WO2004074460A2

WO2004074460A2 - Nucleotide and protein sequences of coc genes and methods based thereon

Info

Publication number: WO2004074460A2
Application number: PCT/US2004/005185
Authority: WO
Inventors: Ali H. Brivanlou; Ignacio Munoz-Sanjuan; Esther Bell; Curtis Altmann
Original assignee: The Rockefeller University
Priority date: 2003-02-19
Filing date: 2004-02-19
Publication date: 2004-09-02
Also published as: WO2004074460A3

Abstract

The present invention provides nucleotide sequences of a neural induction gene, Coco, and amino acid sequences of encoded proteins, as well as derivatives (e.g., fragments) and analogs thereof. Coco is an inhibitor of TGF-β, BMP, and Wnt signal transduction. In a specific embodiment, the Coco protein is a human protein. Antibodies to Coco, its derivatives and analogs, are additionally provided. Methods of production of the Coco proteins, derivatives and analogs, e.g., by recombinant means, are also provided. Therapeutic and diagnostic methods and pharmaceutical compositions are provided.

Description

NUCLEOTIDE AND PROTEIN SEQUENCES OF COCO GENES AND METHODS BASED THEREON

CROSS REFERENCE TO RELATED APPLICATION

[001] This application claims the benefit of United States provisional application

Serial No. 60/448,257, filed February 19, 2003, which is incorporated by reference herein in its entirety. STATEMENT OF GOVERNMENT INTEREST

[002] This invention was made at least in part with Government support under grant number HD32105 awarded by the National Institutes of Health. The Government has certain rights in the invention.

1. TECHNICAL FIELD

[003] The present invention relates to neural induction genes encoding inhibitors of TGF- 3, BMP, or Wnt, in particular to "Coco" gene, and their encoded protein products, as well as derivatives and analogs thereof. Production of Coco proteins, derivatives, and antibodies is also provided. The invention further relates to therapeutic compositions and methods of diagnosis and therapy.

2. BACKGROUND OF THE INVENTION

[004] Patterning of the pre-gastrula embryo and subsequent neural induction post gastrulation are complex and intricate processes of which little is understood. Recent research has begun to elucidate certain aspects of how these processes occur and the molecular mechanisms involved. The earliest decision in neural development, the choice between epidermal or neural fates, is regulated by bone morphogenetic protein (BMP) signaling within the ectoderm. Inhibition of BMPs is sufficient for neural induction (Hemmati-Brivanlou and Melton, 1994, Cell 77:273-281; Wilson and Hemmati-Brivanlou, 1995, Nature 376:331-333; Weinstein and Hemmati-Brivanlou, 1999, Annu Rev Cell Dev Biol. 15:411-433; Harland, 2000:Curr. Opin. Genet. Dev. 10, 357-362). Many BMP inhibitors are expressed exclusively within the organizer of the Xenopus gastrula embryo (Spemann and Mangold, 1924, Wilhelm Roux' Arch. Entw.Mech. Org. 100:599-638 ) and therefore are predicted to act as bonafide endogenous neural inducers (Lamb et al, 1993, Science 262:713-718; Hemmati-Brivanlou et al, 1994, Cell 77:283-295; Sasai et al, 1994, Cell 79:779-790; Bouwmeester et al, 1996, Nature 382:595-601). Other BMP inhibitors are more widely expressed, such as the inhibitory Smads (Hata et al, 1994, Genes Dev. 15:186-197; Casellas and Brivanlou, 1998, Dev. Biol. 198:1-12). [005] BMP, TGF-/3 and wnt signaling are critical to a number of cellular and physiological processes and have been implicated in neural cell induction and differentiation, modulation of epidermal cell induction, growth and or differentiation, bone, cartilage and other connective tissue formation, regulation of cell proliferation, tumorigenesis, metastasis, etc. Thus, proteins that modulate BMP, TGF-/3 and/or wnt activity have diagnostic and therapeutic uses. The present invention provides a novel protein that inhibits BMP, TGF-/3 and wnt signaling.

[006] Citation or identification of any reference in Section 2, or in any other section of this application, shall not be considered an admission that such reference is available as prior art to the present invention.

3. SUMMARY OF THE INVENTION

[007] The present invention relates to a novel protein Coco, and nucleic acids coding therefor, that inhibits BMP (particularly BMP-4), TGF- , and wnt signaling. In specific embodiments, the protein of the invention has an amino acid sequence comprising or consisting of SEQ ID NO: 2 (human Coco), 4 (Xenopus Coco), 6 (a partial sequence of mouse Coco) or 8 (fugu Coco), or a processed product thereof, e.g., a fragment of SEQ ID NO: 2, 4, 6, or 8 produced by proteolytic cleavage, such as, but not limited to removal of a signal peptide. In one example, the products of proteolytic cleavage corresponds to amino acids 1 to 22 and amino acids 23 to the terminus of SEQ FD NO: 4. The invention specifically includes the full length mouse sequence comprising SEQ LD NO: 6. [008] Coco proteins, fragments, derivatives, and variants thereof are collectively referred to herein as "polypeptides of the invention" or "proteins of the invention." Nucleic acid molecules encoding the polypeptides or proteins of the invention, e.g., SEQ ID NO: 1 (encoding human Coco), SEQ ID NO: 3 (encoding Xenopus Coco), SEQ ID NO: 5 (encoding a partial sequence of murine Coco), and SEQ ID NO: 7 (encoding Fugu Coco), are collectively referred to as "nucleic acids of the invention."

[009] The nucleic acids and polypeptides of the present invention are useful as modulating agents in regulating BMP, TGF- and/or wnt signalling. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding a polypeptide of the invention or a biologically active portion thereof. The present invention also provides nucleic acid molecules which are suitable for use as primers or hybridization probes for the detection of nucleic acids encoding a polypeptide of the invention. [0010] The invention features nucleic acid molecules that are at least 30%, 35%,

40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the nucleotide sequence of SEQ FD NO: 1, 3, 5 or 7 or a complement thereof. [0011] The invention features isolated polypeptides or proteins that are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 30%, preferably 35%, 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the nucleotide sequence of SEQ ID NOS.:l, 3, 5 or 7 or a complement thereof, wherein the polypeptides or proteins also exhibit at least one structural and/or functional feature of a polypeptide of the invention, e.g., modulation of BMP, TGF-/3, and/or wnt signaling, antigenicity (i.e, able to bound by an anti-Coco antibody), immunogenicity (i.e, able to produce an anti-Coco antibody when used as an immunogen), induction of neural tissue, bone, cartilage or other connective tissue, modulation of the growth or differentiation of neural tissue, epidermal tissue, inhibition of cell proliferation or transformation, and maintenance or induction of a stem cell state (i.e., self-renewing and at least pluripotent, preferably totipotent). [0012] The invention features nucleic acid molecules comprising or consisting of at least 480, 500, 550, 600, 650, 700, 750, 800, 850, 1000, 1100, 1200, 1300, or 1400 nucleotides of the nucleotide sequence of SEQ ID NO: 1, 3, 5, or 7, or a complement thereof.

[0013] The invention features isolated polypeptides or proteins that are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 25, 50, 75, 100, 150, 200, 250, 300, 400, 450, 500, 550, 600, 650 or more contiguous nucleotides identical to the nucleic acid sequence of SEQ ID NO: 1, 3, 5 or 7, or a complement thereof, wherein the polypeptides or proteins also exhibit at least one structural and/or functional feature of a polypeptide of the invention.

[0014] The invention also features nucleic acid molecules that include a nucleotide sequence encoding a protein having an amino acid sequence that is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, a processed product thereof, or a complement of the nucleic acid, wherein the protein encoded by the nucleotide sequence also exhibits at least one structural and/or functional feature of a polypeptide of the invention. [0015] Also within the invention are nucleic acid molecules that encode a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 or a fragment thereof, including at least 10, 15, 20, 25, 30, 50, 75, 100, 125, 150, 175, or 200 contiguous amino acids of SEQ ID NO: 2, 4, 6 or 8, preferably, wherein the fragment exhibits at least one structural and/or functional feature of a polypeptide of the invention. [0016] The invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, and particularly of a processed product of the polypeptides comprising the amino acid sequences of SEQ ID NO: 2, 4, 6, or 8, e.g., stretches of amino acids that are present within the polypeptide sequences of SEQ LD NO: 2, 4, 6, or 8. Processed products include but are not limited to, the 25 kDa full length peptide of SEQ ID NO: 2, 4, 6, or 8, the 23 kDa protein formed upon cleavage at the signal peptide site at amino acid number 23 of SEQ ID NO: 4, the 18.4 kDa cleavage product formed by the cleavage at amino acid number 59 of SEQ ID NO: 4 or the first RRK site, the 15.6 kDa cleavage product formed by the cleavage at amino acid number 82 of SEQ LD NO: 4 or the second RRK site, and the corresponding fragments of SEQ ID NO: 2, 6, or 8 as determined by amino acid sequence alignment.

[0017] Also within the invention are isolated polypeptides or proteins having an amino acid sequence that is at least about 30%, preferably 40%, 45%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, or processed products thereof, wherein the protein or polypeptide also exhibits at least one structural and/or functional feature of a polypeptide of the invention. [0018] Also within the invention are isolated polypeptides or proteins that are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 30%, preferably, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to the nucleotide sequence encoding SEQ ID NO: 1, 3, 5 or 7, and isolated polypeptides or proteins that are encoded by a nucleic acid molecule having a nucleotide sequence that hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7, or complement thereof, wherein the polypeptides or proteins also exhibit at least one structural and/or functional feature of a polypeptide of the invention.

[0019] The invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, 3, 5, or 7, or a complement thereof, hi one embodiment, the nucleic acid molecules are at least 480, 500, 550, 600, 650, 700, 750, 800, 1000, 1100, 1200, 1300, or 1400 nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7, or a complement thereof, preferably encoding a protein with a Coco function.

[0020] The invention also features nucleic acid molecules that are at least 15, preferably 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 600 or more nucleotides in length and that hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, or 7, or a complement thereof, wherein such nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or functional feature of a polypeptide of the invention.

[0021] In one embodiment, the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a nucleic acid of the invention. In another embodiment, the nucleic acid molecule is a double stranded RNA in which one strand is complementary to a mRNA that encodes a Coco protein of the invention. [0022] Another aspect of the invention provides vectors, e.g., recombinant expression vectors, comprising a nucleic acid molecule of the invention. In another embodiment, the invention provides host cells containing such a vector or engineered to contain and/or express a nucleic acid molecule of the invention, for example, containing a nucleotide sequence that encodes a protein or polypeptide of the invention operably linked to a heterologous promoter. The invention also provides methods for producing a polypeptide of the invention by culturing, in a suitable medium, a host cell of the invention such that a polypeptide of the invention is produced.

[0023] Another aspect of this invention features isolated or recombinant proteins and polypeptides of the invention. Preferred proteins and polypeptides possess at least one biological activity possessed by a corresponding naturally-occurring Coco protein. An activity, a biological activity, or a functional activity of a polypeptide or nucleic acid of the invention refers to an activity exerted by a protein, polypeptide or nucleic acid molecule of the invention on a responsive cell as determined in vivo or in vitro, according to standard techniques.

[0024] For Coco proteins or modulators thereof, biological activities include, e.g.,

(1) the ability to modulate (this term, as used herein, includes, but is not limited to, stabilize, promote, inhibit or disrupt) the development, differentiation, proliferation and/or activity of neurons, nerve tissue, and related cells, e.g., Schwann cells, oligodendrocytes, glial cells, astrocytes, etc. ; (2) the ability to modulate the development and progression of cancer and other hyperproliferative or hypoprohferative diseases; (3) the ability to modulate the development, differentiation, proliferation and/or activity of bone, cartilage, related precursors, such as osteoblasts and chondrocytes, and other connective tissue; (4) the ability to modulate the development, differentiation, proliferation and/or activity of epidermal cells; (5) the ability to maintain embryonic stem cells in an undifferentiated state; (6) the ability to modulate BMP, TGF-/3, and/or wnt signalling; (7) the ability to modulate the development of organs, tissues and/or cells in an embryo and or fetus; and (8) the ability to maintain stem cells in an undifferentiated, self-renewing, pluripotent state. [0025] hi one embodiment, a polypeptide of the invention has an amino acid sequence sufficiently identical to an identified domain of a polypeptide of the invention. As used herein, the term "sufficiently identical" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have or encode a common structural domain and/or common functional activity. For example, amino acid or nucleotide sequences which contain or encode a common structural domain having about 60% identity, preferably 65% identity, more preferably 75%, 85%, 95%, 98% or more identity are defined herein as sufficiently identical.

[0026] In one embodiment, a Coco protein includes a cysteine knot domain, e.g., amino acids 100 through 163 of SEQ ID NO: 4 which has at least nine cysteine residues (see e.g., Figure 6B). In a specific embodiment, a Coco protein has multiple cysteine amino acid residues in the C-terminal region of its polypeptide sequence. In a more specific embodiment, a Coco protein has nine cysteine amino acid residues in the C-terminal region of its polypeptide sequence. In another embodiment, a Coco protein is missing a Cysteine knot domain or is missing one or more cysteines in the C-knot domain (i.e., contains 0, 1, 2, 3, 4, 5, 6, 7, or 8 cysteines in the C-knot domain or portion thereof). [0027] The polypeptides of the present invention, or biologically active portions thereof, can be operably linked to a heterologous amino acid sequence to form fusion proteins, e.g., fusions to an F_c domain to increase the in vivo half life of the protein, or fusions to a peptide tag to facilitate detection and or purification.

[0028] The invention further features antibodies, such as monoclonal or polyclonal antibodies or fragments thereof, that specifically bind a polypeptide of the invention. The antibodies of the invention can be conjugated antibodies comprising, for example, therapeutic or diagnostic agents. For example, the antibodies can be conjugated to a therapeutic moiety such as a chemotherapeutic cytotoxin, e.g., a cytostatic or cytocidal agent (e.g., paclitaxol, cytochalasin B or diphtheria toxin), a thrombotic or anti-angiogenic agent or a radioactive or fluorescent label. Antibodies of the invention include human or humanized antibodies, scFvs, Fab fragments and other antigen-binding antibody forms. In particular embodiments, the antibody binds the Coco cysteine knot domain, but, in specific embodiments, not the cysteine knot domain of these cysteine knot family members such as Cerberus.

[0029] In addition, the polypeptides of the invention or biologically active portions thereof, or antibodies of the invention or modulaters of polypeptides of the invention, can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.

[0030] In another aspect, the present invention provides methods for detecting the presence, activity or expression of a polypeptide of the invention in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of the presence, activity or expression such that the presence activity or expression of a polypeptide of the invention is detected in the biological sample, such methods include in vivo detection and quantitation of Coco proteins or nucleic acids.

[0031] In another aspect, the invention provides methods for modulating activity of a polypeptide of the invention comprising contacting a cell with an agent that modulates (inhibits or stimulates) the activity or expression of a polypeptide of the invention such that activity or expression in the cell is modulated. In one embodiment, the agent is an antibody that specifically binds to a polypeptide of the invention, i another embodiment, the agent ^• is a fragment of a polypeptide of the invention or a nucleic acid molecule encoding such a polypeptide fragment, e.g., missing one or more cysteine residues of the cysteine knot domain. In another embodiment, the agent is a peptidomimetic or other small molecule. [0032] In another embodiment, the agent modulates expression of a polypeptide of the invention by modulating transcription, splicing, or translation of an mRNA encoding a polypeptide of the invention. In another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an mRNA encoding a polypeptide of the invention. In another embodiment, the agent is a double stranded RNA. In yet another emodiment, the agent is a ribozyme.

[0033] The present invention also provides methods to treat, prevent, manage, or ameliorate symptoms in a subject having a disease or disorder characterized by aberrant activity of a polypeptide of the invention or aberrant expression of a nucleic acid of the invention by administering an agent which is a modulator of the activity of a polypeptide of the invention or a modulator of the expression of a nucleic acid of the invention to the subject. In one embodiment, the modulator is a protein of the invention. In another embodiment, the modulator is a nucleic acid of the invention. In other embodiments, the modulator is a peptide, peptidomimetic, or other small molecule. In specific embodiments, the disease or disorder includes a disease or disorder associated with abnormal neural cell induction, growth, or differentiation. In other embodiments, the disease or disorder is associated with abnormal bone, cartilage, or other connective tissue, cell growth, induction, or differentiation, or epidermal cell induction, growth, or differentiation. [0034] The present invention also provides a method of maintaining stem cells, in particular of maintaining embryonic stem cells, particularly mammalian, such as, but not limited to mouse, primate, or human stem cells in an undifferentiated, self-renewing, pluriportent state. In another embodiment, the invention is used in the development of cell- based therapies.

[0035] The present invention further provides diagnostic means and methods. The present invention also provides diagnostic and prognostic assays for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of: (i) aberrant modification or mutation of a gene encoding a polypeptide of the invention, (ii) mis-regulation of a gene encoding a polypeptide of the invention, and (iii) aberrant post- translational modification of the invention wherein a wild-type form of the gene encodes a protein having the activity of the polypeptide of the invention. [0036] In another aspect, the invention provides a method for identifying a compound that binds to or modulates the activity of a polypeptide of the invention, h general, such methods entail measuring a biological activity (e.g., but not limited to, modulation of BMP, TGF-/3, and/or wnt signaling in vitro) of the polypeptide in the presence and absence of a test compound and identifying those compounds which alter the activity of the polypeptide.

[0037] The invention also features methods for identifying a compound that modulates the expression of a polypeptide or nucleic acid of the invention by measuring the expression of the polypeptide or nucleic acid in the presence and absence of the compound. [0038] Other features and advantages of the invention will be apparent from the following detailed description and claims. 3.1 DEFINITIONS

[0039] The term "analog" as used herein refers to a polypeptide that possesses a similar or identical function as Coco, e.g., having an amino acid sequence comprising SEQ ID NO: 2, 4, 6 or 8, a fragment of Coco, an anti-Coco antibody, or antibody fragment (or any other protein identified as a modulator of Coco) but does not necessarily comprise a similar or identical amino acid sequence of Coco, a fragment of Coco, an anti-Coco antibody, or antibody fragment, or possess a similar or identical structure of a Coco, a fragment of Coco, an anti-Coco antibody, or antibody fragment. A polypeptide that has a similar amino acid sequence refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide having an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of Coco, a fragment of Coco, an anti-Coco antibody, or antibody fragment described herein; (b) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding Coco, a fragment of Coco, an anti-Coco antibody, or antibody fragment described herein of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues; and (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the nucleotide sequence encoding Coco, an anti-Coco antibody, or antibody fragment described herein. A polypeptide with similar structure to Coco, a fragment of Coco, an anti-Coco antibody, or antibody fragment described herein refers to a polypeptide that has a similar secondary, tertiary or quaternary structure of Coco, a fragment of Coco, an anti- Coco antibody, or antibody fragment described herein. The structure of a polypeptide can be determined by methods known to those skilled in the art, including but not limited to, X- ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy. [0040] The term "derivative" as used herein refers to a polypeptide that comprises an amino acid sequence of Coco, a fragment of Coco, an anti-Coco antibody, or antibody fragment (or any other protein identified as a modulator of Coco) which has been altered by the introduction of amino acid residue substitutions, deletions or additions. The term "derivative" as used herein also refers to a Coco, a fragment of Coco, an anti-Coco antibody, or antibody fragment (or any other protein identified as a modulator of Coco) which has been modified, e.g, by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, Coco, a fragment of Coco, an anti- Coco antibody, or antibody fragment maybe modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative of Coco, a fragment of Coco, an anti-Coco antibody, or antibody fragment may be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative of Coco, a fragment of Coco, an anti-Coco antibody, or antibody fragment may contain one or more non-classical amino acids. A polypeptide derivative possesses a similar or identical function as Coco, a fragment of Coco, an anti- Coco antibody, or antibody fragment described herein.

[0041] The term "epitopes" as used herein refers to portions of a Coco polypeptide

(or any other protein identified as a modulator of Coco) having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. An epitope having immunogenic activity is a portion of a Coco polypeptide that elicits an antibody response in an animal. An epitope having antigenic activity is a portion of a Coco polypeptide to which an antibody immunospecifically binds as determined by any method well known in the art, for example, by the immunoassays described herein. Antigenic epitopes need not necessarily be immunogenic.

[0042] The term "fragment" as used herein refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least contiguous 80 amino acid residues, at least contiguous 90 amino acid residues, at least contiguous 100 amino acid residues, at least contiguous 125 amino acid residues, at least 150 contiguous amino acid residues, at least contiguous 175 amino acid residues, at least contiguous 200 amino acid residues, or at least contiguous 250 amino acid residues of the amino acid sequence of Coco or an anti-Coco antibody (or any other protein identified as a modulator of Coco).

[0043] An "isolated" or "purified" molecule (e.g., a protein, antibody, peptide, etc.) is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of a protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an protein that is substantially free of cellular material includes preparations of proteins having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein"). When the protein is recombinantly produced, it is also preferably substantially free of culture medium, i.e, culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the protein or other molecule is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e, it is separated from chemical precursors or other chemicals which are involved in the synthesis of the molecule. Accordingly such preparations of the protein or other molecule have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the molecule of interest. In a preferred embodiment, antibodies, proteins and other molecules of the invention or fragments thereof are isolated or purified.

