AU742779B2 - VEGF-B/receptor complex and uses thereof - Google Patents

Description

WO 98/28621 PCT/US97/23533 VEGF-B/RECEPTOR COMPLEX AND USES THEREOF Background of the Invention The present invention relates to a complex of Vascular Endothelial Growth Factor-B (VEGF-B) and the Flt-1 receptor, to methods of using such complexes to induce or antagonize a VEGF-B-mediated cellular response, to assay kits for identifying VEGF-B and/or VEGF-B analogs, and to isolated binding partners, such as antibodies, which bind to VEGF- B/Flt-1 complexes.

Vascular Endothelial Growth Factor (VEGF or VEGF-A; sometimes also referred to as Vascular Permeability Factor or VPF) is an angiogenic growth factor of the PDGF family.

It exerts its effect through two endothelial receptor tyrosine kinases (RTKs), Fit-1 (also known as VEGFR-1) [Shibuya et al., Oncogene, 5:519-524 (1990); de Vries et al., Science, 255:989-991 (1992)] and Flk-1/KDR (also known as VEGFR-2) [Matthews et al., Proc. Natl. Acad. Sci. USA, 88:9026-30 (1991); Terman et al., Biochem. Biopphys. Res.

Comm., 187:1579-86 (1992); Millauer et al., Cell,72:835-46 (1993)]. These receptors appear to play a pivotal role in regulation of endothelial cell growth and differentiation and in maintenance of the functions of the mature endothelium [Shalaby et al., Nature, 376:62-66 (1995); Fong et al., Nature, 376:66-70 (1995)]. VEGF and its high affinity receptors Fit-i and KDR/flk-1 are required for the formation and maintenance of the vascular system as well as for both physiological and pathological angiogenesis.

Placenta growth factor (PlGF) [Maglione et al., Proc.

Natl. Acad. Sci. USA, 88:9267-71 (1991)] is another ligand for the Fit-i RTK [Park et al., J. Biol. Chem., 269:25646-54 i'WO 98/28621 PCT/US97/23533 (1994)]. It is also a member of the PDGF family and is structurally related to VEGF, but its biological function is not presently well understood.

VEGF-B is a distinct growth factor for endothelial cells described in Olofsson et al., "Vascular endothelial growth factor B, a novel growth factor for endothelial cells", Proc. Natl. Acad. Sci. USA, 93:2576-81 (1996). Like VEGF and PlGF, it is a member of the PDGF family of growth factors with which it shares substantial structural similarities, including a pattern of conserved cysteine residues which form disulfide bonds involved in homo- and hetero-dimerization of the molecule. Nevertheless, VEGF-B exhibits only approximately 40 to 45 percent sequence similarity to VEGF and only approximately 30 percent sequence similarity to P1GF. VEGF-B has been found to be co-expressed with VEGF in various tissues and is particularly abundant in heart and skeletal muscle tissue.

It promotes mitosis and proliferation of endothelial cells and appears to have a role in endothelial tissue growth and angiogenesis. VEGF-B may potentiate the mitogenic activity of low concentrations of VEGF both in vitro and in vivo.

The present invention is based on the discovery that VEGF-B is capable of binding to the extracellular domain of Flt-1 receptor tyrosine kinase to form bioactive complexes which mediate useful cell responses and/or antagonize undesired biological activities.

References herein to the amino acid sequence of VEGF-B refer to the sequence for human VEGF-BI.

6 described in Eriksson et al., published PCT Application No. WO 96/26736 (Genbank database accession no. U52819). References to the amino acid or nucleotide sequences of Flt-1 refer to the sequences described by Shibuya et al., Oncogene, 5:519 (1990), (EMBL database accession no. X51602) Binding affinity of VEGF-B and/or VEGF-B analogs for the Flt-1 receptor or analogs thereof is tested according to the procedure described in Lee et al., Proc. Natl. Acad. Sci., 2 93:1988 (1996). A useful method for assaying endothelial cell proliferation is described in Olofsson et al., Proc.

Natl. Acad. Sci. USA, 93:2576-81 (1996).

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

In accordance with one preferred aspect of the invention, the invention relates to a method for identifying a VEGF-B analog having substantially the same binding affinity for a cell surface receptor as VEGF-B, the method comprising the steps of: providing a sample containing a receptor protein selected from the group consisting of: a polypeptide chain comprising an amino acid sequence defined by residues 1-347 of Flt-1, or a VEGF-B-specific receptor analog thereof; (ii) a polypeptide chain having binding affinity for VEGF-B and sharing at least 30% amino acid identity with residues 1-347 of Fit-i; and (iii) a polypeptide chain having binding affinity for VEGF-B and encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising the sequence defined by nucleotides 1-1293 of Flt-1; -3- 7 o 3 17 OCT 2001 contacting said sample of step with a candidate VEGF-B analog; and detecting specific binding between the candidate VEGF-B analog and the receptor protein of step VEGF-B binding to a cell surface receptor is considered to involve a VEGF-B dimer binding to the receptor which causes dimerization of the receptor and autophosphorylation of that receptor followed by intra-cellular signalling and, in appropriate circumstances, a cellular response such as angiogenesis.

The sample containing receptor protein could be, for example, soluble Fit receptor produced naturally in the 3a 1 7 OCT 2001 WO 98/28621 PCT/US97/23533 conditioned medium of cells that normally express the receptor. Tissue samples or tissue fluids shed naturally from cells by proteolytic events also could be used as receptor samples.

The VEGF-B analogs identified by this aspect of the invention may be small molecules, for example proteins or peptides or non-proteinaceous compounds such as DNA or RNA.

The analogs also could include VEGF-B or a derivative (including, but not limited to, a fragement of a VEGF-B monomer or dimer) tagged with a toxin or drug or radioactive isotope which could target Flt-i expressed and upregulated on endothelial cells in tumors. Such molecules could be useful to antagonize or inhibit unwanted VEGF-B induced cellular responses such as tumor-induced angiogenesis or psoriasis or retinopathies by techniques analogous to those described in Kim et al., Nature, 362(6243):841-44 (1993) or Aiello et al., New England Journal of Medicine, 331(22):1480-87 (1994).

One procedure for isolating VEGF-B/Flt-1 complexes involves using fusion proteins of the Fit-i receptor and immunoglobulin G (IgG) followed by Sepharose A binding.

Alternatives to the use of Sepharose A include using ionexchange chromatography, gel filtration or affinity chromatography. Conditioned medium containing receptor/IgG fusion proteins could be allowed to interact with conditioned medium either from cells either transfected with DNA encoding the VEGF-B ligand or analog thereof, or from cells which naturally express the ligand, or with a solution containing a candidate ligand analog.

In accordance with another preferred aspect of the invention, the invention relates to a method for identifying a VEGF-B analog having substantially the same binding affinity for a cell surface receptor as VEGF-B, the method comprising the steps of: 4 WO 98/28621 PCT/US97/23533 providing a sample containing cells that express a surface receptor protein having binding affinity for VEGF-B selected from the group consisting of: a polypeptide chain comprising an amino acid sequence defined by residues 1-347 of Flt-1, or a VEGF-B-specific receptor analog thereof; (ii) a polypeptide chain having binding affinity for VEGF-B and sharing at least 30% amino acid identity with residues 1-347 of Fit-1; and (iii) a polypeptide chain having binding affinity for VEGF-B and encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising the sequence defined by nucleotides 1-1293 of Fit-1; contacting the cells with a candidate VEGF-B analog, and detecting induction of a VEGF-B-mediated cellular response. Examples of such detectable cellular responses include endothelial cell proliferation, angiogenesis, tyrosine phosphorylation of receptors, and cell migration.

The cells which express the cell surface receptor protein may be cells which naturally express the receptor, or they may be cells transfected with the receptor such that the receptor is expressed. Conditioned medium from culturing such cells can be passed over a Sepharose A column or matrix to immobilize the receptor, or they can be immobilized in cellulose disks or absorbed onto plastic, in the form of an ELISA test. A second solution containing conditioned medium from cells expressing the ligand is then passed over such immobilized receptor. If desired, the ligand may be radioactively labelled in order to facilitate measurement of the amount of bound ligand by radioassay techniques. Such an assay can be used to screen for conditions involving overexpression of the Fit-i receptor, i.e. through detection of increased bound radioactivity 5 WO 98/28621 PCT/US97/23533 compared to a control. This methodology can also be used to screen for the presence of competing VEGF-B analogs, i.e.

through detection of decreased bound radioactivity compared to a control indicative of competition between the radioactively labelled VEGF-B ligand used in the test and a non-radioactive putative analog.

Alternatively, the foregoing assay could be reversed by immobilizing the VEGF-B ligand or candidate analog and contacting the immobilized ligand with conditioned medium from cells expressing the receptor.

