JP2006238894A - Fibroblast growth factor-23 molecules and uses thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide novel polypeptides having diagnostic or therapeutic advantages and to identify nucleic acids encoding the polypeptides. <P>SOLUTION: The isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of nucleotide sequence: (a) as set forth in SEQ ID NO: 1; (b) of the DNA insert in ATCC Accession No. PTA-1617; (c) encoding the polypeptide as set forth in SEQ ID NO: 2; (d) that hybridizes to the complement of the nucleotide sequence of any of (a)-(c) under a moderately or highly stringent condition; and (e) that is complementary to the nucleotide sequence of any of (a)-(c). <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

(Field of Invention)
The present invention relates to fibroblast growth factor-23 (FGF-23) polypeptide and nucleic acid molecules encoding the same. The invention also relates to selective binding agents, vectors, host cells and methods for producing FGF-23 polypeptides. The invention further relates to pharmaceutical compositions and methods for diagnosis, treatment, amelioration, and / or prevention of diseases, disorders, and conditions associated with FGF-23 polypeptides.

(Background of the Invention)
Technological advances in the identification, cloning, expression, and manipulation of nucleic acid molecules and the decoding of the human genome have greatly accelerated the discovery of new therapeutic agents. Currently, high-speed nucleic acid sequencing technology can generate sequence information at an unprecedented rate, and combined with computer analysis, allows for the assembly of overlapping sequences and parts of the entire genome and identification of polypeptide coding regions To do. Comparison of the deduced amino acid sequence to a database compilation of known amino acid sequences allows to determine the degree of homology to previously identified sequence and / or structural landmarks. Cloning and expression of the polypeptide coding region of a nucleic acid molecule provides a polypeptide product for structural and functional analysis. Manipulation of the nucleic acid molecule and encoded polypeptide can confer advantageous properties on the product for use as a therapeutic agent.

  Despite significant technological advances in genome research over the past decade, the potential for the development of new therapeutic agents based on the human genome has yet to be realized. A number of genes that encode potentially advantageous polypeptide therapeutics or the polypeptides they encode (which may serve as “targets” to the therapeutic molecule) have not yet been identified.

  Accordingly, it is an object of the present invention to identify novel polypeptides and nucleic acid molecules encoding them that have diagnostic or therapeutic advantages.

(Summary of the Invention)
The present invention relates to novel FGF-23 nucleic acid molecules and encoded polypeptides.

The present invention provides an isolated nucleic acid molecule comprising the following:
(A) the nucleotide sequence shown in SEQ ID NO: 1;
(B) the nucleotide sequence of the DNA insert in ATCC accession number PTA-1617;
(C) a nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 2;
(D) a nucleotide sequence that hybridizes to the complement of any of (a)-(c) under moderately or highly stringent conditions; and (e) any of (a)-(c) Complementary nucleotide sequence,
A nucleotide sequence selected from the group consisting of:

The invention also provides an isolated nucleic acid molecule, the nucleic acid molecule comprising:
(A) a nucleotide sequence encoding a polypeptide that is at least about 70% identical to the polypeptide set forth in SEQ ID NO: 2, wherein the encoded polypeptide is a polypeptide sequence represented by SEQ ID NO: 2 A nucleotide sequence having activity;
(B) the nucleotide sequence shown in SEQ ID NO: 1, the nucleotide sequence of the DNA insert in ATCC Accession No. PTA-1617, or the nucleotide sequence encoding the allelic variant or splice variant of (a);
(C) a region of the nucleotide sequence of SEQ ID NO: 1 encoding a polypeptide fragment of at least about 25 amino acid residues, a region of the nucleotide sequence of the DNA insert in ATCC accession number PTA-1617, a region of the nucleotide sequence of (a) Or a region of the nucleotide sequence of (b), wherein the polypeptide fragment has the activity of the encoded polypeptide shown in SEQ ID NO: 2 or is antigenic;
(D) a region of the nucleotide sequence of SEQ ID NO: 1 comprising a fragment of at least about 16 nucleotides, a region of the nucleotide sequence of the DNA insert in ATCC Accession No. PTA-1617, or any nucleotide of (a)-(c) Region of the sequence;
(E) a nucleotide sequence that hybridizes under moderate or highly stringent conditions to the complement of any of (a)-(d); and (f) any of (a)-(d) A nucleotide sequence complementary to
A nucleotide sequence selected from the group consisting of:

The invention further provides an isolated nucleic acid molecule, the nucleic acid molecule comprising:
(A) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO: 2 having at least one conservative amino acid substitution, wherein the encoded polypeptide is an activity of the polypeptide set forth in SEQ ID NO: 2 Having a nucleotide sequence;
(B) a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 2 having at least one amino acid insertion, wherein the encoded polypeptide has the activity of the polypeptide set forth in SEQ ID NO: 2 Nucleotide sequence;
(C) a nucleotide sequence encoding a polypeptide shown in SEQ ID NO: 2 having at least one amino acid deletion, wherein the encoded polypeptide exhibits activity of the polypeptide shown in SEQ ID NO: 2 Having a nucleotide sequence;
(D) a nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 2 having a C-terminal truncation and / or an N-terminal truncation, wherein the encoded polypeptide is a polypeptide shown in SEQ ID NO: 2 A nucleotide sequence having the activity of:
(E) a nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 2 having at least one modification selected from the group consisting of amino acid substitution, amino acid insertion, amino acid deletion, C-terminal truncation, and N-terminal truncation, Wherein the encoded polypeptide has a nucleotide sequence having the activity of the polypeptide set forth in SEQ ID NO: 2;
(F) the nucleotide sequence of any of (a)-(e) comprising a fragment of at least about 16 nucleotides;
(G) a nucleotide sequence that hybridizes under moderate or highly stringent conditions to the complement of any of (a)-(f); and (h) any of (a)-(e) A nucleotide sequence complementary to
A nucleotide sequence selected from the group consisting of:

The present invention provides an isolated polypeptide, the polypeptide comprising:
(A) the amino acid sequence shown in SEQ ID NO: 2; and (b) the amino acid sequence encoded by the DNA insert in ATCC Accession No. PTA-1617,
An amino acid sequence selected from the group consisting of:

The invention also provides an isolated polypeptide, the polypeptide comprising:
(A) the amino acid sequence set forth in SEQ ID NO: 3 further comprising an amino terminal methionine as required;
(B) the amino acid sequence for the ortholog of SEQ ID NO: 2;
(C) an amino acid sequence that is at least about 70% identical to the amino acid sequence of SEQ ID NO: 2, wherein the polypeptide has the activity of the polypeptide shown in SEQ ID NO: 2;
(D) a fragment of the amino acid sequence shown in SEQ ID NO: 2 comprising at least about 25 amino acid residues, wherein said fragment has the activity of the polypeptide shown in SEQ ID NO: 2 or is antigenic A fragment; and (e) the amino acid sequence set forth in SEQ ID NO: 2, the amino acid sequence encoded by the DNA insert in ATCC Accession No. PTA-1617, or an allelic variant or splice of (a)-(c) The amino acid sequence of the variant,
An amino acid sequence selected from the group consisting of:

The present invention further provides an isolated polypeptide, the polypeptide comprising:
(A) the amino acid sequence shown in SEQ ID NO: 2 having at least one conservative amino acid substitution, wherein the polypeptide has the activity of the polypeptide shown in SEQ ID NO: 2;
(B) the amino acid sequence shown in SEQ ID NO: 2 having at least one amino acid insertion, wherein the polypeptide has the activity of the polypeptide shown in SEQ ID NO: 2;
(C) the amino acid sequence shown in SEQ ID NO: 2 having at least one amino acid deletion, wherein the polypeptide has the activity of the polypeptide shown in SEQ ID NO: 2;
(D) the amino acid sequence shown in SEQ ID NO: 2 having a C-terminal truncation and / or an N-terminal truncation, wherein the polypeptide has the activity of the polypeptide shown in SEQ ID NO: 2; And (e) the amino acid sequence set forth in SEQ ID NO: 2 having at least one modification selected from the group consisting of amino acid substitutions, amino acid insertions, amino acid deletions, C-terminal truncations, and N-terminal truncations, wherein The polypeptide is an amino acid sequence having the activity of the polypeptide represented by SEQ ID NO: 2,
An amino acid sequence selected from the group consisting of:

  A fusion polypeptide comprising the FGF-23 amino acid sequence is also provided.

  The present invention also includes an expression vector comprising an isolated nucleic acid molecule as set forth herein, a recombinant host cell comprising a recombinant nucleic acid molecule as set forth herein, and FGF-23 poly A method of producing a peptide is provided, the method comprising culturing the host cell and optionally isolating the polypeptide so produced.

  Also included in the invention are transgenic non-human animals comprising a nucleic acid molecule encoding an FGF-23 polypeptide. The FGF-23 nucleic acid molecule is introduced into the animal in a manner that allows expression of the FGF-23 polypeptide and increased levels of FGF-23 polypeptide, which may include increased circulating levels. Alternatively, the FGF-23 nucleic acid molecule is introduced into the animal in a manner that inhibits expression of the endogenous FGF-23 polypeptide (ie, creates a transgenic animal carrying the FGF-23 polypeptide gene knockout). The transgenic non-human animal is preferably a mammal, more preferably a rodent (eg, rat or mouse).

  Derivatives of the FGF-23 polypeptides of the present invention are also provided.

  Further provided are selective binding agents (eg, antibodies and peptides) that can specifically bind to the FGF-23 polypeptides of the present invention. Such antibodies and peptides may be agonists or antagonists.

  Also encompassed by the present invention is a pharmaceutical composition comprising a nucleotide, polypeptide, or selective binding agent of the present invention and one or more pharmaceutically acceptable formulations. The pharmaceutical composition is used to provide a therapeutically effective amount of a nucleotide or polypeptide of the invention. The invention also relates to methods of using this polypeptide, nucleic acid molecule, and selective binding agent.

  The FGF-23 polypeptides and FGF-23 nucleic acid molecules of the invention can be used to treat, prevent, ameliorate, and / or detect diseases and disorders, including those listed herein.

  The invention also provides a method of assaying a test molecule to identify a test molecule that binds to an FGF-23 polypeptide. The method includes contacting an FGF-23 polypeptide with a test molecule to determine the extent of binding of the test molecule to the polypeptide. The method further includes determining whether such a test molecule is an agonist or antagonist of the FGF-23 polypeptide. The invention further provides a method of testing the effect of a molecule on the expression of FGF-23 polypeptide or the activity of FGF-23 polypeptide.

  Also encompassed by the invention are methods of modulating the expression of FGF-23 polypeptide and methods of modulating (ie, increasing or decreasing) the level of FGF-23 polypeptide. One method involves administering to an animal a nucleic acid molecule encoding an FGF-23 polypeptide. In another method, a nucleic acid molecule comprising an element that modulates or regulates expression of an 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 invention, an FGF-23 polypeptide can be used to identify its receptor (“FGF-23 polypeptide receptor”). Various forms of “expression cloning” have been used extensively to clone receptors for protein ligands. See, for example, Simonsen and Rodish, 1994, Trends Pharmacol. Sci. 15: 437-41 and Tartaglia et al., 1995, Cell 83: 1263-71. Isolation of the FGF-23 polypeptide receptor is useful for the identification or development of novel agonists and novel antagonists of the FGF-23 polypeptide signaling pathway. Such agonists and antagonists include soluble FGF-23 polypeptide receptors, anti-FGF-23 polypeptide receptor-selective binding agents (eg, antibodies and derivatives thereof), small molecules, and antisense oligonucleotides, Any of these may be useful for the treatment of one or more diseases or disorders, including those disclosed herein.
Thus, the present invention provides the following:
(1) An isolated nucleic acid molecule comprising:
(A) the nucleotide sequence shown in SEQ ID NO: 1;
(B) the nucleotide sequence of the DNA insert in ATCC accession number PTA-1617;
(C) a nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 2;
(D) a nucleotide sequence that hybridizes to the complement of any of (a)-(c) under moderately or highly stringent conditions; and (e) any of (a)-(c) Complementary nucleotide sequence,
An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
(2) An isolated nucleic acid molecule comprising:
(A) a nucleotide sequence encoding a polypeptide that is at least about 70% identical to the polypeptide set forth in SEQ ID NO: 2, wherein the encoded polypeptide is a polypeptide sequence represented by SEQ ID NO: 2 A nucleotide sequence having activity;
(B) the nucleotide sequence shown in SEQ ID NO: 1, the nucleotide sequence of the DNA insert in ATCC Accession No. PTA-1617, or the nucleotide sequence encoding the allelic variant or splice variant of (a);
(C) a nucleotide sequence of SEQ ID NO: 1 encoding a polypeptide fragment of at least about 25 amino acid residues, a DNA insert in ATCC Accession No. PTA-1617, a region of (a) or (b), wherein A region wherein the polypeptide fragment has the activity of the encoded polypeptide set forth in SEQ ID NO: 2 or is antigenic;
(D) the nucleotide sequence of SEQ ID NO: 1, a DNA insert in ATCC Accession No. PTA-1617, or any region of (a)-(c), comprising a fragment of at least about 16 nucleotides;
(E) a nucleotide sequence that hybridizes under moderate or highly stringent conditions to the complement of any of (a)-(d); and (f) any of (a)-(d) A nucleotide sequence complementary to
An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
(3) an isolated nucleic acid molecule, the following:
(A) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO: 2 having at least one conservative amino acid substitution, wherein the encoded polypeptide comprises an activity of the polypeptide set forth in SEQ ID NO: 2 Having a nucleotide sequence;
(B) a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 2 having at least one amino acid insertion, wherein the encoded polypeptide has the activity of the polypeptide set forth in SEQ ID NO: 2 Nucleotide sequence;
(C) a nucleotide sequence encoding a polypeptide shown in SEQ ID NO: 2 having at least one amino acid deletion, wherein the encoded polypeptide exhibits activity of the polypeptide shown in SEQ ID NO: 2 Having a nucleotide sequence;
(D) a nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 2 having a C-terminal truncation and / or an N-terminal truncation, wherein the encoded polypeptide is a polypeptide shown in SEQ ID NO: 2 A nucleotide sequence having the activity of:
(E) a nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 2 having at least one modification selected from the group consisting of amino acid substitution, amino acid insertion, amino acid deletion, C-terminal truncation, and N-terminal truncation, Wherein the encoded polypeptide has the activity of the polypeptide set forth in SEQ ID NO: 2, a nucleotide sequence;
(F) the nucleotide sequence of any of (a)-(e) comprising a fragment of at least about 16 nucleotides;
(G) a nucleotide sequence that hybridizes under moderate or highly stringent conditions to the complement of any of (a)-(f); and (h) any of (a)-(e) A nucleotide sequence complementary to
An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
(4) A vector comprising the nucleic acid molecule according to any one of items 1, 2 and 3.
(5) A host cell comprising the vector according to item 4.
(6) The host cell according to item 5, which is a eukaryotic cell.
(7) The host cell according to item 5, which is a prokaryotic cell.
(8) A process for producing an FGF-23 polypeptide comprising culturing the host cell according to item 5 under conditions suitable for expressing the polypeptide, and, if necessary, Isolating said polypeptide from the culture.
(9) A polypeptide produced by the process according to item 8.
(10) The process according to item 8, wherein the nucleic acid molecule is operably linked to DNA encoding FGF-23 polypeptide other than promoter DNA for native FGF-23 polypeptide. A process comprising promoter DNA.
(11) The isolated nucleic acid molecule according to item 2, wherein the percent identity is selected from the group consisting of GAP, BLASTN, FASTA, BLASTA, BLASTX, BestFit, and Smith-Waterman algorithm. An isolated nucleic acid molecule determined using a computer program.
(12) A process for determining whether a compound inhibits FGF-23 polypeptide activity or FGF-23 polypeptide production, the cell according to any of item 5, item 6, or item 7 Exposing the compound to a compound, and measuring FGF-23 polypeptide activity or FGF-23 polypeptide production in the cell.
(13) An isolated polypeptide comprising:
(A) the amino acid sequence shown in SEQ ID NO: 2; and (b) the amino acid sequence encoded by the DNA insert in ATCC Accession No. PTA-1617,
An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
(14) An isolated polypeptide comprising:
(A) the amino acid sequence shown in SEQ ID NO: 3, and optionally further comprising an amino terminal methionine;
(B) the amino acid sequence for the ortholog of SEQ ID NO: 2;
(C) an amino acid sequence that is at least about 70% identical to the amino acid sequence of SEQ ID NO: 2, wherein the polypeptide has the activity of the polypeptide set forth in SEQ ID NO: 2;
(D) a fragment of the amino acid sequence shown in SEQ ID NO: 2 comprising at least about 25 amino acid residues, wherein the fragment has the activity of the polypeptide shown in SEQ ID NO: 2 or is antigenic A fragment; and (e) the amino acid sequence set forth in SEQ ID NO: 2, the amino acid sequence encoded by the DNA insert in ATCC Accession No. PTA-1617, or an allelic variant or splice of (a)-(c) The amino acid sequence of the variant,
An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
(15) An isolated polypeptide comprising:
(A) an amino acid sequence set forth in SEQ ID NO: 2 having at least one conservative amino acid substitution, wherein the polypeptide has the activity of the polypeptide set forth in SEQ ID NO: 2;
(B) an amino acid sequence set forth in SEQ ID NO: 2 having at least one amino acid insertion, wherein the polypeptide has the activity of the polypeptide set forth in SEQ ID NO: 2;
(C) the amino acid sequence shown in SEQ ID NO: 2 having at least one amino acid deletion, wherein the polypeptide has the activity of the polypeptide shown in SEQ ID NO: 2;
(D) an amino acid sequence set forth in SEQ ID NO: 2 having a C-terminal truncation and / or an N-terminal truncation, wherein the polypeptide has the activity of the polypeptide set forth in SEQ ID NO: 2; And (e) the amino acid sequence set forth in SEQ ID NO: 2 having at least one modification selected from the group consisting of amino acid substitutions, amino acid insertions, amino acid deletions, C-terminal truncations, and N-terminal truncations, wherein The polypeptide has an amino acid sequence having the activity of the polypeptide shown in SEQ ID NO: 2,
An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
(16) An isolated polypeptide encoded by the nucleic acid molecule according to any one of items 1, 2, or 3, wherein the polypeptide is the polypeptide represented by SEQ ID NO: 2. An isolated polypeptide having the activity of:
(17) The isolated polypeptide according to item 14, wherein the percent identity is selected from the group consisting of GAP, BLASTP, FASTA, BLASTA, BLASTX, BestFit, and Smith-Waterman algorithm. An isolated polypeptide determined using a computer program.
(18) A selective binding agent or a fragment thereof that specifically binds to the polypeptide according to any one of items 13, 14, or 15.
(19) The selective binding agent or fragment thereof according to item 18, which specifically binds to a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or a fragment thereof.
(20) The selective binding agent according to item 18, which is an antibody or a fragment thereof.
(21) The selective binding agent according to item 18, which is a humanized antibody.
(22) The selective binding agent according to item 18, which is a human antibody or a fragment thereof.
(23) The selective binding agent according to item 18, which is a polyclonal antibody or a fragment thereof.
(24) The selective binding agent according to item 18, which is a monoclonal antibody or a fragment thereof.
(25) The selective binding agent according to item 18, which is a chimeric antibody or a fragment thereof.
(26) The selective binding agent according to item 18, which is a CDR-grafted antibody or a fragment thereof.
(27) The selective binding agent according to item 18, which is an anti-idiotype antibody or a fragment thereof.
(28) The selective binding agent according to item 18, which is a variable region fragment.
(29) The variable region fragment according to item 28, which is a Fab fragment or a Fab ′ fragment.
(30) A selective binding agent or fragment thereof comprising at least one complementarity determining region having specificity for a polypeptide having the amino acid sequence of SEQ ID NO: 2.
(31) The selective binding agent according to item 18, which is bound to a detectable label.
(32) The selective binding agent according to item 18, which antagonizes the biological activity of FGF-23 polypeptide.
(33) A method for treating, preventing or ameliorating a FGF-23 polypeptide-related disease, condition or disorder, the method comprising administering an effective amount of the selective binding agent according to item 18 to a patient. Including the method.
(34) A selective binding agent produced by immunizing an animal with a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.
(35) A hybridoma that produces a selective binding agent capable of binding the polypeptide according to any one of items 1, 2, or 3.
(36) A method for detecting or quantifying the amount of FGF-23 polypeptide using the anti-FGF-23 antibody or fragment according to item 18.
(37) A composition comprising the polypeptide of any of items 13, 14, or 15 and a pharmaceutically acceptable formulation.
(38) A composition according to item 37, wherein the pharmaceutically acceptable formulation is a carrier, an adjuvant, a solubilizer, a stabilizer, or an antioxidant.
(39) A composition according to item 37, wherein the polypeptide comprises the amino acid sequence represented by SEQ ID NO: 3.
(40) A polypeptide comprising a derivative of the polypeptide according to any one of item 13, item 14, or item 15.
(41) The polypeptide according to item 40, which is covalently modified with a water-soluble polymer.
(42) The polypeptide according to item 41, wherein the water-soluble polymer is polyethylene glycol, monomethoxypolyethylene glycol, dextran, cellulose, poly- (N-vinylpyrrolidone) polyethylene glycol, or propylene glycol homopolymer. A polypeptide selected from the group consisting of a polypropylene oxide / ethylene oxide copolymer, a polyoxyethylated polyol, and polyvinyl alcohol.
(43) A composition comprising the nucleic acid molecule according to any one of items 1, 2 and 3, and a pharmaceutically acceptable formulation.
(44) A composition according to item 43, wherein the nucleic acid molecule is contained in a viral vector.
(45) A viral vector comprising the nucleic acid molecule according to any one of items 1, 2 and 3.
(46) A fusion polypeptide comprising the polypeptide of any one of items 13, 14, or 15 fused to a heterologous amino acid sequence.
(47) The fusion polypeptide according to item 46, wherein the heterologous amino acid sequence is an IgG constant domain or a fragment thereof.
(48) A method for treating, preventing, or ameliorating a medical condition, comprising the polypeptide according to any one of items 13, 14, or 15, or the item 1, the item 2, or the item 3. A method comprising administering to a patient a polypeptide encoded by any of the nucleic acids.
(49) A method for treating, preventing or ameliorating a medical condition, comprising the polypeptide according to any one of items 13, 14 or 15, or the nucleic acid according to any one of items 1, 2, or 3. Administering to the patient an agonist or antagonist of the biological activity of the encoded polypeptide.
(50) A method according to any of items 48 or 49, wherein the medical condition to be treated, prevented or ameliorated is autosomal dominant hypophosphatemic rickets (ADHR).
(51) A method for diagnosing a pathological condition or susceptibility to a pathological condition in a subject, comprising:
(A) a polypeptide according to any one of items 13, 14, or 15 or a polypeptide encoded by a nucleic acid molecule according to any of items 1, 2 or 3 in a sample; Determining the presence or level of expression; and (b) diagnosing the pathological condition or susceptibility to the pathological condition based on the presence or level of expression of the polypeptide,
Including the method.
(52) Device, the following:
(A) a membrane suitable for transplantation; and (b) a cell encapsulated within the membrane, wherein the cell secretes a protein of any of items 13, 14, or 15;
With
The device wherein the membrane is permeable to the protein and impermeable to substances harmful to the cells.
(53) A method for identifying a compound that binds to an FGF-23 polypeptide, comprising:
(A) contacting the polypeptide of any of items 13, 14, or 15 with a compound; and (b) determining the degree of binding of the FGF-23 polypeptide to the compound;
Including the method.
(54) A method according to item 53, further comprising a step of determining the activity of the polypeptide when bound to the compound.
(55) A method for regulating the level of a polypeptide in an animal, comprising the step of administering to the animal the nucleic acid molecule of any of items 1, 2 or 3.
(56) A transgenic non-human mammal comprising the nucleic acid molecule according to any one of items 1, 2, or 3.
(57) A process for determining whether a compound inhibits FGF-23 polypeptide activity or FGF-23 polypeptide production, comprising exposing the transgenic mammal according to item 56 to the compound And measuring FGF-23 polypeptide activity or FGF-23 polypeptide production in said mammal.

(Detailed description of the invention)
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All references cited in this application are expressly incorporated herein by reference.

(Definition)
The terms "FGF-23 gene" or "FGF-23 nucleic acid molecule" or "FGF-23 polynucleotide" refer to the nucleotide sequence set forth in SEQ ID NO: 1, the nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 2, ATCC Nucleic acid molecule comprising or consisting of the nucleotide sequence of the DNA insert under accession number PTA-1617 and the nucleic acid molecule as defined herein.

  The term “FGF-23 polypeptide allelic variant” refers to one of several naturally occurring alternative forms of a gene that occupies a given locus of a chromosome of an organism or population of organisms. .

  The term “FGF-23 polypeptide splice variant” refers to a nucleic acid molecule (usually RNA ).