[0044] An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized, hi a preferred embodiment, nucleic acid molecules encoding proteins of the invention are isolated or purified.

[0045] The term "antibodies or fragments that iimnunospecifically bind to Coco" as used herein refers to antibodies or fragments thereof that specifically bind to a Coco polypeptide or a fragment of a Coco polypeptide and do not non-specifically bind to other polypeptides. Antibodies or fragments that immunospecifically bind to a Coco polypeptide or fragment thereof may have cross-reactivity with other antigens. Preferably, antibodies or fragments that immunospecifically bind to a Coco polypeptide or fragment thereof do not cross-react with other antigens. Antibodies or fragments that immunospecifically bind to a Coco polypeptide can be identified, for example, by immunoassays or other techniques known to those of skill in the art.

[0046] To determine the percent identity of two amino acid sequences or of two nucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e, % identity = number of identical overlapping positions/total number of positions x 100%). In one embodiment, the two sequences are the same length.

[0047] The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength^^ to obtain nucleotide sequences homologous to a nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. [0048] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0049] As used herein, a "therapeutically effective amount" refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder associated with aberrant Coco expression and/or aberrant TGF-/5, BMP or wnt signalling. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of the disease or disorder. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of the disease or disorder. Further, a therapeutically effective amount with respect to a therapeutic agent of the invention means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of such diseases or disorders. [0050] As used herein, a "prophylactically effective amount" refers to that amount of the prophylactic agent sufficient to result in the prevention of the onset, recurrence, or spread of a disease or disorder associated with aberrant Coco expression and/or aberrant TGF-/3, BMP or wnt signalling; including prevention of the recurrence or spread of such disease or disorder. A prophylactically effective amount with respect to a prophylactic agent of the invention means that amount of prophylactic agent alone, or in combination with other agents, that provides a prophylactic benefit in the prevention of such disease or disorder.

[0051] As used herein, the terms "therapeutic agent" and "therapeutic agents" refer to any agent(s) that can be used in the prevention, treatment, or management of a disease or disorder associated with aberrant Coco expression and/or aberrant TGF-/3, BMP or wnt signalling.

[0052] As used herein, the terms "therapies" and "therapy" can refer to any protocol(s), method(s) and or agent(s) that can be used in the prevention, treatment, or management of diseases or disorders associated with aberrant Coco expression and/or aberrant TGF- 5, BMP or wnt signalling.

[0053] As used herein, the terms "prophylactic agent" and "prophylactic agents" refer to any agent(s) that can be used in the prevention of a disease or disorder or the recurrence or spread of a disease or disorder associated with aberrant Coco expression and/or aberrant TGF-/3, BMP or wnt signalling.

[0054] As used herein, a "therapeutic protocol" refers to a regimen of timing and dosing of one or more therapeutic agents.

[0055] As used herein, a "prophylactic protocol" refers to a regimen of timing and dosing of one or more prophylactic agents.

[0056] A used herein, a "protocol" includes dosing schedules and dosing regimens.

[0057] As used herein, "in combination" refers to the use of more than one prophylactic and/or therapeutic agents.

[0058] As used herein, the terms "subject" and "patient" are used interchangeably.

As used herein, a subject is preferably a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkey and human), most preferably a human.

[0059] As used herein, the term "adjunctive" is used interchangeably with "in combination" or "combinatorial." Such terms are also used where two or more therapeutic or prophylactic agents affect the treatment or prevention of the same disease.

[0060] As used herein, the terms "manage", "managing" and "management" refer to the beneficial effects that a subject derives from a prophylactic or therapeutic agent, which does not result in a cure of the disease. In certain embodiments, a subject is administered one or more prophylactic or therapeutic agents to "manage" a disease so as to prevent the progression or worsening of the disease.

[0061] As used herein, the terms "prevent", " preventing" and "prevention" refer to the prevention of the recurrence, spread or onset of a disease in a subject resulting from the administration of a prophylactic or therapeutic agent.

[0062] As used herein, underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its encoded protein product, which is indicated by the name of the gene in the absence of any underscoring or italicizing. For example, "Coco'^'' shall mean the

Coco gene, whereas "Coco" shall indicate the protein product of the Coco gene.

[0063] As used herein, the terms "treat", "treating" and "treatment" refer to the eradication, reduction, modulation, or amelioration of symptons of a disease or disorder.

4. BRIEF DESCRIPTION OF THE FIGURES

[0064] FIGS. lA-C. Identification of Coco, a novel BMP inhibitor. A. Nucleotide sequence and deduced amino acid sequence of Xenopus Coco ("xCoco"). The open reading frame is in bold and the underlined text also indicates primers used for the reverse transcriptase-polymerase chain reaction (RT-PCR). B. Alignment at the amino acid level of Xenopus, fugu, human and mouse Coco and other family members: Cerberus and

Caronte. C. Tree illustrating that the Xenopus, human and mouse homologs of xCoco belong to the Cerberus/Dan gene family. Numbers represent percentage identity with

Xenopus Coco. For details, see Section 6 (Example 1).

[0065] FIGS. 2A-E. Expression pattern of Coco mRNA during Xenopus development (A) Wholemount in situ hybridisation of Coco, Bmp4 and VegT mRNA in the egg. Expression of Coco was also compared to Vgl by RT-PCR (B) Coco is expressed strongly in the animal pole at the 2-cell, 8-cell stages and at stage 8. (C) Coco is strongly expressed maternally and then is downregulated post-gastrula/pre-neurula stages, in contrast

Cerberus is first expressed at stage 9 and then downregulated after the onset of neurulation.

(D) At gastrula Coco is detected at high levels in the ectoderm and marginal zones by whole mount in situ hybridisation and (E) as seen by RT-PCR at much lower levels in the vegetal pole. ODC was used as a loading control for the RT-PCR. * depicts the organizer. For details, see Section 6 (Example 1).

[0066] FIGS. 3A-N. Pheno types of Coco overexpression in Xenopus embryos.

Analysis of injected embryos at gastrula (A-D), neurula (H) and early tadpole stage embryos (E-G, I-N). In A-D, H-N: top panels are uninj ected embryos, lower panels are embryos injected with lng Coco vegetally in 1 cell at the 2-cell stage (A-D, F) or one cell at the 4-cell stage ventrally (G-N). (A) Xbra, brachyury, marker of mesoderm, (B)fgβ, mesoderm, (C) Otx2, organizer and anterior ectoderm, (D) Gsc, Goosecoid, organizer. (E) unmjected control embryo, (F) dorsal animal injection, (G) 1/4 ventral vegetal injected, (H) rx, forebrain, (I) Emxl, dorsal telencephalon, (J) Otx2, forebrain and midbrain, (K) En2, midbrain/hindbrain boundary, (L) Hoxb9, spinal cord, (M) nkx2.5, heart and (N) 12-101 Ab, muscle. The * depicts the extra head structures. A, B, D are vegetal views; C, lateral with animal pole to the top; E-G, I-K, lateral views; H, L, N dorsal views; M, ventral view. In E- N, anterior is to the right. For details, see Section 6 (Example 1). [0067] FIGS. 4A-E. Dorsalization effects of Coco in embryonic explants. (A)

Animal caps injected with 1 ng Coco at the 2-cell stage and analysed for the expression of epidermal, mesodermal and neural markers. (B) Animal caps analysed for neural induction at early tadpole stages. Both the general neural markers, Ncam and nrpl have been induced as well as anterior markers, Otx and XAG. (C) Morphology of the VMZ+Coco explants compared to control DMZ and VMZ. (D) RT-PCR analysis of VMZ explants expressing lng Coco compared to DMZ and VMZ uninjected explants at gastrula stages. The organizer markers chordin and goosecoid are induced in the VMZ+Coco explants. (E) Analysis at tadpole stages. The VMZ+Coco now expresses dorsal molecular markers. ODC was used as a loading control. For details, see Section 6 (Example 1).

[0068] FIGS. 5A-I. Inhibitory effects of Coco on BMP, TGF-B and Wnt signaling.

(A) BMP4 and Coco were injected separately and together into embryos at the 2-cell stage. Animal caps were analysed at gastrula stages for the presence of Xbra and epidermal keratin. Coco blocked the induction of both these markers by BMP4. (B) Wnt8 and Coco were injected into animal caps and the markers Xnr3 and Siamois analysed. Coco blocked the induction of these markers by Wnt8. (C) Inhibition of nodal signaling by Coco. Coco blocked the induction of chordin, Xbra and wnt8 by xNR-1. (D) Inhibition of activin signaling by Coco. (E, F) Direct binding of Coco to BMP4 and xNRl. Flag-tagged Coco was co-injected with HA-BMP4 (E) and HA-xNRl (F). Coco inhibited the activation of both the Wnt8-responsive promoter TOP -Flash (G) and the Bmp response element (H). (I) Ectodermal competence assay. Ectodermal explants uninjected or Coco injected were exposed to activin conditioned media at different stages. Notice that in the presence of Coco, explants are unable to respond to activin from stage 10 onwards.

5. DETAILED DESCRIPTION OF THE INVENTION

[0069] The present inventors have discovered a novel inhibitor of BMP, TGF-/3 and

Wnt signaling, which, as such, plays an important physiological role in the regulation of cell proliferation, nervous tissue formation and differentiation, epidermal tissue growth

(e.g., skin and scar tissue growth) and differentiation, growth and differentiation of bone and cartilage and other connective tissues. The protein is named Coco. The invention encompasses Coco proteins and the nucleic acids encoding them (e.g., Xenopus, human, mouse and fugu Coco, SEQ ID NO: 1, 3, 5, and 7 for nucleic acids and SEQ ID NO: 2, 4, 6, and 8 for proteins), and processed products thereof, respectively. [0070] The present invention provides nucleotide sequences of Coco genes, and amino acid sequences of their encoded proteins. The invention further provides fragments and other derivatives and analogs of Coco proteins, preferably fragments, derivatives and analogs of Coco having one or more Coco biological activities. Nucleic acids encoding such fragments or derivatives are also within the scope of the invention. The invention provides Coco genes and their encoded proteins of many different species. The Coco genes of the invention include Xenopus, human, and mouse and Fugu Coco and related genes (homologs) in other species. In specific embodiments, the Coco genes and proteins are from vertebrates, or more particularly, mammals. In a preferred embodiment of the invention, the Coco genes and proteins are of human origin. Production of the foregoing proteins and derivatives, e.g., by recombinant methods, is provided. [0071] Coco is a gene identified by its ability to regulate the competence of the ectoderm and to regulate ectodermal patterning during gastrulation, i.e, embryogenesis, and to induce the formation of neural tissue. Coco has also been shown to inhibit BMP, TGF- 3 and Wnt signaling. Coco has also been shown to be remarkably upregulated in embryonic stem cells, indicating a role of Coco in maintaining stem cells in a self renewing and undifferentiated state.

[0072] The invention also provides Coco derivatives and analogs of the invention that are functionally active, i.e, they are capable of displaying one or more known functional activities associated with a full-length (wild-type) Coco protein. Such functional activities include, but are not limited to, inhibition of BMP, TGF 3 and Wnt signaling pathways, antigenicity (ability to bind, or to compete with Coco for binding, to an anti- Coco antibody), immunogenicity (ability to generate antibody that binds to Coco), ability to modulate formation or differentiation of neural cells or tissues, modulate growth differentiation of epidermal tissue, bone, cartilage or other connective tissue, modulate cell proliferation, diseases and disorders associated with aberrant expression of Coco and/or aberrant TGF-/3, BMP or Wnt signalling, an ability to modulate formation of mesodermal cells or tissues, and an ability to maintain embryonic stem cells in an undifferentiated state. [0073] The invention further provides fragments (and derivatives and analogs thereof) of Coco that comprise one or more domains of the Coco protein, such as, but not limited to, a cysteine knot domain. [0074] Antibodies to Coco, its derivatives and analogs, are additionally provided.

[0075] The present invention also provides therapeutic and diagnostic methods and compositions based on Coco proteins and nucleic acids and on anti-Coco antibodies. The invention provides for treatment of disorders through the administration of compounds that promote Coco activity (e.g., Coco proteins and functionally active analogs and derivatives (including fragments) thereof; nucleic acids encoding the Coco proteins, analogs, or derivatives and agonists of Coco).

[0076] The invention also provides methods of treatment of such diseases and disorders by administering compounds that antagonize, or inhibit, Coco function (e.g., antibodies, Coco antisense nucleic acids, Coco ribozymes, double stranded Coco RNA). [0077] Methods of modulating the differentiation of embryonic stem cells, animal models, and screening methods for modulators of Coco activity are also provided by the invention.

[0078] For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections that follow. 5.1 ISOLATION OF COCO NUCLEIC ACIDS

[0079] The invention provides the nucleotide sequences of Coco nucleic acids. In specific embodiments, Coco nucleic acids comprise the sequences of SEQ ID NO: 1, 3, 5 and 7, or the coding regions thereof, where the sequence of SEQ ID NO: 7 is a partial sequence of mouse Coco, or nucleotide sequences encoding a Coco protein (e.g., a protein having the sequence of SEQ ID NO: 2, 4, 6, and 8, or processed products thereof, where sequence 8 is a partial sequence of mouse Coco). The invention provides purified nucleic acids consisting of at least 8 nucleotides (i.e, a hybridizable portion) of a Coco sequence; in other embodiments, the nucleic acids consist of at least 25 (continuous) nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, or 200 nucleotides of a Coco sequence, or a full-length Coco coding sequence, hi another embodiment, the nucleic acids are smaller than 35, 200 or 500 nucleotides in length. Nucleic acids can be single or double stranded. [0080] In instances wherein the nucleic acid molecule is a cDNA or RNA, e.g. , mRNA, molecule, such molecules can include a poly A "tail", or, alternatively, can lack such a 3 ' tail. Although cDNA or RNA nucleotide sequences may be depicted herein with such tail sequences, it is to be understood that cDNA nucleic acid molecules of the invention are also intended to include such sequences lacking the depicted poly A tails. [0081] In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 or a portion thereof. A nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize (e.g., under conditions of high stringency), to the given nucleotide sequence thereby forming a stable duplex. [0082] Moreover, a nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence encoding a full length polypeptide of the invention for example, a fragment which can be used as a probe or primer or a fragment may encode a biologically active portion of a polypeptide of the invention. The nucleotide sequence determined from the Coco genes identified herein allows for the generation of probes and primers designed for use in identifying and/or cloning homologues in other cell types, e.g., from other tissues, as well as homologues from other mammals. The probe/primer typically comprises substantially purified oligonueleotide. In one embodiment, the oligonueleotide comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides of the sense or anti-sense sequence of SEQ ID NO: 1, 3, 5 or 7. In another embodiment, the oligonueleotide comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least 400, preferably 450, 500, 530, 550, 600, 700, 800, 900, or 1000 consecutive nucleotides of the sense or antisense sequence of SEQ ID NO: 1, 3, 5 or 7.

[0083] Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts or genomic sequences encoding the same protein molecule encoded by a selected nucleic acid molecule. The probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which mis-express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted. [0084] A nucleic acid fragment encoding a biologically active portion of a polypeptide of the invention can be prepared by isolating a portion of any of SEQ ID NO: 1, 3, 5 or 7 expressing the encoded portion of the polypeptide protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the polypeptide. [0085] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7. [0086] In addition to the nucleotide sequences of SEQ ID NO: 1, 3, 5 or 7, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequence may exist within a population (e.g., the human population). Such genetic polymorphisms may exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes which occur alternatively at a given genetic locus. For example, human Coco has been mapped to the 19pl3.2 chromosome region, and therefore Coco family members can include nucleotide sequence polymorphisms (e.g., nucleotide sequences that vary from SEQ ID NO: 1 that map to this chromosome region), and such sequences represent Coco allelic variants. As used herein, the phrase "allelic variant" refers to a nucleotide sequence that occurs at a given locus or to a polypeptide encoded by the nucleotide sequence. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention. In one embodiment, polymorphisms that are associated with a particular disease and/or disorder are used as markers to diagnose said disease or disorder. In a preferred embodiment, polymorphisms are used as a marker to diagnose abnormal coronary function such as atherosclerosis.

[0087] Moreover, nucleic acid molecules encoding proteins of the invention from other species (homologues), which have a nucleotide sequence which differs from that of the human, mouse, Xenopus or fugu protein described herein are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of a cDNA of the invention can be isolated based on their identity to the human nucleic acid molecule disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.

[0088] In addition to naturally-occurring allelic variants of a nucleic acid molecule of the invention sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation thereby leading to changes in the amino acid sequence of the encoded protein, without altering the biological activity of the protein. For example, one can make nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are not conserved or only semi-conserved among homologues of various species may be non-essential for activity and thus would be likely targets for alteration. Alternatively, amino acid residues that are conserved among the homologues of various species (e.g., mouse and human) maybe essential for activity and thus would not be likely targets for alteration.

[0089] Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding a polypeptide of the invention that contain changes in amino acid residues that are not essential for activity. Such polypeptides differ in amino acid sequence from SEQ ID NO: 2, 4, 6 or 8 yet retain biological activity. In one embodiment, the isolated nucleic acid molecule includes a nucleotide sequence encoding a protein that includes an amino acid sequence that is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8. In another embodiment, the isolated nucleic acid molecule includes a nucleotide sequence encoding a protein that is a mutant with inhibitory activity, e.g., a protein that is missing one or more Cys residues.

[0090] An isolated nucleic acid molecule encoding a variant protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined, for example, using assays described in infra.

[0091] The invention also provides nucleic acids hybridizable to or complementary to the foregoing sequences. In specific aspects, nucleic acids are provided that comprise a sequence complementary to at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of a Coco gene. In a specific embodiment, a nucleic acid that is hybridizable to a Coco nucleic acid (e.g., having sequence SEQ ID NO: 1, 3, 5 or 7), or to a nucleic acid encoding a Coco derivative, under conditions of low stringency is provided. By way of example and not limitation, procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. USA 78:6789-6792): Filters containing DNA are pretreated for 6 h at 40°C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10⁶ cpm ³²P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40°C, and then washed for 1.5 h at 55°C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60°C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68°C and reexposed to film. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations).

[0092] In another specific embodiment, a nucleic acid that is hybridizable to a Coco nucleic acid under conditions of high stringency is provided. By way of example and not limitation, procedures using such conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65 °C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65°C in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20 X 10⁶ cpm of ³²P-labeled probe. Washing of filters is done at 37°C for 1 h in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1X SSC at 50°C for 45 min before autoradiography. As another example, the hybridization conditions are hybridization in 50% formamide, 5X SSC, 5X Denhardt's, 1% SDS, 0.2 mg/ml salmon sperm DNA at 42°C; with wash at 55°C in 0.2 X SSC, 0.1% SDS. Other conditions of high stringency that may be used are well known in the art.

[0093] In another specific embodiment, a nucleic acid that is hybridizable to a Coco nucleic acid under conditions of moderate stringency is provided.

[0094] Nucleic acids encoding derivatives and analogs of Coco proteins and Coco antisense nucleic acids are additionally provided. As is readily apparent, as used herein, a "nucleic acid encoding a fragment or portion of a Coco protein" shall be construed as referring to a nucleic acid encoding only the recited fragment or portion of the Coco protein and not the other contiguous portions of the Coco protein as a continuous sequence. [0095] Fragments of Coco nucleic acids comprising regions conserved between

(with homology to) other Coco nucleic acids, of the same or different species, are also provided. Nucleic acids encoding one or more Coco domains are provided, e.g., encoding amino acids corresponding to those shown in Figure 6B.

[0096] Specific embodiments for the cloning of a Coco gene, presented as a particular example but not by way of limitation, follows:

[0097] For expression cloning (a technique commonly known in the art), an expression library is constructed by methods known in the art. For example, mRNA (e.g., human) is isolated, cDNA is made and ligated into an expression vector (e.g., a bacteriophage derivative) such that it is capable of being expressed by the host cell into which it is then introduced. Various screening assays can then be used to select for the expressed Coco product. In one embodiment, anti-Coco antibodies can be used for selection.

[0098] In another embodiment, polymerase chain reaction (PCR) is used to amplify the desired sequence in a genomic or cDNA library, prior to selection. Oligonueleotide primers representing known Coco sequences can be used as primers in PCR. In a preferred aspect, the oligonueleotide primers represent at least part of the Coco conserved segments of strong homology between Coco of different species; see, e.g., Section 6 infra.) The synthetic oligonucleotides may be utilized as primers to amplify by PCR sequences from a source (RNA or DNA), preferably a cDNA library, of potential interest. PCR can be carried out, e.g., by use of a PCR thermal cycler (e.g., PerkinElmer, Eppendorf, Applied Biosystems) and Taq polymerase (GeneAmp®, Applied Biosystems). The DNA being amplified can include mRNA or cDNA or genomic DNA from any eukaryotic species. One can choose to synthesize several different degenerate primers, for use in the PCR reactions. It is also possible to vary the stringency of hybridization conditions used in priming the PCR reactions, to allow for greater or lesser degrees of nucleotide sequence similarity between the known Coco nucleotide sequence and the nucleic acid homolog being isolated. For cross species hybridization, low stringency conditions are preferred. For same species hybridization, moderately stringent conditions are preferred. After successful amplification of a segment of a Coco homolog, that segment may be molecularly cloned and sequenced, and utilized as a probe to isolate a complete cDNA or genomic clone. This, in turn, will permijt the determination of the gene's complete nucleotide sequence, the analysis of its expression, and the production of its protein product for functional analysis, as described infra, hi this fashion, additional genes encoding Coco proteins and Coco analogs may be identified.

