WO2009081114A1

WO2009081114A1 - Fusion proteins comprising an slrp-derived anti-angiogenic sequence

Info

Publication number: WO2009081114A1
Application number: PCT/GB2008/004187
Authority: WO
Inventors: Paul G. Bishop
Original assignee: The University Of Manchester
Priority date: 2007-12-20
Filing date: 2008-12-19
Publication date: 2009-07-02
Also published as: GB0724832D0

Abstract

Provided are fusion proteins comprising an angiogenic factor-inhibitory moiety and an anti-angiogenic sequence derived from a small leucine-rich repeat proteoglycan (SLRP) family member, as well as nucleic acid molecules encoding the same. The fusion proteins or nucleic acid molecules are of use in the prevention or treatment of pathological conditions associated with new blood vessel growth. The angiogenic factor-inhibitory moiety may be a neutralising antibody, a neutralising antibody fragment, or an angiogenic factor-binding fragment of a cellular receptor such as VEGFR-I.

Description

FUSION PROTEINS COMPRISING AN SLRP-DERIVED ANTI-ANGIOGENIC SEQUENCE

The present invention relates to fusion proteins having anti-angiogenic effects, and to nucleic acids encoding such fusion proteins. The invention also relates to medical uses of such fusion proteins or nucleic acids, and methods of treatment and pharmaceutical compositions utilising these agents.

The short leucine-rich proteoglycans (SLRPs) are a family of proteoglycans sharing a number of structural similarities. The class III are a class of SLRPs the members of which share a number of biological activities, and have been shown to inhibit new blood vessel formation in vitro and in vivo.

The formation of new blood vessels arises primarily as result of angiogenesis (a sprouting outgrowth from existing blood vessels) and in situ vasculogenesis (the differentiation of precursor cells into blood vessel networks). In many contexts new blood vessel formation plays an important role in the supply of oxygen and nutrients to developing or damaged tissues, however there are also many pathological conditions associated with new blood vessel formation.

Examples of diseases associated with new blood vessel formation include cancer, where the development of new blood vessels is associated with tumour growth and propagation, the vasoproliferative retinopathies including proliferative diabetic retinopathy, retinopathy of prematurity and sickle cell retinopathy, 'wet' macular degeneration and other forms of choroidal neovascularisation, psoriasis, and many inflammatory conditions such as arthritis.

Vasoproliferative retinopathies, in which the eye is subject to pathologically increased vascularisation, constitute one of the leading causes of visual impairment and blindness in the western world. Proliferative diabetic retinopathy is a major blinding complication of diabetes. In this condition there is ischaemia of the retina that results in the upregulation of pro-angiogenic growth factors including vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). These growth factors drive an angiogenic response in which blood vessels grow from the pre-existing retinal circulation into the normally avascular vitreous humour of the eye, a process called preretinal neovascularisation. These new blood vessels bleed and cause tractional retinal detachments, this in turn frequently results in blindness. Current treatments for this condition such as laser photocoagulation of the retina or antibody-based therapies are not always effective and can result in severe complications, hi the light of these failings it can be seen that better anti-angiogenic agents and treatments are needed.

hi a first aspect of the invention there is provided a fusion protein comprising an angiogenic factor-inhibitory moiety and an anti-angiogenic sequence derived from a short leucine-rich proteoglycan SLRP. The anti-angiogenic sequence is preferably derived from a class III SLRP.

hi a second aspect of the invention there is provided a nucleic acid encoding a fusion protein in accordance with the first aspect of the invention. Such nucleic acids may be usefully employed in methods of gene therapy.

The present invention is based on the inventors finding that fusion proteins, comprising an anti-angiogenic sequence derived from a SLRP in combination with a moiety derived from another protein that is able to inhibit the angiogenic activity of otherwise pro- angiogenic factors, have potent ability to prevent new blood vessel formation, and are highly suitable for therapeutic uses based on this activity. In particular, these fusion proteins have a notable utility in the prevention of new blood vessel formation associated with pathologies of the eye. The fusion proteins and nucleic acids of the invention may preferably make use of human protein sequences, and may preferably be used in the treatment of human subjects (though it will be appreciated they may also be used in non- human animals).

For the purposes of the present invention, any reference to "a fusion protein" should be taken to exclude naturally occurring proteins. Preferably a fusion protein in accordance with the present invention includes regions derived from two, or more, different proteins. The inventors believe that the properties provided by fusion proteins of the present invention may also be provided by derivatives of such fusion proteins. Thus, though for purposes of the brevity present invention will generally refer to "fusion proteins" these references should, except for where the context requires otherwise, be taken to encompass non-peptide derivatives of such fusion proteins. Examples of suitable derivatives are considered in greater detail elsewhere in the specification.

Fusion proteins in accordance with the invention should also be taken to encompass proteins that have been subject to post-translational modification. In particular references to fusion proteins should be taken to encompass SLRPs having the characteristics required by the first aspect of the invention.