  The term “isolated nucleic acid molecule” refers to (1) at least about 50 percent of proteins, lipids, carbohydrates, or other substances that are naturally found together when total nucleic acid is isolated from a source cell. (2) a nucleic acid molecule of the present invention that is not linked to all or part of a polynucleotide to which an “isolated nucleic acid molecule” is naturally linked, (3) ) Refers to a nucleic acid molecule of the invention that is operably linked to a polynucleotide that is not naturally linked, or (4) a nucleic acid molecule of the invention that does not exist naturally as part of a larger polynucleotide sequence. Preferably, an isolated nucleic acid molecule of the invention is any other contaminating nucleic acid molecule, or other contaminant found in the natural environment (which may be used in polypeptide production, or therapeutic use, diagnostic use). , Which prevents prophylactic or research use).

  The term “nucleic acid sequence” or “nucleic acid molecule” refers to a DNA or RNA sequence. The term includes molecules formed from any of the known base analogs of DNA and RNA, which base analogs include, for example, but are not limited to: 4-acetylcytosine, 8-hydroxy-N6- Methyladenosine, aziridinyl-cytosine, pseudoisocytosine, 5- (carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydro Uracil, inosine, N6-iso-pentenyladenine, 1-methyladenine, 1-methyl pseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcyto 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-ethylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, β-D-mannosylqueosine, 5′-methoxycarbonyl-methyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutoxocin, pseudouracil, queosine ), 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, pseudouracil, Queosine, 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 into a host cell.

  The term “expression vector” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and / or control the expression of inserted heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing (if an intron is present).

  The term “operably linked” is used herein to refer to a method of arrangement of adjacent sequences. Here, the adjacent sequence thus described is configured or assembled to perform its normal function. Thus, flanking sequences operably linked to a coding sequence can result in replication, transcription and / or translation of the coding sequence. For example, if a promoter is capable of directing transcription of a coding sequence, the coding sequence is operably linked to the promoter. A flanking sequence need not be contiguous with the coding sequence, so long as it functions correctly. Thus, for example, an untranslated but transcribed intervening sequence can exist between a promoter sequence and a coding sequence, and the promoter sequence is still considered “operably linked” to the coding sequence. obtain.

  The term “host cell” is used to refer to a transformed cell or a cell that can be transformed with a nucleic acid sequence and then express a selected gene of interest. The term includes this progeny of the parent cell, regardless of whether the progeny is identical in nature or nature to the original parent as long as the selected gene is present.

  The term “FGF-23 polypeptide” refers to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and related polypeptides. Related polypeptides include: FGF-23 polypeptide fragments, FGF-23 polypeptide orthologs, FGF-23 polypeptide variants, and FGF-23 polypeptide derivatives. These have at least one activity of the polypeptide shown in SEQ ID NO: 2. The FGF-23 polypeptide can be a mature polypeptide as defined herein and may or may not have an amino terminal methionine residue depending on the method of preparation.

  The term “FGF-23 polypeptide fragment” includes shortening of the amino terminus (with or without the leader sequence) and / or shortening of the carboxyl terminus of the FGF-23 polypeptide shown in SEQ ID NO: 2. Refers to a polypeptide. The term “FGF-23 polypeptide fragment” also refers to a shortening of the amino and / or carboxyl terminus of an FGF-23 polypeptide ortholog, FGF-23 polypeptide derivative, or FGF-23 polypeptide variant, Alternatively, it refers to a shortening of the amino and / or carboxyl terminus of a polypeptide encoded by an FGF-23 polypeptide allelic variant or FGF-23 polypeptide splice variant. FGF-23 polypeptide fragments can result from alternative RNA splicing or from in vivo protease activity. Membrane-bound forms of FGF-23 polypeptides are also contemplated by the present invention. In preferred embodiments, the shortening and / or deletion comprises 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 100 amino acids. Polypeptide fragments so produced contain about 25 consecutive 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 200 amino acids. . Such FGF-23 polypeptide fragments can optionally include 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 from another species corresponding to the FGF-23 polypeptide amino acid sequence shown in SEQ ID NO: 2. For example, mouse and human FGF-23 polypeptides are considered orthologous to each other.

  The term “FGF-23 polypeptide variant” refers to a substitution of one or more amino acid sequences as compared to the FGF-23 polypeptide amino acid sequence shown in SEQ ID NO: 2 (with or without a leader sequence). FGF-23 polypeptides comprising amino acid sequences having deletions (eg, internal deletions and / or FGF-23 polypeptide fragments), and / or additions (eg, internal additions and / or FGF-23 fusion polypeptides) Say. Variants can exist in nature (eg, FGF-23 polypeptide allelic variants, FGF-23 polypeptide orthologs, and FGF-23 splice variants) or can be artificially constructed. Such FGF-23-polypeptide variants can be prepared from corresponding nucleic acid molecules having a DNA sequence that is altered from the DNA sequence set forth in SEQ ID NO: 1. In preferred embodiments, the variant is 1-3, or 1-5, or 1-10, or 1-15, or 1-20, or 1-25, or 1-50, or 1-75, or Have 1 to 100, or more than 100 amino acid substitutions, insertions, additions and / or deletions, where the substitutions can be conservative or non-conservative, or combinations thereof It can be.

  The term “FGF-23 polypeptide derivative” refers to a chemically modified polypeptide shown in SEQ ID NO: 2, FGF-23 polypeptide fragment, FGF-23 poly as defined herein. It refers to a peptide ortholog or FGF-23 polypeptide variant. The term “FGF-23 polypeptide derivative” also refers to chemically modified FGF-23 polypeptide allelic variants or FGF-23 polypeptide splice variants as defined herein. Refers to the encoded polypeptide.

  The term “mature FGF-23 polypeptide” refers to an FGF-23 polypeptide that lacks a leader sequence. Mature FGF-23 polypeptides also have amino-terminal (with or without leader sequence) and / or carboxyl-terminal proteolytic processing, cleavage of smaller polypeptides from larger precursors, N-linked forms Other modifications of the polypeptide such as glycosylation and / or O-linked glycosylation may be included. An exemplary mature CHL polypeptide is represented by the amino acid sequence of SEQ ID NO: 3.

  The term “FGF-23 fusion polypeptide” refers to the polypeptide shown in SEQ ID NO: 2, FGF-23 polypeptide fragment, FGF-23 polypeptide ortholog, FGF-23 poly as defined herein. A peptide variant or a fusion of one or more amino acids (eg, a heterologous protein or peptide) at the amino terminus or carboxyl terminus of an FGF-23 polypeptide derivative. The term “FGF-23 fusion polypeptide” also refers to the amino acid of a polypeptide encoded by an FGF-23 polypeptide allelic variant or FGF-23 polypeptide splice variant, as defined herein. A fusion of one or more amino acids at the terminus or carboxyl terminus.

  The term “biologically active FGF-23 polypeptide” refers to an FGF-23 polypeptide having at least one activity characteristic of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. Furthermore, the FGF-23 polypeptide can be active as an immunogen; that is, the FGF-23 polypeptide can comprise at least one epitope against which an antibody can be raised.

  The term “isolated polypeptide” refers to a polypeptide of the invention as follows: (1) polynucleotides, lipids, carbohydrates that are naturally found together when isolated from a source cell Or a polypeptide that is separated from at least about 50% of other materials, (2) all or part of the polypeptide to which the “isolated polypeptide” is naturally bound (covalently linked to each other). A polypeptide that is not bound (by action or non-covalent interaction), (3) operably linked to a polypeptide that is not naturally bound (by covalent or non-covalent interaction) Or (4) a non-naturally occurring polypeptide. Preferably, the isolated polypeptide substantially eliminates any other contaminating polypeptide or other contaminant found in its natural environment that interferes with its therapeutic, diagnostic, prophylactic or research use. Not included.

  The term “identity” is determined by comparing sequences of two or more polypeptide molecules, or two or more nucleic acid molecules, as is known in the art. , Say relationship. In the art, “identity” is also determined by the degree of sequence relatedness of a nucleic acid molecule or polypeptide, possibly in this case by a match between a string of two or more nucleotide sequences or two or more amino acid sequences. Meaning). “Identity” refers to the identity match between the shorter of two or more sequences with gap alignment (if any), as located by a particular arithmetic model or computer program (ie, algorithm) Measure percentage.

  The term “similarity” is a related concept, but in contrast to “identity”, “similarity” is a measure of relevance that includes both identity and conservative substitution matches. Say. If the two polypeptide sequences have, for example, 10/20 identical amino acids and the rest are all non-conservative substitutions, the percent identity and percent similarity are both 50%. In the same example, if there are five more positions that are conservative substitutions, the percent identity remains 50%, but the percent similarity is 75% (15/20). Thus, when there are conservative substitutions, the percent similarity between two polypeptides is higher than the percent identity between these two polypeptides.

  The term “naturally occurring” or “native” when used with reference to biological materials such as nucleic acid molecules, polypeptides, host cells, etc., refers to materials that are found in nature and have not been manipulated by humans. . Similarly, “non-naturally occurring” or “non-native” as used herein refers to materials that are not found in nature, or that have been structurally altered or synthesized by humans. .

  The terms “effective amount” and “therapeutically effective amount” each support an observable level of one or more biological activities of the FGF-23 polypeptides set forth herein. Refers to the amount of FGF-23 polypeptide or FGF-23 nucleic acid molecule used.

  As used herein, the term “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” refers to an FGF-23 polypeptide, FGF-23 nucleic acid molecule, as a pharmaceutical composition, Or refers to one or more formulation materials suitable to achieve or enhance delivery of an FGF-23 selective binding agent.

  The term “antigen” is a molecule or part of a molecule that can be bound by a selective binding agent (eg, an antibody) and is further used in an animal to produce an antibody that can bind to an epitope of that antigen. Refers to a molecule or part of a molecule that can be obtained. An antigen can have one or more epitopes.

  The term “selective binding agent” refers to a molecule or molecules that have specificity for an FGF-23 polypeptide. As used herein, the terms “specific” and “specificity” refer to the ability of a selective binding agent to bind to a human FGF-23 polypeptide but not to a human non-FGF-23 polypeptide. Say. However, the selective binding agent also binds an ortholog of a polypeptide as shown in SEQ ID NO: 2 (ie, an interspecies version (eg, mouse FGF-23 polypeptide and rat FGF-23 polypeptide)). It is understood that you get.

  The term “transduction” is used to refer to the transfer of a gene (usually by phage) from one bacterium to another. “Transduction” also refers to the capture and introduction of eukaryotic cell sequences by retroviruses.

  The term “transfection” is used to refer to the uptake of foreign or exogenous DNA by a cell. A cell is then “transfected” when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art and are disclosed herein. For example, Graham et al., 1973, Virology 52: 456; Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratories, 1989); Davis et al., Basic Meth. See Gene 13: 197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

  The term “transformation” as used herein refers to a change in the genetic characteristics of a cell, and a cell has been transformed if it has been modified to contain new DNA. For example, a cell has been transformed when it has been genetically modified from its native state. Following transfection and transduction, the transforming DNA can be recombined with the cell's DNA by physical integration into the cell's chromosomes, can be maintained transiently as a non-replicating episomal element, or as a plasmid. Can be replicated independently. A cell is considered stably transformed if its DNA replicates with the division of the cell.

(Relevance of nucleic acid molecules and / or polypeptides)
It is understood that related nucleic acid molecules include allelic or splice variants of the nucleic acid molecule of SEQ ID NO: 1 and include sequences that are complementary to any of the above nucleotide sequences. A related nucleic acid molecule is also a polypeptide comprising or consisting essentially of substitution, modification, addition, and / or deletion of one or more amino acid residues as compared to the polypeptide of SEQ ID NO: 2. A nucleotide sequence encoding Such related FGF-23 polypeptides can include, for example, the addition and / or deletion of one or more N-linked or O-linked glycosylation sites, or the addition of one or more cysteine residues. / Or may contain deletions.

  Related nucleic acid molecules also have at least about 25 contiguous amino acid residues, or about 50 amino acid residues, or about 75 amino acid residues, or about 100 amino acid residues of the amino acid residues of the FGF-23 polypeptide of SEQ ID NO: 2. Or a fragment of an FGF-23 nucleic acid molecule encoding a polypeptide of more than about 100 amino acid residues.

  Furthermore, related FGF-23 nucleic acid molecules also include those of the FGF-23 nucleic acid molecule of SEQ ID NO: 1 under moderately stringent conditions or highly stringent conditions as defined herein. A complete complementary sequence, or a complete complementary sequence of a molecule encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 2, or a complete complementary sequence of a nucleic acid fragment as defined herein Or a molecule comprising a nucleotide sequence that hybridizes to the fully complementary sequence 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 a cDNA library, genomic library, or synthetic DNA library for related sequences. Regions of the DNA and / or amino acid sequence of FGF-23 polypeptides that exhibit significant identity with known sequences are readily determined using a sequence alignment algorithm as described herein, and these This region can be used to design probes for screening.

The term “highly stringent conditions” refers to conditions designed to allow hybridization of DNA strands whose sequences are highly complementary and to exclude hybridization of significantly mismatched DNA. Say. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of “highly stringent conditions” for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68 ° C., or 0.015 M sodium chloride at 42 ° C., 0 .0015M
Sodium citrate, and 50% formamide. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory, Chapter 1989); Anderson et al., Nucleic Acid Hybrid.

More stringent conditions (eg, higher temperature, lower ionic strength, higher formamide, or other denaturing agents) may be used-however, the rate of hybridization is affected. Other agents can be included in the hybridization and wash buffers for the purpose of reducing non-specific hybridization and / or background hybridization. Examples include 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (NaDodSO ₄ or SDS), Ficoll, Denhardt's solution, sonication Salmon sperm DNA (or another non-complementary DNA), and dextran sulfate, although other suitable agents can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is largely independent of pH. See Anderson et al., Nucleic Acid Hybridization: A Practical Approach, Chapter 4 (IRL Press Limited).

Factors that affect the stability of the DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by those skilled in the art to accommodate these variables and to allow different sequence related DNAs to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following formula:
Tm (° C.) = 81.5 + 16.6 (log [Na +]) + 0.41 (% G + C) −600 / N−0.72 (% formamide)
Where N is the length of the duplex formed, [Na +] is the molar concentration of sodium ions in the hybridization or wash solution, and% G + C is (guanine + cytosine in the hybrid) ) Percentage of base. For imperfectly matched hybrids, the melting temperature decreases by about 1 ° C. for each 1% mismatch.

  The term “moderate” stringent conditions refers to conditions under which DNA duplexes with a greater degree of base pair mismatch can form than can occur under “highly stringent conditions”. Examples of representative “moderately stringent conditions” are 0.015 M sodium chloride, 0.0015 M sodium citrate at 50-65 ° C., or 0.015 M sodium chloride at 37-50 ° C. 0.0015 M sodium citrate, and 20% formamide. As an example, a “moderately stringent” condition of 50 ° C. in 0.015 M sodium ion tolerates a mismatch of about 21%.

  It will be appreciated by those skilled in the art that there is no complete distinction between “highly” stringent conditions and “moderate” stringent conditions. For example, at 0.015M sodium ion (no formamide), the melting temperature of perfectly matched long DNA is about 71 ° C. In washing at 65 ° C. (same ionic strength), this allows about 6% mismatch. To capture the related sequences more remotely, one skilled in the art can simply reduce the temperature or increase the ionic strength.

For oligonucleotide probes up to about 20 nt, a reasonable estimate of the melting temperature in 1M NaCl ^* is provided by:
Tm = 2 ° C per AT base + 4 ° C per GC base pair
^* The sodium ion concentration in 6x sodium citrate (SSC) is 1M. See Suggs et al., Developmental Biology Using Purified Genes, 683 (Brown and Fox (ed.) 1981).

  Highly stringent wash conditions for the oligonucleotide are typically temperatures of 0-5 ° C. below the Tm of the oligonucleotide at 6 × SSC, 0.1% SDS.

  In another embodiment, the related nucleic acid molecule comprises or consists of a nucleotide sequence that is at least about 70 percent identical to the nucleotide sequence as set forth in SEQ ID NO: 1, or in the polypeptide set forth in SEQ ID NO: 2. Comprise or consist essentially of a nucleotide sequence encoding a polypeptide that is at least about 70 percent identical. In preferred embodiments, the nucleotide sequence is about 75 percent, or about 80 percent, or about 85 percent, or about 90 percent, or about 95 percent, 96 percent, 97 percent, 98 percent or 99 percent identical, or the nucleotide sequence is about 75 percent, or about 80 percent, or about 85 percent, or about 90 percent, or about 95 percent of the polypeptide sequence shown in SEQ ID NO: 2. It encodes polypeptides that are percent, 96 percent, 97 percent, 98 percent or 99 percent identical. A related nucleic acid molecule encodes a polypeptide having at least one activity of the polypeptide set forth in SEQ ID NO: 2.

  Differences in the nucleic acid sequence can result in conservative or non-conservative modifications of the amino acid sequence relative to the amino acid sequence of SEQ ID NO: 2.

  Conservative modifications to the amino acid sequence of SEQ ID NO: 2 (and modifications corresponding to the coding nucleotides) produce FGF-23 polypeptides with functional and chemical characteristics similar to these FGF-23 polypeptides. In contrast, substantial alterations in the functional and / or chemical characteristics of the FGF-23 polypeptide can be achieved by selecting substitutions in the amino acid sequence of SEQ ID NO: 2, which substitutions are (a Significant in its effect on the maintenance of the structure of the molecular backbone in the substitution region (eg, sheet conformation or helical conformation), (b) molecular charge or hydrophobicity at the target site, or (c) side chain size Different.

  For example, a “conservative amino acid substitution” is a substitution of a native amino acid residue with a non-native residue that has little or no effect on the polarity or charge of the amino acid residue at that position. May be included. Furthermore, any native residue in the polypeptide can also be replaced with alanine, as previously described for “alanine scanning mutagenesis”.

  Conservative amino acid substitutions also include non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other inverted or inverted forms of amino acid moieties.

Naturally occurring residues can be divided into classes based on common side chain properties:
1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;
2) Neutral hydrophilicity: Cys, Ser, Thr;
3) Acidity: Asp, Glu;
4) Basic: Asn, Gln, His, Lys, Arg;
5) Residues that affect chain orientation: Gly, Pro; and 6) Aromatics: Trp, Tyr, Phe.

  For example, non-conservative substitutions can include exchanging a member of one of these classes for a member from another class. Such substituted residues can be introduced into regions of the human FGF-23 polypeptide that are homologous with non-human FGF-23 polypeptides, or into the non-homologous regions of the molecule.

  In making such changes, the hydrophobicity index of amino acids can be considered. Each amino acid is assigned a hydrophobic hydrophilicity index based on its hydrophobicity and charge characteristics. These hydrophobic hydrophilic indices are: isoleucine (+4.5); valine (+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); Glutamic acid (-3.5); Glutamine (-3.5); Aspartic acid (-3.5); Asparagine (-3.5); (-3.9) and arginine (-4.5).

  The importance of the hydropathic amino acid index in identifying interactive biological functions for proteins is generally understood in the art (Kyte et al., 1982, J. Mol. Biol. 157: 105. -31). It is known that certain amino acids can be used in place of other amino acids with similar hydropathy indices or scores and still maintain similar biological activity. In making changes based on the hydropathic index, substitution of amino acids whose hydropathic index is within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred .

  Similar amino acid substitutions can be made effectively on the basis of hydrophilicity (in particular, the biologically functionally equivalent protein or peptide produced thereby can be used in immunological embodiments as in this case. Is also understood in the art. The largest local average hydrophilicity of a protein correlates with its immunogenicity and antigenicity, ie the biological properties of the protein, as governed by the hydrophilicity of its neighboring amino acids.

  The following hydrophilicity values were assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+ 3.0 ± 1); glutamic acid (+ 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); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8) Tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). When making changes based on similar hydrophilicity values, amino acid substitutions with hydrophilicity values within ± 2 are preferred, those within ± l are particularly preferred, and those within ± 0.5 are preferred Even more particularly preferred. From the primary amino acid sequence, epitopes can also be identified based on hydrophilicity. These regions are also referred to as “epitope core regions”.

  Desired amino acid substitutions (whether 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 an FGF-23 polypeptide, or can increase or decrease the affinity of an FGF-23 polypeptide described herein. Exemplary amino acid substitutions are listed in Table I.

当業者は、配列番号２に記載のポリペプチドの適切な改変体を、周知の技術を使用して、決定し得る。生物学的活性を破壊することなく変化され得る分子の適切な領域を同定するために、当業者は、活性のために重要であるとは考えられない領域を標的化し得る。例えば、同じ種由来かまたは他の種由来の、類似の活性を有する類似のポリペプチドが既知である場合には、当業者は、ＦＧＦ−２３ポリペプチドのアミノ酸配列を、このような類似のポリペプチドと比較し得る。このような比較を用いて、類似のポリペプチド間で保存される分子の残基および部分を同定し得る。このような類似のポリペプチドに対して保存されていない、ＦＧＦ−２３分子の領域における変化が、ＦＧＦ−２３ポリペプチドの生物学的活性および／または構造にさほど不利に影響を与えるようではないことが、理解される。当業者にはまた、比較的保存された領域においてさえ、化学的に類似のアミノ酸を、活性を維持しながら天然に存在する残基と置換し得ることが公知である（保存的アミノ酸残基置換）。従って、生物学的活性または構造のために重要であり得る領域でさえ、生物学的活性を破壊することなく、またはポリペプチド構造に不利に影響を与えることなく、保存的アミノ酸置換に供され得る。

One skilled in the art can determine an appropriate variant of the polypeptide set forth in SEQ ID NO: 2 using well-known techniques. In order to identify appropriate regions of a molecule that can be altered without destroying biological activity, one skilled in the art can target regions that are not considered important for activity. For example, if a similar polypeptide with similar activity, either from the same species or from another species, is known, one skilled in the art can determine the amino acid sequence of the FGF-23 polypeptide as such a similar polypeptide. It can be compared with peptides. Such a comparison can be used to identify residues and portions of the molecule that are conserved among similar polypeptides. Changes in regions of the FGF-23 molecule that are not conserved relative to such similar polypeptides do not appear to adversely affect the biological activity and / or structure of the FGF-23 polypeptide. Is understood. Those skilled in the art also know that even in relatively conserved regions, chemically similar amino acids can be replaced with naturally occurring residues while maintaining activity (conservative amino acid residue substitutions). ). Thus, even regions that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without destroying biological activity or adversely affecting the polypeptide structure. .

  In addition, one of ordinary skill in the art can review structure-function studies that identify residues in similar polypeptides that are important for activity or structure. In view of such a comparison, the importance of amino acid residues in FGF-23 polypeptides that correspond to amino acid residues important for activity or structure in similar polypeptides can be predicted. One skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues of the FGF-23 polypeptide.

  One skilled in the art can also analyze the three-dimensional structure and amino acid sequence with respect to the three-dimensional structure in similar polypeptides. In view of such information, one skilled in the art can predict the alignment of amino acid residues of an FGF-23 polypeptide with respect to its three-dimensional structure. One skilled in the art can choose not to make abrupt changes to the amino acid residues predicted to be present on the surface of the protein. This is because such residues can be involved in important interactions with other molecules. In addition, one skilled in the art can generate test variants that include a single amino acid substitution at each amino acid residue. These variants can be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, if it is discovered that a change to a particular amino acid residue causes disruption, is undesirably reduced, or is an inappropriate activity, variants having such a change are avoided. In other words, based on information gathered from such routine experimentation, one skilled in the art will recognize amino acids whose further substitutions should be avoided either alone or in combination with other mutations. Can be easily determined.

  A number of scientific publications have been devoted to the estimation of secondary structure. Mout, 1996, Curr. Opin. Biotechnol. 7: 422-27; Chou et al., 1974, Biochemistry 13: 222-45; Chou et al., 1974, Biochemistry 113: 211-22; Chou et al., 1978, Adv. Enzymol. Relat. Area Mol. Biol. 47: 45-48; Chou et al., 1978, Ann. Rev. Biochem. 47: 251-276; and Chou et al., 1979, Biophys. J. et al. 26: 367-84. In addition, computer programs are currently available to assist in the estimation of secondary structure. One method for estimating secondary structure is based on homology modeling. For example, two polypeptides or proteins having greater than 30% sequence identity or greater than 40% similarity often have similar structural topologies. Recent growth of protein structure databases (PDBs) has provided enhanced predictability of secondary structure, including the number of possible folds in the structure of a polypeptide or protein. 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 significant number of structures have been analyzed, the structure estimation is dramatically more accurate (Brenner Et al., 1997, Curr.Opin.Struct.Biol.7: 369-76).

  Additional methods for estimating secondary structure are described in “threading” (Jones, 1997, Curr. Opin. Struct. Biol. 7: 377-87; Shippl et al., 1996, Structure 4: 15-19). Analysis "(Bowie et al., 1991, Science, 253: 164-70; Gribskov et al., 1990, Methods Enzymol. 183: 146-59; Gribskov et al., 1987, Proc. Nat. Acad. Sci. USA 84. : 4355-58), and "evolutionary linkage" (see Holm et al., Supra, and Brenner et al., Supra).