[0099] The above-methods are not meant to limit the following general description of methods by which clones of Coco may be obtained.

[00100] Any eukaryotic cell can potentially serve as the nucleic acid source for the molecular cloning of the Coco gene. The nucleic acid sequences encoding Coco can be isolated from vertebrate, mammalian, human, porcine, bovine, feline, avian, equine, canine, murine, amphibia, preferably, Xenopus, fish, e.g., fugu, as well as additional primate sources, insects, plants, etc. The DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library"), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell. (See, for example, Sambrook et al, 2001, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, N.Y.; Ausubel et al, 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.; and Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II, each of which is hereby incorporated by reference in its entirety). Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will contain only exon sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.

[00101] In the molecular cloning of the gene from genomic DNA, DNA fragments are generated, some of which will encode the desired gene. The DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication. The linear DNA fragments can then be separated according to size by standard techniques, including, but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography. [00102] Once the DNA fragments are generated, identification of the specific DNA fragment containing the desired gene may be accomplished in a number of ways. For example, a portion of a Coco (of any species) gene or its specific RNA, or a fragment thereof can be purified and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton, W. and Davis, R., 1977, Science 196:180; Grunstein, M. And Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Those DNA fragments with substantial homology to the probe will hybridize. It is also possible to identify the appropriate fragment by restriction enzyme digestion(s) and comparison of fragment sizes with those expected, according to a known restriction map if such is available. Further selection can be carried out on the basis of the properties of the gene.

[00103] Alternatively, the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, cDNA clones, or DNA clones that hybrid-select the proper mRNAs, can be selected that produce a protein that, e.g., has similar or identical electrophoretic migration, isoelectric focusing behavior, proteolytic digestion maps, inhibition of neural induction activity, substrate binding activity, or antigenic properties as known for Coco. If an antibody to Coco is available, the Coco protein may be identified by binding of labeled antibody to the putatively Coco synthesizing clones, in an ELISA (enzyme-linked immunosorbent assay)- type procedure.

[00104] The Coco gene can also be identified by mRNA selection by nucleic acid hybridization followed by in vitro translation. In this procedure, fragments are used to isolate complementary mRNAs by hybridization. Such DNA fragments may represent available, purified Coco DNA of another species (e.g., Xenopus, mouse, human). Immunoprecipitation analysis or functional assays (e.g., aggregation ability in vitro; binding to receptor; see infra) of the in vitro translation products of the isolated products of the isolated mRNAs identifies the mRNA and, therefore, the complementary DNA fragments that contain the desired sequences. In addition, specific mRNAs may be selected by adsorption of polysomes isolated from cells to immobilized antibodies specifically directed against Coco protein. A radiolabeled Coco cDNA can be synthesized using the selected mRNA (from the adsorbed polysomes) as a template. The radiolabeled mRNA or cDNA may then be used as a probe to identify the Coco DNA fragments from among other genomic DNA fragments.

[00105] Alternatives to isolating the Coco genomic DNA include, but are not limited to, chemically synthesizing the gene sequence itself from a known sequence or making cDNA to the mRNA that encodes the Coco protein. For example, RNA for cDNA cloning of the Coco gene can be isolated from cells that express Coco. Other methods are possible and within the scope of the invention.

[00106] The identified and isolated gene can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as PBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene). The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector that has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and Coco gene may be modified by homopolymeric tailing. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.

[00107] In an alternative method, the desired gene may be identified and isolated after insertion into a suitable cloning vector in a "shot gun" approach. Enrichment for the desired gene, for example, by size fractionization, can be done before insertion into the cloning vector.

[00108] In specific embodiments, transformation of host cells with recombinant

DNA molecules that incorporate the isolated Coco gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.

[00109] The Coco sequences provided by the instant invention include those nucleotide sequences encoding substantially the same amino acid sequences as found in native Coco proteins, and those encoded amino acid sequences with functionally equivalent amino acids, as well as those encoding other Coco derivatives or analogs, as described in Sections 5.6 infra. 5.2 EXPRESSION OF COCO GENES

[00110] The nucleotide sequence coding for a Coco protein or a functionally active analog or fragment or other derivative thereof, can be inserted into an appropriate expression vector, i.e, a vector that contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. The necessary transcriptional and translational signals can also be supplied by the native Coco gene and/or its flanking regions. A variety of host- vector systems may be utilized to express the protein-coding sequence. These include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used. In specific embodiments, the human Coco gene is expressed, or a sequence encoding a functionally active portion of human Coco. In yet another embodiment, a fragment of Coco comprising a domain of the Coco protein is expressed. [00111 ] Any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of nucleic acid sequence encoding a Coco protein or peptide fragment may be regulated by a second nucleic acid sequence so that the Coco protein or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a Coco protein may be controlled by any promoter/enhancer element known in the art. In a specific embodiment, the promoter is not a native Coco gene promoter. Promoters that may be used to control Coco expression include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441- 1445), the regulatory sequences of the metallothionem gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the 3-lactamase promoter (Villa- Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al, 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; plant expression vectors comprising the nopaline synthetase promoter region (Herrera-Estrella et al, Nature 303:209-213) or the cauliflower mosaic virus 35S RNA promoter (Gardner, et al., 1981, Nucl. Acids Res. 9:2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region, which is active in pancreatic acinar cells (Swift et al, 1984, Cell 38:639-646; Ornitz et al,

1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region, which is active in pancreatic beta cells (Hanahan,

1985, Nature 315:115-122), immunoglobulin gene control region, which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region, which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region, which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region, which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al..

1987, Science 235:53-58; alpha 1-antitrypsin gene control region, which is active in the liver (Kelsey et al, 1987, Genes and Devel. 1:161-171), beta-globin gene control region, which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al.,

1986, Cell 46:89-94; myelin basic protein gene control region, which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region, which is active in skeletal muscle (Sani, 1985, Nature 314:283- 286), and gonadotropic releasing hormone gene control region, which is active in the hypothalamus (Mason et al, 1986, Science 234:1372-1378).

[00112] In a specific embodiment, a vector is used that comprises a promoter operably linked to a Coco-encoding nucleic acid, one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene). [00113] In a specific embodiment, an expression construct is made by subcloning a

Coco coding sequence into the EcoRI restriction site of each of the three pGEX vectors (Glutathione S-Transferase expression vectors; Smith and Johnson, 1988, Gene 7:31-40). This allows for the expression of the Coco protein product from the subclone in the correct reading frame. [00114] Expression vectors containing Coco gene inserts can be identified by three general approaches: (a) nucleic acid hybridization, (b) presence or absence of "marker" gene functions, and (c) expression of inserted sequences. In the first approach, the presence of a Coco gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted Coco gene. In the second approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of a Coco gene in the vector. For example, if the Coco gene is inserted within the marker gene sequence of the vector, recombinants containing the Coco insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by assaying the Coco product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the Coco protein in in vitro assay systems, e.g., modulation of neural induction, modulation of epidermal tissue induction, modulation of bone, cartilage or other connective tissue induction, binding with anti-Coco antibody, etc.

[00115] Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, the expression vectors that can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.

[00116] In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered Coco protein may be controlled. Furthermore, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation of proteins). Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce an unglycosylated core protein product. Expression in yeast will produce a glycosylated product. Expression in mammalian cells can be used to ensure "native" glycosylation of a heterologous protein. Furthermore, different vector/host expression systems may effect processing reactions to different extents.

[00117] In other specific embodiments, the Coco protein, fragment, analog, or derivative may be expressed as a fusion, or chimeric protein product (comprising the protein, fragment, analog, or derivative joined via a peptide bond to a heterologous protein sequence (of a different protein)). Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques, e.g. , by use of a peptide synthesizer. [00118] Both cDNA and genomic sequences can be cloned and expressed.

[00119] The host cells of the invention can also be used to produce nonhuman transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a sequence encoding a polypeptide of the invention has been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous sequences encoding a polypeptide of the invention have been introduced into their genome or homologous recombinant animals in which endogenous encoding a polypeptide of the invention sequences have been altered. Such animals are useful for studying the function and/or activity of the polypeptide and for identifying and/or evaluating modulators of polypeptide activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, such as Xenopus, and fish, such as zebra fish and fugu, etc. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, an "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[00120] A transgenic animal of the invention can be created by introducing nucleic acid encoding a Coco polypeptide of the invention (or a homologue thereof) into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the polypeptide of the invention to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, U.S. Patent No. 4,873,191 and in Hogan, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of mRNA encoding the transgene in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying the transgene can further be bred to other transgenic animals carrying other transgenes. [00121] To create an homologous recombinant animal, a vector is prepared which contains at least a portion of a gene encoding a polypeptide of the invention into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the gene. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous gene is functionally disrupted (i.e, no longer encodes a functional protein; also referred to as a "knock out" vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous protein). In the homologous recombination vector, the altered portion of the gene is flanked at its 5' and 3' ends by additional nucleic acid of the gene to allow for homologous recombination to occur between the exogenous gene carried by the vector and an endogenous gene in an embryonic stem cell. The additional flanking nucleic acid sequences are of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see, e.g., Thomas and Capecchi (1987) Cell 51:503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced gene has homologously recombined with the endogenous gene are selected (see, e.g., Li et al. (1992) Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see, e.g., Bradley in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach,

Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined

DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in

Bradley (1991) Current Opinion in Bio/Technology 2:823-829 and in PCT Publication Nos.

WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.

[00122] In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI . For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl.

Acad. Sci. USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science

251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

[00123] Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 385:810-813 and PCT Publication NOS. WO 97/07668 and WO 97/07669.

5.3 IDENTIFICATION AND PURIFICATION OF THE COCO GENE PRODUCTS

[00124] In particular aspects, the invention provides amino acid sequences of Coco, preferably human Coco, and fragments and derivatives thereof, e.g., that comprise an antigenic determinant (i.e, can be recognized by an antibody) or that are otherwise functionally active, as well as nucleic acid sequences encoding the foregoing, e.g. the amino acid sequences of SEQ ID NOs. 2, 4, 6 and 8, and fragments thereof, particularly processed products thereof. "Functionally active" Coco material as used herein, refers to that material displaying one or more known functional activities associated with a full-length (wild-type) Coco protein, e.g., neural induction activity, inhibition of epidermal or mesodermal induction, inhibition of TGF-/3, BMP or Wnt signalling (particularly, BMP-4, Wnt-8 or nodal signalling), binding to a Coco substrate or Coco binding partner, antigenicity (binding to an anti-Coco antibody), immunogenicity, etc.

[00125] In specific embodiments, the invention provides fragments of a Coco protein consisting of at least 6 amino acids, 10 amino acids, 50 amino acids, or of at least 75 amino acids. In other embodiments, the proteins comprise or consist essentially of a cysteine knot domain, or any combination of the foregoing, of a Coco protein. Fragments, or proteins comprising fragments, lacking some or all of the foregoing regions of a Coco protein are also provided. Nucleic acids encoding the foregoing are provided.

[00126] Once a recombinant nucleic acid that expresses the Coco gene sequence is identified, the gene product can be expressed and analyzed. This is achieved by assays based on the physical or functional properties of the product, including radioactive labeling of the product followed by analysis by gel electrophoresis, immunoassay, etc.

[00127] Once the Coco protein is identified, it may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. The functional properties may be evaluated using any suitable assay.

[00128] Alternatively, once a Coco protein produced by a recombinant is identified, the amino acid sequence of the protein can be deduced from the nucleotide sequence of the chimeric gene contained in the recombinant. As a result, the protein can be synthesized by standard chemical methods known in the art (e.g., see Hunkapiller, M., et al, 1984, Nature

310:105-111).

[00129] In another alternate embodiment, native Coco proteins can be purified from natural sources, by standard methods such as those described above (e.g., immunoaffinity purification).

[00130] In a specific embodiment of the present invention, such Coco proteins, whether produced by recombinant DNA techniques or by chemical synthetic methods or by purification of native proteins, include but are not limited to those containing, as a primary amino acid sequence, all or part of the amino acid sequence substantially as depicted in

Figure 6 or SEQ ID NO: 2, 4, 6, or 8, as well as fragments and other derivatives, and analogs thereof, including proteins homologous thereto.

5.4 STRUCTURE OFTHE COCO GENE AND PROTEIN

[00131] The structure of the Coco gene and protein can be analyzed by various methods known in the art. 5.4.1 GENETIC ANALYSIS

[00132] The cloned DNA or cDNA corresponding to the Coco gene can be analyzed by methods including but not limited to Southern hybridization (Southern, E.M., 1975, J. Mol. Biol. 98:503-517), Northern hybridization (see e.g., Freeman et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:4094-4098), restriction endonuclease mapping (Sambrook et al, 2001, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, N.Y.; Ausubel et al, 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.), and DNA sequence analysis. Polymerase chain reaction (PCR; U.S. Patent Nos. 4,683,202, 4,683,195 and 4,889,818; Gyllenstein et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7652-7656; Ochman et al., 1988, Genetics 120:621-623; Loh et al, 1989, Science 243:217-220) followed by Southern hybridization with a Coco-specific probe can allow the detection of the Coco gene in DNA from various cell types. Methods of amplification other than PCR are commonly known and can also be employed. In one embodiment, Southern hybridization can be used to determine the genetic linkage of Coco. Northern hybridization analysis can be used to determine the expression of the Coco gene. Various cell types, at various states of development or activity can be tested for Coco expression. The stringency of the hybridization conditions for both Southern and Northern hybridization can be manipulated to ensure detection of nucleic acids with the desired degree of relatedness to the specific Coco probe used. Modifications of these methods and other methods commonly known in the art can be used.

[00133] Restriction endonuclease mapping can be used to roughly determine the genetic structure of the Coco gene. Restriction maps derived by restriction endonuclease cleavage can be confirmed by DNA sequence analysis.

[00134] DNA sequence analysis can be performed by any techniques known in the art, including but not limited to the method of Maxam and Gilbert (1980, Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger, F., et al, 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463), the use of T7 DNA polymerase (Tabor and Richardson, U.S. Patent No. 4,795,699), or use of an automated DNA sequenator (e.g., Applied Biosystems, Foster City, CA).

5.4.2 PROTEIN ANALYSIS

[00135] The amino acid sequence of the Coco protein can be derived by deduction from the DNA sequence, or alternatively, by direct sequencing of the protein, e.g., with an automated amino acid sequencer. [00136] The Coco protein sequence can be further characterized by a hydrophilicity analysis (Hopp, T. and Woods, K., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824). A hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of the Coco protein and the corresponding regions of the gene sequence that encode such regions.

[00137] Secondary, structural analysis (Chou, P. and Fasman, G., 1974, Biochemistry

13:222) can also be done, to identify regions of Coco that assume specific secondary structures.

[00138] Manipulation, translation, and secondary structure prediction, open reading frame prediction and plotting, as well as determination of sequence homologies, can also be accomplished using computer software programs available in the art.

[00139] Other methods of structural analysis can also be employed. These include but are not limited to X-ray crystallography (Engstom, A., 1974, Biochem. Exp. Biol. 11:7-

13) and computer modeling (Fletterick, R. and Zoller, M. (eds.), 1986, Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, Cold Spring

Harbor Laboratory, Cold Spring Harbor, New York).

5.4.3 GENERATION OF ANTIBODIES TO COCO PROTEINS AND DERIVATIVES THEREOF

[00140] According to the invention, Coco protein, its fragments or other derivatives, or analogs thereof, may be used as an immunogen to generate antibodies that immunospecifically bind such an immunogen. In a specific embodiment, antibodies to a human Coco protein are produced. In another embodiment, antibodies to a domain (e.g., the cysteine knot domain, of a Coco protein are produced. In a specific embodiment, fragments of a Coco protein identified as hydrophilic are used as immunogens for antibody production.

[00141] Various procedures known in the art may be used for the production of polyclonal antibodies to a Coco protein or derivative or analog. In a particular embodiment, rabbit polyclonal antibodies to an epitope of a Coco protein comprising a sequence of SEQ ID NO: 2, 4, 6 or 8, or a subsequence thereof, can be obtained. For the production of antibody, various host animals can be immunized by injection with the native Coco protein, or a synthetic version, or derivative (e.g., fragment) thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, Keyhole limpet hemocyanms, dmitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. [00142] Antibodies of the invention include, but are not limited to, monoclonal antibodies, synthetic antibodies, recombinantly produced antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, and epitope-binding fragments of any of the above. In particular, antibodies used in the methods of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e, molecules that contain an antigen binding site that immunospecifically binds to Coco. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG_\, IgG₂, IgG , IgG , IgAi and IgA₂) or subclass of immunoglobulin molecule.

[00143] The antibodies used in the methods of the invention may be from any animal origin including birds and mammals (e.g., human, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken). Preferably, the antibodies are human or humanized monoclonal antibodies. As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from mice that express antibodies from human genes. [00144] The antibodies used in the methods of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may immunospecifically bind to different epitopes of a Coco polypeptide or may immunospecifically bind to both a Coco polypeptide as well a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., International Publication Nos. WO 93/17715, WO 92/08802, WO 91/00360, and WO 92/05793; Tutt, et al., 1991, J Immunol. 147:60-69; U.S. Patent Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and 5,601,819; and Kostelny et al., 1992, J. Immunol. 148:1547-1553.

[00145] The antibodies used in the methods of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

[00146] The present invention also provides antibodies of the invention or fragments thereof that comprise a framework region known to those of skill in the art. Preferably, the antibody of the invention or fragment thereof is human or humanized. [00147] hi certain embodiments, the antibody to be used with the invention binds to an intracellular epitope, i.e, is an intrabody. An intrabody comprises at least a portion of an antibody that is capable of immunospecifically binding an antigen and preferably does not contain sequences coding for its secretion. Such antibodies will bind antigen intracellularly. h one embodiment, the intrabody comprises a single-chain Fv ("sFv"). sFvs are antibody fragments comprising the V_H and V_L domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the sFv polypeptide further comprises a polypeptide linker between the V_H and V_L domains which enables the sFv to form the desired structure for antigen binding. For a review of sFvs see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer- Verlag, New York, pp. 269-315 (1994). In a further embodiment, the intrabody preferably does not encode an operable secretory sequence and thus remains within the cell (see generally Marasco, WA, 1998, "Intrabodies: Basic Research and Clinical Gene Therapy Applications" SpringeπNew York). Generation of intrabodies is well-known to the skilled artisan and is described, for example, in U.S. Patent Nos. 6,004,940; 6,072,036; 5,965,371, which are incorporated by reference in their entireties herein. Further, the construction of intrabodies is discussed in Ohage and Steipe, 1999, J. Mol. Biol. 291:1119-1128; Ohage et al., 1999, J. Mol. Biol. 291:1129-1134; and Wirtz and Steipe, 1999, Protein Science 8:2245- 2250, which references are incorporated herein by reference in their entireties. Recombinant molecular biological techniques such as those described for recombinant production of antibodies may also be used in the generation of intrabodies. [00148] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[00149] Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. Briefly, mice can be immunized with Coco (either the full length protein or a domain or other fragment thereof) and once an immune response is detected, e.g., antibodies specific for Coco are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. Hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

[00150] Accordingly, monoclonal antibodies can be generated by culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with Coco or fragment thereof with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind Coco. [00151 ] Antibody fragments which recognize specific Coco epitopes may be generated by any technique known to those of skill in the art. For example, Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain. Further, the antibodies of the present invention can also be generated using various phage display methods known in the art. [00152] In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues). The DNA encoding the VH and VL domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector is electroporated in E. coli and the E. coli is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and Ml 3 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to the Coco epitope of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9; Burton et al., 1994, Advances in Immunology 57:191-280; International Application No. PCT/GB91/01134; International Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and WO97/13844; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

[00153] Phage may be screened for Coco binding, or other Coco-related activity, such as modulation of Coco-mediated inhibition of TGF-/3, BMP or Wnt signalling. [00154] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below. Techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in International Publication No. WO 92/22324; Mullinax et al., 1992, BioTechniques 12:864; Sawai et al., 1995, AJRI 34:26; and Better et al., 1988, Science 240:1041 (said references incorporated by reference in their entireties).

[00155] To generate whole antibodies, PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones. Utilizing cloning techniques known to those of skill in the art, the PCR amplified VH domains can be cloned into vectors expressing a VH constant region, e.g., the human gamma 4 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., human kappa or lambda constant regions. Preferably, the vectors for expressing the VH or VL domains comprise an EF-lα promoter, a secretion signal, a cloning site for the variable domain, constant domains, and a selection marker such as neomycin. The VH and VL domains may also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art. [00156] For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use human or chimeric antibodies. Completely human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.

