Treatment of ocular conditions

Cross-reference to related application

This application claims priority from Australian provisional application number 2017902737, the entire contents of which are hereby incorporated in their entirety.

Field of the invention

The present invention relates to methods, compositions and kits for the treatment of ocular conditions. In particular, the methods, compositions and kits are particularly useful for, but not limited to, the treatment of ocular conditions by promoting proliferation and/or migration of corneal epithelial cells.

Background of the invention

The cornea serves a dual function: first, as a key tissue through which light is refracted and transmitted for vision, and second, as a protective barrier to the external environment. The very superficial layer of the cornea consists of a multilayered squamous epithelium and its integrity and transparency is critical for sight. A compromised ocular surface renders the cornea liable to vision-threatening infection, neovascularization, scarring, and melting, with the risk of perforation and loss of the eye.

Resurfacing of the corneal epithelium after a corneal wound forms occurs in three phases: migration of adjacent intact epithelial cells to cover the injured zone, proliferation of the migrated monolayer to reestablish epithelial thickness, and differentiation of the reconstruct epithelium to restore structure and function. The initial stage is characterized by cells attaching and spreading over the denuded substratum which typically occurs by epithelial "sliding." An important aspect of this process is cell attachment to the extracellular matrix (ECM). Most corneal epithelial wounds are promptly repaired via the above-mentioned endogenous processes. However, in some individuals healing is delayed, wounds persevere, and a condition collectively known as persistent epithelial defects (PEDs) can develop. PEDs are characterized by ulcers with delayed (>2 weeks) healing and can recur once healed, particularly when the basement membrane (BM) is lost. They arise from a variety of insults, including trauma, tear-film disorders, neoplasia and its treatment, corneal graft, surgery, chemical and thermal burns, or in neurotrophic, diabetic, and herpetic keratopathies, and can also accompany immunological disorders.

Current therapies for PEDs are often unavailable for routine clinical use. Current therapies are largely based on application of topical antibiotics and nonpreserved tear- substitutes. However, these agents are readily cleared from the ocular surface and require strict compliance to frequent application by the patient for success. Further, "active" therapies for PEDs such as human amniotic membrane (HAM) grafts have antiinflammatory, antiangiogenic, and anti-scarring properties, however, this is a foreign biomaterial that can integrate and remain opaque if not adequately remodeled, or may spontaneously dislodge from the cornea.

There exists a need for new and/or improved methods and compositions for treating corneal wounds.

Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

Summary of the invention In one aspect, the present invention provides a method for treating or preventing a corneal wound in an individual in need thereof, the method comprising administering:

(i) a first peptide capable of binding to and activating a growth factor receptor; and

(ii) a second peptide capable of binding to an integrin, thereby treating a corneal wound in the individual.

Preferably, the first peptide comprises, consists essentially of or consists of an amino acid sequence of a growth factor, or at least a domain of a growth factor, that is capable of binding to and activating its cognate growth factor receptor. More preferably, the growth factor receptor is the insulin like growth factor I receptor (IGF-IR) or epidermal growth factor receptor (EGFR).

In one embodiment, the first peptide comprises, consists essentially of or consists of an amino acid sequence of a full length growth factor. Preferably, the first peptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 22 or 24.

Preferably, the second peptide comprises an integrin binding sequence such as RGD. Typically, the second peptide comprises, consists essentially of or consists of an amino acid sequence of an integrin-binding domain of vitronectin (VN) or fibronectin (FN). The integrin-binding domain of VN may have an amino acid sequence of SEQ ID NO: 23. In one embodiment, the peptide does not comprise an amino acid sequence of the heparin binding domain of VN. In one embodiment, the second peptide comprises, consists essentially of or consists of an amino acid sequence of a full length VN or FN.

In another aspect, the present invention provides a method for increasing the rate of corneal wound healing in an individual, the method comprising administering: (iii) a first peptide capable of binding to and activating a growth factor receptor; and

(iv) a second peptide capable of binding to an integrin, thereby increasing the rate of corneal wound healing in the individual.

In another aspect, the present invention provides a method for treating or preventing a corneal wound in an individual in need thereof, the method comprising administering:

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

(ii) vitronectin (VN) or fibronectin (FN), or at least an integrin-binding domain of VN or FN, thereby treating a corneal wound in the individual.

In any aspect of the invention, the corneal wound is a corneal epithelial wound.

In any aspect of the present invention the growth factor is selected from insulin like growth factor (IGF)-I or epidermal growth factor (EGF).

In any method of the present invention, the method further comprises administering a further peptide capable of binding to and activating a growth factor receptor that is different from the growth factor receptor that the first peptide binds to and activates. In one embodiment, the first peptide and further peptide do not activate the same growth factor receptor. For example, the first peptide may be capable of binding to and activating an IGF-I receptor, and the further peptide may be capable of binding to and activating an EGF receptor.

In addition, in any method of the invention, the method comprises administering two or more growth factors, or domains of two or more growth factors which are capable of binding to their respective cognate growth factor receptors. Preferably, the growth factors are IGF-I and EGF. Typically, the method comprises administering IGF-I and EGF.

In any method of the present invention: (i) a first peptide capable of binding to and activating a growth factor receptor; and

(ii) a second peptide capable of binding to an integrin, are administered simultaneously or sequentially.

In any method of the present invention: (i) a first peptide capable of binding to and activating a growth factor receptor; and

(ii) a second peptide capable of binding to an integrin, are covalently linked. Preferably the covalent linker comprises, consists essentially of or consists of amino acids. More preferably, the amino acids are selected from glycine and serine. In any embodiment of the invention, the covalent linker may be chosen from SEQ ID NO: 25, 26, 27, 28, 29, 30, 31 or 32. In one embodiment, the first peptide and second peptide are covalently linked and comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 18, 19 or 20.

In any method of the present invention, when two or more peptides or growth factors, or domains of two or more growth factors which are capable of binding and activating their respective cognate growth factor receptors, are present they may be covalently linked. Alternatively, only one peptide, growth factor, or at least a domain of a growth factor which is capable of binding to and activating a cognate growth factor receptor, may be covalently linked to a peptide capable of binding to an integrin or at least an integrin-binding domain of VN or FN, and any further growth factor is administered simultaneously or sequentially.

In any method of the present invention, the corneal wound may result from surgical and non-surgical trauma or abrasion, tear-film disorders, severe dry eye, neoplasia and its treatment, corneal graft, diabetic keratopathy, neuropathic keratopathy, thermal or chemical burns, herpetic epithelial keratitis, injury caused by ocular anti-viral or -microbial agents, or immunological disorders. The corneal wound may be a persistent epithelial defect (PED).

The present invention provides use of: (i) a first peptide capable of binding to and activating a growth factor receptor; and

(ii) a second peptide capable of binding to an integrin, in the manufacture of a medicament for the treatment or prevention of a corneal wound in an individual in need thereof. The present invention also provides a pharmaceutical composition comprising:

(i) a first peptide capable of binding to and activating a growth factor receptor; and

(ii) a second peptide capable of binding to an integrin, and

(iii) a pharmaceutically acceptable carrier, excipient or diluent. Typically, the pharmaceutical composition does not contain IGF binding protein 3

(IGFBP-3). In other words, the composition is IGFBP-3 free. Further, in some embodiments the pharmaceutical composition does not contain any IGF binding proteins (IGFBPs). In other words, the composition is IGFBP free.

In any method or use of the invention, an IGFBP-3 is not administered to the individual. Preferably, no IGF binding proteins are administered to the individual.

In another aspect, the present invention provides a chimeric or fusion protein comprising a first peptide joined directly or through a linker to a second peptide, wherein the first peptide is capable of binding to and activating an EGF receptor, and the second peptide is capable of binding to an integrin.

In another aspect, the present invention provides a chimeric or fusion protein comprising a first peptide joined directly or through a linker to a second peptide, wherein the first peptide comprises part of, or all of an amino acid sequence that is the same as, or homologous to an epidermal growth factor (EGF), and the second peptide comprises part of, or all of an amino acid sequence that is the same as, or homologous to the sequence of an vitronectin (VN) or fibronectin (FN).

In this aspect, the first peptide may comprise, consist essentially of or consist of an amino acid sequence of a receptor binding domain of EGF. Typically, the EGF is human EGF. In this aspect, the second peptide may comprise, consist essentially of or consist of an amino acid sequence of an integrin-binding domain of VN or FN. Typically, the VN or FN is human VN or FN.

In this aspect, the chimeric or fusion protein may comprise a secretion signal peptide of VN and amino acids 1 to 64 of VN. More preferably, the chimeric or fusion protein comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 19.

In another aspect, the present invention provides a chimeric or fusion protein comprising a first peptide, second peptide and third peptide joined directly or through a linker, wherein the first peptide comprises part of, or all of an amino acid sequence that is the same as, or homologous to an epidermal growth factor (EGF), the second peptide comprises part of, or all of an amino acid sequence that is the same as, or homologous to the sequence of an vitronectin (VN) or fibronectin (FN), and the third peptide comprises part of, or all of an amino acid sequence that is the same as, or homologous to an IGF.

The IGF may be either IGF-I or IGF-I I. Preferably, the third peptide comprises, consists essentially of or consists of an amino acid sequence of an IGF-I receptor binding domain of either IGF-I or IGF-I I. Typically, the IGF is human IGF.

In this aspect, the chimeric or fusion protein comprises the secretion signal peptide of VN and amino acids 1 to 64 of VN. Preferably, the chimeric or fusion protein comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 20.

In any aspect, within the chimeric or fusion protein the first peptide and second peptide may be arranged such that the first peptide is N-terminal to the second peptide. In one embodiment, the peptides are arranged from N to C-terminal: first peptide and second peptide. In any aspect, within the chimeric or fusion protein the first, second and third peptides may be arranged such that the first peptide is N-terminal to the second peptide, and the second peptide is N-terminal to the third peptide. In one embodiment, the peptides are arranged from N to C-terminal: first peptide, second peptide and third peptide. In yet another aspect, the invention provides a composition comprising a chimeric or fusion protein as broadly described above, optionally a pharmaceutically acceptable carrier, excipient or diluent.

In this aspect, the invention also provides a method of treating or preventing a corneal wound in an individual in need thereof, the method comprising administering to the individual a chimeric or fusion protein as described above, or a composition as described above, thereby treating or preventing a corneal wound in the individual.

In this aspect, the invention further provides the use of a chimeric or fusion protein as described above, or a composition as described above, in the manufacture of a medicament for treating or preventing a corneal wound in an individual in need thereof.

In yet another aspect, the invention also provides a nucleic acid molecule comprising, consisting essentially of or consisting of a nucleotide sequence encoding a chimeric or fusion protein as described above, optionally operatively linked to at least one regulatory element.

In one embodiment, the nucleic acid molecule comprises, consists essentially of or consists of a nucleotide sequence encoding a chimeric or fusion protein comprising, consisting essentially of or consisting of amino acid sequences shown in SEQ ID NO: 22 and 23, optionally further comprising or consisting of amino acid sequences shown in SEQ ID NO: 21 and any one or more of SEQ ID NOs: 25 to 33. In another embodiment, the nucleic acid molecule comprises, consists essentially of or consists of a nucleotide sequence encoding a chimeric or fusion protein comprising, consisting essentially of or consisting of amino acid sequences shown in SEQ ID NO: 22, 23 and 24, optionally further comprising or consisting of amino acid sequences shown in SEQ ID NO: 21 and any one or more of SEQ ID NOs: 25 to 33.

In one embodiment, the nucleic acid molecule comprises, consists essentially of or consists of nucleotide sequences shown in SEQ ID NO: 5 and 6, optionally further comprising, consisting essentially of or consisting of nucleotide sequences shown in SEQ ID NO: 4 and any one or more of SEQ ID NOs: 8 to 16. In another embodiment, the nucleic acid molecule comprises or consists essentially of nucleotide sequences shown in SEQ ID NO: 5, 6 and 7, optionally further comprising, consisting essentially of or consisting of nucleotide sequences shown in SEQ ID NO: 4 and any one or more of SEQ ID NOs: 8 to 16.

The nucleotide sequence may comprise SEQ ID NO: 1 , 2 or 3, or any other nucleotide sequence as shown in Table 3A, or encode for VF003 or VF004 as described herein.

