WO2011027129A1 - Treatment of vasculoproliferative conditions - Google Patents

Abstract

This invention relates to the field of molecular physiology. Specifically, this invention relates to the prevention and/or treatment of vasculoproliferative conditions, especially those of the eye and in the treatment of tumours that exhibit vascular proliferation. Levels of leucine-rich alpha-2-glycoprotein (Lrg1) have been demonstrated to be increased in patients suffering from such conditions and animal models of such conditions. Antagonists of Lrg1 can be used to prevent and/or treat vasculoproliferative conditions.

Description

TREATMENT OF VASCULOPROLIFERATIVE CONDITIONS

FIELD OF THE INVENTION

The invention is in the field of molecular physiology and relates to the use of antagonists of Leucine-rich alpha-2-glycoprotein 1 (Lrgl) for use in the treatment or prevention of vasculoproliferative conditions, particularly in the eye and in the treatment of tumours that exhibit vascular proliferation.

BACKGROUND OF THE INVENTION

Aberrant remodelling of the retinal vasculature is a prominent feature of sight- threatening conditions such as diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, age-related macular degeneration and macular telangiectasia. These vascular changes manifest themselves as both new capillary growth from pre-existing retinal vessels (angiogenesis) and the development of vascular malformations of existing vessels (e.g. telangiectasia). This pathogenic vascular remodelling in these diseases is a major contributing factor to loss of vision.

Similar vascular pathology and dysfunction also accompanies tumour growth, where angiogenesis permits the enlargement and growth of solid tumours.

Substantial resource has been directed towards gaining an understanding of the mechanisms that drive these vascular responses (referred to as angiogenesis, neoangiogenesis, vascular proliferation, vascular remodelling, vascular pathology). Of the various molecules identified as playing an important role in the process, the vascular endothelial growth factors (VEGFs) and their receptors are viewed as critical components. Therapeutic targeting of the VEGF pathway in ocular angiogenesis using the anti-cancer agent Avastin (or the closely related Lucentis) has resulted in improved clinical outcome, at least over the short term, of a number of sight- threatening conditions. However, there is concern over the long-term use of anti- VEGF strategies for the treatment of retinal vascular problems as VEGF has both neuroprotective activities and housekeeping roles such as the maintenance of choroidal fenestration. Moreover, although VEGF is considered to be the principal pro-angiogenic factor in neoangiogenesis, the process requires coordinated crosstalk between many factors, and the biological basis for other vascular changes, such as the formation of dilated and tortuous telangiectatic vessels, is not known.

There is therefore a need to identify alternative therapeutic targets and novel drugs which, in isolation or in combination with existing therapies, may be more effective and possess fewer off-target effects for the treatment of conditions where uncontrolled blood vessel growth and/or remodelling contributes to the disease.

SUMMARY OF THE INVENTION

We have identified Leucine-rich alpha-2-glycoprotein 1 (Lrgl gene identifiers:

HGNC: 29480; Entrez Gene: 116844; Ensembl: ENSG00000171236; UniProtKB: P02750) as a drugable target for the modulation of pathogenic vascular remodelling.

Lrgl was identified in 1977 (Haupt & Baudner, 1977) and its primary structure determined in 1985 (Takahashi et al, 1985). Lrgl is highly evolutionarily conserved between mice and humans, polyclonal antibodies to human Lrgl are commercially available and there are reports of concomitant increases in the level of transforming growth factor beta 1 (TGFpi), TGFp receptor II (TGFpRII) and Lrgl in certain diseases (Sun et al, 1995; Li et al, 1997). Other groups have identified Lrgl as a biomarker of certain diseases (US 2005/0064516; WO 2008/092214) and as a ligand for cytochrome c (US 2007/0184503). Dysfunction of TGFp signalling in endothelial cells leads to the disease hemorrhagic hereditary telangiectasia (HHT). In this group of diseases, which are characterised by vascular abnormalities including telangiectases, mutations in the TGF endothelial accessory receptor endoglin and the ΤβΜ co-receptor ALKl lead to HHTl (McAllister et al. 1994) and HHT2 (Johnson et al. 1995) respectively. TGFp has also been found to be increased in the retina of patients with diabetic retinopathy (Spirin et al., 1999) where vascular remodelling is prevalent. Lrgl expression has also been found to be increased in the plasma of certain tumour patients, suggesting that it may serve as a possible tumour biomarker (Heo et al, 2007; Ferrero et al, 2009; Kakisaka et al, 2007). However, very little is known about the biology of Lrgl .

We have now identified Lrgl as a drugable target for the modulation of pathogenic vascular remodelling, particularly in the eye and in tumours that exhibit

vasculoproliferation.

Using mouse models of retinal disease involving vascular changes, we first determined that, amongst other genes, Lrgl is upregulated in the vessels of these diseased retinas. Increased expression of Lrgl in the retina of these mouse models was then validated by quantitative PCR and western blotting and its retinal distribution confirmed as vascular by in situ hybridisation and immunohistochemistry. These models are standard models of angiogenesis and are applicable to angiogenesis at sites other than the eye.

We then investigated the connection between Lrgl and the TGF signalling pathway.

In endothelial cells TGF signaling can occur through TGFp receptor II associating either with the ubiquitous TGFP type I receptor activin receptor-like kinase 5 (ALK5) or the endothelial cell specific ALKl with the cellular response depending on which pathway predominates. In the case of ALK5 there is under certain conditions increased ECM deposition and cell quiescence whilst with ALKl there is endothelial cell activation manifest as increased migration and proliferation. This differential signalling is partly controlled by the concentration/bioavailability of TGFP and by members of a family of downstream effector proteins called Smads, whereby Smad 2 and 3 are activated by ALK5 and Smad 1 , 5 and 8 by AL 1.

Immunoprecipitation showed that Lrgl associates with both TGFpRII and ALK1, suggesting that Lrgl has a role in connection with these two molecules as part of the TGFP signalling complex.

We hypothesised, therefore, that Lrgl acts as a modulator of TGFp signalling, causing fine-tuning between the ALK1- and ALK5-activated signalling cascades. In support of this, Lrgl knockdown in endothelial cells with siRNA blocks TGFP-mediated increased cell proliferation and reduced Smad5 phosphorylation whilst Lrgl overexpression leads to an enhanced proliferation, downregulation in Smad2 expression and increased Smad5 phosphorylation. These observations therefore reveal one way in which Lrgl may regulate angiogenesis. Also, in a Matrigel angiogenesis assay to investigate the effect of Lrgl on "vessel" formation, the degree of vascular formation, as measured by vessel formation, tube formation and cord formation was significantly increased when conditioned medium from Lrgl overexpressing cells was added and the increased vascularisation correlated with Lrgl protein expression in the medium.

These data are consistent with decreased signalling via the TGFpRII/ALK5 receptor complex pathway and hence a shift towards activation of the vasculopathogenic TGFPRII/ALKI signalling pathway.

This suggests that blocking Lrgl within the TGF signalling complex has the potential to divert TGFp away from pathogenic vascularisation. To test this proposition, we determined whether we could block endothelial Smad5

phosphorylation induced by TGFP with either an anti-Lrgl antibody or by peptide sequences derived from Lrgl that could be expected to compete with Lrgl for binding to ALKl. The anti-Lrgl antibody caused reduction in phosphorylation whilst one of the peptides exhibited a particularly large reduction in phosphorylation.

