WO2005070448A2

WO2005070448A2 - Methods of use for secreted frizzled-related protein 3 (sfrp-3) in the prevention and treatment of disease

Info

Publication number: WO2005070448A2
Application number: PCT/US2005/001886
Authority: WO
Inventors: Kristen Pierce; Amy L. Tsui Collins; Hongbing Zhang; Lewis Thomas Williams; Pierre Alvaro Beaurang; Haishan Lin; Keting Chu; Yan Wang
Original assignee: Five Prime Therapeutics, Inc.
Priority date: 2004-01-21
Filing date: 2005-01-21
Publication date: 2005-08-04
Also published as: WO2005070448A3

Abstract

Compositions containing sFRP-3 polypeptides, polynucleotides, and modulators thereof, such as antibodies thereto, are provided. Such compositions are useful for the treatment, prevention, and diagnosis of diseases. Such diseases include immune and proliferative diseases. Methods for utilizing these polypeptides, polynucleotides, and modulators aredisclosed; they include methods for treating proliferative, inflammatory, and bone and cartilage diseases.

Description

METHODS OF USE FOR SECRETED FRIZZLED-RELATED PROTEIN 3 (SFRP-3) IN THE PREVENTION AND TREATMENT OF DISEASE PRIORITY CLAIM

[001] This application claims the benefit of provisional application 60/538,367, filed in the United States Patent and Trademark Office on January 21, 2004 and provisional application 60/589,785, filed in the United States Patent and Trademark Office on July 22, 2004. The disclosures of both are hereby incorporated by reference. DESCRIPTION OF THE INVENTION Technical Field

[002] This invention generally relates to a novel alternative splice variant of secreted frizzled-related protein-3 (sFRP-3). It provides for polynucleotides, polypeptides, vectors, host cells, fusion molecules, compositions, and kits comprising this splice variant, and methods of making them. It also provides for modulators, including antibodies, of sFRP-3. It further provides methods of using these compositions and methods to promote chondrogenesis, to treat patients with bone diseases such as osteoartl ritis, and to treat patients with cancers, such as breast, lung, and colon cancers.

Background of the Invention

[003] The secreted frizzled related protein family is characterized, in part, by a cysteine-rich N-terminal domain that is similar to the cysteine-rich domain of the frizzled proteins, a family of proteins with seven transmembrane spanning domains. The frizzled proteins bind to a family of secreted signal transduction proteins, the wingless integrated proteins (Wnts), to transduce signals for activation or inhibition of intracellular pathways, such as for the stabilization of intracellular β-catenin levels. Unlike the frizzled proteins, the sFRPs are Wnt antagonists, and as such, can block Wnt-mediated β-catenin stabilization, h addition to the N-terminal cysteine-rich frizzled like domain, sFRPs possess a C-terminal netrin domain. Netrins are extracellular proteins that can control the guidance of central nervous system commissural axons. Proteins with netrin domains have been reported to perform diverse biological roles, including the regulation of Wnt signaling (Banyai et al., 1999).

[004] The human sFRP-3 gene encodes a secreted 36,254 dalton protein comprised of 325 amino acids, which localizes to chromosome 2q32.1 (GeneCard; http://bioinfo. weizmann. ac.il/cards-birι/carddisp?Fl^B&search=fizb&suff=txt). Secreted FRP-3 undergoes post-translational processing that provides it with eight disulfide bonds and N-linked glycosylation (Chong et al, 2002). It also is present in many tissues, including brain, eye, heart, kidney, spleen, and testis (Rattner et al., 1997). Multiple RNA transcripts have been observed in retinal tissue (Rattner et al., 1997). In situ hybridization analysis and immunochemical analysis of human embryos representing different stages of development detected sFRP-3 in developing skeletal structures, specifically, in the developing appendicular skeleton, craniofacial bones, and the epiphyseal ends of the rib cage (Hoang et al., 1996). [005] Secreted sFRP-3 has been found to be associated with, inter alia, inflammatory, proliferative, and bone-related diseases. For example, expression of the secreted frizzled-related protein gene family was shown to be downregulated in human mesothelioma (Lee et al., 2004). However, the cause or effect of its presence in these diseases remains unclear. It would be highly desirable to clarify the function and utility of sFRP-3, so that therapeutic agents can be provided to address diseases and conditions associated with this protein, including those associated with their under expression, over expression, insufficiency, or requirement for inhibition. SUMMARY OF THE INVENTION [006] The invention provides a pharmaceutical composition with a pharmaceutically acceptable carrier and an isolated sFRP-3 polypeptide, for example, one found in the Sequence Listing. The invention also provides a polypeptide encoded by a nucleic acid molecule found in the Sequence Listing or in Table 1, for example, SEQ ID NOS: 15 and 20 - 24. Suitable carriers include, but are not limited to, saline, phosphate buffered saline, and a lipid-based formulation.

[007] The composition can further include an isolated antibody that specifically binds to or interferes with the activity of an sFRP-3 polypeptide, such as, for example, one that specifically binds to or interferes with the polypeptide of SEQ ID NOS: 15, 20 - 24 and 26 - 30, or SEQ ID NOS: 25, 31 - 47, 48 - 50 and 55 - 66. The antibody may also bind to a ligand of the polypeptide. Such an antibody may be a human antibody or a humanized antibody. It may be a polyclonal antibody, a monoclonal antibody, a single chain antibody, an agonist antibody, an antagonist antibody, a neutralizing antibody, and/or an active fragment of any of these. The active fragment can be an antigen binding fragment, an Fc fragment, a cdr fragment, a Nπ fragment, a N fragment, and/or a framework fragment. Antibodies of the invention can include one or more sequences chosen from a variable region, a constant region, a heavy chain, a light chain, and an antigen-binding region of an immunoglobulin. [008] Isolated antibodies of the invention may specifically bind to or interfere with the activity of a polypeptide of the invention, for example, a sequence of at least six contiguous amino acid residues chosen from the Sequence Listing or Table 1. These antibodies may also bind to the polypeptide itself or to a ligand of the polypeptide. These antibodies may also be a human antibody or a humanized antibody. It may be a polyclonal antibody, a monoclonal antibody, a single chain antibody, an agonist antibody, an antagonist antibody, a neutralizing antibody, and/or an active fragment of any of these. The active fragment can be an antigen binding fragment, an Fc fragment, a cdr fragment, a V_H fragment, a N fragment, and/or a framework fragment, and may also include one or more sequence chosen from a variable region, a constant region, a heavy chain, a light chain, and an antigen-binding region of an immunoglobulin.

[009] In another aspect, the invention provides a method of treating or preventing an infection in a subject by providing a composition with a substantially pure sFRP-3 polypeptide or a modulator thereof and administering the composition to the subject. This method can be used to treat bacterial infections, mycoplasma infections, fungal infections, and viral infections. This method can be performed by administering the polypeptide, e.g., sFRP-3, or the modulator, to the subject locally or systemically. This method can be performed by administering a polypeptide having an amino acid sequence from the Sequence Listing. The method can also be performed by administering a polypeptide encoded by a nucleic acid molecule having a nucleotide sequence as shown in the Sequence Listing

[010] The invention also provides a method of inhibiting undesirable proliferative growth in a subject, by providing a composition with a substantially pure sFRP-3 polypeptide or a modulator of sFRP-3, and administering the polypeptide to the subject. This method can be performed by administering the polypeptide locally or systemically. The undesirable growth may be a tumor, for example a tumor of the ovary, colon, rectum, pancreas, breast, kidney, cervix, lung, prostate, or bladder, or a mesothelioma or glioblastoma. This method can be performed by administering a polypeptide having an amino acid sequence from the Sequence Listing. The method can also be performed by administering a polypeptide encoded by a nucleic acid molecule from the Sequence Listing. [Oil] The invention further provides a method of modulating an immune response in a subject by providing a modulator of a sFRP-3 polypeptide having an agonist or an antagonist function, and administering the modulator to the subject. Agonists and antagonists suitable for performing this method include those that interfere with the binding of sFRP-3 to its ligand, as well as those that bind to sFRP-3 or to a binding partner or ligand of sFRP-3. The antagonist may be a small molecule drug, an antisense molecule, a ribozyme, an RNAi molecule and/or an aptamer. [012] Agonists and antagonists suitable for performing this method may be antibodies. They may be polyclonal antibodies, monoclonal antibodies, single chain antibodies, agonist antibodies, antagonist antibodies, neutralizing antibodies, and/or active fragments of any of these. The active fragment can be an antigen binding fragment, an Fc fragment, a cdr fragment, a N_H fragment, a N_L fragment, and/or a framework fragment, and may also include one or more sequence chosen from a variable region, a constant region, a heavy chain, a light chain, and/or an antigen- binding region of an immunoglobulin. Antibodies suitable for performing this method may be conjugated to a toxic compound, e.g., a toxin or a chemotherapeutic agent, or immunoregulatory compound, e.g., IL-2 or IL-15. [013] This method may modulate the immune response by suppressing inflammation, suppressing autoimmune disease, for example, treating rheumatoid arthritis, osteoarthritis, osteoporosis, psoriasis, inflammatory bowel disease, multiple sclerosis, SLE, myocardial infarction, stroke, or fulminant liver failure. [014] The invention provides a method of enhancing immune response to a vaccine in a subject by providing a composition comprising a substantially purified sFRP-3 polypeptide, providing a vaccine composition, and administering the polypeptide and the vaccine composition to the subject. The polypeptide can be administered to the subject prior to, after, or substantially contemporaneously with administering the vaccine. The polypeptide can be an isolated sFRP-3 polypeptide comprising an amino acid sequence of the Sequence Listing. It can be an isolated sFRP-3 polypeptide encoded by a nucleic acid sequence of the Sequence Listing. [015] The invention also provides a vector comprising a nucleic acid molecule of the Sequence Listing or a nucleic acid molecule encoding a polypeptide of the Sequence Lisitng. This vector may also include a regulatory region that regulates the expression of this nucleic acid molecule. The invention further comprises a host cell with any of the vectors of the invention. [016] The invention provides a a pharmaceutical composition with a pharmaceutically acceptable carrier and an isolated sFRP-3 polypeptide, for example, one found in the Sequence Listing, or a polypeptide encoded by a nucleic acid molecule found in the Sequence Listing, which also has a heterologous secretory leader sequence of a secreted protein which is operably linked to an N-terminus of the polypeptide. The polypeptide of this composition may further comprise a fusion partner, e.g., a polymer, such as polyethylene glycol, or a second polypeptide, such as a fragment or the entirety of human serum albumin, and/or an Fc immunoglobulin fraction. BRIEF DESCRIPTION OF THE TABLES AND DRAWINGS [017] Table 1, SFRP-3 Sequence Identification, lists characteristics of the sFRP-3 polypeptides of the invention. Each sFRP-3 polypeptide is identified by the internal reference designation (FP ID), as shown in the first column. The nucleotide sequence identification number for the open reading frame of the nucleic acid sequence (Nl) is shown in the second column. The amino acid sequence identification number for the polypeptide sequence (PI) is shown in the third column. The nucleotide sequence identification number for the entire nucleic acid sequence that contains UTR (NO) is shown in the fourth column. The fifth, column shows an internal reference designation (Reference ID).

[018] Table 2, Pfam Coordinates of SFRP-3 Sequences, lists the novel splice variant, CLN00271203, and currently known forms of sFRP-3 (Reference ID) in the first column. The second column lists the Pfam domains of each polypeptide (Pfam) and the third column lists the coordinates of each Pfam domain, in terms of amino acid residues, beginning with "1" at the N-terminus of the full-length polypeptide. [019] Table 3, Signal Peptide and Transmembrane Coordinates of SFRP-3 Sequences, shows the properties of the present novel polypeptide and other variants. The first column lists the Reference LD. The second column shows that each polypeptide listed is classified by the inventors as a secreted protein based on an internally developed classification system. Table 3 also specifies the result of an internally developed algorithm that predicts whether a sequence is secreted (Treevote), with "1" being a high probability that the polypeptide is secreted and "0" being a low probability that the polypeptide is secreted. This algorithm is constructed on the basis of a number of physical and chemical attributes. The predicted length of each polypeptide, expressed as the number of amino acid residues, is shown in the fourth column (Predicted Protein Length). The fifth column lists a signal peptide (or secretory leader) position of each polypeptide (Signal Peptide Coords.) based on positions of the starting and end amino acid residues. The sixth column lists alternate predictions of the coordinates of the hydrophobic domains of the signal peptide or secretory leader sequences based on the starting and ending amino acid residue positionsof each polypeptide. The seventh column lists the corresponding mature protein coordinates, i.e., the amino acid residues of the mature polypeptide after cleavage of the signal peptide (or secretory leader) sequence of each polypeptide (Mature Protein Coords.). The eighth column shows the corresponding amino acid residue positions of the corresponding mature polypeptide after cleavage of the signal peptide sequence of each polypeptide (Alternate Mature Protein Coords.). In instances where the mature protein start residue overlaps the signal peptide end residue, some of the amino acid residues may be cleaved off, such that the mature protein does not begin at the next sequential amino acid residue following the signal peptide, resulting in a polypeptide with the designated alternate mature protein coordinates. Table 3, column nine, in the tenth column indicates that the listed polypeptides have no transmernbrane regions (TM). Finally, Table 3 lists the coordinates of the non-transmembrane regions (non-TM Coords). [020] Table 4, Annotated SFRP-3 Sequences, compares the present novel sFRP polypeptide with the human proteins in the public National Center for Information Biotechnology (NCBI) database that have the greatest similarity to the full-length polypeptide being claimed. The first column lists the protein identification number of the novel polypeptide (Reference ID). The second column lists the NCBI accession numbers (Public ID). The third column lists the NCBI's annotation of the public sequence (Public Annotation). The fourth column lists the length of the public sequences in number of amino acid residues. The fifth column lists the number of identical amino acid residues between the Reference LD polypeptide and the public sequences in number of amino acid residues (Number of Matches). Finally, the sixth column shows the percent identity between the matching amino acid residues and the amino acid residues of the novel sequence (% ID Over Query Length). For example, the length of the novel polypeptide is 303 amino acid residues. The number of amino acid matches with NP_001454 is 302 amino acid residues. The % LD over Query is 302/303 x 100% = 99.7%. The seventh column shows the percent identity between the matching amino acid residues and the amino acid residues of the public sequence (% ID Over Public Length). For example, NP_001454 is 325 amino acid residues in length, and the length of the match with the novel polypeptide is 302 amino acid residues. Hence, % ID over Public Length is 302/325 x 100% - 93%. [021] Figure 1 shows the amino acid sequence alignment of the novel splice variant of the invention, CLN00271203_5pvl_CLN00271203_3pvl, as compared to three known sFRP-3 sequences from the NCBI database. The asterisks (*) indicate shared amino acid residues. The colons ":" indicate conserved amino acid changes. The hyphens "-"indicate amino acid residues that are missing from the novel sequence. MODES FOR CARRYING OUT THE INVENTION [022] The inventors herein have surprisingly found a novel sFRP-3 polypeptide that is missing exon 3 of previously described sFRP-3 molecules. The cystine-rich frizzled domain is intact, while the netrin domain is shorter than the netrin domains of previously described sFRP-3 molecules. The signal sequence is intact. Both the novel splice variant and sFRP-3 molecules in the public domain find novel methods of uses in the diagnosis, prevention, and therapy of proliferative diseases, such as cancer, and bone and cartilage-related diseases, such as osteoarthritis. The novel splice variant was deected in bone. In vitro evidence suggests that this variant sFRP-3 promotes chondrogenesis, and thus provides an effective therapeutic for osteoarthritis. Definitions

[023] The terminologies used herein have their ordinary meanings. Further, the present invention can be more readily understood in light of the following particular definitions.

[024] The terms "polynucleotide," "nucleotide," "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide sequence," and "nucleotide sequence" are used interchangeably herein to refer to polymeric forms of nucleotides of any length. The polynucleotides can contain deoxyribonucleotides, ribonucleotides, and/or their analogs or derivatives. For example, nucleic acids can be naturally occurring DNA or RNA, or can be synthetic analogs, as known in the art. The terms may encompass genomic DNA, genes, gene fragments, exons, introns, regulatory sequences or regulatory elements (such as promoters, enhancers, initiation and termination regions, other control regions, expression regulatory factors, and expression controls), DNA comprising one or more single-nucleotide polymorphisms (SNPs), allelic variants, isolated DNA of any sequence, and cDNA. The terms also encompass mRNA, tRNA, rRNA, ribozymes, splice variants, antisense RNA, antisense conjugates, RNAi, and isolated RNA of any sequence. The terms additionally encompass recombinant polynucleotides, heterologous polynucleotides, branched polynucleotides, labeled polynucleotides, hybrid DNA/RNA, polynucleotide constructs, vectors comprising the subject nucleic acids, nucleic acid probes, primers, and primer pairs. The polynucleotides can comprise modified nucleic acid molecules, with alterations in the backbone, sugars, or heterocyclic bases, such as methylated nucleic acid molecules, peptide nucleic acids, and nucleic acid molecule analogs, which maybe suitable as, for example, probes if they demonstrate superior stability and/or binding affinity under assay conditions. The terms also encompass single- stranded, double-stranded and triple helical molecules that are either DNA, RNA, or hybrid DNA RNA and that may encode a full-length gene or a biologically active fragment thereof. Biologically active fragments of polynucleotides can comprise regulatory regions that regulate the expression of a gene or can encode the polypeptides herein, as well as anti-sense and RJSTAi molecules. Thus, the full length polynucleotides herein may be treated with enzymes, such as Dicer, to generate a library of short RNAi fragments which are within the scope of the present invention. [025] "Nucleic acid composition" as used herein is a composition comprising a nucleic acid molecule, including one having a nucleotide sequence open reading frame that encodes a polypeptide and is capable, under appropriate conditions, of being expressed as a polypeptide. The term includes, for example, vectors, including plasmids, cosmids, viral vectors (e.g., retrovirus vectors such as lentivirus, adeno irus, and the like), human, yeast, bacterial, PI -derived artificial chromosomes (HAC's, YAC's, BAC's, PAC's, etc), and mini-chromosomes, in vitro host cells, in vivo host cells, tissues, organs, allogenic or congenic grafts or transplants, multicellular organisms, and chimeric, genetically modified, or transgenic animals comprising a subject nucleic acid sequence.