[00157] Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the J_H region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinj ected into blastocysts to produce chimeric mice. The chimeric mice are then be bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., International Publication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, lie. (TreemόhV^'CA), Genpharm (San Jose, CA) and Medarex (Princeton, NJ) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

[00158] A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397, which are incorporated herein by reference in their entirety. Chimeric antibodies comprising one or more CDRs from a non-human species and framework regions from a human immunoglobulin molecule can be produced using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; International Publication No. WO 91/09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology 28(4/5):489- 498; Studnicka et al, 1994, Protein Engineering 7:805; and Roguska et al, 1994, PNAS 91 :969), and chain shuffling (U.S. Patent No. 5,565,332). Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., U.S. Patent No. 5,585,089; and Riechmann et al, 1988, Nature 332:323, which are incorporated herein by reference in their entireties.)

5.4.4 POLYNUCLEOTIDES ENCODING AN ANTIBODY

[00159] The methods of the invention also encompass polynucleotides that encode an antibody of the invention. The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art (e.g., by cloning or amplifying the nucleic acids encoding the heavy to light chain antibody, e.g., from the hybridoma, and sequencing the nucleic acids encoding the heavy and light chains). Such a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR. [00160] Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5 ' ends of the sequence or by cloning using an oligonueleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

[00161 ] Once the nucleotide sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

[00162] In a specific embodiment, one or more of the CDRs is inserted within framework regions using routine recombinant DNA techniques. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., 1998, J. Mol. Biol. 278:457-479 for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds to Coco. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art. 5.4.5 RECOMBINANT EXPRESSION OF AN ANTIBODY

[00163] Recombinant expression of an antibody of the invention, derivative, analog or fragment thereof, (e.g. , a heavy or light chain of an antibody of the invention or a portion thereof or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably, but not necessarily, containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody or a portion thereof, or a heavy or light chain CDR, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., International Publication Nos. WO 86/05807 and WO 89/01036; and U.S. Patent No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.

[00164] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention or fragments thereof, or a heavy or light chain thereof, or portion thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double- chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below. [00165] A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention (see, e.g., U.S. Patent No. 5,807,715). Such host- expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionem promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990, BioTechnology 8:2).

[00166] In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. Other vectors that can be prepared, include, but are not limited to, HA fusions used to measure the interaction of Coco with other proteins (See e.g., Example 6.2.5). pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[00167] In an insect system, Autographa californica nuclear polyhedrosis virus

(AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non- essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

[00168] In mammalian host cells, a number of viral-based expression systems may be utilized, hi cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest maybe ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. Tins chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, 1984, PNAS 8 1:355-359). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:516-544).

[00169] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells.

[00170] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.

[00171] A number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11 :223), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al, 1980, Cell 22:8-17) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, PNAS 11-351; O'Hare et al., 1981, PNAS 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, PNAS 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573; Mulligan, 1993, Science 260:926; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191; May, 1993, TIB TECH 11:155-); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre- Garapin et al., 1981, J. Mol. Biol. 150:1, which are incorporated by reference herein in their entireties.

[00172] The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Grouse et al., 1983, Mol. Cell. Biol. 3:257).

[00173] The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. hi such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, PNAS 77:2197). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

[00174] Once an antibody molecule of the invention has been produced by recombinant expression, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antibodies of the present invention or fragments thereof may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[00175] In another embodiment of the invention (see infra), anti-Coco antibodies and fragments thereof containing the antigen binding domain are Therapeutics. [00176] The present invention encompasses the use of antibodies or fragments thereof recombinantly fused or chemically conjugated (including both covalent and non- covalent conjugations) to a heterologous polypeptide (or portion thereof, preferably to a polypepetide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. For example, antibodies may be used to target heterologous polypeptides to particular cell types, either in vitro or in vivo, by fusing or conjugating the antibodies to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to heterologous polypeptides may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., PCT publication WO 93/21232; EP 439,095; Naramura et al, Immunol. Lett. 39:91-99 (1994); U.S. Patent 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); and Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.

[00177] The present invention further includes compositions comprising heterologous polypeptides fused or conjugated to antibody fragments. For example, the heterologous polypeptides may be fused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab)₂ fragment, or portion thereof. Methods for fusing or conjugating polypeptides to antibody portions are known in the art. See, e.g., U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; EP 307,434; EP 367,166; PCT publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al, Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al, J. Immunol. 154:5590-5600 (1995); and Vil et al, Proc. Natl. Acad. Sci. USA 89:11337- 11341(1992) (said references incorporated by reference in their entireties).

[00178] Additional fusion proteins may be generated through the techniques of gene- shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be employed to alter the activities of antibodies of the invention or fragments thereof (e.g., antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308- 13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). Antibodies or fragments thereof, or the encoded antibodies or fragments thereof, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. [00179] Moreover, the antibodies or fragments thereof can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al, 1984, Cell 37:767) and the "flag" tag. [00180] In other embodiments, antibodies of the present invention or fragments or variants thereof conjugated to a diagnostic or detectable agent. Such antibodies can be useful for detecting, monitoring or prognosing the development or progression of a disease or disorder associated with aberrant Coco expression. Such diagnosis and detection can accomplished by coupling the antibody to detectable substances including, but not limited to various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as but not limited to streptavidin/biotin and avidin/biotin; fluorescent materials, such as but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and

• 1 ^* 1 1 19^ 1 1 aequorin; radioactive materials, such as but not limited to iodme ( I, I, I, I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²h , ^{π ι}In), and technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re,¹⁴² Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Sn; positron emitting metals using various positron emission tomographies, and noradioactive paramagnetic metal ions.

[00181] An antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., one of the radioactive ions described above, such as, but not limited to, alpha-emitters. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (U) ^'(D^'DP)^{' '}cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

[00182] Further, an antibody or fragment thereof may be conjugated to a therapeutic agent or drug moiety that modifies a given biological response. Therapeutic agents or drug moieties are not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, /3-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-a, TNF-/3, AIM I (see, International Publication No. WO 97/33899), AIM II (see, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al, 1994, J. hninunol., 6:1567-1574), and VEGI (see, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, a biological response modifier such as, for example, a lymphokine (e.g., interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), and granulocyte colony stimulating factor ("G-CSF")), or a growth factor (e.g., growth hormone ("GH")). [00183] Moreover, an antibody can be conjugated to therapeutic moieties such as a

• 91 ^" radioactive metal ion, such as alph-emiters such as Bi or macrocychc chelators useful for conjugating radiometal ions, including but not limited to, In, Lu, Y, Ho, Sm, to polypeptides. In certain embodiments, the macrocychc chelator is 1,4,7,10- tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOT A) which can be attached to the antibody via a linker molecule. Such linker molecules are commonly known in the art and described in Denardo et al, Clin Cancer Res. 4(10):2483-90 (1998); Peterson et al. Bioconjug. Chem. 10(4):553-7 (1999); and Zimmermanet al, Nucl Med. Biol. 26(8):943-50 (1999) each incorporated by reference in their entireties.

[00184] Techniques for conjugating therapeutic moieties to antibodies are well known, see, e.g., Arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellsirom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. _{4 /} -506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of

Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer

Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and

Thorpe et al, 1982, Immunol. Rev. 62:119-58.

[00185] Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

[00186] Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

[00187] The foregoing antibodies can be used in methods known in the art relating to the localization and activity of the Coco protein sequences of the invention, e.g., for imaging these proteins, measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc.

[00188] In another embodiment of the invention (see infra) anti-Coco antibodies and fragments thereof containing the binding domain are Therapeutics.

5.4.6 COCO PROTEINS, DERIVATIVES AND ANALOGS

[00189] The invention further provides Coco proteins, and derivatives (including, but not limited to, fragments) and analogs of Coco proteins. In one embodiment, the Coco proteins are encoded by the Coco nucleic acids described in Section 5.1 supra. In particular aspects, the proteins, derivatives, or analogs are of Coco proteins of animals, e.g., frog, mouse, fly, fish, rat, pig, cow, dog, monkey, human, or of plants. In specific embodiments, the Coco proteins comprise an amino acid sequence of SEQ ID NO: 2, 4, 6 or 8. [00190] The production and use of derivatives and analogs related to Coco are within the scope of the present invention. In a specific embodiment, the derivative or analog is functionally active, i.e, capable of exhibiting one or more functional activities associated with a full-length, wild-type Coco protein. As one example, such derivatives or analogs that have the desired immunogenicity or antigenicity can be used, for example, in immunoassays, for immunization, for inhibition of Coco activity, etc. Derivatives or analogs that retain, or alternatively lack or inhibit, a desired Coco property of interest (e.g., binding to a Coco binding partner, neural, bone, or cartilage induction activity, inhibition of mesodermal or epidermal induction, inhibition of cell proliferation or transformed cell phenotype, maintenance of or induction of undifferentiated stem cell phenotype, inhibition of BMP, TGF and Wnt signaling pathways), can be used as inducers, or inhibitors, respectively, of such property and its physiological correlates. A specific embodiment provides a Coco fragment that can be bound specifically by an anti-Coco antibody. Derivatives or analogs of Coco can be tested for the desired activity by procedures known in the art, including but not limited to the assays described infra.

[00191] hi particular, Coco derivatives can be made by altering Coco sequences by substitutions, additions or deletions that provide for functionally equivalent molecules. The Coco derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a Coco protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[00192] In a specific embodiment of the invention, proteins consisting of or comprising a fragment of a Coco protein consisting of at least 10 (continuous) amino acids of the Coco protein is provided. In other embodiments, the fragment consists of at least 20 or 50 amino acids of the Coco protein. In specific embodiments, such fragments are not larger than 35, 100 or 200 amino acids. Derivatives or analogs of Coco include but are not limited to those molecules comprising regions that are substantially homologous to Coco or fragments thereof (e.g., in various embodiments, at least 60% or 70% or 80% or 90% or 95%> identity over an amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art) or whose encoding nucleic acid is capable of hybridizing to a coding Coco sequence, under stringent, moderately stringent, or nonstringent conditions. [00193] The Coco derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations that result in their production can occur at the gene or protein level. For example, the cloned Coco gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al, 2001, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, N.Y.; Ausubel et al, 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. In the production of the gene encoding a derivative or analog of Coco, care should be taken to ensure that the modified gene remains within the same translational reading frame as Coco, uninterrupted by translational stop signals, in the gene region where the desired Coco activity is encoded.

[00194] Additionally, the Coco-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson, C, et al., 1978, J. Biol. Chem 253:6551), use of TAB® linkers (Pharmacia), etc.

[00195] Manipulations of the Coco sequence may also be made at the protein level.

Included within the scope of the invention are Coco protein fragments or other derivatives or analogs that are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.

[00196] hi addition, analogs and derivatives of Coco can be chemically synthesized.

For example, a peptide corresponding to a portion of a Coco protein that comprises the desired domain, or that mediates the desired activity in vitro, can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the Coco sequence. Non- classical amino acids include but are not limited to the D-isomers of the common amino acids, c-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, 7-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t- butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, /3-alanine, fluoro-amino acids, designer amino acids such as 3-methyl amino acids, Cα-methyl amino acids, Nα- methyl ammo aciαs, and ammo acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

[00197] i a specific embodiment, the Coco derivative is a chimeric, or fusion, protein comprising a Coco protein or fragment thereof (preferably consisting of at least a domain or motif of the Coco protein, or at least 10 amino acids of the Coco protein) joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein. In one embodiment, such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising a Coco-coding sequence joined in-frame to a coding sequence for a different protein). Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques, e.g. , by use of a peptide synthesizer. Chimeric genes comprising portions of Coco fused to any heterologous protein-encoding sequences may be constructed. A specific embodiment provides a chimeric protein comprising a fragment of Coco of at least six amino acids. [00198] One useful fusion protein is a GST or HA fusion protein in which the polypeptide of the invention is fused to the C-terminus of GST or HA sequences. Such fusion proteins can facilitate the purification of a recombinant polypeptide of the invention. [00199] In another embodiment, the fusion protein contains a heterologous signal sequence at its N-terminus. For example, the native signal sequence of a polypeptide of the invention can be removed and replaced with a signal sequence from another protein. For example, the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992). Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California). In yet another example, useful prokaryotic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).

[00200] In yet another embodiment, the fusion protein is an immunoglobulin fusion protein in which all or part of a polypeptide of the invention is fused to sequences derived from a member of the immunoglobulin protein family particularly all or part of a constant domain (or F_c fragment of an immunoglubin, e.g., an IgG (see e.g., U.S. Patent No. 5,116,964 by Capon et al.)). The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject. The 'ϊmh unδ^'grόbulm fusion protein can be used to affect the bioavailabihty of a polypeptide of the invention. Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies directed against a polypeptide of the invention in a subject.

[00201] In another specific embodiment, the Coco derivative is a molecule comprising a region of homology with a Coco protein. By way of example, in various embodiments, a first protein region can be considered "homologous" to a second protein region when the amino acid sequence of the first region is at least 30%, 40%, 50%, 60%, 70%), 75%, 80%), 90%), or 95% identical, when compared to any sequence in the second region of an equal number of amino acids as the number contained in the first region or when compared to an aligned sequence of the second region that has been aligned by a computer homology program known in the art. For example, a molecule can comprise one or more regions homologous to a Coco domain or a portion thereof. [00202] A signal sequence of a polypeptide of the invention, for example, amino acids 1 through 22 of SEQ ID NO: 2, 4, 6, or 8, can be used to facilitate secretion and isolation of the secreted protein or other proteins of interest. Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway. Thus, the invention pertains to the described polypeptides having a signal sequence, as well as to the signal sequence itself and to the polypeptide in the absence of the signal sequence (i.e, the cleavage products). In one embodiment, a nucleic acid sequence encoding a signal sequence of the invention can be operably linked in an expression vector to a protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate. The signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by art recognized methods. Alternatively, the signal sequence can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.

[00203] In another embodiment, the signal sequences of the present invention can be used to identify regulatory sequences, e.g., promoters, enhancers, repressors. Since signal sequences are the most amino-terminal sequences of a peptide, it is expected that the nucleic acids which flank the signal sequence on its amino-terminal side will be regulatory sequences which affect transcription. Thus, a nucleotide sequence which encodes all or a portion of a signal sequence can be used as a probe to identify and isolate signal sequences and their flanking regions, and these flanking regions can be studied to identify regulatory elements therein.

[00204] Other specific embodiments of derivatives and analogs are described in the subsection below and examples sections infra.

[00205] The Coco polypeptides of the invention can also be conjugate to a heterologous moiety, e.g., to an antibody.

5.4.7 DERIVATIVES OF COCO CONTAINING ONE OR MORE DOMAINS OF THE PROTEIN

[00206] h a specific embodiment, the invention provides Coco derivatives and analogs, in particular Coco fragments and derivatives of such fragments, that comprise, or alternatively consist of, one or more domains of a Coco protein, including but not limited to the cysteine knot domain, and/or functional (e.g., binding) fragments of any of the foregoing. In particular examples relating to the Xenopus, mouse and human Coco proteins, such domains are disclosed in Section 6, and in FIG. 6B.

[00207] A specific embodiment provides molecules comprising specific fragments of

Coco that are those fragments in the respective Coco protein most homologous to specific fragments of a human or mouse Coco protein. A fragment comprising a domain of a Coco homolog can be identified by protein analysis methods as described in Sections 5 or 6. [00208] In a specific embodiment, a Coco protein, derivative or analog is provided that has a cysteine knot domain (SEQ ID NO: 2, 4, or 8) of a Coco protein. In another specific embodiment, a molecule is provided that lacks one or more domains (or functional portion thereof) of a Coco protein, hi particular examples, Coco protein derivatives are provided that lack a cysteine knot domain or one or more cysteines of the cysteine knot domain, e.g., the mouse protein encoded by the partial mouse sequence provided herein (SEQ ID NO: 5). In another embodiment, a molecule is provided that comprises one or more domains (or functional portion thereof) of a Coco protein, and that has one or more mutant (e.g., due to deletion or point mutation(s)) domains of a Coco protein (e.g., such that the mutant domain has decreased function). By way of example, the domain may be mutant so as to have reduced, absent, or increased neural, bone, or cartilage induction, stem cell maintenance, or inhibition of cell proliferation activity. 5.4.8 ASSAYS OF COCO PROTEINS, DERIVATIVES AND ANALOGS

[00209] The functional activity of Coco proteins, derivatives and analogs can be assayed by various methods. The functional activity of Coco proteins, derivatives, and analogs can also be assayed in order to test the Coco inhibitors.

[00210] For example, in one embodiment, where one is assaying for the ability to bind or compete with wild-type Coco for binding to anti-Coco antibody, various immunoassays known in the art can be used, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody, h another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

[00211] In another embodiment, where a Coco-binding protein is identified, the binding can be assayed, e.g., by means well-known in the art. In another embodiment, physiological correlates of Coco binding to its substrates (signal transduction) can be assayed.

[00212] In another embodiment, a competence assay (e.g., an animal cap assay, such as that set forth in Section 6) is used measure induction of mesodermal markers by Coco. By way of example, mRNA, encoding the protein to be tested for Coco activity, is injected into embryos at various stages of development (e.g., at the 2 or 4-cell stage or at the gastrula stage). Animal cap explants are then harvested at a later stage, e.g., i Xenopus, at stage 8. In certain embodiments, uninjected and Coco-injected explants maybe incubated in, e.g., activin, or another mesoderm inducer, at stages 8, 9, 10 and 11. For example, Xenopus explants maybe harvested at stage 12/13. The embryos are analyzed, using standard methods, for level of expression of the mesodermal markers (e.g., brachy ry (Smith et al, 1991, Cell 67:79-87) andfgβ (Christen and Slack, 1997, Dev. Biol. 192:455-466)), which are expected to be repressed by Coco. In addition, the endogenous ectodermal expression of Otx2 should increase or be induced by a molecule having Coco activity. [00213] In another embodiment, to analyse Coco's activity at a molecular level, fate changes in embryonic explants may be analyzed by RT-PCR for a variety of molecular markers. For example, embryos may be injected with the test molecule, at the 2 cell-stage in the animal caps, and may be analyzed later, in gastrula-staged explants, for the presence of markers for the organizer or endoderm, e.g., pan-neural markers (Ncam and nrpl), anterior-specific markers (Otx and XAG), and Nkx2.5.

[00214] In another embodiment, Coco mRNA (e.g., xCoco mRNA) may be injected into the ventral marginal zone (VMZ) of a Xenopus embryo at the 4-cell stage. The consequence of such injection on cell fate determination may be analyzed in explants isolated at gastrula stages or for morphological changes at tadpole (stage 27) stages to test whether xCoco can neuralize ventral tissue. At gastrula stages, the organizer markers chordin and goosecoid are expected to be weakly induced in the VMZ expressing xCoco, whereas the expression of brachyury is expected to be suppressed. At st27, uninjected VMZ explants are not expected to express the neural markers Ncam, nrpl and Otx (Pannese et al, 1995, Development 121:707-720), nor the cement gland marker XAG. In injected VMZ explants expressing xCoco, all of these markers are expected to be induced, consistent with Coco blocking BMP signals.

[00215] In another embodiment, a Coco protein is co-injected together with RNAs encoding bmp4, Xnrl (nodal-related factor- 1; Hyde and Old, 2000, Development 127:1221-1229), or WntS, or exposed injected caps to activin conditioned media (see Section 6), and the expression of immediate response genes normally activated by these signaling molecules in the ectoderm is monitored. For instance, exposure to BMP4 induces the expression of brachyury and increases epidermal keratin expression. In this assay, Coco should block induction of these markers. Coco should also block Wnt8 induction of Xnr3 and siamois expression (Sokol and Melton, 1992, Nature 351:409-411) and nodal signaling, as detected by the inhibition of the expression of chordin, brachyury and Wnt8 induced by Xnrl.

[00216] In another embodiment, Coco function can be assayed by observing activin signaling inhibition. Activin induces dorsal mesoderm in animal caps, causing them to elongate (Smith et al, 1990, Nature 345:729-731; Sokol and Melton, 1991, Nature 351:409- 411). Embryos may be injected at the 2-cell stage with Coco and explanted animal caps in medium containing activin protein. Both control and Coco injected caps should elongate in "'the presence of activin. Coco should also be unable to suppress the expression of markers induced by activin (chordin, brachyury and Wnt8).

[00217] In another embodiment, a biochemical assay is used to measure the interaction of Coco with Xnrl , BMP4, or Wnt8. By way of example, Coco may be flag-tagged in the C-terminus by standard PCR methods. Coco-flag tagged may be co-injected into embryos at the 2-cell stage with BMP4-HA or Xnrl -HA. Explants are then harvested at stage (st) 10-11. Homogenized explants were tested for direct binding in immuno-precipitation experiments (see Section 6). For example, explants may be co-immunoprecipitated with an anti-HA polyclonal antibody, using antibodies and procedures obtained from Sigma (St. Louis, MO) and then probed with an anti-flag monoclonal antibody (Sigma, St. Louis, MO). Assays can be performed in order to examine the effects of molecules of the invention on neural cell growth, differentiation, induction, or maintenance, bone cell growth, differentiation, induction, or maintenance, cartilage cell growth, induction, differentiation, or maintenance, and epidermal cell growth, induction, maintenance, or differentiation, as well as cell proliferation and transformation and induction or maintenance of stem cell state of stem cells. Such assays are well known in the art.

[00218] In another embodiment, in insect or other model systems, genetic studies can be done to study the phenotypic effect of a Coco mutant that is a derivative or analog of wild-type Coco, e.g., in the non-human transgenic animals as described in Section 5.2, supra.

