MXPA02007619A

MXPA02007619A - Fibroblast growth factor 23 molecules and uses thereof.

Info

Publication number: MXPA02007619A
Application number: MXPA02007619A
Authority: MX
Inventors: Roland Luethy
Original assignee: Amgen Inc
Priority date: 2000-02-15
Filing date: 2001-02-15
Publication date: 2002-12-13
Also published as: CA2398603A1; JP2005508131A; WO2001061007A9; EP1257645A2; WO2001061007A2; WO2001061007A3; AU3976701A

Abstract

The present invention provides Fibroblast Growth Factor 23 (FGF 23) polypeptides and nucleic acid molecules encoding the same. The invention also provides selective binding agents, vectors, host cells, and methods for producing FGF 23 polypeptides. The invention further provides pharmaceutical compositions and methods for the diagnosis, treatment, amelioration, and or prevention of diseases, disorders, and conditions associated with FGF 23 polypeptides.

Description

MOLECULES OF FACTOR 23 GROWTH OF FIBROBLASTS AND ITS USE FIELD OF THE INVENTION The present invention relates to polypeptides of fibroblast growth factor 23 (FGF-23, for its acronym in English) and nucleic acid molecules that code for them. The invention also relates to selective binding agents, vectors, host cells and methods for producing FGF-23 polypeptides. The invention also relates to pharmaceutical compositions and methods for diagnosis, treatment, reduction or to avoid diseases, disorders and conditions associated with FGF-23 polypeptides.

BACKGROUND OF THE INVENTION Technical advances in the identification, cloning, expression and manipulation of nucleic acid molecules and the deciphering of the human genome have greatly accelerated the discovery of novel therapeutic substances. Nucleic acid rapid sequence techniques can now generate sequence information at unprecedented speeds and, together with the analysis REF '"" 40843 computational, allow the assembly of sequences of overlap within the partial and complete genome as well as the identification of regions that code for polypeptides. A comparison of the predicted sequence of 5 amino acids against a database compilation of known amino acid sequences allows a person to determine the degree of homology of previously identified sequences or structural building blocks. The cloning and expression of a nucleic acid molecule that The coding for a polypeptide provides a polypeptide product for structural and functional analysis. The manipulation of the nucleic acid molecules and the encoded polypeptides can confer advantageous properties in a product for use as a substance 15 therapeutic. Despite important technical advances in genome research over the past decade, the potential for the development of novel therapeutic substances based on the human genome has not yet been 20 out to a large extent. Many genes code for potentially beneficial polypeptide therapeutic substances or for these coding polypeptides, which may act as "targets" for therapeutic molecules, which have not yet been identified. Accordingly, it is an object of the invention _____________ ta¡.¿-identify novel polypeptides and nucleic acid molecules that code for them, which have diagnostic or therapeutic benefit.

SUMMARY OF THE INVENTION The present invention relates to novel nucleic acid molecules for FGF-23 and encoded polypeptides. The invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that is selected from the group consisting of: (a) the nucleotide sequence as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 1; (b) the nucleotide sequence of an insert of DNA in ATCC, deposit number PTA-1617; (c) a nucleotide sequence encoding the polypeptide as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2; (d) a nucleotide sequence that hybridizes under conditions of moderate or high stringency, with the complement of any of the items (a) - (c); and (e) a nucleotide sequence complementary to any of the items (a) - (c). The invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence that is selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide that is at least about 70 percent identical to the polypeptide that is set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2; (b) a nucleotide sequence coding for an allelic variant or splicing variant of the nucleotide sequence as set forth in SEQUENCE IDENTIFICATION NUMBER: 1, the nucleotide sequence of the DNA insert in ATCC, deposit number PTA-1617, or in subsection (a); (c) a region of the nucleotide sequence of SEQUENCE IDENTIFICATION NUMBER: 1, the DNA insert in ATCC, deposit number PTA-1617, (a) or (b) encoding a polypeptide fragment of at least about 25 amino acid residues, wherein the polypeptide fragment has an activity of the encoded polypeptide as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, or is antigenic; (d) a region of the nucleotide sequence of the SEQUENCE OF IDENTIFICATION NUMBER: 1, the insert of DNA in ATCC deposit number PTA-1617, or any of the clauses (a) - (c) comprising a fragment of at least about 16 nucleotides; (e) a nucleotide sequence that hybridizes under conditions of moderate or high stringency with the complement of any of the items (a) - (d); and (f) a nucleotide sequence complementary to any of items (a) - (d). The invention further provides an isolated nucleic acid molecule comprising a nucleotide sequence that is selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, thereby minus a conservative amino acid substitution, wherein the encoded polypeptide has a polypeptide activity that is set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2; (b) a nucleotide sequence encoding a polypeptide as set forth in SEQUENCE IDENTIFICATION NUMBER: 2, with at least one amino acid insertion, wherein the encoded polypeptide has a polypeptide activity as set forth in the SEQUENCE OF IDENTIFICATION NUMBER: 2; (c) a nucleotide sequence encoding a polypeptide as set forth in SEQUENCE IDENTIFICATION NUMBER: 2, with at least one amino acid deletion, wherein the encoded polypeptide has a polypeptide activity as set forth in the SEQUENCE OF IDENTIFICATION NUMBER: 2; (d) a nucleotide sequence encoding a polypeptide as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, which has a C or N terminal truncated part, wherein the encoded polypeptide has a polypeptide activity as set forth in the SEQUENCE IDENTIFICATION NUMBER: 2; (e) a nucleotide sequence encoding a polypeptide as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, with at least one modification that is selected from the group consisting of substitutions, insertions, amino acid deletions, cleavage in part C and N-terminal, wherein the encoded polypeptide has a polypeptide activity as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2; (f) a nucleotide sequence of any of (a) - (e), comprising a fragment of at least about 16 nucleotides; (g) a nucleotide sequence that hybridizes under conditions of moderate or high stringency with the complement of any of the items (a) - (f); and (h) a nucleotide sequence complementary to any of items (a) - (e). The present invention provides an isolated polypeptide comprising an amino acid sequence that is selected from the group consisting of: (a) the amino acid sequence as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2; and (b) the amino acid sequence encoded by the DNA insert in ATCC, deposit number PAT 1617. The invention also provides an isolated polypeptide comprising an amino acid sequence that is selected from the group consisting of: (a) the sequence of amino acids as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 3, optionally additionally comprising an amino terminal methionine; (b) an amino acid sequence for an ortholog of SEQUENCE OF IDENTIFICATION NUMBER: 2; (c) an amino acid sequence which is at least about 70 percent identical to the amino acid sequence of SEQUENCE OF IDENTIFICATION NUMBER: 2, wherein the polypeptide has a polypeptide activity as set forth in SEQUENCE OF IDENTIFICATION NUMBER : 2; (d) a fragment of the amino acid sequence set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2 comprising at least about 25 amino acid residues, wherein the fragment has an activity of the polypeptide that is set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, or it is antigenic; and (e) an amino acid sequence for an allelic variant or splicing variant of the amino acid sequence as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, the amino acid sequence encoded by the DNA insert in ATCC, deposit number PTA- 1617, or paragraphs (a) - (c). The invention further provides an isolated polypeptide comprising an amino acid sequence that is selected from the group consisting of: (a) the amino acid sequence as set forth in SEQUENCE IDENTIFICATION NUMBER: 2, with at least one amino acid conservative substitution , wherein the polypeptide has an activity of the polypeptide that is set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2; (b) the amino acid sequence as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, with at least one amino acid insertion, wherein the polypeptide has an activity of the polypeptide set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2; (c) the amino acid sequence as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, with at least one amino acid deletion, wherein the polypeptide has an activity of the polypeptide set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2; (d) the amino acid sequence as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, which has a cut in the C or N-terminal part, wherein the polypeptide has a polypeptide activity that is set forth in SEQUENCE OF IDENTIFICATION NUMBER : 2; and (e) the amino acid sequence as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, with at least one modification that is selected from the group consisting of substitutions, insertions, amino acid deletions, cuts in the C-part and N-terminal , wherein the polypeptide has an activity of the polypeptide that is set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2. Fusion polypeptides comprising amino acid sequences of FGF-23 are also provided. The present invention also provides an expression vector comprising isolated nucleic acid molecules, as set forth herein, recombinant host cells comprising the recombinant nucleic acid molecules as set forth herein, and a method for producing a FGF polypeptide. -23, which comprises culturing the host cells and optionally isolating the polypeptide that is produced in this manner.

A transgenic non-human animal, comprising a nucleic acid molecule encoding a FGF-23 polypeptide is also encompassed by the invention. The nucleic acid molecules for FGF-23 are introduced into the animal from a host that allows the expression of increased levels of a FGF-23 polypeptide, which may include increased circulating levels. Alternatively, the FGF-23 nucleic acid molecules are introduced into the animal in a manner that prevents expression of the endogenous FGF-23 polypeptide (ie, a transgenic animal is generated that possesses a gene blocked for the FGF-23 polypeptide). The transgenic non-human animal is preferably a mammal, and more preferably it is a rodent, such as a rat or a mouse. Derivatives of the FGF-23 polypeptides of the present invention are also provided. In addition, selective binding agents are provided such as antibodies and peptides capable of specifically binding the FGF-23 polypeptides of the invention.

Such antibodies and peptides may be agonists or antagonists. Pharmaceutical compositions comprising nucleotides, polypeptides or selective binding agents of the invention and one or more pharmaceutically acceptable formulation agents are also included by the invention. The pharmaceutical compositions are used to provide therapeutically effective amounts of the nucleotides or polypeptides of the present invention. The invention also relates to methods for using the polypeptides, nucleic acid molecules and selective binding agents. The FGF-23 polypeptides and the nucleic acid molecules of the present invention can be used to treat, prevent, diminish or detect diseases and disorders, including those mentioned herein. The present invention also provides a method for assaying test molecules to identify a test molecule that binds to a FGF-23 polypeptide. The method comprises contacting a FGF-23 polypeptide with a test molecule to determine the degree of binding of the test molecule to the polypeptide. The method further comprises determining whether such molecules are agonists or antagonists of a FGF-23 polypeptide. The present invention further provides a method for detecting the impact of the molecules on the expression of the FGF-23 polypeptide or on the activity of the FGF-23 polypeptide. Methods to regulate expression and modular (ie, increase or decrease) levels of a FGF-23 polypeptide are also encompassed by the invention. One method comprises administering to an animal a nucleic acid molecule encoding a FGF-23 polypeptide. In another method, a nucleic acid molecule comprising elements that regulate or modulate the expression of a FGF-23 polypeptide can be administered. Examples of these methods include gene therapy, cell therapy and antisense therapy, as further described herein. In another aspect of the present invention, the FGF-23 polypeptides can be used to identify receptors thereof ("FGF-23 polypeptide receptors"). Various forms of "expression cloning" have been used extensively to clone receptors for protein ligands. See, for example, Simonsen and Lodish, 1994, Trends Pharmacol. Sci. 15: 437-41 and Tartaglia et al. , 1995, Cell 83: 1263-71. The isolation of a FGF-23 polypeptide receptor is useful for identifying or developing novel agonists and antagonists of the FGF-23 polypeptide signaling pathway. Such agonists and antagonists include soluble FGF-23 polypeptide receptors, receptor-selective binding agents against FGF-23 polypeptide (such as antibodies and derivatives thereof), small molecules and antisense oligonucleotides, any of which can be used to treat one or more diseases or disorders, including those described herein.

BRIEF DESCRIPTION OF THE FIGURES Figures 1A-1B illustrate the nucleotide sequence of the gene for human FGF-23 (SEQUENCE OF IDENTIFICATION NUMBER: 1) and the deduced amino acid sequence of the human FGFR polypeptide (SEQUENCE OF IDENTIFICATION NUMBER: 2). The predicted signal peptide is indicated (underlined); Figures 2A-2G illustrate the amino acid sequence alignment of human FGF-1 (hu FGF-1; SEQUENCE OF IDENTIFICATION NUMBER: 4), human FGF-2 (hu FGF-2; SEQUENCE OF IDENTIFICATION NUMBER: 5), FGF -3 human (hu FGF-3; SEQUENCE OF IDENTIFICATION NUMBER: 6), human FGF-4 (hu FGF-4; SEQUENCE OF IDENTIFICATION NUMBER: 7), human FGF-5 (hu FGF-5; SEQUENCE OF IDENTIFICATION NUMBER: 8), human FGF-6 (hu FGF-6; SEQUENCE OF IDENTIFICATION NUMBER: 9), human FGF-7 (hu FGF-7; SEQUENCE OF IDENTIFICATION NUMBER: 10), human FGF-8 (hu FGF-8; SEQUENCE IDENTIFICATION NUMBER: 11), human FGF-9 (hu FGF-9; SEQUENCE OF IDENTIFICATION NUMBER: 12), human FGF-10 (hu FGF-10; SEQUENCE OF IDENTIFICATION NUMBER: 13), human FGF-11 (hu FGF - 11; IDENTIFICATION SEQUENCE NUMBER: 14), human FGF-12 (hu FGF-12; IDENTIFICATION SEQUENCE NUMBER: 15), human FGF-13 (hu FGF-13; IDENTIFICATION SEQUENCE NUMBER: 16), FGF-14 human (hu FGF-14; IDENTIFIC SEQUENCE ATION NUMBER: 17), human FGF-16 (hu FGF-16; IDENTIFICATION SEQUENCE NUMBER: 18), human FGF-17 (hu FGF-17; IDENTIFICATION SEQUENCE NUMBER: 19), human FGF-18 (hu FGF-18; IDENTIFICATION SEQUENCE NUMBER: 20), human FGF-19 (hu FGF-19; SEQUENCE OF IDENTIFICATION NUMBER: 21), human FGF-23 (hu FGF-23; SEQUENCE OF IDENTIFICATION NUMBER: 22), murine FGF-1 (mu FGF-1; SEQUENCE OF IDENTIFICATION NUMBER: 23), FGF- 2 murine (mu FGF-2; SEQUENCE OF IDENTIFICATION NUMBER: 24), murine FGF-3 (mu FGF-3; SEQUENCE OF IDENTIFICATION NUMBER: 25), murine FGF-4 (mu FGF-4; SEQUENCE OF IDENTIFICATION NUMBER: 26 ), Murine FGF-5 (mu FGF-5; SEQUENCE OF IDENTIFICATION NUMBER: 27), murine FGF-6 (mu FGF-6; SEQUENCE OF IDENTIFICATION NUMBER: 28), murine FGF-7 (mu FGF-7; SEQUENCE OF IDENTIFICATION NUMBER: 29), murine FGF-8 (mu FGF-8; SEQUENCE OF IDENTIFICATION NUMBER: 30), murine FGF-9 (mu FGF-9; SEQUENCE OF IDENTIFICATION NUMBER: 31), murine FGF-10 (mu FGF- 10; SEQUENCE OF IDENTIFICATION NUMBER: 32), FGF-11 m urine (mu FGF-11; SEQUENCE OF IDENTIFICATION NUMBER: 33), murine FGF-12 (mu FGF-12; SEQUENCE OF IDENTIFICATION NUMBER: 34), murine FGF-13 (mu FGF-13; SEQUENCE OF IDENTIFICATION NUMBER: 35), murine FGF-14 (mu FGF-14; SEQUENCE OF IDENTIFICATION NUMBER: 36), murine FGF-15 (mu FGF-15; IDENTIFICATION SEQUENCE NUMBER: 37), rat FGF-16 (rat FGF-16; SEQUENCE OF IDENTIFICATION NUMBER: 38), murine FGF-17 (mu FGF-17; SEQUENCE OF IDENTIFICATION NUMBER: 39); Figure 3 illustrates the expression of mRNA for FGF-23 as detected by hybridization in si tu in the brain and cardiac muscle (heart) of normal adult mouse (contrast staining H & E = hematoxylin and eosin; ISH = hybridization in si you); Figure 4 illustrates the expression of AR? m for FGF-23 as detected by hybridization in si tu in the subcapsular region of the lymph node (lymph node), thymic medulla (thymus), lacuna of the cortical bone of the tibia (tibia) and trabecular bone in the head (head) of a non-expressing transgenic mouse (contrast staining H & E hematoxylin and eosin; ISH = in-situ hybridization); Figure 5 illustrates the expression of AR? m for FGF-23 detected by hybridization in si tu in the liver, spleen, thymic marrow (thymus) and megakaryocytes in the bone marrow (bone marrow) of a transgenic mouse of high expression (staining of H & E = haematoxylin and eosin contrast; ISH = in situ hybridization); Figure 6 illustrates the expression of AR? m for FGF-23, detected by hybridization in si tu in smooth muscle tissue near the prostate (smooth muscle), muscle tissue of the jaw (muscle), chondrocytes in the tibia (tibia) ) and chondrocytes in the vertebrae (vertebrae) of transgenic mice with high expression (contrast staining H & E = hematoxylin and eosin; ISH = hybridization in si tu).

DETAILED DESCRIPTION OF THE INVENTION The section headers used here are for organizational purposes only and are not considered as limiting the subject matter described. All references mentioned in this application are expressly incorporated by reference herein.

Definitions The terms "gene for FGF-23" or "nucleic acid molecule for FGF-23" or "FGF-23 polynucleotide" refer to a nucleic acid molecule comprising or consisting of a nucleotide sequence as set forth in the SEQUENCE IDENTIFICATION NUMBER: 1, a nucleotide sequence encoding the polypeptide as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, a nucleotide sequence of a DNA insert in ATCC, deposit number PTA-1617, and nucleic acid molecules as define in the present. The term "allelic variant of FGF-23 polypeptide" refers to one or several possible alternative forms that occur naturally of a gene occupying a given locus on a chromosome of an organism or a population of organisms. The term "polypeptide splice variant" FGF-23"refers to a nucleic acid molecule, usually RNA, which is generated by alternative processing of intron sequences of an RNA transcript of the amino acid sequence of the FGF-23 polypeptide, as set forth in the SEQUENCE OF IDENTIFICATION NUMBER: 2. The term "isolated nucleic acid molecule" refers to a nucleic acid molecule of the invention that: (1) has been separated from at least 50 percent proteins, lipids, carbohydrates or other materials with which is found naturally when total nucleic acid is isolated from the source cells, (2) is not bound to all or a portion of a polynucleotide to which an "isolated nucleic acid molecule" binds in nature, (3) ) is operably linked to a polynucleotide to which it is not bound in nature, or (4) it does not occur in nature as part of a larger polynucleotide sequence. The nucleic acid of the present invention is substantially free of any other nucleic acid contaminating molecule or other contaminants that are in their natural environment that could interfere with their use in the production of the polypeptide or in its therapeutic, diagnostic, prophylactic or therapeutic use. research. The term "nucleic acid sequence" or "nucleic acid molecule" refers to a DNA or RNA sequence. The term encompasses molecules that are formed from any of the known DNA and RNA base analogues such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylethytosine, pseudoisocytosine, 5- (carboxyhydroxymethyl) uracil, -fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine , 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutoxosine, pseudoouracil, kerosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester of N-acid uracil-5-oxyacetic acid, uracil-5-oxyacetic acid, pseudouracil, kerosine, 2-thiocytosine and 2,6-diaminopurine. The term "vector" is used to refer to any molecule (eg, nucleic acid, plasmid or virus) used to transfer coding information to a host cell. The term "expression vector" refers to a vector that is suitable for the transformation of a host cell and that contains nucleic acid sequences that direct or control the expression of the heterologous nucleic acid insert sequences. The expression includes, but is not limited to procedures such as RNA transcription, translation and splicing, if introns are present. The term "operably linked" is used herein to refer to a distribution of flanking sequences wherein the flanking sequences described in this manner are configured or assembled in a manner that performs their usual function. Thus, a flanking sequence operably linked to a coding sequence may be capable of carrying out the replication, transcription or translation of the coding sequence. For example, the coding sequence is operably linked to a promoter when the promoter is capable of directing the transcription of that coding sequence. A flanking sequence need not be contiguous with the coding sequence, as long as it functions correctly. Thus, for example, the interposed but transcribed untranslated sequences may be present between the promoter sequence and a coding sequence of the promoter sequence can still be considered "operably linked" to the coding sequence. The term "host cell" is used to refer to a cell which has been transformed or is capable of being transformed with a nucleic acid sequence and then expressing a selected gene of interest. The term includes the progeny of the original cell, whether or not the progeny are identical in morphology or in their genetic constitution to the original cell so that the selected gene is present. The term "FGF-23 polypeptide" refers to a polypeptide comprising the amino acid sequence of SEQUENCE OF IDENTIFICATION NUMBER: 2 and related polypeptides. Related polypeptides include the FGF-23 polypeptide fragments, FGF-23 polypeptide orthologs, FGF-23 polypeptide variants, and FGF-23 polypeptide derivatives, which possess at least one activity of the polypeptide as set forth in the SEQUENCE OF IDENTIFICATION NUMBER: 2. FGF-23 polypeptides may be mature polypeptides, as defined herein, and may or may not have an amino terminal methionine residue, based on the method by which they are prepared. The term "FGF-23 polypeptide fragment" refers to a polypeptide comprising a part truncated in the amino-terminal part (with or without a leader sequence) or a cut in the carboxyl-terminal portion of the polypeptide as set forth in the SEQUENCE OF IDENTIFICATION NUMBER: 2. The term "FGF-23 polypeptide fragment" also refers to a terminal amino or carboxy-terminal portion of the FGF-23 polypeptide orthologs, FGF-23 polypeptide derivatives or FGF-23 polypeptide variants, or with the amino or carboxyl terminal cuts of the polypeptides encoded by the allelic variants of the FGF-23 polypeptide or of the splice variants of the FGF-23 polypeptide. FGF-23 polypeptide fragments may result from an alternative RNA splice or from protease activity in vivo. Membrane-bound forms of a FGF-23 polypeptide are also contemplated by the present invention. In preferred embodiments, the cuts or deletions comprise about 10 amino acids, or about 20 amino acids or about 50 amino acids, or about 75 amino acids or about 100 amino acids or more than about 100 amino acids. The polypeptide fragments produced in this manner will comprise about 25 contiguous amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids or about 150 amino acids, or about 200 amino acids, or more than about 200 amino acids. Such FGF-23 polypeptide fragments can optionally comprise an amino terminal methionine residue. It will be appreciated that such fragments can be used, for example, to generate antibodies to FGF-23 polypeptides. The term "FGF-23 polypeptide ortholog" refers to a polypeptide of another species that corresponds to the amino acid sequence of the FGF-23 polypeptide, as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2. For example, FGF-23 polypeptides of mouse and human are considered orthologous to each other. The term "FGF-23 polypeptide variants" refers to FGF-23 polypeptides that comprise amino acid sequences having in the amino acid sequence, one or more substitutions, deletions (such as internal deletions or FGF-23 polypeptide fragments) or additions (such as internal additions or FGF-23 fusion polypeptides) compared to the amino acid sequence of the FGF-23 polypeptide set forth in SEQUENCE IDENTIFICATION NUMBER: 2 (with or without a leader sequence). Variants may be those that occur naturally (eg allelic variants of the FGF-23 polypeptide, FGF-23 polypeptide orthologs and splice variants of the FGF-23 polypeptide), or can be artificially constructed. Such variants of the FGF-23 polypeptide can be prepared from corresponding nucleic acid molecules having a DNA sequence that varies in consequence of the DNA sequence as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 1. In the preferred embodiments, the variants have the 3, or 5, or 10, or 15, or 1 to 20, or 1 to 25, or 1 to 50, or 1 to 75, or 100 , or more than 100 substitutions, insertions, additions or deletions of amino acids, wherein the substitutions may be conservative or non-conservative, or any combination thereof. The term "FGF-23 polypeptide derivatives" refers to the polypeptide as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, FGF-23 polypeptide fragments, FGF-23 polypeptide orthologs or FGF-23 polypeptide variants as define in the present, that they have been modified chemically. The term "FGF-23 polypeptide derivatives" also refers to polypeptides encoded by the allelic variants of the FGF-23 polypeptide of the FGF-23 polypeptide splice variants, as defined herein that have been chemically modified. The term "mature FGF-23 polypeptide" refers to a FGF-23 polypeptide that lacks a leader sequence. A mature FGF-23 polypeptide may also include other modifications such as a proteolytic processing of the amino-terminal part (with or without a leader sequence) or the carboxyl-terminal part, the separation of a smaller polypeptide from a larger precursor, N-linked or O-linked glycosylation and the like. An exemplary mature CHL polypeptide is shown by the amino acid sequence of NUMBER IDENTIFICATION SEQUENCE: 3. The term "FGF-23 fusion polypeptide" refers to the fusion of one or more amino acids (such as a heterologous protein or peptide). ) of the amino or carboxyl terminal portion of the polypeptide, as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, FGF-23 polypeptide fragments, FGF-23 polypeptide orthologs, or polypeptide variants of FGF-23 or FGF-23 derivatives , as defined herein. The term "FGF-23 fusion polypeptide" also refers to a fusion of one or more amino acids in the amino or carboxyl terminal portion of the polypeptide encoded by the allelic variants of the FGF-23 polypeptide or the splice variants of the FGF-23 polypeptide. , as defined herein. The term "biologically active FGF-23 polypeptide" refers to FGF-23 polypeptides having at least one characteristic activity of the polypeptide comprising the amino acid sequence of SEQUENCE IDENTIFICATION NUMBER: 2. In addition, a FGF-23 polypeptide can be active as an immunogen; that is, the FGF-23 polypeptide contains at least one epitope for which antibodies can be generated. The term "isolated polypeptide" refers to a polypeptide of the present invention that: (1) has been separated from at least 50 percent of the polynucleotides, lipids, carbohydrates or other materials with which it occurs naturally when isolates from the source cell, (2) is not bound (by covalent or non-covalent interaction) to all or a portion of a polypeptide to which the "isolated polypeptide is bound in nature, (3) is operably linked (eg, covalent or non-covalent interaction) to a polypeptide with which nature is not bound, or (4) does not occur in nature Preferably, the isolated polypeptide is substantially free of any other contaminating polypeptide or other contaminants that are found In their natural environment and which may interfere with their therapeutic, diagnostic, prophylactic or research use, the term "identity", as it is known in the art, refers to re to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, determined when comparing the sequences. In the "identity" technique it also means the degree of sequence relationship between nucleic acid molecules or polypeptides as the case may be, determined by the coincidence between chains of two or more nucleotide sequences or two or more amino acid sequences. The "identity" measures the percent of identical matches between the smallest of the two or more sequences with alignment separations (if any) resolved by a particular mathematical model or a computer program (ie, "algorithms"). The term "similarity" is a related concept, but in contrast to "identity", "similarity" refers to a measure of relationship which includes both identical coincidences and coincidences by conservative substitution. For example if two polypeptide sequences have 10/20 identical amino acids, and the rest are non-conservative substitutions, then the percent identity and similarity in both cases would be 50%. If in the same example, there were five additional positions where there were conservative substitutions, then the percent identity remains at 50% but the percent similarity would be 70% (15/20). Therefore, in cases where there are conservative substitutions, the percent similarity between two polypeptides will be greater than the percent identity between these two polypeptides. The term "occurring naturally" or "native", when used in relation to biological materials such as nucleic acid molecules, polypeptides, host cells and the like, refers to materials which are found in nature and not They are manipulated by man. Similarly "not occurring naturally" or "non-native" as used herein, refers to a material that is not found in nature or that has been structurally modified or synthesized by man. The terms "effective amount" and "therapeutically effective amount" each refer to the amount of a FGF-23 polypeptide or a nucleic acid molecule for FGF-23 used to support an observable level of one or more biological activities of the polypeptides. FGF-23, as set forth herein. The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein, refers to one or more suitable formulation materials for carrying out or enhancing the delivery of the FGF-23 polypeptide, the nucleic acid molecule for FGF-23 or the selective binding agent of FGF-23, as a pharmaceutical composition. The term "antigen" refers to a molecule or a portion of a molecule capable of binding by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. An antigen can have one or more epitopes. The term "selective binding agent" refers to one or more molecules that have specificity for a FGF-23 polypeptide. As used herein, the terms "specific" and "specificity" refer to the ability of selective binding agents to bind human FGF-23 polypeptides and not to bind to polypeptides other than human FGF-23. However, it will be appreciated that selective binding agents can also bind orthologs of the polypeptide as set forth in SEQUENCE IDENTIFICATION NUMBER: 2, that is, interspecies versions thereof, such as the mouse FGF-23 polypeptides and rat. The term "transduction" is used to refer to the transfer of genes from one bacterium to another, usually by a phage. "Transduction" also refers to the acquisition and transfer of eukaryotic cell sequences by retroviruses. The term "transfection" is used to refer to the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced into the cell membrane. Many transfection techniques are well known in the art and are described herein. See, for example, Graham et al. 1973, Virology 52: 456; Sambrook et al. , Molecular 5 Cloning, A. Laboratory Manual (Cold Spring Harbor Laboratories, 1989); Davis et al. , Basic Methods in Molecular Biology (Elsevier, 1986); and Chu et al. , 1981. Gene 13: 197. Such techniques can be used to introduce one or more portions of exogenous DNA into host cells 10 adequate. The term "transformation" as used herein, refers to a change in the genetic characteristics of the cell, and a cell that has been transformed when it has been modified to contain a DNA 15 new. For example, a cell is transformed when it has been genetically modified with respect to its native state. After transfection or transduction, the transforming DNA can be recombined with that of the cell by physically exchanging it in the chromosome of the cell, and 20 can be transiently maintained as an episomal element without replication, or can be replicated independently as a plasmid. A cell is considered to have been stably transformed when the DNA replicates with the division of the cell. 25 ^ gj £ g Relationship of Nucleic Acid Molecules and Polypeptides It is understood that the related nucleic acid molecules include allelic or splice variants of the nucleic acid molecule of SEQUENCE IDENTIFICATION NUMBER: i, and include sequences which are complementary to any of the above nucleotide sequences. Related nucleic acid molecules also include a nucleotide sequence that 10 encodes a polypeptide comprising or consisting essentially of a substitution, modification, addition or deletion of one or more amino acid residues as compared to the polypeptide of SEQUENCE IDENTIFICATION NUMBER: 2. Such related FGF-23 polypeptides may comprise, 15 for example, an addition or a deletion of one or more glycosylation sites? or O-linked or an addition or deletion of one or more cysteine residues. Related nucleic acid molecules also include fragments of nucleic acid molecule 20 for FGF-23 which encodes a polypeptide of at least about 25 contiguous amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or more than 100 amino acid residues of the FGF-23 polypeptide of the SEQUENCE 25 IDENTIFICATION NUMBER: 2. __M _____ ^ _______ áttíH _____? In addition, the related nucleic acid molecules for FGF-23 also include those molecules which comprise nucleotide sequences that hybridize under conditions of moderate or high stringency as defined herein, with a sequence completely complementary to the nucleic acid molecule for FGF. -23 of the SEQUENCE OF IDENTIFICATION NUMBER: 1, or of a molecule encoding a polypeptide, polypeptide which comprises the amino acid sequence as shown in SEQUENCE OF IDENTIFICATION NUMBER: 2, or a fragment of nucleic acid as defined in the present, or of a nucleic acid fragment encoding a polypeptide as defined herein. Hybridization probes can be prepared using the FGF-23 sequences provided herein to screen cDNAs, genomic or synthetic DNA libraries for related sequences. The regions of the DNA or of the amino acid sequence for the FGF-23 polypeptide that show significant identity with known sequences are easily determined using sequence alignment algorithms as described herein and these regions can be used to design probes for screening or systematic identification. The term "highly stringent conditions" refers to those conditions that are designed to allow the hybridization of DNA strands whose sequences are highly complementary, and to exclude the hybridization of DNAs with significant mismatch. The stringency of hybridization is determined primarily by temperature, ionic strength and the concentration of denaturing agents such as formamide. Examples of "highly stringent conditions" for high hybridization are 0.015M sodium chloride, 0.0015M sodium citrate at 65-68 ° C, or 0.015M sodium chloride, 0.0015M sodium citrate and 50% formamide at 42 ° C. . See Sambrook, Fritsch &; Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory, 1989); Anderson et al. , Nucleic Acid Hybridi sa tion: A Practical Approach Ch. 4 (IRL Press Limited). More stringent conditions (such as higher temperature, lower ionic strength, higher concentration of formamide or other denaturing agent) can also be used - however, the rate of hybridization will be altered. Other agents can be included in the hybridization and wash buffer for the purpose of reducing non-specific or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecylsulfate NaDodS0, (SDS), ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA) and dextran sulfate, although other suitable agents can also be used. The concentration and types of these additives can change without substantially altering the stringency of the hybridization conditions. Hybridization experiments are usually carried out at a pH of 6.8-7.4; however, under typical conditions of ionic strength, the hybridization rate depends very much on the pH. See Anderson et al. , Nucleic Acid Hybridization: A Practical Approach Ch. 4 (IRL Press Limited). Factors that affect the stability of a double strand of DNA include base compositions, length and degree of mismatch of base pairs. Hybridization conditions can be adjusted by those skilled in the art in order to adapt these variables and allow DNAs with different sequence ratios to form hybrids. The melting temperature of a double DNA perfectly matched by the following equation can be determined: Tm (° C) = 81.5 +16.6 (log [Na +]) + 0.41 (% of G + C) - 600 / N - 0.72 (% of formamide), where N is the length of the double chain that is formed, [Na +], and the molar concentration of the sodium ion in the hybridization or washing solution,% of G + C is the percentage of bases (guanine + cytosine) in the hybrid. For hybrids f ~ * - * ¿- - * - - imperfectly matched, the melting temperature is reduced by approximately 1 ° C for every 1% mismatch. The term "conditions of moderate stringency" refers to conditions under which a double DNA strand can be formed with a higher degree of mismatch of base pairs than would occur under "high stringency conditions". Examples of "moderate stringency conditions" are 0.015 M sodium chloride, 0.0015 M sodium citrate at 60-65 ° C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 20% formamide at 37-50 ° C. . As an example, the "moderate stringency conditions" of 50 ° C in 0.015 M sodium ions will allow approximately a mismatch of 21%. It will be appreciated by those skilled in the art that there is no absolute differentiation between "conditions of high stringency" and "conditions of moderate stringency". For example, at a 0.015 M sodium ion concentration (without formamide), the perfectly matched long DNA fusion temperature is about 71 ° C. With a wash at 65 ° C (at the same ionic strength), this may allow approximately a mismatch of 6%. To retain related sequences more distantly, a person skilled in the art can simply decrease the temperature or increase the ionic strength. _ ». . . - r _, t _ .. »_.