In this aspect, the invention further provides a vector including such a nucleic acid molecule, as well as a prokaryotic or eukaryotic cell including such a nucleic acid molecule.

In this aspect, the invention also provides a method of treating a corneal wound in an individual in need thereof, the method comprising administering to the individual a nucleic acid molecule as described above, a vector as described above, or a prokaryotic or eukaryotic cell as described above.

In this aspect, the invention further provides the use of a nucleic acid molecule as described above, a vector as described above, or a prokaryotic or eukaryotic cell as described above, in the manufacture of a medicament for treating a corneal wound in an individual in need thereof.

In any method or use of the invention, the administration of the first and second peptide, or chimeric or fusion protein, does not result in significant ocular phimosis or corneal neovascularisation. In one embodiment, the administration of the first and second peptide, or chimeric or fusion protein, does not result in any clinically observable ocular phimosis or corneal neovascularisation.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings. Brief description of the drawings

Figure 1 : VF001 (VN:IGF-I) purification from Sf9 conditioned media.

Conditioned media from expression cultures was passed onto Q-Sepharose IEX and eluted therefrom using high salt. The subsequent sample was then applied to Ni2+-NTA IMAC resin and eluted with 250 mM Imidazole. Purification samples were interrogated by Silver Stain SDS-PAGE (A) and Western blot (B) using a- VN (red) and a-IGF-l (green) Ab's.

Figure 2. VF003 (VN:EGF) purification from Sf9 conditioned media.

Conditioned media from expression cultures was passed onto Q-Sepharose IEX and eluted therefrom using high salt. The subsequent sample was then applied to Ni2+-NTA IMAC resin and eluted with 250 mM Imidazole. Purification samples were interrogated by Silver Stain SDS-PAGE (A) and Western blot (B) using a- VN (red) and a-EGF (green) Ab's.

Figure 3. VF004 (EGF:VN:IGF-I) purification from Sf9 conditioned media

Conditioned media from expression cultures was passed onto Q-Sepharose IEX and eluted therefrom using high salt. The subsequent sample was then applied to Ni2+-NTA IMAC resin and eluted with 250 mM Imidazole. Purification samples were interrogated by Silver Stain SDS-PAGE (A) and Western blot (B) using a- VN (red) and a-IGF-l (green) Ab's and (C) with a-EGF only (green).

Figure 4. Bovine Serum Albumin (BSA) standard curve for VF001 , VF003 and VF004 quantitation using a BCA assay. BSA standard of 31.25 pg/mL to 2000 Mg/mL were used to generate a standard curve from which test samples were correlated to calculate their protein concentration.

Figure 5. HCE-T cell proliferation (MTS) in response to chimeric and control treatments. Cell proliferation in HCE-T cells was measured after 48 hours in response to chimeric proteins and control treatments by the MTS method. V+l = VN + IGF-I, VF4 = VF4-P03, VN: IGF-I = VF001 , V+E = VN + EGF, VN:EGF = VF003, V+E+l = VN + EGF + IGF-I, EGF:VN: IGF-I = VF004. VF4-P03 was included as a good manufacturing practice (GMP)-grade VF001 reference material that is manufactured using a Pichia pastoris expression system. Data is depicted as the pooled results from three independent experiments with each treatment performed in at least triplicate within each experiment (n=>9). Data were analysed using a One-Way ANOVA with Tukey's post- hoc test and each treatment compared to each other, ns indicates no significant difference while ^* indicates a significant difference to SFM (p<0.05).

Figure 6. HCE-T cell migration (Fence) in response to chimeric and control treatments. Cell migration in HCE-T cells was measured after 48 hours in response to chimeric proteins and control treatments by the fence method. V+l = VN + IGF-I, VF4 = VF4-P03, VN: IGF-I = VF001 , V+E = VN + EGF, VN:EGF = VF003, V+E+l = VN + EGF + IGF-I, EGF:VN: IGF-I = VF004. Data is depicted as the pooled results from three independent experiments with each treatment performed in duplicate within each experiment (n=6). Data were analysed using a One-Way ANOVA with Tukey's post-hoc test and each treatment compared to each other, ns indicates no significant difference, ^* indicates a significant difference to SFM (p<0.05) and # indicates significant difference to the concentration equivalent VN: IGF-I (VF001 ) treatment (p<0.05).

Figure 7. Stained HCE-T cells. Example of image analysis after fixing and staining of migrated HCE-T cells. Figure 8. HCEC cell proliferation (MTS) in response to chimeric and control treatments. Cell proliferation in HCECs was measured after 72 hours in response to chimeric proteins and control treatments by the MTS method. Data is depicted as the pooled results from three independent experiments with each treatment performed in at least triplicate within each experiment (n=>9). Data were analysed using a One-Way ANOVA with Tukey's post-hoc test and each treatment compared to each other. No significant differences were found comparing treatments at equimolar concentrations.

Figure 9. HCEC migration (Fence) in response to chimeric and control treatments. Cell migration in HCECs was measured after 48 hours in response to chimeric proteins and control treatments by the fence method. Data is depicted as the pooled results from three independent experiments with each treatment performed in duplicate within each experiment (n=6). Data were analysed using a One-Way ANOVA with Tukey's post-hoc test and each treatment compared to each other. All treatments were found to induce HCEC migration significantly above Basal medium (not noted on plot), ^* indicates a significant decrease in migration compared to equimolar VF001 (p<0.05) and # indicates significant increase in migration above GM (p<0.05).

Figure 10. Efficacy of best performing test articles as measured by fluorescein staining. Fastest wound healing was noticed with VF003, then VF004, followed by VF001 . Least resolved area was observed with Placebo treatment group. (Images for additional test groups are given in Figure 4). Figure 11. Diffuse areas of fluorescein staining were observed with VF004

(high concentration) and VF003 in both high and low concentrations. This observation is indicative of more rapid healing.

Figure 12. Corneal transparency and clarity as imaged at Day 5: The rate of resolution of corneal transparency and clarity seems almost the same in all groups but is best with VF003 (high concentration). Figure 13. Representative images of fluorescein staining across treatment groups. The most significant change in the epithelial defects were observed at Day 4 and Day 5 with fastest healing observed in the VF003 high and low concentration groups. VF004 and VF001 treatments also showed better re-epithelialization than the placebo (vehicle alone) group.

Figure 14. Representative images of corneal transparency and clarity across treatment groups.

Figure 15. Change in epithelial wound area between Day 0 and Day 5. A predictable dose-response benefit was observed with the high dose treatments performing better than the low doses in all 3 treatment groups.

Figure 16. Corneal epithelial wound healing expressed as a percentage of the initial wound area with time.

Figure 17. Relative changes in wound area across time - day 3.5. Corneal epithelial wound healing commenced from day 2 in the placebo group. However, wound healing rates increased on Day 3 for the VF treatment groups.

Figure 18. Relative changes in wound area across time - day 4. Corneal epithelial wound healing commenced from day 2 in the placebo group. However, wound healing rates increased on Day 3 for the VF treatment groups. On Day 4 reduction in wound area was significantly superior for VF003, VF004 (high dose) and VF001 (high dose) treatments as compared to the placebo group.

Figure 19. Relative changes in wound area across time - day 5. Corneal epithelial wound healing commenced from day 2 in the placebo group. However, wound healing rates increased on Day 3 for the VF treatment groups. On Day 4 and Day 5, reduction in wound area was significantly superior for VF003, VF004 (high dose) and VF001 (high dose) treatments as compared to the placebo group.

Figure 20. Relative healing rates by linear regression. Linear regression of wound areas from Day 0 to Day 5 was used as a measure of healing rates with higher values (steeper slope) indicating faster healing rates. This analysis indicates superior healing rates in VF003 (high and low dose) and VF004 (high) treatment groups. Detailed description of the embodiments

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

All of the patents and publications referred to herein are incorporated by reference in their entirety.

For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

The inventors have found that the presence of various peptides capable of binding to and activating a growth factor receptor and peptides capable of binding to an integrin can stimulate proliferation and/or migration of corneal epithelial cells. This clearly identifies an application in the treatment of corneal wounds. The peptides can exert their effects when covalently linked or when separate, distinct molecules (i.e. not covalently linked). In certain aspects, peptides, fusion and chimeric proteins of the invention have been shown to stimulate primary human corneal epithelial cell migration, increase corneal wound healing and increase the rate of corneal wound healing in vivo.

An aspect of the present invention includes the presence of or use of peptides capable of binding to and activating a growth factor receptor. A growth factor receptor mediates cellular growth, proliferation, and/or cellular differentiation upon activation. Examples of growth factor receptors are the insulin like growth factor I receptor (IGF-IR) or epidermal growth factor receptor (EGFR). Typically, these receptors are activated on the binding growth factors such as IGF-I, IGF-II or EGF.

The IGF-family of proteins is comprised of two growth factor receptor ligands (IGF-I and IGF-II) and six 'classic' IGF binding proteins (IGFBPs). Together these proteins regulate a range of cellular responses throughout the body especially those associated with tissue development and regeneration. The primary action of IGF is mediated by binding to its specific receptor, the insulin-like growth factor I receptor (IGF1 R), which is present on many cell types in many tissues. Binding to the IGF-IR, a receptor tyrosine kinase, initiates intracellular signalling. IGF-I and IGF-II bind to at least two cell surface receptors: the IGF-I receptor (IGF-IR), and the insulin receptor.

IGF-I is one of the most potent activators of the AKT signalling pathway, a stimulator of cell growth and proliferation, and a potent inhibitor of programmed cell death. The IGF-IR signals through multiple pathways including phosphatidylinositol-3 kinase (PI3K) and its downstream partner, the mammalian target of rapamycin (mTOR). Epidermal growth factor (EGF) is a growth factor that stimulates cell growth, proliferation, and differentiation by binding to its cognate receptor EGFR. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface. This stimulates ligand-induced dimerization, activating the intrinsic protein-tyrosine kinase activity of the receptor. The tyrosine kinase activity, in turn, initiates a signal transduction cascade that results in a variety of biochemical changes within the cell that ultimately lead to DNA synthesis and cell proliferation.

The binding of a peptide, domain or growth factor to a cognate receptor may be measured by any routine means in the art including BIAcore analysis (Forbes et al., 2002, Eur J Biochem. 269: 961 -968), ligand radiobinding assays, liquid phase ligand binding assays or solid phase ligand binding assays. Activity of the cognate receptor may be determined by routine means in the art including assessment of downstream signalling pathway activation (ie phosphorylation of Akt) or assessment of PI3K activity. Activation of the IGF-IR or insulin receptor (IR-A or IR-B) may be measured as described in Denley, et al. 2004 Mol. Endocrinol. 18(10): 2502-2512.

In any aspect of the present invention, there is provided a second peptide which comprises, consists essentially of or consists of an amino acid sequence of an integrin- binding domain of vitronectin (VN) or fibronectin (FN).

VN is a multi-functional 75 kDa glycoprotein that is found in the circulation and numerous tissues and forms a major component of the ECM. VN binds to a number of ligands including integrin receptors, heparin, plasminogen, plasminogen activator inhibitor-1 (PAI-1 ), thrombin-anti-thrombin (TAT) complexes, glycosaminoglycans, collagen, complement, and the urokinase plasminogen activator receptor (uPAR) Cellular responses to VN are mediated via a _v integrins (α _ν β3 and a _v Ps) which recognise an Arg-Gly-Asp (RGD) sequence adjacent to the protein's N-terminus.

Fibronectin is a high-molecular weight (~440kDa) glycoprotein of the extracellular matrix that binds to membrane-spanning receptor proteins called integrins. Fibronectin exists as a protein dimer, consisting of two nearly identical monomers linked by a pair of disulfide bonds. In any aspect of the invention, the term a "peptide capable of binding to an integrin" refers to a peptide that includes, but not limited to, an amino acid sequence of VN and FN that mediates binding to an integrin such as α _ν β3 and α _ν β5. The binding of FN or VN to integrins can be determined by any number of known methods including solid-phase binding assays, FN or VN binding assays, or surface plasmon resonance (SPR) on Biacore. Typically, the peptide comprises an integrin binding sequence of RGD. The integrin-binding domain of VN may have an amino acid sequence of SEQ ID NO: 23. In one embodiment, the peptide does not comprise an amino acid sequence of the heparin binding domain of VN. In one embodiment, the first peptide comprises, consists essentially of or consists of an amino acid sequence of a full length VN or FN. In the chimeric or fusion proteins of the present invention, the C-terminal residue of the first peptide may be covalently linked to the N-terminal residue of the second peptide, or the N-terminal residue of the first peptide may be covalently linked to the C- terminal residue of the second peptide. In this arrangement, the first peptide and the second peptide are said to be "directly linked" or "adjacent". A suitable covalent linker is preferably selected from glycine and serine residues.