In accordance with yet another aspect of the invention, the invention relates to a kit for identifying VEGF-B or a candidate VEGF-B analog in a sample, the kit comprising: a receptacle adapted to receive a sample and containing a receptor protein selected from the group consisting of: a polypeptide chain comprising an amino acid sequence defined by residues 1-347 of Flt-1, or a VEGF-B-specific receptor analog thereof; (ii) a polypeptide chain having binding affinity for VEGF-B and sharing at least 30% amino acid identity with residues 1-347 of Fit-1; and (iii) a polypeptide chain having binding affinity for VEGF-B and encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising the sequence defined by nucleotides 1-1293 of Fit-1; and means for detecting interaction of VEGF-B or a candidate VEGF-B analog with the receptor protein contained in the receptacle, wherein the VEGF-B or candidate VEGF-B analog comprises part of a sample received in the receptacle. The detecting means may comprise, for example, means for detecting specific binding interaction of VEGF-B or a VEGF-B analog with the receptor protein or means for detecting induction of a VEGF-B induced cellular response.

6 WO 98/28621 PCT/US97/23533 A still further aspect of the invention relates to an isolated ligand-receptor complex comprising two molecules, one defining the ligand and comprising at least amino acids 1-115 of VEGF-B or a receptor-binding analog thereof, and the second defining the receptor and being selected from the group consisting of: a polypeptide chain comprising an amino acid sequence defined by residues 1-347 of Flt-1, or a VEGF-B-specific receptor analog thereof; (ii) a polypeptide chain having binding affinity for VEGF-B and sharing at least 30% amino acid identity with residues 1-347 of Fit-1; (iii) a polypeptide chain having binding affinity for VEGF-B and encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising the sequence defined by nucleotides 1-1293 of Fit-1.

Preferably the ligand is VEGF-B and the receptor is the Flt-1 receptor which also has binding affinity for VEGF-A and P1GF.

Isolation and purification of the ligands or complexes could be effected by conventional procedures such as immunoaffinity purification using monoclonal antibodies according to techniques described in standard reference works such as Harlow et al., Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press (1988) and/or Marshak et al., Strategies for Protein Purification and Characterization, Cold Spring Harbor Laboratory Press (1996). Suitable antibodies to the individual ligands or to the complexes could be generated by conventional techniques.

A cell-free complex could be used either in vivo or in vitro to compete with VEGF-B binding to a cell surface receptor or to prevent dimerization of the cell-bound receptor after ligand binding. Such a cell-free complex would comprise at least one receptor molecule, for example soluble FLT (sFLT), and a VEGF-B dimer molecule, VEGF-B analog dimer molecule or mixed VEGF-B/VEGF-B analog dimer 7 WO 98/28621 PCT/US97/23533 molecule so that one molecule of the dimer can be bound to the receptor molecule in the complex and the second molecule of the dimer has a free binding site available to bind to a cell surface receptor.

It is also an aspect of the present invention to provide an isolated binding partner having specific binding affinity for an epitope on a ligand-receptor complex comprising VEGF-B protein or an analog thereof in specific binding interaction with the ligand binding domain of a cell surface receptor defined by: a polypeptide chain comprising an amino acid sequence defined by residues 1-347 of Flt-1, or a VEGF-B-specific receptor analog thereof; (ii) a polypeptide chain having binding affinity for VEGF-B and sharing at least 30% amino acid identity with residues 1-347 of Flt-1; or (iii) a polypeptide chain having binding affinity for VEGF-B and encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising the sequence defined by nucleotides 1-1293 of Fit-1; wherein the binding partner has substantially no binding affinity for uncomplexed VEGF-B or VEGF-B analog.

Preferably the binding partner also will have substantially no binding affinity for any uncomplexed form of the cell surface receptor protein or receptor analog thereof. The binding partner may be an antibody which reacts with or recognizes such growth factor/receptor complexes. Either polyclonal or monoclonal antibodies may be used, but monoclonal antibodies are preferred. Such antibodies can be made by standard techniques, screening out those that bind to either receptor or ligand individually.

An additional aspect of the invention relates to the use. of a VEGF-B analog obtained according to the methods described above for antagonizing VEGF-B binding to a cell surface receptor, or 8 WO 98/28621 PCT/US97/23533 (ii) antagonizing induction of a VEGF-B-mediated cellular response.

A preferred VEGF-B analog comprises an antibody having binding specificity for the ligand binding domain of a cell surface receptor having binding affinity for VEGF-B, or (ii) a receptor binding domain of VEGF-B or a receptorbinding analog thereof.

The ligand binding domain of a cell surface receptor having binding affinity for VEGF-B desirably will exhibit at least 30%, preferably at least 35%, amino acid identity with residues 1-347 of Flt-1 and especially preferably will correspond thereto. The receptor binding domain of a VEGF-B analog desirably will exhibit at least 50%, preferably at least 65%, sequence identity with amino acid residues 1-115 of VEGF-B, and especially preferably will correspond thereto.

Yet another aspect of the invention relates to the use of a receptor protein selected from the group consisting of: a polypeptide chain comprising an amino acid sequence defined by residues 1-347 of Flt-1, or a VEGF-B-specific receptor analog thereof; (ii) a polypeptide chain having binding affinity for VEGF-B and sharing at least 30% amino acid identity with residues 1-347 of Flt-1; or (iii) a polypeptide chain having binding affinity for VEGF-B and encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising the sequence defined by nucleotides 1-1293 of Flt-1; in a method for antagonizing: VEGF-B binding to a cell surface receptor, or induction of a VEGF-B-mediated cellular response.

The polypeptide chain competes with the cell surface receptor for VEGF-B and ties up the available VEGF-B, thereby preventing it from effectively interacting with the cell surface receptor and inducing the VEGF-B mediated 9 cellular response. A suitable peptide chain could be a solubilized form of the receptor (sFLT) as described in Kendall et al., Proc. Natl. Acad. Sci., 90:10705-709 (1993).

Additionally, it is an aspect of the invention to provide a method for antagonizing VEGF-B binding to a cell surface receptor, the method comprising the step of providing a protein having binding specificity for the amino acid sequence defined by residues 1-347 of Flt-1 or a VEGF-B receptor binding sequence variant thereof, wherein the protein has at least 50%, and preferably at least 65%, amino acid sequence identity with residues 1-115 of VEGF-B, such that the protein, when provided to a cell expressing the cell surface receptor, is competent to interact specifically with the receptor and thereby substantially inhibits VEGF-B binding to the receptor. The protein may desirably be a VEGF-B analog obtained according to one of the methods described above.

In accordance with a further aspect of the invention, pharmaceutical preparations are provided which comprise such growth factor/receptor complexes.

In yet another aspect of the invention a method is provided for treating a condition associated with overexpression of an Flt-i cell surface receptor, said method comprising administering to a patient suffering from said disease state an effective receptor-binding amount of VEGF-B or a VEGF-B analog obtained according to one of the methods described above.

In yet another aspect of the invention, a method is provided for treating a condition associated with underexpression of an Fit-i cell surface receptor said method comprising administering to a patient in said condition an effective receptor-binding amount of VEGF-B or VEGF-B analogue obtained according to one of the methods described above.

Where the receptor protein comprises a polypeptide chain other than residues 1-347 of Fit-i but which nevertheless exhibits a binding affinity for VEGF-B, it S1 17 CT 2001 03 should exhibit at least 30%, desirably at least preferably at least 65%, particularly preferably at least and especially preferably at least 95%, amino acid identity with residues 1-347 of Flt-1. Useful VEGF-B analogs should exhibit at least 50%, preferably at least particularly preferably at least 90%, and especially preferably at least 95%, sequence identity to VEGF-B.

'L lOa 1 7 CCT M01l WO 98/28621 PCT/US97/23533 Brief Description of the Drawings The invention will be described in further detail hereinafter with reference to illustrative experiments, the results of which are illustrated in the accompanying drawings in which: Figure 1 is an anti-PTyr probed Western blot of Flt-1 immunoprecipitates from Flt-1 expressing NIH3T3 cells stimulated with conditioned media from 293 EBNA cells transfected respectively with expression vectors for human VEGF and VEGF-B; Figures 2(a) and are respectively long and short exposures of SDS-PAGE electrophoresis gels showing binding of 1"S-methionine-labelled murine VEGF-B 1 86 to Flt-1-IgFc fusion protein; Figure 3 is an SDS-PAGE analysis of the binding of

VEGF

1 is, VEGF-B 16 VEGF-Big and VEGF-C to soluble VEGFR-1, VEGFR-2 and VEGFR-3; Figure 4 is a graph of the displacement of [125] -hVEGF 6 from VEGFR-1/Flt-1 by mVEGF-B.

86 using NIH3T3 Flt-1 cells; Figure 5 is a graph of competition on NIH-VEGFR-1/Flt-1 by mVEGF 16 4 Figure 6 shows displacement of VEGF-B, 6 and VEGF-B 1 8 6 from soluble VEGFR-1 by excess VEGF 6 s; Figure 7 is an SDS-PAGE analysis showing proteolytic processing of VEGF-B.

