HK1128479A - Proteins, nucleic acids and medicaments - Google Patents

Description

Protein, nucleic acid and drug

The present invention relates to proteins derived from TGF-beta 3, biologically active fragments of such proteins, and nucleic acids encoding the proteins. The invention also provides derivatives of the proteins or biologically active fragments. The invention also provides medicaments comprising the proteins, fragments, derivatives or nucleic acids of the invention, as well as methods of treatment using the proteins, fragments, derivatives or nucleic acids of the invention.

Transforming growth factor beta (TGF- β) is part of a superfamily of growth factors involved in regulating many cellular processes including proliferation, migration, apoptosis, adhesion, differentiation, inflammation, immunosuppression, and expression of extracellular proteins.

There are 3 mammalian isoforms of TGF-beta, known as TGF-beta 1, TGF-beta 2, and TGF-beta 3.TGF- β is produced by a wide range of cell types including epithelial cells, endothelial cells, hematopoietic cells, neuronal cells and connective tissue cells.

TGF-. beta.s have utility in a variety of different therapeutic contexts, and the TGF-. beta.3 isoform has a number of particularly advantageous therapeutic uses. Due to the therapeutic potential of TGF-. beta.3, its pharmaceutical applications have received much attention. The amino acid sequence of the full-length wild-type TGF-. beta.3 is shown as SEQ ID No. 1, and the cDNA encoding the TGF-. beta.3 is shown as SEQ ID No. 2.

TGF-. beta.3 is known to play a key role in regulating the wound healing response. The activity of TGF- β 3 may affect the rate of wound healing and the extent of scarring resulting from healing.

TGF-beta 3 may also be used to treat fibrotic conditions, pulmonary fibrosis, cirrhosis, scleroderma; angiogenic disorders, restenosis, adhesions, endometriosis, ischemic diseases, bone and cartilage induction, in vitro fertilization, oral mucositis, nephropathy, prevention, reduction or inhibition of scarring, promotion of peripheral and central nervous system neuronal reconnection, prevention, reduction or inhibition of complications of ocular surgery (e.g. LASIK or PRK surgery) or scarring at the back of the eye (e.g. proliferative vitreoretinopathy).

The therapeutic utility of TGF-beta 3 itself has established a recognized need for a source of biologically active TGF-beta 3 protein, and many attempts have been made to produce this valuable protein by recombinant methods. However, the existing methods for producing TGF-. beta.3 are severely limited because the complex protein must be refolded in order to obtain the bioactive molecule.

TGF-. beta.s occur naturally as homodimeric proteins comprising two subunits of 112 amino acids. Each of these TGF- β 3 subunits contains an alpha helix forming domain between residues 58 and 67 of the active peptide fragment. In addition to the alpha helix between residues 58 and 67, each TGF-. beta.3 subunit contains a number of subunit internal bonds, including salt bridges and disulfide bonds.

TGF-. beta.3 is secreted as a potentially inactive precursor molecule of 100-kDa (LTGF-. beta.3). The LTGF- β 3 molecule consists of:

i) a C-terminal 25kDa dimeric signal peptide (active fragment); and

ii) a potentially related peptide (LAP).

LTGF- β is activated by dissociation of LAP from the active fragment. Cleavage of LTGF- β may be mediated by the action of enzymes such as endopeptidases (furin, plasmin and thrombin), or by acidification of the extracellular space. Active TGF- β dimer fragments are stabilized by hydrophobic and ionic interactions that are further enhanced by disulfide bonds between subunits. Each monomer comprises several extended beta strands linked by 3 of 4 internal disulfide bonds and forms a compact structure known as a "cysteine knot".

Due to the complexity of biologically active TGF- β 3 molecules (which, as already shown above, are homodimeric proteins containing 8 intrachain and 1 interchain disulfide bonds), they were originally expressed in eukaryotes. However, the relatively low expression levels possible with eukaryotic expression systems, and the high cost of this approach, mean that the use of microbial hosts is being investigated in order to increase the commercial efficiency of TGF- β 3 production.

A disadvantage of expressing recombinant molecules, such as TGF- β 3, comprising multiple disulfide bonds, with microbial hosts such as e.coli (e.coli) is that the proteins produced are often not correctly folded and often form insoluble inclusion bodies. These inclusion bodies require solubilization followed by renaturation to allow the protein to refold into its native, biologically active conformation. For efficient renaturation of TGF-. beta.3 homodimers, it is necessary to regenerate the correctly oriented covalent disulfide bonds. Given the 9 disulfide bonds, allowing 34,459,245 possible disulfide bond combinations, the probability of forming the correct TGF- β 3 homodimer by a random disulfide bond formation method is very low. It is therefore not surprising that refolding of recombinantly produced TGF- β 3 severely affects its production, as such a fold may take up to 144 hours and typically only about 20% refolding efficiency is achieved.

It is an object of the present invention to obviate or mitigate some of the problems of the prior art. It is an object of certain aspects of the present invention to provide TGF- β 3 (or fragments or derivatives thereof) having improved refolding efficiency compared to wild type TGF- β 3. It is a further object of certain aspects of the invention to provide agents having TGF-beta 3 activity other than wild-type TGF-beta 3. The agents provide a valuable alternative to naturally occurring TGF- β 3.

In a first aspect of the invention there is provided TGF-beta 3, or a fragment or derivative thereof, wherein the alpha helix forming region between amino acid residues 58 and 67 of full length wild type TGF-beta 3 includes at least one alpha helix stabilizing substitution. According to a first aspect of the invention, there is also provided a nucleic acid encoding TGF-. beta.3 or fragments or derivatives thereof.

The inventors of the present invention have surprisingly found that the novel TGF-. beta.3 disclosed in the first aspect of the present invention has the same biological activity as naturally occurring TGF-. beta.3 and a significantly improved efficiency of protein refolding compared to wild type TGF-. beta.3. This increased efficiency of protein refolding constitutes a significant important advantage, as it simplifies the refolding conditions for the production of biologically active TGF-beta 3, and in turn greatly increases the yield of the protein (or fragment or derivative of the protein) that can be produced using prokaryotic protein expression systems.

Without wishing to be bound by any hypothesis, the inventors believe that the introduction of a firm alpha-helix replacement into the alpha-helix forming region may advantageously reduce the flexibility of the alpha-helix formed in this region. This reduced flexibility helps to improve the correct refolding of TGF-beta 3 of the first aspect of the invention for the production of a biologically active protein (or fragment or derivative thereof). The reduced flexibility conferred by the stabilisation of the alpha helix (by substitution of the stabilised alpha helix) is sufficient to increase the yield of correctly refolded TGF-beta 3 (and particularly refolded TGF-beta 3 dimers), however surprisingly the inventors have found that the substitution does not alter the biological activity of TGF-beta 3 of the first aspect of the invention and that the biological and therapeutic efficacy of the TGF-beta 3 is not reduced.

The numbering of amino acid residues in this specification is based on the amino acid sequence of the TGF- β 3 active peptide portion, unless the context requires otherwise. For example, reference to "full-length wild-type TGF-beta 3" is generally used to refer to the amino acid sequence of the active peptide shown in sequence ID No. 1, and a corresponding explanation is provided for the alpha helix forming region between amino acid residues 58 and 67.

One alpha helix-stabilising substitution may preferably comprise a substitution of the glycine residue at position 63 of the full-length wild type TGF-beta 3. However, suitable substitutions may additionally or alternatively include, for example, substitution of one or two threonine at positions 60 or 67 of full-length wild-type TGF-beta 3, or substitution of asparagine at position 66 of full-length wild-type TGF-beta 3. Preferably, the alpha helix-stabilising substitutions used in the present invention do not include a substitution of valine 61.

The "substitution to stabilize the alpha helix" according to the invention is understood to mean the following substitution: wherein a specified amino acid residue present in wild-type TGF-beta 3 is replaced with a residue that is more prone to form an alpha helix. Thus, a replacement amino acid residue that introduces a substitution that stabilizes the alpha helix need not be an amino acid residue that readily stably integrates itself into the alpha helix, but need only have a greater propensity for stable integration than the amino acid residue being substituted. However, it is generally preferred that the replacement amino acid residue introduced as part of the alpha helix-stabilising substitution is one which does favour integration into the alpha helix.

The inventors of the present invention have found that preferred alternative amino acid residues that may be introduced into the alpha helix-stabilising substitution of the first aspect of the invention may be any one or combination of amino acids selected from the group consisting of: alanine, serine, threonine, valine, leucine, isoleucine, methionine and phenylalanine. These preferred alternative amino acid residues are all believed to be suitable for robust alpha-helical substitution of the glycine residue at position 63 of full-length wild-type TGF-beta 3. That is, a replacement amino acid residue selected from the group may be substituted at any position in the alpha helix forming region between amino acid residues 58 and 67, where the replacement amino acid residue may provide a substitution that stabilizes the alpha helix.

Although the above list of amino acid residues represents the preferred residue for substitution to stabilize the alpha helix, it should also be understood that: there are several alternative qualitative and quantitative systems by which the propensity of amino acid residues to contribute to alpha helix formation (and the suitability of the substituted residues for alpha helix stabilisation) can be determined, and suitable substituted amino acid residues for alpha helix stabilisation can be selected with reference to any of these systems and in conjunction with knowledge of the TGF-beta 3 sequence.

By way of example, the qualitative system described by Chou and Fasman identified 5 different classes of amino acid residues based on their propensity to form alpha helices. They are in turn:

forming a strong spiral shape;

forming a weak spiral shape;

a neutral form;

weak helix failure type; and

and (3) a strong spiral failure type.

For the purposes of the present description, the amino acid residues glutamic acid, histidine, tryptophan, lysine, alanine, methionine, valine, isoleucine, leucine, glutamine and phenylalanine may be considered to be helical formations, with glutamine, methionine, alanine and leucine constituting strong helical formations. In contrast, asparagine, glycine, and proline can be considered to constitute helix-disrupted forms, with glycine and proline being strong helix-disrupted forms.

Thus, if the propensity for alpha helix formation is assessed with reference to this qualitative scale, it should be recognized that, although alpha helix-stabilising substitutions may be preferred where amino acid residues are replaced by helix-forming substitutions, suitable alpha helix-stabilising substitutions may alternatively utilise neutral-forming or even helix-disrupting forms, depending on the nature of the amino acid residue to be replaced. For example, where the neutral form of an amino acid residue is the subject of a substitution that stabilizes the alpha helix, a suitable replacement amino acid residue may be a strong helical conformation or a weak helical conformation. In the case where the strong helix-disrupted form is the subject of a substitution that stabilizes the alpha helix, the replacement amino acid residue may be a strong helix-shaped form, a weak helix-shaped form, a neutral form amino acid, or a weak helix-disrupted form.

Accordingly, the replacement of a strong alpha helix according to the invention may comprise the replacement of a strong helix failure mode by a weak helix failure mode, or a neutral mode, or a weak helix formation or a strong helix formation. Alternatively or additionally, suitable replacement of the stabilized alpha helix may include replacement of the weak helix breaking form by a neutral form, a weak helix forming, or a strong helix forming. Alternatively or additionally, suitable alpha helix-stabilizing substitutions may include substitutions of neutral form amino acids by weak or strong helical formation. Alternatively or additionally, suitable displacement of the stabilizing alpha helix may include displacement of a weak helical formation by a strong helical formation.

An alternative assessment of the propensity of an amino acid residue to form an alpha helix, and thus its suitability for use as a replacement moiety to stabilise an alpha helix, is based on any quantitative series known to those skilled in the art.

Examples of such quantitative scales for determining the suitability of amino acid residues for substitutions that stabilize the alpha helix are shown in table 1. The table provides values on the kcal/mol scale reflecting the propensity of amino acids to contribute to the alpha helix. The high values in table 1 are accompanied by a low tendency to form alpha helices.

Thus, when assessing the suitability of an amino acid residue for alpha helix-stabilizing substitution using a quantitative scale (as shown in table 1) as required by the first aspect of the present invention, a suitable alpha helix-stabilizing substitution is one in which the amino acid residue is substituted with an amino acid residue having a greater propensity for helix formation (lower kcal/mol value shown in table 1) than the substituted residue.

Suitable substitutions that may be utilized in accordance with the present invention include those that introduce artificial replacement amino acids. Suitable examples of artificial amino acids that may be advantageously used to stabilize the alpha helix include amino acid residues having alkyl and hydroxyl side chains.

Alanine represents a particularly preferred alternative amino acid residue for substitution suitable for stabilizing the alpha helix. Most preferably, the TGF- β 3 or fragment or derivative thereof of the first aspect of the invention comprises a substitution of the glycine at position 63 of the full length wild type TGF- β 3 with an alanine.

A preferred TGF-. beta.3 of the first aspect of the present invention has an amino acid sequence shown by SEQ ID No. 3 (Gly-63Ala), and a DNA encoding the TGF-. beta.3 has an amino acid sequence shown by SEQ ID No. 4. Fragments or derivatives of TGF-beta 3 of sequence ID No. 3 which contain a substitution of the alanine at position 63 of the full length wild type TGF-beta 3 represent preferred fragments or derivatives of TGF-beta 3 of the first aspect of the invention.

Suitable substitutions may be those in which one or more amino acid residues between 58 and 67 in the full length wild type TGF-beta 3 are replaced by one or more natural or artificial amino acid residues. By way of further illustration, suitable substitutions may include the substitution of a single amino acid residue with one or more replacement residues, or the substitution of more than one amino acid residue with one or more replacement residues. Preferred substitutions may be those in which the number of amino acid residues is conservative, i.e., in which the number of substituted amino acid residues is the same as the number of introduced replacement amino acid residues.

It will also be appreciated that the preferred amino acid residue (or residues) to be replaced may be selected with reference to a qualitative or quantitative scale as discussed above. Thus, amino acids suitable as objects of alpha helix-stabilising substitutions may be classified as helix-disrupting, or preferably strong helix-disrupting, with reference to the qualitative ratings as discussed above. With reference to the quantitative scale shown in table 1, amino acids suitable as subjects for alpha helix-stabilizing substitutions may preferably be those having a helix propensity value greater than or equal to 0.50, more preferably greater than or equal to 0.60, and most preferably a helix propensity value of 1.00.

The inventors believe that TGF-. beta.3, or a biologically active fragment or derivative thereof, of the first aspect of the invention may be used in all circumstances where it is desirable to utilise the biological activity of wild-type TGF-. beta.3. These environments include particularly, but are not limited to, therapeutic uses. Consistent with the described therapeutic uses, the present invention also provides the use of TGF-. beta.3, or fragments or derivatives thereof, according to the first aspect of the invention as a medicament.

It will be appreciated that although it may be preferred that the fragment or derivative of TGF-beta 3 of the first aspect of the invention includes the full length alpha helix forming region containing a substitution to stabilise the alpha helix, this need not necessarily be the case. Suitable fragments or derivatives may include truncated alpha helix forming regions, so long as the truncated alpha helix forming region comprises at least one alpha helix stabilizing substitution.

In a second aspect the invention provides TGF-beta 3 or a fragment or derivative thereof wherein the glycine residue at position 63 of the full length wild type TGF-beta 3 is replaced by proline. The invention also provides a nucleic acid encoding a TGF-beta 3 of the second aspect of the invention, or a fragment or derivative thereof.

The inventors have surprisingly found that the protein of the second aspect of the invention (or a fragment or derivative thereof) has biological activity comparable to wild-type TGF-beta 3. This finding was unexpected because it is believed that the presence of proline in the TGF- β 3 region normally associated with α -helix formation would interfere with the secondary structure of this protein, thereby impairing its biological function. Although the efficiency of refolding TGF-beta 3 of the second aspect of the invention is lower than wild-type TGF-beta 3, the expected impairment of function surprisingly does not occur.

The proteins of the second aspect of the invention (or fragments or derivatives thereof) therefore provide a valuable contribution to the art as they extend all the components of compounds useful to the skilled person which are capable of exerting TGF- β 3 activity. The compounds may be used, for example, in environments where therapeutic use of TGF-beta 3 activity is required.

Preferred TGF-. beta.3 amino acid sequences of the second aspect of the present invention are shown by SEQ ID No. 5 (Gly-63Pro) and the DNA encoding the TGF-. beta.3 is shown by SEQ ID No. 6. Fragments of TGF-beta 3 or derivatives thereof of sequence ID No. 5 containing a substitution of the proline at position 63 in full-length wild-type TGF-beta 3 represent preferred fragments of TGF-beta 3 or derivatives thereof of the second aspect of the invention.

If TGF-beta 3 of the second aspect of the invention, or a fragment or derivative thereof, may be used in an environment in which it is desirable to therapeutically utilise the biological activity of wild-type TGF-beta 3, it will be appreciated that the use of TGF-beta 3 of the second aspect of the invention, or a fragment or derivative thereof, as a medicament is also provided. The inventors believe that the drug may be used in all clinical settings where it is known to utilise the biological activity of TGF- β 3.

In a third aspect of the invention there is provided TGF-beta 3 or a fragment or derivative thereof comprising a substitution of the glutamic acid residue at position 12 of full length wild type TGF-beta 3 and/or a substitution of the arginine residue at position 52 of full length wild type TGF-beta 3. The invention also provides a nucleic acid encoding TGF-beta 3 or a fragment or derivative thereof according to the third aspect of the invention.

It will be appreciated that the third aspect of the invention therefore includes TGF-beta 3 in which the glutamic acid at position 12 of the full length wild type TGF-beta 3 is replaced but the arginine at position 52 of the full length wild type TGF-beta 3 is retained. The third aspect of the present invention also includes TGF-beta 3 in which the glutamic acid at position 12 of full-length wild-type TGF-beta 3 is retained but the arginine at position 52 of full-length wild-type TGF-beta 3 is replaced.