A good determination of the melting temperature in NaCl 1M * for oligonucleotide probes up to approximately 20 nt is given by: Tm = 2 ° C per base pair A-T + 4 ° C per base pair G-C * The concentration of the sodium ion in sodium citrate in 6X saline solution (SSC) is 1M. See Suggs et al. , Developmental Biology Using Purified Genes 683 (Brown and Fox, eds., 1981). The high stringency washing conditions for oligonucleotides are usually at a temperature of 0-5 ° C below the Tm of the oligonucleotide in 6X SSC, 0.1% SDS. In another embodiment, the related nucleic acid molecules comprise or consist of a nucleotide sequence that is at least about 70 percent identical to the nucleotide sequence as shown in SEQUENCE IDENTIFICATION NUMBER: 1, or comprise or consist essentially of a nucleotide sequence encoding a polypeptide that is at least about 70 percent identical to the polypeptide as set forth in SEQUENCE IDENTIFICATION NUMBER: 2. In preferred embodiments, the nucleotide sequences are about 75 percent, or about 80 percent one hundred, or about 85 percent, or about 90 percent, or about 95, 96, 97, 98, or 99 percent identical to the nucleotide sequence as shown in SEQUENCE IDENTIFICATION NUMBER: 1, or the nucleotide sequences they encode for a polypeptide are approximately 75 percent, or approximately 80 percent , or approximately 85 percent, or approximately 90 percent, or approximately 95, 96, 97, 98, or 99 percent identical to the polypeptide sequence as set forth in SEQUENCE IDENTIFICATION NUMBER: 2. Related nucleic acid molecules that encode for polypeptides that possess at least one activity of the polypeptide as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2. Differences in the nucleic acid sequence may result in conservative or non-conservative modifications of the amino acid sequence relative to the sequence of amino acids of the SEQUENCE OF IDENTIFICATION NUMBER: 2. The conservative modifications of the amino acid sequences of the SEQUENCE OF IDENTIFICATION NUMBER: 2 (and the corresponding modifications to the coding nucleotides) will produce a polypeptide having similar functional and chemical characteristics to those of the FGF-23 polypeptides. By contrast, substantial modifications can be made in the functional or chemical characteristics of the FGF-23 polypeptides when selecting substitutions in the amino acid sequence of SEQUENCE OF IDENTIFICATION NUMBER: 2 that differ significantly in their effect in maintaining: (a) the structure of the main molecular structure in the substitution area, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. For example, a "conservative amino acid substitution" may involve a substitution of a native amino acid residue with a non-native residue so that there is little or no effect on the polarity or charge of the amino acid residue in that position. In addition, any native residue in the polypeptide may also be substituted with alanine, as previously described for "alanine scanning mutagenesis". Conservative amino acid substitutions also encompass amino acid residues that do not occur naturally and that are usually incorporated by chemical peptide synthesis instead of by synthesis in biological systems. These include peptide mimetics and other reverse or inverted forms of amino acid portions. The residues that occur naturally can be divided into classes based on their common side chain properties: 1) hydrophobic: norleucine, Met, Ala, Val, Leu, lie; 2) neutral hydrophilic: Cys, Ser, Thr; 3) Acids: Asp, Glu; 4) Basic: Asn, Gln, His, Lys, Arg; 5) residues that alter the orientation of the chain: Gly, Pro; and 6) aromatics: Trp, Tyr, Phe. For example, non-conservative substitutions may involve the exchange of one member of these classes by the member of another class. Such substituted residues can be introduced into regions of human FGF-23 polypeptide that are homologous with non-human FGF-23 polypeptides or in non-homologous regions of the molecule. In making such changes, the hydropathic index of amino acids should be considered. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and its loading characteristics. The hydropathic indexes are: isoleucine (+4.5); valina (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); Alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3) proline (-1.6); histidine (-3.2); glutamate (-3.5) glutamine (-3.5); aspartate (-3.5); asparagine (-3.5) lysine (-3.9); and arginine (-4.5). The importance of the hydropathic amino acid index to confer an interactive biological function on a protein is generally understood in the art (Kyte et al., 1982, J. Mol. Biol. 157: 105-31). It is known that some amino acids can be substituted by other amino acids having a similar hydropathic rating or rating and still retain similar biological activity. When making changes based on the hydropathic index, it is preferred to substitute amino acids whose hydropathic indices are within + 2, those which are within + 1 are particularly preferred, and those that are within + 0.5 are more particularly preferred. . It is also understood in the art that replacement of similar amino acids can be carried out effectively on the basis of hydrophilicity, particularly when the functionally equivalent protein biologically or the peptide thus generated is designed for use in immunological modalities, as in the present case. The highest local average hydrophilicity of a protein, governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, that is, with a biological property of the protein. The following hydrophilicity values have been assigned for these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 + 1); Alanine (-0.5); histidine (-0.5); cystine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based on similar hydrophilicity values, substitution of amino acids whose hydrophilicity values are within +2 is preferred, those which are within +1 being preferred, and those within +0.5 more particularly preferred. One can also identify epitopes of primary amino acid sequences based on hydrophilicity. These regions are also referred to as "epitopic core regions." The desired amino acid substitutions (conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the FGF-23 polypeptide, or to increase or decrease the affinity - of the FGF-23 polypeptides described herein. The exemplary substitutions of amino acids are established in Table I.

Table 1 Amino Acid Substitutions Original Residual Substitutions Preferred Substitutions Ala Val, Leu, Lie Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg lie Leu, Val, Met, Ala, Leu Phe, Norleucine Leu Norleucine, Lie, Lie Val, Met, Ala, Phe Lys Arg, 1, 4-diamino-- Arg butyric, Gln, Asn Met Leu, Phe, lie Leu Phe Leu, Val, Lie, Wing, Leu Tyr Pro Wing Gly Ser Thr, Wing, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, thr, Ser Phe Val lie, Met, Leu, Phe, Leu Ala, Norleucine A person skilled in the art will be able to determine suitable variants of the polypeptide as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2 using well-known techniques. To identify suitable areas of the molecule that can be changed without destroying the biological activity, a person skilled in the art can address areas that are not considered important for the activity. For example, when similar polypeptides with similar activities of the same species or of another species are known, a person skilled in the art can compare the amino acid sequence of a FGF-23 polypeptide with such similar polypeptides. With such a comparison, one can identify waste and portions of r. ... I ... ... the molecules that are conserved between similar polypeptides. It will be appreciated that changes in the areas of the FGF-23 molecule that do not retain such similar polypeptides are less likely to adversely alter the biological activity or structure of a FGF-23 polypeptide. One skilled in the art can also know that, even in relatively conserved regions, one can substitute chemically similar amino acids for residues that occur naturally while retaining activity (conservative amino acid residue substitutions). Therefore, even areas that may be important for biological activity or for structure may be subjected to conservative amino acid substitutions without destroying the biological activity or without adversely altering the structure of the polypeptide. Additionally, a person skilled in the art can review structure and function studies that identify residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a FGF-23 polypeptide that correspond to amino acid residues that are important for activity or structure in similar polypeptides. A person skilled in the art can opt for chemically similar amino acid substitutions for such predicted important amino acid residues of the FGF-23 polypeptides. A person skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, a person skilled in the art can predict the alignment of amino acid residues of the FGF-23 polypeptide with respect to its three-dimensional structure. A person skilled in the art can choose not to make radical changes in the predicted amino acid residues that are on the surface of the protein, since such residues can be related in important interactions with other molecules. In addition, a person skilled in the art can generate test variants containing a single amino acid substitution at each amino acid residue. The variants can be examined using activity assays known to those skilled in the art. Such variants can be used to obtain information about suitable variants. For example, if one discovers that a change in a particular amino acid residue results in an activity that is destroyed, undesirably reduced or inadequate, variants with such a change should be avoided. In other words, based on the information obtained from such routine or systematic experiments, a person skilled in the art can readily determine the amino acids wherein additional substitutions should be avoided either alone or in combination with other mutations. Many scientific publications have been made for the prediction of secondary structure (see Moult, 1996, Curr Opin Opin Biotechnol 7: 422-27; Chou et al. , 1914, Biochemistry 13: 222-45; Chou et al. , 1974, Biochemistry 113: 211-22; Chou et al. , 1978, Adv. Enzymol. Relat. Mol areas. Biol. 47: 45-48; Chou et al. , 1978, Ann. Rev. Biochem. A: 251-276; and Chou et al. , 1979, Biophys. J. 26: 367.84. In addition, computer programs are now available to help predict the secondary structure. A method to predict secondary structure is based on homology modeling. For example, two polypeptides or proteins having a sequence identity greater than 30%, or a similarity greater than 40%, often have similar structural topologies. The recent advance in the structural protein database (PDB) has provided improved prediction capacity in the secondary structure, including the potential number of folds within the structure of a polypeptide or protein. See Holm et al. , 1999, Nucleic Acids Res. 27: 244-47. It has been suggested that there is a limited number of folds in a given polypeptide or protein and that, once a critical number of structures have been resolved, the structural prediction will be markedly more accurate (Brenner et al., 1997, Curr. Opin. Struct. Biol. 7: 369-76). Additional methods for predicting a secondary structure include "sequencing or chain establishment" (Jones, 1997, Curr Opin Struct., Biol. 7: 377-87, Sippl et al., 1996, Structure 4: 15-19). , "profile analysis" (Bowie et al., 1991, Sciences, 253: 164-170, Gribskov et al., 1990, Method Enzymol, 183: 146-59, Gribskov et al., 1987, Proc. Natl. Acad. Sci. USA 84: 4355-58), and "evolutionary link" (See Holm et al., Supra, and Brenner et al., Supra.) Preferred variants of the FGF-23 polypeptide include glycosylation variants wherein has altered the number or type of glycosylation sites compared to the amino acid sequence set forth in SEQUENCE IDENTIFICATION NUMBER: 2. In one embodiment, FGF-23 polypeptide variants comprise a greater or lesser number of glycosylation sites joined to N in comparison with the amino acid sequence that is established in SEQUENCE OF IDENTIFICATION NUMBER: 2. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue less proline. Substitution of the amino acid residues to create this sequence provides a potential new site for the addition of a N-linked carbohydrate chain. Alternatively, substitutions that eliminate this sequence will eliminate an existing chain of N-linked carbohydrate. A rearrangement is also provided. of the N-linked carbohydrate chains wherein one or more N-linked glycosylation sites are eliminated (typically those that occur naturally), and one or more new sites are generated attached to N. Preferred FGF-23 variants Additional ones include cysteine variants, wherein one or more cysteine residues are deleted or substituted with another amino acid (eg serine) as compared to the amino acid sequence set forth in SEQUENCE IDENTIFICATION NUMBER: 2. Cysteine variants are useful when FGF-23 polypeptides must be renatured in a biologically active conformation for example after the isolation of insoluble inclusion bodies. Cysteine variants generally have less cysteine residues than the native protein and typically have an even number to minimize interactions that result from unpaired cysteines. In other embodiments, the related nucleic acid molecules comprise or consist of a non-nucleotide sequence encoding a polypeptide as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2 with at least one insertion of an amino acid and wherein the polypeptide has a polypeptide activity as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, or a nucleotide sequence encoding a polypeptide as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2 with at least one deletion of an amino acid and wherein the polypeptide has a polypeptide activity as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2. Related nucleic acid molecules also comprise or consist of a nucleotide sequence encoding a polypeptide as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, wherein the polypeptide has a truncated carboxyl or amino terminal and furthermore wherein the polypeptide has a polypeptide activity which is set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2. Related nucleic acid molecules also comprise or consist of a nucleotide sequence encoding a polypeptide as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, with at least one modification that is selected from group consisting of substitutions, insertions and deletions of amino acids, cuts in the carboxyl terminal part and cuts in the amino terminal part, and wherein the polypeptide has a polypeptide activity that is established in SEQUENCE OF IDENTIFICATION NUMBER: 2. In addition, the polypeptide comprising the amino acid sequence of the SEQUENCE OF IDENTIFICATION NUMBER: 2, or another polypeptide FGF-23, can be fused to a - homologous polypeptide to form a homologous homodimer or polypeptide to form a heterodimer. Heterologous peptides and polypeptides include, but are not limited to: an epitope to allow detection and isolation of a FGF-23 fusion polypeptide; a transmembrane receptor protein or a portion thereof, such as an extracellular domain or a transmembrane and intracellular domain; a ligand or a portion thereof which binds to a transmembrane receptor protein; an enzyme or a portion thereof which is catalytically active, a polypeptide or peptide which promotes oligomerization such as a leucine zipper domain; a polypeptide or peptide that increases stability such as an immunoglobulin constant region; and a polypeptide which has a therapeutic activity other than the polypeptide comprising the amino acid sequence as set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, or other FGF-23 polypeptide. The fusions may be carried out either in the amino terminal portion or in the carboxyl terminal portion of the polypeptide comprising the amino acid sequence set forth in SEQUENCE OF IDENTIFICATION NUMBER: 2, or other FGF-23 polypeptide. The fusions can be carried out without a binding or adapter molecule, or through a binding or adapter molecule. A linker or adapter molecule can be one or more amino acid residues, typically from about 20 to about 50 amino acid residues. A binding or adapter molecule can also be designed with a separation site for a restriction endonuclease by DNA or for a protease in order to allow separation of the fused portions. It will be appreciated that once constructed, the fusion polypeptides can form derivatives according to the methods described in 10 present. In a further embodiment of the invention, the polypeptide comprising the amino acid sequence of the SEQUENCE OF IDENTIFICATION NUMBER: 2 or other polypeptide FGF-23, is fused to one or more domains of a Fe region of 15 human IgG. The antibodies comprise two functionally independent parts, a variable domain known as "Fab", which binds an antigen, and a constant domain known as "Fe", which is related to effector functions such as complement activation and attack by 20 phagocytic cells. A Fe has a prolonged serum half-life, while the Fab has a short duration. Capón et al. , 1989, Nature 337: 525-31. When they are constructed together with a therapeutic protein, a Fe domain can provide a longer half-life or incorporate 25 functions such as Fe receptor binding, protein binding -______ * ____._.-. ^ _ .., - A, complement fixation and maybe even placental transfer. Table II summarizes the use of certain Fe fusions known in the art.

Table II Fe Fusions with Therapeutic Proteins 10 fifteen twenty 25 In one example, a region of human hinge IgG, CH2 and CH3 can be fused to either the amino terminal or carboxyl terminal portion of the FGF-23 polypeptides using methods known to those skilled in the art. In another example, the hinge human IgG region, CH2 and CH3 can be fused to either the amino terminal or carboxyl terminal portion of a fragment of the FGF-23 polypeptide (e.g., the predicted extracellular portion of the FGF-23 polypeptide) . The resulting FGF-23 fusion polypeptide can be purified by the use of a protein A affinity column. Peptides and proteins fused to a Fe region have been found to exhibit a substantially higher half-life in vivo compared to their non-human counterparts. merged In addition, a fusion to a Fe region allows the dimerization / multimerization of the fusion polypeptide. The Fe region can be a Fe region that occurs naturally, or it can be altered to improve certain qualities, such as therapeutic quality, circulation time or reduced aggregation. The identity and similarity of related nucleic acid molecules and polypeptides is easily calculated by known methods. Such methods include, but are not limited to those described in Computational Molecular Biology (A.M. Lesk, ed., Oxford University Press 1988); Biocomputing: Informatics and Genome Projects (D. W. Smith, ed., Academic Press 1993): Computer Analysis of Sequence Data (Part 1, A.M. Griffin and H.G. Griffin, eds., Humana Press 1994); G. von Heinle, Sequence Analysis in 5 Molecular Biology (Academic Press 1987); Sequence Analysis Primer (M. Gribskov and J. Devereux, eds., M. Stockton Press 1991); and Carillo et al. , 1988, SIAM J. Applied Math. , 48: 1073. Preferred methods to determine identity or 10 Similarities are designed to provide the greatest match between the sequences tested. Methods to determine identity and similarity are described in publicly available computer programs. The preferred computer program methods to determine identity 15 and similarity between two sequences include, but are not limited to, the GCG program package, which includes GAP (Devereux et al., 1984, Nucleic Acids Res. 12: 387; Genetics Computer Group, University of Wisconsin, Madison, Wl), BLASTP, BLASTN and FASTA (Altschul et al., 1990, J. "Mol. Biol. 20 215: 403-10). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (Altschul et al., BLAST Manual (NCB NLM NIH, Bethesda, MD), Altschul et al., 1990 supra). You can also use the well-known Smith Waterman algorithm 25 to determine identity. - | - | * tñt_i '_' ^ μf * - *** - '"• to¿ - * - Certain alignment schemes for aligning two amino acid sequences may result in the pairing of only a short region of the two sequences, and this small aligned region may have a very high sequence identity even though there is no significant relationship between the two full length sequences.As a result, in a preferred embodiment, the selected alignment method (GAP program) will result in an alignment that spans at least 50 contiguous amino acids 10 of the claimed polypeptide. For example, using the GAP computer algorithm (Genetics Computer Group, University of Wisconsin, Madison, Wl), two polypeptides are aligned for which the percent identity of 15 sequence, for optimal matching of their respective amino acids ("the paired chain" determined by the algorithm). A separation gap penalty is used (which is calculated as 3X the average diagonal, the "average diagonal" is the average of the diagonal of the 20 comparison matrix used; the "diagonal" is the rating or the number assigned for each perfect match of amino acids by the particular comparison matrix), and a penalty for extension of separation (which is usually 0. IX the opening punishment of 25 separation) as well as a comparison matrix such as PAM 250 or BLOSUM 62, together with the algorithm. A standard comparison matrix can also be used by the algorithm (see Dayhoff et al., 5 Atlas of Protein Sequence and Structure (Supp. 3 1978) (comparison matrix PAM250), Henikoff et al., 1992, Proc. Natl. Acad Sci USA 89: 10915-19 (comparison matrix BLOSUM 62)). Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, 1970, J. "Mol. Biol. 48: 443-53; Comparison matrix: BLOSUM 62 (Henikoff et al., Supra); Penalty for separation: 12 Punishment for separation length: 4 Similarity threshold: 0 The GAP program is useful with the above parameters The parameters mentioned above are default parameters for polypeptide comparisons (besides not presenting punishment for completion of separations) using the GAP algorithm The preferred parameters for comparison of sequences of nucleic acid molecules include the following: Algorithm: Needleman and Wunsch, supra; Comparison matrix: pairing = +10, mismatch = 0 Punishment by separation: 50 Punishment by length of separation: 3 The GAP program is also useful with the above parameters.The parameters mentioned above are the parameters by omission for comparisons of nucleic acid molecule. Other exemplary algorithms, separation opening punishments, separation extension punishments, comparison matrices and similarity thresholds, including those established in the Manual Program, may be used.

Wisconsin Package, Version 9, September 1997. The particular choices made will be apparent to those skilled in the art and will depend on the specific comparison being made, such as DNA to DNA, protein to protein, or protein to DNA; and additionally, if the comparison is between given pairs of sequences (in which case, GAP or BestFit are generally preferred) or between a sequence and a large sequence database (in which case FASTA or BLASTA are preferred).

Nucleic Acid Molecules Nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of - - a FGF-23 polypeptide can be easily obtained in various ways including, without limitation, chemical synthesis, cDNA screening of a genomic library, screening of an expression library or PCR amplification of cDNA. The recombinant DNA methods used herein are generally those set forth in Sambrook et al. , Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) or Current Protocols in 10 Molecular Biology (Ausubel et al., Eds., Green Publishers Inc. and Wiley and Sons 1994). The invention provides nucleic acid molecules as described herein as well as methods for obtaining such molecules. When a gene has been identified as a species 15 which codes for the amino acid sequence of a FGF-23 polypeptide, all or a portion of that gene can be used as a probe to identify orthologs or related genes for the same species. The probes or primers can be used to screen cDNA libraries 20 from various tissue sources believed to express the FGF-23 polypeptide. In addition, part or all of the nucleic acid molecule having the sequence set forth in SEQUENCE OF IDENTIFICATION NUMBER: 1 can be used to examine a genomic library for 25 identify and isolate a gene that codes for the sequence ^^^^ Sfcj? - of amino acids of a FGF-23 polypeptide. Typically, conditions of moderate or high stringency will be used to screen in order to minimize the number of false positive results obtained from screening. 5 'The nucleic acid molecules encoding the amino acid sequence of the FGF-23 polypeptides can also be identified by expression cloning which uses the detection of positive clones based on a property of the expressed protein. Typically, they are screened 10 nucleic acid libraries by the binding of an antibody or other binding partner (eg, receptor or ligand) to cloned proteins that are expressed and displayed on the surface of a host cell. The antibody or binding partner is modified with a label 15 detectable to identify those cells that express the desired clone. The recombinant expression techniques carried out according to the descriptions set out below can be followed to generate these 20 polynucleotides and to express the encoded polypeptides. For example, by inserting a nucleic acid sequence encoding the amino acid sequence of a FGF-23 polypeptide into an appropriate vector, a person skilled in the art can easily produce large 25 amounts of the desired nucleotide sequence. The sequences can then be used to generate detection probes or amplification primers. Alternatively, a polynucleotide encoding the amino acid sequence of a FGF-23 polypeptide can be inserted into an expression vector. By introducing an expression vector into an appropriate host, the encoded FGF-23 polypeptide can be produced in large quantities. Another method for obtaining a suitable nucleic acid sequence is the polymerase chain reaction (PCR).

In this method, cDNA is prepared from poly (A) + RNA or total RNA using the enzyme reverse transcriptase. Two primers, which are usually complementary to two separate regions of cDNA encoding the amino acid sequence of a FGF-23 polypeptide, then 15 are added to the cDNA together with a polymerase such as Taq polymerase, and the polymerase amplifies the cDNA region between the two primers. Another means for preparing a nucleic acid molecule encoding the amino acid sequence of a FGF-23 polypeptide is chemical synthesis using methods well known to those skilled in the art such as those described by Engels et al. , 1989, Angew. Chem. Intl. Ed. 28: 716-34. These methods include, for example, the phosphotriester, phosphoramidite and H-phosphonate methods for synthesis of 25 nucleic acid. A preferred method for such synthesis ^ gtea ^^^^ »- Chemistry is the synthesis supported in polymer using the standard chemistry of phosphoramidite. Typically, the DNA encoding the amino acid sequence of a FGF-23 polypeptide will be several hundred nucleotides in length. The nucleic acids are larger than about 100 nucleotides and can be synthesized as several fragments using these methods. The fragments can then be ligated together to form the full length nucleotide sequence of a gene for FGF-23. Usually, the DNA fragment encoding the amino terminal part of the polypeptide will have an ATG, which codes for a methionine residue. This methionine may or may not be present in the mature form in the FGF-23 polypeptide, depending on whether the polypeptide produced in the host cell is designed to be secreted from that cell. Other methods known to those skilled in the art can also be used. In some embodiments, nucleic acid variants containing codons have been altered for optimal expression in a FGF-23 polypeptide in a given host cell. Particular alterations of the codons will depend on the FGF-23 polypeptide and the host cell selected for expression. Such "codon optimization" can be carried out by various methods, for example, by selecting codons that are preferred for use in genes highly expressed in a given host cell. Computer algorithms which incorporate codon frequency tables such as "Eco_high.Cod" for codon preference of highly expressed bacterial genes can be used and are provided by the University of Wisconsin Package Version 9.0 (Genetics Computer Group, Madison, WI). ). Other useful codon frequency tables include "Celegans_high. Cod", "Celegans_low. Cod", "Drosophila_high.cod", "Human_high. Cod", "Maize_high. Cod" and "Yeast_high.cod". In some cases, it may be desirable to prepare nucleic acid molecules that code for FGF-23 polypeptide variants. Nucleic acid molecules encoding variants can be generated using site-directed mutagenesis, PCR amplification or other appropriate methods wherein the primer (s) has the desired point mutations (see Sambrook et al., Supra and Ausubel et al., supra for descriptions of mutagenesis techniques). Chemical synthesis can also be used using methods described by Engels et al. , supra to prepare such variants. Other methods known to those skilled in the art may also be used.

Vectors and Guest Cells A nucleic acid molecule is inserted that ratftf? f- r i * i *** - *** - • - - - = - "* • - - **». **. * -. codes for the amino acid sequence of a FGF-23 polypeptide, within an appropriate expression vector using standard ligation techniques. The vector is typically selected to be functional in the particular host cell used (i.e., the vector is compatible with the organelles of the host cell so that gene amplification or gene expression can occur). A nucleic acid molecule encoding the amino acid sequence of a FGF-23 polypeptide can be 10 amplify / express in prokaryotic, yeast, insect (baculovirus systems) and eukaryotic host cells. The selection of the host cell will depend, in part, on whether a FGF-23 polypeptide is to be modified post-translationally (eg, glycosylated or phosphorylated). If so, the yeast, insect or mammalian host cells are preferable. For a review of expression vectors see Meth. Enz. , vol. 185 (D.V. Goeddel, ed., Academic Press 1990). Typically, the expression vectors used 20 in any of the host cells will contain sequences for the maintenance of plasmids and for the cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences" in some embodiments will typically include 25 one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a site of ribosome binding, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Each of these sequences is discussed in the following. Optionally, the vector may contain a "tag" coding sequence ie, a molecule located at the 5 'or 3' end of the sequence encoding the FGF-23 polypeptide; the oligonucleotide sequence encodes for polyHis (such as hexaHis), or another "tag" such as FLAG, HA (influenza virus hemagglutinin) or myc from which commercially available antibodies exist. This tag typically fuses to the polypeptide upon expression of the polypeptide and can serve as a means for affinity purification of the FGF-23 polypeptide from the host cell. Affinity purification can be carried out, for example, by column chromatography using antibodies against the label, as an affinity matrix. Optionally, the label can be separated subsequently - from purified FGF-23 polypeptide by various means such as the use of certain peptidases for separation. The flanking sequences may be homologous (i.e., of the same species or strain as the host cell), heterologous (i.e., of a different species of the species or strain of the host cell), hybrid (i.e., a combination of flanking sequences from more than one source), or synthetic, or flanking sequences may be native sequences which usually function to regulate the expression of the FGF-23 polypeptide. In this manner, the source of a flanking sequence can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, with the proviso that the flanking sequence is functional and can be activated by the organelles of the host cell. The flanking sequences that are used in the vectors of this invention can be obtained by any of several methods well known in the art. Typically, the flanking sequences used herein - different from the flanking sequences of the gene for FGF-23 - have been previously identified by mapping or by restriction endonuclease digestion and thus can be isolated from a tissue source appropriate using the appropriate restriction endonucleases. In some cases, the complete nucleotide sequence of a flanking sequence can be known. Here, the flanking sequence can be synthesized using the methods described herein for the synthesis or cloning of nucleic acid. When all or only a portion of the flanking sequence is known, it can be obtained using PCR or screening of a genomic library with an oligonucleotide or flanking sequence fragment suitable for the same or another species. When the flanking sequence is not known, a fragment of DNA containing a flanking sequence can be isolated from a larger piece of DNA which may contain, for example, a coding sequence or even another gene or genes. Isolation can be carried out by restriction endonuclease digestion to produce an appropriate DNA fragment, followed by isolation using agarose gel purification, Qiagen ™ column chromatography (Chatsworth, CA) or other methods known to those skilled in the art. . The selection of suitable enzymes to carry out this purpose will be readily apparent to a person ordinarily skilled in the art. An origin of replication is typically a part of commercially acquired prokaryotic expression vectors, and auxiliaries of origin in the amplification of the vector in a host cell. Amplification of the vector to a certain number of copies in some cases may be important for optimal expression of a FGF-23 polypeptide. If the vector of choice does not contain an origin of replication site, one can be synthesized chemically in base in a known sequence, and can be ligated into the vector. For example, the origin of replication of the pBR322 plasmid (New England Biolabs, Beverly, MA) is suitable for most gram-negative bacteria and the various origins (eg SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV, its acronym in English) or papillomavirus such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not necessary for mammalian expression vectors (for example, the SV40 origin is often used only because it contains the early promoter). The transcription termination sequence is typically 3 'to the end of a region encoding a polypeptide and serves to finalize transcription. Usually, the transcription termination sequence in prokaryotic cells is a fragment rich in G-C, followed by a poly-T sequence. Although the sequence is easily cloned from a library or even commercially purchased as part of a vector, it can also be easily synthesized using methods for nucleic acid synthesis such as those described herein. A selectable marker gene element encodes a protein necessary for the survival and growth of a host cell that grows in a selective culture medium. Typical selection marker genes encode proteins that: (a) confer resistance to antibiotics or other toxins, for example to ampicillin, tetracycline or kanamycin for prokaryotic host cells; (b) they complement auxotrophic deficiencies of the cell; or (c) provide fundamental nutrients not available from complex media. Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene and the tetracycline resistance gene. A neomycin resistance gene can also be used for selection in prokaryotic and eukaryotic host cells. Other selection genes can be used to amplify the gene to be expressed. Amplification is the procedure in which the genes that are required most for the production of a protein indispensable for growth, are repeatedly placed in tandem within the chromosomes of successive generations of recombinant cells. Examples of selectable markers suitable for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. The mammalian cell transformants are placed under the selection pressure where only the transformants are uniquely adapted to survive by virtue of the selection gene present in the vector. The selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium is successively changed, whereby the amplification of both the selection gene and the DNA encoding a polypeptide is carried out. FGF-23. As a result, increased amounts of the FGF-23 polypeptide are synthesized from the amplified DNA. The ribosome binding site is usually necessary for the initiation of translation of the mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or by a Kozak sequence (eukaryotes). The element is typically located 3 'to the promoter and 5' to the coding sequence of a FGF-23 polypeptide to be expressed. The sequence of Shine-Dalgarno varies, but is typically a polypurine (ie, which has a high content of A-G). Many Shine-Dalgarno sequences have been identified, each of which can be easily synthesized using methods set forth herein and used in a vector - prokaryote A leader sequence or signal can be used to direct a FGF-23 polypeptide outside the host cell. Typically, a nucleotide sequence encoding the signal sequence is placed in the coding region of a nucleic acid molecule for FGF-23, or directly at the 5 'end of a region encoding the FGF-23 polypeptide. Many signal sequences have been identified, and any of these that are functional in the selected host cell can be used in conjunction with a nucleic acid molecule for FGF-23. Therefore, a signal sequence may be homologous (occurring naturally) or heterologous to the nucleic acid molecule for FGF-23. Additionally, a signal sequence can be chemically synthesized using methods described herein. In most cases, the secretion of a FGF-23 polypeptide from the host cell via the presence of a signal peptide will result in the separation of the signal peptide from the secreted FGF-23 polypeptide. The signal sequence may be a component of the vector, or it may be part of a nucleic acid molecule for FGF-23 that is inserted into the vector. Included within the scope of this invention is the use of either a nucleotide sequence encoding a signal sequence for the native FGF-23 polypeptide linked to a region encoding the FGF-23 polypeptide, or a nucleotide sequence encoding for a heterologous signal sequence linked to a region encoding the FGF-23 polypeptide. The heterologous signal sequence selected must be one that is recognized and processed, that is, separated by a signal peptidase, by the host cell. For procarite host cells that do not recognize and process the native FGF-23 polypeptide signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected, for example, from the group of alkaline phosphatase, penicillinase or thermostable enterotoxin II leaders. . For yeast secretion, the signal sequence of the native FGF-23 polypeptide can be replaced by yeast invertase, a-factor or acid phosphatase leaders. In expression in mammalian cells, the native signal sequence is satisfactory, although other mammalian signal sequences may be suitable. In some cases, such as in those where glycosylation is desired in a eukaryotic host cell expression system, one can manipulate the various presequences to improve glycosylation or performance. For example, one can alter the peptidase separation site of a particular signal peptide, or - add prosequences which can also alter glycosylation. The final protein product in position -1 (relative to the first amino acids of the mature protein) one or more additional amino acids incident to expression, which may not have been completely separated. For example, the final protein product may have one or two amino acid residues found at the peptidase separation site, joined at the amino terminal part. Alternatively, the use of some sites of The enzyme separation can result in a slightly truncated form of the desired FGF-23 polypeptide, if the enzyme cuts into such an area within the mature polypeptide. In many cases, the transcription of a nucleic acid molecule is increased by the presence of one or more 15 introns in the vector; this is particularly true when the polypeptide is produced in eukaryotic host cells, especially mammalian host cells. The introns used can occur naturally within the gene for FGF-23 especially where the gene is used as 20 a full length genomic sequence or a fragment thereof. When the intron does not naturally occur within the gene (as is the case for most cDNAs), the intron can be obtained from another source. The position of the intron with respect to the sequences 25 flankers and the gene for FGF-23 is usually ^^^ J ^^ g ^^^^^^^ j ^^ important, since the intron must be transcribed to be effective. Therefore, when a cDNA molecule for FGF-23 is transcribed, the preferred position for the intron is 3 'to the transcription start site and 5' to the poly-A transcription termination sequence. Preferably, the intron or introns will be located side by side (ie, 5 'or 3') of the cDNA, so that it does not interrupt the coding sequence. Any intron of any source, including viral, prokaryotic and eukaryotic organisms (plant or animal), can be used to carry out this invention, provided that it is compatible with the host cell into which it is inserted. Synthetic introns are also included in the present. Optionally, more than one intron can be used in the vector. The expression and cloning vectors of the present invention will typically contain a promoter that is recognized by the host organism and that is operably linked to the molecule encoding the FGF-23 polypeptide. The promoters are non-transcribed sequences that are located towards the 5 'end with respect to the start codon of a structural gene (generally within about 100 to 1000 bp) that controls the transcription of the structural gene. The promoters are conventionally grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient, or a change in temperature. On the other hand, the constitutive promoters initiate the production of a continuous gene product; that is, there is little or no control over the expression of the gene. A large number of promoters, recognized by various potential host cells, are well known. A suitable promoter is operably linked to the DNA encoding the FGF-23 polypeptide by separating the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence within the vector. The promoter sequence for native FGF-23 can be used to direct the amplification or expression of a nucleic acid molecule for FGF-23. However, a heterologous promoter is preferred, if higher transcription with higher yields of the expressed protein compared to the native promoter is allowed, and if it is compatible with the host cell system that has been selected for use. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems; alkaline phosphatase; a tri-trophane (trp) promoter system, and hybrid promoters such as promoter _ ^^^^ ¡! "-« • ** - * '- - tac Other known bacterial promoters are also suitable. Their sequences have been published, thus allowing a person skilled in the art to link them into the desired DNA sequence using linkers or adapters as needed to provide useful restriction sites. Promoters suitable for use with yeast hosts are also well known in the art. Yeast improvers are advantageously used with 10 yeast promoters. Promoters suitable for use with mammalian host cells are well known and include, but are not limited to those that are obtained in the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), viruses from 15 bovine papilloma, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and most preferably simian virus 40 (SV40). Other suitable promoters for mammals include heterologous mammalian promoters, for example, heat shock promoters and actin promoters. Additional promoters which may be of interest for controlling gene expression for FGF-23 include, but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290: 304-10); the CMV promoter; the promoter contained in the repeated sequence 25 long terminal 3 'of Rous sarcoma virus (Yamamoto, et al. al., 1980, Cell 22: 787-97); the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 1444-45); the regulatory sequences of the gene for metallothionine (Brinster et al., 1982, Nature 296: 39-42); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Kamaroff et al., 1978, Proc.Natl, Acad.Sci.U.S.A., 75: 3727-31); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A., 80: 21-25). Also of interest are the following control regions of animal transcripts, which show tissue specificity and have been used in transgenic animals: the control region of the elastase I gene which is active in pancreatic acinar cells (Swift et al. , 1984, Cell 38: 639-46, Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol, 50: 399-409 (1986); MacDonald, 1987, Hepatology 7: 425-515); the insulin gene control region which is active in pancreatic β-cells (Hanahan, 1985, Nature 315: 115-22); the control region of the immunoglobulin gene, which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38: 647-58; Adames et al., 1985, Nature 318: 533-38; Alexander et al., 1987, Mol. Cell, Biol., 1: 1436-44); the control region of the mouse mammary tumor virus which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45: 485-95); the control region of the gene - - for albumin which is active in the liver (Pinker et al., 1987, Genes and Devel. 1: 268-76); the control region of the gene for the fetus-protein, which is active in the liver (Krumlauf et al., 1985, Mol Cell. Biol., 5: 1639-48; Hammer et al., 1987, Science 235: 53-58); the gene control region for antitrypsin-1 to which it is active in the liver (Kelsey et al., 1987, Genes and Devel.1: 161-71); this control region of the ß-globin gene which is active in myeloid cells (Mogram et al., 1985, Nature 315: 338-40, Kollias et al., 1986, Cell 46: 89-94); the control region of the gene for the myelin basic protein which is active in oligodendrocytic cells in the brain (Readhead et al., 1987, Cell 48: 703-12); the control region of the myosin light chain 2 gene, which is active in skeletal muscle (Sani, 1985, Nature 314: 283-86); and the control region of the gonadotropic releasing hormone gene which is active in the hypothalamus (Mason et al., 1986, Science 234: 1372-78). A sequence enhancer can be inserted into the vector to increase the transcription of a DNA encoding a FGF-23 polypeptide of the present invention by higher eukaryotes. The enhancers are cis-acting elements of DNA, where usually a length of 10 to 300 bp acts on the promoter to increase transcription. The breeders are relatively independent of orientation and position. 5 'and 3' have been found with respect to the transcription unit. Several available enhancer sequences of mammalian genes are known (e.g., globin, elastase, albumin, α-feto-protein and insulin). Typically, however, a virus improver will be used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancers are exemplary breeders for activating promoters 10 eukaryotes. Although an enhancer can be spliced into the vector at a position 5 'or 3' to a nucleic acid molecule for FGF-23, it is typically located at a site 5 'to the promoter. The expression vectors of the invention are 15 can build from an initial vector such as a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. When one or more of the flanking sequences described herein are not present in advance in The vector can be obtained individually and ligated in the vector. The methods used to obtain each of the flanking sequences are well known to those skilled in the art. The preferred vectors to carry out 25 this invention are those which are compatible with - - bacterial, insect and mammalian host cells. Such vectors include, for example, pCRII, pCR3 and pcDNA3.1 (Invitrogen, San Diego, CA), pBSII (Stratagene, La Jolla, CA), pET15 (Novagen, Madison, Wl), pGEX (Pharmacia Biotech, Piscataway, NJ ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBacII, Invitrogen), pDSR-a (PCT Publication No. WO 90/14363) and pFastBacDual (Gibco-BRL, Grand Island, NY). Additional suitable vectors include, but are not limited to, cosmids, plasmids or modified viruses but it will be appreciated that the vector system must be compatible with the selected host cell. Such vectors include, but are not limited to plasmids such as those derived from the plasmid Bluescript ™ (a phagemid based on ColEl with a high copy number, Stratagene Cloning Systems, La Jolla CA), PCR cloning plasmids designed to clone PCR products amplified by Taq (for example the TOPOMR team, TA Cloning ™, plasmid derivatives PCR2.1 ™, Invitrogen, Carlsbard, CA), and mammalian, yeast or virus vectors such as a baculovirus expression system (derived from the plasmid pBacPAK, Clontech , Palo Alto, CA). After the vector has been constructed and a nucleic acid molecule encoding a FGF-23 polypeptide has been inserted into the appropriate site of the vector, the completed vector can be inserted into a suitable host cell for amplification or expression of the r * * • * polypeptide. The transformation of an expression vector for a FGF-23 polypeptide into a selected host cell can be carried out by well-known methods including methods such as transfection, infection, calcium chloride, electroporation, microinjection, lipofection, DEAE method. -dextran or other known techniques. The method selected in part will be a function of the type of host cell that is used. These methods and other suitable methods are well known to those skilled in the art and are established, for example, in Sambrook et al. , supra. The host cells can be prokaryotic host cells (such as E. coli), or eukaryotic host cells (such as yeast, insect or vertebrate cells). When cultured under the appropriate conditions, the host cells synthesize a FGF-23 polypeptide which can then be harvested from the culture medium (if secreted into the medium by host cells) or directly from the host cells that produce it (when secrete). The selection of an appropriate host cell will depend on various factors such as the desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and the ease of folding or naturalization in a biologically active Many suitable host cells are known in the art and many are available from the American Type Culture Collection (ATCC), Manassas, VA. Examples 5 include but are not limited to mammalian cells such as Chinese hamster ovary (CHO) cells, CHO DHFR (-) cells (Urlaub et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 97: 4216-20), 293 or 293T human embryonic kidney (HEK) cells, or 3T3 cells. The selection is known in the art 10 of suitable mammalian host cells and methods of transformation, culture, amplification, screening, product elaboration and purification. Other suitable mammalian cell lines are the COS-1 and COS-7 monkey cell lines as well as the CV-1 cell line. The cells Additional exemplary mammalian hosts include primate cell lines and rodent cell lines that include transformed cell lines. Normal diploid cells, cell strains derived from an in vi tro culture of primary tissue as well as primary explants 20 are also suitable. Candidate cells may be genotypically defective in the selection gene, or may contain a selection gene that acts dominantly. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, 25 mouse L-929 cells, 3T3 lines derived from mice _.Jt .- ^ - ^^ _ a < e «_i_acfe.?ah» a.a. - > _._ *, * -_ c ,. ^ ___ ..w- ^ c '-.... (.. yr ~. .... ..:. .-. rr Iry ._...__ .. »&Swiss, Balb-C or NIH and BHK or HaK hamster cell lines Each of these cell lines is known and available to those skilled in the art of protein expression 5 Bacterial cells are similarly useful as host cells suitable for the present invention. For example, the various strains of E. coli (for example HB101, DH5a, DH10 and MC1061) are well known as host cells in the field of biotechnology. 10 use different strains of B in this method. subtilis, Pseudomonas spp., other Bacillus spp. , Streptomyces spp. , and similar. Many strains of yeast cells known to those skilled in the art are also available, 15 as host cells for the expression of the polypeptides of the present invention. Preferred yeast cells include, for example, Sacharomyces cerevisiae and Pichia pastoris. Additionally, when desired, they can be 20 use insect cell systems in the methods of the present invention. Such systems are described, for example, in Kitts et al. , 1993, Biotechniques, 14: 810-17; Lucklow, 1993, Curr. Opin. Biotechnol. 4: 564-72; and Lucklow et al. , 1993, J ". Virol., 67: 4566-79.Preferred insect cells 5 with Sf-9 and Hi5 (Invitrogen).