86 Figure 8 is an SDS PAGE analysis showing plasmin digestion of VEGF-B 1 86 Figure 9 is a schematic diagram showing mutations of VEGF-Bi 7 used for mutational analysis and binding of receptor binding epitope mutants to soluble VEGFR-1; Figures 10a through 10c show an SDS-PAGE analysis of cysteine mutations of VEGF-B; Figure 11 shows an SDS-PAGE analysis of VEGF-B, 7 mutants labelled in the presence of 10 Ag/ml heparin; Figure 12 shows a Northern Blot analysis of RNA from bovine microvascular endothelial (BME) cells incubated in the presence of 50 ng/ml hVEGF-B,.

8 and 11 WO 98/28621 PCTIUS97/23533 Figures 13a and 13b show a zymographic and reverse zymographic analysis of cell extracts prepared from BME cells incubated in the presence of hVEGF-B,, 6 General Methods Cell culture and materials: Sf-9 cells were maintained in Sf-900 II SFM (Gibco BRL, Life Technologies) supplemented with 0.1% pluronic f-68 for suspension growth.

High Five cells (Invitrogen) were maintained in Ex-cell 400 media (JHR Bioscience UK).

293-EBNA, COS-7, 293-T and NIH3T3-Flt-1 cells [Sawano et al., Cell Growth Differentiation 7, 213-21 (1996)] were grown in Dulbecco's minimum essential medium (DMEM) supplemented with 10% fetal calf serum (FCS). NIH3T3 Flt-1 were kept under continuous selection using 200 Ag/ml neomycin.

Bovine adrenal cortex-derived microvascular endothelial (BME) cells were grown in MEM, alpha modification (Gibco AG, Basel, Switzerland) supplemented with 15% donor calf serum on 1.5% gelatin coated tissue culture flasks.

PAE-KDR cells [Waltenberger et al., J. Biol. Chem., 269:26988-95 (1994)] were cultured in Ham's F12 media with

FCS.

Construction of receptor Ig-fusions and Expression Vectors: a) pIg-VEGFR-1.

The expression plasmid pIg-VEGFR-1 coding for the first five Ig-like domains of VEGFR-1 fused to human IgG1 Fc was constructed by ligating a HindIII fragment (coding for the amino acids 1-549 of VEGFR-1) from pLTR Fltl into pIgplus vector (Ingenius). Prior to the cloning the pIgplus vector was digested with XhoI and XbaI, blunted and religated in order to correct the reading frame for the fusion protein production.

12 WO 98/28621 PCT/US97/23533 b) spIg-VEGFR-2.

For the spIg-VEGFR-2 construct, cDNA encoding the first four Ig-like domains of VEGFR-2 was amplified by polymerase chain reaction (PCR) using human fetal lung cDNA library (Clontech) as a template. The primers 5'-atggtacccccaggctcagcatacaaaaagac-3' (SEQ ID NO. 1) and gcgtctagagggtgggacatacacaaccag-3' (SEQ ID NO. 2) were used, and the amplified fragment was cleaved with Kpn-l and Xba-1 and cloned into corresponding sites of signal pig vector (Ingenius).

c) mVEGF-B 1 8 6 pFASTBAC1.

mVEGF-B 1 86 cDNA [Olofsson et al., J. Biol. Chem.

271:19310-19317 (1996)] was cleaved by EcoRI and subcloned into pFASTBAC1 (Gibco BRL Life Technologies). A (His) 6 tag (and an enterokinase site) was introduced at the N-terminus devoid of signal sequence, using PCR with mVEGF-B, 6 pSG5 as a template, and the primers ccagttt-3' (SEQ ID NO. 3) and 5'-caagggcggggcttagagatctagct-3' (SEQ ID NO. 4) (both containing Bgl II sites) were used. The amplified fragment was cleaved with Bgl II and cloned into the Bam HI site in frame with the signal sequence of GP-67, of pAcGP67A, (Pharmingen, d) hVEGF-B 1 8 6 pPIC-9.

hVEGF-B.

8 was amplified by PCR using the forward primer 5'-ggaattccccgcccaggcccctgtc-3' (SEQ ID NO. and the reverse primer 5'-ggaattcaatgatgatgatgatgatgagccccgcccttggc-3' (SEQ ID NO.

6).

The amplified product containing a C-terminal (His) 6 tag was cloned into the EcoRI site of pPIC-9 (Invitrogen) in frame with the alpha mating factor signal sequence.

13 WO 98/28621 PCT/US97/23533 The authenticity of all sequences was verified by sequencing.

Protein Expression and Purification: For baculoviral production using Sf9 and High Five cells, mVEGF-B recombinant plaques were purified and amplified [Summers et al., Tex. Agric. Exp. Stn. Bull.

1555:1-57 (1988)], and the corresponding expressed proteins as well as the Pichia pastoris (strain GS115) expressed hVEGF-Big 6 were purified using Ni-NTA Superflow resin (Qiagen).

For quantitative immunoblots, media from infected insect cells were run together with 1-30 ng purified m(His),VEGF-B 86 as a standard on a reducing 12% SDS-PAGE and blotted with the affinity purified antibody against m (His) 6 VEGF-Bi 6 Transfection, immunoprecipitation and soluble receptor binding: 293-T cells or COS-7 cells were transfected with hVEGF1 6 spSG5, mVEGF-B 167 pSG5, mVEGF-BkEx-spSG5, mVEGF-B 86 VEGFR-1 pig and VEGFR-2 pig using calcium phosphate precipitation. VEGFR-3 EC-Ig pREP7 (obtained from Dr. Katri Pajusola) and hVEGF-CANAC (His) 6 pREP7 [Joukov et al., EMBO J. 16:3898-911 (1997)] were similarly expressed in 293-EBNA cells. The cells expressing the growth factors were metabolically labelled 48 hours post transfection with 100 uCi/ml Promix TM L-35 S (Amersham) for 5-6 hours (unless otherwise stated), and the media were collected. Heparin or 50 g/ml) was added to the labeling medium when indicated. The metabolically labelled media (except from the VEGF transfection) was immuno-depleted of endogenous expressed VEGF and heterodimers for 2 hours with 2jg/ml VEGF antibody MAB 293 (R&D Systems). For the soluble receptors the media was replaced 48 hours post transfection by DMEM containing 0.1% BSA and incubated for additional 12 hours.

14 WO98/28621 PCT/US97/23533 The receptor-Ig fusions (in some cases the amounts were quantified on 10% SDS-PAGE stained with Colloidal Comassie (Novex, San Diego)) and the same volume of media from mock transfected cells were absorbed to protein-A-Sepharose. The metabolically labelled growth factors were incubated with the receptor Ig fusions for 3 hours at 4 0 C and washed with ice-cold binding buffer (PBS 0.5% BSA, 0.02% Tween 20 and ImM PMSF) three times and twice with PBS containing ImM

PMSF.

Antibody Production: Rabbits were immunized with purified m(His) 6

VEGF-B

86 according to standard procedures and the resulting antiserum was collected. Antiserum to mVEGF-B N-terminal peptide was produced as described in [Olofsson et al., J. Biol. Chem.

271:19310-19317 (1996)]. The antisera were affinity purified against m (His) VEGF-B 8 6 covalently bound to CNBr-activated Sepharose CL-4B (Pharmacia).

Example 1 Procedure: Cultures of NIH3T3 cells expressing human Flt-1 receptor protein were first starved in 0.5% fetal calf serum (FCS) in DMEM for 24 hours. The cells were then stimulated for 5 minutes at 37 0 C in conditioned medium from cultures of 293 EBNA cells which had been transfected respectively with pREP7-hVEGF-A or pREP7-hVEGF-B 67 Conditioned medium from 293 EBNA cells transfected with pREP7 alone was used as a negative control (Mock). All conditioned media contained 1 gg/ml heparin. After stimulation, the cells were rinsed in ice-cold PBS containing 0.1 mM sodium orthovanadate and lysed in RIPA buffer containing 2 mM sodium orthovanadate, 1 mg/ml aprotinin and 1 mM PMSF. The lysates were sonicated, clarified by centrifugation and incubated on ice for 2 hours with the anti-flt-1 antibody, SC316 (Santa Cruz). The resulting immune complexes were collected by 15 WO 98/28621 PCT/US97/23533 precipitation with protein A-sepharose beads.

Immunoprecipitates were washed three times with the lysis buffer, separated by electrophoresis on 6% SDS PAGE and transferred to a nitrocellulose filter. The filter was probed with horse-radish peroxide (HRP)-conjugated antiphosphotyrosine antibody RC20H (Transduction Labs) and immunoreactivity detected by ECL (Amersham). The antiphosphotyrosine antibody recognized phosphorylated tyrosine on Flt-1 and enabled observation of autophosphorylation of the Fit-1 receptor.

Results: As can be seen from the accompanying Figure 1, which is an anti-PTyr probed Western blot of Fit-1 immunoprecipitated from Fit-i expressing NIH3T3 cells stimulated with heparin-supplemented conditioned medium from VEGF vector, empty vector or human VEGF-B 1 6 pREP7 vector-transfected 293 EBNA cells, somewhat weak but nevertheless positive tyrosine phosphorylated bands are observed for both VEGF-A and VEGF-B which indicate that both of these ligands cause autophosphorylation of Fit-1. In contrast, the mock lane in the center of the Figure is devoid of autophosphorylation.

