WO2009044158A2

WO2009044158A2 - Inhibitors and uses

Info

Publication number: WO2009044158A2
Application number: PCT/GB2008/003357
Authority: WO
Inventors: Roy Bicknell; Dov Joseph Stekel; John Matthew Jeff Herbert; Victoria Lowdon Heath; Rajeeb Kumar Swain
Original assignee: Cancer Research Technology Limited
Priority date: 2007-10-03
Filing date: 2008-10-03
Publication date: 2009-04-09
Also published as: WO2009044153A2; WO2009044158A3; WO2009044153A3

Abstract

A method of inhibiting tumour angiogenesis in an individual, the method comprising administering to the individual an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ. A method of combating a solid tumour in an individual, the method comprising administering to the individual an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ. The inhibitor may be an antibody, an siRNA molecule, an antisense molecule, or a ribozyme.

Description

INHIBITORS AND USES

The present invention relates to tumour endothelium specific genes and polypeptides, to the use of antibodies that bind these polypeptides for imaging and targeting tumour vasculature, and to the use of inhibitors of these genes/polypeptides for inhibiting angiogenesis in solid tumours.

The endothelium plays a central role in many physiological and pathological processes and it is known to be an exceptionally active transcriptional site. Approximately 1 ,000 distinct genes are expressed in an endothelial cell, although many of them are not endothelial cell specific. In contrast red blood cells were found to express 8, platelets 22 and smooth muscle 127 separate genes (Adams et a/ (1995) Nature 377 (6547 Suppl): 3-174). Known endothelial specific genes attract much attention from both basic research and the clinical community. For example, the endothelial-specific tyrosine kinases Tie, TIE2/TEK, KDR, and flt1 are crucial players in the regulation of vascular integrity, endothelium-mediated inflammatory processes and angiogenesis.

We have previously used an in silico database screening approach to identify endothelial specific genes, and identified four new candidate endothelial specific genes (Huminiecki & Bicknell, 2000).

Ho et al (2003) used data mining and micro-array expression analysis to identify endothelial specific genes, and identified 64 genes that are either specific for endothelial cells or at least 3-fold preferentially expressed in endothelial cells.

Endothelial cells form a single cell layer that lines all blood vessels and regulates exchanges between the blood stream and the surrounding tissues. New blood vessels develop from the walls of existing small vessels by the outgrowth of endothelial cells in the process called angiogenesis. Endothelial cells even have the capacity to form hollow capillary tubes when isolated in culture. Once the vascular system is fully developed, endothelial cells of blood vessels normally remain quiescent with no new vessel formation, with the exception of the formation of new blood vessels in natural wound healing.

However, some tumours attract a new blood supply by secreting factors that stimulate nearby endothelial cells to construct new capillary sprouts. Angiogenesis plays a major role in the progression of solid tumours and is widely recognised as a rate-limiting process in the growth of solid tumours. Tumours that fail to attract a blood supply are severely limited in their growth. Thus the ability to inhibit inappropriate or undesirable angiogenesis may be useful in the treatment of solid tumours.

The development of new blood vessels is essential for both local tumour progression and the development of distant metastases. Tumour angiogenesis involves the degradation of the basement membrane by activated tissue or circulating endothelial precursors, proliferation and migration of endothelial cells, interaction with the extracellular matrix, morphological differentiation, cell adherence and vascular tube formation. Inhibition of tumour angiogenesis is thus a target for anti-tumour therapies, employing either angiogenesis inhibitors alone or in combination with standard cancer treatments. However, targeting anti-tumour agents to the site of angiogenesis depends upon the identification of specific markers of tumour angiogenesis. Indeed, it is now accepted that the growth of solid tumours is dependent on their capacity to acquire a blood supply, and much effort has been directed towards the development of anti-angiogenic agents that disrupt this process. It has also become apparent that targeted destruction of the established tumour vasculature is another avenue for exciting therapeutic opportunities (Neri & Bicknell, 2005). These therapeutic approaches depend upon the identification of specific tumour endothelial markers (TEMs).

In a screen for tumour-specific endothelial markers that might be candidates for anti- angiogenic tumour therapy, St Croix et al (2000) identified 79 genes that were differentially expressed between endothelial cells derived from tumour endothelium and normal colonic mucosa. The expression of 33 of these genes was elevated at least 10- fold in tumour endothelial cells, including 11 known and 14 as-then uncharacterised genes. In situ hybridization on tissue samples confirmed that the expression of eight of the nine uncharacterised genes that were studied in depth were specific for tumour endothelial cells. Moreover, these genes were also expressed on endothelial cells of other tumours including lung and brain tumours. Except for one gene, these genes were also expressed at elevated levels in other angiogenic states such as healing wounds.

Khodarev et al (2003) modelled tumour/endothelial-cell interactions by co-culturing U87 human glioma cells with human umbilical vein endothelial cells (HUVECs). U87 cells induced an 'activated' phenotype in HUVECs, including an increase in proliferation, migration and net-like formation. Activation was observed in co-cultures where cells were either in direct contact or physically separated, suggesting an important role for soluble factor(s) in the phenotypic and genotypic changes observed. Expressional profiling of tumour-activated endothelial cells was evaluated using cDNA arrays and confirmed by quantitative PCR. Matching pairs of receptors/ligands were found to be coordinately expressed, including TGFβRII with TGFB3, FGFRII and cysteine-rich fibroblast growth factor receptor (CRF-1) with FGF7 and FGF12, CCR1 , CCR3, CCR5 with RANTES and calcitonin receptor-like gene (CALCRL) with adrenomedullin. (Khodarev et al (2003) "Tumour-endothelium interactions in co-culture: coordinated changes of gene expression profiles and phenotypic properties of endothelial cells", Journal of Cell Science 116: 1013-1022.)

Seaman et al (2007) compared gene expression patterns of endothelial cells derived from the blood vessels of eight normal resting tissues, five tumours, and regenerating liver. They identified organ-specific endothelial genes as well as 25 transcripts over- expressed in tumour versus normal endothelium, 13 of which were not found in the angiogenic endothelium of regenerating liver. Most of the shared angiogenesis genes were expected to have roles in cell-cycle control, but those specific for tumour endothelium were primarily cell surface molecules of uncertain function (Seaman et al (2007) "Genes that distinguish physiological and pathological angiogenesis." Cancer Cell. 11(6): 539-54).

Nevertheless, there is a need in the art for additional tumour endothelial markers.

We have now identified a number of genes (listed in Table 1) whose expression is highly specific to the tumour endothelium by data-mining public cDNA and SAGE libraries. We have thus identified these genes/polypeptides as novel Tumour Endothelial Markers (TEMs). TEMs are particularly good anticancer drug targets as they can be targeted directly via the blood supply.

Table 1. Newly Identified Tumour Endothelial Markers (TEMs)

Each of the genes/polypeptides listed in Table 1 are ones we have newly identified as having a high degree of tumour endothelial specificity.

For ECSM2 and RHOJ (and for other genes identified in the screening), we have confirmed by RT-PCR that the expression of these genes is highly- or completely- specific for human umbilical vein endothelial cells (HUVECs) and adult human dermal microvascular endothelial cells (HDMECs).

We have found that RHOJ is specifically upregulated in endothelial cells, and that the downregulation of RHOJ using siRNA technology in HUVEC reduced cell growth/proliferation, significantly impaired tube formation on fibrin gels and Matrigef, and inhibited cell migration in scratch wound and chemotaxis assays. The powerful effect of RHOJ downregulation indicates that RHOJ may play an important role in tumour angiogenesis.

We have found that the downregulation of RHBDL6 using siRNA technology in HUVEC reduced cell growth/proliferation, cuased defective tube formation on Matrigef, and inhibited cell migration in scratch wound assays. The powerful effect of RHBDL6 downregulation indicates that RHBDL6 may play an important role in tumour angiogenesis.

By in-situ hybridisation we have also shown that LRRC8C is expressed specifically in the endothelium of squamous cell carcinoma and that PCHD12 is expressed specifically in the endothelium of breast cancer tissue and fibrous histiocytioma tissue, but not in non- tumour control samples.

By in-situ hybridisation we have also shown that ECSM2 is expressed solely in the endothelium of a number of different solid tumours, but not in non-tumour control samples. Furthermore, we have shown that inhibition of ECSM2 using siRNA technology significantly inhibits endothelial cell proliferation, which is an essential component of angiogenesis. The ECSM2 data are not shown herein, but are included in our co- pending PCT application filed on the same day as this application under Attorney Docket No. CRTBV/P41653PC, and which also claims priority from US provisional patent application No. 60/997,477 filed on 3 October 2007.

As a further confirmation, our screening also identified a number of genes, such as ROBO4, ANGPT2, VIM, SPARC, SPHK1 and MED28 that were previously known or putative Tumour Endothelial Markers.

Accordingly, we conclude that each of the nine genes listed in Table 1 genuinely encode TEMs. Therefore, we now consider that each of these genes/polypeptides will be valuable as markers of the tumour endothelium; that antibodies that selectively bind these polypeptides can be used to image and target the tumour endothelium; and that inhibitors of these genes/polypeptides would be therapeutically useful in the inhibition of tumour neoangiogenesis for the treatment of solid tumours.

A first aspect of the invention thus provides a method of inhibiting tumour angiogenesis in an individual in need thereof, the method comprising administering to the individual an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ.

The invention includes the use of an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ in the preparation of a medicament for inhibiting tumour angiogenesis in an individual.

The invention further includes an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ for use in inhibiting tumour angiogenesis in an individual.

It is appreciated that inhibiting tumour angiogenesis is therapeutically beneficial in an individual having a solid tumour. Accordingly, a second aspect of the invention provides a method of combating a solid tumour in an individual, the method comprising administering to the individual an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ. The invention includes the use of an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ in the preparation of a medicament for combating a solid tumour in an individual.

The invention also includes an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ for use in combating a solid tumour in an individual.

By "combating" we include the meaning that the method or the medicament or the inhibitor can be used to alleviate symptoms of the tumour (i.e. the method is used palliatively), or to treat the tumour, e.g. to prevent the (further) growth of the tumour, to prevent the spread of the tumour (metastasis), or to reduce the size of the tumour.

Typically, the tumour is associated with undesirable neovasculature formation and the treatment reduces this to a useful extent. The reduction of undesirable neovasculature formation may halt the progression of the tumour and can lead to a clinically useful reduction of tumour size and growth.

Typically, the individual has a solid tumour which can be treated by inhibiting angiogenesis, i.e. a solid tumour which is associated with new blood vessel production. The term "tumour" is to be understood as referring to all forms of neoplastic cell growth, including tumours of the lung, brain, colon, kidney, prostate and skin as well as tumours of the liver, pancreas, stomach, uterus, ovary, breast, lymph glands and bladder.

VEGF is a well-known TEM, and the anti-VEGF monoclonal antibody Bevacizumab (Avastin™) by Genentech, Inc. was the first angiogenesis inhibitor approved by the FDA for the treatment of solid tumours. Bevacizumab has been investigated for efficacy in treatment in a number of cancers including metastatic or advanced colorectal cancer, breast cancer including recurrent or metastatic breast cancer, lung cancer including advanced non-squamous non-small cell lung cancer, advanced or metastatic renal cell carcinoma, pancreatic cancer, and ovarian cancer.

Accordingly, we consider that cancers including metastatic or advanced colorectal cancer, breast cancer including recurrent or metastatic breast cancer, lung cancer including advanced non-squamous non-small cell lung cancer, advanced or metastatic renal cell carcinoma, pancreatic cancer and ovarian cancer may be treatable by inhibiting angiogenesis using an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE or RHOJ as disclosed herein.

The therapy (treatment) may be on humans or animals. Preferably, the methods of the inventions are used to treat humans. It is appreciated that when the methods are for treatment of non-human mammals, it is preferred if the inhibitor is specific for the homologous gene/polypeptide from the other species. Such "homologous genes and polypeptides" are well known in the art and preferably, although not necessarily, have at least 70%, or at least 80%, or at least 90% sequence identity with the corresponding human sequences.

A third aspect of the invention provides an ex vivo method of inhibiting angiogenesis, the method comprising administering an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ to tissue or cells ex vivo. Typically, this is an. ex vivo method of inhibiting angiogenesis in a model of tumour angiogenesis, such as the various models described below. Thus the cells may be established tumour cell lines or tumour cells that have been removed from an individual. The tissue or cells are preferably mammalian tissue or cells, and most preferably are human tissue or cells. Preferably, the tissue or cells comprise tumour endothelium, or are a model of tumour endothelium.

Suitable inhibitors of the above-listed genes/polypeptides include antibodies that selectively bind to a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ. Other suitable inhibitors of the above-listed polypeptides include siRNA, antisense polynucleotides and ribozyme molecules that are specific for the polynucleotides encoding these polypeptides, and which prevent their expression.

It is appreciated that polynucleotide inhibitors of a gene/polypeptide selected fromRHBDL.6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ may be administered directly, or may be administered in the form of a polynucleotide that encodes the inhibitor. Thus, as used herein, unless the context demands otherwise, by administering to the individual an inhibitor of a gene/polypeptide selected from RHBDL6,

KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ which is a polynucleotide, we include the meanings of administering the inhibitor directly, or administering a polynucleotide that encodes the inhibitor. Similarly, as used herein, unless the context demands otherwise, by a medicament or a composition comprising an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ which is a polynucleotide, we include the meanings that the medicament or composition comprises the inhibitor itself, or comprises a polynucleotide that encodes the inhibitor.

A fourth aspect of the invention provides an inhibitor of the gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ.

A fifth aspect of the invention provides an inhibitor of a gene/polypeptide selected RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ for use in medicine.

A sixth aspect of the invention provides a pharmaceutical composition comprising an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ and a pharmaceutically acceptable carrier, diluent or excipient.

Antibodies Suitable antibodies which bind to the above-listed polypeptides, or to specified portions thereof, can be made by the skilled person using technology long-established in the art. Methods of preparation of monoclonal antibodies and antibody fragments are well known in the art and include hybridoma technology (Kohler & Milstein (1975) "Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-497); antibody phage display (Winter et al (1994) "Making antibodies by phage display technology." Annu. Rev. Immunol. 12: 433-455); ribosome display (Schaffitzel et al (1999) "Ribosome display: an in vitro method for selection and evolution of antibodies from libraries." J. Immunol. Methods 231: 119-135); and iterative colony filter screening (Giovannoni et al (2001) "Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening." Nucleic Acids Res. 29: E27). Further, antibodies and antibody fragments suitable for use in the present invention are described, for example, in the following publications: "Monoclonal Hybridoma Antibodies: Techniques and Application", Hurrell (CRC Press, 1982); "Monoclonal Antibodies: A Manual of Techniques" , H. Zola, CRC Press, 1987, ISBN: 0-84936-476-0; "Antibodies: A Laboratory Manuaf 1^st Edition, Harlow & Lane, Eds, Cold Spring Harbor Laboratory Press, New York, 1988. ISBN 0-87969-314-2; "Using Antibodies: A Laboratory Manuaf 2^nd Edition, Harlow & Lane, Eds, Cold Spring Harbor Laboratory Press, New York, 1999. ISBN 0-87969-543-9; and "Handbook of Therapeutic Antibodies" Stefan Dϋbel, Ed., 1^st Edition, - Wiley-VCH, Weinheim, 2007. ISBN: 3-527-31453-9.

Antibodies that are especially active at inhibiting tumour angiogenesis are preferred for anti-cancer therapeutic agents, and they can be selected for this activity using methods well known in the art and described below.

By an antibody that selectively binds a specified polypeptide we mean that the antibody molecule binds that polypeptide with a greater affinity than for an irrelevant polypeptide, such as human serum albumin (HSA). Preferably, the antibody binds the specified polypeptide with at least 5, or at least 10 or at least 50 times greater affinity than for the irrelevant polypeptide. More preferably, the antibody molecule binds the specified polypeptide with at least 100, or at least 1 ,000, or at least 10,000 times greater affinity than for the irrelevant polypeptide. Such binding may be determined by methods well known in the art, such as one of the Biacore^® systems. Preferably, the antibody molecule selectively binds the specified polypeptide without significantly binding other polypeptides in the body. It is preferred if the antibodies have an affinity for their target polypeptide of at least 10^"7 M and more preferably 10^'8 M, although antibodies with higher affinities, e.g. 10^"9 M, or higher, may be even more preferred.

By an antibody that selectively binds a specific portion of a polypeptide we mean that not only does the antibody selectively bind to the target polypeptide as described above, the antibody molecule also binds the specified portion of the polypeptide with a greater affinity than for any other portion of that polypeptide. Preferably, the antibody binds the specified portion with at least 2, or at least 5, or at least 10 or at least 50 times greater affinity than for any other epitope on the same polypeptide. More preferably, the antibody molecule binds the specified portion with at least 100, or at least 1 ,000, or at least 10,000 times greater affinity than for than for any other epitope on the same polypeptide. Such binding may be determined by methods well known in the art, such as one of the Biacore^® systems. It is preferred if the antibodies have an affinity for their target epitope on the specified polypeptide of at least 10^"7 M and more preferably 10^"8 M, although antibodies with higher affinities, e.g. 10^'9 M, or higher, may be even more preferred. Preferably, the antibody selectively binds the particular specified epitope within the polypeptide and does not bind any other epitopes within that polypeptide. Preferably, when the antibody is administered to an individual, the antibody binds to the target polypeptide or to the specified portion thereof with a greater affinity than for any other molecule in the individual. Preferably, the antibody binds to (a specified portion of) the target polypeptide with at least 2, or at least 5, or at least 10 or at least 50 times greater affinity than for any other molecule in the individual. More preferably, the agent binds the polypeptide (at the specific domain) with at least 100, or at least 1,000, or at least 10,000 times greater affinity than any other molecule in the individual.

The term "antibody" or "antibody molecule" as used herein includes but is not limited to polyclonal, monoclonal, chimaeric, single chain, Fab fragments and fragments produced by a Fab expression library. Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody. The term also includes antibody-like molecules which may be produced using phage-display techniques or other random selection techniques for molecules which bind to the specified polypeptide or to particular regions of it. Thus, the term antibody includes all molecules which contain a structure, preferably a peptide structure, which is part of the recognition site (i.e. the part of the antibody that binds or combines with the epitope or antigen) of a natural antibody. Furthermore, the antibodies and fragments thereof may be humanised antibodies, which are now well known in the art.

By "ScFv molecules" we mean molecules wherein the V_H and V_L partner domains are linked via a flexible oligopeptide. Engineered antibodies, such as ScFv antibodies, can be made using the techniques and approaches long known in the art. The advantages of using antibody fragments, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties, such as better penetration to the target site. Effector functions of whole antibodies, such as complement binding, are removed. Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the fragments. Whole antibodies, and F(ab')₂ fragments are "bivalent". By "bivalent" we mean that the antibodies and F(ab')₂ fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining site.

Antibodies may be produced by standard techniques, for example by immunisation with the appropriate polypeptide or portion(s) thereof, or by using a phage display library. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc) is immunised with an immunogenic polypeptide bearing a desired epitope(s), optionally haptenised to another polypeptide. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to the desired epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are well known in the art.

Monoclonal antibodies directed against entire polypeptides or particular epitopes thereof can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody- producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein- Barr virus. Panels of monoclonal antibodies produced against the polypeptides listed above can be screened for various properties; i.e., for isotype and epitope affinity. Monoclonal antibodies may be prepared using any of the well known techniques which provides for the production of antibody molecules by continuous cell lines in culture.

It is preferred if the antibody is a monoclonal antibody. In some circumstance, particularly if the antibody is going to be administered repeatedly to a human patient, it is preferred if the monoclonal antibody is a human monoclonal antibody or a humanised monoclonal antibody, which are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Suitably prepared non-human antibodies can be "humanised" in known ways, for example by inserting the CDR regions of mouse antibodies into the framework of human antibodies. Humanised antibodies can be made using the techniques and approaches described in Verhoeyen et al (1988) Science, 239, 1534-1536, and in Kettleborough et al, (1991) Protein Engineering, 14(7), 773-783. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non- human residues. In general, the humanised antibody will contain variable domains in which all or most of the CDR regions correspond to those of a non-human immunoglobulin, and framework regions which are substantially or completely those of a human immunoglobulin consensus sequence.

Completely human antibodies may be produced using recombinant technologies. Typically large libraries comprising billions of different antibodies are used. In contrast to the previous technologies employing chimerisation or humanisation of e.g. murine antibodies this technology does not rely on immunisation of animals to generate the specific antibody. Instead the recombinant libraries comprise a huge number of pre- made antibody variants wherein it is likely that the library will have at least one antibody specific for any antigen. Thus, using such libraries, an existing antibody having the desired binding characteristics can be identified. In order to find the good binder in a library in an efficient manner, various systems where phenotype i.e. the antibody or antibody fragment is linked to its genotype i.e. the encoding gene have been devised. The most commonly used such system is the so called phage display system where antibody fragments are expressed, displayed, as fusions with phage coat proteins on the surface of filamentous phage particles, while simultaneously carrying the genetic information encoding the displayed molecule (McCafferty et al, 1990, Nature 348: 552- 554). Phage displaying antibody fragments specific for a particular antigen may be selected through binding to the antigen in question. Isolated phage may then be amplified and the gene encoding the selected antibody variable domains may optionally be transferred to other antibody formats, such as e.g. full-length immunoglobulin, and expressed in high amounts using appropriate vectors and host cells well known in the art. Alternatively, the "human" antibodies can be made by immunising transgenic mice which contain, in essence, human immunoglobulin genes (Vaughan et al (1998) Nature Biotechnol. 16, 535-539).

It is appreciated that when the antibody is for administration to a non-human individual, the antibody may have been specifically designed/produced for the intended recipient species.

The format of displayed antibody specificities on phage particles may differ. The most commonly used formats are Fab (Griffiths et al, 1994. EMBO J. 13: 3245-3260) and single chain (scFv) (Hoogenboom et al, 1992, J MoI Biol. 227: 381-388) both comprising the variable antigen binding domains of antibodies. The single chain format is composed of a variable heavy domain (V_H) linked to a variable light domain (VL) via a flexible linker (US 4,946,778). Before use as a therapeutic agent, the antibody may be transferred to a soluble format e.g. Fab or scFv and analysed as such. In later steps the antibody fragment identified to have desirable characteristics may be transferred into yet other formats such as full-length antibodies.

WO 98/32845 and Soderlind et al (2000) Nature BioTechnol. 18:852-856 describe technology for the generation of variability in antibody libraries. Antibody fragments derived from this library all have the same framework regions and only differ in their CDRs. Since the framework regions are of germline sequence the immunogenicity of antibodies derived from the library, or similar libraries produced using the same technology, are expected to be particularly low (Soderlind et al, 2000). This property is of great value for therapeutic antibodies, reducing the risk that the patient forms antibodies to 1he administered antibody, thereby reducing risks for allergic reactions, the occurrence of blocking antibodies, and allowing a long plasma half-life of the antibody. Thus, when developing therapeutic antibodies to be used in humans, modem recombinant library technology (Soderlind et al, 2001 , Comb. Chem. & High Throughput Screen. 4: 409-416) is now used in preference to the earlier hybridoma technology.

siRNA

Small interfering RNAs are described by Hannon et al. Nature, 418 (6894): 244-51 (2002); Brummelkamp et al., Science 21 , 21 (2002); and Sui et al., Proc. Natl Acad. Sci. USA 99, 5515-5520 (2002). RNA interference (RNAi) is the process of sequence- specific post-transcriptional gene silencing in animals initiated by double-stranded (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific mRNA degradation are typically 21- and 22-nucleotide small interfering RNAs (siRNAs) which, in vivo, may be generated by ribonuclease III cleavage from longer dsRNAs. 21 -nucleotide siRNA duplexes have been shown to specifically suppress expression of both endogenous and heterologous genes (Elbashir et al (2001) Nature 411 : 494-498). In mammalian cells it is considered that the siRNA has to be comprised of two complementary 21 mers as described below since longer double- stranded (ds) RNAs will activate PKR (dsRNA-dependent protein kinase) and inhibit overall protein synthesis.

Duplex siRNA molecules selective for a polynucleotide encoding any of the polypeptides

RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE or RHOJ can readily be designed by reference to its cDNA sequence. For example, they can be designed by reference to the cDNA sequences in the Genbank Accession Nos. listed above. Typically, the first 21-mer sequence that begins with an AA dinucleotide which is at least 120 nucleotides downstream from the initiator methionine codon is selected. The RhJA sequence perfectly complementary to this becomes the first RNA oligonucleotide. The second RNA sequence should be perfectly complementary to the first 19 residues of the first, with an additional UU dinucleotide at its 3' end. Once designed, the synthetic RNA molecules can be synthesised using methods well known in the art.

siRNAs may be introduced into cells in the patient using any suitable method, such as those described herein. Typically, the RNA is protected from the extracellular environment, for example by being contained within a suitable carrier or vehicle. Liposome-mediated transfer, e.g. the oligofectamine method, may be used.

Antisense polynucleotides

Antisense nucleic acid molecules selective for a polynucleotide encoding any of the polypeptides RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE or RHOJ can readily be designed by reference to its cDNA or gene sequence, as is known in the art. Antisense nucleic acids, such as oligonucleotides, are single-stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed. These nucleic acids are often termed "antisense" because they are complementary to the sense or coding strand of the gene. Recently, formation of a triple helix has proven possible where the oligonucleotide is bound to a DNA duplex. It was found that oligonucleotides could recognise sequences in the major groove of the DNA double helix. A triple helix was formed thereby. This suggests that it is possible to synthesise a sequence-specific molecules which specifically bind double-stranded DNA via recognition of major groove hydrogen binding sites. By binding to the target nucleic acid, the above oligonucleotides can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking the transcription, processing, poly(A)addition, replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting RNA degradations.

Antisense oligonucleotides are prepared in the laboratory and then introduced into cells, for example by microinjection or uptake from the cell culture medium into the cells, or they are expressed in cells after transfection with plasmids or retroviruses or other vectors carrying an antisense gene. Antisense oligonucleotides were first discovered to inhibit viral replication or expression in cell culture for Rous sarcoma virus, vesicular stomatitis virus, herpes simplex virus type 1 , simian virus and influenza virus. Since then, inhibition of mRNA translation by antisense oligonucleotides has been studied extensively in cell-free systems including rabbit reticulocyte lysates and wheat germ extracts. Inhibition of viral function by antisense oligonucleotides has been demonstrated ex vivo using oligonucleotides which were complementary to the AIDS HIV retrovirus RNA (Goodchild, J. 1988 "Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides", Proc. Natl. Acad. Sci. (USA) 85(15), 5507-11 ). The Goodchild study showed that oligonucleotides that were most effective were complementary to the poly(A) signal; also effective were those targeted at the 5' end of the RNA, particularly the cap and 5N untranslated region, next to the primer binding site and at the primer binding site. The cap, 5' untranslated region, and poly(A) signal lie within the sequence repeated at the ends of retrovirus RNA (R region) and the oligonucleotides complementary to these may bind twice to the RNA.

Typically, antisense oligonucleotides are 15 to 35 bases in length. For example, 20-mer oligonucleotides have been shown to inhibit the expression of the epidermal growth factor receptor mRNA (Witters et a/., Breast Can∞r Res Treat 53:41-50 (1999)) and 25- mer oligonucleotides have been shown to decrease the expression of adrenocorticotropic hormone by greater than 90% (Frankel et a/., J Neurosurg 91 :261-7 (1999)). However, it is appreciated that it may be desirable to use oligonucleotides with lengths outside this range, for example 10, 11 , 12, 13, or 14 bases, or 36, 37, 38, 39 or 40 bases.

Antisense polynucleotides may be administered systemically. Alternatively, and preferably, the inherent binding specificity of polynucleotides characteristic of base pairing is enhanced by limiting the availability of the polynucleotide to its intended locus in vivo, permitting lower dosages to be used and minimising systemic effects. Thus, polynucleotides may be applied locally to the solid tumour to achieve the desired effect. The concentration of the polynucleotides at the desired locus is much higher than if the polynucleotides were administered systemically, and the therapeutic effect can be achieved using a significantly lower total amount. The local high concentration of polynucleotides enhances penetration of the targeted cells and effectively blocks translation of the target nucleic acid sequences.

It will be appreciated that antisense agents may also include larger molecules which bind to polynucleotides (mRNA or genes) encoding any of the above listed polypeptides and substantially prevent expression of the protein. Thus, antisense molecules which are substantially complementary to the respective mRNA are also envisaged.

The molecules may be expressed from any suitable genetic construct and delivered to the patient. Typically, the genetic construct which expresses the antisense molecule comprises at least a portion of the cDNA or gene operative Iy linked to a promoter which can express the antisense molecule in the cell. Preferably, the genetic construct is adapted for delivery to a human cell.

Ribozvmes Ribozymes are RNA or RNA-protein complexes that cleave nucleic acids in a site- specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity. For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate. This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids. For example, US Patent No 5,354,855 reports that certain ribozymes can act as endonucieases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications, and ribozymes specific for a polynucleotide encoding any of the polypeptides RHBDL6, KCTD15, LRRC8C, PCDH 12, LOC55726, GBP4, IKBKE or RHOJ may be designed by reference to the cDNA sequences listed in the Genbank Accession Nos. given above.

Methods and routes of administering polynucleotide inhibitors, such as siRNA molecules, antisense molecules and ribozymes, to a patient, are described in more detail below.

Further agents that inhibit transcription of the genes encoding any of the above listed polypeptides can also be designed, for example using an engineered transcription repressor described in lsalan et al (Nat Biotechnol, 19(7): 656-60 (2001)) and in Urnov (Biochem Pharmacol, 64 (5-6): 919 (2002)). Additionally, they can be selected, for example using the screening methods described in later aspects of the invention. KCTD15

The gene KCTD15 (potassium channel tetramerisation domain containing 15) encodes a K⁺ channel tetramerisation domain and probably is part of a voltage-gated K⁺ channel. KCTD15 is also known as hypothetical protein MGC2628. KCTD15 was identified by Ballif et a/ (2004, "Phosphoproteomic analysis of the developing mouse brain" MoI. Cell Pmteomics 3(11): 1093-1101).

