WO2000075280A2 - Membre de la famille frzb - Google Patents

Membre de la famille frzb Download PDF

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Publication number
WO2000075280A2
WO2000075280A2 PCT/US2000/015814 US0015814W WO0075280A2 WO 2000075280 A2 WO2000075280 A2 WO 2000075280A2 US 0015814 W US0015814 W US 0015814W WO 0075280 A2 WO0075280 A2 WO 0075280A2
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Prior art keywords
polypeptide
frazzled
seq
polynucleotide
nucleotide sequence
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PCT/US2000/015814
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English (en)
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WO2000075280A3 (fr
Inventor
Michael William Lark
Ian Edward James
Sanjay Kumar
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Smithkline Beecham Corporation
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Priority to EP00941289A priority Critical patent/EP1198469A4/fr
Publication of WO2000075280A2 publication Critical patent/WO2000075280A2/fr
Publication of WO2000075280A3 publication Critical patent/WO2000075280A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production. More particularly, the polynucleotides and polypeptides of the present invention relate to FRZB family, hereinafter referred to as FRAZZLED. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.
  • frizzled family proteins which control developmental patterning. These proteins are members of a large family (referred to as the frizzled family), exemplified by frizzled and smoothened [Moon, et al., Cell 88: 725- 728 (1997)]. Smoothened is a 7 transmembrane protein which associates with the 12 transmembrane protein, patched, to regulate signaling of the soluble agonist, indian hedgehog (Stone, et al., Science 384(14): 129-134 (1996)] .
  • Indian hedgehog and parathyroid hormone-related peptide appear to regulate the differentiation of chondrocytes in mammalian systems [Nortkamp, et al. Science 273(2): 613-622 (1996)].
  • the control of the chondrocyte phenotype could be c ⁇ tically important in the maintenance of cartilage homeostasis in diseases involving both bone and cartilage including osteoarth ⁇ tis, osteoporosis and rheumatoid arth ⁇ tis.
  • a soluble frizzled-related protein subfamily has recently been desc ⁇ bed.
  • Frzb Frzb, F ⁇ tz, frezzled or sFRPs (soluble frizzled related proteins)
  • sFRPs soluble frizzled related proteins
  • Frzb was expressed m chondrocytes in both developing cartilage rudiments and at sites of long bone growth. These authors also desc ⁇ bed the human Frzb homologue and reported that it is 94% identical to the bovine sequence. More recently, several sFRPs have been identified in the mouse [Rattner, et al., PNAS 94: 2859-2863 (1997)]. One member of this subfamily, sFRP-3 is 92% identical to bovine and human Frzb.
  • FRZB family has an established, proven history as therapeutic targets.
  • FRZB family has an established, proven history as therapeutic targets.
  • FRZB family can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, chronic and acute inflammation, arth ⁇ tis, osteoarth ⁇ tis and other osteopenic conditions, Paget's disease, rheumatoid arth ⁇ tis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, pso ⁇ asis), transplant rejection, graft vs.
  • diseases including, but not limited to, chronic and acute inflammation, arth ⁇ tis, osteoarth ⁇ tis and other osteopenic conditions, Paget's disease, rheumatoid arth ⁇ tis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, pso ⁇ asis), transplant rejection, graft vs.
  • ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • the invention relates to FRAZZLED polypeptides and recombinant mate ⁇ als and methods for their production. Another aspect of the invention relates to methods for usmg such
  • FRAZZLED polypeptides and polynucleotides Such uses include the treatment of chronic and acute inflammation, arth ⁇ tis, osteoarth ⁇ tis and other osteopenic conditions, Paget's disease, rheumatoid arth ⁇ tis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, pso ⁇ asis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (e.g., osteoporosis), cancer including bone and cartilage cancers and related tumors (e.g., lymphoprohferative disorders), atherosclerosis, and Alzheimers disease.
  • autoimmune diseases e.g., inflammatory bowel disease, pso ⁇ asis
  • transplant rejection graft vs. host disease
  • infection stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis
  • brain injury AIDS
  • the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with FRAZZLED imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with mapprop ⁇ ate FRAZZLED activity or levels.
  • FRAZZLED refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 or an allelic va ⁇ ant thereof.
  • FRAZZLED activity or FRAZZLED polypeptide activity refers to the metabolic or physiologic function of said
  • FRAZZLED including similar activities or improved activities or these activities with decreased undesirable side-effects. Also included are antigenic and lmmunogenic activities of said FRAZZLED.
  • FRAZZLED gene refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 1 or allelic va ⁇ ants thereof and/or their complements.
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.