Therefore, Smad5 phosphorylation can be inhibited by blocking Lrgl and, because Smad5 is associated with the vasculopathogenic ALKl -activated signalling cascade, this demonstrates that blocking Lrgl has the potential to block that cascade relative to the non-pathogenic, ALK5 -activated alternative cascade.

Taken together, these data suggest that: (a) Lrgl interacts with both TGFpRII and ALKl, promoting directly or through one or more intermediaries the interaction between TGFPRII and ALKl as opposed to ALK5, such that (b) blocking Lrgl will direct the activity of TGFp away from the vasculopathogenic, ALKl -activated signalling cascade and into the non-pathogenic, ALK5 -activated cascade, with the result that (c) Lrgl is a valid drug target for the treatment of pathogenic

vascularisation in the eye and elsewhere. Treatments with various Lrgl -blocking agents, notably peptide fragments of Lrgl, monoclonal antibodies to Lrgl and siR A molecules, can hence be envisaged for ocular and other disorders that involve pathogenic tissue vascularisation. Without being bound by theory, Figure 16 illustrates the role for Lrgl that is suggested by our data.

Additionally, our data suggest that, in contrast to VEGF, Lrgl may be involved only in pathogenic vascularisation in the eye and not in normal developmental

vascularisation or vascular homeostasis. This makes it a potentially superior target to VEGF in terms of avoiding interference with processes that it is not desirable to disrupt. A further attraction of Lrgl as a target is that it is extracellular and hence more easily accessed via systemic therapeutic routes.

Experiments were also conducted to investigate the role of Lrgl ex vivo and in vivo. Our experiments show that angiogenic vessel sprouting is reduced in aortic rings from Lrgl knockout mice as compared to aortic rings from control mice. Also, we found that choroidal neovascularisation (CNV) after retinal injury and retinal

neovascularization following oxygen-induced retinopathy (OIR) was reduced in Lrgl knockout mice as compared to control mice.

As evidence of the role of Lrgl in human pathology, our data shows that Lrgl and TGFp expression are increased in human patients suffering from proliferative diabetic retinopathy, supporting the in vivo data obtained from mice.

We have also demonstrated that antibodies against Lrgl are capable of inhibiting tube formation by human umbilical vein endothelial cells (HUVEC) in Matrigel angiogenesis assays. These data suggest that antibodies against Lrgl will be useful in the treatment or prevention of vasculoproliferative conditions, particularly those of the eye and of tumours that exhibit vasculoproliferation.

Accordingly, the invention provides:

An antagonist of Leucine-rich alpha-2-glycoprotein 1 (Lrgl) for use in the treatment or prevention of a vasculoproliferative condition.

The invention also provides:

A method of identifying antagonists of Lrgl comprising: providing a candidate antagonist, and determining whether or not said candidate antagonist blocks function or activity of Lrgl ; wherein said candidate antagonist is identified as an antagonist of Lrgl if blocking of the function or activity Lrgl is observed.

The invention also provides:

A monoclonal antibody which specifically recognises an epitope within amino acids LI -24 of Appendix 2 or L94-1 17 of Appendix 3 (SEQ ID NO: 3), L169-192 of Appendix 2 or L262-285 of Appendix 3 (SEQ ID NO: 4) or L227-252 of Appendix 2 or L320-345 of Appendix 3 (SEQ ID NO: 5) and blocks the activity of Lrgl.

The invention also provides:

A monoclonal antibody which specifically recognises an epitope within amino acids LI -24 of Appendix 2 or L94-1 17 of Appendix 3 (SEQ ID NO: 3), L169-192 of Appendix 2 or L262-285 of Appendix 3 (SEQ ID NO: 4) or L227-252 of Appendix 2 or L320-345 of Appendix 3 (SEQ ID NO: 5) and blocks the interaction between ALK1 , TGFpRII and/or TGFp and Lrgl .

The invention also provides:

A method for producing such an antibody, comprising: immunising a non-human mammal with an immunogen comprising an epitope within the sequence of LI -24 of Appendix 2 or L94-117 of Appendix 3 (SEQ ID NO: 3), L169-192 of Appendix 2 or L262-285 of Appendix 3 (SEQ ID NO: 4) or L227-252 of Appendix 2 or L320-345 of Appendix 3 (SEQ ID NO: 5) of Lrgl ; and obtaining an antibody preparation from said mammal and deriving therefrom monoclonal antibodies that specifically recognise said epitope.

The invention also provides:

A method for determining what sites within Lrgl can be targeted to block the function or activity of Lrgl, comprising providing peptide fragments of the Lrgl protein; and determining whether or not said each of said peptide fragments blocks the function or activity of Lrgl .

The invention also provides: Use of an antagonist of Lrgl in the manufacture of a medicament for the treatment or prevention of a vasculoproliferative condition.

The invention also provides:

A method of treating a vasculoproliferative condition comprising administering to a patient in need thereof an effective amount of an antagonist of Lrgl

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Low and high power images of retinal vascular remodelling in the RCS rat (20 wks), VLDLR^7" mouse (16 wks), Curlytail-J mouse (13 wks) and RD1 mouse (16 wks). Vessels in retinal flat mounts were stained with anti-collagen IV and anti- claudin-5 antibodies to decorate the vascular basal lamina and endothelial cell junctions respectively.

Figure 2. A. Schematic representation of Lrgl protein and its proposed glycosylation sites. B. Structure of Lrgl protein predicted by ROBETT A (University of

Washington, USA).

Figure 3. A. Quantitative RT PCR analysis of Lrgl expression in whole mouse retina of C57B16 control mice (BL6), VLDL receptor KO mice (VLDLR^"7"), Curlytail-J mice (CT) and the retinal dystrophy 1 mice (RD1). B. Western blot of Lrgl protein expression (top) and semi-quantitation (bottom) from whole retina. C. In situ hybridisation of normal retina showing Lrgl gene expression. D.

Immunohistochemical staining of Lrgl in retinal flat mounts (top) and retinal sections (bottom) showing vascular pattern of expression.

Figure 4. A. Co-immunoprecipitation from GPNT endothelial cell lysates of Lrgl with TGF3RII and ALK1. B. Recombinant HA-tagged Lrgl associates with both TGFp and TGFpRII. C. Co-localisation of Lrgl and TGFPRII expression in GPNT endothelial cells. D. Endothelial cell proliferation assay demonstrating that Lrgl knockdown attenuates TGFP induced proliferation and decreased Smadl/5

phosphorylation (* p<0.05). E. Endothelial cell proliferation assay demonstrating that overexpression of Lrgl enhances TGF induced proliferation and increases Smadl/5 phosphorylation (* p<0.05).

Figure 5. Lrgl blocking peptide derived from the C-terminus inhibits Lrgl/TGFpi induced Smad5 phosphorylation in GPNT endothelial cells (n=3 for each condition, pO.0001). The two right hand lanes are the C-terminal peptide alone, and the three peptides LI -24 of Appendix 2 or L94-1 17 of Appendix 3 (SEQ ID NO: 3), L169-192 of Appendix 2 or L262-285 of Appendix 3 (SEQ ID NO: 4) or L227-252 of Appendix 2 or L320-345 of Appendix 3 (SEQ ID NO: 5) combined.

Figure 6. A. Western blot of Lrgl knockdown in GPNT endothelial cells with siRNA and Lrgl over-expression in GPNT cells. B. Lrgl knockdown reduces TGFp 1 -mediated endothelial cell proliferation (* p<0.0001) and Lrgl over-expression enhances TGF l -mediated endothelial cell proliferation (* p<0.0005) (n=3 for each condition).

