MXPA02002491A - Fibroblast growth factor 19 (fgf 19) nucleic acids and polypeptides and methods of use for the treatment of obesity. - Google Patents

Abstract

The present invention is directed to novel polypeptides belonging to the fibroblast growth factor family and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention. Furthermore, methods of treating obesity are provided.

Description

NUCLEIC ACIDS AND POLYPEPTIDES OF THE GROWTH FACTOR 19 OF FIBROBLASTOS AND METHODS OF USE FOR TREATMENT OF THE OBESITY Field of the Invention The present invention relates in general to the identification and isolation of novel DNA and to the recombinant production of novel polypeptides designated herein as polypeptides of fibroblast growth factor 19 (FGF-19), and to methods, compositions and assays using such polypeptides for the therapeutic treatment of obesity and for producing pharmaceutically active materials having therapeutic and pharmacological properties including those associated with the treatment of obesity.

BACKGROUND OF THE INVENTION Obesity is a chronic disease that is highly prevalent in modern society and is associated not only with a social stigma, but also with a reduced prolongation of life and numerous medical problems, including adverse psychological development. .136361 reproductive disorders such as polycystic disease of the ovaries, dermatological disorders such as infections, varicose veins, Acanthosis nigricans, and eczema, exercise intolerance, diabetes mellitus, insulin resistance, hypertension, hypercholesterolemia, cholelithiasis , osteoarthritis, orthopedic injuries, thromboembolic disease, cancer and coronary heart disease, Rissanen et al., British Medical Journal, 301: 835-837 (1990). Existing therapies for obesity include standard diets and exercise, very low calorie diets, behavioral therapies, pharmacotherapy that involves appetite suppressants, thermogenic drugs, food absorption inhibitors, mechanical devices such as wires for the jaws, laces for the waist and balls, and surgery. Jung and Chong, Clinical Endocrinology, 35: 11-20 (1991); Bray, Am. J. Clin. Nutr., 55: 538S-544 (1992). The fast modified by the shortage of proteins has been reported to be effective in reducing adolescents' weight. Lee et al., Clin. Pediatr., 31: 234-236 (April 1992). The restriction of calories as a treatment for obesity causes the catabolism of the body's proteins to store and produce a negative balance of nitrogen. Diets supplemented with proteins, therefore, have become popular as a means to reduce nitrogen loss during caloric restriction. Because such diets produce only a shortage of moderate nitrogen, a more effective way to preserve lean body mass and stores of proteins is necessary. In addition, the treatment of obesity could be improved if such a regime also leads to an accelerated loss of body fat. Several approaches to such treatment include those described by Eintraub and Bray, Med. Clinics N. Amer., 73: 237 (1989); Nutrition Reviews, 49: 33 (1991). Considering the high prevalence of obesity in our society and the serious consequences associated with it as described above, any therapeutic drug potentially useful in reducing the weight of obese people could have a profound beneficial effect on their health. There is a need in the art for a drug that will reduce the total body weight of the obese subjects towards their ideal body weight without significant adverse side effects and which will help the obese subject to maintain the reduced weight level. It is therefore desirable to provide a treatment regimen that is useful for returning the body weight of the obese subjects to an ideal, normal body weight.

Furthermore, it is desirable to provide a therapy for obesity that leads to the maintenance of reduced body weight over a prolonged period of time. It is also desirable to prevent obesity and, once the treatment has begun, stop the progression or prevent the onset of diseases that are the consequence of, or are side effects for, obesity, such as arteriosclerosis and polycystic ovarian disease. Such treatment methods and related compositions are provided herein. Also provided here are novel proteins and nucleic acids, and methods for selecting modulators thereof. Other methods, treatments and compositions provided herein will become apparent to those skilled in the art.

Brief Description of the Invention A cDNA clone (designated here as DNA49435-1219) has been identified, which encodes a novel polypeptide, which has some sequence similarity to members of the fibroblast growth factor family, designated in the present application as the "fibroblast growth factor 19" (FGF-19).

In one embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FGF-19 polypeptide. In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% identity of the nucleic acid sequence, alternatively at least about 81% identity of the nucleic acid sequence, alternatively to the less about 82% identity of the nucleic acid sequence, alternatively at least about 83% identity of the nucleic acid sequence, alternatively at least about 84% identity of the nucleic acid sequence, alternatively at least about 85% of identity of the nucleic acid sequence, alternatively at least about 86% identity of the nucleic acid sequence, alternatively at least about 87% identity of the nucleic acid sequence, alternatively at least about 88% identity of the sequence of nucleic acid, alternatively at least about 89% id nucleic acid sequence entity, alternatively at least about 90% identity of the nucleic acid sequence, alternatively at least about 91% identity of the nucleic acid sequence, alternatively at least about 92% identity of the sequence of nucleic acid, alternatively at least about 93% identity of the nucleic acid sequence, alternatively at least about 94% identity of the nucleic acid sequence, alternatively at least about 95% identity of the nucleic acid sequence, alternatively to the at least about 96% identity of the nucleic acid sequence, alternatively at least about 97% identity of the nucleic acid sequence, alternatively at least about 98% identity of the nucleic acid sequence, alternatively at least about 99% identity of the nucleic acid sequence with res pect to (a) a DNA molecule encoding a PEACH polypeptide having the sequence of amino acid residues from about 1 or about 23 to about 216, inclusive, of Figure 2 (SEQ ID NO: 2), or (b) ) the complement of the DNA molecule of (a). In another aspect, the isolated nucleic acid molecule comprises (a) a nucleotide sequence encoding a FGF-19 polypeptide having the sequence of amino acid residues from about 1 or about 23 to about 216, inclusive, of the Figure 2 (SEQ ID NO: 2), or (b) the complement of the nucleotide sequence of (a). In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% identity of the nucleic acid sequence, alternatively at least about 81% identity of the nucleic acid sequence, alternatively at least about 82% identity of the nucleic acid sequence, alternatively at least about 83% identity of the nucleic acid sequence, alternatively at least about 84% identity of the nucleic acid sequence, alternatively at least about 85% identity of the nucleic acid sequence, alternatively at least about 86% identity of the nucleic acid sequence, alternatively at least about 87% identity of the nucleic acid sequence, alternatively at least about 88% identity of the nucleic acid sequence, alternatively at least about 89% identity of the nucleic acid sequence, alternatively at least about 90% identity of the nucleic acid sequence, alternatively at least about 91% identity of the nucleic acid sequence, alternatively at least about 92% identity of the nucleic acid sequence, alternatively at least about 93% identity of the nucleic acid sequence, alternatively at least about 94% identity of the nucleic acid sequence, alternatively at least about 95% identity of the nucleic acid sequence, alternatively at least about 96% identity of the nucleic acid sequence, alternatively at least about 97% identity of the nucleic acid sequence , alternatively at least about 98% of identity of the nucleic acid sequence, alternatively at least about 99% identity of the nucleic acid sequence with respect to(a) a DNA molecule having the nucleotide sequence from about 464 or about 530 to about llll, inclusive, of Figure 1 (SEQ ID NO: 1), or (b) the complement of the DNA molecule of (FIG. to) . In another aspect, the isolated nucleic acid molecule comprises (a) the nucleotide sequence from about 464 or about 530 to about 111, inclusive, of Figure 1 (SEQ ID NO: 1), or (b) the complement of the nucleotide sequence of (a). In a further aspect, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% identity of the nucleic acid sequence, alternatively at least about 81% identity of the sequence nucleic acid, alternatively at least about 82% identity of the nucleic acid sequence, alternatively at least about 83% identity of the nucleic acid sequence, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% identity of the nucleic acid sequence, alternatively at least about 86% identity of the nucleic acid sequence, alternatively at least about 87% identity of the nucleic acid sequence, alternatively at least about 88% identity of the nucleic acid sequence, alternative at least about 89% identity of the nucleic acid sequence, alternatively at least about 90% identity of the nucleic acid sequence, alternatively at least about 91% identity of the nucleic acid sequence, alternatively at least about 92 % identity of the nucleic acid sequence, alternatively at least about 93% identity of the nucleic acid sequence, alternatively at least about 94% identity of the nucleic acid sequence, alternatively at least about 95% identity of the nucleic acid sequence, alternatively at least about 96% identity of the nucleic acid sequence, alternatively at least about 97% identity of the nucleic acid sequence, alternatively at least about 98% identity of the nucleic acid sequence, alternatively at least about 99% identity of the nucleic acid sequence with respect to (a) a nucleotide sequence encoding the same mature polypeptide encoded by the human protein cDNA deposited with the ATCC on November 21, 1997, under ATCC Deposit No. 209480 (DNA49435 -1219) or (b) the complement of the nucleotide sequence of (a). In another aspect, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% identity of the nucleic acid sequence, alternatively at least about 81% identity of the sequence of nucleic acid, alternatively at least about 82% identity of the nucleic acid sequence, alternatively at least about 83% identity of the nucleic acid sequence, alternatively at least about 84% identity of the nucleic acid sequence, alternatively to the less about 85% identity of the nucleic acid sequence, alternatively at least about 86% identity of the nucleic acid sequence, alternatively at least about 87% identity of the nucleic acid sequence, alternatively at least about 88% of identity of the nucleic acid sequence, alternatively to the less about 89% identity of the nucleic acid sequence, alternatively at least about 90% identity of the nucleic acid sequence, alternatively at least about 91% identity of the nucleic acid sequence, alternatively at least about 92% identity of the nucleic acid sequence, alternatively at least about 93% identity of the nucleic acid sequence, alternatively at least about 94% identity of the nucleic acid sequence, alternatively at least about 95% identity of the nucleic acid sequence, alternatively at least about 96% identity of the nucleic acid sequence, alternatively at least about 97% identity of the nucleic acid sequence, alternatively at least about 98% identity of the nucleic acid sequence, alternatively at least about 99% identity of the nucleic acid sequence with respect to (a) the coding sequence of the full-length polypeptides of the human protein cDNA deposited with the ATCC on November 21 of 1997 under Deposit No. 209480 of the ATCC (DNA49435-1219) or (b) the complem of the nucleotide sequence of (a). In a preferred embodiment, the isolated nucleic acid molecule comprises (a) the coding sequence of the full length polypeptides of the DNA deposited with the ATCC on November 21, 1997 under Deposit No. 209480 of the ATCC (DNA49435-1219) or (b) the complement of the nucleotide sequence of (a). In another aspect, the invention relates to an isolated nucleic acid molecule which encodes an active FGF-19 polypeptide as defined below, comprising a nucleotide sequence that hybridizes to the complement of the nucleic acid sequence encoding the amino acids 1 or approximately 23 to approximately 216, inclusive, of Figure 2 (SEQ ID NO: 2). Preferably, hybridization occurs under severe washing and hybridization conditions. In still another aspect, the invention relates to an isolated nucleic acid molecule which encodes an active FGF-19 polypeptide as defined below, comprising a nucleotide sequence that hybridizes to the complement of the nucleic acid sequence between about nucleotides 464 or about 530 and about llll, inclusive of Figure 1 (SEQ ID NO: 1). Preferably, hybridization occurs under severe washing and hybridization conditions. In a further aspect, the invention relates to a nucleic acid molecule having at least about 22 nucleotides and which is produced by the hybridization of a test DNA molecule under severe conditions with (a) a DNA molecule encoding a FGF-19 polypeptide having the sequence of amino acid residues from about 1 to about 23 to about 216, inclusive, of Figure 2 (SEQ ID NO: 2), or (b) the complement of the DNA molecule of (a), if the test DNA molecule has at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% of identity of the nucleic acid sequence, alternatively at least about 83% identity of the nucleic acid sequence, alternatively at least about 84% identity d of the nucleic acid sequence, alternatively at least about 85% identity of the nucleic acid sequence, alternatively at least about 86% identity of the nucleic acid sequence, alternatively at least about 87% identity of the sequence of nucleic acid, alternatively at least about 88% identity of the nucleic acid sequence, alternatively at least about 89% identity of the nucleic acid sequence, alternatively at least about 90% identity of the nucleic acid sequence, alternatively to the less about 91% identity of the nucleic acid sequence, alternatively at least about 92% identity of the nucleic acid sequence, alternatively at least about 93% identity of the nucleic acid sequence, alternatively at least about 94% identity of the nucleic acid sequence, alternatively at least about 95% identity of the nucleic acid sequence, alternatively at least about 96% identity of the nucleic acid sequence, alternatively at least about 97% identity of the nucleic acid sequence, alternatively at least about 98% identity of the nucleic acid sequence, alternatively at least about 99% identity of the nucleic acid sequence with respect to (a) or (b), and isolating the DNA from the test DNA.

In another aspect, the invention relates to an isolated nucleic acid molecule comprising (a) a nucleotide sequence encoding a polypeptide that records at least about 80% positives, alternatively at least about 81% positives, alternatively at least about 82 % positive, alternately at least about 83% positive, alternately at least about 84% positive, alternately at least about 85% positive, alternately at least about 86% positive, alternately at least about 87% positive, alternately at least about 88% positive , alternatively at least about 89% positive, alternating at least about 90% positive, alternating at least about 91% positive, alternating at least about 92% positive, alternating at least about 93% positive, alternating at least about 94 % positives, alternatively at least about 95% positives, alternatively at least about 96% positives, alternatively at least about 97% positives, alternatively at least about 98% positives, alternatively at least about 99% positives when compared to the amino acid sequence of the residues of about 1 or about 23 to 216, inclusive, of Figure 2 (SEQ ID NO: 2), or (b) the complement of the nucleotide sequence of (a). In a specific aspect, the invention provides an isolated nucleic acid molecule comprising the DNA encoding a FGF-19 polypeptide without the sequence of the N-terminal signal and / or the initiating methionine, or which is complementary to such a molecule of nucleic acid coding. The signal peptide has been tentatively identified as extending from about position 1 of the amino acids to about position 22 of the amino acids, inclusive, in the sequence of Figure 2 (SEQ ID NO: 2). It is noted, however, that the C-terminal border of the signal peptide may vary, but more likely at no more than about 5 amino acids on either side of the C-terminal border or boundary of the signal peptide as initially identified. herein, wherein the C-terminal boundary or border of the signal peptide can be identified with respect to the criteria routinely employed in the art to identify this type of amino acid sequence element (eg, Nielsen et al., Prot. Eng. 10: 1-6 (1997) and von Heinje et al., Nucí Acids, Res. 14: 4683-4690 (1986)). In addition, it is also recognized that, in some cases, segmentation of a signal sequence from a secreted polypeptide is not completely uniform, leading to more than one of the secreted species. These polypeptides, and the polynucleotides that encode them, are contemplated by the present invention. As such, for the purposes of the present application, the peptide of the FGF-19 polypeptide signal shown in Figure 2 (SEQ ID NO: 2) extends from amino acids 1 to X of Figure 2 (SEQ ID NO: 2). : 2) (SEQ ID NO: 2), wherein X is any amino acid from 17 to 27 of Figure 2 (SEQ ID NO: 2). Therefore, the mature forms of the FGF-19 polypeptide which are encompassed by the present invention include those comprising amino acids X to 216 of Figure 2 (SEQ ID NO: 2), wherein X is any amino acid from 17 to 27 of Figure 2 (SEQ ID NO: 2) and variants thereof as described below. Isolated nucleic acid molecules encoding these polypeptides are also contemplated. Another embodiment is directed to fragments of a polypeptide sequence of FGF-19 which includes the coding sequence that can find use, for example, as hybridization probes or to encode fragments of a FGF-19 polypeptide that can encode optionally a polypeptide comprising an agglutination site for an anti-FGF-19 antibody. Such nucleic acid fragments are usually at least about 20 nucleotides in length, alternatively of at least about 30 nucleotides in length, alternatively of at least about 40 nucleotides in length, alternatively of at least about 50 nucleotides in length, alternatively of at least about 60 nucleotides in length, alternatively of at least about 70 nucleotides in length , alternatively of at least about 80 nucleotides in length, alternatively of at least about 90 nucleotides in length, alternatively of at least about 100 nucleotides in length, alternatively of at least about 110 nucleotides in length, alternatively of at least about 120 nucleotides in length , alternatively of at least about 130 nucleotides in length, alternatively of at least about 140 nucleotides in length, alternatively of at least about 150 nucleotides in length, alternatively of at least about about 160 nucleotides in length, alternatively of at least about 170 nucleotides in length, alternatively of at least about 180 nucleotides in length, alternatively of at least about 190 nucleotides in length, alternatively of at least about 200 nucleotides in length, alternatively of at least about 250 nucleotides in length, alternatively of at least about 300 nucleotides in length, alternatively of at least about 350 nucleotides in length, alternatively of at least about 400 nucleotides in length, alternatively of at least about 450 nucleotides in length, alternatively of at least about 500 nucleotides in length, alternatively of at least about 600 nucleotides in length, alternatively of at least about 700 nucleotides in length, alternatively of at least about 800 nucl. otides of length, alternatively of at least about 900 nucleotides in length, alternatively of at least about 1000 nucleotides in length, wherein in this context the term "approximately" means the length of the nucleotide sequence referred to plus or minus 10% of that referred length. In a preferred embodiment, the fragment of the nucleotide sequence is derived from any coding region of the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1). It was pointed out that novel fragments of a nucleotide sequence encoding the FGF-19 polypeptide can be determined in a routine manner by aligning the nucleotide sequence encoding the FGF-19 polypeptide with other known nucleotide sequences using any of a number of alignment programs of well-known sequences and determining which fragment (s) of the nucleotide sequence encoding the FGF-19 polypeptide is novel (s). All such nucleotide sequences encoding the FGF-19 polypeptide are contemplated herein and can be determined without undue experimentation. FGF-19 polypeptide fragments encoded by these fragments of the nucleotide molecules, preferably those fragments of the FGF-19 polypeptide comprising a binding site for an antibody of FGF-19, are also contemplated. In another embodiment, the invention provides a vector comprising a nucleotide sequence encoding FGF-19 or its variants. The vector can comprise any of the isolated nucleic acid molecules identified hereinabove. A host cell comprising such a vector is also provided. By way of example, the host cells can be CHO cells, E. coli, insect cells infected with the baculovirus, or yeast. A process for producing the FGF-19 polypeptides is further provided and comprises culturing the host cells under conditions suitable for expression of FGF-19 and recovery of FGF-19 from cell culture. In another modality, the invention provides the isolated FGF-19 polypeptide encoded by any of the isolated nucleic acid sequences identified hereinbefore. In a specific aspect, the invention provides the FGF-19 polypeptide of the isolated natural sequence, which in certain embodiments, includes an amino acid sequence comprising the residues from about 1 or approximately 23 to approximately 216 of the Figure 2 (SEQ ID NO: 2). In another aspect, the invention relates to an isolated FGF-19 polypeptide, comprising an amino acid sequence having an amino acid sequence identity of at least about 80%, alternatively at least about 81% identity of the sequence of the amino acids, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% % identity of the amino acid sequence, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% sequence identity of the amino acids, alternatively at least about 93% sequence identity of the amino acids, alternatively at least about 94% sequence identity of the amino acids, alternatively at least about 95% sequence identity of the amino acids, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity, alternatively at least approximately 99% of identida d of the sequence of the amino acids with the sequence of amino acid residues from about 1 to about 23 to about 216, inclusive, of Figure 2 (SEQ ID NO: 2). In a further aspect, the invention relates to an isolated FGF-19 polypeptide comprising an amino acid sequence having an amino acid sequence identity of at least about 80%, alternatively at least about 81% of the identity of the sequence of the amino acids, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% % identity of amino acid sequence, alternativ at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92 % sequence identity of the amino acids, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% identity of the sequence of the amino acids, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity, alternatively at least about 99% amino acid sequence identity with respect to one sequence of amino acids encoded by the cDNA of the human protein deposited with the ATCC on November 21, 1997 under ATCC Deposit No. 209480 (DNA49435-1219). In a preferred embodiment, the isolated FGF-19 polypeptide comprises an amino acid sequence encoded by the human human protein cDNA deposited with the ATCC on November 21, 1997 under ATCC Deposit No. 209480 (DNA49435-1219). In a further aspect, the invention relates to an isolated FGF-19 polypeptide comprising an amino acid sequence that registers at least about 80% and positive, alternately at least about 81% positive, alternately at least about 82% positive, alternately at least about 83% positive, alternately at least about 84% positive, alternately at least about 85% positive, alternating at least about 86% positive, alternatively at least about 87% positive, alternately at least about 88% positive, alternately at least about 89% positive, alternately at least about 90% positive, alternately at least about 91% positive, alternately at least about 92% positive, alternately at less about 93% positive, alternatively at least about 94% positive, alternately at least about 95% positive, alternately at least about 96% positive, alternating at least about 97% positive, alternately at less about 98% positive, al least at least about 99% positive when compared to the amino acid sequence of the residues from about 1 or about 23 to 216, inclusive, of Figure 2 (SEQ ID NO: 2). In a specific aspect, the invention provides an FGF-19 polypeptide isolated without the sequence of the N-terminal signal and / or the start methionine and is encoded by a nucleotide sequence encoding such an amino acid sequence as described herein above. . The processes for producing same are also described herein, wherein these processes comprise culturing a host cell comprising a vector which comprises the appropriate coding nucleic acid molecule under conditions suitable for expression of the FGF-19 polypeptide and recovering the FGF-19 polypeptide from cell culture. In yet another aspect, the invention relates to an isolated FGF-19 polypeptide, comprising the amino acid residue sequence from about 1 or about 23 to about 216, inclusive, of Figure 2 (SEQ ID NO: 2) , or a fragment thereof which is biologically active or sufficient to provide a binding or agglutination site for an anti-FGF-19 antibody, wherein the identification of FGF-19 polypeptide fragments possessing the biological activity or provide an agglutination site for an anti-FGF-19 antibody can be performed in a routine manner using techniques which are well known in the art. Preferably, the FGF-19 fragment retains a qualitative biological activity of a natural FGF-19 polypeptide, including the ability to therapeutically treat obesity. In a still further aspect, the invention provides a polypeptide produced by (i) hybridizing a test DNA molecule under severe conditions with (a) a DNA molecule encoding a FGF-19 polypeptide having the sequence of the amino acid residues from about 1 or about 23 to about 216, inclusive, of Figure 2 (SEQ ID NO: 2), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has a sequence identity of at least about 80%, preferably at least about 80% nucleic acid sequence identity, alternatively at least about 81% identity of the nucleic acid sequence, alternatively at least about 82% of identity of the nucleic acid sequence, alternatively at least about 83% identity of the nucleic acid sequence, alternatively at least about 84% identity ad of the nucleic acid sequence, alternatively at least about 85% identity of the nucleic acid sequence, alternatively at least about 86% identity of the nucleic acid sequence, alternatively at least about 87% identity of the nucleic acid sequence, alternatively at least about 88% identity of the nucleic acid sequence, alternatively at least about 89% identity of the nucleic acid sequence, alternatively at least about 90% identity of the nucleic acid sequence, alternatively at least about 91% identity of the nucleic acid sequence, alternatively at least about 92% identity of the nucleic acid sequence, alternatively at least about 93% identity of the nucleic acid sequence, alternatively at least about 94% identity of the nucleic acid sequence, alternatively at least about 95% identity of the nucleic acid sequence, alternatively at least about 96% identity of the nucleic acid sequence , alternatively at least about 97% d and identity of the nucleic acid sequence, alternatively at least about 98% identity of the nucleic acid sequence, alternatively at least about 99% identity of the nucleic acid sequence with respect to (a) or (b), (ii) culturing a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture.