[026] A "regulatory region" of a polynucleotide is a sequence that aids the expression, including transcription and translation, of a coding sequence to which it is linked. The term includes, e.g., promoters, transcription termination sequences, upstream regulatory domains, polyadenylation signals, leader sequences, and enhancers.

[027] A "vector" is a plasmid that can be used to transfer DNA sequences from one organism to another. An "expression vector" is a cloning vector that contains regulatory sequences that allow transcription and translation of a cloned gene or genes and thus transcribe and clone DNA. Expression vectors can be used to express the polypeptides of the invention and typically include restriction sites to provide for the insertion of nucleic acid sequences encoding heterologous protein or RNA molecules. Artificially constructed "plasmids," i.e., small, independently replicating pieces of extrachromosomal cytoplasmic DNA that can be transfereed from one organism to another, are commonly used as cloning vectors.

[028] The term "host cell" includes an individual cell, cell line, cell culture, or in vivo cell, which can be or has been a recipient of any polynucleotides or polypeptides of the invention, for example, a recombinant vector, an isolated polynucleotide, antibody or fusion protein. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology, physiology, or in total DNA, RNA, or polypeptide complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. Host cells can be prokaryotic or eukaryotic, including mammalian, insect, amphibian, reptile, crustacean, avian, fish, plant and fungal cells. A host cell includes cells transformed, transfected, transduced, or infected in vivo or in vitro with a polynucleotide of the invention, for example, a recombinant vector. A host cell which comprises a recombinant vector of the invention may be called a "recombinant host cell." [029] The term "operably linked" refers to nucleotide sequences that are associated or connected in such a manner that their transcription or translation can be associated or connected, e.g., they can be transcribed or translated together. [030] An "isolated," "purified," or "substantially isolated" polynucleotide, or a polynucleotide in "substantially pure form," in "substantially purified form," in "substantial purity," or as an "isolate," is one that is substantially free of the sequences with which it is associated in nature, or other nucleic acid sequences that do not include a sequence or fragment of the subject polynucleotides. By substantially free is meant that less than about 90%, less than about 80%, less than about 70%, less than about 60%), or less than about 50% of the composition is made up of materials other than the isolated polynucleotide. Where at least about 99% of the total macromolecules is the isolated polynucleotide, the polynucleotide is at least about 99% pure, and the composition comprises less than about 1% contaminant. [031] The terms "polypeptide," "peptide," and "protein," used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include naturally-occurring amino acids, coded and non-coded amino acids, chemically or biochemically modified, derivatized, or designer amino acids, amino acid analogs, peptidomimetics, and depsipeptides, and polypeptides having modified, cyclic, bicyclic, depsicyclic, or depsibicyclic peptide backbones. The term includes single chain protein as well as multimers. The term also includes conjugated proteins, fusion proteins, including, but not limited to, glutathione S-transferase (GST) fusion proteins, fusion proteins with a heterologous amino acid sequence, fusion proteins with heterologous and homologous leader sequences, fusion proteins with or without N-terminal methionine residues, pegolyated proteins, and immunologically tagged, or his-tagged proteins. Also included in this term are variations of naturally occurring proteins, where such variations are homologous or substantially similar to the naturally occurring protein, as well as corresponding homologs from different species. Variants of polypeptide sequences include insertions, additions, deletions, or substitutions compared with the subject polypeptides. The term also includes peptide aptamers.

[032] A "sFRP-3 polypeptide" refers to a full length sFRP-3 polypeptide, or any fragment thereof. The term includes the sequences of the Sequence Listing and tables and fragments of any of these, such as, for example, any one of SEQ ID NOS: 15 and 20 - 24. It also includes a mature polypeptide generated by cleavage of the signal peptide sequence, as shown in the Tables, or active fragments of such polypeptides, for example, those comprising Pfam domains. This term also includes fusion or hybrid molecules containing all or a portion of the full-length polypeptide. [033] An "isolated," "purified," or "substantially isolated" polypeptide, or a polypeptide in "substantially pure form," in "substantially purified form," in "substantial purity," or as an "isolate," is one that is substantially free of the materials with which it is associated in nature or other polypeptide sequences that do not include a sequence or fragment of the subject polypeptides . By substantially free is meant that less than about 90%>, less than about 80%, less than about 70%>, less than about 60%), or less than about 50%) of the composition is made up of materials other than the isolated polypeptide. Where at least about 99% of the total macromolecules is the isolated polypeptide, the polypeptide is at least about 99% pure, and the composition comprises less than about 1%> contaminant. A-s used herein, an "isolated," "purified," or "substantially isolated" polypeptide, or a polypeptide in "substantially pure form," in "substantially purified form," in "substantial purity," or as an "isolate," also refers to recombinant polypeptides, modified, tagged and fusion polypeptides, and chemically synthesized polypeptides, which by virtue or origin or manipulation, are not associated with all or a portion of the materials with which they are associated in nature, are linked to molecules other than that to which they are linked in nature, or do not occur in nature.

[034] A "secreted protein" is one capable of being directed to the endoplasmic reticulum (ER), secretory vesicles, or the extracellular space as a result of a secretory leader, signal peptide, or leader sequence. A "secreted protein" is also one released into the extracellular space, e.g., by exocytosis or proteolytic cleavage, regardless of whether it comprises a signal sequence. A secreted protein may in some circumstances undergo processing to a "mature" polypeptide.

[035] A "secretory leader," "signal peptide," or a "leader sequence," is a sequence of amino acids, typically positioned at the N-terminus of polypeptide, which directs the intracellular trafficking of the polypeptide. Polypeptides which comprise a secretory leader typically also comprise one or more secretory leader cleavage site; a mature polypeptide is generated following cleavage at this site, for example, following extracellular secretion or delivery to an intracellular compartment. Leader sequences can be natural or synthetic, heterologous, or homologous with the protein to which they are attached.

[036] A "fusion partner" is a polypeptide fused in-frame at the N-terminus and/or C- terminus of a therapeutic or prophylactic polypeptide, or internally to a therapeutic or prophylactic polypeptide. For example, the fusion partner may be any human serum albumin, an immunoglobulin F_c fragment, or any fragment thereof.

[037] An "antibody" of the present invention is defined to include active fragments such as the variable region of an immunoglobulin, the antigen binding region, etc. It may comprise a monoclonal antibody, polyclonal antibody, single chain antibody, intrabody, and/or active fragments of any of these. The active fragments may include variable regions from either heavy chains or light chains.

[038] A "polyclonal antibody" a mixture of antibodies of different specificities, as in the serum of an animal immunized to various antigens or epitopes.

[039] A "monoclonal antibody" is an antibody composition having a homogeneous antibody population. The term is not limited with regard to the species or source of the antibody, nor by the manner in which it is made. The term encompasses whole immunoglobulins and immunoglobulin fragments. [040] A "humanized" antibody is an antibody that contains mostly human immunoglobulin sequences. This term is generally used to refer to a non-human immunoglobulin that has been modified to incorporate portions of human sequences, and may include a human antibody that contains entirely human immunoglobulin sequences.

[041] A "single chain antibody" is an Fab fragment comprising only the V domain of a heavy chain linked by a peptide to a V domain of a light chain (Jost et al., J Biol.

Chem. 269:26,267 (1994).

[042] A "framework fragment" is that region of the variable domain that contains relatively invariant sequences and lies between the hypervariable regions. Framework regions provide a protein scaffold for the hypervariable regions.

[043] The "antigen binding fragment (Fab fragment)" is a disulfide-linked heterodimer, each chain of which contains one immunoglobulin constant region (C) domain and one variable region (V) domain; the juxtaposition of the V domains forms the antigen-binding site. The two Fab fragments of an intact immunoglobulin molecule conespond to its two arms, which typically contain light chain regions paired with the V and Cl domains of the heavy chains.

[044] The "fragment crystallizable fragment (Fc fragment)" is the portion of an antibody molecule that interacts with effector molecules and cells. It comprises the carboxy-terminal portions of the immunoglobulin heavy chains. The functional differences between heavy-chain isotypes lie mainly in the Fc fragment.

[045] A complementarity-determining region (cdr) fragment is a portion of the region of an antibody which binds to an antigen.

[046] The "constant region" of an antibody is its effector region, and determines the functional class of the antibody. The constant region of a heavy or light chain is located at or near the carboxyl terminus.

[047] The "variable region" of an antibody is the region that binds to the antigen; it provides antibody specificity. The variable region of a heavy (V_H fragment) or light

(N_L fragment) chain is located at or near the amino terminus.

[048] An "immunoglobulin" is an antibody molecule, i.e., a polypeptide that can respond to a foreign molecule of invading organism, e.g., by binding to it, marking it for destruction, and/or inactivating it. [049] A "heavy chain" is the larger of the two classes of polypeptide chains that combine to form immunoglobulin molecules. The class of the heavy chain determines the class of the immunoglobulin, e.g., IgG, IgA, IgE, IgD, or IgM. [050] A "light chain" is the smaller of the two classes of polypeptide chains that combine to form immunoglobulin molecules. Light chains are generally classified into two classes, kappa and lambda, on the basis of structural differences in their constant regions.

[051] A "vaccine" is a preparation that produces or artificially increases immunity to a particular disease. It may, for example, be comprised of killed microorganisms, living attenuated organisms, or living virulent organisms that is administered to produce or artificially increase immunity to a particular disease. It includes a preparation containing weakened or dead microbes of the kind that cause a particular disease, administered to stimulate the immune system to produce antibodies against that disease.

[052] The term "agonist" refers to a substance that mimics the function of an active molecule. Agonists include, but are not limited to, drugs such as small molecule compounds, hormones, antibodies, and neurotransmitters, as well as analogues and fragments thereof. An "agonist" antibody in reference to the present polypeptides is one that enhances the activity of the present polypeptides, such as, for example, by binding to a molecule that inhibits the activity of the present polypeptides. [053] The term "antagonist" refers to a molecule that competes for the binding sites on a molecule with an agonist, but does not induce an active response. Antagonists include, but are not limited to, drugs such as small molecule compounds, hormones, antibodies, and neurotransmitters, anti-sense molecules, RNAi, small molecules, soluble receptors, as well as analogues and fragments thereof. An "antagonist" antibody in reference to the present polypeptides is one that inhibits the activity of the present polypeptides, such as, for example, by specific binding to such polypeptides. [054] "Active fragments" are those exhibiting activity similar, but not necessarily identical, to an activity of a molecule of the present invention. For example, an entity demonstrates activity when it participates in a molecular interaction with another molecule, or when it has therapeutic value in alleviating a disease condition, or when it has prophylactic value such as in inducing an immune response to the molecule, or when it has diagnostic value in determining the presence of the molecule, such as a active fragment of a polynucleotide that can be detected as unique for the polynucleotide molecule, or that can be used as a primer in PCR. [055] The term "ligand" refers to any molecule that binds to a specific site on another molecule.

[056] The term "receptor" refers to a polypeptide that binds to a specific extracellular molecule and may initiate a cellular response.

[057] The terms "subject," "host," "patient," and "individual," used interchangeably herein, refer to an animal, including, but not limited to, avians, rodents, murines, simians, humans, non-human primates, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian farm animals, mammalian sport animals, and mammalian pets.

[058] A "stem cell" is a pluripotent or multipotent cell with the abilities to self- renew, to remain undifferentiated, and to become differentiated. Stem cells can divide without limit, for at least the lifetime of the animal in which they naturally reside. Stem cells are not terminally differentiated, i.e., they are not at the end of a pathway of differentiation. When a stem cell divides, each daughter cell can either remain a stem cell or it can embark on a course that leads to terminal differentiation. [059] An "embryonic stem cell" is a stem cell that is present in or isolated from an embryo. It can be pluripotent, having the capacity to differentiate into each and every cell present in the organism, or multipotent, with the ability to differentiate into more than one cell type. Embryonic stem cells derived from the inner cell mass of the embryo can act as pluripotent cells when placed into host blastocysts. [060] "Undesirable proliferative growth" is any abnormal cell or tissue growth that does not respond to one or more inhibitory signals present in healthy individuals. It may be benign or malignant. Undesirable proliferative cell growth includes cancer and psoriasis.

[061] A tumor is a solid mass of cells or tissues undergoing uncontrolled proliferative growth.

[062] The term "disease" or "diseases" in a subject or an animal as used herein include diseases, disorders, conditions, syndromes, and infections in the subject. [063] A "pharmaceutically acceptable carrier," "pharmaceutically acceptable diluent," "pharmaceutically acceptable excipient," or "pharmaceutically acceptable vehicle," used interchangeably herein, refer to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type. A phannaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the carrier for a formulation containing polypeptides would not normally include oxidizing agents and other compounds that are known to be deleterious to polypeptides. Suitable carriers include, but are not limited to, water, dextrose, glycerol, saline, ethanol, and combinations thereof. The carrier can contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the formulation. Adjuvants of the invention include, but are not limited to Freunds's, Montanide ISA Adjuvants (Seppic, Paris, France), Ribi's Adjuvants (Ribi ImmunoChem Research, Inc., Hamilton, MT), Hunter's TiterMax (CytRx Corp., Norcross, GA), Aluminum Salt Adjuvants (AUiydrogel - Superfos of Denmark/ Accurate Chemical and Scientific Co., Westbury, NY), Nitrocellulose- Adsorbed Protein, Encapsulated Antigens, and Gerbu Adjuvant (Gerbu Biotechnik GmbH, Gaiberg, Gennany/C-C Biotech, Poway, CA). Topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%_>) in water, or sodium lauryl sulfate (5%>) in water. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents can be added as necessary. Percutaneous penetration enhancers such as Azone can also be included.

[064] "Pharmaceutically acceptable salts" include the acid addition salts (formed with the free amino groups of the polypeptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, maleic, mandelic, oxalic, and tartaric. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, and histidine. [065] The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an "effective amount," that is, a dosage sufficient to produce the desired result or effect in association with a pharmaceutically acceptable carrier. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed, the host, and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host. [066] The term "specifically binds to," in the context of antibody binding, refers to high avidity and/or high affinity binding of an antibody to a specific polypeptide, or more accurately, to an epitope of a specific polypeptide. Antibody binding to such epitope on a polypeptide can be stronger than binding of the same antibody to any other epitopes, particularly other epitopes that can be present in molecules in association with, or in the same sample as the polypeptide of interest. For example, when an antibody binds more strongly to one epitope than to another, adjusting the binding conditions can result in antibody binding almost exclusively to the specific epitope and not to any other epitopes on the same polypeptide, and not to any other polypeptide, which does not comprise the epitope. Antibodies that bind specifically to a subject polypeptide may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10%> or less of the binding shown to the polypeptide of interest). In general, antibodies of the invention bind to a specific polypeptide with a binding affinity of 10^"7 M or greater (e.g., 10^"8 M, 10^"9 M, 10^"10 M, 10^"11 M, etc.). [067] The term "modulate" encompasses an increase or a decrease, a stimulation, inhibition, interference or blockage in the measured activity when compared to a suitable control. "Modulation" of expression levels includes increasing the level and decreasing the level of an mRNA or polypeptide encoded by a polynucleotide of the invention when compared to a control lacking the agent being tested. In some embodiments, agents of particular interest are those which inhibit a biological activity of a subject polypeptide, and/or which reduce a level of a subject polypeptide in a cell, and/or which reduce a level of a subject mRNA in a cell and/or which reduce the release of a subject polypeptide from a eukaryotic cell, hi other embodiments, agents of interest are those that increase a biological activity of a subject polypeptide, and/or which increase a level of a subject polypeptide in a cell, and/or which increase a level of a subject mRNA in a cell and/or which increase the release of a subject polypeptide from a eukaryotic cell.

[068] An agent that "modulates the level of expression of a nucleic acid" in a cell is one that brings about an increase or decrease of at least about 1.25-fold, at least about 1.5-fold, at least about 2-fold, at least about 5-fold, at least about 10-fold, or more in the level (i.e., an amount) of mRNA and/or polypeptide following cell contact with a candidate agent compared to a control lacking the agent.

[069] "Modulating a level of active subject polypeptide" includes increasing or decreasing activity of a subject polypeptide; increasing or decreasing a level of active polypeptide protein; increasing or decreasing a level of mRNA encoding active subject polypeptide, and increasing or decreasing the release of subject polypeptide for a eukaryotic cell. In some embodiments, an agent is a subject polypeptide, where the subject polypeptide itself is administered to an individual. In some embodiments, an agent is an antibody specific for a subject polypeptide. In some embodiments, an agent is a chemical compound such as a small molecule that may be useful as an orally available drug. Such modulation includes the recruitment of other molecules that directly effect the modulation. For example, an antibody that modulates the activity of a subject polypeptide that is a receptor on a cell surface may bind to the receptor and fix complement, activating the complement cascade and resulting in lysis of the cell.

[070] An "agent which modulates a biological activity of a subject polypeptide," as used herein, describes any substance, synthetic, semi-synthetic, or natural, organic or inorganic, small molecule or macromolecular, pharmaceutical or protein, with the capability of altering a biological activity of a subject polypeptide or of a fragment thereof, as described herein. The biological activity can be measured using any assay known in the art.

[071] An agent which modulates a biological activity of a subject polypeptide increases or decreases the activity at least about 10%>, at least about 15%, at least about 20%), at least about 25%, at least about 50%o, at least about 100%), or at least about 2-fold, at least about 5-fold, or at least about 10-fold or more when compared to a suitable control.

[072] "Treatment," "treating," and the like, as used herein, refer to obtaining a desired pharmacologic and/or physiologic effect, covering any treatment of a disease, pathological condition or disorder in a mammal, including a human. The effect may be prophylactic in terms of completely or partially preventing a disorder or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse affect attributable to the disorder. That is, "treatment" includes (1) preventing the disorder from occurring or recurring in a subject who may be predisposed to the disorder but has not yet been diagnosed as having it, (2) inhibiting the disorder, such as arresting its development, (3) stopping or terminating the disorder or at least symptoms associated therewith, so that the host no longer suffers from the disorder or its symptoms, such as causing regression of the disorder or its symptoms, for example, by restoring or repairing a lost, missing or defective function, or stimulating an inefficient process, or (4) relieving, alleviating, or ameliorating the disorder, or symptoms associated therewith, where ameliorating is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, such as inflammation, pain, and/or tumor size. Protein Family Domains

[073] The "Pfam" system is an organization of protein sequence classification and analysis, based on conserved protein domains; it can be publicly accessed in a number of ways, for example, at http://pfam.wustl.edu. Protein domains are portions of proteins that have a tertiary structure and sometimes have enzymatic or binding activities; multiple domains can be connected by flexible polypeptide regions within a protein. Pfam domains can comprise the N-terminus or the C-terminus of a protein, or can be situated at any point in between. The Pfam system identifies protein families based on these domains and provides an annotated, searchable database that classifies proteins into families.