Transgenic animals can also be used to express glycosylated FGF-23 polypeptides. For example, a transgenic animal that produces milk can be used (a cow or goat, for example) and obtain a polypeptide 5 glycosylated present in the animal's milk. Plants can also be used to produce FGF-23 polypeptides, however in general, the glycosylation that occurs in plants is different from that which occurs in mammalian cells, and can result in a glycosylated product 10 which is not suitable for human therapeutic use.

Production of Polypeptide You can grow host cells that 15 comprise the FGF-23 polypeptide expression vector using standard means well known to those skilled in the art. The media will usually contain all the nutrients necessary for the growth and survival of the cells. Suitable media for cell culture of E. coli include, for example, Luria broth (LB) or Terrific broth (TB). Suitable media for eukaryotic cell culture include Roswell Park Memorial Institute media 1640 (RPMI 1640), minimal essential medium (MEM) or medium of 25 Eagle modified by Dulbecco (DMEM, for its acronym in • AMa *, aM * a, i'li * i * - "* - > * - * - *** '- - • * - - - - - - - -« - "and ~ * r ~ ~~ ?. - ~ * ~ r-English), all of which can be supplemented with serum or growth factors as needed for the particular cell line that is cultured. A suitable medium for insect cultures is Grace's medium supplemented with latent yeast, lactalbumin hydrolyzate or fetal bovine serum, as needed. Typically, an antibiotic or other compound useful for the selective growth of transfected or transformed cells is added as a supplement to the medium. The compound to be used will be determined by the selectable marker element present in the plasmid with which the host cell is to be transformed. For example, when the selectable marker element is resistant to kanamycin, the compound added to the culture medium will be kanamycin. Other compounds for selective growth include ampicillin, tetracycline and neomycin. The amount of a FGF-23 polypeptide produced by a host cell can be assessed using standard methods known in the art. Such methods include, without limitation, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, separation by high performance liquid chromatography (HPLC), immunoprecipitation or activity assays such as gel displacement assays. DNA binding.

If a FGF-23 polypeptide has been designed to be secreted from the host cells, most of the polypeptide can be found in the cell culture medium. However, if the FGF-23 polypeptide is not secreted from the host cells, it will be present in the cytoplasm or nucleus (for eukaryotic host cells) or in the cytosol (for host cells of gram-negative bacteria). For a FGF-23 polypeptide that is found in the cytoplasm or host cell nucleus (for eukaryotic host cells) or in the cytosol (for bacterial host cells), the intracellular material (which includes inclusion bodies for gram-negative bacteria) can be extracted. ) of the host cell using any standard technique known to those skilled in the art. For example, host cells can be used to release the periplasmic / cytoplasmic content by a French press, homogenization or sonication followed by centrifugation. If an FGF-23 polypeptide has formed inclusion bodies in the cytosol, the inclusion bodies can often be attached to the inner or outer cell membranes and thus will be found mainly in the sedimented material after centrifugation. The sedimented material can then be treated at extreme pH or with a chaotropic agent such as detergent, guanidine, guanidine derivatives, urea or urea derivatives in the presence of a reducing agent such as dithiothreitol at alkaline pH or tris carboxyethylphosphine at acid pH to release , separate and solubilize inclusion bodies. The solubilized FGF-23 polypeptide can then be analyzed using gel electrophoresis, immunoprecipitation or the like. If it is desired to isolate the FGF-23 polypeptide, isolation can be carried out using standard methods such as those described herein and in Marston et al. , 1990, Meth. Enz. , 182: 264-75. In some cases, a FGF-23 polypeptide may not be biologically active when it is isolated. Various methods can be used to "renature" or convert the polypeptide to its tertiary structure and generate disulfide bonds to restore biological activity. Such methods include exposing the solubilized polypeptide at a pH usually greater than 7 and in the presence of a particular concentration of a chaotrope. The selection of caotrope is very similar to the selections used for solubilization of the inclusion body, but usually the chaotrope is used at a lower concentration and not necessarily the same as the chaotropes used for the solubilization. In most cases, the renaturation / oxidation solution will also contain a reducing agent or reducing agent plus the oxidized form in a specific ratio to generate a particular redox potential that allows disulfide displacement to occur in the formation of the protein cysteine bridges. Some of the commonly used redox couples include cysteine / cystamine, glutathione (GSH) / dithiobis GSH, cupric chloride, dithiothreitol (DTT) / dithiane DTT and 2,2-mercaptoethanol (bME) / dithio-b (ME). In many cases, a co-solvent may be used or it may be necessary to increase the efficiency of the renaturation, and the most common reagents used for this purpose include glycerol, polyethylene glycol of various molecular weights, arginine and the like. If inclusion bodies are not formed to a significant degree upon expression of a FGF-23 polypeptide, then the polypeptide will be found mainly in the supernatant after centrifugation of the cell homogenate. The polypeptide can be further isolated from the supernatant using methods such as those described herein. Purification of a FGF-23 polypeptide from a solution can be carried out using various techniques. If the polypeptide has been synthesized to contain a tag such as hexahistidine (FGF-23 / hexaHis polypeptide) or other small peptide such as FLAG (Eastman Kodak Co., New Haven, CT) or myc (Invitrogen, Carisbad, CA), either in its carboxyl or amino terminal part, can be purified in a one-step procedure by passing the solution through a column of affinity where the column matrix has a high affinity for the label. For example, polyhistidine binds with great affinity and specificity to nickel. In this manner, a nickel affinity column (such as nickel Qiagen ™ columns) can be used for the purification of the FGF-23 / polyHis polypeptide. See, for example, Current Protocols in Molecular Biology § 10.11.8 (Ausubel et al., Eds., Green Publishers Inc. and Wiley and Sons 1993). Additionally, FGF-23 polypeptides can be purified by the use of a monoclonal antibody that is capable of specifically recognizing and binding to a FGF-23 polypeptide. Other methods suitable for purification include, without limitation, affinity chromatography, immunoaffinity chromatography, ion exchange chromatography, molecular sieve chromatography, HPLC, electrophoresis (including native gel electrophoresis), followed by gel elution and preparative isoelectric focusing ( machine / technique "Isoprime", Hoefer Scientific, San Francisco, CA). In some cases, two can be combined or more purification techniques to obtain greater purity. FGF-23 polypeptides can also be prepared by chemical synthesis methods (such as solid-phase peptide synthesis) using techniques known in the art such as those established by Merrifield et al. , 1963, "Am. Chem. Soc. 85: 2149; Houghten et al., 1985, Proc. Natl Acad. Sci. USA 82: 5132; and Stewart and Young, Solid Phase Peptide Synthesis (Pierce Chemical Co. 1984) Such polypeptides can be synthesized with or without a methionine in the amino terminal part.The chemically synthesized FGF-23 polypeptides can be oxidized using methods set forth in these references to form disulphide bridges.The chemically synthesized FGF-23 polypeptides are expected. have biological activity comparable to the corresponding FGF-23 polypeptides produced recombinantly or purified from natural sources and thus can be used interchangeably with a recombinant or wild-type FGF-23 polypeptide Another means to obtain the FGF- polypeptide 23 is by means of purification from biological samples such as tissue or fluid sources in which it naturally encounters the FGF-23 polypeptide. Imation can be carried out using methods for protein purification, as described herein.

The presence of the FGF-23 polypeptide can be monitored during purification, for example, by using an antibody prepared against the recombinantly produced FGF-23 polypeptide or peptide fragments thereof. Many other methods for producing nucleic acids and polypeptides are known in the art, and the methods can be used to produce polypeptides having specificity for the FGF-23 polypeptide. See, for example Roberts et al. , 1997, Proc. Natl. Acad. Sci. 10 U. S. TO . 94: 12297-303, which describes the production of fusion proteins between an mRNA and its encoded peptide. See also Roberts, 1990, Curr. Opin. Chem. Biol. 3: 268-73. Additionally, the patent of E.U.A. No. 469 describes methods for obtaining oligonucleotides capable of 15 carry out a specific biological function. The method involves generating a heterogeneous accumulation of oligonucleotides, each with a 5 'randomized sequence, a previously selected central sequence and a 3' randomized sequence. The heterogeneous accumulated The resulting sample is introduced into a cell population that does not show the desired biological function. The subpopulations of the cells are then subjected to systematic identification to determine those that show a biological function determined in advance. From this subpopulation, 25 isolate the oligonucleotides capable of carrying out the __te ___ a ____ a ... s .... _, ..__ J_A¿ ^ _ »* biological function that is desired. The patents of E.U.A. numbers 5,763,192; 5,814,476; 5,723,323; and 5,817,483 describe methods for making peptides or polypeptides. This is done by making stochastic genes or fragments thereof, and then introducing these genes into host cells that produce one or more proteins encoded by the stochastic genes. The host cells are then screened or systematically identified to identify those clones that produce peptides or polypeptides having the desired activity. Another method for producing peptides or polypeptides is described in PCT / US98 / 20094 (WO99 / 15650) presented by Athersys, Inc. Known as "random activation of gene expression for gene discovery" (RAGE-GD), the procedure involves the activation of endogenous gene expression or overexpression of a gene by recombination methods in si tu. For example, the expression of an endogenous gene is activated or increased by integrating a regulatory sequence into a target cell which is capable of activating the expression of the gene by non-homologous or illegitimate recombination. The target DNA is first subjected to radiation, and the genetic promoter is inserted. The promoter finally locates an interruption in the front of a gene, initiating the transcription of the gene. This results in the expression of the desired peptide or polypeptide. It will be appreciated that these methods can also be used to create comprehensive FGF-23 polypeptide expression libraries, which can then be used for high-throughput phenotypic screening in various assays, such as biochemical assays, cell assays and assays with whole organisms ( for example, plants, mice, etc.).

Synthesis It will be appreciated by those skilled in the art that the nucleic acid and polypeptide molecules described herein may be made by recombinant means or other means.

Selective Union Agents The term "selective binding agent" refers to a molecule that has specificity for one or more FGF-23 polypeptides. Suitable selective binding agents include, but are not limited to antibodies and derivatives thereof, polypeptides and small molecules. Suitable selective binding agents can be prepared using methods known in the art. A selective binding agent for the exemplary FGF-23 polypeptide of the present invention is capable of binding a certain portion of the FGF-23 polypeptide and thereby inhibiting the binding of the polypeptide to a receptor for the FGF-23 polypeptide. Selective binding agents such as antibodies and antibody fragments that bind to FGF-23 polypeptides are within the scope of the present invention. The antibodies can be polyclonal including monospecific polyclonal; monoclonal antibodies (MAb); recombinants; chimeric humanized such as CDR-grafted; human; single chain; or bispecific; as well as fragments; variants or derivatives thereof. Antibody fragments include those portions of the antibody that bind to an epitope of the FGF-23 polypeptide. Examples of such fragments include the Fab and F (ab ') fragments generated by enzymatic cleavage of full-length antibodies. Other binding fragments include those generated by recombinant DNA techniques, such as the expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions. Polyclonal antibodies directed to a FGF-23 polypeptide are generally produced in animals (e.g. rabbits or mice) by means of multiple subcutaneous or intraperitoneal injections of the FGF-23 polypeptide and an adjuvant. It may be useful to conjugate a FGF-23 polypeptide to a carrier protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, or soybean trypsin inhibitor. In addition, aggregation agents such as alum are used to improve the immune response. After the immunization, blood is drawn from the animals and the serum is tested to determine the antibody titer 10 against FGF-23. Monoclonal antibodies directed towards the FGF-23 polypeptides are produced using any method that provides for the production of antibody molecules by continuous cell lines in culture. The Examples of suitable methods for preparing monoclonal antibodies include hybridoma methods of Kohler et al. , 1975, Nature 256: 495-97 as well as the human B lymphocyte hybridoma method (Kozbor, 1984, J. Immunol 133: 3001, Brodeur et al., Monoclonal Antibody Production Techniques 20 and Applications 51-63 (Marcel Dekker, Inc., 1987). Also provided by the invention are hybridoma cell lines that produce monoclonal antibodies reactive with FGF-23 polypeptides. The monoclonal antibodies of the invention are 25 can be modified for use as therapeutic substances. A - "modality" is a "chimeric" antibody in which a portion of the heavy chain (H, for its acronym in English) or light (L, for its acronym in English) is identical or homologous to a corresponding sequence of antibodies derived from a particular species or belonging to a particular class or subclass of antibody, while the rest of the chain (s) are identical or homologous to a corresponding sequence in antibodies derived from another species or belonging to another class or subclass of antibody.

Fragments of such antibodies are also included, insofar as they show the desired biological activity. See the patent of E.U.A. number 4,816,567; Morrison et al. , 1985, Proc. Natl. Acad. Sci. 81: 6851-55. In another embodiment, the monoclonal antibody of the The invention is a "humanized" antibody. Methods for humanizing non-human antibodies are well known in the art. See the patents of E.U.A. Nos. 5,585,089 and 5,693,762. Generally, a humanized antibody has one or more amino acid residues introduced therein starting from 20 from a source that is not human. Humanization can be carried out, for example, using methods described in the art (Jones et al., 1986, Nature 321: 522-25; Riechmann et al., 1998, Nature 332: 323-27; Verhoeyen et al. , 1988, Science 239: 1534-36), by replacing at least a portion 25 of a complementarity determining region (CDR) of ^^^ jj ^ gggl ^^ yy ^^ ^ yggj | ^^^ and ^ B ^^^^ agg ^^^^^^^^^^^^^^^ ttít ^ -t¿tjßa rodent by the regions corresponding to a human antibody. Human antibodies that bind FGF-23 polypeptides are also encompassed by the invention. By utilizing transgenic animals (e.g., mice) that are capable of producing a range of human antibodies in the absence of endogenous immunoglobulin production, such antibodies are produced by immunization with an antigen for the FGF-23 polypeptide (i.e., having at least 6 contiguous amino acids) optionally conjugated with a carrier. See, for example, Jakobovits et al. , 1993, Proc. Natl. Acad. Sci. 90: 2551-55; Jakobovits et al. , 1993, Nature 362: 255-58; Bruggermann et al. , 1993, Year in Im one. 7:33. In one method, such transgenic animals are produced by incapacitating the endogenous loci encoding the immunoglobulin heavy and light chains therein, and by inserting loci encoding the human heavy and light chain proteins within the genome thereof. Partially modified animals, that is, those that have less than the full complement of modifications, are then subjected to crossbreeding to obtain an animal that has all of the desired modifications in the immune system. When an immunogen is administered, these transgenic animals produce antibodies with amino acid sequences of a human (instead of, for example, mouse) and include variable regions which are immunospecific for these antigens. See PCT applications numbers PCT / US96 / 05928 and PCT / US93 / 06926. Additional methods are described in the U.S.A. number 5, 545,807, PCT application numbers PCT / US91 / 245 and PCT / GB89 / 01207, and in European patents numbers 546073B1 and 546073A1. Human antibodies can also be produced by expression of recombinant DNA in host cells or by expression in hybridoma cells, as described herein. In an alternative embodiment, human antibodies can also be produced from phage display libraries (Hoogenboom et al., 1991, J., Mol. Biol. 227: 381; Marks et al. , 1991, J. "Mol. Biol. 222: 581) .These procedures mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophages, and the subsequent selection of phage by binding to an antigen from One such technique is described in PCT application number PCT / US98 / 17364, which describes the isolation of high affinity and functional agonistic antibodies for MPL- and msk- receptors using such an approach Chimeric antibodies, inserted with CDR and humanized ones are typically produced by recombinant methods.The nucleic acids encoding the antibodies are introduced into the host cells and are expressed using materials and methods described herein In a preferred embodiment, the antibodies are produced in mammalian host cells, such as 5 CHO cells, monoclonal antibodies (eg human) can be generated by the expression n recombinant DNA in host cells or by expression in hybridoma cells as described herein. The antibodies against FGF-23 of the invention are 10 can be used in any known assay method, such as competitive binding assays, direct and indirect interposition assays, and immunoprecipitation assays (Sola, Monoclonal Antibodies A Manual of Techniques 147-158 (CRC Press, Inc., 1987)) for the 15 detection and quantification of FGF-23 polypeptides. The antibodies will bind to FGF-23 polypeptides with an affinity that is appropriate for the test method that is used. For diagnostic applications, in some modalities, antibodies against FGF-23 can be labeled 20 with a detectable portion. The detectable portion can be any that is capable of producing, directly or indirectly, a detectable signal. For example, the detectable portion can be a radioisotope, such as 3H, 1C, 32P, 35S, 125I, 99Tc, lxlIn or 67Ga; a fluorescent compound 25 or chemiluminescent such as fluorescein isothiocyanate, rhodamine or luciferin; or an enzyme, such as alkaline phosphatase, β-galactosidase or horseradish peroxidase (Bayer, et al., 1990, Meth., 184: 138-63). Competitive binding assays are based on the ability of a labeled standard (eg, a FGF-23 polypeptide or an immunologically reactive portion thereof) to compete with the test sample analyte (a FGF-23 polypeptide) for binding to a limited amount of antibody against FGF-23. The amount of a FGF-10 23 polypeptide in the test sample is inversely proportional to the amount of standard that binds to the antibodies. To facilitate the determination of the amount of standard that binds, the antibodies typically become insubordinated before or after competition, so that the standard and analyte that binds to the antibodies can be conveniently separated from the standard and analyte that remain without joining Interposition assays (sandwich or sandwich) typically involve the use of two antibodies, or each one capable of binding to a different immunogenic portion, or epitope of the protein to be detected or quantified. In an interposition assay, the test sample analyte typically binds to a first antibody which is immobilized on a solid support and subsequently a second antibody binds to the analyte and najJMÉ.JJj. ^ i, Bi t ... ^. this way an insoluble complex of three parts is formed. See, for example, the patent of E.U.A. number 4,376,110. The second antibody itself can be labeled with a detectable portion (direct interposition assays), or can be measured using an antibody against immunoglobulin that is labeled with a detectable portion (indirect interposition assays). For example, one type of interposition assay is an enzyme-linked immunosorbent assay (ELISA), in which case the detectable portion is an enzyme. Selective binding agents, which include antibodies against FGF-23, are also useful for in vivo imaging. An antibody labeled with a detectable portion can be administered to an animal, preferably in the blood stream, and in the presence of the position of the labeled antibody in the body being tested. The antibody can be labeled with a portion that is detectable in an animal, either by nuclear magnetic resonance, radiology or by other means of detection known in the art. The selective binding agents of the invention, including antibodies, can be used as therapeutic substances. These therapeutic agents are generally agonists or antagonists insofar as they can improve or reduce, respectively, at least one of the biological activities of a FGF-23 polypeptide. In one embodiment, the antagonist antibodies of the invention are antibodies or binding fragments thereof which are capable of specifically binding to a FGF-5 polypeptide and which are capable of inhibiting or eliminating the functional activity of a FGF- polypeptide. 23 in vivo or in vi tro. In preferred embodiments, the selective binding agent, for example, an antagonist antibody will inhibit the functional activity of a FGF-23 polypeptide in at least 10 about 50% and preferably at least about 80%. In another embodiment, the selective binding agent can be an antibody against the FGF-23 polypeptide that is capable of interacting with a binding partner of the FGF-23 polypeptide, (a ligand or receptor) and thus The activity of the FGF-23 polypeptide is inhibited or eliminated in vi tro or in vivo. The selective binding agents, which include antibodies against the FGF-23 polypeptide agonists or antagonists, are identified by screening assays and are well known in the art. The invention also relates to a kit comprising selective binding agents for FGF-23 (such as antibodies) and other reagents useful for detecting the levels or concentrations of the FGF-23 polypeptide in biological samples. Such reagents may include a brand 25 detectable, blocking serum, positive control samples • 2 ^ 2 ^ ¿g¡ and negative and detection reagents.

Microarrays It will be appreciated that DNA microarray technology can be used, in accordance with the present invention. DNA microarrays are high-density and miniature arrays of nucleic acids placed on a solid support, such as glass. Each cell or element within the array contains numerous copies of a single species of nucleic acid that acts as a target for hybridization with a complementary nucleic acid sequence (e.g., mRNA). In profiling the expression using DNA microarray technology, the mRNA is first extracted from a cell or tissue sample and then enzymatically converted to fluorescently labeled cDNA. This material is subjected to hybridization to the microarray and cDNA that does not bind is washed off. The expression of separated genes represented in the array is then visualized by quantification of the amount of labeled cDNA that binds specifically to each target nucleic acid molecule. In this way, the expression of thousands of high-throughput genes can be quantified in a parallel manner in a single sample of biological material.

This high performance expression profiling has a wide range of applications with respect to the FGF-23 molecules of the invention which include, but are not limited to the identification and validation of the genes related to the FGF-23 disease as targets. for therapeutic substances; molecular toxicology of related FGF-23 molecules and inhibitors thereof; stratification of populations and generation of associated markers for clinical trials; and the discovery of improved related small molecule FGF-23 polypeptide drugs that aid in the identification of selective compounds in high throughput screening.

Chemical Derivatives Chemically modified derivatives of the FGF-23 polypeptides can be prepared by those skilled in the art, given what is described herein. The FGF-23 polypeptide derivatives are modified in a manner that is different - either in the type or location of molecules naturally linked to the polypeptide. Derivatives can include molecules that are formed by suppression of one or more chemically linked groups naturally. The polypeptide comprising the amino acid sequence of the SEQUENCE OF IDENTIFICATION NUMBER: 2 or another polypeptide of FGF-23, can be modified by the covalent attachment of one or more polymers. For example, the selected polymer is typically water soluble so that the protein which binds does not precipitate in an aqueous environment, such as a physiological environment. The polymer mixtures are included within the scope of the suitable polymers. Preferably, for therapeutic use of the final product preparation, the polymer will be pharmaceutically acceptable. Each of the polymers can have any molecular weight and can be branched or branched. Each of the polymers typically has an average molecular weight of between about 2 kDa and about 100 kDa (the term "about" indicates that, in the preparations of water-soluble polymers, some molecules will weigh more, some less than the indicated molecular weight). The average molecular weight of each polymer is preferably between about 5 kDa and about 50 kDa, more preferably between about 12 kDa and about 40 kDa, and more preferably between about 20 kDa and about 35 kDa. Suitable water-soluble polymers or mixtures thereof include, but are not limited to, attached or bonded N-carbohydrates, sugars, phosphates, polyethylene glycol (PEG) (which includes the PEG forms that have been used to form protein derivatives, which include monoalkoxy of 1 to 10 carbon atoms or aryloxy-polyethylene glycol), monomethoxy polyethylene glycol, dextran (such as low molecular weight dextran, for example about 6 kD), cellulose or other carbohydrate-based polymers, poly- (N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyethoxylated polyols (for example glycerol) and polyvinyl alcohol. Also encompassed by the present invention are bifunctional crosslinking molecules which can be used to prepare FGF-23 polypeptide multimers covalently linked. In general, the chemical formation of derivatives can be carried out under any suitable condition used to react a protein with a suitable polymeric molecule. Methods for preparing chemical derivatives of polypeptides will generally comprise the steps of: (a) reacting the polypeptide with an activated polymer molecule (such as a reactive ester or an aldehyde derivative of the polymer molecule) under conditions in which the polypeptide comprising the amino acid sequence of the SEQUENCE OF IDENTIFICATION NUMBER: 2 or other polypeptide FGF-23, binds to one or more polymer molecules, and (b) obtain the reaction products. The optimal reaction conditions will be determined based on known parameters and the desired result. For example, the higher the ratio of polymer molecules to the protein, the higher the percentage of bound polymer molecule. In one embodiment, the FGF-23 polypeptide derivative can have a single polymer molecule portion in the amino terminal portion. See, for example, the patent of E.U.A. number 5,234,784. Pegylation (formation of a derivative with PEG) can be carried out specifically using any pegylation reaction known in the art. Such reactions are described, for example, in the following references: Francis et al. , 1992, Focus on Growth Factors 3: 4-10; European Patent Nos. 0154316 and 0401384; and in the U.S. Patent. number 4,179,337. For example, the pegylation can be carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer), as described herein. For the acylation reactions, a selected polymer will have a single reactive ester group. For reductive alkylation, a selected polymer will have a single reactive aldehyde group. A reactive aldehyde is, for example, polyethylene glycol propionaldehyde, which is stable to water, a monoalkoxy of 1 to 10 carbon atoms or aryloxy derivatives thereof (see U.S. Patent No. 5,252,714). In one embodiment, the FGF-23 polypeptides can be chemically coupled to biotin. The biotin / FGF-23 polypeptide molecules are then allowed to bind to avidin, resulting in tetravalent avidin / biotin / FGF-23 polypeptide molecules. The FGF-23 polypeptides can also be covalently coupled to dinitrophenol (DNP) or trinitrophenol (TNP), and the resulting conjugates are precipitated with IgM against DNP or against TNP to form decameric conjugates with a valence of 10. Generally, the conditions that are can alleviate or modulate by the administration of the present FGF-23 polypeptide derivatives include those described herein for FGF-23 polypeptides. However, FGF-23 polypeptide derivatives that are described herein may have additional activities, increased or reduced biological activity, or other characteristics such as increased or decreased half-life, as compared to molecules that do not form derivatives.