In another embodiment, the invention provides chimeric molecules comprising a FGF-19 polypeptide fused to a heterologous polypeptide or amino acid sequence, wherein the FGF-19 polypeptide can comprise any FGF-19 polypeptide, variant or fragment thereof as was described here above. An example of such a chimeric molecule comprises an FGF-19 polypeptide fused to an epitope tag sequence or an Fc region of an immunoglobulin. In another embodiment, the invention provides an antibody as defined below which binds or agglutinates specifically to a FGF-19 polypeptide as described herein above. Optionally, the antibody is a monoclonal antibody, an antibody fragment or a single chain antibody. In still another embodiment, the invention relates to agonists and antagonists of a natural FGF-19 polypeptide as defined below. In a particular embodiment, the agonist or antagonist is an anti-FGF-19 antibody or a small molecule. In a further embodiment, the invention relates to a method of identifying agonists or antagonists for a FGF-19 polypeptide which comprises contacting the FGF-19 polypeptide with a candidate molecule and verifying a biological activity mediated by the FGF-19 polypeptide. Preferably, the FGF-19 polypeptide is a natural FGF-19 polypeptide. In a still further embodiment, the invention relates to a composition of matter comprising a FGF-19 polypeptide, or an agonist or antagonist of a FGF-19 polypeptide as described herein, or an anti-FGF-19 antibody. , in combination with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier. Another embodiment of the present invention is directed to the use of a FGF-19 polypeptide, or an agonist or antagonist thereof as described herein, or an anti-FGF-19 antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the FGF-19 polypeptide, an agonist or antagonist thereof or an anti-FGF-19 antibody. In one embodiment, a method for the selection of a bioactive agent capable of binding to FGF-19 is provided. In one aspect, the method comprises adding a candidate bioactive agent to a sample of FGF-19 and determining the binding or agglutination of the candidate agent for FGF-19, where binding or agglutination indicates a bioactive agent capable of agglutination to FGF- 19 Additional provided herein is a method for the selection of a bioactive agent capable of modulating the activity of FGF-19. In one embodiment, a method is provided, which comprises the steps of adding a candidate bioactive agent to a sample of FGF-19 and determining an alteration in the biological activity of FGF-19, wherein an alteration indicates a bioactive agent capable of Modulate the activity of FGF-19. In one embodiment, the activity of FGF-19 is the reduced absorption of glucose in the cells. In another embodiment, the activity of FGF-19 is the release of the increased leptin from the cells. In a preferred embodiment, the activity of FGF-19 is the reduced absorption of glucose and the release of increased peptin from the cells. Preferably the cells are adipocytes. In still another embodiment, the activity of FGF-19 is the increased oxidation of lipids and carbohydrates. Preferably the cells are the cells of the liver or muscles. In yet another embodiment, the invention provides a method of identifying a FGF-19 receptor. In a preferred embodiment, the method comprises combining the FGF-19 with a composition comprising the material of the cell membrane wherein the FGF-19 forms complexes with a receptor on the cell membrane material, and identify the receptor as a receptor for FGF-19. In one embodiment, the method includes a cross-linking step with the FGF-19 and the receptor. The cell membrane can be from an intact cell or an extract preparation from the cell membrane. In a further aspect of the invention, there is provided a method for inducing the release of leptin from cells, preferably adipocytes. In one embodiment, the method comprises administering FGF-19 to the cells in an amount effective to induce the release of leptin. In the methods provided herein, FGF-19 can be administered as a nucleic acid which expresses FGF-19 or in the form of protein. As described below, FGF-19 can be administered by infusion or in a sustained release formulation. Preferably, FGF-19 is administered to an individual with a pharmaceutically acceptable carrier. Also provided here is a method for inducing a reduction in glucose uptake in cells, preferably adipocyte cells. In one embodiment, the method comprises administering FGF-19 to the cells in an amount effective to induce a reduction in glucose uptake.

In still another aspect of the invention there is provided a method of treating obesity in an individual. In one embodiment the method comprises administering to an individual a composition comprising FGF-19 in an amount effective to treat obesity. In this way, conditions related to obesity can also be treated, such as cardiovascular disease. Also provided herein is a method of reducing total body mass in an individual, comprising administering to the individual an effective amount of FGF-19. In a preferred embodiment, the adiposity (fat) of an individual is reduced. In addition, a method is provided herein for reducing the level of at least one of the triglycerides and free fatty acids in an individual, which comprises administering to the individual an effective amount of FGF-19. Also provided herein is a method for increasing metabolic rate in an individual, which comprises administering to the individual an effective amount of FGF-19. An animal model is also provided here to determine the effects of FGF-19 and modulators thereof under varying conditions and conditions. In one embodiment, an animal, preferably a rodent, is provided, which comprises a genome comprising a transgene encoding FGF-19.

Brief Description of the Drawings Figure 1 shows the nucleotide sequence (SEQ ID NO: 1) of a cDNA containing a nucleotide sequence (nucleotides 464-1111) encoding a natural sequence of FGF-19, wherein the nucleotide sequence (SEQ ID NO: 1) is a clone designated herein as "DNA49435-1219". Also presented in letters with bold and underlined, are the positions of the respective start and stop codons. Figure 2 shows the amino acid sequence (SEQ ID NO: 2) of a natural polypeptide sequence of FGF-19 as derived from the coding sequence of SEQ ID NO: 1. The approximate locations of several of the other important polypeptide domains are also shown. Figures 3A and 3B show bar graphs demonstrating that transgenic MLC-FGF-19 mice weighed less than their non-transgenic littermates (Figure 3A) and have lower circulating leptin levels (Figure 3B). Figure 3A shows the weight of transgenic FGF-19 mice (continuous bars) and non-transgenic (wild type) littermates (dotted bars) at 6 weeks of age during ad libitum feeding (to the left) , after fasts of 6 and 24 hours, and 24 hours after finishing a 24-hour fast (to the right). Figure 3B shows the sera from the same groups of mice presented in Figure 3A in an assay for leptin (vertical bars). Figures 4A-4d are bar graphs showing that transgenic FGF-19 mice have increased food intake and urine output, but have a normal hematocrit count. A group of mice were checked to verify admission of food during ad libitum feeding and 24 hours after finishing a 24-hour fast (Figure 4A), water intake (Figure 4B), urine output (Figure 4C) and the hematocrit count (Figure 4D) where the results for the FGF-19 transgenic mice in each graph are shown by the black bars only and the results for the wild type are shown by the stitch bars. Figure 5 is a bar graph demonstrating that transgenic FGF-19 mice have an increased rate of oxygen consumption. Oxygen consumption is shown for transgenic mice of FGF-19 (black bars only) and wild type (dotted bars) during both light cycles (day or night), following a 24-hour fast and 24 hours after the completion of a 24-hour fast. Figures 6A and 6B are bar graphs showing that transgenic FGF-19 mice (black bars only) have reduced levels of triglycerides (Figure 6A) and free fatty acids (Figure 6B) on wild-type mice (dotted bars). Figures 7A and 7B are bar graphs which demonstrate that the infusion of nontransgenic mice with FGF-19 (black bars only) leads to an increase in the intake of food (Figure 7A) and an increase in the oxygen consumption (Figure 7B) on mice infused with the vehicle lacking FGF-19 (dotted bars), where "n" means night and "d" means day. Figures 8A and 8B are bar graphs indicating that FGF-19 increases the release of leptin from adipocytes (Figure 8A) and reduces the absorption of glucose by adipocytes (Figure 8B). Figure 9 is a bar graph showing the weight of the fat pad of transgenic FGF-19 (shaded bars) or wild-type mice (black bars only) each on a high fat diet (HFD) ) during the course of time, where along the horizontal bar starting on the left, the results are shown at 6 weeks for the epididymis (HFD Ep) and then for retroperitoneal with peri-renal (HFD RP / PR) ), and then at 10 weeks for the epididymis and then for retroperitoneal with peri-renal. Figure 10 is a bar graph showing the glucose tolerance of the transgenic mice of FGF-19 (shaded bars) or wild type (black bars only) over time (both on high fat diets for ten weeks).

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES I. Definitions The terms "polypeptide of FGF-19", "protein of FGF-19"and" FGF-19"when used herein encompass the FGF-19 of the natural sequence and the FGF-19 polypeptide variants (which are further defined herein.) The FGF-19 polypeptide can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant and / or synthetic methods.A "FGF-19 of the natural sequence" comprises a polypeptide having the same amino acid sequence that a FGF-19 derived from nature.Such FGF-19 of the natural sequence can be isolated from nature or can be produced by recombinant and / or synthetic means.The term "FGF-19 of the natural sequence" specifically encompasses the truncated or secreted forms that are naturally present (for example, a sequence of the extracellular domain), variant forms that are naturally present (for example, alternately spliced forms) and allelic variants that are are present in a natural way of the FGF-19. In one embodiment of the invention, FGF-19 of the native sequence is a FGF-19 of the mature or full-length natural sequence comprising amino acids 1 to 216 of Figure 2 (SEQ ID NO: 2). Also, although the FGF-19 polypeptide described in Figure 2 (SEQ ID NO: 2) is shown to start with the methionine residue designated here as position 1 of the amino acid, it is conceivable and possible that another methionine residue located already either upstream or downstream of position 1 of the amino acids in Figure 2 (SEQ ID NO: 2) can be employed as the residue of the starting amino acids for the FGF-19 polypeptide. "FGF-19 variant polypeptide" means an active FGF-19 polypeptide as defined below having at least about 80% identity of the amino acid sequence with the amino acid sequence of (a) residues 1 or approximately 23 to 216 of the FGF-19 polypeptide shown in Figure 2 (SEQ ID NO: 2), (b) X to 216 of the FGF-19 polypeptide shown in Figure 2 (SEQ ID NO: 2), wherein X is any amino acid residue from 17 to 27 of Figure 2 (SEQ ID NO: 2), or (c) another fragment specifically derived from the amino acid sequence shown in Figure 2 (SEQ ID NO: 2). Such variant polypeptides of FGF-19 include, for example, the FGF-19 polypeptides wherein one or more amino acid residues are aggregated, or deleted, at the N and / or C terminus, as well as within one or more internal domains, of the sequence of Figure 2 (SEQ ID NO: 2). Ordinarily, a variant polypeptide of FGF-19 will have at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% identity of the sequence of the amino acids, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89 % identity of the amino acid sequence at least about 90% identity of the amino acid sequence, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% identity of the sequence of the amino acids, alternatively at least about 97% sequence identity of the amino acids, alternatively at least about 98% sequence identity of the amino acids, alternatively at least about 99% sequence identity of the amino acids with (a) residues 1 or approximately 23 to 216 of the FGF-19 polypeptide shown in Figure 2 (SEQ ID NO: 2), (b) X to 216 of the FGF-19 polypeptide shown in Figure 2 (SEQ ID NO: 2), where X is any amino acid residue from 17 to 27 of Figure 2 (SEQ ID NO: 2), or (c) another fragment specifically derived from the amino acid sequence shown in Figure 2 (SEQ ID NO: 2). Variant polypeptides of FGF-19 do not encompass the polypeptide sequence of native FGF-19. Ordinarily, variant FGF-19 polypeptides are at least about 10 amino acids in length, alternatively of at least about 20 amino acids in length, alternatively of at least about 30 amino acids in length, alternatively of at least about 40 amino acids in length, alternatively of at least about 50 amino acids in length, alternatively of at least about 60 amino acids in length, alternatively of at least about 70 amino acids in length, alternatively of at least about 80 amino acids in length, alternatively of at least about 90 amino acids in length, alternatively of at least about 100 amino acids in length, alternatively of at least about 150 amino acids in length, alternatively of at least about 200 amino acids in length, alternatively of at least about 300 amino acids of ngitude, or more. "The percent (%) identity of the amino acid sequence" with respect to the FGF-19 polypeptide sequences identified herein is defined as the percentage of the amino acid residues in a candidate sequence that is identical to the amino acid residues in a sequence of FGF-19, after the alignment of the sequences and the introduction of the gaps, if necessary, to achieve the identity of the maximum percentage sequence, and without considering any of the conservative substitutions as part of the identity of the sequence. Alignment for the purposes of determining the percentage of the identity of the amino acid sequence can be achieved in various ways that are within the skill in the art, for example, using publicly available computer programs such as BLAST programs, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR). Those skilled in the art can determine the appropriate parameters for the measurement of the alignment, including any algorithms necessary to achieve maximum alignment on the total length of the sequences being compared. For the purposes herein, however, the% values of the amino acid sequence identity are obtained as described below using the ALIGN-2 computer program for comparison of the sequences, wherein the source code is complete for the ALIGN-2 program is provided in Table 1 below. The computer program for the comparison of ALIGN-2 sequences was authorized by Genentech, Inc., and the source code shown in Table 1 has been presented with user documentation in the United States Copyright Office. of America, Washington DC, 20559, where it is registered under the United States of America Reserved Rights Register No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California or can be compiled from the source code provided in Table 1. The ALIGN-2 program must be compiled for use on a UNIX operating system , preferably UNIX V4. Digital DO All the comparison parameters of the sequences are set by the ALIGN-2 program and do not vary. For the purposes herein, the% identity of the amino acid sequence of a given amino acid sequence A for, with, or against a given B sequence of amino acids (which may alternatively be expressed as a sequence A of amino acids given to it has or that comprises a certain percentage of identity of the amino acid sequence for, with, or against a given B-amino acid sequence) is calculated as follows: 100 times the fraction X / Y where X is the number of the amino acid residues registered as identical correspondences by the alignment program of the sequence ALIGN-2 in this alignment of the program of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that where the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the% identity of the amino acid sequence of A with respect to B will not be equal to the% identity of the amino acid sequence. the amino acid sequence of B with respect to A. As the examples of the% amino acid sequence identity calculations, Tables 2 and 3 demonstrate how to calculate% identity of amino acid sequence, sequence of amino acids designated "Comparison Protein" with respect to the amino acid sequence designated "PRO". Unless otherwise specified, all% values of the identity of the amino acid sequence used herein are obtained as described above using the computer program for the comparison of ALIGN-2 sequences. However, the% identity of the amino acid sequence can also be determined using the NCBI-BLAST2 sequence comparison program (Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program can be downloaded from http://www.ncbi.nlm.nih.gov or obtained in other ways from the National Institute of Health, Bethesda, MD. The NCBI-BLAST2 program uses several search parameters, where all of these search parameters are set as fault values including, for example, no mask = yes, thread = all, expected presentations = 10, complexity length minimum low = 15/5, value 2 of multiple steps = 0.01, constant for multiple steps = 25, reduction for final alignment = 25 with holes and evaluation matrix = BLOSUM62. In situations where NCBI-BLAST2 is used for comparisons of amino acid sequences, the% identity of the amino acid sequence of a given amino acid sequence A for, with, or against a given amino acid sequence B (which alternatively it can be expressed as a given amino acid sequence A having or comprising a certain% identity of the amino acid sequence for, with, or against a given amino acid sequence B) is calculated as follows 100 times the fraction X / Y where X is the number of the amino acid residues recorded as identical correspondences by the alignment program of the NCBI-BLAST2 sequences in this alignment of the program of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that where the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the% identity of the amino acid sequence of A with respect to B will not be equal to the% identity. of the amino acid sequence of B with respect to A. "FGF-19 variant polynucleotide" or "variant nucleic acid sequence of FGF-19" means a nucleic acid molecule which encodes an active FGF-19 polypeptide as further defined and which has at least about 80% identity of the nucleic acid sequence with either (a) a nucleic acid sequence which encodes residues 1 or approximately 23 to 216 of the polypeptide from FGF-19 shown in Figure 2 (SEQ ID NO: 2), (b) a nucleic acid sequence which encodes amino acids X through 216 of the FGF-19 polypeptide shown in Figure 2 (SEQ ID NO: 2), wherein X is any amino acid residue from 17 to 27 of Figure 2 (SEQ ID NO: 2), or (c) a nucleic acid sequence which encodes another fragment specifically derived from the amino acid sequence shown in Figure 2 (SEQ ID NO: 2). Ordinarily, a variant polynucleotide of FGF-19 will have at least about 80% identity of the nucleic acid sequence, alternatively at least about 81% of the identity of the nucleic acid sequence, alternatively at least about 82% identity of the nucleic acid sequence, alternatively at least about 83% identity of the nucleic acid sequence, alternatively at least about 84% identity of the nucleic acid sequence, alternatively at least about 85% identity of the nucleic acid sequence, alternatively at least about 86% identity of the nucleic acid sequence, alternatively at least about 87% identity of the nucleic acid sequence, alternatively at least about 88% identity of the nucleic acid sequence, alternatively at least about 89% identity of the nucleic acid sequence, alternatively at least about 90% identity of the acid sequence nucleic acid, alternatively at least about 91% identity of the nucleic acid sequence, alternatively at least about 92% identity of the nucleic acid sequence, alternatively at least about 93% identity of the nucleic acid sequence, alternatively at least about 94% sequence identity nucleic acid, alternatively at least about 95% identity of the nucleic acid sequence, alternatively at least about 96% identity of the nucleic acid sequence, alternatively at least about 97% identity of the nucleic acid sequence, alternatively at least about 98% identity of the nucleic acid sequence, alternatively at least about 99% identity of the nucleic acid sequence with either (a) a nucleic acid sequence which encodes residues 1 or approximately 23 to 216 of the FGF-19 polypeptide shown in Figure 2 (SEQ ID NO: 2), (b) a sequence nucleic acid which encodes amino acids X through 216 of the FGF-19 polypeptide shown in Figure 2 (SEQ ID NO: 2), wherein X is any amino acid residue from 17 to 27 of Figure 2 (SEQ ID NO: 2) NO: 2), or (c) a nucleic acid sequence which encodes another fragment specifically derived from the amino acid sequence shown in Figure 2 (SEQ ID NO: 2). Variants of the polynucleotides do not encompass the nucleotide sequence of natural FGF-19. Ordinarily, the variant polynucleotides of FGF-19 are at least about 30 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternatively at least about 240 nucleotides in length, alternatively at least about 270 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 900 nucleotides in length, or more. "The percentage (%) of the identity of the nucleic acid sequence" with respect to the nucleic acid sequences encoding the FGF-19 polypeptide identified herein, is defined as the percentage of the nucleotides in a candidate sequence that are identical with the nucleotides in a nucleic acid sequence encoding the FGF-19 polypeptide, after alignment of the sequences and insertion of the gaps, if necessary, to achieve the identity of the maximum percent sequence. The alignment for the purposes of determining the percent identity of the nucleic acid sequence can be achieved in various ways that are within the experience in the art., for example, using the publicly available computer program, such as the BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign programs (DNASTAR). Those skilled in the art can determine the appropriate parameters by measuring the alignment, including any algorithms necessary to achieve maximum alignment on the total length of the sequences being compared. For the purposes herein, however, the% values of the identity of the nucleic acid sequence are obtained as described below using the computer program for the comparison of the ALIGN-2 sequences, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below. The computer program for the comparison of the ALIGN-2 sequences was authorized by Genentech, Inc., and the source code shown in Table 1 has been presented with the user documentation in the United States Copyright Office. , Washington DC, 20559, where it is registered under the Registry of Rights Reserved No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California or can be compiled from the source code provided in Table 1. The ALIGN-2 program must be compiled for use on a UNIX operating system, preferably UNIX V4. Digital DO All the comparison parameters of the sequences are described by the program ALIGN-2 and do not vary. For the purposes herein, the% identity of the nucleic acid sequence of a given amino acid sequence C for, with, or against a given nucleic acid sequence D (which may be alternatively expressed as a nucleic acid sequence given C having or comprising a certain% identity of the nucleic acid sequence for, with, or against a given nucleic acid sequence D) is calculated as follows: 100 times the fraction W / Z where W is the number of nucleotides registered as the identical correspondences by the alignment program of the ALIGN-2 sequences in this alignment of the program of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that wherein the length of the nucleic acid sequence C is not equal to the length of the nucleic acid sequence D, the% identity of the nucleic acid sequence of C with respect to D will not be equal to the% identity of the nucleic acid sequence. the nucleic acid sequence of D with respect to C. As the examples of the nucleic acid identity% calculations, Tables 4 and 5 demonstrate how to calculate the% identity of the nucleic acid sequence of the nucleic acid sequence designated "Comparison DNA" with respect to the nucleic acid sequence designated "PRO-DNA". Unless specifically stated otherwise, all% of the identity values of the nucleic acid sequence used herein are obtained as described above using the computer program for the comparison of ALIGN-2 sequences. However,% identity of the nucleic acid sequences can also be determined using the NCBI-BLAST2 sequence comparison program (Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program can be downloaded from http://www.ncbi.nlm.nih.gov or obtained in other ways from the National Institute of Health, Bethesda, MD. The NCBI-BLAST2 uses several search parameters, where all of these search parameters are set for fault values that include, for example, no mask = yes, thread = all, expected events = 10, minimum low complexity length = 15/5, multi-step e-value = 0.01, constant for multiple steps = 25, re-direction for final alignment with holes = 25 and evaluation matrix = BLOSUM62. In situations where NCBI-BLAST2 is used for the comparisons of the sequences, the% identity of the nucleic acid sequence of a given nucleic acid sequence C for, with, or against a given nucleic acid sequence D (which alternatively it can be expressed as a given nucleic acid sequence C having or comprising a certain% identity of the nucleic acid sequence for, with, or against a given nucleic acid sequence D) is calculated as follows 100 times the fraction W / Z where W is the number of nucleotides recorded as the identical correspondences by the alignment program of the NCBI-BLAST2 sequences in this alignment of the program of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that wherein the length of the nucleic acid sequence C is not equal to the length of the nucleic acid sequence D, the% identity of the nucleic acid sequence of C with respect to D will not be equal to the% identity of the nucleic acid sequence. the nucleic acid sequence of D with respect to C. In other embodiments, the polynucleotides of the FGF-19 variant are the nucleic acid molecules that encode an active FGF-19 polypeptide and which are capable of hybridization, preferably under severe washing and hybridization conditions, to the nucleotide sequences encoding the full-length FGF-19 polypeptide shown in Figure 2 (SEQ ID NO: 2). Variant polypeptides of FGF-19 may be those that are encoded by a polynucleotide of the FGF-19 variant. The term "positives", in the context of the amino acid sequence identity comparisons performed as described above, includes the amino acid residues in the compared sequences that are not only identical, but also have similar properties. The amino acid residues that register a positive value for an amino acid residue of interest are those that are either identical to the amino acid residue of interest or are a preferred substitution (as defined in Table 6 below) of the amino acid residue of interest. For the purposes here, the% of the value of the positives of a given amino acid sequence A for, with, or against a given amino acid sequence B (which alternatively can be expressed as a given amino acid sequence A having or comprising a certain% of positives for, with, or against a given amino acid sequence B) is calculated as follows 100 times the fraction X / Y where X is the number of amino acid residues that register a positive value as defined above by the alignment program of the NCBI-BLAST2 sequences in this program alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the% positive of A with respect to B will not be equal to the% positive of B with respect to A. "Isolated", when used to describe the various polypeptides described herein, means polypeptides that have been identified and separated and / or recovered from a component of their natural environment. Preferably, the isolated polypeptide is free of association with all the components with which it is naturally associated. The contaminating components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous and non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a sufficient degree to obtain at least 15 residues of the internal or N-terminal amino acid sequence by the use of a rotary cup sequencer, or (2) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, a silver dye. The isolated polypeptides include the polypeptides in situ within the recombinant cells, since at least one component of the natural environment of FGF-19 will not be present. Ordinarily, however, the isolated polypeptide will be prepared by at least one purification step. An "isolated" nucleic acid molecule that encodes a FGF-19 polypeptide is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in the natural source of the acid nucleic acid encoding FGF-19. Preferably, the isolated nucleic acid is free of association with all the components with which it is naturally associated. A nucleic acid molecule encoding the isolated FGF-19 is different from the form or environment in which it is found in nature. The isolated nucleic acid molecules are therefore distinguished from the nucleic acid molecule encoding FGF-19 as it exists in natural cells. However, an isolated nucleic acid molecule encoding a FGF-19 polypeptide includes the nucleic acid molecules encoding FGF-19 contained in cells that ordinarily express FGF-19 where, for example, the acid molecule nucleic is in a chromosomal location different from that of natural cells. The term "control sequences" refers to the DNA sequences necessary for the expression of a coding sequence operably linked in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding or agglutination site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers. The nucleic acid is "operably linked" when it is placed in a functional relationship with other nucleic acid sequences. For example, DNA for a presequence or secretory forward element is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome agglutination site is operably linked to a coding sequence if it is placed to facilitate translation. In general, "operably linked" means that the DNA sequences that are linked are contiguous, and, in the case of a secretory front element, they are contiguous and are in a reading phase. However, breeders do not have to be contiguous. The binding is effected by ligation or binding at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide linkers or linkers are used in accordance with conventional practice.