[074] Sequences encompassed by the invention include, but are not limited to, the polypeptide and polynucleotide sequences of the molecules shown in the tables, figures and Sequence Listing herein, as well as corresponding molecular sequences found at any developmental stage of an organism. Sequences of the invention can comprise genes or gene segments designated in the Sequence Listing, and their gene products, i.e., RNA and polypeptides. They also include variants that are affected in pathological states, such as disease-related mutations or sequences with alterations that lead to pathology.

[075] The polypeptides herein comprise frizzled (Fz) and netrin (NTR) protein family domains ("Pfam"). They comprise one or more frizzled (Fz) domains, also known as cysteine-rich domains, which are protein domains with ten conserved cysteine residues that form five disulphide bridges. This domain is conserved in diverse proteins which include receptor tyrosine kinases. The frizzled genes belong to the seven transmembrane class of receptors, and have in their extracellular region a cysteine-rich domain that has been implicated as the Wnt binding domain. The structure of this domain is known and is composed mainly of α helices (http://pfam.wustl.edu/cgi-bin/getdesc?name=Fz).

[076] The polypeptides herein also comprise one or more netrin NTR/C345C module (NTR) domains, which are known to bind to the metzincin class of zinc 2005/070448 19 proteinases. Netrins are extracellular proteins that control the guidance of central nervous system commissural axons. This domain is present in a number of proteins in addition to netrins, e.g., cobra venom factor and complement proteins C3, C4, and C5 (http://pfam.wustl.edu/cgi-bin/getdesc? name=NTR). Nucleic Acids and Polypeptide Compositions Nucleic Acids [077] The present invention provides novel nucleic acid molecules, novel genes encoding proteins, the encoded proteins, and fragments, complements, and homologs thereof having nucleotide sequences such as any one of those shown in the tables and Sequence Listing, for example, any one of SEQ JO NOS:l - 10, 11, and 51, as well as fusion molecules containing such. Non-limiting embodiments of nucleic acid molecules include genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, siRNA, ribozymes, antisense cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules include splice variants of an mRNA. Nucleic acids can be naturally occurring, e.g. DNA or RNA, or can be synthetic analogs, as known in the art. Such analogs are suitable as probes because they demonstrate stability under assay conditions. A nucleic acid molecule can also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art.

[078] Nucleic acid compositions can comprise a sequence of DNA or RNA, including one having an open reading frame that encodes a polypeptide and is capable, under appropriate conditions, of being expressed as a polypeptide. The nucleic acid compositions also can comprise fragments of DNA or RNA. The term encompasses genomic DNA, cDNA, mRNA, splice variants, antisense RNA, RNAi, siRNA, DNA comprising one or more single-nucleotide polymorphisms (SNP), and vectors comprising nucleic acid sequences of interest.

[079] The nucleic acids of the subject invention can encode all or a part of the subject proteins. Double or single stranded fragments can be obtained from the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, for example by restriction enzyme digestion or polymerase chain reaction (PCR) amplification. The use of the polymerase chain reaction has been described (Saiki et al., 1985) and current techniques have been reviewed 2005/070448 20

(Sambrook et al., 1989; McPherson et al. 2000; Dieffenbach and Dveksler, 1995). For the most part, DNA fragments will be of at least about 5 nucleotides, at least about 8 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 18 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, or at least about 50 nucleotides, at least about 75 nucleotides, or at least about 100 nucleotides. Nucleic acid compositions that encode at least six contiguous amino acids (i.e., fragments of 18 nucleotides or more), for example, nucleic acid compositions encoding at least 8 contiguous amino acids (i.e., fragments of 24 nucleotides or more), are useful in directing the expression or the synthesis of peptides that can be used as immunogens (Lerner, 1982; Shinnick et al., 1983; Sutcliffe et al., 1983).

[080] Nucleic acid molecules of the invention can comprise heterologous nucleic acid sequences, i.e., nucleic acid sequences of any length other than those specified in the Sequence Listing. For example, the subject nucleic acid molecules can be flanked on the 5' and/or 3' ends by heterologous nucleic acid molecules of from about 1 nucleotide to about 10 nucleotides, from about 10 nucleotides to about 20 nucleotides, from about 20 nucleotides to about 50 nucleotides, from about 50 nucleotides to about 100 nucleotides, from about 100 nucleotides to about 250 nucleotides, from about 250 nucleotides to about 500 nucleotides, or from about 500 nucleotides to about 1000 nucleotides, or more in length.

[081] Heterologous sequences of the invention can comprise nucleotides present between the initiation codon and the stop codon, including some or all of the introns that are normally present in a native chromosome. They can further include the 3' and 5' untranslated regions found in the mature mRNA. They can further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 kb, about 2 kb, and possibly more, of flanking genomic DNA at either the 5 ' or 3' end of the transcribed region. Genomic DNA can be isolated as a fragment of 100 kbp or smaller; and substantially free of flanking chromosomal sequence. This genomic DNA flanking the coding region, either 3' or 5', or internal regulatory sequences as sometimes found in introns, may contain sequences required for proper tissue and stage-specific expression.

[082] The sequence of the 5' flanking region can be utilized as promoter elements, including enhancer binding sites that provide for tissue-specific expression and developmental regulation in tissues where the subject genes are expressed, providing 2005/070448 21 promoters that mimic the native pattern of expression. Naturally occurring polymorphisms in the promoter region are useful for determining natural variations in expression, particularly those that may be associated with disease. Promoters or enhancers that regulate the transcription of the polynucleotides of the present invention are obtainable by use of PCR techniques using human tissues, and one or more of the present primers.

[083] Regulatory sequences can be used to identify cis acting sequences required for transcriptional or translational regulation of expression, especially in different tissues or stages of development, and to identify cis acting sequences and transacting factors that regulate or mediate expression. Such transcription or translational control regions can be operably linked to a gene in order to promote expression of wild type genes or of proteins of interest in cultured cells, embryonic, fetal or adult tissues, and for gene therapy (Hooper, 1993).

[084] The invention provides variants resulting from random or site-directed mutagenesis. Techniques for in vitro mutagenesis of cloned genes are known. Examples of protocols for site specific mutagenesis maybe found in Gustin et al., 1993; Barany 1985; Colicelli et al., 1985; Prentki et al., 1984. Methods for site specific mutagenesis can be found in Sambrook et al., 1989 (pp. 15.3-15.108); Weiner et al., 1993; Sayers et al. 1992; Jones and Winistorfer; Barton et al., 1990; Marotti and Tomich 1989; and Zhu, 1989. Such mutated genes can be used to study structure- function relationships of the subject proteins, or to alter properties of the protein that affect its function or regulation. Other modifications of interest include epitope tagging, e.g., with hemagglutinin (HA), FLAG, or c-myc. For studies of subcellular localization, fluorescent fusion proteins can be used.

[085] The invention also provides variants resulting from chemical or other modifications. Modifications in the native structure of nucleic acids, including alterations in the backbone, sugars or heterocyclic bases, have been shown to increase intracellular stability and binding affinity. Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters, and boranophosphates. Achiral phosphate derivatives include 3'-O'-5'-S-phosphorothioate, 3'-S-5'-O- phosphorothioate, 3'-CH -5'-O-phosphonate and 3'-NH-5'-O-phosphoroamidate. Peptide nucleic acids have modifications that replace the entire ribose phosphodiester backbone with a peptide linkage. 2005/070448 22

[086] Sugar modifications are also used to enhance stability and affinity. The a- anomer of deoxyribose can be used, where the base is inverted with respect to the natural /3-anomer. The 2'-OH of the ribose sugar can be altered to form 2'-0-methyl or 2'-O-allyl sugars, which provides resistance to degradation without comprising affinity.

[087] Modification of the heterocyclic bases must maintain proper base pairing. Some useful substitutions include deoxyuridine for deoxythymidine; 5-methyl-2 '- deoxycytidine, and 5-bromo-2'-deoxycytidine for deoxycytidine. 5 propynyl-2'- deoxyuridine and 5 -propynyl-2 '-deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively.

[088] Mutations can be introduced into the promoter region to determine the effect of altering expression in experimentally defined systems. Methods for the identification of specific DNA motifs involved in the binding of transcriptional factors are known in the art, for example sequence similarity to known binding motifs, and gel retardation studies (Blackwell et al., 1995; Mortlock et al., 1996; Joulin and Richard-Foy, 1995).

[089] In some embodiments, the invention provides isolated nucleic acids that, when used as primers in a polymerase chain reaction, amplify a subject polynucleotide, or a polynucleotide containing a subject polynucleotide. The amplified polynucleotide is from about 20 to about 50, from about 50 to about 75, from about 75 to about 10O, from about 100 to about 125, from about 125 to about 150, from about 150 to about 175, from about 175 to about 200, from about 200 to about 250, from about 250 to about 300, from about 300 to about 350, from about 350 to about 400, from about 400 to about 500, from about 500 to about 600, from about 600 to about 700, from about 700 to about 800, from about 800 to about 900, from about 900 to about 1000, from about 1000 to about 2000, from about 2000 to about 3000, from about 3000 to about 4000, from about 4000 to about 5000, or from about 5000 to about 6000 nucleotides or more in length.

[090] The isolated nucleic acids themselves are from about 10 to about 20, from about 20 to about 30, from about 30 to about 40, from about 40 to about 50, from about 50 to about 100, or from about 100 to about 200 nucleotides in length. Generally, the nucleic acids are used in pairs in a polymerase chain reaction, where they are referred to as "forward" and "reverse" primers. [091 ] The subj ect nucleic acid compositions find use in a variety of different investigative applications. Applications of interest include identifying genomic DNA sequence using molecules of the invention, identifying homologs of molecules of the invention, creating a source of novel promoter elements, identifying expression regulatory factors, creating a source of probes and primers for hybridization applications, identifying expression patterns in biological specimens; preparing cell or animal models to investigate the function of the molecules of the invention, and preparing in vitro models to investigate the function of the molecules of the invention. [092] The isolated nucleic acids of the invention can be used as probes to detect and characterize gross alteration in a genomic locus, such as deletions, insertions, translocations, and duplications, e.g., by applying fluorescence in situ hybridization (FISH) techniques to examine chromosome spreads (Andreeff et al., 1999). These nucleic acids are also useful for detecting smaller genomic alterations, such as deletions, insertions, additions, translocations, and substitutions (e.g., SNPs). [093] When used as probes to detect nucleic acid molecules capable of hybridizing with nucleic acids described in the Sequence Listing, the nucleic acid molecules can be flanked by heterologous sequences of any length. When used as probes, a subject nucleic acid can include nucleotide analogs that incorporate labels that are directly detectable, such as radiolabels or fluorescent labels, or nucleotide analogs that incorporate labels that can be visualized in a subsequent reaction. Polypeptides [094] The invention provides novel polypeptides and related polypeptide compositions. Generally, a polypeptide of the invention refers to a polypeptide which has the amino acid sequence set forth in the Sequence Listing. The novel polypeptides of the invention include fragments thereof, and variants, as discussed in more detail below.

[095] In an embodiment, the invention provides an isolated polypeptide comprising an amino acid sequence, wherein the amino acid sequence is chosen from the Sequence Listing or the tables, or a biologically active fragment thereof, or is encoded by a polynucleotide sequence chosen from Sequence Listing or the tables, or a biologically active fragment thereof, such as, for example, any one of SEQ ID NOS: 15 - 30 or any one of SEQ ID NOS: 31 - 47 and 55 - 66.

[096] The proteins of the subject invention have been separated from their naturally occurring environment and are present in a non-naturally occurring environment. In 2005/070448 24 certain embodiments, the proteins are present in a composition where they are more concentrated than in their naturally occurring environment.

[097] The invention provides isolated polypeptides which are substantially free of the materials with which it is associated in nature or other polypeptide sequences that do not include a sequence or fragment of the subject polypeptides. By substantially free is meant that less than about 90%, less than about 80%, less than about 70%, less than about 60%, or less than about 50%> of the composition is made up of materials other than the isolated polypeptide. For example, the isolated polypeptide is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% free of the materials with which it is associated in nature. For example, an isolated polypeptide may be present in a composition wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of the total macromolecules (for example, polypeptides, fragments thereof, polynucleotides, fragments thereof, lipids, polysaccharides, and oligosaccharides) in the composition is the isolated polypeptide. Where at least about 99% of the total macromolecules is the isolated polypeptide, the polypeptide is at least about 99% pure, and the composition comprises less than about 1% contaminant.

[098] Polypeptides of the invention include conjugated proteins, fusion proteins, including, but not limited to, GST fusion proteins, fusion proteins with a heterologous amino acid sequences, fusion proteins with heterologous and homologous leader sequences, fusion proteins with or without N-terminal methionine residues, pegolyated proteins, and immunologically tagged proteins. Also included are variations of naturally occurring proteins, where such variations are homologous or substantially similar to the naturally occurring protein, as well as corresponding homologs from different species.

[099] Alterations of the native amino acid sequence may be accomplished by any of a number of known techniques. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site- specific mutagenesis procedures can be employed to provide an altered gene having 2005/070448 25 particular codons altered according to the substitution, deletion, or insertion required (Walder and Walder, 1986; Bauer et al., 1985; Craik, 1985; and U.S. Patent Nos. 4,518,584 and 4,737,462).

[0100] In some embodiments, a subject polypeptide is present as an oligomer, including homodimers, homotrimers, homotetramers, and multimers that include more than four monomeric units. Oligomers also include heteromultimers, e.g., heterodimers, heterotrimers, heterotetramers, etc. where the subject polypeptide is present in a complex with proteins other than the subject polypeptide. Where the multimer is a heteromultimer, the subject polypeptide can be present in a 1 : 1 ratio, a 1 :2 ratio, a 2: 1 ratio, or other ratio, with the other protein(s). [0101] Oligomers may be formed by disulfide bonds between cysteine residues on different polypeptides, or by non-covalent interactions between polypeptide chains, for example. In other embodiments, oligomers comprise from two to four polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the polypeptides. Such peptides may be peptide linkers (spacers), or peptides that have the property of promoting oligomerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of polypeptides attached thereto, as described in more detail below and in WO 94/10308.

[0102] Polypeptides of the invention can be obtained from naturally-occurring sources or produced synthetically. The sources of naturally occurring polypeptides will generally depend on the species from which the protein is to be derived, i.e., the proteins will be derived from biological sources that express the proteins. The subject proteins can also be derived from synthetic means, e.g., by expressing a recombinant gene encoding a protein of interest in a suitable system or host or enhancing endogenous expression, as described in more detail below. Further, small peptides can be synthesized in the laboratory by techniques well known in the art. [0103] Protein expression systems known in the art can produce fusion proteins that incorporate the polypeptides of the invention. The invention provides an isolated amino acid molecule with a first polypeptide comprising a molecule chosen from the Sequence Listing, or one or more of its biologically active fragments or variants, and a second molecule. This second molecule can facilitate production, secretion, and/or purification. It can confer a longer half-life to the first polypeptide when administered to an animal. Second molecules suitable for use in the invention include, for example, polyethylene glycol (PEG), human serum albumin, F_c, and/or one or more of their fragments. The invention can also provide a nucleic acid molecule with a second nucleotide sequence that encodes a fusion partner. This second nucleotide sequence can be operably linked to the first nucleotide sequence.

[0104] Thus, the invention provides polypeptide fusion partners. They may be part of a fusion molecule, e.g., a polynucleotide or polypeptide, which represents the joining of all of or portions of more than one gene. For example, a fusion protein can be the product obtained by splicing strands of recombinant DNA and expressing the hybrid gene. A fusion molecule can be made by genetic engineering, e.g., by removing the stop codon from the DNA sequence of a first protein, then appending the DNA sequence of a second protein in frame. The DNA sequence will then be expressed by a cell as a single protein. Typically this is accomplished by cloning a cDNA into an expression vector in frame with an existing gene. The invention provides fusion proteins with heterologous and homologous leader sequences, fusion proteins with a heterologous amino acid sequence, and fusion proteins with or without N-terminal methionine residues. The fusion partners of the invention can be either N-terminal fusion partners or C-terminal fusion partners.

[0105] As noted above, suitable fusion partners include, but are not limited to, albumin and F_c. These fusion partners can include any variant of or any fragment of such. Such modified polypeptides can show, e.g., enhanced activity or increased stability. In addition, they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.

[0106] Fusion polypeptides can be secreted from the cell by the incorporation of leader sequences that direct the protein to the membrane for secretion. These leader sequences can be specific to the host cell, and are known to skilled artisans; they are also cited in the references. The invention includes appropriate restriction enzyme sites for vector cloning. In addition to facilitating the secretion of these fusion proteins, the invention provides for facilitating their production. This can be accomplished in a number of ways, including producing multiple copies, employing strong promoters, and increasing their intracellular stability, e.g., by fusion with beta- galactosidase.

[0107] The invention also provides for facilitating the purification of these fusion proteins. Fusion with a selectable marker can facilitate purification by affinity chromatography. For example, fusion with the selectable marker glutathione S- transferase (GST) produces polypeptides that can be detected with antibodies directed against GST, and isolated by affinity chromatography on glutathione-sepharose; the GST marker can then be removed by thrombin cleavage. Polypeptides that provide for binding to metal ions are also suitable for affinity purification. For example, a fusion protein that incorporates His_n, where n is between three and ten, inclusive, e.g., a 6xHis-tag can be used to isolate a protein by affinity chromatography using a nickel ligand.

[0108] The fusion partners of the invention can also include linkers, i.e., fragments of synthetic DNA containing a restriction endonuclease recognition site that can be used for splicing genes. These can include polylinkers, which contain several restriction enzyme recognition sites. A linker may be part of a cloning vector. It may be located either upstream or downstream of the therapeutic protein, and it may be located either upstream or downstream of the fusion partner.