  • Isolated means altered “by the hand of man” from the natural state. If an "isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeox ⁇ bonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to t ⁇ ple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases mclude, for example, t ⁇ tylated bases and unusual bases such as inosine.
  • a va ⁇ ety of modifications has been made to DNA and RNA; thus, "polynucleotide” embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characte ⁇ stic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as o gonucleotides.
  • Polypeptide refers to any peptide or protein comprising two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
  • Polypeptide refers to both short chains, commonly refe ⁇ ed to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred to as proteins. Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids.
  • Polypeptides include ammo acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chains and the ammo or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitmation, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP- ⁇ bosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide de ⁇ vative, covalent attachment of a hpid or pid derivative, covalent attachment of phosphotidyhnositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, my ⁇ stoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arg ylation, and ubiquitmation.
  • Changes in the nucleotide sequence of the va ⁇ ant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in ammo acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a va ⁇ ant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted ammo acid residue may or may not be one encoded by the genetic code.
  • a va ⁇ ant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • Non-naturally occur ⁇ ng variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by compa ⁇ ng the sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” can be readily calculated by known methods, including but not limited to those desc ⁇ bed in
  • Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, j., et al., Nucleic Acids Research 12(1): 387 ( 1984)), BLASTP,
  • Gap Length Penalty 3 Available as: The "gap” program from Genetics Computer Group, Madison WI. These are the default parameters for nucleic acid compa ⁇ sons.
  • Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO: l, wherem said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5 ' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups withm the reference sequence, and wherem said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1
  • NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
  • a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2, that is it may be 100% identical, or it may include up to a certain integer number of ammo acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity.
  • Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups with the reference sequence.
  • the number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of ammo acids in SEQ ID NO:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO:2, or: n n ⁇ x n - (x n • y), wherem n n is the number of amino acid alterations, x n is the total number of ammo acids in SEQ ID NO:2, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., • is the symbol for the multiplication operator, and wherem any non-mteger product of x n and y is rounded down to the nearest integer p ⁇ or to subtracting it from x n .
  • Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO: 2 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherem said alterations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or m one or more contiguous groups withm the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of ammo acids in SEQ ID NO:2 by the integer defining the percent identity divided by 100 and then subtracting that
  • y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0 95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO.2, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity.
  • Such alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of ammo acid alterations for a given % identity is determined by multiplying the total number of amino acids m SEQ ID NO:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO:2, or: n a ⁇ x a - (x a • y), wherein n a is the number of am o acid alterations, x a is the total number of ammo acids in SEQ ID NO:2, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and • is the symbol for the multiplication operator, and wherem any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • the present invention relates to FRAZZLED polypeptides (or FRAZZLED proteins) .
  • the FRAZZLED polypeptides include the polypeptide of SEQ ID NOS:2 and 4; as well as polypeptides comprising the amino acid sequence of SEQ ID NO: 2; and polypeptides comprising the ammo acid sequence which have at least 80% identity to that of SEQ ID NO:2 over its entire length, and still more preferably at least 90%) identity, and even still more preferably at least 95% identity to SEQ ID NO: 2.
  • those with at least 97-99% are highly preferred.
  • FRAZZLED polypeptides having the ammo acid sequence which have at least 80% identity to the polypeptide having the ammo acid sequence of SEQ ID NO:2 over its entire length, and still more preferably at least 90% identity, and still more preferably at least 95% identity to SEQ ID NO:2. Furthermore, those with at least 97-99% are highly preferred.
  • FRAZZLED polypeptide exhibit at least one biological activity of FRAZZLED.
  • the FRAZZLED polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional ammo acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidme residues, or an additional sequence for stability du ⁇ ng recombinant production.
  • Fragments of the FRAZZLED polypeptides are also included in the invention.
  • a fragment is a polypeptide having an amino acid sequence that entirely is the same as part, but not all, of the ammo acid sequence of the aforementioned FRAZZLED polypeptides.
  • fragments may be "free-standing,” or comp ⁇ sed withm a larger polypeptide of which they form a part or region, most preferably as a single continuous region.
  • Representative examples of polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of FRAZZLED polypeptide.
  • Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of FRAZZLED polypeptides, except for deletion of a continuous se ⁇ es of residues that includes the amino terminus, or a continuous se ⁇ es of residues that includes the carboxyl terminus or deletion of two continuous se ⁇ es of residues, one including the ammo terminus and one including the carboxyl terminus.
  • fragments characte ⁇ zed by structural or functional att ⁇ butes such as fragments that comp ⁇ se alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet- formmg regions, turn and turn-forming regions, coil and coil-formmg regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface- forming regions, substrate binding region, and high antigenic index regions.