Figure 7. Effect of Lrgl on HUVEC "vessel" formation in vitro. A. Untreated media or media from control endothelial cell (EC) or from EC overexpressing Lrgl was added to a Matrigel angiogenesis assay. Lrgl conditioned medium enhanced HUVEC "cord" formation. B. Western blot of Lrgl in unconditioned media, GPNT endothelial cell conditioned media (7 days) and conditioned media from GPNT cells over-expressing Lrgl . C. Quantification of Matrigel endothelial cord formation complexity (number of closed vascular circles and total vascular area) following different treatments (n=3 for each condition). Lrgl conditioned media from Lrgl overexpressing cells induced the greatest angiogenic vascular plexus as measured by number of closed vascular circles or total vascular area (p<0.0001). Figure 8. Effect of Lrgl antibody (a commercially available polyclonal antibody to the N-terminal domain of Lrgl) and Lrgl peptides on endothelial Smad5

phosphorylation following treatment with TGFp and Lrgl .

Figure 9. Aortic rings from Lrgl knockout mice display reduced angiogenic vessel sprouting as compared to aortic rings from wild type mice. Representative images of aortic ring angiogenesis (stained green with isolectin B4) demonstrating reduced angiogenic vessel sprouting in aortas from Lrgl KO mice and their quantification. n=30 aortic rings for each group (** p<0.01).

Figure 10. Genes up-regulated in Curlytail-J, RD1 and VLDLR -/- mouse models of retinal disease.

Figure 1 1. A. Fluorescein angiograms (FA) of choroidal neovascularisation induced by laser burns to the retina of WT and Lrgl KO mice. Quantitation of early (B) and late (C) FA showing size of angiogenic growth and leakage respectively recorded at 7 days post lesion. n=10 (** pO.01).

Figure 12. (Left) The brain endothelial cell line GPNT expresses the requisite components for studying the effect of Lrgl on TGFP signalling. P = cell pellet; M = cell media. (Right) TGFp induces Lrgl gene expression in GPNT cells.

Figure 13. Representative images of mouse retinal vasculature (stained red with isolectin B4) at PI 7 following oxygen-induced retinopathy (OIR) demonstrating increased avascular region and decreased neovascular tufts in Lrgl KO mice.

Quantification of (B) avascular region and (C) neovascular tufts in WT and Lrgl KO mice (n=6 and 9 respectively). The avascular region is increased in the Lrgl knockout (* p<0.05), with fewer neovascular tufts visible in the knockout compared with the wild type (** pO.01). Figure 14. Tube formation of Human Umbilical Vein Endothelial Cells (HUVEC) in Matrigel in vitro was reduced following addition of a neutralizing anti -human Lrgl polyclonal antibody compared to irrelevant IgG. Tube formation was measured with regard to A. the number of branch points, tube number and B. total tube length (n = 3, * p<0.05, ** p<0.01).

Figure 15. A. Cross section through a human retina stained for Lrgl. B. Western blot and quantification of Lrgl (n=4) in vitreous samples from non-diabetic patients and patients with proliferative diabetic retinopathy (PDR). C. Western blot and quantification of TGF i in vitreous samples from non-diabetic patients and patients with PDR (** p<0.01).

Figure 16. Schematic of working hypothesis. A. Under normal conditions TGF l signalling is directed predominantly towards the TGFPRII/ALK5/Smad2/3 pathway and Lrgl is sequestered in the basal lamina. B. Under pathogenic conditions increased Lrgl expression results in a redirection of TGFpl signalling towards the

TGFpRII/ALKl/Smadl/5/8 pathway which contributes to vascular remodelling.

DETAILED DESCRIPTION OF THE INVENTION

Blocking Lrgl

Antagonists of the invention block the function of Lrgl . Blocking of Lrgl

encompasses any reduction in its activity or function that results in reduced vasculoproliferative effects, including endothelial cell proliferation, pericyte drop-out, endothelial cell death, vascular remodelling, angiogenesis, telangiectasia, vascular leakage.

For example, blocking of Lrgl may be via blocking its interaction with ALK1 , TGFpRII and/or TGFP, which our data suggest promotes the interaction between TGFpRII and ALK5 rather than ALK1, thus diverting the activity of TGFp into the less-pathogenic AL -5 activated signally cascade and away from the

vasculopathogenic ALK-1 associated cascade. Blocking of Lrgl may also result in reduced bioavailability of TGFp.

Blocking encompasses both total and partial reduction of Lrgl activity or function, for example total or partial prevention of the ALKl-Lrgl , TGFPRII-Lrgl and/or TGFP- Lrgl interactions. For example, a blocking antagonist of the invention may reduce the activity of Lrgl by from 10 to 50%, at least 50% or at least 70%, 80%, 90%, 95% or 99%.

Blocking of Lrgl activity or function can be measured by any suitable means. For example, blocking of the ALKl-Lrgl, TGFpRJI-Lrgl and/or TGFp-Lrgl interaction can be determined by measuring the effect on Smad5 phosphorylation, on the basis that Smad5 phosphorylation is characteristic of the ALK1 activated pathway rather than the ALK5-activated one.

Blocking of Lrgl can also be measured via assays that measure angiogenesis, for example in vitro assays such as vessel growth in Matrigel, vessel growth from aortic rings and in vivo assays such as those that measure retinal angiogenesis (eg laser induced choroidal neovascularisation, oxygen-induced retinopathy).

Blocking may take place via any suitable mechanism, depending for example on the nature (see below) of the antagonist used, e.g. steric interference in any direct or indirect ALKl-Lrgl, TGFPRII-Lrgl and/or TGFp-Lrgl interaction or knockdown of Lrgl expression.

Antagonists of Lrgl Any suitable antagonist may be used according to the invention, for example peptides and peptidomimetics, antibodies, small molecule inhibitors, double-stranded R A, aptamers and ribozymes. Preferred antagonists include peptide fragments of Lrgl , double-stranded RNA, aptamers and antibodies.

Peptides

Peptide antagonists will typically be fragments of Lrgl that compete with full-length Lrgl for binding to TGFPRII and/or ALK1 and hence antagonise Lrgl . Such peptides may be linear or cyclic. Peptide antagonists will typically be from 5 to 50, preferably 10-40, 10-30 or 15-25 amino acids in length and will generally be identical to contiguous sequences from within Lrgl but may have less than 100% identity, for example 95% or more, 90% or more or 80% or more, as long as they retain Lrgl - blocking properties. Blocking peptides can be identified in any suitable manner, for example, by systematic screening of contiguous or overlapping peptides spanning part or all of the Lrgl sequence. Peptidomimetics may also be designed to mimic such blocking peptides.

Double-stranded RNA

Using known techniques and based on a knowledge of the sequence of Lrgl, double- stranded RNA (dsRNA) molecules can be designed to antagonise Lrgl by sequence homology-based targeting of Lrgl RNA. Such dsRNAs will typically be small interfering RNAs (siRNAs), usually in a stem-loop ("hairpin") configuration, or micro-RNAs (miRNAs). The sequence of such dsRNAs will comprise a portion that corresponds with that of a portion of the mRNA encoding Lrg 1. This portion will usually be 100% complementary to the target portion within the Lrgl mRNA but lower levels of complementarity (e.g. 90% or more or 95% or more) may also be used. Aptamers

Aptamers are generally nucleic acid molecules that bind a specific target molecule. Aptamers can be engineered completely in vitro, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications. These characteristics make them particularly useful in pharmaceutical and therapeutic utilities.