[0109] Gene manipulation techniques have enabled the development and use of recombinant therapeutic proteins with fusion partners that impart desirable pharmacokinetic properties. Recombinant human serum albumin fused with synthetic heme protein has been reported to reversibly carry oxygen (Chuang et al., 2002). The long half-life and stability of human serum albumin (HSA) makes it an attractive candidate for fusion to short-lived therapeutic proteins (U.S. Patent No. 6,686,179). [0110] For example, the short plasma half-life of unmodified interferon alpha makes frequent dosing necessary over an extended period of time, in order to treat viral and proliferative disorders. Interferon alpha fused with HSA has a longer half life and requires less frequent dosing than unmodified interferon alpha; the half-life was 18- fold longer and the clearance rate was approximately 140 times slower (Osborn et al., 2002). Interferon beta fused with HSA also has favorable pharmacokinetic properties; its half life was reported to be 36-40 hours, compared to 8 hours for unmodified interferon beta (Sung et al., 2003). A HSA-interleukin-2 fusion protein has been reported to have both a longer half-life and favorable biodistribution compared to unmodified interleukin-2. This fusion protein was observed to target tissues where lymphocytes reside to a greater extent than unmodified interleukin 2, suggesting that it exerts greater efficacy (Yao et al., 2004).

[0111] The Fc receptor of human immunoglobulin G subclass 1 has also been used as a fusion partner for a therapeutic molecule. It has been recombinantly linked to two soluble p75 tumor necrosis factor (TNF) receptor molecules. This fusion protein has been reported to have a longer circulating half-life than monomeric soluble receptors, and to inhibit TNFα-induced proinflammatory activity in the joints of patients with rheumatoid arthritis (Goldenberg, 1999). This fusion protein has been used clinically to treat rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis (Nanda and Bathon, 2004).

[0112] The peptides of the invention, including the fusion proteins, can be modified with or covalently coupled to one or more of a variety of hydrophilic polymers to increase their solubility and circulation half-life. Suitable nonproteinaceous hydrophilic polymers for coupling to a peptide include, but are not limited to, polyalkylethers as exemplified by polyethylene glycol and polypropylene glycol, polylactic acid, polyglycolic acid, polyoxyalkenes, polyvinylalcohol, polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran and dextran derivatives, etc. Generally, such hydrophilic polymers have an average molecular weight ranging from about 500 to about 100,000 daltons, from about 2,000 to about 40,000 daltons, or from about 5,000 to about 20,000 daltons. The peptide can be derivatized with or coupled to such polymers using any of the methods set forth in Zallipsky 1995; Monfardini et al., 1995; U.S. Pat. Nos. 4,791,192; 4,670,417; 4,640,835; 4,496,689; 4,301,144; 4,179,337 and WO 95/34326. [0113] Conjugating biomolecules with polyethylene glycol (PEG), a process known as pegylation, increases the circulating half-life of therapeutic proteins (Molineux, 2002). Polyethylene glycols are nontoxic water-soluble polymers that, owing to their large hydrodynamic volume, create a shield around the pegylated drug, thus protecting it from renal clearance, enzymatic degradation, and recognition by cells of the immune system.

[0114] Pegylated agents have improved pharmacokinetics that permit dosing schedules that are more convenient and more acceptable to patients. This improved pharmacokinetic profile may decrease adverse effects caused by the large variations in peak-to-trough plasma drug concentrations associated with frequent administration and by the immunogenicity of unmodified proteins (Harris et al., 2001). In addition, pegylated proteins may have reduced immunogenicity because PEG-induced steric hindrance can prevent immune recognition (Harris et al., 2001). [0115] Polypeptides of the invention can be isolated by any appropriate means known in the art. For example, convenient protein purification procedures can be employed (e.g., Deuthscher et al., 1990). In general, a lysate can be prepared from the original source, (e.g., a cell expressing endogenous polypeptide, or a cell comprising the expression vector expressing the polypeptide(s)), and purified using HPLC, exclusion chromatography, gel electrophoresis, or affinity chromatography, and the like. [0116] h another aspect, the invention provides a method of making a polypeptide of the invention by providing a nucleic acid molecule that comprises a polynucleotide sequence encoding a polypeptide of the invention, introducing the nucleic acid molecule into an expression system, and allowing the polypeptide to be produced. Briefly, the methods generally involve introducing a nucleic acid construct into a host cell in vitro and culturing the host cell under conditions suitable for expression, then harvesting the polypeptide, either from the culture medium or from the host cell, (e.g., by disrupting the host cell), or both, as described in detail above. The invention also provides methods of producing a polypeptide using cell-free in vitro transcription/translation methods, which are well known in the art, also as provided above. Antibodies [0117] The invention provides an antibody directed to a polypeptide of the Sequence Listing or encoded by a nucleic acid molecule of the Sequence Listing. The invention also provides an antibody specifically binding to and/or interfering with the biological activity of a polypeptide of the Sequence Listing or encoded by a nucleic acid molecule of the Sequence Listing.

[0118] This antibody may be a monoclonal antibody, a polyclonal antibody, a single chain antibody, an Fab fragment, an antibody comprising a backbone of a molecule with an Ig domain, a V_H fragment, a V_L fragment, a cdr fragment, and/or a framework fragment. It may also be a cytotoxic antibody, targeting antibody, an antibody agonist, an antibody antagonist, an antibody that promotes endocytosis of a target antigen, an antibody that mediates antibody dependent cell cytoxicity (ADCC), and/or an antibody that mediates cell-dependent cytotoxicity (CDC). [0119] An antibody of the invention can be a human antibody, a non-human primate antibody, a non-primate animal antibody, a rabbit antibody, a mouse antibody, a rat antibody, a sheep antibody, a goat antibody, a horse antibody, a porcine antibody, a cow antibody, a chicken antibody, a humanized antibody, a primatized antibody, and a chimeric antibody. These antibodies can comprise a cytotoxic antibody with one or more cytotoxic component chosen from a radioisotope, a microbial toxin, a plant toxin, and a chemical compound. The chemical compound can be chosen from doxorubicin and cisplatin.

[0120] In another aspect, the invention provides antibody targets. The polynucleotides and polypeptides of the invention comprise nucleic acid and amino acid sequences that can be recognized by antibodies. A target sequence can be any polynucleotide or amino acid sequence of approximately eighteen or more contiguous nucleotides or six or more amino acids. A variety of comparing means can be used to accomplish comparison of sequence information from a sample (e.g., to analyze target sequences, target motifs, or relative expression levels) with the data storage means. A skilled artisan can readily recognize that any one of the publicly available homology search programs can be used as the search means for the computer based systems of the present invention to accomplish comparison of target sequences and motifs. Computer programs to analyze expression levels in a sample and in controls are also known in the art. A target sequence includes an antibody target sequence, which refers to an amino acid sequence that can be used as an immunogen for injection into animals for production of antibodies or for screening against a phage display or antibody library for identification of binding partners.

[0121] The invention provides target structural motifs, or target motifs, i.e., any rationally selected sequences or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration formed upon the folding of the target motif, or on consensus sequences of regulatory or active sites. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, hairpin structures, promoter sequences, and other expression elements, such as binding sites for transcription factors. [0122] Antibodies of the invention bind specifically to their targets. The term binds specifically, in the context of antibody binding, refers to high avidity and/or high affinity binding of an antibody to a specific polypeptide, or more accurately, to an epitope of a specific polypeptide. Antibody binding to such epitope on a polypeptide can be stronger than binding of the same antibody to any other epitopes, particularly other epitopes that can be present in molecules in association with, or in the same sample as the polypeptide of interest. For example, when an antibody binds more strongly to one epitope than to another, adjusting the binding conditions can result in antibody binding almost exclusively to the specific epitope and not to any other epitopes on the same polypeptide, and not to any other polypeptide, which does not comprise the epitope. Antibodies that bind specifically to a subj ect polypeptide may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10% or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily discernible from the specific antibody binding to a subject polypeptide, e.g., by use of appropriate controls. In general, antibodies of the invention bind to a specific polypeptide with a binding affinity of 10^"7 M or greater (e.g., 10^'8 M, 10^"9 M, 10^**10 M, 10^"11 M, etc.).

[0123] The invention provides antibodies that can distinguish the variant sequences of the invention from cunently known sequences. These antibodies can distinguish polypeptides that differ by no more than one amino acid (U.S. Patent No. 6,656,467). They have high affinity constants, i.e., in the range of approximately 10^"10M, and are produced, for example, by genetically engineering appropriate antibody gene sequences, according to the method described by Young et al., in U.S. Patent No. 6,656,467.

[0124] Antibodies of the invention can be provided as matrices, i.e., as geometric networks of antibody molecules and their antigens, as found in immunoprecipitation and flocculation reactions. An antibody matrix can exist in solution or on a solid phase support.

[0125] Antibodies of the invention can be provided as a library of antibodies or fragments thereof, wherein at least one antibody or fragment thereof specifically binds to at least a portion of a polypeptide comprising an amino acid sequence according to any one of SEQ ID NOS.:15 - 24 and 26 - 30 or any one of SEQ ID NOS: 25, and 31 - 47, and/or wherein at least one antibody or fragment thereof interferes with at least one activity of such polypeptide or fragment thereof. In certain embodiments, the antibody library comprises at least one antibody or fragment thereof that specifically inhibits binding of a subject polypeptide to its ligand or substrate, or that specifically inhibits binding of a subject polypeptide as a substrate to another molecule. The present invention also features coreesponding polynucleotide libraries comprising at least one polynucleotide sequence that encodes an antibody or antibody fragment of the invention. In specific embodiments, the library is provided on a nucleic acid anay or in computer-readable format. [0126] The invention provides a method of making an antibody by introducing an antigen chosen from an isolated nucleic acid molecule comprising at least one polynucleotide sequence chosen from the Sequence Listing; sequences that hybridize to these sequences under high stringency conditions; sequences having at least 80% sequence identity to these sequences, or sequences that hybridize to them under high stringency conditions; complements of any of these sequences; or biologically active fragments of any of the above-listed sequencesor an isolated polypeptide comprising an amino acid sequence, wherein the amino acid sequence is chosen from the Sequence Listing, or a biologically active fragment thereof, or is encoded by a polynucleotide sequence chosen from the Sequence Listing, or a biologically active fragment thereof into an animal in an amount sufficient to elicit generation of antibodies specific to the antigen, and recovering the antibodies therefrom. [0127] Generally, the invention features a method of making an antibody by immunizing a host animal (Coligan, 2002). hi this method, a polypeptide or a fragment thereof, a polynucleotide encoding a polypeptide, or a polynucleotide encoding a fragment thereof, is introduced into an animal in a sufficient amount to elicit the generation of antibodies specific to the polypeptide or fragment thereof, and the resulting antibodies are recovered from the animal. Initial immunizations can be performed using either polynucleotides or polypeptides. Subsequent booster immunizations can also be performed with either polynucleotides or polypeptides. Initial immunization with a polynucleotide can be followed with either polynucleotide or polypeptide immunizations, and an initial immunization with a polypeptide can be followed with either polynucleotide or polypeptide immunizations. [0128] The host animal will generally be a different species than the immunogen, e.g., a human protein used to immunize mice. Methods of antibody production are well known in the art (Coligan, 2002; Howard and Bethell, 2000; Harlow et al., 1998; Harlow and Lane, 1988). The invention thus also provides a non-human animal comprising an antibody of the invention. The animal can be a non-human primate, (e.g., a monkey), a rodent (e.g., a rat, a mouse, a hamster, a guinea pig), a chicken, cattle (e.g., a sheep, a goat, a horse, a pig, a cow), a rabbit, a cat, or a dog. [0129] The present invention also features a method of making an antibody by isolating a spleen from an animal injected with a polypeptide or a fragment thereof, a polynucleotide encoding a polypeptide, or a polynucleotide encoding a fragment thereof, and recovering antibodies from the spleen cells. Hybridomas can be made from the spleen cells, and hybridomas secreting specific antibodies can be selected. [0130] The present invention further features a method of making a polynucleotide library from spleen cells, and selecting a cDNA clone that produces specific antibodies, or fragments thereof. The cDNA clone or a fragment thereof can be expressed in an expression system that allows production of the antibody or a fragment thereof, as provided herein.

[0131] The immunogen can comprise a nucleic acid, a complete protein, or fragments and derivatives thereof, or proteins expressed on cell surfaces. Proteins domains, e.g., extracellular, cytoplasmic, or luminal domains can be used as immunogens. Immunogens comprise all or a part of one of the subject proteins, where these amino acids contain post-translational modifications, such as glycosylation, found on the native target protein, hnmunogens comprising protein extracellular domains are produced in a variety of ways known in the art, e.g., expression of cloned genes using conventional recombinant methods, or isolation from tumor cell culture supernatants, etc. The immunogen can also be expressed in vivo from a polynucleotide encoding the immunogenic peptide introduced into the host animal.

[0132] Polyclonal antibodies are prepared by conventional techniques. These include immunizing the host animal in vivo with the target protein (or immunogen) in substantially pure form, for example, comprising less than about 1% contaminant. The immunogen can comprise the complete target protein, fragments, or derivatives thereof. To increase the immune response of the host animal, the target protein can be combined with an adjuvant; suitable adjuvants include alum, dextran, sulfate, large polymeric anions, and oil and water emulsions, e.g., Freund's adjuvant (complete or incomplete). The target protein can also be conjugated to synthetic carrier proteins or synthetic antigens. The target protein is administered to the host, usually intradermally, with an initial dosage followed by one or more, usually at least two, additional booster dosages. Following immunization, blood from the host is collected, followed by separation of the serum from blood cells. The immunoglobulin present in the resultant antiserum can be further fractionated using known methods, such as ammonium salt fractionation, or DEAE chromatography and the like. [0133] Cytokines can also be used to help stimulate immune response. Cytokines act as chemical messengers, recruiting immune cells that help the killer T-cells to the site of attack. An example of a cytokine is granulocyte-macrophage colony-stimulating factor (GM-CSF), which stimulates the proliferation of antigen-presenting cells, thus boosting an organism's response to a cancer vaccine. As with adjuvants, cytokines can be used in conjunction with the antibodies and vaccines disclosed herein. For example, they can be incorporated into the antigen-encoding plasmid or introduced via a separate plasmid, and in some embodiments, a viral vector can be engineered to display cytokines on its surface.

[0134] The method of producing polyclonal antibodies can be varied in some embodiments of the present invention. For example, instead of using a single substantially isolated polypeptide as an immunogen, one may inject a number of different immunogens into one animal for simultaneous production of a variety of antibodies. In addition to protein immunogens, the immunogens can be nucleic acids (e.g., in the form of plasmids or vectors) that encode the proteins, with facilitating agents, such as liposomes, microspheres, etc, or without such agents, such as "naked" DNA.

[0135] The invention provides a bacteriophage comprising an antibody specifically binding to and or interfering with the biological activity of an isolated nucleic acid molecule comprising at least one polynucleotide sequence of the Sequence Listing; sequences that hybridize to these sequences under high stringency conditions; sequences having at least 80%) sequence identity to the Sequence Listing or sequences that hybridize to them under high stringency conditions; complements of any of these sequences; or biologically active fragments of any of the above-listed sequences; or an isolated polypeptide comprising an amino acid sequence, wherein the amino acid sequence is chosen from the Sequence Listing, or a biologically active fragment thereof, or is encoded by a polynucleotide sequence chosen from Sequence Listing, or a biologically active fragment thereof; or a fragment of such an antibody. The invention further provides a bacterial cell comprising such a bacteriophage. It provides a recombinant host cell that produces such an antibody or a fragment of such an antibody.

[0136] In an embodiment, polyclonal antibodies can be prepared using phage display libraries, which are conventional in the art. hi this method, a collection of bacteriophages displaying antibody properties on their surfaces are made to contact subject polypeptides, or fragments thereof. Bacteriophages displaying antibody properties that specifically recognize the subject polypeptides are selected, amplified, for example, in E. coli, and harvested. Such a method typically produces single chain antibodies, which are further described below.

[0137] Phage display technology can be used to produce Fab antibody fragments, which can be then screened to select those with strong and/or specific binding to the protein targets. The screening can be performed using methods that are known to those of skill in the art, for example, ELISA, immunoblotting, immunohistochemistry, or immunoprecipitation. Fab fragments identified in this manner can be assembled with an Fc portion of an antibody molecule to form a complete immunoglobulin molecule.

[0138] Monoclonal antibodies are also produced by conventional techniques, such as fusing an antibody-producing plasma cell with an immortal cell to produce hybridomas. Suitable animals will be used, e.g., to raise antibodies against a mouse polypeptide of the invention, the host animal will generally be a hamster, guinea pig, goat, chicken, or rabbit, and the like. Generally, the spleen and/or lymph nodes of an immunized host animal provide the source of plasma cells, which are immortalized by fusion with myeloma cells to produce hybridoma cells. Culture supernatants from individual hybridomas are screened using standard techniques to identify clones producing antibodies with the desired specificity. The antibody can be purified from the hybridoma cell supernatants or from ascites fluid present in the host by conventional techniques, e.g., affinity chromatography using antigen, e.g., the subject protein, bound to an insoluble support, e.g., protein A sepharose, etc. [0139] The antibody can be produced as a single chain, instead of the normal multimeric structure of the immunoglobulin molecule. Single chain antibodies have been previously described (i.e., Jost et al., 1994). DNA sequences encoding parts of the immunoglobulin, for example, the variable region of the heavy chain and the variable region of the light chain are ligated to a spacer, such as one encoding at least about four small neutral amino acids, i.e., glycine or serine. The protein encoded by this fusion allows the assembly of a functional variable region that retains the specificity and affinity of the original antibody.