Preferably, however, TGF- β 3 of the third aspect of the invention includes a substitution of the glutamic acid residue at position 12 in full-length wild type TGF- β 3 and a substitution of the arginine residue at position 52 in full-length wild type TGF- β 3.

The inventors have surprisingly found that the protein of the third aspect of the invention also has a biological activity which is comparable to the biological activity of wild-type TGF-beta 3. This finding was unexpected because the substitution of one or both of the glutamic acid residue at position 12 in full-length wild-type TGF- β 3 and/or the arginine residue at position 52 in full-length wild-type TGF- β 3 disrupted the formation of salt bridges within the subunits normally present in wild-type TGF- β 3. This failure to achieve correct salt bridge formation may be expected to reduce the biological activity of TGF-. beta.3 of the third aspect of the invention, since the biological activity of proteins such as TGF-. beta.3 is generally considered to be dependent on their conformation.

Furthermore, the inventors have found that TGF-. beta.3 of the second aspect of the invention exhibits an efficiency of successful refolding which is as high as that observed with wild-type TGF-. beta.3. This finding is highly surprising, since it is believed by those skilled in the art that the absence of subunit internal salt bridge formation that must occur in TGF- β 3 according to the second aspect of the invention can adversely affect the incidence of refolding and thus reduce the yield of biologically active TGF- β 3.

The protein of the third aspect of the invention therefore serves to extend all the components of a compound useful to the skilled person which is capable of exerting TGF- β 3 activity. As noted above, the availability of the compounds is important in environments where therapeutic use of TGF- β 3 activity is required.

The inventors of the present invention have found that either the glutamic acid at position 12 in full-length wild-type TGF- β 3 or the arginine at position 52 in full-length wild-type TGF- β 3 can be substituted by any one of (or any combination of) amino acid residues selected from the following group: serine, alanine, threonine, valine, isoleucine, methionine, phenylalanine, and leucine.

Preferably, serine is used as the replacement amino acid residue for TGF-beta 3 or a fragment or derivative thereof according to the third aspect of the invention. Serine may be used to replace the glutamic acid at position 12 in full-length wild-type TGF-beta 3 or the arginine at position 52 in full-length wild-type TGF-beta 3. Most preferably, serine is used to replace both the glutamic acid at position 12 in full-length wild-type TGF-beta 3 and the arginine at position 52 in full-length wild-type TGF-beta 3.

A first example of a preferred TGF-. beta.3 of the third aspect of the invention is shown in SEQ ID No. 7. The invention encompasses biologically active fragments or derivatives of sequence ID No. 7, which sequence ID No. 7 includes the Glu12-Ser substitution. The cDNA encoding the preferred TGF-. beta.3 is shown in sequence ID No. 8.

A second example of a preferred TGF-. beta.3 according to the third aspect of the invention is shown in SEQ ID No. 9. A biologically active fragment or derivative of sequence ID No. 9 comprising the Arg52-Ser substitution also constitutes a preferred fragment or derivative of the invention. The cDNA encoding the preferred TGF-. beta.3 is shown as sequence ID No. 10.

A third example of a preferred TGF-. beta.3 according to the third aspect of the invention is shown in SEQ ID No. 11. The biologically active fragment or derivative of sequence ID No. 11 comprising the Glu12-Ser substitution and the Arg52-Ser substitution also constitutes a preferred fragment or derivative of the invention. The cDNA encoding the preferred TGF-. beta.3 is shown as sequence ID No. 12.

The inventors believe that TGF-beta 3, or a biologically active fragment or derivative thereof, of the third aspect of the invention may be used in all circumstances where it may be desirable to utilise the biological activity of wild-type TGF-beta 3. These environments include, but are not limited to, therapeutic uses of TGF-beta 3. Accordingly, the invention also provides the use of TGF-beta 3 or fragment or derivative thereof according to the third aspect of the invention as a medicament.

TGF-. beta.3 or a biologically active fragment or derivative thereof of the present invention may be used to treat wounds (including chronic wounds such as ulcers). They are particularly useful in promoting accelerated wound healing, and/or promoting epithelial regeneration of wounds, by preventing, reducing or inhibiting scarring. TGF- β 3 of the invention may also be used to effect prophylaxis or treatment of fibrotic diseases, which may be independently selected from the group comprising pulmonary fibrosis, cirrhosis, scleroderma and glomerulonephritis, pulmonary fibrosis, hepatic fibrosis, dermal fibrosis, muscle fibrosis, radiation fibrosis, kidney fibrosis, proliferative vitreoretinopathy and uterine fibrosis.

TGF-. beta.3 s of the invention may be used to treat scleroderma, angiogenic disorders, restenosis, adhesions, endometriosis, ischemic disease, induction of bone and cartilage, in vitro fertilization, oral mucositis and renal disease. As an example, topical application of wild-type dimeric TGF- β 3 in animal models and in clinic has been shown to accelerate the rate of healing of chronic, non-healing pressure ulcers; reduces the incidence, severity and duration of oral mucositis; and reduces the adverse side effects of radiation gastrointestinal syndrome which results from stem cell damage caused by radiation and chemotherapy in the treatment of cancer. The inventors believe that TGF-beta 3 of the invention, or fragments or derivatives thereof, may be used beneficially in all of these indications.

TGF-. beta.3 of the invention may be used, for example, in the same manner as naturally occurring TGF-. beta.3 in the treatment of a condition which may, for example, be independently selected from: fibrotic diseases, scleroderma, angiogenic disorders, restenosis, adhesions, endometriosis, ischemic diseases, induction of bone and cartilage, in vitro fertilization, oral mucositis, renal disease, prevention, reduction or inhibition of scarring, promotion of peripheral and central nervous system neuronal reconnection, and prevention, reduction or inhibition of complications of ocular surgery (e.g. LASIK or PRK surgery). TGF-. beta.3 s of the invention may be used to treat cleft lip and palate (e.g. in conjunction with surgical repair of this state), and to reduce or inhibit scarring and promote healing of tendons. The disclosed mutant forms of TGF-beta 3 are capable of promoting accelerated wound healing and/or preventing, reducing or inhibiting scarring in the same manner as naturally occurring TGF-beta 3. They may also promote epithelial regeneration at the site of epithelial cell injury.

"TGF-beta 3 of the invention" includes any mutant TGF-beta 3 of any of the first, second or third aspects of the invention. It should be understood that TGF-. beta.3 of the present invention does not include TGF-. beta.1 or TGF-. beta.2. The identity of TGF-. beta.3 may be determined by reference to its sequence or preferably by reference to its biological activity. TGF-beta 3 may therefore be distinguished from TGF-beta 1 or TGF-beta 2 based on being able to reduce scarring at the wound to which TGF-beta 3 is administered. TGF-. beta.3 of the present invention may preferably be non-native TGF-. beta.3.

TGF-beta 3 of any aspect of the invention may be used in the manufacture of a medicament for use in the treatment of any condition in which the use of TGF-beta 3 may be desirable. Such uses include, but are not limited to, treating any of the conditions contemplated in this specification. It may be preferred that TGF- β 3 of the invention is used in the manufacture of a medicament for use in promoting accelerated wound healing, and/or preventing, reducing or inhibiting scarring. The scarring may be associated with a wound and/or fibrotic disease. Medicaments produced with TGF-beta 3 s of the invention may preferably be applied to the skin, or eye (e.g. to accelerate healing of the skin or eye, or to prevent, reduce or inhibit scarring of the skin or eye).

TGF-. beta.3 of the invention may be latent TGF-. beta.3 or active TGF-. beta.3 (e.g., with or without a potentially related peptide).

Unless the context requires otherwise, all references to TGF-beta 3 of the invention are to be taken as including fragments of said TGF-beta 3 or derivatives thereof, wherein said fragments or derivatives are characterised in that they include substitutions (suitably in accordance with the first, second or third aspect of the invention) which distinguish them from fragments of wild type TGF-beta 3 or derivatives thereof whose amino acid sequence is represented by sequence ID number 1 within the contemplation of the invention. Suitable fragments of TGF- β 3 or derivatives thereof of the first, second or third aspects of the invention may comprise at least 10 amino acid residues, preferably at least 40 amino acid residues, more preferably at least 70 amino acid residues, most preferably at least 100 amino acid residues.

Unless the context requires otherwise, references to TGF-beta 3 and fragments of TGF-beta 3 in the present specification also include derivatives of such proteins or fragments.

Without limitation, suitable examples of suitable forms of the derivative may be selected from: a therapeutically effective peptide derivative of TGF-beta 3 (or fragment thereof) of the invention, a therapeutically effective fragment or derivative including or based on the pharmacophore of TGF-beta 3 (or fragment thereof) of the invention, a therapeutically effective peptoid derivative of TGF-beta 3 (or fragment thereof) of the invention, a therapeutically effective D-amino acid derivative of TGF-beta 3 (or fragment thereof) of the invention, a therapeutically effective peptidomimetic based on TGF-beta 3 (or fragment thereof) of the invention, a therapeutically effective peptide analog of TGF-beta 3 (or fragment thereof) of the invention, a therapeutically effective pseudopeptide based on TGF-beta 3 (or fragment thereof) of the invention, a therapeutically effective trans-peptide based on TGF-beta 3 (or fragment thereof) of the invention, a therapeutically effective depsipeptide derivative based on TGF-beta 3 (or fragment thereof) of the invention, a therapeutically effective beta-peptide derivative based on TGF-beta 3 (or fragment thereof) of the invention, and a therapeutically effective beta-peptide derivative based on TGF-beta 3 (or fragment thereof) of the invention -a therapeutically effective retro-peptoid derivative of β 3 (or a fragment thereof).

It will be appreciated that for purposes of the present invention, "TGF-beta 3" may include both monomeric and dimeric forms of TGF-beta 3. The inventors of the present invention have surprisingly found that TGF-. beta.3 of the present invention exerts its biological effects both in monomeric and dimeric form. This is surprisingly in contrast to previous reports of the prior art, which generally held that TGF- β, such as TGF- β 3, can only exert biological activity in the dimeric form. The inventors' discovery that TGF-beta 3 of the present invention may be used in monomeric form provides a great advantage in that the monomeric form may be produced by relatively simple folding techniques (as further discussed below in the examples), thus increasing the rate of production of the biologically active molecule, while also reducing the costs associated with the production of the molecule.

Preferably, the monomeric TGF-. beta.3 of the invention is TGF-. beta.3 shown in SEQ ID Nos. 3, 5, 7, 9 or 11, or a fragment or derivative thereof.

"the agent of the present invention" includes any agent of TGF-. beta.3 of the present invention. The medicament of the invention may additionally or alternatively be a medicament comprising a nucleic acid encoding a TGF-beta 3 of the invention. The medicament includes both the medicament itself (i.e., irrespective of the use of the medicament) and the medicament for a particular therapeutic application (e.g., treating or ameliorating a condition contemplated in the present specification). The agents of the invention are intended to be understood to include agents comprising suitable fragments of TGF-beta 3 of the invention or derivatives thereof, or nucleic acids encoding said fragments or derivatives.

A "method of treatment of the invention" (or "method of the invention") is considered to include any method of treatment which utilises a therapeutically effective amount of a TGF-beta 3 of the invention, or a nucleic acid encoding said TGF-beta 3. The therapeutic methods of the invention are also intended to be understood to include therapeutic methods that utilize a suitable fragment or derivative of TGF-beta 3 of the invention, or nucleic acid encoding such a fragment or derivative.

Medicaments comprising TGF-beta 3 or a biologically active fragment or derivative thereof of the invention may be used to treat wounds, including chronic wounds such as ulcers. They are particularly useful in promoting accelerated wound healing, and/or promoting re-epithelialization at wounds, by preventing, reducing, or inhibiting scarring. TGF- β 3 of the invention may be used to effect prophylaxis or treatment of fibrotic diseases, such as pulmonary fibrosis, cirrhosis and fibrosis, scleroderma, glomerulonephritis, skin fibrosis, radiation fibrosis, kidney fibrosis, proliferative vitreoretinopathy or uterine fibrosis.

TGF- β 3 of the invention may be used to treat a condition independently selected from: scleroderma, angiogenic disorders, restenosis, adhesions, endometriosis, ischemic diseases, induction of bone and cartilage, in vitro fertilization, oral mucositis, nephropathy, pulmonary fibrosis, cirrhosis and fibrosis of the liver, glomerulonephritis, dermal fibrosis, radiation fibrosis, kidney fibrosis and uterine fibrosis. By way of example, topical application of wild-type, dimeric TGF- β 3 in animal models and in clinic has been shown to accelerate the rate of healing of chronic, non-healing pressure ulcers; reducing the incidence, severity and duration of oral mucositis; reducing the adverse side effects of radiation gastrointestinal syndrome resulting from stem cell damage caused by radiation and chemotherapy in the treatment of cancer. The inventors believe that TGF-beta 3 of the invention, or fragments or derivatives thereof, may be used beneficially in all of these indications.

The biological activity exhibited by TGF-beta 3 or fragments or derivatives thereof of the invention may preferably be that of TGF-beta 3, which anti-scarring activity may preferably be studied in vivo.

A therapeutically effective amount of TGF-beta 3 or fragment or derivative thereof of the present invention is an amount sufficient to cause the following requirements:

i) accelerating wound healing and/or inhibiting scarring; or

ii) promoting epithelial regeneration; or

iii) preventing and/or treating fibrotic diseases.

The degree of acceleration of wound healing and/or inhibition of scarring or epithelial regeneration that may be required is evident to, or indeed may be readily determined by, for example, the clinician responsible for caring for the patient. Suitable assessments of the extent to which wound healing is accelerated and/or scarring inhibited or epithelial regeneration is promoted may be determined by a clinician and reference may be made to the suggested methods of measurement described herein.

Suitable TGF-. beta.3 s of the invention, as well as preferred fragments or derivatives of such TGF-. beta.3 s, may be selected with reference to any or all of the considerations described herein.

The ability of TGF-. beta.3 of the present invention to accelerate wound healing is readily understood and/or measured with reference to the properties exhibited by treated wounds. For the present purposes, a "treated wound" can be considered a wound exposed to a therapeutically effective amount of a medicament of the present invention or a wound that has been treated according to a method of the present invention.

The acceleration of healing of a treated wound may be indicated by an increased rate of epithelialization compared to a control wound. Thus, the methods and medicaments of the present invention promote a faster reconstruction of the functional epithelial layer over the wound area than would otherwise be possible.

Alternatively or additionally, the acceleration of healing of a treated wound may be represented by a reduced width at the corresponding time point compared to a control wound. It will be appreciated that this reduction in wound width ensures a relatively fast rate of wound closure (due to the smaller width of the wound to be closed), suggesting the ability of these drugs to accelerate the healing response. A narrower wound may result in a narrower scar that is aesthetically superior to a wider scar.

Thus, accelerated wound healing in the context of the present invention should be taken to include any increase in the rate of wound healing of a treatment as compared to the rate of healing that occurs in a control treated or untreated wound. Preferably, the acceleration of wound healing is assessed in terms of a comparison of the re-epithelialization rates obtained in the treated and control wounds, or in terms of a comparison of the relative widths of the treated and control wounds at corresponding time points. More preferably, accelerated wound healing may be defined as comprising an increased rate of re-epithelialization and a decrease in wound width at the corresponding time point as compared to a control wound.

Preferably, the acceleration of wound healing is promoted to produce a wound healing rate that is at least 5%, 10%, 20%, or 30% greater than the rate of healing that occurs in a control or untreated wound. More preferably, the acceleration of wound healing promotion may result in a healing rate that is at least 40%, 50% or 60% greater than the healing of a control wound. Even more preferably, the accelerated wound healing may result in a healing rate that is at least 70%, 80%, or 90% higher than that occurring in a control wound, and most preferably, the accelerated wound healing may result in a healing rate that is at least 100% higher than that occurring in a control wound.

There is a wide range of wound healing disorders characterized, or at least partially characterized, by an inappropriate failure, delay or obstruction of the normal wound healing response. Thus, the ability of certain methods and agents of the invention to promote accelerated wound healing is useful in the prevention or treatment of these diseases.

It will be appreciated that certain methods and medicaments of the invention are particularly advantageous for the treatment of wounds in patients prone to defects, delays or other forms of injury due to the acceleration of wound healing by promoting activation of the re-epithelialization response (thus increasing the rate of wound closure). For example, it is well known that skin wounds in the elderly exhibit a weaker re-epithelialization response than skin wounds in younger individuals. There are also many other conditions or diseases in which wound healing is associated with delayed or otherwise impaired re-epithelialization. For example, patients with diabetes, combination medication (e.g., due to aging), postmenopausal women, patients prone to stress injury (e.g., paraplegic patients), patients with venous disease, clinically obese patients, patients receiving chemotherapy, patients receiving radiation therapy, patients receiving steroid therapy, or patients with immunocompromised function may all experience wound healing with re-formation of damaged epithelium. In many such cases, the lack of an appropriate re-epithelialization reaction contributes to the development of infection at the wound site, which may in turn contribute to the formation of chronic wounds such as ulcers. It will therefore be appreciated that these patients are particularly likely to benefit from a suitable method or medicament of the invention.

Chronic wounds are perhaps the most important example of a disease associated with delayed wound healing response. A wound is defined as chronic if it does not show any propensity to heal within eight weeks of formation when subjected to appropriate (conventional) therapeutic treatment. Well-known examples of chronic wounds include venous ulcers, diabetic ulcers and pressure sores, but chronic wounds can be triggered at any time by otherwise normal acute injury. In general, chronic wounds can be caused by infection at the wound site, improper wound treatment, or as a result of progressive tissue failure due to venous, arterial, or metabolic vascular disease, compression, radiation injury, or tumor.