By the KCTD15 polypeptide we include the meaning of a gene product of the human KCTD15 gene, including naturally occurring variants thereof. A cDNA sequence corresponding to a human KCTD15 mRNA is found in Genbank Accession No NM_024076. Human KCTD15 polypeptide includes the amino acid sequence found in Genbank Accession No NP_076981 , and naturally occurring variants thereof. The KCTD15 polypeptide sequence from NP_076981 is shown in Figure 5 (SEQ ID No: 1).

According to NP_076981 , the human KCTD15 gene encodes a 234 amino acid residue polypeptide, of which residues 58-145 are a K⁺ channel tetramerisation domain, an N- terminal cytoplasmic tetramerisation domain (T1) of voltage-gated K⁺ channels which encodes molecular determinants for subfamily-specific assembly of alpha-subunits into functional tetrameric channels.

To the best of the inventors' knowledge, KCTD15 has not been associated with the tumour endothelium, or with solid tumours. Indeed, to the best of the inventors' knowledge, neither inhibitors of the KCTD15 gene, such as siRNA, antisense molecules or ribozymes specific for KCTD15, nor inhibitors of the KCTD15 polypeptide, such as antibodies that selectively bind to KCTD15, have been associated with the inhibition of angiogenesis in the tumour endothelium, or with the treatment of solid tumours. Thus the invention includes a method of inhibiting tumour angiogenesis by administering an inhibitor of the gene/polypeptide KCTD15, a method of combating a solid tumour by administering an inhibitor of the gene/polypeptide KCTD15, and the use of an inhibitor of the gene/polypeptide KCTD15 in the manufacture of a medicament for inhibiting tumour angiogenesis or for combating a solid tumour.

Moreover, the inventors are not aware of any suggested therapeutic use of inhibitors of

KCTD15, whether antibodies, siRNA, antisense molecules or ribozymes. Thus the invention includes an inhibitor of the gene/polypeptide KCTD15, an inhibitor of the gene/polypeptide KCTD15 for use in medicine, and a pharmaceutical composition comprising an inhibitor of the gene/polypeptide KCTD15 and a pharmaceutically acceptable carrier, diluent or excipient.

In a preferred embodiment, the antibody that selectively binds KCTD15 binds to the extracellular or cell surface exposed region of KCTD15.

In another embodiment, the antibody that selectively binds KCTD15 binds to the K⁺ channel tetramerisation domain of KCTD15 (residues 58-145).

LRRC8C

The gene LRRC8C (leucine rich repeat containing 8 family, member C) encodes a member of a leucine rich repeat protein family. LRRC8C is also known as FAD158. LRRC8C was identified by Tominaga et al (2004, "The novel gene Fad158, having a transmembrane domain and leucine-rich repeat, stimulates adipocyte differentiation." J Biol Chem 279(33): 34840-34848) and Kubota et al (2004, "LRRC8 involved in B cell development belongs to a novel family of leucine-rich repeat proteins." FEBS Lett 564(1- 2): 147-152). Tominaga et al (2004) predicted that LRRC8C can regulate adipocyte differentiation. Kubota et al (2004) predicted that LRRC8C could be involved in proliferation and activation of lymphocytes and monocytes.

By the LRRC8C polypeptide we include the meaning of a gene product of the human LRRC8C gene, including naturally occurring variants thereof. A cDNA sequence corresponding to a human LRRC8C mRNA is found in Genbank Accession No NM_032270. Human LRRC8C polypeptide includes the amino acid sequence found in Genbank Accession No. NP_115646, and naturally occurring variants thereof. The LRRC8C polypeptide sequence from NP_115646 is shown in Figure 5 (SEQ ID No: 2).

According to NP_115646, the human LRRC8C gene encodes an 803 amino acid residue polypeptide which contains 4 transmembrane domains and 8 leucine rich repeats. Residues 598, 621 , 669, 689, 691 are reported as forming a substrate binding site.

To the best of the inventors' knowledge, LRRC8C has not been associated with the tumour endothelium, or with solid tumours. Indeed, to the best of the inventors' knowledge, neither inhibitors of the LRRC8C gene, such as siRNA, antisense molecules or ribozymes specific for LRRC8C, nor inhibitors of the LRRC8C polypeptide, such as antibodies that selectively bind to LRRC8C, have been associated with the inhibition of angiogenesis in the tumour endothelium, or with the treatment of solid tumours. Thus the invention includes a method of inhibiting tumour angiogenesis by administering an inhibitor of the gene/polypeptide LRRC8C, a method of combating a solid tumour by administering an inhibitor of the gene/polypeptide LRRC8C, and the use of an inhibitor of the gene/polypeptide LRRC8C in the manufacture of a medicament for inhibiting tumour angiogenesis or for combating a solid tumour.

Moreover, the inventors are not aware of any suggested therapeutic use of inhibitors of LRRC8C, whether antibodies, siRNA, antisense molecules or ribozymes. Thus the invention includes an inhibitor of the gene/polypeptide LRRC8C, an inhibitor of the gene/poiypeptide LRRC8C for use in medicine, and a pharmaceutical composition comprising an inhibitor of the gene/polypeptide LRRC8C and a pharmaceutically acceptable carrier, diluent or excipient.

In a preferred embodiment, the antibody that selectively binds LRRC8C binds to one of the extracellular regions of LRRC8C at residues 1-15, 149-261 and 342-803.

In another embodiment, the antibody that selectively binds LRRC8C binds to one of the 8 leucine rich repeat regions

In yet another embodiment, the antibody that selectively binds LRRC8C binds to the substrate binding site (residues 598, 621 , 669, 689 and 691).

PCDH12 The gene PCDHM (protocadherin 12 precursor) encodes a member of the protocadherin gene family, a subfamily of the cadherin superfamily. PCDH12 is also known as vascular endothelial cadherin-2. PCDH 12 has been described in Rampon et al (2005, "Protocadherin 12 (VE-cadherin 2) is expressed in endothelial, trophoblast, and mesangial cells." Exp Cell Res 302(1): 48-60); Ludwig et al (2000, "cDNA cloning, chromosomal mapping, and expression analysis of human VE-Cadherin-2." Mamm Genome 11(11): 1030-1033); Clark et al (2003, "The secreted protein discovery initiative (SPDI), a large-scale effort to identify novel human secreted and transmembrane proteins: a bioinformatics assessment." Genome Res. 13 (10): 2265-2270); and TeIo et al (1998, "Identification of a novel cadherin (vascular endothelial cadherin-2) located at intercellular junctions in endothelial cells." J Biol Chem 273(28): 17565-17572). By the PCDH 12 polypeptide we include the meaning of a gene product of the human PCDH12 gene, including naturally occurring variants thereof. A cDNA sequence corresponding to a human PCDH12 mRNA is found in Genbank Accession No NM_016580. Human PCDH12 polypeptide includes the amino acid sequence found in Genbank Accession No NP_057664 and naturally occurring variants thereof. The PCDH12 polypeptide sequence from NPJD57664 is shown in Figure 5 (SEQ ID No: 3).

The encoded protein consists of an extracellular domain containing 6 cadherin repeat domains (repeats in the extracellular region which are thought to mediate cell-cell contact when bound to calcium), a transmembrane domain and a cytoplasmic tail that differs

^■ from those of the classical cadherins. The gene localises to the region on chromosome

5 where the protocadherin gene clusters reside. The exon organization of this transcript is similar to that of the gene cluster transcripts, notably the first large exon, but no significant sequence homology exists. The function of PCDH 12 has not yet been determined but the mouse homologue of protocadherin 12 does not bind catenins and appears to have no affect on cell migration or growth (Ludwig et al 2000).

According to NPJD57664, PCDH12 encodes an 1184 amino acid residue polypeptide of which residues 1-29 are a signal peptide, and residues 30-1184 represent the mature peptide. Cadherin repeat domains are present at residues 36-236, with Ca²⁺ binding sites at residues 38-39, 93, 95, 127, 129-130, 161 , 163 and 218; at residues 139-344, with Ca²⁺ binding sites at residues 146-147, 203, 205, 236, 238-239, 270, 272 and 326; at residues 363-543, with Ca²⁺ binding sites at residues 365-366, 419, 421, 452, 454- 455, 486, 488 and 539; and at residues 465-693, with Ca²⁺ binding sites at residues 471- 472, 524, 526, 557, 559-560, 625, 627 and 679. Residues 826-895 contain a region of homology to RNA polymerase Rpb1 , domain 1 and residues 989-1103 contain a region of homology to Neisseria meningitidis TspB protein.

PCDH 12 is a cell adhesion molecule which is vasculogenic rather than angiogenic, reported to be expressed in trophoblasts and mesangial cells, and knock-out mice have no observable phenotype (Rampon et al 2005). PCDH12 is expressed in highly vascularised tissues (Ludwig et al 2000) and in the endothelium of multiple tissues (TeIo ef a/ 1998).

To the best of the inventors' knowledge, PCDH12 has not been associated with the tumour endothelium, or with solid tumours. Indeed, to the best of the inventors' knowledge, neither inhibitors of the PCDH12 gene, such as siRNA, antisense molecules or ribozymes specific for PCDH12, nor inhibitors of the PCDH12 polypeptide, such as antibodies that selectively bind to PCDH12, have been associated with the inhibition of angiogenesis in the tumour endothelium, or with the treatment of solid tumours. Thus the invention includes a method of inhibiting tumour angiogenesis by administering an inhibitor of the gene/polypeptide PCDH12, a method of combating a solid tumour by administering an inhibitor of the gene/polypeptide PCDH 12, and the use of an inhibitor of the gene/polypeptide PCDH12 in the manufacture of a medicament for inhibiting tumour angiogenesis or for combating a solid tumour.

Moreover, the inventors are not aware of any suggested therapeutic use of inhibitors of PCDH12, whether antibodies, siRNA, antisense molecules or ribozymes. Thus the invention includes an inhibitor of the gene/polypeptide PCDH 12, an inhibitor of the gene/polypeptide PCDH 12 for use in medicine, and a pharmaceutical composition comprising an inhibitor of the gene/polypeptide PCDH 12 and a pharmaceutically acceptable carrier, diluent or excipient.

Typically, the antibody that selectively binds PCDH 12 binds to the mature peptide (residues 30-1184) and not to the signal peptide.

In a preferred embodiment, the antibody that selectively binds PCDH 12 binds to the extracellular region of PCDH12 at residues 1-715, and more preferably to residues 30- 715

In another embodiment, the antibody that selectively binds PCDH 12 binds to the cadherin repeat domain at residues 36-236, or to the cadherin repeat domain at residues 139-344, the cadherin repeat domain at residues 363-543, the cadherin repeat domain at residues 465-693, or to the Ca²⁺ binding sites therein.

In another embodiment, the antibody selectively binds to the region of PCDH 12 that has homology to RNA polymerase (residues 826-895), or to the region of PCDH12 that has homology to N. meningitidis TspB protein (989-1103).

C12ORF11/ LOC55726 The open reading frame known as human C12ORF11 (chromosome 12 open reading frame 11) encodes hypothetical protein LOC55726. It is also known as sarcoma antigen NY-SAR-95, FLJ10630 and FLJ10637. LOC55726 has been discussed in Olsen et al (2006, "Global, in vivo, and site-specific phosphorylation dynamics in signaling networks" Cell 127(3): 635-648); Lee et al (2003, "Immunomic analysis of human sarcoma" Proc. Natl. Acad. ScL U.S.A. 100(5): 2651-2656); and Bourdon et al (2002, "Genomic and expression analysis of the 12p11-p12 amplicon using EST arrays identifies two novel amplified and overexpressed genes" Cancer Res. 62(21): 6218-6223).

By the LOC55726 polypeptide we include the meaning of a gene product of human C12ORF11, including naturally occurring variants thereof. A cDNA sequence corresponding to a human C12ORF11 mRNA is found in Genbank Accession No NM_018164. Human LOC55726 polypeptide includes the amino acid sequence found, in Genbank Accession No NP_060634 and naturally occurring variants thereof. The LOC55726 polypeptide sequence from NP_060634 is shown in Figure 5 (SEQ ID No: 4).

According to Lee et al (2003) C12ORF11 encodes a sarcoma antigen (NY-SAR-95). However, to the best of the inventors' knowledge, C12ORF11/LOC55726 has not been associated with the tumour endothelium. Moreover, to the best of the inventors' knowledge, neither inhibitors of the C12ORF11 gene, such as siRNA, antisense molecules or ribozymes specific for C12ORF11, nor inhibitors of the LOC55726 polypeptide, such as antibodies that selectively bind to LOC55726, have been associated with the inhibition of angiogenesis in the tumour endothelium. Thus the invention includes a method of inhibiting tumour angiogenesis by administering an antibody that selectively binds the polypeptide LOC55726, a method of combating a solid tumour by administering an antibody that selectively binds the polypeptide LOC55726, and the use of an antibody that selectively binds the polypeptide LOC55726in the manufacture of a medicament for inhibiting tumour angiogenesis or for combating a solid tumour.

Thus the invention includes a method of inhibiting tumour angiogenesis by administering an inhibitor of the gene/polypeptide C12ORF11/LOC55726, a method of combating a solid tumour by administering an inhibitor of the gene/polypeptide C12ORF11/LOC55726, and the use of an inhibitor of the gene/polypeptide C12ORF11/LOC55726 in the manufacture of a medicament for inhibiting tumour angiogenesis or for combating a solid tumour. Moreover, the inventors are not aware of any suggested therapeutic use of inhibitors of C12ORF11/LOC55726, whether antibodies, siRNA, antisense molecules or ribozymes.

Thus the invention includes an inhibitor of the gene/polypeptide C12ORF11/LOC55726, an inhibitor of the gene/polypeptide C12ORF11/LOC55726 for use in medicine, and a pharmaceutical composition comprising an inhibitor of the gene/polypeptide C12ORF11/LOC55726 and a pharmaceutically acceptable carrier, diluent or excipient.

In a preferred embodiment, the antibody that selectively binds LOC55726 binds to a cell- surface exposed region of LOC55726.

LOC55726 does not contain any known domains but does contain a Coiled Coil motif which is a protein domain that forms a bundle of two or three alpha helices and which may be involved in protein interactions. Thus, in another embodiment of the various aspects of the invention, the antibody that selectively binds LOC55726 binds to the coiled coil motif within LOC55726.

GBP4

The gene GBP4 (guanylate binding protein 4) encodes a member of the family of guanylate-binding proteins (GBPs) which are induced by interferon^. GBPs, such as GBP1 , are characterized by their ability to specifically bind guanine nucleotides (GMP₁ GDP, and GTP) and are distinguished from the GTP-binding proteins by the presence of 2 binding motifs rather than 3 (Cheng et al (1991) "Interferon-induced guanylate-binding proteins lack an N(T)KXD consensus motif and bind GMP in addition to GDP and GTP" MoI Cell Biol. 11(9): 4717-25). GBP4 is discussed by Han et al who suggested that mouse GBP4 plays a role in the erythroid differentiation (Han et al (1998) "Cloning, expression, and characterization of a novel guanylate-binding protein, GBP3 in murine erythroid progenitor cells." Biochim Biophys Acta 1384(2): 373-386).

By the GBP4 polypeptide we include the meaning of a gene product of the human GBP4 gene, including naturally occurring variants thereof. A cDNA sequence corresponding to a human GBP4 mRNA is found in Genbank Accession No NM_052941. Human GBP4 polypeptide includes the amino acid sequence found in Genbank Accession No NP_443173, and naturally occurring variants thereof. The GBP4 polypeptide sequence from NP_443173 is shown in Figure 5 (SEQ ID No: 5). According to NP_443173, the human GBP4 gene encodes an 640 amino acid residue polypeptide in which residues 48-289 represent the guanylate-binding protein (GBP) N- terminal domain; residues 60-67 are a G1 box region; residues 62-68, 82-84, 89-90, 115, 196-197 and 254 form a GTP/Mg²⁺ binding site; residues 79-84, 89-96 are a Switch I region; residue 90 forms (part of) the G2 box region; residues 112-115 form (part of) the G3 box region; residues 114-118, 129-135 and 138-140 are a Switch Il region; residues 196-199 form (part of) the G4 box region; residues 252-254 form (part of the) G5 box region; and residues 298-594 represent the Guanylate-binding protein C-terminal domain.

To the best of the inventors knowledge, GBP4 has not been associated with the tumour endothelium, or with solid tumours. Indeed, to the best of the inventors' knowledge, neither inhibitors of the GBP4 gene, such as siRNA, antisense molecules or ribozymes specific for GBP4, nor inhibitors of the GBP4 polypeptide, such as antibodies that selectively bind to GBP4, have been associated with the inhibition of angiogenesis in the tumour endothelium, or with the treatment of solid tumours. Thus the invention includes a method of inhibiting tumour angiogenesis by administering an inhibitor of the gene/polypeptide GBP4, a method of combating a solid tumour by administering an inhibitor of the gene/polypeptide GBP4, and the use of an inhibitor of the gene/polypeptide GBP4 in the manufacture of a medicament for inhibiting tumour angiogenesis or for combating a solid tumour.

Moreover, the inventors' are not aware of any suggested therapeutic use of inhibitors of GBP4, whether antibodies, siRNA, antisense molecules or ribozymes. Thus the invention includes an inhibitor of the gene/polypeptide GBP4, an inhibitor of the gene/polypeptide GBP4 for use in medicine, and a pharmaceutical composition comprising an inhibitor of the gene/polypeptide GBP4 and a pharmaceutically acceptable carrier, diluent or excipient.

In another embodiment, the antibody that selectively binds GBP4 binds to the N-terminal domain (residues 48-289).

In other embodiments, the antibody that selectively binds GBP4 binds to the G1 box region, the G2 box region, the G3 box region, the G4 box region or the G5 box region. In still other embodiments, the antibody that selectively binds GBP4 binds to the GTP/Mg²⁺ binding site (residues 62-68, 82-84, 89-90, 115, 196-197 and 254).

In further embodiments, the antibody that selectively binds GBP4 binds to the Switch I region; or the Switch Il region.

In an alternative embodiment, the antibody that selectively binds GBP4 binds to the C- terminal domain (residues 298-594)

IKBKE

The gene IKBKE (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon) encodes the polypeptide IKBPE (also known as IKKE, IKKI, IKK-i, KIAA0151 , MGC125294, MGC125295 and MGC125297).

By the IKBKE polypeptide we include the meaning of a gene product of the human IKBKE gene, including naturally occurring variants thereof. A cDNA sequence corresponding to a human IKBKE mRNA is found in Genbank Accession No NM_014002. Human IKBKE polypeptide includes the amino acid sequence found in Genbank Accession No NP_054721 , and naturally occurring variants thereof. The IKBKE polypeptide sequence from NP_054721 is shown in Figure 5 (SEQ ID No: 6).

According to NP_054721 , the human IKBKE gene encodes a 716 amino acid residue polypeptide in which residues 9-242 represent a serine/threonine protein kinase catalytic domain, residues 15-16, 18, 21 , 23, 36, 38, 86, 140, 144 and 156-158 form an ATP binding pocket, residues 93, 137, 173-175 and 177-178 form a substrate binding pocket, residues 133-141 and 144 form a catalytic loop, and residues 157-166 and 171-183 form an activation loop.

IKBKE preferentially phosphorylates ser36 rather than ser32 of l-kappa-B-alpha (NFKBIA). Whereas TNFA and IL1B enhance the kinase activity of IKBKA and IKBKB, they do not enhance IKKI kinase activity. (Shimada et al (1999) "IKK-i, a novel lipopolysaccharide-inducible kinase that is related to IkappaB kinases" Int. Immunol.

11(8): 1357-1362). Recombinant IKBKE directly phosphorylates only ser36 of NFKBIA

(Peters et al (2000) "IKKepsilon is part of a novel PMA-inducible IkappaB kinase complex" MoI. Cell 5(3): 513-522). IKBKE has a role in the pathway triggering an antiviral response to viral infection (Sharma et al (2003) "Triggering the interferon antiviral response through an IKK-related pathway" Science 300: 1148-1151). Mice lacking IKBKE produce normal amounts of interferon-beta but are hypersusceptible to viral infection because of a defect in the IFN signaling pathway (TenOever et al (2007) "Multiple functions of the IKK-related kinase IKK-epsilon in interferon-mediated antiviral immunity." Science 315: 1274-1278.

To the best of the inventors' knowledge, IKBKE has not been associated with the tumour endothelium, or with solid tumours. Indeed, to the best of the inventors' knowledge, neither inhibitors of the IKBKE gene, such as siRNA, antisense molecules or ribozymes specific for IKBKE, nor inhibitors of the IKBKE polypeptide, such as antibodies that selectively bind to IKBKE, have been associated with the inhibition of angiogenesis in the tumour endothelium, or with the treatment of solid tumours. Thus the invention includes a method of inhibiting tumour angiogenesis by administering an inhibitor of the gene/polypeptide IKBKE, a method of combating a solid tumour by administering an inhibitor of the gene/polypeptide IKBKE, and the use of an inhibitor of the gene/polypeptide IKBKE in the manufacture of a medicament for inhibiting tumour angiogenesis or for combating a solid tumour.

Moreover, the inventors are not aware of any suggested therapeutic use of inhibitors of IKBKE, whether antibodies, siRNA, antisense molecules or ribozymes. Thus the invention includes an inhibitor of the gene/polypeptide IKBKE, an inhibitor of the gene/polypeptide IKBKE for use in medicine, and a pharmaceutical composition comprising an inhibitor of the gene/polypeptide IKBKE and a pharmaceutically acceptable carrier, diluent or excipient.

In an embodiment, the antibody that selectively binds IKBKE binds to the serine/threonine protein kinase catalytic domain (residues 9-242).

In more specific embodiments, the antibody that selectively binds IKBKE binds to the ATP binding pocket (residues 15-16, 18, 21, 23, 36, 38, 86, 140, 144 and 156-158), the substrate binding pocket (residues 93, 137, 173-175 and 177-178), the catalytic loop (residues 133-141 and 144), or the activation loop (residues 157-166 and 171-183).

RHBDL6 The gene RHBDL6 (rhomboid-like protein 6) encodes at least two isoforms of the polypeptide RHBDL6 (also known as rhomboid 5 homolog 2 (RHBDF2), rhomboid, veinlet-like 6, rhomboid veinlet-like 5 (RHBDL5) and FLJ22341).

Rhomboid-like protein-6 (RHBDL6) is a 7-transmembrane domain containing protein that may regulate the function of other rhomboid proteases. In Drosophila, rhomboid (also known as veinlet) regulates epidermal growth factor signalling and vessel formation in the fly wing. Substrates for rhomboids in vertebrates remain largely unknown (Urban, 2006). RHBDL6 is also discussed in the following references: Suzuki et al (2004) "Sequence comparison of human and mouse genes reveals a homologous block structure in the promoter regions" Genome Res. 14(9): 1711-1718; Puente & Lopez-Otin (2004) "A genomic analysis of rat proteases and protease inhibitors" Genome Res. 14(4): 609-622; and Puente et al (2003) "Human and mouse proteases: a comparative genomic approach" Nat. Rev. Genet. 4(7): 544-558.

By the RHBDL6 polypeptide we include the meaning of a gene product of the human RHBDL6 gene, including naturally occurring variants thereof. Two alternative transcript variants encoding different protein isoforms have been described for the human RHBDL6 gene: RHBDL6 isoform 1 is encoded by transcript variant 1 and RHBDL6 isoform 2 is encoded by transcript variant 2. A cDNA sequence corresponding to human RHBDL6 transcript variant 1 is found in Genbank Accession No NM_024599. Human RHBDL6 isoform 1 polypeptide includes the amino acid sequence found in Genbank Accession No NP_078875, and naturally occurring variants thereof. A cDNA sequence corresponding to human RHBDL6 transcript variant 2 is found in Genbank Accession No NM_001005498. Human RHBDL6 isoform 2 polypeptide includes the amino acid sequence found in Genbank Accession No NP_001005498, and naturally occurring variants thereof. The RHBDL6 isoform 1 and isoform 2 polypeptide sequences from NP_078875 and NP_001005498 are shown in Figure 5 (SEQ ID Nos: 7 and 8, respectively).

According to NP_078875, RHBDL6 isoform 1 is an 856 residue polypeptide having a rhomboid region at residues 651-774. According to NP_001005498, RHBDL6 isoform 2 is an 827 residue polypeptide having a rhomboid region at residues 622-745.

To the best of the inventors' knowledge, RHBDL6, neither isoform 1 nor 2, has not been associated with the tumour endothelium, or with solid tumours. Indeed, to the best of the inventors' knowledge, neither inhibitors of the RHBDL6 gene (transcript variant 1 or 2), such as siRNA, antisense molecules or ribozymes specific for RHBDL6, nor inhibitors of the RHBDL6 polypeptide (isoform 1 or 2), such as antibodies that selectively bind to RHBDL6, have been associated with the inhibition of angiogenesis in the tumour endothelium, or with the treatment of solid tumours. Thus the invention includes a method of inhibiting tumour angiogenesis by administering an inhibitor of the gene/polypeptide RHBDL6 (transcript variant/isoform 1 or 2), a method of combating a solid tumour by administering an inhibitor of the gene/polypeptide RHBDL6, and the use of an inhibitor of the gene/polypeptide RHBDL6 in the manufacture of a medicament for inhibiting tumour angiogenesis or for combating a solid tumour.

Moreover, the inventors are not aware of any suggested therapeutic use of inhibitors of RHBDL6, neither transcript variant/isoform 1 nor 2, whether antibodies, siRNA, antisense molecules or ribozymes.

Thus the invention includes an inhibitor of the gene/polypeptide RHBDL6 (transcript variant/isoform 1 or 2), an inhibitor of the gene/polypeptide RHBDL6 for use in medicine, and a pharmaceutical composition comprising an inhibitor of the gene/polypeptide RHBDL6 and a pharmaceutically acceptable carrier, diluent or excipient.

In a preferred embodiment, the antibody that selectively binds RHBDL6 binds to one of the extracellular regions of RHBDL6. According to TMHMM2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0), the extracellular regions of RHBDL6 isoform 1 are at residues 1-653, 711-719, 769-771 and 827-856, and the extracellular regions of RHBDL6 isoform 2 are at residues 398-624, 682-690, 740-742 and 798-827.

In an embodiment, the antibody that selectively binds RHBDL6 binds to the rhomboid region (residues 651-774 of isoform 1 or residues 622-745 of isoform 2).

RHOJ

The gene RHOJ (Ras homologue gene family, member J) encodes the polypeptide RHOJ (also known as ARHJ, TC10-like protein (TCL)). RHOJ belongs to the Rho family of small GTP-binding proteins. Rho proteins regulate the dynamic assembly of cytoskeletal components for several physiologic processes, such as cell proliferation and motility and the establishment of cell polarity. They are also involved in pathophysiologic process, such as cell transformation and metastasis. According to Vignal et al (2000, "Characterization of TCL, a new GTPase of the Rho family related to TC10 and Cdc42." J. Biol. Chem. 275: 36457-36464), the deduced 214- amino acid human protein contains the canonical G1 , G2, and G3 boxes involved in nucleotide binding, as well as a 2-cysteine box, which is a substrate for geranylation and farnesylation.

By the RHOJ polypeptide we include the meaning of a gene product of the human RHOJ gene, including naturally occurring variants thereof. A cDNA sequence corresponding to a human RHOJ mRNA is found in Genbank Accession No NM_020663. Human RHOJ polypeptide includes the amino acid sequence found in Genbank Accession No NP_065714, and naturally occurring variants thereof. The RHOJ polypeptide sequence from NP_065714 is shown in Figure 5 (SEQ ID No: 9).

According to NP_065714, the human RHOJ gene encodes a 214 amino acid residue polypeptide in which residues 23, 53-54, 57-58, 60, 62, 70, 74-75, 77-79, 85 and 88 form a putative guanine nucleotide exchange factor interaction site; residues 28-35 form (part of) the G1 box region; residues 31-36, 75-76, 78, 134, 136 and 177-178 form a GTP/Mg²⁺ binding site; residues 52-58 are a Switch I region; residue 53 is (part of) the G2 box region; residues 54, 77, 85 and 87 form a putative guanine nucleotide dissociation inhibitor interaction site; residues 54-55, 79 and 85 form a putative GTPase- activating protein interaction site; residues 55-56, 85 and 88 form a putative effector interaction site; residues 75-78 are (part of) the G3 box region; residues 78-79, 85-88, 93-95 are a Switch Il region; residues 133-136 are (part of) the G4 box region; and residues 176-178 are (part of) the G5 box region.

Further details regarding RHOJ are provided below in Example 2.

To the best of the inventors' knowledge, RHOJ has not been associated with the tumour endothelium, or with solid tumours. Indeed, to the best of the inventors' knowledge, neither inhibitors of the RHOJ gene, such as siRNA, antisense molecules or ribozymes specific for RHOJ, nor inhibitors of the RHOJ polypeptide, such as antibodies that selectively bind to RHOJ, have been associated with the inhibition of angiogenesis in the tumour endothelium, or with the treatment of solid tumours. Thus the invention includes a method of inhibiting tumour angiogenesis by administering an inhibitor of the gene/polypeptide RHOJ, a method of combating a solid tumour by administering an inhibitor of the gene/polypeptide RHOJ, and the use of an inhibitor of the gene/polypeptide RHOJ in the manufacture of a medicament for inhibiting tumour angiogenesis or for combating a solid tumour.

Moreover, the inventors are not aware of any suggested therapeutic use of inhibitors of RHOJ, whether antibodies, siRNA, antisense molecules or ribozymes.

Thus the invention includes an inhibitor of the gene/polypeptide RHOJ, an inhibitor of the gene/polypeptide RHOJ for use in medicine, and a pharmaceutical composition comprising an inhibitor of the gene/polypeptide RHOJ and a pharmaceutically acceptable carrier, diluent or excipient.