This data shows that human VEGF-B binds with and induces autophosphorylation of the Fit-i receptor. This indicates that Fit-i also is a receptor for human VEGF-B 67 Example 2 Procedure: Receptor IgG fusion proteins: cDNA encoding the first three immunoglobulin (Ig) loops of Fit-i was spliced to the Fc region of a human IgG heavy chain and cloned in to the vector pREP7 (Invitrogen) to yield the plasmid pREP7 Flt-l-IgFc. pREP7 KDR-IgFc was constructed in a similar fashion. The Flt-1-Ig Fc and KDR- Ig Fc cDNAs used in this experiment were RT-PCR products 16 WO 98/28621 PCT/US97/23533 from a plasmid construct called pBJFltKT3. The resulting plasmids were used to transfect 293 EBNA cells by the calcium phosphate method, and the resulting conditioned medium was harvested 48 hours post-transfection.

Receptor IgG precipitation of 3 S-labeled mVEGF-B,, 6 Plasmid pSJ5 (Stratagene) encoding for murine VEGF-B, 8

VEGF-BB

86 was transfected into COS cells by electroporation, and the cells were labeled for 10 hours with 3 S-methionine and 3 S-cysteine. 35 S-labeled hVEGF-A16S was used as a positive control for receptor binding and was produced in 293 EBNA cells transfected with pREP7 VEGF-A and labeled as described above. About 1 ml of conditioned medium containing Flt-l-IgFc or KDR-IgFc was incubated with 40 Al of a 50% slurry of protein-A sepharose for 1 hour at 4 0 C under continuous agitation. Conditioned medium from mock-transfected cells was used as a negative control. The protein-A sepharose beads were collected by centrifugation, and incubated with 1 ml of conditioned medium containing 35 S-labeled mVEGF-B.

8 or VEGF(-A) in binding buffer (PBS, BSA, 0.02% Tween 20, 1 gg/ml heparin) for 3 hours at room temperature with gentle agitation. The protein-A sepharose beads were collected by centrifugation, washed twice in ice-cold binding buffer and once in 20 mM tris pH 7.5, boiled in SDS sample buffer and electrophoresed on

PAGE.

Results: The results are shown in Figures 2(a) and which are long and short exposures of the SDS-PAGE gels. In the Figures, lane 1 shows immunoprecipitated murine VEGF-B.

6 lane 2 shows Flt-l-Ig, mVEGF-BI 86 lane 3 shows Flt-l-Ig, mock; lane 4 shows mock, mVEGF-B 86 lane 5 shows KDR-Ig, mVEGF-B6s; lane 6 shows KDR-Ig, mock. To determine whether

VEGF-B

1 8 6 is a ligand for Flt-1, plasmid containing cDNA for this factor, as well as plasmid encoding VEGF(-A), or the 17 WO 98/28621 PCT/US97/23533 expression vector alone were transfected into mammalian cells, and the proteins were labeled with 3 "S amino acids.

Conditioned medium from these cells were precipitated with Flt-1-IgFc or KDR-IgFc bound to protein-A sepharose beads.

A 32 kDa band [identified by the upper arrow in Fig. 2(a)] was precipitated from the VEGF-B, 8 conditioned medium with Flt-1-IgFc. This band, which co-migrates with immunoprecipitated VEGF-B 1 8 6 was absent in the Flt-l-IgFc precipitation of mock transfected cells, or precipitation of

VEGF-B,

8 by protein-A sepharose alone. Little precipitation of this 32 kDa band was also found with KDR-IgFc.

The data clearly show the formation of complexes between the murine VEGF-B 86 and the human Flt-1 receptor.

As can also be seen from the Figs. 2(a) and both Flt-1-IgFc and KDR-IgFc additionally precipitated lower molecular weight species, but these three bands also were found in conditioned medium of mock transfected cells, and are considered to represent endogenous factors produced by COS cells, possibly VEGF(-A). Of these, the band indicated by the arrow in brackets may also partially represent VEGF-B related material.

Example 3 Test of VEGF-B binding to VEGFR-1, -2 and/or -3: To determine whether VEGF-B is a ligand for VEGFR-1 -2 or 293T cells were transfected with expression plasmids for VEGFis, m, mVEG, mVEGF-B 1 86 or VEGF-C, and the proteins were metabolically labelled, in the presence of 50 pg/ml heparin for VEGF 65 and VEGF-B 67 and the media was collected.

Conditioned media from all except the VEGF transfection were precleared of endogenous VEGF and VEGF/VEGF-B heterodimers and then the respective proteins were either immunoprecipitated with specific antibodies or bound to soluble receptor Ig fusion proteins containing the first five Ig-like domains of Flt-1 bound to protein A-Sepharose (PAS). The precipitated ligands were analyzed by SDS-PAGE 18 WO 98/28621 PCT/US97/23533 under reducing conditions. Approximately 50 ng of soluble receptor was used for each ligand precipitation. As shown in Fig. 3, both VEGF-B splice isoforms specifically bound to VEGFR-1 but not to VEGFR-2 or Functionality of the VEGFR-2 and VEGFR-3 receptors was confirmed by the binding tests with VEGF and VEGF-C, respectively. This test shows that VEGF-B binds specifically to VEGFR-1/Flt-1, making it the third ligand identified for VEGFR-1, after VEGF and P1GF.

Two bands of 32 kD and 16 kD were precipitated from the mVEGF-B,, conditioned medium with the specific antibody and by VEGFR-1. The 32 kD band corresponds to the glycosylated, secreted form of mVEGF-B 86 [Olofsson et al., J. Biol. Chem.

271:19310-19317 (1996)]. The 16 kD form is apparently a product of proteolytic processing described in further detail hereinafter.

Example 4 Further Examination of VEGF-B/VEGFR-1 Binding: The ability of VEGF-B to bind VEGFR-1 expressed on cell surface was examined using NIH3T3-Fltl cells. Consistent with the data obtained with soluble receptors, conditioned media from mVEGF-B,, 6 -infected, but not mock infected High Five cells, competed for 125 I-VEGF binding to NIH3T3-Fltl as seen in Fig. 4. Half maximum inhibitory concentration for mVEGF-B.

8 6 was estimated using quantitative immunoblots to 3 ng/ml compared to recombinant mVEGF 164 which competed for iodinated hVEGF 1 6 5 at a half maximum inhibitory concentration at 1.5 ng/ml (Figs. 4 and This effect was specific to VEGFR-1 as no competition was observed with PAE-KDR cells.

Thus it is apparent that although VEGF-B 186 like VEGF 121 lacks the C-terminal basic residues found in VEGF 16 s, it nevertheless binds to VEGFR-1 on NIH3T3-Fltl cells. VEGF-B also was found to bind equally well to the VEGFR-1 Ig-fusion containing the three N-terminal Ig-like domains of VEGFR-1 (residues 1-347) as well as to the VEGFR-1 Ig-fusion containing five of the Ig-like domains.

19 WO 98/28621 PCT/US97/23533 Due to aggregation and protein stability problems, purified His-tagged VEGF-B (human and mouse (His) 6

VEGF-B

1 86 also C-terminal deletion mutants covering exons 1-5 of mouse and human (His) 6 VEGF-B) competed only at high concentrations with iodinated VEGF for VEGFR-1 binding.

Example VEGF Competition Studies: mVEGF-B and mVEGF-B 8 expressed in transfected 293-T cells were labelled and precleared as described in the binding test, with the only difference being that 10 Ag/ml heparin was used for mVEGF-B, 7 in the labelling media. 2 4g of recombinant hVEGF.

6 s was added as a cold competitor to the binding reaction when indicated. Equal volumes of metabolically labelled factors were bound to soluble VEGFR-1 or immunoprecipitated with affinity purified N-terminal peptide VEGF-B antibody for 2 hours and washed twice with ice-cold 10mM Tris-HCl pH 8.0, 1% TritonX-100, 25 mM EDTA, 1mM PMSF and twice with PBS containing 1mM PMSF. The precipitates were analyzed by 15% SDS-PAGE. As shown in Fig. 6, the binding of these two forms as well as that of mVEGF-B 6 7 to VEGFR-1 was abolished by excess rhVEGF, thereby indicating that binding of VEGF-B to VEGFR-1 can be competed by excess VEGF. This test result confirms the specificity of the interaction and suggests that the interaction sites for VEGF and VEGF-B on the receptor must be overlapping, partially overlapping or at least in close proximity.

Example 6 Competition for Binding to Cell Surface Receptors: For the competition assays, High five cells were infected with the recombinant virus for native mVEGF-B 186 (mVEGF-B 8 6 pFASTBAC1) and with a mock virus, and the media were harvested 48 hours post infection and immediately used or frozen at -70 0

C.