  • Other preferred fragments are biologically active fragments. Biologically active fragments are those that mediate FRAZZLED activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or lmmunogemc m an animal, especially in a human.
  • va ⁇ ants of the defined sequence and fragments also form part of the present invention.
  • Preferred va ⁇ ants are those that vary from the referents by conservative ammo acid substitutions — i.e., those that substitute a residue with another of like characte ⁇ stics. Typical such substitutions are among Ala, Val, Leu and lie; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr.
  • the FRAZZLED polypeptides of the invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally occumng polypeptides, recombmantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for prepa ⁇ ng such polypeptides are well understood in the art.
  • FRAZZLED polynucleotides include isolated polynucleotides which encode the FRAZZLED polypeptides and fragments, and polynucleotides closely related thereto. More specifically, FRAZZLED polynucleotide of the invention include a polynucleotide comp ⁇ sing the nucleotide sequence contained m SEQ ID NO: 1
  • FRAZZLED polynucleotides further include a polynucleotide comp ⁇ sing a nucleotide sequence that has at least 80%) identity over its entire length to a nucleotide sequence encoding the FRAZZLED polypeptide of SEQ ID NO:2, and a polynucleotide comprising a nucleotide sequence that is at least 80% identical to that of SEQ D NO: 1 over its entire length.
  • polynucleotides at least 90%> identical are particularly preferred, and those with at least 95%> are especially preferred. Furthermore, those with at least 97% are highly preferred and those with at least 98-99% are most highly preferred, with at least 99% being the most preferred.
  • FRAZZLED polynucleotides are also included under FRAZZLED polynucleotides.
  • the invention also provides polynucleotides which are complementary to such FRAZZLED polynucleotides.
  • FRAZZLED of the invention is structurally related to other proteins of the FRZB family, as shown by the results of sequencing the cDNA encoding human FRAZZLED.
  • the cDNA sequence of SEQ ED NO: 1 contains an open reading frame (nucleotide number 171 to 1208) encodmg a polypeptide of 346 amino acids of SEQ ED NO:2.
  • the ammo acid sequence of Table 1 (SEQ ED NO:2) has about 51.1% identity (using FASTA) in 319 amino acid residues with mouse sFRP-3 (A. Ratner et al., Proc. Natl. Acad. Sci. U.S.A.
  • FRAZZLED polypeptides and polynucleotides of the present invention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled in the art.
  • a nucleotide sequence of a human FRAZZLED (SEQ ID NO: 1).
  • An ammo acid sequence of a human FRAZZLED (SEQ ED NO: 2).
  • One polynucleotide of the present invention encoding FRAZZLED may be obtained using standard cloning and screening, from a cDNA library de ⁇ ved from mRNA in cells of human osteoblasts using the expressed sequence tag (EST) analysis (Adams, M.D., et al. Science (1991) 252: 1651- 1656; Adams, M.D.
  • Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA hbra ⁇ es or can be synthesized using well known and commercially available techniques.
  • nucleotide sequence encoding FRAZZLED polypeptide of SEQ ED NO:2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 171 to 1208 of SEQ ED NO:l), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ED NO:2.
  • the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself; the coding sequence for the mature polypeptide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
  • a marker sequence which facilitates pu ⁇ fication of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidme peptide, as provided m the pQE vector (Qiagen, Inc.) and desc ⁇ bed in Gentz et al , Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag.
  • the polynucleotide may also contain non-coding 5' and 3' sequences, such as transc ⁇ bed, non-translated sequences, splicing and polyadenylation signals, ⁇ bosome binding sites and sequences that stabilize mRNA.
  • FRAZZLED va ⁇ ants comp ⁇ sing the amino acid sequence of FRAZZLED polypeptide of Table 2 (SEQ ED NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acid residues are substituted, deleted or added, m any combination.
  • polynucleotides of the present invention is contained in Table 3 (SEQ ED NO: 3) encoding the amino acid sequence of Table 4 (SEQ ID NO: 4).
  • a partial nucleotide sequence of a human FRAZZLED (SEQ ID NO: 3).
  • the present invention further relates to polynucleotides that hyb ⁇ dize to the herein above- desc ⁇ bed sequences.
  • the present invention especially relates to polynucleotides which hyb ⁇ dize under st ⁇ ngent conditions to the here above-desc ⁇ bed polynucleotides.
  • st ⁇ ngent conditions means hyb ⁇ dization will occur only if there is at least 80%, and preferably at least 90%>, and more preferably at least 95%, yet even more preferably 91-99% identity between the sequences.