In an embodiment, the antibody that selectively binds RHOJ binds to the guanine nucleotide exchange factor interaction site (residues 23, 53-54, 57-58, 60, 62, 70, 74-75, 77-79, 85 and 88) of RHOJ.

In other embodiments, the antibody that selectively binds RHOJ binds to the G1 box region, the G2 box region, the G3 box region, the G4 box region or the G5 box region of RHOJ.

In still another embodiment, the antibody that selectively binds RHOJ binds to the GTP/Mg²⁺ binding site (residues 31-36, 75-76, 78, 134, 136 and 177-178) of RHOJ.

In further embodiments, the antibody that selectively binds RHOJ binds to the Switch I region; or the Switch Il region of RHOJ.

In additional embodiments, the antibody that selectively binds RHOJ binds to the putative guanine nucleotide dissociation inhibitor interaction site (residues 54, 77, 85 and 87), the putative GTPase-activating protein interaction site (residues 54-55, 79 and 85), or the putative effector interaction site (residues 55-56, 85 and 88).

Formulations and routes of administration

It is appreciated that the inhibitor will typically be formulated for administration to an individual as a pharmaceutical composition, i.e. together with a pharmaceutically acceptable carrier, diluent or excipient. By "pharmaceutically acceptable" is included that the formulation is sterile and pyrogen free. Suitable pharmaceutical carriers, diluents and excipients are well known in the art of pharmacy. The carriers) must be "acceptable" in the sense of being compatible with the inhibitor and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free; however, other acceptable carriers may be used.

In an embodiment, the pharmaceutical compositions or formulations of the invention are for parenteral administration, more particularly for intravenous administration. In a preferred embodiment, the pharmaceutical composition is suitable for intravenous administration to a patient, for example by injection.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

In an alternative preferred embodiment, the pharmaceutical composition is suitable for topical administration to a patient.

Preferably, the formulation is a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the active ingredient.

The inhibitor may be administered orally or by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.

In human therapy, the inhibitor will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

For example, the inhibitor may be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The inhibitor may also be administered via intracavernosal injection.

Suitable tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycoliate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose

(HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The inhibitor can also be administered parenterally, for example, intravenously, intra- arterially, intraperitoneal^, intrathecal^, intraventricular^, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques.

They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. For oral and parenteral administration to human patients, the daily dosage level of an inhibitor will usually be from 1 to 1,000 mg per adult (i.e. from about 0.015 to 15 mg/kg), administered in single or divided doses.

Thus, for example, the tablets or capsules of the inhibitor may contain from 1 mg to 1 ,000 mg of active agent for administration singly or two or more at a time, as appropriate. The physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.

The inhibitor can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuiiser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1 ,1 ,1,2-tetrafluoroethane (HFA 134A3 or 1 ,1 ,1 ,2,3,3,3- heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuiiser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a antibody and a suitable powder base such as lactose or starch. Such formulations may be particularly useful for treating solid tumours of the lung.

Aerosol or dry powder formulations are preferably arranged so that each metered dose or "puff' contains at least 1 mg of the inhibitor for delivery to the patient. It will be appreciated that the overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.

Alternatively, the inhibitor can be administered in the form of a suppository or pessary, particularly for combating solid colorectal tumours or prostate tumours. The inhibitor may also be administered by the ocular route. For ophthalmic use, the inhibitor can be formulated as, e.g., micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum. Such formulations may be particularly useful for treating solid tumours of the eye, such as retinoblastoma.

The inhibitor may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder, or may be transdermal^ administered, for example, by the use of a skin patch. For application topically to the skin, the inhibitor can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Such formulations may be particularly useful for treating solid tumours of the skin.

For skin cancers, the inhibitors can also be delivered by electroincorporation (El). El occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In El, these particles are driven through the stratum corneum and into deeper layers of the skin. The particles can be loaded or coated with inhibitor or can simply act as "bullets" that generate pores in the skin through which the inhibitor can enter.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier. Such formulations may be particularly useful for treating solid tumours of the mouth and throat.

In an embodiment, when the inhibitor is a polypeptide, such as an antibody, it may be delivered using an injectable sustained-release drug delivery system. These are designed specifically to reduce the frequency of injections. An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.

The antibody can be administered by a surgically implanted device that releases the drug directly to the required site, for example, into the eye to treat ocular tumours. Such direct application to the site of disease achieves effective therapy without significant systemic side-effects.

An alternative method of polypeptide delivery is the ReGeI injectable system that is thermo-sensitive. Below body temperature, ReGeI is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active drug is delivered over time as the biopolymers dissolve.

Polypeptide pharmaceuticals such as antibodies can also be delivered orally. The process employs a natural process for oral uptake of vitamin B₁₂ in the body to co-deliver proteins and peptides. By riding the vitamin Bi₂ uptake system, the protein or peptide can move through the intestinal wall. Complexes are synthesised between vitamin Bi₂ analogues and the drug that retain both significant affinity for intrinsic factor (IF) in the vitamin Bi₂ portion of the complex and significant bioactivity of the drug portion of the complex.

Polynucleotides may be administered by any effective method, for example, parenterally (e.g. intravenously, subcutaneously, intramuscularly) or by oral, nasal or other means which permit the polynucleotides to access and circulate in the patient's bloodstream.

Polynucleotides administered systemically preferably are given in addition to locally administered polynucleotides, but also have utility in the absence of local administration. A dosage in the range of from about 0.1 to about 10 grams per administration to an adult human generally will be effective for this purpose.

The polynucleotide may be administered as a suitable genetic construct as is described below and delivered to the patient where it is expressed. Typically, the polynucleotide in the genetic construct is operatively linked to a promoter which can express the compound in the cell. The genetic constructs of the invention can be prepared using methods well known in the art, for example in Sambrook et al (2001). Although genetic constructs for delivery of polynucleotides can be DNA or RNA, it is preferred if they are DNA.

Preferably, the genetic construct is adapted for delivery to a human cell.

Means and methods of introducing a genetic construct into a cell in an animal body are known in the art. For example, the constructs of the invention may be introduced into cells by any convenient method, for example methods involving retroviruses, so that the construct is inserted into the genome of the cell. For example, in Kuriyama et al (1991, Cell Strυc. and Func. 16, 503-510) purified retroviruses are administered. Retroviral DNA constructs comprising a polynucleotide as described above may be made using methods well known in the art. To produce active retrovirus from such a construct it is usual to use an ecotropic psi2 packaging cell line grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal calf serum (FCS). Transfection of the cell line is conveniently by calcium phosphate co-precipitation, and stable transformants are selected by addition of G418 to a final concentration of 1 mg/ml (assuming the retroviral construct contains a neo^H gene). Independent colonies are isolated and expanded and the culture supernatant removed, filtered through a 0.45 μm pore-size filter and stored at -70⁰C. For the introduction of the retrovirus into tumour cells, for example, it is convenient to inject directly retroviral supernatant to which 10 μg/ml Polybrene has been added. For tumours exceeding 10 mm in diameter it is appropriate to inject between 0.1 ml and 1 ml of retroviral supernatant; preferably 0.5 ml.

Alternatively, as described in Culver et al (1992, Science 256, 1550-1552), cells which produce retroviruses may be injected. The retrovirus-producing cells so introduced are engineered to actively produce retroviral vector particles so that continuous productions of the vector occurred within the tumour mass in situ.

Targeted retroviruses are also available for use in the invention; for example, sequences conferring specific binding affinities may be engineered into pre-existing viral env genes (see Miller & Vile (1995) Faseb J. 9, 190-199, for a review of this and other targeted vectors for gene therapy).

Other methods involve simple delivery of the construct into the cell for expression therein either for a limited time or, following integration into the genome, for a longer time. An example of the latter approach includes liposomes (Nassander et al (1992) Cancer Res. 52, 646-653).

Other methods of delivery include adenoviruses carrying external DNA via an antibody- polylysine bridge (see Curiel (1993) Prog. Med. Virol. 40, 1-18) and transferrin-polycation conjugates as carriers (Wagner et al (1990) Proc. Natl. Acad. ScL USA 87, 3410-3414). In the first of these methods a polycation-antibody complex is formed with the DNA construct or other genetic construct of the invention, wherein the antibody is specific for either wild-type adenovirus or a variant adenovirus in which a new epitope has been introduced which binds the antibody. The polycation moiety binds the DNA via electrostatic interactions with the phosphate backbone. The adenovirus, because it contains unaltered fibre and penton proteins, is internalised into the cell and carries into the cell with it the DNA construct of the invention. It is preferred if the polycation is poiylysine.

In an alternative method, a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecuies into cells is employed. This is accomplished by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, or the chicken homologue conalbumin, or combinations thereof is covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulphide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with DNA constructs or other genetic constructs of the invention independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs). When complexes of transferrin-polycation and the DNA constructs or other genetic constructs of the invention are supplied to the tumour cells, a high level of expression from the construct in the cells is expected.

High-efficiency receptor-mediated delivery of the DNA constructs or other genetic constructs of the invention using the endosome-disruption activity of defective or chemically inactivated adenovirus particles produced by the methods of Cotten et al (1992) Proc. Natl. Acad. Sci. USA 89, 6094-6098 may also be used. This approach appears to rely on the fact that adenoviruses are adapted to allow release of their DNA from an endosome without passage through the lysosome, and in the presence of, for example transferrin linked to the DNA construct or other genetic construct of the invention, the construct is taken up by the cell by the same route as the adenovirus particle. This approach has the advantages that there is no need to use complex retroviral constructs; there is no permanent modification of the genome as occurs with retroviral infection; and the targeted expression system is coupled with a targeted delivery system, thus reducing toxicity to other cell types.

It will be appreciated that "naked DNA" and DNA complexed with cationic and neutral lipids may also be useful in introducing the DNA of the invention into cells of the individual to be treated. Non-viral approaches to gene therapy are described in Ledley (1995, Human Gene Therapy 6, 1129-1144).

Although for solid tumours of specific tissues it may be useful to use tissue-specific promoters in the vectors encoding a polynucleotide inhibitor, this is not essential. This is because the targeted genes are only expressed, or selectively expressed, in the tumour endothelium. Accordingly, expression of gene-specific inhibitors such as siRNA, antisense molecules and ribozymes in the body at locations other than the solid tumour would be expected to have no effect since the genes that they are designed to inhibit are not expressed. Moreover, the risk of inappropriate expression of these inhibitors, in a cell that may express the target polypeptide at a low level, is miniscule compared to the therapeutic benefit to a patient suffering from a solid tumour.

Targeted delivery systems are also known, such as the modified adenovirus system described in WO 94/10323, wherein, typically, the DNA is carried within the adenovirus, or adenovirus-like, particle. Michael et al (1995) Gene Therapy 2, 660-668 describes modification of adenovirus to add a cell-selective moiety into a fibre protein. Mutant adenoviruses which replicate selectively in p53-deficient human tumour cells, such as those described in Bischoff et al (1996) Science 27 '4, 373-376 are also useful for delivering the genetic construct of the invention to a cell. Thus, it will be appreciated that a further aspect of the invention provides a virus or virus-like particle comprising a genetic construct of the invention. Other suitable viruses, viral vectors or virus-like particles include lentivirus and lentiviral vectors, HSV, adeno-assisted virus (AAV) and AAV-based vectors, vaccinia and parvovirus.

Methods of delivering polynucleotides to a patient are well known to a person of skill in the art and include the use of immunoliposomes, viral vectors (including vaccinia, modified vaccinia, adenovirus and adeno-associated viral (AAV) vectors), and by direct delivery of DNA₁ e.g. using a gene-gun and electroporation. Furthermore, methods of delivering polynucleotides to a target tissue of a patient for treatment are also well known in the art.

Methods of targeting and delivering therapeutic agents directly to specific regions of the body, including the brain, are well known to a person of skill in the art. For example, US Patent No 6,503,242 describes an implanted catheter apparatus for delivering therapeutic agents directly to the hippocampus. Methods of targeting and delivering agents to the brain can be used for the treatment of solid tumours of the brain, such as astrocytoma, ganglioma, metastatic adenocarcinoma, glioblastoma and medulioblastoma. In one embodiment, therapeutic agents including vectors can be distributed throughout a wide region of the CNS by injection into the cerebrospinal fluid, e.g., by lumbar puncture (See e.g., Kapadia et al (1996) Neurosurg 10: 585-587). Alternatively, precise delivery of the therapeutic agent into specific sites of the brain can be conducted using stereotactic microinjection techniques. For example, the subject being treated can be placed within a stereotactic frame base (MRI-compatible) and then imaged using high resolution MRI to determine the three-dimensional positioning of the particular region to be treated. The MRI images can then be transferred to a computer having the appropriate stereotactic software, and a number of images are used to determine a target site and trajectory for microinjection of the therapeutic agent. The software translates the trajectory into three-dimensional coordinates that are precisely registered for the stereotactic frame. In the case of intracranial delivery, the skull will be exposed, burr holes will be drilled above the entry site, and the stereotactic apparatus used to position the needle and ensure implantation at a predetermined depth. The therapeutic agent can be delivered to regions of the CNS such as the hippocampus, cells of the spinal cord, brainstem, (medulla, pons, and midbrain), cerebellum, diencephalon (thalamus, hypothalamus), telencephalon (corpus stratium, cerebral cortex, or within the cortex, the occipital, temporal, parietal or frontal lobes), or combinations, thereof. In another embodiment, the therapeutic agent is delivered using other delivery methods suitable for localised delivery, such as localised permeation of the blood-brain barrier. US Patent Application No 2005/0025746 describes delivery systems for localised delivery of an adeno-associated virus vector (AAV) vector encoding a therapeutic agent to a specific region of the brain.

When a therapeutic agent for the treatment of a solid tumour of, for example, the brain, is enocoded by a polynucleotide, it may be preferable for its expression to be under the control of a suitable tissue-specific promoter. Central nervous system (CNS) specific promoters such as, neuron-specific promoters (e.g., the neurofilament promoter (Byrne and Ruddle, 1989) and glial specific promoters (Morii et al, 1991) are preferably used for directing expression of a polynucleotide preferentially in cells of the CNS. Preferably, the promoter is tissue specific and is essentially not active outside the central nervous system, or the activity of the promoter is higher in the central nervous system than in other cells or tissues. For example, the promoter may be specific for the spinal cord, brainstem, (medulla, pons, and midbrain), cerebellum, diencephalon (thalamus, hypothalamus), telencephalon (corpus stratium, cerebral cortex, or within the cortex, the occipital, temporal, parietal or frontal lobes), or combinations, thereof. The promoter may be specific for particular cell types, such as neurons or glial cells in the CNS. If it is active in glial cells, it may be specific for astrocytes, oiigodentrocytes, ependymal cells, Schwann cells, or microglia. If it is active in neurons, it may be specific for particular types of neurons, e.g., motor neurons, sensory neurons, or intemeurons. The promoter may be specific for cells in particular regions of the brain, for example, the cortex, stratium, nigra and hippocampus.

Suitable neuronal specific promoters include, but are not limited to, neuron specific enolase (NSE; Olivia et al (1991); GenBank Accession No: X51956), and human neurofilament light chain promoter (NEFL; Rogaev et al (1992); GenBank Accession No: L04147). Glial specific promoters include, but are not limited to, glial fibrillary acidic protein (GFAP) promoter (Morii et al (1991); GenBank Accession No:M65210), S100 promoter (Morii et al (1991); GenBank Accession No: M65210) and glutamine synthase promoter (Van den et al (1991); GenBank Accession No: X59834). In a preferred embodiment, the gene is flanked upstream (i.e., 5¹) by the neuron specific enolase (NSE) promoter. In another preferred embodiment, the gene of interest is flanked upstream (i.e., 5') by the elongation factor 1 alpha (EF) promoter. A hippocampus specific promoter that might be used is the hippocampus specific glucocorticoid receptor (GR) gene promoter.

Alternatively, for treatment of solid tumours of the heart, Svensson et al (1999) describes the delivery of recombinant genes to cardiomyocytes by intramyocardial injection or intracoronary infusion of cardiotropic vectors, such as recombinant adeno-associated virus vectors, resulting in transgene expression in murine cardiomyocytes in vivo. MeIo et al (2004) review gene and cell-based therapies for heart disease. An alternative preferred route of administration is via a catheter or stent. Stents represent an attractive alternative for localized gene delivery, as they provide a platform for prolonged gene elution and efficient transduction of opposed arterial walls. This gene delivery strategy has the potential to decrease the systemic spread of the viral vectors and hence a reduced host immune response. Both synthetic and naturally occurring stent coatings have shown potential to allow prolonged gene elution with no significant adverse reaction. (Sharif et al, 2004).

It may be desirable to be able to temporally regulate expression of the polynucleotide inhibitor in the cell, although this is not essential for the reasons given above. Thus, it may be desirable that expression of the polynucleotide is directly or indirectly (see below) under the control of a promoter that may be regulated, for example by the concentration of a small molecule that may be administered to the patient when it is desired to activate or, more likely, repress (depending upon whether the small molecule effects activation or repression of the said promoter) expression of the antibody from the polynucleotide. This may be of particular benefit if the expression construct is stable, i.e., capable of expressing the antibody (in the presence of any necessary regulatory molecules), in the cell for a period of at least one week, one, two, three, four, five, six, eight months or one or more years. Thus the polynucleotide may be operatively linked to a regulatable promoter. Examples of regulatable promoters include those referred to in the following papers: Rivera et al (1999) Proc Natl Acad Sci USA 96(15), 8657-62 (control by rapamycin, an orally bioavailable drug, using two separate adenovirus or adeno- associated virus (AAV) vectors, one encoding an inducible human growth hormone (hGH) target gene, and the other a bipartite rapamycin-regulated transcription factor); Magari et al (1997) J CHn Invest 100(11), 2865-72 (control by rapamycin); Bueler (1999) Biol Chem 380(6), 613-22 (review of adeno-associated viral vectors); Bohl et al (1998) Blood 92(5), 1512-7 (control by doxycycline in adeno-associated vector); Abruzzese et al (1996) J MoI Med 74(7), 379-92 (review of induction factors, e.g.. hormones, growth factors, cytokines, cytostatics, irradiation, heat shock and associated responsive elements).

For veterinary use, the inhibitor is typically administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.

Combination therapy According to a National Cancer Institute Press Release dated 14 April 2005, updated 16 June 2005, ("Bevacizumab Combined With Chemotherapy Improves Progression-Free Survival for Patients With Advanced Breast Cancer"), the angiogenesis inhibitor anti- VEGF monoclonal antibody Bevacizumab improves the clinical outcome for a number of solid tumours when administered in combination with standard chemotherapy. Combinations that have been used include bevacizumab in combination with irinotecan, fluorouracil, and leucovorin; bevacizumab in combination with FOLFOX4 (a regimen of oxaliplatin, 5-fluorouracil and leucovorin); bevacizumab in combination with paciitaxel; and bevacizumab in combination with paciitaxel and carbopiatin.

It is therefore appreciated that although the inhibitors of the genes/polypeptides selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, may be clinically effective in the absence of any other anti-cancer compound, it may be advantageous to administer these inhibitors in conjunction with a further anticancer agent.

Accordingly, a seventh aspect of the invention provides a pharmaceutical composition comprising: (i) an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, and (ii) at least one further anticancer agent, and a pharmaceutically acceptable carrier, diluent or excipient.

The further anticancer agent may be selected from alkylating agents including nitrogen mustards such as mechlorethamine (HN₂), cyclophosphamide, ifosfamide, melphalan (L- sarcolysin) and chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulphonates such as busulphan; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazole- carboxamide); antimetabolites including folic acid analogues such as methotrexate (amethopterin); pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); and purine analogues and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6- thioguanine; TG) and pentostatin (2'-deoxycoformycin); natural products including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D)₁ daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C); enzymes such as L-asparaginase; and biological response modifiers such as interferon alphenomes; miscellaneous agents including platinum coordination complexes such as cisplatin (c/s-DDP) and carboplatin; anthracenedione such as mitoxantrone and anthracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH); and adrenocortical suppressant such as mitotane (o,p -DDD) and aminoglutethimide; taxol and analogues/derivatives; cell cycle inhibitors; proteosome inhibitors such as Bortezomib (Velcade^®); signal transductase (e.g. tyrosine kinase) inhibitors such as lmatinib (Glivec^®), COX-2 inhibitors, and hormone agonists/antagonists such as flutamide and tamoxifen.

The clinically used anticancer agents are typically grouped by mechanism of action: Alkylating agents, Topoisomerase I inhibitors, Topoisomerase Il inhibitors, RNA/DNA antimetabolites, DNA antimetabolites and Antimitotic agents. The US NIH/National Cancer Institute website lists 122 compounds

(http://dtp.nci.nih.gov/docs/cancer/searches/standard_mechanism.html), all of which may be used in conjunction with an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ. They include Alkylating agents including Asaley, AZQ, BCNU, Busulfan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, c/s-platinum, clomesone, cyanomorpholino-doxorubicin, cyclodisone, dianhydrogalactitol, fluorodopan, hepsurfam, hycanthone, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, teroxirone, tetraplatin, thio-tepa, triethylenemelamine, uracil nitrogen mustard, Yoshi-864; Anitmitotic agents including allocolchicine, Halichondrin B, colchicine, colchicine derivative, doiastatin 10, maytansine, rhizoxin, taxol, taxol derivative, thiocolchicine, trityl cysteine, vinblastine sulfate, vincristine sulfate; Topoisomerase I Inhibitors including camptothecin, camptothecin, Na salt, aminocamptothecin, 20 camptothecin derivatives, morpholinodoxorubicin; Topoisomerase Il Inhibitors including doxorubicin, amonafide, m- AMSA, anthrapyrazole derivative, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, mitoxantrone, menogaril, N,N-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26, VP-16; RNA/DNA antimetabolites including L-alanosine, 5- azacytidine, 5-fluorouracil, acivicin, 3 aminopterin derivatives, an antifol, Baker's soluble antifol, dichlorallyl lawsone, brequinar, ftorafur (pro-drug), 5,6-dihydro-5-azacytidine, methotrexate, methotrexate derivative, N-(phosphonoacetyl)-L-aspartate (PALA), pyrazofurin, trimetrexate; DNA antimetabolites including, 3-HP, 2'-deoxy-5-fluorouridine, 5-HP₁ alpha-TGDR, aphidicolin glycinate, ara-C, 5-aza-2'-deoxycytidine, beta-TGDR, cyclocytidine, guanazole, hydroxyurea, inosine glycodialdehyde, macbecin II, pyrazoloimidazole, thioguanine, thiopurine. It is preferred if the further anticancer agent is selected from cisplatin, carboplatin, 5- flurouracil, paclitaxel, mitomycin C, doxorubicin, gemcitabine, tomudex, pemetrexed, methotrexate, irinotecan, fluorouracil and leucovorin; oxaliplatin, 5-fluorouracil and leucovorin; and paclitaxel and carboplatin.

An eighth aspect of the invention provides (i) an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, and (ii) at least one further anticancer agent as defined above in the seventh aspect of the invention, for use in medicine.

A ninth aspect of the invention provides a method of combating a solid tumour in an individual, the method comprising administering to the patient (i) an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, in combination with (ii) at least one further anticancer agent as defined above in the seventh aspect of the invention.

Typically, the method comprises administering to the individual a pharmaceutical composition as defined above in the seventh aspect of the invention. However, it is appreciated that the inhibitor of the gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, and the further anticancer agent, may be administered separately, for instance by separate routes of administration. Thus it is appreciated that the inhibitor of the gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ and the at least one further anticancer agent can be administered sequentially or (substantially) simultaneously. They may be administered within the same pharmaceutical formulation or medicament or they may be formulated and administered separately.

This aspect of the invention includes the use of (i) an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, and (ii) at least one further anticancer agent as defined above in the seventh aspect of the invention, in the preparation of a medicament for combating a solid tumour in an individual.

The invention also includes the use of an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, in the preparation of a medicament for combating a solid tumour in an individual who is administered at least one further anticancer agent as defined above in the seventh aspect of the invention. Typically the individual is administered the further anticancer agent at the same time as the medicament, although the individual may have been (or will be) administered the further anticancer agent before (or after) receiving the medicament containing the inhibitor.

The invention further includes the use of at least one further anticancer agent as defined above in the seventh aspect of the invention in the preparation of a medicament for combating a solid tumour in an individual who is administered an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ. Typically the individual is administered the inhibitor at the same time as the medicament, although the patient may have been (or will be) administered the inhibitor before (or after) receiving the medicament containing the further anticancer agent.

The invention also includes (i) an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, and (H) at least one further anticancer agent as defined above in the seventh aspect of the invention, for use in combating a solid tumour in an individual.

Suitable inhibitors of the above-listed genes/polypeptides for the seventh, eighth and ninth aspects of the invention include antibodies that selectively bind to the polypeptides, and siRNA, antisense polynucleotides and ribozyme molecules that are specific for the polynucleotides encoding these polypeptides, as discussed in detail above.

General preferences for the solid tumour and for the individual patient to be treated, and for the further anticancer agent, are as described above. However, when the further anticancer agent has been shown to be particularly effective for a specific tumour type, it is preferred if the inhibitor is used in combination with that further anticancer agent to treat that specific tumour type.

Tumour imaging, detection and diagnosis

In a further embodiment, the antibodies that selectively bind a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ may useful in imaging, for example vascular imaging of tumours. Typically, in this embodiment, the antibody is attached to a detectable moiety. Methods and compounds useful in vascular imaging of tumours are described in our earlier publication WO 02/36771 , incorporated herein by reference.

A tenth aspect of the invention provides compound comprising an antibody that selectively binds a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, and a detectable moiety.

A compound comprising an antibody as defined above and a detectable moiety can be used, in combination with an appropriate detection method, to detect the location of the compound in the individual, and hence to identify the sites and extent of tumour angiogenesis in the individual, as well as inhibiting the angiogenesis in the individual.

By a "detectable moiety" we include the meaning that the moiety is one which, when located at the target site following administration of the compound of the invention into a patient, may be detected, typically non-invasively from outside the body, and the site of the target located. Thus, the compounds of this aspect of the invention are useful in imaging and diagnosis, especially in the imaging and diagnosis of neovasculature of solid tumours.

Typically, the readily detectable moiety is or comprises a radioactive atom which is useful in imaging. Suitable radioactive atoms include technetium-99m or iodine-123 for scintigraphic studies. Other readily detectable moieties include, for example, spin labels for magnetic resonance imaging (MRI) such as iodine-123 again, iodine-131 , indium-111 , fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Clearly, the compound of the invention must have sufficient of the appropriate atomic isotopes in order for the molecule to be detectable.

The radio- or other label may be incorporated in the compound in known ways. For example, if the antibody may be biosynthesised or synthesised by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as ^99mTc, ¹²³1, ¹⁸⁶Rh₁ ¹⁸⁸Rh and ¹¹¹In can, for example, be attached via cysteine residues in the antibody. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Comm. 80, 49-57) can be used to incorporate iodine-123. The reference ("Monoclonal Antibodies in

Immunoscintigraphy", J. F. Chatal, CRC Press, 1989) describes other methods in detail. The invention further includes a pharmaceutical composition comprising a compound according to this aspect of the invention and a pharmaceutically acceptable carrier, diluent or excipient. Preferences for the pharmaceutical composition are as described above.

An eleventh aspect of the invention provides a method of imaging tumour neovasculature in an individual, the method comprising: administering to the individual a compound comprising an antibody that selectively binds a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, and a detectable moiety, and detecting or imaging the location of the detectable moiety in the body.

Preferences for the antibody, the compound and the detectable moiety are as described above.

Typically, the individual has a solid tumour, and the neovasculature of the solid tumour is imaged. This method may be useful, for example, in determining the size of a previously diagnosed solid tumour, the effectiveness of a therapy against the solid tumour, or the extent of metastasis of the tumour. Methods for imaging a detectable moiety in the body are well known in the art, and include PET (positron emission tomography).

A twelfth aspect of the invention provides a method of detecting, diagnosing or prognosing a solid tumour in an individual, the method comprising: administering to the individual a compound comprising an antibody that selectively binds a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, and a detectable moiety, and detecting the presence and/or location of the detectable moiety in the body.

Preferences for the antibody, the compound and the detectable moiety are as described above. Thus, the localisation of the antibody at a particular organ in the body indicates that the individual may have or may be developing a solid tumour at that organ.

Targeted delivery of cytotoxic agents One avenue towards the development of more selective, better anticancer drugs is the targeted delivery of bioactive molecules to the tumour environment by means of binding molecules (for example, human antibodies) that are specific for tumour-endothelial markers. Due to their accessibility and to the therapeutic options that they allow (for example, intraluminal blood coagulation or recruitment of immune cells), vascular markers selectively expressed on tumour blood vessels seem to be ideally suited for ligand-based tumour-targeting strategies, opening new possibilities for the imaging and the therapy of cancer.

Accordingly, a thirteenth aspect of the invention provides a compound comprising an antibody that selectively binds a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, and a cytotoxic moiety.

Typically the cytotoxic moiety is selected from a directly cytotoxic chemotherapeutic agent, a directly cytotoxic polypeptide, a moiety which is able to convert a prodrug into a cytotoxic drug, a radiosensitizer, a directly cytotoxic nucleic acid, a nucleic acid molecule that encodes a directly or indirectly cytotoxic polypeptide or a radioactive atom. Examples of such cytotoxic moieties, as well as methods of making the conjugates comprising the antibody and the cytotoxic moiety, are provided in our earlier publications WO 02/36771 and WO 2004/046191 , incorporated herein by reference.

The cytotoxic moiety may be directly or indirectly toxic to cells in neovasculature or cells which are in close proximity to and associated with neovasculature. By "directly cytotoxic" we include the meaning that the moiety is one which on its own is cytotoxic. By "indirectly cytotoxic" we include the meaning that the moiety is one which, although is not itself cytotoxic, can induce cytotoxicity, for example by its action on a further molecule or by further action on it.