[0140] The invention also provides intrabodies that are intracellularly expressed single-chain antibody molecules designed to specifically bind and inactivate target molecules inside cells. Intrabodies have been used in cell assays and in whole organisms (Chen et al., 1994; Hassanzadeh et al, 1998). Inducible expression vectors can be constructed with intrabodies that react specifically with a protein of the invention. These vectors can be introduced into host cells and model organisms. [0141] The invention also provides "artificial" antibodies, e.g., antibodies and antibody fragments produced and selected in vitro, h some embodiments, these antibodies are displayed on the surface of a bacteriophage or other viral particle, as described above. In other embodiments, artificial antibodies are present as fusion proteins with a viral or bacteriophage structural protein, including, but not limited to, Ml 3 gene III protein. Methods of producing such artificial antibodies are well known in the art (U.S. Patent Nos. 5,516,637; 5,223,409; 5,658,727; 5,667,988; 5,498,538; 5,403,484; 5,571,698; and 5,625,033). The artificial antibodies, selected, for example, on the basis of phage binding to selected antigens, can be fused to a Fc fragment of an immunoglobulin for use as a therapeutic, as described, for example, in US 5,116,964 or WO 99/61630. Antibodies of the invention can be used to modulate biological activity of cells, either directly or indirectly. A subject antibody can modulate the activity of a target cell, with which it has primary interaction, or it can modulate the activity of other cells by exerting secondary effects, i.e., when the primary targets interact or communicate with other cells. The antibodies of the invention can be administered to mammals, and the present invention includes such administration, particularly for therapeutic and/or diagnostic purposes in humans. [0142] The antibodies can be partially human or fully human antibodies. For example, xenogenic antibodies, which are produced in animals that are transgenic for human antibody genes, can be employed to make a fully human antibody. By xenogenic human antibodies is meant antibodies that are fully human antibodies, with the exception that they are produced in a non-human host that has been genetically engineered to express human antibodies (e.g., WO 98/50433; WO 98/24893 and WO 99/53049).

[0143] Chimeric immunoglobulin genes constructed with immunoglobulin cDNA are known in the art (Liu et al. 1987a; Liu et al. 1987b). Messenger RNA is isolated from a hybridoma or other cell producing the antibody and used to produce cDNA. The cDNA of interest can be amplified by the polymerase chain reaction using specific primers (U.S. Patent Nos. 4,683,195 and 4,683,202). Alternatively, a library is made and screened to isolate the sequence of interest. The DNA sequence encoding the variable region of the antibody is then fused to human constant region sequences. The sequences of human constant (c) regions genes are known in the art (Kabat et al., 1991). Human C region genes are readily available from known clones. The choice of isotype will be guided by the desired effector functions, such as complement fixation, or antibody-dependent cellular cytotoxicity. IgGl, IgG3, and IgG4 isotypes, and either of the kappa or lambda human light chain constant regions can be used. The chimeric, humanized antibody is then expressed by conventional methods. [0144] Consensus sequences of heavy (H) and light (L) J regions can be used to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments. C region cDNA can be modified by site directed mutagenesis to place a restriction site at the analogous position in the human sequence.

[0145] A convenient expression vector for producing antibodies is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed, such as plasmids, retroviruses, YACs, or EBV derived episomes, and the like. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The resulting chimeric antibody can be joined to any strong promoter, including retroviral LTRs, e.g., SV-40 early promoter, (Okayama, et al. 1983), Rous sarcoma virus LTR (Gonnan et al. 1982), and Moloney murine leukemia virus LTR (Grosschedl et al. 1985), or native immunoglobulin promoters. [0146] Antibody fragments, such as Fv, F(ab')2, and Fab can be prepared by cleavage of the intact protein, e.g., by protease or chemical cleavage. These fragments can include heavy and light chain variable regions. Alternatively, a truncated gene can be designed, e.g., a chimeric gene encoding a portion of the F(ab') fragment that includes DNA sequences encoding the CHI domain and hinge region of the H chain, followed by a translational stop codon.

[0147] Antibodies may be administered by injection systemically, such as by intravenous injection; or by injection or application to the relevant site, such as by direct injection into a tumor, or direct application to the site when the site is exposed in surgery; or by topical application, such as if the disorder is on the skin, for example. [0148] For in vivo use, particularly for injection into humans, in some embodiments it is desirable to decrease the antigenicity of the antibody. An immune response of a recipient against the antibody may potentially decrease the period of time that the therapy is effective. Methods of humanizing antibodies are known in the art. The humanized antibody can be the product of an animal having transgenic human immunoglobulin genes, e.g., constant region genes (e.g., Grosveld and Kolias, 1992; Murphy and Carter, 1993; Pinkert, 1994; and International Patent Applications WO 90/10077 and WO 90/04036). Alternatively, the antibody of interest can be engineered by recombinant DNA techniques to substitute the CHI, CH2, CH3, hinge domains, and/or the framework domain with the conesponding human sequence (see, e.g., WO 92/02190). Humanized antibodies can also be produced by immunizing mice that make human antibodies, such as Abgenix xenomice, Medarex's mice, or Kirin's mice, and can be made using the technology of Protein Design Labs, Inc. (Fremont, CA) (Coligan, 2002). Both polyclonal and monoclonal antibodies made in non-human animals may be humanized before administration to human subjects. [0149] The antibodies of the present invention may be administered alone or in combination with other molecules for use as a therapeutic, for example, by linking the antibody to cytotoxic agent or radioactive molecule. Radioactive antibodies that are specific to a cancer cell, disease cell, or virus-infected cell may be able to deliver a sufficient dose of radioactivity to kill such cancer cell, disease cell, or virus-infected cell. The antibodies of the present invention can also be used in assays for detection of the subject polypeptides. In some embodiments, the assay is a binding assay that detects binding of a polypeptide with an antibody specific for the polypeptide; the subject polypeptide or antibody can be immobilized, while the subject polypeptide and/or antibody can be detectably-labeled. For example, the antibody can be directly labeled or detected with a labeled secondary antibody. That is, suitable, detectable labels for antibodies include direct labels, which label the antibody to the protein of interest, and indirect labels, which label an antibody that recognizes the antibody to the protein of interest.

[0150] These labels include radioisotopes, including, but not limited to ⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁴1, ¹²⁵1, ¹³¹1, ¹³⁷Cs, ¹⁸⁶Re, ²¹¹At, ²¹²Bi, ²¹³Bi, ²²³Ra, ²⁴¹Am, and ²⁴⁴Cm; enzymes having detectable products (e.g., luciferase, -galactosidase, and the like); fluorescers and fluorescent labels, e.g., as provided herein; fluorescence emitting metals, e.g.,

1 Eu, or others of the lanthamde senes, attached to the antibody through metal chelating groups such as EDTA; chemiluminescent compounds, e.g., luminol, isoluminol, or acridinium salts; and bioluminescent compounds, e.g., luciferin, or aequorin (green fluorescent protein), specific binding molecules, e.g., magnetic particles, microspheres, nanospheres, and the like.

[0151] Alternatively, specific-binding pairs may be used, involving, e.g., a second stage antibody or reagent that is detectably-labeled and that can amplify the signal. For example, a primary antibody can be conjugated to biotin, and horseradish peroxidase-conjugated strepavidin added as a second stage reagent. Digoxin and antidigoxin provide another such pair. In other embodiments, the secondary antibody can be conjugated to an enzyme such as peroxidase in combination with a substrate that undergoes a color change in the presence of the peroxidase. The absence or presence of antibody binding can be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, or scintillation counting. Such reagents and their methods of use are well known in the art. [0152] Nucleic acid, polypeptides, and antibodies of the invention can be provided in the form of anays, i.e., collections of plural biological molecules such as nucleic acids, polypeptides, or antibodies, having locatable addresses that may be separately detectable. Generally, a microarray encompasses use of sub microgram quantities of biological molecules. The biological molecules may be affixed to a substrate or may be in solution or suspension. The substrate can be porous or solid, planar or non- planar, unitary or distributed, such as a glass slide, a 96 well plate, with or without the use of microbeads or nanobeads. As such, the term "microarray" includes all of the devices refened to as microarrays in Schena, 1999; Bassett et al., 1999; Bowtell, 1999; Brown and Botstein, 1999; Chakravarti, 1999; Cheung et al., 1999; Cole et al, 1999; Collins, 1999; Debouck and Goodfellow, 1999; Duggan et al., 1999; Hacia, 1999; Lander, 1999; Lipshutz et al, 1999; Southern, et al., 1999; Schena, 2000; Brenner et al, 2000; Lander, 2001; Steinhaur et al, 2002; and Espejo et al, 2002. Nucleic acid microarrays include both oligonucleotide arrays (DNA chips) containing expressed sequence tags ("ESTs") and anays of larger DNA sequences representing a plurality of genes bound to the substrate, either one of which can be used for hybridization studies. Protein and antibody microanays include anays of polypeptides or proteins, including but not limited to, polypeptides or proteins obtained by purification, fusion proteins, and antibodies, and can be used for specific binding studies (Zhu and Snyder, 2003; Houseman et al., 2002; Schaeferling et al., 2002; Weng et al., 2002; Winssinger et al., 2002; Zhu et al., 2001; Zhu et al. 2001; and MacBeath and Schreiber, 2000).

[0153] All of the immunogenic methods of the invention can be used alone or in combination with other conventional or unconventional therapies. For example, immunogenic molecules can be combined with other molecules that have a variety of antiproliferative effects, or with additional substances that help stimulate the immune response, i.e., adjuvants or cytokines. Embryonic Stem Cells [0154] Embryonic stem cells can be pluripotent; they can differentiate into any of the cells present in the organism. When they divide in vivo, pluripotent stem cells can maintain their pluripotency while giving rise to differentiated progeny. Thus, stem cells can produce replicas of themselves which are pluripotent, and are also able to differentiate into lineage-restricted committed progenitor cells. Stem cells can also reproduce and differentiate in vitro. Embryonic stem cells have been directed to differentiate into cardiac muscle cells in vitro and, alternatively, into early progenitors of neural stem cells, and then into mature neurons and glial cells in vitro (Trounson, 2002).

[0155] Mouse embryonic stem cells are derived from the inner cell mass, the cells which give rise to the embryo. An inner cell mass of a donor mouse at the blastomere stage of development can be transfened into the embryo of a second mouse, and the donor mouse can contribute genes to every organ of the host embryo. Inner cell mass blastomeres can be isolated from the embryo and cultured in vitro to produce ES cell cultures and cell lines. ES cells retain their totipotency in vitro, and each of them can contribute to all the organs of a host embryo, e.g., following injection into the host embryo. ES cell cultures and cell lines can incorporate new DNA as transgenes. Embryonic stem cells can be transfonned with nucleic acids encoding a protein or a fragment of a protein, e.g., a secreted protein or an extracellular domain of a transmembrane protein.

[0156] The activity of the proteins or fragments thereof encoded can be assayed. In this aspect, the gene encoding the protein is expressed, and the modulation of the proliferation and/or differentiation of the stem cell transformed with the gene are observed. Changes a parameter associated with a disease, e.g., the rate of proliferation, the lack of proliferation, and/or differentiation of the genetically-altered stem cells can be compared with the wild type, non-genetically-altered stem cells. [0157] For example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown under culture conditions well known in the art. The vector is introduced into ES cells by transformation methods such as electroporation, liposome delivery, microinjection, and the like, which are well known in the art. The vector can contain genes for a secreted protein, for example, such as growth factors, cytokines, or hormones. The endogenous rodent gene is replaced by the disrupted disease gene through homologous recombination and integration during cell division. Then transformed ES cells are selected and used to study the proliferation and differentiation into various cell types. The differentiated embryonic stem cells can form various cell types and tissues in vitro, such as neural cells, hematopoietic lineages, and cardiomyocytes (Bain et al, 1995; Wiles and Keller, 1991; Klug et α ._; 1996).

[0158] The invention provides a modified embryonic stem cell with a stem cell of an animal and one or more introduced nucleic acid molecule encoding an sFRP-3 polypeptide, wherein the polypeptide comprises the amino acid sequence shown in SEQ ID NOS.: 15, and 20 - 24. The invention also provides an animal genetically modified to express one or more sFRP polypeptide, wherein the polypeptide comprises the amino acid sequence shown in SEQ ID NOS.: 15, and 20 - 24. The invention incorporates PCT/US/03/34811 by reference in its entirety. Screening and Diagnostic Methods Identifying Biological Molecules that Interact with a Polypeptide [0159] Formation of a binding complex between a subject polypeptide and an interacting polypeptide or other macromolecule (e.g., DNA, RNA, lipids, polysaccharides, and the like) can be detected using any known method. Suitable methods include: a yeast two-hybrid system (Zhu et al., 1997; Fields and Song, 1989; U.S. Pat. No. 5,283,173; Chien et al. 1991); a mammalian cell two-hybrid method; a fluorescence resonance energy transfer (FRET) assay; a bioluminescence resonance energy transfer (BRET) assay; a fluorescence quenching assay; a fluorescence anisotropy assay (Jameson and Sawyer, 1995); an immuno logical assay; and an assay involving binding of a detectably labeled protein to an immobilized protein. Detecting mRNA Levels and Monitoring Gene Expression [0160] The present invention provides methods for detecting the presence of sFPR-3 mRNA in a biological sample. The methods can be used, for example, to assess whether a test compound affects sFPR-3 gene expression, either directly or indirectly. The present invention provides diagnostic methods to compare the abundance of an sFPR-3 nucleic acid with that of a control value, either qualitatively or quantitatively, and to relate the value to a normal or abnormal expression pattern. [0161] Methods of measuring mRNA levels are known in the art, as described in for example, WO 97/27317. These methods generally comprise contacting a sample with a polynucleotide of the invention under conditions that allow hybridization and detecting hybridization, if any, as an indication of the presence of the polynucleotide of interest. Detection can be accomplished by any known method, including, but not limited to, in situ hybridization, PCR, RT-PCR, and "Northern" or RNA blotting, or combinations of such techniques, using a suitably labeled subject polynucleotide. A common method employed is use of microanays which can be purchased or customized, for example, through conventional vendors such as Affymetrix. Detecting and Monitoring Polypeptide Presence and Biological Activity [0162] The present invention provides methods for detecting the presence and/or biological activity of a subject polypeptide in a biological sample. The assay used will be appropriate to the biological activity of the particular polypeptide. Thus, e.g., where the biological activity is binding to a second macromolecule, the assay detects protein-protein binding, protein-DNA binding, protein-carbohydrate binding, or protein-lipid binding, as appropriate, using well known assays. Where the biological activity is signal transduction (e.g., transmission of a signal from outside the cell to inside the cell) or transport, an appropriate assay is used, such as measurement of intracellular calcium ion concentration, measurement of membrane conductance changes, or measurement of intracellular potassium ion concentration. [0163] The present invention also provides methods for detecting the presence or measuring the level of a normal or abnormal polypeptide in a biological sample using a specific antibody. The methods generally comprise contacting the sample with a specific antibody and detecting binding between the antibody and molecules of the sample. Specific antibody binding, when compared to a suitable control, is an indication that a polypeptide of interest is present in the sample. [0164] A variety of methods to detect specific antibody-antigen interactions are known in the art, e.g., standard immunohistological methods, immunoprecipitation, enzyme immunoassay, and radioimmunoassay. Briefly, antibodies are added to a cell sample, and incubated for a period of time sufficient to allow binding to the epitope, usually at least about 10 minutes. The antibody may be labeled with radioisotopes, enzymes, fluorescers, chemiluminescers, or other labels for direct detection. Alternatively, specific-binding pairs may be used, involving, e.g., a second stage antibody or reagent that is detectably-labeled, as described above. Such reagents and their methods of use are well known in the art Modulating mRNA and Peptides in Biological Samples [0165] The present invention provides screening methods for identifying agents that modulate the level of a mRNA molecule of the invention, agents that modulate the level of a polypeptide of the invention, and agents that modulate the biological activity of a polypeptide of the invention. In some embodiments, the assay is cell- free; in others, it is cell-based. Where the screening assay is a binding assay, one or more of the molecules can be joined to a label, where the label can directly or indirectly provide a detectable signal.

[0166] In these embodiments, the candidate agent is combined with a cell possessing a polynucleotide transcriptional regulatory element operably linked to a polypeptide- coding sequence of interest, e.g., a subject cDNA or its genomic component; and determining the agent's effect on polynucleotide expression, as measured, for example by the level of mRNA, polypeptide, or fusion polypeptide [0167] In other embodiments, for example, a recombinant vector can comprise an isolated polynucleotide transcriptional regulatory sequence, such as a promoter sequence, operably linked to a reporter gene (e.g., -galactosidase, CAT, luciferase, or other gene that can be easily assayed for expression). In these embodiments, the method for identifying an agent that modulates a level of expression of a polynucleotide in a cell comprises combining a candidate agent with a cell comprising a transcriptional regulatory element operably linked to a reporter gene; and determining the effect of said agent on reporter gene expression. [0168] Known methods of measuring mRNA levels can be used to identify agents that modulate mRNA levels, including, but not limited to, PCR with detectably- labeled primers. Similarly, agents that modulate polypeptide levels can be identified using standard methods for determining polypeptide levels, including, but not limited to an immunoassay such as ELISA with detectably-labeled antibodies. [0169] A wide variety of cell-based assays can also be used to identify agents that modulate eukaryotic or prokaryotic mRNA and/or polypeptide levels. Examples include transformed cells that over-express a cDNA construct and cells transformed with a polynucleotide of interest associated with an endogenously-associated promoter operably linked to a reporter gene. Expression levels are measured and compared in the test and control samples.

[0170] The present invention further provides methods of identifying agents that modulate a biological activity of sFRP-3 polypeptides. The method generally comprises contacting a test agent with a sample containing the subject polypeptide and assaying a biological activity of the subject polypeptide in the presence of the test agent. An increase or a decrease in the assayed biological activity in comparison to the activity in a suitable control (e.g., a sample comprising a subject polypeptide in the absence of the test agent) is an indication that the substance modulates a biological activity of the subject polypeptide. The mixture of components is added in any order that provides for the requisite interaction.

[0171] Accordingly, the present invention provides a method for identifying an agent, particularly a biologically active agent that modulates the level of expression of a nucleic acid in a cell, the method comprising: combining a candidate agent to be tested with a cell comprising a nucleic acid that encodes the polypeptide, and determining the agent's effect on polypeptide expression.