For the purposes of the present disclosure, an "angiogenic factor-inhibitory moiety" should be taken to encompass any moiety that is able to inhibit the angiogenic activity of a factor that would otherwise be expected to promote angiogenesis. Preferably an angiogenic factor that may be inhibited by such a moiety may be a soluble angiogenic factor. More preferably, an angiogenic factor that may be inhibited by such a moiety may be a member of the VEGF subfamily of growth factors. Even more preferably, an angiogenic factor that may be inhibited by such a moiety may be selected from the group consisting of: VEGF and PlGF.

As used in the present specification, references to VEGF should, except for where the context requires otherwise, be taken to encompass all splice variants of this growth factor, including VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206. In particular, references to VEGF and VEGF inhibition should be taken to encompass VEGFl 21 and VEGFl 65 and inhibition of these splice variants.

As used in the present specification, references to PlGF should, except for where the context requires otherwise, be taken to encompass all splice variants of this growth factor, including including PlGFl, PLGF2, P1GF3 and P1GF4. In particular, references to PlGF and PlGF inhibition should be taken to encompass PlGFl and P1GF2 and inhibition of these splice variants. Various moieties capable of inhibiting the activity of angiogenic factors are known to those skilled in the art, and the inventors believe that any suitable moiety may be used in the fusion proteins of the invention. Merely by way of example, and without limitation, suitable angiogenic factor-inhibitory moieties may comprise neutralising antibodies (or function-neutralising fragments of antibodies), such as antibodies capable of binding to an angiogenic factor or its receptors in a manner that inhibits their interaction and signalling; and angiogenic factor-binding fragments of cellular receptors.

Such fragments of cellular receptors constitute preferred angiogenic factor-inhibitory moieties for use in accordance with the present invention. Suitable fragments will contain the angiogenic factor-binding domains of the receptor, and act as inhibitors by competing with the native cellular receptors for binding of angiogenic factors in the extracellular environment. Since binding of these fragments to an angiogenic factor does not give rise to a signal causing angiogenesis, this effectively serves to inhibit angiogenic activity that may otherwise by stimulated by the factor.

In the case that a fragment of a cellular receptor is to be used as an angiogenic factor- inhibitory moiety, it may be preferred to employ a fragment of a receptor capable of binding a member of the VEGF subfamily of growth factors. This family encompasses VEGF (as defined above); VEGF-B; VEGF-C; VEGF-D and PlGF. It is especially preferred that fragments used in accordance with this embodiment of the invention are able to inhibit VEGF and/or PlGF.

Fragments in accordance with this embodiment of the invention may be derived from any suitable member of the VEGF and PlGF receptor family including VEGFR-I (also referred to as Fltl); VEGFR-2 (also referred to as Flkl, KDR or CD309); neuropilin-1 (NRP-I) and neuropilin-2 (NRP-2), Platelet derived growth factor receptor (PDGFR)- alpha and PDGFR-beta. In particular, it may be especially preferred that a fragment to be used in accordance with this embodiment of the invention comprises a fragment of VEGFR-I. It is known that the extracellular portion of VEGFR-I contains seven immunoglobulin- like domains and domains and the second and third immunoglobulin-like domains, are involved in the high affinity binding of VEGF and PlGF. It has been found that the second immunoglobulin-like domain of VEGFR-I is itself sufficient to bind and inhibit VEGF or PlGF, and so a preferred angiogenic factor-inhibitory moiety for use in accordance with the present invention may comprise all or part of the second immunoglobulin-like domain of VEGFR-I (comprising at least the portion of this domain responsible for binding to VEGF or PlGF). However, the second and third immunoglobulin-like domains of VEGFR-I, when acting in combination, exhibit an affinity for VEGFR-I or PlGF that is higher than the affinity of the second domain alone. Thus a preferred angiogenic factor-inhibitory moiety that may be used in accordance with the present invention may comprise the second and/or third immunoglobulin-like domains of VEGFR-I (either in whole or in sufficient part to allow high affinity binging of VEGF or PlGF).

For the purposes of the present disclosure, "an anti-angiogenic sequence derived from a SLRP" may comprise any sequence having anti-angiogenic activity if said sequence corresponds directly to all or part of the amino acid sequence of a SLRP, or if said sequence is derived from the amino acid sequence of a SLRP. This term should be taken to encompass both amino acid sequences and non-amino acid sequences (particularly in the case of derivatives of the fusion proteins of the invention). Preferred anti-angiogenic sequences for use in accordance with the invention are those derived from class III SLRPs.