In one embodiment the cytotoxic moiety is a cytotoxic chemotherapeutic agent. Cytotoxic chemotherapeutic agents are well known in the art. Cytotoxic chemotherapeutic agents, such as anticancer agents, include those listed above with respect to the seventh aspect of the invention.

Various of these cytotoxic moieties, such as cytotoxic chemotherapeutic agents, have previously been attached to antibodies and other targeting agents, and so compounds of the invention comprising these agents may readily be made by the person skilled in the art. For example, carbodiimide conjugation (Bauminger & Wilchek (1980) Methods Enzymol. 70, 151-159) may be used to conjugate a variety of agents, including doxorubicin, to antibodies. Other methods for conjugating a cytotoxic moiety to an antibody can also be used. For example, sodium periodate oxidation followed by reductive alkylation of appropriate reactants can be used, as can glutaraldehyde cross- linking. Methods of cross-linking polypeptides are known in the art and described in WO 2004/046191. However, it is recognised that, regardless of which method of producing a compound of the invention is selected, a determination must be made that the antibody maintains its targeting ability and that the attached moiety maintains its relevant function.

In a further embodiment of the invention, the cytotoxic moiety may be a cytotoxic peptide or polypeptide moiety by which we include any moiety which leads to cell death.

Cytotoxic peptide and polypeptide moieties are well known in the art and include, for example, ricin, abrin, Pseudomonas exotoxin, tissue factor and the like. Methods for linking them to targeting moieties such as antibodies are also known in the art. The use of ricin as a cytotoxic agent is described in Burrows & Thorpe (1993) Proc. Natl. Acad. ScL USA 90, 8996-9000, and the use of tissue factor, which leads to localised blood clotting and infarction of a tumour, has been described by Ran et al (1998) Cancer Res.

58, 4646-4653 and Huang et al (1997) Science 275, 547-550. Tsai et al (1995) Dis.

Colon Rectum 38, 1067-1074 describes the abrin A chain conjugated to a monoclonal antibody. Other ribosome inactivating proteins are described as cytotoxic agents in WO 96/06641. Pseudomonas exotoxin may also be used as the cytotoxic polypeptide moiety

(see, for example, Aiello et a/ (1995) Proc. Natl. Acad. Sci. USA 92, 10457-10461).

Certain cytokines, such as TN Fa and IL-2, may also be useful as cytotoxic agents.

Certain radioactive atoms may also be cytotoxic if delivered in sufficient doses. Thus, the cytotoxic moiety may comprise a radioactive atom which, in use, delivers a sufficient quantity of radioactivity to the target site so as to be cytotoxic. Suitable radioactive atoms include phosphorus-32, iodine-125, iodine-131 , indium-111 , rhenium-186, rhenium-188 or yttrium-90, or any other isotope which emits enough energy to destroy neighbouring cells, organelles or nucleic acid. Preferably, the isotopes and density of radioactive atoms in the compound of the invention are such that a dose of more than 4000 cGy (preferably at least 6000, 8000 or 10000 cGy) is delivered to the target site and, preferably, to the cells at the target site and their organelles, particularly the nucleus. The radioactive atom may be attached to the antibody in known ways. For example EDTA or another chelating agent may be attached to the antibody and used to attach ¹¹¹In or ⁹⁰Y. Tyrosine residues may be labelled with ¹²⁵I or ¹³¹I.

The cytotoxic moiety may be a radiosensitizer. Radiosensitizers include fluoropyrimidines, thymidine analogues, hydroxyurea, gemcitabine, fludarabine, nicotinamide, halogenated pyrimidines, 3-aminobenzamide, 3-aminobenzodiamide, etanixadole, pimonidazoie and misonidazole (see, for example, McGinn et al (1996) J. Natl. Cancer Inst. 88, 1193-11203; Shewach & Lawrence (1996) Invest. New Drugs 14, 257-263; Horsman (1995) Acta Oncol. 34, 571-587; Shenoy & Singh (1992) CHn. Invest. 10, 533-551 ; Mitchell et al (1989) Int. J. Radial Biol. 56, 827-836; lliakis & Kurtzman (1989) Int. J. Radial Oncol. Biol. Phys. 16, 1235-1241 ; Brown (1989) Int. J. Radial Oncol. Biol. Phys. 16, 987-993; Brown (1985) Cancer 55, 2222-2228).

The cytotoxic moiety may be an indirectly cytotoxic polypeptide. In a particularly preferred embodiment, the indirectly cytotoxic polypeptide is a polypeptide which has enzymatic activity and can convert a relatively non-toxic prodrug into a cytotoxic drug. When the targeting moiety is an antibody this type of system is often referred to as ADEPT (Antibody-Directed Enzyme Prodrug Therapy). The system requires that the targeting moiety locates the enzymatic portion to the desired site in the body of the patient (e.g. the site of new vascular tissue associated with a tumour) and after allowing time for the enzyme to localise at the site, administering a prodrug which is a substrate for the enzyme, the end product of the catalysis being a cytotoxic compound. The object of the approach is to maximise the concentration of drug at the desired site and to minimise the concentration of drug in normal tissues (Senter et al (1988) "Anti-tumor effects of antibody-alkaline phosphatase conjugates in combination with etoposide phosphate" Proc. Natl. Acad. ScL USA 85, 4842-4846; Bagshawe (1987) Br. J. Cancer 56, 531-2; and Bagshawe, et al (1988) "A cytotoxic agent can be generated selectively at cancer sites" Br. J. Cancer. 58, 700-703.) Bagshawe (1995) Drug Dev. Res. 34, 220-230 and WO 2004/046191 , both of which are incorporated herein by reference, describe various enzyme/prod rug combinations which may be suitable in the context of this invention.

Typically, the prodrug is relatively non-toxic compared to the cytotoxic drug. Typically, it has less than 10% of the toxicity, preferably less than 1 % of the toxicity as measured in a suitable in vitro cytotoxicity test. It is likely that the moiety which is able to convert a prodrug to a cytotoxic drug will be active in isolation from the rest of the compound but it is necessary only for it to be active when (a) it is in combination with the rest of the compound and (b) the compound is attached to, adjacent to or internalised in target cells.

The further moiety may be one which becomes cytotoxic, or releases a cytotoxic moiety, upon irradiation. For example, the boron-10 isotope, when appropriately irradiated, releases α particles which are cytotoxic (see for example, US 4,348,376 to Goldenberg; Primus et al (1996) Bioconjug. Chem. 7, 532-535).

Similarly, the cytotoxic moiety may be one which is useful in photodynamic therapy such as photofrin (see, for example, Dougherty et a/ (1998) J. Natl. Cancer Inst. 90, 889-905).

The invention further includes a compound according to the thirteenth aspect of the invention for use in medicine. The invention also includes a pharmaceutical composition comprising a compound according to the thirteenth aspect of the invention and a pharmaceutically acceptable carrier, diluent or excipient. Preferences for the formulation of pharmaceutical compositions are as described above.

It is appreciate that the compounds according to the thirteenth aspect of the invention can be used to inhibit tumour angiogenesis in an individual and to treat a solid tumour as discussed above with respect to the first and second aspects of the invention.

Thus, a fourteenth aspect of the invention provides a method of inhibiting tumour angiogenesis in an individual, the method comprising administering to the individual a compound according to the thirteenth aspect of the invention.

A fifteenth aspect of the invention provides method of combating a solid tumour in an individual, the method comprising administering to the individual a compound according to the thirteenth aspect of the invention.

The invention includes the use of a compound according to the thirteenth aspect of the invention in the preparation of a medicament for inhibiting tumour angiogenesis or for combating a solid tumour in an individual in an individual. For the fourteenth and fifteenth aspects of the invention, preferences for the compound, the cytotoxic moiety, the individual to be treated, the types of solid tumour, the routes of administration, and so on are as defined above.

Screening

A sixteenth aspect of the invention provides a method of identifying an agent that may be useful in the treatment of a solid tumour, or a lead compound for the identification of an agent that may be useful in the treatment of a solid tumour, the method comprising: providing a candidate compound that binds a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, or a fragment thereof; and testing the candidate compound in an angiogenesis assay, wherein a candidate compound that inhibits angiogenesis in the assay may be an agent that is useful in the treatment of a solid tumour, or may be a lead compound for the identification of an agent that is useful in the treatment of a solid tumour.

A seventeenth aspect of the invention provides a method of identifying an agent that may be useful in the treatment of a solid tumour, or a lead compound for the identification of an agent that may be useful in the treatment of a solid tumour, the method comprising: providing a candidate compound; determining whether the candidate compound selectively binds to a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and

RHOJ, or a fragment thereof; and testing a candidate compound that selectively binds to the polypeptide or the fragment in an angiogenesis assay, wherein a candidate compound that selectively binds to the said polypeptide or fragment and which inhibits angiogenesis in the assay may be an agent that is useful in the treatment of a solid tumour, or may be a lead compound for the identification of an agent that is useful in the treatment of a solid tumour.

It is appreciated that these methods can be used to identify an anti-angiogenic factor, which may be an anti-cancer agent.

By a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH 12, LOC55726, GBP4, IKBKE and RHO J₁. we include polypeptides having the sequences listed in Figure

5 (SEQ ID Nos: 1-9), and naturally occurring variants thereof. It is appreciated that for the binding assay, it is not necessary to use a polypeptide having 100% sequence identity to the sequences listed in Figure 5 (SEQ ID Nos: 1-9) (whether over the full- length polypeptide or the fragment thereof). Accordingly, in this aspect of the invention it is possible to use a variant polypeptide having at least 80%, more preferably at least 85%, still more preferably at least 90%, yet more preferably at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the sequences listed in Figure 5 (SEQ ID Nos: 1-9). It is preferred if the variant polypeptide has a consecutive region of at least 20 amino acid residues, more preferably at least 50 residues, of the sequence of the polypeptide listed in Figure 5 (SEQ ID Nos: 1-9). Such variants may be made, for example, using the methods of recombinant DNA technology, protein engineering and site-directed mutagenesis which are well known in the art.

The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally. The alignment may alternatively be carried out using the Clustal W program (Thompson et a/., (1994) Nucleic Acids Res 22, 4673-80). The parameters used may be as follows: Fast pairwise alignment parameters: K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM.

It is also appreciated that in order to determine whether a candidate compound binds to a specified polypeptide, it is not necessary to use the entire full-length polypeptide in the binding assay, and fragments of the polypeptide may be usefully employed. Preferably, the fragment is at least 20 amino acid residues in length, and may be between 20 and 50 residues or between 50 and 100 residues or between 100 and 150 residues or between 150 and 200 residues in length, or more.

In an embodiment, the candidate compound may be an antibody that selectively binds a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH 12, LOC55726, GBP4, IKBKE and RHOJ, or a fragment thereof. Suitable antibodies are described above.

In another embodiment, the candidate compound may be a peptide. Suitable peptides that bind to a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, or a fragment thereof; may be identified by methods such as phage display of peptide libraries (Scott & Smith (1990) "Searching for peptide ligands with an epitope library." Science 249: 386-390; Felici et al (1995) "Peptide and protein display on the surface of filamentous bacteriophage." Biotechnol. Annu. Rev. 1: 149-183); and Collins et al (2001) "Cosmix-plexing: a novel recombinatorial approach for evolutionary selection from combinatorial libraries." J. Biotechnol. 74: 317-338); including in vivo panning (Pasqualini et al (1997) "αv integrins as receptors for tumor targeting by circulating ligands. Nature Biotechnol. 15: 542-546), and solid-phase parallel synthesis (Frank (2002) The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports — principles and applications." J. Immunol. Methods 267: 13-26; and Pinilla et al (2003) "Advances in the use of synthetic combinatorial chemistry: mixture-based libraries." Nature Med. 9: 118-122). The dissociation constants of peptides are typically in the micromolar range, although avidity can be improved by multimerization (Terskikh et al (1997) "Peptabody": a new type of high avidity binding protein. Proc. Natl Acad. Sci. USA 94, 1663-1668; and Wrighton et al (1997) "Increased potency of an erythropoietin peptide mimetic through covalent dimerization. Nature Biotechnol. 15, 1261-1265).

In still another embodiment, the candidate compound may be an aptamer, i.e. a single- stranded DNA molecule that folds into a specific ligand-binding structure. Suitable aptamers that bind to a polypeptide selected from RHBDL6, KCTD15, LRRC8C,

PCDH12, LOC55726, GBP4, IKBKE and RHOJ, or a fragment thereof; may be identified by methods such as in vitro selection and amplification (Ellington & Szostak (1992)

"Selection in vitro of single stranded DNA molecules that fold into specific ligand binding structures." Nature 355: 850-852; and Daniels et al (2003) "A tenascin-C aptamer identified by tumor cell SELEX: systematic evolution of ligands by exponential enrichment." Proc. Natl Acad. Sci. USA 100, 15416-15421). The aptamer may be a nuclease-stable 'Spiegelmer' (Helmling, S. et al (2004) "Inhibition of ghrelin action in vitro and in vivo by an RNA-Spiegelmer." Proc. Natl Acad. Sci. USA 101: 13174-13179). Aptamers typically have dissociation constants in the micromolar to the subnanomolar range.

In yet another embodiment, the candidate compound may be a small organic molecule.

Suitable small molecule that bind to a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, or a fragment thereof; may be identified by methods such as screening large libraries of compounds (Beck-Sickinger & Weber (2001) Combinational Strategies in Biology and Chemistry (John Wiley & Sons, Chichester, Sussex); by structure-activity relationship by nuclear magnetic resonance (Shuker et al (1996) "Discovering high-affinity ligands for proteins: SAR by NMR. Science 27 '4: 1531-1534); encoded self-assembling chemical libraries Melkko et al (2004) "Encoded self-assembling chemical libraries." Nature Biotechnol. 22: 568-574); DNA- templated chemistry (Gartner et al (2004) "DNA-templated organic synthesis and selection of a library of macrocycles. Science 305: 1601-1605); dynamic combinatorial chemistry (Ramstrom & Lehn (2002) "Drug discovery by dynamic combinatorial libraries." Nature Rev. Drug Discov. 1: 26-36); tethering (Arkin & Wells (2004) "Small-molecule inhibitors of protein-protein interactions: progressing towards the dream. Nature Rev. Drug Discov. 3: 301-317); and speed screen (Muckenschnabel et al (2004) "SpeedScreen: label-free liquid chromatography-mass spectrometry-based high- throughput screening for the discovery of orphan protein ligands." Anal. Biochem. 324: 241-249). Typically, small organic molecules will have a dissociation constant for the polypeptide in the nanomolar range, particularly for antigens with cavities. The benefits of most small organic molecule binders include their ease of manufacture, lack of immunogenicity, tissue distribution properties, chemical modification strategies and oral bioavailability.

The capability of a candidate compound to bind to or interact with the polypeptide or fragment thereof may be measured by any method of detecting/measuring a protein/protein interaction or other compound/protein interaction, as discussed further below. Suitable methods include methods such as, for example, yeast two-hybrid interactions, co-purification, ELISA, co-immunoprecipitation and surface plasmon resonance methods. Thus, the candidate compound may be considered capable of binding to the polypeptide or fragment thereof if an interaction may be detected between the candidate compound and the polypeptide or fragment thereof by ELISA, co- immunoprecipitation or surface plasmon resonance methods or by a yeast two-hybrid interaction or copurification method. It is preferred that the interaction can be detected using a surface plasmon resonance method. Surface plasmon resonance methods are well known to those skilled in the art. Techniques are described in, for example, O'Shannessy DJ (1994) "Determination of kinetic rate and equilibrium binding constants for macromolecular interactions: a critique of the surface plasmon resonance literature" Curr Opin Biotechnol. 5(1):65-71 ; Fivash et al (1998) "BIAcore for macromolecular interaction." Curr Opin Biotechnol. 9(1):97-101 ; Malmqvist (1999) "BIACORE: an affinity biosensor system for characterization of biomolecular interactions." Biochem Soc Trans. 27(2):335-40.

It is appreciated that screening assays which are capable of high throughput operation are particularly preferred. Examples may include cell based assays and protein-protein binding assays. An SPA-based (Scintillation Proximity Assay; Amersham International) system may be used. For example, an assay for identifying a compound capable of modulating the activity of a protein kinase may be performed as follows. Beads comprising scintillant and a substrate polypeptide that may be phosphorylated may be prepared. The beads may be mixed with a sample comprising the protein kinase and ³²P-ATP or ³³P-ATP and with the test compound. Conveniently this is done in a multi- well (e.g., 96 or 384) format. The plate is then counted using a suitable scintillation counter, using known parameters for ³²P or ³³P SPA assays. Only ³²P or ³³P that is in proximity to the scintillant, i.e. only that bound to the polypeptide, is detected. Variants of such an assay, for example in which the polypeptide is immobilised on the scintillant beads via binding to an antibody or antibody fragment, may also be used.

Other methods of detecting polypeptide/polypeptide interactions include ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods. Fluorescence Energy Resonance Transfer (FRET) methods, for example, well known to those skilled in the art, may be used, in which binding of two fluorescent labelled entities may be measured by measuring the interaction of the fluorescent labels when in close proximity to each other.

A further method of identifying a compound that is capable of binding to the polypeptide or fragment thereof is one where the polypeptide is exposed to the compound and any binding of the compound to the said polypeptide is detected and/or measured. The binding constant for the binding of the compound to the polypeptide may be determined. Suitable methods for detecting and/or measuring (quantifying) the binding of a compound to a polypeptide are well known to those skilled in the art and may be performed, for example, using a method capable of high throughput operation, for example a chip- based method. Technology, called VLSIPS™, has enabled the production of extremely small chips that contain hundreds of thousands or more of different molecular probes. These biological chips or arrays have probes arranged in arrays, each probe assigned a specific location. Biological chips have been produced in which each location has a scale of, for example, ten microns. The chips can be used to determine whether target molecules interact with any of the probes on the chip. After exposing the array to target molecules under selected test conditions, scanning devices can examine each location in the array and determine whether a target molecule has interacted with the probe at that location.

It is appreciated that the identification of a candidate compound that binds to the polypeptide or fragment thereof may be an initial step in the drug screening pathway, and the identified compounds may be further selected e.g. for the ability to inhibit angiogenesis.

By "inhibiting angiogenesis" we include the meaning of reducing the rate or level of angiogenesis. The reduction can be a low level reduction of about 10%, or about 20%, or about 30%, or about 40% of the rate or level of angiogenesis. Preferably, the reduction is a medium level reduction of about 50%, or about 60%, or about 70%, or about 80% reduction of the rate or level of angiogenesis. More preferably, the reduction is a high level reduction of about 90%, or about 95%, or about 99%, or about 99.9%, or about 99.99% of the rate or level of angiogenesis. Most preferably, inhibition can also include the elimination of angiogenesis or its reduction to an undetectable level.

Methods and assays for determining the rate or level of angiogenesis, and hence for determining whether and to what extent a test compound inhibits angiogenesis, are known in the art. For example, US Patent No. 6,225,118 to Grant et al, incorporated herein by reference, describes a multicellular ex vivo assay for modelling the combined stages of angiogenesis namely the proliferation, migration and differentiation stages of cell development. The AngioKit, Catalogue No. ZHA-1000, by TCS CellWorks Ltd, Buckingham MK18 2LR, UK, is a suitable model of human angiogenesis for analysing the anti-angiogenic properties of compounds. The rate or level of angiogenesis can also be determined using the aortic ring assay and the sponge angiogenesis assay described in Example 4.

Assays for endothelial cell proliferation, migration and invasion are also useful as angiogenesis assays. Suitable assays for endothelial cell proliferation and migration are known to a person of skill in the art and are described herein, and preferably are those described in Example 4. Suitable assays for endothelial cell invasion are also known to a person of skill in the art and include the BD BioCoat™ Angiogenesis System for Endothelial Cell Invasion which is available as Catalogue Nos. 354141 and 354142 from BD Biosciences, Bedford, MA, USA.

We also consider that a candidate compound that selectively binds to a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ may inhibit migration of tumour endothelial cells, including bFGF- and VEGF- induced migration, inhibit proliferation of tumour endothelial cells, or invasion of tumour endothelial cells. Accordingly, candidate compounds that show inhibitory activity in the HUVEC migration assay, or that show antiproliferative activity or that show anti-invasive activity in an assay such as the BD BioCoat™ Angiogenesis System for Endothelial Cell Invasion (BD Biosciences, Bedford, MA, USA), may be therapeutically useful in combating solid tumours in which unwanted, undesirable or inappropriate tumour endothelial cell migration, proliferation or invasion contributes to the angiogenesis of neovasculature and hence the pathology of solid tumours.

It is appreciated that these methods may be a drug screening methods, a term well known to those skilled in the art, and the candidate compound may be a drug-like compound or lead compound for the development of a drug-like compound.

The term "drug-like compound" is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 Daltons and which may be water-soluble. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes or the blood:brain barrier, but it will be appreciated that these features are not essential.

The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics. In an embodiment, the identified compound is modified, and the modified compound is tested for the ability to inhibit angiogenesis. Suitable assays for the inhibition of angiogenesis are described above.

It is appreciated that the screening methods can be used to identify agents that may be useful in combating solid tumours. Thus, the screening methods preferably also comprise the further step of testing the identified compound or the modified compound for efficacy in an animal model of cancer, particularly a solid tumour. Suitable models are known in the art and include Lewis lung carcinoma subcutaneous implants in mice (homograft in Black 57 mice) or HT29 xenografts subcutaneous implants in nude mice.

The invention may comprise the further step of synthesising, purifying and/or formulating the identified compound or the modified compound.

The invention may further comprise the step of formulating the compound identified into a pharmaceutically acceptable composition.

Compounds may also be subjected to other tests, for example toxicology or metabolism tests, as is well known to those skilled in the art.

Thus the invention includes a method for preparing an anticancer compound that may be useful in the treatment of a solid tumour, the method comprising identifying a compound using the screening methods described above and synthesising, purifying and/or formulating the identified compound.

Thus, the invention also includes a method of making a pharmaceutical composition comprising the step of mixing the compound identified using the methods described above with a pharmaceutically acceptable carrier.

All of the documents referred to herein are incorporated herein, in their entirety, by reference.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge The invention will now be described in more detail by reference to the following Examples and Figures.

Figure 1 : A Venn diagram pictorial representation of the analyses carried out to predict TEMs. The results of an endothelial and tumour screen are combined to produce putative TEMs.

Figure 2: An overview of the EST-to-gene assignment process. Each EST sequence is BLAST searched against a Refseq mRNA database and the best mRNA is assigned that

EST. In tandem, a mapping of all ESTs and Refseq mRNA to the human genome assigns ESTs to genes based on genome position. A decision tree makes the final assignment based on the quality of alignment and agreement between the two methods.

If the genome position and BLAST result agree, the EST is assigned, if they don't agree but the BLAST result if of high quality (> 92% and > 100 bp alignment) the EST is also assigned. For any other result the EST is removed from the analysis.

Figure 3: Real time PCR was carried out on predicted endothelial genes and results show the power of the bioinformatics models as all genes examined were up-regulated or specific to HUVECs and/or HDMECs.

Figure 4: Real time PCR was carried out on predicted endothelial genes and results show the power of the bioinformatics models as all genes examined were up-regulated or specific to HUVECs and/or HDMECs.

Figure 5: Polypeptide sequence of human KCTD15 (SEQ ID No: 1), LRRC8C (SEQ ID No: 2), PCDH12 (SEQ ID No: 3), LOC55726 (SEQ ID No: 4), GBP4 (SEQ ID No: 5), IKBKE (SEQ ID No: 6), RHBDL6 (SEQ ID Nos: 7 and 8) and RHOJ (SEQ ID No: 9) from the respective Genbank entries mentioned above.

Fig. 6. RhoJ expression in different cell lines. RT-PCR studies showed that RhoJ expression is specific to endothelial cells, but not other cell lines.

Figure 7: lmmunohistochemistry of endothelium in human placenta or heart sections with anti-RhoJ antibody. Human placenta sections were soaked in histosol, ethanol and citrate buffer to remove paraffin and to fix the sections. These were then stained with anti-RhoJ antibody (A) or anti-CD31 antibody as positive control (B). Frozen heart sections were fixed in ice cold acetone and stained with anti-RhoJ antibody (C) or anti- CD31 antibody (D) (4OX magnification).

Figure 8: Development of RhoJ expression after transfection with RhoJ siRNA. HUVEC were transfected with nonbinding siRNA as negative control, RhoJ-1 and RhoJ-2 siRNA. Cells were lysed on day 1-4 after RhoJ siRNA transfection. Lysates were then separated by SDS-PAGE and western blotted using anti-RhoJ and anti-β-actin antibodies.

Figure 9: Expression levels of several RhoJ proteins compared to β-actin. HUVEC were mock transfected (m), transfected with non-binding (n), RhoJ-1 or RhoJ-2 siRNA. Total RNA was then isolated two days after transfection and 5 μg thereof were transcribed into cDNA. Expression levels of β-actin (A) and RhoJ (B) were determined and expression levels of RhoA (C), RhoB (D), RhoJ (E) and Cdc42 (F) relative to the β-actin expression were generated with Q-PCR out of individual dilution series for each protein.

Figure 10: Effect of RhoJ downregulation on cell growth in HUVEC. HUVEC were either mock transfected (B) or transfected with non-binding negative control (C), RhoJ-1 siRNA (D) or Rho-2 siRNA (E). 15'0OO cells were then seeded per well of a 24 well plate in triplicate per time point. On day 1-3 after transfection cells were harvested and counted with a hemocytometer. Results for each sample were plotted together as an average cell number in A and individually in BE with standard deviations added.

Figure 11 : Effect of downregulation of RhoJ by siRNA on tube formation in HUVEC. (A) HUVEC were mock transfected or transfected with siRNA duplexes RhoJ-1 , RhoJ-2 or with a non-binding negative control. Two days after transfection, cells were seeded onto a matrigel layer and photographed after 2, 4, 8 and 24 hours (40X magnification). (B)

Each sample was lysed and western blots performed with RhoJ antibody to show reduced RhoJ level in the RhoJ siRNA transfected cells and with β-actin as a loading control.

Figure 12: Effect of RhoJ downregulation on cell migration. Mock transfected cells or cells transfected with a negative control siRNA, RhoJ-1 or RhoJ-2 siRNA, were seeded into a well of a 6 well plate. (A) The day after seeding, a scratch was created and cell migration observed by taking pictures after 4, 8 and 12 hours (40X magnification). (B) To confirm RhoJ downregulation on the day of the experiment, remaining cells were lysed and a western blot was performed using anti-RhoJ antibody.

Figure 13: Human umbilical vein endothelial cells were transfected with RhoJ specific siRNA D1 or D2, NCD or mock treated with transfection reagent only (Mock) resulting in reduction of RhoJ protein.

Figure 14: Down-regulation of RhoJ expression using siRNA results in results in impaired tube formation in fibrin gels.

Figure 15: Down-regulation of RhoJ expression using siRNA results in results in impaired tube formation on matrigel.

Figure 16: HUVEC with reduced RhoJ fail to migrate in a scatch wound assay.

Figure 17: HUVEC with reduced RhoJ fail to migrate in a chemotaxis assay in a Boyden chamber. HUVEC were transfected with 1 nM of RhoJ specific siRNA D1 or D2, NCD or mock treated with transfection reagent only. (A) Two days after transfection, a Boyden chamber chemotaxis assay was set up. Images represent migrated cells stained with 0.5% crystal violet on polycarbonate filter. Scale bar represents 200μm. (B) Quantification of migrated cells from 12 wells of 3 independent repeats (n=36, error bars represent ± SE).

Figure 18: Down-regulation of RhoJ expression in HUVEC using siRNA results in growth impairment.

Figure 19: Activation of RhoJ by VEGF-A in HUVECs. HUVEC were rested 1 hr and then stimulated with VEGF-A for the times indicated. Active RhoJ was determined by pulldown. A, Results of Western Blotting showing the pull-down of active RhoJ in relation to the lysate. B, Densitometrical quantitation of western blotting results are shown.

Figure 20: RHBDL6 (RHBDF2) siRNA knocks-down mRNA expression without eliciting interferon response. HUVECs were either mock transfected, transfected with non-binding negative duplex or 3 different siRNA duplexes targeting RHBDL6. Total RNA was prepared from the HUVECs 24 hours after transfection and cDNA was prepared. Knock- down of RHBDL6 mRNA was measured by quantitative RT-PCR. Samples were normalized to the expression of Actin.

Figure 21 : RHBDL6 (RHBDF2) knock-down in HUVECS results in defects in tube- formation on matrigel. The HUVECs were either mock transfected, transfected with negative duplex or siRNA duplexes against RHBDL6. 48 hours after transfection, cells were seeded onto a matrigel layer and photographed after 2, 12 and 24 hours. 5OnM of siRNA was used..

Figure 22: RHBDL6 (RHBDF2) knock-down results in migration defects in HUVECS, with HUVECs showing reduced cell migration. The HUVECs were either mock transfected, transfected with negative duplex or siRNA duplexes against RHBDL6. 48 hours after transfection, a scratch was created and cell migration observed by taking pictures at 0, 4, 8 and 20 hours. 5OnM of siRNA was used.

Figure 23: RHBDL6 (RHBDF2) knock-down results in reduced cell proliferation in HUVECS. HUVECs were either mock transfected, transfected with negatve control siRNA duplexes or 3 different siRNA duplexes targeting RHBDL6. 4 hours after transfection, the cells were trypsin ized and plated on 24 well plates at 15000 cells per well. Cells were counted 48, 72 and 96 hrs after transfection to determine the effect of siRNA transfection on cell proliferation. 5OnM of siRNA was used. The cell numbers are represented as a percentage of initial cell numbers (cell numbers at day 0 being 100%).

Figure 24: zRHBDLδ mRNA expression in 1 day old zebrafish embryo by in situ hybridization. The Zebrafish ortholog of RHBDL6 ZRHBDL6 (zRHBDF2) is strongly expressed in endothelial and haematopoietic cells during development. Shown are expression in a 24-hour-old embryo at low magnification (a) and expression in the same embryo focusing on the tail region at high magnification (b). Arrows indicate the position of endothelial and haematopoietic cells.