  Preferred FGF-23 polypeptide variants include glycosylation variants in which the number and / or type of glycosylation sites are altered compared to the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, the FGF-23 polypeptide variant comprises more or fewer N-linked glycosylation sites than the amino acid sequence set forth in SEQ ID NO: 2. N-linked glycosylation sites are characterized by the sequence Asn-X-Ser or Asn-X-Thr, where the amino acid residue marked X can be any amino acid residue other than proline. Substitution of amino acid residues to create this sequence provides a potential new site for the addition of N-linked carbohydrate chains. Alternatively, substitutions that eliminate this sequence will remove an existing N-linked carbohydrate chain. N-linked carbohydrate chain rearrangements in which one or more N-linked glycosylation sites (typically naturally occurring glycosylation sites) are eliminated and one or more new N-linked sites are also created. Provided. Further preferred FGF-23 variants include one or more cysteine residues deleted compared to the amino acid sequence set forth in SEQ ID NO: 2 or substituted with another amino acid (eg, serine). And a modified cysteine. Cysteine variants are useful when the FGF-23 polypeptide must be refolded into a biologically active configuration, for example after isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have the same number of cysteine residues, minimizing interactions resulting from unpaired cysteines.

  In another embodiment, the relevant nucleic acid molecule has at least one amino acid insertion, and the nucleotide that encodes the polypeptide set forth in SEQ ID NO: 2, wherein the polypeptide has the activity of the polypeptide set forth in SEQ ID NO: 2. A polypeptide according to SEQ ID NO: 2 comprising or consisting of this sequence or having at least one amino acid deletion, wherein the polypeptide has the activity of the polypeptide according to SEQ ID NO: 2. Contains or consists of the encoding nucleotide sequence. A related nucleic acid molecule also includes SEQ ID NO: 2, wherein the polypeptide has a carboxyl-terminal and / or amino-terminal truncation, and further wherein the polypeptide has the activity of the polypeptide set forth in SEQ ID NO: 2. It comprises or consists of a nucleotide sequence encoding the described polypeptide. Related nucleic acid molecules also include at least one modification selected from the group consisting of amino acid substitutions, amino acid insertions, amino acid deletions, carboxyl terminal truncations, and amino terminal truncations, wherein the polypeptide is in SEQ ID NO: 2. It comprises or consists of a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 2 having the activity of the described polypeptide.

  Furthermore, the polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or any other FGF-23 polypeptide can be fused to a homologous polypeptide to form a homodimer, or can be fused to a heterologous polypeptide to form a heterodimer. Can be formed. Heterologous peptides and polypeptides include, but are not limited to: epitopes that allow detection and / or isolation of FGF-23 fusion polypeptides; transmembrane receptor proteins or portions thereof (eg, cells An outer domain or transmembrane domain and an intracellular domain); a ligand or portion thereof that binds to a transmembrane receptor protein; a catalytically active enzyme or portion thereof; a polypeptide or peptide that promotes oligomerization (eg, leucine A polypeptide or peptide that increases stability (eg, an immunoglobulin constant region); and a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or a different therapeutic activity than another FGF-23 polypeptide. Polypeptide with .

  Fusions can be made either at the amino terminus or the carboxyl terminus of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or other IL1ra-R polypeptide. The fusion may be direct without the use of a linker or adapter molecule, or via a linker or adapter molecule. The linker or adapter molecule can be one or more amino acid residues, typically from about 20 to about 50 amino acid residues. The linker or adapter molecule can also be designed with a cleavage site for a DNA restriction endonuclease or protease to allow separation of the fused portion. It will be appreciated that once constructed, the derived polypeptide can be derivatized according to the methods described herein.

  In further embodiments of the invention, a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or other FGF-23 polypeptide, is fused to one or more domains of the Fc region of human IgG. The antibody comprises two functionally independent parts: a variable domain known as “Fab” that binds the antigen, and involved in effector functions such as complementary activation and attack by phagocytic cells. Constant domain known as "Fc". Fc has a long serum half-life, while Fab has a short life. Capon et al., 1989, Nature 337: 525-31. When constructed with therapeutic proteins, Fc domains may provide longer half-lives or perform functions such as Fc receptor binding, protein A binding, complement binding, and possibly even placental import. Can be incorporated. Ibid. Table II summarizes the use of certain Fc fusions known in the art.

一例において、ヒトＩｇＧのヒンジ領域、ＣＨ２領域、およびＣＨ３領域は、当業者に公知の方法を使用して、ＦＧＦ−２３ポリペプチドのアミノ末端またはカルボキシル末端のいずれかにおいて、融合し得る。別の例において、ヒトＩｇＧのヒンジ領域、ＣＨ２領域、およびＣＨ３領域は、ＦＧＦ−２３ポリペプチドフラグメント（例えば、ＦＧＦ−２３ポリペプチドの推定細胞外部分）のアミノ末端またはカルボキシル末端のいずれかにおいて、融合し得る。

In one example, the hinge region, CH2 region, and CH3 region of human IgG can be fused at either the amino terminus or the carboxyl terminus of the FGF-23 polypeptide using methods known to those skilled in the art. In another example, the hinge region, CH2 region, and CH3 region of human IgG are either at the amino terminus or the carboxyl terminus of an FGF-23 polypeptide fragment (eg, a putative extracellular portion of an FGF-23 polypeptide), Can merge.

  The resulting FGF-23 fusion polypeptide can be purified by use of a protein A affinity column. Peptides and proteins fused to the Fc region were found to exhibit a substantially longer in vivo half-life than their unfused counterparts. Also, fusion to the Fc region allows for dimerization / multimerization of the fusion polypeptide. The Fc region can be a naturally occurring Fc region or can be altered to improve a particular quality such as therapeutic quality, circulation time, or reduced aggregation.

  The identity and similarity of related nucleic acid molecules and polypeptides can be readily calculated by known methods. Such methods include: Computational Molecular Biology (Edited by AM Rescue, Oxford University Press 1988); 1, AM Griffin and HG Griffin, Humana Press 1994); von Heinle, Sequence Analysis in Molecular Biology (Academic Press 1987); Sequence Analysis Primer (M. Gribskov and J. Develux eds. 198, M. StockJ. Applied Math. 48: 1073, but is not limited thereto.

  Preferred methods for determining identity and / or similarity are designed to give the greatest fit between the sequences tested. Methods for determining identity and similarity are described in publicly available computer programs. A preferred computer program method for determining identity and similarity between two sequences includes the GCG program package (GAP (Devereux et al., 1984, Nucleic Acids Res. 12: 387; Genetics Computer Group, University of Wisconsin, Including, but not limited to, Madison, WI), BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J. Mol. Biol. 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). The well-known Smith Waterman algorithm can also be used to determine identity.

  A specific alignment scheme for aligning two amino acid sequences can result in a match of only a short region of these two sequences, and this small alignment region is even when there is no significant association between the two full-length sequences. May have very high sequence identity. Thus, in a preferred embodiment, the selected alignment method (GAP program) results in an alignment over at least 50 contiguous amino acids of the claimed polypeptide.

For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, Wis.), Two polypeptides whose percentage of sequence identity is to be determined are optimal fits of their respective amino acids (algorithm For the “fit span” determined by Gap opening penalty (which is calculated as 3 times the mean diagonal: “mean diagonal” is the mean of the diagonals of the comparison matrix used; The “corner” is the score or number assigned to each complete amino acid match by a particular comparison matrix) and the gap extension penalty (gap extension).
penalty) (which is usually 0.1 times the cap opening penalty), as well as comparison matrices such as PAM 250 or BLOSUM 62 are used in combination with this algorithm. Standard comparison matrices are also used by this algorithm (Dayhoff et al., 5 Atlas of Protein Sequence and Structure (Appendix 3).
1978) (PAM250 comparison matrix); Henikoff et al., 1992, Proc. Natl. Acad. Sci USA 89: 10915-19 (BLOSUM 62 comparison matrix).

Preferred parameters for polypeptide sequence comparison include the following:
Algorithm: Needleman and Wunsch, 1970, J. Mol. Mol. Biol. 48: 443-53;
Comparison matrix: BLOSUM 62 (Henikoff et al., Supra);
Gap penalty: 12
Gap Length Penalty: 4
Threshold of similarity: 0
This GAP program is useful with the above parameters. The above parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps) using the GAP algorithm.

Preferred parameters for nucleic acid molecule sequence comparison include:
Algorithm: Needleman and Wunsch, supra;
Comparison matrix: conformity = + 10, nonconformity = 0
Gap penalty: 50
Gap length penalty: 3
This GAP program is also useful with the above parameters. The above parameters are default parameters for nucleic acid molecule comparison.

  Other exemplary algorithms, gap opening penalties, gap extension penalties, comparison matrices, and similarity thresholds may be used, including those described in Program Manual, Wisconsin Package, Version 9, September 1997. The particular choice to be made will be apparent to those skilled in the art and the particular comparison to be made (eg, DNA vs. DNA, protein vs. protein, protein vs. DNA); (In this case GAP or BestFit are generally preferred) or between one sequence and a large database sequence (in this case FASTA or BLASTA is preferred).

(Nucleic acid molecule)
Nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of an FGF-23 polypeptide can include various modes (chemical synthesis, screening of cDNA or genomic libraries, expression library screening, and / or PCR amplification of cDNA). But is not limited to these).

The recombinant DNA methods used herein are generally described by Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) and / or Current Protocols in.
Molecular Biology (edited by Ausubel et al., Green Publishers Inc. and Wiley and Sons 1994). The present invention provides nucleic acid molecules as can be described herein, and methods for obtaining such molecules.

  If a gene encoding the amino acid sequence of FGF-23 polypeptide is identified from one species, use all or part of this gene as a probe to identify orthologs or related genes from the same species Can do. This probe or primer can be used to screen cDNA from various tissue sources suspected of expressing the FGF-23 polypeptide. In addition, portions or all of a nucleic acid molecule having a sequence as set forth in SEQ ID NO: 1 can be used to screen a genomic library to identify and isolate a gene encoding the amino acid sequence of an FGF-23 polypeptide. . Typically, moderate or high stringency conditions are used for screening to minimize the number of false positives resulting from this screening.

  Nucleic acid molecules encoding the amino acid sequence of FGF-23 polypeptide can also be identified by expression cloning using detection of positive clones based on the properties of the expressed protein. Typically, nucleic acid libraries are screened by binding antibodies or other binding partners (eg, receptors or ligands) to cloned proteins expressed and displayed on the host cell surface. The antibody or binding partner is modified with a detectable label to identify cells that express the desired clone.

  These polynucleotides can be produced and the encoded polypeptides can be expressed according to recombinant expression techniques performed according to the instructions set forth below. For example, by inserting a nucleic acid sequence encoding the amino acid sequence of an FGF-23 polypeptide into an appropriate vector, one skilled in the art can readily generate large quantities of the desired nucleotide sequence. These sequences can then be used to generate detection probes or amplification primers. Alternatively, a polynucleotide encoding the amino acid sequence of FGF-23 polypeptide can be inserted into an expression vector. By introducing the expression vector into a suitable 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 (typically complementary to two distinct regions of the cDNA encoding the amino acid sequence of the FGF-23 polypeptide) are then added to this cDNA along with a polymerase such as Taq polymerase. And the polymerase amplifies the region between the two primers of the cDNA.

  Another means of preparing nucleic acid molecules encoding the amino acid sequence of FGF-23 polypeptide is described in Engels et al., 1989, Angew. Chem. Intl. Chemical synthesis, using methods well known to those skilled in the art, such as those described by 2nd edition 8: 716-34. These methods include, among others, the phosphotriester, phosphoramidite, and H-phosphonate methods for nucleic acid synthesis. A preferred method for such chemical synthesis is a polymer-supported synthesis using standard phosphoramidite chemistry. Typically, the DNA encoding the amino acid sequence of FGF-23 polypeptide is several hundred nucleotides long. Nucleic acids longer than about 100 nucleotides can be synthesized as several fragments using these methods. These fragments can then be ligated together to form the full length nucleotide sequence of the FGF-23 gene. Usually, the DNA fragment encoding the amino terminus of this polypeptide has an ATG, which encodes a methionine residue. This methionine may or may not be present in the mature form of 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 be used as well.

  In certain embodiments, the nucleic acid variant comprises a codon that has been altered for optimal expression of the FGF-23 polypeptide in a given host cell. The particular codon change will depend on the host cell and FGF-23 polypeptide selected for expression. Such “codon optimization” can be performed by various methods (eg, by selecting preferred codons for use in genes that are highly expressed in a given host cell). A computer algorithm that incorporates a codon frequency table such as “Eco_high.Cod” for codon preference of highly expressed bacterial genes can be used, and 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 .

  In some cases, it may be desirable to prepare nucleic acid molecules that encode FGF-23 polypeptide variants. Nucleic acid molecules encoding the variants can be generated using site-directed mutagenesis, PCR amplification, or other suitable method in which the primer has the desired point mutation (for a description of mutagenesis techniques, see Sambrook et al. , Supra, and Ausubel et al., Supra). Chemical synthesis using the method described by Engels et al., Supra, can also be used to prepare such variants. Other methods known to those skilled in the art can be used as well.

(Vector and host cell)
A nucleic acid molecule encoding the amino acid sequence of the FGF-23 polypeptide is inserted into an appropriate expression vector using standard ligation techniques. The vector is typically selected to be functional in the particular host cell used (ie, the vector is compatible with the host cell machinery such that gene amplification and / or gene expression occurs). Is). Nucleic acid molecules encoding FGF-23 polypeptide amino acid sequences can be amplified / expressed in prokaryotic, yeast, insect (baculovirus-based) and / or eukaryotic host cells. The choice of host cell depends in part on whether the FGF-23 polypeptide is post-translationally modified (eg, glycosylated and / or phosphorylated). If so, yeast host cells, insect host cells, or mammalian host cells are preferred. For a review of expression vectors, see Meth. Enz. , Vol. 185 (DV Goeddel ed., Academic Press 1990).

  Typically, expression vectors used in any host cell contain sequences for plasmid maintenance and for cloning and expression of foreign nucleotide sequences. Such sequences (collectively referred to as “adjacent sequences”), in certain embodiments, typically comprise one or more of the following nucleotide sequences: promoter, one or more enhancer sequences, origin of replication. , Complete intron sequence including transcription termination sequence, donor and acceptor splice sites, sequence encoding leader sequence for polypeptide secretion, ribosome binding site, polyadenylation sequence, nucleic acid encoding expressed polypeptide A polylinker region for selection, as well as a selectable marker element. Each of these sequences is discussed below.

  Optionally, the vector can include a “tag” coding sequence (ie, an oligonucleotide molecule located at the 5 ′ or 3 ′ end of the FGF-23 polypeptide coding sequence); the oligonucleotide sequence can be polyHis ( For example, hexaHis), another “tag” (eg FLAG, HA (hemagglutinin influenza virus)) or myc in which commercially available antibodies are present. This tag is typically fused 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 achieved, for example, by column chromatography using antibodies against the tag as an affinity matrix. If necessary, the tag is subsequently removed from the purified FGF-23 polypeptide by various means such as using a specific peptidase for cleavage.

  The flanking sequence can be homologous (ie, from the same species and / or strain as the host cell), heterologous (ie, from a species other than the host cell species or strain), or hybrid (ie, more than one). It can be a combination of flanking sequences from many sources) or it can be synthetic or the flanking sequences can be native sequences that function normally to regulate FGF-23 polypeptide expression. Thus, the source of flanking sequences can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequences are functional in the host cell machinery. And can be activated by host cell machinery.

  Flanking sequences useful for the vectors of the invention can be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein, other than the FGF-23 gene flanking sequences, have been previously identified by mapping and / or restriction endonuclease digestion and therefore use appropriate restriction endonucleases. And can be isolated from a suitable tissue source. In some cases, the full length nucleotide sequence of the flanking sequence may be known. Here, flanking sequences can be synthesized using the methods described herein for nucleic acid synthesis or cloning.

  If all or part of the flanking sequence is known, use PCR and / or screen genomic libraries with appropriate oligonucleotides and / or flanking sequence fragments from the same or another species Can be obtained. If the flanking sequence is not known, a fragment of DNA containing the flanking sequence can be isolated from a large piece of DNA that can contain, for example, a coding sequence or another gene. Isolation may be restriction endonuclease digestion to produce the appropriate DNA fragment followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, CA), or other known to those skilled in the art It can be achieved by the method. The selection of an appropriate enzyme to accomplish this purpose will be readily apparent to those skilled in the art.

  The origin of replication is typically part of a prokaryotic expression vector purchased commercially, and this origin serves for amplification of the vector in the host cell. Amplification of the vector for a particular copy number may be important in some cases for optimal expression of the FGF-23 polypeptide. If the selected vector does not contain an origin of replication site, it can be chemically synthesized based on known sequences and ligated into the vector. For example, the origin of replication from plasmid pBR322 (New England Biolabs, Beverly, Mass.) Is suitable for most gram-negative bacteria, and various origins (eg, SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV). Or papillomaviruses such as HPV or BPV) are useful for cloning of vectors in mammalian cells. Generally, the origin of replication component is a mammalian expression vector (eg, the SV40 origin is often used just because it contains the early promoter).

  A transcription termination sequence is typically located 3 'to the end of the polypeptide coding region and serves to terminate transcription. Usually, the transcription termination sequence in prokaryotic cells is a GC rich fragment followed by a poly-T sequence. This sequence can be easily cloned from a library or purchased commercially as part of a vector, which is easily synthesized using methods for nucleic acid synthesis as described herein above. obtain.

  The selectable marker gene element encodes a protein necessary for the survival and growth of host cells grown in a selective culture medium. Representative selectable marker genes (a) confer resistance to prokaryotic cells to antibiotics or other toxins (eg, ampicillin, tetracycline, or kanamycin); (b) deficient cellular auxotrophy Or (c) encodes a protein that supplies important nutrients that are not available from the complex medium. 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 expressed gene. Amplification is a process in which genes that are highly required by the production of proteins important for growth are repeated in tandem within the chromosome of the continuous production of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. Mammalian cell transformants are placed under selective pressure, where only transformants are uniquely adapted to survive with a selection gene present in the vector. Selection pressure is the cultivation of transformed cells under conditions where the concentration of the selection factor in the medium changes continuously, thereby leading to amplification of both the selection gene and DNA encoding the FGF-23 polypeptide. Imposed by doing. As a result, increased amounts of FGF-23 polypeptide are synthesized from the amplified DNA.

  The ribosome binding site is usually required for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotic) or a Kozak sequence (eukaryotic). This element is typically placed 3 'to the promoter of the expressed FGF-23 polypeptide and 5' to the coding sequence. The Shine-Dalgarno sequence is variable but is typically a polypurine (ie, high AG content). A number of Shine-Dalgarno sequences have been identified, and each can be easily synthesized and used in prokaryotic vectors using the methods described herein.

  A leader sequence, or signal sequence, can be used to direct the FGF-23 polypeptide from the host cell. Typically, the nucleotide sequence encoding the signal sequence is located in the coding region of the FGF-23 nucleic acid molecule or directly 5 'to the FGF-23 polypeptide coding region. Many signal sequences have been identified, and any of the signal sequences that are functional in the selected host cell can be used in combination with the FGF-23 nucleic acid molecule. Thus, the signal sequence can be homologous (natural) or heterologous to the FGF-23 nucleic acid molecule. In addition, signal sequences can be chemically synthesized using the methods described herein. In most cases, secretion of the FGF-23 polypeptide from the host cell by the presence of the signal peptide results in the removal of the signal peptide from the secreted FGF-23 polypeptide. The signal sequence can be a component of the vector or can be part of an FGF-23 nucleic acid molecule inserted into the vector.

  Use of either a nucleotide sequence encoding a native FGF-23 polypeptide signal sequence linked to an FGF-23 polypeptide coding region or a nucleotide sequence encoding a heterologous signal sequence linked to an FGF-23 polypeptide coding region Is within the scope of the present invention. The selected heterologous signal sequence should be one that is recognized and processed (ie, cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native FGF-23 polypeptide signal sequence, the signal sequence is selected, for example, by a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, or heat stable enterotoxin II leader. Is replaced. For yeast secretion, the native FGF-23 polypeptide signal sequence can be replaced by a yeast invertase, alpha factor, or acid phosphatase leader. For mammalian cell expression, the native signal sequence is satisfactory, but other mammalian signal sequences may be suitable.

  In some cases where glycosylation is desirable in eukaryotic host cell expression systems, various signal peptides can be engineered to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide can be altered or a pro-sequence can be added, which can also affect glycosylation. The final protein product may have one or more additional amino acids associated with expression at position 1 (relative to the first amino acid of the mature protein), which may not be completely removed. For example, the final protein product can have one or two amino acid residues found at the peptidase cleavage site attached to the amino terminus. Alternatively, some enzyme cleavage sites can result in a slightly shortened form of the desired FGF-23 polypeptide when the enzyme is cleaved at such a region within the mature polypeptide.

  In many cases, transcription of a nucleic acid molecule is increased by the presence of one or more introns in the vector; this is when the polypeptide is produced in a eukaryotic host cell (especially a mammalian host cell) This is especially true. The intron used can be naturally present in the FGF-23 gene, especially when the gene used is a full-length genomic sequence or a fragment thereof. If the intron is not naturally present in the gene (for most cDNAs), the intron can be obtained from another source. The position of the intron with respect to the flanking sequences and the FGF-23 gene is generally important because the intron must be transcribed effectively. Thus, when FGF-23 cDNA molecules are transcribed, the preferred position of the intron is 3 'to the transcription start site and 5' to the poly-A transcription termination sequence. Preferably, introns are placed on one or other side (ie 5 'or 3') of the cDNA so as not to interfere with the coding sequence. The invention can be practiced using introns from any source (including viruses, prokaryotes and eukaryotes (plants or animals)) provided that the intron is compatible with the host cell into which it is inserted. It is. Synthetic introns are also included here. If desired, more than one intron can be used in the vector.

  The expression and cloning vectors of the invention typically include a promoter that is recognized by the host organism and is operably linked to a molecule encoding an FGF-23 polypeptide. A promoter is a non-transcribed sequence that is located upstream (i.e., 5 ') with respect to the start codon of a structural gene (generally about 100 to 1000 bp) that controls transcription of the structural gene. Promoters are usually 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 (eg, the presence or absence of nutrients, or a change in temperature). On the other hand, constitutive promoters initiate the production of continuous gene products; that is, little or no control of genes for gene expression. A number of promoters (recognized by a variety of potential host cells) are well known. A suitable promoter is operably linked to DNA encoding the FGF-23 polypeptide by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector. The native FGF-23 promoter sequence can be used to direct the amplification and / or expression of FGF-23 nucleic acid molecules. However, heterologous promoters are preferred if they allow higher production of large transcribed and expressed proteins compared to the native promoter and are compatible with the host cell system selected for use.

  Suitable promoters for use with prokaryotic hosts include β-lactamase and lactose promoter systems; alkaline phosphatase; tryptophan (trp) promoter system; and hybrid promoters (eg, tac promoter). Other known bacterial promoters are also suitable. These sequences have been published so that one skilled in the art can link them to the desired DNA using linkers or adapters required to provide any useful restriction sites. obtain.

  Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used for use with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, polyoma virus, fowlpox virus, adenovirus (eg, Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus , Promoters derived from the genomes of viruses such as hepatitis B virus and most preferred simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters (eg, heat shock promoter and actin promoter).

Additional promoters that may be of interest in controlling FGF-23 gene expression include, but are not limited to: SV40 early promoter region (Bernoist and Chamber, 1981, Nature 290: 304-10); CMV promoter; Promoters contained in the 3 'long terminal repeat of the sarcoma virus (Yamamoto, et al., 1980, Cell 22: 787-97); herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 1444-45); regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296: 39-42); of the β-lactamase promoter Such prokaryotic expression vectors (Villa-Kamarov et al., 1978, Proc. Natl. Acad. Sci. USA, 75: 3727-31); or tac promoter (DeBoer et al., 1983, Proc. Natl.Acad.Sci.U.S.A., 80: 21-25). The following animal transcriptional control regions that exhibit tissue specificity and are utilized in transgenic animals are also of interest: elastase I gene control region 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); insulin gene regulatory region active in pancreatic β cells (Hanahan, 1985, Nature 315: 115-22); immunity active in lymphocytes Globulin gene regulatory region (Grosschedl et al., 1984, Cell 38: 647-58; Adames et al., 1985, Nature 318: 533-38; Alexander et al., 1987, Mol. Cell. Biol., 7: 1436. -44); mouse mammalian tumor virus regulatory region active in testis, breast, lymphocytes and mast cells (Leder et al., 1986, Cell 45: 485-95); Active albumin gene regulatory region (Pinkert et al., 1987, Genes and Devel. 1: 268-76); α-fetoprotein gene regulatory region active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol Hammers et al., 1987, Science 235: 53-58); α1-antitrypsin gene regulatory region active in the liver (Kelsey et al., 1987, Genes and Devel. 1: 161-). 71); β-globin gene regulatory region active in myeloid cells (Mogram et al., 1985, Nature 315: 338-40; Kollias et al., 1986, Cell 46: 89-94); Myelin-based protein gene regulatory region active in neuroglia (Readhead et al., 1987, Cell 48: 703-12); myosin light chain-2 gene regulatory region active in skeletal muscle (Sani, 1985, Nature 314) : 283-86); and the gonadotropin-releasing hormone gene regulatory region active in the hypothalamus (Mason et al., 1986, Science 234: 1372-78).

