutation or truncation of a VN protein or amino acid sequence so that the C-terminal HBD and/or the polyanionic region are absent or at least substantially non-functional.

Techniques such as proteolytic digestion and site directed mutagenesis maybe utilized for this purpose, as are well understood in the art.

In particular embodiments, said at least an integrin-binding domain of VN has an amino acid sequence selected from the group consisting of:

(i) amino acid residues 1 to 459 of VN;

(ii) amino acid residues 1 to 379 of VN;

(iii) amino acid residues I to 346 of VN;

(iv) amino acid residues 1 to 31 1 of VN;

(v) amino acid residues 1 to 1 30 of VN;

(vi) amino acid residues I to 125 of VN;

(vii) amino acid residues 1 to 64 of VN; and (viii) amino acid residues 1 to 52 of VN (all references are to the mature VN sequence (SEQ ID NO:2)).

Additional amino acid sequences which also may be included are selected from the group consisting of:

(ix) amino acid residues 65 to 459 of VN;

(x) amino acid residues 343 to 376 of VN;

(xi) amino acid residues 347 to 459 of VN; and

(xii) amino acid residues 347 to 379 of VN (all references are to the mature VN sequence (SEQ ID NO:2)).

The aforementioned sequences may be used in combination, for example amino acid residues 1 to 130 of VN and amino acid residues 347 to 459 of VN or amino acid residues 1 to 52 of VN and amino acid residues 65 to 459 of VN.

Particular, non-limiting example of chimeric proteins comprising KGF and VN are set forth in FIG. 3, and include: (i) 1 -459 VN:(Gly₄ Ser)₄: l - 163 KGF:Gly Ser Gly₄:6 His; (ii) 1 -3 1 1 VN:(Gly Ser)₄: 1 - 163 KGF:Gly₄ Ser Gly :6 His; (iii) 1 - 125 VN:(Gly₄ Ser)₄: l - 163 KGF:Gly₄ Ser Gly₄:6 His; (iv) 1 -64 VN:(Gly₄ Ser)₄: l - 163 KGF:Gly .Ser Gly₄:6 His; and (v) 1 -64 VN:(GIy₄ Ser)₄:343-376 VN:(Gly₄ Ser)₄: l - 163 KGF:Gly₄ Ser Gly₄:6 His.

in other embodiments, the invention provides isolated protein complexes, such as in the fonn of synthetic chimeric proteins, comprising KGF and VN, or a fragment of VN that comprises at least an integrin-binding domain of VN.

In this context, by "fragment" is meant a domain, sub-sequence or portion of VN. The fragment preferably constitutes less than 500, less than 400, less than 300 or more preferably about 80-280 contiguous amino acids of a mature VN sequence.

The integrin binding domain of VN suitably comprises an RGD sequence

(amino acids 45-47 of a mature VN sequence). Accordingly, in one particular embodiment, the synthetic chimera comprises a VN fragment comprising an RGD sequence.

Preferably, synthetic chimeric proteins as hereinbefore described further comprise a "linker sequence" located between and contiguous with a keratinocyte growth factor sequence and a VN amirio acid sequence. I '

In one embodiment, said linker sequence comprises one or more glycine residues and one or more serine residues.

Particular examples of linker sequences may be selected from; Gly₄ Ser (SEQ ID NO:4); Gly₄ Ser^ (SEQ ID NO:5); (Gly₄ Ser)₃ (SEQ I D NO:6); and (Gly„ Ser)₄ (SEQ ID NO:7). although without limitation thereto.

In another embodiment, the linker sequence includes a Plasmin Cleavage Recognition Site (Sakiyama-Elbert et al, 2001 , FASEB 15:1300-02), such as according to the sequence:

Leu lie Lys Met Lys Pro (SEQ ID NO:8)

In yet another embodiment, the linker sequence includes a Collagenase-3

Cleavage Recognition Site (Kim & Healy, 2003, Biomacromolecules 4: 12 1 4-23), such as according to the sequence:

Gin Pro Gin Gly Leu Ala Lys (SEQ ID NO:9) The invention also extends to use of biologically-active fragments of the synthetic chimeric proteins of the invention and/or to use of biologically-active fragments of the keratinocyte growth factor receptor-binding domains and integrin binding domains exempli ied herein.

In one embodiment, said "biologically-active fragment" has no less than 10%, preferably no less than 25%, more preferably no less than 50% and even more preferably no less than 75%, 80%, 85%, 90%, or 95% of a biological activity of a protein from which it is derived.

In another embodiment, said "biologically-active fragment" has no less than 10%, preferably no less than 25%, more preferably no less than 50% and even more preferably no less than 75%, 80%, 85%, 90%, or 95% of a contiguous amino acid sequence of a protein from which it is derived.

Also contemplated are variant protein complexes of the invention.

Typically, and in relation to proteins, a "variant" protein has one or more amino acids that have been replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the protein (conservative substitutions). It will be appreciated that one or more amino acid residues of a reference sequence, such as keratinocyte growth factor, receptor-binding domain of keratinocyte growth factor, an integrin-binding domain of VN, or one or more corresponding residues present in a synthetic chimeric protein, may be modified or deleted, or additional sequences added, without substantially altering the biological activity of the isolated protein complex of the invention.