In other embodiments, the chimeric or fusion protein includes a linker for linking first, second and/or third peptides. The linker may be any linker able to join two or more peptides, including both amino acid and non-amino acid linkers. Preferably, the linker is non-immunogenic. Suitable linkers may be at least, or equal to, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 and 25 amino acids in length, although any linker that allows each peptide to bind to its cognate growth factor receptor or integrin is contemplated.

In one embodiment, the linker sequence comprises one or more glycine residues and one or more serine residues. Particular examples of linker sequences may be selected from; (Gly ₄ Ser) ₄ (SEQ ID NO:25) _; Gly ₄ Ser Gly ₄ (SEQ ID NO:26); Gly ₄ Ser Gly ₄ Ser Gly ₄ Ser Gly ₄ (SEQ ID NO:27); Gly ₄ Ser (SEQ ID NO:28); Gly ₄ Ser ₃ (SEQ ID NO:29) and (Gly ₄ Ser) ₃ (SEQ ID NO:30), although without limitation thereto.

In another embodiment, the linker sequence includes a Plasmin Cleavage Recognition Site, such as Leu lie Lys Met Lys Pro (SEQ ID NO:31 ). In yet another embodiment, the linker sequence includes a Collagenase-3 Cleavage Recognition Site, such as Gin Pro Gin Gly Leu Ala Lys (SEQ ID NO:32).

In any embodiment according to the invention, there is provided a nucleic acid molecule including a nucleotide sequence encoding a chimeric or fusion protein as broadly described above, optionally operatively linked to at least one regulatory element. Preferably, the nucleic acid is isolated, purified, recombinant or synthetic.

By "operably linked" or "operably connected" is meant that said regulatory nucleotide sequence(s) is/are positioned relative to the 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 tet- 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.

For the purposes of host cell expression, the recombinant nucleic acid may be operably linked to one or more regulatory sequences in an expression vector. An "expression vector" may be either a self-replicating extrachromosomal vector such as a plasm id, or a vector that integrates into a host genome. Numerous types of appropriate expression vectors are known in the art for a variety of host cells. For instance, expression vectors may include viral vectors such as vaccinia, and viral vectors useful in gene therapy. The latter include adenovirus and adenovirus-associated viruses (AAV) such as described in Braun-Falco et al., 1999, Gene Ther. 6: 432, retroviral and lentiviral vectors such as described in Buchshacher et al., 2000, Blood, 2499 and vectors derived from herpes simplex virus and cytomegalovirus.

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), Fe portion of human IgG, maltose binding protein (MBP) and hexahistidine (His ₆ ), 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 system (Qiagen) useful with (His ₆ ) fusion partners and the Pharmacia GST purification system.

As used herein, a reference to a "homologue" of a peptide or polypeptide is a reference to a peptide or polypeptide having an amino acid sequence that shares homology or that is homologous to, or that has identity with the amino acid sequence of the first-mentioned peptide or polypeptide, preferably at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. Sequence identity refers to exact matches between the amino acids of two sequences which are being compared. Such a homologue may derive from a naturally occurring variant or isolate of, for example, an IGF, EGF, VN or FN. Alternatively, it may be a "conservative-substitution" variant of a peptide or polypeptide in which one or more amino acid residues have been changed without altering the overall conformation and function of the peptide or polypeptide; including, but by no means limited to, replacement of an amino acid with one having similar properties.

Amino acids with similar properties are well known in the art. For example, polar/hydrophilic amino acids which may be interchangeable include asparagine, glutamine, serine, cysteine, threonine, lysine, arginine, histidine, aspartic acid and glutamic acid; nonpolar/hydrophobic amino acids which may be interchangeable include glycine, alanine, valine, leucine, isoleucine, proline, tyrosine, phenylalanine, tryptophan and methionine; acidic amino acids which may be interchangeable include aspartic acid and glutamic acid and basic amino acids which may be interchangeable include histidine, lysine and arginine. Preferably such conservative-substitution variants have less than 20, more preferably less than 15, more preferably less than 10, and most preferably less than 5 amino acid changes. The peptides described herein may have conservative substitutions of at least one amino acid residue. Preferably, this conservative substitution does not alter the overall conformation or function of the peptide. Preferably the conservative substitution comprises a replacement of an amino acid with another having one or more similar properties. Table 1 outlines the properties of each of the amino acids.

Table 1

aliphatic

Isoleucine lie 1 hydrophobic

neutral

aliphatic

Leucine Leu L hydrophobic

neutral

polar

Lysine Lys K hydrophilic

charged (+)

hydrophobic

Methionine Met M

neutral

aromatic

Phenylalanine Phe F hydrophobic

neutral

hydrophobic

Proline Pro P

neutral

polar

Serine Ser S hydrophilic

neutral

polar

Threonine Thr T hydrophilic

neutral

aromatic

Tryptophan Trp w hydrophobic

neutral

aromatic

Tyrosine Tyr Y polar

hydrophobic

aliphatic

Valine Val V hydrophobic

neutral

The peptides described herein may have non-, or unnatural amino acids incorporated. Unless otherwise specified, any amino acid may be natural or non-natural / unconventional. Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t- butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6- methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural / non-conventional amino acids contemplated herein is shown in Table 2.

Table 2

Non-conventional Code Non-conventional Code amino acid amino acid a-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-a-methylbutyrate Mgabu L-N-methylarginine Nmarg am inocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyl lysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva

D-glutamic acid Dglu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine NIe D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr a-methyl-aminoisobutyrate Maib

D-valine Dval a-methyl-y-aminobutyrate Mgabu

D-a-methylalanine Dmala a-methylcyclohexylalanine Mchexa D-a-methylarginine Dmarg a-methylcylcopentylalanine Mcpen

D-a-methylasparagine Dmasn a-methyl-a-napthylalanine Manap

D-a-methylaspartate Dmasp a-methylpenicillamine Mpen

D-a-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu

D-a-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg D-a-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-a-methylisoleucine Dmile N-amino-a-methylbutyrate Nmaabu

D-a-methylleucine Dmleu a-napthylalanine Anap

D-a-methyllysine Dmlys N-benzylglycine Nphe

D-a-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln D-a-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn D-a-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu

D-a-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-a-methylserine Dmser N-cyclobutylglycine Ncbut

D-a-methylthreonine Dmthr N-cycloheptylglycine Nchep D-a-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-a-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-a-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe

D-N-methylglutamine Dnmgin N-(3-guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-(1 -hydroxyethyl)glycine Nthr D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-y-aminobutyrate Nmgabu

N-methylcyclohexylalanineNmchexa D-N-methylmethionine Dnmmet D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine Nala D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-(1 -methylpropyl)glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-(1 -methylethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr

L-f-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-a-methylalanine Mala

L-a-methylarginine Marg L-a-methylasparagine Masn

L-a-methylaspartate Masp L-a-methyl-f-butylglycine Mtbug

L-a-methylcysteine Mcys L-methylethylglycine Metg

L-a-methylglutamine Mgln L-a-methylglutamate Mglu

L-a-methylhistidine Mhis L-a-methylhomophenylalanine Mhphe

L-a-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-a-methylleucine MIeu L-a-methyllysine Mlys

L-a-methylmethionine Mmet L-a-methylnorleucine Mnle

L-a-methylnorvaline Mnva L-a-methylornithine Morn

L-a-methylphenylalanine Mphe L-a-methylproline Mpro

L-a-methylserine Mser L-a-methylthreonine Mthr

L-a-methyltryptophan Mtrp L-a-methyltyrosine Mtyr

L-a-methylvaline Mval L-N-methylhomophenylalanine Nmhphe

N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine

1 -carboxy-1 -(2,2-diphenyl-Nmbc

ethylamino)cyclopropane

As used herein, the terms "heterologous protein" or "chimeric or fusion protein" are used to refer to a protein that is composed of functional units, domains, sequences or regions of amino acids derived from different sources or that are derived from the same source and that have been assembled so as to have an organisation that is distinguished from that observed in a molecule from which the unit, domain, sequence or region is derived or related to.

According to any embodiment of the invention, there is provided a method of treating a corneal wound in an individual in need thereof, the method comprising administering to the individual a chimeric or fusion protein as described herein.

The term "wound" refers to an injury, such as an ulcer or lesion, as a result of a disease or disorder, or as a result of an accident, incident or surgical procedure. Further, as used herein a 'corneal wound' includes all ocular surface conditions with impairment of the corneal epithelia that would benefit from a topical treatment that stimulates corneal epithelial cell proliferation and migration. Such conditions may result from surgical and non-surgical trauma or abrasion, tear-film disorders, severe dry eye, neoplasia and its treatment, corneal graft, diabetic keratopathy, neuropathic keratopathy, thermal or chemical burns, herpetic epithelial keratitis, injury caused by ocular anti-viral or -microbial agents, or immunological disorders. For example, the wound may be an abrasion, which is caused by contact of the cornea with foreign bodies (e.g. sand) or contact lenses. The wound may be a corneal wound (including specifically a corneal epithelial wound, together with or without other wound or injury) that is a result of an alkali injury i.e. an alkali-induced wound.

The condition to be treated that results in a corneal wound may therefore result from surgical and non-surgical trauma or abrasion, severe dry eye, diabetic keratopathy, neuropathic keratopathy, thermal or chemical burns, herpetic epithelial keratitis, extensive contact lens wear, injury caused by ocular anti-viral or -microbial agents. Preferably, the treatment stimulates corneal epithelial cell proliferation and/or migration to promote repair and/or healing of the cornea. A corneal wound may also include a persistent epithelial defect (PED). PEDs are characterized by ulcers with delayed (>2 weeks) healing and can recur once healed, particularly when the basement membrane (BM) is lost. They arise from a variety of insults, including trauma, tear-film disorders, neoplasia and its treatment, corneal graft, surgery, chemical and thermal burns, or in neurotrophic, diabetic, and herpetic keratopathies, and can also accompany immunological disorders. Corneal epithelial cells aid in maintaining a stable tear film, and secrete the epithelial basement membrane which is critical in corneal healing. Corneal epithelial cells are constantly turned over as the outermost cells are shed into the tear film. The entire epithelium is turned over in approximately seven to 10 days. This process is accelerated during wound healing and generally leads to rapid healing for corneal injuries that only involve the epithelial cells.

The process of corneal epithelial wound healing can be divided into phases that occur in sequence, but may overlap in time. The three phases are: migration of adjacent intact epithelial cells to cover the injured zone, proliferation of the migrated monolayer to reestablish epithelial thickness, and differentiation of the reconstruct epithelium to restore structure and function. The initial stage is characterized by cells attaching and spreading over the denuded substratum which typically occurs by epithelial "sliding." An important aspect of this process is cell attachment to the extracellular matrix (ECM), which is mediated by integrins. Integrins and their ligands are components of structures known as focal contacts which facilitate cell-to-ECM interactions during wound healing.