As used herein, "aptamer" refers in general to a single or double stranded

oligonucleotide or a mixture of such oligonucleotides, wherein the oligonucleotide or mixture is capable of binding specifically to a target. Oligonucleotide aptamers will be discussed here, but the skilled reader will appreciate that other aptamers having equivalent binding characteristics can also be used, such as peptide aptamers.

In general, aptamers may comprise oligonucleotides that are at least 5, at least 10 or at least 15 nucleotides in length. Aptamers may comprise sequences that are up to 40, up to 60 or up to 100 or more nucleotides in length. For example, aptamers may be from 5 to 100 nucleotides, from 10 to 40 nucleotides, or from 15 to 40 nucleotides in length. Where possible, aptamers of shorter length are preferred as these will often lead to less interference by other molecules or materials.

Non-modified aptamers are cleared rapidly from the bloodstream, with a half-life of minutes to hours, mainly due to nuclease degradation and clearance from the body by the kidneys. Such non-modified aptamers have utility in, for example, the treatment of transient conditions such as in stimulating blood clotting. Alternatively, aptamers may be modified to improve their half life. Several such modifications are available, such as the addition of 2'-fluorine-substituted pyrimidines or polyethylene glycol (PEG) linkages.

Aptamers may be generated using routine methods such as the Systematic Evolution of Ligands by Exponential enrichment (SELEX) procedure. SELEX is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules. It is described in, for example, US 5,654,151, US 5,503,978, US

5,567,588 and WO 96/38579.

The SELEX method involves the selection of nucleic acid aptamers and in particular single stranded nucleic acids capable of binding to a desired target, from a collection of oligonucleotides. A collection of single-stranded nucleic acids (e.g., DNA, RNA, or variants thereof) is contacted with a target, under conditions favourable for binding, those nucleic acids which are bound to targets in the mixture are separated from those which do not bind, the nucleic acid-target complexes are dissociated, those nucleic acids which had bound to the target are amplified to yield a collection or library which is enriched in nucleic acids having the desired binding activity, and then this series of steps is repeated as necessary to produce a library of nucleic acids (aptamers) having specific binding affinity for the relevant target.

Antibodies

The term "antibody" as referred to herein includes whole antibodies and any antigen binding fragment (i.e., "antigen-binding portion") or single chains thereof. An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V_H) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as V_L) and a light chain constant region. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The H and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

An antibody of the invention may be a monoclonal antibody or a polyclonal antibody, and will preferably be a monoclonal antibody. An antibody of the invention may be a chimeric antibody, a CDR-grafted antibody, a nanobody, a human or humanised antibody or an antigen binding portion of any thereof. For the production of both monoclonal and polyclonal antibodies, the experimental animal is typically a non- human mammal such as a goat, rabbit, rat or mouse but may also be raised in other species such as camelids.

Polyclonal antibodies may be produced by routine methods such as immunisation of a suitable animal, with the antigen of interest. Blood may be subsequently removed from the animal and the IgG fraction purified.

Monoclonal antibodies (mAbs) of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology e.g., the standard somatic cell hybridization technique of Kohler and Milstein. The preferred animal system for preparing hybridomas is the murine system. Hybridoma production in the mouse is a very well-established procedure and can be achieved using techniques well known in the art.

An antibody according to the invention may be produced by a method comprising: immunising a non-human mammal with an immunogen comprising full-length Lrgl, a peptide fragment of Lrgl , an epitope within the sequence of LI -24 of Appendix 2 or L94-117 of Appendix 3 (SEQ ID NO: 3), LI 69-192 of Appendix 2 or L262-285 of Appendix 3 (SEQ ID NO: 4) or L227-252 of Appendix 2 or L320-345 of Appendix 3 (SEQ ID NO: 5) of Lrgl or an epitope within other regions of Lrgl ; obtaining an antibody preparation from said mammal; and deriving therefrom monoclonal antibodies that specifically recognise said epitope.

The term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment and an isolated complementarity determining region (CDR). Single chain antibodies such as scFv antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments may be obtained using

conventional techniques known to those of skill in the art, and the fragments may be screened for utility in the same manner as intact antibodies.

An antibody of the invention may be prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for the immunoglobulin genes of interest or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody of interest, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences to other DNA sequences.

An antibody of the invention may be a human antibody or a humanised antibody. The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline

immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

Such a human antibody may be a human monoclonal antibody. Such a human monoclonal antibody may be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

Human antibodies may be prepared by in vitro immunisation of human lymphocytes followed by transformation of the lymphocytes with Epstein-Barr virus.

The term "human antibody derivatives" refers to any modified form of the human antibody, e.g., a conjugate of the antibody and another agent or antibody.

The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

Screening methods as described herein may be used to identify suitable antibodies that are capable of binding toLrgl . Thus, the screening methods described herein may be carried out using an antibody of interest as the test compound.

Antibodies of the invention can be tested for binding to Lrgl by, for example, standard ELISA or Western blotting. An ELISA assay can also be used to screen for hybridomas that show positive reactivity with the target protein. The binding specificity of an antibody may also be determined by monitoring binding of the antibody to cells expressing the target protein, for example by flow cytometry. Thus, a screening method of the invention may comprise the step of identifying an antibody that is capable of binding Lrgl by carrying out an ELISA or Western blot or by flow cytometry. Antibodies having the required binding properties may then be further tested to determine their effects on the activity of Lrgl as described further above.

Antibodies of the invention will have Lrgl antagonist (blocking) properties as discussed above. In one embodiment, a monoclonal antibody specifically recognises an epitope within Lrgl and blocks the activity of Lrgl . In one embodiment, the monoclonal antibody specifically recognises an epitope within Lrgl and blocks the interaction between ALK1, TGFpRII or TGFp and Lrgl. In one embodiment, a monoclonal antibody specifically recognises an epitope within amino acids LI -24 of Appendix 2 or L94-1 17 of Appendix 3 (SEQ ID NO: 3), L169-192 of Appendix 2 or L262-285 of Appendix 3 (SEQ ID NO: 4) or L227-252 of Appendix 2 or L320-345 of Appendix 3 (SEQ ID NO: 5) and blocks the activity of Lrgl. In one embodiment, a monoclonal antibody specifically recognises an epitope within amino acids LI -24 of Appendix 2 or L94-117 of Appendix 3 (SEQ ID NO: 3), LI 69- 192 of Appendix 2 or L262-285 of Appendix 3 (SEQ ID NO: 4) or L227-252 of Appendix 2 or L320-345 of Appendix 3 (SEQ ID NO: 5) and blocks the interaction between ALK1, TGFpRII or TGF and Lrgl.

Antibodies of the invention specifically recognise Lrgl, i.e. epitopes within Lrgl. An antibody, or other compound, "specifically binds" or "specifically recognises" a protein when it binds with preferential or high affinity to the protein for which it is specific but does not substantially bind, or binds with low affinity, to other proteins. The specificity of an antibody of the invention for target protein may be further studied by determining whether or not the antibody binds to other related proteins as discussed above or whether it discriminates between them. For example, an antibody of the invention may bind to human Lrgl but not to mouse or other mammalian Lrgl . Antibodies of the invention will desirably bind to Lrgl with high affinity, preferably in the picomolar range, e.g. with an affinity constant (¾) of lOnM or less, lnM or less, 500pM or less or ΙΟΟρΜ or less, measured by surface plasmon resonance or any other suitable technique.