  Enhancer sequences can be inserted into the vector so as to increase transcription of DNA encoding the FGF-23 polypeptide of the present invention by higher eukaryotes. Enhancers are elements that act on the cis of DNA, usually about 10-300 bp in length, that act on promoters to increase transcription. Enhancers are relative, direction and position independent. These were found 5 'and 3' to the transcription unit. Several enhancer sequences are known that are available from mammalian genes (eg, globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, a virus-derived enhancer is used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancer are exemplary enhancers for eukaryotic promoter activation. Enhancers can be spliced into the vector at the 5 'or 3' position relative to the FGF-23 nucleic acid molecule, but are typically placed 5 'to the promoter.

  The expression vectors of the present invention can be constructed from a starting vector such as a commercially available vector. Such vectors may or may not contain all desired flanking sequences. If one or more of the flanking sequences described herein are not already in the vector, these can be obtained individually and linked to the vector. The methods used to obtain each of the flanking sequences are well known to those skilled in the art.

Preferred vectors for practicing the invention are vectors that are compatible with bacterial, insect, and mammalian host cells. Such vectors include, among others, pCRII, pCR3, and pcDNA3.1 (Invitrogen, San Diego, CA), pBSII (Stratagene, La
Jolla, CA), pET15 (Novagen, Madison, WI), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBacII, InvitroRn, PC number) 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 is understood that these vector systems must be compatible with the selected host cell. Such vectors include, but are not limited to, Bluescript® plasmid derivatives (high copy number ColEl-based phagemids, Stratagene Cloning).
Systems, La Jolla CA), PCR cloning plasmids designed to clone Taq amplified PCR products (eg, TOPO ^™ TA Cloning® Kit, PCR2.1® plasmid derivatives, Invitrogen, Carlsbad, CA) ), And mammalian vectors such as baculovirus expression systems, yeast vectors or viral vectors (pBacPAK plasmid derivatives, Clontech, Palo Alto, CA).

  After the vector is constructed and the nucleic acid molecule encoding the FGF-23 polypeptide is inserted into the appropriate site of the vector, the complete vector can be inserted into a suitable host cell for amplified expression and / or polypeptide expression. Transformation of an expression vector for FGF-23 polypeptide into a selected host cell can be accomplished by transfection, infection, calcium chloride, electroporation, microinjection, lipofectin, DEAE-dextran method, or other known techniques. It can be achieved by known methods including various methods. The method chosen is somewhat related to the type of host cell used. These and other suitable methods are well known to those skilled in the art and are described, for example, in Sambrook et al., Supra.

  The host cell can be a prokaryotic host cell (eg, E. coli) or a eukaryotic host cell (eg, yeast cell, insect cell, or vertebrate cell). If the host cell is cultured under appropriate conditions, it will synthesize FGF-23 polypeptide, which can subsequently be collected from the culture medium (if the host cell secretes the polypeptide into the medium). Or collected directly from host cells that produce the polypeptide (if the polypeptide is not secreted). The selection of an appropriate host cell can be achieved with a variety of factors such as the desired level of expression, polypeptide modifications desirable or necessary for activity (eg, glycosylation or phosphorylation), and ease of folding into biologically active molecules. Depends on factors.

Many suitable host cells are known in the art, and many are available from the American Type Culture Collection (ATCC), Manassas, VA. Examples include, but are not limited to, Chinese hamster ovary cells (CHO), CHO DHFR (−) cells (Urlab)
et al. , 1980, Proc. Natl. Acad. Sci. US. A. 97: 4216-20), human embryonic kidney (HEK) 293 or 293T cells, or mammalian cells such as 3T3 cells. Methods for selection and transformation, culture, amplification, screening, product production, and purification of suitable mammalian host cells are known in the art. Other suitable mammalian cell lines are the monkey COS-1 and COS-7 cell lines, and the CV-1 cell line. Further exemplary mammalian host cells include primate cell lines and rodent cell lines (including transformed cell lines). Also suitable are normal diploid cells, cell lines derived from in vitro cultures of primary tissues, and primary explants. Candidate cells may lack a selection gene, or may contain a selection gene that acts preferentially. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or Hak hamsters. Cell lines. Each of these cell lines is known and available to those skilled in the art of protein expression.

  Similarly, bacterial cells are useful as suitable host cells for the present invention. For example, E.I. Various strains of E. coli (eg, HB101, DH5α, DH10, and MC1061) are well known as host cells in the field of biotechnology. B. subtilis, Pseudomonas spp. , Other Bacillus spp. , Streptomyces spp. Various strains such as can also be used in the method.

  Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the invention. Preferred yeast cells include, for example, Saccharomyces cervisaee and Pichia pastoris.

  Further, if desired, insect cell lines can be utilized in the methods of the invention. Such systems are described, for example, by Kitts et al. 1993, Biotechniques, 14: 810-17; Luclow, 1993, Curr. Opin. Biotechnol. 4: 564-72; and Luclow et al. 1993, J. et al. Virol. 67: 4566-79. Preferred insect cells are Sf-9 and Hi5 (Invitrogen).

  Transgenic animals can also be used to express glycosylated FGF-23 polypeptides. For example, transgenic milk-producing animals (eg, cows or goats) can be used to obtain the glucosylated polypeptides of the present invention in animal milk. Plants can also be used to produce FGF-23 polypeptides, however, in general, glycosylation that occurs in plants differs from that produced in mammalian cells and is not suitable for use in human therapy. Can result in no glycosylated product.

(Polypeptide production)
Host cells containing the FGF-23 polypeptide expression vector can be cultured using standard media well known to those of skill in the art. This medium usually contains all the nutrients necessary for cell growth and survival. E. Suitable media for culturing E. coli cells include, for example, Luria Broth (LB) and / or Territic Broth (TB). Suitable culture media for culturing eukaryotic cells include Roswell Park Memorial Institute medium 1640 (RPMI 1640), Minimal Essential Medium (MEM) and / or Dulbecco's.
Modified Eagle Medium (DMEM), all of which can be supplemented with serum and / or growth factors necessary for the particular cell line being cultured. A suitable medium for insect cells is Grace's medium supplemented with yeastate, lactalbumin hydrolyzate, and / or fetal calf serum as needed.

  Typically, antibiotics or other compounds useful for the selective growth of transfected or transformed cells are added to the medium as a supplement. The compound used can be detected by a selectable marker element present on the plasmid into which the host cell is transformed. For example, if the selectable marker element is kanamycin resistant, the compound added to the culture medium is kanamycin. Other compounds for selective growth include ampicillin, tetracycline, and neomycin.

  The amount of FGF-23 polypeptide produced by the host cell can be assessed using standard methods known in the art. Such methods include, but are not limited to, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, high performance liquid chromatography (HPLC) separation, immunoprecipitation, and / or DNA binding gel shift assays. Active activity assays.

  If the FGF-23 polypeptide is designed to be secreted from the host cell, the majority of the polypeptide can be found in the cell culture medium. However, if the FGF-23 polypeptide is not secreted from the host cell, it is present in the cytoplasm and / or nucleus (for eukaryotic host cells) or in the cytosol (for gram positive bacterial host cells).

  For FGF-23 polypeptides located in the host cytoplasm and / or nucleus (for eukaryotic host cells) or in the cytosol (for bacterial host cells), intracellular substances (inclusion bodies for Gram-positive bacteria) Can be extracted from the host cell using any standard technique known to those of skill in the art. For example, host cells can be lysed to release periplasm / cytoplasmic contents by French press, homogenization, and / or sonication followed by centrifugation.

  If the FGF-23 polypeptide forms inclusion bodies in the cytosol, the inclusion bodies can often be bound to the inner and / or outer cell membranes and are therefore found primarily in the pellet material after centrifugation. The pellet material is then subjected to a surfactant, guanidine, guanidine derivative, at an extreme pH, or at an alkaline pH in the presence of a reducing agent such as dithiothreitol, or at an acidic pH in the presence of triscarboxyethylphosphine. Treatment with a chaotropic agent such as urea or a urea derivative may release, sever and dissolve the inclusion bodies. The solubilized FGF-23 polypeptide can then be analyzed using gel electrophoresis, immunoprecipitation, and the like. If it is desired to isolate an FGF-23 polypeptide, the isolation is described herein and in Marston et al. , 1990, Meth. Enz. 182: 264-75, and can be achieved using standard methods.

  In some cases, the FGF-23 polypeptide may not be biologically active upon isolation. Various methods for “refolding” or converting a polypeptide to its tertiary structure, as well as for generating disulfide bonds, can be used to replicate biological activity. Such methods involve exposing the dissolved polypeptide to a certain pH (usually above 7) and the presence of a specific concentration of chaotropic agent. The choice of chaotropic agent is very similar to the options used for inclusion body lysis, but usually the chaotropic agent is used at low concentrations and is not necessarily the same as the chaotropic agent used for lysis. In many cases, the refolding / oxidation solution also includes a reducing agent, or a specific ratio of reducing agent and its oxidized form, to produce a specific redox potential and result in the formation of cysteine bridges in the protein. Enable. Some of the commonly used redox pairs include cysteine / cystamine, glutathione (GSH) / dithiobis GSH, copper (II) chloride, dithiothreitol (DTT) / dithian DTT, and 2-2 mercaptoethanol (bME). ) / Dithio-b (ME). In many cases, a co-solvent may be used or needed to increase the efficiency of refolding, and more common reagents used for this purpose include glycerol, various molecular weights Examples include polyethylene glycol and arginine.

  If inclusion bodies are not formed to a significant extent in the expression of FGF-23 polypeptide, the polypeptide is found primarily in the supernatant after centrifugation of the cell homogenate. The polypeptide can be further isolated from the supernatant using methods as described herein.

  Purification of FGF-23 polypeptide from solution can be accomplished using a variety of techniques. The polypeptide is a tag such as hexahistidine (FGF-23 polypeptide / hexaHis) or other small peptide (eg, FLAG (Eastman Kodak Co., New Haven, CT) or myc (Invitrogen, Carlsbad, Calif.)). When synthesized to contain at either its carboxy terminus or amino terminus, the column matrix can be purified in one step by passing the solution through an affinity column with high affinity for the tag.

For example, polyhistidine binds with great affinity and specificity for nickel. Thus, a nickel affinity column (eg, Qiagen® nickel column) can be used for the purification of FGF-23 polypeptide / polyHis. For example, Current Protocols
See Molecular Biology § 10.11.8 (Ausubel et al., eds., Green Publishers Inc. and Wiley and Sons 1993).

  Furthermore, FGF-23 polypeptides can be purified through the use of monoclonal antibodies that can specifically recognize and bind to FGF-23 polypeptides.

  Other suitable means for purification include, but are not limited to, affinity chromatography, immunoaffinity chromatography, ion exchange chromatography, molecular sieve chromatography, HPLC, electrophoresis (including native electrophoresis), Subsequent gel elution and preparative isoelectric focusing ("Isoprime" machine / technique, Hoefer Scientific, San Francisco, CA). In some cases, two or more purification techniques may be combined to achieve increased purity.

FGF-23 polypeptides are also described in Merrifield et al. , 1963, J. et al. 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) can be prepared by chemical synthesis methods (eg, solid phase peptide synthesis) using techniques known in the art. Such polypeptides can be synthesized with or without a methionine at the amino terminus. Chemically synthesized FGF-23 polypeptides can be oxidized using the methods described in these references to form disulfide bonds. A chemically synthesized FGF-23 polypeptide is expected to have biological activity comparable to the corresponding FGF-23 polypeptide produced recombinantly or purified from a natural source, Thus, they can be used interchangeably with recombinant or natural FGF-23 polypeptides.

  Another means of obtaining FGF-23 polypeptide is by purification from biological samples such as source tissues and / or fluids in which the FGF-23 polypeptide is naturally found. Such purification can be performed using methods for protein purification as described herein. During purification, the presence of FGF-23 polypeptide can be monitored, for example, using antibodies prepared against recombinantly produced FGF-23 polypeptide or peptide fragments thereof.

  Many additional methods for producing nucleic acids and polypeptides are known in the art, and this method can be used to produce polypeptides having specificity for FGF-23 polypeptides. For example, Roberts et al. , 1997, Proc. Natl. Acad. Sci. U. S. A. 94: 12297-303. This describes the production of a fusion protein between the mRNA and its encoded peptide. Roberts, 1999, Curr. Opin. Chem. Biol. 3: 268-73. In addition, US Pat. No. 5,824,469 describes a method for obtaining oligonucleotides that can perform specific biological functions. This procedure involves generating heterogeneous pools of oligonucleotides, each having a 5 'randomized sequence, a central pre-selected sequence, and a 3' randomized sequence. The resulting heterogeneous pool is introduced into a population of cells that do not exhibit the desired biological activity. A subpopulation of cells is then screened for those exhibiting a predetermined biological function. From that subpopulation, oligonucleotides that can perform the desired biological function are isolated.

  U.S. Pat. Nos. 5,763,192; 5,814,476; 5,723,323; and 5,817,483 describe processes for producing peptides or polypeptides. Describe. This is accomplished by producing stochastic genes or fragments thereof and then introducing these genes into a host cell that produces one or more proteins encoded by the stochastic genes. The host is then screened to identify those clones that produce a peptide or polypeptide having the desired activity.

  Another method for producing peptides or polypeptides is described in Athersys, Inc. PCT / US98 / 20094 (WO 99/15650), filed by A., known as “Random Activation of Gene Expression for Gene Discovery” (RAGE-GD), which process is described in-situ recombination method. By activation of endogenous gene expression or gene overexpression. For example, endogenous gene expression is activated or increased by incorporating regulatory sequences into target cells that can activate gene expression by non-homologous or aberrant recombination. This target DNA is first subjected to radiation and inserted into a gene promoter. This promoter is finally placed in front of the gene and initiates transcription of the gene. This results in the expression of the desired peptide or polypeptide.

  These methods can also be used to generate comprehensive FGF-23 polypeptide expression libraries, which can be followed by various assays (eg, biochemical assays, cellular assays and whole biological assays ( It is understood that it can be used for high-throughput phenotypic screening, for example in plants, mice etc.)).

(Synthesis)
It will be appreciated by those skilled in the art that the nucleic acid and polypeptide molecules described herein can be produced by recombinant and other means.

(Selective binding factor)
The term “selective binding agent” refers to a molecule having 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. Exemplary FGF-23 polypeptide selective binding agents of the invention can bind a specific portion of the FGF-23 polypeptide, thereby inhibiting the binding of the polypeptide to the FGF-23 polypeptide receptor.

  Selective binding agents such as antibodies and antibody fragments that bind to FGF-23 polypeptides are within the scope of the invention. This antibody can be a polyclonal, including monospecific polyclonal; monoclonal (MAb); recombinant; chimeric; humanized (eg, CDR grafted); human; single chain; and / or bispecific; A variant; or a derivative thereof. Antibody fragments include those portions of the antibody that bind to an epitope on FGF-23 polypeptide. Examples of such fragments include Fab and F (ab ') fragments produced by enzymatic cleavage of full length antibodies. Other binding fragments include those produced by recombinant DNA technology (eg, expression of a recombinant plasmid containing a nucleic acid sequence encoding an antibody variable region).

  Polyclonal antibodies directed against FGF-23 polypeptide are generally produced in animals (eg, rabbits or mice) by multiple subcutaneous or intraperitoneal injections of FGF-23 polypeptide and adjuvant. It may be useful to link the FGF-23 polypeptide to a carrier protein, which may be useful in immunized species such as keyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, or soybean trypsin inhibitor. It is immunogenic. Aggregating agents such as alum are also used to enhance the immune response. Following immunization, the animals are bled and the serum is assayed for anti-FGF-23 polypeptide antibody titer.

  Monoclonal antibodies directed against FGF-23 polypeptides are produced using any method provided to produce antibody molecules by continuous cell lines in culture. Examples of suitable methods for preparing monoclonal antibodies include the hybridoma method of Kohler et al., 1975, Nature 256: 495-97, and the human B cell hybridoma method (Kozbor, 1984, J. Immunol. 133: 3001; Brodeur et al. , Monoclonal Antibody Production Technologies and Applications 51-63 (Marcel Dekker, Inc., 1987)). Hybridoma cell lines that produce monoclonal antibodies reactive with FGF-23 polypeptides are also provided by the present invention.

  The monoclonal antibodies of the invention can be modified for use as therapeutic agents. One embodiment is a “chimeric” antibody in which a portion of the heavy chain (H) and / or light chain (L) is derived from a particular species or of a particular antibody class or subclass. Is identical or homologous to the corresponding sequence in the antibody to which it belongs, while the rest of this chain is derived from another species or corresponds in an antibody belonging to another antibody class or subclass Is identical or homologous to the sequence to be Such antibody fragments are also included so long as the antibody fragment exhibits the desired biological activity. U.S. Pat. No. 4,816,567; Morrison et al., 1985, Proc. Natl. Acad. Sci. 81: 6851-55.

In another embodiment, a monoclonal antibody of the invention is a “humanized” antibody. Methods for humanizing non-human antibodies are well known in the art. See U.S. Pat. Nos. 5,585,089 and 5,693,762. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. Humanization is described, for example, 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) using at least a portion of the complementarity determining regions (CDRs) of rodents corresponding to human antibodies. This is done by replacing the region.

  Human antibodies that bind to FGF-23 polypeptides are also encompassed by the present invention. Using transgenic animals (e.g., mice) that can produce a repertoire of human antibodies in the absence of endogenous immunoglobulin products, such antibodies can be expressed as FGF-23 polypeptide antigens (i.e., With at least 6 consecutive amino acids) and, if necessary, coupled to 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 Immuno. See 7:33. In one method, such transgenic animals disable the endogenous loci encoding heavy and light chain immunoglobulins herein, and loci encoding human heavy and light chain proteins. Is inserted into its genome. The partially modified animals (which have modifications that are not fully complemented) are then cross-bred to obtain animals with all of the desired immune system modifications. When the immunogen is administered, these transgenic animals will have antibodies that have human (eg, other than mouse) amino acid sequences (the amino acid sequences include modified regions that are immunospecific for these antigens). Produce. See PCT Application Nos. PCT / US96 / 065928 and PCT / US93 / 06926. Further methods are described in US Pat. No. 5,545,807, PCT application numbers PCT / US91 / 245 and PCT / GB89 / 01207, and European Patents 546073B1 and 546073A1. Human antibodies can also be produced by expression of recombinant DNA in host cells or expression in hybridoma cells as described herein.

  In alternative embodiments, human antibodies can also be produced from phage display libraries (Hoogenboom et al., 1991, J. Mol. Bio. 227: 381; Marks et al., 1991, J. Mol. Biol. 222: 581). These processes mimic immune selection through display of antibody repertoires on the surface of linear bacteriophage and subsequent selection of phage by their binding to selected antigens. One such technique is described in PCT Application No. PCT / US98 / 17364, which uses such an approach to high affinity antibodies for MPL-reporters and msk-reporters. And isolation of functional agonist antibodies is described.

  Chimeric, CDR-grafted, and humanized antibodies are typically produced by recombinant methods. The nucleic acid encoding the antibody is introduced into a host cell and expressed using the materials and procedures described herein. In preferred embodiments, the antibodies are produced in mammalian host cells (eg, CHO cells). Monoclonal (eg, human) antibodies can be produced by expression of recombinant DNA in host cells or expression in hybridoma cells, as described herein.

The anti-FGF-23 antibodies of the invention are used in any known assay method (eg competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays) for the detection and quantification of FGF-23 polypeptides. (Sola, Monoclonal Antibodies: A Manual
of Technologies 147-158 (CRC Press, Inc., 1987)). These antibodies bind to FGF-23 polypeptide with an affinity appropriate for the assay method used.

For diagnostic applications, in certain embodiments, the anti-FGF-23 antibody can be labeled with a detectable moiety. The detectable moiety can be any moiety that can produce a detectable signal, either directly or indirectly. For example, the detectable moiety can be a radioisotope (eg, ³ H, ¹⁴ C, ³² P, ³⁵ S, ¹²⁵ I, ⁹⁹ Tc, ¹¹¹ In, or ⁶⁷ Ga); a fluorescent or chemiluminescent compound (eg, fluorescein) It can be isothiocyanate, rhodamine, or luciferin) or an enzyme (eg, alkaline phosphatase, β-galactosidase, or horseradish peroxidase) (Bayer et al., 1990, Meth. Enz. 184: 138-63).

  Competitive binding assays rely on the ability of a labeled standard (eg, FGF-23 polypeptide, or an immunologically active portion thereof) to test a test sample for binding to a limited amount of anti-FGF-23 antibody. Compete with specimen (FGF-23 peptide). The amount of FGF-23 polypeptide in the test sample is inversely proportional to the amount of standard that binds to the antibody. To facilitate the determination of the amount of standard to bind, these antibodies are typically insolubilized before or after competition, so that standards and analytes that bind to these antibodies remain unbound. And can be conveniently separated from the specimen.

  Sandwich assays typically involve the use of two antibodies, each capable of binding to a different immunogenic portion or epitope of the protein to be detected and / or quantified. In a sandwich assay, the test sample analyte is typically bound by a first antibody that is immobilized on a solid support, after which a second antibody binds to the analyte and forms a soluble three-part complex. Form. See, for example, US Pat. No. 4,376,110. The second antibody itself can be labeled with a detectable moiety (direct sandwich assay) or measured using an anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay). . For example, one type of sandwich assay is an enzyme linked immunosorbent assay (ELISA), where the detectable moiety is an enzyme.

  Selective binding agents, including anti-FGF-23 antibodies, are also useful for in vivo imaging. An antibody labeled with a detectable moiety can be administered to an animal, preferably in the bloodstream, and the presence and location of the labeled antibody in the host is assayed. The antibody can be labeled with any moiety that is detectable in an animal, either by nuclear magnetic resonance, radiology, or other detection means known in the art.

  The selective binding agents of the invention, including antibodies, can be used as therapeutic agents. These therapeutic agents are generally agonists or antagonists, each of which either increases or decreases the biological activity of at least one FGF-23 polypeptide. In one embodiment, an antagonist antibody of the invention is capable of specifically binding to an FGF-23 polypeptide and binding or binding thereof that can inhibit or eliminate the functional activity of an FGF-23 polypeptide in vivo or in vitro. Fragment. In preferred embodiments, the selective binding agent (eg, antagonist antibody) inhibits the functional activity of the FGF-23 polypeptide by at least about 50%, and preferably by at least about 80%. In another embodiment, the selective binding assay can be an anti-FGF-23 polypeptide capable of interacting with an FGF-23 polypeptide binding partner (ligand or receptor), thereby allowing FGF-23 poly (s) in vitro or in vivo. Inhibits or eliminates peptide activity. Selective binding agents, including agonist and antagonist anti-FGF-23 polypeptide antibodies, are identified by screening assays that are well known in the art.

  The invention also relates to kits containing FGF-23 selective binding agents (eg, antibodies) and other agents that are useful for detecting FGF-23 polypeptide levels in biological samples. Such agents can include detectable labels, blocking sera, positive and negative control samples, and detection factors.

(Microarray)
It will be appreciated that DNA microarray technology can be utilized in accordance with the present invention. A DNA microarray is a small, high-density array of nucleic acids placed on a solid support (eg, glass). Each cell or element in the array contains multiple copies of a single nucleic acid species that serve as a label for hybridization with a complementary nucleic acid sequence (eg, mRNA). In expression profiling using DNA microarray technology, mRNA is first extracted from a cell or tissue sample and then enzyme-labeled cDNA is converted to fluorescently labeled cDNA. This material is hybridized to the microarray and unbound cDNA is removed by washing. The expression of the individual genes shown on this array is then visualized by quantifying the amount of labeled cDNA that is specifically bound to target each nucleic acid molecule. In this way, the expression of thousands of genes can be quantified in a high-throughput parallel manner from a single sample of biological material.

  This high-throughput expression profiling has a wide range of applications in connection with the FGF-23 molecules of the present invention, including but not limited to: FGF-23 as a therapeutic target Identification and validation of disease-related genes; molecular toxicity of related FGF-23 molecules and their inhibitors; population of alternative markers for clinical trials and stratification of production; and identification of selected compounds in high-throughput screening Enhance related FGF-23 polypeptide small molecule drug discovery by assisting.

(Chemical derivatives)
Chemically modified derivatives of FGF-23 polypeptides can be prepared by one of ordinary skill in the art when this disclosure is described herein. FGF-23 polypeptide derivatives are modified in different ways, either in the type or position of the molecule that is naturally associated with the polypeptide. Derivatives can include molecules formed by the deletion of one or more naturally linked chemical groups. A polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or other FGF-23 polypeptide can be modified by covalent attachment of one or more polymers. For example, the selected polymer is typically water soluble so that the protein to which it is attached does not precipitate in an aqueous environment (eg, a physiological environment). Mixtures of polymers are included within the scope of suitable polymers. Preferably, for therapeutic use of the target product preparation, the polymer is pharmaceutically acceptable.

  Each of the polymers can have any molecular weight and can be branched or unbranched. Each of the polymers typically has an average molecular weight between about 2 kDa and about 100 kDa (this term “about” indicates that several molecules are shown in the preparation of the water-soluble polymer. Higher molecular weights and some have lower molecular weights). 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 most preferably between about 20 kDa and about 35 kDa.