Non-Human Animals Subjected to Genetic Engineering They are additionally included within the scope of the present invention non-human animals such as mice, rats or other rodents, rabbits, goats, sheep or other farm animals, in which genes encoding the native FGF-23 polypeptide have been disrupted (i.e., have been "blocked") ") so that the level of expression of the FGF-23 polypeptide is significantly reduced or completely suppressed. Such animals can be prepared using techniques and methods such as those described in U.S. Pat. number 5,557,032. The present invention also includes non-human animals such as mice, rats or other rodents; rabbits, goats, sheep or other farm animals, in which the native form of a gene for FGF-23 for that animal, or a heterologous gene for FGF-23 is overexpressed by the animal, so that an animal is generated "transgenic". Such transgenic animals can be prepared using well-known methods such as those described in the US patent. No. 5,489,743 and PCT publication number WO 94/28122. The present invention further includes non-human animals in which the promoter of one or more of the FGF-23 polypeptides of the present invention is activated or inactivated (e.g. by the use of homologous recombination methods) to alter the expression level of one or more native FGF-23 polypeptides.

These non-human animals can be used for the systematic identification of candidate drugs. In such a systematic identification or screening, the impact of a candidate drug on the animal can be measured. For example, candidate drugs can decrease or increase the expression of the gene for FGF-23. In some embodiments, the amount of FGF-23 polypeptide that occurs after exposure of the animal to the candidate medicament can be measured. Additionally, in some modalities, one can detect the actual impact of the candidate drug on the animal. For example, overexpression of a particular gene may result in, or may be associated with, a disease or pathological disorder, in such cases, one may test the ability of a candidate drug to decrease the expression of a gene or its ability to avoid or inhibit a pathological condition. In other examples, the production of a particular metabolic product such as a fragment of a polypeptide may result in, or be associated with, a disease or pathological condition. In such cases, one can test the ability of a candidate drug to decrease the production of such a metabolic product or its ability to prevent or inhibit a pathological condition.

Assay for Other Modulators of FGF-23 Polypeptide Activity In some situations, it may be desirable to identify molecules that are modulators, ie, agonists or antagonists of FGF-23 polypeptide activity. Natural or synthetic molecules that modulate the FGF-23 polypeptide can be identified using one or more screening assays, such as those described in 10 the present. Such molecules can be administered ex vivo or in vivo by injection or by oral delivery, implantation device or the like. The "test molecule" refers to a molecule that is under evaluation to determine the ability to 15 modulate (i.e., increase or decrease) the activity of a FGF-23 polypeptide. Most commonly, a test molecule will interact directly with a FGF-23 polypeptide. However, it is also contemplated that a test molecule also indirectly modulates polypeptide activity 20 FGF-23, for example by altering the expression of the FGF-23 gene or by binding a FGF-23 polypeptide binding partner (e.g., a receptor or ligand). In one embodiment, a test molecule will bind to a FGF-23 polypeptide with an affinity constant of at least about 10"6M, 25 preferably about 108M, more preferably WMttiMiffifYt - -rtí - * - * - * ^ "* - - * .- *» - »-" -. * «- - * - * ^« .. * y- ^ ~~ * ~ » ~ ^ * ja ~ »~ ~ .. ~. preferably about 10"9M, and even more preferably about 10" 10M. Methods for identifying compounds that interact with FGF-23 polypeptides are included by the present invention. In some embodiments, a FGF-23 polypeptide is incubated with a test molecule under conditions that allow interaction of the test molecule with a FGF-23 polypeptide, and the degree of interaction is measured. The test molecule can be subjected to systematic identification in a substantially purified form or in a crude mixture. In some embodiments, a FGF-23 polypeptide agonist or antagonist can be a protein, peptide, carbohydrate, lipid, or small molecular weight molecule that interacts with the FGF-23 polypeptide to regulate its activity. Molecules that regulate FGF-23 polypeptide expression include nucleic acids which are complementary to nucleic acids encoding a FGF-23 polypeptide, or are complementary to nucleic acid sequences which direct or control the expression of FGF- polypeptide 23 and which act as antisense expression regulators. Once a test molecule has been identified, an element that interacts with a FGF-23 polypeptide, 25 the molecule can be further evaluated for determine its ability to increase or decrease the activity of the FGF-23 polypeptide. Measurement of the interaction of the test molecule with the FGF-23 polypeptide can be carried out in various formats, including cell-based binding assays, membrane binding assays, solution phase assays and immunoassays. In general, a test molecule is incubated with a FGF-23 polypeptide for a specified period of time, and the activity of the FGF-23 polypeptide is determined by one or more assays to measure biological activity. A test can also be performed to determine the interaction of the test molecules with the FGF-23 polypeptides directly using polyclonal or monoclonal antibodies in an immunoassay. Alternatively, modified forms of the FGF-23 polypeptides containing epitope tags as described herein can be used in solution and immunoassays. In the case where the FGF-23 polypeptides show biological activity by an interaction with a binding partner (eg, a receptor or a ligand), various in vitro assays can be used to measure the binding of a FGF- polypeptide. 23 to the corresponding binding partner (such as a selective binding agent, receptor or ligand). These assays can be used to screen test molecules to determine their ability to increase or decrease the rate or degree of binding of a FGF-23 polypeptide to its binding partner. In one assay, a FGF-23 polypeptide is immobilized in the wells of a microtiter plate. The radiolabelled FGF-23 polypeptide binding partner (e.g., the binding partner of the iodinated FGF-23 polypeptide) and the test molecule can then be added either one at a time (in any order) or simultaneously to the wells . After incubation, the walls are washed and a radioactivity count is performed using a scintillation counter, to determine the degree to which the binding partner binds to the FGF-23 polypeptide. Typically a molecule will be tested over a range of concentrations, and a series of control wells lacking one or more elements of the test assays can be used to determine the accuracy in the evaluation of the results. An alternative to this method involves inverting the "positions" of the proteins, i.e., immobilizing the binding partner of the FGF-23 polypeptide to the wells of the microtitre plate, incubating with the radiolabelled FGF-23 polypeptide and test molecule, and determining the degree of binding of the FGF-23 polypeptide. See, for example, Current Protocols in Molecular Biology, chap. 18 (Ausubel et al., Eds., Green Publishers Inc. and Wiley and Sons 1995).

As an alternative to radiolabelling, a FGF-23 polypeptide or its binding partner can be conjugated with biotin, and the presence of the biotinylated protein can then be detected using streptavidin linked to an enzyme, such as horseradish peroxidase (HRP, for its acronym in English) or alkaline phosphatase (AP), which can be detected colorimetrically or by fluorescent streptavidin labeling. An antibody directed against a FGF-23 polypeptide or a FGF-23 polypeptide binding partner and which is conjugated to biotin can also be used for detection purposes after incubation of the complex with enzyme bound streptavidin, bound to AP or HRP . A FGF-23 polypeptide or a FGF-23 polypeptide binding partner can also be immobilized by attachment to agarose spheres, acrylic spheres or other types such as inert solid phase substrates. The substrate-protein complex can be placed in a solution containing a complementary protein and the test compound. After incubation, the spheres can be precipitated by centrifugation and the amount of binding between the FGF-23 polypeptide and its binding partner can be determined using methods described herein. Alternatively, the substrate-protein complex can be immobilized in a column with the test molecule and the complementary protein passing through the column. The formation of a complex between the FGF-23 polypeptide and its binding partner can then be determined using any of the techniques described herein (e.g., radiolabeled or antibody binding). Another in vitro assay that is useful for identifying a test molecule which increases or decreases the formation of a complex between a FGF-23 polypeptide binding protein and the FGF-23 polypeptide binding partner is the resonance detector system of surface plasmon such as the BIAcore assay system (Pharmacia, Piscataway, NJ). The BIAcore system is used as specified by the manufacturer. This assay essentially involves the covalent attachment of either the FGF-23 polypeptide or a FGF-23 polypeptide binding partner to a detector chip coated with dextran that is located in a detector. The test compound and the other complementary protein can then be injected, simultaneously or sequentially, into the chamber containing the detector chip. The amount of complementary protein that binds based on the change and the molecular mass that physically relates to the dextran-coated side of the detector chip can be determined, and the change in molecular mass is measured by the detector system. In some cases, it may be desirable to evaluate two or more test compounds together to determine their ability to increase or decrease the formation of a complex between a FGF-23 polypeptide and a binding partner of the FGF-23 polypeptide. In these cases, the assays set forth herein can be easily modified by adding such additional test compounds, simultaneously or subsequent to the first test compound. The rest of the stages in the trial are as set forth herein. In vitro assays such as those described herein can be advantageously used to screen large amounts of compounds to determine an effect on the formation of a complex between the FGF-23 polypeptide and a binding partner of the FGF-23 polypeptide. The assays can be automated to analyze compounds generated in phage display, synthetic peptides and chemical synthesis libraries. Compounds that increase or decrease the formation of a complex between a FGF-23 polypeptide and a FGF-23 polypeptide binding partner can also be examined in cell cultures using cells and cell lines that express the FGF-23 polypeptide or the associated of binding of FGF-23 polypeptide. Cells and cell lines can be obtained from any mammal, but preferably from human or primate, canine or rodent sources. The binding of a FGF-23 polypeptide to the - - Cells expressing the binding partner of the FGF-23 polypeptide on the surface are evaluated in the presence or absence of the test molecules and the degree of binding can be determined, for example, by flow cytometry using a biotinylated antibody to a partner of polypeptide binding FGF-23. Cell culture assays can be used advantageously to further evaluate the compounds that give positive results in the protein binding assays described herein. Cell cultures can also be used to screen the impact of a candidate drug,. For example, candidate drugs can decrease or increase the expression of the FGF-23 gene. In some embodiments, the amount of the FGF-23 polypeptide or a fragment of the FGF-23 polypeptide that is produced can be measured after exposure of the cell culture to the candidate drug. In some modalities, one can detect the actual, impact of the candidate drug on the cell culture. For example, overexpression of a particular gene may have a particular impact on cell culture. In such cases, one can test the ability of the candidate drug to increase or decrease the expression of the gene or its ability to prevent or inhibit a particular impact on cell culture. In other examples, the preparation of a particular metabolic product, such as a fragment of a polypeptide, may result or be associated with a disease or a pathological condition. In such cases, one can test the ability of a candidate drug to decrease the production of such a metabolic product in a cell culture.

Protein Internalization The tat (HIV) protein sequence can be used to internalize proteins in a cell. See, for example, Falwell et al. , 1994, Proc. Natl. Acad. Sci. USES . 91: 664-68. For example, a sequence of 11 amino acids (YGRKKRRQRRR; SEQUENCE OF IDENTIFICATION NUMBER: 40) of the HIV tat protein (called the "protein transduction domain" or TAT PDT) has been described as an element that mediates the release through the cytoplasmic membrane and the nuclear membrane of a cell. See Schwarze et al. , 1999, Science 285: 1569-72; and Nagahara et al. , 1998, Nat. Med. 4: 1449-52. In these procedures, mounts are prepared (recombinant plasmids) bound to FITC (G-G-G-Y-G-R-K-K-R-R-Q-R-R-R labeled with FITC; IDENTIFICATION SEQUENCE NUMBER: 41) which penetrate tissues after their intraperitoneal administration, and the binding of such mounts to the cells is detected by fluorescence activated cell sorting analysis (FACS, for its acronym in English). Cells treated with tat-ß-gal fusion protein will show ß-gal activity. After injection, the expression of such mounts can be detected in many tissues including, liver, kidney, lung, heart and brain tissue. It is considered that such assemblies undergo some degree of denaturation in order to enter the cell, and as such may require renaturation after entering the cell. Therefore, it will be appreciated that the tat protein sequence can be used to internalize a desired polypeptide to a cell. For example, using the tat protein sequence, an FGF-23 antagonist (such as a selective binding agent against FGF-23, a small molecule, a soluble receptor or an antisense oligonucleotide) can be administered intracellularly, to inhibit activity of a molecule FGF-23. As used herein, the term "FGF-23 molecule" refers to both nucleic acid molecules for FGF-23 and FGF-23 polypeptides as defined herein. When desired, the FGF-23 protein itself can also be administered internally to a cell using these methods. See also Straus, 1999, Science 285: 1466-67.

Identification of the Cell Source Using the Polypeptide FGF-23 According to some embodiments of the invention, it may be useful to be able to determine the source of a certain type of cell related to a FGF-23 polypeptide. For example, it may be useful to determine the origin of a disease or pathological condition as an aid in the selection of appropriate therapy. In some embodiments, the nucleic acids encoding a FGF-23 polypeptide can be used as a probe to identify cells described herein by screening nucleic acids from cells with such a probe. In other embodiments, one can use antibodies against the FGF-23 polypeptide to determine the presence of FGF-23 polypeptide in cells and thereby determine whether such cells are of the types described herein.

Compositions of Polypeptide FGF-23 and Administration Therapeutic compositions are within the scope of the present invention. Such pharmaceutical compositions of the FGF-23 polypeptide may comprise a therapeutically effective amount of a FGF-23 polypeptide or a nucleic acid molecule for FGF-23 in admixture with an agent pharmaceutical formulation or physiologically acceptable that is selected for its proper condition with the administration mode. The pharmaceutical compositions may comprise a therapeutically effective amount of one or more selective binding agents for the FGF-23 polypeptide in admixture with a pharmaceutically or physiologically acceptable formulation agent that is selected to suit the mode of administration. The acceptable formulation materials are preferably non-toxic to the receptors at the dosages and concentrations used. The pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, pH, osmolarity, viscosity, transparency, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. . Materials suitable formulation include, but are not limited to amino acids (such as glycine, glutamine, asparagine, arginine or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen sulfite), buffers (such as borate, bircarbonato, Tris-HCl, citrates, phosphates or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides and other carbohydrates (such as glucose, mannose or dextrins), proteins (such as ceric albumin, gelatin or immunoglobulins), coloring agents, flavoring and diluting, emulsifying agents, hydrophilic polymers (such as polyvinyl pyrrolidone), low molecular weight polypeptides, counterions formed res salts (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide), solvents (such as glycerin, propylene or polyethylene glycol) sugar alcohols (such as mannitol or sorbitol) agents that improve the suspension, surfactants or wetting agents (such as Pluronics; PEG; sorbitan esters; polysorbates such as Polysorbate 20 or Polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability-enhancing agents (such as sucrose or sorbitol), tonicity-enhancing agents (such as alkali metal halides - preferably sodium or potassium chloride - or mannitol or sorbitol), delivery vehicles, diluents, excipients or pharmaceutical adjuvants. See Remington's Pharmaceutical . *. * ».» AtJliB t ^ afc ^ .. - - »~.« A »». ^^^^^^^ ¿_ Sciences (18th Ed., AR Gennaro, ed., Mack Publishing Company 1990). The optimum pharmaceutical composition will be determined by a person skilled in the art., on the basis, for example, of the administration route used, the supply format and the dosage that is desired. See, for example, Remington's Pharmaceutical Sciences, supra. Such compositions can alter the physical state, stability, in vivo release rate and clearance rate of the FGF-23 molecule. The carrier or primary carrier in a pharmaceutical composition can be aqueous or non-aqueous in nature. For example, a vehicle or carrier suitable for injection may be water, physiological saline or artificial cerebrospinal fluid, possibly supplemented with other materials common in the compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are additional exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may also include sorbitol or a suitable substitute. In one embodiment of the present invention, the FGF-23 polypeptide compositions can be prepared for storage by mixing the selected composition having the desired degree of purity, with optional formulating agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. In addition, the FGF-23 polypeptide product can be formulated as a lyophilizate using appropriate excipients such as sucrose. The FGF-23 polypeptide pharmaceutical compositions can be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation of the supply through the digestive tract, for example orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. The formulation components are present in concentrations that are acceptable for the site of administration. For example, the buffers to be used to maintain the composition at physiological pH or slightly lower pH are usually within a pH range of from about 5 to about 8. When parenteral administration is contemplated, the therapeutic compositions for use in This invention may be in the form of a pyrogen-free and parenterally acceptable aqueous solution, comprising the desired FGF-23 molecule in a pharmaceutically vehicle.

TMI I J G * f ftirt-rr-if frt - * acceptable. A vehicle particularly suitable for parenteral injection is sterile distilled water in which the FGF-23 molecule is formulated as a sterile isotonic solution suitably preserved. Another additional preparation may involve the formulation of the desired molecule with an agent such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), spheres or liposomes that provide controlled or sustained release of the product which can then be supply by means of deposition injection. Hyaluronic acid can also be used and this can have the effect of promoting sustained duration in the circulation. Another suitable means for the introduction of the desired molecule includes implantable drug delivery devices. In one embodiment, a pharmaceutical composition for inhalation can be formulated. For example, the FGF-23 polypeptide can be formulated as a dry powder for inhalation. FGF-23 polypeptide inhalation solutions or nucleic acid molecule can also be formulated with a propellant for aerosol delivery. In another additional embodiment, the solutions can be nebulized. Pulmonary administration is further described in PCT Publication No. WO 94/20069, which describes the pulmonary delivery of chemically modified proteins.

It is also contemplated that some formulations may be administered orally. In one embodiment of the present invention, FGF-23 polypeptides that are administered in this manner can be formulated with or without those carriers commonly used in the manufacture of compounds of solid dosage forms such as tablets and capsules. For example, a capsule can be designed to release the active portion of the formulation at a point in the intestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to facilitate absorption of the FGF-23 polypeptide. Diluents, flavoring agents, waxes with low melting temperature, vegetable oils, lubricants, agents that improve the suspension, tablet disintegrating agents and binders can also be used. Another pharmaceutical composition may involve an effective amount of FGF-23 polypeptides in a mixture with non-toxic excipients that are suitable for tabletting. By dissolving the tablets in sterile water, or other suitable vehicle, solutions can be made in unit dosage form. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or ^? wfctf ??? ? c c ^^ g ^ i ^^^^^^^ binders such as starch, gelatin or, acacia; or lubricating agents such as magnesium stearate, stearic acid or talc. Additional pharmaceutical compositions of FGF-23 polypeptide will be apparent to those skilled in the art, including formulations involving FGF-23 polypeptides in sustained or controlled delivery formulations. Techniques for the preparation of formulations of various sustained or controlled delivery means, such as liposome carriers, bioerodible microparticles and porous spheres as well as deposition injections are also known to those skilled in the art. See, for example, PCT / US93 / 00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. Additional examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, for example films or microcapsules. Sustained-release matrices may include polyesters, hydrogels, polylactides (US Patent No. 3,773,919 and European Patent No. 058481), copolymers of L-glutamic acid and ethyl L-glutamate (Sidman et al., 1983, Biopolymers 22: 547-56), poly (2-hydroxyethyl methacrylate) (Langer et al. ,;. ^.? i j? »* t?. ~. J ... ^.,. *. »-... .. - - al , 1981, J. Biomed. Mater. Res. 15: 167-277 and Langer, 1982, Chem. Tech. 12: 98-105), ethylene vinylacetate (Langer et al., Supra) or poly-D (-) - 3-hydroxybutyric acid (European Patent No. 133988). Sustained release compositions can also include liposomes which can be prepared by any of the various methods known in the art. See, for example, Eppstein et al. , 1985, PROC. Natl. Acad. Sci. USA 82: 3688-92; and European Patent Nos. 036676, 088046 and 143949. The pharmaceutical composition of FGF-23 to be used for in vivo administration should typically be sterile. This can be carried out by filtration through membranes for filter sterilization. When the composition is lyophilized, sterilization using this method can be carried out before or after lyophilization and dissolution. The composition for parenteral administration can be stored in lyophilized form or in a solution. In addition, parenteral compositions are generally placed in a container having a sterile access port, for example an intravenous solution bag or a bottle having a plug pierceable by a needle for hypodermic injection. Once the pharmaceutical compositions have been formulated, they can be stored in sterile bottles as a solution, suspension, gel, emulsion, solid or as a dehydrated or lyophilized powder. Such formulations may be stored in a ready-to-use form or in a form (eg lyophilized) that requires dissolution prior to administration. In a specific embodiment, the present invention relates to equipment for producing a single-dose administration unit. The kits can each contain both the first container having the dried protein as well as a second container having an aqueous formulation. Also included within the scope of this invention are equipment containing syringes previously filled with a single chamber or with multiple chambers (for example syringes with liquid and lyse-syringes). The effective amount of a pharmaceutical composition of FGF-23 to be used therapeutically will depend, for example, on the context and therapeutic objectives. A person skilled in the art will appreciate that the appropriate dosage levels for treatment in this manner will vary based, in part, on the molecule delivered, the indication for which the FGF-23 molecule is used, the route of administration and the size (body weight, body surface or size of organs) and the condition (age and general health) of the patient. Consequently, the doctor can adjust the dosage and modify the route of administration to obtain the effect optimal therapeutic A typical dosage may vary from about 0.1 μg / kg to about 100 mg / kg or more, based on the factors mentioned above. In other embodiments, the dosage may vary from 0.1 μg / kg to approximately 100 mg / kg; or from 1 μg / kg to approximately 100 mg / kg; or 5 μg / kg to approximately 100 mg / kg. The frequency of dosing will depend on the pharmacokinetic parameters of the FGF-23 molecule in the formulation used. Usually, a physician will administer the composition until a dosage is reached that generates the desired effect. The composition can therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) with respect to time, or as a continuous infusion via an implantation device or catheter. A further adjustment of the appropriate dosage is usually performed by those skilled in the art and is within the scope of the tasks usually performed by them. Appropriate dosages can be determined by the use of appropriate dose and response data. The route of administration of the pharmaceutical composition agrees with the known methods, for example orally; through injection via intracerebral, intraperitoneal, intracerebral (within the parenchyma) intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional; by sustained release systems or by implantation devices. When desired, the compositions can be administered by rapid and continuous injection either continuously by infusion, or by an implantation device. Alternatively or additionally, the composition can be administered locally by implantation of a membrane, sponge or other suitable material in which the desired molecule has been absorbed or encapsulated. When an implantation device is used, it can be implanted in any suitable tissue or organ, and the delivery of the desired molecule can be via diffusion, rapid injection of synchronized release or by continuous administration. In some cases it is desirable to use pharmaceutical compositions of the FGF-23 polypeptide in an ex vivo manner. In such cases, the cells, tissues or organs that have been extracted from the patient are exposed to the pharmaceutical compositions of the FGF-23 polypeptide after which the cells, tissues or organs are again implanted in the patient. In other cases, the FGF-23 polypeptide can be delivered by implanting certain cells that have been engineered using methods such as those described herein to express and secrete the FGF-23 polypeptide. Such cells can be animal or human cells, and can be autologous, heterologous or xenogeneic. Optionally, the cells can be immortalized. To decrease the likelihood of an immune response, the cells can be encapsulated to prevent infiltration of the surrounding tissues. The encapsulation materials are typically biocompatible, they are semi-permeable polymeric membranes or enclosures that allow the release of the protein product (s) but which prevent the destruction of the cells by the patient's immune system or by other damaging factors of the surrounding tissues. As discussed herein, it may be desirable to treat populations of isolated cells (e.g., such as hemocytoblasts, lymphocytes, erythrocytes, chondrocytes, neurons, and the like) with one or more FGF-23 polypeptides. This can be accomplished by exposing the isolated cells to the polypeptide directly, when it is in a form that is permeable to the cell membrane. Additional embodiments of the present invention relate to cells and methods (eg, homologous recombination or other methods of recombinant production) both for the production of therapeutic polypeptides in vitro and for the production and delivery of therapeutic polypeptides by gene therapy or by cell therapy. Homologous recombination methods or other recombination methods can be used to modify a cell containing a FGF-23 gene that is normally transcriptionally silent (not transcribed) or an underexpressed gene, and therefore produce a cell that expresses therapeutically effective amounts of the FGF-23 polypeptides. Homologous recombination is a technique originally developed for target genes to induce or correct mutations in transcriptionally active genes. Kucherlapati, 1989, Prog. In Nucí. Acid Res. & Mol. Biol. 36: 301 The basic technique was developed as a method to introduce specific mutations within specific regions of the mammalian genome (Thomas et al., 1986, Cell 44: 419-28, Thomas and Capecchi, 1987, Cell 51: 503-12, Doetschman et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 8583-87) or to correct specific mutations within defective genes (Doetschman et al., 1987, Nature 330: 576-78). Exemplary homologous recombination techniques are described in the U.S. Patent. No. 5,272,071; European Patent Nos. 9193051 and 505500; PCT / US90 / 07642 and PCT Publication NO. WO 91/09955). Through homologous recombination, the ^^^ j ^^^^ g? ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ is going to be inserted into the genome can be directed to a specific region of the gene of interest by binding it to target DNA. The target DNA is a nucleotide sequence that is complementary (homologous) to a region of the genomic DNA. Small pieces of target DNA that are complementary to a specific region of the genome are contacted with the original strand during the DNA replication procedure. A general property of DNA is that it has been inserted into a cell to hybridize and therefore recombines with other pieces of endogenous DNA through shared homologous regions. If this complementary strand is linked to an oligonucleotide containing a different mutation or sequence or an additional nucleotide, it is also incorporated into the newly synthesized strand as a result of recombination. As a result of the reading verification function, it is possible that the new DNA sequence serves as a template. Thus, the transferred DNA is incorporated into the genome. Attached to these pieces of targeted DNA are regions of DNA that can interact with, or control the expression of a FGF-23 polypeptide, e.g., flanking sequences. For example, a promoter / enhancer element, a suppressor or an exogenous transcription modulator element is inserted into the genome of the proposed host cell in sufficient proximity and orientation to influence the transcription of DNA encoding the desired FGF-23 polypeptide. . The control element controls a portion of the DNA present in the genome of the host cell. In this manner, expression of the desired FGF-23 polypeptide can be obtained not by transfection of DNA encoding the gene for FGF-23 itself, but rather by the use of targeted DNA (containing regions of homology with the endogenous gene). of interest) coupled with DNA regulatory segments that provide the endogenous gene sequence with recognizable signals by transcription of an FGF-23 gene. In an exemplary method, the expression of a desired targeted gene in a cell (i.e., a desired endogenous cellular gene) is altered via homologous recombination in the cell genome at a site selected in advance, by the introduction of DNA that includes minus a regulatory sequence, an exon and a splice donor site. These components are introduced into the chromosomal (genomic) DNA in such a way that, in effect, it results in the production of a new transcription unit (in which the regulatory sequence, elexon and the splice donor site are present in the assembly of DNA and operably linked to the endogenous gene). As a result of the introduction of these components into chromosomal DNA, the expression is altered i £ i 1 -C.l.? rl - tlA ~ A i *. of the desired endogenous gene. The altered expression of the gene, as described herein, encompasses activating a gene (or causing it to be expressed) which is usually not expressed in the cell as obtained, as well as an increase in the expression of a gene that is not expresses at physiologically significant concentrations in the cell as obtained. The modalities further comprise changing the pattern of regulation or induction so that it is different from the pattern of regulation or induction that occurs in the cell as obtained, and reducing (even eliminating) the expression of a gene which is expressed in the cell how is it obtained. One method by which homologous recombination can be used to increase or elicit the production of the FGF-23 polypeptide from the endogenous FGF-23 gene of the cell involves first using homologous recombination to establish a recombination sequence from a system of site-specific recombination (eg, Cre / loxP, FLP / FRT) (Sauer, 1994, Curr Opin. Biotechnol., 5: 521-27; Sauer, 1993, Methods Enzymol., 225: 890-900) the 5 'end with respect to the endogenous genomic coding region for the FGF-23 polypeptide. A plasmid containing a recombination site homologous to the site is placed just towards the 5 'end of the genome encoding region of the polypeptide FGF-23 within the cell line modified together with the appropriate recombinase enzyme. This recombinase causes the plasmid to integrate, via the site of recombination of the plasmid, into the recombination site which is located just upstream of the genomic coding region of the FGF-23 polypeptide in the cell line (Baubonis and Sauer, 1993, Nucleic Acids Res. 21: 2025-29; 0 • Gorman et al., 1991, Science 251: 1351-55). Any flanking sequence that is known to increase transcription (eg, a promoter / promoter, intron, translation enhancer) if properly placed in this plasmid, can be integrated in a way that generates a new or modified transcription unit resulting in a de novo or augmented production of FGF-23 polypeptide from the endogenous cell gene for FGF-23. An additional method for using the cell line in which the site-specific recombination sequence has been placed just towards the 5 'end of the endogenous genomic coding region of the cell for the FGF-23 polypeptide is to use homologous recombination to introduce a second recombination site elsewhere in the genome of the cell line. The appropriate recombinase enzyme is then introduced into a cell line with two recombination sites, which causes a recombination phenomenon (deletion, inversion and translocation) (Sauer, 1994, Curr Opin. Biotechnol., 5: 521-27; Sauer, 1993, Methods Enzymol., 225: 890-900) which can generate a new or modified transcriptional unit resulting in a de novo or increased production of FGF-23 polypeptide from the endogenous gene of the cell for FGF-23 . An additional approach to increase or elicit the expression of the FGF-23 polypeptide from the endogenous gene of the cell for FGF-23 which involves increasing or causing the expression of a gene or genes (for example transcription factors) or decreasing the expression of a gene or genes (e.g., transcriptional repressors) in a manner that results in a de novo or augmented production of FGF-23 polypeptide from the endogenous gene of cell 5 for FGF-23. This method includes introducing a polypeptide that does not occur naturally (eg, a polypeptide comprising a site-specific DNA binding domain fused to a transcription factor domain) within the cell, so that it results at 0 the de novo or increased production of the FGF-23 polypeptide from the endogenous gene of the cell for FGF-23. The present invention also relates to assemblies (recombinant plasmids) of DNA useful in the method for altering the expression of a target gene. In 5 some modalities, exemplary DNA assemblies ^ m ^^^^^ í? they comprise: (a) one or more directed sequences, (b) a regulatory sequence, (c) an exon and (d) an unpaired splice donor site. The directed sequence in the DNA assembly directs the integration of elements (a) - (d) into the target gene in a cell such that elements (b) - (d) are operably linked to the target gene sequences endogenous. In another embodiment, the DNA assembly comprises: (a) one or more directed sequences, (b) a regulatory sequence, (c) an exon, (d) a donor site, 10 splice, (e) an intron and (f) a splice acceptor site, where the directed sequence directs the integration of elements (a) - (f) so that the elements of (b) - (f) are operatively linked to the endogenous gene. The directed sequence is homologous to a site previously selected in 15 the cellular chromosomal DNA in which homologous recombination occurs. In the assembly, the exon is generally 3 'to the regulatory sequence and the splice donor site is 31 relative to the exon. If the sequence of a particular gene is known, As the nucleic acid sequence for the FGF-23 polypeptide presented here, a piece of DNA that is complementary to a region selected from the gene can be synthesized or otherwise obtained, for example by proper restriction of the native DNA in places 25 of specific recognition that unite the region of interest.

IMÜtBltlIÉ? T mi? Tt if ri 'J ¡ni tf? Tti? Rlgfaa °' A "'-» «> --'-- aia' ~ - 't-" t - * - - * - " - - • - -This piece serves as a sequence directed to the insertion in the cell and will hybridize to its homologous region within the genome.If this hybridization occurs during DNA replication, this piece of DNA, and any additional sequence linked to it will act as a fragment of Okazaki and will be incorporated into the newly synthesized daughter strand of DNA.Therefore, the present invention includes nucleotides encoding a FGF-23 polypeptide, nucleotides which can be used as target sequences. contemplates therapy with cells containing FGF-23 polypeptide, for example, the implantation of cells that produce FGF-23 polypeptides This involves implanting cells capable of synthesizing and secreting a biologically active form of the FGF-23 polypeptide. polypeptide FGF-23 pu eden be cells that are natural producers of FGF-23 polypeptides or can be recombinant cells in which the ability to produce FGF-23 polypeptides has been increased by transformation with a gene encoding the desired FGF-23 polypeptide or with a gene which increases the expression of the FGF-23 polypeptide. Such modification can be carried out by means of a suitable vector to deliver the gene and also promote its expression and secretion. In order to minimize the potential immunological reaction in patients administered a FGF-23 polypeptide, which could occur with the administration of a polypeptide of a foreign species, it is preferred that the natural cells producing the FGF-23 polypeptide be of origin human and produce human FGF-23 polypeptide. Likewise, it is preferred that the recombinant cells that produce the FGF-23 polypeptide are transformed with an expression vector that contains a gene encoding a human FGF-23 polypeptide. The implanted cells can be encapsulated to prevent infition of the surrounding tissue. Non-human cells or animal cells can be implanted in patients in semi-permeable biocompatible polymeric enclosures or membranes that allow the release of the FGF-23 polypeptide, but which prevent the destruction of the cells by the patient's immune system or by other tissue damaging factors surrounding. Alternatively, the patient's own cells transported ex vivo to produce FGF-23 polypeptide can be implanted directly into the patient without such encapsulation. Techniques are known to encapsulate living cells, and the preparation of encapsulated cells and their implantation in patients can be carried out in a customary manner. For example, Baetge et al. , (PCT Publication No. WO 95/05452 and PCT / US95 / 09299) describe membrane capsules containing cells engineered for the effective delivery of biologically active molecules. The capsules are biocompatible and are easily recoverable. Capsules encapsulate cells transfected with recombinant DNA molecules comprising DNA sequences encoding biologically active molecules operably linked to promoters that are not subject to down regulation in vivo upon implantation in a mammalian host. The devices provide for the delivery of molecules from living cells at specific sites within a receptor. In addition, see the Patents of E.U.A. Nos. 4,892,538; 5,011,472; and 5,106,627. A system for encapsulating living cells is described in PCT Publication No. WO 91/10425 (Aebischer et al.). See also Publication No. WO 91/10470 (Aebischer et al.); Winn et al. , 1991, Exper, Neurol. 113: 322-29; Aebischer et al. , 1991, Exper. Neurol. 111: 269-75; and Tresco et al. , 1992, ASAIO 38: 17-23. The delivery by gene therapy in vivo and in vi tro of the FGF-23 polypeptides is also considered. An example of a gene therapy technique is to use the gene for FGF-23 (either genomic DNA, cDNA or synthetic DNA) which codes for a FGF-23 polypeptide which can be operably linked to a constitutive or inducible promoter for form a "DNA assembly of gene therapy".