[0172] Agents that decrease a biological activity can find use in treating disorders associated with the biological activity of the molecule. Alternatively, some embodiments will detect agents that increase a biological activity. Agents that increase a biological activity of a molecule of the invention can find use in treating disorders associated with a deficiency in the biological activity. [0173] A variety of different candidate agents can be screened by the above methods. Candidate agents encompass numerous chemical classes, as described herein. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. Numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. For example, random peptide libraries obtained by yeast two-hybrid screens (Xu et al, 1997), phage libraries (Hoogenboom et al., 1998), or chemically generated libraries. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced, including antibodies produced upon immunization of an animal with subject polypeptides, or fragments thereof, or with the encoding polynucleotides. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and can be used to produce combinatorial libraries. Further, known pharmacological agents can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, and amidification, etc, to produce structural analogs. Kits [0174] The present invention provides methods for diagnosing disease states based on the detected presence and/or level of sFRP polynucleotides, polypeptides, or antibodies in a biological sample, and/or the detected presence and/or level of biological activity of the polynucleotide or polypeptide. These detection methods can be provided as part of a kit. Thus, the invention further provides kits for detecting the presence and/or a level of an sFRP-3 polynucleotide, polypeptide, or antibody in a biological sample and/or or the detected presence and/or level of biological activity of the sFRP-3 polynucleotide, polypeptide, or antibody.

[0175] Where the kit provides for polypeptide detection, it can include one or more specific antibodies. In some embodiments, the antibody specific to the sFRP-3 polypeptide is detectably labeled, hi other embodiments, the antibody specific to the polypeptide is not labeled; instead, a second, detectably-labeled antibody is provided that binds to the specific antibody. The kit may further include blocking reagents, buffers, and reagents for developing and/or detecting the detectable marker. The kit may further include instructions for use, controls, and interpretive information. [0176] The present invention provides for kits with unit doses of an active agent, hi some embodiments, the agent is provided in oral or injectable doses. Such kits will comprise containers containing the unit doses and an informational package insert describing the use and attendant benefits of the drugs in treating a condition of interest.

Therapeutic Compositions

[0177] The invention further provides agents identified using a screening assay of the invention, and compositions comprising the agents, subject polypeptides, subject polynucleotides, modulators thereof including antibodies, recombinant vectors, and/or host cells, including pharmaceutical compositions containing such in a pharmaceutically acceptable carrier or excipient for therapeutic administration. The subject compositions can be formulated using well-known reagents and methods. These compositions can include a buffer, which is selected according to the desired use of the agent, polypeptide, polynucleotide, recombinant vector, or host cell, and can also include other substances appropriate to the intended use. Those skilled in the art can readily select an appropriate buffer, a wide variety of which are known in the art, suitable for an intended use. Excipients and Formulations [0178] In some embodiments, sFRP-3 compositions are provided in formulation with pharmaceutically acceptable excipients, a wide variety of which are known in the art (Gennaro, 2000; Ansel et al., 2004; Kibbe et al., 2000). Pharmaceutically acceptable excipients, such as vehicles, adjuvants, earners or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

[0179] In pharmaceutical dosage forms, the compositions of the invention can be administered in the form of their pharmaceutically acceptable salts, or they can also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The subject compositions are formulated in accordance to the mode of potential administration. Administration of the agents can be achieved in various ways, including oral, buccal, nasal, rectal, parenteral, intraperitoneal, intradermal, transdermal, subcutaneous, intravenous, intra-arterial, intracardiac, intraventricular, intracranial, intratracheal, and intrathecal administration, etc., or otherwise by implantation or inhalation. Thus, the subject compositions can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. The following methods and excipients are merely exemplary and are in no way limiting.

[0180] Compositions for oral administration can form solutions, suspensions, tablets, pills, granules, capsules, sustained release formulations, oral rinses, or powders. For oral preparations, the agents, polynucleotides, and polypeptides can be used alone or in combination with appropriate additives, for example, with conventional additives, such as lactose, mannitol, com starch, or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatins; with disintegrators, such as com starch, potato starch, or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives, and flavoring agents. [0181] Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle can contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art (Gennaro, 2003). The composition or fonnulation to be administered will, in any event, contain a quantity of the agent adequate to achieve the desired state in the subject being treated. [0182] The agents, polynucleotides, and polypeptides can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. Other fonnulations for oral or parenteral delivery can also be used, as conventional in the art [0183] The antibodies, agents, polynucleotides, and polypeptides can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. Further, the agent, polynucleotides, or polypeptide composition may be converted to powder form for administration intranasally or by inhalation, as conventional in the art. [0184] Furthermore, the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature. [0185] A polynucleotide, polypeptide, or other modulator, can also be introduced into tissues or host cells by other routes, such as viral infection, microinjection, or vesicle fusion. For example, expression vectors can be used to introduce nucleic acid compositions into a cell as described above. Further, jet injection can be used for intramuscular administration (Furth et al., 1992). The DNA can be coated onto gold microparticles, and delivered intradermally by a particle bombardment device, or "gene gun" as described in the literature (Tang et al., 1992), where gold microprojectiles are coated with the DNA, then bombarded into skin cells. [0186] Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions can be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet, or suppository, contains a predetermined amount of the composition containing one or more agents. Similarly, unit dosage forms for injection or intravenous administration can comprise the agent(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier. Active Agents (or Modulators) [0187] The nucleic acid, polypeptide, and modulator compositions of the subject invention find use as therapeutic agents in situations where one wishes to modulate an activity of a subject polypeptide in a host, particularly the activity of the subject polypeptides, or to provide or inhibit the activity at a particular anatomical site. Thus, the compositions are useful in treating disorders associated with an activity of a subject polypeptide. The following provides further details of active agents of the present invention. Antisense Oligonucleotides [0188] In certain embodiments of the invention, the active agent is an agent that modulates, and generally decreases or down regulates, the expression of sFRP-3 in a host, i.e., antisense molecules. Anti-sense reagents include antisense oligonucleotides (ODN), i.e., synthetic ODN having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA. The antisense sequence is complementary to the mRNA of the targeted gene, and inhibits expression of the targeted gene products. Antisense molecules inhibit gene expression through various mechanisms, e.g., by reducing the amount of mRNA available for translation, through activation of RNase H, or steric hindrance. One or a combination of antisense molecules can be administered, where a combination can comprise multiple different sequences.

[0189] Antisense molecules can be produced by expression of all or a part of the target gene sequence in an appropriate vector, where the transcriptional initiation is oriented such that an antisense strand is produced as an RNA molecule. Alternatively, the antisense molecule is a synthetic oligonucleotide. Antisense oligonucleotides can be chemically synthesized by methods known in the art (Wagner et al., 1993; Milligan et al., 1993). Antisense oligonucleotides will generally be at least about 7, at least about 12, or at least about 20 nucleotides in length, and not more than about 500, not more than about 50, or not more than about 35 nucleotides in length, where the length is governed b;49;fficiency of inhibition, and specificity, including absence of cross-reactivity, and the like. Short oligonucleotides, of from about 7 to about 8 bases in length, can be strong and selective inhibitors of gene expression (Wagner et al., 1996).

[0190] As an alternative to anti-sense inhibitors, catalytic nucleic acid compounds, e.g., ribozymes, or anti-sense conjugates can be used to inhibit gene expression. Ribozymes can be synthesized in vitro and administered to the patient, or can be encoded in an expression vector, from which the ribozyme is synthesized in the targeted cell (WO 9523225; Beigelman et al., 1995). Examples of oligonucleotides with catalytic activity are described in WO 9506764. Conjugates of anti-sense ODN with a metal complex, e.g., terpyridyl Cu(II), capable of mediating mRNA hydrolysis are described in Bashkin et al., 1995. Interfering RNA [0191] h some embodiments, the active agent is an interfering RNA (RNAi), including dsRNAi. RNA interference provides a method of silencing eukaryotic genes. Use of RNAi to reduce a level of a particular mRNA and/or protein is based on the interfering properties of double-stranded RNA derived from the coding regions of a gene. The technique is an efficient high-throughput method for disrupting gene function (O'Neil, 2001). RNAi can also help identify the biochemical mode of action of a drug and to identify other genes encoding products that can respond or interact with specific compounds.

[0192] In one embodiment of the invention, complementary sense and antisense RNAs derived from a substantial portion of the subject polynucleotide are synthesized in vitro. The resulting sense and antisense RNAs are annealed in an injection buffer, and the double-stranded RNA injected or otherwise introduced into the subject, i.e., in food or by immersion in buffer containing the RNA (Gaudilliere et al., 2002; O'Neil et al., 2001; WO99/32619). In another embodiment, dsRNA derived from a gene of the present invention is generated in vivo by simultaneously expressing both sense and antisense RNA from appropriately positioned promoters operably linked to coding sequences in both sense and antisense orientations. Peptides and Modified Peptides [0193] In some embodiments of the present invention, the active agent is a peptide. Suitable peptides include peptides of from about 5 amino acids to about 50, from about 6 to about 30, or from about 10 to about 20 amino acids in length. In some embodiments, a peptide has a sequence of from about 7 amino acids to about 45, from about 9 to about 35, or from about 12 to about 25 amino acids of conesponding naturally-occurring protein. In some embodiments, a peptide exhibits one or more of the following activities: inhibits binding of a subject polypeptide to an interacting protein or other molecule; inhibits subject polypeptide binding to a second polypeptide molecule; inhibits a signal transduction activity of a subject polypeptide; inhibits an enzymatic activity of a subject polypeptide; or inhibits a DNA binding activity of a subject polypeptide.

[0194] Peptides can include naturally-occurring and non-naturally occurring amino acids. Peptides can comprise D-amino acids, a combination of D- and L- amino acids, and various "designer" amino acids (e.g., /3-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids, etc.) to convey special properties. Additionally, peptides can be cyclic. Peptides can include non-classical amino acids in order to introduce particular conformational motifs. Any known non-classical amino acid can be used. Non-classical amino acids include, but are not limited to, 1,2,3,4- tetrahydroisoquinoline-3-carboxylate; (2S,3S)-methylphenylalanine, (2S,3R)-methyl- phenylalanine, (2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine; 2- aminotetrahydronaphthalene-2-carboxylic acid; hydroxy- 1,2,3,4- tetrahydroisoquinoline-3-carboxylate; /3-carboline (D and L); HIC (histidine isoquinoline carboxylic acid); and HIC (histidine cyclic urea). Amino acid analogs and peptidomimetics can be incorporated into a peptide to induce or favor specific secondary structures, including, but not limited to, LL-Acp (LL-3-amino-2- propenidone-6-carboxylic acid), a /3-turn inducing dipeptide analog; /3-sheet inducing analogs; /3-turn inducing analogs; cu-helix inducing analogs; γ-turn inducing analogs; Gly-Ala turn analogs; amide bond isostere; or tretrazol, and the like. [0195] In addition to the foregoing N-tem inal and C-terminal modifications, a peptide or peptidomimetic can be modified with or covalently coupled to one or more of a variety of hydrophilic polymers to increase solubility and circulation half-life of the peptide. Suitable nonproteinaceous hydrophilic polymers for coupling to a peptide include, but are not limited to, polyalkylethers as exemplified by polyethylene glycol and polypropylene glycol, polylactic acid, polyglycolic acid, polyoxyalkenes, polyvinylalcohol, polyvinylpynolidone, cellulose and cellulose derivatives, dextran, and dextran derivatives. Generally, such hydrophilic polymers have an average molecular weight ranging from about 500 to about 100,000 daltons, from about 2,000 to about 40,000 daltons, or from about 5,000 to about 20,000 daltons. The peptide can be derivatized with or coupled to such polymers using any of the methods set forth in Zallipsky, (1995); Monfardini et al., (1995); U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; 4,179,337, or WO 95/34326. Peptide Aptamers [0196] Another suitable agent for modulating an activity of a subject polypeptide is a peptide aptamer. Peptide aptamers are peptides or small polypeptides that act as dominant inhibitors of protein function. Peptide aptamers specifically bind to target proteins, blocking their functional ability (Kolonin and Finley, 1998). Due to the highly selective nature of peptide aptamers, they can be used not only to target a specific protein, but also to target specific functions of a given protein (e.g., a signaling function). Further, peptide aptamers can be expressed in a controlled fashion by use of promoters which regulate expression in a temporal, spatial or inducible manner. Peptide aptamers act dominantly, therefore, they can be used to analyze proteins for which loss-of-function mutants are not available. [0197] Peptide aptamers that bind with high affinity and specificity to a target protein can be isolated by a variety of techniques known in the art. Peptide aptamers can be isolated from random peptide libraries by yeast two-hybrid screens (Xu et al., 1997). They can also be isolated from phage libraries (Hoogenboom et al., 1998) or chemically generated peptides/libraries. Therapeutic Applications: Methods of Use

[0198] Surprisingly, the novel sFRP3 splice variant is expressed differently in tumor tissues than the other known sFRP3 polypeptides, making this polypeptide and its modulators additionally useful for therapeutic applications. [0199] The instant invention provides various therapeutic methods. In some embodiments, methods of modulating, including increasing and inhibiting, a biological activity of a subject protein are provided. In other embodiments, methods of modulating a signal transduction activity of a subject protein are provided. In further embodiments, methods of modulating interaction of a subject protein with another, interacting protein or other macromolecule (e.g., DNA, carbohydrate, lipid), are provided.

[0200] Thus, in an embodiment, the therapeutic compositions herein are administered to subjects for treatment of a proliferative disease such as a tumor or psoriasis. In another embodiment, the therapeutic compositions herein are administered to subjects for modulation of immune related diseases or infections. J-n a further embodiment, the therapeutic compositions herein are administered to subjects for modulation of apoptosis-related diseases. Such compositions are administered either locally or systemically, for example, intranasally or by inhalation, by intravenous, intramusclular, subcutaneous, intrathecal, intraventricular, or intraperitoneal administration.

[0201] As mentioned above, an effective amount of the active agent (e.g., small molecule, antibody specific for a subject polypeptide, a subject polypeptide, or a subject polynucleotide) is administered to the host, where "effective amount" means a dosage sufficient to produce a desired effect or result. In some embodiments, the desired result is at least a reduction in a given biological activity of a subject polypeptide as compared to a control, for example, a decreased level of expression or activity of the subject protein in the individual, or in a localized anatomical site in the individual. In further embodiments, the desired result is at least an increase in a biological activity of a subject polypeptide as compared to a control. [0202] The agents can be provided in unit dosage forms, i.e., physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0203] An effective amount of the active is administered to the host at a dosage sufficient to produce a desired result. In some embodiments, the desired result is at least a reduction in a given biological activity of a subject polypeptide as compared to a control. In other embodiments, the desired result is an increase in the level of the active subject polypeptide (in the individual, or in a localized anatomical site in the individual), as compared to a control. In some embodiments, the desired result is at least a reduction in enzymatic activity of a subject polypeptide as compared to a control. In other embodiments, the desired result is an increase in the level of enzymatically active subject polypeptide (in the individual, or in a localized anatomical site in the individual), as compared to a control. In still other embodiments, the desired result is a decrease in ischemic cardiac injury as compared to a control. A decrease in ischemic cardiac injury may be indicated by a variety of indicia known in the art or described herein.

[0204] Typically, the compositions of the instant invention will contain from less than 1% to about 95%o of the active ingredient, in some embodiments, about 10%o to about 50%. Generally, between about 100 mg and 500 mg of the compositions will be administered to a child and between about 500 mg and 5 grams will be administered to an adult. Administration is generally by injection and often by injection to a localized area. The frequency of administration will be determined by the care given based on patient responsiveness. Other effective dosages can be readily determined by one of ordinary skill in the art through trials establishing dose response curves. [02O5] In order to calculate the amount of therapeutic agent to be administered, those skilled in the art could use readily available information with respect to the amount of agent necessary to have the desired effect. The amount of an agent necessary to increase a level of active subject polypeptide can be calculated from in vitro experimentation. The amount of agent will, of course, vary depending upon the particular agent used.

[0206] Typically, the compositions of the instant invention will contain from less than about 1% to about 99% of the active ingredient, about 10%> to about 90%>, or 20%> to about 80%), or 30% to about 70%, or 40% to about 60%, or about 50%. Generally, between about 100 mg and about 500 mg will be administered to a child and between about 500 mg and about 5 grams will be administered to an adult. [0207] Other effective dosages can be readily determined by one of ordinary skill in the art through routine trials establishing dose response curves, for example, the amount of agent necessary to increase a level of active subject polypeptide can be calculated from in vitro experimentation. Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms, and the susceptibility of the subject to side effects, and preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means. For example, in order to calculate the polypeptide, polynucleotide, or modulator dose, those skilled in the art can use readily available information with respect to the amount necessary to have the desired effect, depending upon the particular agent used.

[0208] The active agent(s) can be administered to the host via any convenient means capable of resulting in the desired result. Administration is generally by injection and often by injection to a localized area. The frequency of administration will be determined by the care given based on patient responsiveness. For example, the agents may be administered daily, weekly, or as conventionally determined appropriate.

[0209] A variety of hosts are treatable according to the subject methods. The host, or patient, may be from any animal species, and will generally be mammalian, e.g., primate sp., e.g., monkeys, chimpanzees, and particularly humans; rodents, including mice, rats and hamsters, guinea pig; rabbits; cattle, including equines, bovines, pig, sheep, goat, canines; felines; etc. Animal models are of interest for experimental investigations, providing a model for treatment of human disease. Proliferative Conditions [0210] In some embodiments, a protein of the present invention is involved in the control of cell proliferation, and an agent of the invention inhibits undesirable cell proliferation. Such agents are useful for treating disorders that involve abnormal cell proliferation, including, but not limited to, cancer, psoriasis, and scleroderma. Whether a particular agent and/or therapeutic regimen of the invention is effective in reducing unwanted cellular proliferation, e.g., in the context of treating cancer, can be determined using standard methods.

[0211] The therapeutic compositions and methods of the invention can be used in the treatment of cancer, i.e., an abnormal malignant cell or tissue growth, e.g., a tumor. In an embodiment, the compositions and methods of the invention kill tumor cells. In an embodiment, they inhibit tumor development. Cancer is characterized by the proliferation of abnormal cells that tend to invade the surrounding tissue and metastasize to new body sites. The growth of cancer cells exceeds that of and is uncoordinated with the normal cells and tissues. In an embodiment, the compositions and methods of the invention inhibit the progression of premalignant lesions to malignant tumors.