Once a suitable antibody has been identified and selected, the amino acid sequence of the antibody may be identified by methods known in the art. The genes encoding the antibody can be cloned using degenerate primers. The antibody may be

recombinantly produced by routine methods.

Epitopes within Lrgl can be identified by methods known in the art and discussed herein, notably by systematic screening of contiguous or overlapping peptides via a "PEPSCAN" approach or by forming antibodies to peptide fragments (see above) shown to block Lrgl . Examples of such peptides within which epitopes can be identified for antibody production are the LI -24 of Appendix 2 or L94-1 17 of Appendix 3 (SEQ ID NO: 3), L169-192 of Appendix 2 or L262-285 of Appendix 3 (SEQ ID NO: 4) and L227-252 of Appendix 2 or L320-345 of Appendix 3 (SEQ ID NO: 5) peptides discussed herein. These and other epitope-containing peptides can be used as immunogens for the generation of antibodies.

Therapeutic Indications

Any condition in which Lrg-1 -mediated vasculoproliferation occurs may in principle be treated, prevented or ameliorated according to the present invention.

"Vasculoproliferation", "vasculoproliferative", "vasculoproliferative conditions" and similar terms as used herein encompass any and all pathologies related to the aberrant or unwanted development of blood vessels or vascular tissue or cells. For example, both pathogenic angiogenesis (the formation of new blood vessels, for example via new capillary growth from existing blood vessels) and vascular malformation (e.g. telangiectasia, the formation of dilated, tortuous and incompetent vessels,

microaneurysms) can be prevented or reduced, as can neovascularisation and vascular endothelial cell proliferation. Also, as is known in the art, neoplastic growth requires the formation of new blood vessels to provide a blood supply to the growing tumour. Tumours in which Lrgl -mediated vasculoproliferation occurs are therefore also conditions which may be treated, prevented or ameliorated according to the present invention.

Preferably, there is no, or minimal effect on normal, e.g. developmental

vascularisation, especially developmental vascularisation in the retina.

Treatment of ocular vasculoproliferative conditions is a preferred embodiment.

Among conditions that can be treated are: diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, macular telangiectasia, age-related macular degeneration or choroidal neovascularisation.

Treatment of tumours, typically solid tumours, can also be effected, in that preventing angiogenesis in tumours derives the tumour of blood supply. Tumour treatment targets include brain, breast, kidney, colorectal, lung, prostate, head and neck, stomach, pancreatic, skin, cervical, bone, ovarian, testicular and liver tumours.

Pharmaceutical Compositions, Dosages and Dosage Regimes

Antagonists of the invention will typically be formulated into pharmaceutical compositions, together with a pharmaceutically acceptable carrier.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

Preferably, the carrier is suitable for parenteral, e.g. intravenous, intramuscular, subcutaneous, intraocular or intravitreal administration (e.g., by injection or infusion). Depending on the route of administration, the modulator may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or more

pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts.

Preferred pharmaceutically acceptable carriers comprise aqueous carriers or diluents. Examples of suitable aqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, buffered water and saline. Examples of other carriers include ethanol, polyols (such as glycerol, propylene glycol,

polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug

concentration.

Pharmaceutical compositions of the invention may comprise additional active ingredients, notably VEGF antagonists as discussed herein.

Also within the scope of the present invention are kits comprising antagonists of the invention and instructions for use. The kit may further contain one or more additional reagents, such as an additional therapeutic or prophylactic agent as discussed above. The antagonists and compositions of the present invention may be administered for prophylactic and/or therapeutic treatments.

In therapeutic applications, modulators or compositions are administered to a subject already suffering from a disorder or condition as described above, in an amount sufficient to cure, alleviate or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in severity of disease symptoms, or an increase in frequency or duration of symptom-free periods. An amount adequate to accomplish this is defined as a "therapeutically effective amount".

In prophylactic applications, formulations are administered to a subject at risk of a disorder or condition as described above, in an amount sufficient to prevent or reduce the subsequent effects of the condition or one or more of its symptoms. An amount adequate to accomplish this is defined as a "prophylactically effective amount".

Effective amounts for each purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject. An example of a condition that may be treated prophylactically in the context of the invention is wet AMD (age-related macular degeneration); one eye may develop the condition before the other, with the first eye being treated once the problem is recognised and the second prophylactically.

A subject for administration of the antagonists of the invention may be a human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Administration to humans is preferred.

An antagonist of the present invention may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Preferred routes of administration for modulators of the invention include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection. Alternatively, an antibody of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration.

A suitable dosage of a modulator of the invention may be determined by a skilled medical practitioner. Actual dosage levels of the active ingredients in the

pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A suitable dose may be, for example, in the range of from about O.^g/kg to about lOOmg/kg body weight of the patient to be treated. For example, a suitable dosage may be from about \ μgfkg to about lOmg/kg body weight per day or from about 10 g kg to about 5 mg kg body weight per day. For intraocular administration, a suitable dosage may be from about ^g - lmg, typically every 28 days.

Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Administration may be in single or multiple doses. Multiple doses may be administered via the same or different routes and to the same or different locations. Alternatively, doses can be via a sustained release formulation, in which case less frequent administration is required. Dosage and frequency may vary depending on the half-life of the antagonist in the patient and the duration of treatment desired.

As mentioned above, modulators of the invention may be co-administered with one or other more other therapeutic agents. For example, the other agent may be an analgesic, anaesthetic, immunosuppressant or anti-inflammatory agent; or a VEGF antagonist.

Combined administration of two or more agents may be achieved in a number of different ways. Both may be administered together in a single composition, or they may be administered in separate compositions as part of a combined therapy. For example, the one may be administered before, after or concurrently with the other.

Combination Therapies

As noted above, Lrgl antagonists of the invention may be administered in

combination with any other suitable active compound. In particular, because antagonism of both Lrgl and VEGF will reduce pathogenic vascularisation, Lrgl antagonists, notably anti-VEGF antibodies such as Avastin and/or Lucentis and/or receptor-based VEGF traps such as Aflibercept. The following Examples illustrate the invention. EXAMPLES

1. Gene expression analysis of abnormal retinal vessels.

There are various animal models of retinal disease that, despite having distinct genetic and cellular origins, exhibit an aberrant vascular response incorporating not only angiogenesis but also other vascular changes such as telangiectasia (dilated, tortuous and incompetent vessels). In order to gain new insight into the biological basis of retinal vascular remodelling we conducted a study in four of these models (Fig 1) whereby we investigated the differential expression of genes in pathogenic retinal microvessels compared to microvessels from normal controls. Isolated and purified microvessel fragments from the retinae of wild type (WT) mice, retina dystrophy (RD) 1 mice, Curlytail (CT) mice and very-low density lipoprotein receptor (VLDLR) knock-out mice at time points corresponding to stages when vascular anomalies were present were investigated. Microarray gene expression analysis (Affymetrix) on isolated RNA from the three mouse models revealed 63 genes common to all that were either up- or down-regulated in the microvessels from diseased retina (Fig. 10). Of the 63 genes differentially expressed in the retinal vasculature of the three mouse models of vascular remodelling, leucine-rich alpha-2-glycoprotein-l (Lrgl) was ranked the most significant (following FDR analysis). Lrgl is a secreted glycoprotein (Fig 2) of the leucine-rich repeat family of proteins which are involved in protein- protein interactions, signalling and cell adhesion and development.