The promoter can be homologous or etherologous to the endogenous gene for FGF-23, provided that it is active in the cell or type of tissue in which the assembly is to be inserted. Other components of the gene therapy DNA assembly optionally may include DNA molecules designed for site-specific integration (e.g., endogenous sequences useful for homologous recombination), tissue-specific promoters, enhancers or gene expression blockers, DNA capable of providing a selective advantage over an original cell, DNA molecules useful as labels for identifying transformed cells, negative selection systems, cell-specific binding agents (such as for example for cell targeting), specific internalization factors of cell, transcription factors that improve the expression of a vector and vectors that allow the production of the vector. In this way, a gene therapy DNA assembly (either ex vivo or in vivo) can be introduced into the cells using viral or non-viral vectors. A means for introducing the gene therapy DNA assembly is by means of viral vectors, as described herein. Some vectors, such as retroviral vectors, will supply the assembly of DNA to the chromosomal DNA of the cells, and the gene can be integrated into the chromosomal DNA.

Other vectors will function as episomes, and the DNA assembly of gene therapy will remain in the cytoplasm. In other additional embodiments, regulatory elements may be included for the controlled expression of the gene for FGF-23 in the target cell. Such elements are activated in response to an appropriate effector. In this way, the therapeutic polypeptide can be expressed when desired. A conventional control means involves the use of dimerizers or small molecule rapists to dimerize chimeric proteins containing a small molecule binding domain and a domain capable of initiating a biological process such as a protein that binds to DNA or a protein of transcription activation (see PCT publications Nos. WO 96/41865, WO 97/31898 and WO 97/31899). The dimerization of the proteins can be used to initiate the transcription of the transgene. An alternative regulatory technology uses a method to store proteins that are expressed from the gene of interest within the cell as an aggregate or grouped. The gene of interest is expressed as a fusion protein that includes a conditional aggregation domain that results in retention of the aggregated protein in the endoplasmic reticulum. The stored proteins are stable and inactive inside the cell. However, proteins can be released when a drug is administered (for U-? ifaj. ± * t - * ..... * ^. ^ .. example a small molecule ligand) that removes the conditional aggregation domain and in this way specifically separates the aggregates or clusters so that the proteins can be secreted from the cell. See Aridor et al. , 2000, Science 287: 816-17 and Rivera et al. , 2000, Science 287: 826-30. Another means of adequate control of gene activation includes, but is not limited to, the systems described herein. Mifepristone (RU486) is used as a progesterone antagonist. The binding of a modified progesterone receptor from a ligand binding domain to a progesterone antagonist activates transcription to form a dimer of two transcription factors and then passes to the nucleus to bind DNA. The ligand-binding domain is modified to eliminate the ability of the receptor to bind to the natural ligand. The modified steroid hormone receptor system is further described in the U.S. Patent. No. 5,364,791 and PCT Publication Nos. WO 96/40911 and WO 97/10337. Another additional control system uses ecdysone (a steroid hormone from the fruit fly) which binds and activates an ecdysone receptor (cytoplasmic receptor). The receptor then goes to the nucleus to bind to a specific DNA response element (a promoter of the gene that responds to ecdysone). The receiver of ecdysone includes a transactivation domain, a DNA binding domain, a ligand binding domain to initiate transcription. The ecdysone system is further described in the U.S. Patent. No. 5,514,578 and 5 in PCT Publication Nos. WO 97/38117, WO 96/37609 and WO 93/03162. Another control means uses a transactivator controllable by positive tetracycline. This system involves a DNA binding domain of mutated 10 tet repressor protein (the mutated tet R-4 amino acid changes, resulting in a transactivating protein regulated by reverse tetracycline, that is, it binds to a tet operator in the presence of tetracycline ) linked to a polypeptide which activates transcription. Such systems are described in the patents of the U.S.A. Nos. 5,464,758, 5,650,298 and 5,654,168. Additional expression control systems and assemblies (recombinant plasmids) of nucleic acid are described in U.S. Pat. Nos. 5,741,679 and 20 5,834,186 for Innovir Laboratories Inc. Gene therapy in vivo can be carried out by introducing the gene encoding the FGF-23 polypeptide into cells by means of local injection of a nucleic acid molecule for FGF -23 or by another vector of 25 appropriate viral or non-viral supply. Hefti, 1994, Neurobiology 25: 1418-35. For example, a nucleic acid molecule encoding a FGF-23 polypeptide can be contained in an adeno-associated virus vector (AAV) for delivery to target cells (see, eg, Johnson, PCT Publication No. WO 95/34670; PCT Application No. PCT / US95 / 07178). The recombinant AAV genome usually contains inverted terminal AW reverse sequences flanking a DNA sequence encoding a FGF-23 polypeptide operably linked to a functional promoter as well as polyadenylation sequences. Suitable alternative viral vectors include, but are not limited to, retroviruses, adenoviruses, herpes simplex viruses, lentiviruses, hepatitis viruses, parvoviruses, papovaviruses, poxviruses, alphaviruses, coronaviruses, rhabdoviruses, paramyxoviruses and papilloma virus vectors. The Patent of E.U.A. No. 5,672,344 discloses an in vivo virus mediated gene transfer system that involves a recombinant neurotrophic HSV-1 vector. The Patent of E.U.A. No. 5,399,346 provides examples of a method for providing a patient with therapeutic protein by delivery of human cells which have been treated in vi tro to insert a segment of DNA encoding a therapeutic protein. Additional methods and materials for the practice of gene therapy techniques are described in U.S. Pat. No. 5,631,236 (related to adenoviral vectors), 5,672,510 (related to retroviral vectors), and 5,635,399 (related to retroviral vectors that express cytokines). Non-viral delivery methods include, but are not limited to, liposome-mediated transfer, naked DNA delivery (direct injection), receptor-mediated transfer (ligand-DNA complex), electroporation, calcium phosphate precipitation and microparticle bombardment (for example, gene gun). Gene therapy materials as well as methods may also include inducible promoters, tissue-specific promoters-enhancers, DNA sequences designed for site-specific integration, DNA sequences capable of providing a selective advantage over the original cell, labels to identify transformed cells, negative selection system and expression control systems (safety measures), cell-specific binding agents (for cell targeting), cell-specific internalization factors and transcription factors to improve expression by a vector as well as vector manufacturing methods. Such additional methods and materials for the practice of gene therapy techniques are described in U.S. Pat. Nos. 4, 970,154 (which relates to electroporation techniques), 5,679,559 (which describes a system containing lipoprotein for the delivery of genes), 5,676,954 (related to liposame carriers), 5,593,875 (which describes methods for transfection with calcium phosphate) and 4,945,050 (which describes a process in which biologically active particles are driven into cells at a rate by which particles penetrate the surface of cells and are incorporated into the interior of cells), and PCT Publication No. WO 96 / 40958 (related to nuclear ligands). It is also contemplated that gene therapy with FGF-23 or cell therapy may further include the delivery of one or more additional polypeptides in the same cell or in different cells. Such cells can be introduced separately into the patient, or the cells can be contained in a single implantable device, such as an encapsulating membrane, described above, or the cells can be modified separately, by means of viral vectors. One way to increase the endogenous expression of the FGF-23 polypeptide in a cell via gene therapy is to insert one or more enhancer elements in the promoter for the FGF-23 polypeptide, wherein the enhancer elements can serve to increase the transcriptional activity of the gene for FGF-23. The enhancer elements used will be selected based on the tissue in which one wishes to activate the gene - the enhancer elements are known to confer promoter activation in that tissue that is selected. For example, if a gene encoding a FGF-23 polypeptide is going to be "activated" on T lymphocytes, the lck promoter enhancer element can be used. Here, the portion of the transcriptional element to be added can be inserted into a DNA fragment containing the FGF-23 polypeptide promoter (and optionally can be inserted into a vector and in the flanking sequences 51 or 3 ') using standard cloning techniques. This assembly, known as a "homologous recombination assembly", can then be introduced into the desired cells either ex vivo or in vivo. Gene therapy can also be used to decrease the expression of the FGF-23 polypeptide by modifying the nucleotide sequence of the endogenous promoter. Such modification is typically carried out via homologous recombination methods. For example, a DNA molecule containing all or a portion of the gene promoter for FGF-23 selected for inactivation can be engineered to remove or replace pieces of the promoter that regulate transcription. For example, the TATA sequence or the binding site of a promoter transcriptional activator can be suppressed using standard or conventional molecular biology techniques; such suppression can inhibit the promoter activity and in this way the transcription of the corresponding gene for FGF-23 is repressed. The deletion of the TATA sequence from the transcription activator binding site in the promoter can be carried out by generating a DNA assembly comprising all or the relevant portion of the FGF-23 polypeptide promoter (for the same species or species). related to the gene for FGF-23 to be regulated), in which one or more of the nucleotides of the TATA sequence or of the transcriptional activator binding site is mutated by means of substitution, deletion or insertion of one or more nucleotides. As a result, the TATA sequence or the activator binding site have decreased activity or become completely inactive. This assembly, which typically will contain at least about 500 DNA bases corresponding to the native (endogenous) 51 and 3 'DNA sequences adjacent to the promoter segment that has been modified, can be introduced into appropriate cells (ex vivo or in vivo). live) directly or by means of a viral vector, as described herein. Typically, the integration of the assembly into the genomic DNA of the cells will be via homologous recombination, in wherein the 5 'and 3' DNA sequences in the promoter assembly can serve to help integrate the modified promoter region via hybridization to the endogenous chromosomal DNA.

Therapeutic Uses The nucleic acid molecules for FGF-23, polypeptides, agonists and antagonists thereof can be used to treat, diagnose, diminish or prevent many diseases, disorders or conditions including those mentioned herein. FGF-23 polypeptide agonists and antagonists include those molecules that regulate the activity of the FGF-23 polypeptide and increase or decrease at least one activity of the mature form of the FGF-23 polypeptide. The agonists or antagonists can be cofactors such as a protein, peptide, carbohydrate, lipid or small molecular weight molecule that interacts with the FGF-23 polypeptide and thus regulates its activity. Potential agonists or antagonists of the polypeptide include antibodies that react with the soluble or membrane-bound forms of FGF-23 polypeptides that comprise part or all of the extracellular domains of the proteins. Molecules that regulate FGF-23 polypeptide expression typically include nucleic acids encoding FGF-23 polypeptide that can act as antisense expression regulators. The nucleic acid molecules for FGF-23, the polypeptides, agonists and antagonists thereof of the present invention are useful for the same purposes for which members of the FGF polypeptide family are known to be useful. Therefore, the FGF-23 polypeptides of this invention are potent mitogens for various cells of mesodermal, ectodermal and endodermal origin, including fibroblasts, cornea and vascular endothelial cells, granulocytes, adrenal cortex cells, chondrocytes, myoblasts, vascular smooth muscle cells, lens epithelial cells, melanocytes, keratinocytes, oligodendrocytes, astrocytes, osteoblasts and hematopoietic cells. These biological activities include the ability to stimulate the proliferation of vascular endothelial cells and allow endothelial cells to penetrate the basement membrane. Consistent with these properties, the FGF-23 polypeptides of this invention can stimulate angiogenesis and promote wound healing (i.e., facilitate repair or replacement of diseased tissue damage resulting from burns, traumatic damage, surgery or ulcers). These polypeptides can also induce mesoderm formation and modulate neuronal cell differentiation, adipocytes and musculoskeletal cells. The polypeptides can also be used to prevent or decrease skin aging due to sun exposure by stimulating the growth of keratinocytes. In addition, the polypeptides of this invention can be used to maintain organs prior to transplantation or to nourish cultures of primary cells and tissues. In addition, these polypeptides can be used to prevent hair loss since members of the FGF family activate hair-forming cells and promote the growth of melanocytes. They can also be used to stimulate the growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines. FGF-23 has been linked to the autosomal human dominant genetic disease, hypophosphatemic rickets (ADHR) (The ADHR Consortium, 2000, Nature Genetics 26: 345-48). Accordingly, the nucleic acid molecules for FGF-23, the polypeptides, agonists and antagonists thereof of the present invention can be used to treat, diagnose, diminish or prevent ADHR. It has been shown that the gene for FGF-23 is much more closely related to human FGF-21 (Yamashita et al., 2000, Biochem. Biophys., Res. Commun. 277: 494-98), a gene which is expressed in more abundant in the liver and at lower concentrations in the thymus (Nishimura et al., 2000, Biochim, Biophys, Acta 1492: 203-06). Accordingly, the nucleic acid molecules of FGF-23, polypeptides and agonists and antagonists thereof can be used to treat, diagnose, diminish or prevent diseases, disorders or conditions that are related to the liver or thymus. A list, which is not exhaustive, of other diseases, disorders or conditions which can be treated, diagnosed, diminished or avoided with the nucleic acid molecules for FGF-23, polypeptides, agonists and antagonists thereof of the present invention include: skin lesions, bullous epidermalysis, alopecia with male pattern, gastric ulcer, duodenal ulcer, erosive gastritis, esophagitis, esophageal reflux disease, inflammatory bowel disease, radiation toxicity induced by radiation or chemotherapy, hyaline membrane disease, necrosis of the respiratory epithelium, emphysema, pulmonary inflammation, pulmonary fibrosis, liver cirrhosis, fulminant hepatic failure, viral hepatitis and diabetes. The agonists or antagonists of the FGF-23 polypeptide function can be used (simultaneously or sequentially) in combination with one or more cytokines, TO. ÍIA * Í? U..ii- *? ** growth factors, antibiotics, anti-inflammatories or chemotherapeutic agents as appropriate for the condition being treated. Other diseases caused or mediated by undesirable concentrations of FGF-23 polypeptides are encompassed within the scope of the invention. Undesirable concentrations include excessive concentrations of FGF-23 polypeptides and subnormal concentrations of FGF-23 polypeptides.

Uses of the Nucleic Acids and Polypeptides of FGF-23 The nucleic acid molecules of the invention (which include those which in themselves do not encode biologically active polypeptides) can be used to map the gene positions for FGF-23 and related genes on chromosomes. The mapping can be performed by techniques known in the art, such as PCR amplification and in-situ hybridization. The nucleic acid molecules for FGF-23 (which include those which in themselves do not code for biologically active polypeptides), can be useful as hybridization probes in diagnostic assays to prove, qualitatively or quantitatively, the presence of an acid molecule nucleic acid for FGF-23 in mammalian tissue or body fluid samples. Other methods may also be used where it is desirable to inhibit the activity of one or more FGF-23 polypeptides. Such inhibition can be carried out by nucleic acid molecules which are complementary and which hybridize with the expression control sequences (triple helix formation) or with mRNA for FGF-23. For example, antisense DNA or RNA molecules, which have a sequence that is complementary to at least a portion of a gene for FGF-23 can be introduced into the cell. Antisense probes can be designed by available techniques using the sequence of the FGF-23 gene described herein. Typically, each antisense molecule will be complementary to the start site (5 'end) of each gene for selected FGF-23. When the antisense molecule then hybridizes with the corresponding FGF-23 mRNA, translation of this mRNA is prevented or reduced. Antisense inhibitors provide information regarding the decrease or absence of a FGF-23 polypeptide in a cell or organism. Alternatively, gene therapy can be used to create a dominant negative inhibitor or one or more FGF-23 polypeptides. In this situation, a DNA encoding a mutant polypeptide of each selected FGF-23 polypeptide can be prepared and introduced into the cells of a patient using viral or non-viral methods, as described herein. Each of such mutants is typically designed to compete with the endogenous polypeptide in its biological role. In addition, a FGF-23 polypeptide, whether biologically active or not, can be used as an immunogen, ie, the polypeptide contains at least one epitope for which antibodies can be generated. Selective binding agents that bind to a FGF-23 polypeptide (as described herein) can be used for in vivo or in vitro diagnostic purposes including, but not limited to, use in labeled form to detect presence of the FGF-23 polypeptide in a body fluid or in a cell sample. Antibodies can also be used to prevent, treat or diagnose many of the diseases and disorders that include those mentioned herein. Antibodies that can bind to a FGF-23 polypeptide in a manner that decreases or blocks at least one characteristic activity of a FGF-23 polypeptide, or that can bind to a polypeptide to increase at least one activity characteristic of a FGF-23 polypeptide (included by increase in the pharmacokinetics of FGF-23 polypeptide). The FGF-23 polypeptides of the present invention can be used to clone FGF-23 polypeptide receptors, using an expression cloning strategy. The radiolabelled FGF-23 polypeptide (125I) or affinity / activity tagged FGF-23 polypeptide (such as a Fe fusion or an alkaline phosphatase fusion) can be used in binding assays to identify a cell type or cell line or tissue expressing receptors for the FGF-23 polypeptide. RNA isolated from such cells or tissues can be converted to cDNA, clone into a mammalian expression vector and transfect into mammalian cells (such as COS or 293 cells) to create an expression library. A radiolabelled or labeled FGF-23 polypeptide can then be used as an affinity ligand to identify and isolate from this library the subset of cells expressing receptors for the FGF-23 polypeptide on its surface. The DNA of these cells can then be isolated and transfected into mammalian cells to create a secondary expression library in which the fraction of cells expressing receptors for the FGF-23 polypeptide is many times larger than the original library. This enrichment procedure can be repeated again and again until a single recombinant clone containing a receptor for the FGF-23 polypeptide is isolated. The isolation of the receptors for the FGF-23 polypeptide is useful to identify or develop novel agonists and antagonists of the FGF-23 polypeptide signaling. Such agonists and antagonists include soluble receptors for the FGF-23 polypeptide, antibodies against the FGF-23 polypeptide receptor, small molecules or antisense oligonucleotides, and can be used to treat, prevent or diagnose one or more of the diseases or disorders described at the moment. A cDNA deposit encoding the human FGF-23 polypeptide, subcloned into the pGEM-t vector and having accession number No. PTA-1617, was made to the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209 on March 31, 2000. The following examples are designed for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

Example 1: Cloning of the gene for the human FGF-23 polypeptide To isolate cDNA sequences encoding the human FGF-23 polypeptide, BLAST investigations are performed based on homology of a human genome database. A putative coding sequence that shares homology with the fibroblast growth factor family (FGF) in a clone is identified i ??? -? Jiijaifk? M **. ^. ..y ** ..... * ^ - - * • »*«. *. t & amp; .i.? yk? JÉ of the human genome (GenBank, Access No. AC008012). The putative coding sequence consists of three potential exons separated by introns of 6.6 kb and 1.87 kb. This sequence is used to design gene-specific oligonucleotides for the identification of cDNA sources and the generation of cDNA clones, using various PCR strategies. Many cDNA libraries are analyzed in amplification reactions containing 10 pmol each of the amplimers (amplification primers) (5'-CTAT-CCCAATGCCTCCCCACTG-3 '; SEQUENCE OF IDENTIFICATION NO: 42 and 5' -CGCCCCTGACCACCCC- TAATG-3 •; ID SEQUENCE NO: 43) and PCR Ready-To-Go spheres (Pharmacia, Piscataway, NJ), in a total reaction volume of 250 μl. The reactions are carried out at 95 ° C for 5 minutes during a cycle; 95 ° C for 30 seconds, 68 ° C for 15 seconds and 72 ° C for 1 minute for 35 cycles; and 72 ° C for 7 minutes during a cycle. A PCR product of the expected size (616 bp) is identified in many of the cDNA libraries, including libraries derived from the T25 colon tumor (random priming), fetal mesentery (primer with oligo-dT), fetal gall bladder (priming randomly) and fetal heart (primed with oligo-dT). The PCR product generated from the fetal mesentery cDNA library is subclone using the TopoTA 4.0 cloning kit (Invitrogen) and four clones are sequenced to verify that the clones contain the predicted cDNA sequence for FGF-23. The fetal mesentery cDNA library is screened for further amplification experiments to isolate full-length cDNA sequences coding for the FGF-23 polypeptide. The fetal mesentery cDNA library is prepared as follows. Total RNA is extracted from human fetal mesentery using standard or conventional RNA extraction procedures and AR? poly-A + from this AR? total using standard procedures. AD? C are synthesized baited with oligo-dT from this AR? poly-A + using the Superscript Plasmid System for AD? c synthesis and the Plasmid Cloning kit (Gibco-BRL) according to the procedures suggested by the manufacturer. The resulting AD? C is digested with the restriction endonucleases Sal I and? Ot I, and then ligated into pSPORT-1. The ligation products are transformed into E. coli using standard techniques and the bacterial transformants are selected on culture plates containing ampicillin. The AD? C library consists of all or a subset of these transformants. The 5 'RACE and 3' RACE reactions are performed in order to generate a full length AD? C frequency for the FGF-23 polypeptide. To isolate cDNA sequences corresponding to the 5 'end of the cDNA sequence for the FGF-23 polypeptide, 5' RACE is performed using the Smart RACE cDNA amplification kit (Clontech), the primed human fetal mesentery cDNA library randomly in pSPORTl, and the primers 5 '-GTGTGGA-ATTGTGAGCGGATAAC-3' (SEQUENCE OF IDENTIFICATION NO: 44) and 5 '-CTGATGGGGTGCGCCA- TCCACA-3' (SEQUENCE OF IDENTIFICATION NO: 45). The reactions are carried out at 94 ° C for 1 minute for one cycle; 94 ° C for 5 seconds, 68 ° C for 10 seconds and 72 ° C for 3 minutes for 35 cycles; and 72 ° C for 7 minutes for one cycle. The hosted PCR is performed using a portion of the 5 'amplification product RACE (diluted 1/100) and the primers 5' -CTATGACCATGATTACG- CCAAGC-3 '(SEQUENCE OF IDENTIFICATION NO: 46) and 5' -C- ATTCTTGTGGATCTGCAGGTG-GT -3 '(SEQUENCE OF IDENTIFICATION NO: 47). The hosted PCR reactions are performed at 94 ° C for 5 minutes for one cycle; 94 ° C for 15 seconds, 68 ° C for 15 seconds and 72 ° C for 3 minutes, for 30 cycles; and 72 ° C for 7 minutes for one cycle. The amplification products are analyzed by agarose gel electrophoresis and a prominent 200 bp PCR product is isolated and subcloned using the TopoTA 4.0 cloning kit. The sequencing analysis of the clones - isolated indicates that the 5 'PCR product did not extend the known sequence. Additional 5 'RACE experiments are performed to isolate the additional cDNA sequences corresponding to the 5' end of the cDNA sequence for the FGF-23 polypeptide. Additional 5 'RACE experiments are performed using the Advantage-2 PCR (Clontech) kit, a Marathon ™ human heart cDNA library (Clontech), and the primers 5'- CTGATGGGGTGCGCCATCCAC-A-3 '(SEQUENCE OF 10 ID NO: 45) and API (Clontech). Reactions are performed at 94 ° C for 30 seconds for one cycle and 94 ° C for 5 seconds and 68 ° C for 4 minutes for 30 cycles. Hosted PCR is performed using a portion of the 5 'RACE amplification product (diluted 1/100), and primers 5'- 15 C-A-T-T-C-T-T-G-T-G-G-A-T-C-T-G-C-A-G-G-T-G-G-T-3' (SEQUENCE OF IDENTIFICATION NO: 47) and AP2 (Clontech). The hosted PCR reactions are performed at 94 ° C for 30 seconds for one cycle and at 94 ° C for 30 seconds, 68 ° C for 4 minutes for 30 cycles. The products of Amplification is analyzed by agarose gel electrophoresis and its most prominent PCR product (350 bp) is isolated and subcloned using the TopoTA 4.0 cloning kit. Sequence analyzes of the isolated clones indicate that the 5 'PCR product extends in sequence 25 known for approximately 143 bp. -feí ^ H ^^ feÍ ^ lttMffitfMti rAiif '? ifr ^^ fr ^' ** 8"^ ^ ^ '^^ i? i ^ £ M ^^' ^^^^ fej ^^ tea? * ^ ** ™ '- - * * * - ** «i * iag ^ tf» * »* i ^^ - To isolate cDNA sequences corresponding to the 3' end of the cDNA sequence for the FGF-23 polypeptide, 3 'RACE using the Smart RACE cDNA amplification kit (Clontech), the human fetal mesentery cDNA library randomly primed in pSPORTl, and the 5' primers -CGGCCTCCTGTTCACAGGAGC-TC-3 '(SEQUENCE OF IDENTIFICATION NO: 48) and 5 '-CGGGCCTCTTCGCTATTACGC-3' (IDENTIFICATION SEQUENCE NO: 49) Reactions are carried out at 94 ° C for 1 minute for one cycle, 94 ° C for 5 seconds, 68 ° C for 10 seconds and 72 ° C for 3 minutes for 35 cycles, and 72 ° C for 7 minutes for one cycle The hosted PCR is performed using a portion of the 5 'amplification product RACE (diluted 1/100) and the primers 5' -GCGCCGAGGACAACAGCCCGA-3 '(SE ID NO: 50) and 5 '-T-G-G-C-G-A-A-A-G-G-G-G-G-A-T-G-T-G-C-T-G-3' (SEQUENCE OF IDENTIFICATION NO: 51). The hosted PCR reactions are performed at 94 ° C for 5 minutes per one cycle; 94 ° C for 15 seconds, 68 ° C for 15 seconds and 72 ° C for 3 minutes for 30 cycles; and 72 ° C for 7 minutes for one cycle. The amplification products are analyzed by agarose gel electrophoresis and a prominent 650 bp PCR product is isolated and subcloned using the TopoTA 4.0 cloning kit. The sequencing analysis of the isolated clones indicated in you. i ^^^^^ * ^^^^^^^^ £ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^; -AceeJJC.J fe - - the 3 'PCR product extends to the known sequence by 433 bp, including the poly-A region. A contiguous sequence is generated that appears to contain the full-length open reading frame for the FGF-23 gene using the sequence derived from the initial PCR amplification and the 5 'and 3' RACE amplifications. Sequence analysis of this consensus sequence indicates that the gene for FGF-23 comprises an open reading frame of 753 bp which codes for a protein of 251 amino acids (Figure 1A-1B). Sequence analysis also shows that the FGF-23 polypeptide shares homology with the fibroblast growth factor (FGF) family. Figures 2A-2G illustrate the amino acid sequence alignment of human FGF-1 (FGF-1 hu; SEQUENCE OF IDENTIFICATION NO: 4); Human FGF-2 (FGF-2 hu; ID SEQUENCE NO: 5), human FGF-3 (FGF-3 hu; ID SEQUENCE NO: 6), human FGF-4 (FGF-4 hu; SEQUENCE OF IDENTIFICATION NO : 7); Human FGF-5 (FGF-5 hu; ID SEQUENCE NO: 8), human FGF-6 (FGF-6 hu; ID SEQUENCE NO: 9), human FGF-7 (FGF-7 hu; SEQUENCE OF IDENTIFICATION NO : 10), human FGF-8 (FGF-8 hu; IDENTIFICATION SEQUENCE NO: 11), human FGF-9 (FGF-9 hu; IDENTIFICATION SEQUENCE NO: 12), human FGF-10 (FGF-10 hu; IDENTIFICATION SEQUENCE NO: 13), human FGF-11 (FGF-11 hu; IDENTIFICATION SEQUENCE Ji i ^ fcft? AJfi_i4ftfrrtiif'fralrttf ^ * - ^^ NO: 14), human FGF-12 (hu FGF-12; SEQUENCE OF IDENTIFICATION NO: 15), human FGF-13 (FGF-13 hu; SEQUENCE OF IDENTIFICATION NO : 16), human FGF-14 (FGF-14 hu; IDENTIFICATION SEQUENCE NO: 17), human FGF-16 (FGF-16 hu; IDENTIFICATION SEQUENCE NO: 18), human FGF-17 (FGF-17 hu; IDENTIFICATION SEQUENCE NO: 19), human FGF-18 (FGF-18 hu; IDENTIFICATION SEQUENCE NO: 20), human FGF-19 (FGF-19 hu; IDENTIFICATION SEQUENCE NO: 21), human FGF-23 (FGF -23 hu; IDENTIFICATION SEQUENCE NO: 22), murine FGF-1 (FGF-1 mu ID SEQUENCE NO: 23), Murine FGF-2 (FGF-2 mu ID SEQUENCE NO: 24), Murine FGF-3 (FGF-3 mu ID SEQUENCE NO: 25), Murine FGF-4 (FGF-4 mu SEQUENCE OF IDENTIFICATION NO: 26), Murine FGF-5 (FGF-5 mu SEQUENCE OF IDENTIFICATION NO: 27), FGF-6 murine (FGF-6 mu SEQUENCE OF IDENTIFICATION NO: 28), Murine FGF-7 (FGF-7 mu ID SEQUENCE NO: 29), Murine FGF-8 (FGF-8 mu ID SEQUENCE NO: 30), Murine FGF-9 (FGF-9 mu ID SEQUENCE NO: 31), murine FGF-10 (FGF-10 mu; SEQUENCE OF IDENTIFICATION NO: 32), murine FGF-11 (FGF-11 mu; SEQUENCE OF IDENTIFICATION NO: 33), murine FGF-12 (FGF-12 mu; SEQUENCE OF IDENTIFICATION NO: 34), murine FGF-13 (FGF-13 mu; SEQUENCE OF IDENTIFICATION NO: 35), murine FGF-14 (FGF-14 mu; SEQUENCE ID NO: 36), murine FGF-15 (FGF-15 mu; SEQUENCE OF IDENTIFICATION NO: 37), rat FGF-16 (Rat FGF-16; IDENTIFICATION SEQUENCE NO: 38), murine FGF-17 (FGF-17 mu; SEQUENCE OF IDENTIFICATION NO: 39). For the amino acid sequence analysis shown in Figures 2A-2G, the gene for FGF-23 appears to be closely related to murine FGF-15 and human FGF-19. The regionally restricted pattern of FGF-15 in the developing nervous system suggests that FGF-15 may play an important role in regulating cell division and pattern formation within specific regions of the embryonic brain, spinal cord and sensory organs (McWhirter et al., 1997, Development 124: 3221-32). Accordingly, the nucleic acid molecules for FGF-23, the polypeptides and agonists and antagonists thereof may be useful for the diagnosis or treatment of diseases involving the nervous system in development. Human FGF-19, which is expressed in fetal cartilage, skin and retina, gallbladder and adult and a line of colon adenocarcinoma cells, maps to a region of chromosome 11 that is associated with an osteoporosis-pseudoglioma syndrome. skeletal and retinal defects (Xie et al., 1999, Cytokine 11: 729-35). Accordingly, the FGF-23 nucleic acid molecules, the polypeptides and agonists and antagonists thereof may be useful for the diagnosis or treatment of diseases involving the skeletal system or the retina.

»You < á & < It is proved that the gene for FGF- 23 is more closely related to human FGF-21 (Yamashita et al., 2000, Biochem Biophys Res. Commun. 277: 494-98), a gene which is more abundantly expressed in the liver and at lower levels in the liver. thymus (Nishimura et al., 2000, Biochim Biophys Acta 1492: 203-06) Consequently, the nucleic acid molecules for FGF-23, the polypeptides and agonists and antagonists thereof can be useful for diagnosis or treatment of diseases that involve the liver or thymus.