The term "antibody" is used in the broadest sense and specifically covers, for example, unique anti-FGF-19 monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies), anti-FGF-19 antibody compositions with specificity of polyepitopes, anti-FGF-19 single chain antibodies, and anti-FGF-19 antibody fragments (see below). The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for the possible mutations that are naturally present, which can be present in smaller quantities. The "severity" of the hybridization reactions can be readily determined by a person of ordinary skill in the art, and is generally an empirical calculation dependent on the length of the probe, the wash temperature, and the concentration of the salt. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of the denatured DNA for annealing when the complementary strands are present in an environment below its melting temperature. The higher the desired degree of homology between the probe and the hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures may tend to make the reaction conditions more severe, while lower temperatures lead to less severe conditions. For additional details and explanation of the severity of the hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). "Severe conditions" or "high severity conditions", as defined herein, may be identified by those that: (1) employ a lower ionic strength and a high temperature for washing, for example 0.015M sodium chloride / citrate sodium 0.0015 M / 0.1% sodium dodecyl sulfate at 50 ° C; (2) employ during denaturation a denaturing agent, such as formamide, for example, 50% formamide (v / v) with 0.1% bovine serum albumin / 0.1% Ficoll / 1% polyvinylpyrrolidone / buffer 50 mM sodium phosphate pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, DNA from salmon sperm subjected to the action of sound (50 μg / ml), 0.1% SDS, and 10% dextran sulfate at 42 ° C, washed at 42 ° C in 0.2 x SSC (sodium chloride / sodium citrate) and 50% formamide at 55 ° C, followed by a high severity wash consisting of 0.1 x SSC containing EDTA at 55 ° C. "Moderately severe conditions" can be identified as described by Sambrook et al., In Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of wash solution and hybridization conditions (for example, temperature, ionic strength and concentration and% SDS) less severe than those described above. An example of moderately severe conditions is overnight incubation at 37 ° C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x of the Denhardt's solution, 10% dextran sulfate, and 20 mg / ml of sperm DNA from salmon subjected to shearing, denaturation, followed by washing the filters in 1 x SSC at approximately 37- 50 ° C. The skilled artisan will recognize how to adjust the temperature, the ionic intensity, etc., when necessary to adapt factors such as the length of the probe and the like. The term "epitope tagging" when used herein, refers to a chimeric polypeptide comprising a FGF-19 polypeptide fused to a "tag polypeptide". The polypeptide of the tag has enough residues to provide an epitope against which an antibody can be made, which is still short enough such that it does not interfere with the activity of the polypeptide to which it is fused. The polypeptide of the tag is preferably also quite original so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues). When used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding or agglutination specificity of a heterologous protein (an "adhesin") with the effector functions of the immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the specificity of the desired agglutination which is different from the recognition and agglutination site of an antibody antigen (ie, it is "heterologous"), and a constant domain sequence of immunoglobulin. The adhesin part of an immunoadhesin molecule is typically a contiguous amino acid sequence comprising at least one agglutination site of a receptor or a ligand. The constant domain sequence of the immunoglobulin in the immunoadhesin can be obtained from any immunoglobulin, such as the subtypes IgG-1, IgG-2, IgG-3, or IgG-4, IgA (including IgA-1 and IgA-2) , IgE, IgD or IgM. "Active" or "activity" for the purposes herein refers to the form (s) of FGF-19 which retain a biological and / or immunological activity of the native FGF-19 or that is naturally present, wherein the "biological" activity refers to a biological function (either inhibitory or stimulatory) caused by a native FGF-19 or that is naturally present, different from the ability to induce the production of an antibody against an antigenic epitope possessed by a native FGF-19 or that is naturally present and an "immunological" activity refers to the ability to induce the production of an antibody to an antigenic epitope possessed by a native FGF-19 or that is naturally present . A preferred biological activity includes any one or more of the following activities: increasing the metabolism (or metabolic rate) in an individual, reducing the body weight of an individual, reducing the adiposity in an individual, reducing the absorption of glucose in the adipocytes, increase the release of leptin from adipocytes, reduce triglycerides in an individual, and reduce free fatty acids in an individual. It is understood that some of the activities of FGF-19 are directly induced by FGF-19 and some are induced indirectly, however, each is the result of the presence of FGF-19 and otherwise could not have the same result in the absence of FGF-19. The term "antagonist" is used in the broadest sense, and includes any molecule that blocks, inhibits, or partially or totally neutralizes a biological activity of a naturally occurring FGF-19 polypeptide described herein. In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that minimizes a biological activity of a naturally occurring FGF-19 polypeptide described herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or variants of amino acid sequences of the natural FGF-19 polypeptides, peptides, small organic molecules, etc. Methods for identifying agonists or antagonists of a FGF-19 polypeptide may comprise contacting a FGF-19 polypeptide with a candidate agonist or antagonist molecule and measuring the detectable change in one or more biological activities associated with the FGF polypeptide. -19. "Treatment" refers to both therapeutic and prophylactic treatment or preventive measures, where the object is to prevent or delay (decrease) the condition or pathological disorder located as target. Those in need of treatment include both those who already have the disorder and those prone to have the disorder or those in whom the disorder is to be prevented. "Chronic" administration refers to the administration of the agent (s) in a continuous mode as opposed to an acute mode, to maintain the therapeutic effect (activity) for a prolonged period of time. The "intermittent" administration is the treatment that is not done consecutively without interruption, but rather is cyclic in nature.

A "mammal" for treatment purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo animals, animals for sporting activities, or pets, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is a human being. An "individual" is any subject, preferably a mammal, more preferably a human being. "Obesity" refers to a condition whereby a mammal has a Body Mass Index (BMI), which is calculated as weight (kg) by height2 (meters), of at least 25.9. Conventionally, those with a normal weight have a BMI of 19.9 to less than 25.9. Obesity here can be due to any cause, either genetic or environmental. Examples of disorders that can lead to obesity or be the cause of obesity include overfeeding and bulimia, polycystic ovarian disease, craniopharyngioma, Prader-Willi syndrome, Frohlich syndrome, diabetes Type II, subjects deficient in GH, short stature variable normal, Turner syndrome, and other pathological conditions that show reduced metabolic activity or a reduction in the expenditure of resting energy as a percentage of fat-free mass total, for example, children with acute lymphoblastic leukemia. "Conditions related to obesity" refer to conditions which are the result of, or which are aggravated by obesity, such as, but not limited to dermatological disorders such as infections, varicose veins, Acanthosis nigricans, and eczema, exercise intolerance, diabetes mellitus, insulin resistance, hypertension, hypercholesterolemia, cholelithiasis, osteoarthritis, orthopedic injuries, thromboembolic disease, cancer, and coronary heart disease (or cardiovascular disease), particularly those cardiovascular conditions associated with elevated levels of triglycerides and free fatty acids in an individual. The administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. "Carrier" as used herein includes pharmaceutically acceptable carriers, excipients, or stabilizers, which are not toxic to the cell or mammal that is exposed thereto, at the dosages and concentrations employed. Frequently the physiologically acceptable carrier is a buffered solution of aqueous pH. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; the low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar or saccharide alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN ™, polyethylene glycol (PEG), and PLURONICS ™. "Antibody fragments" comprise a portion of an intact antibody, preferably the variable or agglutination region of the intact antibody antigen. Examples of the antibody fragments include the Fab, Fab ', F (ab') 2, and Fv fragments; "diabodies" (fragments of small antibodies with two antigen binding sites); linear antibodies (Zapata et al., Protein Eng. 8 (10): 1057-1062 [1995]); Single-chain antibody molecules; and multispecific antibodies formed from the antibody fragments. Papain digestion of antibodies produces two identical antigen agglutination fragments, called "Fab" fragments, each with a unique antigen binding site, and a residual "Fc" fragment, a designation that reflects the ability to crystallize easily. The pepsin treatment produces an F (ab ') 2 fragment that has two antigen combining sites and is still capable of cross-linking the antigen. "Fv" is the minimal antibody fragment which contains a binding site and recognition of the complete antigen. This region consists of a dimer of a variable domain of a light chain and a heavy chain, in a non-covalent, narrow association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer a binding specificity of the antigen to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind the antigen, albeit at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab fragments differ from the Fab 'fragments by the addition of a small amount of residues at the carboxy terminus of the CH1 domain of the heavy chain that includes one or more cysteines from the antibody's articulation region. Fab'-SH is the designation here for Fab 'in which the cysteine residue (s) of the constant domains carry a free thiol group. F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments which have articulation cysteines between them. Other chemical bonds or couplings of the antibody fragments are also known. The "light chains" of antibodies (immunoglobulins) of any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), eg, IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The "sFv" or "single chain Fv" antibody fragments comprise the VH and VL domain of the antibodies, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which makes it possible for the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). The term "diabodies" refers to small antibody fragments with two antigen binding sites, such fragments comprise the heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). Using a linker that is too short to allow pairing or pairwise clustering between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. "Diabodies" or fragments of small antibodies with two antigen binding sites are described more fully, for example, in EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993). An "isolated" antibody is one which has been identified and separated and / or recovered from a component of its natural environment. The contaminating components of their natural environment are the materials which could interfere with therapeutic or diagnostic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to a degree greater than 95% by weight of the antibody as determined by the Lowry method, and more preferably greater than 99% by weight, (2) to a sufficient degree to obtain at least 15 residues of the N-terminal or internal amino acid sequence by the use of a rotating cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, the silver dye. The isolated antibody includes the antibody in situ within the recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, the isolated antibody will be prepared by at least one purification step.

The word "tag" when used here, refers to a detectable composition or compound which is directly or indirectly conjugated to the antibody to generate a "labeled" antibody. The label may be detectable by itself (eg, radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, it may catalyze the chemical alteration of a compound or composition of the substrate which is detectable. By "solid phase" is meant a non-aqueous matrix to which the antibody of the present invention can be adhered. Examples of the solid phases covered here include those formed partially or entirely of glass (for example, controlled pore size glass), polysaccharides (for example, agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase may comprise the cavity of a test plate; in others it is a purification column (for example, an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149. A "liposome" is a small vesicle composed of several types of lipids, phospholipids and / or the surfactant which is useful for the delivery of a drug (such as a polypeptide of FGF-19 or the antibody therefor). The components of the liposome are commonly distributed in a bilayer formation, similar to the lipid arrangement of the biological membranes. A "small molecule" is defined herein to have a molecular weight below about 500 Daltons.

Table 1 / * * * C-C increased from 12 to 15 * Z is the average of EQ * B is the average of ND * correspondence with stop element is _M; stop-stop = 0; correspondence J (associated) = 0 * / #define -M -8 / * value of a correspondence with a stop element * /? nt. d-ar261 [26] =. { / * A B CD E F G H I J K L M N O P Q R S T U V W X Y Z * / / * A * /. { 2, 0, -2, 0, 0, -4, 1, -1, -1, 0, -1, -2, -1, 0, Ü, 1, 0, -2, 1, 1, 0, 0, -6, 0, -3, 0.}. , / * B * /. { 0, 3, -4, 3, 2, -5, 0, 1, -2, 0, 0, -3, -2, 2, M, -1, 1, 0, 0, 0, 0, -2 , -5, 0, -3, 1.}. , / * C * / í -2, -4, 15, -5, -5, -4, -3, -3, -2, 0, -5, -6, -5, -4, M, - 3, -5, -4, 0, -2, 0, -2, -8, 0, 0, -5} , / * D * /. { 0, 3, -5, 4, 3, -6, 1, 1, -2, 0, 0, -4, -3, 2, -M, -1, T > , -1, 0, 0, 0, -2, -7, 0, -4, 2.}. , / * E * /. { 0, 2, -5, 3, 4, -5, 0, 1, -2, 0, 0, -3, -2, 1, M, -1, 2, -1, 0, 0, 0, - 2, -7, 0, -4, 3.}. , / * p *. { - *, -5, -4, -6, -5, 9, -5, -2, 1, 0, -5, 2, 0,, M, -5, -5, -4, -3, - 3, 0, -1, 0, 0, 7, -5} , l * G * l. { 1, 0, -3, 1, 0, -5, 5, -2, -3, 0, -2, -4, -3, 0, JvL -1, -1, -3, 1, 0, 0 , -1, -7, 0, -5, 0.}. 5 l * H * l. { -1, 1, -3, 1, 1, -2, -2, 6, -2, 0, 0, -2, -2, 2, _M, 0, 3, 2, -1, -1, 0 , -2, -3, 0, 0, 2.}. , 1 * 1 * 1 í-1, -2, -2, -2, -2, 1, -3, -2, 5, 0, -2, 2, 2, -2, M, -2, - 2, -2, -1, 0, 0, 4, -5, 0, -1, -2} , l * J * l. { or. o, o, o, o, o, o, o, o, o, o, o, o, o, o o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o, o , l * K * /. { -1, 0, -5, 0, 0, -5, -2, 0, -2, 0, 5, -3, ü, 1, _M, -1, 1, 3, 0, 0, 0, - 2, -3, 0, -4, 0.}. , 1 * 1. * I. { -2, -3, -6, -4, -3, 2, -4, -2, 2, 0, -3, 6, 4, -3, M, -3, -2, -3, -3 , -1, 0, 2, -2, 0, -1, -2} , l * M * l. { -1, -2, -5, -3, -2, 0, -3, -2, 2, 0, 0, 4, 6, -2, M, -2, -1, 0, -2, - 1, O, 2, -4, 0, -2, -1} , l * N * l. { 0, 2, -4, 2, 1, -4, 0, 2, -2, 0, 1, -3, -2, 2, _ív -1, 1, 0, 1, 0, 0, -2, -4, 0, -2, 1.}. ,? / ** po **? . { _H M, _H_M._ ._M, _M, _H M, _H - H_H_H M, o, H M, H M M, M, M, H H M. M.}. ,. { 1, -r, -3, -1, -1, -5, -1, 0, -2, 0, -1, -3, -2, -1, _ T6, 0, 0, 1, 0, 0, -1, -6, 0, -3, 0.}. , ~ ~ / * Q * /. { 0, 1, -5, 2, 2, -5, -l, 3, -2, 0, 1, -2, -1, 1, _M, 0, 4, 1, -1, -1, 0, -2, -5, 0, -4, 3), / * R * /. { -2, 0, -4, -1, -1, -4, -3, 2, -2, 0, 3, -3, 0, 0, M, 0, 1, 6, 0, -1, 0 , -2, 2, 0, -4, 0.}. , / * S * /. { 1, 0, 0, 0, 0, -3, 1, -1, -1, 0, 0, -3, -2, 1, _M¡ 1, -1, 0, 2, 1, 0, -1 , -2, 0, -3, 0.}. ,? * t *? \ l, O, -2, 0.0, -3, 0, -1, 0, 0, 0, -1, -1, 0, M, O, -1, -1, 1, 3, 0, 0, -5, 0, -3, 0.}. ,? *? *? IO, 0, 0, 0, 0, 0. O, O, O, O, O, O, O, O, _M, D, O, O, O, O, O, O, O, O, O, 01,? / ** wv *? . { O, -2, -2, -2, - ?, -1, -1, -2, 4, O, -2, 2, 2, -2, M, -1, -2, -2, -1 O, O, 4, -6, O, -2, -2} ,. { -6, -5, -8, -7, -7, O, -7 -3, -5, O, -3, -2, -4, -4, M, O, O, O, O, O , O, O, O, O, O, O.}. / * * / / * * * * // IO, O, O, O, O, O, O, O, O, O, O, O, O, M, O, O, ü, O, O, O, O, O, O, O, O.}. , Y . { -3, -3, O, -4, -4, 7, -5, O, -1, O, -4, -1, -2, -2, M, -5, -4, -4, - 3, -3, O, -2, O, O, 10, -4} ,? * z *? . { O, 1, -5, 2, 3, -5, 0, 2, -2, O, O, -2, -1, 1, _M, T > , 3, 0, 0, 0, 0, -2, -6, 0, -4, 4.}. }; Page 1 of day.h Table 1 (cont.) / * * / Iinclude < stdio.h >; #include < cty? e.h > #define MAXJMP 16 / * maximum jumps in one diag * / #defitte MAXGAP 24 / * do not continue to penalize gaps larger than this * / #def? ne JMPS 1024 / * maximum jumps in one way * / #def? ne MX 4 / * save if there are at least MX-1 bases since the last jump * / #define DMAT 3 / * value of equalized bases * / #define DMIS 0 / * penalty for unmatched bases * / #define DINSO 8 / * penalty for a gap * / #define DINS1 1 / * penalty per base * / #define PINSO 8 / * penalty for a gap * / #defme P1NS1 4 / * penalty for waste * / struct jmp. { short níMAXJMPl; / * jump size (neg for delay) * / unsigned short xpvIAXJ P.}.; / * Base number of the jump in sec. x * / / * limits of sec. to 2 * 16 -1 * / struct diag. { int value / * value in the last jump * / long detour; / * deviation from the previous block * / short Jump index / * current jump index * / struct jmp jp; / * jump list * /} : struct path. { int / * number of leading spaces * / short nTJMPS]; / * jump size (hollow) * / int xfJMPSJ; / * loe. of the jump (last element before the gap) * /}; char * ofile; / * output log name * / char * namex [2]: / * names of sec: getseqs () * / char * prog. / * nom ibbrree < d "e prog- for messages error * / char * secx [2]; / * secs:: getseqsQ * / int dniax; / * best diag: nw () * / ipt dmaxO; / * final diag * / int dna; / * fix if dna: principal () * / int endgaps; / * fix if holes are penalized at the ends * / int gapx, gapy; / * total gaps in sequences * / int IenO, lenl; / * reduction of sec. * / int ngapx, ngap; / * total size of the gaps * / int smax; / * max value: nw () * / int * xbm; / * bit map for correspondence * / long detour; / * common detour in jump record * / struct diag * d ?; / * retained diagonals * / struct patn / * stopped path for the sequences * / char * ccaallfkocO, * malloc (), * strcpy (=; char * getseqO, * g_calloc0; Page 1 of nw. h Table 1 (Cont) / * Needleman-Wunsch Alignment Program * * use: file 1 file 2 of the programs * where file 1 and file 2 are two protein sequences or two DNA * Sequences can be uppercase or lowercase and may contain ambiguities * Any lines that begin with ';', * > 'or' < 'are ignored * The maximum length of a file is 65535 (limited by a short x with no signal in the jump structure) * A sequence with 1/3 or more of its elements ACGTÚ is supposed to be DNA * The output is in the file "align.out" * Page 2 of nw.c for / * update the penalty for the in x seq;; I * update the penalty for the in and seq;; * favor new ongong envelope * / si (final holes 1 1 ndelx <MAXGAP). { yes (coll [yy-l] - insO> = delx). { delx = coll [yy-l] - (insO + insl); ndelx = 1; } as well . { delx - = ins 1; ndelx + +; } as well . { yes (colllyy-1] - (insO + insl) > = delx). { delx = coll [yy-l] - (insO + insl); ndelx = 1; } also ndelx + +; / * take the maximum value, it is done favoring * my over any dei and delx about dely * / Page 3 of nw.c 65 Page 4 of nw.c Table 1 (cont) I * * * print (0) - only routine visible outside this module * * static: * getmatO - trace the best route again, count the equalities: print () * pr alignO - print alignment of what is described in the pfj array: printO * dumpbiockO - empty the memory of a block of lines with numbers: stars: pr_align () * numsO - remove a line number: dumpblock () * putline0 - delete a line (name, [num], seq, [num]): dumpblock () * starsO - - place a line of stars: dumpblock () * stripnameO - remove any path and prefix from a sequence name #include "nw.h" #def? ne SPC 3 #defu? e P LINE 256 / * maximum output line * / #defineP_SPC 3 / * space between name or num. and sec. * / extern day [26] [26]; int olen; / * set the length of the output line * / FILE / * output file * / printO print. { int lx, ly, first hollow, last hollow; / * overlay * / log); lenl); pr_align0; } Table 1 (Cont) I * * trace the best route again, count the equalities * / _ static get at getmat (lx, ly, first hollow, last hollow) int lx, ly; / * "core" (minus the final gaps) * / int first gap, last gap / * rear rear overlap * / int pm, jO, il, sizO, sizl; char outx [32]; double pct; register nO, ni; register char * p0, * pl; / * provide the total equalities, record * / iO = il = sizO = sizl = 0; pO = seqx 0] + pp [l]. ssppcc;; pl = seqx "1J + p] pj j. spc; nO = ppf 1 .spc: + - f; ni = pp [0 .spc + 1; Page 2 of n print. c Table 1.Cont) .getmat fprint (fx, "<hollows in the first sequence:% d", gapx); yes (gapx). { (void) frintf (outx, "(% d% s% s)", ngapx. (dna)? "baseM:" residueH, (ngapx = = 1)? "H:" s "); rmtfíf ^ s3, outx ); static nm; / * Equalities in the kernel - to verify * / static lmax; / * lengths of file names removed * / static / * jump index for a path * / static / * number at the start of the current line * / static / * number of current elements - for gap formation * / static s? izzfr; static char / * ptr for the current element * / static char / * ptr for the next output character slot * / static char LINE]; / * output line * / static char starp INE]; / * fix by starsO * / / * * print the alignment of what is described in the path of the structure ppfj * / static pr_align pr_align0 int nn; / * character count * / int plus; register i; for (i = 0, lmax = 0; i <2; i + +). { nn = stripname (namex [i]); if (nn> Imax) lmax = nn; Page 3 of nwprint.c Table 1 (cont) for (nn = nm = 0, more = 1; more;). { .pr_align for (i = more = 0; i <2; i ++). { / * * Do you have more than this sequence? * / if (! * ps [i]) continue; more ++; gum / * * empty the memory of a block of lines, including numbers, stars: pr_align () estatic dumpblock dumpblockO register i; for (i = 0; i <2; i ++) * po [i] - = '\ 0 ,; Page 4 of n print.c Table 1 (cont) .dumpblock / * * delete a number line: dumpblock () * / static nums (ix) nu s int rx; / * index in the line of the retention sequence out [] * /. { char? linerP_LINE]; register Us; register char * pn, px, py; / * * delete a line (name, [num], sec, [num]): dumpblock () * / static putline (ix) putline int ix:. { Page 5 of nwprint.c Table 1 (Cont) .putline hit register char * px; for (px = namexfix), i = 0; * px & * px! = ':'; px ++, i ++) (void) putc (* px, faith); for (; i < lmax + P SPC; i ++) (void) putc ('', faith); / * these are counted from 1: * nip is the current element (from 1) * ncf | is the number at the beginning of the current line * / for (px = out [ix]; * px; px ++) (void) putc (* px &0x7F, faith); (void) putc ('\ n', faith); / * * place a line of stars (the sequences always in out [0], out [l]): dumpblock () * / static starsO stars bit i; register char * pO, * pl, ex, * px; if (! * out [0J 1 1 (* out [0] = '' & * (pq [0]) = ") 1 1 outrill | (* outrjl] === • '& & (po [?]) == ") 1 1 returp; px = star; for (i = lmax + P SPC; 1; i-) * px + -f = p; for (pO = = ouuttJrOOJ] ,, ppll = : oouutt [rli]];; ** pp00 & amp; & ** ppll;; pi OH-, pl ++) { i (isalpha (* p0) &? sa-pha (* pl)) { yes (xbm pO-'A'J & xbmPpl-'A ')) {. ex nm also if (! dna & _day [* p0 -?'] [* pl- ' A '] > 0) cx =' also cx = also cx = '* px ++ = ex; * px ++ = V; + px = * \ 0'; Page 6 of nwprint.c Table 1 (cont) / * * prefix or via withdrawal of pn, returns len: pr alignO static stripname (? N) strpname char * pn; / * file name (can be a path) * /. { register char * px, * py; py); return str in (pn); Table 1 (cont) * cleanupO - delete any tmp file * getsegO - read in the sequence, set dna, len, max fill * g_cal? OcO - call with error verification * readjmpsO - give the good jumps, from the file tmp if necessary writejmpsO - write an array full of hops for a tmp file: nw () * / #include "nw.h" #include < sys / ftle.h > char * i "/ tmp? lomgXXXXXX, '; / * tmp file for jmps * / FILE * tj. int cleanupO; / * delete file tmp * / long lseekO; / * * remove any tmp file if it is lost * / cleanurXi) cleanup int i; . { if (fj) (void) unlink (jname); exit (i); } I * * read, return ptr to the sequence, adjust dna, len, maxlen * skip the lines starting with,, '< ', or' > '* sequence in uppercase or lowercase letters * / char * getseqO-Ue, len) get seq char * file; / * file name * / int * len; / * seq len * / char line [1024], * pseq; register char * px, * py; int natgc, tlen; FILE * fp; if ((íp = fopen (file, "r")) === 0). { fpnntf (stderr, "% s: can not read% s \ n", prog, file); left (l); prog. tlen + 6, file); Page 1 of nwsubr.c Table 1 (Contd) .getseq py = pseq + 4; * len = tlen; rewind (fp); although (fgetsflínea, 1024, fp)). { if (* line = ';' | f line == '<' 1 1 * line = '>') continue; for (px = line; * px! = '\ n'; px ++). { yes (isupper (* px)) * px ++ = * px; also if islower (* px)) * py ++ = toupper (* px); if (index (, rATGCU ,,, * O and l ») natgc ++;.}. * py ++ = -.O '; * py = W; (void) fclose (fb); dna = natgc > (tlen / 3); return (pseq + 4);.}. char * g_calloc (msg, nx, sz) g.calloc char * msg: / * program, call routine * / int nx, sz; / * number and size of the elements * / { char * px, * callocO; if ((px = calloc (unsigned) nx, (unsigned) sz)): 0). { prog. msg, nx, sz); return (px); I * * provide the final hops from dxfj or tmp file, adjust pp [], reset dmax: main () * / readjmpsO readjmps Neither fd = -l; int siz, iO, il; register i, j, xx; yes (5). { (void) fclose (fj); yes ((fd = openQname, OR RDONLY, 0)) < 0) í ~? Rintf (stderr, "% s: can not openO% s \ n", prog, jname); eanup (l); } for (i = iO = il = 0, dmaxO = dmax, xx = lenO;; i ++). { although (1). { for (j = dx [dmax] .ijmp; j > = 0 & &dx [dmax] .jp.xfj] > = xx; j-) Page 2 of nwsubr.c break; prOl.nFiO] = i; pf I. ?fi? j = i; my Page 3 of nwsubr.c Table 1 (Contd) I * * Write a jump structure deviation filled with a previous one (if one exists): nwO * / writejmps (ix) writejmps int íx; . { char * mktemp; yam); ívoid) fwrite ((char *) & dx [ix] .jp, sizeof (struct jmp), 1, fj); (void) fwrite ((char *) & dx [ixj. ofíset, sizeof (dxpx] .offset), 1, fj); Page 4 of nwsubr.c Table 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) Protein of XXXXXYYYYYYY (Length = 12 amino acids) Comparison % identity of the amino acid sequence = (the number of amino acid residues that correspond identically between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) = divided by 15 = 33.3% Table 3 PRO XXXXXXXXXX (Length = 10 amino acids) Protein of XXXXXYYYYYYZZYZ (Length = 15 amino acids) Comparison % identity of the amino acid sequence = (the number of amino acid residues that correspond identically between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) = divided by 10 = 50% Table 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) DNA of NNNNNNLLLLLLLLL (Length = 16 nucleotides) Comparison % identity of the nucleic acid sequence = (the number of nucleotides corresponding identically between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the nucleic acid sequence of PRO-DNA) = 6 divided by 14 = 42.9% Table 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides; DNA of NNNNLLLW (Length = 9 nucleotides) Comparison % identity of the nucleic acid sequence = (the number of nucleotides corresponding identically between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the nucleic acid sequence of PRO-DNA) = 4 divided by 12 = 33.3% II. Compositions and Methods of the Invention A. Total length FGF-19 polypeptide The present invention provides isolated and newly identified nucleotide sequences encoding the polypeptides referred to in the present application as FGF-19 (or also UNQ334). In particular, the cDNA encoding a FGF-19 polypeptide has been identified and isolated, as described in further detail in the following Examples. It is noted that the proteins produced in the separate expression rounds can be given different PRO numbers but the UNQ number is unique for any given DNA and encoded protein, and will not be changed. However, for purposes of simplicity, in the present specification the protein encoded by DNA49435-1219 as well as all additional natural homologs and variants, included in the preceding definition of FGF-19 (also sometimes referred to as PR0533), will be referred to as "FGF-19", regardless of its origin or mode of preparation. As described in the following Examples, a cDNA clone designated here as DNA49435-1219 has been deposited with the ATCC. The actual nucleotide sequence of the clone can be easily determined by the skilled artisan by sequencing the deposited clone using routine methods in the art. The predicted amino acid sequence can be determined from the nucleotide sequence using routine experience. For the FGF-19 polypeptide and the nucleic acid coding described herein, the Applicants have identified what is believed to be the best identifiable reading frame with the sequence information available at the present time. Using the computer program for the alignment of the ALIGN-2 sequence referred to above, it has been found that the FGF-19 of the full length natural sequence (shown in Figure 2 and SEQ ID NO: 2) has a certain identity of the amino acid sequence with AF007268_1. Accordingly, it is currently believed that the FGF-19 polypeptide described in the present application is a newly identified element of the fibroblast growth factor protein family and may possess one or more biological and / or immunological activities or properties. typical of this protein family.