Specific mutations in mature VN (SEQ ID NO:2) that are contemplated by the present invention include: (i) T50A; (ii) T57A; (iii) T50E; (iv) T57E; ,(v) S378E; (vi) S378A; and (v) S362E.

In one embodiment, a protein_, variant shares at least 70%, preferably at least

80% and more preferably at least 90%, 95%, 98%, or 99% sequence identity with a reference amino acid sequence.

Preferably, sequence identify is measured over at least 60%, more preferably over at least 75%, more preferably over at least 90%, or more preferably over at least 95%, 98% or substantially the full length of the reference sequence.

In order to determine percent sequence identity, optimal alignment of amino acid and/or nucleotide sequences may be conducted by computerised implementations of algorithms (Geneworks program by Intel ligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, WI, USA) or by inspection and the best alignment (i. e. , resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul el al. ( 1997, Nucl. Acids Res. 25:3389-402).

In another example, "sequence identity" may be understood to mean the

"match percentage" calculated by the DNAS1S computer program (Version 2.5 for windows; available from Hitachi Software engineering Go., Ltd., South San Francisco, California, USA).

A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995- 1999). t he invention also contemplates derivatives of a receptor-binding domain of keratinocyte growth factor, an integrin-binding domain of VN or an isolated protein complex comprising the same.

As used herein, "derivative" proteins of the invention have been altered, for example by addition, conjugation or complexing with other chemical moieties or by post-translational modification techniques as are well understood in the art

"Additions" of amino acids may include fusion of the polypeptides or variants thereof with other polypeptides or proteins. The other protein may, by way of example, assist in the purification of the protein. For instance, these include a polyhistidine tag, maltose binding protein, green fluorescent protein (GFP), Protein A or glutathione S-transferase (GST).

Other derivatives contemplated by the invention include, but are not limited to, modification to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the polypeptides, fragments and variants of the invention. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by aeylation with acetic anhydride; acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; amidination with methylacetimidate; carbamoylation of amino groups with cyanate; pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH₄; reductive alkyiation by reaction with an aldehyde followed by reduction with NaBH^ and trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS).

The carboxyl group may be modified by carbodiimide activation via O- acylisourea formation followed by subsequent derivitization, by way of example, to a corresponding amide.

The guanidine group of arginine residues may be modified by formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

Sulphydryl groups may be modified by methods such as performic acid oxidation to cysteic acid; formation^' of mercurial derivatives using 4- 0700

14 chloromercuriphenylsulphonic acid, 4-chIoromercuribenzoate; 2-chloromercuri-4- nitrophenol, phenylmercury chloride, and other mercurials; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; carboxymethylation with iodoacetic acid or iodoacetamide; and carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified, for example, by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides, or by oxidation with N-bromosuccinimide.

Tyrosine residues may be modified by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

The imidazole ring of a histidine residue may be modified by N- carbethoxylation with diethylpyrocarbonate or by alkylation with iodoacetic acid derivatives.

Examples of incorporating non-natural amino acids and derivatives during peptide synthesis include, but are not limited to, use of 4-amino butyric acid, 6- aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3- hydroxy-6-methylheptanoic acid, t-butylglycine, norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine, and/or D-isomers of amino acids.

An example of methods suitable for chemical derivatization of proteins is provided in Chapter 1 5 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et. ai, John Wiley & Sons NY ( 1995-2001 ).

Isolated protein complexes, and individual protein components thereof, (inclusive of fragments, variants, derivatives, and homologs) may be prepared by any suitable procedure known to those of skill in the art.

In one embodiment, proteins of the invention are produced by chemical synthesis. Chemical synthesis techniques are well known in the art, although the skilled person may refer to Chapter 18 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et. al, John Wiley & Sons NY ( 1995-200 1 ) for examples of suitable methodology.

In another embodiment, proteins may be prepared as recombinant proteins. T AU2011/000700

15

While production of recombinant proteins is well known in the art, the skilled person may refer to standard protocols as for example described in Sambrook et al., MOLECULAR CLONING A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 1 7: CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons, Inc. 1 95-1999), in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. 1995- 1999), in particular Chapters 1 , 5 and 6. -

In one embodiment, a recombinant protein is produced by a method including the steps of:

(i) preparing an expression construct which comprises a nucleic acid encoding said protein, operably linked to one or more regulatory nucleotide sequences in an expression vector;

(ii) transfecting or transforming a host cell with the expression construct; and

(iii) expressing the recombinant protein in said host cell.

An "expression vector" may be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome.

By "operably linked" or "operably connected^" is meant that said regulatory nucleotide sequence(s) is/are positioned relative to the recombinant nucleic acid of the invention to initiate, regulate or otherwise control transcription of the nucleic acid, or translation of a protein encoded by the nucleic acid.

Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.

Typically, said one or more regulatory nucleotide sequences may include, but are not limited to. promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, splice donor/acceptor sequences, and. enhancer or activator sequences.