Expression of mRNA for FGF-23 The expression of FGF-23 is analyzed by RT-PCR. A total RNA is prepared from several human fetal tissues using standard techniques. Mixtures of template and primer are prepared using 2 μg of total RNA and 50 ng of random primer (Gibco-BRL) in a volume of 12 μl. The mixtures are heated at 70 ° C for 10 minutes and then cooled in ice. Reverse transcription is performed by adding 4 μl of the 5X first chain buffer (Gibco-BRL), 2 μl of 0.1 M DTT and 1 μl of 10 mM dNTP to the template-primer mixture, the reaction mixture is heated to 37 ° C. ° C for 2 minutes, add 1 μl of Superscript II RT (Gibco-BRL) and then incubate the reaction mixture at 37 ° C for 1 hour. Differences in RNA concentration and cDNA conversion efficiency are normalized by performing PCR amplifications on each cDNA sample using specific primers for glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a gene expected to be expressed approximately at same level in all the tissues that are going to be examined. Control PCR amplifications are performed using the 5 'amplimers -TCCACCACCCTGTTGCTGTAG-3' (SEQUENCE OF IDENTIFICATION NO: 52) and 5'-GACCA-CA-GTCCATGCCATCACT-3 '(SEQUENCE OF IDENTIFICATION NO: 53) and PCR spheres Ready- To-Go (Amersham Pharmacia Biotech, Piscataway, NJ). The reactions are carried out at 95 ° C for 1 minute for one cycle; 92 ° C for 30 seconds, 55 ° C for 45 seconds and 72 ° C for 1 minute for 25 cycles; and 72 ° C for 5 minutes per one cycle. The control reaction products are analyzed on 2% agarose gels, the relative intensities of the control products are determined and the concentration of the cDNA samples is adjusted so that the cDNA samples generate G3PDH bands of equal intensity. The expression analysis of FGF-23 is carried out using standardized cDNA samples in terms of concentration. The expression of FGF-23 in tá ^. ^ .. * act¿ * ^ * ^ lc - «^ l tfci, .j jat.j.i..j nj-ra ai'jt '; PCR amplifications containing the 5'-C-T-A-T-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-T-G-3 'amplimers (SEQUENCE OF ID NO: 54) and 5 '-C-G-C-C-C-C-T-G-A-C-C-C-C-C-C-T-A-A-T-G-3' (SEQUENCE OF IDENTIFICATION NO: 55) and spheres 5 PCR Ready-To-Go. The reactions are carried out at 95 ° C for 5 minutes per one cycle; 95 ° C for 30 seconds, 68 ° C for 30 seconds and 72 ° C for 1 minute for 30 cycles; and 72 ° C for 7 minutes for one cycle. The PCR products are analyzed on 2% agarose gels and determined 10 the relative intensities of the PCR products (using the purest PCR product as a point of comparison). The results of this analysis are shown in Table III. 15 Table III Relative expression of FGF-23 twenty 25 i ni ii 'ífWiiMiÉiliiiÉÉiiiií ii tj? i ^ íi? f- ^ k¡ ^? i ^' '*' -'-''-- v ^^ - --- * - * »-« * * • ** ^ "- • *** • < * • * ¿** * - * - Tissue Relative expression level Spinal cord +++ Bladder +++ Adrenal + Bone +/- Placenta ++ Intestine ++ ++ Mesentery ++ Lung ++ Thymus +/- Pancreas + Umbilical cord blood +++ Uterus +/- Heart ++ Testicles +++ Eye - The expression of mRNA for FGF-23 in Northern blots is analyzed. Human tissues are screened by Northern blot (Clontech) with a suitable restriction fragment isolated from a cDNA clone for the human FGF-23 polypeptide. The probe is marked with 32P-dCTP using standard techniques. Northern blots are prehybridized for 2 hours at 42 ° C in hybridization solution (5X SSC, 50% deionized formamide, 5X Denhardt's solution, SDS 0. 5% and 100 mg / ml of denatured salmon sperm DNA), and then hybridized at 42 ° C overnight in fresh hybridization solution containing 5 ng / ml of the marked probe. After hybridization, the filters are washed twice for 10 min at room temperature in 2X SSC and 0.1% SDS and then twice for 30 minutes at 65 ° C in 0.1 X SSC and 0.1% SDS. The spots are then exposed to autoradiography. Expression of mRNA for FGF-23 in normal adult mouse tissue and in transgenic mouse tissue of three weeks of age with high expression level and without expression (see Example 5) is localized by in situ hybridization. The tissues of normal and adult embryonic mice are fixed by immersion, embedded by paraffin and cut at 5 μm. Hybridization is done in your own using standard techniques. The cut tissues are hybridized overnight at 60 ° C in hybridization solution containing an antisense riboprobe labeled with 33P complementary to the human FGF-23 gene. The riboprobe is obtained by transcription in vi tro of a clone containing cDNA sequences for human FGF-23 using standard techniques. After hybridization, the sections are treated with RNAseA to digest the unhybridized probe and washed in SSC 0. IX at 55 ° C for 30 minutes. The slices are then immersed in NTB-2 emulsion (Kodak, Rochester, NY), exposed for 3 weeks at 4 ° C, revealed and subjected to contrast staining with hematoxylin and eosin. The morphology of the tissue and the hybridization signal are analyzed Ici éeáto. simultaneously using dark and conventional field illumination for brain samples, gastrointestinal system (parotid, submandibular and sublingual gland, esophagus, stomach, duodenum, jejunum, ileum, proximal and distal colon 5, liver and pancreas), cardiopulmonary system (heart, lung, trachea and blood vessels); hematolymphoid system (lymph nodes, spleen, thymus and bone marrow), urinary system (kidney and bladder), endocrine system (adrenal gland, gland) 10 thyroid and pituitary), reproductive system (testicle, prostate and gonad, ovary, uterus and oviduct, placenta); and skeletal muscle system (bone, skeletal muscle, skin and adipose tissue). The embryos of normal mice in E18 and E14.5 with placenta were also analyzed by 15 hybridization in si tu. A low to moderate and diffuse expression of FGF-23 is detected in the tissues of normal adult mice. A RNase protection assay is carried out in a limited sample of mouse tissues in which 20 detects a diffuse signal by in-situ hybridization to determine if this signal is due to non-specific binding. The results of the RNAse protection assay indicate that FGF-23 is expressed only weakly in heart and brain and is not detected in liver, thyroid / parathyroid and stomach. These 25 results suggest that the diffuse signal detected by Hybridization in if your is most likely due to non-specific binding, especially in epithelial cell types. Since the RNase protection assay indicates that FGF-23 is weakly expressed in heart and brain, the low signal that is observed in these tissues by hybridization in if your can be real. In the brain, a low expression is usually observed in most of the neurons that include areas of the thalamus, caudate putamen, septum and hypothalamus. However, moderate marking is observed in the 10 granular hippocampus and pyramidal cells, the neocortex (figure 3) and the piriform cortex. A low expression is found in the ependyma and the choroid plexus. Both the cardiac and skeletal muscles also show low levels of FGF-23 expression. In cardiac muscle, it is 15 evidenced a diffuse low signal in the left and right ventricles with a slightly larger signal in the atrium (figure 3). A low diffuse signal is also found in skeletal muscle. No expression is found in embryos of 20 mouse E14.5 or E18. None of the transgenic 3-week-old transgenic mice showed diffuse non-specific signal that was detectable in normal adult mice. In transgenic mice that do not express, a strong expression of FGF-23 in cells is detected 25 dispersed in the lymph nodes (Figure 4), bone marrow thymic (figure 4) and bone (figure 4). Although positive identification of the labeled cells is not possible, expression in bone appears to be in mesenchymal cells spread in the lagoon part and trabecula in the bones of the hind limbs, vertebrae, ribs and nasal cavities. In transgenic mice with a high level of expression, the distribution of the labeled cells is more widespread. In the ligand, a strong expression of FGF-23 is observed in the hepatocytes (figure 5). Strong labeling is also detected in scattered cells of the thymic marrow (Figure 5), as is observed in non-expressing transgenic mice. However, in transgenic mice with a high level of expression, well-labeled cells are also found in the red pulp of the spleen (Figure 5), the smooth muscle adjacent to the prostate gland (Figure 6) and the striated muscle of the mandible. (figure 6). Strong expression is also detected in some metakaryocytes identified in bone marrow and in many chondrocytes and in the back joints and vertebrae (Figure 6).

Example 3: Production of FGF-23 polypeptides A. Expression of FGF-23 polypeptides in bacteria rf.t ^ i ii PCR is used to amplify template DNA sequences encoding a FGF-23 polypeptide using primers corresponding to the 5 'and 3' ends of the sequence. Amplified DNA products can be modified to contain restriction enzyme sites that allow insertion into expression vectors. The PCR products are gel purified and inserted into expression vectors using standard recombinant DNA methodology. An exemplary vector, such as pAMG21 10 (ATCC No. 98113) containing the lux promoter and a gene coding for kanamycin resistance is digested with Bam Hl and Nde I for directional cloning of the inserted DNA. The ligated mixture is transformed into an E. coli host cell by electroporation and the transformants are selected by 15 resistance to kanamycin. The plasmid DNA of the selected colonies is isolated and subjected to DNA sequencing to confirm the presence of the insert. The transformed host cells are incubated in 2xYT medium containing 30 μg / ml kanamycin at 30 ° C, before 20 of his induction. The expression of the gene is induced by the addition of N- (3-oxohexanoyl) -di-homoserine lactone to a final concentration of 30 ng / ml followed by incubation either at 30 ° C or 37 ° C for six hours. The expression of the FGF-23 polypeptide is evaluated by centrifugation of the culture, 25 resuspension and lysis of bacterial sediments and the jMJfcj aaa Í- - • tf ^ -. i ^ é, A Ía Áá?, ^^ ?? Analysis of the host cell proteins by SDS-polyacrylamide gel electrophoresis. Inclusion bodies containing the FGF-23 polypeptide are purified as follows. Bacterial cells are pelleted by centrifugation and resuspended in water. The cell suspension is lysed by sonication and centrifuged at 195,000 xg for 5 to 10 minutes. The supernatant is discarded and the pellet is washed and transferred to a homogenizer. The pellet is homogenized in 5 ml of a Percoll solution (75% Percoll liquid and 0.15 M NaCl) until it is uniformly suspended and then diluted and centrifuged at 21,600 xg for 30 minutes. The gradient fractions containing the inclusion bodies are recovered and accumulated. Isolated inclusion bodies are analyzed by SDS-PAGE. A single band is cut from the gel on an SDS-polyacrylamide gel corresponding to the FGF-23 polypeptide produced in E. coli, and the N-terminal amino acid sequence is determined essentially as described by Matsudaira et al. , 1987, J. Biol. Chem. 262: 10-35.

B. Expression of the FGF-23 polypeptide in mammalian cells PCR is used to amplify template DNA sequences that encode a FGF-23 polypeptide using primers corresponding to the 5 'and 3' ends of the sequence. The amplified DNA products can be modified to contain restriction enzyme sites that allow insertion into expression vectors. The PCR products are gel purified and inserted into expression vectors using standard recombinant DNA methodology. An exemplary expression vector, pCEP4 (Invitrogen, Carisbad, CA), which contains an Epstein-Barr virus origin of replication can be used for the expression of FGF-23 polypeptides in 293-EBNA-1 cells. The amplified and gel-purified PCR products are ligated into the pCEP4 vector and introduced into 293-EBNA cells by lipofection. The transfected cells are selected in 100 μg / ml hygromycin and the resulting cultures resistant to drugs are grown to confluence. The cells are then cultured in serum-free medium for 72 hours. The conditioned medium is removed and expression of the FGF-23 polypeptide is analyzed by SDS-PAGE. The expression of the polypeptide can be detected FGF-23 by staining with silver. Alternatively, a FGF-23 polypeptide is produced as a fusion protein with an epitope tag such as an IgG constant domain or a FLAG epitope, which can be detected by Western blot analysis using antibodies for the peptide tag. The FGF-23 polypeptides can be cut from an SDS-polyacrylamide gel, or the FGF-23 fusion proteins are purified by affinity chromatography to the epitope tag and subjected to amino acid sequence analysis.

N terminal as described herein.

C. Purification of FGF-23 polypeptide from mammalian cells Mounts (recombinant plasmids) for expression of the FGF-23 polypeptide are introduced into 293 EBNA or CHO cells either by lipofection or by calcium phosphate protocol. To carry out functional studies on the FGF-23 polypeptides that are produced, large quantities of conditioned medium are generated from an accumulated clone of 293 EBNA selected by hygromycin. The cells are grown in 500 cm triple Nunc flasks, up to 80% confluence before switching to serum free medium one week before harvesting the medium. The conditioned medium is harvested and frozen at -20 ° C until the protein is going to be purified. The conditioned medium is purified by affinity chromatography as described in the following.

The medium is reheated and then passed through a 0.2 μm filter. A protein G column is equilibrated with PBS at pH 7.0, and then loaded with the filtered medium. The column is washed with PBS until the absorbance at A280 reaches an initial value. The FGF-23 polypeptide is eluted from the column with 0.1 M glycine hydrochloride at pH 2.7 and immediately neutralized with 1 M Tris-HCl to pH 8.5. Fractions containing the FGF-23 polypeptide are pooled, dialysed in PBS and stored at -70 ° C. Separation of factor Xa from the fusion polypeptide of human FGF-23 polypeptide-Fe, the purified protein by affinity chromatography is dialyzed in 50 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl 2 at pH 8.0. The restriction protease factor Xa is added to the dialyzed protein at 1/100 (w / w) and the sample is digested overnight at room temperature.

Example 4: Production of antibodies against FGF-23 polypeptide Antibodies against the FGF-23 polypeptides can be obtained by immunization with purified protein or with FGF-23 peptides produced by biological or chemical synthesis. Suitable methods for generating antibodies include those described in Hudson and Bay, - Practical Immunology (2nd ed., Blackwell Scientific Publications). In a procedure for the preparation of antibodies, animals (typically mice or rabbits) are injected with an FGF-23 antigen (such as a FGF-23 polypeptide), and those with sufficient serum titre levels, as determined by ELISA, are selected for hybridoma production. The spleens of the immunized animals are harvested and prepared as single cell suspensions from which the splenocytes are recovered. Splenocytes are fused to mouse myeloma cells (such as Sp2 / 0-Agl4 cells), and incubated first in DMEM with 200 U / ml penicillin, 200 μg / ml streptomycin sulfate and 4 mM glutamine, and then they are incubated in a HAT selection medium (hypoxanthine, aminopterin and thymidine). After selection, tissue culture supernatants are harvested from each fusion well and tested for antibody production against FGF-23 by ELISA. Alternative methods can also be used to obtain antibodies against FGF-23 such as immunization of transgenic mice harboring human Ig loci for human antibody production, and screening of synthetic antibody libraries, such as those generated by mutagenesis of a variable domain. from antibody.

Example 5: Expression of FGF-23 polypeptide in transgenic mice To determine the biological activity of the FGF-23 polypeptide, an assembly (recombinant plasmid) encoding the FGF-23 polypeptide is prepared under the control of the ApoE promoter (TH00-026). It is expected that expression of the gene for FGF-23 will cause pathological changes in the transgenic mouse that may be indicative of the function of the FGF-23 polypeptide. A distinctive phenotype is produced in 3-week-old BDF1 mice after transfer of the THO0-026 mount where their littermates show unusually high numbers of smaller animals than their species. Although not all of the expressing mice are smaller and some of their littermates that do not express the gene are smaller in size, the proportion of smaller sized animals is higher among the mice expressing the gene. All of the smaller animals include many small animals that do not express the gene, are taken before weaning for examination. The skulls of all the mice that express the gene shorten and are more rounded and the lower jaw develops properly. As a result, all mice expressing the gene have protruding lower teeth. This condition is evident in an external examination and a radiographic evaluation. In addition, it has been found that the two mice with the highest expression have low concentrations of serum phosphorus and serum calcium. However, no other signs of rickets are observed - such as inadequate mineralization, overgrowth of the cartilage of the epiphysis, disorganized organization of the cartilage and overgrowth of the capillaries. { Pathologic Basis of Disease (Cotran ed., 1994)) -. Bone morphology in the smallest animals that express the gene is not different from that of smaller animals that do not express it and both types of small animals differ from their littermates that are not smaller animals. Partial hepatectomy was performed in 22 DNA positive mice and 4 DNA negative mice. All the mice subjected to hepatectomy were subjected to blood extraction and serum calcium, phosphorus and alkaline phosphatase determinations were made. Tests were also performed on the animals to determine the outstanding lower tooth phenotype. Groups of mice that were evaluated include: controls or controls, animals that express the phenotype macroscopically, animals with high non-phenotypic expression level and animals that they express the gene in a moderate way. All phenotypic mice are found to be animals that express the gene strongly or very strongly. Serum calcium levels are decreased to some extent in phenotypic mice compared to the other groups, but this difference is not statistically significant. However, it was found that serum phosphorus concentrations are significantly decreased in animals that express the phenotypic gene and in animals with high non-phenotypic gene expression compared to animals that moderately express the gene and controls . Serum alkaline phosphatase (ALP) is significantly elevated in phenotypic groups versus controls and animals that exhibit moderate expression (see Table IV). The variability in the phenotype of the animals expressing the gene may be due to genetic variation in the BDF1 mice, an exogamic line that is a cross between strains of C57 / B6 and DBA mice.

Table IV Calcium, phosphorus and serum ALP in THOO-026 transgenic mice Although the present invention has been described in terms of the preferred embodiments, it is understood that modifications and modifications will occur to those skilled in the art. Therefore, the claims are intended to cover all such variations 15 equivalents that are within the scope of the invention as claimed. It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the 20 present description of the invention.

LIST OF SEQUENCES < 110 > Luethy, Roland Yang, Robert Suggs, Sidney V. Saroei, Ildi or < 120 > Molecules of fibroblast growth factor 23 and its use < 130 > 01 - 004 < U40 > 60/182, 442 < 141 > 2000 -02 - 15 «= 160:» 54 < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 753 < 212 > DNA < 213 > Homo sapiens «220 > < 221 > CDS < 222 > (1) .. (753) < 220 > < 221 > sig peptide < 222 > (1) . - (72) < 400 > 1 atg ttg ggg gcc cgc etc agg etc tgg gtc tgt gcc ttg tgc age gtc 48 Met Leu Gly Ala Arg Leu Arg Leu Trp Val Cyß Ala Leu Cys Ser Val 1 5 10 15 tgc age atg age gtc etc aga gcc tat ecc aat gcc tec cca ctg etc 96 Cys Ser Met Ser Val Leu Arg Wing Tyr Pro Asn Wing Pro Pro Leu Leu 20 25 30 ggc tec age tgg ggt ggc ctg ate drops ctg tac here gcc ac gcc agg 144 Gly Ser Ser Trp Gly Gly Leu lie Ris Leu Tyr Thr Wing Thr Wing Arg 35 40 45 aac age tac falls ctg cag ate drops aag aat ggc cat gtg gat ggc gca 192 Asn Ser Tyr His Leu Gln He His Lys Asn Gly My Val Asp Gly Wing 50 55 60 ecc cat cag acc ate tac agt gcc ctg atg ate aga tea gag gat gct 240 Pro His G n Thr He Tyr Be Ala Leu Met He Arg Ser Glu Aßp Ala 65 70 75 80 ggc ttt gtg gtg att ac ggt gtg atg age aga aga tac etc tgc atg 288 Gly Val val He Thr Gly Val Met Ser Arg Arg Tyr Leu Cys Met 85 90 95 gat ttc aga ggc aac att ttt gga tea falls tat tcc gac ccg gag aac 336 Asp Phe Arg Gly Aen He Phe Gly Ser His Tyr Phe Asp Pro Glu Asn 100 105 110 tgc agg ttc caá falls cag acg ctg gaa aac ggg tac gac gtc tac falls 384 lllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll --- > - ^^ a. ... .. . .....?.? i ..