Figure 25: Functional requirement of RHBDL6 in development of zebrafish embryonic vasculature. (A) Knockdown of zRHBDLβ protein expression using a morpholino antisense oligonucleotide results in reduced inter somatic vessels (ISV) in the embryo as seen by Fli-1 expression (black arrows). Shown are a control embryo (untreated) and embryos treated with 0.125nM and 0.5nM morpholino RHBDL6 antisense oligonucleotide. (B) The percentage of embryos with normal FIM expression following treatment demonstrates that the morpholino effect on ISV formation is concentration dependent. 53 out of 56 (95%) embryos treated with 0.5nM RHBDL6 antisense oligonucleotide, 32 out of 35 (91 %) embryos treated with 0.25nM RHBDL6 antisense oligonucleotide and 22 out of 35 (63%) embryos treated with 0.125nM RHBDL6 antisense oligonucleotide showed abnormal vasculature.

Figure 26: In-situ hybridisation shows (A) LRRC8C expression in endothelial cells of squamous cell carcinoma tissue (B) PCDH 12 expression in endothelial cells of breast cancer tissue (160331 A) and (C) PCDH 12 expression in endothelial cells of malignant fibrous histiocytoma tissue taken from the abdomen of a 67 year old male. Vessels were stained with Ulex Eururopeaus Agglutinin (UEA1) conjugated with rodhamin (red), and LRRC8C (A) or PCDH 12 (B-D) LNA biotin probes were detected with avidin conjugated with fluorescein (green). The figures show overlapping signals from UEA1 and the gene specific probes (yellow) confirming an endothelial cell expression pattern. Less expression of LRRC8C and PCDH 12 was detected in matching normal tissues (data not shown).

Example 1 : A Method for Accurate Expressed Sequence Tag to Gene Assignment and a Novel Statistical Analysis of Differential Gene Expression Across Multiple cDNA Libraries Applied to the Identification of Endothelial and Tumour Endothelial Genes.

Summary

In this study, differential gene expression analysis using complementary DNA (cDNA) libraries has been improved by the introduction of an accurate method of assigning

Expressed Sequence Tags (ESTs) to genes and a novel maximum likelihood statistical scoring of differential gene expression between two pools of cDNA libraries. These methods were applied to the latest available cell line and bulk tissue cDNA libraries in a two-step screen to predict novel tumour endothelial markers. Initially, endothelial cell lines were subtracted in silico from non-endothelial cell lines to identify endothelial genes. Subsequently, a second bulk tumour versus normal tissue subtraction was employed to predict tumour endothelial markers.

From an endothelial cDNA library analysis, 431 genes were found to be significantly up- regulated in endothelial cells with a False Discovery Rate adjusted q-value of 0.01 or less, and 104 of these were expressed only in endothelial cells. Combining the cDNA library data with the latest Serial Analysis of Gene Expression (SAGE) library data derived a complete list of 459 genes preferentially expressed in endothelium. 27 genes were predicted tumour endothelial markers in multiple tissues based on the second bulk tissue screen.

This ability to accurately assign an EST to a gene, statistically measure differential expression between two pools of cDNA libraries and predict putative tumour endothelial markers before entering the laboratory represents a significant advance.

Background

Study aim

The growth and survival of tumours is dependent on their ability to obtain a blood supply and damage inflicted on the tumour endothelium has been shown to effectively eradicate tumours [1]. It follows that the discovery of widely expressed tumour endothelial markers promises much clinical benefit [2]. The aim of this study was to apply novel bioinformatic methods to the latest public expression data repositories, with an emphasis on cDNA library analysis, to create an up-to-date list of putative endothelial genes and to predict tumour endothelial markers that are potential anti-cancer targets.

Previous work employing in silico analysis to identify tissue specific genes

Previous studies [3-15] have employed cDNA or SAGE libraries to predict the transcriptional profiles of tissues of interest that were subsequently confirmed by experimental analysis. Our analysis [8] employed a cDNA subtractive Basic Local Alignment Search Tool (BLAST) [16] algorithm to predict endothelial specific genes. This approach required cross-referencing of the results to SAGE libraries to confidently predict endothelial expression due to a large number of false positives associated with the BLAST method of EST-to-gene assignment. In one study [7], Unigene's Digital Differential Display (DDD) tool was employed to predict endothelial genes, which is reliant on Unigene clusters. DDD requires at least 1 ,000 EST sequences from a cDNA library to be clustered into Unigene clusters for valid statistical analysis and can measure statistical significance accurately between only two libraries [15]. This 1 ,000 sequence limit of DDD can remove small, but often potentially relevant, cDNA libraries from an analysis.

Improving cDNA library analysis We aimed to improve both the statistical analysis and EST-to-gene assignment methods used in subtractive in silico cDNA differential gene expression analyses. To eliminate the cDNA library false positive discovery rate of our previous study [8], EST-to-gene assignment was improved by combining human genome BLAST Like Alignment Tool (BLAT) [17], alignment data with a new BLAST protocol. Further, a new maximum likelihood ratio test was developed that is based upon the intrinsic variability of cDNA library counts and represents a maximally powerful approach to analyze this type of data. This method alleviated the need for the 1 ,000 Unigene cluster limit of the DDD tool, enabled any size cDNA library to be analysed and accurately determined differential expression across more than two cDNA libraries.

In silico Tumour Endothelial Marker prediction

A two-step analysis was performed to predict tumour endothelial markers (TEMs). The first stage identified endothelial genes by comparing the expression patterns of genes between endothelial and non-endothelial cell lines. The second stage involved a comparison of bulk tumour and bulk normal cDNA libraries to identify genes up-regulated in tumours. Putative TEMs are genes that were both endothelial and preferentially expressed in tumours. The Venn diagram in Figure 2 summarises the analysis.

Materials and Methods

Construction of databases

A large part of this study involved the collection and processing of data in the public domain with speed and accuracy, in particular the creation and use of a Relational Database Management System (RDBMS) MySQL database called dbestlibraries. The database was central to all processes in tandem with Perl scripts, which were written for the import of data, assignment of EST-to-gene symbols and the accurate calculation of the FDR-adjusted q-value results.

Data was collected from Genbank flat files (release 154) downloaded from the NCBI ftp://ftp.ncbi.nih.gov/genbank/ that supplied all cDNA library data imported into the database. 10,788 libraries containing 8,003,786 ESTs were imported into the database.

Information concerning 29,367 human reference sequence project mRNAs and gene predictions were downloaded from release 14 of the Reference sequence project at

(ftp://ftp.ncbi.nih.gov/refseq). Finally, all information relating to Refseq sequences was downloaded into the database from ftp://ftp.ncbi.nlm.nih.gov/gene/DATA/. Selection of EST library pools

The CGAP library finder: (http://cgap.nci.nih.gov/Tissues/LibraryFinder) was used as a tool for choosing which libraries to compare in tumour and endothelial screens. Additional endothelial cDNA libraries were discovered, using a Perl script to parse raw Genbank flat files, which identified libraries with keywords such as "cell lines" and "endothelial". Normalised or subtracted libraries were excluded from this analysis.

Normal versus tumour tissue screen

Bulk tumour and normal cDNA libraries for six organs were chosen using the CGAP library browser. The combined algorithm was employed to perform virtual subtraction hybridisation between tumour and normal libraries of the same organ. All results were imported into the dbestlibraries database. Results with an FDR-adjusted q-value of 0.01 were significant.

cDNA library screen (EST-to-αene assignment)

To perform in silico virtual subtraction, two different protocols for assigning an EST to a gene were combined for greatest accuracy. The first protocol took advantage of the almost complete human genome by using genome address to assign an EST to a gene. A genome address of a gene or EST is the physical base pair position it occupies on a chromosome. Both cDNA pools and all Refseq mRNAs were aligned to the human genome using BLAT to generate genome addresses. The BLAT alignment genome addresses, were clustered using a Perl algorithm called the Jake cluster algorithm to identify EST sequences that overlapped with a gene and to assign them. To save processing time using BLAT, the human genome addresses of Refseq genes and ESTs were downloaded from the University of California Santa Cruz (UCSC) table browser page (http://genome.ucsc.edu/cgi-bin/hgTables). This file contained the pre-processed BLAT output [17]. BLAT is designed to rapidly align DNA sequences that are 95% identical or more, over at least 40 base pairs.

For the second method of assigning an EST to a gene, each EST from both cDNA library pools was collected as a FASTA sequence and BLAST searched against a database of all Refseq mRNAs. An expectation cut-off of 1 was employed and the -v and -b BLAST options were set to 1. This ensured that only the best mRNA that matched the EST was returned in the BLAST results. A Perl script algorithm (Jake cluster) was constructed to combine the results of the genome BLAT address with the BLAST search method. If the genome address assignment agreed with the BLAST result, then the EST was assigned to the gene; if they disagreed, only a high quality BLAST result allowed EST-to-gene assignment (>= 92% identity, >= 100 bp alignment).

Combining cDNA and SAGE library analysis

For Experiments 2 and 3 described in the results section, the cDNA analysis was combined with a SAGE library analysis for endothelial gene prediction. The SAGEmap xProfiler tool at NCBI was used for this: http://www.ncbi.nlm.nih.gov/projects/SAGE/index.cgi?cmd=expsetup. No SAGE analyses were carried out for the tumour screen, as there were insufficient bulk tumour or bulk normal libraries SAGE libraries available.

The SAGE experiments were performed using a fold difference factor of 10 and a 0% coefficient of variance cut off. Only genes with a p-value of 0.9 or more were considered significant. In pool "A" (endothelial cell line pool) there were 10 SAGE libraries containing 427,254 SAGE transcripts. In pool "B", the normal non-endothelial pool, there were 11 normal non-endothelial libraries with 329,470 transcripts. For the cancer cell line non-endothelial pool [8], there were 24 SAGE libraries consisting of 733,461 transcripts. As the cancer cell line non-endothelial pool was twice the size of the normal non-endothelial pool, more genes were significantly up-regulated in the former due to pool size and statistics.

Statistical methods

We now describe a statistical methodology for the comparison of two groups of cDNA libraries to enable the discovery of differentially expressed genes. The method combines a generalised maximum likelihood ratio test with a False Discovery Rate procedure (FDR) in order to provide a robust list of differentially expressed genes. The analyses extend our earlier work, which identified differentially expressed genes in a single group of cDNA libraries [15].

As described in [15], we consider the expression of gene j in a set of cDNA libraries.

There are two groups of libraries: m libraries from non-endothelial cell lines, and n libraries from endothelial cell lines. We let Ni : 1< i < m be the number of ESTs sequenced in each non-endothelial cell line library, and Nm+i : 1< i < n be the number of ESTs sequenced in each of the endothelial cell line library. For each gene j, let xi,j be the number of copies of associated ESTs in library i.

For each gene, we compare two hypotheses concerning its frequency of expression in the libraries, using a generalised likelihood test. Under the null hypothesis, the gene is not differentially expressed and we would expect its frequency to be identical in both the non-endothelial and endothelial cell libraries. In contrast, under the alternative hypothesis, the gene is differentially expressed, and so we would expect the frequency to be different in the non-endothelial and endothelial cell lines.

In both cases, as long as the number of copies of ESTs from the gene is small relative to the total number of ESTs sequenced in the library, the distribution of the gene is well approximated by a Poisson distribution. Under the null hypothesis, the frequency is fj, then for library i, the number of ESTs is approximately distributed as a Poisson variable with parameter fiNi. Thus the likelihood of the observed data is

The maximum likelihood estimate of ^ under the null hypothesis can be found by solving:

= 0

2) #,

And the solution ^Jj is given by:

m+n f (0) ' = m+n

3) Equation 3 is simply the proportion of ESTs for the gene of interest among all ESTs in all of the libraries. Thus under the null hypothesis, the likelihood of the data ' " is given by equation 1 w ,i;tthk- J ^~ Jj

For the alternative hypothesis, the frequency of gene transcripts is different in the non- endothelial and endothelial cell line libraries. By a similar argument, we derive

( f I frequencies for each gene ^J in the non-endothelial libraries ^KJj ' and the endothelial

libraries ( ^κjf^j(2)) which is given by:

/ . ^Xm+ij /^• (2) _ __£=1

Z_J m+i

5)

Equations 4 and 5 are very similar to equation 3, and simply represent the proportion of ESTs for the gene of interest among all ESTs in hypothesis, the likelihood of the data ^κ > is given by:

R, = iog(/_y ⁽¹⁾ /fj^m)±x_u + iog(//²⁾ /fj^m)∑^χ _m+u 7) '=' '='

Equation 7 can be explained very simply: there are two terms, one for the non- endotheiial libraries and one for the endothelial libraries. Each term is the log ratio of the frequency of the gene in the relevant libraries and the overall frequency of the gene, multiplied by the total number of ESTs for that gene in the relevant libraries. The equation is very similar to the R statistic derived in Stekel et a/, 2000 [15].

9 R

Under Wilke's Theorem, ^J is distributed as a ^ distribution with a 1 degree of freedom. Therefore it is straightforward to compute a p-value for each gene. However, when analyzing all genes in the library in order to find those that are most differentially expressed, it is essential to combine the p-value with a False Discovery Rate Procedure [19]. Thus the results we present are the FDR-adjusted q-values.

A definition of terms: m = the number of non-endothelial cell line libraries n = the number of endothelial cell line libraries l'^J = the number of transcript copies of gene j in cDNA library i

N 1 = the total number of clones sequenced in the cDNA library i

JC "ⁱ⁺ⁱ.^j = the number of copies of gene j in the m + i'th cDNA library

N m^+ι = the total number of clones sequenced in the m + i'th cDNA library

/, = the frequency of gene j

Computation of the statistics To compute the FDR adjusted q-values for a given data set, we calculate the R-values for all genes. We then compute the p-values for every gene using the Chi squared distribution of 2R. The genes were then ordered according to the p-values, ranked from smallest to highest. Each p-value was adjusted by multiplying it by the number of genes in the analysis and dividing by its rank position (The smallest p-value is rank position 1). To derive the q-value, the list of ranked values was stepped through, comparing p-value and its adjusted value and always selecting the lowest.

Cell isolates and extraction of RNA

Human umbilical vein endothelial cells (pooled HUVEC), adult human dermal microvascular endothelial cells (HDMEC), human bronchial epithelial cells (HBEC) and adult human Epidermal keratinocytes were obtained from TCS Cellworks (Botolph Claydon, UK). Cells were grown in their appropriate growth media and supplements) according to manufacturers instructions and RNA extracted at passage 2-3. Human lung fibroblasts (MRC-5) were obtained from American Type Culture Collection (Manassas, VA) and cultured in DMEM containing 10% FCS. All cells were grown at 37°C in a humidified atmosphere of 5% CO₂ in air.

Cryopreserved human hepatocytes (TCS Cellworks) were thawed in Leibovitch L15 medium (Invitrogen, Paisley, UK), centrifuged and resuspended in fresh media, RNA was extracted after 30 minutes incubation at 37⁰C in 5% CO₂. Cryopreserved human peripheral blood lympbocytes were obtained from TCS Cellworks, after thawing they were washed in PBS and used immediately for RNA extraction.

Quantitative PCR

Total RNA was extracted from cells in culture using TRI reagent (Sigma, Dorset, UK) cDNA was prepared using a high capacity cDNA archive kit (Applied Biosystems, Cheshire, UK). The Universal ProbeLibrary system (Roche) was used for real time PCR analysis. Reactions were performed in triplicate using Absolute QPCR mix (ABgene, Epsom, UK) according to the manufacturer's instructions using 10ng cDNA.

Reactions were performed in a Rotor-GENE RG30000 thermocycler (Corbett Research, UK) using the following cycling conditions; 95⁰C for 10 minutes followed by 40 cycles of 95⁰C for 15 seconds and 6O⁰C for 1 minute. The appropriate housekeeper genes were determined as described by Vandesompele et al [47] using the software geNorm. For the cell type screen FLOT2, Ubiquitin C and B-Actin were used. The raw data was analysed using a method described by Pfaffl [48].

List of abbreviations BLAST Basic Local Alignment Search Tool

BLAT BLAST Like Alignment Tool cDNA Complementary DNA

DDD Digital Differential Display

EST Expressed Sequence Tag FDR False Discovery Rate

FDR-adjusted False Discovery Rate adjusted

GFP Green Fluorescent Protein

HBEC Human bronchial epithelial cells

HDMECs Human dermal micro-vascular endothelial cells HUVECs Human umbilical vein endothelial cells

MRC-5 Human lung fibroblasts PCR Polymerase Chain Reaction

RDBMS Relational Database Management System

Refseq Reference Sequence Project

SAGE Serial Analysis of Gene Expression TEM Tumour Endothelial Marker

Results

Development of an algorithm for EST-to-gene assignment

We have developed a new algorithm for assigning an EST to a gene that takes advantage of the almost complete human genome and combines it with a BLAST analysis to achieve an accurate result. Initially, two EST pools and all Reference sequence project (Refseq) mRNA sequences were aligned to the human genome using BLAT. Sequences occupying an ambiguous position in the genome were removed. The aligned sequences were then collected into Peri data structures and a custom-clustering algorithm (Jake cluster) assigned each EST to a gene or gene prediction based on their overlapping genome position. In the BLAST analysis, each EST was BLAST searched against a Refseq database of all mRNA and gene predictions. Only the best mRNA hit from the BLAST analysis was assigned to an EST. The BLAT and BLAST results were cross-referenced and accurate EST-to-gene assignments were made based on the following decision tree:

If genome BLAT mapping and BLAST results agreed, then that gene was assigned. If the results disagreed, then the BLAST result was accepted only if the alignment was of high quality that is greater than or equal to 100 base pairs with at least 92% identity.

A pictorial representation of the analysis is shown in Figure 3. The approach was able to assign ESTs to a gene even when the single pass cDNA sequencing of an EST was of low quality. Thus, first finding an unambiguous position in the genome that overlaps with a gene and then searching with BLAST to find the best gene, it was able to assign an EST to a gene. Further, using a high quality BLAST alignment alone for the assignment gives this approach the ability to also assign a gene that lies in a gap in the human genome sequence.

Validation of the EST-to-gene assignment algorithm The results of the BLAST subtraction method used in our previous work [8] were compared to those of the algorithm developed here. Using a custom relational database that we developed, cDNA libraries were collected and divided into 2 pools (endothelial and non-endothelial cells respectively) and formatted into BLAST databases. The same data were used in this experiment as used in the earlier study because the EST-to-gene BLAST protocol was dependent on an expectation value. The expectation value was optimised in the earlier work for predictive capacity by performing trial runs. Expectation values are dependent on the size of a BLAST database and so it was important to use the same data. This was possible for the endothelial cell lines as the exact 11 ,117 ESTs were collected. However, for the non-endothelial pool the EST count had increased from 173,137 to 178,653 as a result of further EST sequencing. The expectation value for the non-endothelial pool was kept at 10e-20 as the larger pool size made the expectation value more stringent and less likely to deliver false positive hits.

Although there was a good agreement between the old BLAST and combined method EST assignments for some genes, a problem with searching mRNA queries against an EST database is that any EST is able to hit more than one gene using an expectation value cut-off. In reality this is not possible, as an EST is derived from a single transcript derived from a single gene. Table 2 shows the number of EST sequences that hit more than one gene for both EST-to-gene assignment methods. From the endothelial pool using the earlier [8] method there were 5,228 from the 11 ,117 ESTs that were assigned to more than one gene using an expectation cut-off of 10e-30. The ambiguous assignment means that it is not possible to know which EST-to-gene assignment was correct without manual inspection and as such failed. Assuming the remaining 5,889 ESTs that hit only one gene were correctly assigned, this amounted to a 53% success rate. For the new, combined algorithm there were no EST sequences assigned to more than one gene and the success rate for EST-to-gene assignment from the endothelial pool was 91%.

Table 2: Comparison of the two EST-to-gene assignment methods. The new method of EST-to-gene assignment improved accuracy enabling a higher percentage of ESTs to be unambiguously assigned compared to the Huminiecki and Bicknell (2000) method [8].

Testing of statistical significance To measure statistical significance of differential gene expression using cDNA libraries there is a Digital Differential Display (DDD) tool available at Unigene. This tool employs the Fisher exact test [18] to measure statistical significance (P > 0.05) between two libraries. According to [15] the statistics used by DDD are not valid for measuring statistical significance across multiple cDNA libraries, but only between two cDNA libraries. A further requirement of DDD is that it is only valid for cDNA libraries that contain at least 1 ,000 sequences collected into Unigene clusters. Several endothelial cell line libraries used in this study contained less than 1 ,000 sequences and comparisons between more than two libraries were required. For these reasons, the DDD tool and Unigene clusters were of no use in this analysis.

The statistics in the analyses used here combine a generalised maximum likelihood ratio test with a False Discovery Rate (FDR) that accounts for the different size of the cDNA library pools. During cDNA library construction, bacterial colonies are picked at random from agar plates for single pass sequencing of the EST insert. This process is random and can be modelled by a Poisson distribution. To derive the appropriate statistical method, two hypotheses were compared with each other. The NULL hypothesis states there is no difference in gene expression between two cDNA library pools and any differences in gene expression are due to sampling errors from the picking of colonies. Alternatively, the difference in gene expression could be due to a genuine biological effect. The maximum likelihood ratio statistic (R-statistic) is derived by dividing the likelihood of seeing the data under the null hypothesis into the likelihood of seeing the data under the alternative hypothesis.

A p-value can be derived from the R-statistic as 2R is Chi square distributed. It should be noted that multiple testing on all genes in the human genome and using a p-value would result in many false positives. To account for multiple testing errors, a False Discovery Rate adjusted (FDR-adjusted) procedure was employed [19]. A q-value of 0.01 represents 1% false discovery rate and means that 10 in 1000 significantly differentially expressed genes were false positives. A q-value of 0.01 was considered to be significant.

Application of the statistics to the analysis

Applying the new analysis and statistics to our previous data [8], 14 genes were predicted to be significantly endothelial specific and a further 160 were significantly up- regulated in endothelial cells. Table 3 lists the 14 predicted significantly endothelial specific genes.

Table 3: Endothelial predicted genes from the original cDNA libraries analysed with the new gene assignment tool and statistical methods. 14 genes were predicted as significantly endothelial specific. A further 160 genes were predicted as showing significantly upregulated endothelial expression (q-value <= 0.01) but were not endothelial specific (i.e. had EST hits in the non-endothelial pool). With the new analysis there was no longer a need to cross reference to SAGE libraries for accurate prediction.

It is of interest to compare the 16 predicted endothelial genes listed in Table 7 of our previous analysis [8] with those found here. Three of the original 16 genes were no longer predicted as significantly endothelial and Table 4 summarises the results. RAMP2 had no ESTs in either pool; COL4A1 was up-regulated in endothelial cells but not to significance with a q-value of 0.5. In contrast, RASIP1 was endothelial specific with a single EST found in the endothelial pool but absent from the non-endothelial pool. However, the q-value of 0.36 was again not statistically significant. Table 4: Listing of the genes from Table 7 of Huminiecki and Bicknell (2000), and comparison with the new analysis. 13 of the 16 genes were significantly endothelial, however, non-endothelial hits to known endothelial genes showed that the choice of non- endothelial cell lines could be improved, q-values in bold denote a significance threshold of <= 0.01.

Three genes identified as endothelial specific in the original analysis were not found to be so here. ROBO4 hit the EST [GenBank:AA577940] from the library NCI_CGAP_HSC1 that is a flow-sorted and non-normalized bone marrow cDNA library. EST accession [GenBank:AI380234] hit CD93 that is from a B-cell, chronic lymphocytic leukaemia flow-sorted cell line (NCI_CGAP_CLL1), while vWF hit a non-endothelial EST from the NCI_CGAP_Br4 library [GenBank:AA721546]. The last library was prepared from micro-dissected normal breast duct tissue and in view of the extensive literature showing restriction of von Willibrand factor expression to endothelium, is presumably from endothelial contamination of the dissected tissue. In subsequent analyses the non- endothelial pool was refined to exclude such hits.

Current data with the new algorithm and statistics. Experiment 1. Employing the new EST assignment algorithm and the novel statistical method, a similar subtractive screen to [8] was carried out but this time with the most recent publicly available data. In our earlier 2000 study there were 11 ,117 endothelial EST sequences. This has now increased to 31 ,114 and 64% of the currently available endothelial cell data was new. Table 5 (below) lists the 30 endothelial cell libraries used.

In view of aberrant gene expression by carcinoma lines arising from genetic instability and endothelial contamination of libraries isolated by FACS sorting or micro-dissection, we constructed a non-endothelial pool with no carcinoma cell, flow sorted or micro- dissected lines of 136,336 ESTs from 208 Genbank normal, non-endothelial cDNA libraries (Experiment 1 ).

From a cDNA library endothelial subtraction analysis alone (Experiment 1 ), there were 431 genes that were significantly up-regulated in endothelial cell lines. Of these, 104 genes showed an endothelial specific profile (Table 6, below), as transcripts were absent in the non-endothelial pool. The gene with the most significant endothelial specific profile was the metallo-proteinase gene MMP1 , a surprising result as literature suggests this gene is widely expressed [20, 21]. It is worthy of note however, that MMP1 is also up- regulated in endothelial cells according to SAGE library analysis in Experiments 2 and 3. This analysis predicted ROBO4, CD93 and VWF as endothelial specific genes.

cDNA and SAGE library analysis combined. Experiment 2

For the second experiment, the data from the cDNA analysis of Experiment 1 was combined with SAGE in the same way as the 2000 analysis. The SAGE analysis used the latest endothelial libraries against a pool of normal non-endothelial libraries. There were 10 endothelial cell line SAGE libraries containing 427,254 tags and 11 normal non- endothelial libraries of 329,470 tags. 74% of the SAGE library data was new since 2000 and submitted to SAGEmap [22]. The SAGE library screen was very similar to the cDNA library approach as it involved comparing two pools of SAGE library cell lines using the SAGEmap xProfiler tool. Only genes with at least 10x the number of transcripts per million tags was considered significant with a p-value of 0.9 or more. Results are presented in Table 7, below, that lists 27 endothelial genes. cDNA and SAGE library analysis combined (including carcinoma cell line cDNA data). Experiment 3

Although cancer, micro-dissected and sorted libraries in non-endothelial cell lines were thought to contaminate and invalidate the analysis, there exist many of these libraries in the public domain. Thus, to maximise the chance of predicting a comprehensive set of endothelial genes, a final experiment (Experiment 3) was performed using non- endothelial libraries that included cancer, micro-dissected and sorted libraries (178,653 ESTs and 733,461 SAGE tags). The SAGEmap xProfiler analysis was again combined with a cDNA library subtraction. 58 endothelial genes were predicted from this analysis (Table 8, below).

A comprehensive set of in silico predicted endothelial genes

Combining the results of all three analyses gave a non-redundant list of 459 genes preferentially expressed at a statistically significant level in endothelial cells.

Experimental validation of the endothelial gene prediction

Real time PCR was carried out on predicted endothelial genes to examine the predictive power of the in silico analyses. A random selection of 12 genes (ECSM2, MMP1 , SOX18, ERG, RHOJ, APLN, MMRN2, STAB1 , LYL1 , ELTD1 , EFEMP1 and BMX) was PCR amplified from human umbilical vein endothelial cells (HUVECs), human dermal micro-vascular endothelial cells (HDMECs) and a selection of normal primary, non- endothelial isolates; human lung fibroblasts (MRC-5), human bronchial epithelial cells (HBEC), adult human epidermal keratinocytes, peripheral blood lymphocytes and hepatocytes. Total RNA was extracted and real-time PCR was performed to measure differential expression of these genes between the cell types. Figures 4 and 5 show the power of the bioinformatics models as all genes examined were either highly up- regulated or completely specific to HUVECs and/or HDMECs.

Tumour endothelial marker prediction

Following the prediction of endothelial genes, a second screen was performed to identify genes up-regulated in tumours or foetal tissue. Bulk tissue cDNA libraries that contain endothelium were used. The subtraction procedure carried out compared bulk tumour with bulk normal cDNA libraries from the same organ or tissue. The analysis involved six tissues, namely, lung, brain, colon, kidney, prostate and skin. Three foetal tissues (lung, brain and kidney) were also screened since foetal tissues, like tumours, have active angiogenesis. Specifically, 237 brain tumour bulk tissue libraries containing 140,621 ESTs were used versus brain normal libraries; 24 brain foetal bulk tissue libraries containing 69,862 ESTs were used versus brain normal libraries; 302 brain normal bulk tissue libraries containing 100,554 ESTs were used versus brain tumour/foetal libraries; 178 lung tumour bulk tissue libraries containing 108,107 ESTs were used versus lung normal libraries; 10 lung foetal bulk tissue libraries containing 112,690 ESTs were used versus lung normal libraries; 91 lung normal bulk tissue libraries containing 82,757 ESTs were used versus lung tumour/foetal libraries; 7 kidney bulk tumour tissue libraries containing 38,519 ESTs were used versus kidney normal libraries; 5 kidney bulk foetal tissue libraries containing 2,605 ESTs were used versus kidney normal libraries, 5 kidney bulk normal tissue libraries containing 72,476 ESTs were used versus kidney tumour/foetal libraries; 131 prostate bulk tumour tissue libraries containing 19,125 ESTs were used versus prostate normal libraries; 132 prostate bulk normal tissue libraries containing 68,480 ESTs were used versus prostate tumour libraries; 6 skin bulk tumour tissue libraries containing 12,484 ESTs were used versus skin normal libraries; 4 skin bulk normal tissue libraries containing 33,218 ESTs were used versus skin tumour libraries; 557 colon bulk tumour tissue libraries containing 143,025 ESTs were used versus colon normal libraries; and 134 colon bulk normal tissue libraries containing 37,269 ESTs were used versus colon tumour libraries to find differentially expressed genes.