20 WO 98/28621 PCT/US97/23533 Recombinant mVEGF 164 (obtained from Dr. Herbert Weich) or hVEGF 16 5 (R&D systems or Peprotech) were labelled with 125I using the lodo-Gen reagent (Pierce) and purified by gel filtration on PD-10 columns (Pharmacia). The specific activities were 2.2x10 5 cpm/ng and 1.0x10 s cpm/ng for mVEGF and hVEGF, respectively. For binding analysis PAE-KDR and NIH3T3-Fltl cells were seeded in 24 well plates coated with 0.2% gelatin, grown to confluence, washed twice with ice-cold binding buffer (Ham's F12, 0.5mg/ml BSA, 10 mM Hepes pH 7.4 for PAE-KDR and DMEM, 0.5 mg/ml BSA, 10 mM Hepes pH 7.4 for NIH3T3 Fltl) and incubated in triplicate with 0.5 ng/ml [12 5 "I-VEGF in binding buffer containing increasing amounts of unlabelled VEGF or media from VEGF-B or mock infected insect cells. After incubation for 2 hours at 4 0 C, the cells were washed three times with ice-cold binding buffer and twice with PBS containing 0.5 mg/ml BSA and lysed in 0.5 M NaOH. The solubilized radioactivity was measured using a gamma counter. Fig. 4 shows displacement of [125s] -hVEGFi 65 from VEGFR-1/Fltl by mVEGF-B 8 6 using NIH 3T3 Flt-1 cells. Fig. 5 shows competition on NIH-VEGFR-1/Flt-1 by mVEGF 16 4 In an analagous test, mVEGF-B 18 6 was found not to compete with 12 5 I]-VEGF for VEGFR-2 on PAE-KDR cells.

Competition analysis using purified recombinant (His) 6

VEGF-BE

8 6 indicated that only a minor portion of the protein is biologically active, since the native (own signal sequence) unpurified VEGF-B 8 6 competed far more efficiently with iodinated VEGF for VEGFR-1 binding.

Example 7 Proteolytic Processing of VEGF-Bis 6 mVEGF-B 86 expressed in COS cells is modified by O-linked glycosylation, which increases the apparent molecular weight from 25 kDa of the intracellular form to 32 kDa in the secreted form. As noted above, when mVEGFB.

8 was expressed in 293-T cells, a faster form migrating as a 16 kDa band 21 WO 98/28621 PCT/US97/23533 appeared in addition to the 32 kDa form. This band was also observed in conditioned media from COS cells when the cells were labeled for a longer period. The following test was carried out to compare the migration of dimers formed by mVEGF-B 8 6 to mVEGF-B 67 and a C-terminal truncated form expressed in 293-T cells and their ability to bind the sVEGFR-1. The mVEGF-B exon 1-5 mutant containing a C-terminal Kemptide motif [Mohanraj et al., Protein Expression Purification, 8:175-82 (1996)] (mVEGF-BkExipSG5, was produced by polymerase chain reaction (PCR).

VEGF-B

167 and VEGF-BI, 6 were expressed in 293-T cells. The cells were metabolically labelled. Mock transfected and VEGF-B 67 transfected cells were labelled in the presence of 10 Ag/ml heparin. The collected media were precleared of VEGF and heterodimers, and were immunoprecipitated with an affinity purified N-terminal VEGF-B peptide antibody or bound to VEGFR-1. The bound ligands were analyzed by SDS-PAGE under non-reducing conditions. The results are shown in Fig. 7.

It can be seen that mVEGF-B, 6 migrates as three different dimeric polypeptides the shortest being 34 kDa, an intermediate form of 48 kDa and the full length form of The 34 kDa band migrates slightly slower than mVEGF-BkE.is, indicating that the putative cleavage site is more C-terminal, presumably in the beginning of the translated exon 6A [Olofsson et al., J. Biol. Chem.

271:19310-19317 (1996)].

The test clearly shows that the longer VEGF-B.

8 isoform undergoes proteolytic processing which results in a shorter form containing the receptor binding epitopes for VEGFR-1.

The functional aspects of this proteolytic processing of

VEGF-B

86 are not fully understood. Since the VEGF-B.

8 isoform is readily secreted from cells, the proteolytic processing does not appear to be a way of regulating the release or availability of the protein.

22 WO 98/28621 PCT/US97/23533 As can be seen from Fig. 7, the relative intensity of the VEGF-B signal compared between the immunoprecipitated forms and the receptor-bound forms shows that the strength of the signal seems to correlate with the level of processed subunits in the dimers, thereby indicating that processing leads to an increased affinity for the receptor. The 48 kDa band is believed to consist of a dimer between a processed (16 kDa) and a full length (32 kDa) monomer. The 34 kDa band consists of a dimer between two processed monomers of 16 kDa each. It is significant that both these dimers which comprise the 16 kDa analog produced by processing of VEGF-B, bind better to the VEGFR-1 receptor than the c60 kDa dimer which is made up of two full length 32 kDa monomers.

Example 8 Plasmin Cleavage: The following test was run to determine whether a full length form of VEGF-BI 1 6 expressed in COS cells could be cleaved by the addition of plasmin, and if this affects the VEGFR-1 binding. This could be a physiological mechanism at the site of basement membrane degradation in the angiogenesis process.

COS-7 cells were transfected with mVEGF-B 86 metabolically labelled for 75 minutes, and the collected media was precleared of VEGF and heterodimers. The media was then incubated at 37 0 C with 0.1 U/ml plasmin (Boehringer Mannheim) for the time periods of 0, 5, 15, 30 and minutes. The reaction was stopped by addition of 1 mM PMSF and 0.1 casein units of aprotinin. The media were immunoprecipitated by the affinity purified N-peptide VEGF-B antibody and also bound to VEGFR-1 Ig. The precipitated proteins were analyzed by SDS-PAGE under reducing conditions. The results are shown in Fig. 8.

Concominant with the reduced amounts of the full length form is the appearance a 15 kDa fragment followed by a secondary fragment of 12 kDa. Thus plasmin cleavage does 23 WO 98/28621 PCT/US97/23533 occur, but evidently does not give rise to the same fragment as the endogenous proteolytic processing of VEGF-B 1 8 6 described above. Nevertheless, this N-teminal fragment is fully capable of interacting with sVEGFR-1, suggesting that VEGF-B is similar to VEGF, in that the recepetor binding epitopes are contained in the N-terminal fragment which is resistent to proteases such as plasmin.

Example 9 Mutational Analysis of Receptor Epitopes: Crystal structure determination and mapping of the VEGFR-2 epitope for VEGF has pointed to a number of hot spot amino acid residues, with the most important residues for the ligand-receptor interaction being Ile-46, Ile-83, Glu-64, Phe-17, Gln-79, Pro-85, Ile-43 and Lys-84 [Muller et al., Proc. Natl. Acad. Sci. USA 94:7192-7 (1997)]. The extent to which these residues are involved in VEGFR-1 binding is less clear. By charged amino acid to alanine scan mutagenesis [Keyt et al., J. Biol. Chem., 271:5638-5646 (1996)] the VEGFR-1 binding epitope in VEGF was proposed to involve a stretch of acidic residues (Asp-63, Glu-64 and Glu-67). These amino acid residues are conserved in VEGF-B (Asp-63, Asp-64 and Glu-67) and to a lesser extent in P1GF.

In order to analyze whether the acidic amino acid residues which are conserved between VEGF and VEGF-B and which have been implicated in VEGF/VEGFR-1 binding, are also are the major determinants for VEGF-B/VEGFR-1 binding, Asp63 Asp64 and Glu67 were mutated into alanines. The mutation scheme is illustrated in Fig. 9, which is a schematic illustration of the wildtype VEGF-B forms and the different mutants.

The putative receptor epitope mutants of VEGF-B 6 were expressed in transfected 293-T cells and metabolically labelled in the presence of 50 g/ml heparin. In order to study the VEGF-B homodimers, endogenous VEGF and VEGF heterodimers formed by VEGF and overexpressed VEGF-B were 24 WO 98/28621 PCT/US97/23533 immunodepleted with VEGF antibodies (MAB 293 from R&D Systems). The VEGF-B mutants were either immunoprecipitated with affinity purified N-terminal peptide antibody or bound to soluble VEGFR-1 Ig. The precipitates were analyzed by SDS-PAGE under reducing conditions. From the results it is apparent that neither mutation of two first acidic residues nor the mutation of all three acidic amino acid residues abolished VEGF-B binding to VEGFR-1. Thus, this data based upon either mutation of all three charged amino acids to alanines or on mutation of only the first two charged amino acid residues into alanines, indicates that the conserved acidic residues are not the major contributors to the binding of VEGF-B to VEGFR-1.

Example Mutational Analysis of Conserved Cysteines in VEGF-B 16 To examine the contribution of the conserved cysteines to dimer formation of VEGF-B and test the structural prediction based upon the anti-parallel covalent VEGF dimer model, cysteine-51 (Cys 2) and cysteine-60 (Cys 4) were mutated to serine residues. The cysteine to serine mutants in mVEGF-B 6 7 pSG5 were generated by M13-based in vitro single stranded mutagenesis employing the helper phage M13K07 [Viera et al., Methods Enzymol., 153:3-11 (1987)] and the dut- ung- E.coli strain RZ1032 [Kunkel et al., Methods Enzymol. 154:367-382 (1987)]. Mutations were carried out both as single mutants (C2S and C4S) and as a double mutant (C2S,C4S). The mutation scheme is illustrated in Fig. 9.