[00219] In addition, assays that can be used to detect or measure the ability to inhibit, or alternatively promote, neural induction are described in Section 5.8.8. [00220] Other methods will be known to the skilled artisan and are within the scope of the invention.

5.5 THERAPEUTIC USES

[00221] The invention provides for treatment or prevention of various diseases and disorders by administration of a therapeutic compound (termed herein "Therapeutic"). Such "Therapeutics" include but are not limited to: Coco proteins and functionally active analogs and derivatives (including fragments) thereof (e.g., as described hereinabove); antibodies, and antigen binding fragments thereto (as described hereinabove); peptidomimetics; nucleic acids encoding the Coco proteins, analogs, or derivatives (e.g., as described hereinabove); Coco antisense nucleic acids, double stranded RNA, and ribozymes and Coco agonists and antagonists, e.g., small molecules. Disorders involving neural tissue growth and/or differentiation, epidermal tissue growth and/or differentiation, bone or * carϊifage^'or other connective tissue growth and/or differentiation, hyper or hypo cell proliferation, or other disease or disorder associated with abnormal Coco expression and/or abnormal TGF-β, BMP, or Wnt signalling are treated or prevented by administration of a

Therapeutic that promotes Coco function. Disorders in which neural cell induction, growth, differentiation or maintenance is desired or in which an inhibition of epidermal, bone or cartilege cell induction, growth, differentiation or maintenance, is desired, or inhibition of cell proliferation or a transformed cell phenotype is desired, are treated or prevented by administration of a Therapeutic that induces, increases or upregulates Coco function.

Diseases and disorders in which epidermal bone or cartilege cell growth, induction, differentiation, or maintenance, is desired, or inhibition of neural cell growth, induction, differentiation or maintenance is desired are treated or prevented by administration of a

Therapeutic that reduces or inhibits Coco function. The above is described in detail in the subsections below.

[00222] Generally, administration of products of a species origin or species reactivity

(in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, a human Coco protein, derivative, analog, or nucleic acid, or an antibody to a human Coco protein, preferably a human or humanized antibody, is therapeutically or prophylactically admimstered to a human patient.

[00223] Additional descriptions and sources of Therapeutics that can be used according to the invention are found in Sections 5.1 through 5.7 herein..

[00224] In another embodiment, administration of a therapeutic of the invention may be used to modulate TGF-β, BMP, or Wnt signalling. In particular, embodies, BMP-4,

Wnt-8 or nodal signaling is modulated.

5.5.1 TREATMENT AND PREVENTION OF DISORDERS INVOLVING NEURAL CELL GROWTH, DIFFERENTIATION, OR INDUCTION.

[00225] The methods and compositions of the present invention are useful in the treatment, prevention, management or amelioration of symptoms of a variety of diseases and disorders involving neural cell growth, differentiation, maintenance, or induction. In one embodiment, the invention relates to the treatment of diseases and disorders involving a deficiency in neural cell growth, differentiation, maintenance, or induction by administration of a Therapeutic that promotes and/or mimics Coco activity or function.

Diseases and disorders involving a deficiency in neural induction or neural cell growth, maintenance, and differentiation are treated or prevented by administration of a Therapeutic that promotes (i.e, increases or supplies) Coco function. Examples of such a Therapeutic include, but are not limited to, Coco proteins, dervatives, or fragments that are functionally active, particularly that are active in neural induction (e.g., as demonstrated in in vitro assays or in animal models or in Xenopus embryos), and nucleic acids encoding a Coco protein or functionally active derivative or fragment thereof (e.g., for use in gene therapy). Other Therapeutics that can be used, e.g., Coco agonists, can be identified using in vitro assays or animal models, or assays in Xenopus, examples of which are described infra. [00226] Diseases and disorders involving a decreased level of neural cell growth, maintenance, or induction, that can be treated or prevented by promoting Coco function, include, but are not limited to, degenerative disorders, growth deficiencies, hypoprohferative disorders, physical trauma, lesions, wounds, or to promote regeneration in degenerated, lesioned, or injured tissues, etc. hi a specific embodiment, nervous system disorders are treated. In another specific embodiment, a disorder that is not of the nervous system is treated.

[00227] In one embodiment, such diseases and disorders are treated or prevented by the administration of a Therapeutic that promotes Coco activity, expression, or function. In another embodiment, a Therapeutic of the invention mimics the activity of a Coco protein. In yet another embodiment, a Therapeutic of the invention promotes the expression and or translation of Coco proteins.

[00228] h a more specific embodiment, the Therapeutic of the invention is a Coco protein, fragment or agonist thereof, that can promote neural cell growth, differentiation, maintenance, or induction. In another more specific embodiment, the Therapeutic of the invention is a Coco protein or fragment thereof, that is able to modulate the activity of a BMP, TGF-/3, or Wnt signalling pathway. In yet another more specific embodiment, the Therapeutic of the invention is a Coco protein or fragment thereof, that is able to decrease or inhibit the activity of a BMP, TGF-/5, or Wnt signalling pathway, thereby promoting neural cell growth, differentiation, maintenance, or induction. In a more specific embodiment, the Coco protein or fragment of the invention has an amino acid sequence corresponding to SEQ ID NO: 2, 4, 6, or 8, or a fragment thereof, hi another more specific embodiment, the Therapeutic of the invention comprises the cysteine knot domain of a Coco protein, or a fragment thereof, and is active in neural induction, h another specific embodiment, the Therapeutic of the invention promotes Coco function and is a derivative or analog comprising a domain of a Coco protein (e.g., the cysteine knot domain) that has been mutated so as to be dominantly active.

[00229] In another embodiment of the invention, a Therapeutic of the invention is a peptidomimetic of the Coco protein that is active in neural induction. In a more specific embodiment, said peptidomimetic mimics the activity of Coco. In another embodiment, the peptidomimetic of the invetion mimes the activity of Coco but also has an enhanced neural induction activity, hi one specific embodiment, the peptidomimetic of the invention inhibits or decreases the activity of BMP, TGF-β, and/or Wnt, thereby increasing neural induction activity.

[00230] The diseases or disorders related to neural cell growth, differentiation, maintenance, or induction that can be treated or prevented include, but are not limited to, the amelioration of symptoms of diseases and disorders associated with damage to, degeneration of, or defects in nervous tissue, such as, but not limited to, spinal cord, brain, peripheral nervous system tissue, etc., in a patient (preferably a mammal, more preferably a human) having such a disease or disorder. The Therapeutics of the invention are also used in methods for treating injuries to nervous tissue, such as spinal cord injuries, or traumatic brain injury in a patient having such an injury.

[00231] Lesions that may be treated according to the present invention include but are not limited to the following lesions:

[00232] (i) traumatic lesions, including lesions caused by physical injury or associated with surgery;

[00233] (ii) ischemic lesions, in which a lack of oxygen results in cell injury or death, e.g., myocardial or cerebral infarction or ischemia, or spinal cord infarction or ischemia;

[00234] (iii) malignant lesions, in which cells are destroyed or injured by malignant tissue;

[00235] (iv) infectious lesions, in which tissue is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis;

[00236] (v) degenerative lesions, in which tissue is destroyed or injured as a result of a degenerative process, including but not limited to nervous system degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis;

[00237] (vi) lesions associated with nutritional diseases or disorders, in which tissue is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration;

[00238] (vii) lesions associated with systemic diseases including but not limited to diabetes or systemic lupus erythematosus; [00239] (viii) lesions caused by toxic substances including alcohol, lead, chemotherapeutic agents or other toxins; and

[00240] (ix) demyelinated lesions of the nervous system, in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

[00241] Nervous system lesions that may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the lesions of either the central (including spinal cord, brain) or peripheral nervous systems.

[00242] Promotion of Coco function can also have uses in vitro, e.g., to expand neural cells in vitro, e.g., to grow cells/tissue in vitro prior to administration to a patient

(preferably a patient from which the cells were derived), etc.

[00243] Therapeutics that are useful according to this embodiment of the invention for treatment of a disorder may be selected by testing for biological activity in promoting the survival or differentiation of cells (see also Section 5.9). For example, in a specific embodiment relating to therapy of the nervous system, a Therapeutic that elicits one of the following effects may be useful according to the invention:

[00244] (i) increased sprouting of neurons in culture or in vivo;

[00245] (ii) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or

[00246] (iii) decreased symptoms of neuron dysfunction in vivo.

[00247] Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased sprouting of neurons may be detected by methods set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-82) or Brown et al. (1981, Ann. Rev.

Neurosci. 4:17-42); and increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured.

[00248] In other embodiments in which decreased neural cell growth, differentiation, maintenance or induction is desired, a disease or disorder is treated or prevented by administration of a Therapeutic of the invention that inhibits Coco function. [00249] In other embodiments, therapeutics of the invention are co-administered with one or more non-Coco based therapeutics, e.g., therapies that are not entirely sufficient to treat, manage, and/or ameliorate the symptoms of a disease or disorder. 5.5.2 EPIDERMAL DISORDERS

[00250] The methods and compositions of the present invention are useful in the treatment, prevention, management or amelioration of symptoms of a variety of epidermal and related disorders. In one embodiment, this invention relates to the treatment of diseases and disorders involving deficiency in epidermal cell growth, differentiation, maintenance, induction, or function, by administration of a Therapeutic that inhibits Coco function. Examples of such a Therapeutic include, but are not limited to, Coco antibodies, Coco sense, and dsRNA. Other Therapeutics that can be used, e.g., Coco antagonists, can be identified using in vitro assays or animal models, or assays in Xenopus, examples of which are described infra.

[00251] In one embodiment, epidermal diseases and disorders are treated or prevented by the administration of a Therapeutic that decreases Coco activity, expression, or function. In another embodiment, a Therapeutic of the invention decreases or inhibits the activity of a Coco protein. In another embodiment of the invention, the inhibition of Coco activity with a Therapeutic of the invention inhibits or abolishes the activity of a Coco protein, thereby promoting the activity of BMP, TGF- 3, or Wnt. In yet another embodiment, a Therapeutic of the invention prevents or inhibits the expression and/or translation of Coco proteins.

[00252] In another embodiment, the Therapeutic of the invention is an antibody that binds a Coco protein, or fragment thereof, said Coco protein or fragment having an amino acid sequence corresponding to SEQ ID NO: 2, 4, 6, or 8. In a more specific embodiment, said antibody or Therapeutic decreases the activity of Coco and thereby promotes the activity of BMP, TGF-/3, and/or Wnt. h another embodiment of the invention, epidermal growth or differentiation is enhanced due to the inhibition of Coco activity using said Coco antibody as a Therapeutic.

[00253] In another specific embodiment, the Therapeutic of the invention is used to treat a number of disorders that are related to wound healing, including but not limited to, idiopathic pulmonary fibrosis, scleroderma, keloids, and other disorders that share the characteristics of fibrosis. Other diseases or disorders that can be treated or prevented include, but are not limited to, epidermal hyperprohferation, psoriasis, scar tissue formation, allergic contact dermatitis, systemic sclerosis, atopic dermatitis, leprosy, congestive heart failure, chronic obstructive pulmonary disease, liver cirrohsis, and kidney diseases. [00254] In other embodiments in which decreased epidermal cell growth, differentiation, maintenance or induction is desired, a disease or disorder is treated or prevented by administration of a Therapeutic of the invention that promotes Coco function. [00255] In other embodiments, therapeutics of the invention are co-administered with one or more non-Coco based therapeutics, e.g., therapies that are not entirely sufficient to treat, manage, and/or ameliorate the symptoms of a disease or disorder. [00256] Inhibition of Coco function can also have uses in vitro, e.g., to expand epidermal cells in vitro, e.g., to grow cells/tissue in vitro prior to administration to a patient (preferably a patient from which the cells were derived), etc.

5.5.3 BONE AND CARTILEGE DISORDERS

[00257] The methods and compositions of the present invention are useful in the treatment, prevention, management or amelioration of symptoms of a variety of bone and cartilege and related disorders. In one embodiment, this invention relates to the treatment of diseases and disorders involving deficiency in bone or cartilege cell growth, differentiation, maintenance, induction, or function, by administration of a Therapeutic that inhibits Coco function. Examples of such a Therapeutic include, but are not limited to, Coco antibodies, Coco sense, and dsRNA. Other Therapeutics that can be used, e.g., Coco antagonists, can be identified using in vitro assays or animal models, or assays in Xenopus, examples of which are described infra.

[00258] In one embodiment, epidermal diseases and disorders are treated or prevented by the administration of a Therapeutic that decreases Coco activity, expression, or function. In another embodiment, a Therapeutic of the invention decreases or inhibits the activity of a Coco protein. In another embodiment of the invention, the inhibition of Coco activity with a Therapeutic of the invention inhibits or abolishes the activity of a Coco protein, thereby promoting the activity of BMP, TGF-/3, or Wnt. In yet another embodiment, a Therapeutic of the invention prevents or inhibits the expression and/or translation of Coco proteins.

[00259] In another embodiment, the Therapeutic of the invention is an antibody that binds a Coco protein, or fragment thereof, said Coco protein or fragment having an amino acid sequence corresponding to SEQ ID NO: 2, 4, 6, or 8. In a more specific embodiment, said antibody or Therapeutic decreases the activity of Coco and thereby promotes the activity of BMP, TGF-β, and/or Wnt. In another embodiment of the invention, bone or cartilege cell growth or differentiation is enhanced due to the inhibition of Coco activity using said Coco antibody as a Therapeutic, hi other embodiments, a disease or disorder is treated or pro vented using a Therapeutic of the invention that promotes Coco activity, for example when decreased bone or cartilege cell growth, differentiation, maintenance or induction is desired.

[00260] hi another specific embodiment, the Therapeutic of the invention is used to treat or prevent a number of disorders such as Padget's disease, osteoarthritis, osteonecrosis, osteochondritis, osteoporosis, osteomalacia, bone and cartilege injury, congenital hip dysplasia, epiphyseal dysplasia, chondrodysplasias, Perthes disease, Kashin- Bek disease, joint hypermobility, cancers of bone and cartilege, etc. [00261] hi other embodiments in which decreased bone or cartilege cell growth, differentiation, maintenance or induction is desired, a disease or disorder is treated or prevented by administration of a Therapeutic of the invention that promotes Coco function. [00262] In other embodiments, therapeutics of the invention are co-administered with one or more non-Coco based therapeutics, e.g., therapies that are not entirely sufficient to treat, manage, and/or ameliorate the symptoms of a disease or disorder. [00263] Promotion of Coco function can also have uses in vitro, e.g., to expand bone or cartilege cells in vitro, e.g., to grow cells/tissue in vitro prior to administration to a patient (preferably a patient from which the cells were derived), etc. 5.5.4 CELL PROLIFERATION DISORDERS

[00264] Coco also has a role in modulating cell proliferation and cell transformation

(i.e., transformation to a cell lacking contact inhibition, such as a cancer cell). Accordingly, Therapeutics of the invention, particularly Therapeutics that promote Coco function maybe useful in treating or preventing hyperproliferative active disorders, particularly cancers. [00265] Cancers and related disorders that can be treated or prevented by methods and compositions of the present invention include but are not limited to the following: Leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erytliroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrδm's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas such as but not limited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, hposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer; adrenal cancer such as but not limited to pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer such as but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers such as but limited to Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; eye cancers such as but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and cilliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers such as but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers such as but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers such as but not limited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, hposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers such as but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancers such as adenocarcinoma; cholangiocarcinomas such as but not limited to pappillary, nodular, and diffuse; lung cancers such as non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers such as but not limited to renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and/ or uterer); Wilms' tumor; bladder cancers such as but not limited to transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., Inc., United States of America)

[00266] Accordingly, the methods and compositions of the invention are also useful in the treatment or prevention of a variety of cancers or other abnormal proliferative diseases, including (but not limited to) the following: carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Berketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal orignin, including fibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscarama, and osteosarcoma; and other tumors, including melanoma, xenoderma pegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer and teratocarcinoma. It is also contemplated that cancers caused by aberrations in apoptosis would also be treated by the methods and compositions of the invention. Such cancers may include but not be limited to follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes. In specific embodiments, malignancy or dysproliferative changes (such as metaplasias and dysplasias), or hyperproliferative disorders, are treated or prevented in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus. In other specific embodiments, sarcoma, melanoma, or leukemia is treated or prevented.

[00267] In some embodiments, therapy by administration of one or more

Therapeutics of the invention is combined with the administration of one or more therapies such as, but not limited to, chemotherapies, radiation therapies, hormonal therapies, and/or biological therapies/immunotherapies.

[00268] In a specific embodiment, the methods of the invention encompass the administration of one or more angio genesis inhibitors such as but not limited to: Angiostatin (plasminogen fragment); antiangiogenic antithrombin III; Angiozyme; ABT- 627; Bay 12-9566; Benefm; Bevacizumab; BMS-275291; cartilage-derived inhibitor (CDI); CAI; CD59 complement fragment; CEP-7055; Col 3; Combretastatin A-4; Endostatin (collagen XVIII fragment); fibronectin fragment; Gro-beta; Halofuginone; Heparinases; Heparin hexasaccharide fragment; HMV833; Human chorionic gonadotropin (hCG); IM- 862; Interferon alpha/beta/gamma; Interferon inducible protein (IP-10); Interleukin- 12; Kringle 5 (plasminogen fragment); Marimastat; Metalloproteinase inhibitors (TDVIPs); 2- Methoxyestradiol; MMI 270 (CGS 27023 A); MoAb IMC-1C11; Neovastat; NM-3; Panzem; PI-88; Placental ribonuclease inhibitor; Plasminogen activator inhibitor; Platelet factor-4 (PF4); Prinomastat; Prolactin 16kD fragment; Proliferin-related protein (PRP); PTK 787/ZK 222594; Retinoids; Solimastat; Squalamine; SS 3304; SU 5416; SU6668; SU11248; Tetrahydrocortisol-S; tetrathiomolybdate; thahdomide; Thrombospondin-1 (TSP- 1); TNP-470; Transforming growth factor-beta (TGF-b); Vasculostatin; Vasostatin (calreticulin fragment); ZD6126; ZD 6474; farnesyl transferase inhibitors (FTI); and bisphosphonates.