The anti-angiogenic activity of an angiogenic factor-inhibitory moiety, or of an anti- angiogenic sequence derived from a SLRP may be determined with reference to any one of a number of well-defined models of angiogenesis. For reference, the ability of such moieties or sequences to inhibit angiogenesis may be assessed in an in vitro capillary morphogenesis assay (sometimes also referred to as an endothelial cell network forming assay), such as the assay described elsewhere in the present specification. Merely by way of example, the ability of a moiety or sequence of interest to reduce network length or the number of loops formed in such an assay by at least 10% (or preferably 20%, or more) should be considered to indicate suitable anti-angiogenic activity. An alternative approach to assessment of anti-angiogenic activity in such a model may be to consider a moiety or sequence of interest capable of reducing network length or the number of loops formed by a statistical difference with a P value of 0.05 or less (as compared to a suitable control) as possessing suitable anti-angiogenic activity. Although they do not wish to be bound by any hypothesis, the inventors believe that suitable anti-angiogenic fragments may preferably achieve their activity through an integrin-dependent mechanism.

An anti-angiogenic sequence derived from a SLRP (and preferably a class III SLRP) may comprise at least three residues (such as amino acid residues) either corresponding directly to, or derived from, the amino acid sequence of a SLRP. Preferably, such a sequence may comprise at least 5, 10, 15 or 20 residues corresponding to, or derived from, the amino acid sequence of a SLRP. A suitable sequence may even comprise the whole of a SLRP. For the purposes of the present disclosure, the class III SLRPs may be considered to comprise opticin, epiphycan or mimecan (also referred to as osteoglycin). The amino acid sequences of the human forms of these class III SLRPs are shown in Sequence ID Nos. 1, 2 and 3 respectively.

An anti-angiogenic sequence may be considered to be derived from a class III SLRP if it shares at least 60% sequence homology with a class III SLRP, and more preferably 70, 80, or 90% sequence homology. Even more preferably, an anti-angiogenic sequence derived from a class III SLRP may share at least 60% sequence identity with a class III SLRP, and more preferably at least 70, 80, or 90% sequence identity.

It may be preferred that an anti-angiogenic sequence derived from a class III SLRP further comprises a sequence capable of retaining the fusion protein at a site of interest. This retention sequence may comprise all, some, or none of the sequence that confers anti-angiogenic activity. A preferred retention sequence may be capable of causing retention of the fusion protein in the vitreous humour of the eye (in particular on the vitreous collagen fibrils). "Retention" in the context of the present invention may be considered to be exhibited in the event that at least 5% of a fusion protein administered to a specified site is still present at said site eight weeks, or more, after such administration. The use of a sequence having retention activity of this sort confers a number of notable advantages. One advantage is that fusion proteins retained in the vitreous humour of the eye do not cross into the retina, and thus do not give rise to unwanted effects in this tissue. This is important, since, as well as being an important contributor to harmful preretinal neovascularisation, VEGF is also important for providing survival signals to retinal neural cells and vascular endothelium that are necessary for the function of these cells. As these cells are typically compromised in many conditions (such as diabetes) in which it is wished to administer inhibitors of VEGF, there are considerable concerns that such inhibitors may compound the pre-existing retina damage if they can access retina tissue. This concern has led many to consider existing VEGF inhibitors, such as Ranibizumab and Bevacizumab to be unsatisfactory for use in the eye, since, when administered by intravitreal injection they rapidly diffuse into the retina.

The retention of fusion proteins in accordance with this embodiment of the invention in the vitreous humour also means that an effective "depot" of proteins having anti- angiogenic activity may be retained at the site where their activity is required. This may allow the fusion proteins to have a long duration of action, and avoid the need for frequent administration. In contrast, agents such as Ranibizumab and Bevacizumab typically require re-administration every four to six weeks. Since administration is by means of intravitreal injection it will be appreciated that this is impractical as a long-term therapy for chronic conditions such as proliferative diabetic retinopathy.

Preferred anti-angiogenic sequences in accordance with any or all of the embodiments described above may be derived from the leucine-rich repeat (LRR) region of a SLRP. A preferred anti-angiogenic sequence may comprise all or part of the LRR of a class III SLRP. A particularly preferred sequence comprises substantially the whole of the LRR of opticin.

The LRR of opticin is linked to a glycosylated serine/threonine rich region (located N- terminal to the LRR) that contains a number of substitutions with O-linked oligosaccharides. This glycosylated region does not appear to be found in other SLRPs, and its inclusion in fusion proteins of the invention provides a number of advantages. The glycosylated region contributes to the solubility of opticin, and also of fusion proteins comprising this region. The glycosylated region constitutes a relatively "flexible" sequence, which allows fusion proteins incorporating this sequence to take up conformations in which the angiogenic factor-inhibitory moiety is readily accessible to soluble angiogenic factors while the SLRP-derived portion remains bound to extracellular matrix components by virtue of a retention signal. Furthermore, the glycosylation of this region (and the inventors believe the presence of O-linked oligosaccharides in particular) helps to prevent access of fusion proteins comprising this sequence into the retina (where their anti-angiogenic activity may have undesirable consequences). Accordingly, it is preferred that fusion proteins of the invention comprise all or part of this glycosylated sequence from opticin, most preferably in combination with the LRR of opticin.