  • Polynucleotides of the invention which are identical or sufficiently identical to a nucleotide sequence contained m SEQ ED NO: 1 or a fragment thereof (including that of SEQ ED NO:3), may be used as hyb ⁇ dization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding FRAZZLED polypeptide and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence simila ⁇ ty to the FRAZZLED gene.
  • Such hyb ⁇ dization techniques are known to those of skill in the art.
  • these nucleotide sequences are 80%> identical, preferably 90% identical, more preferably 95% identical to that of the referent.
  • the probes generally will comp ⁇ se at least 15 nucleotides. Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will range between 30 and 50 nucleotides.
  • a polynucleotide encoding FRAZZLED polypeptide including homologs and orthologs from species other than human, comp ⁇ ses the steps of screening an approp ⁇ ate library under stingent hyb ⁇ dization conditions with a labeled probe having the SEQ ED NO: 1 or a fragment thereof (including that of SEQ ED NO: 3), and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
  • hyb ⁇ dization techniques are well known to those of skill in the art.
  • FRAZZLED polynucleotides of the present invention further include a nucleotide sequence comp ⁇ sing a nucleotide sequence that hyb ⁇ dize under st ⁇ ngent condition to a nucleotide sequence having SEQ ED NO: 1 or a fragment thereof (including that of SEQ ED NO:3).
  • polypeptide comp ⁇ sing amino acid sequence encoded by nucleotide sequence obtained by the above hyb ⁇ dization condition are as defined above or, alternatively, conditions under overnight incubation at
  • polynucleotides and polypeptides of the present invention may be employed as research reagents and mate ⁇ als for discovery of treatments and diagnostics to animal and human disease
  • the present invention also relates to vectors which comp ⁇ se a polynucleotide or polynucleotides of the present mvention, and host cells which are genetically engineered with vectors of the invention and to the production of polypeptides of the invention by recombinant techniques.
  • Cell-free translation systems can also be employed to produce such proteins using RNAs de ⁇ ved from the DNA constructs of the present mvention.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
  • Introduction of polynucleotides into host cells can be effected by methods desc ⁇ bed in many standard laboratory manuals, such as Davis et al , BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al., MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed., Cold Sp ⁇ ng Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, NN. (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, canonic pid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • approp ⁇ ate hosts include bacte ⁇ al cells, such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtihs cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosoph ⁇ a S2 and Spodoptera Sf cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.
  • bacte ⁇ al cells such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtihs cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosoph ⁇ a S2 and Spodoptera Sf cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • Such systems include, among others, chromosomal, episomal and virus-de ⁇ ved systems, e.g., vectors de ⁇ ved from bacte ⁇ al plasmids, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SN40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors de ⁇ ved from combinations thereof, such as those de ⁇ ved from plasmid and bacte ⁇ ophage genetic elements, such as cosmids and phagemids.
  • vectors de ⁇ ved from bacte ⁇ al plasmids, from bacte ⁇ ophage from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses,
  • the expression systems may contain control regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used.
  • the approp ⁇ ate nucleotide sequence may be inserted mto an expression system by any of a va ⁇ ety of well-known and routine techniques, such as, for example, those set forth m Sambrook et al, MOLECULAR CLONING, A LABORATORY
  • approp ⁇ ate secretion signals may be incorporated into the desired polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals
  • the polypeptide be produced at the surface of the cell.
  • the cells may be harvested p ⁇ or to use in the screening assay If FRAZZLED polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced mtracellularly, the cells must first be lysed before the polypeptide is recovered.
  • FRAZZLED polypeptides can be recovered and pu ⁇ fied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, amon or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for pu ⁇ fication. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured du ⁇ ng isolation and or pu ⁇ fication. Diagnostic Assays
  • This invention also relates to the use of FRAZZLED polynucleotides for use as diagnostic reagents. Detection of a mutated form of FRAZZLED gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over-expression or altered expression of FRAZZLED. Individuals carrying mutations in the FRAZZLED gene may be detected at the DNA level by a va ⁇ ety of techniques.
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, u ⁇ ne, saliva, tissue biopsy or autopsy mate ⁇ al.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques p ⁇ or to analysis.
  • RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in compa ⁇ son to the normal genotype. Point mutations can be identified by hyb ⁇ dizmg amplified DNA to labeled FRAZZLED nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denatu ⁇ ng agents, or by direct DNA sequencing.
  • an array of o gonucleotides probes comp ⁇ sing FRAZZLED nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
  • Array technology methods are well known and have general applicability and can be used to address a va ⁇ ety of questions m molecular genetics including gene expression, genetic linkage, and genetic va ⁇ abihty. (See for example: M.Chee et al, Science, Vol 274, pp 610-613 (1996)).