The capacity for a peptide, chimeric or fusion protein of the invention to promote corneal wound healing can be determined in vitro by a number of routine methods. Suitable assay systems used to determine corneal wound healing include mechanically wounding confluent epithelial cells by passing a pipette tip through the monolayer. The rate of repair (% wound recovery) is monitored using an inverted phase-contrast microscope and wound recovery compared with cells grown on uncoated dishes over a given period of time. Alternatively, a fenestrated/barrier assay may be employed. In these experiments, epithelial cells are resuspended in media and dispensed into adjacent chambers within a l-Dish (ibidi, Munich, Germany). When cells reach confluence, the insert is removed to reveal a gap of 500 Im. Cells are incubated in the presence or absence of various factors and monitored in a live cell imager for cell movement across the clear zone over 24 hours. This method to measure wound healing or migration is analogous to the Fence method as described in Example 4 herein. Another assay to measure epithelial wound healing is the Biostation assay in which epithelial cells are grown to confluence in 96-well plates and a 96-pin wound- making tool (WoundMaker; Essen Bioscience) is used to simultaneously create a precise and reproducible wound in each well. Epithelial cell proliferation may be determined by any number of routine methods in the art including a colorimetric 5-bromo-2-deoxyuridine (BrdU) ELISA which detects BrdU incorporation into newly synthesized DNA, the MTS method as shown in Example 3, or the MTT assay. The MTS method of assessing proliferation is based on the reduction of MTS tetrazolium compound by viable cells to generate a coloured formazan product that is soluble in cell culture media. The MTT assay measures conversion of methyl-thiazolyldiphenyl-tetrazolium bromide (MTT) by mitochondrial enzymes.

Symptoms of a corneal wound may include blurred vision, eye pain or stinging and burning in the eye, feeling like something is in your eye (may be caused by a scratch or something in your eye), light sensitivity, redness of the eye, swollen eyelids, watery eyes or increased tearing. An individual in need of treatment by a method, use, chimeric or fusion protein, or composition of the invention may display one or more of these symptoms. Clinically, corneal wounds may be diagnosed by physical eye examination. Obvious signs include evidence of penetrating trauma, infection, and significant vision loss. In corneal abrasion, the pupil is typically round and central, and conjunctival injection may be present. Ciliary spasm causing miosis, pain, and ciliary flush (injection of ciliary vessels surrounding the cornea) may indicate traumatic iritis. A corneal opacity or infiltrate may occur with corneal ulcers or infection.

Fluorescein staining can help to identify a corneal epithelial wound. A drop of topical anesthetic (proparacaine 0.5%) is applied directly into the eye or on a fluorescein strip. The patient's lower lid is pulled down, and the fluorescein strip is lightly touched to the bulbar conjunctiva. The dye spreads over the cornea as the patient blinks, and stains any exposed basement membrane of the epithelium. In normal light, an abrasion may stain yellow. Illumination with cobalt blue light shows the defect as green. Traumatic corneal wounds typically have linear or geographic shapes. If contact lenses are involved, the abrasion may have several punctate lesions that coalesce into a round, central defect. A branching (dendritic) appearance suggests herpetic keratitis. Multiple vertical lines on the superior cornea suggest a foreign body under the upper eyelid. The existence of, or improvement in, treatment of or prevention of a corneal wound may be by any clinically or biochemically relevant method of the individual or a biopsy therefrom. For example, a parameter measured may be epithelial cell migration or proliferation, or lack of yellow light staining in the presence of Fluorescein. Alternatively, existence of, or improvement in, treatment may also result in a reduction or lessening of one or more symptoms as described herein. As used herein, "preventing" or "prevention" is intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are also well known by physicians. In some embodiments, the methods or uses of the invention may prevent or delay a corneal wound from developing into a persistent epithelial defect.

The terms "treatment" or "treating" of a subject includes the application or administration of peptides, chimeric or fusion protein, or composition of the invention to a subject with the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition. The term "treating" refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being. The phrase 'therapeutically effective amount' generally refers to an amount of one or more peptides, chimeric or fusion proteins of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. As used herein, the disease or disorder is a corneal wound, preferably a corneal epithelial wound.

A peptide, chimeric or fusions protein, or composition of the present invention may be present in an ophthalmic composition, which is a composition suitable for administration to the eye. Examples of ophthalmic compositions according to the invention are suspensions, ointments, sustained release formulations, gels or solutions suitable for application as an eye drop. Alternatively, the composition may be loaded onto, or impregnated into, a contact lens or other suitable biomaterial. Preferably, the pharmaceutical compositions according to the present invention will be formulated for topical administration or for sustained release delivery. Preferably, the composition of the present invention is in a form suitable for administration to the eye. Aqueous solutions are generally preferred, based on ease of formulation, as well as a subject's ability to easily administer such compositions by means of instilling one to two drops of the solutions in the affected eyes. However, the compositions may also be suspensions, viscous or semi-viscous gels, or other types of solid or semi-solid compositions, or those appropriate for sustained release.

Any of a variety of carriers may be used in the compositions of the present invention including water, mixtures of water and water-miscible solvents, such as Ci to C ₇ alkanols, vegetable oils or mineral oils comprising from 0.5 to 5% non-toxic water- soluble polymers, gelling products, such as gelatin, alginates, pectins, tragacanth, karaya gum, xanthan gum, carrageenin, agar and acacia, and their derivatives, starch derivatives, such as starch acetate and hydroxypropyl starch, cellulose and its derivatives and also other synthetic products, such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxide, preferably cross-linked polyacrylic acid, such as neutral Carbopol, or mixtures of those polymers, naturally- occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan mono-oleate. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above.

The composition according to the present invention may comprise at least one gelling agent. Gelling agents suitable for use in pharmaceutical compositions are well known to those of ordinary skill in the art and include, for example, xanthan gum and its derivatives, carbomer and its derivatives, acrylate based copolymers and cross polymers, sodium polyacrylate and its derivatives, cellulose and its derivatives, and starch and agar and their derivatives. The selection of the gelling agent according to the present invention is important in providing a clear gel. The amount of gelling agent added to the composition may be readily determined by one of ordinary skill in the art with a minimum of experimentation, and will depend upon factors known to those skilled in the art, such as the properties of the gelling agent and the desired properties of the pharmaceutical composition.

Additional ingredients that may be included in the pharmaceutical composition of the invention include tonicity enhancers, preservatives, solubilizers, stabilizers, nontoxic excipients, demulcents, sequestering agents, pH adjusting agents, co-solvents and viscosity building agents. For the adjustment of the pH, preferably to a physiological pH, buffers may especially be useful. The pH of the present solutions should be maintained within the range of between 4 to 8, preferably 6 to 7.5. It will be understood by a person of ordinary skill in the art that any pH that is compatible with the ocular surface is suitable. Suitable buffers may be added, such as boric acid, sodium borate, potassium citrate, citric acid, sodium bicarbonate, TRIS, disodium edetate (EDTA) and various mixed phosphate buffers (including combinations of Na ₂ HP0 ₄ , NaH ₂ P0 ₄ and KH ₂ P0 ₄ ) and mixtures thereof. Generally, buffers will be used in concentrations ranging from about 0.05 to 0.5 M.

Tonicity is adjusted if needed typically by tonicity enhancing agents. Such agents may, for example, be of ionic and/or non-ionic type. Examples of ionic tonicity enhancers are alkali metal or earth metal halides, such as, for example, CaCI ₂ , KBr, KCI, LiCI, Nal, NaBr or NaCI, Na ₂ S0 ₄ or boric acid. Non-ionic tonicity enhancing agents are, for example, urea, glycerol, sorbitol, mannitol, propylene glycol, or dextrose. The aqueous solutions of the present invention are typically adjusted with tonicity agents to approximate the osmotic pressure of normal lachrymal fluids. In certain embodiments, the compositions of the invention additionally comprise a preservative. A preservative may typically be selected from a quaternary ammonium compound such as benzalkonium chloride (N-benzyl-N-(C ₈ -Ci ₈ alkyl)-N. N- dimethylammonium chloride), benzoxonium chloride or the like. Examples of preservatives different from quaternary ammonium salts are alkyl-mercury salts of thiosalicylic acid, such as, for example, thiomersal, phenylmercuric nitrate, phenylmercuric acetate or phenylmercuric borate, sodium perborate, sodium chlorite, parabens, such as, for example, methylparaben or propylparaben, sodium benzoate, salicylic acid, alcohols, such as, for example, chlorobutanol, benzyl alcohol or phenyl ethanol, guanidine derivatives, such as, for example, chlorohexidine or polyhexamethylene biguanide, sodium perborate, Germal™ or sorbic acid. Preferred preservatives are quaternary ammonium compounds, in particular benzalkonium chloride or its derivative such as Polyquad (see US patent number 4,407,791 ), alkyl- mercury salts and parabens. Where appropriate, a sufficient amount of preservative is added to the ophthalmic composition to ensure protection against secondary contaminations during use caused by bacteria and fungi.

In other embodiments, the compositions of this invention do not include a preservative. Such formulations would be particularly useful for subjects who wear contact lenses.

The composition of the invention may additionally require the presence of a solubilizer, in particular if the active or the inactive ingredients tends to form a suspension or an emulsion. A solubilizer suitable for an above concerned composition is for example selected from the group consisting of tyloxapol, fatty acid glycerol polyethylene glycol esters, fatty acid polyethylene glycol esters, polyethylene glycols, glycerol ethers, a cyclodextrin (for example alpha-, beta- or gamma-cyclodextrin, e.g. alkylated, hydroxyalkylated, carboxyalkylated or alkyloxycarbonyl-alkylated derivatives, or mono- or diglycosyl-alpha-, beta- or gamma-cyclodextrin, mono- or dimaltosyl-alpha-, beta- or gamma-cyclodextrin or panosyl-cyclodextrin), polysorbate 20, polysorbate 80 or mixtures of those compounds. A specific example of an especially preferred solubilizer is a reaction product of castor oil and ethylene oxide, for example the commercial products Cremophor EL ^® or Cremophor RH40 ^® . Reaction products of castor oil and ethylene oxide have proved to be particularly good solubilizers that are tolerated extremely well by the eye. Another preferred solubilizer is selected from tyloxapol and from a cyclodextrin. The concentration used depends especially on the concentration of the active ingredient. The amount added is typically sufficient to solubilize the active ingredient. The compositions may comprise further non-toxic excipients, such as, for example, emulsifiers, wetting agents or fillers, such as, for example, the polyethylene glycols designated 200, 300, 400 and 600, or Carbowax designated 1000, 1500, 4000, 6000 and 10000. The amount and type of excipient added is in accordance with the particular requirements and it will be understood by a person of ordinary skill in the art what types and amounts of excipients and other additives may be present in a composition such that the composition is compatible with the eye. Other compounds may also be added to the compositions of the present invention to increase the viscosity of the carrier. Examples of viscosity enhancing agents include, but are not limited to: polysaccharides, such as hyaluronic acid and its salts, chondroitin sulfate and its salts, dextrans, various polymers of the cellulose family; vinyl polymers; and acrylic acid polymers.

The pharmaceutical compositions of the present invention may contain other active ingredients that are effective in the treatment of wounds e.g. growth factors, cleansers and antibiotics. Generally, these active ingredients and treatments are provided in a combined amount effective to promote the healing of a wound. This may involve administering the composition of the present invention and the active ingredient/treatment at the same time or at times close enough such that the administration results in an overlap of the desired effect. Alternatively, the composition of the present invention may precede or follow other treatments. The composition may be administered in any way that is deemed suitable by a person of ordinary skill in the art. The pharmaceutical composition may be administered topically.

The composition of the invention may be administered in single or multiple doses and for any length of time until the wound is either completely healed or until the desired level of wound healing has been achieved. The person of ordinary skill in the art will recognise that the dosage amount, dosage regime and length of treatment will depend on factors such as, for example, the wound type, the location of the wound and the health of the subject. In the case of chemical injuries, the treatment required will depend on factors such as the extent of the ocular surface damaged, the degree of intraocular penetration by the chemical agent, and the concentration and nature of the agent involved. In one embodiment, the composition is administered every half hour or hourly, up to, for example, eight times a day.

The kit or "article of manufacture" may comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a peptide, chimeric or fusion protein, or pharmaceutical composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the peptide, chimeric or fusion protein, or pharmaceutical composition is used for treating the condition of choice. In one embodiment, the label or package insert includes instructions for use and indicates that the therapeutic composition can be used to treat a corneal wound.