Constitutive promoters (such as CMV. RSV, adenovirus, SV40, and human elongation factor promoters) and inducible/repressible promoters (such as let- 11 000700

16 repressible promoters and IPTG-, metallothionine- or ecdysone-inducible promoters) are well known in the art and are contemplated by the invention. It will also be appreciated that promoters may be hybrid promoters that combine elements of more than one promoter.

The expression construct may also include a fusion partner (typically provided by the expression vector) so that the recombinant protein of the invention is expressed as a fusion polypeptide with said fusion partner; The main advantage of fusion partners is that they assist identification and/or purification of said fusion protein.

Well known examples of fusion partners include, but are not limited to, glutathione-S-transferase (GST), Fc portion of human IgG, maltose binding protein (MBP), and hexahistidine (HlSe). which are particularly useful for isolation of the fusion protein by affinity chromatography. For the purposes of fusion protein purification by affinity chromatography, relevant matrices for affinity chromatography are glutathione-, amylose-, and nickel- or cobalt-conjugated resins respectively. Many such matrices are available in "kit" form, such as the QIAexpress^{1 M} system (Qiagen) useful with (HIS₆) fusion partners and the Pharmacia GST purification system.

in some cases, the fusion partners also have protease cleavage sites, such as for Factor X_a or Thrombin, which allow the relevant protease to partially digest the fusion protein of the invention and thereby liberate the recombinant polypeptide of the invention therefrom. The liberated protein can then be isolated from the fusion partner by subsequent chromatographic separation.

Fusion partners according to the invention also include within their scope "epitope tags", which are usually short peptide sequences for which a specific antibody is available. Well known examples of epitope tags for which specific monoclonal antibodies are readily available include c-myc, haemagglutinin and FLAG tags.

Suitable host cells for expression may be prokaryotic or eukaryotic, such as Escherichia coli (ΏΗ5α for example;, yeast cells, Sf9 cells utilized with a baculovirus expression system, CHG cells, COS, CV- 1 , NIH 3T3 and 293 cells, although without limitation thereto. Expression constructs may also include one or more selection marker nucleotide sequences that confer transformed host cell resistance to a selection agent. Selection markers useful for the purposes of selection of transformed bacteria include bla, kanR and tetR while transformed eukarvotic cells may be selected by markers such as hygromyein, G41 8 and puromycin, although without limitation thereto.

With regard to introducing genetic material into host cells, the terms "transforming¹' and "transfecting" are used generally to describe introduction of genetic material into a host cell. There are many well known methods for introducing foreign genetic material into a host cell including, but not limited to, calcium phosphate precipitation, electroporation, delivery by lipofectamtne, lipofectin and other lipophilic agents, calcium phosphate precipitation,^■ DEAE- Dextran transfection, microparticle bombardment, microinjection, and protoplast fusion. ·

The invention provides an isolated nucleic acid that encodes a synthetic chimeric protein of the invention, including variants and homologs thereof.

The term "nucleic acid" as used herein designates single-or double-stranded mR A, RNA, cRNA, RNAi, and DNA, inclusive of cD A and genomic DNA and DNA-RNA hybrids.

• A "polynucleotide'^" is a nucleic acid having ^'eighty (80) or more contiguous nucleotides, while an "oligonucleotide" has less than eighty (80) contiguous nucleotides.

A "probe" may be a single or double-stranded oligonucleotide or polynucleotide, suitably labeled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.

A "primer" is usually a single-stranded oligonucleotide, preferably having 15-

50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid "template" and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or Sequenase .

Synthetic nucleic acids of the invention may be produced by chemical synthetic approaches or by recombinant methods that utilize nucleic acid sequence amplification techniques, or a combination thereof, as, are well known in the art. Chemically synthesized primers and oligonucleotides, synthesizers and associated technologies useful according to the present invention are typically available in most laboratories or may be purchased from commercial sources.

Suitable nucleic acid amplification techniques are wel l known to the skilled person, and include polymerase chain reaction (PCR) and ligase chain reaction (LCR) as for example described in Chapter 1 5 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995- 1 999); strand displacement amplification (SDA) as for example described in U.S. Patent 5,422,252; rolling circle replication (RCR) as for example described in Liu et al. ( 1 996, J. Am. Chem. Soc. 1 18: 1587-94), International application WO 92/01 813 and International Application WO 97/19193; nucleic acid sequence-based amplification (NASBA) as for example described by Sooknanan et al. (1994, Biotechniques 1 7: 1077-80); and Q-β replicase amplification as for example described by Tyagi et al. ( 1996, Proc. Natl. Acad. Sci. USA 93:5395-400), although without limitation thereto.

A preferred nucleic acid sequence ampli fication technique is PCR.

As used herein, an "amplification product" refers to a nucleic acid product generated by a nucleic acid amplification technique.

In producing and expressing nucleic acids of the invention, it will also be appreciated that advantage may be taken with respect to codon sequence redundancy, such that the nucleic acids exemplified herein may be readily modified without changing an amino acid sequence encoded thereby.

In particular embodiments, nucleic acids may be optimized according to preferred ''codon usage^*' of a host cell to be used for recombinant expression, as is well known in the art. This can effectively "tailor" a nucleic acid for optimal expression in a particular organism, or cells thereof, where preferential codon usage affects protein expression.

