WO2003004045A2

WO2003004045A2 - Methods for identifying therapeutic agents for treating diseases involving wnt polypeptides and wnt receptors

Info

Publication number: WO2003004045A2
Application number: PCT/CA2002/001016
Authority: WO
Inventors: Marcia L. Macdonald; Yigal P. Goldberg; Michael R. Hayden
Original assignee: University of British Columbia; Xenon Genetics Inc
Current assignee: University of British Columbia; Xenon Pharmaceuticals Inc
Priority date: 2001-07-05
Filing date: 2002-07-05
Publication date: 2003-01-16
Anticipated expiration: 2004-01-05
Also published as: AU2002317105A1; WO2003004045A3

Abstract

Mutations in Wnt receptor genes, such as FZD4, associated with hereditary human visual disorders, such as familial exudative vitreoretinopathy ('FEVR'), are disclosed along with methods of use of Wnt and/or Wnt receptor genes and proteins, including in assays for therapeutic agents useful in treating such diseases and/or ameliorating their effects as well as methods of diagnosing diseases and disorders caused by mutations in these genes.

Description

METHODS FOR IDENTIFYING THERAPEUTIC AGENTS FOR TREATING DISEASES INVOLVING WNT POLYPEPTIDES AND WNT RECEPTORS

This application claims priority of U.S. Provisional Applications 60/303,285, filed 5 July 2001 , 60/340,409, filed 29 October 2001 , and 60/360,352, filed 28 February 2002, the disclosures of all of which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the field of Wnt and Wnt receptor genes and proteins disclosed herein, and mutations in those genes/proteins which are associated with certain human disorders and diseases, uses of such mutations in the diagnosis of diseases, and assays for identification of therapeutic agents useful in treating those diseases, and to therapeutic agents that modulate expression or activity of Wnt and/or Wnt receptor genes and proteins.

BACKGROUND OF THE INVENTION

Familial exudative vitreoretinopathy (FEVR) [OMIM 133780, "OMIM" = "Online Mendelian Inheritance in Man"] is a bilateral disorder of the retinal vasculature, characterized by an abrupt cessation of the growth of peripheral retinal capillaries (Criswick and Schepens, 1969). The phenotype is variable and in severe cases, the formation of fibrovascular vitreous membranes that cause retinal traction may result in the displacement of the macula, the presence of retinal folds, and/or the detachment of the retina, any of which may cause a visual-acuity decrease sufficient to result in legal blindness. Patients with mild forms of the disease are asymptomatic, and their only disease-related abnormality is an arc of avascular retina in the extreme temporal periphery. (Downey et al. 2001. Am. J. Hum. Genet. 68) Autosomal dominant inheritance is the most common form of this disease, although recessive and X-linked pedigrees are also seen. Several genetic loci have been identified, but despite considerable investigation, no genes have been confirmed to be directly associated with the autosomal dominant form of the disease. A single locus designated EVR1 was established at 11 q13-23 (Li et al. 1992; Muller et al. 1994). This locus was further refined to approximately 200 kb on 11 q14 in a recent report involving seven small families of Japanese origin. (Kondo, H. et al. 2001. Delineation of the critical interval for the familial exudative vitreoretinopathy gene by linkage and haplotype analysis. Hum Genet. May;108(5):368-75.) A bioinformatics analysis of this region did not reveal any positional candidate genes, suggesting that an unlinked family was used to define the distal boundary by the polymorphic marker GATA30G01. Locus EVR2 was designated based on the finding, in the X-linked form, of mutations in the gene for Norrie disease at Xp11.4-p11.3 (Chen et al 1993z; Shastry et al. 1995). A new locus, EVR3, was recently designated in the report of Bamashmus et al. (2000 Br. J. Opthalmol. 84:358-363) at 11 p12-13, located about 30 cM from EVR1.

There remains a great need to identify the underlying genetic basis of FEVR. The need goes beyond the specific needs of diagnosing and treating patients affected by this disease. For example, a gene involved in this disorder would also be a suitable drug target for treating many related forms of retinopathy, such as retinopathy of prematurity (ROP), diabetic retinopathy, and others, and other diseases related to a deficiency/excess of angiogenesis and vasculogenesis, not the least of which is cancer and neoplasia. The cellular processes that underlie vascularization of the human retina, as they are known, are set out in Hughes, S. et al. 2000. Invest. Opthalmol. Vis. Sci. 41 (5):1271 - 1228. Disorders relating to any of these cellular processes, whether occurring at the retina, in neurological tissue or elsewhere in the body, should be treatable if the underlying genetic basis of FEVR can be determined. When the FEVR gene is identified it will lead to a broad class of genes/proteins which are implicated in FEVR and disease processes related to FEVR. It is an object of this invention to provide other genes and proteins with overlapping specificity to FEVR protein and related ligands that may be used to target therapeutic agents for intervention in such disease processes, and which may also be useful in treating diseases including various angiogenic based disorders.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method for identifying a modulator of frizzled-polypeptide biological activity, comprising:

(a) contacting an agent with a frizzled polypeptide under conditions promoting said contacting and said biological activity; and

(b) determining a difference in the biological activity of said frizzled polypeptide compared to when said agent is not present,

thereby identifying said agent as a modulator of frizzled polypeptide biological activity.

In specific embodiments, such modulation may be an increase or a decrease in the biological activity. In a preferred embodiment, the biological activity is the binding of a ligand, preferably a Wnt polypeptide, most preferably a Wnt polypeptide of Table 1.

In another preferred embodiment, the frizzled polypeptide is a human frizzled polypeptide, preferably other than FZD4, most preferably a member selected from one or more of FZD1 , FZD2, FZD3, FZD5, FZD6, FZD7, FZD8, FZD9 and FZD10.

In a preferred embodiment of such a method, the frizzled polypeptide is part of a lipid bilayer, preferably wherein the frizzled polypeptide is part of a cell membrane, and most preferably wherein said frizzled polypeptide is part of an intact cell. In an additional preferred embodiment, said cell expresses a frizzled polypeptide on the surface of said cell, especially wherein the cell is a recombinant cell, most preferably wherein this recombinant cell express a frizzled polypeptide on its surface as a result of engineering, including where the recombinant cell does not express a frizzled polypeptide absent said engineering, especially where the engineering is genetic engineering.

In a further aspect, the present invention relates to a method for identifying an agent useful in treating a Wnt-related disease, comprising:

(a) contacting an agent with a Wnt-receptor under conditions promoting a biological activity of said Wnt-receptor and conditions promoting said contacting; and

(b) determining a difference in said biological activity compared to when said agent is not present,

thereby identifying said agent as a modulator of Wnt-receptor biological activity.

In specific embodiments, such modulation may be an increase or a decrease in the biological activity. In a preferred embodiment, the biological activity is the binding of a ligand, preferably a Wnt polypeptide, most preferably a Wnt polypeptide of Table 1. In another preferred embodiment, the Wnt receptor is a Wnt-receptor of Table 2. In a most preferred embodiment, the Wnt-polypeptide is a Wnt polypeptide of Table 1 and the Wnt-receptor is a Wnt-receptor of Table 2.

In a preferred embodiment of such a method, the Wnt-receptor is part of a lipid bilayer, preferably wherein the Wnt-receptor is part of a cell membrane, and most preferably wherein said Wnt-receptor frizzled polypeptide is part of an intact cell. In an additional preferred embodiment, said cell expresses a Wnt-receptor on the surface of said cell, especially wherein the cell is a recombinant cell, most preferably wherein this recombinant cell express a Wnt-receptor on its surface as a result of engineering, including where the recombinant cell does not express a Wnt- receptor absent said engineering, especially where the engineering is genetic engineering. In a further aspect, the present invention relates to a method for treating a Wnt-linked disease in a patient afflicted therewith comprising administering to said patient an effective amount of an agent that modulates Wnt-receptor activity, wherein such agent has such modulating activity in an of the foregoing assay methods, preferably wherein the agent was first shown to have Wnt-receptor modulating activity using such a method. In a further preferred embodiment, the disease is a retinopathy, such as familial exudative vitreoretinopathy (FEVR).

In preferred embodiments, the disease is selected from the group consisting of vascular disease, angiogenic disorders, retinopathies and cancer. In a preferred embodiment, said agent is an antibody that binds to a frizzled-polypeptide, preferably wherein said frizzled polypeptide is a human frizzled-polypeptide, more preferably wherein said human frizzled polypeptide is other than FZD4, and most preferably wherein said frizzled polypeptide is a member selected from the group consisting of FZD1 , FZD2, FZD3, FZD5, FZD6, FZD7, FZD8, FZD9 and FZD10.

In another preferred embodiment, the agent is a gene product encoded by and FZD gene, especially a polypeptide, most preferably a polypeptide that is one or more of of FZD1 , FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9 and FZD10. In one preferred embodiment, the agent is a polypeptide that comprises the amino acid sequence of SEQ ID NO: 2, 4 or 6. Such agents may be utilized in any combination.

The present invention also relates to a method for producing a product comprising identifying an agent according to any of the screening assay methods disclosed herein and where the product is the data collected with respect to said agent as a result of said method and wherein said data is sufficient to convey the chemical structure and/or properties of said agent. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 sets forth a large part of the family tree of the FEVR proband.

Figure 2 shows the effects of a number of mutations in the FEVR sequence. Figure 2A shows the effects of a 6 bp deletion producing superimposed sequences; 2B a 2 bp deletion producing superimposed sequences; 2C a 1033 A>G mutation (meaning "a" is replaced by "G" at residue 1033 of the nucleotide sequence of FZD4); 2D a502 C>T mutation; 2E a 97 C>T mutation; 2F a 766 A>G mutation. For such mutations in amino acid sequences, standard one-letter code is employed.

Figure 3A sets forth a comparison of known members of the human frizzled gene family. The FZD4 M493-W494del mutation found in FEVR patients is included for comparison. The highly conserved nature of the region where the mutation is found is self-evident Conserved amino acids are outlined in black and functionally conserved amino acids are in gray. Variable amino acids are not outlined. The mutation in FEVR patients is identified as a two amino acid mutation at site M493-W494del. Figure 3B is a comparison of the FZD4 L501fsX533 mutation with the normal sequence around the mutation site.

Figure 4A sets forth a comparison of known wild-type FZD4 homologs from a variety of eukaryotes. The FZD4 M493-W494del mutation found in FEVR patients is included for comparison. The highly conserved nature of the region where the mutation is found is self-evident Conserved amino acids are outlined in black and functionally conserved amino acids are in gray. Variable amino acids are not outlined. The mutation in FEVR patients is identified as a two amino acid mutation at site M493-W494del. Figure 4B is an alignment of the FZD4 M342V variation with sequences from FZD4 homologs from other eukaryotes. The FZD4 homologs shown are human FEVR FZD4 (FEVR), human (hFZD4), mouse (mFZD4), rat (rFZD4), chicken (gallus gallus) frizzled- 4 (gFz-4), Xenopus laevis (XFz4), zebrafish (Danio rerio) frizzled protein) (Zfz4), zebrafish (Danio rerio), Frizzled X (Zfzx), Caenorhabditis elegans wingless protein receptor (Cfz2), and Drosophila (Dfz2)

Figure 5A shows a sequence alignment for the P33S mutation (proline replaced by serine at residue 33) for a number of eukaryotes. Figure 5B shows an alignment the P168S mutation. Figure 5C is an alignment for the I256V change for the ROP5 mutation.