The mutants and wildtype VEGF-B 167 were expressed alone or in different combinations as co-transfections and metabolically labelled in 293-T cells, and the media were precleared from VEGF and heterodimers. The results are shown in Figs. 10a-c. The media were either immunoprecipitated with the affinity purified N-terminal VEGF-B antibody and analyzed under both non-reducing conditions (Fig. 10a) and reducing conditions (Fig. 10b) or 25 WO 98/28621 PCT/US97/23533 bound to soluble VEGFR-1 Ig (Fig. 10c) As can be seen from Fig. 10b, all the mutants were expressed in approximately same amounts.

It was found that VEGF-B 86 is cleaved, most likely C-terminal of the region identical in the two splice variants, which is encoded by exons 1-5 and contains the cysteine knot as well as the receptor binding epitopes.

Wildtype VEGF-Ba 6 migrated under non-reducing conditions as two bands 42 kDa and 46 kDa, however only the 46 kDa form was capable of binding to the VEGFR-1 (compare Figs. 2A and 3C). The 42 kD band is believed to correspond to dimers joined together by aberrant disulfide bridges, since these doublet bands are not seen with VEGF-B 8 6 or VEGF-BkEx-s, which lack the additional eight cysteines found in the C-terminal part of VEGF-B 167 The single mutant C4S gave rise to monomers. Also some dimers migrating as a 42kDa band were observed which were unable to bind to VEGFR-1. Surprisingly the C2S mutant, although partly produced as monomers, could still form dimers capable of receptor binding. Cotransfection of the single mutants (C2S+C4S) led to increased amounts of the 46kDa band regaining receptor binding, indicating that the dimerization impairment can be complemented by establishing a disulfide link between the non-mutated cysteins similar to VEGF [Potgens et al., J.

Biol. Chem., 269:32879-85 (1994)]. Co-transfection of a single mutant with the double mutant failed to complement.

Some of the VEGF-B 16 mutants were expressed as above, and cells were labelled in the presence of 10 g/ml heparin.

The collected media were incubated with VEGFR-1 Ig, and the bound ligands were subjected to SDS-PAGE under non-reducing conditions. The results are shown in Fig. 11. It can be seen that the C4S mutant and the double mutant C2SC4S showed residual receptor binding which is explainable by the interactions of the soluble receptor to the monomers.

26 WO98/28621 PCT/US97/23533 Thus, the mutational analysis of conserved cysteines which contribute to the formation of VEGF-B dimers indicates a structural conservation with VEGF and PDGF.

Example 11 Biological Response to VEGF-B: RT-PCR analysis using specific primers based on the bovine VEGFR-1 sequence shows that VEGFR-1 mRNA is expressed by bovine adrenal cortex-derived microvascular endothelial (BME) and bovine aortic endothelial (BAE) cells. To determine the biological response of VEGF-B on endothelial cells, replicate filters containing 5 ig/lane of total cellular RNA prepared from confluent monolayers of BME cells incubated in the presence of 50 ng/ml hVEGF-BI.

6 were hybridized with ["P]-labelled cRNA probes. BME cells [Furie et al., J. Cell Biol., 98:1033-41 (1984)] were grown in MEM alpha modification (Gibco AG, Basel, Switzerland) supplemented with 15% donor calf serum on 1.5% gelatin coated tissue culture flasks. The cytokine was added to confluent monolayers of BME cells to which fresh complete medium had been added 24 hours previously. Total cellular RNA was prepared after time periods of 0, 1, 3, 9, 24 and 48 hours using Trizol reagent (Life Technologies AG, Basel, Switzerland). Northern blots, UV-cross linking and methylene blue staining of filters, in vitro transcription, hybridization and post hybridization washes were carried out as described in [Pepper et al., J. Cell Biol., 111:743-55 (1990)]. The 3 P-labelled cRNA probes were prepared from bovine u-PA [Kratzschmar et al., Gene, 125:177-83 (1993)], human t-PA [Fisher et al., J. Biol. Chem., 260:11223-30 (1985)] and bovine PAI-1 [Pepper et al., J. Cell Biol., 111:743-55 (1990)] cDNAs as described in [Pepper et al., J.

Cell Biol., 111:743-55 (1990); Pepper et al., J. Cell Biol., 122:673-84 (1993)]. The results are shown in Fig. 12. RNA integrity and uniformity of loading were determined by staining the filters with methylene blue after transfer and 27 WO 98/28621 PCT/US97/23533 cross-linking (lower panel of the figure); 28S and 18S ribosomal RNAs are shown.

The Northern blot analysis showed that VEGF-B 6 ng/ml) increased steady state levels of urokinase type plasminogen activator (u-PA) and plasminogen activator inhibitor 1 (PAI-1) mRNAs in BME cells. That is, the test showed that endothelial cells responded to VEGF-B by inducing PAI-1 mRNA and u-PA mRNA. Thus, binding of VEGF-B to its receptor on endothelial cells stimulates the activity of u-PA as well as of PAI-1, which are important modulators of extracellular matrix degradation and cell adhesion and migration.

Example 12 Zymography and Reverse Zymography: Cell extracts prepared from BME cells incubated in the presence of hVEGF-B.

6 at concentrations of 0, 1, 3, 10, and 100 ng/ml, VEGF at a concentration of 30 ng/ml, or recombinant human bFGF (155 amino acid form obtained from Dr. P. Sarmientos) at a concentration of 30 ng/ml, were subjected to zymography and reverse zymography as follows.

Confluent monolayers of BME cells in 35 mm gelatin coated tissue culture dishes were washed twice with serum free medium and the cytokines were added in serum free medium containing trasylol (200 KIU/ml). After 15 hours incubation time, cell extracts were prepared and analysed by zymography and reverse zymography as described in Vassalli et al., J.

Exp. Med., 159:1653-68 (1984) and in Pepper et al., J. Cell Biol., 111:743-55 (1990). The results are shown in Figs.

13a-b and indicate that recombinant hVEGF-B.

8 increases u-PA and PAI-1 activity in BME cells. The Fig. 13a shows a zymographic analysis and Fig. 13b shows a reverse zymography analysis of cell extracts from BME cells. It can be seen that VEGF-B,,, induces a dose-dependent increase in u-PA and PAI-1 activity in the BME cells. The apparent lack of induction of PAI-1 activity by VEGF used as a control, 28 WO 98/28621 PCT/US97/23533 reflects rapid sequestration of PAI-1 into a complex with VEGF-induced tPA. This complex is observed by zymography of the culture supernatant of VEGF-treated cells. In contrast to VEGF [Pepper et al., Biophys. Res. Commun., 189:824-31 (1992)], VEGF-B 186 did not increase t-PA activity. The test showed that endothelial cells responded to VEGF-B by increasing synthesis of u-PA and PAI-1 and the resultant protein activities. However, the kinetics of PAI-1 induction were more rapid (within 1 hour) and transient (maximal effect observed at 3 hours) than those of u-PA (induced after 9 hours and sustained for up to 48 hours).

This is in agreement with what has been observed for bFGF and VEGF [Pepper et al., J. Cell Biol., 111:743-55 (1990); Pepper et al., Biochem. Biophys. Res. Commun., 181, 902-906 (1991); Mandriota et al., J. Biol. Chem., 270:9709-16 (1995)].

Examples 11 and 12 show that recombinant hVEGF-B 186 increases steady-state levels of u-PA and PAI-1 mRNAs in BME cells. In similar testing, VEGF-B 1 8 6 also induced PAI-1 but not u-PA mRNA in BAE cells.

Usefulness The formation of complexes between Flt-i tyrosine kinase receptors and VEGF-B and/or VEGF-B analogs may be used as a treatment for disease states characterized by overexpression of the Fit-i receptor by administering to a patient suffering from such a disease state an effective Fit-i receptor binding or receptor antagonizing amount of VEGF-B or a VEGF-B analog. An example of such a disease state characterized by overexpression of the Fit-i receptor is hemangioendothelioma. The Fit-1 receptor also is overexpressed in various tumors [Warren et al., J. Clin.

Invest., 95(4):1789-97 (1995); Hatva et al., Amer. J.

Pathology, 146(2):368-78 (1995)] The formation of complexes between VEGFR-1 and VEGF-B or a VEGF-B analog may also be useful in treating states characterized by 29 WO 98/28621 PCT/US97/23533 underexpression on a Flt-1 receptor. Such states may include normal adult endothelium or states which require increased blood vessel formation. The amount to be administered in a given case will depend on the characteristics of the patient and the nature of the disease state and can be determined by a person skilled in the art by routine experimentation.

The VEGF-B or VEGF-B analog may suitably be administered intravenously or by means of a targeted delivery system analogous to the systems heretofore used for targeted delivery of VEGF or FGF. Examples of such systems include use of DNA in the form of a plasmid [Isner et al., Lancet, 348:370 (1996)] or use of a recombinant adenovirus [Giordano et al., Nature Medicine, 2:534-39 (1996)]. VEGF-B could also be provided in protein form by techniques analogous to those described for VEGF [Bauters et al., The American Physiological Society, pp H1263-271 (1994); Asahara et al., Circulation, 91:2793 (1995)] or through use of a defective herpes virus [Mesri et al., Circulation Research, 76:161 (1995)]. Small molecule VEGF-B analogues could be administered orally. Other standard delivery modes, such as sub-cutaneous or intra-peritoneal injection, could also be used.