It will be appreciated that the methods and medicaments of the present invention which accelerate wound healing can be applied to existing treatments of chronic wounds with the aim of promoting their healing. These methods and medicaments can promote re-epithelialization of chronic wounds, leading to healing and termination of the disease. Preferred methods and medicaments of the invention (e.g., those using TGF-beta 3 including sequence ID numbers 3, 5, 7, 9, or 11) also inhibit scarring associated with wound healing. Prevention of scarring is particularly advantageous in these situations, as chronic wounds can typically extend over a relatively large portion of the patient's body.

Alternatively or in addition to its use in existing chronic wound therapy, suitable methods and medicaments of the invention may be used to prevent acute wounds in patients with a propensity for impaired wound healing to progress to chronic wounds. Because suitable methods and agents of the invention promote epithelial coverage of the injury site, they reduce the likelihood of infection of the treated wound. Likewise, such promotion of re-epithelialization may be beneficial in the treatment of chronic wounds resulting from other conditions such as diabetes or venous disease.

Another group of patients that may benefit particularly from the methods and medicaments of the invention are those patients with a low immune system (e.g., patients undergoing chemotherapy or radiation therapy or suffering from HIV infection). It is widely recognized that immunocompromised patients may not be able to develop a normal inflammatory response after wound generation, which wounds tend to be associated with poor healing outcomes. These patients may benefit from suitable methods and drug treatments of the present invention.

TGF- β 3 of the invention (such as those comprising sequence ID numbers 3, 5, 7, 9 or 11) promotes accelerated wound healing, whilst the ability to prevent, reduce or inhibit scarring is also useful in a more general clinical setting. Examples of these further benefits may be judged with reference to wound healing for primary, secondary or tertiary healing as described below.

For the purposes of the present invention, primary healing therapy may be considered to involve closure of opposing edges of the wound with surgical means (e.g., sutures, adhesive tape or staples). Primary healing treatments are typically used for surgical incisions or other treatments that clean wounds, and are associated with minimal levels of tissue loss. The skilled person realizes that as TGF- β 3 s of the invention (e.g. those comprising sequence ID numbers 3, 5, 7, 9 or 11) can reduce wound width, they also facilitate the joining of opposing edges of the wound, which may be beneficial in wound healing for primary healing. Moreover, these methods or medicaments (as described further below) result in the prevention, reduction, or inhibition of scarring that may otherwise occur with such healing. The inventors believe that treatment in this manner can have an effect on the macroscopic and microscopic appearance of the treated wound scarring; macroscopic scars may be less noticeable and fuse with the surrounding skin, and microscopic scars may exhibit more regeneration of normal skin structures.

For the purposes of the present invention, secondary healing therapy may be considered to include wound closure during the wound healing process without direct surgical intervention. Wounds to be treated for secondary healing may be continuously cared for (e.g. dressing and repacking of wounds and application of suitable drugs), but it is a natural process of granulation tissue formation and re-epithelialization, resulting in wound closure. It will be appreciated that TGF- β 3 s of the invention (e.g. those comprising sequence ID numbers 3, 5, 7, 9 or 11) are useful in promoting secondary healing wound healing as they increase the rate of re-epithelialisation compared to that which occurs in control wounds.

Tertiary healing therapy may be considered to involve surgical closure of a previously open wound, allowing at least partial granulation tissue formation and re-epithelialization. The characteristics of the preferred methods and medicaments of the present invention that make them suitable for primary or secondary healing therapy applications are also beneficial in promoting tertiary healing in treating wounds.

The use of TGF- β 3 (e.g. sequence ID nos. 3, 5, 7, 9 or 11) of the invention to promote re-epithelialization as part of their promotion of accelerated wound healing while inhibiting scarring is also particularly effective in the treatment of wounds associated with transplantation procedures. Treatment with these methods and agents of the present invention is beneficial to both the graft donor site (where it can assist in the reconstruction of a functional epithelial layer while preventing, reducing, or inhibiting scarring) and the graft recipient site (where the anti-scarring effect of treatment inhibits scarring while accelerated healing promotes integration of the graft tissue). The present inventors believe that the methods and medicaments of the present invention provide advantages in terms of implantation with skin, artificial skin or skin substitutes.

The present inventors have found that the methods and medicaments of the invention, which utilize TGF- β 3 (including sequence ID numbers 3, 5, 7, 9 or 11), when administered prior to wound creation or when a wound has formed, promote accelerated wound healing and inhibit scarring.

The inventors have found that the methods or medicaments of the invention using TGF- β 3 (e.g. those comprising sequence ID numbers 3, 5, 7, 9 or 11) promote epithelial regeneration. Promoting epithelial regeneration in the context of the present invention is understood to include any increase in the rate of epithelial regeneration as compared to regeneration occurring in control treated or untreated epithelia.

The rate of epithelial regeneration achieved using a suitable method or agent of the invention can be readily compared to the rate achieved with control treated or untreated epithelia, and this can be done using any suitable model of epithelial regeneration known in the art. For example, the rate of regeneration at an experimental epithelial injury site of known area can be compared using well-known mouse, rat, rabbit or pig in vivo models, such as those described in Tomlinson & Ferguson (2003), Davidson et al (1991), and Paddock et al (2003).

The inventors do not wish to be bound by any hypothesis, and believe that the promotion of epithelial regeneration by TGF-. beta.3 of the invention is mediated by the promoting effect of epithelial cell migration. Epithelial cells (whose migration has been promoted) are thus able to reconstitute and regenerate damaged epithelium more rapidly than untreated epithelia.

Understandably, promotion of epithelial regeneration using TGF- β 3 of the invention may be useful to induce effective re-epithelialization in cases where the re-epithelialization response is impaired, inhibited, delayed, or otherwise deficient. Promotion of epithelial regeneration may also be effective in accelerating the rate of a defective or normal epithelial regeneration response in a patient experiencing epithelial injury.

In many cases, the re-epithelialization reaction of the body may be defective. For example, defects in skin re-epithelialization may be associated with conditions such as pemphigus, Hailey-Hailey disease (familial benign pemphigus), Toxic Epidermal Necrolysis (TEN)/Lyell's syndrome, epidermolysis bullosa, cutaneous leishmaniasis, and actinic keratosis. Defects in lung re-epithelialization may be associated with primary pulmonary fibrosis (IPF) or pulmonary interstitial disease. Defects in ocular re-epithelialization may be associated with conditions such as partial limbal stem cell loss or corneal erosion. Defects in re-epithelialization of the gastrointestinal tract or colon may be associated with conditions such as chronic anal fissure (anal fissure), ulcerative colitis or Crohn's disease, and other inflammatory bowel diseases.

As stated above, TGF-beta 3 of the invention may be used to prevent, reduce or otherwise inhibit scarring. This inhibition of scarring can be achieved in any body area and in any tissue or organ, including skin, eyes, nerves, tendons, ligaments, muscles and oral cavity (including lips and palate), as well as internal organs (e.g., liver, heart, brain, abdominal cavity, pelvic cavity, thoracic cavity, intestinal and reproductive tissues). In the skin, treatment can improve the sub-ocular and microscopic appearance of scar flesh; the scar under the flesh eye may be less visible and fuse with the surrounding skin, and the collagen fibers in the scar under the microscope may have a morphology and tissue more similar to that of the surrounding skin. Preventing, reducing or inhibiting scarring in the context of the present invention should be understood to include preventing, reducing or inhibiting scarring to any degree as compared to the level of scarring produced by a control treated or untreated wound (as defined elsewhere in the specification). Except where the context requires additional reference, the "prevention", "reduction" or "inhibition" of scarring may be taken as an all-proven equivalent mechanism in anti-scarring activity.

The prevention, reduction or inhibition of skin scarring achieved using the methods and medicaments of the present invention may be assessed and/or measured by the microscopic, or preferably macroscopic, appearance of the treated scar compared to the appearance of the untreated scar. More preferably, prevention, reduction, or inhibition of scarring may be assessed according to the microscopic and macroscopic appearance of the treated scar. For the purposes of the present invention, a "treated scar" can be defined as a scar that forms when a treated wound heals, while an "untreated scar" can be defined as a scar that forms when an untreated wound, or a wound treated with placebo or standard care, heals. A suitable scar comparison may preferably be matched to the scar being treated based on the age, location, size and patient of the scar.

The extent of scarring and hence the magnitude of any scarring prevention, reduction or inhibition can be assessed in accordance with any of a number of parameters, as follows, when considering the sub-ocular appearance of scarring resulting from treatment of a wound.

Suitable parameters for macroscopic scar assessment may include:

i) the color of the scar. As mentioned above, scars typically can be hyperpigmented or hyperpigmented relative to the surrounding skin. Inhibition or reduction of scarring is indicated when the pigmentation of the treated scar is closer to the scar-free skin than the pigmentation of the untreated scar. Likewise, scars may be redder than the surrounding skin. In this case, inhibition or reduction of scar formation is indicated when the red color of the treated scar fades earlier, more completely, or more closely resembles the appearance of the surrounding skin than the untreated scar.

ii) height of scar. Scars can typically be raised or lowered compared to the surrounding skin. Inhibition or reduction of scarring is indicated when the height of the treated scar is closer to scarless skin (i.e., no elevation and reduction) than the height of the untreated scar.

iii) the surface texture of the scar. Scars may have a relatively smoother surface (producing a "shiny" appearance) or be rougher than the surrounding skin. Inhibition or reduction of scarring is indicated when the surface texture of the treated scar is closer to the scar-free skin than the surface texture of the untreated scar.

iv) the stiffness of the scar. The abnormal composition and structure of scars means that they are generally harder than the intact skin surrounding the scar. In this case, inhibition or reduction of scar formation is indicated when the hardness of the treated scar is closer to scar-free skin than the hardness of the untreated scar.

Preferably, the treated scar will indicate prevention, inhibition or reduction of scar formation when assessed according to at least one parameter assessed macroscopically as described above. More preferably, the treated scar will indicate prevention, inhibition or reduction of scarring according to at least two of these parameters, even more preferably at least three parameters and most preferably all four parameters. A comprehensive assessment of scar formation can be made using, for example, Visual analog scoring (Visual analog Scale) or numerical ratings.

Suitable parameters for microscopic scar assessment may include:

i) thickness of extracellular matrix (ECM) fibers. Scars typically contain finer ECM fibers than the surrounding skin. This property is even more pronounced in the case of keloids and hypertrophic scars. Inhibition or reduction of scarring is indicated when the ECM fiber thickness of the treated scar is closer to that of scar-free skin than the ECM fiber thickness of the untreated scar.

ii) orientation of the ECM fibres. ECM fibers found in scars tend to exhibit a higher degree of alignment with one another than those found in scar-free skin (often with random orientation, known as "basket network"). ECM of pathological scars, such as keloids and hypertrophic scars, can exhibit more irregular orientation, often forming large "eddies" or "pockets" of ECM molecules. Thus, inhibition or reduction of scarring is indicated when the ECM fiber orientation of the treated scar is closer to the ECM fiber orientation found in scar-free skin than the ECM fiber orientation in the untreated scar.

iii) ECM components of the scar. The components of the ECM molecules present in the scar show differences from those found in normal skin, and the ECM of the scar presents a reduced amount of elastin. Thus, inhibition or reduction of scarring is indicated when the ECM fibrous component of the dermal scar of the treated scar is closer to this fibrous component found in scar-free skin than the component found in the untreated scar.

iv) cellular structure of the scar. Scars tend to contain relatively fewer cells than scarless skin. It can therefore be understood that inhibition or reduction of scar formation is indicated when the cellular structure of the treated scar is closer to that of scar-free skin than the cellular structure of the untreated scar.

Preferably, the treated scar will indicate prevention, inhibition or reduction of scar formation when assessed according to at least one parameter assessed microscopically as described above. More preferably, a treated scar will indicate prevention, inhibition or reduction of scarring assessed according to at least two of these parameters, even more preferably at least three parameters, and most preferably all four parameters.

Prevention, inhibition or reduction of scarring of the treated wound may be further assessed according to suitable parameters for use as follows:

i) macroscopic clinical assessment of scars, in particular assessment of scars in individuals;

ii) evaluation of scar picture images;

iii) evaluation of the silicone mold or a positive gypsum model made from a scarred silicone mold; and

iv) microscopic evaluation of the scar, e.g. histological analysis of the scar microstructure.

Understandably, prevention, inhibition, or reduction of scarring of a treated wound may be indicated by improvement in one or more of these suitable parameters, which may be combined according to different assessment schemes (e.g., reduction, inhibition, or improvement of at least one parameter used in a macroscopic assessment and at least one parameter used in a microscopic assessment) where prevention, inhibition, or reduction is assessed according to a number of parameters.

Prevention, reduction, or inhibition of scarring may be indicated by an improvement in one or more parameters, which indicates that the scar treated is closer to scarless skin than the untreated or control scar, depending on the parameters selected.

Suitable parameters for clinical measurements and assessments of scars may be selected based on a variety of measurements or assessments, including those described by Beausang et al (1998) and van Zuijen et al (2002).

Typically, suitable parameters may include:

1. assessment of scar score according to Visual Analogue Score (VAS).

Prevention, reduction, or inhibition of scarring is indicated by a decrease in the VAS score of the treated scar when compared to the control scar. Suitable VAS's for use in scar assessment can be based on the method described by Beausang et al (1998).

2. Height of the scar, width of the scar, circumference of the scar, area of the scar, or volume of the scar.

The height and width of the scar can be measured directly from the individual, for example, by using a manual measuring device such as a caliper. The width, perimeter and area of the scar can be measured directly from the individual or from a photographic image analysis of the scar. Other non-invasive methods and apparatus are also known to those skilled in the art and may be used to study suitable parameters, including silicone modeling, ultrasound, optical three-dimensional profiling, and high resolution magnetic resonance imaging.

Prevention, reduction, or inhibition of scarring is indicated by a reduction in the height, width, area, or volume, and any combination thereof, of the treated scar as compared to the untreated scar.

3. The appearance and/or color of the scar is compared to the surrounding scar-free skin.

The appearance or color of the treated scar can be compared to the appearance or color of the surrounding scar-free skin, and the difference (if any) compared to the appearance and color of the untreated scar and the difference between the appearance and color of the scar-free skin. This comparison can be made on the basis of a visual assessment of the respective scarred and scarless skin. The appearance of a scar can be compared to scarless skin depending on whether the scar is brighter or darker than the scarless skin. The respective colors of the scar and the skin can be perfectly matched, slightly mismatched, significantly mismatched, or significantly mismatched to each other.

Alternatively or additionally to visual assessment, there are a number of non-invasive colorimetric devices that are capable of providing data on the pigmentation of scar and scarless skin, as well as the redness of the skin (which can be an indication of the extent of vascularity present in the scar or skin). Examples of such devices include minoltachrronameter CR-200/300; labscan 600; lange Micro Colour; skin Spectrometer (Derma Spectrometry), laser-Doppler flow meter (laser-Doppler flow meter) and intracutaneous analytical Spectrophotometry (SIA).

The magnitude of the difference between the appearance or color of the treated scar and the scar-free skin is smaller than the difference between the untreated scar and the scar-free skin, indicating prevention, reduction, or inhibition of scar formation.

4. Deformation and mechanical properties of scars

The deformation of the scar can be assessed by visual comparison of scar and scar-free skin. A suitable comparison may classify the selected scar as being non-deformed, slightly deformed, moderately deformed, or severely deformed.

The mechanical properties of scars can be assessed using a number of non-invasive methods and devices based on suction, pressure, torsion, tension and acoustics. Suitable examples include known devices that can be used to assess the mechanical properties of a scar, including direct-reading indentation durometers, skin elastometers, reviscometers, viscoelastic skin analyzers, Dermaflex, sclerometers, skin torquemeters, and elastometers.

A reduction in the deformation caused by a treated scar when compared to the deformation caused by an untreated scar indicates prevention, reduction or inhibition of scar formation. It will also be appreciated that the prevention, reduction or inhibition of scarring may be indicated by the mechanical properties of the treated scar being more similar to that of scar-free skin than an untreated scar.

5. Contour of scar and texture of scar

The contour of the scar can be studied by visual assessment. Suitable parameters to consider in this assessment include whether the scar and surrounding skin are flush, slightly raised, slightly jagged, hypertrophic scar or keloid. The texture of a scar can be assessed by the appearance of the scar, which can also be by a visual assessment of whether the scar is, for example, matte or shiny or has a rough or smooth appearance when compared to scarless skin.

In addition, the texture of a scar can be assessed by whether the scar has the same texture as scarless skin (normal texture), and is palpable, firm, or hard compared to scarless skin. The texture of the scar can also be assessed according to the Hamilton scale (Hamilton scale) (described by Crowe et al, 1998).

In addition to the techniques described above, there are a number of non-invasive topographic devices that use optical or mechanical methods to assess the contour and/or texture of a scar. Such assessment can be made on the body of the individual, or for example on an impression of a silicone mold of a scar, or a positive plaster model made from such an impression.

Prevention, reduction, or inhibition of scarring is indicated if the treated scar has a contour and texture that is more comparable to the scar-free skin than the untreated scar.

Photo evaluation

Independent lay panel (Independent lay panel)

Photographic assessment of treated and untreated scars can be performed by an independent expert panel with standardized and calibrated photographs of scars. Scars can be assessed by independent laypersons to provide categorical scoring data (e.g., "better", "worse", or "no difference" for a particular treated scar as compared to an untreated scar) and quantitative data obtained using Visual Analogue Scoring (VAS) based on the method described by Beausang et al (1998). Such data may be retrieved using suitable software and/or electronic systems as described in applicant's co-pending patent applications.