Figure 6 is a schematic of the domain structure of FZD4 with the cysteine-rich domain near the N-terminus and the M493-W494 deletion.

Figure 7 illustrates the two exon genomic structure of FZD4. Domains marked in black encode open reading frames. Shown at the top is the genomic arrangement of exons. The middle panel illustrates a potential alternative transcript of FZD4 resulting from alternative splicing. The bottom panel illustrates the approximate location of a mutation directly associated with FEVR.

Figure 8 shows that the 2^nd ATG is closer to the optimal Kozak consensus C-C-[AG]-C-C-A-T-G-G context for initiation of translation and is the first ATG in mouse and rat FZD4 mRNA. Putative start codons are indicated by italics.

Figure 9 shows a proposed mechanism of action of FZD4.

DEFINITIONS

In accordance with the present invention, the term "DNA segment" refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the segment and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector. Such segments are provided in the form of an open reading frame uninterrupted by internal nontranslated sequences, or introns, which are typically present in eukaryotic genes. Sequences of non-translated DNA may be present downstream from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.

The term "coding region" refers to that portion of a gene which either naturally or normally codes for the expression product of that gene in its natural genomic environment, i.e., the region coding in vivo for the native expression product of the gene. The coding region can be from a normal, mutated or altered gene, or can even be from a DNA sequence, or gene, wholly synthesized in the laboratory using methods well known to those of skill in the art of DNA synthesis.

In accordance with the present invention, the term "nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides. Generally, DNA segments encoding the proteins provided by this invention are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon.

The term "expression product" means that polypeptide or protein that is the natural translation product of the gene and any nucleic acid sequence coding equivalents resulting from genetic code degeneracy and thus coding for the same amino acid(s). The term "fragment," when referring to a coding sequence, means a portion of DNA comprising less than the complete coding region whose expression product retains essentially the same biological function or activity as the expression product of the complete coding region.

The term "promoter" means a region of DNA involved in binding of RNA polymerase to initiate transcription. As used herein, reference to a DNA sequence includes both single stranded and double stranded DNA. Thus, the specific sequence, unless the context indicates otherwise, refers to the single strand DNA of such sequence, the duplex of such sequence with its complement (double stranded DNA) and the complement of such sequence.

"Isolated" in the context of the present invention with respect to polypeptides (or polynucleotides) means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living organism is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

As used herein, the term "frizzled" refers to the genes, and encoded polypeptides and RNAs of Table 2. The term "frizzled gene" or "frizzled polynucleotide" refers to any of the genes of Table 2 and the term "frizzled polypeptide" means any polypeptide receptor of table 2. When following one of the latter terms, the term "biological activity" refers to the activity of a frizzled gene or polypeptide and includes binding of a ligand to a frizzled polypeptide and/or a modulation of the activity of a gene, not necessarily a frizzled gene, that directly or indirectly effects a change in the biological activity of a frizzled polypeptide, especially where the change in biological activity is a change in the binding of a ligand, preferably a Wnt ligand, most preferably a Wnt ligand of Table 1 , by said frizzled polypeptide. The term "frizzled biological activity" refers to the activity of any frizzled gene or gene product, either RNA or polypeptide, such as a change in expression of the gene or a change in the activity of the polypeptide. Unless stated otherwise, the terms "activity" and "biological activity" are understood to be synonymous. In all cases, the term "frizzled polypeptide, " "frizzled protein" and "frizzled receptor" shall be construed as being synonymous, unless expressly stated to be otherwise. The term "Wnt receptor" in Table 2 includes all frizzled family members, including some, such as SMOH, which are not known to bind Wnt ligands. The term "Wnt receptor" as used in this specification includes such frizzled family members as required by context herein. In addition, in any case where the singular is used, the plural shall be understood to be also implied, unless specifically stated otherwise. As used herein, the terms "portion," "segment," and "fragment," when used in relation to polypeptides, refer to a continuous sequence of residues, such as amino acid residues, which sequence forms a subset of a larger sequence. For example, if a polypeptide were subjected to treatment with any of the common endopeptidases, such as trypsin or chymotrypsin, the oligopeptides resulting from such treatment would represent portions, segments or fragments of the starting polypeptide. When used in relation to a polynucleotides, such terms refer to the products produced by treatment of said polynucleotides with any of the common endonucleases.

In accordance with the present invention, the term "percent identity" or "percent identical," when referring to a sequence, means that a sequence is compared to a claimed or described sequence after alignment of the sequence to be compared (the "Compared Sequence") with the described or claimed sequence (the "Reference Sequence"). The Percent Identity is then determined according to the following formula: Percent Identity = 100 [1 -(C/R)] wherein C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence wherein (i) each base or amino acid in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Compared Sequence and (ii) each gap in the Reference Sequence and (iii) each aligned base or amino acid in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference; and R is the number of bases or amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid.

If an alignment exists between the Compared Sequence and the Reference Sequence for which the percent identity as calculated above is about equal to or greater than a specified minimum Percent Identity then the Compared Sequence has the specified minimum percent identity to the Reference Sequence even though alignments may exist in which the hereinabove calculated Percent Identity is less than the specified Percent Identity.

The term "conservative amino acid substitutions" is defined herein as exchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides:

Asp, Asn, Glu, Gin;

III. Polar, positively charged residues:

His, Arg, Lys;

IV. Large, aliphatic, nonpolar residues:

Met Leu, lie, Val, Cys

V. Large, aromatic residues:

Phe, Tyr, Trp DETAILED DESCRIPTION OF THE INVENTION

This invention relates to the discovery that Wnt and Wnt receptor (including frizzled family) genes and proteins are important targets for therapeutic intervention in a wide variety of diseases, particularly diseases related to angiogenesis and ocular diseases. The invention is based on the discovery that mutations in the Frizzled-4 gene ("fzd4'), a Wnt receptor, are responsible for a hereditary human disorder, familial exudative vitreoretinopathy ("FEVR"). In accordance with the present invention there is disclosed the first reported phenotype for a frizzled mutation in humans.

The therapeutic applications of this invention have implications substantially beyond the rare clinical disorder of FEVR. It is to be understood from this invention that the human Wnt and Wnt receptor polypeptides, including the human Frizzled-4 protein ("FZD4"), are involved in FEVR and in many pathologically related forms of retinopathy, such as retinopathy of prematurity (ROP), diabetic retinopathy, and others, and other pathologically related diseases involving a deficiency/excess of angiogenesis and vasculogenesis in neurological and/or other tissues, not the least of which is cancer and neoplasia. Therefore, this invention now teaches therapeutic targets and agents for a wide variety of diseases including FEVR, retinopathies, angiogenic and vascularization disorders in neurological tissues and throughout the body, including cancer and neoplasia. These therapeutic agents may be compounds, such as small organic molecules, which modulate Wnt and/or Wnt receptor activity or expression and thereby provide therapeutic benefit to a person in need thereof.

Tables 1 and 2 summarize the Wnt and Wnt receptor family members that are useful in this invention. Based on the FZD4 finding, other members of the Wnt and Wnt receptor family, such as the frizzled gene family, are implicated in human disease processes, and are also suitable targets for therapeutic intervention in human disease processes. In order to illustrate various embodiments of the instant invention, the following disclosure employs a member of the Wnt and Wnt receptor family, preferably the frizzled gene family, most preferably FZD4, as the basis for identifying frizzled gene or polypeptide, preferably FZD4, modulating agents. In general the composition and processes described below also applies to other Wnt and Wnt receptor genes and proteins.

Identification of a frizzled modulating agent

In a preferred embodiment of such a method, the Wnt-receptor is part of a lipid bilayer, preferably wherein the Wnt-receptor is part of a cell membrane, and most preferably wherein said Wnt-receptor frizzled polypeptide is part of an intact cell. In an additional preferred embodiment, said cell expresses a Wnt-receptor on the surface of said cell, especially wherein the cell is a recombinant cell, most preferably wherein this recombinant cell express a Wnt-receptor on its surface as a result of engineering, including where the recombinant cell does not express a Wnt- receptor absent said engineering, especially where the engineering is genetic engineering.

As disclosed elsewhere herein, such process may be by either in vitro or in vivo means. In a preferred embodiment, said disease is a retinopathy, most preferably familial exudative vitreoretinopathy (FEVR).

In another aspect, the present invention relates to a process for treating a disease in a patient afflicted therewith comprising administering to said patient an effective amount of an agent that modulates the biological activity of frizzled genes or polypeptides, preferably FZD4, wherein such agent was first identified as having such activity by process as disclosed herein. .

Such disease may be any of those as disclosed herein, such as vascular disease, angiogenic disorders, retinopathies and cancer, preferably a retinopathy, most preferably familial exudative vitreoretinopathy (FEVR).

For testing in non-human animals, the present invention relates to a process for identifying an agent having therapeutic activity in ameliorating the symptoms of a disease comprising:

(a) contacting a compound with a source of frizzled biological activity,

(b) determining a change in the frizzled biological activity in the presence of said compound compared to when said compound is not present,

(c) administering said agent to a non-human animal model of disease,

thereby identifying an agent having said therapeutic activity.

In preferred embodiments, the animal model demonstrates the effect of

FZD4 specific modulators. Preferred rodent models include an mouse model of ROP and diabetic retinopathy (Smith LE, Wesolowski E, McLellan A,

Kostyk SK, D'Amato R, Sullivan R, D'Amore PA. Oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci. 1994 Jan;35(1 ):101 -11); an ROP rat model (Reynaud X, Dorey CK. Extraretinal neovascularization induced by hypoxic episodes in the neonatal rat. Invest Ophthalmol Vis Sci. 1994 Jul;35(8):3169-77) or a rat retinal vein occlusion model (Hayashi A, Kim HC, de Juan E Jr. Alterations in protein tyrosine kinase pathways following retinal vein occlusion in the rat. Curr Eye Res. 1999 Mar;18(3):231 -9).

The present invention also finds use in inducing ocular angiogenesis. Preferred rodent models for tumor growth and angiogenesis include mouse tumour xenografts (Lyden D, Young AZ, Zagzag D, Yan W, Gerald W, O'Reilly R, Bader BL, Hynes RO, Zhuang Y, Manova K, Benezra R. Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts. Nature. 1999 Oct 14;401 (6754):670-7. and O'Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell. 1997 Jan 24;88(2):277-85.). Using modern methods of peptide synthesis, amino acid substitutions can be made anywhere within the sequence of the peptide epitopes and such substitutions are by no means limited to the sequences disclosed herein. Such substitutions may be of a conservative nature, for example, where one amino acid is replaced by an amino acid of similar structure and characteristics, such as where a hydrophobic amino acid is replaced by another hydrophobic amino acid. Even more conservative would be replacement of amino acids of the same or similar size and chemical nature, such as where leucine is replaced by isoleucine. In studies of sequence variations in families of naturally occurring homologous proteins, certain amino acid substitutions are more often tolerated than others, and these are often show correlation with similarities in size, charge, polarity, and hydrophobicity between the original amino acid and its replacement, and such is the basis for defining "conservative substitutions."