VEGF-B protein/Fit-1 receptor complexes also can be used to produce antibodies. The antibodies may be either polyclonal antibodies or monoclonal antibodies. In general, conventional antibody production techniques may be used to produce antibodies to VEGF-B/Flt-1 complexes. For example, specific monoclonal antibodies may be produced via immunization of fusion proteins obtained by recombinant DNA expression. Both chimeric and humanized antibodies and antibody fragments to the VEGF-B/Receptor complex are expressly contemplated to be within the scope of the invention. Labelled monoclonal antibodies, in particular, should be useful in screening for medical conditions characterized by overexpression or underexpression of the 30 WO 98/28621 PCTIUS97/23533 Fit-i receptor. Examples of such conditions include endothelial cell tumors of blood and lymphatic vessels, for example, hemangioendothelioma.

In one preferred embodiment of a diagnostic/prognostic means according to the invention, either the antibody, the growth factor or the receptor is labelled, and one of the three is substrate-bound, such that the antibody-complex interaction can be established by determining the amount of label attached to the substrate following binding between the antibody and the growth factor/receptor complex. In a particularly preferred embodiment of the invention, the diagnostic/prognostic means may be provided as a conventional ELISA kit.

The foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting. Since modifications of the described embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed broadly to include all variations falling within the scope of the appended claims and equivalents thereof.

31- WO 98/28621 PCT/US97/23533 The following references provide technical background information and are hereby incorporated herein by reference: References Ferrara, N. Davis-Smyth, T. (1997) Endocrine Reviews 18, 4-25.

Risau, W. (1997) Nature 386, 671-4.

Folkman, J. (1995) Nature Medicine 1, 27-31.

Barleon, Sozzani, Zhou, Weich, H. A., Mantovani, A. Marme, D. (1996) Blood 87, 3336-43.

Clauss, Weich, Breier, Knies, Rockl, W., Waltenberger, J. Risau, W. (1996) J. Biol. Chem. 271, 17629-34.

Fong, G. Rossant, Gertsenstein, M. Breitman, M.

L. (1995) Nature 376, 66-70.

Shalaby, Rossant, Yamaguchi, T. Gertsenstein, Wu, X. Breitman, M. L. Schuh, A. C. (1995) Nature 376, 62-66.

Shalaby, Ho, Stanford, W. Fischer, K. D., Schuh, A. Schwartz, Bernstein, A. Rossant, J.

(1997) Cell 89, 981-90.

Carmeliet, Ferreira, Breier, Pollefeyt, S., Kieckens, Gertsenstein, Fahrig, Vandenhoeck, A., Harpal, Ebenhardt, Declercq, Pawling, Moons, Collen, Risau, W. Nagy, A. (1996) Nature 380, 435-439.

Ferrara, Carver-Moore, Chen, Dowd, Lu, L., O'Shea, K. Powell-Braxton, Hilan, K. J. Moore, M.

W. (1996) Nature 380, 438-442.

Pepper, M. Ferrara, Orci, L. Montesano, R.

(1991) Biochem. Biophys. Res. Commun. 181, 902-906.

Chapman, H. A. (1997) Curr. Opin. Cell Biol. 9, 714-724.

Bacharach, Itin, A. Keshet, E. (1992) Proc. Natl.

Acad. Sci. USA 89, 10686-10690.

Maglione, Guerriero, Viglietto, Delli-Bovi, P.

Persico, M. G. (1991) Proc. Natl. Acad. Sci. USA 88, 9267-9271.

Olofsson, Pajusola, Kaipainen, Von Euler, G., Joukov, Saksela, Orpana, Pettersson, R. F., Alitalo, K. Eriksson, U. (1996) Proc. Natl. Acad. Sci. USA 93, 2576-2581.

32 WO 98/28621 PCTUS97/23533 Grimmond, Lagercrantz, J. Drinkwater, Silms, Townson, Pollock, Gotley, Carson, Rakar, S., Nordenskjold, Ward, Hayward, N. Weber, G. (1996) Genome Res. 6, 124-131.

Joukov, Pajusola, Kaipainen, Chilov, D., Lahtinen, Kukk, Saksela, Kalkkinen, N. Alitalo, K. (1996) EMBO J. 15, 290-298.

Lee, Gray, Yuan, Louth, Avraham, H. Wood, W. (1996) Proc. Natl. Acad. Sci. USA 93, 1988-1992.

Orlandini, Marconcini, L. Ferruzzi, R. Oliviero, S.

(1996) Proc. Natl. Acad. Sci. USA 93, 1167S-11680.

Yamada, Nezu, Shimane, M. Hirata, Y. (1997) Genomics 42, 483-8.

Park, J. Chen, H. Winer, Houck, K. A. Ferrara, N. (1994) J. Biol. Chemn. 269, 25646-25G54.

Olofsson, Pajusola, von Euler, Chilov, D., Alitalo, K. Eriksson, U. (1996) J. Biol. Chemn. 271, 19310-19317.

Keyt, B. Nguyen, H. Berleau, L. Duarte, C. M., Park, Chen, H. Ferrara, N. (1996) J. Biol. Chem. 271, 5638-5646.

Potgens, A. Lubsen, N. van Altena, M. C., Vermeulen, Bakker, Schoenmakers, J. Ruiter, D.

J. de Waal, R. M. (1994) J. Biol. Chem. 269, 32879-85.

Muller, Y. Li, Christinger, H. Wells, J. A., Cunningham, B. C. de Vos, A. M. (1997) Proc. Natl. Acad.

Sci. USA 94, 7192-7.

Sawano, Takahashi, Yamaguchi, Aonuma, M. Shibuya, M. (1996) Cell Growth Differentiation 7, 213-21.

Furie, M. Cramer, E. Naprstek, B. L. Silverstein, S. C. (1984) J. Cell Biol. 98, 1033-41.

Mohanraj, Wahlsten, J. L. Ramakrishnan, S. (1996) Protein Expression Purification 8, 175-82.

Viera, J. Messing, J. (1987) Production of single-stranded plasmid DNA. Methods Enzymol. 153, 3-11.

Kunkel, Roberts, J.D. Zakour, R.A. (1987) Rapid and efficient site-specific mutagenesis without phenotype selection. Methods Enzymiol. 154, 367-382.

Summers, M.D. Smith, G.E. (1988) A manual of methods for baculovirus vectors and insect cell culture procedures. Tex.

Agric. Exp. Stn. Bull. 1555, 1-57.

33 WO 98/28621 PTU9133 PCTIUS97/23533 Joukov, V. Sorsa, Kumnar, V. Jeltsch, M., Claesson-Welsh, Cac, Saksela, Kalkkinen, N.& Alitalo, K. (1997) EMB30 J. 16, 3898-911.

Vassalli, J. Dayer, J. Wohiwend, A. Belin, D.

(1984) J. Exp. Med. 159, 1653-68.

Pepper, M. Belin, Montesano, Orci, L.& Vassalli, J. D. (1990) LT. Cell Biol. 111, 743-55.

Kratzschmar, Haendler, Kojima, Rifkin, D. B.

Schleuning, W. D. (1993) Gene 125, 177-83.

Fisher, Wailer, E. Grossi, Thompspon, D., Tizard, R. Schleuning, W. D. (1985) J. Biol. Chemn. 260, 11223-30.

Pepper, M. Vassalli, J. Orci, L. Montesano, R.

(1993) Exp. Cell Res. 204, 356-363.

Mandriota, S. Seghezzi, Vassalli, Ferrara, Wasi, Mazzieri, Mignatti, P. Pepper, M. S.

'(1995) J. Biol. Chemn. 270, 9709-9716.

Davis-Smyth, Chen, H. Park, Presta, L. G. Ferrara, N. (1996) EMBO J. 15, 4919-27.

Barleon, Totzke, Herzog, Blanke, Krernmer, Siemeister, Marne, ID. Martiny-Baron, G. (1997) J.

Biol. Chemn. 272, 10382-8.

Wiesmann, Fuh, Christinger, H. Eigenbrot, C., Wells, J. A. de Vos, A. M. (1997) Cell 91, 695-704.

Plouet, Moro, Bertagnolli, Coldeboeuf, N., Mazarguil, Clamens, S. Bayard, F. (1997) J. Biol.

Chem. 272, 13390-13396.

Park, Keller, G.A. Ferrara, N. (1993) Mol. Biol.

Cell 4, 1317-1326.

Takahashi, T. Shibuya, M. (1997) Oncogene 14, 2079-2089.

Seetharam, Gotoh, Maru, Neufeld, G., Yamaguchi, S. Shibuya, M. (1995) Oncogene 10, 135-147.

DiSalvo, Bayne, M. Conn, Kwok, P. Trivedi, P. G. Soderman, ID. Palisi, P. Sullivan, K. A. Thomas, K. A. (1995) J. Biol. Chem. 270, 7717-7723.