Therefore, the invention includes synthetic nucleic acids that are homologous to the nucleic acids exemplified herein.

In one embodiment, nucleic acid homologs share at least 70%, preferably at. least 80%, more preferably at least 90%, and even more preferably at least 95% sequence identity with a nucleic acid encoding any one of the synthetic chimeric protein constructs described herein.

Preferably, sequence identify is measured over at least 70%, more preferably at least 80%, even more preferably at least 90%, 95% or advantageously over substantially the full length of the encoding nucleic acid of the invention.

In another embodiment, nucleic acid homologs hybridize to a nucleic acid encoding any one of the synthetic chimeric protein constructs described herein under high stringency conditions.

"Hybridize and hybridization" is used herein to denote the pairing of at least partly complementary nucleotide sequences to produce a DNA-D A, RNA-RNA or DNA-RNA duplex. Hybridized sequences o.ccur through base-pairing between complementary purines and pyrimidines as is well known in the art.

In this regard, it will be appreciated that modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines (thiouridine and methylcytosine) may also engage in base pairing.

"Stringency", as used herein, refers to temperature and ionic strength conditions, and presence or absence of certain organic solvents and/or detergents during hybridisation. The higher the stringency, the higher will be the required level of complementarity between hybridizing nucleotide sequences.

"Stringent conditions" designates those conditions under which only nucleic acid having a high frequency of complementary bases will hybridize.

Reference herein to high stringency conditions includes and encompasses:

(i) from at least about 3 1 % v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0. 1 5 M salt for hybridisation at 42°C, and at least about 0.01 M to at least about 0. 15 M salt for washing at 42°C;

(ii) 1 % BSA, 1 mM EDTA, 0.5 M NaHP0₄ (pH 7.2). 7% SDS for hybridization at 65°C, and (a) 0. 1 x SSC, 0. 1 % SDS; or (b) 0.5% BSA, 1 m EDTA, 40 mM NaHP0₄ (pH 7.2), 1 % SDS for washing at a temperature in excess of 65°C for about one hour; and

(iii) 0.2 x SSC, 0.1 % SDS for washing at or above 68°C for about 20 minutes. In general, washing is carried out at T_m = 69.3 + 0.41 (G + C) % - 12°C. In general, the T_m of a duplex D A decreases by about 1 °C with every increase of ,1 % in the number of mismatched bases.

^"Notwithstanding the above, stringent conditions are well known in the art, such as described in Chapters 2.9 and 2. 10 of CURRENT PROTOCOLS ΓΝ MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995- 1999), and in particular at pages 2.9.1 through 2.9.20.

The invention also contemplates antibodies against a synthetic chimeric protein of the invention, inclusive of chimeric proteins, or fragments, variants and/or derivatives thereof. Antibodies of the invention may be polyclonal or monoclonal. Well-known protocols applicable to antibody production, purification and use may be found, for example, in Chapter 2 of Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons NY, 1991 - 1994) and Harlow, E. & Lane, D. Antibodies: A Laboratory Manual. Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988.

Generally, antibodies of the invention bind to or conjugate with a polypeptide, fragment, variant or derivative of the invention. For example, the antibodies may comprise polyclonal antibodies. Such antibodies may be prepared for example by injecting a polypeptide, fragment, variant or derivative of the invention into a production species, which may include mice or rabbits, to obtain polyclonal antisera. Methods of producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols which may be used are described for example in Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons NY, 1991 - 1994), and in Harlow, E. & Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988.

In lieu of the polyclonal antisera obtained in the production species, monoclonal antibodies may be produced using the standard method as for example, described by ohler & Milstein ( 1975, Nature 256:495-97), or by more recent modifications thereof as, for example, described in Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY (John Wi ley & Sons NY, 1991 - 1994) by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with one or more of the polypeptides, fragments, variants or derivatives of the invention.

The invention also includes within its scope antibodies which comprise Fc or Fab fragments of the polyclonal or monoclonal antibodies referred to above. Alternatively, the antibodies may comprise single chain Fv antibodies (scFvs) against the proteins of the invention. Such scFvs may be prepared, for example, in accordance with the methods described respectively in U.S. Patent 5,091 ,5 13, European Patent 239,400 or the article by Winter & Milstein ( 1991 , Nature 349:293- 99).

Labels may be associated with the antibody or antibody fragment.

The label may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorophore, a chemi!uminescent molecule, a lanthanide ion such as

Europium (Eu³⁴), a radioisotope, and a direct visual label. In the case of a direct visual label, use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.

A large number of enzymes useful as labels are disclosed in U.S. Patent

4,366,2 1 , U.S. Patent 4,843,000 and U.S. Patent 4,849,338. Enzyme labels useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, b-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like. The enzyme label may be used alone or in combination with a second enzyme in solution. ■

By way of example, the fluorophore may be fluorescein isothiocyanate

(FITC), Oregon green, tetramethylrhodamine isothiocyanate (TRITL), allophycocyanin (APC) and R-Phycoerythrin (RPE), although without limitation thereto.