Suitable water soluble polymers or mixtures thereof include, but are not limited to: N-linked or O-linked carbohydrates, sugars, phosphates, polyethylenes, glycols (PEG) (which are mono- (C _1- C ₁₀ ), including forms of PEG used to derive proteins including alkoxy- or aryloxy-polyethylene glycol), monomethoxy-polyethylene glycol, dextran (eg, low molecular weight dextran of about 6 kD) Cellulose, or other carbohydrate-based polymers, poly- (N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), and Vinyl alcohol. Also encompassed by the present invention are bifunctional cross-linking molecules that can be used to prepare covalently linked FGF-23 polypeptide multimers.

  In general, chemical induction can be performed under any suitable conditions used to react proteins with activated polymer molecules. A method for preparing a chemical derivative of a polypeptide generally includes the following steps: (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or other FGF-23 polypeptide comprises one or more polypeptides Reacting a polypeptide with an activated polymer molecule (eg, a reactive ester or aldehyde derivative of the polymer molecule) under conditions that bind to the polymer molecule, and (b) obtaining a reaction product. Optimal reaction conditions are determined based on known parameters and the desired result. For example, the greater the ratio of polymer molecules to protein, the greater the proportion of bound polymer molecules. In one embodiment, the FGF-23 polypeptide derivative may have an amino-terminal single polymer molecular moiety. See, for example, US Pat. No. 5,234,784.

The pegylation of a polypeptide can be performed 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 US Pat. No. 4,179,337. For example, pegylation can be performed via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or a similar reactive water-soluble polymer) as described herein. For the acylation reaction, the selected polymer should have a single reactive ester group. For reductive alkylation, the selected polymer should have a single reactive aldehyde group. The reactive aldehyde is, for example, polyethylene glycol propionaldehyde, which is water soluble or mono C ₁ -C ₁₀ alkoxy or an aryloxy derivative thereof (see US Pat. No. 5,252,714). .

  In another embodiment, the FGF-23 polypeptide can be chemically conjugated to biotin. The biotin / FGF-23 polypeptide molecule can then bind to avidin, yielding a tetravalent avidin / biotin / FGF-23 polypeptide molecule. The FGF-23 polypeptide can also be covalently linked to dinitrophenol (DNP) or trinitrophenol (TNP), and the resulting conjugate is precipitated with anti-DNP or anti-TNP-IgM 10 Form a body conjugate.

  In general, conditions that can be alleviated or modulated by administration of an FGF-23 polypeptide derivative of the present invention include the conditions described herein for FGF-23 polypeptides. However, the FGF-23 polypeptide derivatives disclosed herein have increased or decreased additional activity, enhanced or decreased biological activity or other properties (eg, compared to non-derivatized molecules). Half-life).

(Gene engineered non-human animal)
Also included within the scope of the present invention are non-human animals such as mice, rats, or other rodents; rabbits, goats, sheep, or other domestic animals, where the native FGF-23 polypeptide is The encoding gene is disrupted (ie, “knocked out”) so that the expression level of the FGF-23 polypeptide is significantly reduced or completely destroyed. Such animals can be prepared using techniques and methods as described in US Pat. No. 5,557,032.

  The present invention further includes non-human animals such as mice, rats, or other rodents; rabbits, goats, sheep, or other domestic animals, the native form of the animal's FGF-23 gene or heterologous FGF. Any of the −23 genes is overexpressed by the animal, thereby creating a “transgenic” animal. Such transgenic animals can be prepared using well-known methods such as those described in US Pat. No. 5,489,743 and PCT Publication No. WO 94/28122.

  The present invention further includes non-human animals, wherein the promoter of one or more FGF-23 polypeptides of the present invention is activated (eg, by using homologous recombination methods), Or inactivated, altering the level of expression of one or more native FGF-23 polypeptides.

  These non-human animals can be used for drug candidate screening. In such screening, the influence of drug candidates on animals can be measured. For example, the drug candidate can decrease or increase the expression of the FGF-23 gene. In certain embodiments, the amount of FGF-23 polypeptide produced can be measured after exposing an animal to a drug candidate. Further, in certain embodiments, the actual effects of drug candidates on animals can be detected. For example, overexpression of a particular gene causes or is associated with a disease state or pathological state. In such cases, the ability of drug candidates to reduce gene expression or the ability to prevent or inhibit pathological conditions can be tested. In another example, the production of a specific metabolite, such as a fragment of a polypeptide, causes or is associated with a disease state or pathological state. In such cases, the ability of drug candidates to reduce the production of such metabolites or the ability to prevent or inhibit pathological conditions may be tested.

Assay for other modifiers of FGF-23 polypeptide activity
In some situations it may be desirable to identify molecules (eg, agonists or antagonists) that are modulators of FGF-23 polypeptide activity. Natural or synthetic molecules that modulate FGF-23 polypeptides can be identified using one or more screening assays, as described herein. Such molecules can be administered by injection, or oral delivery, implantation device, etc., either in an ex vivo or in vivo manner.

“Test molecule” refers to a molecule that evaluates its ability to modulate (ie, increase or decrease) the activity of an FGF-23 polypeptide. Most commonly, the test molecule interacts directly with the FGF-23 polypeptide. However, the test molecule also indirectly inhibits FGF-23 polypeptide activity, for example, by affecting FGF-23 gene expression or by binding to an FGF-23 polypeptide binding partner (eg, receptor or ligand). It is also intended that it can be adjusted in an automated manner. In one embodiment, the test molecule has an affinity constant of at least about 10 ⁻⁶ M, preferably about 10 ⁻⁸ M, more preferably about 10 ⁻⁹ M, and even more preferably about 10 ⁻¹⁰ M. (Affinity
bind to the FGF-23 polypeptide.

  Methods for identifying compounds that interact with FGF-23 polypeptides are encompassed by the present invention. In certain embodiments, the FGF-23 polypeptide is incubated with the test molecule under conditions that allow the test molecule to interact with the FGF-23 polypeptide, and the extent of interaction is measured. Test molecules can be screened in substantially purified form or in a crude mixture.

  In certain embodiments, the FGF-23 polypeptide agonist or antagonist can be a protein, peptide, carbohydrate, lipid, or low molecular weight molecule that interacts with the FGF-23 polypeptide to render its activity. Adjust. The molecule that regulates expression of the FGF-23 polypeptide is complementary to a nucleic acid encoding the FGF-23 polypeptide or is complementary to a nucleic acid sequence that directs or controls expression of the FGF-23 polypeptide. It includes certain nucleic acid molecules and nucleic acid molecules that act as antisense regulators of expression.

  Once a test compound is identified when interacting with an FGF-23 polypeptide, the molecule can be further evaluated for the ability to increase or decrease FGF-23 polypeptide activity. Measurement of the interaction between a test molecule and an FGF-23 polypeptide can be performed in several formats, including cell-based binding assays, membrane binding assays, liquid phase assays, and immunoassays. In general, a test molecule is incubated with a FGF-23 polypeptide for a specified period of time, and FGF-23 polypeptide activity is determined by one or more assays to measure biological activity.

  The interaction of the test molecule with the FGF-23 polypeptide can also be assayed directly using polyclonal or monoclonal antibodies in an immunoassay. Alternatively, modified forms of FGF-23 polypeptides that include an epitope tag as described herein can be used in solutions and immunoassays.

  In the case where an FGF-23 polypeptide exhibits biological activity through interaction with a binding partner (eg, a receptor or ligand), various in vitro assays can be performed with the corresponding binding partner (eg, a selective binding agent). , Receptor, or ligand) can be used to measure binding of FGF-23 polypeptide. These assays can be used to screen test molecules for the ability to increase or decrease the rate and / or extent of binding of FGF-23 polypeptide to a binding partner. In one assay, FGF-23 polypeptide is immunized in the wells of a microtiter plate. The radiolabeled FGF-23 polypeptide binding partner (eg, iodinated FGF-23 polypeptide binding partner) and the test compound are then placed in the well either one at a time (in any order) or simultaneously. Can be added. Following incubation, the wells are washed and a scintillation counter is used to count the radioactivity and determine the extent to which the binding partner binds to the FGF-23 polypeptide. Typically, molecules are tested over a range of concentrations, and a series of control wells that lack one or more elements of the test assay can be used for accuracy of evaluation of results. An alternative to this method is to reverse the step of reversing the “position” of the protein (ie, immunizing the FGF-23 polypeptide binding partner against the microtiter plate wells, the test molecule and the radiolabeled FGF. Incubating the -23 polypeptide and determining the extent of FGF-23 polypeptide binding). See, eg, Current Protocols in Molecular Biology, Chapter 18 (Edited by Ausubel et al., Green Publishers Inc. and Wiley and Sons 1995).

  As an alternative to radioactive labeling, the FGF-23 polypeptide or its binding partner can be conjugated with biotin, and the presence of the biotinylated protein can then be determined using an enzyme such as wasabi peroxidase (HRP) or alkaline phosphatase ( AP)) (which are detected colorimetrically) can be detected using streptavidin or can be detected by fluorescent tagging of streptavidin. Antibodies against FGF-23 polypeptide or FGF-23 polypeptide binding partners (which are conjugated to biotin) are also detected following incubation of the complex with enzyme-linked streptavidin linked to AP or HRP. Can be used for any purpose.

  The FGF-23 polypeptide or FGF-23 polypeptide binding partner can be immobilized by attachment to agarose beads, acrylic beads, or other types of such inert solid phase substrates. The substrate-protein complex can be placed in a solution containing the complementary protein and the test compound. After incubation, these beads can be precipitated by centrifugation and the amount of binding between the FGF-23 polypeptide and its binding partner can be assessed using the methods described herein. Alternatively, the substrate-protein complex can be immobilized in the column using test molecules and complementary proteins that pass through the column. Complex formation between the FGF-23 polypeptide and its binding partner can then be assessed using any of the techniques described herein (eg, radiolabeling or antibody binding).

  Another in vitro assay useful for identifying test molecules that increase or decrease the formation of complexes between FGF-23 polypeptide binding proteins and FGF-23 polypeptide binding partners is the surface plasmon resonance detector system ( For example, BIAcore assay system (Pharmacia, Piscataway, NJ)). The BIAcore system is utilized as specified by the manufacturer. This assay essentially involves covalent binding of either FGF-23 polypeptide or FGF-23 polypeptide binding partner to a dextran-coated sensor chip (which is present in the detector). The test compound and other complementary proteins can then be injected into the chamber containing the sensor chip, either simultaneously or sequentially. The amount of complementary protein that binds can be assessed based on a change in the mass of the molecule physically related to the dextran-coated side of the sensor chip, the change in the mass of the molecule being measured by the detector system.

  In some cases, two or more test compounds can be evaluated together for their ability to increase or decrease the formation of a complex between an FGF-23 polypeptide and an FGF-23 polypeptide binding partner. May be desired. In these cases, the assays described herein can be readily modified by adding such additional test compounds either simultaneously with or following the first test compound. The remainder of the steps in this assay are described herein.

  In vitro assays (eg, those described herein) are for screening large numbers of compounds for effects on the formation of complexes between FGF-23 polypeptides and FGF-23 polypeptide binding partners. Can be used advantageously. These assays can be automated to screen compounds generated in phage display, synthetic peptides, and chemical synthesis libraries.

  Compounds that increase or decrease the formation of a complex between FGF-23 polypeptide and FGF-23 polypeptide binding partner also express either FGF-23 polypeptide or FGF-23 polypeptide binding partner F Cells and cell lines can be used to screen in cell culture. The cells and cell lines can be obtained from any mammal, but are preferably derived from human or other primate, canine or rodent sources. The binding of the FGF-23 polypeptide to the cells expressing the FGF-23 polypeptide binding partner on its surface is assessed in the presence or absence of the test compound, and the extent of binding is, for example, FGF- It can be determined by flow cytometry using a biotinylated antibody against the 23 polypeptide binding partner. Cell culture assays can be advantageously used to further evaluate compounds scored as positive in the protein binding assays described herein.

  Cell cultures can also be used to screen the effects of drug candidates. For example, the drug candidate can decrease or increase the expression of the FGF-23 gene. In certain embodiments, the amount of FGF-23 polypeptide or FGF-23 polypeptide fragment produced can be measured after exposure of the cell culture to the drug candidate. In certain embodiments, the actual impact of drug candidates on the cell culture can be detected. For example, overexpression of a particular gene can have a particular effect on cell culture. In such cases, the ability of a drug candidate to increase or decrease gene expression or its ability to prevent or inhibit a particular effect on cell culture can be tested. In other examples, the production of specific metabolites (such as fragments of a polypeptide, etc.) can cause or be associated with a disease or pathological condition. In such cases, the ability of drug candidates to reduce the production of such metabolites in cell culture can be tested.

(Internalization protein)
The tat protein sequence (from HIV) can be used to internalize proteins into cells. For example, Falwell et al., 1994, Proc. Natl. Acad. Sci. U. S. A. 91: 664-68. For example, the sequence of 11 amino acids of the HIV tat protein (YG-R-K-K-R-R-Q-R-R-R; SEQ ID NO: 3) (referred to as "protein transduction domain" or TAT PDT Have been described as mediating delivery across the cytoplasmic and nuclear membranes of cells. Schwarze et al., 1999, Science 285: 1569-72; and Nagahara et al., 1998, Nat. Med. 4: 1449-52. In these procedures, the FITC construct (FITC-labeled GGGGGY-G-R-K-K-R-R-Q-R-R-R; SEQ ID NO: 4) Penetrating the tissue following administration) is prepared, and binding of such constructs to the cells is detected by fluorescence cytometric separation (FACS) analysis. Cells treated with the tat-β-gal fusion protein show β-gal activity. Following injection, the expression of such constructs can be detected in many tissues (liver, kidney, lung, heart and brain tissue). It is believed that such constructs undergo some degree of denaturation to enter the cell and as such may require refolding following transfer into the cell.

  Thus, it is understood that the tat protein sequence can be used to internalize the desired polypeptide into the cell. For example, using tat protein sequences, FGF-23 antagonists (eg, anti-IL-lra-R selective binding agents, small molecules, soluble receptors, or antisense oligonucleotides) inhibit the activity of FGF-23 molecules. To do so, it can be administered intracellularly. As used herein, the term “FGF-23 molecule” refers to both FGF-23 nucleic acid molecules and FGF-23 polypeptides, as described herein. If desired, the FGF-23 protein itself can also be administered internally to the cells using these procedures. See also Strauss, 1999, Science 285: 1466-67.

(Identification of cell sources using FGF-23 polypeptides)
In accordance with certain embodiments of the invention, it may be useful to be able to determine the source of a particular cell type associated with an FGF-23 polypeptide. For example, it may be useful to determine the origin of a disease or pathological condition as an aid in selecting an appropriate treatment. In particular embodiments, nucleic acids encoding FGF-23 polypeptides are used as probes to identify the cells described herein by screening cellular nucleic acids with such probes. obtain. In other embodiments, an anti-FGF-23 polypeptide antibody can be used to test for the presence of FGF-23 polypeptide in a cell, and thus such a cell is a cell of the type described herein. Or not.

(FGF-23 polypeptide composition and administration)
Therapeutic compositions are within the scope of the present invention. Such FGF-23 polypeptide pharmaceutical compositions receive a therapeutically effective amount of a FGF-23 polypeptide or FGF-23 nucleic acid molecule selected pharmaceutically or physiologically to be compatible with the mode of administration. It can be included in a mixture with possible prescription drugs. A pharmaceutical composition comprises a mixture of a therapeutically effective amount of one or more FGF-23 polypeptide selective binding agents with a pharmaceutically or physiologically acceptable prescription drug selected to be compatible with the mode of administration. May be included.

  Acceptable formulation materials are preferably non-toxic to recipients at the dosages and concentrations used.

  A pharmaceutical composition may modify, maintain or permeate, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissociation or release, adsorption, or permeation of the composition. Formulation materials for storage may be included. Suitable formulation materials include, but are not limited to: amino acids (eg, glycine, glutamine, asparagine, arginine, or lysine), antibacterial agents, antioxidants (eg, ascorbic acid, sodium sulfite, Or sodium hydrogen-sulfite), buffers (eg, borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids), bulking agents (eg, mannitol) Or glycine), chelating agents (eg, ethylenediaminetetraacetic acid (EDTA)), complexing agents (eg, caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), bulking agents, Sugars, disaccharides, and other carbohydrates Eg glucose, mannose or dextrin), protein (eg serum albumin, gelatin or immunoglobulin), colorant, fragrance and diluent, emulsifier, hydrophilic polymer (eg polyvinylpyrrolidone), low molecular weight polypeptide, salt Forming counterion (eg, sodium), preservative (eg, benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvent (eg, glycerin) , Propylene glycol, or polyethylene glycol), sugar alcohols (eg, mannitol or sorbitol), suspending agents, surfactants or wetting agents (eg, pluronic); PEG Sorbitan ester; polysorbate (eg, polysorbate 20 or polysorbate 80); triton; tromethamine; lecithin; cholesterol or tyloxapol), stability enhancer (eg, sucrose or sorbitol), tonicity enhancer (eg, alkali halide) Metals (preferably sodium chloride or potassium chloride), or mannitol, sorbitol), delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. Remington's Pharmaceutical Sciences 18th edition, A.R. R. See Gennaro, Mack Publishing Company 1990.

  The optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. Such compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the FGF-23 molecule.

The primary vehicle or carrier in the pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for infusion can be water, saline solution, or artificial cerebrospinal fluid, and can be supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin is a further exemplary vehicle. Other exemplary pharmaceutical compositions comprise Tris buffer at about pH 7.0-8.5 or acetate buffer at about pH 4.0-5.5, which further comprises sorbitol or suitable substitutes. Can be included. In one embodiment of the invention, the FGF-23 polypeptide composition may be selected from any prescription drug (Remington's Pharmaceuticals) having a desired degree of purity.
Sciences, supra) can be prepared for storage in the form of a lyophilized cake or an aqueous solution. Furthermore, the FGF-23 polypeptide product can be formulated as a lyophilizate using appropriate excipients such as sucrose.

  The FGF-23 polypeptide pharmaceutical composition may be selected for parenteral delivery. Alternatively, these compositions can be selected for delivery via inhalation or gastrointestinal tract (eg, oral). The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

  The formulation ingredients are present in concentrations acceptable at the site of administration. For example, a buffer is used to maintain the composition at a physiological pH or slightly lower pH (typically within a pH range of about 5 to about 8).

  When intended for parenteral administration, a therapeutic composition for use in the present invention comprises a pyrogen-free parenterally acceptable, comprising the desired FGF-23 molecule in a pharmaceutically acceptable vehicle. It can be in the form of an aqueous solution. A particularly suitable vehicle for parenteral injection is sterile distilled water, where the FGF-23 molecule is formulated as a sterile isotonic solution and stored appropriately. Yet another preparation involves the desired molecule and drug (eg, injectable microspheres, bio-erodible particles, polymer compounds (eg, polylactic acid or polyglycolic acid), beads or liposomes). Which provides controlled or sustained release of the product that can then be delivered via a cumulative injection. Hyaluronic acid can also be used, and this can have the effect of promoting duration in the circulation. Other suitable means for introduction of the desired molecule include implantable drug delivery devices.

  In one embodiment, the pharmaceutical composition can be formulated for inhalation. For example, the FGF-23 polypeptide can be formulated as a dry powder for inhalation. Inhalation solutions of FGF-23 polypeptide or nucleic acid molecules can also be formulated with a propellant for aerosol delivery. In yet another embodiment, the solution can be nebulized. Pulmonary administration is further described in PCT Publication No. WO94 / 20069, which describes pulmonary delivery of chemically modified proteins.

It is also contemplated that certain formulations can be administered orally. In one embodiment of the invention, the FGF-23 polypeptide administered in this manner is with or without a carrier conventionally used in the preparation of solid dosage forms (eg, tablets or capsules). Can be prescribed. For example, capsules have maximized bioavailability and pre-systemic degradation (pre-systemic degradation).
It can be designed to release the active part of the formulation at a point in the gastrointestinal tract when degradation is minimized. Additional agents can be included to facilitate absorption of the FGF-23 polypeptide. Diluents, fragrances, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be used.

  Another pharmaceutical composition may contain an effective amount of FGF-23 polypeptide in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. Solutions can be prepared in unit dosage form by dissolving the tablets in sterile water or another suitable vehicle. Suitable excipients include, but are not limited to: an inert diluent (eg, calcium carbonate, sodium carbonate or sodium bicarbonate, lactose, or calcium phosphate); or a binder (eg, starch, gelatin or Acacia); or lubricant (eg, magnesium stearate, stearic acid or talc).

  Additional FGF-23 polypeptide pharmaceutical compositions will be apparent to those of skill in the art and include formulations comprising FGF-23 polypeptide in a sustained or controlled delivery formulation. Techniques for formulating various other sustained or controlled delivery means (eg, liposomal carriers, bioerodable microparticles or porous beads and accumulating injections) are also known to those skilled in the art. See, for example, PCT / US93 / 00829, which describes controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions.

  Further examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles (eg, films or microcapsules). Sustained release matrices include polyesters, hydrogels, polylactides (US Pat. No. 3,773,919 and European Patent No. 058881), copolymers of L-glutamic acid and γ ethyl-L-glutamate (Sidman et al., 1983, Biopolymers). 22: 547-56), poly (2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15: 167-277 and Langer, 1982, Chem. Tech. 12: 98-105). Mention may be made of ethylene vinyl acetate (Langer et al., Supra) or poly-D (-)-3-hydroxybutyric acid (European Patent 133988). Sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, eg, Epstein 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 used for in vivo administration typically must be sterile. This can be achieved by filtration through sterile filtration membranes. If the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. In addition, parenteral compositions are generally placed in a container having a sterile access port (eg, a vial having a stopper pierceable by an intravenous solution bag or hypodermic needle).

  Once the pharmaceutical composition is formulated, it can be stored as a solution, suspension, gel, emulsion, solid, or a dry or lyophilized powder in a sterile vial. Such formulations can be stored either in a ready-to-use form or in a form that requires reconstitution prior to administration (eg, lyophilized).

  In certain embodiments, the present invention relates to kits for generating single dose dosage units. The kits can each include both a first container having a dry protein and a second container having an aqueous formulation. Also included within the scope of the present invention are kits that include single and multi-chambered pre-filled syringes (eg, liquid syringes and dispersion syringes).

  The effective amount of a pharmaceutical composition of FGF-23 used therapeutically will depend, for example, on the nature and purpose of the treatment. Thus, the appropriate dosage level for treatment will depend, in part, on the molecule being delivered, the indication in which the FGF-23 molecule is used, the route of administration, and the size of the patient (weight, body surface, or It will be appreciated by those skilled in the art that it varies depending on the size of the organ) and condition (age and general health). Thus, a clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. A typical dosage can range from about 0.1 μg / kg to about 100 mg / kg or more, depending on the factors described above. In other embodiments, the dosage may range from 0.1 μg / kg to about 100 mg / kg; or 1 μg / kg to about 100 mg / kg; or 5 μg / kg to about 100 mg / kg.

  The frequency of dosing will depend on the pharmacokinetic parameters of the FGF-23 molecule in the formulation used. Typically, the clinician will administer the composition until a dosage is reached that achieves the desired effect. Thus, the composition may be administered as a single long-term dose, as two or more doses (which may or may not contain the same amount of the desired molecule), or as a continuous infusion through an implantation device or catheter. As can be administered. Further improvements in appropriate dosages are routinely made by those skilled in the art and are within the scope of work routinely performed by those skilled in the art. Appropriate dosages can be ascertained through the use of appropriate dose-response data.

  The route of administration of the pharmaceutical composition follows known methods such as: for example, orally; intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial Via injection by the portal vein, or intralesional route; by the sustained release system; or by the implantation device. If desired, these compositions can be administered by bolus injection, can be administered continuously by infusion, or can be administered by an implantation device.

  Alternatively or additionally, the composition can be administered locally via implantation of a membrane, sponge, or other suitable material in which the desired molecule is absorbed or encapsulated. If an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed release bolus or continuous administration.

  In some cases, it may be desirable to use FGF-23 polypeptide pharmaceutical compositions in an ex vivo manner. In such instances, the cells, tissues, or organs removed from the patient are added to the FGF-23 polypeptide pharmaceutical composition after these cells, tissues, or organs are subsequently implanted back into the patient. Be exposed.

  In other cases, the FGF-23 polypeptide uses a method as described herein to transplant specific cells that are genetically engineered to express and secrete the FGF-23 polypeptide. Can be delivered. Such cells can be animal or human cells and can be autologous, heterologous, or xenogeneic. If necessary, the cells can be immortalized. To reduce the chance of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissue. The encapsulating material is typically a biocompatible semi-permeable polymeric enclosure or membrane that allows release of the protein product, but does not allow other products from the patient's immune system or surrounding tissue. Prevent cell destruction by harmful factors.

  As discussed herein, it may be desirable to treat an isolated cell population (eg, stem cells, lymphocytes, erythrocytes, chondrocytes, neurons, etc.) with one or more FGF-23 polypeptides. possible. This can be accomplished by exposing the isolated cells directly to the polypeptide if it is in a form that is permeable to the cell membrane.

  Further embodiments of the invention provide cells and methods (eg, homologous recombination and / or other methods) for both in vitro production of therapeutic polypeptides and production and delivery of therapeutic polypeptides by gene therapy or cell therapy. Recombinant production method). Homologous recombination and other methods of recombination can be used to modify cells that contain the normally transcriptionally silent FGF-23 gene (ie, the underexpressed gene), thereby providing a therapeutically effective amount of Cells expressing the FGF-23 polypeptide can be produced.