Cys Arg Phe Gln His Gln Thr Leu Glu Asn Gly Tyr Asp Val Tyr His 115 120 125 tet ect cag tat falls ttc ctg gtc agt ctg ggc cgg gcg aag aga gcc 432 Ser Pro Gln Tyr His Phe Leu Val Ser Leu Gly Arg Wing Lye Arg Wing 130 135 140 ttc ctg cca ggc atg aac cca CCC ccg tac tec cag ttc ctg tec cgg 4T0 Phe Leu Pro Gly Met Asn Pro Pro Pro Tyr Ser Gln Phe Leu Ser Arg 145 150 155 160 agg aac gag ate CCC cta att falls tcc aac acc CCC ata cca cgg cgg 528 Arg Asn Glu He Pro Leu He His Phe Asn Thr Pro He Pro Arg Arg 165 170 175 falls acc cgg age gcc gag gac gac teg gag cgg gac ecc ctg aac gtg 576 Hiß Thr Arg Ser Wing Glu Asp Asp Ser Glu Arg Asp Pro Leu Asn Val 180 185 190 ctg aag CCC cgg gcc cgg atg acc ccg gcc ccg gec tec tgt tea cag 624 Leu Lys Pro Arg Ala Arg Met Thr Pro Wing Pro Wing Ser Cye Ser Gln 195 200 205 gag etc ccg age gcc gac gac aac age ccg atg gcc agt gac cca tta 672 Glu Leu Pro Ser Wing Glu Asp Asn Ser Pro Met Wing Ser Asp Pro Leu 210 215 220 ggg gtg gtc a99 ggc ggt cga gtg aac acg drops gct 999 gga acg ggc 720 Gly Val Val Arg Gly Gly Arg Val Asn Thr His Wing Gly Gly Thr Gly 225 230 235 240 ccg gaa ggc tgc cgc ecc tcc gcc aag ttc ate 753 Pro Glu Gly Cys Arg Pro Phe Ala Lys Phe He 245 250 < 210 > 2 < 211 > 251 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Met Leu Gly Ala Arg Leu Arg Leu Trp Val Cys Ala Leu Cys ser Val 1 5 10 15 Cys Ser Met Ser Val Leu Tyr Pro Asn Ala be Pro Leu Leu 20 25 30 Gly Ser Ser Trp Gly Gly Leu He His Leu Tyr Thr Ala Thr Ala Arg 35 40 45 Asn Ser Tyr His Leu Gln He His Lys Asn Gly His Val Asp Gly Wing 50 55 60 Pro His Gln Thr He Tyr Ser Ala Leu Met He Arg Ser Glu Asp Ala 65 70 75 80 Gly Phe Val Val He Thr Gly Val Met Ser Arg Arg Tyr Leu Cys Met 85 90 95 Asp Phe Arg Gly Asn He Phe Gly Ser His Tyr Phe Asp Pro Glu Asn j-t-n-f? trli ^^^ ** 8 * ^ * J? uA Í? Lit * dt? Tt¿ .Í.? . 100 105 110 Cye Arg Phe Gln His Gln Thr Leu Glu Asn Gly Tyr Asp Val Tyr His 115 120 125 Ser Pro Gln Tyr His Phe Leu Val Ser Leu Gly Arg Ala Lys Arg Ala 130 135 140 Phe Leu Pro Gly Met Asn Pro Pro Pro Tyr Ser Gln Phe Leu Ser Arg 145 150 155 160 Arg Asn Glu He Pro Leu He His Phe Asn Thr Pro He Pro Arg Arg 165 170 175 His Thr Arg Ser Wing Glu Asp Asp Ser Glu Arg Asp Pro Leu Aan Val 180 185 190 Leu Lys Pro Arg Wing Arg Met Thr Pro Wing Pro Wing Ser Cye Ser Gln 195 200 205 Glu Leu Pro Ser Wing Glu Asp Asn Ser Pro Met Wing Ser Asp Pro Leu 210 215 220 Gly val Val Arg Gly Gly Arg Val Asn Thr His Wing Gly Gly Thr Gly 225 230 235 240 Pro Glu Gly Cys Arg Pro Phe Ala Lys Phe He 245 250 < 210 > 3 < 211 > 227 < 212 > PRT c213 > Homo sapiens < 400 > 3 Tyr Pro Asn Ala Ser Pro Leu Leu Gly Ser Ser Trp Gly Gly Leu He 1 5 10 15 His Leu Tyr Thr Ala Thr Ala Arg Asn Ser Tyr His Leu Gln He His 20 25 30 Lys Asn Gly His Val Asp Gly Ala Pro His Gln Thr He Tyr Ser Ala 35 40 45 Leu Met He Arg Ser Glu Asp Ala Gly Phe Val Val He Thr Gly Val 50 55 60 Met Ser Arg Arg Tyr Leu Cys Met Asp Phe Arg Gly Asn He Phe Gly 65 70 75 80 Ser His Tyr Phe Asp Pro Glu Asn Cys Arg Phe Gln Hie Gln Thr Leu 85 90 95 Glu Aen Gly Tyr Asp Val Tyr His Ser Pro Gln Tyr His Phe Leu Val 100 105 110 Ser Leu Gly Arg Ala Lys Arg Ala Phe Leu Pro Gly Met Pro Pro 115 120 125 Pro Tyr Ser Gln Phe Leu Ser Arg Arg Asn Glu He Pro Leu He His tf ^ fc 130 135 140 Phe Asn Thr Pro He Pro Arg Arg His Thr Arg Ser Wing Glu Aep Asp 145 150 15S 160 Ser Glu Arg Asp Pro Leu Asn Val Leu Lys Prc Arg Wing Arg Met Thr 165 170 175 Pro Wing Pro Wing Cye Ser Gln Glu Leu Pro Ser Wing Glu Asp Asn 1B0 185 190 Ser Pro Met Wing Ser Asp Pro Leu Gly Val Val Arg Gly Arg Val 195 200 205 Asn Thr His Wing Gly Gly Thr Gly Pro Glu Gly Cys Arg Pro Phe Wing 210 215 220 Lys Phe He 225 < 210 > 4 < 211 > 155 c212 > PRT c213 > Homo sapiens < 400 > 4 Met Wing Glu Gly Glu He Thr Thr Phe Thr Wing Leu Thr Glu Lye Phe 1 5 10 15 Aen Leu Pro Pro Gly Asn Tyr Lye Lys Pro Lys Leu Leu Tyr Cye Ser 20 25 30 Asn Gly Gly His Phe Leu Arg He Leu Pro Asp Gly Thr Val Asp Gly 35 40 45 Thr Arg Asp Arg As Asp Glp His He Gln Leu Gln Leu Ser Ala slu 50 55 60 Ser Val Gly Glu Val Tyr He Lys Ser Thr Glu Thr Gly Gln Tyr Leu 65 70 75 80 Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu 85 90 95 Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105 110 He Ser Lys Lys His Wing Glu Lys Asn Trp Phe Val Gly Leu Lys Lys 115 120 125 Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Wing 130 135 140 He Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155 < 210 > 5 < 211 > 155 < 12 > PRT < 213 > Homo sapiens < 400 > 5 Met Ala Ala Gly Be He Thr Thr Leu Pro Ala Leu Pro Glu Aep Gly 1 5 10 15 Gly Ser Gly Wing Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30 Tyr Cye Lys Asn Gly Gly Phe Phe Leu Arg He His Pro Asp Gly Arg 35 40 45 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gln Leu 50 55 60 Gln Wing Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cye Wing Asn 65 70 75 80 Arg Tyr Leu Wing Met Lyß Glu Asp Gly Arg Leu Leu Wing Ser Lye Cyß 85 90 95 Val Thr Aap Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Aen Asn Tyr 100 105 110 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125 Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys 130 135 140 Wing He Leu Phe Leu Pro Met Ser Wing Lys Ser 145 150 155 < 210 > 6 < 211 > 239 < 212 > PRT c2l3 > Ho or sapiens e400 > 6 Met Gly Leu He Trp Leu Leu Leu Leu Ser Leu Leu Glu Pro Gly Trp 1 5 10 15 Pro Wing Wing Gly Pro Gly Wing Arg Leu Arg Arg Asp Wing Gly Gly Arg 20 25 30 Gly Gly Val Tyr Glu His Leu Gly Gly Wing Pro Arg Arg Arg Lys Leu 35 40 45 Tyr Cys Wing Thr Lys Tyr His Leu Gln Leu His Pro Ser Gly Arg Val 50 55 60 Asn Gly Ser Leu Glu Asn Ser Wing Tyr Ser He Leu Glu He Thr Wing 65 70 75 80 Val Glu Val Gly He Val Wing He Arg Gly Leu Phe Ser Gly Arg Tyr 85 90 95 Leu Ala Met Asn Lys Arg Gly Arg Leu Tyr Ala Ser Glu His Tyr be 100 105 110 Wing Glu Cys Glu Phe Val Glu Arg He Hxs Glu Leu Gly Tyr Asn Thr 115 120 125 Tyr Ala Ser Arg Leu Tyr Arg Thr Val Being Ser Tnr Pro Gly Wing Arg 130 '135 140 Arg Gln Pro Being Wing Glu Arg Leu Trp Tyr Val Ser Val Asn Gly Lys 145 150 155 160 Gly Arg Pro Arg Arg Gly Phe Lys Thr Arg Arg Thr Gln Lys Ser Ser 165 170 175 Leu Phe Leu Pro Arg Val Leu Asp His Arg Asp His Glu Met Val Arg 180 185 190 Gln Leu Gln Ser Gly Leu Pro Arg Pro Pro Gly Lys Gly Val Gln Pro 195 200 205 Arg Arg Arg Gln Lyn Gln Ser Pro Asp Asn Leu Glu Pro Ser His 210 215 220 Val Gln Wing Ser Arg Leu Gly Ser Gln Leu Glu Wing Ser Wing HIG 225 230 235 < 210 > 7 < 211 > 206 < 212 > PRT < 213 > Homo sapiens < 400 > 7 Met Ser Gly Pro Gly Thr Wing Wing Val Wing Leu Leu Pro Wing Val Leu 1 5 10 15 Leu Wing Leu Leu Wing Pro Trp Wing Gly Arg Gly Wing Wing Wing Pro 20 25 30 Thr Wing Pro Aen Gly Thr Leu Glu Wing Glu Leu Glu Arg Arg Trp Glu 35 40 45 Ser Leu Val Ala Leu Ser Leu Ala Arg Leu Pro Val Ala Ala Gln Pro 50 55 60 Lys Glu Ala Ala Val Gln Ser Gly Ala Gly Asp Tyr Leu Leu Gly He 65 70 75 80 Lys Arg Leu Arg Arg Leu Tyr Cys Asn Val Gly He Gly Phe His Leu 85 90 95 Gln Ala Leu Pro Asp Gly Arg He Gly Gly Ala His Wing Asp Thr Arg 100 105 110 Asp Ser Leu Leu Glu Leu Ser Pro Val Glu Arg Val Val Be He 115 120 125 Phe Gly Val Wing Ser Arg Phe Phe Val Wing Met Ser Ser Lys Gly Lye 130 135 140 Leu Tyr Gly Ser Pro Phe Phe Thr Asp Glu Cye Thr Phe Lys Glu He 145 150 155 160 Leu Leu Pro Asn Asn Tyr Asn Wing Tyr Glu Ser Tyr Lys Tyr Pro Gly 165 170 175 Met Phe lie Ala Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn Arg 180 185 190 Val Ser Pro Thr Met Lys Val Thr His Phe Leu Pro Arg Leu 195 200 205 < 210 > 8 < 211 > 268 c212 > PRT < 213 > Ho or sapiens < 400 > 8 Met Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe Ser His Leu He Leu 1 5 10 15 Be Wing Trp Wing His Gly Glu Lys Arg Leu Wing Pro Lys Gly Gln Pro 20 25 30 Gly Pro Wing Wing Thr Asp Arg Asn Pro He Gly Being Being Arg Gln 35 40 45 Being Being Being Wing Met Being Ser Being Wing Being Ser Ser Pro Ala 50 55 60 Wing Being Leu Gly Being Gln Gly Being Gly Leu Glu Gln Being Being Phe Gln 65 70 75 80 Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly 85 90 95 He Gly Phe Hiss Leu Gln He Tyr Pro Asp Gly Lys Val Asn Gly Ser 100 105 110 His Glu Wing Asn Met Leu Ser Val Leu Glu He Phe Wing Val Ser Gln 115 120 125 sly He Val sly He Arg sly Val Phe Ser Asn Lys Phe Leu Wing Met 130 135 140 Ser Lys Lys Gly Lys Leu His Wing Being Wing Lys Phe Thr Asp Asp Cys 145 150 155 160 Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Wing Ser 165 170 175 Wing He His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Wing Leu 180 185 190 Asn Lys Arg Gly Lys Wing Lys Arg Gly Cys Ser Pro Arg Val Lye Pro 195 200 205 Gln His He Ser Thr His Phe Leu Pro Arg Phe Lye Gln Ser Glu Gln 210 215 220 Pro Glu Leu Ser Phe Thr Val Thr Val Pro Glu Lys Lys Asn Pro Pro 22S 230 235 240 rti i *? i lfi flütir nn?? IMIÍ Ser Pro He Lys Ser Lys He Pro Leu Ser Wing Pro Arg Lys Asn Thr 245 250 255 Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 260 265 < 210 > 9 < 211 208 < 212 > PRT < 213 > Homo sapiens < 400 > 9 Met Ala Leu Gly Gln Lys Leu Phe He Thr Met Ser Arg Gly Ala Gly 1 5 10 15 Arg Leu Gln Gly Thr Leu Trp Wing Leu Val Phe Leu Gly He Leu Val 20 25 30 Gly Met Val Val Pro Ser Pro Wing Gly Thr Arg Wing Asn Asn Thr Leu 35 40 45 Leu Asp Ser Arg Gly Trp Gly Thr Leu Leu Ser Arg Ser Arg Ala Gly 50 55 60 Leu Wing Gly Glu He Wing Gly Val Asn Trp Glu Ser Gly Tyr Leu Val 65 70 75 80 Gly He Lys Arg Gln Arg Arg Leu Tyr Cys Asn Val Gly He Gly Phe 85 90 95 His Leu Gln Val Leu Pro Aep Gly Arg He Ser Gly Thr Hie Glu Glu 100 105 110 Asn Pro Tyr Ser Leu Leu Glu He Ser Thr Val Glu Arg Val Val 115 120 125 Ser Leu Phe Gly Val Arg Ser Ala Leu Phe Val Ala Met Aen Ser Lys 130 135 140 Gly Arg Leu Tyr Wing Thr Pro Ser Phe Gln slu Glu Cys Lye Phe Arg 145 150 155 160 Glu Thr Leu Leu Pro Asn Asn Tyr Asn Wing Tyr Glu Ser Asp Leu Tyr 165 170 175 Gln Gly Thr Tyr He Wing Leu Ser Lys Tyr Gly Arg Val Lys Arg sly 180 185 190 Ser Lys Val Ser Pro He Met Thr Val Thr His Phe Leu Pro Arg He 195 200 205 < 210 > 10 c211 > 194 < 212 > PRT < 213 > Homo sapiens < 400 > 10 Met His Lys Trp He Leu Thr Trp He Leu Pro Thr Leu Leu Tyr Arg 1 5 10 15 Be Cys Phe His He He Cys Leu Val Gly Thr He Ser Leu Cys Wing 20 25 30 Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Asn Cys Ser 35 40 45 Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp He 50 55 60 Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg He Asp 65 70 7S 80 Lys Arg Gly Lys Val Lyß sly Thr Gln Glu Met Lyß Asn Aßn Tyr Asn 85 90 95 He Met Glu He Arg Thr Val Wing Val Gly He Val Wing lie Lye Gly 100 105 110 Val Glu Ser Glu Phe Tyr Leu Wing Met Asn Lys Glu Gly Lyß Leu Tyr 115 120 125 Wing Lys Lys Glu Cys Aßn Glu Asp Cys Asn Phe Lyß Glu Leu He Leu 130 135 140 Glu Asn His Tyr Asn Thr Tyr Wing Ser Wing Lys Trp Thr His Asn Gly 145 150 155 160 Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly He Pro Val Arg Gly 165 170 175 Lys Lys Thr Lys Lys Glu Gln Lys Thr Wing His Phe Leu Pro Met Wing 180 185 190 He Thr < 210 > 11 < 211 > 233 < 212 > PRT < 13 > Homo sapiens < 400 > 11 Met Gly Ser Pro Arg Ser Ala Leu Ser Cys Leu Leu Leu His Leu Leu 1 5 10 15 Val Leu Cys Leu Gln Wing Gln Glu Gly Pro Gly Arg Gly Pro Wing Leu 20 25 30 Gly Arg Glu Leu Wing Being Leu Phe Arg Wing Gly Arg slu Pro Gln Gly 35 40 45 Val Ser Gln Gln His Val Arg Glu Gln Ser Leu Val Thr Asp Gln Leu 50 55 60 Ser Arg Arg Leu He Arg Thr Tyr Gln Leu Tyr Ser Arg Thr Ser Gly 65 70 75 80 Lys His Val Gln Val Leu Wing Asn Lys Arg He Asn Wing Met Wing Glu 85 90 95 Asp Gly Asp Fro Phe Ala Lys Leu He Val Glu Thr Asp Thr Phe Gly 100 105 110 Ser Arg Val Arg Val Arg Gly Wing Glu Thr Gly Leu Tyr He Cys Met 115 120 125 Asn Lys Lys Gly Lye Leu He Wing Lys Ser Asn Gly Lys Gly Lys Asp 130 135 140 Cys Val Phe Thr Glu He Val Leu Glu Asn Asn Tyr Thr Ala Leu Gln 145 150 155 160 Asn Ala Lys Tyr Glu sly Trp Tyr Met Wing Phe Thr Arg Lys Gly Arg 165 170 175 Pro Arg Lys Gly Ser Lys Thr Arg Gln His Gln Arg Glu Val Hie Phe 180 185 190 Met Lys Arg Leu Pro Arg Gly His His Thr Thr Glu Gln Ser Leu Arg 195 200 205 Phe Glu Phe Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu Arg Gly Ser 210 215 220 Gln Arg Thr Trp Wing Pro Glu Pro Arg 225 230 < 210 > 12 e211 > 208 < 212 > PRT < 213 > Homo sapiens < 400 > 12 Met Wing Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gln Asp Wing 1 5 10 15 val Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp Ser Pro Val Leu 20 25 30 Leu Ser Asp His Leu Gly Gln Ser Glu Ala Gly Gly Leu Pro Arg Gly 35 40 45 Pro Wing Val Thr Asp Leu Asp His Leu Lys Gly He Leu Arg Arg Arg 50 55 60 Gln Leu Tyr Cye Arg Thr Gly Phe His Leu Glu He Phe Pro Asn Gly 65 70 75 80 Thr lie Gln Gly Thr Arg Lys Aep His Being Arg Phe Gly He Leu Glu 85 90 95 Phe He Ser He Wing Val Gly Leu Val Ser He Arg Gly Val Asp Ser 100 105 110 Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu 115 120 125 Lys Leu Thr Gln Glu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn Trp 130 135 140 Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr Lys His Val Asp Thr Gly Arg 145 150 155 160 Arg Tyr Tyr Val Ala Leu Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr 165 170 175 Arg Thr Lys Arg His Gln Lyß Phe Thr His Phe Leu Pro Arg Pro Val 180 185 190 Aep Pro Asp Lys Val Pro Glu Leu Tyr Lye Aep He Leu Ser Gln Ser 195 195 205 < 210 > 13 < 211 > 208 < 212 > PRT < 213 > Homo sapiens < 400 > 13 Met Trp Lys Trp He Leu Thr His Cys Wing Ser Wing Phe Pro Hie Leu 1 5 10 15 Pro Gly Cys Cys Cys Cys Cye Phe Leu Leu Leu Phe Leu Val Ser Ser 20 25 30 val Pro Val Thr Cys Gln Ala Leu Gly sln Asp Met Val Ser Pro slu 35 40 45 Wing Thr Asn Being Ser Being Ser Phe Ser Ser Pro Ser Wing Gly 50 55 60 Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg 65 70 75 80 Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly 85 90 95 Lys Val Ser Gly Thr Lye Lyß Glu Asn Cye Pro Tyr Ser He Leu Glu 100 105 110 He Thr Ser Val Glu He Gly Val Val Ala Val Lys Ala He Aßn Ser 115 120 125 Asn Tyr Tyr Leu Wing Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys 130 135 140 Glu Phe Asn Aen Asp Cys Lys Leu Lye slu Arg He Glu Glu Asn Gly 145 150 155 160 Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln Hie Asn Gly Arg sln Met 165 170 175 Tyr Val Wing Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr 180 185 190 Arg Arg Lys Asn Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 195 200 205 c210 > 14 < 211 > 225 < 212 > PRT < 213 >; Homo sapiene < 400 > 14 Met - Ala Ala Leu Ala Ser Ser Leu He Arg Gln Lyß Arg slu Val Arg 1 5 10 15 Glu Pro sly Gly Ser Arg Pro Val Ser Wing Gln Arg Arg Val Cye Pro 20 25 30 Arg sly Thr Lys be Leu Cys Gln Lys Gln Leu Leu He Leu Leu Ser 35 40 45 Lys Val Arg Leu Cy Gly Gly Arg Pro Wing Arg Pro Asp Arg Gly Pro 50 55 60 Glu Pro Gln Leu Lys Gly He Val Thr Lys Leu Phe Cys Arg Gln sly 65 70 75 80 Phe Tyr Leu Gln Wing Asn Pro Aep Gly Ser He Gln sly Thr Pro Glu 85 90 95 Asp Thr Ser Being Phe Thr Hie Phe Asn Leu He Pro Val Gly Leu Arg 100 105 110 Val Val Thr He Gln Ser Ala Lys Leu Gly His Tyr Met Wing Met Asn 115 120 125 Wing Glu Gly Leu Leu Tyr Ser Ser Pro Hiß Phe Thr Wing Olu Cys Arg 130 135 140 Phe Lys Glu Cys Val Phe Glu Aen Tyr Tyr Val Leu Tyr Ala Ser Wing 145 150 155 160 Leu Tyr Arg Gln Arg Arg Ser Gly Arg Wing Trp Tyr Leu Gly Leu Aep 165 170 175 Lys slu sly Gln Val Met Lys Gly Aßn Arg Val Lys Lys Thr Lys Wing 180 185 190 Wing Wing His Phe Leu Pro Lys Leu Leu Glu Val Wing Met Tyr Gln Glu 195 twenty 0 205 Pro Ser Leu His Ser Val Pro Glu Ala Pro Ser Ser Ser Pro Pro Ala 210 215 220 Pro 225 < 210 > fifteen i AAA 'x- ^ ^ -' - »- .. ...... i..i» -iaa.i.i ?? i. «RtniSi < 211 > 243 < 212 > PRT < 213 > Homo sapiens < 400 > 15 Met Ala Ala Ala He Ala Ser Ser Leu He Arg Gln Lys Arg Oln Ala 1 5 10 15 Arg Glu Ser Asn Ser Aep Arg Val Ser Ala Ser Lye Arg Arg Ser Ser 20 25 30 Pro Ser Lys Asp Gly Arg Ser Leu Cys Glu Arg His Val Leu Gly Val 35 40 45 Phe Ser Lys Val Arg Phe Cys Ser Gly Arg Lys Arg Pro Val Arg Arg 50 55 60 Arg Pro Glu Pro Gln Leu Lys Oly He Val Thr Arg Leu Phe Ser Oln 65 70 75 80 Gln Gly Tyr Phe Leu Gln Met His Pro Asp Gly Thr He Asp Gly Thr 85 90 95 Lys Asp Glu Asn Ser Asp Tyr Thr Leu Phe Asn Leu He Pro Val Gly 100 105 110 Leu Arg Val Val Ala He Gln Gly Val Lys Ala Ser Leu Tyr Val Ala 115 120 125 Met Asn Gly Glu Gly Tyr Leu Tyr Ser As Asp Val Phe Thr Pro Glu 130 135 140 Cys Lys Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val He Tyr Ser 145 150 155 160 Ser Thr Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp Phe Leu Gly 165 170 175 Leu Asn Lys Glu Gly Gln He Met Lyss Gly Aen Arg Val Lyss Lyss Thr 180 185 190 Lyss Pro Ser Ser Hie Phe Val Pro Lys Pro He Glu Val Cys Met Tyr 195 200 twenty 5 Arg Glu Leu Ser Pro Glu His Gln Lys Glu Gly He Gly Arg Ser Arg Lys Ser 210 215 220 Ser Gly Thr Pro Gly Gly Thr Asn Met Val Lys Val Asn Gln Asp 225 230 235 240 Ser Thr < 210 > 16 c211 > 245 < 212 > PRT < 213 > Homo sapiens < 400 > 16 Met Ala Ala Ala lie Ala Ser Ser Leu lie Arg Gln Lys Arg Gln Ala i? .a? i? i? -? i? A * L ~~? Xt ^ .- ^ uL.y? ¿.t * - - ^ tac .... f ^ Jm 10 15 Arg Glu Arg Glu Lys Ser Asn Wing Cys Lye Cye Val Ser Ser Pro 20 25 30 Lys Gly Lys Thr Ser Cys Asp Lys A3n Lys Leu Asn Val Phe Ser Arg 35 40 45 Val Lys Leu Phe Gly Ser Lys Lys Arg Arg Arg Arg Arg Pro Glu Pro 50 55 60 Gln Leu Lys Gly He Val Thr Lys Leu Tyr Ser Arg Gln Gly Tyr His 65 70 75 80 Leu Gln Leu Gln Wing Asp Gly Thr He Asp Gly Thr Lys Asp Glu Asp 85 90 95 Be Thr Tyr Thr Leu Phe Asn Leu He Pro Val Gly Leu Arg Val Val 100 105 110 Wing He Gln Gly Val Gln Thr Lys Leu Tyr Leu Ala Met Asn Ser Glu 115 120 125 Gly Tyr Leu Glu Leu Tyr Ser Thr Phe Cys Lys Thr Pro Glu Phe Lys Ser Glu 130 135 140 Val Phe Glu Val Thr Tyr Tyr Assn Tyr Tyr Ser Ser Met He 145 150 155 160 Arg Gln Gln Gln Ser Gly Arg Gly Trp Tyr Leu Gly Leu Asn Lys Glu 165 170 175 Gly Glu He Met Lys Gly Aen His Val Lye Lyß Aßn Lye Pro Ala Wing 180 185 190 His Phe Leu Pro Lys Pro Leu Lyg Val Wing Met Tyr Lys Glu Pro Ser 195 200 205 Leu His Asp Leu Thr Glu Phe Ser Arg Ser Gly Ser Gly Thr Pro Thr 210 215 220 Lys Ser Arg Ser Val Ser Gly Val Leu Asn Gly Gly Lys Ser Met Ser 225 230 235 240 His Asn Glu Ser Thr 245 < 210 > 17 < 211 =. 247 < 212 > PRT < 213 > Homo sapiens < 400 > 17 Met Ala Ala Ala He Ala Be Gly Leu He Arg Gln Lys Arg Gln Ala 1 5 10 15 Arg Glu Gln His Trp Asp Arg Pro Be Wing Ser Arg Arg Arg Ser Ser 20 25 30 Pro Ser Lys Aen Arg Gly Leu Cyn Asn Gly Asn Leu Val Asp He Phe > A aaaBwif J L i iitiii1 faw c -???? E.íífea- ^ A * 35 40 45 Ser Lys Val Gly Leu Arg Phe Lys He Lys Arg Arg Arg Arg Gln Leu 50 55 60 Asp Leu Pro Gln Lys Gly Val I Thr Arg Leu Tyr Cys Arg Gln sly 65 70 75 80 Tyr Tyr Leu sln Met His Pro Asp Gly Ala Leu Asp Gly Thr Lys Asp 85 90 95 Asp Ser Thr Asn Ser Thr Leu Phe Asn Leu He Pro Val Gly Leu Arg 100 105 110 Val Val Wing He Gln Gly Val Lys Thr Gly Leu Tyr He Wing Met Asn 115 120 125 Gly Glu Gly Tyr Leu Tyr Pro Ser Glu Leu Phe Thr Pro Glu Cys Lyß 130 135 140 Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val He Tyr Ser Ser Met 145 150 155 160 Leu Tyr Arg Gln Gln Glu Ser Gly Arg Wing Trp Phe Leu Gly Leu Aen 165 170 175 Lys Glu Gly Gln Wing Met Lyß Gly Asn Arg Val Lys Lys Thr Lyß Pro 180 185 190 Wing Wing Phe Leu Pro Lys Pro Leu Glu Val Wing Met Tyr Arg Glu 195 200 205 Pro Ser Leu Hie Aßp Val Gly Glu Thr Val Pro Lys Pro Gly Val Thr 210 215 220 Pro Ser Lys Ser Thr Ser Ala Ser Ala He Met Aßn Gly Gly Lyß Pro 225 230 235 240 Val Asn Lys Ser Lys Thr Thr 245 < 210 > 18 < 211 > 207 < 212 > PRT < 2 3 > Homo ßapiens 400 > 18 Met Wing Glu Val Gly Gly Val Phe Wing Ser Leu Asp Trp Asp Leu His 1 5 10 15 Gly Phe Being Being Leu Gly Asn Val Pro Leu Wing Asp Being Pro Gly 20 25 30 Phe Leu Asn Glu Arg Leu Gly Gln He Glu Gly Lye Leu Gln Arg Gly 35 40 45 Ser Pro Thr Asp Phe Wing His Leu Lys Gly He Leu Arg Arg Arg Gln 50 55 60 Leu Tyr Cys Arg Thr Gly Phe His Leu Glu He Phe Pro Asn Gly Thr 65 70 75 80 Val His Gly Thr Arg His Asp His Ser Arg Phe Gly He Leu Glu Phe 85 90 95 I Am Being Leu Wing Val Gly Leu I Have Being Arg Gly Val Asp Being Gly 100 105 110 Leu Tyr Leu Gly Met Asn Glu Arg Gly Glu Leu Tyr Gly Ser Lys Lys 115 120 125 Leu Thr Arg Glu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn Trp Tyr 130 135 140 Asn Thr Tyr Ala Ser Thr Leu Tyr Lys Hie Ser Asp Ser Glu Arg Gln 145 150 155 160 Tyr Tyr Val Wing Leu Asn Lys Asp Gly Ser Pro Arg Glu Gly Tyr Arg 165 170 175 Thr Lys Arg His Gln Lye Phe Thr Hiß Phe Leu Pro Arg Pro Val Asp 180 185 190 Pro Ser Lye Leu Pro Ser Met Ser Arg Aep Leu Phe His Tyr Arg 195 200 205 c210 > 19 < 211 > 207 < 212 > PRT < 213 > Homo sapiens < 400 > 19 Met Tyr Ser Ala Pro Ser Ala Cys Thr Cys Leu Cys Leu His Phe Leu 1 5 10 15 Leu Leu Cys Phe Gln Val Val Val Leu Val Ala slu slu Asn Val Asp 20 25 30 Phe Arg He His Val Glu Asn Gln Thr Arg Ala Arg Asp Asp Val Ser 35 40 45 Arg Lys Gln Leu Arg Leu Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys 50 55 60 His He Gln Val Leu Gly Arg Arg He Ser Wing Arg Gly Glu Asp Gly 65 70 75 80 Aep Lye Tyr Ala Gln Leu Leu Val Glu Thr Asp Thr Phe Gly Ser Gln 85 90 95 Val Arg He Lye sly Lys Glu Thr slu Phe Tyr Leu Cye Met Asn Arg 100 105 110 Lye Gly Lys Leu Val Gly Lys Pro Asp Gly Thr Ser Lys Glu Cys Val 115 120 125 Phe He Glu Lys Val Leu Glu Aßn Aan Tyr Thr Ala Leu Met Ser Wing 130 135 140 Lys Tyr Ser Gly Trp Tyr Val Gly Phe Thr Lys Lys Gly Arg Pro Arg 145 150 155 160 Lys Gly Pro Lye Thr Arg Glu Asn Gln Gln Asp Val His Phe Met Lys 165 170 175 Arg Tyr Pro Lys Gly sln Pro Glu Leu Gln Lye Pro Phe Lye Tyr Thr 180 185 190 Thr Val Thr Lys Arg Ser Arg Arg He Arg Pro Thr His Pro Wing 195 200 205 < 210 > 20 < 211 > 216 < 212 > PRT < 213 > Homo sapiene < 400 > 20 Met Arg Ser Gly Cys Val Val Val HIE Val Trp He Leu Ala Gly Leu 1 5 10 15 Trp Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Aep Ala Gly Pro 20 25 30 His Val His Tyr Gly Trp Gly Asp Pro He Arg Leu Arg Hiß Leu Tyr 35 40 45 Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg He Arg Wing 50 55 60 Asp Gly Val Val Asp Cys Wing Arg Gly Gln Ser Wing His Ser Leu Leu 65 70 75 80 Glu He Lys Ala Val Ala Leu Arg Thr Val Ala He Lys Gly Val His 85 90 95 Ser Val Arg Tyr Leu Cys Met Gly Wing Asp Gly Lys Met Gln Gly Leu 100 105 110 Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu He Arg Pro 115 120 125 Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser 130 135 140 Leu Ser Be Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu 145 150 155 160 Pro Leu Ser Hiß Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro 165 170 175 Glu Asp Leu Arg Gly His Leu Glu As Asp Met Phe Ser Ser Pro Leu 180 185 190 Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Wing 195 200 2 05 Val Arg Ser Pro Ser Phe Glu Lye 210 215 < 210 > 21 < 211 > 233 < 212 > PRT < 213 > Homo sapiens < 400 > 21 Met Ser Val Leu Arg Wing Tyr Pro Asn Wing Pro Pro Leu Leu Gly Ser 1 5 10 15 Ser Trp Gly Gly Leu He His Leu Tyr Thr Wing Thr Wing Arg Asn Ser 20 25 30 Tyr His Leu Gln He His Lys Asn Gly His Val Asp Gly Ala Pro His 35 40 45 Gln Thr He Tyr Be Ala Leu Met He Arg Ser Glu Asp Ala Gly Phe 50 55 60 Val Val He Thr Gly Val Met Ser Arg Arg Tyr Leu Cys Met Asp Phe 65 70 75 80 Arg Gly Asn He Phe Gly Ser His Tyr Phe Aßp Pro Glu Asn Cys Arg 85 90 95 Phe Gln His Gln Thr Leu Glu Aßn Gly Tyr Aßp Val Tyr Hiß Ser Pro 100 105 110 Gln Tyr His Phe Leu Val Ser Leu Gly Arg Ala Lyß Arg Ala Phe Leu 115 120 125 Pro Gly Met Asn Pro Pro Pro Tyr Ser Gln Phe Leu Ser Arg Arg Asn 130 135 140 Glu He Pro Leu He Hiß Phe Asn Thr Pro He Pro Arg Arg His Thr 145 150 155 160 Arg Ser Ala Glu Aßp Aßp Ser Glu Arg Asp Pro Leu Asn Val Leu Lys 165 170 175 Pro Arg Wing Arg Met Thr Pro Wing Pro Wing Ser Cys Ser Gln Glu Leu 180 185 190 Pro Ser Wing Glu Asp Asn Ser Pro Met Wing Ser Asp Pro Leu Gly Val 195 20 0 205 Val Arg Gly Gly Arg Val Asn Thr His Wing Gly Gly Thr Gly Pro Glu 210 215 220 Gly Cys Arg Pro Phe Wing Lys Phe He 225 230 «210:» 22 < 211 > 155 < 212 > PRT < 213 > Mus musculus < 400 > 22 Met Wing Glu Gly Glu He Thr Thr Phe Wing Wing Leu Thr Glu Arg Phe 1 S 10 15 t * l -i..a- ». -.... t ^^ tfriaitártiifr '' * "***» '"- - • - *" - "- ti-tfutel Asn Leu Pro Leu Gly Asn Tyr Lyß Lyß Pro Lyß Leu Leu Tyr Cys Ser 20 25 30 Asn Gly Gly His Phe Leu Arg He Leu Pro Asp Gly Thr Val Asp Gly 35 40 45 Thr Arg Asp Arg As Asp Gln His He sln Leu sln Leu Ser Ala slu 50 55 60 Be Wing Gly Glu Val Tyr He Lys Gly Thr Glu Thr Gly Gln Tyr Leu 65 70 75 80 Wing Met Asp Thr Glu Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asp Glu 85 90 95 Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105 110 Thr Ser Lys Lys His Wing Glu Lys Asn Trp Phe Val Gly Leu Lys Lyß 115 120 125 Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Wing 130 135 140 He Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155 c210 > 23 < 211 > 154 < 212 > PRT < 213 > Mus musculus < 400 > 23 Met Ala Ala Ser Gly He Thr Ser Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15 Gly Ala Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu Tyr 20 25 30 Cys Lys Asn Gly Gly Phe Phe Leu Arg He His Pro Asp Gly Arg Val 35 40 45 Asp Gly Val Arg Glu Lys Ser Asp Pro His Val Lyß Leu Gln Leu Gln 50 55 60 Wing Glu Glu Arg Gly Val Val Ser He Lyß Gly Val Cyß Wing Asn Arg 65 70 75 80 Tyr Leu Wing Met Lye Glu Aep Gly Arg Leu Leu Wing Ser Lye Cye Val 85 90 95 Thr Glu Glu Cyß Phe Phe Phe Glu Arg Leu Glu Ser Aßn Asn Tyr Asn 100 105 110 Thr Tyr Arg Ser Arg Lys Tyr Ser Ser Trp Tyr Val Wing Leu Lys Arg 115 '120 125 Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys Wing 130 135 140 .ce.- ^ ubSÁ.eH. a i. . ^ ... ± Á He Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 < 210 > 24 < 211 > 245 < 212 > PRT < 213 > Muß mueculus < 400 > 24 Met Gly Leu He Trp Leu Leu Leu Leu Ser Leu Leu Glu Pro Ser Trp 1 5 10 15 Pro Thr Thr Gly Pro Gly Thr Arg Leu Arg Arg Asp Wing Gly Gly Arg 20 25 30 Gly Gly Val Tyr Glu His Leu Gly Gly Wing Pro Arg Arg Arg Lys Leu 35 40 45 Tyr Cys Wing Thr Lys Tyr His Leu Gln Leu His Pro Ser Oly Arg Val 50 55 60 Asn Oly Ser Leu Glu Asn Ser Wing Tyr Ser He Leu slu He Thr Wing 65 70 75 80 Val Glu Val Gly Val Val Ala He Lys Gly Leu Phe Ser sly Arg Tyr 85 90 95 Leu Wing Met Asn Lys Arg Gly Arg Leu Tyr Wing Being Asp Hie Tyr Asn 100 105 110 Wing Glu Cys Glu Phe Val Glu Arg He His Glu Leu Gly Tyr Asn Thr 115 120 125 Tyr Wing Being Arg Leu Tyr Arg Thr Gly Being Ser Gly Pro Gly Wing Gln 130 135 140 Arg Gln Pro Gly Wing Gln Arg Pro Trp Tyr Val Ser Val Aen Gly Lys 145 150 155 160 Gly Arg Pro Arg Arg Gly Phe Lys Thr Arg Arg Thr Gln Lys Ser Ser 165 170 175 Leu Phe Leu Pro Arg Val Leu Gly His Lys Asp His Glu Met Val Arg 180 185 190 Leu Leu Gln Ser Ser sln Pro Arg Ala Pro Gly Glu Gly Ser Gln Pro 195 200 205 Arg sln Arg Arg Gln Lys Lys Gln Ser Pro sly Asp His Gly Lys Met 210 215 220 Glu Thr Leu Ser Thr Arg Wing Thr Pro Ser Thr Gln Leu His thr Gly 225 230 235 240 Gly Leu Ala Val Wing 245 < 210 > 25 c211 > 202 ..ie .. - ^^^. A. ^ .. > .- ...... ^ aihtoü,, tos.abaüfca m "- • * • '-? fr? Wflfflifi < 212 > PRT < 213 > Mus musculus < 400 > 25 Met Ala Lys Arg Gly Pro Thr Thr Gly Thr Leu Leu Pro Arg Val Leu 1 5 10 15 Leu Wing Leu Val Val Wing Leu Wing Asp Arg Gly Thr Wing Wing Pro Asn 20 25 30 Gly Thr Arg Hxs Wing Glu Leu Gly His Gly Trp Asp Gly Leu Val Wing 35 40 45 Arg Ser Leu Wing Arg Leu Pro Val Wing Wing Gln Pro Pro Gln Wing Wing 50 55 60 to Arg Ser Gly Wing Gly Asp Tyr Leu Leu Gly Leu Lys Arg Leu Arg 65 70 75 60 Arg Leu Tyr Cys Asn Val Gly He Gly Phe His Leu Gln Val Leu Pro 85 90 95 Asp Gly Arg He Gly Gly Val His Wing Asp Thr Arg Asp Ser Leu Leu 100 105 110 Glu Leu Ser Pro Val Gln Arg Gly Val Val Ser He Phe Gly Val Ala 115 120 125 Being Arg Phe Phe Val Wing Met Ser Being Arg Gly Lys Leu Phe Gly Val 130 135 140 Pro Phe Phe Thr Asp Glu Cys Lys Phe Lys Olu He Leu Leu Pro Asn 145 150 155 160 Asn Tyr Asn Wing Tyr Glu Wing Tyr Wing Tyr Pro Gly Met Phe Met Wing 165 170 175 Leu Ser Lys Asn Gly Arg Thr Lys Lys Gly Asn Arg Val Ser Pro Thr 180 185 190 Met Lye Val Thr Hiß Phe Leu Pro Arg Leu 195 200 210 > 26 < 211 > 264 «: 212 > PRT < 213 > Mus musculus < 400 > 26 Met Ser Leu Ser Leu Leu Phe Leu He Phe Cys Ser His Leu He His 1 5 10 15 Be Wing Trp Wing Hie Gly Glu Lys Arg Leu Thr Pro Glu Gly Gln Pro 20 25 30 Wing Pro Pro Arg Asn Pro Gly Asp Being Gly Ser Arg Gly Arg Ser 35 40 45 Being Wing Thr Phe Being Being Ser Wing Being Ser Pro Val Wing Wing Ser 50 55 60 k? úul. = .StA? .ÍA? , AB »« jt? ^^ M ... «_ j .j. »..en.Mj« a '»jja Aj Pro Gly Ser Gln Gly Ser Gly Ser Glu His Ser Ser Phe Gln Trp Ser 65 70 75 80 Pro Ser 01y Arg Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly He Gly 85 90 95 Phe His Leu Gln He Tyr Pro Asp Gly Lys Val Asn Gly Ser His Glu 100 105 110 Wing Ser Val Leu Ser He Leu Glu He Phe Wing Val Ser Gln Gly He 115 120 125 Val Gly He Arg Gly val Phe Ser Asn Lys Phe Leu Ala Met Ser Lye 130 135 140 Lys Gly Lye Leu His Wing Being Wing Lys Phe Thr Asp Asp Cys Lys Phe 145 150 155 160 Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Wing Ser Wing He 165 170 175 Hie Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Wing Ala Leu Aen Lye 180 185 190 Arg Gly Lys Wing Lys Arg Gly Cys Ser Pro Arg Val Lys Pro Gln His 195 200 205 Val Ser Thr His Phe Leu Pro Arg Phe Lye Gln Ser Glu Gln Pro slu 210 215 220 Leu Ser Phe Thr Val Thr Val Pro Glu Lys Lys Pro Pro Val Lye 225 230 235 240 Pro Lys Val Pro Leu Ser Gln Pro Arg Arg Pro Pro Pro Val Lys 245 250 255 Tyr Arg Leu Lys Phe Arg Phe Gly 260 < 210 > 27 < 211 > 208 < 212 PRT < 213 > Mus musculus < 400 > 27 Met Ala Leu Gly Gln Arg Leu Phe He Thr Met Ser Arg w / Ala Gly 1 5 10 15 Arg Val Gln Gly Thr Leu Gln Ala Leu Val Phe Leu Gly Val Leu Val 20 25 30 Gly Met Val Val Pro Ser Pro Wing Gly Wing Arg Wing Asn Gly Thr Leu 35 40 45 Leu Asp Ser Arg Oly Trp Gly Thr Leu Leu Ser Arg Ser Arg Wing Gly 50 55 60 Leu Wing Gly Glu He Ser Gly Val Asn Trp Glu Ser Gly Tyr Leu val 65 70 75 80 Gly He Lys Arg Gln Arg Arg Leu Tyr Cys Asn Val Gly He Gly Phe 85 90 95 His Leu Gln Val Pro Pro Asp Gly Arg He Ser Gly Thr Hxs Glu Glu 100 105 110 Asn Pro Tyr Ser Leu Leu Glu He Ser Thr Val Glu Arg Gly Val Val 115 120 125 Ser Leu Phe Gly Val Lys Ser Wing Leu Phe He Wing Met Asn Ser Lys 130 135 140 Gly Arg Leu Tyr Thr Thr Pro Ser Phe Hia Aßp Glu Cys Lys Phe Arg 145 150 155 160 Glu Thr Leu Leu Pro Asn Asn Tyr Asn Wing Tyr Glu Being Asp Leu Tyr 165 170 175 Arg Gly Thr Tyr He Wing Leu Ser Lys Tyr Gly Arg Val Lyß Arg Gly 180 185 190 Ser Lys Val Ser Pro He Met Thr Val Thr His Phe Leu Pro Arg He 195 200 205 < 210 > 28 < 211 > 194 < 212 > PRT < 213 > Mus musculus < 400 > 28 Met Arg Lys Trp He Leu Thr Arg He Leu Pro Thr Leu Leu Tyr Arg 1 5 10 15 Ser Cys Phe His Leu Val Cys Leu Val Gly Thr He Ser Leu Ala Cys 20 25 30 Asn Asp Met Ser Pro Glu Gln Thr Ala Thr Ser Val Asn Cye Ser Ser 35 40 45 Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly sly Asp He 50 55 60 Arg Val Arg Arg Leu Phe Cys Arg thr sln Trp Tyr Leu Arg He Asp 65 70 75 80 Lys Arg Gly Lys Val Lye Gly Thr Gln Glu Met Lys Asn Ser Tyr Asn 85 90 95 He Met Glu He Arg Thr Val Wing Val Gly He Val Wing He Lys Gly 100 105 110 Val Glu Ser Glu Tyr Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr 115 120 125 Wing Lye Lye Glu Cys Asn Glu Aßp Cyß Asn Phe Lye Glu Leu He Leu 130 135 140 &, A _. ^ j > , ^. * .. »se > Att «^ &4jafaa * 3rit ^ .¡..jlt. i.