[0212] Cancer encompasses carcinomas, which are cancers of epithelial cells, and are the most common forms of human cancer; carcinomas include squamous cell carcinoma, adenocarcinoma, melanomas, and hepatomas. Cancer also encompasses sarcomas, which are tumors of mesenchymal origin, and includes osteo genie sarcomas, leukemias, and lymphomas. Cancers can have one or more than one neoplastic cell type. Some characteristics that can, in some instances, apply to cancer cells are that they are morphologically different from normal cells, and may appear anaplastic; they have a decreased sensitivity to contact inhibition, and may be less likely than normal cells to stop moving when sunounded by other cells; and they have lost their dependence on anchorage for cell growth, and may continue to divide in liquid or semisolid surroundings, whereas normal cells must be attached to a solid surface to grow.

[0213] Treatment herein refers to obtaining a desired pharmacologic and/or physiologic effect, covering any treatment of a pathological condition or disorder in a mammal, including a human. The effect may be prophylactic in tenns of completely or partially preventing a disorder or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse affect attributable to the disorder. That is, "treatment" includes (1) preventing the disorder from occurring or recurring in a subject who may be predisposed to the disorder but has not yet been diagnosed as having it, (2) inl ibiting the disorder, such as anesting its development, (3) stopping or terminating the disorder or at least symptoms associated therewith, so that the host no longer suffers from the disorder or its symptoms, such as causing regression of the disorder or its symptoms, for example, by restoring or repairing a lost, missing or defective function, or stimulating an inefficient process, or (4) relieving, alleviating, or ameliorating the disorder, or symptoms associated therewith, where ameliorating is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, such as inflammation, pain, and/or tumor size. [0214] The polynucleotides, polypeptides, and antibodies described above can be used to treat cancer. In an embodiment, a fusion protein or conjugate can additionally comprise a rumor-targeting moiety. Suitable moieties include those that enhance delivery of an therapeutic molecule to a tumor. For example, compounds that selectively bind to cancer cells compared to normal cells, selectively bind to tumor vasculature, selectively bind to the tumor type undergoing treatment, or enhance penetration into a solid tumor are included in the invention. Tumor targeting moieties of the invention can be peptides. Nucleic acid and amino acid molecules of the invention can be used alone or as an adjunct to cancer treatment. For example, a nucleic acid or amino acid molecules of the invention may be added to a standard chemotherapy regimen. It may be combined with one or more of the wide variety of drugs that have been employed in cancer treatment, including, but are not limited to, cisplatin, taxol, etoposide, Novantrone (mitoxantrone), actinomycin D, camptohecin (or water soluble derivatives thereof), methotrexate, mitomycins (e.g., mitomycin C), dacarbazine (DTIC), and anti-neoplastic antibiotics such as doxorubicin and daunomycin.

[0215] Drugs employed in cancer therapy may have a cytotoxic or cytostatic effect on cancer cells, or may reduce proliferation of the malignant cells. Drugs employed in cancer treatment can also be peptides. A nucleic acid or amino acid molecules of the invention can be combined with radiation therapy. A nucleic acid or amino acid molecules of the invention may be used adjunctively with therapeutic approaches described in De Vita et al., 2001. For those combinations in which a nucleic acid or amino acid molecule of the invention and a second anti-cancer agent exert a synergistic effect against cancer cells, the dosage of the second agent maybe reduced, compared to the standard dosage of the second agent when administered alone. A method for increasing the sensitivity of cancer cells comprises co-administering a nucleic acid or amino acid molecule of the invention with an amount of a chemotherapeutic anti-cancer drug that is effective in enhancing sensitivity of cancer cells. Co-administration may be simultaneous or non-simultaneous administration. A nucleic acid or amino acid molecule of the invention may be administered along with other therapeutic agents, during the course of a treatment regimen. In one embodiment, administration of a nucleic acid or amino acid molecule of the invention and other therapeutic agents is sequential. An appropriate time course may be chosen by the physician, according to such factors as the nature of a patient's illness, and the patient's condition.

[0216] The invention also provides a method for prophylactic or therapeutic treatment of a subject needing or desiring such treatment by providing a vaccine, that can be administered to the subject. The vaccine may comprise one or more of a polynucleotide, polypeptide, or modulator of the invention, for example an antibody vaccine composition, a polypeptide vaccine composition, or a polynucleotide vaccine composition, useful for treating cancer, proliferative, inflammatory, immune, metabolic, bacterial, or viral disorders.

[0217] For example, the vaccine can be a cancer vaccine, and the polypeptide can concomitantly be a cancer antigen. The vaccine may be an anti-inflammatory vaccine, and the polypeptide can concomitantly be an inflammation-related antigen. The vaccine may be a viral vaccine, and the polypeptide can concomitantly be a viral antigen. In some embodiments, the vaccine comprises a polypeptide fragment, comprising at least one extracellular fragment of a polypeptide of the invention, and/or at least one extracellular fragment of a polypeptide of the invention minus the signal peptide, for the treatment, for example, of proliferative disorders, such as cancer. In certain embodiments, the vaccine comprises a polynucleotide encoding one or more such fragments, administered for the treatment, for example, of proliferative disorders, such as cancer. Further, the vaccine can be administered with or without an adjuvant.

[0218] Vaccine therapy involves the use of polynucleotides, polypeptides, or agents of the invention as immunogens for tumor antigens (Machiels et al., 2002). For example, pep tide-based vaccines of the invention include unmodified subject polypeptides, fragments thereof, and MHC class I and class Il-restricted peptide (Knutson et al., 2001), comprising, for example, the disclosed sequences with universal, nonspecific MHC class Il-restricted epitopes. Peptide-based vaccines comprising a tumor antigen can be given directly, either alone or in conjunction with other molecules. The vaccines can also be delivered orally by producing the antigens in transgenic plants that can be subsequently ingested (U.S. Patent No. 6,395,964). [0219] In some embodiments, antibodies themselves can be used as antigens in anti- idiotype vaccines. That is, administering an antibody to a tumor antigen stimulates B cells to make antibodies to that antibody, which in turn recognize the tumor cells [0220] Nucleic acid-based vaccines can deliver tumor antigens as polynucleotide constructs encoding the antigen. Vaccines comprising genetic material, such as DNA or RNA, can be given directly, either alone or in conjunction with other molecules. Administration of a vaccine expressing a molecule of the invention, e.g., as plasmid DNA, leads to persistent expression and release of the therapeutic immunogen over a period of time, helping to control unwanted tumor growth.

[0221] In some embodiments, nucleic acid-based vaccines encode subject antibodies. In such embodiments, the vaccines (e.g., DNA vaccines) can include post- transcriptional regulatory elements, such as the post-transcriptional regulatory acting RNA element (WPRE) derived from Woodchuck Hepatitis Virus. These post- transcriptional regulatory elements can be used to target the antibody, or a fusion protein comprising the antibody and a co-stimulatory molecule, to the tumor microenvironment (Peril et al., 2003).

[0222] Besides stimulating anti-tumor immune responses by inducing humoral responses, vaccines of the invention can also induce cellular responses, including stimulating T-cells that recognize and kill tumor cells directly. For example, nucleotide-based vaccines of the invention encoding tumor antigens can be used to activate the CD 8 cytotoxic T lymphocyte arm of the immune system. [0223] In some embodiments, the vaccines activate T-cells directly, and in others they enlist antigen-presenting cells to activate T-cells. Killer T-cells are primed, in part, by interacting with antigen-presenting cells, i.e., dendritic cells. In some embodiments, plasmids comprising the nucleic acid molecules of the invention enter antigen- presenting cells, which in turn display the encoded tumor-antigens that contribute to killer T-cell activation. Again, the tumor antigens can be delivered as plasmid DNA constructs, either alone or with other molecules.

[0224] In further embodiments, RNA can be used. For example, dendritic cells can be transfected with RNA encoding tumor antigens (Heiser et al., 2002; Mitchell and Nair, 2000). This approach overcomes the limitations of obtaining sufficient quantities of tumor material, extending therapy to patients otherwise excluded from clinical trials. For example, a subject RNA molecule isolated from tumors can be amplified using RT-PCR. h some embodiments, the RNA molecule of the invention is directly isolated from tumors and transfected into dendritic cells with no intervening cloning steps.

[0225] In some embodiments the molecules of the invention are altered such that the peptide antigens are more highly antigenic than in their native state. These embodiments address the need in the art to overcome the poor in vivo immunogenicity of most tumor antigens by enhancing tumor antigen immunogenicity via modification of epitope sequences (Yu and Restifo, 2002).

[0226] Another recognized problem of cancer vaccines is the presence of preexisting neutralizing antibodies. Some embodiments of the present invention overcome this problem by using viral vectors from non-mammalian natural hosts, i.e., avian pox viruses. Alternative embodiments that also circumvent preexisting neutralizing antibodies include genetically engineered influenza viruses, and the use of "naked" plasmid DNA vaccines that contain DNA with no associated protein. (Yu and Restifo, 2002).

[0227] All of the immunogenic methods of the invention can be used alone or in combination with other conventional or unconventional therapies. For example, immunogenic molecules can be combined with other molecules that have a variety of antiproliferative effects, or with additional substances that help stimulate the immune response, i.e., adjuvants or cytokines. [0228] For example, in some embodiments, nucleic acid vaccines encode an alphaviral replicase enzyme, in addition to tumor antigens. This recently discovered approach to vaccine therapy successfully combines therapeutic antigen production with the induction of the apoptotic death of the tumor cell (Yu and Restifo, 2002). [0229] In some embodiments, a protein of the present invention is involved in the control of cell proliferation, and an agent of the invention inhibits undesirable cell proliferation. Such agents are useful for treating disorders that involve abnormal cell proliferation, including, but not limited to, cancer, psoriasis, and scleroderma. Whether a particular agent and/or therapeutic regimen of the invention is effective in reducing unwanted cellular proliferation, e.g., in the context of treating cancer, can be determined using standard methods. For example, the number of cancer cells in a biological sample (e.g., blood, a biopsy sample, and the like), can be determined. The tumor mass can be determined using standard radiological or biochemical methods. [0230] The polynucleotides, polypeptides, and modulators of the present invention find use in immunotherapy of hyperproliferative disorders, including cancer, neoplastic, and paraneoplastic disorders. That is, the subject molecules can conespond to tumor antigens, of which 1770 have been identified to date (Yu and Restifo, 2O02). Immunotherapeutic approaches include passive immunotherapy and vaccine therapy and can accomplish both generic and antigen-specific cancer immunotherapy.

[0231] Passive immunity approaches involve antibodies of the invention that are directed toward specific tumor-associated antigens. Such antibodies can eradicate systemic tumors at multiple sites, without eradicating normal cells. In some embodiments, the antibodies are combined with radioactive components, as provided above, for example, combining the antibody's ability to specifically target tumors with the added lethality of the radioisotope to the tumor DNA. [0232] Useful antibodies comprise a discrete epitope or a combination of nested epitopes, i.e., a 10-mer epitope and associated peptide multimers incorporating all potential S-mers and 9-mers, or overlapping epitopes (Dutoit et al., 2002). Thus a single antibody can interact with one or more epitopes. Further, the antibody can be used alone or in combination with different antibodies, that all recognize either a single or multiple epitopes.

[0233] Neutralizing antibodies can provide therapy for cancer and proliferative disorders. Neutralizing antibodies that specifically recognize a protein or peptide of the invention can bind to the protein or peptide, e.g., in a bodily fluid or the extracellular space, thereby modulating the biological activity of the protein or peptide. For example, neutralizing antibodies specific for proteins or peptides that play a role in stimulating the growth of cancer cells can be useful in modulating the growth of cancer cells. Similarly, neutralizing antibodies specific for proteins or peptides that play a role in the differentiation of cancer cells can be useful in modulating the differentiation of cancer cells. Inflammation and Immunity [0234] In other embodiments, e.g., where the subject polypeptide is involved in modulating inflammation or immune function, the invention provides agents for treating such inflammation or immune disorders. For example, neutralizing antibodies can provide immunosuppressive therapy for inflammatory and autoimmune disorders. Neutralizing antibodies can be used to treat disorders such as, for example, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, transplant rejection, and psoriasis. Neutralizing antibodies that specifically recognize a protein or peptide of the invention can bind to the protein or peptide, e.g., in a bodily fluid or the extracellular space, thereby modulating the biological activity of the protein or peptide. For example, neutralizing antibodies specific for proteins or peptides that play a role in activating immune cells are useful as immunosuppressants. Chondrogenesis and Bone Disease [0235] Secreted FRPs, including sFRP-3, have been reported to be expressed in developing bone osteoblasts, where they regulate Wnt signaling, thus regulating bone formation during growth and development (Westendorf et al., 2004). Secreted FRP-3 has been shown to regulate both the proliferation and the differentiation of developing bone (Chung et al., 2004; Westendorf et al, 2004).

[0236] Accordingly, the invention provides a method of treating cartilage and bone diseases, including those of metabolic, inflammatory, and traumatic origin. Secreted frizzled-related proteins have been reported to be expressed by osteoblasts and to modulate osteoclast formation (Hauser et al., 2004). Secreted frizzled-related proteins have also been shown to be involved in skeletal and joint patterning in embryogenesis (Loughlin et al., 2004). They have been implicated as determining factors in the achievement of mature bone mass (Loughlin et al., 2004). A variant sFRP-3, specifically, an SNP with an Arg324Gly substitution, has been shown to possess a diminished ability to modulate Wnt signaling pathways (Loughlin et al., 2004). This reduction in Wnt modulation creates a predisposition to osteoarthritis by altering the development or stability of cartilage or bone in weight-bearing joints (Loughlin et al., 2004). Accordingly, the invention provides a method of treating bone and cartilage diseases.

[0237] The invention also specifically provides a method of treating bone diseases with a pathogenesis that involves abnormal phosphaturia. Secreted FRPs, as regulators of the Wnt signaling pathway, have been demonstrated to modulate phosphaturia (Bemdt et al., 2003). Phosphorous, as well as magnesium and calcium, is transported between the bones and the blood, kidney, and gastrointestinal tract using a variety of active and passive transport mechanisms. Where a polypeptide of the invention is involved in modulating phosphaturia, an agent of the invention is useful for treating conditions or disorders relating to chondrogenesis and/or bone disease. Disorders Related to Cell Death [0238] The expression of sFRP-3 has been reported to conelate with apoptosis-related gene expression (Schumann et al., 2000). Where a polypeptide of the invention is involved in modulating cell death, an agent of the invention is useful for treating conditions or disorders relating to cell death (e.g., DNA damage, cell death, apoptosis). Cell death-related indications that can be treated using the methods of the invention to reduce cell death in a eukaryotic cell, include, but are not limited to, cell death associated with Alzheimer's disease, Parkinson's disease, rheumatoid arthritis, autoimmune thyroiditis, septic shock, sepsis, stroke, central nervous system inflammation, intestinal inflammation, osteoporosis, ischemia, reperfusion injury, cardiac muscle cell death associated with cardiovascular disease, polycystic kidney disease, cell death of endothelial cells in cardiovascular disease, degenerative liver disease, multiple sclerosis, amyotropic lateral sclerosis, cerebellar degeneration, ischemic injury, cerebral infarction, myocardial infarction, acquired immunodeficiency syndrome (AIDS), myelodysplastic syndromes, aplastic anemia, male pattern baldness, and head injury damage. Also included are conditions in which DNA damage to a cell is induced by external conditions, including but not limited to irradiation, radiomimetic drugs, hypoxic injury, chemical injury, and damage by free radicals. Also included are any hypoxic or anoxic conditions, e.g., conditions relating to or resulting from ischemia, myocardial infarction, cerebral infarction, stroke, bypass heart surgery, organ transplantation, and neuronal damage, etc.

[0239] Apoptosis, or programmed cell death, is a regulated process leading to cell death via a series of well-defined morphological changes. Programmed cell death provides a balance for cell growth and multiplication, eliminating unnecessary cells. The default state of the cell is to remain alive. A cell enters the apoptotic pathway when an essential factor is removed from the extracellular environment or when an internal signal is activated. Genes and proteins of the invention that suppress the growth of tumors by activating cell death provide the basis for treatment strategies for hyperproliferative disorders and conditions.

[0240] Apoptosis can be assayed using any known method. Assays can be conducted on cell populations or an individual cell, and include morphological assays and biochemical assays. Procedures to detect cell death based on the TUNEL method are available commercially, e.g., from Boehringer Mannheim (Cell Death Kit) and Oncor (Apoptag Plus).

[0241] Such stimulatory properties render sFRP-3 polypeptides and modulators thereto useful for the treatment, prevention, and diagnosis of diseases. Secreted FRP- 3 and modulators thereof, such as antibodies thereto, may be used as therapeutic proteins or therapeutic targets in the treatment of diseases involved in the malfunction of the immune system, including inflammatory and autoimmune diseases, such as rheumatoid arthritis and osteoarthritis, psoriasis, inflammatory bowel disease, and multiple sclerosis. Antibodies against these proteins or small molecules inhibiting these proteins or their receptors could also be used to treat inflammatory and autoimmune diseases. These proteins may also be used as immunotherapeutic agent for treatment of cancers and infectious diseases, and in vaccines. They may be used as therapeutic protein to treat cancers, such as by inducing apoptosis. [0242] The description herein are put forth to provide those of ordinary skill in the art with a complete disclosure of how to make and how to use the present invention, and is not intended to limit the scope of what the inventors regard as their invention, nor is it intended to represent that the experiments set forth are all or the only experiments performed.

[0243] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. [0244] Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. Moreover, advantages described in the body of the specification, if not included in the claims, are not, per se, limitations to the claimed invention.

[0245] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Moreover, it must be understood that the invention is not limited to the particular embodiments described, as such may, of course, vary. Further, the terminology used to describe particular embodiments is not intended to be limiting, since the scope of the present invention will be limited only by its claims.

[0246] With respect to ranges of values, the invention encompasses each intervening value between the upper and lower limits of the range to at least a tenth of the lower limit's unit, unless the context clearly indicates otherwise. Further, the invention encompasses any other stated intervening values. Moreover, the invention also encompasses ranges excluding either or both of the upper and lower limits of the range, unless specifically excluded from the stated range.