[00269] Additional examples of anti-cancer agents that can be used in the various embodiments of the invention, including pharmaceutical compositions and dosage forms and kits of the invention, include, but are not limited to: acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine, ambomycin, ametantrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan, cactinomycin, calusterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, cirolemycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decarbazine, decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin hydrochloride, erbulozole, esorubicin hydrochloride, estramustine, estramustine phosphate sodium, etanidazole, etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, flurocitabine, fosquidone, fostriecin sodium, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, ilmofosine, interleukin 2 (including recombinant interleukin 2, or rIL2), interferon alpha-2a, interferon alpha-2b, interferon alpha-nl, interferon alpha-n3, interferon beta-I a, interferon gamma-I b, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin, mitomycin, mitosper, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nitrosoureas, nocodazole, nogalamycin, ormaplatin, oxisuran, paclitaxel, pegaspargase, peliomycin, pentamustine, peplomycin sulfate, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, plomestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, riboprine, rogletimide, safmgol, safmgol hydrochloride, semustine, simtrazene, sparfosate sodium, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, talisomycin, tecogalan sodium, tegafur, teloxantrone hydrochloride, temoporfm, teniposide, teroxirone, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, toremifene citrate, trestolone acetate, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tubulozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, zorubicin hydrochloride. Other anti-cancer drugs include, but are not limited to: 20-epi-l,25 dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecypenol, adozelesin, aldesleukin, ALL-TK antagonists, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anti-dorsalizing morphogenetic protein- 1, antiandrogens, antiestrogens, antineoplaston, aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators, apurinic acid, ara-CDP-DL-PTBA, arginine deaminase, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivatives, balanol, batimastat, BCR/ABL antagonists, benzochlorins, benzoylstaurosporine, beta lactam derivatives, beta-alethine, betaclamycin B, betulinic acid, bFGF inhibitor, bicalutamide, bisantrene, bisaziridinylspermme, bisnafide, bistratene A, bizelesin, breflate, bropirimine, budotitane, buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives, canarypox IL-2, capecitabine, carboxamide-amino-triazole, carboxyamidotriazole, CaRest M3, CARN 700, cartilage derived inhibitor, carzelesin, casein kinase inhibitors (ICOS), castanospermme, cecropin B, cetrorelix, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, clomifene analogues, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analogue, conagenin, crambescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentanthraquinones, cycloplatam, cypemycin, cytarabine ocfosfate, cytolytic factor, cytostatin, dacliximab, decitabine, dehydrodidemnin B, deslorelin, dexamethasone, dexifosfamide, dexrazoxane, dexverapamil, diaziquone, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxamycin, diphenyl spiromustine, docetaxel, docosanol, dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emitefur, epirubicin, epristeride, estramustine analogue, estrogen agonists, estrogen antagonists, etanidazole, etoposide phosphate, exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunorunicin hydrochloride, forfenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfam, heregulin, hexamethylene bisacetamide, hypericin, ibandronic acid, idarubicin, idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridones, imiquimod, immunostimulant peptides, insulin-like growth factor- 1 receptor inhibitor, interferon agonists, interferons, interleukins, iobenguane, iododoxorubicin, ipomeanol, iroplact, irsogladine, isobengazole, isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, leukemia inhibiting factor, leukocyte alpha interferon, leuprolide+estrogen+progesterone, leuprorelin, levamisole, liarozole, linear polyamine analogue, lipophihc disaccharide peptide, lipophihc platinum compounds, lissoclinamide 7, lobaplatin, lombricine, lometrexol, lonidamine, losoxantrone, lovastatin, loxoribine, lurtotecan, lutetium texaphyrin, lysofylline, lytic peptides, maitansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, menogaril, merbarone, meterelin, methioninase, metoclopramide, MIF inhibitor, mifepristone, miltefosine, mirimostim, mismatched double stranded RNA, mitoguazone, mitolactol, mitomycin analogues, mitonafide, mitotoxin fibroblast growth factor-saporin, mitoxantrone, mofarotene, molgramostim, monoclonal antibody, human chorionic gonadotrophin, monophosphoryl lipid A+myobacterium cell wall sk, mopidamol, multiple drug resistance gene inhibitor, multiple tumor suppressor 1 -based therapy, mustard anticancer agent, mycaperoxide B, mycobacterial cell wall extract, myriaporone, N-acetyldinaline, N-substituted benzamides, nafarelin, nagrestip, naloxone+pentazocine, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxide antioxidant, nitruUyn, O6-benzylguanine, octreotide, okicenone, oligonucleotides, onapristone, ondansetron, ondansetron, oracin, oral cytokine inducer, ormaplatin, osaterone, oxaliplatin, oxaunomycin, paclitaxel, paclitaxel analogues, paclitaxel derivatives, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase, peldesine, pentosan polysulfate sodium, pentostatin, pentrozole, perflubron, perfosfamide, perillyl alcohol, phenazinomycin, phenylacetate, phosphatase inl ibitors, picibanil, pilocarpine hydrochloride, pirarubicin, piritrexim, placetin A, placetin B, plasminogen activator inhibitor, platinum complex, platinum compounds, platinum-triamine complex, porfimer sodium, porfiromycin, prednisone, propyl bis-acridone, prostaglandin J2, proteasome inhibitors, protein A-based immune modulator, protein kinase C inhibitor, protein kinase C inhibitors, microalgal, protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, purpurins, pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene conjugate, raf antagonists, raltitrexed, ramosetron, ras farnesyl protein transferase inhibitors, ras inhibitors, ras-GAP inhibitor, retelliptine demethylated, rhenium Re 186 etidronate, rhizoxin, ribozymes, RII retinamide, rogletimide, rohitukine, romurtide, roquinimex, rubiginone Bl, ruboxyl, safmgol, saintopin, SarCNU, sarcophytol A, sargramostim, Sdi 1 mimetics, semustine, senescence derived inhibitor 1, sense oligonucleotides, signal transduction inhibitors, signal transduction modulators, single chain antigen binding protein, sizofiran, sobuzoxane, sodium borocaptate, sodium phenylacetate, solverol, somatomedin binding protein, sonermin, sparfosic acid, spicamycin D, spiromustine, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-cell division inhibitors, stipiamide, stromelysin inhibitors, sulfinosine, superactive vasoactive intestinal peptide antagonist, suradista, suramin, swainsonine, synthetic glycosaminoglycans, tallimustine, tamoxifen methiodide, tauromustine, taxol, tazarotene, tecogalan sodium, tegafur, tellurapyrylium, telomerase inhibitors, temoporfm, temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine, thaliblastine, thahdomide, thiocoraline, thioguanine, thrombopoietin, thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid stimulating hormone, tin ethyl etiopurpurin, tirapazamine, titanocene bichloride, topsentin, toremifene, totipotent stem cell factor, translation inhibitors, tretinoin, triacetyluridine, triciribine, trimetrexate, triptorelin, tropisetron, turosteride, tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, urogenital sinus-derived growth inhibitory factor, urokinase receptor antagonists, vapreotide, variolin B, vector system, erythrocyte gene therapy, velaresol, veramine, verdins, verteporfin, vinorelbine, vinxaltine, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb, and zinostatin stimalamer. Preferred additional anticancer drugs are 5-fluorouracil and leucovorin.

[00270] The invention also encompasses administration of a Therapeutic of the invention in combination with radiation therapy comprising the use of x-rays, gamma rays and other sources of radiation to destroy the cancer cells. In preferred embodiments, the radiation treatment is administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. In other preferred embodiments, the radiation treatment is administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass. [00271 ] Cancer therapies and their dosages, routes of administration and recommended usage are known in the art and have been described in such literature as the Physician 's Desk Reference (56^th ed., 2002). 5.5.5 GENE THERAPY

[00272] In a specific embodiment, nucleic acids comprising a sequence encoding a

Coco protein or functional derivative thereof, are administered to promote Coco function, by way of gene therapy. Gene therapy refers to therapy performed by the administration of a nucleic acid to a subject. In this embodiment of the invention, the nucleic acid produces its encoded protein that mediates a therapeutic effect by promoting Coco function. [00273] Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below. [00274] For general reviews of the methods of gene therapy, see Goldspiel et al,

1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215). Methods commonly known in the art of recombinant DNA technology that can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.

[00275] h a preferred aspect, the Therapeutic comprises a Coco nucleic acid that is part of an expression vector that expresses a Coco protein or fragment or chimeric protein thereof in a suitable host, h particular, such a nucleic acid has a promoter operably linked to the Coco coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, a nucleic acid molecule is used in which the Coco coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the Coco nucleic acid (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438). [00276] Delivery of the nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

[00277] In a specific embodiment, the nucleic acid is directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering it in linkage to a peptide that is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (that can be used to target cell types specifically expressing the receptors), etc.

[00278] In another embodiment, a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated April 16, 1992 (Wu et al); WO 92/22635 dated December 23, 1992 (Wilson et al); WO92/20316 dated November 26, 1992 (Findeis et al.); WO93/14188 dated July 22, 1993 (Clarke et al), WO 93/20221 dated October 14, 1993 (Young)). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

[00279] In a specific embodiment, a viral vector that contains the Coco nucleic acid is used. For example, a retroviral vector can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA. The Coco nucleic acid to be used in gene therapy is cloned into the vector, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129- 141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114. [00280] Adenoviruses are other viral vectors that can be used in gene therapy.

Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503 present a review of adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al, 1991, Science 252:431-434; Rosenfeld et al, 1992, Cell 68:143-155; and Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234.

[00281] Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300. [00282] Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

[00283] In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell- mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al, 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.

[00284] The resulting recombinant cells can be delivered to a patient by various methods known in the art. In a preferred embodiment, epithelial cells are injected, e.g., subcutaneously. In another embodiment, recombinant skin cells may be applied as a skin graft onto the patient. Recombinant blood cells (e.g., cord blood cells,hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

[00285] Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; embryonic stem cells, various stem or progenitor cells, in particular, hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc. [00286] In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

[00287] In an embodiment in which recombinant cells are used in gene therapy, a

Coco nucleic acid is introduced into the cells such that it is expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells that can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention. Such stem cells include, but are not limited to, embryonic stem cells, hematopoietic stem cells (HSC), stem cells of epithelial tissues such as the skin and the lining of the gut, embryonic heart muscle cells, liver stem cells (PCT Publication WO 94/08598, dated April 28, 1994), and neural stem cells (Stemple and Anderson, 1992, Cell 71:973-985).

[00288] Epithelial stem cells (ESCs) or keratinocytes can be obtained from tissues such as the skin and the lining of the gut by known procedures (Rheinwald, 1980, Meth. Cell Bio. 21 A:229). In stratified epithelial tissue such as the skin, renewal occurs by mitosis of stem cells within the germinal layer, the layer closest to the basal lamina. Stem cells within the lining of the gut provide for a rapid renewal rate of this tissue. ESCs or keratinocytes obtained from the skin or lining of the gut of a patient or donor can be grown in tissue culture (Rheinwald, 1980, Meth. Cell Bio. 21 A:229; Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771). If the ESCs are provided by a donor, a method for suppression of host versus graft reactivity (e.g., irradiation, drug or antibody administration to promote moderate immunosuppression) can also be used.

[00289] With respect to hematopoietic stem cells (HSC), any technique that provides for the isolation, propagation, and maintenance in vitro of HSC can be used in this embodiment of the invention. Techniques by which this may be accomplished include (a) the isolation and establishment of HSC cultures from bone marrow cells isolated from the future host, or a donor, or (b) the use of previously established long-term HSC cultures, which may be allogeneic or xenogeneic. Non-autologous HSC are used preferably in conjunction with a method of suppressing transplantation immune reactions of the future host/patient. In a particular embodiment of the present invention, human bone marrow cells can be obtained from the posterior iliac crest by needle aspiration (see, e.g., Kodo et al., 1984, J. Clin. Invest. 73:1377-1384). In a preferred embodiment of the present invention, the HSCs can be made highly enriched or in substantially pure form. This enrichment can be accomplished before, during, or after long-term culturing, and can be done by any techniques known in the art. Long-term cultures of bone marrow cells can be established and maintained by using, for example, modified Dexter cell culture techniques (Dexter et al, 1977, J. Cell Physiol. 91:335) or Witlock-Witte culture techniques (Witlock and Witte, 1982, Proc. Natl. Acad. Sci. USA 79:3608-3612). [00290] In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.

[00291 ] Additional methods that can be adapted for use to deliver a nucleic acid encoding a Coco protein or functional derivative thereof described supra.

5.5.6 TARGETED REDUCTION OF COCO GENE EXPRESSION

5.5.6.1 ANTISENSE REGULATION OF COCO EXPRESSION

[00292] In a specific embodiment, Coco function is inhibited by use of Coco antisense nucleic acids. The present invention provides the therapeutic or prophylactic use of nucleic acids of at least six nucleotides that are antisense to a gene or cDNA encoding

Coco or a portion thereof. A Coco "antisense" nucleic acid as used herein refers to a nucleic acid capable of hybridizing to a portion of a Coco RNA (preferably mRNA) by virtue of some sequence complementarity. The antisense nucleic acid may be complementary to a coding and/or noncoding region of a Coco mRNA. Such antisense nucleic acids have utility as Therapeutics that inhibits Coco function, and can be used in the treatment or prevention of disorders as described supra in Sections 5.8.1 to 5.8.4.

[00293] The antisense nucleic acids of the invention can be oligonucleotides that are double-stranded or single-stranded, RNA or DNA or a modification or derivative thereof, which can be directly administered to a cell or can be produced intracellularly by transcription of exogenous, introduced sequences.

[00294] In a specific embodiment, the Coco antisense nucleic acids provided by the instant invention can be used to promote regeneration or growth (larger size).

[00295] The invention further provides pharmaceutical compositions comprising an effective amount of the Coco antisense nucleic acids of the invention in a pharmaceutically acceptable carrier, as described infra.

[00296] In another embodiment, the invention is directed to methods for inhibiting the expression of a Coco nucleic acid sequence in a prokaryotic or eukaryotic cell comprising providing the cell with an effective amount of a composition comprising a Coco antisense nucleic acid of the invention.

[00297] Coco antisense nucleic acids and their uses are described in detail below.

5.5.6.2 COCO ANTISENSE NUCLEIC ACIDS

[00298] The Coco antisense nucleic acids are of at least six nucleotides and are preferably oligonucleotides (ranging from 6 to about 50 oligonucleotides). In specific aspects, the oligonueleotide is at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 200 nucleotides. The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded. The oligonueleotide can be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonueleotide may include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO 88/09810, published December 15, 1988) or blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134, published April 25, 1988), hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549). [00299] In a preferred aspect of the invention, a Coco antisense oligonueleotide is provided, preferably of single-stranded DNA. In a most preferred aspect, such an oligonueleotide comprises a sequence antisense to the sequence encoding a binding domain of a Coco protein, most preferably, of a human Coco protein. The oligonueleotide may be modified at any position on its structure with substituents generally known in the art. [00300] The Coco antisense oligonueleotide may comprise at least one modified base moiety that is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5 '-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil- 5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. [00301] In another embodiment, the oligonueleotide comprises at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

[00302] In yet another embodiment, the oligonueleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof. [00303] In yet another embodiment, the oligonueleotide is an α-anomeric oligonueleotide. An α-anomeric oligonueleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual /3-units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641).

[00304] The oligonueleotide may be conjugated to another molecule, e.g. , a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

[00305] Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al, 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc. [00306] In an alternative embodiment, the Coco antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector can be introduced in vivo such that it is taken up by a cell, within which cell the vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the Coco antisense nucleic acid. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the Coco antisense RNA can be by any promoter known in the art to act in mammalian, preferably human, cells. Such promoters can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304- 310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionem gene (Brinster et al, 1982, Nature 296:39-42), etc. [00307] The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a Coco gene, preferably a human Coco gene. However, absolute complementarity, although preferred, is not required. A sequence "complementary to at least a portion of an RNA," as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded Coco antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with a Coco RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex. [00308] In another embodiment, the Coco antisense oligonueleotide is a 2'-0- methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al, 1987, FEBS Lett. 215:327-330).

5.5.6.3 RIBOZYMES

[00309] In a specific embodiment, the Coco antisense oligonueleotide comprises catalytic RNA, or a ribozyme. Ribozyme molecules designed to catalytically cleave target gene mRNA transcripts can also be used to prevent translation of target gene mRNA and, therefore, expression of target gene product (see, e.g., PCT International Publication WO 90/11364, published October 4, 1990; Sarver et al., 1990, Science 247:1222-1225). [00310] Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. (For a review, see Rossi, 1994, Current Biology 4, 469-471). The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage event. The composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see, e.g., U.S. Patent No. 5,093,246, which is incorporated herein by reference in its entirety. [00311] While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy target gene mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Myers, 1995, Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, New York, (see especially Figure 4, page 833) and in Haseloff and Gerlach, 1988, Nature, 334, 585-591, which is incorporated herein by reference in its entirety.

[00312] Preferably the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the target gene mRNA, i.e, to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

[00313] The ribozymes of the present invention also include RNA endoribonucleases

(hereinafter "Cech-type ribozymes") such as the one that occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and that has been extensively described by Cech and collaborators (Zaug, et al, 1984, Science, 224, 574-578; Zaug and Cech, 1986,

Science, 231, 470-475; Zaug, et al, 1986, Nature, 324, 429-433; published International patent application No. WO 88/04300 by University Patents Inc.; Been and Cech, 1986, Cell,

47, 207-216). The Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place.

The invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in the target gene.

[00314] As in the antisense approach, the ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells that express the target gene in vivo. A preferred method of delivery involves using a

DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous target gene messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

5.5.6.4 TARGETED HOMOLOGOUS RECOMBINATION OF COCO GENES

[00315] Endogenous target gene expression can also be reduced by inactivating or

"knocking out" the target gene or its promoter using targeted homologous recombination (e.g., see Smithies, et al, 1985, Nature 317, 230-234; Thomas and Capecchi, 1987, Cell 51, 503-512; Thompson, et al, 1989, Cell 5, 313-321; each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional target gene (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous target gene (either the coding regions or regulatory regions of the target gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene. Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target gene (e.g., see Thomas and Capecchi, 1987, Cell 51, 503-512; Thompson, et al, 1989, Cell 5, 313-321). However this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors.

5.5.6.5 TRIPLE HELIX MOLECULES

[00316] Alternatively, endogenous target gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target gene (i.e, the target gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the target gene in target cells in the body. (See generally, Helene, 1991, Anticancer Drug Des., 6(6), 569-584; Helene, et al, 1992, Aim. N.Y. Acad. Sci., 660, 27-36; and Maher, 1992, Bioassays 14(12), 807-815). [00317] Nucleic acid molecules to be used in triplex helix formation for the inhibition of transcription should be single stranded and composed of deoxynucleotides. The base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex. Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC⁺ triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, nucleic acid molecules may be chosen that are purine-rich, for example, contain a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.

[00318] Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5 '-3', 3 '-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex. [00319] In instances wherein the antisense, ribozyme, and/or triple helix molecules described herein are utilized to inhibit mutant gene expression, it is possible that the technique may so efficiently reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles that the possibility may arise wherein the concentration of normal target gene product present may be lower than is necessary for a normal phenotype. In such cases, to ensure that substantially normal levels of target gene activity are maintained, therefore, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity may, be introduced into cells via gene therapy methods such as those described, below, in Section 5.9.2 that do not contain sequences susceptible to whatever antisense, ribozyme, or triple helix treatments are being utilized. Alternatively, in instances whereby the target gene encodes an extracellular protein, it may be preferable to co-administer normal target gene protein in order to maintain the requisite level of target gene activity. [00320] Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules, as discussed above. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

5.5.6.6 RNA INTERFERENCE

[00321] In certain embodiments, an RNA interference (RNAi) molecule is used to decrease Coco expression. RNA interference (RNAi) is defined as the ability of double-stranded RNA (dsRNA) to suppress the expression of a gene corresponding to its own sequence. RNAi is also called post-transcriptional gene silencing or PTGS. Since the only RNA molecules normally found in the cytoplasm of a cell are molecules of single-stranded mRNA, the cell has enzymes that recognize and cut dsRNA into fragments containing 21-25 base pairs (approximately two turns of a double helix). The antisense strand of the fragment separates enough from the sense strand so that it hybridizes with the complementary sense sequence on a molecule of endogenous cellular mRNA. This hybridization triggers cutting of the mRNA in the double-stranded region, thus destroying its ability to be translated into a polypeptide. Introducing dsRNA corresponding to a particular gene thus knocks out the cell's own expression of that gene in particular tissues and/or at a chosen time. [00322] Double-stranded (ds) RNA can be used to interfere with gene expression in mammals (Wianny & Zernicka-Goetz, 2000, Nature Cell Biology 2:70-75; incorporated herein by reference in its entirety). dsRNA is used as inhibitory RNA or RNAi of the function of Coco to produce a phenotype that is the same as that of a null mutant of Coco

(Wianny & Zernicka-Goetz, 2000, Nature Cell Biology 2:70-75). hi particular embodiments, the invention provides ds RNA molecules in which one strand comprises at least 19, 20, 21, or 25 nucleotides of the Coco mRNA nucleotide sequence and the other strand is its reverse complement. In another embodiment, the invention provides expression vectors for expression of RNA containing "hairpin" structures in which the stem of the hair pin is double stranded RNA in which one of the strands has at least 19, 20, 21 or 25 contiguous nucleotides from the Coco mRNA nucleotide sequence.

5.5.7 THERAPEUTIC USE OF COCO ANTISENSE NUCLEIC ACIDS, RIBOZYMES, DOUBLE STRANDED RNA, AND TRIPLE HELIX MOLECULES

[00323] The Coco antisense nucleic acids, ribozymes, triple helix or dsRNA molecules of the invention can be used to treat (or prevent) disorders of a cell type that expresses, or preferably overexpresses, Coco, h a specific embodiment, such a disorder is a neural induction disorder. In a preferred embodiment, a single-stranded DNA antisense

Coco oligonueleotide or double stranded Coco RNA is used.

[00324] Cell types that express or overexpress Coco RNA can be identified by various methods known in the art. Such methods include but are not limited to hybridization with a Coco-specific nucleic acid (e.g. by Northern hybridization, dot blot hybridization, in situ hybridization), observing the ability of RNA from the cell type to be translated in vitro into Coco, immunoassay, etc. In a preferred aspect, primary tissue from a patient can be assayed for Coco expression prior to treatment, e.g., by immunocytochemistry or in situ hybridization.

[00325] Pharmaceutical compositions of the invention (see Section 5.8.9), comprising an effective amount of a Coco antisense nucleic acid, ribozyme, triple helix or dsRNA molecule in a pharmaceutically acceptable carrier, can be administered to a patient having a disease or disorder that is of a type that expresses or overexpresses Coco RNA or protein.

[00326] The amount of Coco antisense nucleic acid, ribozyme, triple helix or dsRNA molecule that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Where possible, it is desirable to determine in vitro the cytotoxicity of the amount of the Coco antisense nucleic acid, ribozyme, triple helix or dsRNA molecule in a selected cell or tissue to be treated, and then in useful animal model systems prior to testing and use in humans.

[00327] hi a specific embodiment, pharmaceutical compositions comprising Coco antisense nucleic acids, ribozymes or triple helix molecules are administered via liposomes, microparticles, or microcapsules. In various embodiments of the invention, it may be useful to use such compositions to achieve sustained release of the Coco antisense nucleic acids, ribozymes, triple helix molecules or dsRNA. In a specific embodiment, it may be desirable to utilize liposomes targeted via antibodies to specific tissue antigens (e.g., such as a tumor antigen, see Leonetti et al, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2448-2451; Renneisen et al, 1990, J. Biol. Chem. 265:16337-16342).

[00328] Additional methods that can be adapted for use to deliver a Coco antisense nucleic acids, ribozymes, triple helix molecules or dsRNA are described herein.