By screening each tissue independently, the analysis was able to identify genes that were putatively up-regulated in a tissue specific fashion. Special attention was taken in choosing normal tissue libraries, to ensure that they contained no active angiogenesis (e.g. foetal libraries were avoided for the normal tissue pools). Genes that were both selectively or preferentially expressed in tumour or foetal tissues and preferentially expressed in endothelial cells constituted predicted TEMs. 27 genes were chosen as being potential TEMs based on the specific or/and significant up-regulation in multiple tissues (Table 9).

Discussion

Identifying genes of interest

There exists great interest in the identification of tissue specific genes as they often perform a unique function within that cell type. In the past, tissue specific genes were sought using a range of molecular subtraction techniques employing mRNA/cDNA from the cell type of interest and a putative 'control' cell. Examples of such techniques include subtractive hybridisation, PCR display and PCR select. These approaches have been highly successful but remain laborious and expensive. Recent approaches have included selective insertional gene trapping or FACs sorting of cell lineages labelled with GFP in e.g. zebrafish followed by gene chip analysis. Both techniques have been used to identify endothelial genes [23, 24], for example in zebrafish the endothelium and precursors were labelled with FIi promoter GFP. Nevertheless, such techniques are still expensive and laborious.

In silico analysis An alternative is to analyse computationally the vast amount of expression data now available in the public domain. We performed such an analysis in 2000 [8] that identified several previously unknown endothelial genes including Robo4, the endothelial roundabout guidance gene. A critical finding in the earlier analysis was the need to cross reference a cDNA with a SAGE analysis to achieve accurate prediction of expression. A complementary approach by Ho [7] combined cDNA and SAGE library database mining with microarray analysis. Virtual subtraction was carried out on data in the public domain using available tools to identify putative endothelial genes. These genes were then micro-arrayed and probed with RNA samples from a selection of cultured endothelial and non-endothelial cell types. A comparison of results is made below.

Introduction of new data gave a better analysis

We became aware that these earlier computational techniques could be improved and there are several cogent reasons for repeating such an analysis now. Firstly, the vast increase in expression data available now compared to 2000, in particular the number of ESTs in the endothelial libraries has more than doubled. Secondly, following the publication of the human genome, we developed a new technique that combines a BLAST search with genome BLAT alignments to increase the accuracy of EST-to-gene assignment. This removes the ambiguity of EST-to-gene assignment and consequent inaccuracies of the results present in our earlier analysis. Finally, we developed a novel maximum likelihood statistic analysis that can identify differentially expressed genes across multiple cDNA libraries. Using these improvements to cDNA library analysis and the inclusion of the latest SAGE library data, we have derived what we consider to be a near definitive set of endothelial specific genes.

cDNA library analysis improvements We previously showed that endothelial genes could not be reliably predicted by using cDNA library analysis alone. Two possible explanations for this were. 1) Computationally, the EST-to-gene assignment was inaccurate for some genes with the BLAST protocol chosen and 2) there was no statistical analysis applied to the EST counts in order to determine the significance of the differential gene expression.

Repeating our analysis on the cell lines used in our earlier [8] study validated the new approach. The new analysis proved the critical importance of accurate EST-to-gene assignment to enable a successful analysis using cDNA libraries alone. The new method produced a successful assignment of 91% of the ESTs compared with 53% for the earlier study. Using the 2007, as apposed to the 2000 data, gave 31,114 assigned endothelial ESTs and a success rate of 94%. It should be noted that this success rate is also dependent on the quality of cDNA libraries, but comparing like for like, the new algorithm improved the accuracy of assignment by 38%. In order to identify differentially expressed genes between two pools of cDNA libraries convincingly, it is essential to employ a rigorous statistical analysis. The method described here makes use of the intrinsic variability associated with cDNA library measurements and represents the most powerful statistical analysis possible associated with that model. We note that the test is more appropriate than a t-test, and more powerful than non-parametric statistics such as the Mann-Whitney test. Differential expression of cDNA libraries can be performed on line at the CGAP and Unigene. However, DDD was not used in these analyses as it does not employ the maximal statistics test and only performs differential expression between cDNA libraries that have at least 1000 EST sequences clustered into Unigene. In contrast, the maximum likelihood statistics used in these analyses can be applied to cDNA libraries of any size and the EST-to-gene assignment does not rely on Unigene clusters.

Comparison of endothelial genes with previous work

It is of interest to compare the results of this analysis with two previous bioinformatic analyses to identify endothelial genes, those of Huminiecki and Bicknell [8] and Ho et al. [7]. In our earlier (2000) study [8], 16 genes were predicted as endothelial by a combined SAGE and cDNA library analysis. From the 16 genes, 13 were also predicted as significantly endothelial in this study. The three genes that differed between the two analyses were COL4A1, RAMP and RASIP1. In the new analysis RASIP1 was endothelial specific but not to significance, COL4A1 was expressed in both cDNA library pools and RAMP was not expressed in either pool. It is interesting that ECSM2 was the most endothelial specific gene in both the Huminiecki and Bicknell [8] and Ho et al [7] studies and was predicted as endothelial here but it was not ranked first, ROBO4 and MMP1 ranked higher. Real time PCR (Figure 5) and in-situ hybridisation (data not shown) show extreme endothelial specificity for ECSM2 and its lower ranking is simply due to fewer ESTs, i.e. it is expressed at a lower level in the cDNA libraries. A comparison with the endothelial genes found in this study with that of Ho et al [7] reveals 30 of the 49 genes were predicted as significantly (q-value <=0.01) up-regulated in endothelial cells. A further 5 genes were endothelial specific but not to significance (q- value > 0.01). 14 genes failed to show significant or specific expression in endothelial cells. Interestingly, the second ranked endothelial gene from the [7] analysis, SHE, showed only a single endothelial EST in this analysis. We conclude that although tissue specific genes can be predicted by cDNA analysis alone, it is advisable to use as many data sources as possible in order to derive a comprehensive list of genes. Finally, our results show that it is better to use normal cell isolates than carcinoma cell lines or libraries derived from micro-dissected or FACS sorted cells for this type of analysis, since several characterised endothelial genes hit ESTs in these non-endothelial libraries (Table 4: VWF, ROBO4 and CDH5).

Extended analysis to find TEMs We extended the analysis to identify which of the endothelial genes were expressed in tumours but not normal tissue. This was achieved by combining the endothelial screen with an analysis that compared gene expression between tumour and normal bulk tissue libraries from several organs. A gene was a predicted TEM if it was preferentially expressed in endothelial cells and tumour tissues but absent in normal tissues. A list of 27 promising new TEMs based on these analyses is given in Table 9. Each cell in Table 9 represents the result for a tumour/foetal screen for a particular organ. Ultimately, we wanted to find genes that showed tumour and foetal specific expression in all or most of the organs at a statistically significant level. Cells with bold, dotted-underlined text represent this type of result, having 0 ESTs in the normal pool and a q-value of less than 0.01. Genes showing significant and specific expression in multiple organs (brain, skin, kidney and foetal lung) were PLOD3 and THRAP4. However, these genes showed expression in normal tissue for several other organs. Likewise some genes were significantly up-regulated in tumours in multiple organs but were not specific to tumours (cells with double-underlined text). These genes, although putative TEMs, were considered of least therapeutic value, as some expression was evident in normal tissues and as such could not be used to specifically target tumours. Investigation of a subset of predicted TEMs

As endothelium comprises less than 5% of tumour tissue, it was hypothesised that genes with a tumour specific although not statistically significantly different expression could still be a TEM. Such cells are shown in Table 9 with single-underlined text. The most promising TEMs from Table 9 were selected based on little or no expression in normal tissue across all or multiple organs (cells with single-underlined and bold, dotted- underlined text, respectively). Of these angiopoietin 2 (ANGPT2), protocadherin 12 (PCDH 12) and leucine rich repeat containing 8 family, member C (LRRC8C) had expression profiles totally restricted to tumour or foetal tissues. ANGPT2, in these in silico results, was restricted to renal and colon tumour tissue in adults and lung in embryos that is supported by the current literature that indicates ANGPT2 is associated with tumour endothelium and tumour progression [25-28]. In contrast, leucine rich repeat containing 8 family member C was not found to be a TEM in the literature but a gene responsible for adipocyte differentiation [29]. A list of 9 putative novel TEMs with the best tumour profile is listed in Table 1 , above. This table excludes genes that already have substantial literature (e.g. angiopoietin2) as possible or actual TEMs. Another gene with an interesting literature is mediator of RNA polymerase Il transcription subunit 28 homolog (S. cerevisiae, MED28): research has shown MED28 to be significantly up- regulated in tumours, its over expression is able to stimulate cellular proliferation and its expression is up-regulated by endothelial cells when exposed to tumour media [30, 31].

TEM experimental validation

To experimentally validate the nine newly identified TEMs (Table 1), in situ hybridisation and immunostaining are the most definitive direct methods. However, the sensitivity to optimisation of the first makes it tricky for high throughput analysis. The second requires antibodies and the time to prepare these slows progress. It is the possible that recently developed phage antibody technology may overcome the delay in TEM validation. Therefore to validate our approach at this time, the next section describes the analyses and literature search results for previously predicted tumour endothelial markers.

Validation of TEM prediction based on known TEMs

Delta4 has been cited to have endothelial specific expression [32-34] and to be up- regulated in tumour vessels [32, 35]. In this study, Delta4 (DLL4) was endothelial specific but was expressed at a very low level in endothelial cell cultures. Delta4 matched one EST from the endothelial pool and none from the non-endothelial pool, with an FDR-adjusted q-value of 0.28. Even though this gene is not statistically significantly up-regulated in endothelial cells, it shows some evidence of being endothelial specific as there are no ESTs found from the non-endothelial pool. DLL4 was found in brain and colon tumour tissues. However, the expression was not specific or significant in tumours. Thus, in our analysis DLL4 was not a predicted TEM. GPR124 (TEM5) was previously identified as a putative TEM using custom SAGE libraries analysis [14]. In the current analysis, GPR124 failed to match any endothelial ESTs from the 31,114 EST in the endothelial pool. From the non-endothelial pool, GPR124 did match a single EST [GenBank: BF325872] from the AN0041 cDNA library derived from a normal amniotic fluid cell line. These results suggest that GPR124 is only expressed at a low level in normal tissue and is absent or at a very low level in cultured endothelial cells. In contrast, GPR124 was predicted as significantly and specifically up-regulated in multiple tumour tissues. Thus, GPR124 appears to be a tumour but not a tumour endothelial marker. TEM1 (endosialin or CD 248) [14] has a count of 1 and 2 ESTs for the endothelial and non-endothelial pools respectively. The FDR-adjusted q-value for this gene was 0.61 , a non-significant value. One EST from the non-endothelial pool, accession [GenBank: CN484271] was from a primary human ocular pericyte cDNA library. This agrees with experimental findings of MacFadyen et al [36, 37] that have shown that endosialin is expressed by fibroblasts and a subset of pericytes associated with tumour vessels but not by tumour endothelium.

Several groups have independently reported ROBO4 as a TEM [38-40]. In this study ROBO4 was highly endothelial specific, both from the in silico and experimental analyses. In the tumour screen, ROBO4 was seen to be tumour specific in brain and kidney tumour tissues but not at a statistically significant level. Thus ROBO4 was predicted as a tumour endothelial marker, but not in all tumour types. In this case our analysis may be under predictive, as experimentally ROBO4 has been found to be a strong TEM [39, 40]. This also demonstrates the absolute need for experimental verification of bioinformatics predictions. Numerous studies have reported SPARC to be up-regulated in endothelial cells, to have a role in tissue remodelling and be linked to tumour progression [41-44]. Our analysis strongly predicted SPARC to be a TEM. SPARC was up-regulated in endothelial cells with a significant q-value of 8.4 x 10-10 and also significantly up-regulated in brain, colon, kidney and prostate tumour tissue. VIM was significantly up-regulated in multiple tumour tissues and endothelial cells. It is an abundant intermediate filament protein and a secreted form of VIM has been shown to be the antigen for the endothelial cell-specific antibody PAL-E [45]. The integrin receptor α2β1 interacts with intracellular endothelial VIM and plays a role in endothelial cell to collagen adhesion [46]. Therefore, a combination of experimental and in silico evidence predicts VIM as a TEM. As above angiopoietin 2 has tumour/foetal specific profile from the in silico analysis this is confirmed by current literature [25-28].

There is evidence both for and against the use of cDNA analyses for the prediction of TEMs. If TEM1 , TEM5 and DLL4 are true TEMs then this technique is not 100% reliable. In contrast, the successful prediction of ROBO4, ANGPT2, VIM and SPARC shows that these methods do have the ability to predict a validated TEM. The novel predictions of this analysis await validation.

Conclusions

New cDNA library data is continually been submitted to Genbank and the amount of relevant information that can be mined is increasing. cDNA library analysis has been improved in this work by more accurate EST-to-gene assignment and the best possible statistics applied to the data. Using these tools on the latest data sets will lead to the prediction of new biologically and therapeutically important genes. This is enhanced by the statistics as they enable the inclusion of cDNA libraries of all sizes.

We have shown that these methods accurately predict the identity of endothelial and tumour endothelial genes by comparing our results with that of known genes. For example, ROBO4 has consistently been shown to be highly endothelial specific and was ranked second in this work. Known TEMs were also successfully predicted as shown by the identification of SPARC and Angiopoeitin 2.

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Table 5: Endothelial cDNA libraries available at Genbank that were used in this study. 21 new libraries have been submitted since our previous analysis [8]. The 30 combined libraries incorporate 31 ,114 endothelial ESTs.

Table 6: The 104 genes in the human genome with the most endothelial-specific expression profile predicted by applying the new analysis to the latest cDNA libraries.

Table 7: Combining cDNA and SAGE library analysis for endothelial gene prediction (normal non-endothelial libraries). 27 genes were predicted to be endothelial specific using a combined SAGE and cDNA library analysis of the latest libraries. The genes are sorted in descending order according to the number of non-endothelial library hits. Experimentally well-characterised endothelial genes are highlighted in bold.

CO 00

Table 8: Combining cDNA and SAGE library analysis for endothelial gene prediction (tumour, microdissected, sorted non-endothelial libraries)

CD CO

O

Table 9: Potential Tumour Endothelial Markers.58 endothelial specific genes were predicted by SAGE-CGAP xProfiler. All SAGE and cDNA non-endothelial cell libraries, including those from transformed cell lines and those produced by tissue micro-dissection or cell sorting, were used in this analysis

Gene Brain colon kidney Lung skin prostate Foetal kidney Foetal lung Foetal brain

SPHK1 D 0.861 1 U 0.5720 U 0.3810 U 0.332 0 U 0.222 0 -- -- U 0.4310 U 0.83 1 1

KCTD15 U 0.3320 U 0.6210 D 0.4202 U 0.086 0 U 0.453 3 -- D 0.7902 U0.01.13.Q U 0.21 20

LRRC8C U 0.3320 U 0.6210 -- -- -- U 0.41 1 q U 0.3320 --

PCDH12 U 0.4310 U 0.5230 U 0.3810 -- -- -- -- U 0.2330 --

SPARC U 00015531 U 001826 U Q 00378 D 0.1821 29 U 0.71 12 27 U 00032 ?5 M 00068 U 0.445029 U 0.442831

ANGPT2 -- U 0.5230 U 0.1330 -- -- -- -- U 0.4310 --

VIM U 0003739 D 0.753611 U 000359 U 0.8750 36 LJ 00023 11 D 0.702 10 U 0.0749 D 0.534036 U 000 449

O CO BGN U 000614 U 0.95287 U 0.8723 U 0.1421 6 U.Q.M.Z o U 0.551 1 D 0.7903 D 0.8576 D 0.81 24

C12orf11 U 0.1150 U 0.4640 D 0.6314 D 0.370 1 U 0.31 1 0 U 0.41 1 0 D 0.7904 U 0.8421 U 0.21 20

C16orf30 U 0.0760 U 0.4050 D 0.201 12 D 0.370 1 -- D 0.540 2 U 0.791 12 U 0.04131 --

ECOP U 0.00270 U 0.4640 D 0.3703 U 0.593 1 U 0.851 2 D 0.550 1 D 0.7903 U 0.8421 --

ECSM2 U 0.1640 U 0.4640 -- U 0.593 1 -- U 0.701 2 -- U 0.2330 --

ERG -- -- D 0.7713 U 0.332 0 -- U 0.41 1 0 D 0.7903 U 0.0480 U 0.34 10

GBP4 U 0.4310 U 0.6210 U 0.0350 U 0.377 2 U 0.31 1 0 D 0.540 2 -- D 0.2302 --

IKBKE U 0.0760 U 0.5720 U 0.3810 D 0.592 3 -- -- U 0.7163 --

LOC653949 U 0.0760 U 0.57101 «0,00320 D 0.373 6 D 0.530 1 D 0.551 8 -- D 0.4346 U 0.34 10

2 27 predicted TEMs that were significantly endothelial were also up-regulated or specific to tumours. Three foetal tissues were also screened as they contain regions of active angiogenesis. The information held in each cell is as follows: e.g., U 0.21 3 0 (space delimited): U/D = gene was Up or Down-regulated in the tissue; 0.21 = FDR q-value; 3 0 = EST counts, 3 tumour bulk tissue ESTs and 0 normal bulk tissue ESTs. 5 Single-underlined text denotes genes showing tumour or foetal specific expression (no counts in normal tissue) but not at a statistically significant level.

Double-underlined text denotes genes that showed statistically significant (q-value <= 0.01) differential expression but not specific (some expression seen in normal tissue).

Bold and dotted-underlined text is for genes that were both significantly (q-value <= 0.01) and specifically (no counts in normal tissue) 10 up-regulated in tumour or foetal tissues.

Example 2A: Function of the Small GTPase RHOJ in endothelial cells Abstract

Rho GTPases are cellular switches and part of multiple signalling pathways within the cell. In their activated GTP-bound state, these proteins stimulate important cellular processes such as the reorganisation of the actin cytoskeleton, cellular traffic, endo- and exocytosis, migration or adhesion to other cells as well at the adhesion to the extracellular matrix. All of these processes play a pivotal role in angiogenesis.

Because we found that RhoJ was specifically upregulated in endothelial cells, its exact function in HUVEC was investigated. RhoJ expression was knocked down using two different siRNA duplexes and the effect on cell behaviour was determined using growth, tube formation and scratch wound assays. The kinetics of RhoJ downregulation were determined by performing a western blot with cell lysates of siRNA transfected cells isolated on days 1-4 after transfection. The specifity and degree of RhoJ downregulation relative to β-actin was confirmed with quantitative PCR. Open reading frames of RhoJwt and RhoJQ79L, a constitutive active mutant, were cloned into pEGFP-N1 and pcDNA my c-H is vectors, and the generated plasmids were used to image RhoJ localisation in

293T cells or in HUVEC and to induce RhoJ overexpression in HUVEC. The effect of

RhoJ overexpression was then investigated by performing tube formation and scratch wound assays.

RhoJ downregulation in HUVEC reduced cell growth, significantly affected tube formation and inhibited cell migration in the scratch wound assay. The powerful effect of RhoJ downregulation indicates that RhoJ may play an important role in tumour angiogenesis.

Introduction

Angiogenesis

The formation of new blood vessels results from two different processes: vasculogenesis, where precursor cells (angioblasts) differentiate into endothelial cells and form a primitive vascular network; and angiogenesis, the development of vessels of an elementary, pre-existing network into a complex system. Although both processes are found in the adult, primarly angiogenesis occurs and leads to functional and mature vessels [1]. Angiogenesis in adult tissues plays an important role in wound healing, physiological growth of organs and in the female reproductive cycle. In normal tissue it is highly regulated and newly formed endothelial cells remain quiescent. In pathologically changed areas, as seen in cancer, diabetic retinal neovascularisation, arthritis, psoriasis and other chronic inflammation as well as in prolonged bleeding during the menstrual cycle, increased angiogenesis with mainly disorganised and non-functional vessels are observed [2;3].

Angiogenesis occurs in response to autocrine or paracrine produced growth factors, hypoxia, mechanical or inflammatory stimuli. It is highly regulated by the environment and different cellular proteins and in the case of a loss of function of these proteins, pathologies can emerge [5]. The first step of angiogenesis involves vasodilatation and increased permeability of existing vessels, followed by the loss of plasma proteins. The leaked proteins provide a new basement membrane, which allows the seeding of endothelial cells after their migration from existing vessels into the interstitial space. To facilitate migration, the vascular basement membrane is locally degraded by matrix metalloproteinases (MMP) produced by endothelial cells, and the endothelial cells lose their close contact with their surrounding pericytes. Pericytes are normally adjacent to endothelial cells on the outside of the vessel and inhibit their proliferation. This inhibition is lost when pericytes cease to contact the endothelial cells. Endothelial cells then form a sprout into the interstitial space, which turns into a migration column by ongoing proliferation. Ultimately the migration column may join preexisting vessels, recruit new pericytes and generate a new basal lamina [2,A].

The movement and rearrangement of cells plays a pivotal role during angiogenesis. For retraction and migration the actin cytoskeleton and cell-cell contacts, as well as cell- matrix contacts need to be disrupted and reformed.

Rho GTPase family

Rho family GTPases are cellular switches, which cycle between GTP-bound active and GDPbound inactive forms. In their active GTP-bound state, Rho GTPases bind to their effector proteins and transmit signals within the cell. The structure of GTP- and GDP- forms differ in the position of two loops which protrude from the proteins surface - these are known as switch region I and Il [8]. The position of these loops are important for GTPase binding proteins to recognise the correct state of the GTPase. NMR structure studies have shown that the CRIB (Cdc42/Rac-lnteractive binding) motif of some Cdc42- and Rac-binding proteins interact with switch I and that the CRIB motif is necessary for a strong binding to the GTPase. For example, two CRIB containing protein which bind to Cdc42 are the Wiskott-Aldrich-syndrome protein (WASP) and the p21 -activated kinase-1 (PAK-1). However, switch I and Il are not the only regions being recognised by other proteins, for instance, the C-terminal regions of the Rho GTPases [6].

Another structural characteristic of the Rho GTPase family is a CAAX (C: cysteine, A: aliphatic aminoacid, X: any aminoacid) motif at their C-terminus, where the cysteine is post-translationally modified with a geranylgeranyl lipid. The lipid modification is crucial for the insertion into the membrane and determines the expression area within the cell. Furthermore it localises the GTPase at the potential interaction site for their effector proteins [6;7].

The GTP-GDP exchange in Rho GTPases is regulated by guanine nucleotide exchange factors (GEFs), which enhance the replacement of GDP with GTP, and GTPase- activating proteins (GAPs), which promote the hydrolysis of GTP to GDP. Although Rho proteins have an intrinsic GTPase-activity by themselves, by interacting with GAPs the GTP hydrolysis proceeds faster and the GTPase converts into the inactive state [3].

Besides GEFs and GAPs, GDP dissociation inhibitors (GDIs) play an important role in regulating GTPase activity. Rho GDIs repress Rho GTPase function by different mechanisms. First, they stabilize the GDP-bound inactive state by inhibiting the activation through GEFs, and second, they inhibit GTP hydrolysis in the active state of the GTPase. Third, besides regulating the activation of Rho GTPases, GDIs form complexes with the small GTPases, such that the GDI covers the lipid-part of the Rho protein and prevents the insertion of the GTPase into a membrane. Thus GDIs sequester Rho proteins in the cytosol, away from membrane-bound effector proteins [9, 10].

The Rho GTPase family consists of 20 different low molecular weight (between 20-30 kDa) proteins, which can be divided into five main subfamilies: the Rho-like, Rac-like, Cdc42-like, Rnd and RhoBTB groups. Among these proteins RhoA, Rac1 and Cdc42 are the most extensively studied Rho family members [11 ;12]. Initially, the Rho family was identified due to its role in regulating the actin cytoskeleton. It was shown that RhoA signalling was involved in the formation of stress fibres and focal adhesions, Rac in the formation of focal contacts and lamellipodia and Cdc42 forfilopodia formation [13]. Rho GTPases mainly regulate the assembly and disassembly of the actin cytoskeleton, but play a role in many other cellular processes, such as cell polarity, gene transcription, cell cycle and vesicular transport. Rho proteins thus take part in several physiological and pathological processes such as cell proliferation, cell movement, establishment of cell polarity, metastasis and cell transformation [14].

Rho proteins are involved in a wide variety of signalling pathways and are activated in response to extracellular stimulation by growth factors, hormones, cytokines or adhesion molecules. They can be activated by G-protein-coupled receptors (GPCR), receptor tyrosine kinases (RTK), cytokine receptors, some cell adhesion proteins such as integrins and cadherins and members of the Immunoglobulin superfamily [15; 16]. Rho proteins are not activated by the receptors themselves, but via the regulation of GEF and GAP activity. Signalling may occur through non selective GEFs and GAPs, which activate several different Rho proteins. Some transmembrane receptors activate different GEFs and GAPs, which means receptors can influence multiple Rho GTPases [16]. In addition to the activation through transmembrane receptors, crosstalk between different Rho proteins may occur. Rho proteins are able to repress GAPs or stimulate GEFs, and thus regulate other family members [12].

The activation of Rho GTPases leads to the mobilisation of different effector proteins, such as kinases or other Rho GTPase effector proteins. Rho proteins mainly activate kinases by interfering with the autoinhibitory interactions. Upon binding to the kinase, Rho GTPases cause the removal of autoinhibitory domain and expose the kinase domain to possible downstream substrates for phosphorylation. In addition, active GTP- Rho may also induce oligomerisation of effector molecules or induce the formation of a complex of proteins able to activate a signalling pathway. Beside the interaction of GTPases with effector proteins, there are also interactions with phosphatidylinositol- phosphate signalling. Some of the interactions between Rho proteins and their downstream molecules are detailed in references [6, 12 and 17]. Cdc42, the most closely related Rho protein to RhoJ that has been extensively studied, activates a number of effectors, which affect multiple aspects of the actin cytoskeleton [6].

Rho GTPases have an important role in endothelial cell behaviour and in endothelial migration, endothelial barrier function and permeability. Endothelial cell migration consists of four different steps: lamellipodium extension, formation of new adhesions, cell body contraction and tail detachment. All steps except tail detachment are known to be regulated by Rho GTPases. For instance, Rac is responsible for lamellipodial extension and is activated by chemotactic stimulation of VEGF, cytokines or by the extracellular matrix. Rac activates its downstream proteins, which leads to accumulation of actin at the leading edge of the cell and it is also required for the formation of focal adhesion complexes within lamellipodia. Focal adhesion complexes mediate the attachment of the cell migrating into the new environment. Furthermore, Rac is involved in the disassembly of these adhesion complexes. RhoA particularly mediates cell body contraction after its activation by vascular endothelial growth factor (VEGF), sphingosine- 1 -phosphate and shear stress. RhoA activation leads to cell body contraction via Rho- kinases (ROCK) and MLC phosphorylation and Cdc42 plays an important role in filopodia formation. It initiates actin polymerisation for filopodial extension. Receptors on these extensions detect chemical changes in the environment, upon which the cell can respond and modulate the directions of its migration [13], [17].

Endothelial barrier function is mainly mediated by adherens junctions and Rho GTPases in endothelial cells are also involved in adhesion to cells or to the extracellular matrix. Adherens proteins, primariy Vascular endothelial (VE)-cadherin, are responsible for the maintenance of endothelial barrier function, but are also involved in migration and cell survival. Rho, Rac and Cdc42 act as downstream proteins of VE-cadherin-mediated signalling and therefore regulate endothelial permeability. Even though some impacts of the Rho GTPases are known, their exact roles remain to be elucidated [13].

To investigate the function of Rho GTPases multiple bacterial toxins which selectively, covalently bind to some Rho GTPases and activate or inactivate them, are used. One commonly used toxin is C3 exoenzyme from Clostridium botulinum, which disrupts the actin cytoskeleton by ribosylation of RhoA, RhoB and RhoC. Since toxins can often affect more than one Rho GTPase, their activity cannot always be ascribed to their inhibition or activation of a single Rho GTPase [10; 17]. To investigate the exact role of a single Rho GTPase, dominant active and dominant-negative mutants are often used.

Thus, to determine RhoJ function, the constitutively active mutant RhoJQ79L and the wildtype RhoJwt were used. Since Toledo et a/ (2003) found that the dominant-negative

RhoJ was degraded after transfection into HeLa cells, we used siRNA to downregulate

RhoJ, rather than using overexpressing the dominant-negative form to knockout RhoJ function. RhoJ / TC10 like (TCP

Vignal et al (2000), who initially cloned and characterized RhoJ/TCL, demonstrated that RhoJ is most closely related to TC10 and Cdc42 Rho GTPases [19]. The amino- terminus and the effector loop are particularly highly conserved. Like TC10 and Cdc42, activated RhoJ also binds to the CRIB-motif of WASP and PAK1. RhoJ as well as Cdc42 can influence the actin cytoskeleton. Constitutively active RhoJQ79L protein expression in REF-52 fibroblasts results in strong actin accumulation at a single point in the cell and in the reduction of stress fibers and filopodial extensions [19]. Additionally, expression of constitutively active RhoJ protein leads to the formation of large intracytoplasmic vesicles. Besides the changes in the cytoskeleton, cell morphology is altered too, such that RhoQ79L transfected REF-52 cells show an elongated appearance with long and thin extensions at their border. This observation contrasts with the effect of overexpressing TC10, which results in the formation of large cellular protrusions. Thus despite their close relatedness, RhoJ and TC10 show different cellular functions [19].