  • the diagnostic assays offer a process for diagnosing or determining a susceptibility to chronic and acute inflammation, arth ⁇ tis, osteoarth ⁇ tis and other osteopenic conditions, Paget's disease, rheumatoid arth ⁇ tis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, pso ⁇ asis), transplant rejection, graft vs.
  • host disease infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (e.g., osteoporosis), cancer including bone and cartilage cancers and related tumors (e.g , lymphoprohferative disorders), atherosclerosis, and Alzheimers disease, can be diagnosed by methods comprising determining from a sample de ⁇ ved from a subject an abnormally decreased or increased level of FRAZZLED polypeptide or FRAZZLED mRNA.
  • Decreased or increased expression can be measured at the RNA level using any of the methods well known m the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to determine levels of a protein, such as an FRAZZLED polypeptide, in a sample de ⁇ ved from a host are well-known to those of skill in the art.
  • Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis, lmmunocytochemistry and ELISA assays.
  • the present invention relates to a diagonostic kit for a disease or suspectabihty to a disease, particularly chronic and acute inflammation, arth ⁇ tis, osteoarth ⁇ tis and other osteopenic conditions, Paget's disease, rheumatoid arth ⁇ tis, septicemia, autoimmune diseases
  • inflammatory bowel disease e.g., inflammatory bowel disease, pso ⁇ asis
  • transplant rejection graft vs. host disease
  • infection stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, ADDS, metabolic and other bone diseases (e.g., osteoporosis), cancer including bone and cartilage cancers and related tumors (e.g., lymphoprohferative disorders), atherosclerosis, and Alzheimers disease, which comprises:
  • FRAZZLED polynucleotide preferably the nucleotide sequence of SEQ ED NO: 1, or a fragment thereof ;
  • the nucleotide sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hyb ⁇ dize with a particular location on an individual human chromosome
  • the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendehan Inhe ⁇ tance in Man (available on line through Johns Hopkins University Welch Medical Library).
  • genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinhe ⁇ tance of physically adjacent genes).
  • linkage analysis coinhe ⁇ tance of physically adjacent genes.
  • the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
  • polypeptides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies lmmunospecific for the FRAZZLED polypeptides.
  • the term "lmmunospecific" means that the antibodies have substantiall greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the p ⁇ or art.
  • Antibodies generated against the FRAZZLED polypeptides can be obtained by admmiste ⁇ ng the polypeptides or epitope-bea ⁇ ng fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell lme cultures can be used.
  • Examples include the hyb ⁇ doma technique (Kohler, G. and Milstem, C, Nature (1975) 256:495-497), the t ⁇ oma technique, the human B-cell hyb ⁇ doma technique (Kozbor et al , Immunology Today (1983) 4:72) and the EBN-hyb ⁇ doma technique (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985) Techniques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can also be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms including other mammals, may be used to express humanized antibodies.
  • the above-desc ⁇ bed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to pu ⁇ fy the polypeptides by affinity chromatography.
  • Antibodies against FRAZZLED polypeptides may also be employed to treat chronic and acute inflammation, arth ⁇ tis, osteoarth ⁇ tis and other osteopenic conditions, Paget's disease, rheumatoid arth ⁇ tis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, pso ⁇ asis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (e.g., osteoporosis), cancer including bone and cartilage cancers and related tumors (e.g., lymphoprohferative disorders), atherosclerosis, and Alzheimers disease, among others.
  • autoimmune diseases e.g., inflammatory bowel disease, pso ⁇ asis
  • transplant rejection graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other
  • Another aspect of the mvention relates to a method for inducing an immunological response m a mammal which comp ⁇ ses inoculating the mammal with FRAZZLED polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from chronic and acute inflammation, arth ⁇ tis, osteoarth ⁇ tis and other osteopenic conditions, Paget's disease, rheumatoid arth ⁇ tis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, pso ⁇ asis), transplant rejection, graft vs.
  • a mammal which comp ⁇ ses inoculating the mammal with FRAZZLED polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from chronic and acute inflammation, arth ⁇ tis, osteoarth ⁇ tis and other osteopenic conditions, Paget's disease, rheumatoid arth ⁇ tis, septicemia,
  • Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering FRAZZLED polypeptide via a vector directing expression of FRAZZLED polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
  • composition which, when introduced into a mammalian host, induces an immunological response m that mammal to a FRAZZLED polypeptide wherem the composition comp ⁇ ses a FRAZZLED polypeptide or FRAZZLED gene.
  • the vaccine formulation may further comprise a suitable carrier.