Expert panel

Alternatively or additionally, the photographic assessment of treated and untreated scars may be performed by a panel of expert assessors with standardized and calibrated photographs of the scars to be assessed. Preferably, the panel of experts consists of a suitable person skilled in the art, for example an orthopaedic surgeon and a scientist adapted to the background.

Such assessment may provide categorized data, such as the image comparisons described above or according to a period of time for selected treated and untreated scars.

Suitable assessments performed may include:

for the purposes of the present invention, the best scar identification is considered to be the scar that most closely resembles the surrounding skin. Once the best scar is identified, the magnitude of the difference between scars can be considered, for example, as a slight or significant difference between scars. Further parameters considered include the earliest time a difference between scars is detected after scar formation, the time when a difference between scars is most apparent after formation (or alternatively the difference continues to be found at the last assessment time point), and whether a better scar consistently remains better.

It is also considered whether a scar will consistently be redder than the other and whether the redness will fade after the identified time point (or continue after the last time point) and, if so, at what time after scar formation. The panel may also consider at what time after formation any difference in red color becomes noticeable, and at what time after formation the difference in red color is most noticeable.

The panel of experts may also consider whether one scar, treated or untreated, is consistently whiter than the other or whiter than scarless skin. If the difference in whiteness is perceptible, the time after scarring when the difference can be detected, the time when the difference is most apparent and the time when the difference disappears are taken into account.

A further parameter that the panel can assess is the texture of the treated and untreated scars. In comparing treated and untreated scars, the panel of experts can consider which scar has the best skin texture, the earliest time any difference that exists after scar formation can be detected, the time when any difference is most apparent after formation, and the time when any difference disappears.

Comparison of treated and untreated scars further assesses which scar is narrowest and which scar is shortest. The shape of the scar and the proportion of the edges of the scar that are distinct from the surrounding skin may also be considered. As previously mentioned, visual assessments, and color assessments of the presence, extent, and location of hyperpigmentation are also contemplated.

As noted above, one method of comparing the properties of treated and untreated scars is by microscopic evaluation. Microscopic assessment of scar properties can typically be performed with histological sections of the scar. The process of microscopic assessment and measurement of scar may take into account classification data based on the following suitable parameters:

1. and (4) reconstructing epidermis. Of particular concern is the degree of recovery of the web ridges (rete ridges) and the thickness of the recovered epidermis.

2. Angiogenesis and inflammation. The number of vessels present, the size of the vessels present, and the signs of inflammation may be considered, including assessing any level of inflammation present.

3. Collagen tissue. In assessing collagen tissue, reference may be made to the orientation of the collagen fibres present in the scar, the density of these fibres and the thickness of the collagen fibres in the papillary and reticular dermis.

4. Visual Analogue Score (VAS) assessment of collagen tissue of papillary dermis and reticular dermis may also provide a useful indicator of scar properties.

5. Other characteristics may be considered when assessing the microscopic properties of the scar, including an increase or decrease in the scar relative to the surrounding scar-free skin and the protrusion or significance of the scar at the normal dermal interface.

6. It can be seen that the assessment described above yields scar score data that provides an indication as to whether a treated scar is better, worse or undifferentiated as compared to a control, untreated or other suitable comparison scar.

In addition to categorical data, image analysis can be used in conjunction with suitable visualization techniques to generate quantitative data (preferably related to the above references). Examples of suitable visual techniques that can be used to assess scar properties are specific histological stains or immunological markers, wherein the degree of staining or marking present can be quantified by image analysis.

Quantitative data relating to the following parameters can be efficiently and easily generated:

1. width, height, elevation, volume and area of the scar.

2. Thickness and extent of epithelium (e.g., area of epidermis present in scar or proportion with epidermis covering wound).

3. The number, size, area (i.e., cross-section), and location of the blood vessels.

4. The extent of inflammation, the number, location and population/type of inflammatory cells present.

5. Collagen tissue, collagen fiber thickness and collagen fiber density.

By a change in any of the parameters considered above, prevention, reduction or inhibition of scarring may be indicated such that the treated scar more closely resembles scar-free skin than a control or untreated scar (or other suitable comparator).

The assessments and parameters in question are suitable for comparing the effect of the peptides in animals or humans with control, placebo or standard of care treatments. To investigate the significance of the results, appropriate statistical tests can be used to analyze the data sets generated from the different treatments.

Preferably, prevention, reduction or inhibition of scarring may be indicated according to one or more parameters. More preferably, prevention, reduction or inhibition of scarring may be indicated based on clinical (i.e., subject under observation) parameters and photographic parameters. Even more preferably, prevention, reduction or inhibition of scarring may be indicated based on clinical parameters, photographic parameters and microscopically assessed parameters (e.g., histological parameters). Most preferably, prevention, reduction or inhibition of scarring is indicated based on the clinical VAS score, the layperson panel VAS score, and the score of the reticular dermis (from the photographic image) and the microscopic VAS score.

The use of the appropriate methods and medicaments of the present invention enables rapid improvement in the aesthetic appearance of the damaged area thus treated. Aesthetic considerations are important in a number of clinical situations, particularly when the wound is formed on a significant part of the body, such as the face, neck and hands. Thus, inhibition of scarring (preferably in combination with accelerated wound healing) at these sites where it is desired to improve the aesthetic characterisation of the scar formed represents a preferred embodiment of the invention.

In addition to its aesthetic effect, scarring of the skin results in a number of adverse consequences that afflict the patient undergoing such scarring. For example, dermal scarring is associated with diminished bodily and mechanical function, especially in the case of a contractive scar (e.g., hypertrophic scar) and/or in the case of a scar that forms across a joint. In these cases, the mechanical properties of the scar forming skin change and the effect of scar retraction, in contrast to scarless skin, greatly limit the movement of the joint (articulation) so affected. Thus, a preferred embodiment is the use of suitable medicaments and methods of the invention for preventing, reducing or inhibiting scarring of wounds that cover a joint of the body (preferably also accelerating healing of such wounds). Another preferred embodiment is the use of suitable medicaments and methods of the invention to promote accelerated wound healing and/or to prevent, reduce or inhibit scarring of wounds with an increased risk of forming shrinkage scars.

The extent of scarring, and thus the aesthetic appearance or other damage caused by scarring, can also be affected by a number of factors, such as the tension at the wound formation site. For example, it is known that skin under relatively high tension (e.g., across the chest or associated with tension lines) tends to scar more severely than other parts of the body. Thus in preferred embodiments, suitable medicaments and methods of the invention are used to promote accelerated healing of wounds and/or to prevent, reduce or inhibit scarring of wounds at sites of high skin tension. Many surgical procedures are available for scar repair to rearrange wounds and scars so as to reduce the strain to which they are subjected. Perhaps the best known of these is "zig-zag" in which two V-shaped pieces of skin sheet tissue are transposed to rotate the tensioning wire. Thus in a more preferred embodiment, such medicaments and methods of the invention are used to promote accelerated healing of a wound and/or to prevent, reduce or inhibit scarring of a wound during surgical repair of a disfiguring scar.

Pathological scarring has more significant adverse consequences than relatively severe normal scarring. Common examples of pathological scars include hypertrophic scars and keloids. It is recognized that certain types of wounds or certain individuals are prone to develop pathological scarring. For example, a black caribbean, japanese or mongolian race, or those with a family history of pathological scars may be considered to have an increased risk of developing hypertrophic scars or keloids. Wounds in children, especially children's burns, are also associated with increased hypertrophic scarring. Accordingly, a preferred embodiment of the present invention is the use of suitable medicaments and methods to promote accelerated healing of wounds and/or to prevent, reduce or inhibit scarring of wounds with an increased risk of pathological scarring.

Although individuals who have experienced pathological scarring may experience an additional excessive tendency to scarring, hypertrophic scars or keloids often must be surgically repaired clinically, with the attendant risk of subsequent pathological scarring. Thus, a further preferred embodiment of the present invention is the use of suitable medicaments and methods for promoting accelerated wound healing and/or preventing, reducing or inhibiting scarring of a wound resulting from surgical repair of pathological scarring.

It is recognized that wounds resulting from burns (which for the purposes of the present invention may also include burns involving hot liquids or gases) can spread over a large area on an individual suffering from such pain. Thus, burns can produce scarring that covers a large portion of the patient's body, increasing the risk that the resulting scarring covers areas of high aesthetic importance (e.g., the face, neck, arms, or hands) or areas of mechanical importance (particularly areas that cover or surround the joints). Children are often subjected to burns caused by hot liquids (e.g. a knocked-over pot, pot or the like) and, due to the relatively small body size of the child, may especially cause extensive injuries covering large parts of the body. A further preferred embodiment of the invention is the use of suitable medicaments and methods to promote accelerated healing of wounds and/or to prevent, reduce or inhibit scarring of wounds resulting from burns.

As noted above, wound healing in response to burns is often associated with adverse scarring results, such as hypertrophic scarring. A further consequence of the relatively large size of burns is that they are particularly susceptible to complications such as infection and dehydration due to a lack of functional epithelial layer. From the foregoing, it will be appreciated that suitable medicaments and methods of the invention may be used in the treatment of burns to reduce the level of scarring arising from the wound and/or to accelerate the reconstruction of a functional epithelial barrier.

The inventors have found that the medicaments and methods of the invention using TGF-beta 3 of the invention promote re-epithelialization. These methods and drugs are therefore particularly effective in the treatment of all injuries involving damage to the epithelial layer. Examples of such lesions are, but are not limited to, skin lesions where the epithelium is damaged. It will be appreciated that such methods and medicaments of the invention may also be used for other types of wounds in which the epithelium is damaged, for example injuries involving the respiratory tract epithelium, the digestive tract epithelium or the epithelium surrounding internal tissues or organs, such as the peritoneal epithelium.

Wound healing involving the peritoneum (the epithelium covering the internal organs and/or the interior of the body cavity) often initiates adhesions. This adhesion is a common result of surgery involving gynecological or intestinal tissue. The inventors believe that the ability of the methods and medicaments of the invention (e.g., those comprising TGF-beta 3 listed in sequence ID nos. 3, 5, 7, 9 or 11) to accelerate peritoneal regeneration to reduce scarring reduces the incidence of improper interconnection of the peritoneal components to one another, thereby reducing the incidence of adhesions. Thus, the use of such methods and medicaments of the invention to prevent intestinal or gynecological adhesions from forming represents a preferred embodiment of the invention. In fact, the use of the methods and medicaments of the present invention in the healing of any wound involving the peritoneum is a preferred embodiment.

The methods or medicaments of the invention may be used prophylactically, for example at a site where no wound is present but otherwise scarring occurs or a chronic wound forms. By way of example, the medicament of the present invention may be administered to a site suffering from a wound caused by selective manipulation (e.g. surgery), or a site believed to have an increased risk of trauma. Preferably, the agent of the invention may be administered to the site at about the time of the wound or immediately prior to wound formation (e.g., during up to 6 hours prior to wound formation), or the agent may be administered at an earlier time prior to the wound (e.g., up to 48 hours prior to wound formation). The skilled artisan understands that the most preferred time for administration prior to wound formation will be determined by a number of factors including the formulation and route of administration of the selected drug, the dosage of the administered drug, the size and nature of the wound formation, and the biological condition of the patient (determined, for example, by the age, health, and propensity of the patient to develop healing complications or adverse scarring). Prophylactic use of the methods and medicaments of the invention is a preferred embodiment of the invention, and in the case of surgical wounds, is particularly preferred for promoting accelerated wound healing and/or preventing, reducing or inhibiting scarring.

If the methods and medicaments of the present invention are administered after wound formation, accelerated healing of the wound and/or inhibition of scarring can also be promoted. Preferably, such administration should be performed as soon as possible after the wound has formed, but the agents of the invention are capable of promoting accelerated wound healing and/or preventing, reducing or inhibiting scarring at any time up to the completion of the healing process (i.e. even if the wound has partially healed, the methods and medicaments of the invention may also be used to accelerate wound healing and/or prevent, reduce or inhibit scarring in the remaining non-healed portion). Understandably, the "window" in which the methods and medicaments of the invention can be used to promote accelerated wound healing and/or to prevent, reduce or inhibit scarring depends on the nature of the wound in question (including the extent to which the lesion occurs, the size of the lesion area). Thus, in the case of a large injury, the methods and medicaments of the invention may be administered relatively late in the healing response, but still promote accelerated wound healing and/or prevent, reduce or inhibit scarring. Preferably, the methods and medicaments of the present invention may be administered, for example, within the first 24 hours after wound formation, but may still promote accelerated wound healing and/or prevent, reduce or inhibit scarring if administered within ten days or more after the wound has occurred.

The methods and medicaments of the present invention may be administered one or more times as needed in order to promote accelerated wound healing and/or to prevent, reduce or inhibit scarring. For example, a therapeutically effective amount of the drug may be administered to the wound as often as necessary until the healing process is complete. By way of example, the medicament of the invention may be administered to the wound once a day or twice a day at least the first three days after the formation of the wound.

Most preferably, the methods and medicaments of the present invention are administered both before and after wound formation. The inventors have found that the agents of the invention are particularly effective in promoting accelerated wound healing and/or in preventing, reducing or inhibiting scarring when applied immediately prior to wound formation and then daily for a period of time following the wound.

For the purposes of this specification, "agent" or "agent of the invention" refers to biologically or therapeutically active TGF- β 3 of the invention; and/or biologically or therapeutically active TGF-beta 3 fragments of the invention; and/or biologically or therapeutically active TGF-beta 3 derivatives of the invention. The agents of the invention may also include nucleic acids encoding TGF-beta 3 (or fragments or derivatives thereof) of the invention. Understandably, all of these agents can be incorporated into a medicament according to the present invention and can be used in the methods or applications of the present invention.

Understandably, the amount of the inventive medicament applied to the wound depends on a number of factors, such as the biological activity and bioavailability of the agent present in the medicament, among other factors, the nature of the agent and the mode of administration of the medicament. Other factors that determine the appropriate therapeutic amount of drug may include:

A) the half-life of the agent in the subject.

B) The particular condition to be treated (e.g., acute or chronic wound).

C) The age of the individual.

The frequency of administration is also influenced by the above-mentioned factors, in particular the half-life of the selected agent in the treated individual.

Generally, when the medicament of the present invention is used to treat an existing wound, the medicament should be administered as soon as the wound occurs (or in the case where the wound does not appear immediately, such as in an internal body site, the wound is administered once it has been diagnosed). Treatment with the methods or medicaments of the invention should be continued until the healing process has been accelerated and/or scarring has been prevented, reduced or inhibited until satisfactory by the clinician.

The frequency of administration depends on the biological half-life of the agent used. Typically, a cream or ointment containing the agents of the present invention should be applied to the target tissue to maintain the concentration of the agent on the wound at a level suitable to produce a therapeutic effect. This requires once daily or even several daily administrations.

The medicament of the present invention may be administered by any route which achieves the desired wound healing promoting and/or scar formation preventing, reducing or inhibiting effect, but preferably the medicament is administered topically at the wound site.

The present inventors have found that promoting accelerated wound healing and/or preventing, reducing or inhibiting scarring may be achieved by administering an agent of the present invention by injection at the wound site. For example, in the case of dermal wounds, the agents of the invention may be administered by intradermal injection. Thus, preferred medicaments of the invention comprise injectable solutions of the agents of the invention (e.g., injection around the edges of epithelial lesions or potential lesions). Suitable formulations for use in embodiments of the invention are considered below.

Alternatively or additionally, the medicament of the invention may also be administered in topical form to promote accelerated wound healing and/or to prevent, reduce or inhibit scarring. Such application may be effected as part of the initial and/or subsequent care of the wound area.

The inventors have found that promoting accelerated healing of a wound and/or preventing, reducing or inhibiting scarring is particularly improved by the topical application of the agents of the invention to a wound (or in the case of prophylactic application to the tissue or site where the wound is formed).

The compositions or medicaments containing the agents of the invention may take many different forms, depending inter alia on the manner in which they are used. Thus, for example, they may be in the form of liquids, ointments, creams, gels, hydrogels, powders or aerosols. All these compositions are suitable for topical application to wounds and are the preferred means of administering the agents of the invention to an individual (human or animal) in need of treatment.

The agents of the invention may be provided in the form of sterile dressings or patches that may be used to cover wounds or other sites of epithelial damage to be treated.

Understandably, the excipients of the compositions comprising the agents of the present invention should be well tolerated by the patient so that the agents are released against the wound. Such excipients are preferably biodegradable, biosoluble, bioabsorbable, and/or non-inflammatory.

The medicaments and compositions comprising the agents of the present invention may be used in a number of ways. Thus, for example, to promote accelerated wound healing and/or to prevent, reduce or inhibit scarring, the compositions may be applied to the interior and/or periphery of a wound. If the composition is applied to an "existing" wound, the pharmaceutically acceptable excipient will be relatively "mild", i.e., the excipient is biocompatible, biosoluble and non-inflammatory.

The agents of the invention, or nucleic acids encoding such agents (further contemplated below), may be incorporated into a sustained or delayed release device. These devices may be placed or inserted subcutaneously, for example, and the agent or nucleic acid may be released over days, weeks, or even months. Such devices are particularly useful for patients who need to promote accelerated wound healing and/or to prevent, reduce or inhibit scarring over an extended period of time, such as those suffering from chronic wounds. The device is particularly advantageous when used for administration of pharmaceutical agents or nucleic acids which typically require frequent administration (e.g., at least daily administration by other routes).

The daily dose of the agent of the invention may be administered in a single application (e.g. daily application of a topical formulation or daily injection). Alternatively, the agents of the invention may need to be administered two or more times a day. Further alternatively, a sustained release device may be used to provide optimal dosing of the agents of the present invention to patients who do not require administration of repeated doses.