5.5.8 DEMONSTRATION OF THERAPEUTIC OR PROPHYLACTIC UTILITY

[00329] The Therapeutics of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays that can be used to determine whether administration of a specific Therapeutic is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a Therapeutic, and the effect of such Therapeutic upon the tissue sample is observed. In one embodiment, where the patient has a malignancy, a sample of cells from such malignancy is plated out or grown in culture, and the cells are then exposed to a Therapeutic. A Therapeutic that promotes neural induction is selected for therapeutic use in vivo. Many assays standard in the art can be used to assess such neural induction; for example, neural induction can be assayed by ectodermal fate asaays using radioactive PCR monitoring cell fate markers, overall embryonic phenotype, inhibition of the BMP pathway in animal cap co-injection experiments, ability of Coco RNA to inhibit the activity of recombinant BMP protein on dissociated ectodermal cells in culture, by measuring the inhibition of a reporter encoding a BMP-4 responsive luciferase or other detectable report, competence assays (e.g., animal cap assay), by direct cell count, by detecting changes in transcriptional activity of known neural genes such as pan-neural markers (Ncam and nrpl), anterior-specific markers (Otx and XAG), and Nkx2.5, by trypan blue staining for cell viability, by visual assessment (e.g., light-microscopic examination) for differentiation based on changes in morphology, etc. [00330] In another embodiment, a Therapeutic is indicated for use that exhibits the desired effect, e.g., upon a patient cell sample, for example, promoting growth or differentiation of neural cells from a patient.

[00331] In another specific embodiment, a Therapeutic is indicated for use in treating cell injury or a degenerative disorder that exhibits in vitro promotion of the growth of the affected cell type of the patient.

[00332] In various specific embodiments, in vitro assays can be carried out with representative cells of cell types involved in a patient's disorder, to determine if a

Therapeutic has a desired effect upon such cell types.

[00333] In other specific embodiments, the in vitro assays described supra can be carried out using a cell line, rather than a cell sample derived from the specific patient to be treated, in which the cell line is derived from or displays characteristic(s) associated with the neural induction disorder desired to be treated or prevented, or is derived from the cell type upon which an effect is desired, according to the present invention.

[00334] Compounds for use in therapy can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc. For in vivo testing, prior to administration to humans, any animal model system known in the art may be used.

5.5.9 THERAPEUTIC/PROPHYLACTIC ADMINISTRATION AND COMPOSITIONS

[00335] The invention provides methods of treatment (and prophylaxis) by administration to a subject of an effective amount of a Therapeutic of the invention. In a preferred aspect, the Therapeutic is substantially purified. The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human. In a specific embodiment, a non-human mammal is the subject.

[00336] Formulations and methods of administration that can be employed when the

Therapeutic comprises a nucleic acid are described herein; additional appropriate formulations and routes of administration can be selected from among those described hereinbelow.

[00337] Various delivery systems are known and can be used to administer a

Therapeutic of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the Therapeutic, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a Therapeutic nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absoφtion through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Admimsfration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. [00338] In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.

[00339] In another embodiment, the Therapeutic can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

[00340] In yet another embodiment, the Therapeutic can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailabihty, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e, the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

[00341] Other controlled release systems are discussed in the review by Langer

(Science 249:1527-1533 (1990)).

[00342] In a specific embodiment where the Therapeutic is a nucleic acid encoding a protein Therapeutic, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide that is known to enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid Therapeutic can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

[00343] The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the Therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of admimsfration. [00344] In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. [00345] The Therapeutics of the invention can be formulated as neutral or salt forms.

Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[00346] The amount of the Therapeutic of the invention that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[00347] Suppositories generally contain active ingredient in the range of 0.5%. to

10% by weight; oral formulations preferably contain 10% to 95% active ingredient.

[00348] The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the foπn prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

5.5.10 ADDITIONAL USE OF INHIBITION OF COCO FUNCTION TO INHIBIT NEURAL INDUCTION

[00349] Inhibition of Coco function (e.g., by administering a compound that inhibits

Coco function as described above), also has utility in the inhibition of neural induction. Thus, inhibition of Coco function can be carried out to delay or prevent the onset of neural induction, in vivo or in vitro.

[00350] Thus, for example, a Coco antagonist (e.g., anti-Coco antibody, Coco antisense nucleic acids, etc.) can be administered to a subject to inhibit or prevent neural induction, h one embodiment, a Coco antagonist is applied topically, e.g., in a cream or gel, to the skin of the subject. In another embodiment, a Coco antagonist is injected, e.g., intradermally, intraperitoneally, or intramuscularly.

[00351] In a specific embodiment, a Coco antagonist is contacted with cells grown in culture, e.g., by addition of the antagonist to the culture medium or by adsorption of the antagonist to the culture plate or flask prior to seeding of the cells, in order to inhibit or delay neural induction. For example, such a method can be carried out in order to lengthen the time that cells can be kept alive in vitro, e.g., in order to facilitate conducting studies of the toxicity of a compound (e.g., a lead drug candidate) upon such cells, to study the effect of a molecule upon cell function, and, generally, to study the function of such cells. Such cells include but are not limited to neurons of the central nervous system (e.g., hippocampal, hypothalmic) or peripheral nervous system, glial cells, fibroblasts, kidney cells, liver cells, heart cells, muscle cells, endothelial cells, melanocytes, and hematopoietic cells such and T and B lymphocytes, macrophages, granulocytes, and mast cells. [00352] In vitro assays of neural induction are well known in the art and described herein in Sections 5.7 and 5.8.8, supra, and can be used to screen potential Coco antagonists prior to use in this aspect of the invention. 5.6 DIAGNOSIS AND SCREENING

[00353] Coco proteins, analogues, derivatives, and subsequences thereof, Coco nucleic acids (and sequences complementary thereto), anti-Coco antibodies, have uses in diagnostics. Such molecules can be used in assays, such as immunoassays, to detect, prognose, diagnose, or monitor various conditions, diseases, and disorders affecting Coco expression, or monitor the treatment thereof. In particular, such an immunoassay is carried out by a method comprising contacting a sample derived from a patient with an anti-Coco antibody under conditions such that immunospecific binding can occur, and detecting or measuring the amount of any immunospecific binding by the antibody. In a specific aspect, such binding of antibody, in tissue sections, can be used to detect aberrant Coco localization or aberrant (e.g., low or absent) levels of Coco. In a specific embodiment, antibody to Coco can be used to assay in a patient tissue or serum sample for the presence of Coco where an aberrant level of Coco is an indication of a diseased condition. By "aberrant levels," is meant increased or decreased levels relative to that present, or a standard level representing that present, in an analogous sample from a portion of the body or from a subject not having the disorder.

[00354] The immunoassays that can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.

[00355] Coco genes and related nucleic acid sequences and subsequences, including complementary sequences, can also be used in hybridization assays. Coco nucleic acid sequences, or subsequences thereof comprising about at least 8 nucleotides, can be used as hybridization probes. Hybridization assays can be used to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with aberrant changes in Coco expression and/or activity as described supra. In particular, such a hybridization assay is carried out by a method comprising contacting a sample containing nucleic acid with a nucleic acid probe capable of hybridizing to Coco DNA or RNA, under conditions such that hybridization can occur, and detecting or measuring any resulting hybridization. [00356] In specific embodiments, diseases and disorders involving neural, epidermal, bone, or cartilege cell growth, differentiation, maintenance, or induction or cell hypo or hyper prolierationcan be diagnosed, or their suspected presence can be screened for, or a predisposition to develop such disorders can be detected, by detecting decreased (or increased) levels of Coco protein, Coco RNA, or Coco functional activity (e.g., neural induction activity, suppression of mesodermal induction, etc.), or by detecting mutations in Coco RNA, DNA or protein (e.g., translocations in Coco nucleic acids, truncations in the Coco gene or protein, changes in nucleotide or amino acid sequence relative to wild-type Coco) that cause decreased (or increased) expression or activity of Coco. Such diseases and disorders include but are not limited to those described in Section 5.8. By way of example, levels of Coco protein can be detected by immunoassay, levels of Coco RNA can be detected by hybridization assays (e.g., Northern blots, dot blots), Coco neural induction activity can be measured by neural induction assays commonly known in the art, Coco binding to a binding partner can be done by binding assays commonly known in the art, translocations and point mutations in Coco nucleic acids can be detected by Southern blotting, RFLP analysis, PCR using primers that preferably generate a fragment spanning at least most of the Coco gene, sequencing of the Coco genomic DNA or cDNA obtained from the patient, etc.

[00357] In a preferred embodiment, levels of Coco mRNA or protein in a patient sample are detected or measured, in which decreased levels indicate that the subject has, or has a predisposition to developing, a neural cell growth disorder; in which the decreased levels are relative to the levels present in an analogous sample from a portion of the body or from a subject not having the neural induction disorder, as the case may be. [00358] In another specific embodiment, diseases and disorders involving a deficiency in neural induction or in which neural induction is desirable for treatment, are diagnosed, or their suspected presence can be screened for, or a predisposition to develop such disorders can be detected, by detecting increased levels of Coco protein, Coco RNA, or Coco functional activity (e.g., neural induction activity, suppression of induction of mesodermal markers, induction of ectodermal markers, etc.), or by detecting mutations in Coco RNA, DNA or protein (e.g., translocations in Coco nucleic acids, truncations in the gene or protein, changes in nucleotide or amino acid sequence relative to wild-type Coco) that cause increased expression or activity of Coco. Such diseases and disorders include but are not limited to those described in Section 5.8. By way of example, levels of Coco protein, levels of Coco RNA, Coco neural induction activity, Coco binding activity, and the presence of translocations or point mutations can be determined as described above. [00359] In a specific embodiment, levels of Coco mRNA or protein in a patient sample are detected or measured, in which altered levels indicate that the subject has, or has a predisposition to developing, a neural, bone, epidermal, cartilege or cell proliferation disorder; in which the altered levels are relative to the levels present in an analogous sample from a portion of the body or from a subject not having the neural induction disorder, as the case may be.

[00360] Kits for diagnostic use are also provided, that comprise in one or more containers an anti-Coco antibody, and, optionally, a labeled binding partner to the antibody. Alternatively, the anti-Coco antibody can be labeled (with a detectable marker, e.g., a chemiluminescent, enzymatic, fluorescent, or radioactive moiety). A kit is also provided that comprises in one or more containers a nucleic acid probe capable of hybridizing to Coco RNA. In a specific embodiment, a kit can comprise in one or more containers a pair of primers (e.g., each in the size range of 6-30 nucleotides) that are capable of priming amplification [e.g., by polymerase chain reaction (see e.g., Innis et al., 1990, PCR Protocols, Academic Press, Inc., San Diego, CA), ligase chain reaction (see EP 320,308) use of Q/3 replicase, cyclic probe reaction, or other methods known in the art] under appropriate reaction conditions of at least a portion of a Coco nucleic acid. A kit can optionally further comprise in a container a predetermined amount of a purified Coco protein or nucleic acid, e.g., for use as a standard or control.

5.7 SCREENING FOR COCO AGONISTS AND ANTAGONISTS

[00361] Coco nucleic acids, proteins, and derivatives also have uses in screening assays to detect molecules that specifically bind to Coco nucleic acids, proteins, or derivatives and thus have potential use as agonists or antagonists of Coco, in particular, molecules that thus affect neural induction. In a preferred embodiment, such assays are performed to screen for molecules with potential utility as drugs or lead compounds for drug development, for the treatment or prevention of diseases and disorders involving neural cell growth, differentiation, or maintenance, bone or cartilage cell growth, differentiation, or maintenance, and cell hyper/hypo proliferation. The invention thus provides assays to detect molecules that specifically bind to Coco nucleic acids, proteins, or derivatives. For example, recombinant cells expressing Coco nucleic acids can be used to recombinantly produce Coco proteins in these assays, to screen for molecules that bind to a Coco protein. Molecules (e.g., putative binding partners of Coco) are contacted with the Coco protein (or fragment thereof) under conditions conducive to binding, and then molecules that specifically bind to the Coco protein are identified. Similar methods can be used to screen for molecules that bind to Coco derivatives or nucleic acids. Methods that can be used to carry out the foregoing are commonly known in the art. [00362] By way of example, diversity libraries, such as random or combinatorial peptide or nonpeptide libraries can be screened for molecules that specifically bind to Coco. Many libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phage display libraries), and in vitro translation-based libraries. [00363] Examples of chemically synthesized libraries are described in Fodor et al.,

1991, Science 251:767-773; Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski, 1994, Bio/Technology 12:709-710; Gallop et al, 1994, J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al, 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al, 1992, Biotechniques 13:412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.

[00364] Examples of phage display libraries are described in Scott and Smith, 1990,

Science 249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian, R.B., et al.,

1992, J. Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/18318 dated August 18, 1994. [00365] In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058 dated April 18, 1991; and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.

[00366] By way of examples of nonpeptide libraries, a benzodiazepine library (see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91 :4708-4712) can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA 91:11138-11142). [00367] Screening the libraries can be accomplished by any of a variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390; Fowlkes et al, 1992; BioTechniques 13:422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al, 1994, Cell 76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellington et al., 1992, Nature 355:850- 852; U.S. Patent No. 5,096,815, U.S. Patent No. 5,223,409, and U.S. Patent No. 5,198,346, all to Ladner et al.; Rebar and Pabo, 1993, Science 263:671-673; and PCT Publication No. WO 94/18318. [00368] hi a specific embodiment, screening can be carried out by contacting the library members with a Coco protein (or nucleic acid or derivative) immobilized on a solid phase and harvesting those library members that bind to the protein (or nucleic acid or derivative). Examples of such screening methods, termed "panning" techniques are described by way of example in Parmley and Smith, 1988, Gene 73 :305-318; Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and in references cited hereinabove.

[00369] hi another embodiment, the two-hybrid system for selecting interacting proteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used to identify molecules that specifically bind to a Coco protein or derivative.

[00370] In addition, Xenopus, Zebrafish, and Drosophila can be used as a model system in order to detect genes that phenotypically interact with Coco. For example, overexpression of xCoco produces neurahzation and repression of the mesodermal markers brachyury and fgf8 as described herein . Mutagenesis of the Xenopus, zebrafish, Drosophila, and Mouse genome can be performed, followed by selecting progeny in which the mutagenesis has resulted in suppression or enhancement of the neurahzation phenotype; the mutated genes in such animals are likely to encode proteins that interact/bind with Coco.

5.8 ANIMAL MODELS

[00371] The invention also provides animal models.

[00372] In one embodiment, animal models for diseases and disorders involving neural cell growth, differentiation, or maintenance, bone or cartilage cell growth, and cell proliferation (e.g., as described in Section 5.8) are provided. Such an animal can be initially produced by promoting homologous recombination between a Coco gene in its chromosome and an exogenous Coco gene that has been rendered biologically inactive (preferably by insertion of a heterologous sequence, e.g., an antibiotic resistance gene). In a preferred aspect, this homologous recombination is carried out by transforming embryo- derived stem (ES) cells with a vector containing the insertionally inactivated Coco gene, such that homologous recombination occurs, followed by injecting the ES cells into a blastocyst, and implanting the blastocyst into a foster mother, followed by the birth of the chimeric animal ("knockout animal") in which a Coco gene has been inactivated (see Capecchi, 1989, Science 244:1288-1292). The chimeric animal can be bred to produce additional knockout animals. Such animals can be mice, hamsters, sheep, pigs, cattle, etc., and are preferably non-human mammals. In a specific embodiment, a knockout mouse is produced.

[00373] Such knockout animals are expected to develop or be predisposed to developing diseases or disorders involving neural induction and thus can have use as animal models of such diseases and disorders, e.g., to screen for or test molecules for the ability to inhibit or promote neural induction and thus treat or prevent such diseases or disorders. [00374] In a different embodiment of the invention, transgenic animals that have incoφorated and express a functional Coco gene have use as animal models of diseases and disorders involving deficiencies in neural induction or in which neural induction is desired. Such animals can be used to screen for or test molecules for the ability to promote neural induction and thus treat or prevent such diseases and disorders. 5.9 USE IN STEM CELL MAINTENANCE.

[00375] The present inventors have determined that Coco is upregulated in undifferentiated embryonic stem cells. Accordingly, the invention provides methods of maintaining stem cells in an undifferentiated state by contacting the stem cells with a molecule of the invention that promotes Coco function. The stem cells are preferably embryonic stem cells but may also be adult (i.e., non-embryonic stem cells). The stem cells are preferably mammalian, including, but not limited to, mouse, rat, porcine, equine, ovine, and, particularly, primate, more particularly, human, but also including, fish, and avians, particularly, chicken and quail, etc.

[00376] A molecule of the invention that promotes Coco function can be administered to the cells by including the molecule in the cell culture medium used to culture the stem cells, expressing the molecule in the stem cells themselves and/or expressing the molecule in feeder cells upon which the stem cells are cultivated, hi a preferred embodiment, the stem cells are cultured in the measured of a molecule of the invention that promotes Coco function but in the absence of feeder cells. In a more preferred embodiment, the stem cells are cultured in the presence of a molecule of the invention but in the absence of serum or any other component derived from animal tissue or fluids (except for components that may be recombinantly expressed in cultured animal cells).

[00377] Stem cells cultured according to methods of the invention have uses in research and in the development of replacement cells/tissues for therapeutic use. In certain embodiments, the stem cells have been transfected with a heterologous coding sequence or have been engineered to contain a coding sequence or preferably linked to a heterologous promoter. 6. EXAMPLE 1 : ECTODERMAL FATE SPECIFICATION AND

COMPETENCE BY COCO, A NOVEL MATERNAL BMP, TGFB AND WNT INHIBITOR

6.1 INTRODUCTION

[00378] This example discloses the identification and characterization of Coco, a novel member of the Cerberus/Dan family of BMP inhibitors, which is involved in neural induction downstream of BMP inhibition. In particular, this example discloses the expression, biochemical and embryological characterization of Coco during ectodermal fate specification in Xenopus laevis. Because of its embryological activities, the gene has been named "Coco," after the Spanish word meaning "head." The example demonstrates that Coco acts in the ectoderm to block BMP and TGF/3 signals to regulate ectodermal fate specification and competence prior to the onset of neural induction during late gastrulation.

6.2 MATERIALS AND METHODS

6.2.1 CDNA MICRO ARRAY ANALYSIS

[00379] A cDNA microarray analysis of Xenopus gastrula genes differentially regulated by Smad7 in animal caps was performed. One of the clones upregulated in the assay, 51-B6, was chosen for further evaluation and found to encode Coco, a novel member of the Cerberus/Dan family of BMP inhibitors, as disclosed hereinbelow.

6.2.2 EMBRYO INJECTIONS AND PREPARATION OF RNA

[00380] Xenopus Coco (xCoco) RNA was made by linearising with Ascl and transcribing using the mMessage mMachine in vitro SP6 transcription kit from Ambion. Embryos were injected in either the animal pole or the ventral vegetal/VMZ with lng of RNA and staged according to the methods of Nieuwkoop and Faber, 1967, Normal Table of Xenopus laevis (Daudin). Amsterdam: North Holland Publishing Company.

6.2.3 REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION (RT-PCR) ANALYSIS

[00381 ] Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on samples from animal and DMZ/VMZ explants according to the methods of Wilson and Melton, 1994, Curr Biol. 4, 676-686. Omithine decarboxylase (ODC) was used as a loading control. Oligonueleotide primers that were used had sequences corresponding to SEQ ID NO: 13, and 14.

6.2.4 WHOLEMOUNT IN SITU HYBRIDISATION

[00382] Wholemount in situ hybridisations were carried out as described (Harland,

1991, Methods in Cell Biology 36:675-678). In situ probes were made according to previously described methods: brachyury (Smith et al, 1991, Cell 67:79-87), emxl (Pannese et al, 1998, Mech Dev. 73, 73-83), en2 (Hemmati-Brivanlou et al, 1991, Development lll:715-724),yg S (Christen and Slack, 1997, Dev Biol. 192, 455-466), Goosecoid (Cho et al, 1991, Cell 67, 1111-1120), hb9 (Wright et al, 1990, Development 109, 225-234), nkx2.5 (Raffm et al, 2000, Dev. Biol. 218:326-340), pax6 (Alimann et al, 1997, Dev. Biol. 185:119-123) and rx (Mathers et al, 1997, Nature 387:603-607). The xCoco in situ probe was made by linearising with Sail and transcribing with T3. The colour substrate used was BMpuφle (Boehringer Mannheim). Once the colour had developed sufficiently, the reaction was stopped with PBT (PBS and 0.1 %> Tween 20) and embryos were fixed in 4% paraformaldehyde (PFA)/PBT. Embryos to be sectioned were embedded in 20% gelatin PBS, then fixed overnight in 4% PFA at 4°C. Sections were cut at 100 μm using a vibratome.

6.2.5 INTERACTION OF COCO WITH XNR1, BMP4, WNT8 AT A BIOCHEMICAL LEVEL

[00383] xCoco was flag-tagged in the C-terminus by standard PCR methods. RNA was made by linearising with Ascl and transcribing with SP6. Coco-flag tagged was co-injected into embryos at the 2-cell stage with BMP4-HA or Xnrl -HA. Explants were harvested at stage (st) 10-11. The homogenized explants were subjected to co- mmunoprecipitation with an anti-HA polyclonal antibody from Sigma (St. Louis, MO) and then probed with an anti- flag monoclonal antibody (Sigma).

6.2.6 INHIBITION OF WNT8 AND BMP4 PROMOTER ACTIVITY BY COCO.

[00384] Injections were made in the animal pole of 4 cell stage embryos with 25 pg of reporter gene DNA, 10 pg Wnt8 RNA or 100 pg BMP4 RNA(Hata et al, 2000, Genes Dev. 15, 186-197), with or without addition of 1 ng Coco RNA. Embryos were recovered at stage 9 for TOP -FLASH activity, and stage 10.5 for BRE activity. Luciferase transcription assays were performed with the Luciferase Assay (Promega Coφ., Madison, WI) as described.