  • FRAZZLED polypeptide may be broken down m the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, mtradermal etc. injection).
  • parenteral administration include aqueous and non-aqueous ste ⁇ le injection solutions which may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes which render the formulation mstonic with the blood of the recipient; and aqueous and non-aqueous ste ⁇ le suspensions which may include suspending agents or thickening agents.
  • the formulations may be presented m unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requi ⁇ ng only the addition of the ste ⁇ le liquid carrier immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • the FRAZZLED polypeptide of the present invention may be employed in a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the FRAZZLED polypeptide of the present invention.
  • polypeptides of the invention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical hbra ⁇ es, and natural product mixtures.
  • agonists or antagonists may be natural or modified substrates, ligands, enzymes, receptors, etc., as the case may be, of the polypeptide of the present invention; or may be structural or functional mimetics of the polypeptide of the present invention. See Co gan et al, Current Protocols in Immunology l(2):Chapter 5 (1991).
  • FRAZZLED polypeptides are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate FRAZZLED polypeptide on the one hand and which can inhibit the function of FRAZZLED polypeptide on the other hand.
  • agonists are employed for therapeutic and prophylactic purposes for such conditions as chronic and acute inflammation, arth ⁇ tis, osteoarth ⁇ tis and other osteopenic conditions, Paget's disease, rheumatoid arth ⁇ tis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, pso ⁇ asis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases
  • osteoporosis e.g., osteoporosis
  • cancer including bone and cartilage cancers and related tumors (e.g., lymphoprohferative disorders), atherosclerosis, and Alzheimers disease.
  • Antagonists may be employed for a va ⁇ ety of therapeutic and prophylactic purposes for such conditions as chronic and acute inflammation, arth ⁇ tis, osteoarth ⁇ tis and other osteopenic conditions, Paget's disease, rheumatoid arth ⁇ tis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, pso ⁇ asis), transplant rejection, graft vs.
  • screening procedures may involve using approp ⁇ ate cells which express the FRAZZLED polypeptide or respond to FRAZZLED polypeptide of the present invention.
  • Such cells include cells from mammals, yeast, Drosophila or E coll.
  • Cells which express the FRAZZLED polypeptide (or cell membrane containing the expressed polypeptide) or respond to FRAZZLED polypeptide are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
  • the ability of the cells which were contacted with the candidate compounds is compared with the same cells which were not contacted for FRAZZLED activity.
  • the FRAZZLED cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of FRAZZLED mRNA and protein in cells.
  • an enzyme linked immunosorbent assay ELISA
  • ELISA enzyme linked immunosorbent assay
  • monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents (i.e. antagonists or agonists) which may inhibit or enhance the production of FRAZZLED from suitably manipulated cells or tissues.
  • the FRAZZLED protein may be used to identify membrane bound or soluble hgand or receptors through standard ligand/receptor binding techniques known in the art.
  • gand binding and crosshnkmg assays m which the FRAZZLED is labeled with a radioactive isotope (e.g., 1251), chemically modified (e.g., biotinylated), or fused to a peptide sequence suitable for detection or pu ⁇ fication, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids).
  • a source of the putative receptor cells, cell membranes, cell supernatants, tissue extracts, bodily fluids.
  • Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy.
  • these binding assays can be used to identify agonists and antagonists of FRAZZLED which compete with the binding of FRAZZLED to its receptors or ligands.
  • the above binding assays can be used to identify cells which respond biologically to FRAZZLED.
  • Cells which respond to FRAZZLED may show changes in mtracellular signal transduction pathways and in gene expression. These changes can be used in screens for agonists or antagonists which mimic or inhibit the action of FRAZZLED, respectively.
  • the assays may simply test binding of a candidate compound wherein adherence to the cells bea ⁇ ng the FRAZZLED polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor.
  • these assays may test whether the candidate compound results in a signal generated by activation of the FRAZZLED polypeptide, using detection systems appropriate to the cells bea ⁇ ng the FRAZZLED polypeptide.
  • Inhibitors of activation are generally assayed m the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
  • the assays may simply comprise the steps of mixing a candidate compound with a solution containing a FRAZZLED polypeptide to form a mixture, measu ⁇ ng FRAZZLED activity in the mixture, and comparing the FRAZZLED activity of the mixture to a standard.
  • the FRAZZLED cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of FRAZZLED mRNA and protein in cells.
  • an ELISA may be constructed for measuring secreted or cell associated levels of FRAZZLED protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of FRAZZLED (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • the FRAZZLED protein may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. These include, but are not limited to, hgand binding and crosshnkmg assays in which the FRAZZLED is labeled with a radioactive isotope (e.g., 1251), chemically modified (e.g., biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy.