Thus, amino acids may be described chemically as hydrophobic, polar, acidic, or basic. Less conservative substitutions might involve the replacement of one amino acid by another that has similar characteristics but is somewhat different in size, such as replacement of an alanine by an isoleucine residue. Highly nonconservative replacements might involve substituting an acidic amino acid for one that is polar, or even for one that is basic in character. However, such radical substitutions cannot be dismissed as potentially ineffective since chemical effects are not totally predictable and radical substitutions might well give rise to serendipitous effects not otherwise predictable from simple chemical principles.

Of course, such substitutions may involve structures other than the common L-amino acids. Thus, D-amino acids might be substituted for the L- amino acids commonly found in the antigenic peptides of the invention and yet still be encompassed by the disclosure herein. In addition, amino acids possessing non-standard R groups (i.e., R groups other than those found in the common 20 amino acids of natural proteins) may also be used for substitution purposes to produce immunogens and immunogenic polypeptides according to the present invention.

Therapeutic agents identified according to the methods herein are4 conveniently used as compositions, or formulations, wherein a compound identified according to one of the screening methods of the invention is present in a composition also comprising a carrier, such carrier including any pharmaceutically usable excipient or diluent. Methods well known in the art for making formulations are found in, for example, Remington: The Science and Practice of Pharmacy, (19th ed.) ed. A.R. Gennaro AR., 1995, Mack Publishing Company, Easton, PA. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for agonists of the invention include ethylenevinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, or example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

The present invention also relates to a process that comprises a method for producing a product comprising identifying an agent according to one of the disclosed processes for identifying such an agent (i.e., the therapeutic agents identified according to the assay procedures disclosed herein) wherein said product is the data collected with respect to said agent as a result of said identification process, or assay, and wherein said data is sufficient to convey the chemical character and/or structure and/or properties of said agent. For example, the present invention specifically contemplates a situation whereby a user of an assay of the invention may use the assay to screen for compounds having the desired enzyme modulating activity and, having identified the compound, then conveys that information (i.e., information as to structure, dosage, etc) to another user who then utilizes the information to reproduce the agent and administer it for therapeutic or research purposes according to the invention. For example, the user of the assay (user 1 ) may screen a number of test compounds without knowing the structure or identity of the compounds (such as where a number of code numbers are used the first user is simply given samples labeled with said code numbers) and, after performing the screening process, using one or more assay processes of the present invention, then imparts to a second user (user 2), verbally or in writing or some equivalent fashion, sufficient information to identify the compounds having a particular modulating activity (for example, the code number with the corresponding results). This transmission of information from user 1 to user 2 is specifically contemplated by the present invention.

Description of human Frizzled-4 (FZD4) gene, protein and FEVR associated mutations

Gene Name: FZD4 Full Name: Frizzled, Drosophila, Homolog Of, 4

GenBank Accession Nos.: AK025791 , AB032417, NM-012193, AB032417, AK025516, AB032417, AB054881

OMIM: 604579

Chromosome: 11 q14-q21

Selected model organism protein similarities: H. sapiens FZD4 100 % / 536 aa; M.musculus SMOH 27 % / 466 aa, R.norvegicus Fz1 45 % / 570 aa, D.melanogaster fz 42 % / 483 aa, C.elegans Cfz2 39 % / 478 aa.

Tissue Expression: cDNA sources:Adipose, Aorta, Bone, Brain, Breast, Gall bladder, Germ Cell, Heart, Kidney, Lung, Lymph, Parathyroid, Placenta, Prostate, Skin, Smooth muscle, Stomach, Uterus, Whole embryo, breast, colon, colon-normal, head-neck, head-normal, heart, kidney, lung, lung- normal, lung-tumor, muscle (skeletal), ovary, small intestine.

FZD4 encodes a deduced 537-amino acid protein that has a cysteine- rich domain in the N-terminal extracellular region, 2 cysteine residues in the second and third extracellular loops, 2 N-linked glycosylation extracellular sites, and the S/T-X-V motif in the C terminus. Amino acid sequence identity with other FZD proteins ranged from 39 to 52% in the N terminus to 42 to 69% in the transmembrane domains. Northern blot analysis revealed expression of a 7.7-kb transcript of FZD4 in large amounts in adult heart, skeletal muscle, ovary, and fetal kidney, in moderate amounts in adult liver, kidney, pancreas, spleen, and fetal lung, and in small amounts in placenta, adult lung, prostate, testis, colon, fetal brain, and liver. Expression was also strong in HeLa S3 cells but not in several other cancer cell lines. Kirikoshi et al. (1999) determined that the FZD4 gene contains 2 exons. By FISH, they mapped the FZD4 gene to chromosome 11 q14-q21. (See: Kirikoshi, H.; Sagara, N.; Koike, J.; Tanaka, K.; Sekihara, H.; Hirai, M.; Katoh, M. Molecular cloning and characterization of human Frizzled-4 on chromosome 11q14-q21. Biochem. Biophys. Res. Commun. 264: 955-961 (1999)). FZD4 has not previously been linked to human diseases, either by mutation or by aberrant expression or biological activity. A human EST fragment of FZD4 was said to have a proposed utility in diagnosis of certain cancers, e.g., breast cancer and colorectal cancer (see: WO 99/26960) although no experimental data was provided. In mouse, large deletions of chromosome 7 that encompass Fzd4 and possibly many other genes resulted in defects in thymus gland development and cardiac disorders. Both disorders were said to result from defects in the cardiac neural crest during embryogenesis, and did not relate to retinopathy of any kind, (see: DeRossi et al, Genesis 27:64-75 (2000))

This present invention provides the correct nucleotide sequence determined from a cDNA (SEQ ID NO: 1) and amino acid sequence as determined from the cDNA (SEQ ID NO: 2) of the human FZD4 gene and protein, respectively. This sequence corresponds to hypothetical cDNAs generated from the human genomic database having accession numbers XM-006150 and gi:13639810.

Figure 8 illustrates an alternate methionine translation initiation site that may be active in some cells of the body. The second ATG is closer to the optimal Kozak consensus C-C-AG-C-C-A-T-G-G (SEQ ID NO: 10) context for initiation of translation. The second ATG is the basis for numbering the FZD4 protein in GenBank (for example, Accession no. NM-012193). For consistency, the same numbering is used in the figures of this specification.

Figure 7 illustrates the 2 exon genomic structure of FZD4. Domains marked in black encode open reading frames. Figure 7 (top) sets out the basic genomic arrangement of exons. Intron 1 is completely spliced out of the translated mRNA for the full length active protein. Figure 7 (middle) illustrates a potential alternative transcript of FZD4 resulting from alternative splicing. In the alternate transcript, a failure of the splicing event at the Exon 1 /Intron 1 boundary leads to an open reading frame from Exon 1 that continues briefly into Intron 1 before terminating. The resulting gene product encodes a portion of the extra-cellular domain of FZD4 but terminates prior to the transmembrane domain. It is believed that this alternative transcript could be involved in regulating expression or activity of FZD4.

All figures in this specification employ standard nucleic acid and amino acid symbols that are well known and widely used in the art.

Identification of agents that modulate other Wnt or Wnt receptor family members

In accordance with the present invention, Wnt and Wnt receptors are useful therapeutic targets for various disorders that are angiogenic based and/or those related directly or indirectly to angiogenesis, as disclosed herein. The screening methods disclosed herein for frizzled receptors, such as the FZD4 receptor, are readily applied to other members of the Wnt receptor family.

Wnt Receptors useful in the methods of the invention include Fz (drosophila) frizzled domain containing, Sizzled (frog), Crescent (frog, chicken), C19orf3 (also called RGS19IP1), and Notch proteins. Such Wnt receptors may be soluble, intracellular or on cell membranes. Tables 3 and 4 summarize the Wnt and Wnt receptor, respectively, family members that are useful in this invention.

The genes and proteins of the respective family members are important targets for therapeutic intervention related to various angiogenic related disorders. The OMIM and GenBank nucleotide and protein reference numbers of the respective Wnts and Wnt receptors are shown in Table 1 and 4, respectively. These reference numbers provide those skilled in the art with simple and direct access to gene and protein sequences for uses according to the invention herein.

A number of the Wnts have been reviewed by Cadigan and Nusse (Genes & Dev. 11 :3286-3305, 1997). The following is a brief review of Wnts and Wnt receptors and Wnt modulators based on OMIM and OMIM related information. Members of the 'frizzled' (FZ) gene family (see OMIM 601766) encode 7-transmembrane domain proteins that are receptors for Wnt (Wnt Receptor, see OMIM 164975) signaling proteins. The Frizzled proteins have on their extracellular region a cysteine-rich domain that has been implicated as the Wnt binding domain. This region has a characteristic spacing of ten cysteines, which has also been identified in FrzB (a secreted antagonist of Wnt signaling) and Smoothened (another 7TMR, which is involved in the hedgehog signaling pathway). Similarity between the cysteine-rich domain of Frizzled and several receptor tyrosine kinases, which have roles in development have been found. These include the muscle-specific receptor tyrosine kinase (MuSK), the neuronal specific kinase (NSK2), and ROR1 and ROR2 (Saldanha et al. Protein Sci 1998 Aug;7(8):1632-5).

Wang et al. (1997) showed that Drosophila cells, as well as human cells, when transfected with an FZD3 expression construct, bind the wg protein. In mouse, the wg-homologous Wnt1 gene (164820) is involved in early development of a large domain of the central nervous system encompassing much of the midbrain and rostral metencephalon (OMIM 601766). Sagara et al. (1998) cloned fetal lung cDNAs encoding FZD1 (OMIM 603408), FZD2 (OMIM 600667), and FZD7 (OMIM 603410). The predicted 647-amino acid FZD1 protein contains a signal peptide, a cysteine-rich domain in the N-terminal extracellular region, 7 transmembrane domains, and a C-terminal PDZ domain-binding motif. FZD1 shares 77% and 74% protein sequence identity with FZD2 and FZD7, respectively. Members of the 'frizzled' gene family encode 7-transmembrane domain proteins that are receptors for Wnt signaling proteins. However, unlike many other Fz family members, FDZ6 does not contain a C-terminal PDZ domain-binding motif. The frizzled- dependent signaling cascade comprises several branches whose differential activation depends on specific Wnt ligands, frizzled receptor isoforms, and the cellular context. In Xenopus embryos, the canonical beta-catenin (116806) pathway contributes to the establishment of the dorsal-ventral axis. A different branch, referred to as the planar cell polarity pathway, is essential for cell polarization during elongation of the axial mesoderm by convergent extension. Winklbauer et al. (2001) demonstrated that a third branch of the cascade is independent of dishevelled (see 601365) function and involves signaling through trimeric G proteins and protein kinase C (PKC, see 176960) (OMIM 6034.10). The selection of intracellular signaling cascade may be determined by different C-terminal motifs in FZD proteins.