Within the cell, RhoJ was found to be localized to vesicles of the endocytic pathway, namely the early and sorting endosomes and at the plasma membrane [7]. However, RhoJ was not found associated with lysosomes, recycling and late endosomes, the Golgi apparatus, nor secretory vesicles and was not found in the cytosol. In addition Toledo et al (2003) showed that RhoJ plays an important role in the endocytic pathway. By suppressing RhoJ, the regulation of the cellular transport of Transferrin (Tf) in HeLa cells was impaired and Tf distribution was visible throughout the cytoplasm compared wit HeLa cells transfected with control siRNA, which showed a local Tf accumulation in juxtanuclear recycling endosomes. In addition, Tf release studies indicated a slower exocytosis in RhoJ repressed cells, whereas the Tf intemalisation itself was not affected Ul

Another pivotal role of RhoJ was described by Nishizuka et al (2003) who showed RhoJ upregulation during adipocyte differentiation [20]. Further experiments indicated that RhoJ overexpression enhanced the differentiation and reduced RhoJ expression inhibited the differentiation of NIH-3T3 fibroblasts into adipocytes. Although TC10 is involved in adipocyte differentiation, TC10 mRNA expression occurs later in this process and therefore implies different roles of RhoJ and TC10 in adipocyte differentiation [20]. Nevertheless, the physiological role of RhoJ has not been well defined.

Using bioinformatic approaches, we have found that RhoJ mRNA expression is highly endothelial specific, which we then validated by quantitative PCR (Figure 6). Due to the restricted endothelial expression of RhoJ and the fact that other Rho GTPases such as RhoA and Rac1 are crucial for cell migration and maintaining endothelial barrier function, the role of RhoJ in aspects of endothelial cell function was examined. Thus in order to understand the role of RhoJ in endothelial cell biology and in angiogenesis the effects of both overexpressing and inhibiting the expression of RhoJ was examined in a number of in vitro assays including cell movement, tube formation and cell growth.

Materials and Methods

Where specific protocols are not provided, standard laboratory protocols well known in the art were followed.

Cell lines

Human umbilical vein endothelial cells (HUVEC, TCS Cell Works Ltd., Buckingham, UK) were used for the transfection and for growth and migration studies. Human embryonic kidney cells 293T (293T) were used to examine the anti-RhoJ antibody binding efficiency.

DNA inserts for mammalian expression of the protein

Oligonucleotides used to produce cDNA inserts

Oligonucleotides were generated by Alta Bioscience (University of Birmingham, UK).

Oligonucleotides used in quantitative PCR studies

Oligonucleotides were produced by Eurogentec (Seraing, Belgium) or Roche Applied Science (Bell Lane, UK). Corresponding probes were part of a probe library from Exiqon (Vedbaek, Denmark) [21].

Ribonucleic acid (RNA)

Small interfering RNA duplexes were generated by Eurogentec (Seraing, Belgium).

Antibodies

Cell Cell culture media used lines

HUVEC Large Vessel Endothelial Cell Basal Medium + Large Vessel Endothelial Cell Growth Supplement (5Ox) + Antibiotic Supplement (Gentamicin/ AmphotericinB) + 10% FCS

293T DMEM + 10% FCS + 0.01% Penicillin/ Streptomycin + 0.01% Glutamate

Cells were cultured at 37°C in a CO₂-incubator (Sanyo, Loughborough, UK) with 5% CO₂. Before plating HUVEC, 10 ml of 0.1% Gelatine/PBS solution was added onto the culture dishes, incubated for 30 minutes and then aspirated.

Transfection of siRNA

One day prior to transfection 6.9 x 10⁵ cells were seeded onto a 10 cm culture dish. Cells were washed twice with PBS the next day, covered with 3.2 ml Optimem and 800 μl of the transfection mix (prepared as described below) and tilted for mixing. After an incubation of 4 hours at 37°C, the transfection mix was replaced by Large Vessel Endothelial Cell Basal Medium (Supplement + 10% FCS, without antibiotics). Cell lysis and growth assays were performed 48 hours after transfection.

For the transfection mix, in separate tubes 10 μl of siRNA duplexes (20 μM) were mixed with 670 μl Optimem and 12 μl Lipofectamine 2000 with 108 μl Optimem. As negative control, a mock transfection and a negative control duplex (binds no known sequence, Eurogentech, Seraing, Belgium) were used. Both mixtures were incubated at RT for 10 minutes before 120 μl of the Lipofectamine 2000 mix was added to the siRNA mixture. This was incubated at RT for a further 10 minutes before adding to cells.

Generation of the RhoJ-insert by PCR For a final volume of 100 μl, 83.5 μl water, 10 μl of 10x cloned Pfu reaction buffer (Stratagene, La JoIIa, USA), 2 μl of dNTP (1OmM, Bioline, London, UK), 0.5 μl of each Oligonucleotide (100 μM) and 1 μl of PfuTurbo DNA Polymerase ( 2.5 U/μl, Stratagene) were mixed with 2.5 μl of RhoJ template (HUVEC cDNA at 50 ng/ml or for Q79L a plasmid generously supplied by Philippe Fort, CRBM/CNRS Montpellier, France) in this order. As negative control the template was omitted. PCR was performed by denaturation for 2 minutes at 95°C, followed by 30 cycles on 95°C for 30 seconds, 30 seconds on 50⁰C and 2 minutes on 72°C. Finally the PCR product was incubated on 72°C for 10 minutes and cooled down to 4°C. PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Crawley, UK).

siRNA annealing and labelling siRNA duplexes were annealed using the siRNA annealing protocol (Eurogentec), according to the manufacturer's instructions.

Quantitative PCR

Quantitative PCR was performed using Exiqon system setup (Vedbaek, Denmark) [21] and Rotor- Gene RG-3000 (Corbett Research Limited, Sidney, Australia). For a final volume of 25 μl per sample 12.5 μl qPCR Mastermix (Abgene, Epsom, UK), 1 μl of the forward and the reverse primer (10 μM), 0.25 μl of the Exiqon probe, 0.25 μl of deionised sterile water and 10 μl of cDNA were mixed. For each cDNA, dilution series were prepared according to the cDNA amount of the individual protein. Samples were then heated at 95°C for 10 minutes followed by 40 cylces at 95°C for 15 seconds and 60⁰C for 45 seconds and analysed with Rotor-Gene 6 software (Corbett Research Limited).

RhoJ-EGFP expression in living HUVEC

RhoJwt-EGFP and RhoJQ79L-EGFP constructs were transfected into HUVEC. As a control, pEGFP-N1 was used. Two days after transfection and six hours prior to visualisation, HUVEC were seeded onto a gelatine coated cover slip in a six well plate. Cover slips were washed with PBS, covered with medium and viewed with a Zeiss Axiovert 200M microscope Zeiss, Welwyn Garden City, UK). Images were captured using Slidebook 4 software (Olympus, London, UK).

Immunohistochemistrv on human placenta and human heart When placenta embedded in paraffin slides was used, the slides were immersed three times in histosol (National diagnostics) for 5 minutes, followed by a an immersion in 100% ethanol for 5 minutes, once in 70% ethanol and then slides were washed in distilled water. Afterwards slides were heated to the boiling point in citrate buffer (0.1 M pH 6), incubated in the hot solution for 5 minutes, reheated to the boiling point and plunged into cold water. When frozen sections were used, slides were immersed in ice cold acetone for 10 minutes and air dried for another 10 minutes. All sections were then washed with PBS/Triton (0.1%) solution and blocked with 2.5% horse serum (Vector laboratories) for 30 minutes at RT. After adding 150 μl of primary antibody and an incubation for 30 minutes at RT (alternatively overnight at 4°C), slides was rinsed three times with PBS/Triton (0.1%). 5 drops of secondary antibody was added and incubated for 30 minutes at RT, rinsed three times with PBS/Triton (0.1%) and once with deionised water. Slides were then dried and covered with peroxidase substrate AEC (Vector laboratories), incubated for 30 minutes at RT in the dark and washed with deionised water. After an immersion in haematoxyiin for 30 seconds, slides were washed with tap water and covered with DAKO Cytomatation Ultra Mount (DAKO Cytomation). Slides were viewed with a Leica DME microscope (Leica, Milton Keynes, UK).

Cvtospin and Immunohistochemistrv with RhoJ transfected 293T cells and HUVEC 2 x 10⁴ cells in 200 μl PBS were added to a cytospin tube and spun at 60 g for 10 minutes (Shandon Cytospin 3, Thermo, Cheshire, UK). After air drying, slides were fixed in methanol (-20⁰C) for 10 minutes, air dried and stained as described above.

RhoJ Protein expression after siRNA transfection

1.75 x 10⁴ HUVEC were seeded on a gelatine coated well of a 6 well plate and transfected with siRNA. 4 plates were prepared to visualise the development of protein expression over 4 days. On day 1-4 after transfection, cells were lysed and SDS-PAGE and a western blot were accomplished. Proliferation assay by direct cell counting

4 hours after siRNA transfection 1.5 x 10⁴ HUVEC in 1 ml medium were seeded per well of a 24 well plate. 3 plates were prepared to count the cells on day 1-3 after transfection. Medium was replaced daily and cells were counted with a hemocytometer.

Matrigel tube formation assay

After matrigel was thawed on ice overnight, 200 μl of matrigel was added and spread in each well of a 6 well plate. The plate then was incubated for 10 minutes at 37⁰C to allow solidification. During the incubation HUVEC were trypsinized and counted. 4 x 10⁵ cells in 2 ml Large Vessel Endothelial Cell Basal Medium containing Large Vessel Endothelial Cell Growth Supplement (5Ox) and 10% FCS were seeded onto the matrigel. After 2, 4, 8 and 24 hours pictures were taken with Leica DM IL microscope using USB 2.0 2M XLi camera.

Scratch wound assay

3.5 x 10⁵ HUVEC were seeded on a gelatine coated well of a 6 well plate. The following day with a 200 μl pipette tip a scratch in the middle of the well was made. Cells were washed twice in PBS and 2 ml medium containing mitomycin C (2.5 μg/ml) was added. Mitomycin C was added to ensure that cells can no longer divide. Pictures were immediately taken using Leica DM IL microscope. Other pictures were taken 4, 8 and 12 hours after creating the scratch.

Results

Cloning of RhoJwt and RhoJQ79L into pcDNA 3.1 B(-) mvc-His and pEGFPNI plasmids To generate RhoJwt and RhoJQ79L myc-His tagged expression constructs and RhoJwt and RhoJQ79LEGFP fusion proteins, RhoJwt and RhoJQ79L open reading frames were amplified by PCR and digested with BamHI and Xhol restriction enzymes. pcDNA myc- His and pEGFP-N1 were used as vectors, and digested with the same enzymes. Inserts and vectors were then ligated. For confirmation of the cloning, full length plasmids without the inserts and plasmids containing either RhoJwt or RhoJQ79L were digested with BamHI and Xhol and run on an agarose gel. The constructed RhoJ plasmid gave both a band representing the linear vector fragment at 5 kb and a band of 0.6 kb, which represented the RhoJ insert (data not shown). The parental vectors were digested as control and showed a single band at 5 kb. To confirm the exact cloning and to exclude any PCR generated mutations, sequencing was performed, which verified that the constructs were correct. Both plasmids were then used for transfection studies in HUVEC and 293T cells.

Verifying of RhoJ recognition on western blots by anti-RhoJ antibody To determine the specificity of the anti-RhoJ antibody, 293T cells were transfected with pEGFPNI and pEGFP-N1 / RhoJwt; Transfected cells were lysed and a western blot was performed using anti-RhoJ and anti-EGFP antibodies. The RhoJ-EGFP fusion protein is recognised by both antibodies, while EGFP is recognised only by the anti-GFP antibody (Figure 8). To examine RhoJ expression in HUVEC and 293T cells, lysates of nontransfected HUVEC and 293T cells were prepared and western blotted with anti- RhoJ antibody. Anti-β-actin antibody was used to confirm equal loading. No RhoJ expression was found in 293T cells, but was observed in HUVEC.

Anti-RhoJ staining on human tissue sections and on RhoJ transfected 293T cells To examine whether the anti-RhoJ antibody is suitable for immunohistochemistry to determine in vivo localisation of RhoJ, this antibody was tested with 293T cells transfected with pEGFP-N1 , pEGFP-N1 /RhoJwt, pcDNA myc-His and pcDNA myc- His/RhoJwt constructs. Cells were attached to glass slides by performing cytospins, fixed with methanol and stained using anti-RhoJ antibody as primary antibody. An enzyme linked anti-mouse secondary antibody was used and substrate was added. The slides were counterstained with haematoxylin. 293T cells did not express RhoJ and therefore, cells transfected only with parental vectors pEGFP-N1 or pcD A myc-His could not be stained. However, some of the cells expressing RhoJ showed strong staining at the plasma membrane. Nevertheless, not all transfected cells were stained, negative cells may not have been transfected with plasmid or the expression level of RhoJ may be too low for immunodetection (data not shown). RhoJ expression in 293T cells transfected with vectors was confirmed by performing cell lysis followed by a western blot (data not shown). Cells transfected with the parental vectors showed no RhoJ expression.

Because endothelial cells constitutively express RhoJ, and RhoJ expression has been reported in the heart [19], RhoJ recognition by the anti-RhoJ antibody was investigated on untransfected HUVECs and on endothelial cells of human tissue sections. Immunohistochemistry was performed using paraffin embedded fixed placenta and frozen heart sections. As a positive control, anti-CD31 antibody was used, which stained endothelial cells on frozen and paraffin embedded sections (Figure 7). In contrast, on sections treated with anti-RhoJ antibody, no staining was detected. Although RhoJ expression in HUVEC was seen by western blotting, anti-RhoJ antibody did not stain cytospun cells (data not shown). This result suggests that the anti-RhoJ antibody recognises RhoJ on cells, but only when protein expression is high.

RhoJwt and RhoJQ79L expression in HUVEC

To determine the subcellular localisation of RhoJ in HUVEC, HUVEC were transfected with RhoJwt-EGFP and RhoJQ79L-EGFP fusion constructs using Transpass D2 as a transfection reagent. As a control the pEFGP-N1 vector expressing EGFP alone was used. Cells were then grown on a gelatine coated slide and live cells were examined using fluorescence microscopy. In contrast to the EGFP control, which showed a cytosolic and possibly a nuclear distribution, RhoJ expressing HUVEC showed a punctuate localisation within the cytoplasm (data not shown). These results are consistent with the findings of Toledo ef a/ (2003) that RhoJ is membrane bound and localises in early and sorting endosomes as well as in the plasma membrane.

RhoJ downregulation using siRNA

In order to examine the role of RhoJ in endothelial cell biology, siRNA technology was used to specifically knockdown the expression of RhoJ in HUVEC. siRNA duplexes for this purpose were designed according to the criteria determined by Reynolds ef a/ (2004) [22;23]. To investigate kinetics of RhoJ knockdown in HUVEC, western blots were performed using HUVEC lysates prepared on day 1-4 after siRNA transfection. siRNA duplexes RhoJ-1 and RhoJ-2 were used to knock down RhoJ mRNA and as negative control a siRNA, which does not bind to any known sequences was used. Cells were lysed on day 1 , 2, 3 and 4 after transfection and western blots were performed using both RhoJ antibody and actin as control for loading. Cells transfected with the negative control showed a constant RhoJ expression over all 4 days. Cells transfected with both RhoJ siRNA duplexes gradually decreased RhoJ protein levels on day 1 and 2 after transfection until no expression could be detected on day 3 and 4 (Figure 8). Assays to look at the role of RhoJ in endothelial cell function were performed on day 2 or 3.

mRNA expression in transfected HUVEC

Bioinformatic analysis of endothelial cDNA libraries suggest that in addition to RhoJ, RhoB and Cdc42 are highly upregulated proteins. RhoB is the most highly expressed Rho GTPase in endothelial cells in general and Cdc42 is closely related to RhoJ [12]. To ensure that mRNA of other Rho family proteins was not affected, RhoA, RhoB and Cdc42 were chosen for examination. To determine the binding specificity of siRNA duplexes to RhoJ and the degree of the mRNA protein downregulation they induced, total RNA of siRNA transfected cells was isolated on the second day after transfection. Prior to RNA isolation, HUVEC were mock transfected, and transfected with RhoJ-1 , RhoJ-2, non-binding negative control siRNA duplexes. Total RNA was then used for generation of cDNA, with which quantitative PCR (Q-PCR) was performed. Expression levels of β- actin, RhoA, RhoB, RhoJ and Cdc42 were determined. Individual dilution series of the produced cDNA were prepared. Using the dilution series one standard curve per protein sample was calculated and values within the curve were taken for further analysis. Expression levels of the Rho proteins were then evaluated relative to β- actin expression. The results showed a slight downregulation of β-actin and RhoJ in cells transfected with RhoJ-1 and RhoJ-2 siRNA compared to mock transfected cells or cells transfected with the non-binding negative control. RhoJ expression in siRNA transfected cells was fivefold lower than in mock transfected cells for RhoJ-1 and tenfold for RhoJ-2 (Figure 9). This indicates that the siRNA duplexes do downregulate RhoJ mRNA, and suggest that RhoJ-2 is more effective than RhoJ-1. In contrast, RhoB and Cdc42 expression compared to β-actin were upregulated in RhoJ siRNA transfected cells. Again, RhoJ-2 showed a stronger effect than RhoJ-1. However, mRNA level of RhoA was not significantly affected in differently treated cells. Using RhoJ-1 and RhoJ-2 siRNA duplexes, RhoJ is specifically downregulated and does not affect other Rho family members.

Impact of siRNA knockdown of RhoJ protein on cell growth

We also examined the effect of RhoJ downregulation on HUVEC cell growth. Cells were transfected with either siRNA duplex RhoJ-1 , RhoJ-2 or with the non-binding negative control. A mock transfection was also performed as a negative control. 15,000 cells per well of a 24 well plate were seeded in triplicate. Cells were harvested and counted on day 1 -3 after transfection using a haemocytometer. In cells where RhoJ downregulation was induced, cells showed a reduced growth on day two and three after transfection compared with the negative control and the mock transfected cells. Cells transfected with RhoJ-2 siRNA reduced their growth even more than RhoJ-1 transfected cells (Figure 10). Thus, RhoJ is pivotal for cell growth and its downregulation leads to a reduction in cell number. This was observed in two independent experiments. Effect of RhoJ downregulation on tube formation by siRNA

Tube formation was investigated on Matrigel™, a basement membrane preparation containing Laminin, Collagen IV and Entactin [24]. It stimulates differentiation and the formation of capillary like structures in endothelial cells. Tube formation on Matrigel™ is endothelial specific and has not been observed in other cell lines, such as human dermal fibroblast or human smooth muscle cells [25]. To determine the effects of RhoJ downregulation on tube formation in HUVEC₁ HUVEC were trypsinized and seeded onto a Matrigel™ layer on the second day after transfection. Tube formation was then observed by taking pictures after 2, 4, 8 and 24 hours (Figure 11A). To confirm RhoJ downregulation on the day of the assay, remaining cells were lysed and a western blot was performed, which showed downregulation of RhoJ in the RhoJ si RNA transfected cells. The β-actin showed equal loading in each lane (Figure 11 B). RhoJ downregulation affected tube formation. At 8 hours after seeding the RhoJ downregulated cells formed fewer and thinner connections between nodes of cells, thereby showing that cells transfected with RhoJ-2 display a stronger phenotype than cells transfected with RhoJ-1 siRNA. This observation was supported by the Q-PCR results, where RhoJ-2 siRNA downregulates RhoJ more effective than RhoJ-1. After 24 hours, some cells generated connections with other cells, but the majority accumulated in loose clusters without any network formation. However, at this point of time the control samples produced a well organised cell network. This was observed in three independent experiments. The powerful effect on tube formation by RhoJ downregulation reflects the important role of RhoJ in angiogenesis.

Effect on cell migration after RhoJ downregulation by siRNA

We also evaluated the effect of RhoJ downregulation on migration using HUVEC transfected with RhoJ-1 , RhoJ-2 and a negative control duplex. Additionally, a mock transfection was also performed. Three days after transfection cells were seeded into a 6 well plate and a scratch was created through the middle of the confluent plate. Mitomycin C, an alkylating agent, which suppresses the synthesis of nucleic acids [26], was added to the medium to prevent cells from dividing and allow migration only. Pictures were taken immediately after having made the scratch and after 4, 8 and 12 hours. RhoJ knockdown in transfected cells was confirmed by performing a cell lysis followed by a western blot. RhoJ downregulated cells covered the created scratch later than the mock transfected cells and cells transfected with the negative control duplex. After 12 hours, in the latter case the scratch had almost disappeared, whereas RhoJ downregulated cells showed weaker migration only at the edge of the scratch and were not able to cover the scratched area (Figure 12). This effect was observed in at least three independent experiments. These results strongly suggest that RhoJ is involved in cell movement and support the importance of RhoJ in angiogenesis.

Discussion

Rho GTPases are part of many different signalling pathways and in their active GTP- bound state these cellular switches bind to their effector proteins and transmit signals, that induce various processes within the cell. For instance, Rho GTPases are involved in the reorganisation of the actin cytoskeleton, the formation of filopodial-like structures, cell migration, and adhesion to the extracellular matrix and to adjacent cells. Due to the fact that we have found RhoJ, a Rho GTPase which is closely related to Cdc42 and TC10, to be specifically expressed in endothelial cells, various aspects of RhoJ function in angiogenesis were investigated. Impacts on endothelial cell behaviour as a result of RhoJ downregulation were observed using growth, tube formation and scratch wound assays. The effect of overexpression of RhoJwt and RhoJQ79L, a constitutively active mutant, in endothelial cells were also analysed with the tube formation and scratch wound assays.

Two different siRNA duplexes RhoJ-1 and RhoJ-2 downregulated RhoJ in HUVEC cells. Strong RhoJ knockdown was observed after 2 and 3 days after transfection of siRNA by western blot. Therefore, assays to investigate the impact of RhoJ knockdown were performed on these days. Reverse transcription and Q-PCR assays showed that the RhoJ siRNA duplexes strongly down regulated RhoJ mRNA. Also we found actin mRNA to be slightly downregulated, RhoB and Cdc42 mRNA slightly upregulated and RhoA mRNA levels unchanged. These results show, that RhoJ downregulation is specific and that the three other tested Rho GTPase family member were not downregulated by either of the RhoJ specific siRNA duplexes. Effects of the RhoJ siRNA duplexes on cell behaviour can therefore be primarily ascribed to RhoJ downregulation. Using siRNA transfection to determine gene function, off-target effects are often observed. The off- target effects appear to be due to the upregulation of interferon-stimulated genes, which are normally responsible for antiviral, antiproliferative or pro-apoptotic effects [27]. To control for this, assays using siRNA were also performed using cells transfected with a non-binding duplex as negative control. Because cells transfected with the negative control have never shown any difference to mock transfected cells, it is unlikely that off- target effects could account for changes in cell behaviour induced by the RhoJ siRNA.

The mRNA upregulation of the closely related Rho GTPase Cdc42 may indicate that endothelial cells are compensating for the loss of RhoJ by upregulating expression of Cdc42, since RhoJ may bind to the same downstream proteins as Cdc42 such as WASP and PAK [19]. Accordingly, RhoJ is likely to be part of an intricate signalling system affecting the cytoskeleton.

siRNA induced RhoJ downregulation in endothelial cells reduced cell growth, affected tube formation and slowed cell migration. The powerful effect seen in all these assays supports RhoJ as a key player in endothelial cell signalling or in endothelial cell behaviour in general. Impaired cell growth suggest that RhoJ may be an important regulator of the cell cycle. This assumption is supported by the fact that many Rho GTPases play important roles in mitosis, for instance, Cdc42 regulates spindle assembly in Xenopus eggs [28]. Another possibility is that the loss of RhoJ may arrest cells in earlier phases of the cell cycle such as G1 , S or in G2. Previous studies have shown the regulation of cell cycling and apoptosis by multiple GTPases [3]. Cdc42, a close relative to RhoJ, was found to be one of the key players in the regulation of the cell cycle and was first identified as a cell division mutant in Saccharomyces cerevisae [29] In fact, cell cycle progression through G1 was stimulated by microinjection of Cdc42, Rho and Rac into quiescent fibroblasts [30]. Cell growth may also be affected because of a loss of survival signals or by induction of apoptosis. If RhoJ is involved in signalling pathways, which maintain cell survival or prevent apoptosis, its downregulation could lead to cell death. This was observed in neurons, where the loss of Rho GTPases results in increased apoptosis and it was also found that the activity of Rac was crucial for survival of the cultured neurons [31].

For the tube formation of endothelial cells on Matrigel™, cytoskeletal reorganisation is necessary. If this ability is impaired, less tube formation is a probable result. Therefore, impaired tube formation observed in RhoJ downregulated cells may due to a loss of cytoskeletal regulation. Because cell-cell contacts are mediated by integrins and cadherins, which in turn are regulated by Rho GTPases [32], the disruption of this regulation by inhibition of RhoJ may lead to reduced cell-cell contacts which would in turn affect tube formation and angiogenesis. The scratch wound assay showed reduced migration of RhoJ siRNA downregulated cells. This result indicates that the coordinated regulation of the actin cytoskeleton required for cell movement might be impaired by RhoJ downregulation. Several Rho GTPases are involved in separate steps of migration, and RhoJ could play a role in lamellipodium extension, formation of new adhesions, cell body contraction or tail detachment. Migration is also dependent on loosening and regeneration of cell adhesion to the extracellular matrix as well as to other cells, another site of possible RhoJ function.

The fact that VEGF regulates many aspects of endothelial cell behaviour including growth, chemotaxis and permeability [34] and that RhoJ is highly specific for endothelial cells, suggests, that RhoJ may be part of the VEGF pathway. If RhoJ is part of the VEGF signalling pathway, its downregulation would affect angiogenesis and therefore cell growth, tube formation and migration in the scratch wound assay. Without wishing to be bound by theory, we consider that our observations support the hypothesis that RhoJ participates in VEGF signalling.

Rho GTPases regulate endocytosis [11] and because RhoJ was found in early and in sorting endosomes and in Tf internalisation [7], RhoJ may also regulate VEGF receptor internalisation and recycling. This is supported by the fact that Rho proteins are involved in epidermal growth factor (EGF) and low-density lipoprotein (LDL) receptor internalisation and sorting [35].

As loosening of adhesion is important for efficient cell migration, the effect of up- or downregulting RhoJ expression in adhesion assays should also be investigated. These assays involve seeding cells on a suitable ECM like collagen IV, fibronectin or iaminin containing plates. After an incubation and a washing step, the remaining amount and therefore adherent cells can be assessed by staining with a fluorescent dye [36].

The scratch wound assay assesses cell movement or cytokinesis, it would be useful to extend these studies by determining the role of RhoJ in chemotaxis. This would be assessed by determining the movement of cells through semi-permeable membranes in response to the addition of different growth factors. Of particular interest would be chemotaxis in response to VEGF₁ thus elucidating potential connections of RhoJ and VEGF. To determine the role of RhoJ in blood vessel development in vivo, RhoJ could be knocked out in the mouse (Mus musculus) or zebrafish (Dario rend). Zebrafish would be a good model, since its embryos are transparent and develop in water and transgenic fish have been developed with fluorescent marked endothelial cells. There is one RhoJ orthologue in D. rerio which could be knocked down using established morpholino technology. This would allow the role of RhoJ in blood vessel development to be investigated. RhoJ knockout may affect correct migration or growth and thus leads to disorganised or underdeveloped vessels [37, 38]. The generation of mouse knockouts would be a good approach to determine the role of RhoJ in blood vessel development in mammals. These experiments may further support the role of RhoJ in human cancer angiogenesis.

In conclusion, we found that RhoJ, a small Rho GTPase and specifically expressed in endothelial cells, plays an important role in assays to determine cell growth, movement and tube formation, all processes required for angiogenesis.

References for Example 2A

[1] Carmeliet (2000) Mechanisms of angiogenesis and arteriogenesis. Nat. Med. 6: 389- 395.

[2] Bergers & Benjamin (2003) Tumorigenesis and the angiogenic switch. Nat.Rev. Cancer 3: 401-410.

[3] Fryer & Field (2005) Rho, Rac, Pak and angiogenesis: old roles and newly identified responsibilities in endothelial cells. Cancer Lett. 229: 13-23. [4] Milkiewicz et al (2006) Regulators of angiogenesis and strategies for their therapeutic manipulation. Int.J.Biochem.Cell Biol. 38: 333-357.

[5] S.Suchting, P. Heal, K.Tahtis, L.M.Stewart, and R.Bicknell, Soluble Robo4 receptor inhibits in vivo angiogenesis and endothelial cell migration. FASEB J. 19 (2005) 121-123.

[6] A.L.Bishop and A.Hall, Rho GTPases and their effector proteins. Biochem.J. 348 Pt 2 (2000) 241-255.

[7] De et al (2003) The GTP/GDP cycling of rho GTPase TCL is an essential regulator of the early endocytic pathway. Mol.Biol.Cell 14 4846-4856.

[8] M.Wherlock and H.Mellor, The Rho GTPase family: a Racs to Wrchs story. J. Cell Sci. 115 (2002) 239-240. [9] C.DerMardirossian and G.M.Bokoch, GDIs: central regulatory molecules in Rho GTPase activation. Trends Cell Biol. 15 (2005) 356-363.

[10] E.Dransart, B.OIofsson, and J.Cherfils, RhoGDIs revisited: novel roles in Rho regulation. Traffic. 6 (2005) 957- 966. [11] Qualmann & Mellor (2003) Regulation of endocytic traffic by Rho GTPases. Biochem.J. 371: 233-241.

[12] Burridge & Wennerberg(2004) Rho and Rac take center stage. Cell 116: 167-179.

[13] Rolfe et a/ (2005) Rho and vascular disease. Atherosclerosis 183: 1-16. [14] S.Etienne-Manneville and A.Hall, Rho GTPases in cell biology. Nature 420 (2002) 629-635.

[15] R.A.Cerione, Cdc42: new roads to travel. Trends Cell Biol. 14 (2004) 127-132.

[16] M.R.Schiller, Coupling receptor tyrosine kinases to Rho GTPases-GEFs what's the link. Cell Signal.2006). [17] AJ.Ridley, Rho GTPases and cell migration. J.Cell Sci. 114 (2001) 2713-2722. [18] Not used

[19] Vignal et al (2000) Characterization of TCL, a new GTPase of the rho family related to TC10 andCcdc42. J.Biol.Chem. 275 (2000) 36457-36464.