The amino acid sequence of a preferred fusion protein in accordance with the invention is shown in Sequence ID No. 4, below:

MRAWIFFLLCLAGRALAAPLAIYIFISDTGRPFVEMYSEIPEIIHMTEGRELVIPCRV

TSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKT

NYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKN

KRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHI

YDKAFITVKVTSLAPATSISPAKSTTAPGTPSSNPTMTRPTTAGLLLSSQPNHGLPT

CLVCVCLGSSVYCDDIDLEDIPPLPRRTAYLYARFNRISRIRAEDFKGLTKLKRIDL

SNNLISSIDNDAFRLLHALQDLILPENQLEALPVLPSGIEFLDVRLNRLQSSGIQPAA

FRAMEKLQFLYLSDNLLDSIPGPLPLSLRSVHLQNNLIETMQRDVFCDPEEHKHTR

RQLEDIRLDGNPINLSLFPSAYFCLPRLPIGRFT

Sequence ID No. 4

Sequence ID No. 4 is produced on fusion of a number of "sub-sequences", including a signal peptide; the second immunoglobulin-like domain VEGFR-I; the third immunoglobulin-like domain of VEGFR-I; the serine/threonine-rich region of opticin; and the LRR of opticin. Each of these regions is described in further detail below. The first of these sequences is Sequence ID No. 5, the BM40 signal peptide incorporated into the pCEP-Pu/AC7 expression vector:

MRAWIFFLLCLAGRALAAPLA Sequence ID No. 5

The second sequence is selected from within the second immunoglobulin-like like domain of VEGFR-I (comprising the whole or part of this domain). The sequence of this domain is shown in Sequence ID No. 6, below:

IYIFISDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGK

RIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTI

Sequence ID No. 6

The third sequence is selected from within the third immunoglobulin-like like domain of VEGFR-I (comprising the whole or part of this domain). The sequence of this domain is shown in Sequence ID No. 7, below:

IDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQ SNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHR Sequence ID No. 7

The serine/threonine region of opticin that may be O-glycosylated and confers various advantages set out above represents a fourth sequence that may preferably be incorporated in a fusion protein according to the invention. The sequence of this region is set out in Sequence ID No. 8 below:

SLAPATSISPAKSTTAPGTPSSNPTMTRPTT Sequence ID No. 8

A fifth sequence that may be incorporated in fusion proteins in accordance with the invention is set out in Sequence ID No. 9 below. This sequence serves as a linker between biologically functional regions of opticin. The sequence is sensitive to cleavage by matrix metalloproteinases (such as MMP2, MMP9 and MMP 13) and it may be preferred that substitutions be made within this sequence to remove amino acid residues associated with proteinase cleavage.

AGLLLSSQPNHG Sequence ID No. 9

Finally, a preferred sixth sequence to be incorporated in a fusion protein of the invention may comprise part (or more preferably substantially all) of the LRR region of opticin, shown as Sequence ID No. 10 below.

LPTCLVCVCLGSSVYCDDIDLEDIPPLPRRTAYLYARFNRISRIRAEDFKGLTKXKR

IDLSNNLISSIDNDAFRLLHALQDLILPENQLEALPVLPSGIEFLDVRLNRLQSSGIQ

PAAFRAMEKLQFLYLSDNLLDSIPGPLPLSLRSVHLQNNLIETMQRDVFCDPEEHK

HTRRQLEDIRLDGNPINLSLFPSAYFCLPRLPIGRFT

Sequence ID No. 10

A nucleic acid encoding a fusion protein in accordance with the present invention may be readily produced in the light of the sequence information provided above. An example of a DNA sequence encoding this preferred fusion protein (with details of the amino acids encoded) is set out in Sequence ID No. 11 (shown in the Sequence Information section at the end of the specification). It will be appreciated that pCEP-Pu/AC7 represents a preferred vector to be used in the expression of such nucleic acids.

It will be recognised from the preceding pages that the fusion proteins of the invention (and nucleic acids encoding such fusion proteins) are particularly well suited to therapeutic use. This gives rise to a third aspect of the invention, in which there is provided a method of inhibiting blood vessel formation in a subject, the method comprising administering to the subject an effective amount of a fusion protein in accordance with the first aspect of the invention or nucleic acid in accordance with the second aspect of the invention to inhibit blood vessel formation. In a fourth aspect of the invention there is provided a method of preventing and/or treating a pathological condition associated with new blood vessel formation, the method comprising administering to a subject an effective amount of a fusion protein in accordance with the first aspect of the invention or nucleic acid in accordance with the second aspect of the invention, wherein the blood vessel formation is inhibited and the pathological condition is prevented and/or treated.