[0247] Unless defined otherwise, the meanings of all technical and scientific terms used herein are those commonly understood by one of ordinary skill in the art to which this invention belongs. One of ordinary skill in the art will also appreciate that any methods and materials similar or equivalent to those described herein can also be used to practice or test the invention. Further, all publications mentioned herein are incorporated by reference.

[0248] It must be noted that, as used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a subject polypeptide" includes a plurality of such polypeptides and reference to "the agent" includes reference to one or more agents and equivalents thereof known to those skilled in the art, and so forth. [0249] Further, all numbers expressing quantities of ingredients, reaction conditions, % purity, polypeptide and polynucleotide lengths, and so forth, used in the specification and s, are modified by the term "about," unless otherwise indicated. Accordingly, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties of the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits, applying ordinary rounding techniques. Nonetheless, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain enors from the standard deviation of its experimental measurement. Example Example 1 : SFRP-3 Gene Expression [0250] The level of gene expression was examined in individual normal and cancer tissue samples. Some normal samples were taken from regions adjacent to cancer tissue. The relative gene expression level in cancer and nonnal tissue was analyzed based on the threshold cycle in quantitative real-time PCR. The expression of each sample (cancer or normal tissue) was normalized to its own internal control 18S rRNA expression and represented by l/2^ΔCt. ΔCt for cancer tissue equals to 2^Ct(gene-^C)" ^Ct(18S-^C) and ΔCt for normal tissue equals 2^ctfe^ene-ⁿ>-^Ct(18S-ⁿ⁾ for normal tissue. [0251] The present inventors also intenogated a proprietary oncology database from GeneLogic, using Affymetrix TJ133 chip probe IDs that conesponded to certain of the sequences studied herein to determine the expression of the sequences in normal tissues and in cancer tissues.

[0252] Secreted FRP-3 was expressed at higher levels in malignant spleen and cervix than in their normal counterparts. Secreted FRP-3 was also expressed at higher levels, although to a lesser extent, in malignant breast, colon, rectum, and intestine. In the pancreas, sFRP-3 gene expression is lower in normal than in malignant tissue. References

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Table 2. Pfam Coordinates of SFRP-3 Sequences

Table 3. Signal Peptide and Transmembrane Coordinates of SFRP-3 Sequences

Table 4. Annotated SFRP-3 Sequences

INDUSTRIAL APPLICABILITY

[0376] The inventors herein have identified a novel sFRP-3 splice variant. The present invention as described herein provides newly identified polypeptides, isolated polynucleotides encoding such, expression vectors, recombinant host cells, fusion molecules, as well as modulators of or antagonists to the isolated polynucleotides and polypeptides. These novel sFRP-3 splice variant molecules have various industrial applications, in particular, they are useful in treating proliferative diseases such as cancer, for example, breast, lung, and colon cancers. In vitro evidence suggests that this novel sFRP-3 splice variant promotes chondrogenesis, and thus provides a therapeutic treatment for diseases of bone and cartilage, for example, osteoarthritis.

Claims

Claim 1. A phannaceutical composition comprising a pharmaceutically acceptable carrier and an isolated polypeptide, wherein the polypeptide is a sFRP-3 polypeptide.

Claim 2. The composition of claim 1 , wherein the polypeptide comprises an amino acid sequence and the amino acid sequence is chosen from SEQ ID NOS: 15,

20 - 24.

Claim 3. The composition of claim 1 , wherein the polypeptide comprises an amino acid sequence and the amino acid sequence is chosen from SEQ ID NOS: 48 -

50, and 55 - 66.

Claim 4. The composition of claim 1 , wherein the polypeptide is encoded by a nucleic acid molecule comprising a polynucleotide sequence chosen from SEQ ID

NOS: l and 6 - 10.

Claim 5. The composition of claim 1, wherein the pharmaceutically acceptable carrier or excipient is selected from saline, phosphate buffered saline, and a lipid- based formulation.

Claim 6. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an isolated antibody, wherein the antibody specifically binds to or interferes with the activity of a sFRP-3 polypeptide.

Claim 7. The composition of claim 6, wherein the pharmaceutically acceptable carrier or excipient is chosen from saline, phosphate buffered saline, and a lipid based formulation.

Claim 8. The composition of claim 6, wherein the polypeptide comprises an amino acid sequence chosen from SEQ ID NOS: 15, 20 - 24, and 26 - 30.

Claim 9. The composition of claim 6, wherein the polypeptide comprises an amino acid chosen from SEQ ID NOS: 25, 31 - 47, 48 - 50, and 55 - 66.

Claim 10. The composition of claim 6, wherein the antibody binds to the polypeptide.

Claim 11. The composition of claim 6, wherein the antibody binds to a ligand of the polypeptide.

Claim 12. The composition of claim 6, wherein the antibody is a human antibody or a humanized antibody.

Claim 13. The composition of claim 6, wherein the antibody is chosen from a polyclonal antibody, a monoclonal antibody, a single chain antibody, an agonist antibody, an antagonist antibody, a neutralizing antibody, and an active fragment of any of these.

Claim 14. The composition of claim 13, wherein the active fragment is chosen from an antigen binding fragment, an Fc fragment, a cdr fragment, a N_H fragment, a

V_L fragment, and a framework fragment.

Claim 15. The composition of claim 6, wherein the antibody comprises at least one sequence chosen from a variable region of an immunoglobulin, a constant region of an immunoglobulin, a heavy chain of an immunoglobulin, a light chain of an immunoglobulin, and an antigen-binding region of an immunoglobulin.

Claim 16. An isolated antibody which specifically binds to or interferes with the activity of a polypeptide, wherein the polypeptide comprises a sequence of at least six contiguous amino acid residues chosen from SEQ ID ΝOS: 15, 20 - 24, and 26 - 29, or from SEQ ID ΝOS: 25, 31 - 50, and 55 - 66.

Claim 17. The antibody of claim 16, wherein the antibody binds to the polypeptide.

Claim 18. The antibody of claim 16, wherein the antibody binds to a ligand of the polypeptide.

Claim 19. The antibody of claim 16, wherein the antibody is a human antibody or a humanized antibody.

Claim 20. The composition of claim 16, wherein the antibody is a chosen from a polyclonal antibody, a monoclonal antibody, a single chain antibody, an agonist antibody, an antagonist antibody, a neutralizing antibody, and active fragments of any of these.

Claim 21. The composition of claim 20, wherein the active fragment is chosen from an antigen binding fragment, an Fc fragment, a cdr fragment, a N_H fragment, a

N_L fragment, and a framework fragment.

Claim 22. The composition of claim 16, wherein the antibody comprises at least one sequence chosen from a variable region of an immunoglobulin, a constant region of an immunoglobulin, a heavy chain of an immunoglobulin, a light chain of an immunoglobulin and an antigen-binding region of an immunoglobulin.

Claim 23. A method of determining the presence of the nucleic acid molecule of one or more of SEQ ID ΝOS. 1, 11, and 51, or a complement thereof in a sample comprising: (a) providing a complement to the nucleic acid molecule or providing a complement to the complement of the nucleic acid molecule; (b) allowing the molecule to interact with the sample; and (c) determining whether interaction has occuned.

Claim 24. A method of determining the presence of a specific antibody to the polypeptide of one or more of SEQ LD NOS.: 15, 20 - 47 in a sample, comprising: (a) providing the polypeptide; (b) allowing the polypeptide to interact with a specific antibody in the sample, if present; and (c) determining whether interaction has occurred.

Claim 25. A method of determining the presence of the polypeptide of one or more of SEQ ID NOS.: 15-50 in a sample, comprising: (a) providing an antibody that specifically binds to or interfere with the activity of the polypeptide; (b) allowing the antibody to interact with the polypeptide in the sample, if any; and (c) determining whether interaction has occurred.

Claim 26. A method of treating or preventing an infection in a subject, comprising: (a) providing a composition comprising a substantially pure sFRP-3 polypeptide; and (b) administering the polypeptide to the subject.

Claim 27. The method of claim 26, wherein the infection is selected from a bacterial infection, a mycoplasma infection, a fungal infection, and a viral infection.

Claim 28. The method of claim 26, wherein administering the polypeptide to the subject comprises administering the polypeptide locally or systemically.

Claim 29. The method of claim 26, wherein the polypeptide comprises an amino acid sequence chosen from among SEQ ID NOS: 15, 20 - 24.

Claim 30. The method of claim 26, wherein the polypeptide comprises an amino acid sequence chosen from SEQ ID NOS: 48 - 50 and 55 - 66.

Claim 31. A method of inhibiting undesirable proliferative growth in a subject, comprising: (a) providing a substantially pure therapeutic agent, wherein the therapeutic agent is a sFRP-3 polypeptide or a modulator thereof; and (b) administering the therapeutic agent to the subject; wherein the modulator specifically binds to or interferes with activity of the polypeptide.

Claim 32. The method of claim 31 , wherein the undesirable proliferative growth is a tumor.

Claim 33. The method of claim 32, wherein the tumor is chosen from a tumor of the ovary, colon, pancreas, mesothelioma, breast, kidney, cervix, and bladder.

Claim 34. The method of claim 31 , wherein the modulator is an antibody.

Claim 35. The method of claim 31 , wherein the modulator is chosen from a small molecule drug, an antisense molecule, a ribozyme, an RNAi molecule, and an aptamer.

Claim 36. The method of claim 34, wherein the antibody binds to a ligand that specifically binds the polypeptide.

Claim 37. The method of claim 31 , wherein the therapeutic agent is a polypeptide and the polypeptide comprises an amino acid sequence chosen from SEQ ID NOS: 15 and 20 - 24.

Claim 38. The method of claim 31 , wherein the therapeutic agent is a polypeptide and the polypeptide comprises an amino acid sequence chosen from SEQ ID NOS: 48 - 50 and 55 - 66.

Claim 39. The method of claim 31, wherein the modulator binds to or interferes with the activity of a polypeptide that comprises an amino acid sequence chosen from SEQ ID NOS: 15, 20 - 24, 25, 26 - 30, 31 -47, 48 - 50, and 55 - 66.

Claim 40. The method of claim 32, wherein the tumor is chosen from an ovarian tumor, a colorectal tumor, a pancreatic tumor, a mesothelioma, a breast tumor, a kidney tumor, a cervical tumor, a bladder tumor, a prostate tumor, a lung tumor, and a glioblastoma.

Claim 41. A method of treatment of a breast tumor in a subj ect, comprising: (a) providing a substantially pure therapeutic agent , wherein the therapeutic agent is a sFRP-3 polypeptide or a modulator thereof; and (b) administering the therapeutic agent to the subject; wherein the modulator specifically binds to or interferes with the activity of the sFRP-

3 polypeptide.

Claim 42. A method of treatment of a lung tumor in a subject, comprising: (a) providing a substantially pure therapeutic agent, wherein the therapeutic agent is a sFRP-3 polypeptide or a modulator thereof; and (b) administering the therapeutic agent to the subject; wherein the modulator specifically binds to or interferes with activity of the sFRP-3 polypeptide.

Claim 43. A method of treatment of a prostate tumor in a subject, comprising: (a) providing a substantially pure therapeutic agent, wherein the therapeutic agent is a sFRP-3 polypeptide or a modulator thereof; and (b) administering the therapeutic agent to the subject; wherein the modulator specifically binds to or interferes with activity of the sFRP-3 polypeptide.

Claim 44. A method of treatment of a colorectal tumor in a subject, comprising: (a) providing a substantially pure therapeutic agent , wherein the therapeutic agent is a sFRP-3 polypeptide or a modulator thereof; and (b) administering the therapeutic agent to the subject; wherein the modulator specifically binds to or interferes with activity of the sFRP-3 polypeptide.

Claim 45. A method of treatment of a pancreatic tumor in a subj ect, comprising: (a) providing a substantially pure therapeutic agent, wherein the therapeutic agent is a sFRP-3 polypeptide or a modulator thereof; and (b) administering the therapeutic agent to the subject; wherein the modulator specifically binds to or interferes with activity of the sFRP-3 polypeptide.

Claim 46. A method of treatment of a bladder tumor in a subj ect, comprising: (a) providing a substantially pure therapeutic agent , wherein the therapeutic agent is a sFRP-3 polypeptide or a modulator thereof; and (b) administering the therapeutic agent to the subject; wherein the modulator specifically binds to or interferes with activity of the sFRP-3 polypeptide.

Claim 47. A method of treatment of a cervical tumor in a subject, comprising: (a) providing a substantially pure therapeutic agent, wherein the therapeutic agent is a sFRP-3 polypeptide or a modulator thereof; and (b) administering the therapeutic agent to the subject; wherein the modulator specifically binds to or interferes with activity of the sFRP-3 polypeptide.

Claim 48. A method of treatment of a kidney tumor in a subject, comprising: (a) providing a substantially pure therapeutic agent, wherein the therapeutic agent is a sFRP-3 polypeptide or a modulator thereof; and (b) administering the therapeutic agent to the subject; wherein the modulator specifically binds to or interferes with activity of the sFRP-3 polypeptide.

Claim 49. A method of treatment of an ovarian tumor in a subject, comprising: (a) providing a substantially pure therapeutic agent, wherein the therapeutic agent is a sFRP-3 polypeptide or a modulator thereof; and (b) administering the therapeutic agent to the subject; wherein the modulator specifically binds to or interferes with activity of the sFRP-3 polypeptide.

Claim 50. A method of treatment of a mesothelioma in a subject, comprising: (a) providing a substantially pure therapeutic agent , wherein the therapeutic agent is a sFRP-3 polypeptide or a modulator thereof; and (b) administering the therapeutic agent to the subject; wherein the modulator specifically binds to or interferes with activity of the sFRP-3 polypeptide.

Claim 51. A method of treatment of a glioblastoma in a subj ect, comprising: (a) providing a substantially pure therapeutic agent, wherein the therapeutic agent is a sFRP-3 polypeptide or a modulator thereof; and (b) administering the therapeutic agent to the subject; wherein the modulator specifically binds to or interferes with activity of the sFRP-3 polypeptide.

Claim 52. The method of any of claims 41-51, wherein the sFRP-3 polypeptide comprises an amino acid sequence and the amino acid sequence is chosen from SEQ

ID NOS: 15, 20 - 24, 52 - 54, and 55 - 66.

Claim 53. The method of any of claims 41-51, wherein the modulator is an antibody.

Claim 54. The method of any of claims 41-51 , wherein the modulator is chosen from a small molecule drug, an antisense molecule, a ribozyme, an RNAi molecule, and an aptamer.

Claim 55. A method of modulating an immune response in a subject, comprising: (a) providing a substantially pure therapeutic agent, wherein the therapeutic agent is a sFRP-3 polypeptide or a modulator thereof; and (b) administering the therapeutic agent to the subject.

Claim 56. The method of claim 55, wherein the modulator specifically binds to or interferes with the activity of the sFRP-3 polypeptide.

Claim 57. The method of claim 55, wherein the therapeutic agent is a polypeptide, and the polypeptide comprises an amino acid sequence chosen from SEQ

ID NOS: 15, 20 - 24, 48 - 50, and 55 - 66.

Claim 58. The method of claim 55, wherein the modulator is an antibody.

Claim 59. The method of claim 55, wherein the modulation of immune response is the suppression of inflammation or suppression of an autoimmune disease.

Claim 60. A method of enhancing immune response to a vaccine in a subject comprising: (a) providing a composition comprising a substantially purified sFRP-3 polypeptide; (b) providing a vaccine composition; and (c) administering the polypeptide and the vaccine composition to the subject.

Claim 60. An isolated sFRP-3 polypeptide comprising an amino acid sequence chosen from SEQ ID NOS: 15 - 24.

Claim 61. An isolated sFRP-3 polypeptide comprising an amino acid sequence, chosen from SEQ ID NOS: 25 - 47.

Claim 62. An isolated nucleic acid molecule comprising a nucleotide sequence which encodes the polypeptide of claim 60 or claim 61.

Claim 63. An isolated nucleic acid molecule chosen from SEQ ID NOS: 1 - 10, and 51.

Claim 64. A vector comprising the nucleic acid molecule of claim 62 or claim 63 and a regulatory region, which regulates the expression of the nucleic acid molecule.

Claim 65. A host cell comprising the vector of claim 64, the nucleic acid molecule of claim 62 or claim 63, or the polypeptide of claim 60 or claim 61.

Claim 66. The composition of any of claim 1, claim 2, or claim 3, wherein the polypeptide further comprises a heterologous secretory leader, wherein the heterologous secretory leader is operably linked to an N-terminus of the polypeptide, and wherein the secretory leader comprises a leader sequence of a secreted protein.

Claim 67. The composition of any of claim 1 , claim 2, or claim 3, wherein the polypeptide further comprises a fusion partner.

Claim 68. The polypeptide of claim 67, wherein the fusion partner is a polymer.

Claim 69. The polypeptide of claim 68, wherein the polymer is a second polypeptide chosen from all or a fragment of human serum albumin, and Fc.

Claim 70. The polypeptide of claim 68, wherein the polymer is polyethylene glycol.

Claim 71. A method for treatment of a bone or cartliage disease in a subject comprising: (a) providing a substantially pure therapeutic agent, wherein the therapeutic agent is a sFRP-3 polypeptide or a modulator thereof; and (b) administering the therapeutic agent to the subject; wherein the modulator specifically binds to or interferes with activity of the sFRP-3 polypeptide.

Claim 72. The method of claim 71, wherein the sFRP-3 polypeptide comprises an amino acid sequence chosen from SEQ ID NOS: 15, 20 - 24, 52 - 54, and 55 - 66.

Claim 73. The method of claim 71 , wherein the modulator is an antibody.

Claim 74. The method of claim 71, wherein the modulator is chosen from a small molecule drug, an antisense molecule, a ribozyme, an RNAi molecule, and an aptamer.

Claim 75. The method of claim 71, wherein the bone disease is osteoarthritis.

Claim 76. The method of claim 71, wherein the bone disease is osteoporosis.

Claim 77. A modified embryonic stem cell comprising a stem cell of an animal and one or more introduced nucleic acid molecule encoding an sFRP-3 polypeptide, wherein the polypeptide comprises an amino acid sequence chosen from SEQ ID

NOS.: 15 and 20 - 24.

Claim 78. An animal genetically modified to express one or more sFRP polypeptide, wherein the polypeptide comprises an amino acid sequence chosen from

SEQ ID NOS.: 15 and 20 - 24.