B. FGF-19 Variants In addition to the FGF-19 polypeptides of the full length natural sequence described herein, it is contemplated that FGF-19 variants can be prepared. The FGF-19 variants can be prepared by introducing the appropriate nucleotide changes into the FGF-19 DNA, and / or by the synthesis of the desired FGF-19 polypeptide. Those skilled in the art will appreciate that amino acid changes can alter the post-translational processes of FGF-19, such as changing the position number of glycosylation sites or altering the binding characteristics of the membrane. Variations in the FGF-19 of the full-length, natural sequence, or in several of the FGF-19 domains described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations. described, for example, in the US Patent No. 5,364,934. The variations may be a substitution, deletion or insertion of one or more codons encoding FGF-19 that lead to a change in the amino acid sequence of FGF-19 when compared to the natural sequence of FGF-19. Optionally the variation is by the substitution of at least one amino acid with any other amino acids in one or more of the domains of FGF-19. Guidance in determining which amino acid residue can be inserted, substituted or deleted without adversely affecting the desired activity, can be found by comparing the sequence of FGF-19 with that of known, homologous protein molecules, and minimizing the number of changes in the sequence of amino acids made in regions of high homology. The amino acid substitutions can be the result of the replacement of an amino acid with another amino acid having similar chemical and / or structural properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. The insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The allowed variation can be determined by inserting, deleting or substituting amino acids periodically in the sequence and testing the resulting variants to verify the activity exhibited by the mature or full-length natural sequence. Fragments of the FGF-19 polypeptides are provided herein. Such fragments can be truncated at the N-terminus or the C-terminus, or they can lack internal residues, for example, when compared to a full length natural protein. Certain fragments lack the amino acid residues that are not essential for a desired biological activity of the FGF-19 polypeptide. FGF-19 fragments can be prepared by any of a number of conventional techniques. Fragments of the desired peptides can be chemically synthesized. An alternative approach involves the generation of FGF-19 fragments by enzymatic dissolution, for example, by treating the protein with a known enzyme to segment the proteins at the sites defined by the particular amino acid residues, or by dissolving the DNA with the appropriate restriction enzymes and isolating the desired fragment. Yet another suitable technique involves the isolation and amplification of a DNA fragment encoding a desired polypeptide fragment by the polymerase chain reaction (PCR). The oligonucleotides defining the desired terminals of the DNA fragment are used in the 5 'and 3' primers in the PCR. Preferably, fragments of the FGF-19 polypeptide share at least one biological and / or immunological activity with the natural FGF-19 polypeptide shown in Figure 2 (SEQ ID NO: 2). In particular embodiments, conservative substitutions of interest are shown in Table 6 under the heading of preferred substitutions. If such substitutions lead to a change in biological activity, then more substantial changes, termed exemplary substitutions in Table 6, or as further described below with reference to the amino acid classes, are introduced and the products selected.

Table 6 Residual Substitutions Original Substitutions Preferred Exemplary Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) be Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; wing wing His (H) asn; gln; lys; arg arg He (I) leu; val; met; to; phe; norleucine leu Leu (L) ñorleucina; ile; val; met; to; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; to; tyr leu Pro (P) wing wing Ser (S) thr thr Thr (T) be Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; to be phe Val (V) ile; leu; met; phe; to; norleucina leu Substantial modifications in the function or immunological identity of the FGF-19 polypeptide are effected by the selection of substitutions that differ significantly in their effect in maintaining (a) the structure of the polypeptide backbone in the area of substitution, for example, as a helical or sheet conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. The residues that are naturally present are divided into groups based on the properties of the common side chain: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cis, ser, thr; (3) acids: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence the orientation of the chain: gly, pro; and (6) aromatics: trp, tyr, phe.

Non-conservative substitutions will cover the exchange of an element of one of these classes by another class. Such substituted residues may also be introduced at the conservative substitution sites or, more preferably, at the remaining (non-conserved) sites. Variations can be made using methods known in the art such as oligonucleotide-mediated mutagenesis (site-directed), alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Cárter et al., Nucí. Acids Res. , 13 ^: 4331 (1986); Zoller et al., Nucí. Acids Res. , 1_0: 6487 (1987)], cassette mutagenesis [Wells et al., Gene, 3_4: 315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317: 415 (1986)] or other known techniques can be performed on the cloned DNA to produce the DNA of the FGF-19 variant. The analysis of amino acids by scanning can also be used to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are the relatively small neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Alanine is typically a preferred amino acid of exploration among this group because it removes the side chain beyond the beta carbon and is less likely to alter the conformation of the variant's backbone.

[Cunningham and Wells, Science, 244: 1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. In addition, it is often found in both exposed and buried positions [Creighton, The Proteins, (W.H. Freeman &Co., N.Y.); Chotia, J. Mol. Biol., 150: 1 (1976)]. If the alanine substitution does not produce adequate amounts of the variant, an isoteric amino acid may be used.

C. Modifications of FGF-19 Covalent modifications of FGF-19 are included within the scope of this invention. One type of the covalent modification includes reacting the targets of the amino acid residues of an FGF-19 polypeptide with an organic derivatizing agent that is capable of reacting with the selected side chains or the N- or C-terminal residues of the FGF. -19. Derivatization with bifunctional agents is useful, for example, for the cross-linking of FGF-19 to a water-insoluble support matrix or surface for use in the purification method of anti-FGF-19 antibodies., and vice versa. Commonly used crosslinking agents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional idodosters, including disuccinimidyl esters such as 3,3'-dithiobis (succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3- [(p-azidophenyl) dithio ] propioimidate. Other modifications include the deamidation of the glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, the hydroxylation of proline and lysine, the phosphorylation of the hydroxyl groups of the seryl and threonyl residues, the methylation of the a-amino groups of the side chains of lysine, arginine, and histidine [TE Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman &; Co., San Francisco, pp. 79-86 (1983)], the acetylation of the N-terminal amine, and the amidation of any C-terminal carboxyl group. Another type of covalent modification of the FGF-19 polypeptide included within the scope of this invention comprises altering the natural glycosylation configuration of the polypeptide. "Alteration of the natural glycosylation configuration" is proposed for the purposes herein to mean the deletion of one or more portions of carbohydrates found in the FGF-19 of the natural sequence (either by removing the implicit glycosylation site or deleting the glycosylation by chemical and / or enzymatic means), and / or the addition of one or more glycosylation sites that are not present in the FGF-19 of the natural sequence. In addition, the phrase includes qualitative changes in the glycosylation of natural proteins, which involve a change in the nature and proportions of various carbohydrate moieties present. The addition of the glycosylation sites to the FGF-19 polypeptide can be effected by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the FGF-19 of the natural sequence (for the O-linked glycosylation sites). The amino acid sequence of FGF-19 can optionally be altered by means of changes at the DNA level, particularly by mutation of the DNA encoding the FGF-19 polypeptide at the preselected bases such that the codons are generated which are will translate into the desired amino acids. Other means of increasing the number of carbohydrate moieties on the FGF-19 polypeptide are by the chemical or enzymatic binding of the glycosides to the polypeptide. Such methods are described in the art, for example, in WO 87/05330 published on September 11, 1987, and Aplin and Wriston, CRC Crit. Rev. Biochem., Pp. 259-306 (1981). Removal of the carbohydrate moieties present on the FGF-19 polypeptide can be effected chemically or enzymatically or by mutational substitution of codons encoding the amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are already known in the art and are described, for example, by Ha imuddin, et al., Arch. Biochem. Biophys., 259: 52 (1987) and by Edge et al., Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of the carbohydrate moieties on the polypeptides can be achieved by the use of a variety of endo- and exo-glucosidases as described by Thotakura et al., Meth. Enzymol., 138: 350 (1987). Another type of covalent modification of FGF-19 comprises linking the FGF-19 polypeptide to one of a variety of non-proteinaceous polymers, for example, polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner described in U.S. Pat.

Nos. 4,460,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The FGF-19 of the present invention can also be modified in a manner to form a chimeric molecule comprising FGF-19 fused to another, heterologous polypeptide or amino acid sequence. In one embodiment, sa chimeric molecule comprises a fusion of FGF-19 with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed on the amino or carboxyl terminus of FGF-19. The presence of sforms labeled with the epitope of FGF-19 can be detected using an antibody against the tag polypeptide. Also, the provision of the epitope tag makes it possible for FGF-19 to be easily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. The various polypeptides of the tag and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) labels; the FL-tag polypeptide HA and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8: 2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies for the same [Evan et al., Molecular and Cellular Biology, 5: 3610-3616 (1985)]; and the glycoprotein D (gD) label of Herpes simplex virus and its antibody [Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)]. Other tag polypeptides include the Flag peptide [Hopp et al., BioTechnology, 6: 1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255: 192-194 (1992)]; an α-tubilin epitope peptide [Skinner et al., J. Biol. Chem., 266: 15163-15166 (1991)]; and the peptide tag of the T7 gene 10 protein [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87: 6393-6397 (1990)]. In an alternative embodiment, the chimeric molecule may comprise a fusion of FGF-19 with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), sfusion could be to the Fc region of an IgG molecule. Ig fusions preferably include the substitution of a soluble form (deleted or inactivated transmembrane domain) of a FGF-19 polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the joint, CH2 and CH3, or the joint, CH1, CH2 and CH3 regions, of an IgG1 molecule. For the production of immunoglobulin fusions see also US Patent No. 5,428,130 issued June 27, 1995.

D. Preparation of FGF-19 The following description relates mainly to the production of FGF-19 by culturing cells transformed or transfected with a vector containing the FGF-19 nucleic acid. Of course, it is contemplated that alternative methods, which are well known in the art, can be employed to prepare FGF-19. For example, the sequence of FGF-19, or portions thereof, can be produced by the synthesis of direct peptides using solid phase techniques [see, for example, Stewart et al., Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc., J35_: 2149-2154 (1963)]. The synthesis of proteins in vi tro can be carried out using manual techniques or by automation. Automated synthesis can be effected, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using the manufacturer's instructions. Several portions of FGF-19 can be chemically synthesized separately and combined using chemical or enzymatic methods to produce full-length FGF-19. 1. Isolation of the DNA encoding FGF-19 The DNA encoding FGF-19 can be obtained from a cDNA library prepared from the tissue believed to possess the FGF-19 RNA and expressed on a detectable label. Accordingly, human FGF-19 DNA can conveniently be obtained from a cDNA library prepared from human tissue, as described in the Examples. The gene encoding FGF-19 can also be obtained from a genomic library or by known synthetic methods (for example automated nucleic acid synthesis). Libraries can be selected with probes (such as antibodies to FGF-19 or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. The selection of the cDNA or the genomic library with the probe can be carried out using standard procedures, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate FGF-19 encoding the gene is to use the PCR methodology [Sambrook et al., Supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)]. The following examples describe the techniques for selecting a cDNA library. The nucleotide sequences selected as the probes should be of sufficient length and sufficiently unambiguous so that false positives are minimized. The oligonucleotide is preferably labeled such that it can be detected during hybridization to the DNA in the library that is selected. Labeling methods are well known in the art, and include the use of radiolabels similar to ATP labeled with 32P, biotinylation or labeling of enzymes. Hybridization conditions, including moderate severity and high severity, are provided in Sambrook et al., Supra. Sequences identified in such library selection methods can be compared and aligned with other known sequences deposited and available in public databases such as GenBank or other databases of private sequences. The identity of the sequence (either at the level of the amino acids or at the level of the nucleotides) within the defined regions of the molecule or through the full-length sequence can be determined using the methods known in the art and as described here . The nucleic acid having the protein coding sequence can be obtained by choosing the selected genomic or cDNA libraries using the deduced amino acid sequence described here for the first time, and, if necessary, using the conventional primer extension methods as it was described in Sambrook et al., supra, to detect the precursors and process intermediate mRNA compounds that may not have to have been reverse transcribed in the cDNA. 2. Selection and Transformation of the Host Cells The host cells are transformed or transfected with the expression or cloning vectors described herein for the production of FGF-19 and cultured in the modified conventional nutrient medium when appropriate to induce the promoters, select the transformants, or amplify the genes that encode the desired sequences. The culture conditions, such as the medium, temperature, pH and the like, can be selected by those skilled in the art without undue experimentation. In general, the principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., Supra. The methods of transfection of eukaryotic cells and the transformation of prokaryotic cells are known to the artisan with ordinary experience, for example the electroporation mediated by CaCl2, CaP0, and by the liposomes. Depending on the host cell used, the transformation is effected using standard techniques appropriate for such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., Supra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used for the transformation of certain plant cells, as described by Shaw et al., Gener 23: 315 (1983) and WO 89/05859 published on June 29, 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52: 456-457 (1978) can be employed. The general aspects of transfections of the host system of the mammalian cell have been described in U.S. Pat. No. 4,399,216. Transformations in the yeast are typically carried out according to the method of Van Zolingen et al., J. Bact., 130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, fusion of bacterial protoplasts with intact cells, or polycations, for example, polybrene, polyornithine, may also be used. For various techniques for the transformation of mammalian cells, see Keown et al., Methods in Enzymology, 185: 527-537 (1990) and Mansour et al., Nature, 336: 348-352 (1988). Suitable host cells for the cloning or expression of DNA in the vectors herein include prokaryotic, yeast, or higher eukaryotic cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, eg, Enterobacteriaceae such as E. coli. Several strains of E. coli are publicly available, such as strain MM294 from E. coli K12 (ATCC 31,446); X1776 from E. coli (ATCC 31,537); W3110 strain of E. coli (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and b. licheniformis (for example, B. licheniformis 41P described in DD 266,710 published April 12, 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting. Strain W3110 is a preferred host or a preferred original host particularly because it is a common host strain for fermentations of the recombinant DNA product. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, the W3110 strain can be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts that include strain E. coli W3110 1A2, which has the complete tonA genotype; strain W3110 9E4 from E. coli, which has the complete tonA ptr3 genotype; Strain W3110 27C7 from E. coli (ARCC 55,244), which has the genotype tonA ptr 3 phoA E15 (argF-lac) 169 degP opmT kan 'complete; the strain W3110 37D6 of E. coli, which has the full genotype tonA ptr 3 phoA The 5 (argF-lac) 169 degP opmT rbsl ilvG kan 'complete; strain W3110 40B4 from E. coli, which is strain 37D6 with a mutation of the degP deletion not resistant to kanamycin, and an E. coli strain having the mutant periplasmic protease described in U.S. Pat. No. 4,946,783 issued August 7, 1990. Alternatively, in vitro cloning methods, for example, PCR or other reactions of the nucleic acid polymerase, are suitable. In addition to prokaryotes, eukaryotic microbes such as fungi or filamentous yeasts are suitable cloning or expression hosts for the vectors encoding FGF-19. Saccharomyces cerevisiae is a lower eukaryotic host microorganism commonly used. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 published May 2, 1985); Kluyveromyces hosts (US Patent No. 4,943,529; Fleer et al., Bio / Technology, 9: 968-975 (1991)) such as, for example, K. Lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al. , J. Bacteriol., 154 (2): 737-742 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. wal tii (ATCC 56,500 ), K. drosophilarum (ATCC 36.906; Van der Berg et al., Bio / Technology, 8: 135 (1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia Pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28: 265-278 [1998]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76: 5259-5263 [1979]); Schwanniomyces such as Schawanniomyces occidentalis (EP 394,538 published October 31, 1990), and filamentous fungi such as, for example, Neurospora, Penicilli um, Tolypocladium (WO 91/00357 published January 10, 1991), and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys., Res. Commun., 112: 284-289 [1983]; Tilburn et al., Gene, 26: 205-221 [1983]; Yelton et al. al., Proc. Nati, Acad. Sci. USA, 81: 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J.,: 475-479 [1985]). Methylotropic yeasts are suitable here and include, but are not limited to, yeasts capable of growing in methanol selected from the genera consisting of Hansenula, Candida, Kloechera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts can be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982). Suitable host cells for expression of the Glycosylated FGF-19 are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Examples of useful mammalian host cell lines include COS cells and those of Chinese hamster ovaries (CHO). More specific examples include the CV1 line of monkey kidney transformed by SV40 (COS-7, ATCC CRL 1651); the human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); the cells of the ovaries of the Chinese hamster / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.