2. Validation of Lrgl overexpression observed in microarray data.

The increased expression of Lrgl in the retina of the mouse models was validated first by quantitative PCR. mRNA from whole retina of WT, RD1, CT and VLDLR-/- mice was extracted and subjected to quantitative real time PCR (qRTPCR). As indicated by microarray analysis the qRTPCR demonstrated that there was a significant (p<0.05) increase in transcript expression of Lrgl in the three models of retinal vascular pathology when compared to control mice (Fig 3 A). To establish that the increase in mRNA translated into increased protein expression we next isolated retinas at identical time-points to the gene expression studies and prepared the tissue for protein analysis by western blot (Fig 3 B). Semi-quantification of the western blot data (n > 3) by densitometric analysis compared to a housekeeping protein (GAPDH) revealed significant increases in Lrgl protein expression (p<0.05). To determine the distribution of Lrgl in the retinal vasculature and to establish whether other cells of the retina express Lrgl we carried out in situ hybridisation and immunohistochemistry to detect Lrgl mRNA and protein respectively. In normal mice Lrgl mRNA (Fig 3C) and protein (Fig 3D) were expressed predominantly by the vasculature.

3. Lrgl associates with the TGF receptors TGF RII and ALK1.

Virtually nothing is known regarding the biology of Lrgl . Several reports have described concomitant increases in the level of expression of TGFpi, T R-II and Lrgl in a number of diseases (Sun et al., 1995; Li et al., 2007). This is particularly germane as dysfunction of TGF signalling in endothelial cells leads to the disease Hemorrhagic Hereditary Telangiectasia (HHT). In this group of diseases, which are characterised by vascular abnormalities including telangiectasis, mutations in the TGFp endothelial accessory receptor endoglin and the TGF type I receptor AL 1 lead to HHT1 (McAllister et al. 1994) and HHT2 (Johnson et al. 1995) respectively. Moreover, we have pilot data in VLDLR-/- mice to the effect that TGF mRNA increases significantly in retinal tissue, but not RPE or microvessels. Of further relevance, TGFp has been found to be increased in the retinas of patients with diabetic retinopathy (Spirin et al., 1999) where vascular remodelling is prevalent.

In endothelial cells TGFp signaling can occur through TGFp receptor II associating either with the ubiquitous TGFp type I receptor activin receptor-like kinase 5 (ALK5) or ALKl, which is expressed primarily in endothelial cells, with the cellular response depending on which pathway predominates. In the case of ALK5 there is increased ECM deposition and cell quiescence whilst with ALKl there is endothelial cell activation manifest as increased migration and proliferation. This differential signalling is partly controlled by the concentration/bioavailability of TGFp and by members of a family of downstream effector proteins called Smads, whereby Smads 2 and 3 associate with ALK5, and Smads 1, 5 and 8 with ALKl . We have explored the connection between Lrgl and the TGF signalling pathway. We first established that a rat brain endothelial cell line (GPNT) expressed both Lrgl , TGF RII as well as other components of TGFp signalling (see appendix 1). We demonstrated that

immunoprecipitation of Lrgl from GPNT cell lysates resulted in co-precipitation of the receptors TGF RII and ALKl (Fig 4A). Similarly, immunoprecipitation of either TGFpRII or ALKl resulted in co-precipitation of Lrgl indicating that Lrgl associates with both receptors (Fig 4A). We have also shown that HA-tagged recombinant Lrgl protein from bacteria associates with TGFPRII and TGFp (Fig 4B). In addition, immunocytochemical visualisation of Lrgl and TGFpRII expression on GPNT cells demonstrates co-localisation (Fig 4C). We hypothesise, therefore, that Lrgl acts as a modulator of TGFP signalling causing fine-tuning between TGFPRII and the ALKl and ALK5 activated signalling cascades. We also have shown that TGF induces Lrgl gene expression in GPNT cells suggesting a possible feedback mechanism (Fig. 16).

4. Lrgl modifies TGFp signalling through differential Smad

phosphorylation.

To establish whether Lrgl affects TGFP-mediated vascular endothelial cell responses we next knocked down Lrgl in GPNT cells with siRNA and determined its effects on TGFp-mediated cell proliferation. In control cells TGFp induces a significant increase (p<0.05) in endothelial cell proliferation (70% confluent cells) over a 2 hour period. Lrgl knockdown in GPNT endothelial cells with siRNA blocks this TGFp-mediated increase in cell proliferation (Fig 6A and B). This correlated with a reduction in Smad5 phosphorylation (Fig 4D). Conversely, in GPNT cells transfected with the Lrgl gene we show that Lrgl overexpression leads to enhanced endothelial cell proliferation in response to TGFP (Fig 6A and B). This enhanced response correlates with a down-regulation in Smad2 expression and increased Smad5 phosphorylation (Fig 4E). These data are consistent with decreased signalling via the TGFPRII/ALK5 receptor complex pathway and hence a shift towards activation of the

vasculopathogenic TGFPRII/ALKl signalling pathway.

5. Lrgl conditioned medium enhances angiogenesis in vitro.

Having established that Lrgl modifies TGFp signalling in endothelial cells and affects TGFp-mediated cell proliferation we next determined whether Lrgl impacts on angiogenesis using a standard in vitro angiogenesis assay. Human umbilical vein endothelial cells (HUVEC) were grown in Matrigel and subjected to unconditioned growth media, media conditioned by GPNT cells (which constitutively secrete Lrgl) and media conditioned by GPNT cells over-expressing Lrgl . Control media contained no Lrgl whilst GPNT and Lrgl over-expressing GPNT media contained moderate and high levels of Lrgl respectively (Fig 7B). The degree of vascular formation was greatest when conditioned medium from Lrgl over-expressing cells was added (Fig 7A and C). The increased vascularisation correlated with Lrgl protein expression in the medium.

6. Peptide sequence L227-252 of Appendix 2 or L320-345 of Appendix 3 derived from Lrgl modifies TGFp signalling in GPNT cells.

We next established whether we could block endothelial Smad5 phosphorylation induced by TGFP with either an anti-Lrgl antibody or by peptide sequences derived from Lrgl. Peptides derived from the leucine-rich repeat regions of the Lrgl sequence (Ll-24 and L169-192 of Appendix 2 or L94-117 and L262-285 of Appendix 3), which are believed to be involved in protein-protein interactions, and from the highly conserved leucine-rich C-terminal domain (L227-252 of Appendix 2 or L320-345 of Appendix 3) were generated (Appendix 2). Lrgl over-expression in GPNT cells did not result in Smad5 phosphorylation. Treatment of control cells with 5 ng/ml TGFB results in a significant increase in Smad5 phosphorylation. In the Lrgl over- expressing cells the effect of TGFp on Smad5 phosphorylation is significantly enhanced. When the Lrg-1 over-expressing endothelial cells were co-treated with the anti-Lrgl polyclonal antibody there was a decrease in the level of Smad5

phosphorylation suggesting that the antibody is capable of interfering with Lrgl interactions. Co-treatment with the peptides had variable effects with peptide LI -24 of Appendix 2 (L94-117 of Appendix 3) having no effect on Smad5 phosphorylation, peptide LI 69- 192 of Appendix 2 (L262-285 of Appendix 3) had a partial effect whilst peptide L227-252 of Appendix 2 (L320-345 of Appendix 3) (Fig 5) had a dramatic inhibitory effect. Combination of all three peptides almost completely abolished TGF mediated Smad5 phosphorylation. These data support the hypothesis that Lrgl modifies TGF mediated signalling and that Lrgl antagonists can be used as therapeutic agents.