The invention also provides pharmaceutical compositions that comprise an isolated protein complex of the invention, inclusive of variants and derivatives thereof.

Such isolated protein complex may Be in any form, inclusive of synthetic chimeric proteins of the invention, although without limitation thereto. Pharmaceutical compositions Of the invention may be used to promote or otherwise facilitate cell migration, tissue regeneration and wound healing. Alternatively, pharmaceutical compositions may be administered to prevent tumour metastasis by preventing or inhibiting tumour cell migration to a secondary site.

The composition may be used in therapeutic or prophylactic treatments as required. For example, pharmaceutical compositions may be applied in the form of therapeutic or cosmetic preparations for skin repair, wound healing, healing of burns and other dermatological treatments.

In this regard, pharmaceutical compositions may be administered in association with, or as a component of, a biomaterial, biopolymer, inorganic material such as hydroxyapatite or derivates thereof, surgical implant, prosthesis, wound or bum dressing, compress, bandage, or the like suitably impregnated, coated or otherwise comprising the pharmaceutical composition.

• Suitably, the pharmaceutical composition comprises an appropriate pharmaceutically-acceptable carrier, diluent or excipient.

Preferably, the pharmaceutically-acceptable carrier, diluent or excipient is suitable for administration to mammals, and more preferably, to humans.

By "pharmaceutically-acceptable carrier, diluent or excipient" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium .sulfate, vegetable oils, synthetic oils, polyols, algihic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water.

A useful reference describing pharmaceutically acceptable carriers, diluents and exeipienls is Remington's Pharmaceutical Sciences (Mack Publ ishing Co. N.J. USA, 1991 ).

Any safe route of administration may be employed for providing a patient with the composition of the invention. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal, and the like may be employed. .

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches, and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids, and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may-be effected by using other polymer matrices, liposomes and/or microspheres.

The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically-effective. The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial response in a patient over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated, inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.

With regard to pharmaceutical compositions for wound healing, particular reference is made to U.S. Patent 5,936,064 and International Publication WO 99/62536.

Pharmaceutical compositions of the invention may also include expression vectors such as viral vectors such as vaccinia, and viral vectors useful in gene therapy. The fatter include adenovirus and adenovirus-associated viruses (AAV) such as described in Braun-Falco et al. ( 1999, Gene Ther. 6:432-41 ), retroviral and lentiviral vectors such as described in Buchshacher et al. (2000, Blood 95:2499-504) and vectors derived from herpes simplex virus and cytomegalovirus. A general overview of viral vectors useful in endocrine gene therapy is provided in Stone et al. (2000, J. Endocrinol. 164: 103- 18).

The present invention may also utilize specific expression vectors which target gene expression to epidermal cells, such as described in U.S. Patent 5,958,764 and for in vivo wound healing applications, such as described in U.S. Patent 5,962,427.

The invention provides methods of treatment using isolated protein complexes, inclusive of synthetic chimeric proteins of the invention. These methods are particularly aimed at therapeutic and/or prophylactic treatment of mammals, and more particularly, humans.

However, therapeutic uses according to the invention may also be applicable to mammals such as domestic and companion animals, performance animals such as horses, camels and greyhounds, livestock, laboratory animals and animals used as sources of cells, organs and tissues for xenotransplantation.

The invention also contemplates methods of cosmetic treatment where isolated protein complexes, inclusive of synthetic chimeric proteins of the invention, are administered to improve or enhance skin quality or skin appearance.

Such treatments may include prevention or remediation of skin disorders such as psoriasis and hypertrophic scarring that result from aberrant skin cell proliferation.

Alternatively, methods of treatment are contemplated whereby tumour metastasis is prevented or inhibited by blocking tumour cell migration to a secondary- site. In addition, methods of treating cancer by blocking cell proliferation also contemplated.

In particular embodiments, therapeutic and/or prophylactic treatments may utilize an isolated protein complex, inclusive of synthetic chimeric proteins of the invention, in association with, or as a component of, a biomaterial, biopolymer, inorganic material such as fluorohydroxyapatite, surgical implant, prosthesis, wound or burn dressing, compress, bandage, or the like suitably impregnated, coated or· otherwise comprising the isolated protein complex.

Such methods include administration of pharmaceutical compositions as hereinbefore defined^ and may be by way of microneedle injection into specific tissue sites, such as described in U.S. Patent 6,090,790, topical creams, lotions or sealant dressings applied to wounds, burns or ulcers, such as described in U.S. Patent 6,054, 122 or implants which release the composition such as described in International Publication WO 99/47070. Gene therapy is also applicable in this regard, such as according to methods set forth i n U.S. Patent 5,929,040 and U.S. Patent 5,962,427.

There also exist methods by which skin cells can be genetically modified for the purpose of creating skin substitutes, such as by genetically engineering desired growth factor expression (Supp et al , 2000, J. Invest. Dermatol. 1 14:5- 13). An example of a review of this field is provided in Bevan et al. ( 1999, Biotechnol. Gent. Eng. Rev. 16:231 -56).