Reprod. , 23: 243-251 (1980)); the cells of the human lung (W138, ATCC CCL 75); the cells of the human liver (Hep G2, HB 8065); and the mouse mammary tumor (MMT 06052, ATCC CCL51). The selection of the appropriate host cell is considered to be within the skill in the art. 3. Selection and Use of a Replicable Vector Nucleic acid (eg, cDNA or genomic DNA) encoding FGF-19 can be inserted into a replicable vector for cloning (amplification of DNA) or for expression. Several vectors are publicly available. The vector, for example, may be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence can be inserted into the vector by a variety of methods. In general, the DNA is inserted into an appropriate restriction endonuclease site (s) using techniques known in the art. The components of the vectors generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a terminator sequence. the transcript The construction of suitable vectors containing one or more of these components employ the standard bonding techniques which are known to the skilled artisan. FGF-19 can be produced recombinant not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which can be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the DNA encoding FGF-19 that is inserted into the vector. The sequence of the signal may be a sequence of the prokaryotic signal selected, for example, from the group of alkaline phosphatase, penicillinase, Ipp, or the stable exterotoxin forward elements with heating. For the secretion of the yeast the sequence of the signal can be, for example, the forward element of the invertase of the yeast, the front element of the alpha factor (including the front elements of the factor a of Saccharomyces and Kluyveromyces, the latter described in US Patent No. 5,010,182, or the front element of the acid phosphatase, the front element of the glucoamylase of C. albicans (EP 362,179 published April 4, 1990), or the signal described in WO 90/13646 published on 15 November 1990. In the expression of mammalian cells, the sequences of the mammalian signal can be used to direct the secretion of the protein, such as the signal sequences from the secreted polypeptides of the same species or from the same species. related species, as well as the viral secretory front elements, both the expression and cloning vectors contain the nucleic acid sequence that makes it possible for the vector is replicated in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeasts, and viruses. The origin of replication of plasmid pBR322 is suitable for Gram-negative bacteria, the origin of plasmid 2μ is suitable for yeast, and several viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in the cells of mammals.

The expression and cloning vectors will typically contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, eg, ampicillin, neomycin, methotrexate, or tetracycline, (b) auxotrophic complement deficiencies, or (c) the supply of critical nutrients not available from the complex medium, for example, the gene encoding the Bacilli D-alanine racemase. An example of suitable selectable markers for mammalian cells are those that are capable of identifying the cells competent to receive the nucleic acid encoding FGF-19, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77: 4216 (1980). A suitable selection gene for use in yeast is the trpl gene present in plasmid YRp7 of the yeast [Stinchcomb et al., Nature, 282: 39 (1979); Kingsman et al., Gene, 7: 141 (1979); Tschemper et al., Gene, 10: 157 (1980)]. The trpl gene provides a selection marker for a mutant strain of the yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)]. Expression and cloning vectors usually contain a promoter operably linked to the nucleic acid sequence encoding FGF-19 to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are also already known. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al., Nature, 275: 615 (1978); Goeddel et al., Nature, 281: 544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res. , 8: 4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983)]. Promoters for use in bacterial systems will also contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding FGF-19. Examples of promoter sequences suitable for use with yeast hosts include promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255: 2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg., 7: 149 (1968); Holland, Biochemistry, 17: 4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate utase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other yeast promoters, which are inducible promoters that have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocitochrome C, acid phosphatase, the degrading enzymes associated with metabolism of nitrogen, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and the enzymes responsible for the use of maltose and galactose. Vectors and promoters suitable for use in the expression of the yeast are further described in EP 73,657. The transcription of FGF-19 from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, bird poxvirus (UK 2,211,504 published on 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, bird sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and Simian Virus 40 ( SV40), of heterologous mammalian promoters, for example, the actin promoter or an immunoglobulin promoter, and thermal shock promoters, provided that such promoters are compatible with the host cell systems. The transcription of a DNA encoding FGF-19 by the higher eukaryotes can be increased by the insertion of an enhancer sequence into the vector. The enhancers are cis-acting elements of the DNA, usually approximately 10 to 300 bp, which act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, an enhancer will be used from a virus of eukaryotic cells. Examples include the SV40 enhancer on the last side of the replication origin (100-270 bp), the cytomegalovirus initial promoter enhancer, the polyoma enhancer on the last side of the origin of replication, and the adenovirus enhancers. The enhancer can be sliced in the vector at a position 5 'or 3' with respect to the coding sequence of FGF-19, but is preferably located at a 5 'site of the promoter.

Expression vectors used in eukaryotic host cells (yeast cells, fungi, insects, plants, animals, humans, or nucleated cells of other multicellular organisms) will also contain sequences necessary for the termination of transcription and to stabilize the mRNA. Such sequences are commonly available from the 5 'and, occasionally 3' end regions of the viral or eukaryotic DNAs or cDNAs. These regions contain the nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the FGF-19 encoding the mRNA. Still other methods, vectors, and host cells suitable for adaptation to the synthesis of FGF-19 in the culture of recombinant vertebrate cells are described in Gething et al., Nature, 293: 620-625 (1981); Mantei et al., Nature, 281: 40-46 (1979); EP 117,060; and EP 117,058. 4. Detection of Gene Expression / Amplification Expression and / or amplification of the gene can be measured in a sample directly, for example, by the conventional Southern blotting procedure to quantitate mRNA transcription [Thomas, Proc. Natl. Acad. Sci, 77: 5201-5205 (1980)], the dot blotting procedure (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or protein-DNA duplexes, may be employed. The antibodies in turn can be labeled and the assay can be carried out where the duplex is bound to a surface, so that during the duplex formation on the surface, the presence of the antibody bound to the duplex can be detected. The expression of the gene, alternatively, can be measured by immunological methods, such as the immunohistochemical staining of the sections of the cells or tissue and the culture assay of the cells or body fluids, to directly quantify the expression of the gene product. . Antibodies useful for immunohistochemical staining and / or assay of sampling fluids can be either monoclonal or polyclonal, and can be prepared in any mammal. Conveniently, the antibodies can be prepared against a FGF-19 polypeptide of the native sequence or against a synthetic peptide based on the DNA sequences provided herein or against the exogenous sequence fused to the FGF-19 DNA and encoding an antibody epitope. specific.

. Purification of the Polypeptide The forms of FGF-19 can be recovered from the culture medium or lysates of the host cell. If they are attached to the membrane, they can be released from the membrane using a suitable detergent solution (for example Triton-X 100) or by enzymatic cleavage. Cells used in the expression of FGF-19 can be altered or broken by various physical or chemical means, such as the liquefied-thaw cycle, the sound application, the mechanical breakdown, or agents for cell lysate. It may be desirable to purify FGF-19 from the polypeptides or proteins of the recombinant cells. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion exchange column; the precipitation with ethanol; the reverse phase HPLC; chromatography on silica or a cation exchange resin such as DEAE; chromatofocusing; SDS-PAGE; precipitation with ammonium sulfate; the use of filtration in a gel, for example, Sephadex G-75; Protein A Sepharose columns to remove contaminants such as IgG; and the chelation columns of the metals to join the forms labeled with the epitope of the FGF-19. Various methods of purifying the protein can be employed and such methods are already known in the art and are described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The selected purification step (s) will depend, for example, on the nature of the production process used and the particular FGF-19 produced.

E. Uses for FGF-19 The nucleotide sequences (or their complement) that encode FGF-19 have several applications in the art of molecular biology, including uses as hybridization probes, in the conformation of gene maps and chromosomes and in the generation of RNA and antisense DNA. The nucleic acids of FGF-19 will also be useful for the preparation of the FGF-19 polypeptides by the recombinant techniques described herein. The FGF-19 gene of the full length natural sequence (SEQ ID NO: 1), or portions thereof, can be used as hybridization probes for a cDNA library to isolate the FGF-19 cDNA in length total or to still isolate other cDNAs (for example, those encoding the naturally occurring variants of FGF-19 or FGF-19 of other species) which have a desired sequence identity with respect to the FGF-sequence 19 described in Figure 1 (SEQ ID NO: 1). Optionally, the length of the probes will be from about 20 to about 50 bases. Hybridization probes can be derived from the at least partially novel regions of the nucleotide sequence of SEQ ID NO: 1 wherein these regions can be determined without undue experimentation or from genomic sequences including promoters, enhancer elements and the introns of FGF-19 of the natural sequence. By way of example, a screening method will comprise isolating the coding region of the FGF-19 gene using the known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes can be labeled by a variety of labels, including radionucleotides such as 32P or 35S, or enzymatic labels such as alkaline phosphatase linked to the probe by means of the avidin / biotin binding systems. Labeled probes having a sequence complementary to that of the FGF-19 gene of the present invention can be used to select libraries of human cDNA, genomic DNA or mRNA to determine which elements of such libraries the probe hybridizes. Hybridization techniques are described in further detail in the following Examples. Any EST sequences described in the present application can be used in a similar manner as probes, using the methods described herein. Other useful fragments of the FGF-19 nucleic acid include sense or antisense oligonucleotides comprising a single-stranded nucleic acid sequence (either ARJST or DNA) capable of binding to the target sequences of the FGF mRNA. -19 (sense) or DNA of FGF-19 (antisense). Sense or antisense oligonucleotides, according to the present invention, comprise a fragment of the DNA coding region of FGF-19. Such a fragment comprises at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive a sense or antisense oligonucleotide, based on a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen (Cancer Res. 48: 2659, 1988) and van der Krol et al. (BioTechniques 6: 958, 1988).

The binding of the sense or antisense oligonucleotides to the target sequences of the nucleic acid leads to the formation of duplexes that block the transcription or translation of the target of the sequence by one of several means, including the improved degradation of the duplexes, the premature termination of transcription or translation, or by other means. Antisense oligonucleotides can thus be used to block the expression of FGF-19 proteins. Sense or antisense oligonucleotides comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar bonds), such as those described in WO 91/06692) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar bonds are stable in vivo (ie, capable of resisting enzymatic degradation) but retain the specificity of the sequence to be capable of binding to the targets of the nucleotide sequences. Other examples of the sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to the organic moieties, such as those described in WO 90/10048, and other portions that increase the affinity of the oligonucleotide for a target sequence of the nucleic acid, such as poly- (L-lysine). Still further, intercalating agents, such as ellipticine, and alkylating agents or metal complexes can be attached to the sense or antisense oligonucleotides to modify the binding or agglutination specificities of the sense or antisense oligonucleotides for the targets. of the nucleotide sequences. Sense or antisense oligonucleotides can be introduced into a cell containing the target sequence of the nucleic acid by any method of genetic transfer, including, for example, transfection of DNA mediated by CaP04-, electroporation, or by the use of the transfer vectors of genes such as the Epstein-Barr virus. In a preferred method, a sense or antisense oligonucleotide is inserted into a suitable retroviral vector. A cell containing the target sequence of the nucleic acid is contacted with the recombinant retroviral vector, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine M-MuLV retrovirus, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated as DCT5A, DCT5B and DCT5C (see WO 90 / 13641).

Sense or antisense oligonucleotides can be introduced into a cell containing the target sequence of the nucleotides by the formation of a conjugate with a binding or agglutination molecule of the ligand, as described in WO 91/04753. Suitable binding or agglutination molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the binding or agglutination molecule of the ligands does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or to block the entry of the sense or antisense oligonucleotide or its version. conjugated in the cell. Alternatively, a sense or antisense oligonucleotide can be introduced into a cell containing the nucleic acid target sequence by the formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase. The probes can also be employed in PCR techniques to generate a group of sequences for the identification of closely related FGF-19 coding sequences. The nucleotide sequences encoding an FGF-19 can also be used to construct hybridization probes by mapping the genes encoding FGF-19 and for the genetic analysis of individuals with genetic disorders. The nucleotide sequences provided herein can be mapped with respect to a chromosome and the specific regions of a chromosome using known techniques, such as in situ hybridization, the analysis of the links against the known chromosomal markers, and the selection of Hybridization with libraries. When the coding sequences for FGF-19 encode a protein which binds to another protein (eg, where FGF-19 is a receptor), FGF-19 can be used in assays to identify other proteins or molecules involved in the interaction of the union or agglutination. By such methods, inhibitors of ligand / receptor binding interaction can be identified. The proteins involved in such binding interactions can also be used to select the peptide or inhibitors of small molecules or agonists of the binding or agglutination interaction. Also, the FGF-19 receptor can be used to isolate the correlating ligand (s). Selection tests can be designed to find the forward compounds that mimic the biological activity of a natural FGF-19 or a receptor for FGF-19. Such screening assays will include assays that are capable of high throughput screening of the chemical libraries, making them particularly suitable for the identification of candidates for small molecule drugs. The contemplated small molecules include synthetic organic or inorganic compounds. Assays can be carried out in a variety of formats, including protein-protein binding assays, biochemical selection assays, immunoassays and cell-based assays, which are well characterized in the art. The nucleic acid encoding FGF-19 or its modified forms can also be used to generate either transgenic animals or "knock out" animals which, in turn, are useful in the development and selection of therapeutically useful reagents. A transgenic animal (e.g., a mouse or rat) is an animal that has cells that contain a transgene, such a transgene was introduced into the animal or an ancestor of the animal in a prenatal stage, for example, an embryonic stage. A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, the cDNA encoding FGF-19 can be used to clone the genomic DNA encoding FGF-19 according to the established techniques and the genomic sequences used to generate the transgenic animals that contain the cells which express the DNA that encodes FGF-19. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically, particular cells could be targeted for the incorporation of the FGF-19 transgene with tissue-specific enhancers. Transgenic animals that include a copy of a transgene encoding FGF-19 introduced into the germline of the animal at an embryonic stage can be used to examine the effect of increased expression of the DNA encoding FGF-19. Such animals can be used as test animals for the reagents that are thought to confer protection against, for example, the pathological conditions associated with their overexpression. According to this facet of the invention, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals carrying the transgene, could indicate a potential therapeutic intervention for the pathological condition. Alternatively, non-human homologs of FGF-19 can be used to construct the "knock out" animal of FGF-19 which has a defective or altered gene encoding FGF-19 as a result of homologous recombination between the gene endogenous coding for FGF-19 and altered genomic DNA encoding FGF-19 introduced into an embryonic propelling cell of the animal. For example, the cDNA encoding FGF-19 can be used to clone the genomic DNA encoding FGF-19 according to established techniques. A portion of the genomic DNA encoding FGF-19 can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to verify integration. Typically, several kilobases of the unaltered flanking DNA (both 5 'and 3' ends) are included in the vector [see for example, Thomas and Capecchi, Cell, 51: 503 (1987) for a description of recombination vectors homologs]. The vector is introduced into a line of the embryonic driving cell (for example, by electroporation) and the cells in which the introduced DNA has been recombined homologously with the endogenous DNA are selected [see for example, Li et al, Cell, 69 : 915 (1992)]. The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see for example, Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implanted in a suitable pseudopregnant female adoptive animal and the embryo brought to term to create a "knock out" animal. Progeny that assemble the homologously recombined DNA in their propellant cells can be identified by standard techniques and used to reproduce animals in which all the cells of the animal contain the homologously recombined DNA. The "knock out" animals can be characterized, for example, by their ability to defend against certain pathological conditions and by their own development of the pathological conditions due to the absence of the FGF-19 polypeptide. The nucleic acid encoding the FGF-19 polypeptides can also be used in gene therapy. In the applications of gene therapy, the genes are introduced into the cells to achieve an in vivo synthesis of a therapeutically effective gene product, for example for the replacement of a defective gene. "Genetic therapy" includes both conventional gene therapy where a final effect is achieved by a single treatment, such as the administration of genetic therapeutic agents, which involves the one-time administration or the repeated administration of a therapeutically effective DNA or mRNA. The antisense RNAs and DNAs can be used as therapeutic agents to block the expression of certain genes in vivo. It has also been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted absorption by the cell membrane. (Za ecnik et al., Proc. Natl. Acad. Sci. USA 83: 4143-4146 [1986]). The oligonucleotides can be modified to improve their absorption, for example by replacing their negatively charged phosphodiester groups with non-charged groups. There are a variety of techniques available to introduce nucleic acid into viable cells. The techniques may vary depending on whether the nucleic acid is transferred to the cells cultured in vitro, or in vivo in the cells of the proposed host. Suitable techniques for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the method of calcium precipitation, etc. Currently preferred in vivo gene transfer techniques include transfection with viral vectors (typically retroviral) and protein-liposome-mediated transfection of the viral coat (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]). ). In some situations it is desirable to provide the source of the nucleic acid with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the cell target, a ligand for a receptor on the cell target, etc. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis can be used for targeting and / or facilitating absorption, for example capsid proteins. or fragments thereof, typical for a particular cell type, antibodies for proteins which undergo internalization in cyclization, proteins that target intracellular localization and improve intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem., 262, 4429-4432 (1987).; and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). For a review of the protocols for gene labeling and gene therapy see Anderson et al., Science 256, 808-813 (1992). The FGF-19 polypeptides described herein may also be employed as molecular weight markers for purposes of protein electrophoresis. The nucleic acid molecules encoding the FGF-19 polypeptides or fragments thereof described herein are useful for the identification of the chromosome. In this regard, there is an increasing need to identify new chromosome markers, since relatively few chromosome marker reagents are currently available, based on the actual sequence data. Each nucleic acid molecule of FGF-19 of the present invention can be used as a chromosome marker. The FGF-19 polypeptides and the nucleic acid molecules of the present invention can also be used for tissue typing, wherein the FGF-19 polypeptides of the present invention can be expressed in one tissue when compared to another. The nucleic acid molecules of FGF-19 will find use to generate probes for PCR, Northern analysis, Southern analysis and Western analysis. The FGF-19 polypeptides and the modulators thereof described can also be used as therapeutic agents. The FGF-19 polypeptides and modulators thereof of the present invention can be formulated according to known methods to prepare compositions that are pharmaceutically useful, whereby the FGF-19 product thereof is combined in a mixed with a pharmaceutically acceptable carrier vehicle. Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16 / ed., Osol, A. Ed. (1980). ), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to patients or recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate or other organic acids; antioxidants include ascorbic acid, low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN ™, PLURONICS ™ or PEG. The formulations that are to be used for in vivo administration must be sterile. This is easily effected by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The therapeutic compositions herein are generally placed in a container having a sterile access opening, for example, an intravenous solution bag or small vial having a stopper pierceable by a hypodermic injection needle. The route of administration is according to known methods, for example injection or infusion by the intravenous, intraperitoneal, intracerebral, intramuscular, infraocular, intraarterial or intralesional routes, topical administration, or by sustained release systems. The dosages and desired drug concentrations of the pharmaceutical compositions of the present invention can be varied depending on the particular use contemplated. The determination of the appropriate dosage or route of administration is within the ordinary experience of a physician. Animal experiments provide a reliable guide for the determination of effective doses for human therapy. The escalation of the interspecies of the effective doses can be carried out following the principles established by Mordenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York 1989, p. 42-96. When the in vivo administration of a FGF-19 polypeptide is employed or an agonist or antagonist thereof is employed, the amounts of the normal dosage may vary from about 10 ng / kg to 100 mg / kg of mammalian body weight or more per day, preferably approximately 1 μg / kg / day to 10 mg / kg / day, depending on the route of administration. The guide for the dosages and particular methods of supply is provided in the literature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. It is anticipated that different formulations will be effective for different treatment compounds and different disorders, that administration to target an organ or tissue, for example, may require delivery in a manner different from that for another organ or tissue. Where the sustained release administration of a FGF-19 polypeptide or modulator is desired in a formulation with release characteristics suitable for the treatment of any disease or disorder that requires administration of the FGF-19 polypeptide or modulator, microencapsulation is contemplated. The microencapsulation of recombinant proteins for sustained release has been successfully effected with human growth hormone (rhGH), interferon- (rhIFN-), interleukin-2, and MN rgp 120. Johnshon et al., Nat.Med. , 2: 795-799 (1996); Yasuda, Biomed. Ther., 27: 1221-1223; Hora et al., Bio / Technology, 8: 755-758 (1990); Cleland, "Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems", in Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman, eds, (Plenum Press: New York, 1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat. No. 5,654,010. The sustained release formulations of these proteins were developed using the polylactic-coglycolic acid polymer due to its biocampatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic and glycolic acids can be quickly discarded within the human body. In addition, the degradability of this polymer can be adjusted from months to years depending on its molecular weight and composition. Lewis, "Controlled release of biactive agents from lactide / glycolide polymer", in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drugs Delivery Systems (Marcel Dekker: New York, 1990), p. 1-41 The therapeutic agents and compositions comprising the FGF-19 provided herein can be used in various applications. Applications include the treatment of an individual with obesity or a condition associated with obesity. In one aspect, FGF-19 is administered to an individual in need of an effective amount to treat the condition. Preferably, the condition is one which requires that at least one of the following be treated: an increase in metabolism, a reduction in body weight, a reduction in body fat, a reduction in triglyceride levels, a reduction in free fatty acids, an increase in the release of glucose from the adipocytes and / or an increase in the release of leptin from the adipocytes. Each of these parameters can be measured by standard methods, for example, by measuring oxygen consumption to determine metabolic rate, using scales to determine weight, and measuring size to determine fat. In addition, the presence and amount of triglycerides, free fatty acids, glucose and leptin can be determined by standard methods. Each of these parameters is later exemplified in the specific examples. FGF-19 and compositions comprising FGF-19 are preferably used in vivo. However, as described below, the administration may be in vivo such as in the methods described below for the selection of modulators of FGF-19. Although, it is understood that modulators of FGF-19 can also be identified by the use of animal models and patient samples. This invention encompasses the methods of selecting the compounds to identify those that equal or enhance the FGF-19 polypeptide (agonists) or prevent or inhibit the effect of the FGF-19 polypeptide (antagonists). The agonists and antagonists are referred to as modulators here. Selection tests for candidates for the antagonist drug are designed to identify compounds that bind or complex with the FGF-19 polypeptides encoded by the genes identified herein., or that otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins. Such screening assays will include assays that are capable of high-throughput screening of chemical libraries, making them particularly suitable for the identification of small molecule drug amounts. Assays can be performed in a variety of formats, including protein-protein binding assays, biochemical selection assays, immunoassays, and cell-based assays, which are well characterized in the art. All assays for antagonists are common because they may require contacting the candidate drug with an FGF-19 polypeptide encoded by a nucleic acid identified herein under the conditions and for a sufficient time to allow these two components to interact.