In one embodiment, the pharmaceutical excipient used for administration of the agents of the present invention may be a liquid and suitable pharmaceutical compositions will take the form of a solution. In another embodiment, the pharmaceutically acceptable excipient is a solid, and suitable compositions take the form of a powder or tablet. In a further embodiment, the agents of the present invention may be formulated as part of a pharmaceutically acceptable transdermal patch.

Solid excipients may include one or more substances that may act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders, or tablet disintegrating agents; or can be used as capsule material. In powders, the excipient is a finely divided solid which is mixed with the finely divided medicament of the invention. In tablets, the agents of the invention are mixed in suitable proportions with excipients having the necessary compression characteristics, and compressed in the desired shape and size. Preferably, the powders and tablets contain up to 99% of the agent of the invention. Suitable solid excipients include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone, low melting waxes and ion exchange resins.

Liquid vehicles can be used to prepare solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The pharmaceutical agents of the present invention may be dissolved or suspended in a pharmaceutically acceptable liquid excipient, such as water, an organic solvent, a pharmaceutically acceptable oil or fat, or a mixture thereof. The liquid excipient may contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickeners, colors, viscosity modifiers, stabilizers or osmo-modifiers. Suitable examples of liquid excipients for oral and parenteral administration include water (partially containing additives as above, e.g., cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration, the excipient may also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid excipients are advantageous in sterile liquid form compositions for parenteral use. The liquid vehicle of the pressurized composition may be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

Sterile solutions or suspensions of the liquid pharmaceutical compositions may be used, for example, by intramuscular, intrathecal, epidural, intraperitoneal, intradermal, intrastromal (corneal) or subcutaneous injection. Sterile solutions can also be administered intravenously. The medicaments of the present invention may be prepared as sterile solid compositions which may be dissolved or suspended at the time of administration in sterile water, saline or other suitable sterile injectable medium. Excipients tend to include necessary and inert binders, suspending agents, lubricants and preservatives.

Where it is desired to administer the agents of the invention in an orally ingestible form, it will be appreciated that the agent selected will preferably be one having an increased degree of resistance to degradation. For example, the agents of the present invention may be protected (e.g., using the techniques described above) so as to reduce their rate of degradation in the digestive tract.

The compositions of the agents of the present invention are suitable for promoting accelerated wound healing and/or preventing, reducing or inhibiting scarring within the cornea. Corneal wounds may be caused by ocular trauma resulting from accidental injury (as contemplated above) or from surgical procedures (e.g., laser surgery of the cornea). In this case the preferred medicament of the invention is in the form of eye drops.

The agents of the present invention may be applied to a range of "internal" wounds (i.e. wounds occurring within the body, not on the external surface). Thus, for example, the medicaments of the invention may be formulated for inhalation for application to wounds created by the lung or other respiratory epithelium.

It is known, for example, that methods routinely used in the pharmaceutical arts (e.g., in vivo experimentation, clinical trials, etc.), can be used to establish specific formulations of compositions comprising the agents of the present invention and the precise treatment regimen to be administered for such compositions (e.g., daily dosages of active agents and frequency of administration).

The agents of the invention are capable of promoting accelerated wound healing and/or preventing, reducing or inhibiting the formation of scarring in a suitable daily dosage depending on a number of factors including, but not limited to, the nature of the wound tissue, the area and/or depth of the wound being treated, the severity of the wound, and the presence or absence of predisposing factors for the formation of pathological or chronic scars.

By way of example, the total amount of active agent that can be administered to a wound or epithelial injury site by local injection is preferably about 50ng/100 μ L per cm of linear wound or epithelial injury area. Such a dose may be administered once a day for three days, thus providing a total dose of 150 ng/cm of linear wound or epithelial lesion.

In the case of topical application to the site of an acute wound or epithelial injury, a suitable amount of active agent may preferably be about 100ng/cm². Such a dose may be administered once a day for a period of three days, thus providing a total dose of 300ng/cm²A wound or epithelial injury.

By way of further example, a preferred amount of active agent administered daily to a wound or epithelial injury site may be about 50ng/cm of linear wound or epithelial injury (if administered by injection) or about 100ng/cm²A wound or epithelial injury (if applied topically).

By way of further example, the amount of active agent that can be administered to a wound or epithelial injury site in a single incidence therapy may preferably be about 50-200ng/cm of linear wound or epithelial injury (if administered by injection), or about 100-300ng/cm²A wound or epithelial injury (if applied topically).

The amount of agent of the invention required to treat a wound or other epithelial injury site is typically from 1pg to 1mg of agent per cm of linear wound or epithelial injury per 24 hours, although this number may be corrected up or down depending on the factors outlined above. The agent may preferably be provided in the form of a solution of the agent in the range 1pg/100 μ L to 1mg/100 μ L, 100 μ L of such solution being applied per cm of linear wound or epithelial lesion over a 24 hour period.

More preferably, the agent is administered in a solution of 10pg/100 μ L to 100 μ g/100 μ L, 100 μ L of such solution being administered per centimeter of linear wound or epithelial lesion over a 24 hour period.

Most preferably, the agent is administered in a solution of 1 ng/100. mu.L to 1000 ng/100. mu.L, 100. mu.L of such solution being administered per cm of linear wound or epithelial lesion over a 24 hour period.

Generally, compositions comprising the agents of the invention should be formulated such that the agent concentration when administered to a wound is between 0.79pM and 0.79mM per cm of linear wound or epithelial lesion. Preferably, the agent is provided at a concentration of 7.9pM to 0.079mM per cm.

The concentration of the agent of the invention (e.g. peptides of sequence ID numbers 3 to 8) administered may be 0.79pM to 0.79 mM. Preferably, the agent of the invention may be administered at a concentration of 7.9pM to 0.079 mM. Most preferably, the agent of the invention may be administered at a concentration of 0.79nM to 0.79. mu.M.

Purely by way of example, when administered by intradermal injection and administered at 100 μ L per cm of linear wound margin, an injectable solution containing an agent of the invention (e.g., TGF- β 3 of sequence ID No. 3, 5, 7, 9 or 11) at a concentration of 10pg/100 μ L to 100 μ g/100 μ L is suitable for use in promoting accelerated dermal wound healing and/or inhibiting scarring.

In the case of TGF-. beta.3 of sequence ID No. 3, the preferred dose administered to the wound is about 1 ng/100. mu.L to 1000 ng/100. mu.L, with about 100. mu.L of this solution being administered per cm of linear wound margin.

In the case of TGF-. beta.3 of sequence ID No. 5, the preferred dose administered to the wound is about 1 ng/100. mu.L to 1000 ng/100. mu.L, with about 100. mu.L of this solution being administered per cm of linear wound margin.

In the case of TGF-. beta.3 of sequence ID No. 7, the preferred dose administered to the wound is about 1 ng/100. mu.L to 1000 ng/100. mu.L, with about 100. mu.L of this solution being administered per cm of linear wound margin.

In the case of TGF-. beta.3 of sequence ID No. 9, the preferred dose administered to the wound is from about 1 ng/100. mu.L to 1000 ng/100. mu.L, with about 100. mu.L of this solution being administered per cm of linear wound margin.

In the case of TGF-. beta.3 of sequence ID No. 11, the preferred dose administered to the wound is about 1 ng/100. mu.L to 1000 ng/100. mu.L, with about 100. mu.L of this solution being administered per cm of linear wound margin.

The agents of the invention may be used as monotherapy (e.g. by administering the agents of the invention alone) to promote accelerated wound healing and/or to prevent, reduce or inhibit scarring. Alternatively, the medicaments or methods of the invention may be used in conjunction with other compounds or treatments that promote wound healing or inhibit scarring. Suitable treatments that can be used as part of these combination therapies are well known to those skilled in the art.

The inventors have found that TGF-. beta.3 of the invention may be advantageously formulated in the presence of a sugar. Such sugars may be reducing or non-reducing sugars and/or their phosphate esters or their phosphate derivatives. Examples of these sugars may be selected from, but are not limited to, those selected from the group consisting of maltose, mannose, trehalose, arabinose, mannitol, sucrose, fructose, dextrose, and glucose. Preferred sugars may be selected from the group consisting of maltose and trehalose.

Understandably, peptides comprising TGF-beta 3 of the invention may represent advantageous agents for administration by cellular expression techniques that include nucleic acid sequences encoding these molecules. These methods of cell expression are particularly suitable for medical applications where long-term therapeutic effects of the peptide are required, for example where it is desired to enhance a defective wound healing response over a long period of time. It is particularly preferred that TGF-. beta.3 administered by cellular expression includes those peptides defined by sequence ID Nos. 3, 5, 7, 9 or 11 or fragments or derivatives thereof. Nucleic acids encoding these peptides are listed in sequence ID Nos. 4, 6, 8, 10 or 12.

Many known methods of administering the peptide agents of the present invention to tissues such as wounds have the following disadvantages: due to the short half-life of the peptide agent in vivo, it is difficult to achieve sustained levels of the agent of the present invention at the treatment site over the course of even a few days. The short half-life of the agent is due to a number of reasons, including:

(i) by degradation by proteases and the like.

(ii) Clearance by the bound protein.

(iii) Is bound by extracellular matrix molecules and inhibits the activity of the agent.

Moreover, agents for promoting accelerated wound healing and/or preventing, reducing or inhibiting scarring need to be administered within suitable excipients and are often provided in the form of compositions comprising the agent and the excipient. As discussed, preferably these excipients are non-inflammatory, biocompatible, bioabsorbable and must not degrade or inactivate (storage or use) the agent. However, it is often difficult to provide a satisfactory excipient to deliver an agent to tissue having a wound to be treated.

A convenient way of eliminating or reducing these problems is to provide a therapeutically effective amount of an agent of the invention to the area to be treated by means of gene therapy.

In a fourth aspect, the invention provides a delivery system for gene therapy techniques, the delivery system comprising a DNA molecule encoding a peptide selected from the group consisting of: sequence ID No. 3, sequence ID No. 5, sequence ID No. 7, sequence ID No. 9 and sequence ID No. 11, which DNA molecule can be transcribed to result in the expression of the selected peptide.

A fifth aspect of the invention provides the use of a delivery system as defined in the preceding paragraph for the manufacture of a medicament for use in promoting accelerated wound healing and/or preventing, reducing or inhibiting scarring.

A sixth aspect of the invention provides the use of a delivery system as defined above for the manufacture of a medicament for use in promoting epithelial regeneration.

In a seventh aspect the present invention provides a method of promoting accelerated wound healing and/or preventing, reducing or inhibiting scarring, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of a delivery system as defined in the ninth aspect of the invention.

In an eighth aspect the invention provides a method of promoting epithelial regeneration, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of a delivery system as defined in the ninth aspect of the invention.

Due to the degeneracy of the genetic code, it is apparent that the nucleic acid sequences encoding agents suitable for use in the present invention may be different or variable and do not substantially affect the sequence of the encoded product to provide a functional variant thereof. The sequences which can be used for possible nucleic acids encoding the peptides defined by sequence ID numbers 3, 5, 7, 9 or 11 will be apparent to the skilled person, who can refer to the examples provided by sequence ID numbers 4, 6, 8, 10 or 12, respectively.

The delivery system of the present invention is well suited to achieve sustained levels of the agents of the present invention at a wound site over a longer period of time than is possible with most conventional delivery systems. The agent of the invention suitable for promoting accelerated wound healing and/or inhibiting scarring may be expressed continuously from cells at the wound site which have been transformed by a DNA molecule as disclosed in the fourth aspect of the invention. Thus, even though the agents of the present invention have a very short half-life in vivo, a therapeutically effective amount of the agent may be expressed continuously from the treated tissue.

Furthermore, the delivery system of the present invention can be used to provide DNA molecules (and thus the agents of the present invention) without the need to use conventional pharmaceutical excipients, such as those required in ointments or creams that contact wounds.

The delivery system of the invention preferably enables the expression of the DNA molecule (when the delivery system is administered to a patient) to produce a peptide defined by the group consisting of sequence ID numbers 3, 5, 7, 9 or 11 or a fragment or derivative of such a peptide. The DNA molecule may be contained within a suitable vector to form a recombinant vector. The vector may be, for example, a plasmid, cosmid or phage. The recombinant vector is well suited for the delivery system of the present invention for transforming cells with DNA molecules.

The recombinant vector may also include other functional elements. For example, a recombinant vector can be designed such that the vector is autonomously replicating in the nucleus of a cell. In this case, elements that induce DNA replication are necessary within the recombinant vector. Alternatively, the recombinant vector may be designed such that the vector and the recombinant DNA molecule are integrated into the genome of the cell. In such cases, a DNA sequence that facilitates targeted integration (e.g., by homologous recombination) is desirable. The recombinant vector may also contain DNA encoding a gene that serves as a selectable marker during cloning.

The recombinant vector may further contain a promoter or regulator for controlling the expression of the gene, if necessary.

The DNA molecule may, but need not, be integrated into the DNA of the cells of the individual to be treated. Undifferentiated cells can be stably transformed, resulting in the production of genetically altered daughter cells. In this case, it is desirable to regulate the expression of the treated individual, for example with specific transcription factors, gene activators or more preferably with inducible promoters, which can transcribe genes in response to specific signals found in the wound. Alternatively, the delivery system may be designed to facilitate unstable or transient transformation of undifferentiated cells in the treated individual. In this case, the importance of expression regulation becomes less because the expression of the DNA molecule will be terminated when the transformed cells die or cease to express the protein (ideally when achieving promotion of accelerated wound healing and reduction of scar formation).

The delivery system may provide the individual with a DNA molecule that is not integrated into the vector. For example, the DNA molecule may be incorporated into a liposome or a viral particle. Alternatively, a "naked" DNA molecule may be inserted into a cell of an individual by suitable means, such as direct endocytic uptake.

DNA molecules can be transferred into cells of an individual by transfection, infection, microinjection, cell fusion, protoplast fusion, or particle gun bombardment. For example, transfer can be performed by gene gun transfection with coated gold particles, liposomes containing DNA molecules, viral vectors (e.g., adenovirus), and by direct provision of DNA uptake (e.g., endocytosis) by direct application of plasmid DNA to a local wound or injection.

Cellular expression of the agents of the invention may be by cells at the margins of the atraumatic area surrounding the wound, or alternatively by cells introduced therapeutically into the wound (e.g. cultured endogenous or exogenous cells associated with the wound healing response).

Understandably, cells that are therapeutically induced to promote accelerated wound healing and/or to prevent, reduce or inhibit scarring can be manipulated in vitro such that they can express increased levels of the agents of the present invention and then introduced into the wound area. Preferably, the cells are cells cultured in vitro for the preparation or manufacture of artificial skin or skin substitutes for use in promoting wound healing. More preferably, the cells are autologous cells, although any suitable cells may understandably be used.

Accordingly, in a ninth aspect the invention provides a medicament comprising a cell induced to express an agent of the invention.

The induction of cellular expression of an agent of the invention may be achieved by means of the incorporation into the cell of a nucleic acid which allows expression of an agent suitable for use in the invention.

The invention will now be further described by way of example with reference to the following experimental protocols and studies and to the accompanying drawings, in which:

table 1 shows values indicating the propensity of amino acid residues involved in the alpha helical structure;

table 2 shows details of the names used in referring to the mutant TGF-. beta.3 of the invention;

table 3 shows the efficiency of refolding wild type TGF-. beta.3 and TGF-. beta.3 of the invention;

table 4 compares the biological activity of wild-type TGF-. beta.3 and Gly63-Ala (a TGF-. beta.3 protein of the invention), as assessed by cytostatic assays;

table 5 shows the reactant concentrations used in the in vivo wound healing study;

table 6 shows the reactant concentrations used in the in vivo wound healing study;

FIG. 1 shows a chromatogram of TGF-. beta.3 'wild type' on a phenyl-sepharose column;

FIG. 2 shows a chromatogram of TGF-. beta.3 'wild-type' monomers and dimers in a UNO-S1 column;

FIG. 3 shows a comparison of TGF-beta 3 mutant protein and 'wild-type' TGF-beta 3 by Coomassie blue stained SDS-PAGE (note that replacement of buffers for Gly63-Ala and Gly63-Pro mutant proteins results in some sample loss, and therefore the actual concentration added to the gel will be less than the stated 3. mu.g);

FIG. 4 shows a template for an incisional wound;

figure 5 shows the average macroscopic score on day 3 of treatment of incisive wounds (a and B) with wild-type TGF- β 3 or TGF- β 3 of the invention, where "×" indicates significantly enhanced healing compared to untreated wounds (p < 0.05);

FIG. 6 is microscopic average wound width on day 3 of treatment of incised wounds (C and D) with ` wild type ` and mutant TGF-. beta.3 proteins;

FIG. 7 shows a template for incisive wounds;

figure 8 illustrates the visual scar score (day 70) for wound treatment with 'wild type' TGF-beta 3, Gly63-Ala and Gly 63-Pro;

figure 9 illustrates a macroscopic scar image (day 70) of wounds treated with 'wild type' TGF- β 3, Gly63-Ala and Gly 63-Pro;

figure 10 illustrates the macroscopic scar score (day 70) for wounds treated with 'wild-type' TGF- β 3, Glu12-Ser and the double serine mutant (Glu12-Ser and Arg52-Ser), where "+" indicates significantly reduced scar formation (p <0.05) compared to placebo treated wounds.

FIG. 11 illustrates the macroscopic scar image (day 70) of wounds treated with 'wild-type' TGF- β 3, Glu12-Ser, and the double serine mutant (Glu12-Ser and Arg 52-Ser);

FIG. 12 illustrates representative microscopic scar images (70 days post-trauma) of wounds treated with ` wild-type ` TGF- β 3, Gly63-Pro, and Gly63-Ala muteins; and

FIG. 13 illustrates representative microscopic scar images of wounds treated with Glu12-Ser and the double serine mutant (Glu12-Ser and Arg52-Ser) 70 days post-trauma.