Also contemplated is "seeding" a recipient with transfected or transformed cells, such as described in International Publication WO 99/ 1 1 789.

These methods can be used to stimulate cell migration and thereby facilitate or progress wound and burn healing, repair of skin lesions such as ulcers, tissue replacement and grafting such as by in vitro culluring of autologous skin, re- epithelialization of internal organs such as kidney and lung and repair of damaged nerve tissue.

Skin replacement therapy has become well known in the art, and may employ use of co-cultured epithelial/keratinocyte cell lines, for example as described in ehe el al. ( 1 99. Arch. Dermatol. Res. 291 :600-05) or in vitro culture of primary (usually autologous) epidermal, dermal and/or keratinocyte cells. These techniques may also utilize engineered biomaterials and synthetic polymer "scaffolds".

Examples of reviews of the field in general are provided in Terskikh &

Vasiliev ( 1999, Int. Rev. Cytol. 1 88:41 -72) and Eaglestein & Falanga ( 1998, Cutis 62: 1 -8).

More particularly, the production , of replacement oral mucosa useful in craniofacial surgery is described in Izumi -et al. (2000, J. Dent. Res. 79:798-805). Fetal keratinocytes and dermal fibroblasts can be expanded in vitro to produce skin for grafting to treat skin lesions, such as described in Fauza et al. ( 1 98, J. Pediatr. Surg. 33:357-61 ), while skin substitutes from dermal and epidermal skin elements cultured in vitro on hyaluronic acid-derived biomaterials have been shown to be potentially useful in the treatment of burns (Zacchi el al , 1998, J. Biomed. Mater. Res. 40: 1 87-94).

Polymer scaffolds are also contemplated for the purpose of facilitating replacement skin engineering, as for example described in Sheridan et al. (2000, .). Control Release 64:91 - 102) and Fauza el al. (1 998, J. Pediatr. Surg. 33:357-61 ), as are microspheres as . agents for the delivery of skin cells to wounds and burns (LaFrance & Armstrong, 1999, Tissue Eng. 5: 153-70).

The invention contemplates use of isolated protein complexes, inclusive of synthetic chimeric proteins of the invention, to identify, screen, design or otherwise produce agonists or antagonists of complexes comprising keratinocyte growth factor and vitronectin. Such agents may be a "mimetic". The term "mimetic " is used herein to refer to molecules that are designed to resemble particular functional regions of proteins or peptides, and includes within its scope the terms "agonist", "analogue" and "antagonist" as are well understood in the art.

In one embodiment, agonists are produced that mimic the binding of the keratinocyte. growth factor receptors and VN receptors by KGF:VN complexes. Such molecules may have utility as stimulators of cell migration such as required for wound healing, skin regeneration and the like.

In another embodiment, antagonists are produced that prevent or inhibit the binding of the keratinocyte growth factor receptors and integrin receptors by GF:VM complexes. Such molecules have utility as inhibitors of cell migration and/or cell proliferation and thereby constitute useful anti-tumour agents and also in treatments of skin disorders such as psoriasis and hypertrophic scarring that result from aberrant cell proliferation.

The aforementioned mimetics, agonists, antagonists and analogues may be peptides, polypeptides or other organic molecules, preferably small organic molecules, with a desired biological activity and half-li fe.

Computer-assisted structural database searching is becoming increasingly utilized as a procedure for identifying mimetics. Database searching methods which, in principle, may be suitable for identifying mimetics, may be found in International Publication WO 94/1 82.32 (directed to producing H IV antigen mimetics), U.S. Patent 5,752,019 and International Publication WO 97/41526 (directed to identifying EPO mimetics).

Other methods include a variety of biophysical techniques which identify molecular interactions. These allow for the screening of candidate molecules according to whether said candidate molecule affects formation of KGF:V1M complexes, for example. Methods applicable to potentially useful techniques such as competitive radioligand binding assays (see, Upton el ai, 1999, Endocrinology 140:2928-3 1 for a relevant method), analytical ultracentrifugation, microcalorimetrys surface plasmon resonance, and optical biosensor-based methods are provided in Chapter 20 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coiigan et al , (John Wiley & Sons, 1997).

So that the present invention may be more readily understood and put into practical effect, the skilled person is referred.to the following non-limiting examples.

EXAMPLES

EXAMPLE 1

KGF: VN chimeras stimulate cell migration and proliferation

Isolated human keratinocyte and fibroblast cells (P I and P3 respectively) were applied to the inner chamber of a seeding ring in culture wells pre-treated with varying doses of a VN:KGF chimera, and controls. After a 4-hour period of attachment, the seeding ring was removed and cells were allowed to migrate outward in response to the pre-bound treatments over a period of 24 hours for keratinocytes and 48 hours for fibroblasts. Cell assay data were pooled from at least 3 separate experiments, each with triplicate independent tests with results expressed as percentage above SG/SFM (negative control) and shown in FIG. 4A and 4B. Errorbars indicate SEM. SG = Stripped Greens media, SFM = Serum-free media (both are negative controls). With reference to experiments utilizing isolated human skin keratinocytes, the VN-.KGF chimera demonstrated functional equivalence to equimolar combinations of individual components (VN+ GF), indicating proper protein expression, purification and processing. Experiments testing the response of isolated skin fibroblasts indicated that the VN:KGF chimera induced cell migration significantly (p = <0.05) above equimolar combinations of individual components (VN+KGF).