The kit may comprise (a) a peptide, chimeric or fusion protein, or pharmaceutical composition; and (b) a second container with a second active principle or ingredient contained therein. The kit in this embodiment of the invention may further comprise a package insert indicating that a peptide, chimeric or fusion protein, or pharmaceutical composition and other active principle can be used to treat a corneal wound. Alternatively, or additionally, the kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

EXAMPLES

Example 1

This study was conducted to generate VF001 (VN: IGF-I), VF003 (VN:EGF) and VF004 (EGF:VN: IGF-I) chimeric proteins using cloning techniques. Methods

Human VN, IGF-I and EGF gene DNA sequences (NCBI accession # AF382388, # X03563 and # AY548762 respectively) were codon-optimised for expression in S. frugiperda and synthesised by GeneArt (Regensburg, Germany). The coding sequences were then cloned into the ρΙΒΛ/5-His expression vector (Invitrogen) incorporating a poly-histidine affinity tag to aid in purification. A truncated section of VN comprising the first 249 nucleotides (coding for the secretion signal peptide and amino acids 1 -64 of the mature protein sequence) was amplified by polymerase chain reaction (PCR) simultaneously incorporating restriction enzyme sites for insertion into the expression vector. A nucleotide sequence encoding a (Gly ₄ Ser) ₄ amino acid linker was inserted via site-directed mutagenesis PCR between protein domain coding sequences and a smaller (Gly ₄ SerGly ₄ ) linker inserted 5' of the poly-histidine tag was also inserted by the same methods. Further rounds of site-directed mutagenesis PCR were used to delete superfluous restriction enzymes sites in the final coding sequence.

The resulting constructs were arranged as follows: 1. VF001 - VNsig:VN(1-64):(Gly ₄ Ser)4:IGF-l:(Gly ₄ SerGly4):HiS6

The DNA and protein sequences according to VF001 are as follows: DNA: atggctcctctgcgtcctctgctgatcctggctctgctggcttgggtggccctggctgac caggag tcttgcaagggacgttgcaccgagggtttcaacgtggacaagaagtgccagtgcgacgag ctgtgctc ctactaccagtcctgctgcaccgactacaccgctgagtgcaagcctcaggtgacccgtgg tgacgtgtt caccatgcccgaggacgagtacactgtgtacgacgacggcgaggag aataataataattccaataat ggtggttccggtggtggtggttccggtggtggtggatccggXccXgagacccXgXgcggX gctgaacX cgtggacgctctgcagttcgtgtgcggtgaccgcggtttctacttcaacaagcccaccgg ttac ggttcctcctctcgtcgtgctcctcagaccggtatcgtggacgagtgctgcttccgttct tgcgac ctgcgtcgcctggagatgtactgcgctcccctgaagcctgctaagtccgctggiggiggi ggiicc aaaaafaafaafoatcatGaecatcaccattga

Protein:

MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTD YTAECKPQVTRGDVFTMPEDEYTVYDDGEEGGGGSGGGGSGGGGSGGGGSG P ET LCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMY

Legend: VN Signal; VN; IGF-I; Linker, 6xHis tag

2. VF003 - VNsig:VN(1-64):(Gly ₄ Ser)4:EGF:(Gly ₄ SerGly ₄ ):His ₆

The DNA and protein sequences according to VF003 are as follows:

DNA: atggctcctctgcgtcctctgctgatcctggctctgctggcttgggtggccctggctgac caggag tcttgcaagggacgttgcaccgagggtttcaacgtggacaagaagtgccagtgcgacgag ctgtgctc ctactaccagtcctgctgcaccgactacaccgctgagtgcaagcctcaggtgacccgtgg tgacgtgtt caccatgcccgaggacgagtacactgtgtacgacgacggcgaggag ααΐαα tgg tggttccgg tgg t ggtggttccggtggtggtggttccggtggtggtggatcc\aactccgactccgaatgccc cctgtcccacg lacggttactgcctgcacgacggtgtctgcatgtacatcgaggctctggacaagtacgct tgcaactgcg

^ggtgggctacatcggcgagcgttgccagtaccgtgacctgaagtggtgggagctgc gte tggtggt ggttccggaggtggtggtcatcatcaccatcaccaUga Protein:

MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAE CKPQVTRGDVFTMPEDEYTVYDDGEEGGGGSGGGGSGGGGSGGGG5|NSDSECPL

SHDGYCLHDGVCMYIEALDKYACNCWGYIGERCQYRDLKWWELRGGGGSGGGGI HHHHH-

Legend: VN Signal; |EGF|; VN; Linker, 6xHis tag

3. VF004 - VNsig:-EGF:(Gly ₄ Ser)3Gly ₄ :VN(1-64):(Gly ₄ Ser)4:IGF

l:(Gly ₄ SerGly ₄ ):His ₆

The DNA and protein sequences according to VF004 are as follows:

DNA: atggctcctctgcgtcctctgctgatcctggctctgctggcttgggtggccctggcta

Itccgaatgccccctgtcccacgacggttactgcctgcacgacggtgtctgcatgtacat cgaggctctg gacaagtacgcttgcaactgcgtggtgggctacatcggcgagcgttgccagtaccgtgac ctgaagtg |g t g g g a g ct g eg i\ggcgg tgg tggttctgg tgg tggtggttccgg tgg tgg tggttccgg tgg tgg tgg tg. accagqaqtcttqcaaqqqacqttqcaccqaqqqtttcaacqtqqacaaqaaqtqccaqt qcqacqa gctgtgctcctactaccagtcctgctgcaccgactacaccgctgagtgcaagcctcaggt gacccgtgg tgacgtgttcaccatgcccgaggacgagtacactgtgtacgacgacggcgaggag aataataataatt ccggtggtggtggttccggtggtggtggttccggtggtggtggatccggXccXgagaccc XgtgcggX gctgaactcgtggacgctctgcagttcgtgtgcggtgaccgcggtttctacttcaacaag ccca ccggttacggttcctcctctcgtcgtgctcctcagaccggtatcgtggacgagtgctgct tccgtt cttgcgacctgcgtcgcctggagatgtactgcgctcccctgaagcctgctaagtccgctg gfggf ggtggttccggaggtggtggtcatcatcaccatcaccaiiga

Protein: MAPLRPLLILALLAWVALANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVv^

IGYIGERCQYRDLKWWELRIGGGGSGGGGSGGGGSGGGGDQESCKGRCTEGFNVD KKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPEDEYTVYDDGEEGGGGSGG GGSGGGGSGGGGSGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTG

Legend: VN Signal; |EGF|; VN; IGF-I; Linker, 6xHis tag

A sequence of 3 amino acids may be included between the VN signal and EGF to ensure that no EGF sequence is cleaved. An example of a 3 amino acid sequence is GTS (e.g. nucleotide sequence is gggacgtct).

DNA sequences according to the VN signal, EGF, VN, IGF-I, linkers and the 6XHis tag are shown below in Table 3A. Protein sequences according to the VN signal, EGF, VN, IGF-I, linkers and the 6XHis tag are shown below in Table 3B.

Table 3A: DNA sequences

gtggtgggagctgcgtlgflf tggtggtggttccggaggtggtggmm catcaccatcaccattga

VF004 atggctcctctgcgtcctctgctgatcctggctctgctggcttgggtg SEQ ID NO:3

(EGF:VN: IG gccctggct|aactccgactccgaatgccccctgtcccacgacggt

F-l) Itactgcctgcacgacggtgtctgcatgtacatcgaggctctggac

laagtacgcttgcaactgcgtggtgggctacatcggcgagcgttgc lcagtaccgtgacctgaagtggtgggagctgcgtLqfflfCflfflffflfflffflffl f.- tctgg tgg tgg tggttccgg tgg tggtggttccgg tgg tgg tgg fga ccaggagtcttgcaagggacgttgcaccgagggtttcaacgtgg acaagaagtgccagtgcgacgagctgtgctcctactaccagtcct gctgcaccgactacaccgctgagtgcaagcctcaggtgacccgt ggtgacgtgttcaccatgcccgaggacgagtacactgtgtacgac gac £ C a££a ggtggtggtggttccggtggtggtggttccggt ggtggtggttccggtggtggtggatccggXccXgagacccXgtg cggtgctgaactcgtggacgctctgcagttcgtgtgcggtga ccgcggtttctacttcaacaagcccaccggttacggttcctcc tctcgtcgtgctcctcagaccggtatcgtggacgagtgctgct tccgttcttgcgacctgcgtcgcctggagatgtactgcgctcc c c t g a a g c c t g c t a a g t c c g c t gg tgg tgg tgg t tccggagg tg gfggfcatcatcaccatcaccattga

VN signal atggctcctctgcgtcctctgctgatcctggctctgctggcttgggtg SEQ ID NO:4 gccctggct

EGF aactccgactccgaatgccccctgtcccacgacggttactgcctg SEQ ID NO:5 cacgacggtgtctgcatgtacatcgaggctctggacaagtacgct tgcaactgcgtggtgggctacatcggcgagcgttgccagtaccgt gacctgaagtggtgggagctgcgt

VN gaccaggagtcttgcaagggacgttgcaccgagggtttcaacgt SEQ ID N0:6 ggacaagaagtgccagtgcgacgagctgtgctcctactaccagt cctgctgcaccgactacaccgctgagtgcaagcctcaggtgacc cgtggtgacgtgttcaccatgcccgaggacgagtacactgtgtac gacgacggcgaggag

IGF-I ggtcctgagaccctgtgcggtgctgaactcgtggacgctctgcagt SEQ ID NO:7 tcgtgtgcggtgaccgcggtttctacttcaacaagcccaccggtta cggttcctcctctcgtcgtgctcctcagaccggtatcgtggacgagt gctgcttccgttcttgcgacctgcgtcgcctggagatgtactgcgct cccctgaagcctgctaagtccgct

Linker ggtggtggtggttccggtggtggtggttccggtggtggtggttccgg SEQ ID NO:8 tggtggtggatcc

Linker ggtggtggtggttccggaggtggtggt SEQ ID NO:9

Linker ggcggtggtggttctggtggtggtggttccggtggtggtggttccgg SEQ ID NO:10 tggtggtggt

Linker ggtggtggtggttcc SEQ ID NO:11

Linker ggtggtggtggttcctcctcc SEQ ID NO:12

Linker ggtggtggtggttccggtggtggtggttccggtggtggtggttcc SEQ ID NO:13

Linker ctgatcaagatgaagccc SEQ ID NO:14

Linker cagccccagggcctggccaag SEQ ID NO:15

6XHis tag catcatcaccatcaccat SEQ ID NO:16

IGF-II SEQ ID NO:17 gcttaccgccccagtgagaccctgtgcggcggggagctggtgga caccctccagttcgtctgtggggaccgcggcttctacttcagcagg cccgcaagccgtgtgagccgtcgcagccctggcatcgttgagga gtgctgtttccgcagctgtgacctggccctcctggagacgtactgtg ctacccccgccaagtccgagtaa

Table 3B: Protein sequences

Name Protein sequence SEQ ID NO

VF001 MAPLRPLLILALLAWVALADQESCKGRCTEGFNV SEQ ID NO:18 (VN:IGF-I) DKKCQCDELCSYYQSCCTDYTAECKPQVTRGDV

FTMPEDEYTVYDDGEE GGGGSGGGGSGGGGS GGGGSG PETLCGAELVDALQFVCGDRGFYFNK PTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMY CAPLKPAKSAGGGGSGGGGHHHHHH-

VF003 MAPLRPLLILALLAWVALADQESCKGRCTEGFNV SEQ ID NO:19

DKKCQCDELCSYYQSCCTDYTAECKPQVTRGDV

(VN:EGF)

FTMPEDEYTVYDDGEE GGGGSGGGGSGGGGS

GGGGS NSDSECPLSHDGYCLHDGVCMYIEALDK

YACNCVVGYIGERCQYRDLKWWELRGGGGSGG

GGHHHHHH-

VF004 MAPLRPLLILALLAWVALANSDSECPLSHDGYCL SEQ ID NO:20

(EGF:VN:IG HDGVCMYIEALDKYACNCVVGYIGERCQYRDLK

F-l) WWELR GGGGSGGGGSGGGGSGGGGDQESCK

GRCTEC FNVDKKCQCDELCSYYQSCCTDYTAEC

KPQVTRGDVFTMPEDEYTVYDDGEE GGGGSGG GGSGGGGSGGGGSG PETLCGAELVDALQFVCG DRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSC DLRRLEMYCAPLKPAKSAGGGGSGGGGHHHHH

H-

VN signal MAPLRPLLILALLAWVALA SEQ ID NO:21

EGF NSDSECPLSHDGYCLHDGVCMYIEALDKYACNC SEQ ID NO:22

VVGYIGERCQYRDLKWWELR

VN DQESCKGRCTEGFNVDKKCQCDELCSYYQSCC SEQ ID NO:23

TDYTAECKPQVTRGDVFTMPEDEYTVYDDGEE

IGF-I GPETLCGAELVDALQFVCGDRGFYFNKPTGYGS SEQ ID NO:24

SSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPA

KSA

Linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO:25 Linker GGGGS GGGG SEQ ID NO:26

Linker GGGGS GGGGSGGGGS GGGG SEQ ID NO:27

Linker GGGGS SEQ ID NO:28

Linker GGGGS SS SEQ ID NO:29

Linker GGGGS GGGGSGGGGS SEQ ID NO:30

Linker L I KM KP SEQ ID NO:31

Linker Q P QG LAK SEQ ID NO:32

6XHis tag H H H H H H- SEQ ID NO:33

IGF-II AYRP S ETLC GG E LVDTLQ FVCG D RG FYFS RPAS SEQ ID NO:34

RVS RRS P G IVE ECC FRSC D LALLETYCATPAKS E

The DNA sequence of all constructs was verified to ensure that the fidelity of the desired DNA sequences were maintained. Clones in the ρΙΒΛ/5-His vector were used to transfect Sf9 insect cells and transiently- expressed secreted protein was detected in the conditioned media, as assessed by immunoblotting. Briefly, the samples were resolved on SDS-PAGE under reducing conditions and the proteins were transferred onto a nitrocellulose membrane using a semi-dry transfer method. The membrane was interrogated with poly-clonal anti-VN, anti-IGF-l and anti-EGF antibodies, and the target protein species were then visualized using enhanced chemiluminescence following the manufacturer's instructions (GE Healthcare, Buckingham- shire, UK).