  Homologous recombination is a technique originally developed to target genes to induce or correct mutations in transcriptionally active genes (Kucherrapti, Prog. In Nucl. Acid Res. And Mol. Biol.,). 36: 301, 1989). The basic technique is as a method for introducing specific mutations into specific regions of the mammalian genome (Thomas et al., Cell, 44: 419-428, 1986; Thomas and Capecchi, Cell, 51: 503-512). 1987; Doetschman et al., Proc. Natl. Acad. )It has been developed. Exemplary homologous recombination techniques are described in US Pat. No. 5,272,071, European Patent Nos. 913051, 505500; PCT / US90 / 07642, International Publication No. WO 91/09955.

  Through homologous recombination, the DNA sequence to be inserted into the genome can be directed to a specific region of the gene of interest by joining this DNA sequence to the targeting DNA. Targeting DNA is a nucleotide sequence that is complementary (homologous) to a genomic DNA region. A piece of targeted DNA that is complementary to a specific region of the genome is brought into contact with the parent strand during the DNA replication process. It is a general property of DNA inserted into cells to hybridize and thus recombine with other pieces of endogenous DNA through shared homologous regions. If this complementary strand is conjugated to an oligonucleotide containing a mutation or a different sequence or additional nucleotides, it will also be incorporated into the newly synthesized strand as a result of recombination. As a result of the proofreading function, the new DNA sequence can serve as a template. Thus, the transferred DNA is integrated into the genome.

  Bound to these pieces of targeting DNA are DNA regions (eg, flanking sequences) that can interact with or control the expression of the FGF-23 polypeptide. For example, the promoter / enhancer element, suppressor, or exogenous transcriptional regulatory element is intended to be in a sufficiently proximal and sufficient orientation to affect transcription of the DNA encoding the desired FGF-23 polypeptide. Inserted into the genome. Control elements control some DNA present in the host cell genome. Thus, expression of the desired FGF-23 polypeptide is not by transfection of DNA encoding the FGF-23 gene itself, but by DNA that provides a recognizable signal for transcription of the FGF-23 gene to the endogenous gene sequence. It can be achieved by the use of targeting DNA combined with regulatory segments, including regions homologous to the endogenous gene of interest.

  In an exemplary method, expression of a desired targeted gene in a cell (ie, a desired endogenous cellular gene) is preselected by introduction of DNA comprising at least regulatory sequences, exons and splice donor sites. Altered through homologous recombination into the cell genome at the selected site. These components are in effect responsible for the production of new transcription units, where the regulatory sequences, exons and splice donor sites present in the DNA construct are operably linked to the endogenous gene. It is introduced into the chromosomal (genomic) DNA in a resulting manner. As a result of the introduction of these components into the chromosomal DNA, the expression of the desired endogenous gene is altered.

  Altered gene expression, as described herein, activates (or causes to be expressed) a gene that is normally silent (not expressed) in the resulting cell, and Increasing the expression of genes that are not expressed at a physiologically important level in the cell when obtained. This embodiment further modifies the pattern of regulation or induction to be different from the pattern of regulation or induction that occurs in the cell when obtained, and reduces the expression of genes expressed in the cell when obtained (Including removing).

  One way in which homologous recombination can be used to increase or produce FGF-23 polypeptide production from a cell's endogenous FGF-23 gene is, first, site-specific using homologous recombination. Recombinant sequences from recombination systems (eg, Cre / loxP, FLP / FRT) (Sauer, Current Opinion In Biotechnology, 5: 521-527, 1994; and Sauer, Methods Enzymology, 225: 890-900, 1993). Placing upstream (ie, 5 ') of the endogenous FGF-23 polypeptide coding region of the cell's genome. A plasmid containing a recombination site homologous to the site located immediately upstream of the genomic FGF-23 polypeptide coding region is introduced into the modified cell line with the appropriate recombinase enzyme. The recombinase enzyme causes the plasmid to be integrated into the recombination site located immediately upstream of the genomic FGF-23 polypeptide coding region in the cell line via the recombination site of the plasmid (Baubonis and Sauer , Nucleic Acids Res., 21: 2025-2029, 1993; and O'Gorman et al., Science, 251: 1351-1355, 1991). Any flanking sequences known to increase transcription (eg, enhancers / promoters, introns or translational enhancers), when properly placed in this plasmid, will create a new or modified transcription unit. Incorporated in a manner, results in de novo FGF-23 polypeptide production or increased FGF-23 polypeptide production from the endogenous FGF-23 gene of the cell.

A further method for using a cell line in which a site-specific recombination sequence is located immediately upstream of the endogenous genomic FGF-23 polypeptide coding region of the cell uses a second homologous recombination, The introduction of recombination sites elsewhere in the genome of this cell line. The appropriate recombinase enzyme is then introduced into the two recombination site cell line, resulting in a new transcription unit or modification that results in de novo or increased FGF-23 polypeptide production from the endogenous FGF-23 gene of the cell. Recombination events (deletions, inversions, or translocations) are generated (Sauer, Current Opinion In Biotechnology, 5; 521-27, 1994; and Sauer, Methods)
In Enzymology, 225: 890-900, 1993).

  Further approaches to increase or cause expression of FGF-23 polypeptide from the cell's endogenous FGF-23 gene are de novo or increased FGF-23 from the cell's endogenous FGF-23 gene. Increasing or causing the expression of a gene (eg, a transcription factor) and / or decreasing the expression of a gene (eg, a transcriptional repressor) in a manner that results in polypeptide production. This method allows non-naturally occurring polypeptides (eg, polypeptides comprising a site-specific DNA binding domain fused to a transcription factor domain) to be de novo or increased FGF from the endogenous FGF-23 gene of the cell. Including introduction into a cell such that -23 polypeptide production occurs.

  The present invention further relates to DNA constructs useful in methods of altering target gene expression. In certain embodiments, exemplary DNA constructs include: (a) one or more targeting sequences; (b) regulatory sequences; (c) exons; and (d) unpaired splice donor sites. . The targeting sequence in this DNA construct is such that the incorporation of elements (a)-(d) into the target gene in the cell is operably linked to the sequence of the endogenous target gene. Orient. In another embodiment, the DNA construct comprises: (a) one or more targeting sequences, (b) regulatory sequences, (c) exons, (d) splice donor sites, (e) introns, and (F) a splice acceptor site; wherein the targeting sequence directs the incorporation of elements (a)-(f) such that the elements of (b)-(f) are operably linked to an endogenous gene. To do. This targeting sequence is homologous to a preselected site of cellular chromosomal DNA where homologous recombination occurs. In this construct, the exon is generally 3 'to the regulatory sequence and the splice donor site is 3' to the exon.

  If the sequence of a particular gene (eg, the FGF-23 nucleic acid sequence presented herein) is known, a piece of DNA complementary to a selected region of this gene can be, for example, native. It can be synthesized or otherwise obtained by appropriate restriction of specific recognition sites bound to the region of interest of DNA. This piece serves as a targeting sequence upon uptake into the cell and hybridizes to its homologous region in the genome. If this hybridization occurs during DNA replication, the DNA piece, and any additional sequence attached to the DNA piece, acts as an Okazaki fragment and is incorporated into the daughter strand of the newly synthesized DNA. . Thus, the present invention encompasses nucleotides that encode FGF-23 polypeptides that can be used as targeting sequences.

  FGF-23 polypeptide cell therapy (eg, transplantation of cells that produce FGF-23 polypeptide) is also contemplated. This embodiment involves transplanting cells capable of synthesizing and secreting a biologically active form of FGF-23. Such soluble FGF-23 polypeptide-producing cells can be cells that are natural producers of FGF-23 polypeptides, or have the ability to produce FGF-23 polypeptides with the desired FGF-23 molecule. It can be a recombinant cell that has been enhanced by transforming with the gene that encodes it or a gene that enhances expression of the FGF-23 polypeptide. Such modifications can be achieved by a vector suitable for delivering the gene and for promoting its expression and secretion. In patients receiving FGF-23 polypeptide, natural cells producing FGF-23 polypeptide can be used to minimize potential immunological reactions, such as may occur by administration of a foreign species polypeptide. It is preferred that is of human origin and that human FGF-23 polypeptides are produced. Similarly, it is preferred that recombinant cells producing FGF-23 polypeptide are transformed with an expression vector comprising a gene encoding human FGF-23 polypeptide.

  The transplanted cells can be encapsulated to avoid infiltration of surrounding tissue. A biocompatible cell that allows human or non-human animal cells to release FGF-23 polypeptide but prevents destruction of the cell by the patient's immune system or by other detrimental factors from surrounding tissues. The patient can be implanted within a semi-permeable polymer foreskin or membrane. Alternatively, the patient's own cells transformed ex vivo to produce FGF-23 polypeptide can be transplanted directly into the patient without such encapsulation.

  Techniques for encapsulating viable cells are known in the art, and preparation of encapsulated cells and transplantation into these patients can be routinely accomplished. For example, Baetge et al. (PCT Publication Nos. WO95 / 105452 and PCT / US94 / 09299) describe membrane capsules containing genetically engineered cells for effective delivery of biologically active molecules. These capsules are biocompatible and easily recoverable. These capsules encapsulate cells transfected with a recombinant DNA molecule comprising a DNA sequence encoding a biologically active molecule operably linked to a promoter, and these cells are directed to a mammalian host. Not exposed to in vivo down-regulation during transplantation. This device provides for delivery of molecules from viable cells to specific sites within the recipient. See also U.S. Pat. Nos. 4,892,538, 5,011,472, and 5,106,627. A system for encapsulating viable cells is described in PCT Publication No. WO 91/10425 (Aebischer et al.). PCT Publication No. WO 91/10470 (Aebischer et al.); Winn et al., Expert. Neurol. 113: 322-329, 1991, Aebischer et al., Exper. Neurol. 111: 269-275, 1991; and Tresco et al., ASAIO, 38: 17-23, 1992.

  In vivo and in vitro gene therapy delivery of FGF-23 polypeptides is also envisioned. One example of a gene therapy technique is the FGF-23 gene (either genomic DNA, cDNA, and / or synthetic DNA) that encodes an FGF-23 polypeptide that can be operably linked to a constitutive or inducible promoter. To form a “gene therapy DNA construct”. This promoter may be homologous or heterologous to the endogenous FGF-23 gene provided that the promoter is active in the cell or tissue type into which the construct is inserted. Other components of the gene therapy DNA construct include DNA molecules designed for site-specific incorporation (eg, endogenous sequences useful for homologous recombination), tissue-specific promoters, enhancers, as appropriate. , Or silencers, DNA molecules that may provide superior selective advantages over parental cells, DNA molecules useful as labels for identifying transformed cells, negative selection systems, cell-specific binding agents (eg, cell targets Cell-specific internalization factors, and transcription factors to enhance expression by the vector, as well as factors that allow the production of the vector.

  The gene therapy DNA construct can then be introduced into cells (either ex vivo or in vivo) using viral or non-viral vectors. One means for introducing gene therapy DNA constructs is by viral vectors as described herein. Certain vectors (eg, retroviral vectors) deliver the DNA construct to the chromosomal DNA of the cell, and this gene can be incorporated into the chromosomal DNA. Other vectors function as episomes and the gene therapy DNA construct remains in the cytoplasm.

In yet other embodiments, the regulatory element may be involved in the controlled expression of the FGF-23 gene in the target cell. Such elements are turned on in response to the appropriate effector. In this manner, the therapeutic polypeptide can be expressed when desired. One conventional control means is a small molecule that is used to dimerize a chimeric protein (eg, a DNA binding protein or a transcriptional activation protein) that includes a small molecule binding domain and a domain that can initiate a biological process. Includes the use of dimerizers or rapalog (PCT publication numbers WO 96/41865, WO 97/31898, and WO
97/31899). Protein dimerization can be used to initiate transgene transcription.

  Alternative regulatory techniques use methods of storing proteins expressed as aggregates or clusters from the gene of interest within the cell. The gene of interest is expressed as a fusion protein, which contains a conditional aggregation domain that results in retention of the aggregated protein within the endoplasmic reticulum. Stored proteins are stable in the cell and are inactive. However, these proteins can be released by administering a drug (eg, a small molecule ligand) that removes the conditional aggregation domain and thereby specifically destroys this aggregate or cluster, resulting in these Of the protein can be secreted from the cell. See Aridol et al., Science 287: 816-817, 2000, and Pivera et al., Science 287, 826-830, 2000.

  Other suitable control means or gene switches include, but are not limited to, the systems described herein. Mifepristone (RU486) is used as a progesterone antagonist. By binding the modified progesterone receptor ligand binding domain to a progesterone antagonist, transcription by formation of a dimer of two transcription factors is activated, which is then passed into the nucleus of the binding DNA. This ligand binding domain is modified to eliminate the ability of the receptor to bind to its natural ligand. Modified steroid hormone receptor systems are further described in US Pat. No. 5,364,791 and PCT Publication Nos. WO96 / 40911 and WO97 / 10337.

  Yet another control system uses ecdysone (a fruit fly steroid hormone), which binds to and activates the ecdysone receptor (cytoplasmic receptor). This receptor then translocates to the nucleus and binds a specific DNA response element (a promoter from an ecdysone responsive gene). The ecdysone receptor includes a trans-acting domain, a DNA binding domain, and a ligand binding domain to initiate transcription. The ecdysone system is further described in US Pat. No. 5,514,578 and PCT Publication Nos. WO 97/38117, WO 96/37609, and WO 93/03162.

  Another control means uses a positive tetracycline controllable transactivator. This system is a mutant tet repressor protein DNA binding domain linked to a polypeptide that activates transcription (mutant tet R-4 amino acid changes that result in a reverse tetracycline-regulated transactivator protein, i.e., in the presence of tetracycline. , To the tet operator). Such systems are described in US Pat. Nos. 5,464,758, 5,650,298, and 5,654,168.

  Additional expression control systems and nucleic acid constructs are available from Innovir Laboratories Inc. U.S. Pat. Nos. 5,741,679 and 5,834,186.

  In vivo gene therapy involves introducing a gene encoding an FGF-23 polypeptide into a cell via local injection of FGF-23 nucleic acid molecules or by other suitable viral or non-viral delivery vectors. (Hefti, Neurobiology, 25: 1418-1435, 1994). For example, a nucleic acid molecule encoding an FGF-23 polypeptide can be included in an adeno-associated virus (AAV) vector for delivery to targeted cells (eg, Johnson, PCT Publication No. WO95 / 34670; and PCT application number PCT / US95 / 07178). A recombinant AAV genome typically includes an AAV inverted terminal repeat flanking a DNA sequence encoding an FGF-23 polypeptide operably linked to a functional promoter and polyadenylation sequence.

  Alternative suitable viral vectors include retrovirus vectors, adenovirus vectors, herpes simplex virus vectors, lentivirus vectors, hepatitis virus vectors, parvovirus vectors, papovavirus vectors, poxvirus vectors, alphavirus vectors, coronaviruses Examples include, but are not limited to, vectors, rhabdovirus vectors, paramyxovirus vectors, and papillomavirus vectors. US Pat. No. 5,672,344 describes an in vivo virus-mediated gene transfer system comprising a recombinant neurotrophic HSV-1 vector. US Pat. No. 5,399,346 provides an example of a process for providing a therapeutic protein to a patient by delivery of human cells that have been treated in vitro to insert a DNA segment encoding the therapeutic protein. Additional methods and materials for carrying out gene therapy techniques include US Pat. No. 5,631,236 (including adenoviral vectors); US Pat. No. 5,672,510 (including retroviral vectors); and US Pat. No. 5,635,399 (including retroviral vectors expressing cytokines).

  Non-viral delivery methods include liposome-mediated transfer, naked DNA delivery (direct injection), receptor-mediated transfer (ligand-DNA complex), electroporation, calcium phosphate precipitation, and microparticle bombardment (eg, gene gun). However, it is not limited to these. Gene therapy materials and methods also include inducible promoters, tissue-specific enhancer-promoters, DNA sequences designed for site-specific uptake, DNA sequences, traits that can provide selective advantages over parental cells Expression by labels, negative selection and expression control systems (safety measures), cell-specific binding factors (for cell targeting), cell-specific internalization factors, and vectors to identify transformed cells Mention may be made of transcription factors for enhancing the expression, as well as methods of vector production. Such additional methods and materials for the implementation of gene therapy techniques are described in US Pat. No. 4,970,154 (including electroporation technology), US Pat. No. 5,679,559 (for gene delivery). US Pat. No. 5,676,954 (including liposomal carrier), US Pat. No. 5,593,875 (describing a method for calcium phosphate transfection), US Pat. No. 4,945,050 (describes a process in which biologically active particles are driven in cells at a rate that allows the particles to penetrate the surface of the cells and be taken inside the cells), And PCT Publication No. WO 96/40958 (including nuclear ligands).

  It is also contemplated that FGF-23 gene therapy or cell therapy can further include delivery of one or more additional polypeptides in the same or different cells. Such cells can be introduced separately into the patient, or these cells can be contained within a single implantable device (eg, the encapsulating membrane described above) or the cells can be viral vectors Can be modified separately.

  A means for increasing endogenous FGF-23 polypeptide expression in cells by gene therapy is to insert one or more enhancer elements into the FGF-23 polypeptide promoter, wherein the enhancer element is It can act to increase the transcriptional activity of the FGF-23 polypeptide gene. The enhancer element used is selected based on the tissue in which it is desired to activate the gene; enhancer elements known to confer promoter activation in this tissue are selected. For example, the lck promoter enhancer element can be used when the gene encoding the FGF-23 polypeptide is “turned on” in T cells. Here, the functional portion of the added transcription element can be inserted into a fragment of DNA containing the FGF-23 polypeptide promoter using standard cloning techniques (and optionally, a vector, 5 ' And / or can be inserted into the 3 ′ flanking sequence). This construct (known as a “homologous recombination construct”) can then be introduced into the desired cells, either ex vivo or in vivo.

  Gene therapy can also be used to reduce FGF-23 polypeptide expression by altering the nucleotide sequence of the endogenous promoter. Such modification is typically accomplished via homologous recombination methods. For example, a DNA molecule comprising all or part of the promoter of the FGF-23 gene selected for inactivation can be engineered to remove and / or replace a piece of promoter that regulates transcription. For example, the TATA box and / or binding site of a promoter's transcriptional activator can be deleted using standard molecular biology techniques; such a deletion can inhibit promoter activity; This suppresses transcription of the corresponding FGF-23 gene. Deletion of the TATA box or transcriptional activator binding site in the promoter comprises a DNA construct comprising all or related portions of an FGF-23 polypeptide promoter (from the same or related species as the FGF-23 gene to be regulated) Wherein one or more TATA boxes and / or nucleotides of the transcriptional activator binding site are mutated via substitution, deletion and / or insertion of one or more nucleotides. Is done. As a result, the TATA box and / or activator binding site is reduced in activity or completely inactivated. The construct also typically includes at least about 500 bases of DNA corresponding to the native (endogenous) 5 'and 3' DNA sequences adjacent to the modified promoter segment. This construct can be introduced into appropriate cells (either ex vivo or in vivo), either directly or via viral vectors as described herein. Typically, incorporation of this construct into the cellular genomic DNA is via homologous recombination, where the 5 ′ and 3 ′ DNA sequences in the promoter construct are altered to endogenous chromosomal DNA via hybridization. Act to assist in the incorporation of different promoter regions.

(Therapeutic use)
FGF-23 nucleic acid molecules, polypeptides, and agonists and antagonists thereof are used to treat, diagnose, ameliorate, or prevent a number of diseases, disorders or conditions, including those cited herein. obtain.

  FGF-23 polypeptide agonists and antagonists include molecules that modulate FGF-23 polypeptide activity and increase or decrease the activity of at least one mature form of FGF-23 polypeptide. An agonist or antagonist can be a cofactor (eg, protein, peptide, carbohydrate, lipid or low molecular weight molecule) that interacts with and thereby regulates its activity. Potent polypeptide agonists or antagonists include antibodies that react with either soluble or membrane-bound forms of FGF-23 polypeptides that contain some or all of the extracellular domain of this protein. Molecules that modulate FGF-23 polypeptide expression typically include nucleic acids encoding FGF-23 polypeptides that can serve as antisense regulators of expression.

  The FGF-23 nucleic acid molecules, polypeptides, and agonists and antagonists thereof of the present invention are useful for the same purposes for which FGF family member polypeptides are known to be useful. Therefore, the FGF-23 polypeptide of the present invention is a cell of mesoderm origin, ectoderm origin and various cells of endoderm origin (fibroblasts, corneal and vascular endothelial cells, granulocytes, adrenocortical cells) , Including chondrocytes, myoblasts, vascular smooth muscle cells, lens epithelial cells, melanocytes, keratinocytes, oligodendrocytes, astrocytes, osteoblasts and hematopoietic cells). Among these biological activities includes the ability to stimulate the proliferation of vascular endothelial cells and allow the endothelial cells to penetrate the basement membrane. Consistent with these properties, the FGF-23 polypeptides of the present invention can stimulate angiogenesis and promote wound healing (ie, in diseased tissue caused by burns, trauma, injury, surgery or ulcers). Promotes the repair or replacement of damage). These polypeptides can also reduce mesoderm formation and regulate neuronal, adipocyte and skeletal muscle cell differentiation. These polypeptides can also be used to prevent or ameliorate skin aging caused by solar radiation by stimulating keratinocyte proliferation. Furthermore, the polypeptides of the present invention can be used to maintain organs prior to transplantation or to support the culture of primary cells and tissues. Furthermore, these polypeptides can be used to prevent alopecia since FGF family members activate hair-forming cells and promote melanocyte proliferation. They can also be used to stimulate the growth and differentiation of hematopoietic and bone marrow cells when used in combination with other cytokines.

  FGF-23 is associated with a human autosomal dominant genetic disorder, hypophosphatemic rickets (ADHR) (The ADHR Consortium, 2000, Nature Genetics 26: 345-48). Accordingly, the FGF-23 nucleic acid molecules, polypeptides, and agonists and antagonists thereof of the present invention can be used to treat, diagnose, ameliorate or prevent ADHR.

  The FGF-23 gene has been shown to be most closely related to human FGF-21 (Yamashita et al., 2000, Biochem. Biophys. Res. Commun. 277: 494-98), and this human FGF-23 gene is The gene that is most abundantly expressed in the liver and expressed at lower levels in the thymus (Nishimura et al., 2000, Biochim. Biophys. Acta 1492: 203-06). Thus, FGF-23 nucleic acid molecules, polypeptides and their agonists and antagonists can be used to treat, diagnose, ameliorate or prevent diseases, disorders or conditions involving the liver or thymus.

  A non-exclusive list of other diseases, disorders or conditions that can be treated, diagnosed, remedied or prevented using the FGF-23 nucleic acid molecules, polypeptides and agonists and antagonists thereof of the present invention includes: skin Wound, epidermolysis bullosa, androgenetic alopecia, gastric ulcer, duodenal ulcer, erosive gastritis, esophagitis, esophageal reflux disease, inflammatory bowel disease, radiation or chemotherapy-induced enterotoxicity, pulmonary hyaline, necrosis of respiratory epithelium, Emphysema, lung inflammation, pulmonary fibrosis, cirrhosis, fulminant liver failure, viral hepatitis and diabetes.

  An agonist or antagonist of FGF-23 polypeptide function may be combined with one or more cytokines, growth factors, antibiotics, anti-inflammatory agents and / or chemotherapeutic agents (concurrently or as appropriate) for the condition being treated. Or subsequently) can be used.

  Other diseases caused by or mediated by undesirable levels of FGF-23 polypeptide are included within the scope of the present invention. Undesirable levels include excessive levels of FGF-23 polypeptide and subnormal levels of FGF-23 polypeptide.

(Use of FGF-23 nucleic acid and FGF-23 polypeptide)
The nucleic acid molecules of the invention (including nucleic acid molecules that themselves do not encode biologically active polypeptides) can be used to map the chromosomal location of the FGF-23 gene and related genes. Mapping can be performed by techniques known in the art, such as PCR amplification and in situ hybridization.

  FGF-23 nucleic acid molecules (including nucleic acid molecules that themselves do not encode biologically active polypeptides) are qualitative or quantitative for the presence of FGF-23 nucleic acid molecules in mammalian tissue or body fluid samples. It may be useful as a hybridization probe in a diagnostic assay for testing with either.

  Other methods can also be used where it is desired to inhibit the activity of one or more FGF-23 polypeptides. Such inhibition may be effected by nucleic acid molecules that are complementary to and hybridize to expression control sequences (triple helix formation) or FGF-23 mRNA. For example, antisense DNA or RNA molecules (which have a sequence that is complementary to at least a portion of the FGF-23 gene) can be introduced into cells. Antisense probes can be designed by available techniques using the sequences of the FGF-23 gene disclosed herein. Typically, each such antisense molecule is complementary to the start site (5 'end) of each selected FGF-23 gene. If the antisense molecule then hybridizes to the corresponding FGF-23 mRNA, translation of the mRNA is prevented or reduced. Antisense inhibitors provide information related to the reduction or absence of FGF-23 polypeptide in a cell or organism.