In order to assess the proliferation inducing potential of the VN:KGF chimera, isolated human keratinocyte and fibroblast cells (PI and P3. respectively) were seeded into wells pre-treated with varying doses of VN .KGF chimera, and controls at a density of 15,000 cells/cm². The cells were allowed to proliferate over 72 and 48 hours for keratinocytes and fibroblasts respectively after which the culture media was removed and the culture plates snap frozen at -80°C . Upon thawing of the plates, a mixture of cell lysis and GR-dye (Invitrogen, CYQUANT kit) was added to each well and incubated at room temperature for 5 minutes. The plates were then interrogated for fluorescence by excitation at 485 nm and absorbance was read at 520 nm. Cell assay data were pooled from at least 3 separate experiments; each with triplicate independent tests with results expressed as percentage above SG/SFM (negative control) and shown in FIG. 4C and 4D. Error bars indicate SEM. SG = Stripped Greens media. SFM = Serum-free media (both are negative controls). Experiments testing proliferation in isolated human skin keratinocytes demonstrated that the VN:KGF chimera functioned in equivalence to equimolar combinations of individual components (VN+KGF). Proliferation experiments utilising isolated skin fibroblasts indicated that the VN:KGF chimera (1 50 nM) induced cell proliferation significantly (p = O.05) above equimolar combinations of individual components and tetrameric VN:IGFBP-3:IGF-i :EGF complex.

EXAMPLE 2

VN: KGF signaling

In order to assess the impact of VN:KGF chimeric proteins on ERK 1 /2 and A T signaling pathways, the CELISA (Millipore) kit was used. Briefly, 20,000 primary keratinocytes or 10,000 primary fibroblasts were seeded into the wells of a 96 well black-bottomed fluorescence plate and allowed to grow overnight at 37 °C. The cells were then washed 2x with serum free media (SFM) and incubated in SFM overnight to serum starve the cells. After approximately 16 hours, serum starvation media was replaced with 100 μί of protein treatments as follows: VN only ( 1 5 nM, equal to 1 125 ng/mL), VN ( 15 nM, equal to 1 125 ng/mL) and KGF (50 nM, equal to 820.4 ng/mL) and VN:KGF (50 nM, equal to 13 18.9 ng/mL). Cells were exposed to protein treatments for time points of 10 minutes, 30 minutes and 60 minutes, after which treatment solutions were replaced with 4% formaldehyde in TBS to fix the cells. Wells were then interrogated for levels of activated (phosphorylated) ERK 1 /2 and AKT as a proportion of total ERK 1 /2 and AKT respectively using antibody- based (ELISA) methods following the manufacturers instructions. Chimeric treatments promote similar levels of relative activation and demonstrate similar trends over time as respective controls (FIG. 5).

EXAMPLE 3

Synthetic chimeric vitronectin .keratinocyte growth factor proteins

Provided herein are examples of synthetic chimeric proteins of the invention, in the form of VN:KGF chimeras.

The synthetic chimeric proteins include any full-length or truncated forms of VN fused with keratinocyte growth factor, with or without amino acid residue modifications. In addition, VN and the keratinocyte growth factor may be fused with or without the various peptide linkers.

A series of chimeric expression constructs are designed in which various lengths of the VN protein are linked to the full-length mature GF protein, or at least a domain of the KGF protein capable of binding a keratinocyte growth factor receptor. In each case, the VIM segments are preferably linked to the KGF sequence via a linker, for example, a Gly₄ Ser (SEQ ID NO:4) linker, a Gly Ser3 (SEQ ID NO:5) linker, a (Gly₄ Ser)₃ (SEQ ID NO:6) linker, or a (Gly₄ Ser)₄ (SEQ ID NO:7) linker.

Exemplary synthetic chimeric proteins include, but are not limited to:

A) l -459VN:[Linker; e.g. , (G₄S)₄]: 1 - 163KGF: [Linker; e.g. , G₄SG₄]:6H

B) 1 -31 l VN:[Linker; e.g. , (G₄S)₄]: l - 163KGF:[Linker; e.g., G₄SG₄]:6H

C) l - 125VN:[Linker; e.g. , (G₄S)₄]: l - 163KGF:[Linker; c^., G₄SG₄] :6H

D) l -64VN:[Linker; e.g., (G₄S)₄]: l - 163KGF:[Linker; e.g. , G₄SG₄] :6H

E) 1 -64V -.[Linker; e.g., (G₄S)₄]:343-376VN:[Linker; e.g., (G₄S)₄]: 1 - 163KGF:[Linker; e.g. , G₄SG₄]:6H

Throughout the specification the -aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All computer programs, algorithms, patent and scienti fic literature referred to herein are incorporated herein by reference.

Claims

1. An isolated protein complex in the form of a synthetic chimeric protein, comprising an amino acid sequence of:

(i) keratinocyte growth factor (KGF), or at least a domain of GF which is capable of binding a keratinocyte growth factor receptor; and

(ii) one or more domains of vitronectin (VN) comprising at least an integrin-binding domain of VN.