Details of sequences of particular interest are provided in the "sequence information" section.

Experimental protocol and results

1. Production, refolding and purification of TGF-. beta.3 of the first and second aspects of the invention.

1.1 production of cDNA

Total RNA from human incisive wounds (sampled on day 5 post-wound) was treated with DNA-free (Ambion) to remove any contaminating DNA. Using total RNA as a template, cDNA for TGF-. beta.3 was generated by reverse transcriptase-polymerase chain reaction (RT-PCR). The reaction mixture of RT-PCR is prepared fromQRT-PCR Core Reagent Kit, one-step method (Stratagene). 1 microgram of RNA was added to 50. mu.L of a solution containing: one-step QRT-PCR buffer, 0.2mM dNTP, 3.5mM MgCl₂1. mu.L of StrataScript reverse transcriptase, 2.5 units of Taq polymerase, 0.4. mu.M sense primer (5 'GAT ATA CCA TGG CTT TGG ACA CCA ATT ACT ACTGC 3') and 0.4. mu.M sense primer (5 '-CAG CCG GAT CCG GTC GAC TCA GCTACA TTT ACA AGA C3'). The reaction takes place in a thermocycler (Hybaid PCR Expresses) and is carried out according to the following conditions: 45 ℃ for 30 minutes, 95 ℃ for 10 minutes, followed by 40 cycles of 95 ℃ for 30 seconds, 65 ℃ for 1 minute, and 72 ℃ for 1 minute. The final step was 10 minutes at 72 ℃. The PCR samples were checked for band size by 2% (w/w) agarose gel electrophoresis and purified by Wizard PCR PrepKit (Promega).

1.2 construction of plasmids

The pET-3d vector is derived from the pBR322 vector and comprises the T7 promoter under the control of LacUV5 and an ampicillin resistance marker geneThus, the method is simple and easy to operate. The cDNA fragment of TGF-. beta.3 (generated in section 3.2) was subcloned into pET-3d at Nco I and Bam HI sites (5 '-3', respectively). The resulting ligation was then transformed into XL10Gold cells (Stratagene) and subjected to colony PCR analysis to locate clones containing the insert. The final clone was grown and used QiaprepSpin miniprep kit (Qiagen) plasmid DNA was extracted into water. The plasmid was sequenced and verified with the T7 promoter primer (5'-TAA TAC GAC TCACTA TAG GG-3') and the T7 terminator primer (5'-GCT AGT TAT TGC TCA GCG G-3').

1.3 site-directed mutagenesis

The ` wild type ` TGF-. beta.3 construct obtained from section 1.2 was subjected to site-directed mutagenesis to generate two mutant constructs encoding TGF-. beta.3 muteins. The names and nucleic acid sequence changes of the constructs/mutants are summarized in table 2. The mutagenized nucleotide positions are shown in the sequence information section.

The in vitro directed mutagenesis method is based on Stratagene's QuickA kit for directed mutagenesis. 100ng of plasmid (from part 1.2) was added to a solution containing: 2.5 μ L10 xQuickMulti-reaction buffer, Quick solution, 100ng of mutagenic primer, 1mL dNTP mix, Pfu Turbo DNA polymerase (Stratagene), supplemented with double distilled water to a final volume of 2.5 mL. The reaction takes place in a thermocycler (Hybaid PCR Expresses) and is carried out under the following conditions: 95 ℃ for 1 minute, 30 cycles of 95 ℃ for 1 minute, 55 ℃ for 1 minute, and a final step of 65 ℃ for 2 minutes. Once the thermal cycling was complete, the reaction was placed on ice for 2 minutes to bring the temperature below 37 ℃. mu.L of DpnI restriction enzyme (10U/. mu.L) was added to each reaction and mixed thoroughly. The reaction mixture was centrifuged (with Sorvall Biofuge, 10,000rpm/min) and the digested doublet was incubated at 37 deg.CParent double-stranded DNA. mu.L of activated XLI-Blue E.coli (Stratagene) and 2. mu.L of beta-ME cocktail (Stratagene) were added to 1-5. mu.L of DpnI-treated DNA per mutagenesis reaction. The suspension was mixed and incubated on ice for 30 minutes. The suspension was heated in a 42 ℃ water bath for 30 seconds. The mixture was incubated on ice for a further 2 minutes. 0.5mL of preheated (42 ℃) NZY + broth was added to each cell suspension. The transformation broth was incubated with shaking at 225-250rpm for 1 hour. mu.L, 10. mu.L and 100. mu.L of each mutagenized transformation broth were plated on LB agar plates containing 100. mu.g/mL ampicillin (Sigma), 80. mu.g/mL of 5-bromo-4-chloro-3-indole-. beta. -D-galactopyranoside (X-gal, Stratagene) and 20mM of isopropyl. beta. -D-thiogalactopyranoside (IPTG, Sigma) and incubated at 37 ℃ for 18 hours. Blue colonies contained mutated plasmids. A single colony of each mutant was picked from agar and inoculated on 10mL of LB medium containing 100. mu.g/mL ampicillin. By usingSpin miniprep kit (Qiagen) isolates plasmids. The plasmid was sequenced using the pQE for and pQE Rev primers and the correct mutations were verified.

1.4 transformation and cloning

mu.L (50 ng/. mu.l) of plasmid DNA (from fractions 1.2 and 1.3) was added to 1mL of cold (4 ℃ C.) competent E.coli BL21(DE3) pLysS Singles^TMCells (Novagen). After 20 minutes the cells were incubated in a water bath at 42 ℃ for 30 seconds to heat shock. mu.L of Psi medium was added to the cell/plasmid mixture and shaken for 90 minutes at 37 ℃.50 μ L and 100 μ L aliquots were spread onto LB agar plates containing 100 μ g/mL ampicillin (Sigma) and incubated at 37 ℃ for 18 hours. The single colonies were cultured and produced cell cryopreserved and stored at-80 ℃. Plasmid DNA of the cell frozen stock was analyzed to verify correct transformation.

1.5 expression

The frozen transformed E.coli cells (from 1.4 parts) were recovered in ampoules and inoculated into flasks containing 100mL of LB medium and 100. mu.g/mL of ampicillinForest sealed Erlenmeyer flask. The flask was incubated with shaking at 37 ℃ overnight. 5mL of this overnight culture was added to a 2L Erlenmeyer flask (500mL LB medium/and 100. mu.g/mL ampicillin) and incubated at 37 ℃ with shaking. 2mL of broth samples were taken every hour and the growth and expression of TGF-. beta.3 "wild type" and mutant proteins (post-induction) were followed. Growth was determined by spectrophotometric measurement of the absorption at a wavelength of 600 nm. When the measured uptake was 0.6Abs, the cells were induced to express "wild-type" and mutant TGF- β 3 protein by addition of isopropyl β -D-thiogalactopyranoside (IPTG, Sigma) to a final concentration of 1 mM. The cultures were incubated for an additional 4 hours. A0.5 mL sample of the broth was pelleted by centrifugation (Sorvall Biofuge10,000rpm for 10min) and the supernatant discarded. The pellet was resuspended in 50. mu.L of Sodium Dodecyl Sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE) sample buffer and heated in a water bath at 95 ℃ for 10 minutes. mu.L of the sample was applied to SDS-PAGE. SDS-PAGE and Coomassie blue staining (as described in A.T Andrews (1986)) withMighty Small SE 245 Dual Gel case (Amersham). The gel thickness was 1mm and contained 15% (v/v) polyacrylamide gel.

1.6 cell Collection and isolation of Inclusion bodies

Cells from fraction 1.5 were pelleted by centrifugation at 5000g for 10min in a Hettich Rotina 46R centrifuge with 4315 rotor heads. Cell disruption and recovery of insoluble (inclusion body) TGF-. beta.3 protein was performed at 4 ℃. Cells were suspended in 50mL 100mM Tris/HCl (Sigma) and 10mM EDTA (Sigma) at pH 8.3 and sonicated with Sanjo Soniprep 150. 0.2% (w/w) Triton X-100(Sigma) was added and the suspension was stirred for 1 hour. The suspension was centrifuged at 15,000g for 40 minutes. The pellet was resuspended in 50mL of 100mM Tris/HCl pH 8.3 and 10mM EDTA before centrifugation at 12,000g for 40 minutes.

1.7 solubilization of Inclusion bodies

The precipitate from fraction 1.6 was resuspended in 40mL 8M Urea 1% (w/w) DL-Dithiothreitol (DTT) and broken up in a Heidolph Diax 900 homogenizer. The suspension was capped and stirred for 1 hour to solubilize the inclusion bodies, reducing the TGF-beta 3 "wild type" and the mutant proteins to their monomeric form. The suspension was then centrifuged at 15,000g for 30 minutes. The supernatant was dialyzed to change the buffer from 8M urea (ICN Biomedical) to 10% (v/v) acetic acid. Coli protein dissolved in 8M urea precipitated out of solution when the buffer was exchanged to 10% (v/v) acetic acid. 1% (w/w) DTT (Sigma) was added to the suspension, capped and stirred for 30 min to reduce any disulfide bonds formed between TGF-beta 3 monomers during buffer exchange. The suspension was centrifuged at 12,000g for 40 minutes to separate soluble and insoluble proteins. Samples taken from the urea solubilization and buffer exchange steps (acetic acid soluble and insoluble material) were then analyzed by SDS-PAGE.

1.8 Ultrafiltration

10% (v/v) acetic acid soluble material from fraction 1.7 was ultrafiltered on a 10kDa membrane in Vivoflow50 (Vivascience). The aim of this was to reduce the volume of the 10% (v/v) acetic acid suspension to 3mL and to remove low molecular weight proteins (<10 kDa).

1.9 gel filtration

Samples from fraction 1.7 were chromatographed on a Hiprep 26/60 Sephacryl S-100 high resolution column (Amersham, 320mL) at a flow rate of 10% (v/v) acetic acid of 1.5 mL/min. The denatured TGF- β 3 fraction (eluting between 100 and 140 minutes) containing the monomer was collected.

1.10 Freeze drying

The collection fraction containing the denatured monomer TGF-. beta.3 was freeze-dried using an IEC Lyoprep-3000 freeze-dryer to remove acetic acid and water from the sample.

1.11 refolding

Lyophilized monomeric TGF-. beta.3 from 1.10 fractions was solubilized in 8M urea containing 10mM DTT until a final TGF-. beta.3 concentration of 10mg/mL was reached. While stirring, the TGF-. beta.3 solution was added dropwise to the refolding solution (1M 3- (-pyrido) -1-propanesulfonate (NDSB-201), 20% (v/v) dimethyl sulfoxideSulfone (DMSO, Sigma), 2% (w/v)3- (3-Cholamidopropyl) dimethylammonio-1-propanesulfonate (CHAPS), 1M NaCl (Sigma), 1% (w/v) reduced glutathione (GSH, Sigma) and 0.05MBase (Sigma) pH 9.3) up to a final concentration of TGF-. beta.3 of 0.2 mg/mL. It is important to maintain the pH in the range of 9.2-9.4 with concentrated NaOH/HCl. The solution was sealed with a Parafilm (Parafilm) perforated to allow oxidation of the monomer TGF-. beta.3 and stirred at 8 ℃. After 144 hours 15,000g of the solution were centrifuged for 40 minutes to remove the precipitate formed and the pH was adjusted to pH 3.5 with glacial acetic acid. The supernatants contained disulfide-linked dimeric TGF-beta 3, which was determined by SDS-PAGE (non-reducing) and Western blotting. SDS-PAGE was performed as described in section 2.1.

For Western blotting, samples were loaded onto a 1mm thick 15% (v/v) polyacrylamide gel. Once the electrophoresis was completed, the proteins in the gel were electrophoretically transferred to nitrocellulose membrane (Sigma) using a TE22Western blotting apparatus (Pharmacia) according to the instructions of the instruction manual. Non-specific binding sites on nitrocellulose membranes were blocked with blocking buffer (5% (w/v) skim milk powder, 1% (v/v) polyoxyethylene sorbitan monolaurate (Tween20, Sigma) in phosphate buffered saline (Invitrogen)). The nitrocellulose membrane was then washed with wash buffer (PBS, 0.1% Tween 20). The nitrocellulose membrane was then incubated for 1 hour with primary antibody (MAB643(R & D system)) diluted 1:500 with PBS containing 0.1% (v/v) Tween 20. The nitrocellulose membrane was washed again and then incubated for 1 hour with a secondary antibody to goat anti-mouse antibody (Abcam) diluted 1:3000 with PBS containing 0.1% (v/v) Tween 20. The nitrocellulose membrane was washed once last, and then the antigen-antibody complex was developed by adding ECL reagent (Amersham). The nitrocellulose membrane was exposed to X-ray film in a dark room before being immersed in a developer, fixative and stop solution. The nitrocellulose membrane is then dried. Refolded TGF-beta 3 'wild type' and mutant monomeric proteins show different levels of dimer formation. The percentage of correctly folded dimers recovered from other incorrectly refolded or non-dimeric TGF-beta 3 proteins is shown in Table 3.

Interestingly, amino acid substitutions within the alpha helix of the TGF-. beta.3 protein had the greatest effect on refolding yields (dimer formation). Replacing glycine with arginine to stabilize the alpha helix increases refolding yields from 20% to 50%. In contrast, replacement of the glycine alpha helix disruption with proline results in a much lower percentage of dimer formation. This suggests that the alpha helix plays an important role in the formation of a correctly refolded TGF-. beta.3 molecule. Substitution of the amino acids involved in the formation of the 'salt bridge' does not affect refolding yields.

1.12 hydrophobic interaction chromatography

Fraction 1.11 of the renaturation solution is concentrated to 50mL by ultrafiltration using a 10kDa (molecular weight cut-off) membrane on Vivoflow50 (Vivascience). The renaturation solution was diluted 1:1 with a solution containing 2M ammonium sulphate (Sigma) and 10% (v/v) acetic acid. A2 ml Biorad column packed with phenyl-sepharose fast flow rate (Amersham) was equilibrated with buffer A (1.0M ammonium sulphate and 10% (v/v) acetic acid). 20mL of the diluted renaturation solution was added to the column at a flow rate of 1mL/min (this flow rate was used throughout the process). The column was then washed with buffer A until the absorbance reading at 280nm reached baseline level (10 mL). Then 100% buffer B (10% (v/v) acetic acid 30% (v/v) isopropanol) was added to the column. The first peak was collected, which contained monomeric and dimeric forms of the TGF-. beta.3 protein (see FIG. 1).

1.13 cation exchange chromatography

Cation exchange chromatography was used to separate the dimeric TGF-beta 3 protein from the monomers. A2 mLUNO-S1 column (Biorad) was equilibrated with buffer A (containing 10% (v/v) acetic acid, 30% (v/v) isopropanol). The fraction collected from fraction 3.12 was applied to a UNO-S1 column at a flow rate of 1 mL/min. The column was then washed with buffer A until the absorbance reading at 280nm reached baseline level (5 min). The linear gradient was run for 5 min, ending with a mixture of 60% buffer A and 40% buffer B (10% (v/v) acetic acid, 30% (v/v) isopropanol and 1M NaCl). The addition of the buffer mixture was maintained for an additional 15 minutes. Monomeric TGF-. beta.3 was eluted from the column 10 minutes after sample injection. The second linear gradient was run for an additional 5 minutes, ending with 100% buffer B for 10 minutes. Dimeric TGF- β 3 was eluted from the column 30 min after sample injection (see figure 2).

1.14 Ultrafiltration/diafiltration

1.13 fractions containing purified monomeric and dimeric TGF-. beta.3 molecules were ultrafiltered/diafiltered to exchange the buffer with 20mM acetic acid, 20% (v/v) isopropanol, and the samples were concentrated to about 10mg/mL (TGF-. beta.3 concentration determined by UV spectrophotometer). Vivoflow50(Vivascience) with a 10kDa cut-off was used to change the buffer and concentrate the samples.

2. In vitro characterization of TGF-beta 3 of the invention

2.1 SDS-PAGE analysis of purified TGF-beta 3 of the invention

Purified TGF-. beta.3 muteins (from fraction 1.14) were assessed by SDS-PAGE to determine purity and molecular weight. Due to the low pH of the TGF-. beta.3 mutant samples from 3.14 fractions, the buffer was exchanged with SDS-PAGE sample buffer using a protein desalting elution column (Pierce). Mu.g of purified TGF-. beta.3 mutein (reduced or non-reduced samples), 3. mu.g of 'wild-type' TGF-. beta.3 positive control (reduced and non-reduced) and 10. mu.L of Invitrogen Mark 12 molecular weight standard were added to polyacrylamide gels (10% -20% (v/v) gradient of acrylamide). Once the electrophoresis was complete, the gel was stained with coomassie blue.

The ` wild type ` TGF-. beta.3 and Gly63-Ala muteins were run to the same position on the gel as expected (about 13kDa for reduced samples and about 25kDa for non-reduced samples). Interestingly, none of the Gly63-Pro mutein bands were detected in the non-reducing gel but in the reducing gel. Since Gly63-Pro refolding efficiency was very low (< 1%), multiple refolding species below the detection level of coomassie (>1 μ g) could be produced. However, when these species were reduced, the concentration of the reduced Gly63-Pro monomer was above the Coomassie staining detection limit of 1 μ g, so Gly63-Pro could be seen in the reducing gel. The reduced Gly63-Pro protein band position was slightly higher than the ` wild type ` TGF-. beta.3 band position. This was expected because the substitution of glycine with proline at position 63 made the Gly63-Pro mutein larger in molecule than the 'wild-type' TGF-beta 3 (see figure 3).