The Int oncogenes, including Int1 , were first identified as targets for insertional activation by the mouse mammary tumor virus (MMTV) in mammary carcinomas. Nusse et al. (1991 ) proposed that the INT1 gene be termed Wnt because it was both an INT gene and a homolog of the Drosophila 'wingless' gene. The WNTs are a family of secreted glycoproteins that have been shown to be involved in a variety of developmental processes in many organisms. The prototype of the family is the Drosophila protein 'wingless' which acts as a segment polarity gene during embryogenesis and later participates in pattern formation of other body parts. Gavin et al. (1990) isolated 7 murine Wnt family members; Wolda and Moon (1992) isolated 7 Xenopus Wnt family members. McMahon (1992) discussed the Wnt family of developmental regulators, with particular reference to mouse mammary gland and the development of mouse mammary tumors. (OMIM 164820).

The Wnt gene family consists of structurally related genes encoding secreted signaling molecules that have been implicated in oncogenesis and in several developmental processes, including regulation of cell fate and patterning during embryogenesis. A Wnt gene was first defined as a protooncogene, intl (WNT1 ; 164820), by Nusse and Varmus (1982). The designation 'Wnt' was derived from 'wingless' and 'int.' Wnt genes have been identified in many organisms.

There are at least 2 families of secreted inhibitors of WNT signaling: the secreted frizzled-related protein family (e.g., SFRP1 ; 604156), all of which have an N-terminal cysteine-rich domain, and the Dickkopf (German for 'big head,' or 'stubborn') family (see DKK1 ; 605189) (OMIM 605186). The signaling function of the WNT/frizzled pathway is antagonized by secreted frizzled-related proteins (e.g. SFRP1 ; 604156), which bind to either WNTs or frizzled receptors (OMIM 606570). WNT-mediated simulation of 'frizzled' receptors (e.g., FZD2; 600667) can activate the beta-catenin (CTNNB1 ; 116806) pathway, mitogen-activated protein kinase (MAPK) cascades, and G protein-dependent pathways (Ref: OMIM 604256 SFRP4). By searching an EST database, Krupnik et al. (1999) identified cDNAs encoding DKK1 , DKK2 (605415), DKK3 (605416), DKK4 (605417), and a DKK3-related protein they designated 'soggy,' or SGY (605418). Functional analysis determined that DKK1 , like DKK4, blocks Xenopus Wnt8, Wnt3a, and Wnt2b, but not Dsh or Fz8, induction of a secondary axis in frog embryos, indicating that DKKs antagonize WNT function upstream of WNT receptors (OMIM 605189).

Hsieh et al. (Nature 398: 431 -436, 1999) identified an EST from human retina encoding WIF1 (WNT inhibitory factor-1). Binding analysis showed that WIF1 can form a specific, high-affinity complex with frog Wnt8 (see 601396) and Drosophila 'wingless' (see 164820) (WIF1 OMIM 605186).

Secreted frizzled, Sizzled (szl), has homology to the extracellular cysteine-rich domain of frizzled receptors is capable of inhibiting Xenopus wntδ (Salic et al. Development 1997 Dec; 124(23) :4739-48). Crescent has also been described as a Wnt antagonist in the same family as frzb-1 , sizzled, and sfrp-2 by Pera et al. (Mech Dev 2000 Sep;96(2): 183-95).

Stone et al. (1996) reported that human and rat SMOH appear to be 7- transmembrane G protein-coupled receptors with 4 glycosylation sites and a putative extracellular amino terminus 203-205 amino acids long which includes 13 cysteines and can bind a polypeptide ligand (OMIM 601500).

Finch et al. (1997) isolated cDNAs encoding a predicted 313-amino acid protein. They designated the protein FRP (frizzled-related protein) because it contains a cysteine-rich domain of approximately 110 residues that is 30 to 40% identical to the putative ligand-binding domain of FZ proteins, but lacks the 7-transmembrane motif that anchors FZ proteins to the plasma membrane (OMIM 604156). The secreted frizzled-related proteins (FRPs or SFRPs) appear to act as soluble modulators of Wnt signaling by competing with membrane-bound frizzled receptors for the binding of secreted Wnt ligands, or, alternatively, as novel ligands for as yet unidentified receptors (OMIM 604157, Rattner et al. Proc Natl Acad Sci U S A. 1997 Apr 1 ;94(7):2859-63).

Screening assays may be performed with a Wnt, Wnt receptor, or a Wnt-binding fragment of a Wnt receptor. The term Wnt receptor is defined to include the Wnt-binding fragment of the receptor.

In accordance with the foregoing, the present invention includes screening assays, such as where a large number of compounds are to be screened for activity in modulating Wnt and/or Wnt receptor biological activity. As to all such assays as disclosed herein, such modulation may include either an increase or a decrease in Wnt and/or Wnt receptor biological activity. "Wnt and/or Wnt receptor biological activity" as used herein is a very broad term that relates to all the directly or indirectly measurable and identifiable biological activities of the Wnt and/or Wnt receptor gene and protein. Relating to the purified Wnt and/or Wnt receptor protein, Wnt and/or Wnt receptor biological activity includes, but is not limited to, all those biological processes, interactions, or binding of ligands, proteins, membrane components or other compounds (such as small organic compounds), binding behavior, binding- activity relationships, pKa, pD, enzyme kinetics, stability, and functional assessments of the protein. Relating to Wnt and/or Wnt receptor biological activity in cell fractions, reconstituted cell fractions or whole cells, these activities include, but are not limited to the ligand or antibody binding behavior and all measurable consequences of this effect, such measurement of any signaling cascade, membrane composition and behavior, cell growth, development or behavior and other direct or indirect effects of Wnt and/or Wnt receptor activity. Relating to Wnt and/or Wnt receptor genes and transcription, Wnt and/or Wnt receptor biological activity includes the rate, scale or scope of transcription of genomic DNA to generate Wnt and/or Wnt receptor mRNA or its alternate transcripts; the effect of regulatory proteins on such transcription, the effect of modulators of such regulatory proteins on such transcription; plus the stability and behavior of mRNA transcripts, post-transcription processing, mRNA amounts and turnover, and all measurements of translation of the mRNA into polypeptide sequences. Relating to Wnt and/or Wnt receptor biological activity in organisms, this includes but is not limited biological activities which are identified by their absence or deficiency in disease processes or disorders caused by aberrant Wnt and/or Wnt receptor biological activity in those organisms. Broadly speaking, Wnt and/or Wnt receptor biological activity can be determined by all these and other means for analyzing biological properties of proteins and genes that are known in the art.

Those skilled in the art are able to identify measurable biological activities of Wnt and/or Wnt receptor which can be usefully incorporated into low or high throughput screening assays. Some possible assays are described herein for illustration purposes. Based on these teachings, other embodiments of Wnt and/or Wnt receptor screening assays will be directly reduced to practice.

In accordance with one aspect of the present invention there is provided an assay for activity of Wnt receptor comprising using members of the Wnt family as ligands to measure binding of radiolabeled ligands to cell membrane fragments containing the receptor. In a preferred embodiment, Wnt11 is the ligand. Other Wnt family ligands may also be used. These assays are similar to typical G-protein coupled receptors (GPCRs) assays, with which Wnt receptors shares at least some sequence elements. When ligand-receptor complexes are passed through a small pore filter, the receptor bound radiolabel remains on the filter and the rest pass through. Small molecule agonists or antagonists of ligand-receptor interaction are identified by the resulting increase or decrease radiolabel on the filter. SPA (scintillation proximity assay) provides a simplified alternative to the filtration technique. In this method, receptors are adsorbed to beads containing a scintillant. Binding of the radiolabelled ligand to the receptor is detectable by scintillation counting. Agonists or antagonists of ligand-receptor binding are identified by increases or decreases in scintillation. In some cases, the membrane bound form of Wnt receptor may be preferred, alternatively, soluble domains may be preferred. Protein-protein interactions of Wnt receptor can also provide a fruitful source of assays. Compounds which modulate these interactions are potential therapeutic agents according to this invention. For example, the extra-cellular domain of Wnt receptor in a nucleic acid construct could be transfected into yeast, and tested in a standard yeast 2 hybrid assay. Drosophila wingless protein or other Wnt ligands would be useful controls. A further exemplary assay was set out in Hsieh, et al. 1999. PNAS USA 96:3546-3551 where Wnt/Frizzled interactions were studied using IgG fusion proteins containing the extra-cellular domain of frizzled 8. Protein A sepharose was used for affinity purification of the frizzled-lgG fusion protein and Wnt8 coprecipitated with the frizzled-lgG fusion protein in a solution binding assay. A frizzled-lgG fusion protein can also be used in a solid phase enzyme-linked binding assay to measure affinity for Wnt-alkaline phosphatase fusion proteins. An important aspect of the instant invention is that a mutation identified in FEVR patients, namely M493-W494del, occurs at the same site as the mutation reported in the human Smoothened (SMO - see OMIM *601500)) gene homolog reported in Xie, J. et al. 1998. Nature 391 :90-92. Surprisingly, the SMO mutation appears to be a gain of function mutation whereby the mutation prevents the inhibition of this protein by a regulating protein, leading to constitutive signaling. Alternately, the mutation may disrupt the formation of a polar pocket that keeps the receptor in a latent state. The inventors hereto recognize that the FEVR mutation may also be a "gain of function" mutation. As such, it remains to be determined whether agonists or antagonists of normal frizzled, such as FZD4, or other Wnt receptor biological activity are preferred for treatment of any one specific disease. Additionally, it is useful to employ the mutant FZD4 or other Wnt receptor gene/protein in screening assays to identify agonists or antagonists thereof.

The putative intracellular signaling pathways of Wnt and/or Wnt receptor also suggests measurable Wnt and/or Wnt receptor biological activities that are suitable for low or high throughput screening assays. Wnt and/or Wnt receptor may stimulate one or more of three known signaling pathways, or other pathways. For example, since the activated FZD4 intracellular domain should bind to Disheveled protein, disheveled expression, binding or behavior would indicate the effect of modulation of FZD4. Other disheveled interacting proteins such as beta-catenin, Gsk3 or GBP may also respond to modulation of FZD4 activity. In another pathway, FZD4 modulates Protein Kinase C (PKC) activity. PKC activation can be measured by various ways, including auto (trans) phosphorylation of PKC or by migration of PKC from the cytoplasm to the plasma membrane. (See Sheldahl et al; Sakai N, Sasaki K, Ikegaki N, Shirai Y, Ono Y, Saito N. J Cell Biol. 1997 Dec 15; 139(6): 1465-76; Spudich A, Meyer T, Stryer L Cell Motil. Cytoskeletoh. 1992;22(4):250-6.) In yet another pathway, phosphorylation of a synthetic peptide by CAMKII can be measured as an outcome of FZD4 modulation. See Sheldahl et al. 1999. Curr. Biol. 9:695-698; and Kuhl, K., 2000. J.B.C. 275(17):12701 -12711. Since phosphorylation/dephosphorylation is key in some of these pathways, agonists and antagonists of Wnt receptor can be identified by measuring the level or rate of phosphorylation of various cellular components. In a related screening assay, Bhanot et al. 1996. Nature. 382:225-230, measured Drosophila (Dfz2) protein activity by measuring levels of the downstream protein Armadillo.