In a fifth aspect of the invention there is provided a method of inhibiting undesirable endothelial cell proliferation or migration, comprising contacting an endothelial cell with an amount of a fusion peptide in accordance with the invention sufficient to inhibit or reduce endothelial cell proliferation or migration.

The fusion proteins or nucleic acids of the invention (and pharmaceutical compositions or therapeutic methods utilising such fusion proteins or nucleic acids) may be used to inhibit new blood vessel formation associated with numerous pathological conditions. As explained elsewhere in the present disclosure, these include pathological conditions of the eye, particularly proliferative retinopathies, and more particularly proliferative diabetic retinopathy. The formation of new blood vessels by angiogenesis also plays a role in the development or progression of a range of other pathological conditions, such as cancer, psoriasis and wound healing. Accordingly the fusion proteins or nucleic acids of the invention (and pharmaceutical compositions or therapeutic methods utilising such fusion proteins or nucleic acids) may be used to inhibit new blood vessel formation associated with these conditions.

It will be appreciated that the fusion proteins of the invention are of particular benefit in uses in which localised delivery and/or retention of anti-angiogenic agents is desirable, since they are able to bind to the extracellular matrix thereby limiting their systemic absorption and potential toxicity. Examples of conditions in which such properties are beneficial include psoriasis and certain cancers. Methods in accordance with these third and fourth and fifth aspects of the invention are suitable for use in the inhibition of blood vessel formation at sites throughout the body, or associated with a wide range of pathological conditions. However, they are particularly suitable for use in methods in which it is wished to inhibit blood vessel formation in the eye. In a particularly preferred embodiment these methods may be used to prevent and/or treat a vasoproliferative retinopathy, such as proliferative diabetic retinopathy. As will be appreciated, it may be preferred to use a fusion protein comprising a sequence capable of causing its retention in the eye (or a nucleic acid encoding such a fusion protein), in accordance with this embodiment of the invention.

When new blood vessel formation is to be inhibited in the eye of a subject, a preferred means by which the fusion protein or nucleic acid of the invention may be administered is via intravitreal injection.

Various formulations suitable for use in accordance with these methods of the invention will be apparent to those skilled in the art. In a sixth aspect of the invention there is provided a pharmaceutical composition comprising a fusion protein in accordance with the first aspect of the invention, or a nucleic acid molecule in accordance with the second aspect of the invention, in a pharmaceutically acceptable diluent, excipient or carrier.

In a seventh aspect the invention also provides the use of a fusion protein in accordance with the first aspect of the invention, or a nucleic acid molecule in accordance with the second aspect of the invention, as a medicament. The fusion proteins or nucleic acid molecules may be in accordance with any of the embodiments described in the present specification. Such medicaments may be used in the inhibition of blood vessel formation, and particularly in the prevention and/or treatment of pathological conditions associated with new blood vessel formation. The medicaments may also be used in inhibiting undesirable endothelial cell proliferation or migration.

The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of fusion protein according to the first aspect of the invention, or a nucleic acid according to the second aspect of the invention, and optionally a pharmaceutically acceptable vehicle. In one embodiment, the amount of the fusion protein or nucleic acid is an amount from about 0.01 mg to about 800 mg. In another embodiment, the amount of the fusion protein or nucleic acid is an amount from about 0.01 mg to about 500 mg. In another embodiment, the amount of the fusion protein or nucleic acid is an amount from about 0.01 mg to about 250 mg. In another embodiment, the amount of the fusion protein or nucleic acid is an amount from about 0.1 mg to about 100 mg. In another embodiment, the amount of the fusion protein or nucleic acid is an amount from about 0.1 mg to about 20 mg.

As has been suggested earlier in the specification, it will be appreciated that the advantages and therapeutic benefits provided by the fusion proteins of the invention will also be obtainable from derivatives of such fusion proteins. Thus the present invention should also be taken to encompass agents that are (either in whole or in part) derived from the fusion proteins disclosed herein. These are suitable for use in accordance with all aspects and embodiments of the invention, except for where the context requires otherwise.

Derivatives of fusion proteins of the invention may include derivatives that increase or decrease the half-lives of such fusion proteins in vivo. Examples of derivatives that increase the half-life of fusion proteins of the invention include modified proteins in which enzyme cleavage motifs have been removed by amino acid deletion and/or substitution, peptoid derivatives of fusion proteins of the invention, D-amino acid derivatives of fusion proteins of the invention and peptide-peptoid hybrids.

Fusion proteins of the invention may be subject to degradation by a number of means (such as protease activity in biological systems). Such degradation may limit the bioavailability of the fusion proteins and hence their ability to inhibit new blood vessel formation. There are many examples of well-established techniques by which peptide derivatives that have enhanced stability in biological contexts can be designed and produced. Such peptide derivatives may have improved bioavailability as a result of increased resistance to protease-mediated degradation. Preferably a peptide derivative or analogue suitable for use according to the invention is more protease-resistant than the fusion protein of the invention from which it is derived. Suitable methods by which protease-resistance may be conferred include protection, substitution or modification of residues present in fusion proteins of the invention. Protease-resistance of a derivative and the fusion protein from which it is derived may be evaluated by means of well-known protein degradation assays. The relative values of protease resistance for the derivative and fusion protein (or glycoprotein or proteoglycan) may then be compared.