2.2 cytostatic assays comparing the biological Activity of TGF-beta 3 of the invention and wild-type TGF-beta 3

The cytostatic assay (A Meager, 1991) is an in vitro bioactivity test of TGF-. beta.molecules. The colorimetric assay is based on the growth inhibitory effect of TGF-beta molecules on Mink Lung Epithelial Cells (MLEC). 100 μ L of a cell suspension containing the following was added to each well of a 96-well tissue culture plate: 1 x 10⁴MLEC cells/mL and complete medium (dmem (Invitrogen), 0.01M Hepes buffer (Invitrogen), 2mM L-glutamine (Invitrogen), L-arginine (Invitrogen), L-asparagine (Invitrogen), 100 units/mL penicillin (Invitrogen), 50 μ g/mL streptomycin (Invitrogen) and 5% fetal bovine serum (Invitrogen)). Passing through a temperature of 37 ℃ and 5% CO₂After overnight incubation, 100. mu.L of serial dilutions (10pg/mL to 500pg/mL) of dimeric TGF-beta 3 mutant samples (from 3.14 fractions) were added to the plates. Control wells were added with 200. mu.L of complete medium and 10% (v/v)0.25M maltose. The plate was incubated for a further 120 hours, 50. mu.L of 2mg/mL3- [4, 5-dimethylthiazol-2-yl ]]-2, 5-diphenyltetrazolium bromide: thiazolium blue (MTT, Sigma) was added to each well. The plates were incubated for an additional 4 hours and the medium was removed. mu.L of 0.05M HCl (BDH), anhydrous isopropanol (BDH) was added to each well, and the resulting solubilized formazan (formazan) was then labeled with a fully automatic quantitative plotter plate reader (Victor)²1420) Quantitative determination at 570 nm.

Gly63-Ala and TGF-. beta.3 of the present invention have an inhibitory effect on MLEC cells at a concentration of 0 to 500 pg/mL. As can be seen from Table 4, the IC of the Gly63-Ala mutein₅₀IC of 34pg/mL versus ` wild type ` TGF-. beta.3₅₀It was 26 pg/mL.

2.3 amino acid sequence analysis

50 microliter purified ` wild type ` and mutant TGF-. beta.3 samples from the 3.14 fraction were vacuum dried and then treated with 20. mu.L of a solution containing 50mM NH₄HCO₃And 10% (v/v) acetonitrile. 20 μ g sequencing grade trypsin (Promega) was resuspended in 10 μ L resuspension buffer (Promega) supplied with the kit so that the trypsin concentration was 2 μ g/. mu.L. The resultant was then treated with 50mM NH₄HCO₃And 10% (v/v) acetonitrile to a final trypsin concentration of 0.2. mu.g/. mu.L. Digestion was carried out overnight by adding trypsin in a ratio of 1:20(w/w) to wild type' and mutant TGF-. beta.3 proteins. Digestion was terminated by addition of formic acid (Fluka) to a final concentration of 0.1% (v/v). The sample was then diluted to 1 pmole/. mu.L. Peptides were analyzed by nano-flow RPLC-MS method (Critical System, Dionex on line Q-ToF2, Trace). Chromatography was performed on a 75 μm C18 column (LC pad) using a 45 min gradient from 5% (v/v) acetonitrile to 55% (v/v) acetonitrile. MS analysis takes the form of data-dependent analysis in which the instrument measures the m/z of the peptide ions eluting from the LC and selects the appropriate ions for MS-MS analysis, using collision-induced dissociation to fragment the peptide ions to provide sequence information.

3 in vivo Properties of TGF-. beta.3 of the invention

The biological effects of refolding active 'wild type' and mutant TGF- β 3 proteins on incisive wound and incisional wound healing (3 days post-wound) and scarring (70 days post-wound) were examined in adult male mice.

3.1 comparison of wound healing Effect of wild-type TGF-. beta.3 and TGF-. beta.3 of the present invention

Males (Sprague Dawley) were anesthetized with halothane and the dorsal hairs were shaved. The wound site was marked as shown in fig. 4 using standard stencil and skin marking ink. Samples were diluted to the concentrations specified in Table 4 with sterile excipient buffer containing 0.25M maltose (Sigma), 0.002% (v/v) acetic acid and 0.33% (v/v) isopropanol. All samples were sterile filtered and endotoxin free. 4 mice were used for each treatment group. 100 μ L samples (table 4) from each treatment group were injected intradermally into the sites a and B of the marked wounds (except for non-treated (non-experimental) mice). Wound sites a and B were punch biopsies. All animals were individually housed in cages. The animals received two doses of the sample after 24 hours. The wounds were photographed 3 days later and analyzed using a Visual Analogue scoring system (modified from Beausang, E et al 1998). Statistical analysis of the data was performed using the Mann WhitneyU/Student T test. Values with p <0.05 were considered significant.

3.2 assessment of wound healing of day 3 incisive wounds treated with wild-type TGF-. beta.3 or TGF-. beta.3 of the invention Using visual analogue scoring with naked eye

Incisive wounds were examined 3 days later using Visual Analogue Scale (VAS). In this 10 point score, 0 points represent well-healed wounds and 10 points represent poorly-healed wounds. And (3) displaying data:

treatment with 50ng/100 μ L or 100ng/100 μ L of wild-type TGF- β 3 reduced the VAS score (i.e., improved the visual appearance of the wound) compared to untreated (uninvolved controls). Treatment with a dose of 100ng/100 μ L significantly improved (p <0.05) the appearance of the wound, i.e. accelerated healing, compared to untreated (non-experimental control).

Treatment with either 50ng/100 μ L or 100ng/100 μ L of Gly63-Ala mutant reduced the VAS score compared to untreated (non-experimental controls) and was comparable to wounds treated with wild-type TGF-. beta.3, i.e., did not impair healing.

Treatment with either 50ng/100 μ L or 100ng/100 μ L of Gly63-Pro mutant reduced VAS scores compared to untreated (uninvolved controls) and was comparable to wounds treated with wild-type TGF-. beta.3, i.e., did not impair healing.

3.3 microscopic assessment of wound Width on day 3 of incisional wounds treated with wild-type and mutant TGF-beta 3 proteins

Incision wound width was evaluated microscopically after 3 days. All wounds treated with wild-type and mutant proteins of TGF-beta 3 showed comparable wound widths to placebo and untreated (non-experimental) controls, demonstrating that the TGF-beta 3 mutein had no adverse effects on healing (FIG. 6).

3.4 'Effect of wild type' and mutant TGF-. beta.3 proteins on wound scarring (wound day 70)

Males (Sprague Dawley) were anesthetized with halothane and the dorsal hairs were shaved. The wound site was marked as shown in fig. 7 with a standard template and skin marking ink. Samples were diluted with sterile vehicle buffer containing 0.25M maltose (Sigma), 0.002% (v/v) acetic acid and 0.33% (v/v) isopropanol to the concentrations specified in Table 5. All samples were sterile, endotoxin free and pyrogen free. 4 mice were used for each treatment group. 100 μ L samples (table 6) from each treatment group were injected intradermally into the sites a and B of the marked wounds (except for non-treated (non-experimental) mice). Incisions of a total depth of 1cm length were made at wound sites a and B with a number 11 surgical blade. All animals were individually housed in cages. The animals received two doses of the sample after 24 hours. Scar formation after 70 days was photographed and analyzed using a visual analogue scoring system with naked eyes (modified from Beausang, E et al 1998). The wound was excised and placed in 10% buffered saline before processing into a wax lump. The wax block was cut into 5 μm serial sections and placed on a glass slide. The slides were stained and analyzed by Masson Trichrome (Massons Trichrome). Statistical analysis of the data was performed using the Mann Whitney U/Student T test. Values with p <0.05 were considered significant.

3.5 evaluation of day 70 incisional wounds Using macroscopic VAS

Incisive wounds were examined after 70 days using a macroscopic VAS system. Score 10 indicates a very bad scar and score 0 is normal skin (figure 8). VAS analysis of wounds at day 70 showed:

'wild type' TGF-. beta.3 (at 50 ng/100. mu.L and 100 ng/100. mu.L doses) reduced scarring compared to placebo-treated and untreated wounds. The reduction in scar formation was statistically significant for both doses compared to the placebo-treated wounds (p < 0.05).

Gly63-Ala mutants (at doses of 50 ng/100. mu.L and 100 ng/100. mu.L) reduced scarring compared to placebo-treated and untreated wounds. The reduction of scar formation with the 50ng/100 μ L dose was statistically significant (p <0.05) compared to the placebo treated wounds.

Gly63-Pro mutant (at doses of 50 ng/100. mu.L and 100 ng/100. mu.L) reduced scarring compared to placebo-treated and untreated wounds.

Glu12-Ser mutant (at 50 ng/100. mu.L and 100 ng/100. mu.L doses) reduced scarring compared to placebo-treated and untreated wounds. The reduction of scar formation with the 50ng/100 μ L dose was statistically significant (p <0.05) compared to the placebo treated wounds.

The double serine mutants (Glu12-Ser and Arg52-Ser) reduced scarring at the doses of 50 ng/100. mu.L and 100 ng/100. mu.L compared to placebo-treated and untreated wounds.

3.6 microscopic evaluation of incisive wounds on day 70

The macroscopic effects recorded using the VAS scoring system were confirmed by histological analysis. Representative examples of tissue sections are shown in fig. 12 and 13. The tissue micrographs show that the addition of the TGF-beta 3 protein of the invention elicits similar improvements as seen in "wild-type" TGF-beta 3. The collagen fibers in the protein-induced scar of the present invention have a morphology and tissue similar to surrounding normal skin.

4. Conclusion

The flexibility of the alpha helix (between amino acid residues 58-67) affects the formation of the functional, correctly refolded dimeric TGF-beta 3 during refolding. The efficiency of refolding is greatly improved by replacing the glycine at position 63 with alanine to stabilize the alpha helix, whereas the opposite effect is obtained by replacing the glycine at position 63 with proline to destabilize the alpha helix.

Substitution of glycine at position 63 with alanine or proline did not alter wound healing compared to 'wild type' TGF- β 3.

Gly63-Ala and Gly63-Pro reduced scarring compared to placebo-treated and untreated wounds.

The formation of a 'salt bridge' (between Arg52 and Glu 12) did not alter TGF- β 3 refolding efficiency.

Compared to placebo-treated and untreated wounds, Glu12-Ser type and the bisserine mutant reduced scarring.

5. Preferred protocols for the production of monomeric or dimeric TGF-beta 3 of the invention

Preferred conditions for producing correctly refolded monomeric TGF-. beta.3 s of the invention are as follows:

0.7M 2- (cyclohexylamino) ethanesulfonic acid (CHES), 2mM reduced glutathione, 0.4mM oxidized glutathione (GSSG), 0.12mg/mL TGF-. beta.3, pH 9.5, 2-8 ℃.

30mM taurodeoxycholate, 0.7M CHES, 2mM GSH, 0.4mM GSSG, 0.12mg/mL TGF-. beta.3, pH 9.5, 2-8 ℃.

1M NDSB-201, 2mM reduced Glutathione (GSH), 2mM oxidized glutathione (GSSG), 0.12mg/mL TGF-. beta.3, pH 9.5, 2-8 ℃.

0.7M CHES, 2mM reduced Glutathione (GSH), 2mM oxidized glutathione (GSSG), 0.12mg/mL TGF-. beta.3, pH 9.5, 2-8 ℃.

30mM taurodeoxycholate plus 1M NDSB-221, 2mM reduced Glutathione (GSH), 2mM oxidized glutathione (GSSG), 0.12mg/mL TGF-. beta.3, pH 9.5, 2-8 ℃.

30mM taurodeoxycholate plus 0.7M CHES, 2mM reduced Glutathione (GSH), 2mM oxidized glutathione (GSSG), 0.12mg/mL TGF-. beta.3, pH 9.5, 2-8 ℃.

30mM taurodeoxycholate, 0.7M CHES, 2mM GSH, 2mM GSSG, 0.12mg/mL TGF-. beta.3, pH 9.5, 2-8 ℃.

In general, TGF-beta 3 of the invention may be folded into a dimeric biologically active form by a method comprising:

adding the dissolved, unfolded monomeric TGF- β 3 to a solution comprising:

(i)2- (cyclohexylamino) ethanesulfonic acid (CHES) or a functional analog thereof; and

(ii) a low molecular weight sulfhydryl/disulfide redox system; and

the growth factors are incubated in the solution until dimeric biologically active TGF-beta 3 is formed.

Preferred conditions for producing correctly refolded dimeric TGF-beta 3 of the invention are as follows:

0.7M 2- (cyclohexylamino) ethanesulfonic acid (CHES), 2mM reduced glutathione, 0.4mM oxidized glutathione (GSSG), 0.12mg/mL TGF-. beta.3, pH 9.5, 2-8 ℃.

30mM taurodeoxycholate, 0.7M CHES, 2mM GSH, 0.4mM GSSG, 0.12mg/mL TGF-. beta.3, pH 9.5, 2-8 ℃.

30mM taurodeoxycholate, 0.7M CHES, 2mM GSH, 2mM GSSG, 0.12mg/mL TGF-. beta.3, pH 9.5, 2-8 ℃.

Preferred conditions of the experiment

5.1 vector cloning and host cell transformation

The pET-24d vector is derived from the pBR322 vector and contains a T7 promoter controlled by LacUV5 and a kanamycin resistance marker gene.

DNA encoding TGF-. beta.3 of the invention can be digested with 0.75. mu.L Nco1(New England Biolabs) and 0.75. mu.L BamH1(New England Biolabs) with 1 XBamH 1 buffer (New England Biolabs) in 15. mu.L reaction solution (Nuclear Free Water, Novagen) at 37 ℃ for 4 hours. 1 microliter of the pET-24d plasmid (Novagen) was digested in the same manner. The digested cDNA and large plasmid fragments were purified on agarose Gel and recovered using the SpinPrep Gel DNA extraction kit (Novagen).

The purified cDNA and plasmid fragments were ligated using T4 ligase kit (Novagen). The ligated cDNA/plasmid was transformed into HMS 174(DE3) (Novagen HMS 174(DE3) transformation kit). Transformants were selected by plating on Luria Broth (LB) agar plates containing 50. mu.g/mL kanamycin (Invitrogen). Suitable clones are selected for restriction digestion and/or expression.

5.2 clonal screening for product expression

Clones were cultured in shake flask medium of 1/2 concentration of "Terrific Broth" (6g/L phytone peptone (Becton Dickinson), 12g/L yeast extract (Becton Dickinson), 2g/L glycerol (JT Baker), 1.16g/L potassium dihydrogenphosphate (JT Baker), 6.25g/L dipotassium hydrogenphosphate (JT Baker), distilled water in an appropriate amount to 1 liter) and grown in exponential growth phase OD₆₀₀Induced with 1mM isopropyl-. beta. -D thiogalactopyranoside (IPTG) at 0.65-0.85. After 3 hours of IPTG addition, post-induction samples were taken and analyzed for induction and expression by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples from suitable clones are described inNovex 12% Bis-TrisGel, 1.0mm (Invitrogen), run at 120 milliamps and 200 volts for about 40-50 minutes, and then stain with Coomassie blue. Thus, expression of TGF-. beta.3 of the present invention was induced in these cultures.

5.3 frozen stock of cells

Clones were grown to OD in shake flasks of Terrific Broth at 1/2 concentration₆₀₀About 1 and was preserved as a glycerol stock by adding glycerol to 20% (v/v). 1.2mL of broth was aliquoted into 12X 2mL cryovials (containing 0.3mL of glycerol) and then stored at-70 ℃.

5.4 TGF-. beta.3 Gene sequence confirmation

Culture samples for cell cryopreserved were obtained before addition of glycerol and used for plasmid isolation using the Qiagen MiniPrep Kit. The isolated plasmids were sequenced and verified with the T7 promoter primer (5'-TAA TAC GAC TCA CTA TAG GG-3') and the T7 terminator primer (5'-GCTAGT TAT TGC TCA GCG G-3').

5.5 seed culture

The appropriate clones selected were inoculated into 2 liter sealed (baffled) Erlenmeyer flasks containing 500mL HySoy medium (12g/L peptone (Quest International), 24g/L yeast extract (Becton Dickinson), 10g/L NaCl (Sigma) and 10g/L glycerol (Sigma) and 50. mu.g/mL kanamycin, flasks incubated at 37 ℃ with shaking at 200rpm, and periodic sampling for OD₅₅₀. When the OD of the culture reached 3.21U/mL (after 7 hours), the cell broth was used to inoculate a 150L fermentor (100L working volume).

5.6 fermentation

900 ml of cell broth (from 3.6 parts) were used to inoculate a 150L fermenter (WHE) containing 90L of batch medium (0.6g/L K)₂HPO₄、0.4g/L KH₂HPO₄、1.25g/LNH₄SO₄12g/L peptone, 24g/L yeast extract and 10g/L glycerol). The operating parameters of the fermentation were controlled as follows: a temperature setpoint of 37 ℃; the pH setpoint was 7.0 (maintained with 4N ammonium hydroxide and 4N phosphoric acid) and Dissolved Oxygen (DO) was initially calibrated to 100%. The vessel discharge pressure was 7psi, the agitation speed and air flow rate were 200-. DO was maintained above 20% by adjusting the parameters set for fermentation in the following priority order: stirring speed (max 400rpm), aeration (max 1.5vvm), oxygen uptake (max 33.3Ipm) and back pressure (max 12 psi). Foaming was controlled with Pluronic L-61 (25% v/v). When the OD of the culture reached 10U/mL, glycerol (50% v/v) was supplemented at an initial flow rate of 45 mL/min. When the OD value reached 40U/mL, IPTG was added to a final concentration of 0.2mM for cell induction.