The interaction of Wnt receptor with intracellular proteins thus suggests a process for identifying a Wnt receptor modulating agent comprising contacting a compound with a polypeptide in the presence of an intracellular Wnt receptor-interacting protein, preferably a protein containing a PDZ domain, and determining a difference in binding of said intracellular protein to said Wnt receptor polypeptide compared to when said compound is not present, thereby identifying a Wnt receptor modulating agent.

In another aspect of this invention, the protein Wnt11 , for example, is implicated in the related ocular disease known as neovascular inflammatory vitreoretinopathy (VRNI), by virtue of its location in a locus identified for autosomal dominant neovascular inflammatory vitreoretinathy (VRNI [MIM 193235] (Sheffield, V. C; Kimura, A. E.; Folk, J. C; Bennett, S. R.; Streb, L M.; Nichols, B. E.; Stone, E. M. The gene for autosomal dominant neovascular inflammatory vitreoretinopathy maps to 11 q13. (Abstract) Am. J. Hum. Genet. 51 (suppl.): A35 only, 1992: and Stone, E. M.; Kimura, A. E.; Folk, J. C; Bennett, S. R.; Nichols, B. E.; Streb, L M.; Sheffield, V. C. Genetic linkage of autosomal dominant neovascular inflammatory vitreoretinopathy to chromosome 11q13. Hum. Molec. Genet. 1 : 685-689, 1992.) The VRNI locus contains WNT11 , a candidate ligand for FZD4. In this respect, the present invention relates to a method for identifying an agent having therapeutic activity in ameliorating Wnt11 -linked diseases comprising :

(a) contacting a compound with a source of Wnt11 biological activity,

(b) determining a change in the Wnt11 biological activity in the presence of said compound compared to when said compound is not present,

thereby identifying an agent having said therapeutic activity.

In preferred embodiments of this modified invention, the Wnt11 -linked disease may be one of vascular disease, angiogenic disorders, retinopathies and cancer, or may include a neurological disease or disease of the eye, including age related macular degeneration. Preferably, the disease is neovascular inflammatory vitreoretinopathy (VRNI).

In preferred embodiments of this modified invention, the Wnt11 biological activity being measured is any measurable biological activity of wnt11 , but preferably its FZD4 binding activity, as measured in a protein/protein binding assay.

Identification of modulators of Wnt activity that affects the interactions of specific Wnt proteins with their receptors would be useful in this invention.

The modulators may inhibit the binding of Wnt to its receptor after incubation (e.g., by competitive or noncompetitive inhibition), or they could potentiate or stabilize the binding.

Examples of such screening approaches include protein-protein binding assays in which the level of binding of Wnt to its receptor, or a biological consequence of such binding, is measured. Where cells not normally expressing Wnt receptors are transformed with a Wnt receptor, and the effects of Wnt on the cells are measured. Such cells may be transformed with the Wnt receptor of choice. Expression can be detected using a Western blot method. Other methods may be employed which are more suitable for high throughput screening applications. For example, labelled antibodies may be used to directly visualize levels in multi-well format screen.

Alternatively, the assays may simply detect the degree of binding between Wnt ligands and Wnt receptors, and not the biological consequences of such binding. For example, cells expressing a selected Wnt receptor may be plated in the wells of a 96-well plate and contacted with a solution containing reporter-labeled Wnt (e.g., radiolabeled of fluorescently-tagged) in the presence and absence of a test compound (i.e., a putative modulator of Wnt/receptor interactions). The effect of the test compound on the extent of binding between Wnt and Wnt receptor is measured, and the compound is identified as effective if its effect on the extent of binding is above a threshold level (e.g., a several-fold difference in binding level between control and experimental samples). In yet another embodiment it is a to one-fold or greater difference and preferably statistically-significant.

In another embodiment, the putative modulator compound can alternatively be added after the cells had been incubated with labeled Wnt. In a screen for inhibitors of binding, the system is assayed for a decrease in the signal reflecting bound labeled Wnt, or an increase in the signal reflecting labeled Wnt in solution.

The screen may also be employed to screen for modulators of

Wnt/receptor interactions. For example, test compounds may be added to the wells (either during or after incubation with labeled Wnt), and the wells then contacted with unlabeled Wnt. Test compounds in wells where the unlabelled Wnt is less effective at displacing the bound labeled Wnt are selected for more detailed examination of ability to potentiate Wnt/receptor binding. Assays such as described above may also be used to determine the relationship between different Wnt proteins and different receptors. For example, the ligand concentration dependence of binding may be used in measurement of the relative affinities of selected Wnt receptors with selected ligands, and ligands with a selected affinity for the receptor can be examined further using, e.g., in vitro or in vivo assays. In this manner, one of skill in the art can identify which Wnt protein(s) is optimally paired with which receptor(s).

In cases where the Wnt ligand has been matched to a specific Wnt receptor, the receptor/ligand pair can be used in a screening assay. For example, the pair may be used in a binding assay to screen for compounds which are effective to modulate the binding of the specific ligand or its receptor. These methods enable the identification of compounds with two general types of activities: (i) those which act generally, e.g., on a class of Wnt/Wnt receptor pairs, to disrupt or facilitate binding, and (ii) those which act selectively disrupt or facilitate the binding between a selected Wnt ligand and its receptor, but not between other Wnt ligands and their receptors.}

Wnt and/or Wnt receptor assays can also be developed based on standard assays well known to those in the art including RNA expression or stability assays, amount or level of Wnt receptor protein in a cell, and the like. Many other types of screening assays can also be employed. Some of these may not actually use human Wnt receptor genes/proteins, but may use homologs or variants of homologs found in other species.

Based on the foregoing, modulators of Wnts and Wnt receptors should be useful in treating a wide variety of diseases, herein sometimes called Wnt and Wnt receptor-linked diseases. These diseases include any disease that is related to the cellular processes that underlie vascularization of the human retina (set out in Hughes, S. et al. 2000. Invest. Opthalmol. Vis. Sci. 41 (5): 1271 - 1228) and which are now treatable by modulators of Wnt and/or Wnt receptor. Thus the therapeutic applications of this invention have implications substantially beyond the rare clinical disorder of FEVR. This invention now teaches therapeutic targets and agents for a wide variety of diseases including FEVR, retinopathies, angiogenic and vascularization disorders in neurological tissues and throughout the body, including cancer and neoplasia.

Specific diseases of interest include Retinopathy of Prematurity (ROP), Coat's disease, Norrie disease, retinal angiomatosis, ocular toxocariasis, retinoblastoma, diabetic retinopathy, sickle cell retinopathy, Eales disease, incontinentia pigmenti, other ischemic eye conditions (generally including arterial occlusive disease, venous occlusive disease, and vasculitis), neovascular inflammatory vitreoretinopathy (VRNI), retinitis pigmentosa, persistent hyperplastic primary vitreous (PHPV), congential retinal folds, congenital retinal nonattachment, retinal dysplasia, microphthalmia, anophthalmia, and other abnormalities associated with some FEVR patients such as abnormalities of platelet aggregation with arachidonic acid, collagen and epinephrine and abnormal bleeding times (see Chaudhuri et al. 1983. Br. J. Ophthalmol. 167:755-758; and Friedrich et al. 1989. Br. J. Ophthalmol. 73:477-478). Wnt receptor-linked diseases may include ALS, stroke, acute spinal cord ischemia, and ischemic neuropathy affected by vascular growth or function. Other related diseases are set out in Jampol et al. 1994. Surv. Ophthalmol. 38:519-540.

Because the Wnt receptor FZD4 has now been directly linked to an angiogenic disorder, modulators of Wnts and Wnt receptors would also be useful in treating any of the other angiogenic disorders, for examples those set out in Liau, G. et al. 2001. DDT 6(13): 689-696.

Additionally, modulators of FZD4, other Wnt receptors and/or Wnt genes and proteins are useful for treating other diseases involving the physiological and pathological processes of neovascularization. As such, these modulators are useful for treating diseases, conditions and disorders including the following neovascularization indications: 1 ) Physiological neovascularization, including female estrus cycle, pregnancy, wound healing, collateral formation, exercise-induced hypertrophy; 2) Indications that may require stimulation of neovascularization, including induction of collateral vessel formation (including myocardial ischemia, peripheral ischemia, cerebral ischemia), coronary artery disease, peripheral vascular disease, stroke, wound healing, engraftment subsequent to organ transplantation such as islet cell tranplantation, fracture and tendon repair, reconstructive surgery, tissue engineering, restenosis, hair loss, decubitus and stasis ulcers, gastrointestinal ulcers, placental insufficiency, aseptic necrosis, pulmonary and systemic hpertension, vascular dementia, Alzheimer's Disease, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); thyroid psuedocyst and lymphoedema; 3) Indications that may require inhibition of neovascularization such as cancer tumour growth and metastasis, neoplasms, ocular neovascularization (including age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, choroidal neovascularization), rheumatoid arthritis, synovitis, bone and cartilage destruction, pannus growth, osteophyte formation, osteomyelitis, psoriasis, obesity, haemangiomas, Kaposi's sarcoma, atherosclerosis (including atherosclerotic plaque rupture), endometriosis, warts, excess hair growth, scar keloids, allergic oedema, dysfunctional uterine bleeding, follicular cysts, ovarian hyperstimulation, endometriosis, cardiac work overload, osteomyelitis, inflammatory and infectious processes (hepatitis, pneumonia, glumerulonephtritis), asthma, nasal polyps, transplantation, liver regeneration, leukomalacia, thyroiditis, thyroid enlargement, lymphoproliferative disorders, haematologic malignancies; 4) indications that may require vascular remodelling, including vascular malformations, psoriasis, pre-eclampsia; and 5) indications that may require inhibition of neovascularization and/or a decrease of vascular permeability, including respiratory distress, ascites, peritoneal sclerosis (dialysis patients), adhesion formation (abdominal surgery) pulmonary hypertension, diabetes, and metastatic spreading.

The present invention also relates to therapeutic agents, regardless of molecular size or weight, effective in treating and/or diagnosing and/or preventing any of the diseases disclosed herein, preferably where such agents have the ability to modulate activity and/or expression of the FZD4, other Wnt receptors and/or Wnts disclosed herein (see Table 2 for Wnt receptors and Table 1 for Wnt polypeptides), and most preferably where said agents have been determined to have such activity through at least one of the screening assays disclosed according to the present invention.

In accordance with the processes disclosed elsewhere herein, test compounds are conveniently compiled into libraries of such compounds, and a key object of the screening assays of the invention is to select which compounds are relevant from libraries having hundreds of thousands, or millions of compounds having unknown therapeutic efficacy.

Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, Wl). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods. In one aspect the present invention relates to agents capable of modulating the activity and/or expression of the Wnt and/or Wnt receptor as disclosed herein, such as where said Wnt and/or Wnt receptor is present on a cell, especially where said modulating ability was first identified using an assay of comprising Wnt and/or receptor or a gene encoding Wnt and/or receptor, or an assay which measures Wnt and/or receptor activity according to this disclosure. As used herein the term "capable of modulating" refers to the characteristic of such an agent whereby said agent has the effect of changing the overall biological activity of Wnt and/or Wnt receptor, either by increasing or decreasing said activity, under suitable conditions of temperature, pressure, pH and the like so as to facilitate such modulation to a point where it can be detected either qualitatively or quantitatively and wherein such modulation may occur in either an in vitro or in vivo environment. In addition, while the term "modulation" is used herein to mean a change in activity, more specifically either an increase or decrease in such activity, the term "activity" is not to be limited to specific enzymatic activity alone (for example, as measured in units per milligram or some other suitable unit of specific activity) but includes other direct and indirect effects of the protein, including increases in enzyme activity due not to changes in specific enzyme activity but due to changes (i.e., modulation) of expression of polynucleotides encoding and expressing said Wnt and/or Wnt receptor enzyme. Wnt and/or Wnt receptor activity may also be influenced by agents which bind specifically to substrates of Wnt and/or Wnt receptor. Thus, the term "modulation" as used herein means a change in Wnt and/or Wnt receptor activity regardless of the molecular genetic level of said modulation, be it an effect on the enzyme per se or an effect on the genes encoding the enzyme or on the RNA, especially mRNA, involved in expression of the genes encoding said enzyme. Thus, modulation by such agents can occur at the level of DNA, RNA or enzyme protein and can be determined either in vivo or ex vivo. "First identified" as used herein, means the first establishment, confirmation or proof of utility, in this case as a modulator of the Wnt and/or Wnt receptor target gene or protein.

In specific embodiments thereof, said assay is any of the assays disclosed herein according to the invention. In addition, the agent(s) contemplated by the present disclosure includes agents of any size or chemical character, either large or small molecules, including proteins, such as antibodies, nucleic acids, either RNA or DNA, and small chemical structures, such as small organic molecules.

In other aspects, the present invention contemplates agents wherein said agent is useful in treating, preventing and/or diagnosing a disease or condition which is identified as being Wnt and/or Wnt receptor related according to this invention.

In one such embodiment, the present invention relates to a method for treating a Wnt-linked, or Wnt-related, disease, such as any disease mediated by Wnt receptors or Wnt polypeptides, in a patient afflicted therewith comprising administering to said patient an effective amount of an agent that modulates Wnt-receptor activity, wherein such agent has such modulating activity in an of the foregoing assay methods, preferably wherein the agent was first shown to have Wnt-receptor modulating activity using such a method. In a further preferred embodiment, the disease is a retinopathy, such as familial exudative vitreoretinopathy (FEVR).

In preferred embodiments, the disease is selected from the group consisting of vascular disease, angiogenic disorders, retinopathies and cancer. In a preferred embodiment, said agent is an antibody that binds to a frizzled-polypeptide, preferably wherein said frizzled polypeptide is a human frizzled-polypeptide, more preferably wherein said human frizzled polypeptide is other than FZD4, and most preferably wherein said frizzled polypeptide is a member selected from the group consisting of FZD1 , FZD2, FZD3, FZD5, FZD6, FZD7, FZD8, FZD9 and FZD10. In another preferred embodiment, the agent is a gene product encoded by and FZD gene, especially a polypeptide, most preferably a polypeptide that is one or more of of FZD1 , FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9 and FZD10. In one preferred embodiment, the agent is a polypeptide that comprises the amino acid sequence of SEQ ID NO: 2, 4 or 6. Such agents may be utilized in any combination.

The present invention also relates to a method for producing a product comprising identifying an agent according to any of the screening assay methods disclosed herein and where the product is the data collected with respect to said agent as a result of said method and wherein said data is sufficient to convey the chemical structure and/or properties of said agent.

In another aspect, the invention provides a method for computationally identifying a compound for modulating Wnt and/or Wnt receptor genes and proteins. The method involves (a) determining the active site of a protein selected from those disclosed herein (i.e. through X-Ray crystallography or other techniques); and (b) through computational modelling, identifying a compound which interacts with the active site, thereby identifying a compound, or its analog, as a compound which is useful for modulating a Wnt or Wnt receptor gene or protein and thereby treating a disease or disorder disclosed herein.

Combinatorial and Medicinal Chemistry

Typically, a screening assay, such as a high throughput screening assay, will identify several or even many compounds which modulate the activity of the assay protein. The compound identified by the screening assay may be further modified before it is used in humans as the therapeutic agent. Typically, combinatorial chemistry is performed on the modulator, to identify possible variants that have improved absorption, biodistribution, metabolism and/or excretion, or other important therapeutic aspects. The essential invariant is that the improved compounds share a particular active group or groups which are necessary for the desired modulation of the target protein. Many combinatorial chemistry and medicinal chemistry techniques are well known in the art. Each one adds or deletes one or more constituent moieties of the compound to generate a modified analog, which analog is again assayed to identify compounds of the invention. Thus, as used in this invention, therapeutic compounds identified using an Wnt and/or Wnt receptor screening assay of the invention include actual compounds so identified, and any analogs or combinatorial modifications made to a compound which is so identified which are useful for treatment of the disorders claimed herein.

Pharmaceutical Preparations and Methods of Administration

The compounds that are determined to affect Wnt and/or Wnt receptor gene expression or Wnt and/or Wnt receptor activity can be administered to a patient at therapeutically effective doses to treat or ameliorate the diseases suggested herein. A therapeutically effective dose refers to that amount of the compound sufficient to result in treatment or amelioration of symptoms of the disease, as specifically determined by those skilled in the art.

Effective Dose

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅o/ ED₅₀. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

Formulations and Use

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi- dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

Other Embodiments of the Invention

Biological modulators of frizzled polypeptide biological activity include frizzled polypeptide antibodies and anti-idiotypic antibodies (including Fab fragments), nucleotide constructs that inhibit expression of a frizzled polypeptide gene (transcription factor inhibitors, antisense and ribozyme molecules, or gene or regulatory sequence replacement constructs), or promote expression of a frizzled polypeptide (e.g., expression constructs in which frizzled polypeptide coding sequences are operatively associated with expression control elements such as promoters, promoter/enhancers, etc.). The invention also relates to host cells and animals genetically engineered to express human frizzled polypeptide (or mutants thereof) that may be administered to patients either in vivo or ex vivo. Some of these compositions are suggested in PCT Publication WO 99/26960 (Inventor Shyjan, A.); however the instant disclosure now provides the correct full length nucleotide and protein sequences for use in these techniques, and additionally provides important new uses for these compositions that were not previously known or understood.

Frizzled polypeptide protein fragments themselves may be used as therapeutic agents. Frizzled polypeptide protein products (especially soluble derivatives) such as peptides corresponding to functional domains of frizzled polypeptide and fusion protein products (especially frizzled polypeptide-lg fusion proteins, i.e., fusions of frizzled polypeptide or a domain of the frizzled polypeptide to an IgFc), antibodies and anti-idiotypic antibodies (including Fab fragments), antagonists or agonists (including compounds that modulate signal transduction which may act on downstream targets in the frizzled polypeptide signal transduction pathway) can be used for therapy of the retinopathies and other diseases disclosed herein. For example, the administration of an effective amount of soluble frizzled polypeptide, a functional domain of frizzled polypeptide or frizzled polypeptide functional domain-lgFc fusion protein or an anti-idiotypic antibody (or its Fab) that mimics a frizzled polypeptide functional domain would "mop up" or "neutralize" endogenous ligands such as the Wnt family proteins, and prevent or reduce binding and receptor activation, leading to treatment or amelioration of the diseases disclosed herein.

Antibodies and antibody fragments that specifically recognize one or more epitopes of frizzled polypeptide, or epitopes of conserved variants of frizzled polypeptide, or peptide fragments of the frizzled polypeptide are also encompassed by the invention. Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

As is known to those skilled in the art, techniques which generate fully humanized antibodies are generally preferred because they are generally less immunogenic and have a longer half-life when administered to humans. Leading available techniques include the UltiMab Human Antibody Development System(sm) featured by Medarex, Inc. (Princeton, NJ) which employs a transgenic mouse having a full suite of human genes for antibodies; and the Xenomouse technology of Abgenix, Inc. (Fremont, CA) which uses genetically engineered strains of mice in which mouse antibody gene expression is suppressed and functionally replaced with human antibody gene expression, while leaving intact the rest of the mouse immune system.

These mice can be induced to generate fully humanized polyclonal antibodies, which are then converted to large-scale production using hybridoma technology. In one embodiment, the method includes immunizing a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene, with a purified or enriched preparation of the frizzled polypeptide protein, or a fragment thereof. B cells (e.g., splenic B cells) of the animal are then obtained and fused with myeloma cells to form immortal, hybridoma cells that secrete human monoclonal antibodies against the frizzled polypeptide protein.

Humanized antibodies may also be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non-immunogenic portion (i.e. chimeric antibodies) (Robinson, R. R. et al., International Patent Publication PCT/U.S.86/02269; Akira, K. et al.,

European Patent Application 1 84, 1 87; Taniguchi, M., European Patent

Application 1 71 ,496; Morrison, S. L. et al., European Patent Application 1 73,494; Neuberger, M. S. et al., PCT Application WO 86/01 533;

Cabilly, S. et al., European Patent Application 1 25,023; Better, M. et al..

Science 240: 1 041 -1 043 ( 1 988); Liu, A. Y. et al. Proc. Natl. Acad. Sci.

USA 84:3439-3443 ( 1 987); Liu, A. Y. et al., J. Immunol. 1 39:3521 -

3526 (1 987); Sun, L. K. et al., Proc. Natl. Acad. Sci. USA 84:21 4-21 8 ( 1 987); Nishimura, Y. et al., Cane. Res. 47:999-1005 (1 987); Wood, C. R. et al., Nature 314:446-449 (1985)); Shaw et al., J. Natl. Cancer Inst. 80: 1553-1559 (1988)). General reviews of "humanized" chimeric antibodies are provided by Morrison, S. L. (Science, 229: 1202-1207 (1985)) and by Oi, V. T. et al., BioTechniques 4:214 (1986). Suitable "humanized" antibodies can alternatively be produced by CDR or CEA substitution (Jones, P. T. et al., Nature 321 :552-525 (1986); Verhoeyan et al., Science 239: 1534 (1988); Beidler, C. B. et al., J. Immunol. 141 :4053-4060 (1988)).

For the production of antibodies, various host animals may be immunized by injection with the frizzled polypeptide, an frizzled polypeptide peptide (e.g., one corresponding the a functional domain of the receptor), truncated frizzled polypeptide polypeptides (frizzled polypeptide in which one or more domains has been deleted), functional equivalents of the frizzled polypeptide or mutants of the frizzled polypeptide. Such host animals may include but are not limited to rabbits, mice, goats and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals.

There are various expression systems that can be used for the production of whole antibodies and antibody fragments. These include bacterial or mammalian cell culture and transgenic animals or plants. The expression system of choice is determined by the intended application and the desired yield, as is known by those skilled in the art. For example, animal cell culture and transgenic expression systems are desirable if glycosylation of the antibody is required, whereas bacterial expression systems are more efficient for production of unglycosylated antibodies, Fab fragments and the like. See Chad, HE and Chamow, SM. 2001. Curr. Opin. Biotech. 12: 188- 193.