Purification of the chimeric proteins was based on Ni-NTA Superflow Agarose (QIAGEN, Australia) affinity chromatography performed according to the manufacturer's instructions. The chimeric proteins were monitored throughout the purification process by SDS-PAGE and Western blot as above. Clarified culture media was loaded onto a 20 mL Q-Sepharose Superflow (GE Healthcare, Buckinghamshire, UK) column equilibrated with _dd H ₂ 0 and bound material was eluted with 100 mL of 50 mM NaH ₂ P0 ₄ , 600 mM NaCI, 10 mM Imidazole, pH 8.5 in order to concentrate the sample. The complete Q- Sepharose eluate was diluted 1 :1 with 50 mM NaH ₂ P0 ₄ , 10 mM Imidazole pH 8.5 to reduce the NaCI concentration to 300 mM. The diluted sample was then loaded onto a 2 ml_ Ni-NTA Superflow Agarose column equilibrated with 50 mM NaH ₂ P0 ₄ , 10 mM Imidazole, 300 mM pH 8.5. After loading, the column was washed with 30 ml_ of equilibration buffer containing 20 mM Imidazole. Bound proteins were eluted with 50 mM NaH ₂ P0 ₄ , 250 mM Imidazole, 300 mM pH 8.5. Elution fractions (1 ml_) were analysed by Silver Stained SDS-PAGE and immunoblotting. Consequently, fractions containing pure (>95%) preparations of immunoreactive protein of the expected MW for each chimera were pooled and independently precipitated by the addition of 40% w/v ammonium sulphate and incubation with rotation for 1 hr at room temperature. Samples were then pelleted by centrifugation and the supernatant discarded. Protein pellets were resuspended in PBS and quantified using the BCA protein assay kit (Peirce). Pure preparations of the chimeric proteins were stored at -80°C.

Example 2 This study was conducted to generate sufficient quantities of VF001 (VN: IGF-I),

VF003 (VN:EGF) and VF004 (EGF:VN: IGF-I) chimeric proteins in Sf9 (insect) cells.

Methods

Materials

Sf9 cells (Invitrogen) were transfected with expression vectors encoding VF001 , VF003 and VF004 constructs. These cells were cultured in SF900-II SFM (Gibco - Thermo Fisher Scientific). Suspension cultures were undertaken using flat bottom vent cap Erlenmeyer flasks of varying volumes sourced from Corning. For purification of the expressed proteins, Q-Sepharose resin was sourced from GE Healthcare while Ni2+- NTA resin was purchased from QIAGEN. Protein quantitation was performed using a BCA kit from Pierce (Thermo Fisher Scientific).

Culture of Sf9 cells transfected with VF001 , VF003 and VF004 expression vectors for protein expression.

Both VF003 and VF004 are currently only available in the Sf9 insect expression system. Transfected cells for each construct were revived and cultured as per standard procedures (Van Lonkhuyzen et al., 2007, Growth Factors, 25(5): 295-308). Briefly, Sf9 cell stocks of VF001 , VF003 and VF004 were thawed, added to T25 flasks containing 5 mL of SF-900II SFM (Gibco - Thermo Fisher Scientific) and incubated at 28°C until confluent. Cells were then dislodged by cell scraper and sloughing before transfer to T75 flasks in 12 mL SF-900II SFM. Again, cells were incubated until confluent and subsequently split 1 :5 into 5x T75 flasks. Upon confluence in the 5x T75 flasks, cells were again harvested and combined into a 100 mL suspension culture in a 250 mL Erlenmeyer flask and cultured at 180 rpm for a minimum of 3 days. Once the suspension culture reached ~8-10 x 10 ⁶ cells/mL it was transferred to a 1 L Erlenmeyer flask and the volume increased to 500 mL. The 500 mL cultures were maintained to produce expression conditioned media through collection and cell passage back to ~2 x 106 cells/mL 3 times weekly. Media collection was performed by aliquoting the cultures into 50 mL disposable sterile centrifuge tubes and pelleting cells at 1 ,500 rpm over 5 minutes. The supernatant (expression conditioned media) was retained and immediately frozen. A proportion of cells sufficient to seed a new culture at ~2 x 10 ⁶ cells/mL was mixed with 1 L of new media and the culture replaced into the incubator with shaking as described above.

Purification of VF001 , VF003 and VF004 from expression culture conditioned media

Accrued conditioned media (~3 L) was thawed and passed through a Q- Sepharose ion-exchange column to which all protein bound under the media conditions. Elution with 50 mM NaH2PO4, 600 mM NaCI, pH 8.0 was performed to concentrate proteins ~60 fold. The protein concentrate was then applied to equilibrated Ni ²⁺ -NTA agarose (QIAGEN) to which the expressed protein bound via the 6x His tag. The resin was washed with 50 mM NaH ₂ PO ₄ , 300 mM NaCI, 20 mM Imidazole, pH 8.0 to remove non-specific bound proteins and elution of expressed proteins was performed with 50 mM NaH ₂ PO ₄ , 300 mM NaCI, 250 mM Imidazole, pH 8.0. Samples (see Table 3 for sample preparation) from throughout the purification process were interrogated via SDS-PAGE and Western blot for purity. The samples identified to be harbouring pure protein were precipitated by the addition of >40% ammonium sulphate (w/v) with mixing (RT for 2 hrs) and centrifugation. The resulting protein pellets were resuspended in 1 mL PBS for each protein. The samples were quantitated via BCA assay and frozen at - 80°C for use in cell-based assays. Table 4. Sam le preparation for SDS-PAGE and Western blotting

Results

Culture of Sf9 cells transfected with VF001 , VF003 and VF004 expressing construct and purification of expressed protein therefrom.

Following the procedures set out above, Sf9 cells transfected with expression vectors encoding VF001 , VF003 and VF004 constructs were propagated and protein expressed within suspension cultures successfully. Conditioned media collected from expression cultures was immediately frozen at -80°C to limit proteolytic degradation. Each construct was subjected to the same purification protocol and were successfully purified. Figure 1 shows the results of the VF001 (VN: IGF-I) purification procedure. The samples demonstrate a prominent band of the expected molecular weight (MW - 27 kDa) in the first Q-Sepharose elution sample (Q-E1 ) which is seen to be depleted in the Ni2+-NTA flow-through (F/T). While a very small amount of this band can be seen in the Ni2+-NTA wash (Ni-W), there is an obvious high density band in Ni2+-NTA elution (Ni- E ^* ) samples 1 -6 (Figure 1A).

Western blotting confirms that the above-mentioned band/s are immunoreactive to both a-VN and a-IGF-l Ab's (Figure 1 B). Thus, it was concluded that the elution samples contained VF001 and the Silver Stain SDS-PAGE demonstrated adequate purity of the samples. A similar result was observed for VF003 (VN:EGF) where by the target protein (again ~27 kDa) was observed in the Q-E1 , depleted in Ni-F/T and in high amounts within Ni-E1 -Ni-E5 (Figure 2 A). In Western blots, this ~27 kDa band was immunoreactive to a-VN Ab's, however, probing with a-EGF failed to return reactivity even to the EGF standard (Figure 2 B). Purity was deemed adequate in Silver Stained SDS-PAGE (Figure 2 A). Purification of VF004 also resulted in a similar manner with target protein (~35 kDa) eluting from QSepharose in fraction 1 , binding strongly to Ni2+- NTA (as evidenced by a depletion in Ni-F/T) and eluting therefrom in high imidazole conditions (present in Ni-E1 - Ni-E6) as seen in Figure 3A. Probing with a-VN, a-IGF-l and a-EGF (in this case at a lower dilution compared to that used for the VF003 blot) Ab's in Western blots showed the ~35 kDa bands were immunoreactive to each Ab and can thus be assumed to be VF004 (Figures 3B & C).

After purification, the positive elution samples for each construct were precipitated with the addition of >40% ammonium sulphate and resuspended in PBS. These samples were then diluted 1 :9 (10 μΙ_ sample concentrate + 90 μΙ_ PBS) and analysed for protein content using the BCA method (Table 5). This was performed as per the manufacturer's instructions and used bovine serum albumin (BSA) to generate a protein standard curve (Figure 4) and VF4-P03 (1 .27 pg/pL) as an internal control. The results are detailed in Table 5, and demonstrate that the assay was run in an accurate manner as evidenced by the recorded concentration for VF4-P03 being as expected (1 .27 pg/pL). The pure samples of VF001 , VF003 and VF004 gave final concentrations of 14.39 pg/pL, 1 1 .99 g/ L and 9.58 g/ L respectively. As each sample is in a volume of 1 mL this gave a total yield of 14.39 mg of VF001 , 1 1 .99 mg VF003 and 9.58 mg VF004, in each case from 3 L of Sf9 conditioned media.

Table 5. Protein quantification of purification samples

Conclusion

Using the expression constructs for VF001 , VF003 and VF004, recombinant proteins were successfully expressed and purified using IEX and IMAC methods. A total of 14.39 mg VF001 , 1 1.99 mg VF003 and 9.58 mg VF004, in each case from 3 L of Sf9 conditioned media, was generated and this is sufficient for forthcoming functional cell- based studies as described in the Examples below.

Example 3

This study was conducted to assess the mitogenic potential of VF001 (VN: IGF-I), VF003 (VN:EGF) and VF004 (EGF:VN: IGF-I) in corneal epithelial cells. Methods Materials

Purified preparations of VF001 , VF003 and VF004 were generated previously as detailed in Examples 1 and 2. Native human vitronectin (VN) was sourced from Promega, while IGF-I was purchased from Gro-Pep and EGF from Thermo-Fisher Scientific (Invitrogen). The Human Corneal Epithelial cell line HCE-T was obtained from the Queensland Eye Institute, Brisbane under the permission of Dr. Damien Harkin. HCE-T cells were cultured in DMEM (high-glucose, + L-glutamine, + pyruvate) with the addition of 10% FCS (Thermo-Fisher Scientific - Gibco) and 1 % Penicillin/Streptomycin (Thermo- Fisher Scientific - Invitrogen). In developing proliferation assays, MTS reagent was used (Promega).