Cao, Chen, Zhou, Chiang, Anand-Apte, Weatherbee, J. Wang, Fang, Flanagan, J. G.

Tsang, M. (1996) J. Biol. Chemn. 271, 3154-3162.

Stefansson, S. &Lawrence, D. A. (1996) Nature 383, 441-443.

34 WO 98/28621 PCTIUS97/23533 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: KORPELAINEN, Eija OLOFSSON, Birgitta GUNJI, Yuji ERIKSSON, Ulf ALITALO, Kari (ii) TITLE OF INVENTION: VEGF-B/RECEPTOR COMPLEX AND USES THEREOF (iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Evenson, McKeown, Edwards Lenahan PLLC STREET: 1200 G Street, Suite 700 CITY: Washington STATE: DC COUNTRY: USA ZIP: 20005 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: WO FILING DATE: 19-DEC-1997

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 60/033,697 FILING DATE: 20-DEC-1996 (viii) ATTORNEY/AGENT INFORMATION: NAME: EVANS, Joseph D.

REGISTRATION NUMBER: 26,269 REFERENCE/DOCKET NUMBER: 1064/43148PC (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (202) 628-8800 TELEFAX: (202) 628-8844 35 WO 98/28621 PCT/US97/23533 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATGGTACCCC CAGGCTCAGC ATACAAAAAG AC 32 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GCGTCTAGAG GGTGGGACAT ACACAACCAG INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 62 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATCGAGATCT TCATCACCAT CACCATCACG GAGATGACGA TGACAAACCT GTGTCCCAGT TT 62 36 WO 98/28621 PCTIUS97/23533 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CAAGGGCGGG GCTTAGAGAT CTAGCT 26 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GGAATTCCCC GCCCAGGCCC CTGTC INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GGAATTCAAT GATGATGATG ATGATGAGCC CCGCCCTTGG C 41 37

Claims (23)

1. A method for identifying a VEGF-B analog having binding affinity for a VEGF-B cell surface receptor, said method comprising the steps of: providing a sample containing a receptor protein selected from the group consisting of: a polypeptide chain comprising an amino acid sequence defined by residues 1-347 of Flt-1, or a VEGF-B-specific receptor analog thereof; (ii) a polypeptide chain having binding affinity for VEGF-B and sharing at least 90% amino acid identity with residues 1-347 of Flt-1; and (iii) a polypeptide chain having binding affinity for VEGF-B and encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising the sequence defined by nucleotides 1-1293 of Flt-1; contacting said sample of step with a candidate VEGF-B analog having an amino acid sequence different from VEGF-B but at least 90% identical to amino acids 1-115 of VEGF-B; and detecting specific binding between said candidate VEGF-B analog and the receptor protein of step

2. A method for identifying a VEGF-B analog having binding affinity for a VEGF-B cell surface receptor, said method comprising the steps of: providing a sample containing cells that express a surface receptor protein having binding affinity for VEGF-B selected from the group consisting of: a polypeptide chain comprising an amino acid sequence defined by residues 1-347 of Flt-1, or a VEGF-B-specific receptor analog thereof; (ii) a polypeptide chain having binding affinity for VEGF-B and sharing at least 90% amino acid identity with residues 1-347 of Flt-1; and (iii) a polypeptide chain having binding affinity 387 i^ n\ 1 72 Ol for VEGF-B and encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising the sequence defined by nucleotides 1-1293 of Flt-1; contacting said cells with a candidate VEGF-B analog having an amino acid sequence different from VEGF-B but at least 90% identical to amino acids 1-115 of VEGF-B, and detecting induction of a VEGF-B-mediated cellular response.

3. A method according to claim 2, wherein said VEGF-B- mediated cellular response detected in step is endothelial cell proliferation.

4. A method according to claim 2, wherein said VEGF-B- mediated cellular response is angiogenesis.

An isolated ligand-receptor complex comprising two molecules, one of said molecules defining said ligand and comprising at least amino acids 1-115 of VEGF-B or a receptor-binding analog of VEGF-B having at least 50% amino acid sequence identity to amino acids 1-115 of VEGF-B or a receptor-binding amino acid sequence variant or xenogeneic homolog thereof, and a second of said molecules defining said receptor and being selected from the group consisting of: a polypeptide chain comprising an amino acid sequence defined by residues 1-347 of Flt-1, or a VEGF-B-specific receptor analog thereof; (ii) a polypeptide chain having binding affinity for VEGF-B and sharing at least 30% amino acid identity with residues 1-347 of Flt-1; (iii) a polypeptide chain having binding affinity for VEGF-B and encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising the sequence defined by nucleotides 1-1293 of Fit-1. 1 o v

6. A complex according to claim 5, wherein said receptor also has binding affinity for VEGF.

7. Use of a VEGF-B analog obtained according to the method of any one of claims 1 to 4 in a method for antagonizing VEGF-B binding to a cell surface receptor, or (ii) antagonizing induction of a VEGF-B-mediated cellular response.

8. A use according to claim 7, wherein said VEGF-B analog comprises an antibody having binding specificity for the ligand binding domain of a cell surface receptor defined by amino acids 1-347 of Flt-1 or a VEGF-B-specific receptor analog thereof.

9. Use of a receptor protein selected from the group consisting of: a polypeptide chain comprising an amino acid sequence defined by residues 1-347 of Flt-1, or a VEGF-B-specific receptor analog thereof; (ii) a polypeptide chain having binding affinity for VEGF-B and sharing at least 90% amino acid identity with residues 1-347 of Flt-1; or (iii) a polypeptide chain having binding affinity for VEGF-B and encoded by a nucleic acid that hybridizes. under stringent conditions with a nucleic acid comprising the sequence defined by nucleotides 1-1293 of Flt-1; in a method for antagonizing: VEGF-B binding to a cell surface receptor, or induction of a VEGF-B-mediated cellular response.

A method for antagonizing VEGF-B binding to a cell surface receptor, said method comprising the step of providing a protein having binding specificity for the amino acid sequence defined by residues 1-347 of Flt-1 or a VEGF-B eceptor binding sequence variant thereof, said protein 7' 40 (44) 17 having at least 90% amino acid sequence identity with residues 1-115 of VEGF-B, such that said protein, when provided to a cell expressing said cell surface receptor, is competent to interact specifically with said receptor, thereby substantially inhibiting VEGF-B binding to said receptor.

11. A method according to claim 9, wherein said protein is a VEGF-B analog obtained according to the method of any one of claims 1 to 4.

12. A method for treating a condition associated with overexpression of an Flt-1 cell surface receptor, said method comprising administering to a patient suffering from said disease state an effective receptor-binding amount of a VEGF-B antagonist, wherein said VEGF-B antagonist comprises a VEGF-B analog obtained according to the method of any one of claims 1 to 4, or an antibody to VEGF-B.

13. A method for treating a condition associated with underexpression of an Fit-i cell surface receptor, said method comprising administering to a patient in said state an effective receptor binding amount of VEGF-B or a VEGF-B agonist, said VEGF-B agonist comprising a VEGF-B analog obtained according to the method of any one of claims 1 to 4.

14. A VEGF-B analog selected from the group consisting of a receptor-binding 16 kDa fragment produced by proteolytic processing of VEGF-B, a receptor-binding fragment produced by plasmin digestion of VEGF-B, a receptor-binding exon mutant fragment containing a C-terminal Kemptide motif, and dimers comprising at least one of said receptor-binding fragments.

A VEGF-B analog according to claim 14, which comprises a dimer of two 16 kDa receptor-binding fragments obtained by proteolytic processing of VEGF-B. 41 17 hI7

16. A VEGF-B analog according to claim 14, which is a dimer of a full-length VEGF-B monomer and a 16 kDa receptor-binding fragment obtained by proteolytic processing of VEGF-B.

17. An isolated polynucleotide encoding a VEGF-B analog according to any one of claims 14 to 16.

18. A method of preventing or treating endothelial tissue growth or angiogenesis comprising the step of administering to a patient in need thereof an effective amount of a VEGF-B analog, according to claim 1 or claim 2.

19. Use of a VEGF-B analog, according to claim 1 or claim 2, for the preparation of a medicament for the treatment of endothelial tissue growth or angiogenesis.

A VEGF-B analog, according to any one of claims 1, 2 or 14, substantially as herein described with reference to the examples and figures.

21. An isolated ligand-receptor complex, according to claim 5, substantially as herein described with reference to the examples and figures.

22. Use of a receptor protein, according to claim 9, substantially as herein described with reference to the examples and figures.

23. A method for antagonising VEGF-B binding to a cell surface receptor, according to claim 10, substantially H:\evonnee\Keep\Speci\56136.98.doc 17/10/01 42 as herein described with reference to the examples and figures. Dated this 17th day of October 2001 LUDWIG INSTITUTE FOR CANCER RESEARCH and HELSINKI UNIVERSITY LICENSING LTD., OY By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:\evonnee\Keep\Speci\56136.98.doc 17/10/01 43