  Alternatively, gene therapy can be used to create dominant negative inhibitors of one or more FGF-23 polypeptides. In this situation, DNA encoding a variant polypeptide of each selected FGF-23 polypeptide can be prepared and using any of the viral or non-viral methods described herein. It can be introduced into the patient's cells. Each such variant is typically designed to compete with an endogenous polypeptide in its biological role.

  Furthermore, FGF-23 polypeptides (whether biologically active or not) can be used as immunogens, ie these polypeptides contain at least one epitope against which antibodies can be elicited. . Selective binding agents that bind to FGF-23 polypeptides (as described herein) can be used for in vivo and in vitro diagnostic purposes, such as body fluid samples or cells. This includes, but is not limited to, use in a labeled form to detect the presence of FGF-23 polypeptide in a sample. These antibodies can also be used to prevent, treat, or diagnose a number of diseases and disorders, including those listed herein. These antibodies can bind to the FGF-23 polypeptide or bind to and bind to the polypeptide so as to reduce or block at least one activity characteristic of the FGF-23 polypeptide. At least one activity characteristic may be increased (including by increasing the pharmacokinetics of the FGF-23 polypeptide).

The FGF-23 polypeptides of the present invention can be used to clone FGF-23 polypeptide receptors using expression cloning strategies. A radiolabeled ( ¹²⁵ iodine) FGF-23 polypeptide or an affinity / activity-tagged FGF-23 polypeptide (eg, Fc fusion or alkaline phosphatase fusion) is used in a binding assay to produce an FGF-23 polypeptide receptor. Cell types or cell lines or tissues that express can be identified. RNA isolated from such cells or tissues can be converted to cDNA, cloned into a mammalian expression vector, and transfected into a mammalian cell (eg, COS or 293 cell) to produce an expression library. Make it. The radiolabeled or tagged FGF-23 polypeptide is then used as an affinity ligand to identify and identify from this library a subset of cells expressing FGF-23 polypeptide receptor on its surface. Can be isolated. DNA can then be isolated from these cells and transfected into mammalian cells to produce a secondary expression library (where the fraction of cells expressing the FGF-23 polypeptide receptor is the original library). Many times higher). This enrichment process can be repeated iteratively until a single recombinant clone containing the FGF-23 polypeptide receptor is isolated. Isolation of the FGF-23 polypeptide receptor is useful for identifying or developing new agonists and antagonists of the FGF-23 polypeptide signaling pathway. Such agonists and antagonists include soluble FGF-23 polypeptide receptors, anti-FGF-23 polypeptide receptor antibodies, small molecules or antisense oligonucleotides, and these are the diseases described herein. Alternatively, one or more of the disorders can be used to treat, prevent, or diagnose.

  Deposit of cDNA encoding human FGF-23 polypeptide, subcloned into pGET-t vector and having accession number PTA-1617, at American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209 I went on March 31, 2000.

  The following examples are intended for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way.

(Example 1: Cloning of human FGF-23 polypeptide gene)
To isolate the cDNA sequence encoding the human FGF-23 polypeptide, a BLAST search based on homology of the human genome database was performed. A putative coding sequence sharing homology with the fibroblast growth factor (FGF) family was identified in a human genomic clone (GenBank accession number AC008012). This putative coding sequence consisted of three potential exons separated by 6.6 kb and 1.87 kb introns. Using this sequence, gene-specific oligonucleotides were designed using various PCR strategies for the identification of cDNA sources and for the generation of cDNA clones.

  Multiple cDNA libraries were prepared in a total reaction volume of 250 μl, each with 10 pmol of amplimer (5′-C-T-A-T-C-C-C-A-A-T-G-C-C. -T-C-C-C-C-A-C-T-G-3 '(SEQ ID NO: 42) and 5'-C-G-C-C-C-C-T-G-A-C- C-A-C-C-C-C-T-A-A-T-G-3 ′ (SEQ ID NO: 43)), and Ready-To-Go PCR beads (Pharmacia, Piscataway, NJ) The reaction was analyzed. Reaction, 95 ° C for 5 minutes in 1 cycle; 95 ° C for 30 seconds, 68 ° C for 15 seconds, and 72 ° C for 1 minute in 35 cycles; and 72 ° C for 7 minutes in 1 cycle went. PCR products of the expected size (616 bp) were generated from a number of cDNA libraries (colon tumor T25 (random primed), fetal mesentery (oligo-dT primed), fetal gallbladder (random prime) And libraries derived from fetal heart (primed with oligo-dT). The PCR product produced from the fetal mesenteric cDNA library was subcloned using the TopoTA 4.0 cloning kit (Invitrogen), and the four clones were sequenced, and these clones were putative FGF-23. It was verified to contain a cDNA sequence. This fetal mesenteric cDNA library was selected for further amplification experiments to isolate the full-length cDNA sequence encoding the FGF-23 polypeptide.

A fetal mesenteric cDNA library was prepared as follows. Total RNA was extracted from human fetal mesentery using standard RNA extraction procedures, and poly-A ⁺ RNA was selected from this total RNA using standard procedures. Oligo-dT primed cDNA was synthesized from this poly-A ⁺ RNA using the Superscript Plasmid System (Gibco-BRL) for cDNA synthesis and plasmid cloning kits according to the manufacturer's suggested protocol. The resulting cDNA is digested with the appropriate restriction endonucleases Sal I and Not I and then ligated into pSPORT-1. The ligation product is purified using standard techniques. E. coli were transformed and bacterial transformants were selected on culture plates containing ampicillin. The cDNA library was composed of all or a subset of these transformants.

  Both 5'RACE and 3'RACE reactions were performed to generate a full-length cDNA sequence for FGF-23 polypeptide. To isolate the cDNA sequence corresponding to the 5 'end of the cDNA sequence for FGF-23 polypeptide, 5' RACE was randomly primed human fetal mesentery in Smart RACE cDNA amplification kit (Clontech), pSPORT1. CDNA library as well as primers (5'-GTGTGGTGAATGATTGTGTGATGAGCGCGGAT) -A-A-C-3 '(SEQ ID NO: 44), and 5'-C-T-G-A-T-G-G-G-G-T-G-C-G-C-C-A -TC-CACA-3 '(SEQ ID NO: 45)). The reaction was run at 94 ° C for 1 minute for 1 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 1 cycle. . Nested PCR was performed using a portion of the 5 ′ RACE amplification product (diluted 1/100) as well as the primer (5′-C-TA-T-G-A-C-C-A-T). -GAT-TAC-CGCC-CAAAGG-3 '(SEQ ID NO: 46), and 5'-CATT-CT -T-G-T-G-G-A-T-C-T-G-C-A-G-G-T-G-G-T-3 '(SEQ ID NO: 47)) It was. Nested PCR reaction, 1 cycle for 5 minutes at 94 ° C; 30 cycles for 94 seconds for 15 seconds, 68 ° C for 15 seconds and 72 ° C for 3 minutes; and 1 cycle for 7 minutes at 72 ° C I went there. The amplified product was analyzed by agarose gel electrophoresis and a 200 bp prominent PCR product was isolated and subcloned using the TopoTA 4.0 cloning kit. Sequencing analysis of the isolated clones showed that the 5 'PCR product did not expand the known sequence.

In order to isolate additional cDNA sequences corresponding to the 5 ′ end of the cDNA sequence for FGF-23 polypeptide, further 5 ′ RACE experiments were performed. Further 5 ′ RACE experiments were performed using the Advantage-2 PCR kit (Clontech), the Marathon ^™ human heart cDNA library (Clontech) and the primers (5′-C-T-G-A-T-G-G-G-G- T-G-C-G-C-C-A-T-C-C-A-C-A-3 '(SEQ ID NO: 45) and AP1 (Clontech)). The reaction was carried out at 94 ° C. for 30 seconds for 1 cycle and 94 ° C. for 5 seconds and 68 ° C. for 4 minutes for 30 cycles. Nested PCR was performed using a portion of 5 ′ RACE amplification product (diluted 1/100) as well as primer (5′-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 ′ (SEQ ID NO: 45) and AP2 (Clontech)). Nested PCR reactions were performed at 94 ° C. for 30 seconds for 1 cycle, 94 ° C. for 30 seconds, and 68 ° C. for 4 minutes for 30 cycles. The amplified product was analyzed by agarose gel electrophoresis and the most prominent PCR product (350 bp) was isolated and subcloned using the TopoTA 4.0 cloning kit. Sequencing analysis of the isolated clones showed that the 5 ′ PCR product expanded the known sequence at approximately 143 bp.

  To isolate the cDNA sequence corresponding to the 3 ′ end of the cDNA sequence for FGF-23 polypeptide, 3′RACE was randomly primed human fetal mesentery in the Smart RACE cDNA amplification kit (Clontech), pSPORT1. CDNA library, as well as primers (5'-CGGGCCTCTCTCTGTCATCACAGG AGG) -C-T-C-3 '(SEQ ID NO: 48), and 5'-C-G-G-G-C-C-T-C-T-C-G-C-T-A-T -TACGC-3 '(SEQ ID NO: 49)). The reaction was run at 94 ° C for 1 minute for 1 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 1 cycle. . Nested PCR was performed using a portion of the 5 ′ RACE amplification product (diluted to 1/100) as well as the primer (5′-GCGCCCAGGAGGAC) A-A-C-A-G-C-C-C-G-A-3 ′ (SEQ 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 '(SEQ ID NO: 51)) The nested PCR reaction was performed at 94 ° C. 5 cycles per 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 1 cycle. Analyzed by gel electrophoresis and isolated a 650 bp prominent PCR product using the TopoTA 4.0 cloning kit, Was subcloned. Sequence analysis of the clones isolated indicated that 3'PCR product at 433bp (including poly -A region) is enlarged to known sequences.

  A contiguous sequence that appeared to contain the full length open reading frame for the FGF-23 gene was generated using the initial PCR amplification and sequences derived from 5 'and 3' RACE amplification. Sequence analysis of this consensus sequence showed that the FGF-23 gene contains a 753 bp open reading frame (FIGS. 1A-1B) encoding a 251 amino acid protein.

  Sequence analysis also showed that the FGF-23 polypeptide shares homology with the fibroblast growth factor (FGF) family. 2A-2G illustrate the following amino acid sequence alignments: human FGF-1 (hu FGF-1; SEQ ID NO: 4), human FGF-2 (hu FGF-2; SEQ ID NO: 5), human FGF-3 ( hu FGF-3; SEQ ID NO: 6), human FGF-4 (hu FGF-4; SEQ ID NO: 7), human FGF-5 (hu FGF-5; SEQ ID NO: 8), human FGF-6 (hu FGF-6; SEQ ID NO: 9), human FGF-7 (hu FGF-7; SEQ ID NO: 10), human FGF-8 (hu FGF-8; SEQ ID NO: 11), human FGF-9 (hu FGF-9; SEQ ID NO: 12), Human FGF-10 (hu FGF-10; SEQ ID NO: 13), human FGF-11 (hu FGF-11; SEQ ID NO: 14), human FGF-12 (hu FGF-12; SEQ ID NO: 15), human FGF-13 hu FGF-13; SEQ ID NO: 16), human FGF-14 (hu FGF-14; SEQ ID NO: 17), human FGF-16 (hu FGF-16; SEQ ID NO: 18), human FGF-17 (hu FGF-17; SEQ ID NO: 19), human FGF-18 (hu FGF-18; SEQ ID NO: 20), human FGF-19 (hu FGF-19; SEQ ID NO: 21), human FGF-23 (hu FGF-23; SEQ ID NO: 22), Mouse FGF-1 (mu FGF-1; SEQ ID NO: 23), mouse FGF-2 (mu FGF-2; SEQ ID NO: 24), mouse FGF-3 (mu FGF-3; SEQ ID NO: 25), mouse FGF-4 ( mu FGF-4; SEQ ID NO: 26), mouse FGF-5 (mu FGF-5; SEQ ID NO: 27), mouse FGF-6 (mu FGF-6; SEQ ID NO: 28), mouse Us FGF-7 (mu FGF-7; SEQ ID NO: 29), mouse FGF-8 (mu FGF-8; SEQ ID NO: 30), mouse FGF-9 (mu FGF-9; SEQ ID NO: 31), mouse FGF-10 ( mu FGF-10; SEQ ID NO: 32), mouse FGF-11 (mu FGF-11; SEQ ID NO: 33), mouse FGF-12 (mu FGF-12; SEQ ID NO: 34), mouse FGF-13 (mu FGF-13; SEQ ID NO: 35), mouse FGF-14 (mu FGF-14; SEQ ID NO: 36), mouse FGF-15 (mu FGF-15; SEQ ID NO: 37), rat FGF-16 (rat FGF-16; SEQ ID NO: 38), Mouse FGF-17 (mu FGF-17; SEQ ID NO: 39).

  From the amino acid sequence analysis shown in FIGS. 2A-2G, the FGF-23 gene appears to be closely related to mouse FGF-15 and human FGF-19. The locally restricted pattern of FGF-15 expression in the developing nervous system is important for FGF-15 to regulate cell division and pattern within specific regions of the embryonic brain, spinal cord and sensory organs (McWilter et al., 1997, Development 124: 3221-32). Thus, FGF-23 nucleic acid molecules, polypeptides and their agonists and antagonists may be useful for the diagnosis or treatment of diseases involving the developing nervous system. Human FGF-19, expressed in fetal cartilage, skin and retina, adult gallbladder and colon adenocarcinoma cell lines, is a region of chromosome 11 associated with osteoporotic pseudoglioma syndrome, a skeletal and retinal defect. Mapping (Xie et al., 1999, Cytokine 11: 729-35). Thus, FGF-23 nucleic acid molecules, polypeptides and their agonists and antagonists can be useful for diagnosing or treating diseases involving the skeletal system or the retina.

  The FGF-23 gene has been shown to be most closely related to human FGF-21 (Yamashita et al., 2000, Biochem. Biophys. Res. Commun. 277: 494-98), It is the gene that is most abundantly expressed in the liver and is expressed at a lower level in the thymus (Nishimura et al., 2000, Biochim. Biophys. Acta 1492: 203-06). Thus, FGF-23 nucleic acid molecules, polypeptides and their agonists and antagonists may be useful for the diagnosis or treatment of diseases involving the liver or thymus.

(Example 2: FGF-23 mRNA expression)
The expression of FGF-23 was analyzed by RT-PCR. Total RNA was prepared from various human fetal tissues using standard techniques. Template and primer mixtures were prepared with 2 μg total RNA and 50 ng random primer (Gibco-BRL) in a volume of 12 μl. The mixture was heated to 70 ° C. for 10 minutes and then cooled on ice. Reverse transcription was performed using 4 μl of 5 × first strand buffer (Gibco-BRL), 2 μl of 0.1 M DTT and 1 μl of 10 mM.
This is done by adding dNTPs to the template-primer mixture, warming the reaction mixture to 37 ° C. for 2 minutes, adding 1 μl Superscript II RT (Gibco-BRL), and then incubating the reaction mixture at 37 ° C. for 1 hour. It was.

  Using primers specific for glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a gene expected to express differences in RNA concentration and cDNA conversion efficiency at approximately the same level in all tissues tested And normalized by performing control PCR amplification on each cDNA sample. Control PCR amplification was performed using the amplimer 5'-TCCCAACCCCACTCGTGTTGATCATGG- 3 ′ (SEQ ID NO: 52) and 5′-GAC-C-A-C-A-G-T-C-C-A-T-G-G-C-A-T-C-A -C-T-3 '(SEQ ID NO: 53) and Ready-To-Go PCR beads (Amersham Pharmacia Biotech, Piscataway, NJ) were used. The reaction was carried out as follows: 95 ° C for 1 minute for 1 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. One cycle for minutes. The control reaction product was analyzed on a 2% agarose gel, the relative intensity of the control product was calculated, and the concentration of the cDNA sample was adjusted so that the cDNA sample produced a band of equal intensity G3PDH. FGF-23 expression analysis was performed using cDNA samples with standardized concentrations.

  The expression of FGF-23 was determined by the expression of amplimer 5′-C-T-A-T-C-C-C-A-A-T-G-C-C-T-C-C-C-C-A-C -T-G-3 '(SEQ ID NO: 54) and 5'-C-G-C-C-C-C-T-G-A-C-C-A-C-C-C-C-T- Analyze in PCR amplification, including AATG-3 ′ (SEQ ID NO: 55) as well as Ready to Go PCR beads. Reaction, 1 cycle at 95 ° C for 5 minutes; 30 cycles at 95 ° C for 30 seconds, 68 ° C for 30 seconds, and 72 ° C for 1 minute; and 72 ° C for 7 minutes in 1 cycle, carry out. The PCR product was analyzed on a 2% agarose gel and the relative intensity of the PCR product was estimated (using a weak PCR product as a baseline). The results of this analysis are shown in Table III.

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FGF-23 mRNA expression is analyzed by Northern blot. Multiple human tissue Northern blots (Clontech) are probed with the appropriate restriction fragments isolated from human FGF-23 polypeptide cDNA clones. The probe is labeled with ³² P-dCTP using standard techniques.

  Northern blots were prehybridized for 2 hours at 42 ° C. in hybridization solution (5 × SSC, 50% deionized formamide, 5 × Denhart solution, 0.5% SDS, and 100 mg / ml denatured salmon sperm DNA). Then hybridize overnight at 42 ° C. in fresh hybridization solution containing 5 mg / ml labeled probe. After hybridization, the filter was washed twice in 2 × SSC and 0.1% SDS for 10 minutes at room temperature and then in 0.1 × SSC and 0.1% SDS for 30 minutes at 65 ° C. Wash twice. The blot is then exposed to autoradiography.

Expression of FGF-23 mRNA in normal adult mouse tissues and 3 week old high-expressing and non-expressing transgenic mouse tissues (see Example 5) was localized by in situ hybridization. A panel of normal embryo and adult mouse tissue was fixed by immersion, embedded in paraffin, and sectioned at 5 μm. In situ hybridization was performed using standard techniques. The sectioned tissue was hybridized overnight at 60 ° C. in a hybridization solution containing a ³³ P-labeled antisense riboprobe complementary to the human FGF-23 gene. The riboprobe is obtained by in vitro transcription of clones containing human FGF-23 cDNA sequence using standard techniques.

  After hybridization, sections were treated with RNase A to digest unhybridized probe and washed in 0.1 × SSC at 55 ° C. for 30 minutes. The sections were then immersed in NTB-2 emulsion (Kodak, Rochester, NY), exposed to 4 ° C. for 3 weeks, developed, and counterstained with hematoxylin and eosin. Histomorphology and hybridization signals were determined in the brain, gastrointestinal system (parotid, submandibular, and sublingual glands; esophagus, stomach, duodenum, jejunum, ileum; proximal and distal colon; liver and prostate) , Cardiopulmonary system (heart, lung, trachea, and blood vessels); vascular lymphatic system (lymph node, spleen, thymus, and bone marrow), urinary system (kidney and bladder), endocrine system (adrenal, thyroid, and pituitary), genital organs Analyze simultaneously by dark and standard illumination for the system (testis, prostate, and gland; ovary, uterus, and fallopian tube; placenta) and the musculoskeletal system (bone, skeletal muscle, skin, and adipose tissue). Normal mouse embryos at E18 and E14.5 with placenta were also analyzed by in situ hybridization.

  Low to moderate, diffuse expression of FGF-23 was detected through normal adult mouse tissue. RNAase protection assays were performed on a limited sample of mouse tissues to detect signals that diffused in situ in these tissues and to determine whether this signal was due to non-specific binding. The results of the RNAase protection assay showed that FGF-23 was only slightly expressed in the heart and brain and not at all in the liver, thyroid / parathyroid, and stomach. These results suggest that the scattered signal detected by in situ hybridization, especially in epithelial cell types, is mostly due to non-specific binding. Since the RNAase protection assay showed that FGF-23 is slightly expressed in the heart and brain, the low signal observed in these tissues by in situ hybridization may be real. In the brain, generally low expression was seen in most neurons, including the thalamus region, caudate putamen, septum, and hypothalamic regions. However, moderate labeling was shown in hippocampal granules and cone cells, neocortex (FIG. 3), and piriform cortex. Low expression was found in the upper garment and choroid plexus. Both myocardium and skeletal muscle also showed low levels of FGF-23 expression. In the myocardium, a low degree of scattered signal is evident through the left and right ventricles with a somewhat larger signal in the chamber (FIG. 3). A low diffusing signal was also found in skeletal muscle.

  No expression was found in either E14.5 or E18 mouse embryos.

  None of the littermates of 3 week old transgenic mice showed diffuse nonspecific signals detected in normal adult mice. In non-expressing transgenic mice, strong FGF-23 expression was detected in scattered cells in lymph nodes (FIG. 4), thymic medulla (FIG. 4) and bone (FIG. 4). Although positive identification of labeled cells was not possible, expression in bone appears to be present in mesenchymal cells that scatter in hiatus and trabeculae in hindlimb bones, vertebrae, ribs, and nasal bones. In highly expressed transgenic mice, the distribution of labeled cells was more extensive. In the liver, strong FGF-23 expression was found in hepatocytes (FIG. 5). Similar to that observed in non-expressing transgenic mice, strong labeling was also detected in scattered cells of the thymic medulla (FIG. 5). However, in highly expressed transgenic mice, well-labeled cells are also present in the red spleen of the spleen (FIG. 5), the smooth muscle adjacent to the prostate (FIG. 6), and the striated muscle of the jaw (FIG. 6). ). Strong expression was also detected in several identified megakaryocytes in bone marrow and chondrocytes in the hind limbs and vertebrae (FIG. 6).

Example 3: Production of FGF-23 polypeptide
A. Expression of FGF-23 polypeptide in bacteria
PCR is used to amplify the template DNA sequence encoding the FGF-23 polypeptide. For this PCR, primers corresponding to the 5 ′ and 3 ′ ends of this sequence are used. The amplified DNA product can be modified to include a restriction enzyme site to allow insertion into the expression vector. The PCR product is gel purified and inserted into an expression vector using standard recombinant DNA methodology. A representative vector such as pAMG21 (ATCC No. 98113) containing a gene encoding the lux promoter and kanamycin resistance is digested with BamHI and NdeI for directional cloning of the inserted DNA. The ligated mixture was electroporated by E. coli. E. coli host strains are transformed and transformants are selected for kanamycin resistance. Plasmid DNA from the selected colonies is isolated and subjected to DNA sequencing to confirm the presence of the insert.

  Transformed host cells are incubated at 30 ° C. in 2 × YT medium containing 30 μg / mL kanamycin before introduction. Gene expression is induced by addition of N- (3-oxohexanoyl) -dl-homoserine lactone to a final concentration of 30 ng / mL followed by 6 hours incubation at 30 ° C or 37 ° C. Expression of FGF-23 polypeptide is assessed by culture, resuspension of bacterial pellets and centrifugation of lysates, and analysis of 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 pelleted by centrifugation at 195,000 × g for 5-10 minutes. The supernatant is discarded and the pellet is washed and transferred to a homogenizer. The pellet is homogenized in 5 mL of Percoll solution (75% liquid Percoll and 0.15 M NaCl), then diluted and centrifuged at 21,600 × g for 30 minutes until uniformly suspended. Gradient fractions containing inclusion bodies are collected and pooled. Isolated inclusion bodies are analyzed by SDS-PAGE.

  E. A single band on an SDS polyacrylamide gel corresponding to the FGF-23 polypeptide produced by E. coli was excised from the gel, and the N-terminal amino acid sequence was essentially analyzed by Matsudaira et al., 1987, J. MoI. Biol. Chem. 262: 10-35.

B. Expression of FGF-23 polypeptide in mammalian cells
PCR is used to amplify the template DNA sequence encoding the FGF-23 polypeptide. For this PCR, primers corresponding to the 5 ′ and 3 ′ ends of this sequence are used. The amplified DNA product can be modified to include a restriction enzyme site to allow insertion into the expression vector. The PCR product is gel purified and inserted into an expression vector using standard recombinant DNA methodology. A representative expression vector containing Epstein-Barr virus origin of replication, pCEP4 (Invitrogen, Carlsbad, CA) can be used for expression of FGF-23 polypeptide in 293-EBNA-1 cells. Amplified and gel purified PCR products are ligated into the pCEP4 vector and introduced into 293-EBNA cells by lipofectin. Transfected cells are selected in 100 μg / mL hygromycin and the resulting drug resistant cultures are grown to confluence. The cells are then cultured for 72 hours in serum-free medium. Conditioned medium is removed and FGF-23 polypeptide expression is analyzed by SDS-PAGE.

  FGF-23 polypeptide expression can be detected by silver staining. Alternatively, the FGF-23 polypeptide is generated as a fusion protein with an epitope tag (eg, an IgG constant domain or FLAG epitope) that can be detected by Western blot analysis using an antibody against the peptide tag.

  FGF-23 polypeptides can be excised from SDS-polyacrylamide gels, or FGF-23 fusion proteins are purified by affinity chromatography against epitope tags and subjected to N-terminal amino acid sequence analysis as described herein. .

C. Purification of FGF-23 polypeptide from mammalian cells
The FGF-23 polypeptide expression construct is introduced into 293 EBNA cells or CHO cells using either the lipofectin or calcium phosphate protocols.