  • a radioactive isotope e.g. 1251
  • chemically modified e.g., biotinylated
  • these binding assays can be used to identify agonists and antagonists of FRAZZLED which compete with the binding of FRAZZLED to its receptors, if any. Standard methods for conducting screening assays are well understood m the art.
  • FRAZZLED polypeptide antagonists include antibodies or, m some cases, ohgonucleotides or proteins which are closely related to the ligands, substrates, enzymes, receptors, etc., as the case may be, of the FRAZZLED polypeptide, e.g., a fragment of the ligands, substrates, enzymes, receptors, etc.; or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.
  • the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for FRAZZLED polypeptides; or compounds which decrease or enhance the production of FRAZZLED polypeptides, which comprises:
  • This invention provides methods of treating abnormal conditions such as, chronic and acute inflammation, arth ⁇ tis, osteoarth ⁇ tis and other osteopenic conditions, Paget's disease, rheumatoid arth ⁇ tis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, pso ⁇ asis), transplant rejection, graft vs.
  • abnormal conditions such as, chronic and acute inflammation, arth ⁇ tis, osteoarth ⁇ tis and other osteopenic conditions, Paget's disease, rheumatoid arth ⁇ tis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, pso ⁇ asis), transplant rejection, graft vs.
  • FRAZZLED polypeptide activity a host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, bram injury, AEDS, metabolic and other bone diseases (e.g., osteoporosis), cancer including bone and cartilage cancers and related tumors (e.g., lymphoprohferative disorders), atherosclerosis, and Alzheimers disease, related to both an excess of and insufficient amounts of FRAZZLED polypeptide activity.
  • FRAZZLED polypeptide If the activity of FRAZZLED polypeptide is in excess, several approaches are available.
  • One approach comp ⁇ ses admmiste ⁇ ng to a subject an inhibitor compound (antagonist) as hereinabove desc ⁇ bed along with a pharmaceutically acceptable earner in an amount effective to inhibit the function of the FRAZZLED polypeptide, such as, for example, by blocking the binding of ligands, substrates, enzymes, receptors, etc., or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • soluble forms of FRAZZLED polypeptides still capable of binding the hgand, substrate, enzymes, receptors, etc. in competition with endogenous FRAZZLED polypeptide may be administered. Typical embodiments of such competitors comprise fragments of the FRAZZLED polypeptide.
  • soluble forms of FRAZZLED polypeptides still capable of binding the hgand in competition with endogenous FRAZZLED polypeptide may be administered. Typical embodiments of such competitors comprise fragments of the FRAZZLED polypeptide.
  • expression of the gene encoding endogenous FRAZZLED polypeptide can be inhibited using expression blocking techniques. Known such techniques involve the use of antisense sequences, either internally generated or separately administered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, FL (1988). Alternatively, ohgonucleotides which form triple helices with the gene can be supplied. See, for example, Lee et al, Nucleic Acids Res
  • o gomers can be administered per se or the relevant ohgomers can be expressed in vivo.
  • o gomers can be administered per se or the relevant ohgomers can be expressed in vivo.
  • a therapeutically effective amount of a compound which activates FRAZZLED polypeptide i.e., an agonist as desc ⁇ bed above, combination with a pharmaceutically acceptable earner, to thereby alleviate the abnormal condition.
  • gene therapy may be employed to effect the endogenous production of FRAZZLED by the relevant cells in the subject.
  • a polynucleotide of the invention may be engineered for expression m a replication defective retroviral vector, as discussed above.
  • the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest.
  • These producer cells may be administered to a subject for eng eenng cells in vivo and expression of the polypeptide in vivo.
  • Peptides such as the soluble form of FRAZZLED polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical earner.
  • a suitable pharmaceutical earner Such formulations compnse a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or exc ⁇ ient.
  • earners include but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Formulation should suit the mode of administration, and is well withm the skill of the art.
  • the invention further relates to pharmaceutical packs and kits comp ⁇ sing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection.
  • Other injection routes such as subcutaneous, intramuscular, or mtrape ⁇ toneal, can be used.
  • Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
  • oral administration may also be possible. Administration of these compounds may also be topical and/or localized, m the form of salves, pastes, gels and the like. The dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner.
  • Suitable dosages are in the range of 0.1-100 ⁇ g kg of subject. Wide vanations m the needed dosage, however, are to be expected m view of the va ⁇ ety of compounds available and the diffe ⁇ ng efficiencies of va ⁇ ous routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Na ⁇ ations in these dosage levels can be adjusted using standard empincal routines for optimization, as is well understood m the art.