In binding or agglutination assays, the interaction is binding or agglutination and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, the FGF-19 polypeptide encoded by the gene identified herein or the candidate drug is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments or attachments. The non-covalent attachment or binding is generally carried out by coating the solid surface with a solution of the FGF-19 polypeptide and drying it. Alternatively, an immobilized antibody, for example, a monoclonal antibody, specific for the FGF-19 polypeptide to be immobilized, can be used to bind it to a solid surface. This test is carried out by adding the non-immobilized component, which can be labeled by a detectable label, to the immobilized component, for example, the coated surface containing the fixed component. When the reaction is supplemented, the components that did not react are removed, for example, by washing, and the complexes fixed on the solid surface are detected. When the non-immobilized component originally carries a detectable label, detection of the immobilized label on the surface indicates that complex formation has occurred. Where the non-immobilized component does not originally carry a label, complex formation can be detected, for example, by using a labeled antibody that binds specifically to the immobilized complex. If the candidate compound interacts with, but does not bind to, a particular FGF-19 polypeptide encoded by a gene identified herein, its interaction with these polypeptides can be assessed by well-known methods for detecting protein-protein interactions. Such assays include traditional approaches, such as, for example, cross-linking, co-immunoprecipitation, and co-purification by means of gradient or chromatographic columns. In addition, protein-protein interactions can be verified using a yeast-based genetic system described by Fields et al. (Fields and Song, Nature (London), 340: 245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA, 88: 9578-9582 (1991)) as described by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one that acts as the DNA binding domain, the other that functions as the transcription-activation domain. The yeast expression system described in the preceding publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two-hybrid proteins, one in which the target protein is fused to the domain of GAL4 DNA binding, and another, in which the candidate activation proteins are fused to the activation domain. The expression of a GALl-laci reporter gene. under the control of a promoter activated with GAL4 depends on the reconstitution of GAL4 activity by means of the protein-protein interaction. Colonies containing the interaction polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete set (MATCHMAKER ™) for the identification of protein-protein interactions between two specific proteins using the two-hybrid techniques is commercially available from Clontech. This system can also be extended to map the protein domains involved in the specific protein interactions as well as the precisely identified amino acid residues that are crucial for these interactions. Compounds that interfere with the interaction of a gene encoding a FGF-19 polypeptide identified herein and other intra- or extra-cellular components, can be tested as follows: usually a reaction mixture is prepared containing the product of the gene and the intra- or extra-cellular component under the conditions and for a time that allows the interaction and the union of the two products. To test the ability of a candidate compound to inhibit binding or agglutination, the reaction is performed in the absence and presence of the test compound. In addition, a placebo can be added to a third reaction mixture, to serve as a positive control. The binding or agglutination (complex formation) between the test compound and the intra- or extra-cellular component present in the mixture is verified as described here above. The formation of a complex in the control reaction (s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner. . For the evaluation of the antagonists, the FGF-19 polypeptide can be added to a cell in the company of the compound to be selected for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the FGF polypeptide. -19 indicates that the compound is an antagonist for the FGF-19 polypeptide. Alternatively, antagonists can be detected by combining the FGF-19 polypeptide and a potential antagonist with the FGF-19 polypeptide receptors attached to the membrane or recombinant receptors under the conditions appropriate for a competitive inhibition assay. The FGF-19 polypeptide can be labeled, such as by radioactivity, such that the number of FGF-19 polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist. The gene encoding the receptor can be identified by the numerous methods known to those skilled in the art, for example, the extension of the ligand and the FACS classification. Coligan et al., Current Protocols in Immun. , 1 (2): Chapter 5 (1991). Preferably, expression cloning is employed wherein the polyadenylated RNA is prepared from a cell responsive to the FGF-19 polypeptide and a cDNA library created from this RNA is divided into groups and used to transfect the cells of COS or other cells that are not responsive to the FGF-19 polypeptide. Transfected cells that are grown on glass slides are exposed to the labeled FGF-19 polypeptide. The FGF-19 polypeptide can be labeled by a variety of means including iodination or the inclusion of a recognition site for a site-specific protein kinase. Following the fixation and incubation, the slides are subjected to autoradiographic analysis. Positive groups are identified and subgroups are prepared and retransfected using a reselection process and interactive subgroup, eventually producing a single clone that encodes the presumed receptor. As an alternative approach for the identification of the receptor, the tagged FGF-19 polypeptide can be linked by photoaffinity with the preparations of the extract or cell membrane that express the receptor molecule. The crosslinked material is resolved by PAGE and exposed to an X-ray film. The tagged complex containing the receptor can be cleaved, resolved into fragments of the peptide, and subjected to microsequencing of the protein. The amino acid sequence obtained from microsequencing could be used to design a set of degenerate oligonucleotide probes to select a cDNA library to identify the gene encoding the putative receptor. In another assay for the antagonists, the mammalian cells or a membrane preparation expressing the receptor could be incubated with the FGF-19 polypeptide in the presence of a candidate compound. The ability of the compound to improve or block this interaction could then be measured. More specific examples of potential antagonists include an oligonucleotide that binds to immunoglobulin fusions with the FGF-19 polypeptide, and, in particular, the antibodies including, without limitation, the poly- and monoclonal antibodies and the antibody fragments, the single-chain antibodies, the anti-idiotypic antibodies, and the chimeric or humanized versions of such antibodies or fragments , as well as human antibodies and antibody fragments. Alternatively, a potential antagonist may be a closely related protein, for example, a mutated form of the FGF-19 polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the FGF-19 polypeptide. In one embodiment here, where competitive binding assays are performed, the FGF receptor 4 or an antibody to FGF-19 is used as a competitor. Another potential FGF-19 polypeptide antagonist is an antisense RNA or DNA construct prepared using the antisense technology, wherein, for example, an antisense RNA or DNA molecule acts to directly block translation of the mRNA by hybridization to the target of mRNA and prevent translation of the protein. The antisense technology can be used to control the expression of the gene through triple helix formation or antisense DNA or RNA, both of which are based on the binding or agglutination of a polynucleotide to DNA or RNA. For example, the coding portion 51 of the polynucleotide sequence, which encodes the mature FGF-19 polypeptides, is used to design an antisense RNA oligonucleotide from about 10 to 40 base pairs in length. An oligonucleotide in DNA is designed to be complementary to a region of the gene involved in transcription (triple helix - see Lee et al., Nucí Acids Res., 6: 3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan et al., Science, 251: 1360 (1991)), whereby transcription and production of the FGF-19 polypeptide is prevented. The antisense RNA oligonucleotide is hybridized to the mRNA in vivo and the translation of the blocks of the mRNA molecule into the FGF-19 polypeptide (antisense-Okano, Neurochem., 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expressión (CRC Press: Boca Raton, FL, 1988) The oligonucleotides described above can also be delivered to the cells in such a way that the antisense RNA or DNA can be expressed in vivo to inhibit the production of the FGF-19 polypeptide. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation start site, for example, between the positions of about -10 and +10 of the nucleotide sequence of the target gene, are preferred.The potential antagonists include the molecules small sites that bind to the active site, the receptor binding site, or the growth factor or other relevant binding site of the FGF-19 polypeptide, by which blocks the normal biological activity of the FGF-19 polypeptide. Examples of the small molecules include, but are not limited to, peptides or peptide-like molecules, small, preferably soluble peptides, and organic or inorganic compounds other than peptidyl, synthetic. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific segmentation of RNA. The ribozymes act by the specific hybridization of the target sequence of the complementary RNA, followed by the endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For additional details see, for example, Rossi, Current Biology, 4: 469-471 (1994), and PCT publication No. WO 97/33551 (published September 18, 1997). The nucleic acid molecules in the triple helix formation used to inhibit transcription must be single stranded and composed of deoxynucleotides. The base composition of these oligonucleotides is designed in such a way that it promotes triple helix formation by means of the Hoogsteen base pair formation rules, which generally require the dimensionable stretches of the purines or pyrimidines on a strand of a duplex For additional details see, for example, PCT publication No. WO 97/33551, supra. These small molecules can be identified by any of one or more of the selection assays described hereinbefore and / or by any other selection techniques well known to those skilled in the art. It will be appreciated that all of the assays provided herein can be used to select a wide variety of candidate bioactive agents. The term "candidate bioactive agent", "candidate agent" or "candidate drug" or grammatical equivalents as used herein, describe any molecule, eg, protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, purine analog, etc. , which will be tested for bioactive agents that are capable of directly or indirectly altering either the phenotype of cellular activity or the expression of a sequence of FGF-19, including both the nucleic acid sequences and the protein sequences . The candidate agents can encompass numerous chemical classes, although they are typically organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons (d). The small molecules are further defined here having a molecular weight between 50 d and 2000 d. In another embodiment, small molecules have a molecular weight of less than 1500, or less than 1200, or less than 1000, or less than 750, or less than 500 d. In one embodiment, a small molecule as used herein has a molecular weight of about 100 to 200 d. The candidate agents comprise the functional groups necessary for the structural interaction with the proteins, particularly the hydrogen bonding, and typically include at least one group of amine, carbonyl, hydroxyl or carboxyl, preferably at least two of the functional chemical groups. Candidate agents often comprise heterocyclic or cyclic carbon structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules which include peptides, saccharides, fatty acids, steroids, pyrimidines, derivatives, structural analogs or combinations thereof. Peptides are particularly preferred. Candidate agents are obtained from a wide variety of sources including libraries of natural or synthetic compounds. For example, numerous media are available for the random or targeted synthesis of a wide variety of organic compounds and biomolecules, including the expression of the oligonucleotides at random. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or are easily produced. Additionally, libraries and compounds produced naturally or synthetically are easily modified through conventional chemical, physical and biochemical means. Known pharmacological agents can be subjected to random or directed chemical modifications, such as acylation, alkylation, esterification, to id, to produce structural analogues. In a preferred embodiment, the candidate bioactive agents are proteins. By "protein" is meant here at least two covalently linked amino acids, which include the proteins, polypeptides, oligopeptides and peptides. The protein can be composed of the amino acids that are naturally present and the peptide bonds, or the synthetic peptidomimetic structures. Therefore "amino acids", or "amino acid residue", as used herein, means both synthetic and naturally occurring amino acids. For example, homo-phenylalanine, citrulline and norleucine are considered as amino acids for the purposes of the invention. The "amino acids" also include the imino acid residues such as proline and hydroxyproline. The side chains can be of either (R) or (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L configuration. If the side chains that are present in a non-naturally occurring manner are used, substituents other than amino acids can be used, for example to prevent or retard degradations in vivo. .

In a preferred embodiment, the candidate bioactive agents are the proteins that are naturally present or the fragments of the proteins that are naturally present. Thus, for example, cell extracts containing the proteins, or the random or targeted solutions of proteinaceous cell extracts, can be used. In this way libraries of prokaryotic or eukaryotic proteins can be made by selection in the methods of the invention. Particularly preferred in this invention are the libraries of the proteins of bacteria, fungi, viruses, and mammals, with the latter being preferred, and the human proteins which are especially preferred. In a preferred embodiment, the candidate bioactive agents are peptides from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids that are preferred, and from about 7 to about 15 which are particularly preferred. The peptides may be dilutions of the proteins that are naturally present as described above, random peptides, or "changed" random peptides. By the "random" or grammatical equivalents herein it is meant that each nucleic acid and peptide consists essentially of nucleotides and random amino acids, respectively. Since generally these random peptides (or nucleic acid, described below) are chemically synthesized, they can incorporate any nucleotides or amino acids at any position. The synthetic process can be designed to generate the proteins or nucleic acid at random, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomly acting bioactive proteinaceous agents. In one modality, the library is totally random, without preferences of the sequence or constants in any position. In a preferred embodiment, the library is changed. That is, some positions within the sequence are either kept constant, or are selected from a limited number of possibilities. For example, in a preferred embodiment, the nucleotides or amino acid residues are randomly distributed within a defined class, for example, of hydrophobic amino acids, hydrophobic residues, sterically changed residues (either small or large), towards the creation of domains of nucleic acid binding, the creation of cysteines, for crosslinking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or for purines, etc. In a preferred embodiment, the candidate bioactive agents are the nucleic acids. By "nucleic acid" or "oligonucleotide" or grammatical equivalents, we mean at least two nucleotides covalently linked together. A nucleic acid for the present invention will generally contain the phosphodiester linkages, although in some cases, as described below, nucleic acid analogs are included, which may have alternative backbones, comprising, for example, phosphoramide (Beaucage et al. al., Tetrahedron 49 (10): 1925 (1993) and the references therein, Letsinger, J. Org. Chem. 35: 3800 (1970), Sprinzl et al., Eur. J. Biochem. 81: 579 (1977). ), Letsinger et al., Nucí Acids Res. 14: 3487 (1986), Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110: 4470 (1988). ), and Pauwels et al., Chemica Scripta 26: 141 91986), phosphorothioate (Mag et al., Nucleic Acids Res. 19: 1437 (1991), and US Patent No. 5,644,048), phosphorodithioate (Briu et al. , J. Am. Chem. Soc. 111: 2321 (1989), O-methylphosphoramidite bonds (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford Uníversity Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114: 1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31: 1008 (1992); Nielsen, Nature, 365: 566 (1993); Carlsson et al., Nature 380: 207 (1996), all of which are incorporated for reference). Other analog nucleic acids include those with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92: 6097 (1995); nonionic backbones (US Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863 Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30: 423 (1991), Letsinger et al., J. Am. Chem. Soc. 110: 4470 (1988), Letsinger et al., Nucleoside & Nucleotide 13: 1597 (1994), Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. YS Sanghui and P. Dan Cook, Mesmaeker et al., Bioorganic &Medicinal Chem. Lett. : 395 (1994), Jeffs et al., J. Biomolecular NMR 34:17 (1994), Tetrahedron Lett. 37: 743 (1996)) and different skeletons of ribose, including those described in US Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7 of the ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. YS Sanghui and P. Dan Cook, Mesmaeker. in one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp.169-176). Various analogs of nucleic acids are described in Rawls, C & E News June 2, 1997 page 35. All of these references are expressly incorporated herein for reference. These modifications of the ribose-phosphate backbone can be made to facilitate the addition of additional portions such as labels, or to increase the stability and half-life of such molecules in physiological environments. In addition, mixtures of the nucleic acids and analogues that are naturally present can be made. Alternatively, mixtures of different nucleic acid analogs, and mixtures of nucleic acids and analogs that are naturally present can be made. The nucleic acids may be single-stranded or double-stranded, as specified, or contain portions of the sequence of both single-stranded and double-stranded. The nucleic acid can be DNA, either genomic or cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine , guanine, inosine, xatanina, hypoxatanina, isocitosina, isoguanina, etc. As generally described above for proteins, the candidate bioactive agents of nucleic acids may be nucleic acids that are naturally present, random nucleic acids, or "changed" random nucleic acids. For example, solutions of prokaryotic or eukaryotic genomes can be used as described above for proteins. In a preferred embodiment, the candidate bioactive agents are organic chemical portions, a wide variety of which are available in the literature. In a preferred embodiment, as described above, selections can be made on the individual genes and the products of the genes (proteins). In a preferred embodiment, the gene or protein has been identified as will be described later in the Examples as a differentially expressed gene associated with particular tissues and therefore conditions related to these tissues. Therefore, in one embodiment, the selections are designed for the first candidate candidates found that can bind to FGF-19, and then these agents can be used in assays that evaluate the candidate agent's ability to modulate the activity of FGF-19. . Thus, as will be appreciated by those skilled in the art, there are a number of different tests which can be operated.

The selection of agents that modulate the activity of FGF-19 can also be done. In a preferred embodiment, the methods for the selection of a bioactive agent capable of modulating the activity of FGF-19 comprise the steps of adding a candidate bioactive agent to a sample of FGF-19 and determining an alteration in the biological activity of the FGF- 19 The "modulation of FGF-19 activity" includes an increase in activity, a reduction in activity, or a change in the type or class of activity present. Accordingly, in this embodiment, the candidate agent must also bind to FGF-19 (although this may not be necessary), and alter its biological or biochemical activity as defined herein. The methods include both in vitro screening methods, as generally described above, and the in vivo selection of cells for alterations in the presence, expression, distribution, activity or amount of FGF-19. Thus, in this embodiment, the methods comprise the combination of a sample and a candidate bioactive agent, and evaluate the effect on the activity of FGF-19. By "FGF-19 protein activity" or the grammatical equivalents herein, at least one of the biological activities of the FGF-19 protein is understood as described above.

In a preferred embodiment, the activity of the FGF-19 protein is increased; in another preferred embodiment, the activity of the FGF-19 protein is reduced. Accordingly, bioactive agents that are antagonists are preferred in some embodiments, and bioactive agents that are agonists may be preferred in other embodiments. In one aspect of the invention, cells containing the FGF-19 sequences are used in the drug screening assays by evaluating the effect of the drug candidates on FGF-19. The cell type includes normal cells, tumor cells, and adipocytes. Methods of evaluating FGF-19 activity such as changes in glucose uptake, leptin release, metabolism, triglyceride and free fatty acid levels, body weight and body fat, and they are known in the art and are exemplified later in the examples. In a preferred embodiment, the methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising FGF-19. Preferred cell types include almost any cell. The cells contain a nucleic acid, preferably recombinant, which encodes an FGF-19 protein. In a preferred embodiment, a library of candidate agents is tested on a plurality of cells. In one aspect, the assays are evaluated in the presence or absence or subsequent exposure to physiological signals, for example hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutic substances, radiation, carcinogenesis, or other cells (ie cell-cell contacts). In another example, determinations are determined at different stages of the cell cycle process. The sequences of FGF-19 provided herein can also be used in diagnostic methods. Overexpression of FGF-19 may indicate an abnormally high metabolic rate and underexpression may indicate that the person is prone to obesity. In addition, a sample from a patient can be analyzed to verify mutated or dysfunctional FGF-19. In general, such methods include comparing a sample from a patient and comparing the expression of FGF-19 with that of a control.

F. Anti-FGF-19 Antibodies The present invention further provides anti-FGF-19 antibodies. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific antibodies, and heteroconjugate antibodies. 1. Polyclonal Antibodies Anti-FGF-19 antibodies can comprise polyclonal antibodies. The methods of preparing the polyclonal antibodies are already known to the skilled artisan. Polyclonal antibodies can be enhanced in a mammal, for example, by one or more injections of an immunization agent and, if desired, an auxiliary. Typically, the immunization and / or auxiliary agent will be injected into the mammal by multiple intraperitoneal or subcutaneous injections. The immunization agent can include the FGF-19 polypeptide or a fusion protein thereof. It may be useful to conjugate the immunization agent with a protein known to be immunogenic in the mammal that is immunized. Examples of such immunogenic proteins include but are not limited to haemocyanin from the marine lapa, serum albumin, bovine thyroglobulin, and the soybean trypsin inhibitor. Examples of auxiliaries that may be employed include Freund's complete assistant and MPL-TDM assistant (Monophosphoryl Lipid A, synthetic trehalose dicorinomycolate). The immunization protocol can be selected by a person skilled in the art without undue experimentation. 2. Monoclonal Antibodies Anti-FGF-19 antibodies can be monoclonal antibodies, alternatively. Monoclonal antibodies can be prepared using the hybridoma methods, such as those described by Kohler and Milstein, Nature, 256: 495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunization agent to produce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunization agent. Alternatively, lymphocytes can be immunized in vitro. The immunization agent will typically include the FGF-19 polypeptide or a fusion protein thereof. In general, any peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if mammalian sources other than human sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) p. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat myeloma or mouse cell lines are used. The hybridoma cells can be cultured in a suitable culture medium which preferably contains one or more substances that inhibit the growth or survival of immortalized, unfused cells. For example, if the original or paternal cells lack the hypoxanthine guanine phosphoribosyl transferase enzyme (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine ("HAT medium"), such substances prevent the growth of cells deficient in HGPRT. Preferred immortalized cell lines are those that efficiently fuse, support stable high level expression of the antibody by the cells producing the selected antibodies, and are sensitive to a medium such as the HAT medium. The most preferred immortalized cell lines are the murine myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. The human-mouse and human myelone heteromyeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) p. 51-63]. The culture medium in which the hybridoma cells are cultured can then be evaluated for the presence of the monoclonal antibodies directed against FGF-19. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as the radioimmunoassay (RIA) or the enzyme-linked immunosorbent assay (ELISA). Such assays and techniques are already known in the art. The binding affinity of monoclonal antibodies, for example, can be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem. 107: 220 (1980).