Peptoid derivatives of fusion proteins of the invention may be readily designed from knowledge of the structure of the fusion protein. Commercially available software may be used to develop peptoid derivatives according to well-established protocols.

Retropeptoids, (in which all amino acids are replaced by peptoid residues in reversed order) are also able to mimic a high-affinity binding proteins. A retropeptoid is expected to bind in the opposite direction in the ligand-binding groove, as compared to a peptide or peptoid-peptide hybrid containing one peptoid residue. As a result, the side chains of the peptoid residues are able point in the same direction as the side chains in the original fusion protein.

A further embodiment of a modified form of fusion proteins of the invention comprises D-amino acids. In this case the order of the amino acid residues is reversed as compared to that found in the original fusion protein.

The invention will now be further described with reference to the following Experimental Results section and Figures, in which:

Figure 1 shows the effect of full length opticin on generation of a capillary network; Figure 2 shows the effect of the N-terminal region of opticin on generation of a capillary network; and

Figure 3 shows the effect of the LRR of opticin on generation of a capillary network.

Experimental Results

The ability of peptides derived from various regions of the class III SLRP opticin to inhibit new blood vessel formation by angiogenesis was investigated in a network formation (angiogenesis) assay.

Briefly human umbilical vein endothelial cells (HUVECs) were placed on Matrigel and covered with growth medium supplemented with FGF-2. Under these conditions the endothelial cells align themselves into networks and this represents a capillary morphogenesis assay. The effects of the following peptides on capillary network formation were investigated:

i) full length purified recombinant opticin (prepared as previously described in

Hindson et al. Invest Ophthalmol. Vis Sci. 2005: 46: 4417) ii) the N-terminal region of opticin (consisting of the first 58 amino acids of the mature bovine opticin protein expressed as a recombinant protein in bacteria and purified) iii) the LRR region of opticin (sequence ID 10, expressed by 293 cells in co-culture experiments)

The effects of these peptides were quantified by measuring total network length or number of complete loops formed per field of view.

The effects of full length opticin and the N-terminal region of opticin may be compared with reference to Figures 1 and 2 respectively. Data shown in these Figures are the results of triplicate experiments. Figure 1 illustrates the results of experiments investigating the effects of full length recombinant opticin on the formation of networks by HUVECs grown on Matrigel.

Part A of Figure 1 shows representative photographs of HUVEC networks formed on culture with opticin at a concentration of: i) OnM (control); ii) 5OnM; and iii) 10OnM. The scale bar shown in i) is 500μm.

Part B of Figure 1 illustrates a dose-response curve in which the total length of HUVEC networks produced in culture on Matrigel is investigated at a range of concentrations of full length opticin.

Part C of Figure 1 illustrates a dose-response curve in which the number of complete loops formed by HUVEC cultured on Matrigel is investigated at a range of concentrations of full length opticin.

In parts B and C of Figure 1 the error bars indicate standard deviation, while "+" indicates p<0.02, "*" indicates pO.Ol and "^Λ" indicates p<0.005.

Figure 2 illustrates the results of experiments investigating the effects of the N-terminal fragment of opticin on the formation of networks by HUVECs grown on Matrigel.

Part A of Figure 2 shows representative photographs of HUVEC networks formed on culture with the N-terminal fragment of opticin at a concentration of: i) OnM (control); ii) 5OnM; and iii) 10OnM. The scale bar shown in i) is 500μm.

Part B of Figure 2 illustrates a dose-response curve in which the total length of HUVEC networks produced in culture on Matrigel is investigated at a range of concentrations of the N-terminal fragment of opticin. The error bars indicate standard deviation

Part C of Figure 2 illustrates a dose-response curve in which the number of complete loops formed by HUVEC cultured on Matrigel is investigated at a range of concentrations of the N-terminal fragment of opticin. Once more, the error bars indicate standard deviation.

These results show that full-length opticin inhibited network formation at concentrations of 75 nM and above, whereas the N-terminal fragment of opticin had no effect on the network at concentrations up to 100 nM.

Since the intact opticin molecule does have anti-angiogenic properties, but these are not present in the N-terminal domain it can be seen that these properties must be elicited by other parts of the molecule.

That the anti-angiogenic properties of opticin may be localised to the LRR of this molecule was illustrated by the following experiment.