2. The isolated protein complex of Claim 1 , wherein the one or more domains of VN comprising at least an integrin-binding domain of VN do not include a C- terminal heparin-binding domain (HBD).

3. The isolated protein complex of Claim 2, comprising amino acids 1 -346 of a mature VN sequence (SEQ ID NO:2)-.

4. The isolated protein complex of Claims 1 -3, wherein the integrin-binding domain is an a_v integrin-binding domain.

5. The isolated protein complex of Claim 4, wherein the integrin-binding domain is an α_νβ3 integrin-binding domain or an _νβ₅ integrin-binding domain.

6. The isolated protein complex of Claim I , wherein the one or more domains of VN comprising at least an integrin-binding domain of VN comprise amino acids 1 - 31 1 of a mature VN sequence (SEQ ID NO:2).

7. The isolated protein complex of Claim 1 , wherein the one or more domains of VN comprising at least an integrin-binding domain of VN comprise amino acids 1 - 1 25 of a mature VN sequence (SEQ ID NO:2).

8. The isolated protein complex of Claim 1 , wherein the one or more domains of VN comprising at least an integrin-binding domain of VN comprise amino acids 1 -64 of a mature VN sequence (SEQ ID NO:2).

9. The isolated protein complex of Claim 1 , wherein the one or more domains of VN comprising at least an integrin-binding domain of VN comprise amino acids 1 -52 of a mature VN sequence (SEQ ID NO:2).

10. The isolated protein complex of Claim 9, wherein the one or more domains of VN comprising at least an integri.n_:binding domain of VN do not comprise a poiyanionic amino acid sequence corresponding to residues 53-64 of a mature VN protein (SEQ ID NO:2).

11. The isolated protein complex of any one of the preceding claims, further comprising an additional fragment of VN.

12. The isolated protein complex of any one of the preceding claims, further comprising at least one linker sequence.

13. The isolated protein complex of Claim 12, wherein the linker sequence comprises a protease cleavage site.

14. The isolated protein complex of Claim 12, wherein the linker sequence is selected from the group consisting of: <

(i) Gly₄ Ser (SEQ ID NO:4);

(ii) Gly₄Ser₃(SEQIDNO:5);

(iii) (Gly₄ Ser)₃ (SEQ ID NO:6);

(iv) (Gly Ser)₄(SEQlDNO:7);

(v) Leu lie Lys Met Lys Pro (SEQ ID NO:8); and

(vi) Gin Pro Gin Gly Leu Ala Lys (SEQ ID NO:9).

15. The isolated protein complex of Claim 1, wherein said synthetic chimeric protein comprises an amino acid sequence of 1-459 VN:(Gly₄ Ser)₄: 1 -163 KGF:Gly₄ Ser Gly₄:6 His (SEQ ID NO: 10), 1-311 VN:(Gly₄ Ser)₄: 1-163 .GF:Gly₄ Ser GIy₄:6 His (SEQ ID NO:l I), 1-125 VN:(Gly₄ Ser)₄:l-163 GF:Gly Ser Gly₄:6 His (SEQ ID NO: 12), 1-64 VN:(Gly₄ Ser)₄:l-163 KGF:Gly₄ Ser Gly₄:6 His (SEQ ID NO: 13), or 1-64 VN:(Gly₄ Ser)₄:343-376 VN:(Gly₄ Ser)₄: 1-163 KGF:Gly Ser Gly₄:6 His (SEQ ID NO: 14).

16. An isolated nucleic acid encoding the isolated protein complex of any one of the preceding claims.

17. A genetic construct, comprising the isolated nucleic acid of Claim 16 operably linked to one or more regulatory nucleotide sequences in a vector.

18. The genetic construct of Claim 17, which is an expression construct, wherein the isolated nucleic acid is operably linked to a promoter.

19. A host cell, comprising the genetic construct of Claim 17.

20. A pharmaceutical composition, comprising the isolated protein complex of any one of Claims 1-15 and a pharmaceutically-acceptable carrier, diluent or excipient.

21 . A surgical implant, scaffold or prosthesis impregnated, coated or otherwise comprising the isolated protein complex of any one of Claims 1 - 15.

22. A wound or burn dressing, comprising the isolated protein complex of any one of Claims 1 - 15.

23. A method of promoting cell migration and/or proliferation, including the step of using the isolated protein complex of any one of Claims 1 - 1 5 to bind both a keratinocvte growth factor receptor and an integrin receptor expressed by a cell to thereby induce, augment or otherwise promote migration and/or proliferation of said cell.

24. The method of Claim 23, wherein the isolated protein complex is administered to an animal to promote cell migration and/or proliferation in situ.

25. The method of Claim 23, for prophylactically or therapeutically to induce, augment or otherwise promote epithelial cell migration and/or proliferation to thereby facilitate wound healing in situ.

26. The method of Claim 24 of Claim 25, wherein the animal is a human.

27. The method of Claim 23, wherein the isolated protein complex is administered to one or more cells or tissues in vitro.