Assessment of the mitogenic potential of VF001 , VF003 and VF004 in corneal epithelial cells

The expressed and purified proteins were functionally assessed in cell proliferation assays based on the MTS method. Briefly, proteins (VF001 , VF003 and VF004, along with controls including VN alone, IGF-I alone, EGF alone, VN+IGF-I, VN+EGF, VN+IGF-I+EGF and M-VF4-P03 (GMP-grade VF001 ) were added to the wells of a 96 well plate at various doses (15 nM, 50 nM and 150 nM for controls and 50 nM, 150 nM and 450 nM for VF001 , VF003, VF004 and M-VF4-P03 - GMP-grade VF001 ) and incubated for a period of 3 hours (See Table 6 for sample concentration summary). Serum starved (4 hours) and harvested (TrypLE) HCE-T cells were added to pre- incubated proteins/protein mixtures in plate wells and incubated at 37°C for 48 hours, after which MTS reagent was added and cells incubated for a further 2 hours to allow for product development. The plate/s were subsequently read at 490 nm using a plate reader and absorbance's used to quantify cell proliferation in response to treatments. Triplicate experiments were performed with each holding a minimum of 3 replicates per experiment (n=>9). Sample concentrations used in the assays are shown in Table 6. Table 6: Sample concentrations used in cell-based functional assays

Results

Following the procedures set out above, HCE-T cells were treated with various concentrations of test articles including chimeric proteins and controls. Following 48 hours of treatment in SFM, MTS reagent was added to the well to assess cell proliferation. The results of this assay are depicted in Figure 5 and are expressed as corrected (SFM control subtracted) absorbance readings. The assays were performed in a 'solution phase' where treatments were added to wells in a 2x concentration and cells added on top in an equal volume in SFM, the wells were not washed between treatment addition and cell seeding. VF4-P03 was included as a good manufacturing practice (GMP)-grade VF001 reference material that is manufactured using a Pichia pastoris expression system.

As expected IGF-I promoted HCE-T proliferation to a significant degree over the negative control (SFM) at all concentrations tested (15 nM, 50 nM and 150 nM). Interestingly, VN alone was inhibitory in function. The combination of VN + IGF-I (V+l in figure) produced similar results to IGF-I alone (not significantly different at any concentration) while VF001 as VF4-P03 (GMP-grade VF001 ) (VF4 in figure) and insect expressed VN: IGF-I also produced cell proliferation to a similar extent when compared to IGF-I alone, indicating no effect on function of the IGF-I portion by the attached VN domain. It was not expected that EGF would stimulate HCE-T cell proliferation and the data support this as EGF alone and VN + EGF (V+E in figure) were not observed to enhance proliferation above SFM except for the V+E 15 nM treatment. This observed lack of stimulation was replicated in VF003 (VN:EGF in figure) treatments as no concertation was observed to be significantly greater than SFM.

However, the addition of IGF-I to the VN + EGF combination (V+E+l in figure) rescued the stimulatory effect at all concentrations and the treatments were indeed equivalent to IGF-I alone. Furthermore, VF004 (EGF:VN: IGF-I in figure) replicated this observation whereby all concentrations tested were equivalent to IGF-I alone and the VF001 treatments (VF4 and VN: IGF-I) while stimulating significant cell proliferation above SFM. Conclusion

The insect produced chimeric proteins VF001 , VF003 and VF004 were assessed for their ability to induce cell proliferation in HCE-T cells by the MTS method. The results showed that VF001 and VF004 were equivalent to each other and the IGF-I alone control. Thus, it is concluded that the chimeric proteins containing IGF-I retain the full function of the IGF-I domain. VF003 (VN:EGF) was unable to stimulate cell proliferation to the same degree however, this was not unexpected and the migration data outlined in Example 3 may provide a beneficial aspect to the inclusion of EGF in the chimeric constructs.

Example 4

This study was conducted to assess the motogenic potential of VF001 (VN: IGF- I), VF003 (VN:EGF) and VF004 (EGF:VN: IGF-I) in corneal epithelial cells.

Methods

Materials

Purified preparations of VF001 , VF003 and VF004 were generated previously as detailed in Examples 1 and 2. Native human vitronectin (VN) was sourced from Promega, while IGF-I was purchased from Gro-Pep and EGF from Thermo-Fisher Scientific (Invitrogen). The Human Corneal Epithelial cell line HCE-T was obtained from the Queensland Eye Institute, Brisbane under the permission of Dr. Damien Harkin. HCE-T cells were cultured in DMEM (high-glucose, + L-glutamine, + pyruvate) with the addition of 10% FCS (Thermo Fisher Scientific - Gibco) and 1 % Penicillin/Streptomycin (Thermo Fisher Scientific - Invitrogen). Migration fence rings were sourced from Aix Scientific (Germany). In developing migration assays, PFA and Crystal Violet (Sigma Aldrich) were used.

Assessment of the motogenic potential of VF001 , VF003 and VF004 in corneal epithelial cells The expressed and purified proteins were assessed in cell migration assays based on the Fence method. Briefly, fence seeding rings (Aix Scientific) were then inserted into each well and allowed to seal over 1 hr. Serum starved (4 hours) HCE-T cells were harvested using TryPLE (Thermo Fisher Scientific), seeded into the inner chamber at 4 x 10 ⁴ cells/well in SFM+10 pg/mL Mitomycin-C and allowed to attach over 2 hours at 37°C. Fence rings were then removed and the wells washed 2x with SFM before proteins (VF's along with controls including VN alone, VN+IGF-I, VN+EGF, VN+EGF+IGF-I and M-VF4-P03 (GMP-grade VF001 reference standard) in SFM were added to appropriate wells and the plates incubated at 37°C for 48 hours to allow outward migration. After the migration period, the media was aspirated and cells fixed with paraformaldehyde followed by staining with crystal violet (20 minutes in each solution), followed by washing with PBS and air drying away from light. Each well was photographed using a Nikon SMZ-745T stereomicroscope (using a DS-Fi2 camera and DS-U3 control unit) and the images interrogated for the overall area of the cell-occupied space using the inbuilt NIS-Elements software. Protein treatments that stimulate migration will result in cells spreading further from the original seeding area. Triplicate experiments were performed with each holding 2 replicates per experiment (n=6). Sample concentrations used in the assays are shown in Table 7.

Table 7: Sample concentrations used in cell-based functional assays

Results

Assessment of the motogenic potential of VF001 , VF003 and VF004 in corneal epithelial cells.

HCE-T cell migration was performed using the Fence method to assess the ability of treatments to stimulate outward migration of cells over a surface. After the migration period cells were fixed, stained and imaged. The image was analysed for the area taken up by the cells as a complete colony with the boundary tracing the outer reaches of cells (see Figure 7 for example of analysis image). Area data were collected and the average SFM result was subtracted from each other treatment within each individual experiment and these corrected data pooled together from each experiment for the final result as shown in Figure 6.

Interestingly, the VN alone treatments were ineffectual in stimulating migration for the HCE-T cells. Like most cell lines, HCE-T proliferation and migration will be attachment dependent. In the absence of serum in the assay media or the inclusion of an ECM protein such as VN, cells will not attach to the plastic surface of the microtitre plates. Consequently, an IGF-I control was not included. The combination of VN and IGF-I were found to be potent stimulators of HCE-T migration at all concentrations and such responses were replicated with all treatments containing IGF-I - VF4-P03, VN: IGF- I/VF001 , VN + EGF + IGF-I and EGF:VN: IGFI/ VF004 with each concentration of these treatments resulting in migration significantly (p<0.05) above SFM. In addition, the lowest concentrations of VN + EGF and VN:EGF/VF003 produced responses significantly (p<0.05) above SFM, however, higher doses did not maintain this level of stimulation. Comparison of the VF proteins showed that VF4-P03, VF001 and VF004 were similar to each other at respective concentrations. Furthermore, although VF003 results were lower than those observed with VF001 , only at 450 nM was a significant (p<0.05) reduction in response detected.

Conclusion

The Fence assay was chosen for migration as this better represented the desired action of cells regenerating in vivo, that is across a surface rather than through a membrane/tissue as would have been assessed using a Transwell® system for example. The data from the assay demonstrate that VN alone is unable to stimulate HCE-T migration, however, the addition of IGF-I as a combination treatment or in the form of a chimeric protein such as VF001 or VF004 resulted in significant increases in migratory responses.

Example 5 This study was conducted to assess the motogenic potential of VF001 (VN: IGF-

I), VF003 (VN:EGF) and VF004 (EGF:VN: IGF-I) in verified primary human corneal epithelial cells to even more accurately represent the in vivo environment.

Methods

Materials Purified preparations of VF001 , VF003 and VF004 were generated as detailed in

Examples 1 through 2. GMP-grade VF001 Drug Substance, M-VF4-P03, was provided by Eurogentec. Native human vitronectin (VN) was sourced from Promega, while IGF-I was purchased from Gro-Pep and EGF from Thermo-Fisher Scientific (Invitrogen). Primary Human Corneal Epithelial Cells (HCEC) were obtained from the American Type Culture Collection (ATCC, #PCS-700-010). HCECs were cultured in Corneal Epithelial Basal Medium (ATCC, #PCS-700-030) supplemented with Corneal Epithelial Growth Kit (ATCC, #PCS-700-040) and 1 % Penicillin/Streptomycin (Thermo-Fisher Scientific - Invitrogen). In developing proliferation assays, MTS reagent was used (Promega). Migration fence rings were sourced from Aix Scientific (Germany). In developing migration assays, PFA and Crystal Violet (Sigma Aldrich) were used. Accutase Cell Dissociation Solution was purchased from Thermo Fisher Scientific.

The VF001 , VF003 and VF004 proteins were functionally assessed in primary HCEC proliferation assays based on the MTS method. A basal medium formulation of Corneal Epithelial Basal Medium supplemented with Apo-transferrin, Epinephrine, Extract-P, hydrocortisone and L-glutamine (lacking rh Insulin and CE growth factors) was employed as the 'serum-free' negative control and all treatments were prepared in this formulation (termed; Basal medium). Briefly, proteins (VF001 , VF003 and VF004, along with controls including VN alone, IGF-I alone, EGF alone, VN+IGF-I, VN+EGF, VN+IGF-I+EGF and M-VF4-P03 (Internal VF001 Drug Substance Reference Batch) were added to the wells of a 96 well plate at various doses (15 nM for controls and 15 nM, 50 nM, 150 nM and 450 nM for VF001 , VF003, VF004 and M-VF4-P03) and incubated for a period of 3 hours (See Table 8 for sample concentration summary).

Serum starved (3 hours) and harvested (TrypLE) HCECs were added to pre- incubated proteins/protein mixtures in plate wells and incubated at 37°C for 72 hours, after which MTS reagent was added and cells incubated for a further 2 hours to allow for product development. The plate/s were subsequently read at 490 nm using a plate reader and absorbance's used to quantify cell proliferation in response to treatments. Triplicate experiments were performed with each holding a minimum of 3 replicates per experiment (n=>9). Table 8. Sample concentrations used in proliferation assays.

Sample Molarity (nM) Concentration ( g/mL)

The VF001 , VF003 and VF004 proteins were further assessed in HCEC migration assays based on the Fence method. Briefly, fence seeding rings (Aix Scientific) were inserted into each well and allowed to seal over 1 hr at 37°C. Serum starved (3 hours) HCECs were harvested using TryPLE (Thermo Fisher Scientific), seeded into the inner chamber at 4 x 10 ⁴ cells/well in basal medium and allowed to attach over 2 hours at 37°C. Fence rings were then removed and the wells washed 2x with Basal medium before proteins (VF001 , VF003 and VF004 along with controls including VN alone, VN+IGF-I, VN+EGF, VN+EGF+IGF-I and M-VF4-P03 (VitroGro ECM, as a reference standard) in Basal medium (See Table 9 for sample concentration summary) were added to appropriate wells and the plates incubated at 37°C for 48 hours to allow outward migration. After the migration period, the media was aspirated and cells fixed with paraformaldehyde followed by staining with crystal violet (20 minutes in each solution), followed by washing with PBS and air drying away from light. Each well was photographed using a Nikon SMZ-745T stereomicroscope (using a DS- Fi2 camera and DS-U3 control unit) and the images interrogated for the overall area of the cell-occupied space using the inbuilt NIS-Elements software. Protein treatments that stimulate migration resulted in cells spreading further from the original seeding area. Triplicate experiments were performed with each holding 2 replicates per experiment (n=6). Table 9. Sample concentrations used in migration assays.