For certain indications, Wnt and/or Wnt receptor genes may themselves be used as therapeutic agents. Wnt and/or Wnt receptor genes can be incorporated into gene-therapy vehicles for administration to a person in need thereof. Such vehicles are well known to those skilled in the art including naked DNA, viral, and non-viral (i.e. lipid based) vehicles.

Diagnostic and pharmacogenomic applications

Nucleic acid sequencing of Wnt and/or Wnt receptor gene in a patient can provided valuable information for predictive diagnosis of angiogenic based or other related disorders. Based on the disclosure herein, those skilled in the art can develop nucleic acid sequencing/analysis compositions methods and kits that are suitable for diagnosis of the disorders. Any method of determining the Wnt and/or Wnt receptor gene sequence can be used in the method of this invention, including full length or partial gene sequencing, probe based assays, RFLP and all other techniques known to those in the art.

In addition, ocular diseases, angiogenic diseases and other disorders may also be found to result from frizzled gene mutations, especially mutations of the FZD4 gene. It is therefore important for diagnosticians to return to disorders with related phenotypes to identify if FZD4 is the causative mutation. These disorders include Retinopathy of Prematurity (ROP), Coat's disease, Norrie disease, retinal angiomatosis, diabetic retinopathy, neovascular inflammatory vitreoretinopathy (VRNI), persistent hyperplastic primary vitreous (PHPV), and congenital retinal folds. Diagnostic gene sequence of fzd4 can then be used to confirm or reject such a diagnosis.

Furthermore, sequence analysis of the Wnt and/or Wnt receptor gene can also be used to predict drug responsiveness, susceptibility to side-effects of drugs, and, importantly, it is useful for designing clinical trials. The Wnt and/or Wnt receptor polymorphisms or mutations disclosed herein can be correlated to a patient response database in order to generate a prognostic database for aiding selection of an appropriate therapeutic regime for a patient. Wnt and/or Wnt receptor proteins or peptides, Wnt and/or Wnt receptor fusion proteins, Wnt and/or Wnt receptor nucleotide sequences (oligonucleotides for probes, sequencing primers or PCR primers), antibodies, antagonists and agonists can be useful for the detection of mutant Wnt and/or Wnt receptors or inappropriately expressed Wnt and/or Wnt receptors for the diagnosis of the diseases disclosed herein.

Such data are readily used to develop diagnostic procedures for diseases and the risk of diseases as disclosed herein as relating to frizzled gene activity, especially FZD4 gene activity. Thus, the nucleotide sequences for Wnt and/or Wnt-related disorders are useful in developing probes for use in the diagnostic methods disclose elsewhere herein. Such probes comprise nucleotide sequences of at least 15, possibly at least 30, preferably at least 50 and most preferably at least 100 nucleotides in length that are specific for mutated portions of the FZD4 gene sequence, such as the sequence of SEQ ID NO: 4, and which hybridize to sequences obtained from a patient suspected of having an FZD4-related disease, or otherwise being at risk thereof, so that such hybridization shows the presence of a mutation in the FZD4 gene of said patient.

Additionally, the antibodies of the invention may be used in the detection of a frizzled polypeptide, such as the FZD4 polypeptide, in a biological sample and may, therefore, be utilized as part of a diagnostic or prognostic technique whereby patients may be tested for abnormal forms or amounts of the frizzled protein, such as FZD4. Such antibodies may also be utilized in conjunction with, for example, compound screening schemes, as described, above, for the evaluation of the effect of test compounds on expression and/or activity of the FZD4 gene product. Additionally, the invention provides compositions, kits and methods for the diagnosis of patients suspected of having, or having a predisposition to, FEVR, retinopathy, cancer, and the like.

Because the purpose of assaying for such agents as are identified by the processes already described according to the present invention is for use as a therapeutic agent in treating, preventing, or otherwise ameliorating the symptoms of diseases as disclosed herein as being related to, or caused in whole or in part by, mutations in a Wnt or Wnt receptor gene, the present invention further relates to processes diagnosing the presence of such a disease, especially at its earliest stages, or the risk to a patient of developing such diseases. Thus, in another aspect, the present invention relates to a process for diagnosing the presence of a disease in a patient suspected of being afflicted therewith comprising detecting a mutation in a Wnt or Wnt receptor gene in the genome of said patient. In a preferred embodiment, the present invention relates to such diagnostic processes where the mutation is the mutation in the nucleotide sequence as depicted in SEQ ID NO: 3.

In particular embodiments of such a process, the mutation is detected in a sample of DNA taken from said patient. Such a sample may be obtained in any manner commonly used to obtain such sample, such as by blood sample, biopsy or other methods. Further in accordance with the processes of the invention, such detecting may be accomplished in vivo, such as where an in situ procedure is used to detect the presence of a mutated Wnt or Wnt receptor gene or nucleotide sequence in a patient. In addition, the diseases that can be so detected are not limited to FEVR but include all manner of neurological diseases and syndromes, especially those involving the visual pathway. In particular embodiments of such a diagnostic process, the disease is one or more of a group consisting of vascular disease, angiogenic disorders, retinopathies and cancer. In preferred embodiments, the disease is a neurological disease, or a disease of the eye, preferably a retinopathy, most preferably familial exudative vitreoretinopathy (FEVR). In accordance with the foregoing, the present invention relates to a process for diagnosing the presence of an Wnt or Wnt receptor linked disease in a patient suspected of being afflicted therewith comprising detecting a mutation in a Wnt or Wnt receptor gene in the genome of said patient.

In preferred embodiments, the invention relates to such a process wherein said mutation is detected in a sample of DNA taken from the patient, most preferably wherein the mutation is the mutation of SEQ ID NO: 3 or SEQ ID NO: 5.

Many different techniques are known for detecting the presence or absence of such a mutation. In preferred embodiments, said detecting is accomplished by determining the ability of a nucleic acid probe comprising at least 15 contiguous nucleotides that are complementary to a mutated portion of the sequence of SEQ ID NO: 1 , most preferably wherein said probe comprises at least 30 contiguous nucleotides, especially where the probe comprises at least 50 contiguous nucleotides. In an especially preferred embodiment, the probe comprises at least 100 nucleotides contiguous nucleotides.

The processes disclosed herein are also useful in determining the risk of a patient in developing one or more of the above diseases, so that the present invention also relates to a process for determining a patient's risk of developing a disease where said patient is suspected to be at risk thereof, comprising detecting a mutation in a Wnt or Wnt receptor gene in the genome of said patient. Such detection may, in keeping with the foregoing, be by either in vitro or in vivo means. In a preferred embodiment, said disease is a retinopathy, most preferably familial exudative vitreoretinopathy (FEVR). In a preferred embodiment, the mutation is a mutation in the gene corresponding to SEQ ID NO: 1.

The invention is described in more detail in the following non-limiting examples. It is to be understood that these methods and examples in no way limit the invention to the embodiments described herein and that other embodiments and uses will no doubt suggest themselves to those skilled in the art.

Example 1

A mutation in FZD4 associated with autosomal dominant familial exudative vitreoretinopathy (FEVR)

Clinical description of a FEVR family and linkage to 11 q13-23 (EVR1 ).

A previously undescribed Canadian proband with adFEVR (Vl:9) presented with bilateral total retinal detachment and a microphthalmic eye a few weeks after birth. At a subsequent examination in 2000 she had light perception vision in one eye only and was not expected to maintain this vision. The retina in both eyes was dysplastic, suggesting onset of the retinal complications of FEVR in utero. The mother has total retinal detachment in one eye and subnormal vision in her other eye. The maternal grandmother and a maternal aunt are affected but are completely asymptomatic, demonstrating the full range of severity of this disease. The maternal grandmother was born in Ontario and is a member of a large pedigree that has been previously described. (Canney & Oliver 1976; Gow & Oliver 1971 ). Additional family members were recruited, resulting in a family collection of 81 individuals, 27 of whom were diagnosed by local ophthalmologists with FEVR on the basis of fundoscopic findings and a fundus fluorescein angiogram when necessary.

DNA was initially collected from eight family members of the proband to assess whether the disease in this family maps to the region of 11 q13-23 to which linkage to adFEVR has been identified. (Muller et al. 1994; Price et al. 1996). Relationships of most of the family members and the proband are illustrated in Figure 1. Genotyping

For all experiments, genomic DNA was extracted from blood samples from the 81 subjects by a standard salt extraction method. DNA was genotyped using fluorescently-labeled repeat-containing microsatellite markers. Original primer sequences for the markers were obtained from the Genome Database. We placed the sequence GTTTCTT (SEQ ID NO: 9) at the 5' end of the reverse primers to promote adenylation of the 3' end of the forward strand, (Brownstein et al. 1996) and labeled the 5' end of the forward primers with one of the fluorophores 6-FAM, HEX, or TAMRA. (Wenz et al. 1998). Each reaction mixture (15uL) contained 10ng genomic DNA, 50mM KCI, 10mM Tris-HCL, pH 8.0, 2.5mM MgCI₂, 0.25μM dNTPs, 0.33μM each primer, and 0.6U AmpliTaq Gold DNA polymerase (Applied Biosystems). The thermal cycling profile consisted of 30 cycles of 94^SC for 15s, 55^eC for 15s, and 72^aC for 15s. PCR products were resolved using an Applied Biosystems Prism 3100 Genetic Analyzer running GeneMapper software (Applied Biosystems) for allele identification.

Linkage analysis

Two-point linkage analysis was performed using the MLINK program from the FASTLINK software package. (Cottingham et al. 1993) Haplotype constructions were carried out using GENEHUNTER software. (Kruglyak, 1996) The disease allele frequency was assumed to be 0.0001 (Muller et al. 1994; Price et al. 1996) with penetrance at 99.9%. Marker allele frequencies were estimated from the genotypes of the founders in the pedigree. We ordered markers according to the UCSC Golden Path sequence maps and the Marshfield Center for Medical Genetics maps.

We used nine DNA markers (six Genethon (Dib et al. 1996) markers

(D11S937, D11S1362, D11S1887, D11S4082, D11S1780, and D11S1311) one CHLC marker (D11S2002) and two Marshfield (Browman, 1998) markers

(D11 S896, D11 S873)) that occur near the previously mapped EVR1 locus in genotyping studies to show that the trait of FEVR was consistent with linkage to chromosome 11 q13-23. Information about markers and physical clones was obtained from the Genome Database and GenBank.

Two-point linkage analysis gave positive LOD scores for all markers within the region examined. Recombination with the proximal marker, D11 S1887, and distal marker, D11 S837, were seen in Vl:70 and V:007

(marked with asterisks in Figure 1 ), respectively, providing centromeric and telomeric boundaries for the disease gene.

Fine linkage mapping

The interval between D1 1S1887 and D11 S873 corresponds to a genetic size of ~5cM, according to the Marshfield Center for Medical Genetics map. To substantiate this finding and increase the resolution of the interval, we performed fine mapping with an additional 24 markers, including several new dinucleotide-repeat markers identified from repeats on BAC clones.

New repeat markers relevant for fine mapping were AP000654CA1 ; CA2AP001528; AC024304CA1 ; AC072050CA1 ; AP000756CA1 ; AP000676CA1 ; CA2AC023888; CA1AC011088; AC018775CA1. The physical locations of the markers were evaluated by the UCSC Golden Path sequence mapping database and the Marshfield Center for Medical Genetics genetic mapping database.