  To conduct functional studies on the generated FGF-23 polypeptide, a large amount of conditioned medium is generated from a pool of hygromycin-selected 293 EBNA clones. The cells are cultured in 500 cm Nunc Triple Flaks until they are 80% confluent and then switched to serum-free medium one week before harvesting the medium. Conditioned medium is collected and frozen at −20 ° C. until protein is purified.

Conditioned medium is purified by affinity chromatography as described below. The medium is thawed and then passed through a 0.2 μm filter. The protein G column was equilibrated to pH 7.0 with PBS and then filtered media was loaded. The column absorption of _{A 280} is washed with PBS until a baseline. 0.1M
The FGF-23 polypeptide is eluted from the column with Glycine-HCl at pH 2.7 and immediately neutralized with 1M Tris-HCl at pH 8.5. Fractions containing FGF-23 polypeptide were pooled, dialyzed in PBS and stored at -70 ° C.

For Factor Xa cleavage of the human FGF-23 polypeptide-Fc fusion polypeptide, the affinity chromatographically purified protein is dialyzed at pH 8.0 in 50 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl ₂ . 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 anti-FGF-23 polypeptide antibody
Antibodies against FGF-23 polypeptides can be obtained by immunizing with purified proteins or FGF-23 peptides generated by biological or chemical synthesis. Suitable procedures for generating antibodies include those described in Hudson and Bay, Practical Immunology (2nd edition, Blackwell Scientific Publicization).

  In one procedure for antibody production, animals (typically mice or rabbits) are injected with FGF-23 antigen (eg, FGF-23 polypeptide) and determined by ELISA for hybridoma production. Animals with sufficient serum titer are selected. The spleen of the immunized animal is collected and prepared as a single cell suspension from which the spleen cells are collected. Spleen cells are fused to mouse myeloma cells (eg, Sp2 / 0-Ag14 cells), first incubated in DMEM (containing 200 U / mL penicillin, 200 μg / mL streptomycin sulfate, and 4 mM glutamine), then HAT selection medium ( Incubate in hypoxanthine, aminopterin and thymidine). After selection, tissue culture supernatant is removed from each fusion well and tested for anti-FGF-23 antibody production by ELISA.

  Another procedure can also be used to obtain anti-FGF-23. This procedure includes, for example, immunization of transgenic mice carrying the human Ig locus for production of human antibodies, and screening of synthetic antibody libraries (eg, libraries generated by mutagenesis of antibody variable domains). is there.

(Example 5: Expression of FGF-23 polypeptide in transgenic mice)
To assess the biological activity of FGF-23 polypeptide, a construct encoding FGF-23 polypeptide is prepared under the control of the ApoE promoter (TH00-026). Expression of the FGF-23 gene was expected to cause pathological changes in transgenic mice, which is information regarding the function of FGF-23 polypeptide.

  A unique phenotype occurred in 3 week old BDF1 mice following transfer of TH00-026 constructs to littermates with an abnormally high number of runts. Not all of the expressed bodies were runted, and some non-expressing littermates were runted, but the ratio of runts was higher among the expressing mice. All runts, including many non-expressed runts, were dissected before weaning for testing. The skulls of all expressing mice were shortened and more rounded and the mandible was properly developed. As a result, all expressing mice had protruding lower teeth. This condition is evident by appearance inspection and radiographic evaluation. In addition, the two highest expressing mice were found to have low serum phosphorus levels and low serum calcium levels. However, other signs of rickets—for example, poor mineralization, epiphyseal overgrowth, disordered organization of cartilage, and overgrowth of capillaries (Pathological Basis of Disease (Cotraned., 1994)). Was not observed. The bone morphology in the expressed runt was not different from that in the non-expressed runt, and both types of runts were different from the non-run litter.

  Partial hepatectomy was performed on 22 DNA positive mice and 4 DNA negative mice. All hepatectomized mice were bled and serum calcium, phosphorus, and alkaline phosphatase measurements were performed. This animal was also examined for protruding lower teeth. The groups of mice evaluated included: controls, macroscopic phenotypic expressors, non-phenotypic high expressers, and moderate expressors. All phenotypic mice were found to be strong or very strong. Serum calcium levels in phenotypic mice were somewhat lower than those in the other groups, but this difference is not statistically significant. However, serum phosphorus levels were found to be significantly lower in phenotypic and non-phenotypically high expressers than moderate expressers and controls. Serum alkaline phosphatase (ALP) was significantly elevated in phenotype groups versus controls and moderate expressors (see Table IV). Variability in the phenotype of the expressor may be due to genetic changes in outbred BDF1 mice, which are crosses between C57 / B6 and DBA mouse strains.

本発明は、好ましい実施形態の観点で記載されているが、変化および修飾が当業者に思い浮かぶことが理解される。従って、添付の特許請求の範囲は、請求された本発明の範囲内で生じるこのような全ての等価な変化を網羅することが意図されている。

Although the present invention has been described in terms of preferred embodiments, it will be understood that variations and modifications will occur to those skilled in the art. Accordingly, the appended claims are intended to cover all such equivalent changes that occur within the scope of the claimed invention.

1A-1B show the nucleotide sequence of human FGF-23 gene (SEQ ID NO: 1) and the deduced amino acid sequence of human FGFR polypeptide (SEQ ID NO: 2). Shows the putative signal peptide (underlined); 1A-1B show the nucleotide sequence of human FGF-23 gene (SEQ ID NO: 1) and the deduced amino acid sequence of human FGFR polypeptide (SEQ ID NO: 2). Shows the putative signal peptide (underlined); 2A-2G show the following amino acid sequence alignments: human FGF-1 (hu FGF-1; SEQ ID NO: 4), human FGF-2 (hu FGF-2; SEQ ID NO: 5), human FGF3 (hu FGF- 3; SEQ ID NO: 6), human FGF-4 (hu FGF-4; SEQ ID NO: 7), human FGF-5 (hu FGF-5; SEQ ID NO: 8), human FGF-6 (hu FGF-6; SEQ ID NO: 9) ), Human FGF-7 (hu FGF-7; SEQ ID NO: 10), human FGF-8 (hu FGF-8; SEQ ID NO: 11), human FGF-9 (hu FGF-9; SEQ ID NO: 12), human FGF- 10 (hu FGF-10; SEQ ID NO: 13), human FGF-11 (hu FGF-11; SEQ ID NO: 14), human FGF-12 (hu FGF-12; SEQ ID NO: 15), human FGF-13 (hu FGF-13; SEQ ID NO: 16), human FGF-14 (hu FGF-14; SEQ ID NO: 17), human FGF-16 (hu FGF-16; SEQ ID NO: 18), human FGF-17 (hu FGF-17; sequence) No. 19), human FGF-18 (huFGF-18; SEQ ID NO: 20), human FGF-19 (hu FGF-19; SEQ ID NO: 21), human FGF-23 (hu FGF-23; SEQ ID NO: 22), mouse FGF -1 (mu FGF-1; SEQ ID NO: 23), mouse FGF-2 (mu FGF-2; SEQ ID NO: 24), mouse FGF-3 (mu FGF-3; SEQ ID NO: 25), mouse FGF-4 (mu FGF) -4; SEQ ID NO: 26), mouse FGF-5 (mu FGF-5; SEQ ID NO: 27), mouse FGF-6 (mu FGF-6; SEQ ID NO: 28), mouse F GF-7 (mu FGF-7; SEQ ID NO: 29), mouse FGF-8 (mu FGF-8; SEQ ID NO: 30), mouse FGF-9 (mu FGF-9 SEQ ID NO: 31), mouse FGF-10 (mu) FGF-10; SEQ ID NO: 32), mouse FGF-11 (mu FGF-11; SEQ ID NO: 33), mouse FGF-12 (mu FGF-12; SEQ ID NO: 34), mouse FGF-13 (mu FGF-13; sequence) No. 35), mouse FGF-14 (mu FGF-14; SEQ ID NO: 36), mouse FGF-15 (mu FGF-15; SEQ ID NO: 37), rat FGF-16 (rat FGF-16; SEQ ID NO: 38), mouse FGF-17 (mu FGF-17; SEQ ID NO: 39). 2A-2G show the following amino acid sequence alignments: human FGF-1 (hu FGF-1; SEQ ID NO: 4), human FGF-2 (hu FGF-2; SEQ ID NO: 5), human FGF3 (hu FGF- 3; SEQ ID NO: 6), human FGF-4 (hu FGF-4; SEQ ID NO: 7), human FGF-5 (hu FGF-5; SEQ ID NO: 8), human FGF-6 (hu FGF-6; SEQ ID NO: 9) ), Human FGF-7 (hu FGF-7; SEQ ID NO: 10), human FGF-8 (hu FGF-8; SEQ ID NO: 11), human FGF-9 (hu FGF-9; SEQ ID NO: 12), human FGF- 10 (hu FGF-10; SEQ ID NO: 13), human FGF-11 (hu FGF-11; SEQ ID NO: 14), human FGF-12 (hu FGF-12; SEQ ID NO: 15), human FGF-13 (hu FGF-13; SEQ ID NO: 16), human FGF-14 (hu FGF-14; SEQ ID NO: 17), human FGF-16 (hu FGF-16; SEQ ID NO: 18), human FGF-17 (hu FGF-17; sequence) No. 19), human FGF-18 (huFGF-18; SEQ ID NO: 20), human FGF-19 (hu FGF-19; SEQ ID NO: 21), human FGF-23 (hu FGF-23; SEQ ID NO: 22), mouse FGF -1 (mu FGF-1; SEQ ID NO: 23), mouse FGF-2 (mu FGF-2; SEQ ID NO: 24), mouse FGF-3 (mu FGF-3; SEQ ID NO: 25), mouse FGF-4 (mu FGF) -4; SEQ ID NO: 26), mouse FGF-5 (mu FGF-5; SEQ ID NO: 27), mouse FGF-6 (mu FGF-6; SEQ ID NO: 28), mouse F GF-7 (mu FGF-7; SEQ ID NO: 29), mouse FGF-8 (mu FGF-8; SEQ ID NO: 30), mouse FGF-9 (mu FGF-9 SEQ ID NO: 31), mouse FGF-10 (mu) FGF-10; SEQ ID NO: 32), mouse FGF-11 (mu FGF-11; SEQ ID NO: 33), mouse FGF-12 (mu FGF-12; SEQ ID NO: 34), mouse FGF-13 (mu FGF-13; sequence) No. 35), mouse FGF-14 (mu FGF-14; SEQ ID NO: 36), mouse FGF-15 (mu FGF-15; SEQ ID NO: 37), rat FGF-16 (rat FGF-16; SEQ ID NO: 38), mouse FGF-17 (mu FGF-17; SEQ ID NO: 39). 2A-2G show the following amino acid sequence alignments: human FGF-1 (hu FGF-1; SEQ ID NO: 4), human FGF-2 (hu FGF-2; SEQ ID NO: 5), human FGF3 (hu FGF- 3; SEQ ID NO: 6), human FGF-4 (hu FGF-4; SEQ ID NO: 7), human FGF-5 (hu FGF-5; SEQ ID NO: 8), human FGF-6 (hu FGF-6; SEQ ID NO: 9) ), Human FGF-7 (hu FGF-7; SEQ ID NO: 10), human FGF-8 (hu FGF-8; SEQ ID NO: 11), human FGF-9 (hu FGF-9; SEQ ID NO: 12), human FGF- 10 (hu FGF-10; SEQ ID NO: 13), human FGF-11 (hu FGF-11; SEQ ID NO: 14), human FGF-12 (hu FGF-12; SEQ ID NO: 15), human FGF-13 (hu FGF-13; SEQ ID NO: 16), human FGF-14 (hu FGF-14; SEQ ID NO: 17), human FGF-16 (hu FGF-16; SEQ ID NO: 18), human FGF-17 (hu FGF-17; sequence) No. 19), human FGF-18 (huFGF-18; SEQ ID NO: 20), human FGF-19 (hu FGF-19; SEQ ID NO: 21), human FGF-23 (hu FGF-23; SEQ ID NO: 22), mouse FGF -1 (mu FGF-1; SEQ ID NO: 23), mouse FGF-2 (mu FGF-2; SEQ ID NO: 24), mouse FGF-3 (mu FGF-3; SEQ ID NO: 25), mouse FGF-4 (mu FGF) -4; SEQ ID NO: 26), mouse FGF-5 (mu FGF-5; SEQ ID NO: 27), mouse FGF-6 (mu FGF-6; SEQ ID NO: 28), mouse F GF-7 (mu FGF-7; SEQ ID NO: 29), mouse FGF-8 (mu FGF-8; SEQ ID NO: 30), mouse FGF-9 (mu FGF-9 SEQ ID NO: 31), mouse FGF-10 (mu) FGF-10; SEQ ID NO: 32), mouse FGF-11 (mu FGF-11; SEQ ID NO: 33), mouse FGF-12 (mu FGF-12; SEQ ID NO: 34), mouse FGF-13 (mu FGF-13; sequence) No. 35), mouse FGF-14 (mu FGF-14; SEQ ID NO: 36), mouse FGF-15 (mu FGF-15; SEQ ID NO: 37), rat FGF-16 (rat FGF-16; SEQ ID NO: 38), mouse FGF-17 (mu FGF-17; SEQ ID NO: 39). 2A-2G show the following amino acid sequence alignments: human FGF-1 (hu FGF-1; SEQ ID NO: 4), human FGF-2 (hu FGF-2; SEQ ID NO: 5), human FGF3 (hu FGF- 3; SEQ ID NO: 6), human FGF-4 (hu FGF-4; SEQ ID NO: 7), human FGF-5 (hu FGF-5; SEQ ID NO: 8), human FGF-6 (hu FGF-6; SEQ ID NO: 9) ), Human FGF-7 (hu FGF-7; SEQ ID NO: 10), human FGF-8 (hu FGF-8; SEQ ID NO: 11), human FGF-9 (hu FGF-9; SEQ ID NO: 12), human FGF- 10 (hu FGF-10; SEQ ID NO: 13), human FGF-11 (hu FGF-11; SEQ ID NO: 14), human FGF-12 (hu FGF-12; SEQ ID NO: 15), human FGF-13 (hu FGF-13; SEQ ID NO: 16), human FGF-14 (hu FGF-14; SEQ ID NO: 17), human FGF-16 (hu FGF-16; SEQ ID NO: 18), human FGF-17 (hu FGF-17; sequence) No. 19), human FGF-18 (huFGF-18; SEQ ID NO: 20), human FGF-19 (hu FGF-19; SEQ ID NO: 21), human FGF-23 (hu FGF-23; SEQ ID NO: 22), mouse FGF -1 (mu FGF-1; SEQ ID NO: 23), mouse FGF-2 (mu FGF-2; SEQ ID NO: 24), mouse FGF-3 (mu FGF-3; SEQ ID NO: 25), mouse FGF-4 (mu FGF) -4; SEQ ID NO: 26), mouse FGF-5 (mu FGF-5; SEQ ID NO: 27), mouse FGF-6 (mu FGF-6; SEQ ID NO: 28), mouse F GF-7 (mu FGF-7; SEQ ID NO: 29), mouse FGF-8 (mu FGF-8; SEQ ID NO: 30), mouse FGF-9 (mu FGF-9 SEQ ID NO: 31), mouse FGF-10 (mu) FGF-10; SEQ ID NO: 32), mouse FGF-11 (mu FGF-11; SEQ ID NO: 33), mouse FGF-12 (mu FGF-12; SEQ ID NO: 34), mouse FGF-13 (mu FGF-13; sequence) No. 35), mouse FGF-14 (mu FGF-14; SEQ ID NO: 36), mouse FGF-15 (mu FGF-15; SEQ ID NO: 37), rat FGF-16 (rat FGF-16; SEQ ID NO: 38), mouse FGF-17 (mu FGF-17; SEQ ID NO: 39). 2A-2G show the following amino acid sequence alignments: human FGF-1 (hu FGF-1; SEQ ID NO: 4), human FGF-2 (hu FGF-2; SEQ ID NO: 5), human FGF3 (hu FGF- 3; SEQ ID NO: 6), human FGF-4 (hu FGF-4; SEQ ID NO: 7), human FGF-5 (hu FGF-5; SEQ ID NO: 8), human FGF-6 (hu FGF-6; SEQ ID NO: 9) ), Human FGF-7 (hu FGF-7; SEQ ID NO: 10), human FGF-8 (hu FGF-8; SEQ ID NO: 11), human FGF-9 (hu FGF-9; SEQ ID NO: 12), human FGF- 10 (hu FGF-10; SEQ ID NO: 13), human FGF-11 (hu FGF-11; SEQ ID NO: 14), human FGF-12 (hu FGF-12; SEQ ID NO: 15), human FGF-13 (hu FGF-13; SEQ ID NO: 16), human FGF-14 (hu FGF-14; SEQ ID NO: 17), human FGF-16 (hu FGF-16; SEQ ID NO: 18), human FGF-17 (hu FGF-17; sequence) No. 19), human FGF-18 (huFGF-18; SEQ ID NO: 20), human FGF-19 (hu FGF-19; SEQ ID NO: 21), human FGF-23 (hu FGF-23; SEQ ID NO: 22), mouse FGF -1 (mu FGF-1; SEQ ID NO: 23), mouse FGF-2 (mu FGF-2; SEQ ID NO: 24), mouse FGF-3 (mu FGF-3; SEQ ID NO: 25), mouse FGF-4 (mu FGF) -4; SEQ ID NO: 26), mouse FGF-5 (mu FGF-5; SEQ ID NO: 27), mouse FGF-6 (mu FGF-6; SEQ ID NO: 28), mouse F GF-7 (mu FGF-7; SEQ ID NO: 29), mouse FGF-8 (mu FGF-8; SEQ ID NO: 30), mouse FGF-9 (mu FGF-9 SEQ ID NO: 31), mouse FGF-10 (mu) FGF-10; SEQ ID NO: 32), mouse FGF-11 (mu FGF-11; SEQ ID NO: 33), mouse FGF-12 (mu FGF-12; SEQ ID NO: 34), mouse FGF-13 (mu FGF-13; sequence) No. 35), mouse FGF-14 (mu FGF-14; SEQ ID NO: 36), mouse FGF-15 (mu FGF-15; SEQ ID NO: 37), rat FGF-16 (rat FGF-16; SEQ ID NO: 38), mouse FGF-17 (mu FGF-17; SEQ ID NO: 39). 2A-2G show the following amino acid sequence alignments: human FGF-1 (hu FGF-1; SEQ ID NO: 4), human FGF-2 (hu FGF-2; SEQ ID NO: 5), human FGF3 (hu FGF- 3; SEQ ID NO: 6), human FGF-4 (hu FGF-4; SEQ ID NO: 7), human FGF-5 (hu FGF-5; SEQ ID NO: 8), human FGF-6 (hu FGF-6; SEQ ID NO: 9) ), Human FGF-7 (hu FGF-7; SEQ ID NO: 10), human FGF-8 (hu FGF-8; SEQ ID NO: 11), human FGF-9 (hu FGF-9; SEQ ID NO: 12), human FGF- 10 (hu FGF-10; SEQ ID NO: 13), human FGF-11 (hu FGF-11; SEQ ID NO: 14), human FGF-12 (hu FGF-12; SEQ ID NO: 15), human FGF-13 (hu FGF-13; SEQ ID NO: 16), human FGF-14 (hu FGF-14; SEQ ID NO: 17), human FGF-16 (hu FGF-16; SEQ ID NO: 18), human FGF-17 (hu FGF-17; sequence) No. 19), human FGF-18 (huFGF-18; SEQ ID NO: 20), human FGF-19 (hu FGF-19; SEQ ID NO: 21), human FGF-23 (hu FGF-23; SEQ ID NO: 22), mouse FGF -1 (mu FGF-1; SEQ ID NO: 23), mouse FGF-2 (mu FGF-2; SEQ ID NO: 24), mouse FGF-3 (mu FGF-3; SEQ ID NO: 25), mouse FGF-4 (mu FGF) -4; SEQ ID NO: 26), mouse FGF-5 (mu FGF-5; SEQ ID NO: 27), mouse FGF-6 (mu FGF-6; SEQ ID NO: 28), mouse F GF-7 (mu FGF-7; SEQ ID NO: 29), mouse FGF-8 (mu FGF-8; SEQ ID NO: 30), mouse FGF-9 (mu FGF-9 SEQ ID NO: 31), mouse FGF-10 (mu) FGF-10; SEQ ID NO: 32), mouse FGF-11 (mu FGF-11; SEQ ID NO: 33), mouse FGF-12 (mu FGF-12; SEQ ID NO: 34), mouse FGF-13 (mu FGF-13; sequence) No. 35), mouse FGF-14 (mu FGF-14; SEQ ID NO: 36), mouse FGF-15 (mu FGF-15; SEQ ID NO: 37), rat FGF-16 (rat FGF-16; SEQ ID NO: 38), mouse FGF-17 (mu FGF-17; SEQ ID NO: 39). 2A-2G show the following amino acid sequence alignments: human FGF-1 (hu FGF-1; SEQ ID NO: 4), human FGF-2 (hu FGF-2; SEQ ID NO: 5), human FGF3 (hu FGF- 3; SEQ ID NO: 6), human FGF-4 (hu FGF-4; SEQ ID NO: 7), human FGF-5 (hu FGF-5; SEQ ID NO: 8), human FGF-6 (hu FGF-6; SEQ ID NO: 9) ), Human FGF-7 (hu FGF-7; SEQ ID NO: 10), human FGF-8 (hu FGF-8; SEQ ID NO: 11), human FGF-9 (hu FGF-9; SEQ ID NO: 12), human FGF- 10 (hu FGF-10; SEQ ID NO: 13), human FGF-11 (hu FGF-11; SEQ ID NO: 14), human FGF-12 (hu FGF-12; SEQ ID NO: 15), human FGF-13 (hu FGF-13; SEQ ID NO: 16), human FGF-14 (hu FGF-14; SEQ ID NO: 17), human FGF-16 (hu FGF-16; SEQ ID NO: 18), human FGF-17 (hu FGF-17; sequence) No. 19), human FGF-18 (huFGF-18; SEQ ID NO: 20), human FGF-19 (hu FGF-19; SEQ ID NO: 21), human FGF-23 (hu FGF-23; SEQ ID NO: 22), mouse FGF -1 (mu FGF-1; SEQ ID NO: 23), mouse FGF-2 (mu FGF-2; SEQ ID NO: 24), mouse FGF-3 (mu FGF-3; SEQ ID NO: 25), mouse FGF-4 (mu FGF) -4; SEQ ID NO: 26), mouse FGF-5 (mu FGF-5; SEQ ID NO: 27), mouse FGF-6 (mu FGF-6; SEQ ID NO: 28), mouse F GF-7 (mu FGF-7; SEQ ID NO: 29), mouse FGF-8 (mu FGF-8; SEQ ID NO: 30), mouse FGF-9 (mu FGF-9 SEQ ID NO: 31), mouse FGF-10 (mu) FGF-10; SEQ ID NO: 32), mouse FGF-11 (mu FGF-11; SEQ ID NO: 33), mouse FGF-12 (mu FGF-12; SEQ ID NO: 34), mouse FGF-13 (mu FGF-13; sequence) No. 35), mouse FGF-14 (mu FGF-14; SEQ ID NO: 36), mouse FGF-15 (mu FGF-15; SEQ ID NO: 37), rat FGF-16 (rat FGF-16; SEQ ID NO: 38), mouse FGF-17 (mu FGF-17; SEQ ID NO: 39). FIG. 3 shows the expression of FGF-23 mRNA detected by in situ hybridization in the brain and myocardium (heart) of normal adult mice (H & E = hematoxylin and eosin counterstaining; ISH = in situ hybridization). FIG. 4 shows the subcapsular of lymph nodes (lymph nodes), thymic medulla (thymus), cortical hiatus from the tibia (tibia), and cancellous (head) in the head of transgenic mice that are not expressed In the region, the expression of FGF-23 mRNA detected by in situ hybridization is shown (H & E = hematoxylin and eosin counterstain; ISH = in situ hybridization); FIG. 5 shows the expression of FGF-23 mRNA detected by in situ hybridization in the liver, spleen, thymic medulla (thymus), and megakaryocytes in the bone marrow (bone marrow) of high-expressing transgenic mice (H & E = hematoxylin) And eosin counterstain; ISH = in situ hybridization). FIG. 6 shows in smooth muscle tissue (smooth muscle), jaw muscle tissue (muscle), chondrocytes in the tibia (tibia), and chondrocytes in the vertebra (vertebrae) in the vicinity of the prostate of a high-expression transgenic mouse. Shows the expression of FGF-23 mRNA detected by in situ hybridization (H & E = hematoxylin and eosin counterstain; ISH = in situ hybridization).

Claims (2)

An isolated nucleic acid molecule comprising:
(A) the nucleotide sequence shown in SEQ ID NO: 1;
(B) the nucleotide sequence of the DNA insert in ATCC accession number PTA-1617;
(C) a nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 2;
(D) a nucleotide sequence that hybridizes to the complement of any of (a)-(c) under moderately or highly stringent conditions; and (e) any of (a)-(c) Complementary nucleotide sequence,
An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:   A method of diagnosing a pathological condition or susceptibility to a pathological condition in a subject.

Images