Sample Molarity (nlUI) Concentration ( g/mL)

Monolayers of HCECs were cultured in growth media (Corneal Epithelial Basal Medium + Corneal Epithelial Growth Kit + 1 % Pen/Strep) within T75 flasks (x6, one for each treatment; Basal medium, Growth medium, VF001 , VF4-P03, VF003 and VF004 each at 50 nM) until cultures were ~80% confluent. Cells were then serum-starved for 3 hours in Basal medium after which treatments were added. Each treatment was applied to an individual T75 flask of HCECs and the cells incubated with treatments for 72 hours. At the completion of the treatments period, cells were harvested using Accutase Cell Dissociation Solution at RT and held separately per treatment. Following counting using Trypan Blue exclusion and a haemocytometer, the cells were fixed in ice-cold Fix buffer on ice for 1 hr. Once fixed, cells were pelleted, washed 2x with DPBS, split into seven (7) equal aliquots per treatment and stained with primary Ab's; a-Pax-6 (1 :50), oc- CK3 (1 : 100), OC-CK12 (1 : 100) and a-CK13 (1 :200) in ice-cold Stain Buffer for 1 hr on ice. Samples of 'unstained' control cell preparations were included for each treatment, these underwent all incubation, wash and centrifugation steps but were incubated in the absence of primary Ab.

Subsequent to staining, cell preparations were again washed with DPBS and oc- Pax-6 samples resuspended in ice-cold Stain buffer and kept on ice for FACS analysis. Unconjugated primary Ab preparations (a-CK3, a-CK12 and a-CK13) were resuspended in ice-cold Stain Buffer containing dilutions of secondary Ab; goat oc- mouse AF488 conjugated (a-CK3 and a-CK13) or goat a-rabbit BV421 (a-CK12) at 1 : 1 ,000 and incubated on ice for 1 hr. 'Secondary only' samples for each Ab were included here, to this point the samples had undergone all incubation, wash and centrifugation steps in the absence of primary Ab. Secondary incubated samples were pelleted and washed with DPBS, finally resuspended in Stain Buffer and kept on ice for FACS analysis.

The FACS Celesta (BD Biosciences) was initiated and prepared as per the manufacturer and facilitator instructions. Cell preparations were run on the machine with detection for side and forward scatter (SSC and FSC) along with appropriate lasers (AF488 and BV421 compatible lasers). Laser intensities were optimised to contain the whole cell fraction within detection parameters and for unstained cells to display fluorescence intensities of ~10 ² units.

Results Mitogenic potential of VF001 , VF003 and VF004 in primary corneal epithelial cells

HCECs were treated with various concentrations of test articles including chimeric proteins and controls according to the above defined methods. Following 72 hours of treatment with test articles and controls in Basal medium, MTS reagent was added to the wells to measure cell proliferation. The results of this assay are depicted in Figure 8 and are expressed as corrected (Basal medium control subtracted) absorbance readings. The assays were performed in a 'solution phase' where treatments were added to wells in a 2x concentration and cells added on top in an equal volume in Basal medium, the wells were not washed between treatment addition and cell seeding. Dissimilar to observations with HCE-T cells, the vitronectin (VN) control stimulated limited proliferation in these primary cells, however, it was the least potent treatments outside of Basal medium alone. As expected IGF-I promoted HCEC proliferation while the combination of VN + IGF-I (V+l in figure) produced similar results to a slightly lesser extent. VF4-P03 batch control and VF001 produced cell proliferation equivalent to IGF-I alone, indicating no clear synergistic effects for the IGF-1 and Vitronectin portions of VF001 in this analysis. It was not expected that EGF would stimulate HCEC cell proliferation and the data supported this as EGF alone and VN + EGF stimulated responses similar to VN alone. VF003 responses were improved over EGF alone and VN+EGF, however were still trailing those of IGF-I, VN+IGF-I, VF4-P03 and VF001 . The addition of IGF-I to the VN + EGF combination was not observed to improve upon VF003, however, VF004 treatments at 15 nM and 150 nM were seen to be close to levels recorded for potent stimulators (IGF-I, VN+IGF-I, VF4-P03 and VF001 ). These results confirm that the most potent stimulant of proliferation is VF001 , although, in this case, VF003 and VF004 were comparatively stronger.

Motogenic potential of VF001 , VF003 and VF004 in primary corneal epithelial cells.

HCEC migration was performed using the fence method to assess the ability of treatments (as per Table 9) to stimulate outward migration of cells over a surface. After the migration period, cells were fixed, stained and imaged. The image was analysed for the area taken up by the cells as a complete colony with the boundary tracing the outer reaches of cells. Area data were collected and the average Basal medium result was subtracted from each other treatment within each individual experiment and these corrected data pooled together from each experiment for the final result as shown in Figure 9. All treatments were found to be significantly above the Basal medium control. Interestingly, the VN alone treatment did not stimulate high levels of migration and was, as per the proliferation assay, the least effective treatment within the experiments. In contrast, the combination of VN and IGF-I was found to be a potent stimulator of HCEC migration to the extent of being significantly above GM (#). Even more potent than the VN+IGF-I combination was the VF001 treatments, providing the greatest migration stimulus of the experiments. As VF001 afforded the greatest proliferative response in the experiments above, analysis here was made on the basis of comparison with molar equivalent VF001 treatments. All treatments except VF004 50 nM were found to be significantly below the VF001 levels ( ^* ), indicating once more that VF001 offers the greatest potential in stimulating HCEC cell motility. These observations are in line with those made for HCE-T cells, however, in this case there is a marked superiority of VF001 over the other candidates.

Example 6

This study was conducted to assess corneal wound healing of VF001 (VN: IGF-I), VF003 (VN:EGF) and VF004 (EGF:VN:IGF-I) in a mouse model.

Study aim/rationale

To evaluate 3 candidate products in a mouse corneal epithelial wound healing model. These 3 candidates have been designed to promote wound closure by stimulating corneal epithelial cell proliferation and migration. Material and methods

Experimental design

Total animais: 7 groups, S C57 mice/group lye dr p ap^ ^

Animals

Species: Mouse (Mus musculus)

Strains or Stocks: C57BL/6J Age: 7-9 weeks old

Weight or Size: 20-25 g

Sex: Male

Source: Invivos, Singapore

Study Details

Animal preparation and anesthesia

Experimentation on mice were performed in accordance with the statement for the use of animals in ophthalmic and vision research approved by the Association for Research in Vision and Ophthalmology. The guidelines of the Animal Ethics Committee of the Singhealth Singapore Association for Assessment and Accreditation of Laboratory Animal Care were also satisfied. Mice were sedated by intraperitoneal injection of ketamine (80 mg /kg body weight) and Xylazine (10 mg/kg) combination. 1 -2 drops of 1 % xylocaine were applied for topical anesthesia to reduce the discomfort.

Induction of corneal epithelial defect:

Animals were anesthetized and the depth of anaesthesia monitored by limb withdrawal using toe pinching. One drop of 1 % Xylocaine (local aesthetic) eye drop was applied. The central cornea was marked by a trephine 2 mm in diameter and the epithelium peeled off using forceps under a dissecting microscope. The corneal epithelial defect was only done in one of the eyes whilst the contralateral eye stays untouched. Wound closure was assessed by fluorescein staining.

Test articles:

Test articles 1 , 2 and 3 were supplied and tested at low concentration (28 μg/mL) and high concentration (280 μg /ml_) where they remained frozen (<-20°C) and thawed only immediately prior to use. Drug labeling details:

Treatment groups

35 mice were randomized to 7 groups (n=5 mice/group) Treatment Route-Topical application

Animals received either placebo (vehicle alone), VF001 (low and high), VF003 (low and high) and VF004 (low and high) in both eyes via topical route twice daily for 6 days starting from Day 0.

Clinical examination:

Visual inspection of all eyes after treatment and signs for any conjunctival irritation, inflammation or infection at cornea was examined daily.

Slit lamp microscopy imaging:

Slit-lamp imaging will be performed 2x daily from Day 1 -3 (to capture any fast healing reaction from the tested solutions), and once daily on Days 0, 4 and 5. The key variable to be evaluated was the corneal wound area. After central cornea wounding, cornea was photographed by fluorescein staining under cobalt blue light at different time points. The wound area was expressed as a percentage of total wounded corneal area. On Day 6, animals were sacrificed and ocular tissues were collected and stored for H&E staining.

Formulation

Formulations comprise 3 distinct recombinant fusion proteins generated using yeast (Pichia pastoris) expression system. Each contain a 64 amino acid domain derived from vitronectin which includes a collagen-binding domain and a cell attachment site (RGD integrin binding motif). These test articles differ in the covalently linked growth factor (either IGF-1 or EGF or both IGF-1 and EGF). All protein-based test articles were formulated at a low concentration (28 μg/mL) and high concentration (280 μg/mL) in Dulbecco's PBS pH 7.2. Consequently, Dulbecco's PBS represents the vehicle or negative control for this study.

VF001 : Vitronectin (1 -64) - (Gly ₄ Ser) ₄ - IGF-1

VF003: Vitronectin (1 -64) - (Gly ₄ Ser) ₄ - EGF

VF004: EGF - (Gly ₄ Ser) ₄ - Vitronectin (1 -64) - (Gly ₄ Ser) ₄ - IGF-1

DPBS: 2.67 mM Potassium Chloride (KCI), 1 .47 mM Potassium Phosphate monobasic (KH ₂ PO ₄ ), 138 mM Sodium Chloride (NaCI), 8.06 mM Sodium Phosphate dibasic (Na ₂ HPO ₄ -7H ₂ O).

Detailed day by day experimental schedule

Animal procedure schedule:

Statistical analysis

Wound healing area was quantified by ImageJ software. Comparisons at each time point between control and test groups were conducted using Student t-test. Significance was set at 0.05 level.

Results

Epithelial Defect as per Fluorescein Staining: 1 . The area of epithelial defect after 5 days post-induction of corneal wounds seems smallest with VF003 treatments, then VF004, followed by VF001 . The group treated with Dulbecco's PBS (Placebo/vehicle) shows the least effect in resolving the epithelial defect as reflected in the area stained with fluorescein at days 4 and 5. (Figures 10 & 13).

2. Area of fluorescein staining is uneven in these groups: VF004 (high concentration) and with VF003 in both high and low concentrations, which indicates wound healing is faster in the VF003 treatment groups and the VF004 high dose group. (Figure 1 1 ).

Rate of Corneal Wound Healing:

1 . Significant reduction in size of wounded corneal area (fluorescein staining) is most obvious at Day 3 in all groups (Figure 13). There is also evidence of wound healing between 2 time points within the same day. Consistent with the overall reduction in size of fluorescein staining area, VF003 is most effective in reducing and progressing corneal wound healing (Figures 16, 17, 18 & 19). High dose VF004 and VF001 also showed increased healing rates relative to the placebo group (Figure 20).

2. The rate of resolution of corneal transparency and clarity seems almost the same in all groups but is best with VF003 (high concentration). (Figures 12 & 14).

Safety:

1 . All test articles tested non-toxic (negative fluorescein staining) when applied to the contralateral eye.

Clinical examination 1 . No ocular phimosis detected in all groups.

2. New vessels were only superficial located at the corneal edge. This was observed for the entire duration of the study (Day 1 -5) in all treatment groups. However, corneal neovascularization was more pronounced in VF001 treated eyes (no scoring was done), both low and high concentration on day 4 & 5. This observation was based on clinical examination during imaging.

Summary and comments

Significant reduction in the size of wounded corneal area (assessed by fluorescein staining) is obvious at Day 3 in all groups. There is also evidence of progress of wound healing between 2 time points within the same day. There is a predictable dose-response benefit with the high dose treatments performing better than the low doses in all 3 treatment groups. However, VF003 is most effective in reducing and progressing corneal wound healing.

The study results show that corneal epithelial wound healing (as measured by area reduction) started from day 2 for the placebo group, however the rate of change for the placebo group decreased following Day 2. The corneal wound healing was slow between Day 0 and Day 2. However, between Day 3 and Day 5 the VF001 , VF003 and VF004 treatments showed more rapid reductions in wound areas. At Day 4 and Day 5, statistically significant differences in mean wound area was observed between VF001 , VF003 and VF004 treatments and the placebo group. Of note, VF003 (high and low dose), VF004 (high dose) and VF001 (high dose) performed better than the placebo.