5.7 Collection

4 hours after induction, the fermentor was cooled to 10 ℃ and the gas flow and agitation rate were reduced to 0.3vvm and 100rpm, respectively. Control of foam and pH was terminated and the back pressure was adjusted to 3 psi. The culture was collected by continuous centrifugation at 10 ℃ using a Westfalia CSA8 continuous centrifuge. The centrifuge was run at 15,000rpm with a flow rate of 3 liters per minute and cell slurry was collected.

5.8 cell lysis and IB recovery

The fermented cell bodies (from fraction 5.7) were diluted 1:5 with lysis buffer (6.1g/L TrizmaBase (Tris), 3.7g/L ethylenediaminetetraacetic acid (EDTA), 58.44g/L NaCl and 10g/L Triton X-100, pH 8.0) and resuspended in a hand-held homogenizer. The resuspended cell bodies were passed twice through a high-pressure homogenizer (parameters: pressure 10,000 psig; flow rate 450 mL/min; temperature 15 ℃). The homogenized cell lysate is then centrifuged (barrel centrifuge, fixed angle rotor) at 5,000Xg for 20 minutes at 4 ℃. The supernatant was removed, leaving the insoluble (inclusion bodies) TGF-. beta.3. Inclusion Body (IB) pellets were resuspended in wash buffer (6.1g/L Tris and 3.72g/L EDTA, pH 8.0) using a hand-held homogenizer and centrifuged (4 ℃, 5,000Xg20 min).

5.9 Inclusion body solubilization

The pellet from fraction 5.8 was diluted 1:10 with lysis buffer (6.1g/L Tris, 15.4g/L DL-Dithiothreitol (DTT) and 360.4g/L urea, pH 8.0) and resuspended in a hand-held homogenizer. The suspension was capped and stirred at room temperature for 60-75 minutes to solubilize the inclusion bodies and reduce TGF-. beta.3 to the monomeric form. Before the second incubation period of 60-75 minutes, the pH of the resuspended pellet was adjusted to pH 9.4-9.6 with NaOH/acetic acid.

5.10 clarification/Ultrafiltration and diafiltration

The dissolved material from fraction 5.9 was purified, concentrated and diafiltered in a Tangential Flow Filtration (TFF) system (Millipore). Initial clarification and concentration was accomplished using a pre-treated clarified TFF membrane (Millipore Pellicon 1000kDa, regenerated cellulose, mesh V). The purified TGF-. beta.3 was collected in the permeate. Switch to an ultrafiltration/diafiltration (UF/DF) membrane (Millipore Pellicon 5kDa, regenerated cellulose, mesh C) followed by washing of TGF-. beta.3 in 6 displacement volumes (diavolme) of lysis buffer (6.1g/L Tris, 15.4g/L DTT and 360.4g/L urea, pH 9.5).

5.11 Ultrafiltration/hydrophobic interaction chromatography

Selected refolding solutions were ultrafiltered (membrane flat plate microporous Pellicon 5kDa, 0.1 m)²Regenerated cellulose, screen) was concentrated 5 times. The pH of the concentrated refolded material was adjusted to pH 2.5-2.8 with glacial acetic acid prior to 1:1 dilution in dilution buffer (2.72g/L sodium acetate, 264.28g/L ammonium sulfate, 100g/L acetic acid and 210.7g/L arginine hydrochloride, pH 3.3). Butyl Sepharose 4 fast flow column (Amersham, bed height 16cm) was equilibrated with four column volumes of buffer A (2.72g/L sodium acetate, 132.14g/L ammonium sulfate and 100g/L acetic acid, pH 3.3). The refolded material was filtered through a 0.22 μ M membrane (Millipak filter) before being applied to the butyl sepharose column at a flow rate of 100cm/hr (this flow rate was used throughout the process). The column was then washed with four column volumes of buffer a. TGF-. beta.3 protein was eluted from the column with buffer B (2.72g/L sodium acetate, 100g/L acetic acid and 300g/L ethanol, pH 3.3). The first peak containing monomeric and dimeric forms of the TGF-beta 3 protein is collected prior to separation of the monomeric and dimeric proteins.

Sequence information

TGF-beta 3 (SEQ ID NO: 1)

ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADT

THSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS

Mutant TGF-. beta.3 "Gly 63-Ala" (SEQ ID NO. 3)

ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADT

THSTVLALYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS

Mutant TGF-. beta.3 "Gly 63-Pro" (SEQ ID NO: 5)

ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADT

THSTVLPLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS

Mutant TGF-. beta.3 "Glu 12-Ser" (SEQ ID No. 7)

ALDTNYCFRNLSENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADT

THSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS

Mutant TGF-. beta.3 "Arg 52-Ser" (SEQ ID NO. 9)

ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLSSADT

THSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS

Mutant TGF-. beta.3 "Glu 12-Ser/Arg 52-Ser" (SEQ ID NO. 11)

ALDTNYCFRNLSENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLSSADT

THSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS

Sequence ID No. 2-DNA encoding wild-type human TGF-. beta.3

GCT TTG GAC ACC AAT TAC TGC TTC CGC AAC TTG GAG GAG AAC TGC TGT GTG CGC

CCC CTC TAC ATT GAC TTC CGA CAG GAT CTG GGC TGG AAG TGG GTC CAT GAA CCT

AAG GGC TAC TAT GCC AAC TTC TGC TCA GGC CCT TGC CCA TAC CTC CGC AGT GCA

GAC ACA ACC CAC AGC ACG GTG CTG GGA CTG TAC AAC ACT CTG AAC CCT GAA GCA

TCT GCC TCG CCT TGC TGC GTG CCC CAG GAC CTG GAG CCC CTG ACC ATC CTG TAC

TAT GTT GGG AGG ACC CCC AAA GTG GAG CAG CTC TCC AAC ATG GTG GTG AAG TCT

TGT AAA TGT AGC

DNA encoding Gly63-Ala mutant of SEQ ID No. 4

GCT TTG GAC ACC AAT TAC TGC TTC CGC AAC TTG GAG GAG AAC TGC TGT GTG CGC

CCC CTC TAC ATT GAC TTC CGA CAG GAT CTG GGC TGG AAG TGG GTC CAT GAA CCT

AAG GGC TAC TAT GCC AAC TTC TGC TCA GGC CCT TGC CCA TAC CTC CGC AGT GCA

GAC ACA ACC CAC AGC ACG GTG CTG GCA CTG TAC AAC ACT CTG AAC CCT GAA GCA

TCT GCC TCG CCT TGC TGC GTG CCC CAG GAC CTG GAG CCC CTG ACC ATC CTG TAC

TAT GTT GGG AGG ACC CCC AAA GTG GAG CAG CTC TCC AAC ATG GTG GTG AAG TCT

TGT AAA TGT AGC

DNA encoding Gly63-Pro mutant of SEQ ID NO. 6

GCT TTG GAC ACC AAT TAC TGC TTC CGC AAC TTG GAG GAG AAC TGC TGT GTG CGC

CCC CTC TAC ATT GAC TTC CGA CAG GAT CTG GGC TGG AAG TGG GTC CAT GAA CCT

AAG GGC TAC TAT GCC AAC TTC TGC TCA GGC CCT TGC CCA TAC CTC CGC AGT GCA

GAC ACA ACC CAC AGC ACG GTG CTG CCA CTG TAC AAC ACT CTG AAC CCT GAA GCA

TCT GCC TCG CCT TGC TGC GTG CCC CAG GAC CTG GAG CCC CTG ACC ATC CTG TAC

TAT GTT GGG AGG ACC CCC AAA GTG GAG CAG CTC TCC AAC ATG GTG GTG AAG TCT

TGT AAA TGT AGC

DNA encoding Glu12-Ser mutant of SEQ ID No. 8

GCT TTG GAC ACC AAT TAC TGC TTC CGC AAC TTG TCG GAG AAC TGC TGT GTG CGC

CCC CTC TAC ATT GAC TTC CGA CAG GAT CTG GGC TGG AAG TGG GTC CAT GAA CCT

AAG GGC TAC TAT GCC AAC TTC TGC TCA GGC CCT TGC CCA TAC CTC CGC AGT GCA

GAC ACA ACC CAC AGC ACG GTG CTG GGA CTG TAC AAC ACT CTG AAC CCT GAA GCA

TCT GCC TCG CCT TGC TGC GTG CCC CAG GAC CTG GAG CCC CTG ACC ATC CTG TAC

TAT GTT GGG AGG ACC CCC AAA GTG GAG CAG CTC TCC AAC ATG GTG GTG AAG TCT

TGT AAA TGT AGC

DNA encoding Arg52-Ser mutant of SEQ ID No. 10

GCT TTG GAC ACC AAT TAC TGC TTC CGC AAC TTG GAG GAG AAC TGC TGT GTG CGC

CCC CTC TAC ATT GAC TTC CGA CAG GAT CTG GGC TGG AAG TGG GTC CAT GAA CCT

AAG GGC TAC TAT GCC AAC TTC TGC TCA GGC CCT TGC CCA TAC CTC AGC AGT GCA

GAC ACA ACC CAC AGC ACG GTG CTG GGA CTG TAC AAC ACT CTG AAC CCT GAA GCA

TCT GCC TCG CCT TGC TGC GTG CCC CAG GAC CTG GAG CCC CTG ACC ATC CTG TAC

TAT GTT GGG AGG ACC CCC AAA GTG GAG CAG CTC TCC AAC ATG GTG GTG AAG TCT

TGT AAA TGT AGC

DNA encoding Glu12-Ser/Arg52-Ser mutant of SEQ ID No. 12

GCT TTG GAC ACC AAT TAC TGC TTC CGC AAC TTG TCG GAG AAC TGC TGT GTG CGC

CCC CTC TAC ATT GAC TTC CGA CAG GAT CTG GGC TGG AAG TGG GTC CAT GAA CCT

AAG GGC TAC TAT GCC AAC TTC TGC TCA GGC CCT TGC CCA TAC CTC AGC AGT GCA

GAC ACA ACC CAC AGC ACG GTG CTG GGA CTG TAC AAC ACT CTG AAC CCT GAA GCA

TCT GCC TCG CCT TGC TGC GTG CCC CAG GAC CTG GAG CCC CTG ACC ATC CTG TAC

TAT GTT GGG AGG ACC CCC AAA GTG GAG CAG CTC TCC AAC ATG GTG GTG AAG TCT

TGT AAA TGT AGC

TABLE 1 grades of alpha helix tropism based on experimental studies of proteins and peptides

TABLE 2

Mutant name	Amino acid substitution	Influence on the structure
			Gly63-Ala	Substitution of glycine at position 63 with alanine	Stabilized alpha helix
Gly63-Pro	Substitution of glycine at position 63 with proline	Destabilization of alpha helices
			Glu12-Ser	Replacement of glutamic acid at position 12 by serine	Prevention of salt bridge formation
Arg52-Ser	Substitution of arginine for serine at position 52	Prevention of salt bridge formation
			Glu12-Ser and Arg52-Ser	Double substitution of glutamic acid at position 12 with serine and arginine at position 52 with serine	Prevention of salt bridge formation

TABLE 3 efficiency of refolding wild-type TGF-. beta.3 and TGF-. beta.3 of the invention

Mutant TGF-beta 3	Percentage of correctly folded dimer
		Gly63-Ala	50
"wild type" TGF-beta 3	20
		Gly63-Pro	1
Glu12-Ser	20
		Glu12-Ser and Arg52-Ser	20

TABLE 4 evaluation of biological Activity of wild-type TGF-. beta.3 and Gly63-Ala (a TGF-. beta.3 protein of the invention) Using a cell growth inhibition assay

Protein	IC50
		"wild type" TGF-beta 3	26pg/mL
Gly63-Ala	34pg/mL

TABLE 5 wound site treatment

Group of	Treatment/sample	Concentration (ng/100. mu.L)
			A	"wild type" TGF-beta 3	50
B	"wild type" TGF-beta 3	100
			C	Gly63-Ala	50
D	Gly63-Ala	100
			E	Gly63-Pro	50
F	Gly63-Pro	100
			G	0.25M maltose (placebo control)	N/A
H	Untreated (unexperienced control)	N/A

TABLE 6 wound site treatment

Group of	Treatment/sample	Concentration (ng/100/. mu.L)
			A	"wild type" TGF-beta 3	50
B	"wild type" TGF-beta 3	100
			C	Gly63-Ala	50
D	Gly63-Ala	100
			E	Gly63-Pro	50
F	Gly63-Pro	100
			G	Glu12-Ser	50
H	Glu12-Ser	100
			I	Glu12-Ser and Arg52-Ser	50
J	Glu12-Ser and Arg52-Ser	100
			K	0.25M maltose (placebo)	N/A
L	Not tested	N/A

Claims (29)

1. TGF- β 3 or a fragment or derivative thereof, wherein the alpha helix forming domain between amino acid residues 58 and 67 of full length wild type TGF- β 3 comprises at least one alpha helix stabilizing substitution.
2. 2.TGF- β 3, or a fragment or derivative thereof, according to claim 1, wherein the glycine residue at position 63 of full-length, wild-type TGF- β 3 is replaced with an alpha helix-stabilising amino acid residue.
3. 3.TGF- β 3, or a fragment or derivative thereof, of claim 1 or claim 2, wherein the alpha helix-stabilising substitution comprises introduction of a residue selected from alanine, serine, threonine, valine, leucine, isoleucine, methionine and phenylalanine.
4. 4. TGF- β 3 or a fragment or derivative thereof according to claim 3, wherein the glycine residue at position 63 of the full length wild type TGF- β 3 is replaced by alanine.
5. 5. TGF- β 3 according to claim 4, comprising sequence ID No. 3 or a fragment or derivative thereof.
6. 6. TGF- β 3 or a fragment or derivative thereof of any preceding claim, which does not comprise a substitution of a valine residue at position 61 of full-length wild-type TGF- β 3.
7. TGF- β 3 or a fragment or derivative thereof, wherein the glycine residue at position 63 of the full length wild type TGF- β 3 is replaced by proline.
8. 8. TGF- β 3 according to claim 7, comprising sequence ID No. 5 or a fragment or derivative thereof.
9. TGF- β 3 or a fragment or derivative thereof comprising a substitution of a glutamic acid residue at position 12 of full length wild type TGF- β 3 and/or a substitution of an arginine residue at position 52 of full length wild type TGF- β 3.
10. 10. TGF- β 3 or fragments or derivatives thereof according to claim 9, wherein the one or more substitutions is with an amino acid residue selected from: serine, alanine, threonine, valine, isoleucine, methionine, phenylalanine, and leucine.
11. 11. TGF- β 3 or a fragment or derivative thereof of claim 9 or claim 10, wherein the glutamic acid residue at position 12 of full-length, wild-type TGF- β 3 is replaced with serine.
12. 12. TGF- β 3 or a fragment or derivative thereof according to any one of claims 9 to 11, wherein the arginine residue at position 52 of full-length wild-type TGF- β 3 is replaced with serine.
13. TGF- β 3 or a fragment or derivative thereof, said TGF- β 3 selected from: sequence ID number 7, sequence ID number 9, and sequence ID number 11.
14. 14. A monomeric TGF- β 3 or a fragment or derivative thereof according to any one of claims 1 to 13.
15. 15. Dimeric TGF- β 3 or a fragment or derivative thereof according to any one of claims 1 to 13.
16. 16. Use of a TGF-beta 3 or fragment of TGF-beta 3 or a TGF-beta 3 derivative according to any preceding claim as a medicament.
17. 17. Use of TGF- β 3 or a fragment or derivative thereof according to any one of claims 1 to 15 in the manufacture of a medicament for accelerating wound healing and/or preventing, reducing or inhibiting scarring.
18. 18. Use of TGF- β 3 or a fragment or derivative thereof according to any one of claims 1 to 15 in the manufacture of a medicament for promoting epithelial regeneration.
19. 19. The use according to claim 17 or claim 18, wherein the medicament is for use on the skin.
20. 20. The use according to claim 17 or claim 18, wherein the medicament is for the eye.
21. 21. Use of TGF- β 3 or a fragment or derivative thereof according to any one of claims 1 to 15 in the manufacture of a medicament for the prevention and/or treatment of fibrotic disease.
22. 22. The use according to claim 22, wherein the fibrotic disease is selected from: pulmonary fibrosis, liver fibrosis, scleroderma, skin fibrosis, muscle fibrosis, radiation fibrosis, kidney fibrosis, proliferative vitreoretinopathy and uterine fibrosis.
23. 23. Use of TGF- β 3 or a fragment or derivative thereof according to any one of claims 1 to 15 in the manufacture of a medicament for the treatment of an angiogenic disorder, restenosis, adhesion, endometriosis, an ischemic disease, oral mucositis and renal disease.
24. 24. Use of TGF- β 3 or a fragment or derivative thereof according to any one of claims 1 to 15 in the manufacture of a medicament for inducing bone and cartilage or in vitro fertilisation.
25. 25. A nucleic acid encoding a TGF- β 3 or fragment or derivative thereof according to any one of claims 1 to 15.
26. 26. A method of accelerating wound healing and/or preventing, reducing or inhibiting scarring, the method comprising providing a therapeutically effective amount of TGF- β 3 or a fragment or derivative thereof according to any one of claims 1 to 13 to a patient in need thereof.
27. 27. A method of preventing and/or treating a fibrotic disease, the method comprising providing to a patient in need of such prevention and/or treatment a therapeutically effective amount of TGF- β 3 or a fragment or derivative thereof according to any one of claims 1 to 13.
28. 28. A method according to claim 26 or claim 27, wherein the TGF- β 3 or fragment or derivative thereof is provided to the skin of the patient.
29. 29. A method according to claim 26 or claim 27, wherein the TGF- β 3 or fragment or derivative thereof is provided to the eye of the patient.