Synthetic chimeric proteins comprising epidermal growth factor and vitronectin
Isolated protein complexes are provided comprising growth factors such as IGF-I, IGF-II, EGF, bFGF, KGF, VEGF or PDGF, or at least domains thereof that enable binding to and activation of both a growth factor receptor, and an integrin receptor-binding domain of vitronectin or fibronectin. These protein complexes may be in the form of oligo-protein complexes or single, synthetic proteins where the growth factor and vitronectin or fibronectin sequences are joined by a linker sequence. In particular forms, vitronectin or fibronectin sequences do not include a heparin binding domain and/or polyanionic domain. Also provided are uses of these protein complexes for stimulating or inducing cell migration and/or proliferation which may have use in wound healing, tissue engineering, cosmetic and therapeutic treatments such as skin replacement and 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.
1. An isolated protein complex in the form of a synthetic chimeric protein, comprising an amino add sequence of:
(i) a growth factor selected from epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and keratinocyte growth factor (KGF), or at least a domain of said growth factor which is capable of binding a cognate growth factor receptor; and
(ii) vitronectin (VN) or a fragment of VN comprising at least an integrin-binding domain, wherein said at least an integrin-binding domain comprises the sequence RGD and facilitates cell attachment.
2. The isolated protein complex of claim 1 , wherein said VN or said fragment of VN comprising at least an integrin-binding domain does not comprise a heparin-binding domain (HBD).
3. The isolated protein complex of claim 1 , wherein the integrin-binding domain is an α v integrin-binding domain.
4. The isolated protein complex of claim 3 , wherein the integrin-binding domain is an α v β 3 integrin-binding domain or an α v β 5 integrin-binding domain.
5. The isolated protein complex of claim 1 , wherein said VN or said fragment of VN comprising at least an integrin-binding domain of VN does not comprise a polyanionic amino acid sequence corresponding to residues 53-64 of a mature VN protein (SEQ ID NO:2).
6. The isolated protein complex of claim 1 , comprising amino acids 1-459 of a mature VN sequence (SEQ ID NO:2).
7. The isolated protein complex of claim 1 , wherein the fragment of VN comprises amino acids 1-311 of a mature VN sequence (SEQ ID NO:2).
8. The isolated protein complex of claim 1 , wherein the fragment of VN comprises amino acids 1-125 of a mature VN sequence (SEQ ID NO:2).
9. The isolated protein complex of claim 1 , wherein the fragment of VN comprises amino acids 1-64 of a mature VN sequence (SEQ ID NO:2).
10. The isolated protein complex of claim 1 , wherein the fragment of VN comprises amino acids 1-52 of a mature VN sequence (SEQ ID NO:2).
11. The isolated protein complex of claim 1 , which does not comprise an IGFBP amino acid sequence.
12. The isolated protein complex of claim 1 , 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)
Gly 4 Ser;
(SEQ ID NO: 4)
(ii)
Gly 4 Ser 3 ;
(SEQ ID NO: 5)
(iii)
(Gly 4 Ser) 3 ;
(SEQ ID NO: 6)
(iv)
(Gly 4 Ser) 4 ;
(SEQ ID NO: 26)
(v)
Leu Ile Lys Met Lys Pro;
(SEQ ID NO: 7)
and
(vi)
Gln Pro Gln Gly Leu Ala Lys.
(SEQ ID NO: 8)
15. The isolated protein complex of claim 1 , wherein said synthetic chimeric protein comprises an amino acid sequence selected from the group consisting of:
(i) 1-64 VN:(Gly 4 Ser) 4 :1-53 EGF:Gly 4 Ser Gly 4 :6 His (SEQ ID NO:27);
(ii) 1-64 VN:(Gly 4 Ser) 4 :1-146 bFGF:Gly 4 Ser Gly 4 :6 His (SEQ ID NO:28); and
(iii) 1-64 VN:(Gly 4 Ser) 4 :1-163 KGF:Gly 4 Ser Gly 4 :6 His (SEQ ID NO:29).
16. An isolated nucleic acid encoding the isolated protein complex of claim 1 .
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. An isolated host cell, comprising the genetic construct of claim 17 .
20. A pharmaceutical composition, comprising the isolated protein complex of claim 1 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 claim 1 .
22. A wound or burn dressing, comprising the isolated protein complex of claim 1 .
23. A method of treating dermatological wounds, including the step of using the isolated protein complex of claim 1 to bind both a growth factor receptor and an integrin receptor expressed by a cell to thereby induce, augment or otherwise promote migration and/or proliferation of an epithelial 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 24 , wherein the animal is a human.
26. The method of claim 23 , wherein the isolated protein complex is administered to one or more cells or tissues in vitro.