After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as the ascites in a mammal. The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or the ascites fluid by conventional immunoglobulin purification methods such as, for example, protein A-Sepharose, hydroxylaparite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. The DNA encoding the monoclonal antibodies of the invention can be isolated and easily sequenced using conventional methods (for example, using oligonucleotide probes that are capable of specifically binding to the genes encoding the light and heavy chains of murine antibodies. ). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed in the expression vectors, which are transfected into the host cells such as the COS cells of the simian, the cells of the Chinese hamster ovaries (CHO), or the myeloma cells that they do not otherwise produce the immunoglobulin protein, to obtain the synthesis of the monoclonal antibodies in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence for the constant domains of the light and heavy chain, human, in place of the homologous murine sequences [U.S. No. 4,816,567; Morrison et al., Supra] or by the covalent attachment to the immunoglobulin coding sequence of all or a part of the coding sequence for a polypeptide other than immunoglobulin. Such a polypeptide other than immunoglobulin can be replaced by the constant domains of an antibody of the invention, or can be substituted by the variable domains of a combination site of the antigen of an antibody of the invention to create a chimeric bivalent antibody. The antibodies can be monovalent antibodies. The methods of preparation of the monovalent antibodies are well known in the art. For example, one method involves the recombinant expression of the immunoglobulin light chain and the modified heavy chain. The heavy chain is truncated generally at any point in the Fc region to prevent cross-linking of the heavy chain. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted to prevent cross-linking. The in vi tro methods are also suitable for the preparation of monovalent antibodies. The digestion of the antibodies to produce the fragments thereof, particularly the Fab fragments, can be effected using routine techniques known in the art. 3. Human and Humanized Antibodies Anti-FGF-19 antibodies may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2 or other antigen binding subsequences. of antibodies) which contain the minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (the receptor antibody) in which the residues of a region of complementary determination (CDR) of the recipient or patient are replaced by the residues of a CDR of a non-human species (donor antibody) like the mouse, the rat or the rabbit, which have a desired specificity, affinity and capacity. In some cases, the residues of the Fv structure of the human immunoglobulin are replaced by the corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the antibody of the recipient or patient nor in the imported CDR or structure sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the All FR regions are those of a consensus sequence of human immunoglobulin. Optimally humanized antibodies will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr.Op. Struct .Biol. , 2: 593-596 (1992)]. Methods for the humanization of non-human antibodies are already known in the art. Generally, a humanized antibody has one or more amino acid residues introduced therein from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from a "import" variable domain. The humanization can be carried out essentially following the method of Winter and collaborators [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 329: 1534-1536 (1988)], replacing the CDR or CDRs sequences of the rodent with the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from the analogous sites in the rodent's antibodies.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J ^ Mol. Biol., 222: 581 (1991)]. The techniques of Colé et al., And Boerner et al., Are also available for the preparation of human monoclonal antibodies (Colé et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985) and Boerner. et al., J. Immunol., 147 (1): 86-95 (1991).] Similarly, human antibodies can be made by introducing the human immunoglobulin sites into the transgenic animals, for example, the mice in the which the endogenous immunoglobulin genes have been partially or completely inactivated During the stimulation, the production of human antibodies is observed, which closely resembles that observed in humans in all aspects, including the rearrangement of the genes, the coupling or assembly and the antibody repertoire.This approach is described, for example, in US Patent Nos. 5,545,807; 5,545,806; 5,569,825, 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio / Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).4. Bispecific Antibodies Bispecific antibodies are preferably human or humanized, monoclonal antibodies, which have binding specificities for at least two different antigens. In the present case, one of the specificities of the binding is for FGF-19, the other is for any other antigen, and preferably for a protein or receptor or subunit receptor of the cell surface. Methods of making bispecific antibodies are already known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two light chain / heavy chain pairs of the immunoglobulin, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305: 537-539 (1983 )]. Because of the randomization of immunoglobulin light and heavy chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually effected by the steps of affinity chromatography. Similar procedures are described in WO 93/08829, published May 13, 1993, and in Traunecker et al., EMBO J., 10: 3655-3659 (1991). The variable domains of the antibodies with the desired binding or agglutination specificities (antibody-antigen combining sites) can be fused to the immunoglobulin constant domain sequences. The fusion preferably is with a constant domain of the immunoglobulin heavy chain, comprising at least a portion of the CH2, and CH3, joint regions. It is preferred to have the first constant region of the heavy chain (CH1) containing the site necessary for the binding or agglutination of the light chain in at least one of the fusions. The DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into the separate expression vectors and are cotransfected in a suitable host organism. For additional details of the generation of bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be designed to maximize the percentage of heterodimers which are recovered from the recombinant cell culture. The preferred interface comprises at least a portion of the CH3 region of a constant domain of the antibody. In this method, one or more side chains of smaller amino acids from the interface of the first antibody molecule are replaced with larger side chains (eg, tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large lateral chain (s) are created on the interface of the second antibody molecule by replacing the side chains of large amino acids with smaller ones. (for example of alanine or threonine). This provides a mechanism for increasing the performance of the heterodimer over unwanted end products such as homodimers. Bispecific antibodies can be prepared as full-length antibodies or fragments of antibodies (for example bispecific antibodies of F (ab ') 2). The techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical binding or binding. Brennan et al., Science 229: 81 (1985) describes a method wherein the intact antibodies are proteolytically cleaved to generate the F (ab ') 2 fragments. These fragments are reduced in the presence of a complexing agent with dithiol, sodium arsenite, to stabilize the neighboring dithiols and prevent the formation of intermolecular disulfide. The generated Fab 'fragments are then converted to thionitrobenzoate derivatives (TNB). One of the Fab'-TNB derivatives is then reconverted to Fab '-thiol by the reduction with mercapetylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of the enzymes. The Fab 'fragments can be recovered directly from E. coli and chemically bound to form the bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describes the production of a fully humanised F (ab ') 2 bispecific antibody molecule. Each Fab 'fragment was secreted separately from E. coli and subjected to direct chemical binding in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against the targets of human breast tumor. Various techniques for making and isolating bispecific antibody fragments directly from the recombinant cell culture have also been described. For example, bispecific antibodies have been produced using the leucine closure elements. Kostelny et al., J. Immunol. 148 (5): 1547-1533 (1992). Peptides of the leucine closure element of the Fos and Jun proteins were linked to the Fab 'portions of two different antibodies for gene fusion. The homodimers of the antibodies were reduced in the region of articulation to form the monomers and then reoxidized to form the heterodimers of the antibodies. This method can also be used for the production of the homodimers of the antibodies. The technology of "small antibody fragments with two antigen binding sites" described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker which is too short to allow grouping as pairs between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the domains of another fragment, whereby two antigen binding sites are formed. Another strategy for making bispecific antibody fragments by the use of single chain Fv dimers (sFv) has also been reported. See, Gruber et al., J. Immunol. 152: 5368 (1994). Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60 (1991). Exemplary bispecific antibodies can bind to two different epitopes on a FGF-19 polypeptide given herein. Alternatively, a branch of the anti-FGF-19 polypeptide can be combined with a branch which binds to a trigger or activation molecule on a leukocyte such as the T cell receptor molecule (e.g., CD2, CD3, CD28). , or B7), or the Fc receptors for IgG (Fc? RIII) (DC16) to focus the cell defense mechanisms for the cell to express the particular FGF-19 polypeptide. Bispecific antibodies can also be used to localize cytotoxic agents to cells which express a particular FGF-19 polypeptide. These antibodies possess a binding branch of FGF-19 and a branch which binds to a cytotoxic agent or a chelator of the radionuclide, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds to the FGF-19 polypeptide and further binds tissue factor (TF).

. Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two antibodies covalently linked. Such antibodies, for example, have been proposed to target cells of the immune system to unwanted cells [U.S. Pat. No. 4,676,980], and for the treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using the methods known in the chemistry of synthetic proteins, including those involving the crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or forming a thioether linkage. Examples of suitable reagents for this purpose include iminothiolate. and methyl-4-mercaptobutyrimidate and those described, for example, in U.S. Pat. No. 4,676,980. 6. Design of the Effector Function It may be desirable to modify the antibody of the invention with respect to effector function, to improve, for example, the effectiveness of the antibody in the treatment of cancer. For example, the cysteine residue (s) can be introduced into the Fc region, whereby formation of the interchain disulfide bond in this region is allowed. The homodimeric antibody thus generated may have an enhanced internalization capacity and / or an extermination of the cells mediated by complement and increased antibody-dependent cellular cytoxicity (ADCC). See Caron et al., J. Exp. Med., 176: 1191-1195 (1992) and Shopes, J. Immunol. , 148: 2918-2922 (1992). Homodimeric antibodies with improved antitumorigenic activity can also be prepared using the heterobifunctional crosslinkers as described in Wolff et al., Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be designed which has double Fc regions and therefore can have enhanced ADCC capacity and complement lysis. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989). 7. Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, the toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof). , or a radioactive isotope (i.e., a radioconjugate). Chemotherapeutic agents useful in the generation of such immunoconjugates have already been described above. The enzymatically active toxins and the fragments thereof that can be used include the diphtheria A chain, the active non-binding fragments of the diphtheria toxin, the exotoxin A chain (from Pseudomonas aeruginosa), the ricin chain A, the chain of abrin A, the chain of modeccin A, alpha-sarcin, the proteins of Aleuri tes fordii, the proteins of diantine, the proteins of Phytolaca americana (PAPI, PAPII, and PAP-S), the inhibitor of the momordica charantia, curcinia, crotina, the inhibitor of sapaonaria officinalis, gelonin, mitogeline, restrictocin, fenomycin, enomycin, and trichothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 18eRe. The conjugates of the antibody and the cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), the bifunctional derivatives of the imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexandiamine), bis-diazonium derivatives (such as bis- (p-diazonium benzoyl) -ethylenediamine), diisocyanates (such as 2,6-tolienium diisocyanate), and bis-active fluorine compounds (such as 1, 5 difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). The l-isothiocyanatobenzyl-3-methyldiethylenetriaminpentaacetic acid labeled with carbon 14 (MX-DTPA) is an exemplary chelating agent for the conjugation of the radionucleotide to the antibody. See WO94 / 11026. In another embodiment, the antibody can be conjugated to a "receptor" (such as streptavidin) for use in pre-location as a target of the tumor, wherein the antibody-receptor conjugate is administered to the patient, followed by removal of the conjugate. unbound from the circulation using a clearing or purifying agent and then the administration of a "ligand" (eg, avidin) which is conjugated to a cytotoxic agent (eg, a radionucleotide). 8. Immunoliposomes The antibodies described herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Patents Nos. 4,485,045 and 4,544,454. Liposomes with improved circulation time are described in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and phosphatidylethanolamine derived with PEG (PEG-PE). The liposomes are extruded through filters of the defined pore size to give liposomes with the desired diameter. The Fab 'fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) by means of a disulfide exchange reaction. . A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposomes. See Gabizon et al., J. National Cancer Inst., 81 (19): 1484 (1989). 9. Pharmaceutical Compositions of Antibodies Antibodies that specifically bind to a FGF-19 polypeptide identified herein, as well as other molecules identified by the screening assays described hereinbefore, can be administered for the treatment of various disorders in the form of the compositions Pharmaceutical If the FGF-19 polypeptide is intracellular and the whole antibodies are used as inhibitors, internalization antibodies are preferred. However, lipofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into the cells. Where the antibody fragments are used, the smaller inhibitory fragment that binds specifically to the binding domain of the target protein is preferred. For example, based on the sequences of the variable region of an antibody, the molecules of the peptides can be designated which retain the ability to bind the target protein sequence. Such polypeptides can be chemically synthesized and / or produced by recombinant DNA technology. See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993). The formulation herein may also contain more than one active compound when necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or additionally, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. Such molecules are suitably present in combination, in amounts that are effective for the purpose proposed. The active ingredients can also be entrapped in the microcapsules prepared, for example, by coacervation or interfacial polymerization techniques, for example, hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacrylate) microcapsules, respectively, in the delivery systems of the colloidal drugs (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences, supra. The formulations that are to be used for in vivo administration must be sterile. This is easily effected by filtration through sterile filtration membranes. Sustained-release preparations can be prepared. Suitable examples of the sustained release preparations include the semipermeable matrices of the solid hydrophobic polymers. Which contains the antibody, such matrices are in the form of shaped articles, for example, films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate), or poly (vinyl alcohol)), polylactides (US Pat. No. 3,773,919), L-glutamic acid copolymers Y ? ethyl-L-glutamate, non-degradable vinyl acetate-ethylene, degradable glycolic acid-lactic acid copolymers such as LUPRON DEPOT ™ (injectable microspheres composed of the lactic acid-glycolic acid copolymer and leuprolide acetate), and the acid poly-D- (-) - 3-hydroxybutyric acid. Although polymers such as vinyl acetate-ethylene and lactic acid-glycolic acid make it possible to release the molecules for more than 100 days, certain hydrogels release the proteins for shorter periods of time. When the encapsulated antibodies remain in the body for a prolonged period of time, they can be denatured or aggregated as a result of exposure to moisture at 37 ° C, leading to a loss of biological activity and possible changes in immunogenicity. Rational strategies can be contemplated for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be the formation of the intermolecular SS bond through thio-disulfide exchange, the stabilization can be achieved by modifying the sulfhydryl residues, lyophilizing from the acid solutions, controlling the moisture content, using the appropriate additives, and developing the specific polymer matrix compositions.

G. Uses for Anti-FGF-19 Antibodies The anti-FGF-19 antibodies of the invention have various utilities. For example, anti-FGF-19 antibodies can be used in diagnostic assays for FGF-19, for example, by detecting their expression in specific cells, tissues, or serum. Various diagnostic assay techniques known in the art can be used, such as binding or competitive agglutination assays, indirect sandwich assays and immunoprecipitation assays carried out in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies : A Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in diagnostic assays can be labeled with a detectable portion. The detectable portion must be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable portion can be a radioisotope, such as 3H, 1C, 32P, 35S, or 125I, a fluorescent or chemiluminescent compound, such as a fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as phosphatase alkaline, beta-galactosidase or horseradish peroxidase. Any method known in the art for conjugating the antibody with the detectable portion can be employed, including those methods described by Hunter et al., Nature, 144; 945 (1962); Davis et al., Biochemistry, 13: 1014 (1974); Pain et al., J. Immunol. Meth., 40: 219 (1981); and Nygren, J. Histochem. And Cytochem., 30: 407 (1982). Anti-FGF-19 antibodies are also useful for affinity purification of FGF-19 from natural sources or culture of recombinant cells. In this process, antibodies against FGF-19 are immobilized on a suitable support, such as a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody is then contacted with a sample containing the FGF-19 to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all of the material in the sample except FGF-19, which is attached to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the FGF-19 from the antibody. The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. All patent and literature references cited in the present specification without being incorporated herein for reference in their entirety.

EXAMPLES Commercially available reagents referred to in the examples were used in accordance with the manufacturer's instructions unless otherwise indicated. The source of these cells identified in the following examples, and throughout the specification, by the access numbers of the ATCC, is the American Type Culture Collection, Manassas, VA.

EXAMPLE 1 Isolation of the cDNA Clones Encoding a Human FGF-19 Accession number AF007268 of the EST sequence, a murine fibroblast growth factor (FGF-15) was used to investigate several databases »of Public ESTs (for example, GenBank, Dayhoff, etc.). The investigation was carried out using the BLAST or BLAST2 computer program [Altschul et al. , Methods in Enzymology, 266: 460-480 (1996)] as a comparison of the sequences of the ECD protein for a translation of 6 frames or structures of the EST sequences. The research led to success with GenBank EST AA220994, which has been identified as a neuronal 937230 precursor of STRATAGENE NT2. The sequence of AA220994 is also referred to here as DNA47412. Based on the DNA47412 sequence, the oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) to be used as probes to isolate a clone from the full-length coding sequence for the FGF-19. The forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of approximately 100-1000 bp in length. The sequences of the probe are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. To select several libraries for a full-length clone, the DNA of the libraries was selected by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology, supra, with the primer pair of PCR. A positive library was then used to isolate the clones encoding the gene of interest using the oligonucleotides from the probe and one of the pairs of the primers. PCR primers (forward and reverse) were synthesized: 5'-forward PCR primer-ATCCGCCCAGATGGCTACAATGTGTA-3 '(SEQ ID NO: 3), and 5'-rear PCR primer -CCAGTCCGGTGACAAGCCCAAA-3' (SEQ ID NO: 4) .

Additionally, a synthetic oligonucleotide hybridization probe was constructed from the sequence DNA47412 which had the following nucleotide sequence: Hybridization probe 5'-GCCTCCCGGTCTCCCTGAGCAGTGCCAAACAGCGGCAGTGTA-3 '(SEQ ID NO: 5). The RNA for the construction of the cDNA libraries was isolated from the tissue of the human fetal retina. The cDNA libraries for isolating cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with the oligo dT containing a NotI site, bound with truncated ends to the Sali hemicinase-treated adapters, cleaved with NotI, appropriately sized by electrophoresis with a gel, and cloned in a defined orientation in a vector of suitable cloning (such as pRKB or PRKD; pRK5B is a precursor of pRK5D not containing the Sfil site; see, Holmes et al., Science, 253: 1278-1280 (1991)) at the unique Xhol and NotI sites. Sequencing of the isolated clones as described above, gave the DNA sequence for a full-length FGF-19 polypeptide (designated here as DNA49435-1219 [Figure 1, SEQ ID NO: 1]) and the protein sequence derivative for this polypeptide of FGF-19. The full-length clone identified above contained a single open reading frame with an apparent translation start site at positions 464-466 of the nucleotide and a stop signal at positions 1112-1114 of the nucleotide (Figure 1, SEQ ID. NO: l). The predicted polypeptide precursor is 216 amino acids in length, has a calculated molecular weight of about 24,003 daltons and an estimated pl of about 6.99. The analysis of the full length FGF-19 sequence shown in Figure 2 (SEQ ID NO: 2) makes evident the presence of a variety of the important polypeptide domains as shown in Figure 2, wherein the given locations for these important polypeptide domains are approximately as described above. The formation of chromosome maps makes it evident that the nucleic acids encoding FGF-19 have a coordinate assignment for chromosome Hql3.1, band ql3.1, in humans. Clone DNA49435-1219 has been deposited with the ATCC on November 21, 1997 and is assigned to the ATCC with deposit No. 209480.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), using the alignment analysis of the ALIGN-2 sequence of the full-length sequence shown in Figure 2 (SEQ ID NO: 2), evidenced the identity of the sequence between the amino acid sequence of FGF-19 and the following Dayhoff sequences: AF007268_1, S54407, P_W52596, FGF2_XENLA, P_W53793, AB002097_1, P_R27966, HSU67918 1, S23595, and P R70824.

EXAMPLE 2 Use of FGF-19 as a Hybridization Probe The following method describes the use of a nucleotide sequence encoding FGF-19 as a hybridization probe. The DNA comprising the full-length coding sequence or the mature FGF-19 is employed as a probe to select homologous DNAs (such as those encoding the variants that are naturally present in FGF-19) in the libraries of the CDNA from human tissue or genomic libraries of human tissue. Hybridization and washing of the filters containing any DNA from the library is carried out under the following high severity conditions. The hybridization of the radiolabelled FGF-19 derived probe to the filters is carried out in a 50% formamide solution, 5xSSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x of the solution of Denhardt, and 10% dextran sulfate at 42 ° C for 20 hours. The filters are washed in an aqueous solution of O.lx SSC and 0.1% SDS at 42 ° C. DNAs having an identity of the desired sequence with the DNA encoding the FGF-19 of the full length natural sequence can then be identified using standard techniques known in the art.

EXAMPLE 3 Expression of FGF-19 in E. coli This example illustrates the preparation of a non-glycosylated form of FGF-19 by recombinant expression in E. coli. The DNA sequence encoding FGF-19 is initially amplified using the selected PCR primers. The primers must contain the restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector. A variety of expression vectors can be employed. An example of a suitable vector is pBR322 (derived from E. coli, see Bolivar et al., Gene, 2:95 (1977)) which contains the genes for resistance to ampicillin and tetracycline. The vector is diluted with the restriction enzyme and dephosphorylated. The sequences amplified by PCR are then ligated into the vector. The vector will preferably include sequences which encode an antibiotic resistance gene, a trp promoter, a polyhis front element (including the first six STII codons, the polyhis sequence, and the enterokinase cleavage site), the coding region of FGF-19, the lambda transcriptional terminator, and an ArgU gene. The binding or ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al., Supra. Transformants are identified by their ability to grow on LB plates and then antibiotic-resistant colonies are selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing. The selected clones can be grown overnight in the liquid culture medium such as the LB broth supplemented with antibiotics. The overnight culture can be used subsequently to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is activated. After culturing the cells for several more hours, the cells can be collected by centrifugation. The cellular microspheres obtained by centrifugation can be solubilized using the various agents known in the art, and the solubilized FGF-19 protein can then be purified using a metal chelation column under conditions that allow tight binding of the protein. FGF-19 can be expressed in E. coli in a labeled form of poly-His, using the following procedure. The DNA encoding FGF-19 is initially amplified using the selected PCR primers. The primers will contain the restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences that provide the beginning of the reliable and efficient translation, the rapid purification on a column of metal chelation, and proteolytic removal with enterokinase. The poly-His-tagged sequences, amplified by PCR, are then bound or bound in an expression vector, which is used to transform the E. coli host based on strain 52 (W3110 fuhA (tonA) Ion galE rpoHts ( htpRts) clpP (lacIq) Transformants are first grown in LB containing 50 mg / ml carbenicillin at 30 ° C with agitation until an OD600 of 3-5 is reached.The cultures are then diluted 50-100 times in the CRAP medium (prepared by mixing 3.57 g (NH4) 2S04, 0.71 g of sodium citrate * 2H20, 1.07 g of KCl, 5.36 g of Difco yeast extract, 5.36 g of Dheffield hycase SF in 500 ml of water, as well as 110 mM MPOS, pH 7.3, 0.55% (w / v) glucose and 7 mM MgSO4) and were grown for about 20-30 hours at 30 ° C with shaking.The samples are removed to verify expression by the analysis of SDS-PAGE, and the volumetric culture is centrifuged to convert the cells into microspheres. res are frozen until purification and refolding. The paste of E. coli from 0.5 to 1 1 of the fermentations (6-10 g of the microspheres) is resuspended in 10 volumes (w / v) in guanidine 7 M, 20 mM Tris, buffer 8. Sodium sulfite solid and tetrathionate sodium are added to make the final concentrations of 0.1M and 0.02M, respectively, and the solution is stirred overnight at 4 ° C. This step leads to a denatured protein with all the cysteine residues blocked by sulfitolization. The solution is centrifuged at 40,000 rpm in a Beckman Ultracentrifuge for 30 minutes. The supernatant is diluted with 3-5 volumes of the buffer of the metal chelate column (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify it. The clarified extract is loaded onto a Ni-NTA Qiagen metal chelate column equilibrated in the metal chelate column buffer. The column is washed with additional buffer containing 50 mM imidazole (Calbiochem., Utrol grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole. The fractions containing the desired protein are pooled and stored at 4 ° C. The concentration of the protein is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence. The proteins are refolded by diluting the sample slowly in the newly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. The folding volumes are chosen so that the concentration of the final protein is between 50 and 100 micrograms / ml. The folding solution is gently stirred at 4 ° C for 12-36 hours. The refolding reaction is stopped by the addition of TFA to a final concentration of 0.4% (pH of about 3). Before further purification of the protein, the solution is filtered through a 0.22 micron filter and acetonitrile is added to a final concentration of 2-10%. The refolded protein is subjected to chromatography on a Poros Rl / H reversed phase column using a mobile shock absorber of 0.1% TFA with elution with a gradient of acetonitrile from 10 to 80%. The aliquots of the fractions with an A280 absorbance are analyzed on SDS polyacrylamide gels and the fractions containing the homogeneous refolded protein are pooled. In general, appropriately refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since these species are mostly compact with their hydrophobic interiors protected from interactions with the phase resin. reverse. Aggregate species are usually eluted at higher acetonitrile concentrations. In addition to the resolution of the wrongly folded forms from the desired shape, the inverted phase step also removes the endotoxin from the samples. The fractions containing the desired folded FGF-19 polypeptide are pooled and the acetonitrile removed using a gentle stream of nitrogen directed into the solution. The proteins are formulated in 20 mM Hepes, pH 6.8, with 0.14 M sodium chloride and 4% mannitol by dialysis or by filtration in a gel using the GF Superfine resins (Pharmacia) balanced in the buffer of the formulation and filtered in a manner sterile.