7. Aortic rings from Lrgl knockout mice display reduced angiogenic vessel sprouting.

The role of Lrgl in angiogenesis in vivo was then examined. Thoracic aortas were removed from P14 Lrgl knock-out mice or wild type littermate controls sacrificed by cervical dislocation and immediately transferred to a culture dish containing ice-cold serum-free OPTI-MEM (Invitrogen). The peri-aortic fibroadipose tissue was carefully removed with fine microdissecting forceps. One millimeter long aortic rings were sectioned and embedded in a rat tail collagen I gel (1.5 mg/ml) prepared in DMEM at pH 7.4. The collagen gels containing the aortic rings were kept at 37°C in 96 well plates for 7 days. Each well contained endothelial cell basal medium supplemented with 2.5% FCS, 100 U/ml penicillin and 100 μ^ηιΐ streptomycin. Images were taken with an Olympus microscope.

The number of angiogenic vessels sprouting from each aortic ring was quantified. Aortic rings isolated from both mice heterozygous and homozygous for the Lrgl gene knockout exhibited significantly reduced angiogenic vessel sprouting compared to aortic rings from wild type mice (p<0.01) (Fig 9).

8. Choroidal neovascularisation (CNV) after retinal injury is reduced in Lrgl knockout mice.

Bruch's membrane was ruptured by laser at three locations surrounding the optic nerve in each eye of Lrgl knock-out mice or wild-type littermate controls. The CNV lesions at Bruch's membrane rupture sites were measured 1 week after laser treatment by in vivo fundus fluorescein angiography (FA). Fluorescein was delivered through intraperitoneal injection. Early and late-phase fundus angiograms were obtained at an interval of 7 minutes. The early phase angiogram was obtained 90 seconds after injection indicating the size of choroidal neovascularisation. The late phase angiogram demonstrates leakage from choroidal neovascular membrane,

The FA clearly showed that choroidal neovascularisation was reduced in the Lrgl knockout mice (Fig 11 A). Quantification of the choroidal neo vasculature revealed that the size of the area of angiogenic growth and leakage was significantly reduced in the Lrgl knockout as compared to the wild type mice (Fig 11 B and C) (** pO.01).

9. Retinal neovascularisation following oxygen-induced retinopathy (OIR) is reduced in Lrgl knockout mice.

P7 Lrgl knock-out mice and wild-type littermate controls with nursing mothers were subjected to hyperoxia (75% oxygen) for 5 days, which leads in the neonates to significant inhibition of retinal vessel development. On PI 2, mice were returned to normoxia whereupon the hypoxic avascular retina triggers both normal vessel regrowth and pathological neovascularisation, which reaches a peak at PI 7. Retinas were isolated, fixed and subjected to whole mount immunostaining using isoIectin-B4 (Fig 13 A). Vascular regrowth was quantified by comparing the avascular area to total retinal area. Neovascularisation was quantified by manually measuring the area of neovascular tufts.

The size of the avascular region was found to be significantly increased in the retinas of Lrgl knockout mice (* p<0.05) (Fig 13B). Also, the number of neovascular tufts was significantly reduced in the Lrgl knockout mice as compared to the wild type mice (** p<0.002) (Fig 13C).

10. Tube, cord and vessel formation of Human Umbilical Vein Endothelial Cells (HUVEC) in Matrigel in vitro is reduced by the addition of a polyclonal anti-Lrgl antibody.

In vitro tube formation assays were carried out in Matrigel using Human Umbilical Vein Endothelial Cells (HUVEC). 96- well plates were coated with 60 μΐ of Matrigel per well. Each well was treated with 100 μΐ of EGM2 medium containing 15,000 HUVEC in the presence of 100 nM of anti-human polyclonal Lrgl antibody (raised against the whole Lrgl glycoprotein), 100 nM isotype IgG or equivalent volume of antibody elution buffer for 16 hours at 37°C, 5% C0₂. Cells were washed and fixed. Tube formation was significantly reduced by the addition of a neutralizing anti-human Lrgl polyclonal antibody, compared with the addition of antibody elution buffer (p<0.01) or compared with the addition of an irrelevant IgG antibody (p<0.05). Tube formation was measured by the number of branch points (Fig 14 A) tube number (Fig 14A) and total tube length (Fig 14B).

11. Lrgl and TGF expression in the vitreous humour is increased in human patients suffering from proliferative diabetic retinopathy (PDR). Immunohistochemical analysis of a human retina was conducted, with staining for Lrgl detected in the retinal vasculature (Fig 15 A). Samples of vitreous humour were obtained from non-diabetic patients and patients suffering from PDR. The presence of Lrgl in the vitreous samples was determined using western blotting and quantified by densitometric analysis (Fig 15B). Lrgl was significantly increased in the vitreous of patients suffering from PDR compared to non-diabetic patients (p<0.01). The presence of TGFp in the vitreous samples was also determined by western blotting and quantified as for Lrgl (Fig 15C). TGFp was also significantly increased in patients suffering from PDR (pO.01).

The present inventors have therefore demonstrated that reduced Lrgl expression is associated with a reduced angiogenic response to retinal trauma. Lrgl and TGFp have also been shown to be up-regulated in patients suffering from PDR, a condition characterised by an increase in retinal neovascularisation. This supports the hypothesis that Lrgl is involved in stimulating vasculoproliferation via TGFP- mediated signalling, and that Lrgl antagonists, especially antibodies, can be used as therapeutic agents to combat undesired vascular proliferation.

Partial sequence alignment of mouse and human Lrg1, arranged to illustrate the leucine-rich repeats (red), and the highly conserved C-terminal domains (green).

Leucine-rich α-2-glycoprotein 1 (Lrgl) exhibited the greatest fold change in the remodelled retinal vessels. Aligned amino acid sequence of human and mouse Lrgl. In red are the leucine rich repeat regions and in green is the human C-terminal domain region used as a blocking peptide. REFERENCES

Isolation and characterization of an unknown, leucine-rich 3.1-S-alpha2- glycoprotein from human serum: Haupt H, Baudner S; Hoppe Seylers Z Physiol Chem. 1977 Jun; 358(6): 639-46. (Title translated from original German)

Periodicity of leucine and tandem repetition of a 24-amino acid segment in the primary structure of leucine-rich alpha 2-glycoprotein of human serum: Takahashi N, Takahashi Y, Putnam FW; Proc Natl Acad Sci USA. 1985 Apr;82(7): 1906-10.

Differentially expressed genes in TGF-beta 1 sensitive and resistant human hepatoma cells: Sun D, Kar S, Carr BI; Cancer Lett. 1995 Feb 10; 89(l):73-9.

Expression of TGF-betas and TGF-beta type II receptor in cerebrospinal fluid of patients with idiopathic normal pressure hydrocephalus: Li X, Miyajima M, Jiang C, Arai H; Neurosci Lett. 2007 Feb 14; 413(2):141-4. Epub 2006 Dec 27.

Identification of putative serum glycoprotein biomarkers for human lung adenocarcinoma by multilectin affinity chromatography and LC-MS MS: Heo SH, Lee SJ, Ryoo HM, Park JY, Cho JY.; Proteomics. 2007 Dec;7(23):4292-302.

Increased expression of one isoform of leucine-rich alpha-2-glycoprotein in peritoneal fluid of women with uterine leiomyomas: Ferrero S, Gillott DJ, Remorgida V, Anserini P, Ragni N, Grudzinskas JG; Arch Gynecol Obstet. 2009 Mar;279(3):365-71. Epub 2008 Jul 30.