HUVECs were seeded onto Matrigel containing 293 EBNA cells transfected with the opticin LRR region (Sequence ID 10) in the pCEPpU/AC7 vector or the empty pCEPpU/AC7 vector. These cells were then stimulated with FGF2 and incubated for 24 hours. The results obtained are shown in Figure 3, in the following panels:

A) Representative photographs of HUVEC networks formed on co-culture with either empty vector (i) or LRR fragment (ii).

B) Dose-response of total length of HUVEC network on Matrigel.

C) Dose-response of number of complete loops formed by HUVEC on Matrigel.

Scale bar = 500 μm. Error bars = standard deviation. * P<0.01. Data shown are representative of triplicate experiments.

These results clearly indicate that the LRR of opticin is able to inhibit angiogenesis. Sequence Information

Sequence ID No.1

ASLPRKERKRREEQMPREGDSFEVLPLRNDVLNPDNYGEVIDLSNYEELTDYGDQLPEVKVTSLA PATSISPAKSTTAPGTPSSNPTMTRPTTAGLLLSSQPNHGLPTCLVCVCLGSSVYCDDIDLEDIP PLPRRTAYLYARFNRISRIRAEDFKGLTKLKRIDLSNNLISSIDNDAFRLLHALQDLILPENQLE ALPVLPSGIEFLDVRLNRLQSSGIQPAAFRAMEKLQFLYLSDNLLDSIPGPLPLSLRSVHLQNNL IETMQRDVFCDPEEHKHTRRQLEDIRLDGNPINLSLFPSAYFCLPRLPIGRFT

Sequence ID No.2

APTLESINYDSETYDATLEDLDNLYNYENIPVGKVEIEIATVMPSGNRELLTPPPQPEKAQEEEE EEESTPRLIDGSSPQEPEFTGVLGPHTNEDFPTCLLCTCISTTVYCDDHELAIPPLPKNTAYFYS RFNRIKKINKNDFASLSDLKRIDLTSNLISEIDEDAFRKLPQLRELVLRDNKIRQELPTTLTFID ISNNRLGRKGIKQEAFKDMYDLHHLYLTDNNLDHIPLPLPENLRALHLQNNNILEMHEDTFCNVK NLTYIRKALEDIRLDGNPINLSKTPQAYMCLPRLPVGSLV

Sequence ID No.3

KPAPPTQQDSRIIYDYGTDNFEESIFSQDYEDKYLDGKNIKEKETVIIPNEKSLQLQKDEAITPLPPKK ENDEMPTCLLCVCLSGSVYCEEVDIDAVPPLPKESAYLYARFNKIKKLTAKDFADIPNLRRLDFTGN LIEDIEDGTFSKLSLLEELSLAENQLLKLPVLPPKLTLFNAKYNKIKSRGIKANAFKKLNNLTFLYLD HNALESVPLNLPESLRVIHLQFNNIASITDDTFCKANDTSYIRDRIEEIRLEGNPIVLGKHPNSFICLKR

LPIGSYF

Sequence ID No.11 atgagggcctggatcttctttctcctttgcctggccgggagggctctggcagccccgcta

M R A W I F F L L C L A G R A L A A P L gcaatctatatatttattagtgatacaggtagacctttcgtagagatgtacagtgaaatc

A I Y I F I S D T G R P F V E M Y S E I cccgaaattatacacatgactgaaggaagggagctcgtcattccctgccgggttacgtca

P E I I H M T E G R E L V I P C R V T S cctaacatcactgttactttaaaaaagtttccacttgacactttgatccctgatggaaaa

P N I T V T L K K F P L D T L I P D G K cgcataatctgggacagtagaaagggcttcatcatatcaaatgcaacgtacaaagaaata

R I I W D S R K G F I I S N A T Y K E I gggcttctgacctgtgaagcaacagtcaatgggcatttgtataagacaaactatctcaca

G L L T C E A T V N G H L Y K T N Y L T catcgacaaaccaatacaatcatagatgtccaaataagcacaccacgcccagtcaaatta

H R Q T N T I I D V Q I S T P R P V K L cttagaggccatactcttgtcctcaattgtactgctaccactcccttgaacacgagagtt

L R G H T L V L N C T A T T P L N T R V caaatgacctggagttaccctgatgaaaaaaataagagagcttccgtaaggcgacgaatt

Q M T W S Y P D E K N K R A S V R R R I gaccaaagcaattcccatgccaacatattctacagtgttcttactattgacaaaatgcag

D Q S N S H A N I F Y S V L T I D K M Q aacaaagacaaaggactttatacttgtcgtgtaaggagtggaccatcattcaaatctgtt

N K D K G L Y T C R V R S G P S F K S V aacacctcagtgcatatatatgataaagcattcatcactgtgaaggtgactagcctcgct

N T S V H I Y D K A F I T V K V T S L A cctgcaaccagcatcagtcccgccaagagcactacggctccagggacaccctcgtcaaac P A T S I S P A K S T T A P G T P S S N cccacgatgaccagacctactacagcagggctgctactgagttcccagcccaaccatggt