Glu Asn His Tyr Asn Thr Tyr Ala Be Wing Lys Trp Thr His Ser Gly 145 150 155 160 Gly Glu Met Phe Val Ala Leu Aen Gln Lys Gly He Pro Val Lys Gly 165 170 175 Lys Lys Thr Lys Lys Glu Gln Lys Thr Wing His Phe Leu Pro Met Wing 180 185 190 He Thr < 21-0 > 29 < 211 > 268 < 212 > PRT < 213 > Mus musculus < 400 > 29 Met Gly Ser Pro Arg Ser Ala Leu Ser Cys Leu Leu Leu Hie Leu Leu 1 5 10 15 Val Leu Cys Leu Gln Wing Gln Val Arg Wing Wing Gln Lys Arg Gly 20 25 30 Pro Gly Wing Gly Asn Pro Wing Asp Thr Leu Gly Gln Gly Hie Glu Asp 35 40 45 Arg Pro Phe Gly Gln Arg Ser Arg Wing Gly Lys Asn Phe Thr Asn Pro 50 55 60 Wing Pro Asn Tyr Pro Glu Glu Gly Ser Lys Glu Gln Arg Asp Ser Val 65 70 75 80 Leu Pro Lys Val Thr Oln Arg His Val Arg slu Gln Ser Leu Val Thr 85 90 95 Asp Gln Leu Be Arg Arg Leu He Arg Thr Tyr Gln Leu Tyr Ser Arg 100 105 110 Thr Ser Gly Lys His Val Gln Val Leu Ala Aßn Lys Arg He Asn Wing 115 120 125 Met Wing Glu Asp Gly Asp Pro Phe Wing Lys Leu He Val Glu Thr Asp 130 135 140 Thr Phe Gly Ser Arg Val Arg Val Arg Gly Wing Glu Thr Gly Leu Tyr 145 150 155 160 He Cys Met Asn Lye Lye Gly Lye Leu He Wing Lys Ser Asn Gly Lys 165 170 175 Gly Lys Asp Cys Val Phe Thr Glu He Val Leu Glu Asn Asn Tyr Thr 180 185 190 Wing Leu Gln Asn Wing Lys Tyr Glu Gly Trp Tyr Met Wing Phe Thr Arg 195 200 205 Lys Gly Arg Pro Arg Lys Gly Ser Lys Thr Arg Gln His Gln Arg Glu 210 215 220 Val His Phe Met Lys Arg Leu Pro Arg Gly Hie His Thr Thr Glu Gln 225 230 235 240 Ser Leu Arg Phe Glu Phe Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu 245 250 255 Arg Gly Ser Gln Arg Thr Trp Wing Pro Glu Pro Arg 260 265 < 210 > 30 «211 > 208 < 212 > PRT < 213 > Mus musculus < 400 > 30 Met Wing Pro Leu Gly Glu Val Gly Ser Tyr Phe Gly Val Gln Asp Wing 1 5 10 15 Val Pro Phe Gly Aen Val Pro Val Leu Pro Val Aßp Ser Pro Val Leu 20 25 30 Leu Asn Asp His Leu Gly sln Ser Glu Ala Gly Gly Leu Pro Arg Gly 35 40 45 Pro Wing Val Thr Asp Leu Asp His Leu Lys Gly He Leu Arg Arg Arg 50 55 60 Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Glu He Phe Pro Aan Gly 65 70 75 80 Thr He Gln Gly Thr Arg Lys Aßp His Ser Arg Phe Gly He Leu Glu 85 90 95 Phe He Ser He Wing Val Gly Leu Val Ser He Arg Gly Val Asp Ser 100 105 110 Gly Leu Tyr Leu sly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu 115 120 125 Lys Leu Thr Gln Glu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn Trp 130 135 140 Tyr Asn Thr Tyr Ser Ser Aen Leu Tyr Lys Hie Val Asp Thr Gly Arg 145 150 155 160 Arg Tyr Tyr Val Ala Leu Aen Lyß Aep Gly Thr Pro Arg Glu Gly Thr 165 170 175 Arg Thr Lys Arg Hiß Gln Lye Phe Thr Hie Phe Leu Pro Arg Pro Val 180 IBS 190 Asp Pro Aap Lys Val Pro Glu Leu Tyr Lys Asp He Leu Ser Gln Ser 195 200 2 05 < 210 > 31 f • * - * - * • "'• ^ jÉJi-JüiMüMiihM'" '' '»' - * - * * * - ** - * ° trr ^ j * 'fB'i? mk £ ee ^ ga | ££ ^^ i < 211 > 209 < 212 > PRT < 213 > Mus musculus < 400 > 31 Met Trp Lys Trp He Leu Thr Hxß Cys Ala Ser Ala Phe Pro His Leu 1 5 10 15 Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser Phe 20 25 30 Pro Val Thr Cye Gln Ala Leu Gly Gln Asp Met Val Ser Gln slu Ala 35 40 45 Thr Asn Cys Ser Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser Wing 50 S5 60 Gly Arg Hiß Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp 65 70 75 80 Arg Arg Leu Phe Ser Phe Thr Lys Tyr Phe Leu Thr He Glu Lye Asn 85 90 95 Gly Lys Val Ser Gly Thr Lys Asn Glu Asp Cys Pro Tyr Ser Val Leu 100 105 110 Glu He Thr Ser Val Glu He Gly Val Val Ala Val Lys Ala He Asn 115 120 125 Ser Asn Tyr Tyr Leu Wing Met Aen Lyß Lyß Gly Lye Leu Tyr Gly Ser 130 135 140 Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Aen 145 150 155 160 Gly Tyr Asn Thr Tyr Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln 165 170 175 Met Tyr Val Wing Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys 180 185 190 Thr Arg Arg Lys Asn Thr Ser Wing His Phe Leu Pro Met Thr He Gln 195 200 205 Thr < = 210 > 32 < 213 > Mus musculus < 400 > 32 Met Ala Ala Ala Ala Be Ser Leu He Arg Gln Lys Arg Glu Val Arg 1 5 10 15 Glu Pro Gly Gly Be Arg Pro Val Be Ala Gln Arg Arg Val Cys Pro 20 25 30 Arg Gly Thr Lys Ser Leu Cye Gln Lys Gln Leu Leu Leu Leu Ser 35 40 45 Lys Val Arg Leu Cys Gly Gly Arg Pro Thr Arg Gln Asp Arg Gly Pro 50 55 60 Glu Pro Gln Leu Lys Gly He Val Thr Lys Leu Phe Cys Arg Gln sly 65 70 75 80 Phe Tyr Leu Gln Wing Asn Pro Asp Gly Ser He Gln sly Thr Pro Glu 85 90 95 Asp Thr Ser Ser Phe Thr Hie Phe Asn Leu He Pro Val Gly Leu Arg 100 105 110 Val Val Thr He Gln Ser Wing Lys Leu Oly His Tyr Met Wing Met Asn 115 120 125 Wing Glu Gly Leu Leu Tyr Ser Ser Pro His Phe Thr Wing Glu Cys Arg 130 135 140 Phe Lys Glu Cys Val Phe Glu Asn Tyr Tyr Val Leu Tyr Ala Ser Wing 145 150 155 160 Leu Tyr Arg Gln Arg Arg Ser Gly Arg Wing Trp Tyx Leu Gly Leu Asp 165 170 175 Lyß Glu Gly Arg Val Met Lys Gly Asn Arg Val Lys Lys Thr Lys Wing 180 185 190 Wing Wing His Phe Val Pro Lys Leu Leu Glu Val Wing Met Tyr Arg Glu 195 200 205 Pro Ser Leu His Ser Val Pro Glu Thr Ser Pro Ser Ser Pro Pro Wing 210 215 220 HlS 225 < 210 > 33 < 211 > 243 < 212 > PRT < 213 > Mus musculus «400 > 33 Met Wing Wing Wing Wing Being Ser Leu He Arg Gln Lys Arg Gln Wing 1 5 10 15 Arg Glu Ser Asn Ser Aep Arg Val Ser Wing Ser Lye Arg Arg Ser Ser 20 25 30 Pro Ser Lys Asp Gly Arg Ser Leu Cys Glu Arg Hie Val Leu Gly Val 35 40 45 Phe Ser Lys Val Arg Phe Cys Ser Gly Arg Lys Arg Pro Val Arg Arg 50 55 60 Arg Pro Glu Pro sln Leu Lys Gly He Val Thr Arg Leu Phe Ser Gln 65 70 75 80 Gln sly Tyr Phe Leu Gln Met His Pro Asp Gly Thr He Asp Gly Thr ?? i? á ** ¿- JAi. »Yy.j ,? . , lr.y .-- &. ~ rt.ak,, ...... To M. * Uiály? llLyl. -_. - < - r. ... Mt.? ., 85 90 95 Lys Asp Glu Asn Ser Asp Tyr Thr Leu Phe Asn Leu He Pro Val Gly 100 105 110 Leu Arg Val Val Ala He Gln sly Val Lys Ala Ser Leu Tyr Val Ala 115 120 125 Met Asn Gly Glu Gly Tyr Leu Tyr Ser Ser Asp Val Phe Thr Pro Glu 130 135 140 Cys Lys Phe Lys Glu Ser Val Phe Glu Asp Tyr Tyr Val He Tyr Ser 145 150 15S 160 Ser Thr Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp Phe Leu Gly 165 170 175 Leu Asn Lys Glu Gly Gln He Met Lys Gly Asn Arg Val Lys Lys Thr 180 185 190 Lys Pro Ser Ser His Phe Val Pro Lys Pro He Glu Val Cys Met Tyr 195 200 205 Arg Glu Pro Ser Leu Hie Glu He Gly Glu Lys sln Oly Arg Ser Arg 210 215 220 Lys Be Ser Gly Thr Pro Thr Met Aen Gly Gly Lye Val Val Asn Gln 225 230 235 240 Asp Ser Thr < 210 > 34 «211 > 245 < 212 > PRT c213 > Mus musculus < 400 > 34 Met Thr Wing Wing Wing Being Ser Leu He Arg Gln Lys Arg Gln Wing 1 5 10"15 Arg Glu Arg Glu Lys Ser Asn Wing Cys Lys Cys Val Ser Ser Pro Ser 20 25 30 Lys Gly Lye Thr Ser Cyß Aep Lye Asn Lyß Leu Asn Val Phe Ser Arg 35 40 45 Val Lys Leu Phe Gly Ser Lye Lyß Arg Arg Arg Arg Pro Glu Pro 50 55 60 Gln Leu Lys Gly He Val Thr Lys Leu Tyr Ser Arg Gln sly Tyr Hie 65 70 75 80 Leu sln Leu Gln Ala Asp Gly Thr He Asp Gly Thr Lys Asp Glu Asp 85 90 95 Ser Thr Tyr Thr Leu Phe Asn Leu He Pro Val Gly Leu Arg Val Val 100 105 110 Ala He Gln Gly Val Gln Thr Lys Leu Tyr Leu Ala Met Asn Ser slu 115 120 125 Gly Tyr Leu Tyr Thr Ser Glu His Phe Thr Pro Glu Cys Lys Phe Lys 130 135 140 Glu Ser Val Phe Glu Asn Tyr Tyr Val Thr Tyr Ser Ser Met He Tyr 145 150 155 160 Arg Gln Gln Gln Ser Gly Arg Gly Trp Tyr Leu Gly Leu Asn Lys Glu 165 170 175 Gly Glu He Met Lys Gly Asn Hiss Val Lys Lys Asn Lys Pro Ala Ala 180 185 190 Hie Phe Leu Pro Lye Pro Leu Lyss Val Ala Met Tyr Lys Glu Pro Ser 195 200 205 Leu His Asp Leu Thr Glu Phe Ser Arg Ser Gly Ser Gly Thr Pro Thr 210 215 220 Lys Ser Arg Ser Val Ser Gly Val Leu Asn Gly Gly Lys Ser Met Ser 225 230 235 240 His Asn Glu Ser Thr 245 «210 > 35 < 211 > 247 < 212 > PRT < 213 > Mus musculus «400 > 35 Met Ala Ala Ala Ala Be Ala Ser Gly Leu He Arg Gln Lyß Arg Gln Ala 1 5 10 15 Arg Glu Gln His Trp Asp Arg Pro Ser Ala Ser Arg Arg Arg Ser Ser 20 25 30 Pro Ser Lys Asn Arg sly Leu Phe Asn sly Asn Leu Val Asp He Phe 35 40 45 Ser Lys Val Arg He Phe Gly Leu Lys Lys Arg Arg Leu Arg Arg Gln 50 55 60 Asp Pro sln Leu Lys Gly He Val Thr Arg Leu Tyr Cys Arg Gln Gly 65 70 75 80 Tyr Tyr Leu Gln Met His. Pro Asp Gly Ala Leu Aep Gly Thr Lys Asp 85 90 95 Asp Ser Thr Asn Ser Thr Leu Phe Asn Leu He Pro Val Gly Leu Arg 100 105 110 Val Val Ala He Gln Gly Val Lys Thr Gly Leu Tyr He Ala Met Asn 115 120 125 Gly Glu Gly Tyr Leu Tyr Pro Ser Glu Leu Phe Thr Pro Glu Cys Lys 130 135 140 Phe Lys Glu Ser Val Phe Glu Asn Tyr Val Tyr Tyr Ser Ser Met 145 150 155 160 Leu Tyr Arg Gln sln Glu Ser Oly Arg Wing Trp Phe Leu Oly Leu Asn 165 170 175 Lys Glu Gly Gln Val Met Lye Gly Asn Arg Val Lys Lys Thr Lys Pro 180 185 190 Ala Ala His Phe Leu Pro Lys Pro Leu Glu Val Ala Met Tyr Arg Glu 195 200 205 Pro Ser Leu His Asp Val Gly Glu Thr Val Pro Lys Wing Gly Val Thr 210 215 220 Pro Ser Lys Ser Thr Ser Ala Wing Wing He Met Asn sly Gly Lye Pro 225 230 235 240 Val Asn Lys Cys Lys Thr Thr 245 < 210 > 36 < 211 218 < 212 = > PRT < 213 > Mus musculus < 400 > 36 Met Ala Arg Lys Trp Asn Gly Arg Ala Val Ala Arg Ala Leu Val Leu 1 5 10 15 Wing Thr Leu Trp Leu Wing Val Ser Gly Arg Pro Leu Wing Gln Gln Ser 20 25 30 Gln Ser Val Ser Asp Glu Asp Pro Leu Phe Leu Tyr Gly Trp Gly Lys 35 40 45 He Thr Arg Leu Gln Tyr Leu Tyr Ser Wing Gly Pro Tyr Val Ser Asn 50 55 60 Cys Phe Leu Arg He Arg Ser Asp Gly Ser Val Asp Cye Glu Glu Aep 65 70 75 80 Without Aen Glu Arg Aen Leu Leu Glu Phe Arg Ala Val Ala Leu Lye Thr 85 90 95 He Wing He Lys Asp Val Ser Ser Val Arg Tyr Leu Cye Met Wing 100 105 110 Asp Gly Lys He Tyr Gly Leu He Arg Tyr Ser Glu Glu Asp Cys Thr 115 120 125 Phe Arg Glu Glu Met Asp Cys Leu Gly Tyr Asn Gln Tyr Arg Ser Met 130 135 140 Lys His His Leu His He He Phe He Slen Ala Lys Pro Arg Glu Gln 145 150 155 160 Leu Gln Asp Gln Lys Pro Ser Asn Phe He Pro Val Phe His Arg Ser 165 170 175 Phe Phe Glu Thr Gly Asp Gln Leu Arg Ser Lys Met Phe Ser Leu Pro 180 185 190 Leu Glu Ser Asp Ser Met Asp Pro Phe Arg Met Val Glu Asp Val Aep 195 200 205 His Leu Val Lys Ser Pro Ser Phe Glr. Lys 210 215 < 210 > 37 < 211 > 207 < 212 > PRT < 213 > Rattus norvegicus < 400 > 37 Met Wing Glu Val Gly Gly Val Phe Wing Ser Leu Asp Trp Asp Leu Gln 1 5 10 15 Gly Phe Ser Ser Leu Gly Asn Val Pro Leu Wing Asp Ser Pro Gly 20 25 30 Phe Leu Asn Glu Arg Leu Gly Gln He Glu Gly Lys Leu Gln Arg Gly 35 40 45 Ser Pro Thr Asp Phe Ala Hiß Leu Lys Gly He Leu Arg Arg Arg Gln 50 55 60 Leu Tyr Cys Arg Thr Gly Phe His Leu Glu He Phe Pro Asn Gly Thr 65 70 75 80 Vai His Gly Thr Arg His Asp His Ser Arg Phe Gly He Leu Glu Phe 85 90 95 He Ser Leu Ala Val Gly Leu He Ser He Arg Gly Val Asp Ser Gly 100 105 110 Leu Tyr Leu Gly Met Asn Glu Arg Gly Glu Leu Phe Gly Ser Lyß Lye US 120 125 Leu Thr Arg Glu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn Trp Tyr 130 135 140 Asn Thr Tyr Ala Ser Thr Leu Tyr Lys His Ser Asp Ser Glu Arg Gln 145 150 1SS 160 Tyr Tyr Val Ala Leu Asn Lyß Asp Gly Ser Pro Arg Glu Gly Tyr Arg 165 170 175 Thr Lyß Arg His Oln Lys Phe Thr Hiß Phe Leu Pro Arg Pro Val Aep 180 185 190 Pro Ser Lys Leu Pro Ser Met Ser Arg Asp Leu Phe Arg Tyr Arg 195 200 205 < 210 > 38 < 211 > 207 < 212 > PRT < 213 > Mus musculus < 400 > 38 Met Tyr Ser Ala Pro Ser Ala Cys Thr Cys Leu Cys Leu His Phe Leu 1 5 10 15 Leu Leu Cys Phe Gln Val Gln Val Leu Ala Wing Glu Glu Asn Val Asp 20 25 30 Phe Arg He His Val Glu Asn Gln Thr Arg Ala Arg Asp Asp val be 35 40 45 Arg Lys Gln Leu Arg Leu Tyr sln Leu Tyr Ser Arg Thr Ser sly Lye 50 SS 60 His He Gln al Leu Gly Arg Arg He Ser Wing Arg Gly Glu Asp Gly 65 70 75 80 Asp Lys Tyr Ala sln Leu Leu Val Glu Thr Asp Thr Phe Gly Ser Gln 85 90 95 Val Arg He Lys Gly Lys Glu Thr Glu Phe Tyr Leu Cys Met Asn Arg 100 105 110 Lye Gly Lye Leu Val Gly Lyß Pro Aßp Gly Thr Ser Lye Glu Cys Val 115 120 125 Phe He Glu Lys Val Leu Glu Aßn Aen Tyr Thr Ala Leu Met Ser Wing 130 135 140 Lys Tyr Ser Gly Trp Tyr Val Gly Phe Thr Lys Lys Gly Arg Pro Arg 145 150 155 160 Lys Gly Pro Lys Thr Arg Glu Asn Gln Qln Asp Val His Phe Met Lys 165 170 175 Arg Tyr Pro Lys Gly Gln Wing Glu Leu Gln Lys Pro Phe Lys Tyr Thr 180 185 190 Thr Val Thr Lys Arg Ser Arg Arg He Arg Pro Thr His Pro Gly 195 200 205 < 210 > 39 < 211 > 11 < 212 > PRT < 213 > Human immunodeficieney virus type 1 < 400 > 39 Tyr Gly Arg Lyß Lys Arg Arg Gln Arg Arg Arg 1 5 10 < 210 > 40 < 211 > 15 «: 212th PRT < 2 3 > artificial sequence < 220 > < 223 > artificial sequence description: internalization domain derived from HIV tat protein < 400 > 40 sly Gly Gly Gly Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg . ^ ^ ^ ^^^^ g ^^ 10 15 c210 41 < 211 > 22 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > description of artificial oligonucleotide sequence; PCR primer < 400 > 41 ctatcccaat gcctccccac tg 22 < 210 > 42 «211 > 21 «= 212 > DNA < 2i3 > artificial sequence < 220 > «223 > description of artificiaholigonucleotide sequence; PCR primer < 400 > 42 cgcccctgac cacccctaat g 21 < 210 > 43 < 211 > 23 < 212 > DNA c2 artificial sequence < 220 > < 223 > description of artificiaholigonucleotide sequence; 5 'RACE primer e400 > 43 gtgcggaatt gtgagcggat aac 23 < 210 > 44 < 13 > artificial sequence < 220 > < 223? description of artificiaholigonucleotide sequence; 5 'RACE primer < 400 > 44 ctgatggggt gcgccatcca ca 22 < 211 > 23 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > description of artificiaholigonucleotide sequence; hosted PCR primer < 400 > 45 ctatgaccat gattacgcca age 23 < 210 > 46 < 211 > 24 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > description of artificiaholigonucleotide sequence; hosted PCR primer < 400 > 46 cattcttgtg gatctgcagg tggt 24 < 210 > 47 < 211 > 23 «212? DNA < 2i3 > artificial sequence < 220 > < 2 3 description of artificiaholigonucleotide sequence; 3 'RACE primer < 400 > 47 cggcctcctg ttcacaggag etc 23 < 210 > 48 < 211 > 21 < 212 > DNA < 2i3 > artificial sequence c220 > < 223 > description of artificiaholigonucleotide sequence; 3 'RACE primer < 400 > 48 cgggcctctt cgctattacg c 21 < 210 > 49 < 211 > 21 < 212 > DNA < 213 > artificial sequence 220 > < 223 > ddeessccrr iippcciiónn ddee sseeccuueeincia artificiaholigonucleótido; hosted PCR primer < 400 > 49 gcgccgagga caacagcccg a 21 < 210 > 50 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > description of artificlaholigonucleotide sequence; hosted PCR primer < 400 > 50 tggcgaaagg gggatgtgct g 21 < 210 > 51 < 211 > 21 < 212 > DNA c2i3 > artificial sequence < 220 > < 223 > description of artificiaholigonucleotide sequence; PCR primer < 400 > 51 tccaceaccc tgttgctgta g 21 < 210 > 52 < 211 > 22 < 212 > DNA < 2i3 > artificial sequence < 220 > < 223 > description of artificiaholigonucleotide sequence; PCR primer c400 > 52 gaccacagtc catgccatca ct 22 < 210 > 53 < 211 > 22 < 212 > DNA < i3 > artificial sequence < 220 > < 223 > description of artificiaholigonucleotide sequence; PCR primer < 400 > 53 ctatcccaat gcctccccac tg 22 < 210 > 54 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > description of artificiaholigonucleotide sequence; PCR primer c4007 54 cgcccctgac cacccctaat g 21 trß «i

Claims

CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. An isolated nucleic acid molecule, comprising a nucleotide sequence characterized in that it is selected from the group consisting of: (a) the nucleotide sequence as set forth in SEQUENCE OF IDENTIFICATION NO: 1; (b) the nucleotide sequence of the DNA insert in ATCC, deposit No. PTA-1617; (c) a nucleotide sequence encoding the polypeptide as set forth in the SEQUENCE OF ID NO: 2; (d) a nucleotide sequence that hybridizes under moderate or high stringency conditions with the complement of any of the sequences of part (a) - (c); and (e) a nucleotide sequence complementary to any of the sequences of part (a) - (c). 2. An isolated nucleic acid molecule, comprising a nucleotide sequence characterized in that it is selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide that is at least about 70 percent identical to the polypeptide as set forth in SEQ ID NO: 2, wherein the encoded polypeptide has an activity of the polypeptide that is established in the IDENTIFICATION SEQUENCE NO: 2; (b) a nucleotide sequence coding for an allelic variant or splicing variant of the nucleotide sequence as set forth in SEQUENCE OF IDENTIFICATION NO: 1, the nucleotide sequence of the DNA insert in ATCC, deposit No. PTA-1617, or of subsection (a); (c) a region of the nucleotide sequence of SEQUENCE OF IDENTIFICATION NO: 1, the insert of DNA in ATCC, deposit No. PTA-1617, (a), or (b) that codes for a polypeptide fragment of at least 25 amino acid residues, wherein the polypeptide fragment has an activity of the encoded polypeptide, as set forth in SEQUENCE OF IDENTIFICATION NO: 2, or is antigenic; (d) a region of the nucleotide sequence of SEQUENCE OF IDENTIFICATION NO: 1, the DNA insert in ATCC, deposit No. PTA-1617, or any of the sequences of part (a) - (c) comprising a fragment of at least about 16 nucleotides; (e) a nucleotide sequence that hybridizes, under moderate or high stringency conditions, with the ^, i > ^^ tafcs ^^ rt ,, - ^ ife «.. atote ^ .. complement of any of the sequences of items (a) - (d), - and (f) a nucleotide sequence complementary to any of the sequences of part (a) - (d). 3. An isolated nucleic acid molecule, comprising a nucleotide sequence, characterized in that it is selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide as set forth in SEQUENCE OF IDENTIFICATION NO: 2, with at least one amino acid conservative substitution, wherein the encoded polypeptide has a polypeptide activity as set forth in SEQUENCE OF IDENTIFICATION NO: 2; (b) a nucleotide sequence encoding a polypeptide as set forth in the SEQUENCE OF ID NO: 2, with at least one amino acid insertion, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQUENCE OF IDENTIFICATION NO: 2; (c) a nucleotide sequence encoding a polypeptide as set forth in SEQUENCE OF IDENTIFICATION NO: 2, with at least one deletion of an amino acid, wherein the encoded polypeptide has a polypeptide activity as set forth in the SEQUENCE OF ID NO: 2; - - (d) a nucleotide sequence coding for a polypeptide as set forth in SEQUENCE OF IDENTIFICATION NO: 2, which has a cut in the C or N-terminal part, wherein the encoded polypeptide has a polypeptide activity as set forth in IDENTIFICATION SEQUENCE NO: 2; (e) a nucleotide sequence encoding a polypeptide, as set forth in SEQUENCE OF IDENTIFICATION NO: 2, with at least one modification selected from the group consisting of substitutions, insertions and deletions of amino acids, cuts in the part C or N terminal, wherein the encoded polypeptide has an activity of the polypeptide that is set forth in SEQUENCE OF IDENTIFICATION NO: 2; (f) a nucleotide sequence of any one of items (a) - (e) comprising a fragment of at least about 16 nucleotides; (g) a nucleotide sequence that hybridizes under moderate or high stringency conditions with the complement of any of the sequences of part (a) - (f), - and (h) a nucleotide sequence complementary to any of the sequences of the subparagraphs (a) - (e). 4. The vector comprising the nucleic acid molecule according to any of claims 1, 2 or 3. 5. The host cell, characterized in that it comprises the vector according to claim 4. 6. The host cell according to with claim 5, characterized in that it is a eukaryotic cell. 7. The host cell, according to claim 5, characterized in that it is a prokaryotic cell. A method for producing a FGF-23 polypeptide, which comprises culturing the host cell according to claim 5 under conditions suitable for expressing the polypeptide, and optionally isolating the polypeptide from the culture. 9. The polypeptide, further characterized in that it is produced by the method according to claim 8. The method according to claim 8, further characterized in that the nucleic acid molecule comprises a promoter DNA different from the promoter DNA for the polypeptide Native FGF-23 operably linked to the DNA encoding the FGF-23 polypeptide. 11. The isolated nucleic acid molecule according to claim 2, further characterized - * vr ti1" ^ ¡D ^ i? ¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡Jj ^^ because the percent identity is determined using a computer program that is selected from the group consisting of GAP, BLASTN, FASTA, BLASTA, BLASTX, BestFit and the algorithm from Smith- aterman. 12. A method for determining whether a compound inhibits the activity of the FGF-23 polypeptide or the production of the FGF-23 polypeptide, characterized in that it comprises exposing a compound according to any one of claims 5, 6 or 7 to the compound, and measuring the activity of the FGF-23 polypeptide or the production of the FGF-23 polypeptide in such cells. 13. An isolated polypeptide, comprising the amino acid sequence characterized in that it is selected from the group consisting of: (a) the amino acid sequence as set forth in SEQUENCE OF IDENTIFICATION NO: 2; and (b) the amino acid sequence encoded by the DNA insert in ATCC, deposit No. PTA-1617. 14. An isolated polypeptide, comprising the amino acid sequence characterized in that it is selected from the group consisting of: (a) the amino acid sequence as set forth in SEQUENCE OF IDENTIFICATION NO: 3, optionally additionally comprising an amino methotine terminal; ^^ l ^^^^ j (b) an amino acid sequence for an ortholog of SEQUENCE OF IDENTIFICATION NO: 2; (c) an amino acid sequence which is at least about 70 percent identical to the amino acid sequence of SEQ ID NO: 2, wherein the polypeptide has a polypeptide activity that is set forth in the SEQUENCE OF IDENTIFICATION NO : 2; (d) a fragment of the amino acid sequence that is set forth in SEQUENCE OF IDENTIFICATION NO: 2, comprising at least about 25 amino acid residues, wherein the fragment has an activity of the polypeptide that is established in the SEQUENCE OF IDENTIFICATION NO : 2, or it is antigenic; and (e) an amino acid sequence for an allelic variant or splicing variant of the amino acid sequence that is set forth in SEQUENCE OF IDENTIFICATION NO: 2, the amino acid sequence is encoded by the DNA insert in ATCC, deposit No. PTA-1617, or from subsections (a) - (c). 15. An isolated polypeptide comprising the amino acid sequence characterized in that it is selected from the group consisting of: (a) the amino acid sequence as set forth in SEQUENCE OF IDENTIFICATION NO: 2, with at least ffmni-fírM-Tf ^ 8 ^^^ - a substitution of a conservative amino acid, wherein the polypeptide has an activity of the polypeptide that is set forth in SEQUENCE OF IDENTIFICATION NO: 2; (b) the amino acid sequence as set forth in SEQUENCE OF IDENTIFICATION NO: 2, with at least one amino acid insertion, wherein the polypeptide has a polypeptide activity as set forth in SEQUENCE OF IDENTIFICATION NO: 2; (c) the amino acid sequence as set forth in SEQUENCE OF IDENTIFICATION NO: 2 with at least one amino acid deletion, wherein the polypeptide has an activity of the polypeptide which is set forth in SEQUENCE OF IDENTIFICATION NO: 2; (d) the amino acid sequence, as set forth in SEQUENCE OF IDENTIFICATION NO: 2, which has a cut in the C or N-terminal part, wherein the polypeptide has a polypeptide activity which is set forth in the IDENTIFICATION SEQUENCE NO: 2; and (e) the amino acid sequence, as set forth in SEQUENCE OF IDENTIFICATION NO: 2, with at least one modification that is selected from the group consisting of substitutions, insertions and deletions of amino acids, cuts in part C and N terminal, wherein the polypeptide has a polypeptide activity that is set forth in SEQUENCE OF IDENTIFICATION NO: 2. - • fA "- 16. An isolated polypeptide, encoded by the nucleic acid molecule according to any of claims 1, 2 or 3, characterized in that the polypeptide has an activity of the polypeptide that is established in the SEQUENCE OF IDENTIFICATION NO: 2. 17. The polypeptide isolated, according to claim 14, characterized in that the identity percent is determined using a computer program that is selected from the group consisting of GAP, BLASTP, FASTA, BLASTA, BLASTX, BestFit, and the Smith-Waterman algorithm. 18. A selective binding agent or a fragment thereof, characterized in that it specifically binds the polypeptide according to any one of claims 13, 14 or 15. 19. The selective binding agent or a fragment thereof, in accordance with the claim 18, characterized in that it specifically binds to the polypeptide comprising the amino acid sequence as set forth in SEQUENCE OF IDENTIFICATION NO: 2 or a fragment thereof. 20. The selective binding agent, according to claim 18, further characterized in that it is an antibody or a fragment thereof. 21. The selective bonding agent, in accordance with claim 18, further characterized in that it is a humanized antibody. 22. The selective binding agent, according to claim 18, further characterized in that it is a human antibody or a fragment thereof. 23. The selective binding agent, according to claim 18, further characterized in that it is a polyclonal antibody or a fragment thereof. 24. The selective binding agent, according to claim 18, further characterized in that it is a monoclonal antibody or fragment thereof. 25. The selective binding agent, according to claim 18, further characterized in that it is a chimeric antibody or fragment thereof. 26. The selective binding agent, according to claim 18, further characterized in that it is an antibody grafted with CDR or a fragment thereof. 27. The selective binding agent, according to claim 18, further characterized in that it is an anti-idiotypic antibody or a fragment thereof. 28. The selective binding agent, according to claim 18, further characterized in that it is a variable region fragment. 29. The variable region fragment according to claim 18, further characterized in that it is a Fab or Fab1 fragment. 30. A selective binding agent or fragment thereof, characterized in that it comprises at least one complementarity determining region with specificity for a polypeptide having the amino acid sequence of SEQUENCE OF IDENTIFICATION NO: 2. 31. The binding agent selective, according to claim 18, further characterized in that it binds to a detectable label. 32. The selective binding agent, according to claim 18, further characterized in that it antagonizes the biological activity of the FGF-23 polypeptide. 33. A method for treating, preventing or reducing a disease, condition or disorder related to the FGF-23 polypeptide, characterized in that it comprises administering to a patient an effective amount of a selective binding agent, in accordance with claim 18. 34. A selective binding agent, characterized in that it is produced by immunizing an animal with a polypeptide comprising an amino acid sequence of SEQUENCE OF IDENTIFICATION NO: 2. 35. A hybridoma, characterized in that it produces a selective binding agent that is capable of binding a polypeptide, according to any of claims 1, 2 or 3. 36. A method for detecting or quantifying the amount of FGF-23 polypeptide, characterized in that it uses the antibody against FGF-23 or a fragment thereof, according to claim 18. 37. A composition, characterized in that it comprises the polypeptide in accordance with any of claims 13, 14 or 15, and a pharmaceutically acceptable formulation agent. 38. The composition according to claim 37, further characterized in that the pharmaceutically acceptable formulation agent is a carrier, adjuvant, solubilizer, stabilizer or antioxidant. 39. The composition according to claim 37, further characterized in that the polypeptide comprises the amino acid sequence as set forth in SEQUENCE OF IDENTIFICATION NO: 3. 40. A polypeptide, characterized in that it comprises a derivative of the polypeptide in accordance with Any of claims 13, 14 or 15. 41. The polypeptide according to claim 40, further characterized in that it is covalently modified with a water-soluble polymer. 42. The polypeptide according to claim 41, further characterized in that the water-soluble polymer is selected from the group consisting of - polyethylene glycol, monomethoxy polyethylene glycol, dextran, cellulose, poly- (N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols and polyvinyl alcohol. 43. A composition, characterized in that it comprises a nucleic acid molecule according to any one of claims 1, 2 or 3, and a pharmaceutically acceptable formulation agent. 44. The composition according to claim 43, further characterized in that the nucleic acid molecule is contained in a viral vector. 45. A viral vector, characterized in that it comprises a nucleic acid molecule according to any of claims 1, 2 or 3. 46. A fusion polypeptide, characterized in that it comprises the polypeptide according to any of claims 13, 14 or 15, fused to a heterologous amino acid sequence. 47. The fusion polypeptide according to claim 46, further characterized in that the heterologous amino acid sequence is a constant domain of IgG or a fragment thereof. 48. A method for treating, avoiding or reducing a medical condition, characterized in that it comprises administering to a patient the polypeptide according to any of claims 13, 14 or 15, or the polypeptide encoded by the nucleic acid according to any one of claims 1, 2 or 3. 49. A method for treating, preventing or decreasing a medical condition, characterized in that it comprises administering to a patient an agonist or antagonist of the biological activity of the polypeptide according to any of claims 13, 14 or 15, or of the polypeptide encoded by the nucleic acid according to any of the claims 1, 2 or 3. 50. The method according to any of claims 48 or 49, characterized in that, in addition to the medical condition being treated, it is avoided or decreased, it is autosomal dominant hypophosphatemic rickets (ADHR, for its acronym in English). ). 51. A method for diagnosing a pathological condition or susceptibility to a pathological condition in a subject, the method is characterized in that it comprises: (a) determining the presence or amount of expression of the polypeptide according to any of claims 13, 14 or 15, or the polypeptide encoded by the nucleic acid molecule according to any of claims 1, 2 or 3, in a sample; Y . ^. ^^ ^ t ^. ^ .., ^ - (b) diagnosing a pathological condition or susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide. 52. A device, characterized in that it comprises: (a) a membrane suitable for implantation; and (b) cells encapsulated within the membrane, wherein the cells secrete a protein, according to any of claims 13, 14 or 15; and the membrane is permeable to protein and impermeable to materials harmful to cells. 53. A method for identifying a compound which binds to a FGF-23 polypeptide, the method is characterized in that it comprises: (a) contacting the polypeptide according to claims 13, 14 or 15, with a compound; and (b) determining the degree of binding of the FGF-23 polypeptide with the compound. 54. The method according to claim 53, further characterized in that it comprises determining the activity of the polypeptide when it is bound to the compound. 55. A method for modulating the levels of a polypeptide in an animal, characterized in that it comprises HfrA¿ * fc J. ttf »^ **" ^ - administering to the animal the nucleic acid molecule according to any of claims 1, 2 or 3. 56. A transgenic non-human mammal, characterized in that it comprises the nucleic acid molecule according to any of claims 1, 2 or 3. 57. A method for determining whether a compound inhibits the activity of the FGF-23 polypeptide or the production of the FGF-23 polypeptide, the method is characterized in that it comprises exposing a transgenic mammal according to claim 56 to the compound and measuring the activity of the FGF-23 polypeptide or the production of FGF-23 polypeptide in the mammal.