Definition of the FEVR minimal genomic region by haplotype analysis

Haplotype construction using the 33 polymorphic markers revealed five proximal and four distal recombinations. Of the nine individuals, five were affected and four were unaffected. The additional data narrowed the boundaries to approximately 1.55MB (based on the April freeze UCSC Golden Path Human Genome Browser) and indicated that the FEVR gene was located in the genomic region delimited by markers CA2AP001528 and CA2AC023888. Mutation Analysis

The FEVR interval contains only two known genes, FZD4 and FLJ22104, and numerous expressed sequence tags. All exons of FZD4 and FLJ22104 were analyzed for mutations in 2 patients with FEVR and 1 unaffected family member by sequence analysis of PCR-amplified fragments.

We detected a 6nt deletion in the coding sequence of FZD4 in Vl:9 and V:86 that resulted in a loss of nt 1479-1484 (GTGGAT) and deletion of a methionine and tryptophan (M493-W494del). (The assignment of deleted nucleotides applies the guidelines proposed by den Dunnen and Anonarakis (arbitrarily assigning the change to the most 3' nt. This mutation may also be described as loss of nt 1477-1482, however the corresponding amino acid deletion is not changed (i.e. M493-W494del). The mutation was absent in the unaffected family member sequenced. Tryptophan 494 is conserved in putative FZD4 homologs (Figure 3) and other members of the human frizzled/smoothened membrane receptor family (Figure 4). Its position in the seventh transmembrane domain is analogous to the position of the activating Smoothened W535L mutation reported in sporadic basal-cell carcinoma.(Xie et al. 1998). We developed PCR-RFLP and fluorescence capillary electrophoresis assays and showed that the mutation segregated with disease and was absent in the 59 unaffected chromosomes in the family and 94 chromosomes from other Caucasian individuals.

We also defined the exon-intron boundaries of FLJ22104 by aligning the genomic sequence contained in a genomic contig (Genbank accession number NT-009184) and the cDNA sequences in Genbank (accession numbers NM-022918, AK000684, AK024853). Intronic primer pairs were designed for each of the exons on the basis of the genomic sequence using the Primer3 program. No mutations or polymorphisms were identified.

In each case exons and splice-site junctions were amplified from 16 ng genomic DNA in a standard 60μL PCR reaction volume using Platinum Taq (Invitrogen). PCR conditions were 35 cycles of 30s each of 94^SC, 57^QC, and 72^SC. The PCR-amplification products were purified on Qiagen spin columns according to the manufacturer's instructions. The purified samples were sequenced in both directions with dye terminators (Applied Biosystems). Sequence reactions were electrophoresed on ABI automated sequencers (Applied Biosystems), and the resultant traces were analyzed by the Staden software package and compared against normal sequences (Genbank). The mutation was described according to the nomenclature established by den Dunnen and Antonarakis (1996), and numbering was started from the initiating ATG codon as annotated in the GenBank reference sequence.

Confirmation of mutation in other patients:

We used PCR-RFLP analysis and fluorescence capillary electrophoresis to screen the remainder of the family and a control population for the presence of the FZD4 mutation observed in the patients. The genomic DNA was amplified by PCR using a 6-FAM-labeled forward primer (for fluorescence detection) or an unlabeled forward primer (for PCR-RFLP). The forward primer was 5'- TGGTGGGCATCACTTCAGG-3' (SEQ ID NO: 7), and the reverse primer was 5'-GCCTTTTCCAGGCTTCACC-3' (SEQ ID NO: 8). For detection of the mutation, the PCR product was incubated with Λ/spl (5U) in a total volume (15μL) for 1 h at 37^SC then resolved on 3% agarose gels. The presence of the deletion removes an Λ/spl site. Alternately, the PCR products were resolved without digestion using an Applied Biosystems Prism 3100 Genetic Analyzer. The wild-type allele is 140bp and the mutant allele is 134bp. (Data not shown). The results indicated that the mutation segregated with disease and was absent in the 59 unaffected chromosomes in the family and 94 chromosomes from other Caucasian individuals. Example 2

Generation of a fully humanized monoclonal antibody against a frizzled polypeptide

To generate fully human monoclonal antibodies to a frizzled polypeptide, HuMab mice can be immunized with a preparation of a frizzled polypeptide selected from the list in Table 2, or a fragment thereof, which has been purified or enriched according to any standard method. HuMab mice contain a human immunoglobulin gene miniloci that encodes unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous mu and kappa chain loci (Lonberg, N. et al. (1994) Nature. 368(6474): 856- 859.). These mice exhibit reduced expression of mouse IgM or kappa, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGkappa monoclonals.

Preferably mice are 6 - 16 weeks of age upon the first immunization. Initial immunization is intraperitoneal (IP) with antigen in complete Freund's adjuvant, followed by every other week IP immunizations (up to a total of 6) with antigen in incomplete Freund's adjuvant. The immune response can be monitored over the course of the immunization protocol with plasma samples obtained by retroorbital bleeds. The plasma can be screened, for example by ELISA or flow cytometry, and mice with sufficient titers of anti-frizzled polypeptide human immunoglobulin can be used for fusions. Mice can be boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected that 2-3 fusions for each antigen are needed for success.

For generation of hybridomas producing human monoclonal antibodies to frizzled polypeptide, the mouse splenocytes are first isolated and fused with PEG to a mouse myeloma cell line based upon standard protocols. The resulting hybridomas are then screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice are fused to one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2 X 10⁵ in flat bottom microtiter plate, followed by a two week incubation in selective medium containing 20% fetal calf serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L- glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2- mercaptoethanol, 50 units/ml penicilliin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and 1 X HAT (Sigma; the HAT is added 24 hours after the fusion). After two weeks, cells are cultured in medium in which the HAT is replaced with HT. Individual wells are then screened by ELISA for human anti-frizzled polypeptide monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, medium is observed usually after 10-14 days. The antibody secreting hybridomas are replated, screened again, and if still positive for human IgG, anti-frizzled polypeptide antibodies, can be subcloned at least twice by limiting dilution. The stable subclones are then cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.

To purify human anti-frizzled polypeptide antibodies, selected hybridomas can be grown in two-litre spinner-flasks for monoclonal antibody purification. Supematants can be filtered and concentrated before affinity chromatography with protein A-sepharose. (Pharmacia, Piscataway, NJ). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD₂so using 1.43 extinction co-efficient. The monolconal antibodies can be aliquoted and stored at -80°C until required for use according to the methods of the invention. Table 1. Wnt Polypeptides

Table 2. Wnt Receptors

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Claims

WHAT IS CLAIMED IS:

1. A method for identifying a modulator of frizzled-polypeptide biological activity, comprising:

2. The method of claim 1 wherein said difference in the biological activity is an increase in the biological activity.

3. The method of claim 1 wherein said difference in the biological activity is a decrease in the biological activity.

4. The method of claim 1 wherein said biological activity is the binding of a ligand.

5. The method of claim 4 wherein said ligand is a Wnt polypeptide.

6. The method of claim 5 wherein said Wnt polypeptide is a Wnt polypeptide of Table 1.

7. The method of claim 1 wherein said frizzled polypeptide is a human frizzled polypeptide.

8. The method of claim 7 wherein said frizzled polypeptide is other than FZD4.

9. The method of claim 8 wherein said frizzled polypeptide is a member selected from the group consisting of FZD1 , FZD2, FZD3, FZD5, FZD6, FZD7, FZD8, FZD9 and FZD10.

10. The method of claim 1 wherein said frizzled polypeptide is part of a lipid bilayer.

11. The method of claim 1 wherein said frizzled polypeptide is part of a cell membrane.

12. The method of claim 1 wherein said frizzled polypeptide is part of an intact cell.

13. The method of claim 12 wherein said cell expresses a frizzled polypeptide on the surface of said cell.

14. The method of claim 12 wherein said cell is a recombinant cell.

15. The method of claim 14 wherein said recombinant cell express a frizzled polypeptide on its surface as a result of engineering.

16. The method of claim 15 wherein said recombinant cell does not express a frizzled polypeptide absent said engineering.

17. The method of claim 15 or 16 wherein said engineering is genetic engineering.

18. A method for identifying an agent useful in treating a Wnt-related disease, comprising:

19. The method of claim 18 wherein said difference in the biological activity is an increase in the biological activity.

20. The method of claim 18 wherein said difference in the biological activity is a decrease in the biological activity.

21. The method of claim 18 wherein said biological activity is the binding of a ligand.

22. The method of claim 21 wherein said ligand is a Wnt polypeptide.

23. The method of claim 22 wherein said Wnt polypeptide is a Wnt polypeptide of Table 1.

24. The method of claim 18 wherein said Wnt-receptor is a Wnt- receptor of Table 2.

25. The method of claim 23 wherein said Wnt-receptor is a Wnt- receptor of Table 2.

26. The method of claim 18 wherein said Wnt-receptor is part of a lipid bilayer.

27. The method of claim 18 wherein said Wnt-receptor is part of a cell membrane.

28. The method of claim 18 wherein said Wnt-receptor is part of an intact cell.

29. The method of claim 28 wherein said cell expresses a Wnt-receptor on the surface of said cell.

30. The method of claim 28 wherein said cell is a recombinant cell.

31. The method of claim 30 wherein said recombinant cell express a frizzled polypeptide on its surface as a result of engineering.

32. The method of claim 31 wherein said recombinant cell does not express a frizzled polypeptide absent said engineering.

33. The method of claim 31 or 32 wherein said engineering is genetic engineering.

34. A method for treating a Wnt-linked disease in a patient afflicted therewith comprising administering to said patient an effective amount of an agent that modulates Wnt-receptor activity, wherein such agent has such modulating activity in a method of claim 1 -33.

35. The method of claim 34 wherein said agent was first shown to have Wnt-receptor modulating activity using a method of claim 1-31.

36. The method of claim 34 wherein said disease is selected from the group consisting of vascular disease, angiogenic disorders, retinopathies and cancer.

37. The method of claim 34 wherein said agent is an antibody that binds to a frizzled-polypeptide.

38. The method of claim 37 wherein said frizzled polypeptide is a human frizzled-polypeptide.

39. The method of claim 38 wherein said human frizzled polypeptide is other than FZD4.

40. The method of claim 38 wherein said frizzled polypeptide is a member selected from the group consisting of FZD1 , FZD2, FZD3, FZD5, FZD6, FZD7, FZD8, FZD9 and FZD10.

41. The method of claim 34 wherein said disease is a retinopathy.

42. The method of claim 34 wherein said disease is familial exudative vitreoretinopathy (FEVR).

43. The method of claim 34 wherein said agent is a gene product encoded by the FZD4 gene.

44. The method of claim 43 wherein said agent is a polypeptide.

45. The method of claim 44 wherein said polypeptide comprises the amino acid sequence of SEQ ID NO: 2, 4 or 6.

46. A method for producing a product comprising identifying an agent according to the method of claim 1 -33 wherein said product is the data collected with respect to said agent as a result of said method and wherein said data is sufficient to convey the chemical structure and/or properties of said agent.