P T M T R P T T A G L L L S S Q P N H G ctgcccacctgcctggtctgcgtgtgcctcggttcctctgtgtattgcgatgacattgac

L P T C L V C V C L G S S V Y C D D I D ctagaggacattcctcctcttcctcggaggactgcctacctgtatgcacgcttcaaccgc

L E D I P P L P R R T A Y L Y A R F N R atcagccgtatcagggccgaagacttcaaagggctgacaaagttgaagaggattgacctc

I S R I R A E D F K G L T K L K R I D L tccaacaacctcatttcctccatcgataatgatgccttccgcctgctacatgccctccag

S N N L I S S I D N D A F R L L H A L Q gacctcatcctcccagagaaccagttggaagctctgcccgtgctgcccagtggcattgag

D L I L P E N Q L E A L P V L P S G I E ttcctggatgtccgcctaaatcggctccagagctcggggatacagcctgcagccttcagg

F L D V R L N R L Q S S G I Q P A A F R gcaatggagaagctgcagttcctttacctgtcagacaacctgctggattctatcccgggg

A M E K L Q F L Y L S D N L L D S I P G cctttgcccctgagcctgcgctctgtacacctgcagaataacctgatagagaccatgcag

P L P L S L R S V H L Q N N L I E T M Q agagacgtcttctgtgaccccgaggagcacaaacacacccgcaggcagctggaagacatc

R D V F C D P E E H K H T R R Q L E D I cgcctggatggcaaccccatcaacctcagcctcttccccagcgcctacttctgcctgcct

R L D G N P I N L S L F P S A Y F C L P cggctccccatcggccgcttcacgtag

R L P I G R F T -

Claims

1. A fusion protein comprising an angiogenic factor-inhibitory moiety and an anti- angiogenic sequence derived from a small leucine-rich repeat proteoglycan (SLRP) family member.

2. A fusion protein according to claim 1, wherein the angiogenic factor- inhibitory moiety is able to inhibit a member of the VEGF subfamily of growth factors.

3. A fusion protein according to claim 2, wherein the angiogenic factor-inhibitory moiety is able to inhibit VEGF and/or PlGF.

4. A fusion protein according to any preceding claim, wherein the angiogenic factor- inhibitory moiety is selected from the group consisting of: a neutralising antibody; a neutralising antibody fragment; and an angiogenic factor-binding fragment of a cellular receptor.

5. A fusion protein according to any preceding claim, wherein the angiogenic factor- binding comprises a fragment of VEGFR-I, VEGFR-2, neuropilin-1, neuropilin-2, platelet derived growth factor receptor alpha or platelet derived growth factor receptor beta.

6. A fusion protein according to claim 5, wherein the angiogenic factor-binding moiety comprises a fragment of VEGFR-I.

7. A fusion protein according to claim 6, wherein the angiogenic factor-binding moiety comprises the second and third immunoglobulin-like domains of VEGFR-I.

8. A fusion protein according to any preceding claim, wherein the anti-angiogenic sequence derived from a SLRP is derived from a class III SLRP.

9. A fusion protein according to any preceding claim, wherein the anti-angiogenic sequence is derived from the leucine-rich repeat region of a SLRP.

10. A fusion protein according to any preceding claim, wherein the anti-angiogenic sequence derived from a class IH SLRP is derived from opticin.

11. A fusion protein according to claim 10, wherein the anti-angiogenic sequence derived from a SLRP comprises the leucine-rich repeat region of opticin.

12. A fusion protein according to any preceding claim, wherein the anti-angiogenic sequence derived from a class III SLRP comprises a sequence capable of retaining the fusion protein at a site of interest.

13. A fusion protein according to claim 12, wherein the anti-angiogenic sequence derived from a class III SLRP comprises a sequence capable of retaining the fusion protein in the vitreal humour.

14. A nucleic acid encoding a fusion protein according to any one of claims 1 to 13.

15. A method of inhibiting blood vessel formation in a subject, the method comprising administering to the subject an effective amount of a fusion protein according to any one of claims 1 to 13 or a nucleic acid to claim 14 to inhibit blood vessel formation.

16. A method of preventing or treating a pathological condition associated with new blood vessel formation, the method comprising administering to a subject an effective amount of a fusion protein according to any one of claims 1 to 13 or a nucleic acid to claim 14, wherein the blood vessel formation is inhibited and the pathological condition is prevented or treated.

17. A method according to claim 15 or claim 16, wherein the blood vessel formation is associated with a vasoproliferative retinopathy.

18. A method according to claim 17, wherein the blood vessel formation is associated with proliferative diabetic retinopathy.

19. A method according to claim 16, wherein the blood vessel formation is associated with a condition selected from the group consisting of: cancer, psoriasis and wound healing.

20. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 13 or a nucleic acid to claim 14, in a pharmaceutically acceptable diluent, excipient or carrier.