Potential diagnostic biomarkers in serum of idiopathic pulmonary arterial hypertension: Zhang J, Zhang Y, Li N, Liu Z, Xiong C, Ni X, Pu Y, Hui R, He J, Pu J.; Respir Med. 2009 Aug 22. [Epub ahead of print] Endoglin, a TGF-beta binding protein of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1. McAllister A, Grogg KM, Johnson DW, Gallione CJ, Baldwin MA, Jackson CE, Helmbold EA, Markel DS, McKinnon WC, Murrell J, et al. Nat Genet. 1994 Dec;8(4):345-51.

A second locus for hereditary hemorrhagic telangiectasia maps to chromosome 12. Johnson DW, Berg JN, Gallione CJ, McAllister KA, Warner JP, Helmbold EA, Markel DS, Jackson CE, Porteous ME, Marchuk DA. Genome Res. 1995

Aug;5(l):21-8.

Basement membrane and growth factor gene expression in normal and diabetic human retinas. Spirin KS, Saghizadeh M, Lewin SL, Zardi L, Kenney MC, Ljubimov AV. Curr Eye Res. 1999 Jun;18(6):490-9.

Plasma proteomics of pancreatic cancer patients by multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis (2D-DIGE): up-regulation of leucine-rich alpha-2-glycoprotein in pancreatic cancer. Kakisaka T, Kondo T, Okano T, Fujii K, Honda K, Endo M, Tsuchida A, Aoki T, Itoi T, Moriyasu F, Yamada T, Kato H, Nishimura T, Todo S, Hirohashi S. J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Jun l;852(l-2):257-67. Epub 2007 Feb 1.

US 2005/0064516 WO 2008/092214

US 2007/0184503

Claims

An antagonist of Leucine-rich alpha-2-glycoprotein 1 (Lrgl) for use in the treatment or prevention of a vasculoproliferative condition.

A Lrgl antagonist according to claim 1 , wherein said antagonist blocks the interaction between:

(a) activin receptor-like kinase 1 (ALKl) and Lrgl;

(b) Lrgl and TGFp Receptor II (TGFpRII); and/or

(c) Lrgl and TGFp, in the TGFp signalling complex.

A Lrgl antagonist according to claim 2, wherein said blocking by said antagonist reduces the interaction between ALK and Lrgl , thereby reducing the interaction between ALKl and TGFp Receptor II (TGFPRII) and promoting the interaction between TGFpRII and activin receptor-like kinase 5 (ALK5) such that the effect of TGFP in the ALKl -activated signalling cascade is reduced relative to the effect of TGFP in the ALK5 -activated signalling cascade.

A Lrgl antagonist according to any one of the preceding claims, which comprises an antibody, a double-stranded RNA, an aptamer, or a peptide or peptidomimetic that blocks Lrgl function.

An antagonist peptide according to claim 4, which is a fragment of Lrgl .

6. An antagonist peptide fragment according to claim 5, which comprises one or more of sequences LI -24 (SEQ ID NO: 3), L169-192 (SEQ ID NO: 4), and L227-252 (SEQ ID NO: 5).

7. An antagonist peptide fragment according to claim 6, which comprises or consists of amino acids 227-252 of Lrgl .

8. An antagonist antibody according to claim 4, which is a monoclonal antibody.

9. An antagonist monoclonal antibody according to claim 8, which specifically recognises an epitope within the sequence of LI -24 (SEQ ID NO: 3), LI 69- 192 (SEQ ID NO: 4) or L227-252 (SEQ ID NO: 5) of Lrgl.

10. An antagonist monoclonal antibody according to claim 9, which specifically recognises an epitope within L227-252 (SEQ ID NO: 5) of Lrgl .

11. An antagonist double-stranded RNA according to claim 4, which is a short interfering RNA (siRNA) or microRNA (miR A).

12. A Lrgl antagonist according to any one of the preceding claims for use in the treatment of a disorder wherein said vasculoproliferative condition comprises neovascularisation, vascular endothelial cell proliferation, angiogenesis, telangiectasia or microaneurysms.

13. A Lrgl antagonist according to any one of the preceding claims, for use in the treatment of a vasculoproliferative condition of the eye.

14. A Lrgl antagonist according to claim 13, for use in the treatment of a disorder of the eye that is selected from diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, macular telangiectasia, age-related macular degeneration or choroidal neovascularization.

15. A Lrgl antagonist according to any one of claims 1 to 12, for use in the

treatment of a tumour that exhibits vasculoproliferation.

16. A Lrgl antagonist according to claim 15, for use in the treatment of a tumour that is selected from brain tumour, breast tumour, kidney tumour, colorectal tumour, lung tumour, prostate tumour, head and neck tumours, stomach tumour, pancreatic tumour, skin tumour, cervical tumour, bone tumour, ovarian tumour, testicular tumour and liver tumours.

17. A Lrgl antagonist according to any one of the preceding claims, for use in combination with an antiangiogenic compound.

18. A Lrgl antagonist of claim 17, wherein the antiangiogenic compound is an antagonist of vascular endothelial growth factor (VEGF).

19. A Lrgl antagonist according to claim 18, wherein said VEGF antagonist is an anti-VEGF antibody.

20. A method of identifying antagonists of Lrgl comprising:

(a) providing a candidate antagonist, and

(b) determining whether or not said candidate antagonist blocks the function or activity of Lrgl ; wherein said candidate antagonist is identified as an antagonist of Lrgl if blocking of the function or activity of Lrgl is observed.

21. A method according to claim 20, wherein the Lrg 1 antagonist blocks the interaction between:

(a) activin receptor-like kinase 1 (ALK1) and Lrgl;

(b) Lrgl and TGFp Receptor II (TGFpRII); and/or

(c) Lrgl and TGFp.

22. A monoclonal antibody which specifically recognises an epitope within amino acids Ll-24 (SEQ ID NO: 3), L169-192 (SEQ ID NO: 4) or L227-252 (SEQ ID NO: 5) and blocks Lrgl activity.

23. A monoclonal antibody which specifically recognises an epitope within amino acids Ll-24 (SEQ ID NO: 3), L169-192 (SEQ ID NO: 4) or L227-252 (SEQ ID NO: 5) and blocks the interaction between:

(a) ALK1 and Lrgl ;

(b) Lrgl and TGFp Receptor II (TGFpRII); and/or

(c) Lrgl and TGFp.

24. A method for producing an antibody according to claim 22 or 23, comprising:

(a) immunising a non-human mammal with an immunogen comprising an epitope within the sequence of Ll-24 (SEQ ID NO: 3), L169-192 (SEQ ID NO: 4) or L227-252 (SEQ ID NO: 5) of Lrgl ; and (b) obtaining an antibody preparation from said mammal and deriving therefrom monoclonal antibodies that specifically recognise said epitope.

25. A method for determining what sites within Lrgl can be targeted to block the function or activity of Lrgl, comprising:

(a) providing peptide fragments of the Lrgl protein; and

(b) determining whether or not said each of said peptide fragments blocks the function or activity of Lrgl .

26. A method according to claim 25, further comprising obtaining antibodies or aptamers that specifically recognise peptide fragments found in step (b) to block function or activity of Lrgl ; and optionally further comprising determining whether or not said antibodies or aptamers block the function or activity of Lrgl .

27. Use of an antagonist of Lrgl in the manufacture of a medicament for the

treatment or prevention of a vasculoproliferative condition.

28. A method of treating a vasculoproliferative condition comprising

administering to a patient in need thereof an effective amount of an antagonist of Lrgl .