  • Polypeptides used in treatment can also be generated endogenously in the subject, m treatment modalities often referred to as "gene therapy" as desc ⁇ bed above.
  • cells from a subject may be engineered with a polynucleotide, such as a D ⁇ A or R ⁇ A, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
  • Example 1 A partial clone encoding FRAZZLED (EST # 2105409) was identified through a search of a commercial EST database using the ammo acid sequence of a previously identified member of the Fnzzled family, Frzb. This clone was then fully sequenced and the full length sequence of this clone shared 73.8% identity with human Frzb (Hoang, et al., J. Biol. Chem. 271(42): 26131-26137 (1996)]. The clone encoding FRAZZLED was found in an osteoblast cell library.
  • This gene is also expressed in chondrosarcoma, osteosarcoma, osteoclastoma, synovial fibroblasts, hodgkin's lymphoma, ovary, uterus, fetal lung, adipose and pancreatic tumor.
  • Two ESTs corresponding to this gene from Soares ⁇ hHMPu SI cD ⁇ A hbranes are found in the public EST database.
  • FRAZZLED in situ hybridization
  • Anti- peptide antibodies were raised to unique sequences in FRAZZLED and their selectivity for FRAZZLED was confirmed using either peptides (by ELISA) or baculovirus- expressed whole protein (by Western blotting).
  • the peptide sequences used to raise polyclonal antibodies are as follows:
  • N-Gln-Glu-Gln-Arg-Arg-Thr-Val-Gln-Asp-Lys-Lys-Lys-Thr-Ala-C (SEQ ID NO:5) (QEQRRTVQDKKKTA - amino acids 292-305)
  • FRAZZLED FRAZZLED homologue
  • FRAZZLED also appears to be highly expressed in these tissues, and in chondrocytes associated with fissuring in OA cartilage. Therefore, it may play a similar role in human cartilage and/or bone. All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

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Abstract

L'invention concerne des polypeptides et polynucléotides FRZB, ainsi que des méthodes de production de ces polypeptides par des techniques de recombinaison. L'invention concerne également des méthodes d'utilisation de ces polypeptides et polynucléotides FRZB dans la mise au point de protocoles de traitement des inflammations chroniques et aiguës, de l'arthrite, de l'ostéoarthrite et autres affections ostéopéniques, de la maladie osseuse de Paget, de l'arthrite rhumatoïde, de la septicémie, des maladies auto-immunes (les maladies intestinales inflammatoires, le psoriasis, par exemple), du rejet des transplantations, de la réaction du greffon contre l'hôte, des infections, de l'apoplexie, de l'ischémie, du syndrome de l'atteinte respiratoire aiguë, des troubles rénaux, de la resténose, des lésions cérébrales, du SIDA, des troubles métaboliques et autres troubles osseux (l'ostéoporose, par exemple), du cancer y compris le cancer des os et du cartilage et des tumeurs liées (les affections lymphoprolifératives, par exemple), de l'athérosclérose, de la maladie d'Alzheimer, entre autres; et dans la mise au point de dosages de diagnostic relatifs à ces pathologies.
PCT/US2000/015814 1999-06-08 2000-06-08 Membre de la famille frzb WO2000075280A2 (fr)

Priority Applications (1)

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EP00941289A EP1198469A4 (fr) 1999-06-08 2000-06-08 Membre de la famille frzb, frazzled

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007090872A2 (fr) * 2006-02-09 2007-08-16 Novartis Ag Agents de liaison de la proteine-4 apparentee a frizzled (sfrp-4)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0887406A3 (fr) * 1997-05-22 2003-03-05 Smithkline Beecham Corporation Un membre de la famille FRZB, frazzled
EP0879881A1 (fr) * 1997-05-23 1998-11-25 Smithkline Beecham Corporation Gene humain similair à proteine secreté frizb (ATG-1639)
EP1012262A4 (fr) * 1997-08-12 2002-11-06 Human Genome Sciences Inc Proteine humaine hflp

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [Online] 19 May 1997 HILLIER ET AL.: 'Generation and anaylsis of 280,000 human expressed sequence tags', XP002935753 Database accession no. AA194152 & GENOME RES. vol. 6, no. 9, 1996, pages 807 - 828 *
See also references of EP1198469A2 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007090872A2 (fr) * 2006-02-09 2007-08-16 Novartis Ag Agents de liaison de la proteine-4 apparentee a frizzled (sfrp-4)
WO2007090872A3 (fr) * 2006-02-09 2007-11-29 Novartis Ag Agents de liaison de la proteine-4 apparentee a frizzled (sfrp-4)

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