6.2.7 COMPETENCE ASSAY

[00385] Embryos were injected at the 2-cell stage with xCoco RNA and then animal cap explants were excised at stage 8 and cultured. Activin was added to uninjected and xCoco-inj ected explants at stages 8, 9, 10 and 11. Explants that were beginning to heal were carefully reopened. All explants were harvested at stage 12/13 and analyzed for the induction of mesodermal markers by standard methods of RT-PCR. 6.3 RESULTS AND DISCUSSION

[00386] Coco (SEQ ID NO: 3) encodes a 25 kD protein (SEQ JO NO: 4) with a predicted secretory signal sequence (FIG. 1 A) with closest similarity to Cerberus and Caronte (FIGS. IB, C). The homology among these family members is low and resides mainly in the spacing of the 9 cysteines and the core domain (FIG. IB). Using the Xenopus Coco nucleotide and amino acid sequences in searches of the National Center for Biotechnology Information (NCBI, National Library of Medicine, Bethesda, MD) and Celera (Rockville, MD) databases, human, mouse and fugu homologs were identified with closest homology to Xenopus Coco (FIGS. IB, C). Human and mouse Coco map to 19pl3.2 and 8, respectively, which are syntenic map locations. Mouse Coco is a partial sequence lacking the 5' region. Human Coco was assembled from an EST (Genbank BC025333) and from genomic searches, as no full-length cDNA had been reported. [00387] Expression of Xenopus Coco was analyzed by in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR). xCoco is expressed maternally and is down regulated after gastrulation, being very weakly expressed at neurula stages (FIGS. 2A, B). At pre-gastrula stages xCoco is expressed in the animal pole exclusively (FIG. 2A, left panel). At the gastrula stage, xCoco mRNA transcripts are detected in both the dorsal marginal zone (DMZ; including the organizer, see *) and the ventral marginal zone (VMZ) and at very high levels in the animal cap ectodenn (FIGS. 2 A, C). Coco is therefore the only known BMP inhibitor expressed maternally. By contrast, Cerberus is expressed zygotically between stages 9 and 13 (Bouwmeester et al, 1996; FIG. 2B ) and is restricted to the anterior endoderm of the organizer (Bouwmeester et al, 1996; FIG. 2C). In view of the maternal expression of xCoco, its widespread expression within the ectoderm and the rapid decline in xCoco mRNA levels following gastrulation, xCoco's function was evaluated in the context of BMP and TGF- signalling during ectodermal patterning. [00388] As a first test for xCoco's biological activities, its mRNA was injected into embryos at either the 2 or 4-cell stage. The initial analysis was done at gastrula stages, where Coco is expressed throughout the ectoderm and marginal zones. At gastrula stages, both brachyury (Smith et al, 1991, Cell 67:79-87) and g S (Christen and Slack, 1997, Dev. Biol. 192:455-466) are expressed in a ring of mesodermal cells around the vegetal pole (FIGS. 3A, B, top panels). After injection of Coco in 1 of 2 cells vegetally (where xCoco is not normally expressed - see FIG. 2), both markers are repressed (FIGS. 3 A, B), suggesting that Coco can inhibit mesoderm formation. xCoco expands the size of the endogenous organizer, as judged by the increase in expression of Otx2 (FIG. 3C) and Gsc (FIG. 3D; see also Cho et al, 1991, Cell 67: 1111-1120). In addition, the endogenous ectodermal expression of Otx2 is increased (FIG. 3C, lower panel) and there are ectopic ectodermal patches of Otx2 expression on the contralateral sides of the embryo (data not shown), suggesting that Coco acts in cell non-autonomous manner.

[00389] Analysis at later stages showed that overexpression of xCoco in the animal pole results in embryos with expanded anterior structures and ectopic cement glands (compare FIGS. 3E with 3F, see*). By contrast, overexpression of Coco venfrally results in posterior truncations and the induction of an extra "head-like"structure (75% of injected embryos have this phenotype; FIG. 3G, see*). Very infrequently these extra "heads" also contain a single eye (5% of cases; data not shown). Molecular analysis of these ectopic structures shows that they contain forebrain and midbrain tissue, as shown by the ectopic expression of the forebrain markers Rx (FIG. 3H; Mathers et al, 1997, Nature 387, 603-607), Emxl (FIG. 31; Pannese et al, 1998, Mech Dev. 73, 73-83), Otx2 (FIG. 3J, top panel and lower panel), and the midbrain marker En2 (Hemmati-Brivanlou et al, 1991, Development 111, 715-724; FIG. 3K). En2 expression is detected where the ectopic head meets the body of the embryo (see *, FIG. 3K, lower panel). By contrast, expression of Hoxb9, a marker of spinal cord (Wright et al, 1990, Development 109, 225-234; FIG. 3L) was not detected. In addition, the heart marker Nkx2.5 was strongly induced around the extra cement gland (Raffin et al, 2000, Dev Biol. 218, 326-340; FIG. 3M). However, ectopic hearts in the xCoco injected embryos were not detected, possibly due to the lack of endoderm formation in xCoco injected embryos.

[00390] These phenotypes, i.e, expanded anterior structures and secondary axis containing head-like structures, are consistent with an inhibitory activity of BMP and Wnt signaling by xCoco (Bouwmeester et al, 1996, Nature 382, 595-601; Glinka et al, 1998, Nature 391, 357-362). In order to further analyse xCoco's activity at a molecular level, fate changes in embryonic explants were analyzed by RT-PCR for a variety of molecular markers. When embryos were injected at the 2 cell-stage in the animal caps, markers for the organizer or endoderm in gastrula-stage explants were not detected. However, by stage 21, pan-neural markers (Ncam and mrpl) and anterior-specific markers (Otx and XAG) are induced in the explants (FIG. 4A), suggesting that xCoco can neuralize ectodermal explants, consistent with an inhibition of BMP signaling (Wilson and Hemmati-Brivanlou, 1995, Annu Rev Cell Dev Biol. 15, 411-433). Nkx2.5 was similarly induced in this assay (FIG. 4A). Next, xCoco mRNA was injected into the VMZ at the 4-cell stage and analyzed its effects on cell fate determination in VMZ explants isolated at gastrula stages or for moφhological changes at tadpole (stage 27) stages to test whether xCoco can neuralize ventral tissue (FIGS. 4b, C, D). At gastrula, the organizer markers chordin and goosecoid were weakly induced in the VMZ expressing xCoco, whereas the expression of brachyury was suppressed (FIG. 4C), consistent with the in vivo results where xCoco blocks mesoderm formation and dorsalizes the embryo. At stage 27, the moφhology of the VMZ+xCoco explants were similar to that of the dorsal marginal zone (DMZ) explants (FIG. 4B), and the injected explants contained anterior neural tissue and cement glands. At st27 VMZ explants do not express the neural markers Ncam, n l and Otx (Pannese et al, 1995, Development 121, 707-720), nor the cement gland marker XAG. hi VMZ explants expressing xCoco all of these markers are now induced (FIG. 4D), consistent with Coco blocking BMP signals.

[00391] In order to demonstrate the inhibitory interactions of xCoco with BMPs,

TGF/3 members and Wnts, xCoco was co-injected for animal cap assays together with RNAs encoding bmp4, Xnrl (nodal-related factor- 1; Hyde and Old, 2000, Development 127, 1221-1229), or Wnt8, or injected caps were exposed to activin conditioned media (FIG. 5), and monitored for the expression of immediate response genes normally activated by these signaling molecules in the ectoderm. For instance, exposure to BMP4 induces the expression of brachyury and increases epidermal keratin expression. In this assay, xCoco blocked induction of these markers (FIG. 5A). In similar assays xCoco could also block Wnt8 induction of Xnr3 and siamois expression (FIG. 5B; Sokol and Melton, 1992, Nature 351, 409-411) and nodal signaling, as detected by the inhibition of the expression of chordin, brachyury and Wnt8 induced by Xnrl (FIG. 5C).

[00392] Finally, xCoco was analyzed and found to inhibit activin signaling. Activin induces dorsal mesoderm in animal caps, causing them to elongate (Smith et al, 1990, Nature 345:729-731; Sokol and Melton, 1991, Nature 351:409-411). Embryos at the 2-cell stage were injected with xCoco and explanted animal caps in medium containing activin protein. Both control and xCoco injected caps elongated in the presence of activm (not shown). In addition, xCoco was unable to suppress the expression of markers induced by activin (chordin, brachyury and Wnt8; FIG. 5D), suggesting that xCoco is unable to block activin signaling in this assay. However, as described below, xCoco is able to change the temporal responsiveness of the explants to activm protein, where it changes the competence of the ectoderm to respond to mesoderm-inducing signals.

[00393] Based on the expression and biological activities of xCoco, the endogenous role of xCoco is to regulate fate determination in the ectoderm through an inhibition of TGF/3 signals. In order to test whether xCoco can directly interact with TGF/3s, tagged xCoco and BMP4 or Xnrl constructs were co-injected into animal caps and tested for direct binding in immuno-precipitation experiments (FIGS. 5E and 5F). Although not wishing to be bound by a particular theory, these experiments demonstrated that the inhibition of BMP4 and Xnrl signaling by xCoco likely takes place through direct binding. [00394] The maternal expression of xCoco makes it a unique gene among the large family of BMP inhibitors. The widespread expression of xCoco in the ectoderm and the timing of the decline of xCoco mRNA levels, which coincides with the loss of competence of ectodermal cells to respond to mesoderm-inducing signals (Green et al, 1990, Development 108:229-238), indicate that a function of xCoco is to inhibit mesoderm-inducing signals operating in the ectoderm until the end of gastrulation. [00395] Mesoderm induction has been shown to be mediated by TGF/3 signals (Smith et al, 1989, Development 107 Suppl: 149-159). For example, nodal signaling patterns the mesoderm and must be blocked to allow the anterior ectoderm to be patterned (Thisse et al, 2000, Nature 403:425-428). The ectoderm is competent to respond to TGF/3 and become mesoderm until stage 12, both in vivo and in vitro (Green et al, 1990, Development 108: 229-238). Therefore, prior to the decision to become epidermal versus neural, ectodermal cells must inhibit mesoderm-inducing signals. Amongst the TGF 3 inhibitors, xCoco is the only inhibitor whose expression is consistent with such a role. By contrast, the other two known nodal inhibitors, antivin (Tanegashima et al, 2000, Mech Dev. 99:3-14) and Cerberus (Bouwmeester et al, 1996, Nature 382:595-601), are not expressed during those stages in the ectoderm.

[00396] The presence of xCoco in the ectoderm was investigated at specific stages to determine whether it could inhibit mesoderm formation or affect the competence of the ectodeπn to respond to mesoderm-inducing signals. Animal cap explants can respond to activin and become mesoderm, although the responsiveness of the explants declines over time until stage 12, where they are no longer able to respond (Green et al, 1990, Nature 382:595-601). Whether xCoco RNA would alter this responsiveness temporally or qualitatively was tested, by exposing dissected caps to activin at different time points (FIG. 5G). The responsiveness of control explants progressively declines with age. Indeed, animal caps expressing xCoco responded differently to activin over time (FIG. 5G). Notably, when activin was added to xCoco injected caps at or after stage 10, no mesoderm induction was observed compared to control explants. This result strongly suggests xCoco changes the responsiveness of the ectoderm to mesoderm inducing signals. [00397] This example demonstrates that Coco is a novel BMP, TGF/3 and Wnt inhibitor, whose expression and biological activities are consistent with xCoco acting to regulate the competence of the ectoderm, to ensure proper ectodermal patterning during gastrulation. xCoco expression in the ectoderm might also act to lower overall levels of BMP signals, so that additional BMP inhibitors expressed in the organizer can induce the formation of the nervous system. In this light, xCoco's expression in the entire ectodermal region prior to gastrulation might act to prevent fate specification in the ectoderm and ensure the maintenance of the stem-cell like properties exhibited by ectodermal cells. Furthermore, the mouse and human homologs of xCoco are also over-expressed in embryonic stem-cells as determined by RT-PCR analysis, indicating that this potent new inhibitor might fulfill similar functions during mammalian embryogenesis. [00398] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[00399] All references cited herein are incoφorated herein by reference in their entirety and for all puφoses to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incoφorated by reference in its entirety for all puφoses.

[00400] The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

Claims

WHAT IS CLAIMED IS:

1. A purified protein which comprises the amino acid sequence of SEQ ID NO: 2, 4, 6, or 8.

2. The protein of claim 1 which comprises the amino acid sequence of SEQ ID NO: 2.

3. A purified protein encoded by a nucleic acid hybridizable, under stringent conditions, to a nucleotide sequence consisting of the coding region of SEQ ID NO: 1, 3, 5, or 7, and having neural induction activity.

4. A processed product of the protein of claim 1 or 3.

5. A purified derivative or analog of the protein of claim 1, which displays one or more biological activities.

6. The derivative or analog of claim 5 wherein said one or more biological activities include being bound by an antibody that specifically binds a protein having the amino acid sequence of SEQ ID NO: 2, 4, 6, or 8.

7. A purified fragment of a protein of claim 1 or 3 comprising a cysteine knot domain.

8. A protein comprising an amino acid sequence that has at least 60% identity to a domain of the protein of claim 1, in which the percentage identity is determined over an amino acid sequence of identical size to the domain, and that has neural induction activity.

9. A protein comprising an amino acid sequence that has at least 90% identity to a domain of the protein of claim 1, in which the percentage identity is determined over an amino acid sequence of identical size to the domain, and that has neural induction activity.

10. A chimeric protein comprising a fragment of the protein of claim 1 or 3 consisting of at least 6 amino acids fused via a covalent bond to an amino acid sequence of a second protein which is different from the protein of claim 1 or 3.

11. The chimeric protein of claim 10 in which the fragment is bound by an antibody that specifically binds a protein having the amino acid sequence of SEQ ID NO: 2, 4, 6, or 8 and wherein said second protein is the Fc domain of an immunoglobulin.

12. An antibody, or antigen binding fragment, specifically binds a protein having an amino acid sequence of SEQ ID NO: 2, 4, 6, or 8.

13. The antibody of claim 12 which is monoclonal.

14. The antibody of claim 12, wherein said antibody is human or humanized.

15. An isolated nucleic acid comprising a nucleotide sequence encoding a protein comprising an amino acid sequence of SEQ ID NO: 2, 4, 6, or 8 and/or the complement thereof.

16. The isolated nucleic acid of claim 15 in which the protein comprises the amino acid sequence of SEQ ID NO: 4.

17. An isolated nucleic acid which hybridizes, under stringent conditions, to a nucleotide sequence consisting of the coding region of SEQ ID NO: 1, 3, 5, or 7, and encoding a protein with neural induction activity.

18. An isolated nucleic acid comprising a nucleotide sequence encoding an at least 10 amino acid fragment of the protein of claim 1.

19. The isolated nucleic acid of claim 18 in which said fragment is functionally active.

20. An isolated nucleic acid comprising a nucleotide sequence encoding the chimeric protein of claim 10.

21. A recombinant cell comprising the nucleic acid of claim 15, in which said nucleotide sequence is operably linked to a heterologous promoter.

22. A recombinant cell comprising the nucleic acid of claim 17, in which said nucleotide sequence is operably linked to a heterologous promoter.

23. A method of producing a Coco protein, said method comprising culturing the recombinant cell of claim 21, under conditions such that the encoded protein is expressed by the cell, and recovering the expressed encoded protein.

24. A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 1 or 3; and a pharmaceutically acceptable carrier.

25. The pharmaceutical composition of claim 24 in which the protein is a human protein.

26. A pharmaceutical composition comprising a therapeutically effective amount of the fragment of claim 7; and a pharmaceutically acceptable carrier.

27. A pharmaceutical composition comprising a therapeutically effective amount of the chimeric protein of claim 11; and a pharmaceutically acceptable carrier.

28. A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen binding fragment thereof of claim 12; and a pharmaceutically acceptable carrier.

29. A pharmaceutical composition comprising a therapeutically effective amount of the nucleic acid of claim 15; and a pharmaceutically acceptable carrier.

30. A pharmaceutical composition comprising a therapeutically effective amount of the recombinant cell of claim 21; and a pharmaceutically acceptable carrier.

31. A method of treating or preventing a disease or disorder, characterized by insufficient neural cell growth, differentiation, or maintenance in a subject, said method comprising administering to a subject in which such freatment or prevention is desired a therapeutically effective amount of a molecule that promotes Coco function.

32. The method according to claim 31 in which the subject is a human.

33. The method according to claim 31 in which the molecule that promotes Coco function is selected from the group consisting of a Coco protein, a Coco derivative or analog that is active in promoting neural cell growth, differentiation, or maintenance, a nucleic acid encoding a Coco protein, and a nucleic acid encoding a Coco derivative or analog that is active in promoting neural cell growth, differentiation, or maintenance.

34. The method according to claim 31 in which the disease or disorder is selected from the group consisting of degenerative disorders, growth deficiencies, hypoprohferative disorders, physical trauma, lesions, and wounds.

35. An isolated oligonueleotide consisting of at least six nucleotides, and comprising a sequence complementary to at least a portion of an RNA transcript encoding the protein of claim 1 or 3, which oligonueleotide hybridizes to the RNA transcript under moderately stringent conditions.

36. A pharmaceutical composition comprising the oligonueleotide of claim 35; and a pharmaceutically acceptable carrier.

37. A method of inhibiting the expression of a nucleotide sequence encoding a Coco protein in a cell, said method comprising providing the cell with an effective amount of the oligonueleotide of claim 35.

38. An isolated double-stranded RNA molecule consisting of at least 21 nucleotides, and comprising on one strand an at least 21 nucleotide sequence complementary to a portion of an RNA transcript encoding the protein of claim 1 or 3.

39. A pharmaceutical composition comprising the double-stranded RNA of claim 38; and a pharmaceutically acceptable carrier.

40. A method of inhibiting the expression of a nucleotide sequence encoding a Coco protein in a cell, said method comprising providing the cell with an effective amount of the double-stranded RNA of claim 38.

41. A method of diagnosing a disease or disorder characterized by an aberrant level of Coco RNA or protein in a subject, comprising measuring the level of Coco RNA or protein in a sample derived from the subject, in which an increase or decrease in the level of Coco RNA or protein, relative to the level of Coco RNA or protein found in an analogous sample not from a patient having the disease or disorder indicates the presence of the disease or disorder in the subject.

42. A kit comprising in one or more containers a molecule selected from the group consisting of an anti-Coco antibody, a nucleic acid probe capable of hybridizing to a Coco RNA, or a pair of nucleic acid primers capable of priming amplification of at least a portion of a Coco nucleic acid.

43. A kit comprising in a container a therapeutically effective amount of the protein of claim 1 or 3.

44. A method of increasing cell growth in an animal or a plant comprising inhibiting Coco expression or activity in said animal or plant.

45. A method of identifying a molecule that specifically binds to a ligand selected from the group consisting of a Coco protein, a fragment of a Coco protein comprising a domain of the protein, and a nucleic acid encoding the protein or fragment, comprising

(a) contacting said ligand with a plurality of molecules under conditions conducive to binding between said ligand and the molecules; and

(b) identifying a molecule within said plurality that specifically binds to said ligand.

46. A recombinant non-human animal that is the product of a process comprising introducing a nucleic acid encoding at least a domain of a Coco protein into a non-human animal or ancestor thereof.

47. A recombinant non-human animal in which a Coco gene has been inactivated by a method comprising introducing a nucleic acid into a non-human animal or an ancestor thereof, which nucleic acid comprises a non-Coco sequence flanked by Coco genomic sequences that promote homologous recombination.

48. A method of of treating, managing, or ameliorating the symptoms of a disease or disorder characterized by insufficient bone cell growth, differentiation, and/or maintenance in a patient suffering therefrom, said method comprising administering to said patient a therapeutically effective amount of a molecule that inhibits Coco activity or expression.

49. A method of treating, managing or ameliorating symptoms of a disease or disorder characterized by insufficient epidermal cell growth, differentiation or maintenance in a patient suffering therefrom, said method comprising administering to said patient a therapeutically effective amount of a molecule that inhibits Coco activity or expression.

50. A method of treating, managing or ameliorating the symptoms of a disease or disorder characterized by excess epidermal cell growth, differentiation which or maintenance in a patient suffering therefrom, said method comprising administering to said patient a therapeutically effective amount of a molecule that promotes Coco activity expression.

51. A method of treating cancer in a patient suffering therefrom, said method comprising administering to said patient a therapeutically effective amount of a molecule that promotes Coco activity or expression.

52. A method of maintaining embryonic stem cells in an undifferentiated, self- renewing state, said method comprising contacting embryonic stem cell in culture with a molecule that promotes Coco function thereby maintaining said embryonic stem cells in an undifferentiated, self-renewing state.

53. A method of inliibiting TGF-/3, BMP, or Wnt signal transduction in a cell, said method comprising contacting said cell with an amount of a molecule that promotes Coco activity or expression sufficient to inhibit signal transduction elicited by TGF-/3, BMP or Wnt.