[20] Nishizuka et al (2003) Crucial role of TCL/TC1 Obeta L, a subfamily of Rho GTPase, in adipocyte differentiation. J.Biol.Chem. 278 15279-15284.

[21] Mouritzen et al (2005). Probe Library: A new method for faster design and execution of quantitative real-time PCR. Nature Methods 2(4), 313-316

[22] P.Sandy, A.Ventura, and T.Jacks, Mammalian RNAi: a practical guide. Biotechniques 39 (2005) 215-224. [23] Reynolds et al (2004) Rational siRNA design for RNA interference. Nat.Biotechnol. 22 326-330.

[24] Beckton Dickinson Biosciences. 2006. Ref Type: Internet Communication

[25] Kubota et al (1988) Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J.Cell Biol. 107: 1589-1598.

[26] Mitomycin C Information Sheet, Sigma Aldrich. 2006. Ref Type: Internet Communication

[27] Sledz & Williams, RNA interference and double-stranded-RNA-activated pathways. Biochem. Soc.Trans. 32 (2004) 952-956. [28] Narumiya & Yasuda, Rho GTPases in animal cell mitosis. Curr.Opin.Cell Biol. 18 (2006) 199-205.

[29] Hall (2005) Rho GTPases and the control of cell behaviour. Biochem.Soc.Trans. 33: 891-895.

[30] M.F.Olson, A.Ashworth, and A.Hall, An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1. Science 269 (1995) 1270-1272.

[31] Loucks et al (2006) Rho family GTPase inhibition reveals opposing effects of mitogen-activated protein kinase kinase/extracellular signalregulated kinase and Janus kinase/signal transducer and activator of transcription signaling cascades on neuronal survival. J.Neurochem. 97 957-967. [32] G.Bazzoni & E.Dejana, Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiol Rev. 84 (2004) 869-901. [33] C.I.Dubreuil, M.J.Winton, and LMcKerracher, Rho activation patterns after spinal cord injury and the role of activated Rho in apoptosis in the central nervous system. J.Cell Biol. 162 (2003) 233-243.

[34] S.Cebe-Suarez, A.Zehnder-Fjallman, and K.Ballmer-Hofer, The role of VEGF receptors in angiogenesis; complex partnerships. Cell MoI. Life Sci. 63 (2006) 601-615.

[35] Kaneko et al (2005) Rho mediates endocytosis of epidermal growth factor receptor through phosphorylation of endophilin A1 by Rho-kinase. Genes Cells 10 973-987.

[36] Adhesion Assays, Cell Bio Labs. 2006. Ref Type: Internet Communication

[37] Saias-Vidal et al (2005) Genomic annotation and expression analysis of the zebrafish Rho small GTPase family during development and bacterial infection. Genomics 86: 25-37.

[38] Kidd & Weinstein (2003) Fishing for novel angiogenic therapies. BrJ. Pharmacol. 140: 585-594.

Example 2B: Further Studies on the role of RHOJ in angiogenesis

We conducted further studies on the role of RHOJ in angiogenesis using standard procedures to supplement the studies described in Example 2A.

Figure 13 shows HUVECs transfected with two RhoJ specific siRNAs D1 or D2, NCD or mock treated with transfection reagent only (Mock) resulting in a reduction of RhoJ protein. These data confirm, complement and support the data in Figure 8.

Figure 14 shows that the down-regulation of RhoJ expression using siRNA results in impaired tube formation in fibrin gels.

Figure 15 shows that down-regulation of RhoJ expression using siRNA results in impaired tube formation on matrigel. These data confirm, complement and support the data in Figure 11.

Figure 16 shows that HUVECs with reduced RhoJ fail to migrate in a scatch wound assay. These data confirm, complement and support the data in Figure 12.

To determine the role of RhoJ in chemotaxis, assays were conducted using a 48-well modified Boyden chamber with 8μm pore size polycarbonate nucleopore filters. Filters were coated with 0.1 % gelatin and placed over a lower chamber containing 30 μl per well of HUVEC media containing 10% fetal bovine serum also with large vessel endothelial cell growth supplement as the chemoattractant factor. Cells were rested in serum-free media for 30 minutes before the assay. Cells were harvested and 2 x 10⁴ cells were seeded per well of the upper chamber in 50 μl basal media of M199 and 4 mM glutamine containing 1 % fetal bovine serum. After 5 hours incubation at 37°C with 5% CO2, the filters were removed, fixed in 100% methanol and stained with 0.5% Crystal Violet in PBS overnight at room temperature. The filters were then washed with tap water and placed onto a glass slide and then non-migrated cells were wiped away with a wet cotton swab. Cells that migrated through the pores towards the chemoattractant were viewed under Leica DM 1000 light microscope using a USB 2.0 2M XIi camera. The number of cells migrated towards the chemoattractant in one quarter of the well area were counted for the 12 replicate of each condition. The mean number of cells migrated and standard error were calculated. The results are provided in Figure 17 which shows that HUVECs with reduced RhoJ fail to migrate in a chemotaxis assay in a Boyden chamber. Since VEGF regulates endothelial cell chemotaxis, these data further support the participation of RhoJ in VEGF signalling.

Figure 18: Down-regulation of RhoJ expression in HUVEC using siRNA results in growth impairment, i.e. reduced cell proliferation. These data confirm, complement and support the data in Figure 10.

We also investigated the activation of RhoJ by VEGF-A in HUVECs. HUVEC were rested for 1 hour and then stimulated with VEGF-A for the times indicated in Figure 19. Active RhoJ was determined by pull-down. Figure 19A shows the results of Western blotting showing the pull-down of active RhoJ in relation to the lysate. Figure 19B shows the densitometries! quantitation of the Western blotting.

Example 3: Effects of RHBDL6 knockdown

We studied the role of RHBDL6 in angiogenesis using standard procedures.

A: Effects ofRHBDLβ knockdown on interferon response

Figure 20 shows RHBDL6 (RHBDF2) siRNA knocks-down mRNA expression without eliciting an interferon response. B: Effects ofRHBDLβ knockdown on HUVEC tube formation

Figure 21 shows that RHBDL6 (RHBDF2) knock-down in HUVECs results in defects in tube-formation on matrigel, supporting the importance of RHBDL6 in angiogenesis

C; Effects ofRHBDLβ knockdown on HUVEC migration

Figure 22 shows that RHBDL6 (RHBDF2) knock-down results in migration defects in HUVECs. Since migration is an essential component of angiogenesis, this further supports the importance of RHBDL6 in angiogenesis.

D: Effects ofRHBDLβ knockdown on HUVEC proliferation

Methods

The effect of RHBDLΘ-specific siRNA on the proliferation of HUVEC cells was assessed based on the protocol described by Nishiwaki et a/ (2003, "Introduction of short interfering RNA to silence endogenous E-selectin in vascular endothelium leads to successful inhibition of leukocyte adhesion". Biochem Biophys Res Commun 310: 1062- 6). 5 x 10⁵ cells in a 10 cm tissue culture plate were treated with 0.2% lipofectamine and 12.5 nM RHBDL6-specific siRNA in a total volume of 6 ml of optimem for 4 h at 37⁰C. After 4 h the transfection media was removed and replaced with fresh tissue culture media (DMEM, 10% foetal calf serum, 1 ng/ml FGF).

Results

By cell counting, we show that RHBDL6-specific siRNA significantly inhibits HUVEC proliferation (Figure 23). Similar results were seen with three different siRNAs. Since endothelial proliferation is an essential component of angiogenesis, inhibitors of endothelial proliferation are usually anti-angiogenic.

E: Effects ofRHBDLβ knockdown on zebrafish vasculature during embryogenesis

Figure 24 shows zRHBDL.6 mRNA expression in 1 day old zebrafish embryo by in situ hybridization (high and low magnification). Strong expression can be seen in both endothelial and haematopoietic cells during development. Figure 25 shows morpholino knock-down of RHBDL6 in zebrafish embryos results in defective vasculature during embryogenesis. Knockdown of RHBDL6 resulted in reduced inter somatic vessels in the embryo as seen by Fii-1 expression (black arrows) and the effect was dependent upon the concentration of morpholino antisense oligonucleotide. These data suggest that RHBDL6 is important for the development of embryonic vasculature.

Example 4: LRRC8C and PCDH11 are specifically expressed in tumour vasculature Methods

Paraffin embedded common cancer arrays and matching normal tissues arrays were used for the experiment. Paraffin was removed by washing the slides three times with histoclear and then re-hydrated by incubation in a series of ethanol, water and then PBS. Tissues were then fixed for 10 minutes with 4% paraformaldehyde and then digested with 0.2% trypsin for ten minutes at 37^°C. Endogenous biotin was blocked by incubation with Biotin avidin blocking kit (Vectorlabs). Tissues were then dehydrated with a series of ethanol. Once the slides were completely dry, the hybridisation mix (50% formamide, 2X SSC, 10% dextran sulphate, 5mM sodium phosphate, 2nM LNA 5' biotin labelled probe (Exiqon)) was applied. The slides where sealed with hybridisation chambers (invitrogen) then incubated at 80^°C for 75 seconds and overnight at 37'C in a humidified chamber. On day two, the chambers were carefully removed, and the slides rinsed with 2x SSC- 0.1% Tween 20, then washed with 0.1x SSC for 30 minutes in a rocking incubator at a temperature of 68-72 ^°C depending on the Tm of the probe. The slides were then washed in PBST, blocked with FCS-PBST 1 :20 for 20 minutes and incubated for 1 hour with fluorescein avidin (Vectorlabs). The slides were then washed with PBST and blocked again with FCS-PBST 1 :20 for 20 minutes and then incubated for 1 hour with Ulex europeaus agglutinin I (UEAI) conjugated with rhodamine (Vectorlabs). After washing the slides were mounted with vectashield containing DAPI (vectorlabs) and analysed using the confocal microscope.

UEA1 is known to be a good marker for human endothelial cells. Therefore, dual labelling of the tissue samples with UEA1 (red) and gene specific LNA biotin labelled probes (Green) was carried out to evaluate endothelial expression of the target genes.

Results Figure 26 shows overlapping signals from UEA1 and gene specific probes for LRRC8C and PCDH11, confirming that LRRC8C and PCDH11 are specifically expressed in tumour endothelium. Thus as tumour endothelium specific markers, LRRC8C and PCDH 11 are important anticancer targets.

Example 5: Testing antibodies against KCTD15, LRRC8C, PCDH12, LOC55726, GBP4 and IKBKE in angiogenesis assays

A: Antibodies against KCTD15, LRRC8C, PCDH12, LOC55726 GBP4 and IKBKE inhibit formation of vessel sprouts in the aortic ring assay

The role of KCTD15, LRRC8C, PCDH12, LOC55726, GBP4 and IKBKE in angiogenesis is investigated using the rat aortic ring assay. Segments of rat aorta are embedded in Matrigel and treated with antibodies that selectively bind to KCTD15, LRRC8C, PCDH12, LOC55726, GBP4 and IKBKE. The sprouting vessels are allowed to develop over five days before scoring, typically by three independent observers. Inter-scorer reliability can be assessed to show the degree of consistency between independent scorers. Treatment of the aortic rings with the antibodies is expected to result in a decrease in sprouting of vessels from the aortic segment.

B; Antibodies against KCTD15, LRRCBC, PCDH12, LOC55726, GBP4 and IKBKE inhibit formation of vessel sprouts in vivo.

The ability of antibodies that selectively bind to KCTD15, LRRC8C, PCDH12, LOC55726, GBP4 and IKBKE to inhibit angiogenesis in vivo is tested using a sponge angiogenesis assay (Hori et al (1996) "Differential effects of angiostatic steroids and dexamethasone on angiogenesis and cytokine levels in rat sponge implants" Br. J. Pharmacol. 118(7): 1584-1591).

There are differences expected between the sponges from the mice who are injected with bFGF alone (controls) and those who are injected with both bFGF and the antibodies. The major expected difference is that there are fewer vessel numbers in the antibody treated sponges compared to the controls.

C; Antibodies against KCTD15, LRRC8C, PCDH12, LOC55726, GBP4 and IKBKE inhibit migration of primary human vascular endothelial cells. A primary human vascular endothelial cell (HUVEC) migration assay is performed using the BD BioCoat™ Angiogenesis System for Endothelial Cell Migration which is available as Catalog No. 354143 from BD Biosciences, Bedford, MA, USA. Instructions for using this kit can be found on the Internet at http://www.bdbiosciences.com/discoveryjabware/ Products/drug_discovery/insert_systems/angiogenesis_system/pdf/Endothelial_Cell_Mig ration_lnstruct.pdf.

This system uses a 24-multiwell insert system and consists of a BD Falcon FluoroBlok PET membrane with 3 micron pore size coated uniformly on the top side with fibronectin. Quantitation of cell migration is achieved by post-labelling of cells with the fluorescent dye Calcein AM and measuring the fluorescence of migrating cells in a fluorescence plate reader. The FluoroBlok membrane effectively blocks the passage of light from 490- 700 nm at >99% efficiency meaning labelled cells that have not migrated are blocked from detection.

Both bFGF and VEGF are known to stimulate migration of endothelial cells (Cross & Claesson-Welsh, 2001 Trends Pharmacol Sci. 22(4): 201-207). The antibodies that selectively bind to KCTD15, LRRC8C, PCDH 12, LOC55726, GBP4 and IKBKE are expected to inhibit migration of HUVEC cells induced by either bFGF or VEGF.

D; Antibodies against KCTD15, LRRC8C, PCDH12, LOC55726, GBP4 and IKBKE inhibit endothelial cell proliferation.

Primary human vascular endothelial cells (HUVEC) are seeded on to multi-well plates in full growth media containing treatment (increasing concentrations of antibodies that selectively bind to KCTD15, LRRC8C, PCDH12, LOC55726, GBP4 and IKBKE, or 100 μg/ml human IgG), or no treatment as a control. After four days incubation at 37°C, the cells are washed in PBS and detached from the wells by addition of 1 ml trypsin solution. After all the cells have detached, 400 μl of the cell suspension is transferred to 19.6 ml lsoton buffer (Beckman Coulter), and the number of cells in each sample is determined in a Coulter Particle Count and Size Analyser (Beckman Coulter). The experiment is typically carried out in triplicate, and replicated three times.

Incubation in the presence of increasing concentrations of antibodies that selectively bind to KCTD15, LRRC8C, PCDH12, LOC55726, GBP4 and IKBKE is expected to have an increasingly strong antiproliferative effect on HUVEC cells compared with untreated cells.

Example 6: Treatment a solid tumour in an animal model A mouse model of a solid tumour (e.g. either a Lewis lung carcinoma subcutaneous homograft implant in Black 57 mice or an HT29 subcutaneous xenograft implant in nude mice) is treated with intravenous infusions of saline solutions of a pharmaceutical composition comprising antibodies that selectively bind to a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726. -GBP4, IKBKE and RHOJ. The infusions are administered weekly for a time of 2 to 4 months. The tumour regresses in the animal model compared to the controls.

Claims

1. A method of inhibiting tumour angiogenesis in an individual, the method comprising administering to the individual an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ.

2. Use of an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ in the preparation of a medicament for inhibiting tumour angiogenesis in an individual.

3. An inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ for use in inhibiting tumour angiogenesis in an individual.

4. A method according to Claim 1 or a use according to Claim 2 or an inhibitor according to Claim 3 wherein the individual has a solid tumour.

5. A method of combating a solid tumour in an individual, the method comprising administering to the individual an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ.

6. Use of an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ in the preparation of a medicament for combating a solid tumour in an individual.

7. An inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ for use in combating a solid tumour in an individual.

8. A method according to Claim 4 or 5, or a use according to Claim 4 or 6, or an inhibitor according to Claim 4 or 7, wherein the solid tumour is a tumour of the lung, brain, colon, kidney, prostate, skin, liver, pancreas, stomach, uterus, ovary, breast, lymph glands or bladder.

9. A method or a use or an antibody according to any of the preceding claims wherein the individual is a human.

10. An ex vivo method of inhibiting angiogenesis, the method comprising administering an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15,

LRRC8C, PCDH 12, LOC55726, GBP4, IKBKE and RHOJ to tumour endothelial cells, or to a tumour angiogenesis model, ex vivo.

11. A method or a use or an inhibitor according to any of the preceding claims wherein the inhibitor is an antibody that specifically binds a polypeptide selected from

RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ.

12. A method or a use or an inhibitor according to Claim 11 wherein the antibody is a monoclonal antibody.

13. A method or a use or an inhibitor according to Claim 11 wherein the antibody is a humanised antibody.

14. A method or a use or an inhibitor according to Claim 11 wherein the antibody is a single-chain antibody.

15. A method or a use or an inhibitor according to any of Claim 1-10 wherein the inhibitor is an siRNA molecule, an antisense molecule, or a ribozyme specific for a gene selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, or a polynucleotide encoding said siRNA molecule, antisense molecule or ribozyme.

16. An isolated antibody that selectively binds a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ.

17. An antibody that selectively binds a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ for use in medicine.

18. A pharmaceutical composition comprising an antibody that selectively binds a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ and a pharmaceutically acceptable carrier, diluent or excipient.

19. An isolated siRNA molecule, an antisense molecule, or a ribozyme specific for a gene selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ.

20. A polynucleotide that encodes the siRNA molecule, antisense molecule, or ribozyme of Claim 19.

21. A vector comprising the polynucleotide of Claim 20.

22. A cell or cell line comprising the polynucleotide of Claim 20 or the vector of Claim 21.

23. An siRNA molecule, an antisense molecule, or a ribozyme specific for a gene selected from RHBDL6, KCTD15, LRRC8C, PCDH 12, LOC55726, GBP4, IKBKE and RHOJ, or a polynucleotide encoding said siRNA molecule, antisense molecule or ribozyme, for use in medicine.

24. A pharmaceutical composition comprising an siRNA molecule, an antisense molecule, or a ribozyme specific for a gene selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, or a polynucleotide encoding said siRNA molecule, antisense molecule or ribozyme, and a pharmaceutically acceptable carrier, diluent or excipient.

25. A compound comprising an antibody that selectively binds a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, and a detectable moiety.

26. A compound according to Claim 25 wherein the detectable moiety comprises iodine-123, iodine-131 , indium-111 , fluorine-19, carbon-13, nitrogen-15, oxygen-17, technitium-99m, gadolinium, manganese or iron.

27. A compound according to Claim 25 or 26 for use in diagnosis.

28. A pharmaceutical composition comprising a compound according to Claim 25 or 26 and a pharmaceutically acceptable carrier, diluent or excipient.

29. A method of imaging tumour neovasculature in the body of an individual the method comprising: administering to the individual a compound according to Claim 25 or 26; and imaging the detectable moiety in the body.

30. A method according to Claim 29 wherein the tumour endothelium comprises neovasculature.

31. A method according to Claim 29 or 30 wherein the individual has a solid tumour.

32. A method of detecting, diagnosing or prognosing a solid tumour in an individual, the method comprising: administering to the individual a compound according to Claim 25 or 26; and detecting the presence of the detectable moiety in the individual.

33. A method according to any of Claims 29-32 further comprising the step of detecting the location of the compound in the individual.

34. A compound comprising an antibody that selectively binds a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH 12, LOC55726, GBP4, IKBKE₁ and RHOJ, and a cytotoxic moiety.

35. A compound according to Claim 34 wherein the cytotoxic moiety is selected from a directly cytotoxic chemotherapeutic agent, a directly cytotoxic polypeptide, a moiety which is able to convert a prodrug into a cytotoxic drug, a radiosensitizer, a directly cytotoxic nucleic acid, a nucleic acid molecule that encodes a directly or indirectly cytotoxic polypeptide or a radioactive atom.

36. A compound according to Claim 35 wherein the radioactive atom is any one of phosphorus-32, iodine-125, iodine-131 , indium-111 , rhenium-186, rhenium-188 or yttrium-90.

37. A compound according to any of Claims 34-36 for use in medicine.

38. A pharmaceutical composition comprising a compound according to any of Claims 34-36 and a pharmaceutically acceptable carrier, diluent or excipient.

39. A method of inhibiting tumour angiogenesis in an individual the method comprising administering to the individual a compound according to any of Claims 34-36.

40. Use of a compound according to any of Claims 34-36 in the preparation of a medicament for inhibiting tumour angiogenesis in an individual.

41. A compound according to any of Claims 34-36 for use in inhibiting tumour angiogenesis in an individual.

42. A method according to Claim 39 or a use according to Claim 40 or an compound according to Claim 41 wherein the individual has a solid tumour.

43. A method of combating a solid tumour in an individual, the method comprising administering to the individual a compound according to any of Claims 34-36.

44. Use of a compound according to any of Claims 34-36 in the preparation of a medicament for combating a solid tumour in an individual.

45. A compound according to any of Claims 34-36 for use in combating a solid tumour in an individual.

46. A method or a use or a compound according to any of Claims 42-45, wherein the solid tumour is a tumour of the lung, brain, colon, kidney, prostate, skin, liver, pancreas, stomach, uterus, ovary, breast, lymph glands or bladder.

47. A method or a use or a compound according to any of Claims 29-33 and 39-45 wherein the individual is a human.

48. A pharmaceutical composition comprising an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and

RHOJ, and at least one further anticancer agent, and a pharmaceutically acceptable carrier, diluent or excipient.

49. A composition comprising an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, and a further anticancer agent, for use in medicine.

50. A method of combating a solid tumour in an individual, the method comprising administering to the patient an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, in combination with a further anticancer agent.

51. Use of an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, in combination with at least one further anticancer agent, in the preparation of a medicament for combating a solid tumour in an individual.

52. Use of an inhibitor of a gene/polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, in the preparation of a medicament for combating a solid tumour in an individual who is administered at least one further anticancer agent.

53. A composition or a method or a use according to any of Claims 48-52 wherein the at least one further anticancer agent is selected from cisplatin; carboplatin; 5-flurouracil; paclitaxel; mitomycin C; doxorubicin; gemcitabine; tomudex; pemetrexed; methotrexate; irinoteca, fluorouracil and leucovorin; oxaliplatin, 5-fluorouracil and leucovorin; and paclitaxel and carboplatin.

54. A method or a use according to any of Claims 50-53 wherein the solid tumour is a tumour of the lung, brain, colon, kidney, prostate, skin, liver, pancreas, stomach, uterus, ovary, breast, lymph glands or bladder.

55. A method or a use according to any of Claims 50-54 wherein the individual is a human.

56. A composition or a method or a use according to any of Claims 48-54 wherein the inhibitor is an antibody as defined in any of Claims 11-14.

57. A composition or a method or a use according to any of Claims 48-54 wherein the inhibitor is an siRNA molecule, an antisense molecule, or a ribozyme specific for a gene selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, or a polynucleotide encoding said siRNA molecule, antisense molecule or ribozyme.

58. A method or a use or an inhibitor or a compound or a composition according to any of preceding Claims 11-14, 16-18, 25-47 or 56 wherein the antibody that selectively binds to the polypeptide RHBDL6 binds to RHBDL6 isoform 1.

59. A method or a use or an inhibitor or a compound or a composition according to Claim 58 wherein the antibody binds to an extracellular region of RHBDL6 isoform 1 (residues 1-653, 711-719, 769-771 and 827-856).

60. A method or a use or an inhibitor or a compound or a composition according to any of preceding Claims 11-14, 16-18, 25-47 or 56 wherein the antibody that selectively binds to the polypeptide RHBDL6 binds to RHBDL6 isoform 2.

61. A method or a use or an inhibitor or a compound or a composition according to Claim 60 wherein the antibody binds to an extracellular region of RHBDL6 isoform 2

(residues 398-624, 682-690, 740-742 and 798-827).

62. A method or a use or an inhibitor or a compound or a composition according to any of preceding Claims 11-14, 16-18, 25-47 or 56 wherein the antibody that selectively binds to the polypeptide RHBDL6 binds to the rhomboid region (residues 651-774 of isoform 1 or residues 622-745 of isoform 2).

63. A method or a use or an inhibitor or a compound or a composition according to any of preceding Claims 11-14, 16-18, 25-47 or 56 wherein the antibody that selectively binds to the polypeptide KCTD15 binds to a cell surface exposed region of KCTD15.

64. A method or a use or an inhibitor or a compound or a composition according to any of preceding Claims 11-14, 16-18, 25-47 or 56 wherein the antibody that selectively binds to the polypeptide KCTD15 binds to the K⁺ channel tetramerisation domain of KCTD15 (residues 58-145).

65. A method or a use or an inhibitor or a compound or a composition according to any of preceding Claims 11-14, 16-18, 25-47 or 56 wherein the antibody that selectively binds to the polypeptide LRRC8C binds to an extracellular region of LRRC8C (residues 1-25, 149-261 or 342-803)

66. A method or a use or an inhibitor or a compound or a composition according to any of preceding Claims 11-14, 16-18, 25-47 or 56 wherein the antibody that selectively binds to the polypeptide LRRC8C binds to a leucine rich repeat region of LRRC8C.

67. A method or a use or an inhibitor or a compound or a composition according to any of preceding Claims 11-14, 16-18, 25-47 or 56 wherein the antibody that selectively binds to the polypeptide PCDH 12 binds to the extracellular region of PCDH 12 (residues 30-715).

68. A method or a use or an inhibitor or a compound or a composition according to Claim 67 wherein the antibody that selectively binds to the polypeptide PCDH12 binds to the cadherin repeat domain at residues 36-236, or to the cadherin repeat domain at residues 139-344, the cadherin repeat domain at residues 363-543, the cadherin repeat domain at residues 465-693, or to the Ca²⁺ binding sites therein.

69. A method or a use or an inhibitor or a compound or a composition according to any of preceding Claims 11-14, 16-18, 25-47 or 56 wherein the antibody that selectively binds to the polypeptide LOC55726 binds to the coiled coil motif within the polypeptide.

70. A method or a use or an inhibitor or a compound or a composition according to any of preceding Claims 11-14, 16-18, 25-47 or 56 wherein the antibody that selectively binds to the polypeptide GBP4 binds to a region of GBP4 selected from the the N- terminal domain (residues 48-289), to the G1 box region, the G2 box region, the G3 box region, the G4 box region, the G5 box region, the GTP/Mg²⁺ binding site (residues 62-68, 82-84, 89-90, 1 15, 196-197 and 254), the Switch I region, the Switch Ii region and the C- terminal domain (residues 298-594).

71. A method or a use or an inhibitor or a compound or a composition according to any of preceding Claims 11-14, 16-18, 25-47 or 56 wherein the antibody that selectively binds to the polypeptide IKBKE binds to a region of IKBKE selected from the serine/threonine protein kinase catalytic domain (residues 9-242), the ATP binding pocket (residues 15-16, 18, 21 , 23, 36, 38, 86, 140, 144 and 156-158), the substrate binding pocket (residues 93, 137, 173-175 and 177-178), the catalytic loop (residues 133-141 and 144) and the activation loop (residues 157-166 and 171-183).

72. A method or a use or an inhibitor or a compound or a composition according to any of preceding Claims 11-14, 16-18, 25-47 or 56 wherein the antibody that selectively binds to the polypeptide RHOJ binds to a region of RHOJ selected from the guanine nucleotide exchange factor interaction site (residues 23, 53-54, 57-58, 60, 62, 70, 74-75, 77-79, 85 and 88), the G1 box region, the G2 box region, the G3 box region, the G4 box region, the G5 box region, the GTP/Mg²⁺ binding site (residues 31-36, 75-76, 78, 134, 136 and 177-178), the Switch I region, the Switch Il region, the putative guanine nucleotide dissociation inhibitor interaction site (residues 54, 77, 85 and 87), the putative GTPase-activating protein interaction site (residues 54-55, 79 and 85), and the putative effector interaction site (residues 55-56, 85 and 88).

73. A method of identifying an agent that may be useful in the treatment of a solid tumour, or a lead compound for the identification of an agent that may be useful in the treatment of a solid tumour, the method comprising: providing a candidate compound that binds a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, or a fragment thereof; and testing the candidate compound in an angiogenesis assay, wherein a candidate compound that inhibits angiogenesis in the assay may be an agent that is useful in the treatment of a solid tumour, or may be a lead compound for the identification of an agent that is useful in the treatment of a solid tumour.

74. A method of identifying an agent that may be useful in the treatment of a solid tumour, or a lead compound for the identification of an agent that may be useful in the treatment of a solid tumour, the method comprising: providing a candidate compound; determining whether the candidate compound selectively binds to a polypeptide selected from RHBDL6, KCTD15, LRRC8C, PCDH12, LOC55726, GBP4, IKBKE and RHOJ, or a fragment thereof; and testing a candidate compound that selectively binds to the polypeptide or the fragment in an angiogenesis assay, wherein a candidate compound that selectively binds to the said polypeptide or fragment and which inhibits angiogenesis in the assay may be an agent that is useful in the treatment of a solid tumour, or may be a lead compound for the identification of an agent that is useful in the treatment of a solid tumour.

75. A method according to Claims 73 or 74 wherein the candidate compound is an antibody, a peptide, an aptamer or a small organic molecule.

76. A method according to any of Claims 73-75 wherein the angiogenesis assay is an aortic ring assay or a sponge angiogenesis assay.

77. A method according to any of Claims 73-76 wherein the angiogenesis assay is an assay of endothelial cell proliferation, migration and/or invasion.

78. A method according to any of Claims 73-77 wherein the identified compound is modified, and the modified compound is tested for the ability to inhibit angiogenesis.

79. A method according to any of Claims 73-78 wherein the identified compound or the modified compound is tested for efficacy in an animal model of a solid tumour.

80. A method according to Claim 79 wherein the animal model is a mouse model of a solid tumour.

81. A method according to any of Claims 73-80 further comprising the step of synthesising, purifying and/or formulating the identified compound or the modified compound.

82. A method for preparing an anticancer compound that may be useful in the treatment of a solid tumour, the method comprising identifying a compound using the method according to any of Claims 73-80 and synthesising, purifying and/or formulating the identified compound.

83. A method of making a pharmaceutical composition comprising mixing the compound identified using a method according to any of Claims 73-80 with a pharmaceutically acceptable carrier, excipient or diluent.