EXAMPLE 4 Expression of FGF-19 in mammalian cells This example illustrates the preparation of a potentially glycosylated form of FGF-19 by recombinant expression in mammalian cells. The vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as the expression vector. Optionally, the FGF-19 DNA is ligated or bound in pRK5 with the selected restriction enzymes to allow insertion of the FGF-19 DNA using the binding methods as described in Sambrook et al., Supra. The resulting vector is called pRK5-FGF-19. In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in a medium such as DMEM supplemented with fetal bovine serum and optionally nutrient components and / or antibiotics. Approximately 10 μg of the pRK5-FGF-19 DNA are mixed with about 1 μg of the DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31: 543 (1982)] and dissolved in 500 μl of Tris- 1 mM HCl, 0.1 mM EDTA, 0.227 M CaCl2. To this mixture, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaP04 are added dropwise, and a precipitate is allowed to form for 10 minutes. minutes at 25 ° C. The precipitate is suspended and added to the 293 cells and allowed to settle for approximately four hours at 37 ° C. The culture medium is completely aspirated and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with the serum free medium, fresh medium is added and the cells are incubated for about 5 days. Approximately 24 hours after the transfections, the culture medium is removed and replaced with the culture medium (alone) or the culture medium containing 200 μCi / ml of 35S-cysteine and 200 μCi / ml of 35S-methionine. After about 12 hours of incubation, the conditioned medium is collected, concentrated on a rotary filter, and loaded on a 15% SDS gel. The processed gel can be dried and exposed to the film for a selected period of time to reveal the presence of the FGF-19 polypeptide. The cultures containing the transfected cells may undergo further incubation (in the serum free medium) and the medium is tested in the selected bioassays. In an alternative technique, FGF-19 can be introduced into 293 cells temporarily using the dextran sulfate method described by Somparyrac et al., Proc. Natl. Acad. Sci., 12: 7575 (1981), 293 cells were grown at maximum density in a spinner and 700 μg of pRK5-FGF-19 DNA is added. The cells are first concentrated from the spinning vessel by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cellular microspheres for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with the tissue culture medium, and 5 μg / ml of bovine insulin and 0.1 are reintroduced into the rotating vessel containing the tissue culture medium. μg / ml of bovine transferrin. After about four days, the conditioned medium is centrifuged and filtered to remove the cells and debris. The sample containing the expressed FGF-19 can then be concentrated and purified by any selected method, such as dialysis and / or column chromatography. In another embodiment, FGF-19 can be expressed in CHO cells. PRK5-FGF-19 can be transfected into CHO cells using known reagents such as CaP04 or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with the culture medium (alone) or the medium containing a radiolabel such as 35 S-methionine. After determining the presence of the FGF-19 polypeptide, the culture medium can be replaced with the serum free medium. Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is collected. The medium containing the expressed FGF-19 can then be concentrated and purified by any selected method. The FGF-19 tagged with the epitope can also be expressed in the host CHO cells. FGF-19 can be subcloned out of the vector pRK5. The subclone insert may undergo PCR to fuse a frame with a selected epitope tag such as a poly-his tag on a Baculovirus expression vector. The insert of FGF-19 labeled with poly-his can then be subcloned into an SV40 driven vector containing a selection marker such as DHFR for the selection of stable clones. Finally, the CHO cells can be transfected (as described above) with the SV40 driven vector. The labeling can be done, as described above, to verify the expression. The culture medium containing the FGF-19 labeled with expressed poly-His can then be concentrated and purified by any selected method, such as by affinity chromatography of the chelate-Ni2 +. FGF-19 can also be expressed in CHO and / or COS cells by a method of temporary expression or in CHO cells by another stable expression method. Stable expression in CHO cells is effected using the following procedure. The proteins are expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e.g., the extracellular domains) of the respective proteins are fused to a sequence of the IgG1 constant region containing the joint, the CH2 and the CH2 domains and / or it is a form labeled with poly-His. Following PCR amplification, the respective DNAs are subcloned into a CHO expression vector using standard techniques as described in Ausubel et al. , Current Protocols of Molecular Biology, Unit 3.16, Jophn Wiley and Sons (1997). CHO expression vectors are constructed to have compatible 5 'and 3' restriction sites of the DNA of interest to allow transport of the cDNAs. The vector used for expression in CHO cells is as described in Lucas et al., Nucí. Acids Res. 24: 9 (1774-1779 (1996), and utilizes the SV40 initial promoter / enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR) .The expression of DHFR allows selection for stable maintenance of the plasmid a continuation of transfection Twelve micrograms of the desired plasmid DNA are introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect® (Qiagen), Dosper® or Fugene® (Boehringer Mannheim). The cells are grown as described in Lucas et al., Supra. Approximately 3 x 10 ~ 7 cells are frozen in an ampoule for growth and additional production as described below. The ampoules containing the plasmid DNA are thawed by placement in a water bath and mixed by swirling. The contents are transferred with a pipette to a centrifuge machine tube containing 10 ml of the medium and centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated and the cells are resuspended in 10 ml of the selective medium (0.2 μm filtered PS20 with 5% diafiltered 0.2 μm fetal bovine serum). The cells were then fed as aliquots to a 100 ml centrifuge machine containing 90 ml of the selective medium. After 1-2 days, the cells are transfected into a 250 ml centrifuge machine filled with 150 ml of the selective growth medium and incubated at 37 ° C. After another 3 days, the centrifuges of 250 ml, 500 ml and 2000 ml are seeded with 3 x 10 5 cells / ml. The cell medium is exchanged with the fresh medium by centrifugation and resuspension in the production medium. Although any suitable CHO medium can be employed, a production medium described in U.S. Pat. No. 5,122,469, issued June 16, 1992, can actually be used. A 3-liter centrifugal production machine is seeded at 1.2 x 106 cells / ml. On day 0, the numerical pH of the cells is determined. On day 1, the centrifugal machine is sampled and spraying with filtered air is started. On day 2, the centrifugal machine is sampled, the temperature is changed to 33 ° C, and 30 ml of the glucose of 500 g / 1 and 0.6 ml of the 10% antifoam are taken (for example, the emulsion of polydimethylsiloxane at 35%, Medical Grade Emulsion from Dow Corning 365). From beginning to end of production, the pH is adjusted when it is necessary to keep it around 7.2. After 10 days, or until the viability has been reduced below 70%, the cell culture is collected by centrifugation and filtration through a 0.22 μm filter. The filtrate was stored either at 4 ° C or immediately loaded on columns for purification. For constructs labeled with poly-His, the proteins are purified using a Ni-NTA column (Qiagen). Before purification, imidazole is added to the conditioned medium at a concentration of 5 mM. The conditioned medium is pumped on a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, the buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml / min., at 4 ° C. After loading, the column is washed with the additional equilibration buffer and the protein eluted with the equilibration buffer containing the 0.25 M imidazole. The highly purified protein is subsequently desalted in a storage buffer containing 10 mM Hepes, NaCl 0.14 M and 4% mannitol, pH 6.8, with a Superfine (Pharmacia) G25 column of 25 ml and stored at -80 ° C. The immunoadhesin constructs (containing Fc) are purified from the conditioned medium as follows. The conditioned medium is pumped on a 5 ml Protein A column (Pharmacia) which has been equilibrated in the 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with the equilibrium buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by the collection of 1 ml fractions in tubes containing 275 μl of the 1 M Tris buffer, pH 9.

The highly purified protein is subsequently desalted in the storage buffer as described above for proteins labeled with poly-His. The homogeneity is evaluated by the polyacrylamide gels of SDS and by the sequencing of the N-terminal amino acids by Edman degradation.

EXAMPLE 5 Expression of FGF-19 in Yeast The following method describes the recombinant expression of FGF-19 in yeast. First, the expression vectors of the yeast are constructed for the intracellular production or the secretion of FGF-19 from the ADH2 / GAPDH promoter. The DNA encoding the FGF-19 and the promoter is inserted into the appropriate restriction enzyme sites in the selected plasmid to direct the intracellular expression of FGF-19. For secretion, the DNA encoding FGF-19 can be cloned into the selected plasmid, along with the DNA encoding the ADH2 / GAPDH promoter, a FGF-19 signal peptide or another peptide from the mammalian signal. , or, for example, an alpha factor of the yeast or the forward sequence / secretory signal of the invertase, and the linker sequences (if necessary) for the expression of FGF-19. Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmids described above and cultured in the selected fermentation medium. Transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with the Coomassie Blue dye. The recombinant FGF-19 can be subsequently isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using the selected cartridge filters. The FGF-19 containing the concentrate can be further purified using the selected column chromatography resins.

EXAMPLE 6 Expression of FGF-19 in Insect Cells Infected with Baculovirus The following method describes the recombinant expression of FGF-19 in insect cells infected with baculovirus. The sequence encoding FGF-19 is fused upstream of an epitope tag contained within the baculovirus expression vector. Such epitope tags include the poly-his tags and the immunoglobulin tags (similar to the IgG Fc regions). A variety of plasmids can be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding the FGF-19 or the desired portion of the FGF-19 coding sequence such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein it is extracellular, it is amplified by PCR with the complementary primers for the 5 'and 3' regions. The 5 'primer can incorporate the flanking restriction enzyme sites (selected). The product is then diluted with those restriction enzymes selected and subcloned into the expression vector.

The recombinant baculovirus is generated by cotransfection of the previous plasmid and the DNA of the virus BaculoGold ™ (Pharmingen) in the cells of Spodoptera frugiperda ("Sf9") (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28 ° C, the released viruses are collected and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994). The FGF-19 labeled with expressed poly-his can then be purified, for example, by affinity chromatography of the chelate-Ni2 + as follows. The extracts are prepared from the Sf9 cells infected with the recombinant virus as described by Rupert et al. , Nature, 362: 175-179 (1993). Briefly, Sf9 cells were washed, resuspended in the buffer for sound application (25 ml Hepes, pH 7.9; 12.5 mM MgCl2; EDTA 0.1 mM; 10% glycerol; 0.1% of NP-40; 0.4 M KCl), and the sound is applied twice for 20 seconds on ice. The materials to which the sound has been applied are cleared by centrifugation, and the supernatant is diluted 50 times in the charge buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through of a 0.45 μm filter. The Ni2 + -NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 ml, washed with 25 ml of water and equilibrated with 25 ml of the loading buffer. The filtered cell extract is loaded onto the column at 0.5 ml per minute. The column is washed to the base line A2so with the load absorber, at which point the collection of the fraction by points begins. The column is then washed with a secondary wash buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 6.0), which binds not specifically to the protein. After reaching baseline A28o again, the column is developed with a gradient of Imidazole from 0 to 500 nM in the secondary wash buffer. The fractions of one ml are collected and analyzed by SDS-PAGE and silver staining or Western blot analysis with the Ni2 + -NTA conjugate for alkaline phosphatase (Qiagen). Fractions containing eluted Hisio tagged FGF-19 are pooled and dialyzed against the charge buffer. Alternatively, the purification of FGF-19 labeled with IgG (or labeled with Fc)) can be effected using known chromatography techniques, including for example, column chromatography of Protein A or G protein.

EXAMPLE 7 Preparation of the Antibodies Binding to FGF-19 This example illustrates the preparation of the monoclonal antibodies which can bind specifically to FGF-19. Techniques for producing monoclonal antibodies are already known in the art and are described, for example, in Goding, supra. Immunogens that can be employed include purified FGF-19, fusion proteins containing FGF-19, and cells expressing recombinant FGF-19 on the cell surface. The selection of the immunogen can be made by the skilled artisan without undue experimentation. Mice, such as Balb / c, are immunized with the FGF-19 immunogen emulsified in the complete Freund's assistant and injected subcutaneously and intraperitoneally in an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in the MPL-TDM helper (Ribi Immunochemical Research, Hamilton, MT), and injected into the carnosity of the animal's hind legs. The immunized mice are then boosted 10 to 12 days later with the additional immunogen emulsified in the selected auxiliary. After this, for several weeks, the mice can also be reinforced with additional immunization injections. Serum samples can be obtained periodically from mice by retro-orbital bleeding for testing in ELISA assays to detect anti-FGF-19 antibodies. After an adequate concentration of the antibodies has been detected, the animals "positive" for the antibodies can be injected with a final intravenous injection of FGF-19. Three to four days later, the mice are sacrificed and the spleen cells are collected. Spleen cells are then fused (using 35% polyethylene glycol) for a selected murine myeloma cell line such as P3X63AgU.l, available from the ATCC, No. CRL 1597. The fusions generate the hybridoma cells which are then placed in tissue culture plates. 96 cavities containing the HAT medium (hypoxanthine, aminopterin, and thymidine) to inhibit proliferation of unfused cells, myeloma hybrids, and hybrids of spleen cells. The hybridoma cells will be selected in a ELISA to verify the reactivity against FGF-19. The determination of the "positive" hybridoma cells that secrete the desired monoclonal antibodies against FGF-19 are within the skill in the art. The positive hybridoma cells can be injected intraperitoneally into the syngeneic Balb / c mice to produce the ascites containing the anti-FGF-19 monoclonal antibodies. Alternatively, the hybridoma cells can be grown in containers for tissue culture or spinning bottles. The purification of the monoclonal antibodies produced in the ascitos can be effected using the precipitation with the ammonium sulfate, followed by gel exclusion chromatography. Alternatively, affinity chromatography based on the binding of the antibody to protein A or protein G may be employed.

EXAMPLE 8 Purification of the FGF-19 Polypeptides Using the Specific Antibodies The natural or recombinant FGF-19 polypeptides can be purified by a variety of standard techniques in the art of protein purification. For example, the pro-FGF-19 polypeptide, the mature FGF-19 polypeptide, or the pre-FGF-19 polypeptide is purified by immunoaffinity chromatography using antibodies specific for the FGF-19 polypeptide of interest. In general, an immunoaffinity column is constructed by covalently linking the antibody of the anti-FGF-19 polypeptide to an activated chromatographic resin. Polyclonal immunoglobulins are prepared from immune serum either by precipitation with ammonium sulfate or by purification on immobilized protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Similarly, monoclonal antibodies are prepared from mouse ascites fluid by precipitation with ammonium sulfate or chromatography on immobilized Protein A. The partially purified immunoglobulin is covalently attached to a chromatographic resin such as SHEPAROSE ™ activated with CnBr (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derived resin is washed according to the manufacturer's instructions. Such an immunoaffinity column is used in the purification of the FGF-19 polypeptide by preparing a fraction of the cells containing the FGF-19 polypeptide in a soluble form. This preparation is derived by the solubilization of the whole cell or a subcellular fraction obtained by means of differential centrifugation by the addition of the detergent or by other methods well known in the art. Alternatively, the soluble FGF-19 polypeptide containing a signal sequence can be secreted in a useful amount into the medium in which the cells are grown. A preparation containing the soluble FGF-19 polypeptide is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of the FGF-19 polypeptide (e.g., high ion intensity buffers in the presence of the detergent). Then, the column is eluted under conditions that alter the binding of the antibody / FGF-19 (e.g., a low pH buffer such as about pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and the FGF-19 polypeptide is collected.

EXAMPLE 9 Drug Selection This invention is particularly useful for the selection of the compounds using the FGF-19 polypeptide or the binding fragment thereof in any of a variety of drug screening techniques. The FGF-19 polypeptide or fragment used in such a test can be free in solution, fixed to a solid support, carried on a cell surface, or localized intracellularly. A method of drug selection utilizes eukaryotic or prokaryotic host cells which are stably transformed with the recombinant nucleic acids expressing the FGF-19 polypeptide or a fragment thereof. The drugs are selected against such transformed cells in competitive binding assays. Such cells, either in fixed or viable form, can be used for standard binding assays. One can measure, for example, the formulation of the complexes between the FGF-19 polypeptide or a fragment and the agent that is tested. Alternatively, the decrease in complex formation between the FGF-19 polypeptide and its target cell or other target receptors caused by the agent being tested can be examined. Thus, the present invention provides screening methods for drugs or any other agents which may affect a disease or disorder associated with the FGF-19 polypeptide. These methods comprise contacting such an agent with a FGF-19 polypeptide or fragment thereof and for evaluating (I) the presence of a complex between the agent and the FGF-19 polypeptide or a fragment, or (ii) to verify the presence of a complex between the FGF-19 polypeptide or a fragment and the cell, by methods well known in the art. In such competitive binding assays, the FGF-19 polypeptide or fragment thereof is typically labeled. After the appropriate incubation, the free FGF-19 fragment or polypeptide is separated from that present in the bound form, and the amount of the free tag or that has not formed a complex is a measure of the particular agent's ability to to bind to the FGF-19 polypeptide or to interfere with the cell / polypeptide complex of FGF-19. Another technique for drug selection provides a high throughput screening for compounds that have a suitable binding affinity for a polypeptide and is described in detail in WO 84/03564, published September 13, 1984. In summary, the large numbers of different test compounds of the small peptides are synthesized on a solid substrate, such as plastic pins or some other surface. When applied to a FGF-19 polypeptide, the peptide test compounds are reacted with the FGF-19 polypeptide and washed. The bound FGF-19 polypeptide is detected by methods well known in the art. The purified FGF-19 polypeptide can also be coated directly on the plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support. This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the FGF-19 polypeptide compete specifically with a test compound for binding to the FGF-19 polypeptide or fragments of FGF-19. the same. In this manner, antibodies can be used to detect the presence of any polypeptide which shares one or more antigenic determinants with the FGF-19 polypeptide.

EXAMPLE 10 Design of a Rational Drug The goal of rational drug design is to produce the structural analogues of the biologically active polypeptide of interest (ie, a FGF-19 polypeptide) or of small molecules with which they interact, for example, the agonists, antagonists, or inhibitors. Any of these examples can be used to adapt to drugs which are more active or stable forms of the FGF-19 polypeptide or which improve or interfere with the function of the FGF-19 polypeptide in vivo (cotéjese, Hodgson, Bio / Technology, 9 19-21 (1991)). In one method, the three-dimensional structure of the FGF-19 polypeptide, or of a FGF-19 inhibitor-polypeptide complex, is determined by x-ray crystallography, by computer modeling or, more typically, by a combination of the two methods. Both the shape and charges of the FGF-19 polypeptide must be ascertained to envision the structure and determine the site (s) of activity of the molecule. Less frequently, useful information regarding the structure of the FGF-19 polypeptide can be obtained by modeling based on the structure of the homologous proteins. In both cases, the relevant structural information is used to design molecules similar to the analogous FGF-19 polypeptide or to identify efficient inhibitors. Useful examples of the rational drug design may include molecules which have improved activity or stability as shown by Braxton and Wells, Biochemistry, 31: 7796-7801 (1992) or which act as inhibitors, agonists, or antagonists of natural peptides as shown by Athauda et al., J, Biochem., 113: 742-746 (1993).

It is also possible to isolate a target-specific antibody, selected by the functional assay, as described above, and then to resolve its crystal structure. This method, initially, produces a pharmaceutical core upon which the design of the subsequent drug may be based. It is possible to derive the crystallography of the proteins together by the generation of the anti-idiotypic antibodies (anti-ids) for a pharmacologically active, functional antibody. Like a mirror image of an image in the mirror, the binding site or agglutination of the anti-ids could be expected to be an analogue of the original receptor. The anti-id could then be used to identify and isolate the peptides from the banks of the chemically or biologically produced peptides. The isolated peptides could then act as a pharmacological core. By virtue of the present invention, sufficient quantities of the FGF-19 polypeptide may be available to perform such analytical studies as X-ray crystallography. In addition, knowledge of the amino acid sequence of the FGF-19 polypeptide provided herein will provide a guide for those people who use computer modeling techniques instead of or in addition to X-ray crystallography.

EXAMPLE 11 Investigation of Weight, Leptin Levels, Urine Production, Oxygen Consumption, and Levels of Triglycerides and Free Fatty Acids in Transgenic FGF-19 Mice As described here, FGF-19 has been identified recently as an element of a growing family of secreted proteins related to the growth factor of fibroblasts. FGF-19 has been characterized herein as interacting with receptor 4 of FGF and it does not seem to act as a mitogen. To further investigate the functions of this protein, transgenic mice have been generated which express human FGF-19. In particular, the cDNA encoding human FGF-19 was cloned into a plasmid containing the promoter for the myosin light chain. This promoter is sufficient for the specific transcription of the transgene muscles. An acceptor or donor of the splice was also included 5 'with respect to the cDNA of FGF-19 to increase the level of expression and a donor and acceptor of the splice with a signal of addition of poly A was included 3' with respect to the cDNA of the FGF -19 to increase the level of transcription and to provide a transcription termination site. The DNA that encompasses the MLC promoter, the acceptor and donor of the 5 'splice, the cDNA of FGF-19 and the acceptor and donor of the 3' splice and the transcription termination site (the transgene) was released from the bacterial vector sequences using the enzymes of appropriate restriction and purified after fractionation by size on the agarose gels. The purified DNA was injected into a pronucleus of the fertilized mouse eggs and the transgenic mice generated and identified as described (Genetic Modification of Animáis; Tim Stewart; In Exploring Genetic Mechanisms pp.565-598; 1997 Eds M Singer and P Berg; University Science Books; Sausalito, Calif). The mice were 6 weeks of age for the measurements described below for water intake, food intake, urine output and hematocrit count. The measurements of leptin, triglycerides and free fatty acids were on the same animals at 8 weeks of age. As the results described below show, these mice demonstrate increased food intake and increased metabolic rate as evidenced by their oxygen consumption rate. Despite increased food intake, these mice weigh significantly less than their non-transgenic littermates. This reduced body weight seems to be a consequence of reduced adiposity such as leptin which correlates closely with adipose tissue mass in humans and rodents and which is reduced in transgenic mice. In further support of this, the transgenic mice have a normal linear growth as evaluated by measurements of the length of the nose to the hip or hind quarter. They are normal with respect to the values of body temperature, body (bone length) and hematology. In a manner consistent with increased food intake, the transgenic mice have an increased urine yield. Because the mice do not appear to drink more and are not dehydrated as determined by a normal hematocrit count, the increased urine yield may be derived from increased food metabolism. Because FGF-19 reduces adiposity without altering muscle mass or the formation of long bones, FGF-19 is indicated as an effective therapeutic element in the treatment of obesity and related conditions.

More particularly, the MLC-FGF-19 transgenic mice were weighed at various instants under different fasting and feeding conditions. More particularly, the groups of transgenic female FGF-19 mice and their non-transgenic littermates were weighed at 6 weeks of age during ad libitum feeding, after 6 and 24 hours of fasting and 24 hours after the completion of a fast of 24 hours. As shown in Figure 3A, under all conditions, transgenic FGF-19 mice (continuous bars) weighed less than their non-transgenic, wild-type littermates (dotted bars). Figure 3B shows the sera from the same groups of mice depicted in Figure 3A, evaluated for leptin. Reduced leptin in transgenic FGF-19 mice is consistent with lower body weights (Figure 3A) that are due to reduced adiposity. A group of 6-week-old transgenic mice was verified in the admission of food (Figure 4A), the admission of water (Figure 4B), the yield of urine (Figure 4C) and the hematocrit count (Figure 4D). As can be seen, transgenic FGF-19 mice (continuous bars) consume more food than their wild type littermates, but do not drink more. Although there is no change in water consumption, the transgenic mice produce more urine (Figure 4C). Despite the increase in urine production, the transgenic mice do not appear to be dehydrated as evidenced by the normal hematocrit count (Figure 4D). The reduction in body weight (Figure 3) with an increase in food consumption (Figure 4) could be explained as an increase in metabolic rate. The metabolic rate was determined by the measurement of oxygen consumption. As shown in Figure 5, transgenic FGF-19 mice have an increased metabolic rate during both light cycles, following a 24-hour fast and 24 hours after the end of a 24-hour fast. Obesity and high levels of triglycerides and free fatty acids are risk factors for cardiovascular disease. As FGF-19 reduces one of the risk factors for cardiovascular disease (obesity (Figure 3)), it was investigated whether FGF-19 could also reduce other risk factors. As can be seen in Figure 6, the level of triglycerides and free fatty acids (FFA) is also lower in FGF-19 transgenic mice.

EXAMPLE 12 Infusion of FGF-19 leads to an Increase in the Absorption of Foods and an Increase in Oxygen Consumption To confirm that the effects observed in the transgenic FGF-19 mice were caused by the FGF-19 protein, Groups of non-transgenic FvB mice were infused with the recombinant FGF-19 (1 mg / kg / day, iv) supplied by an osmotically driven implanted pump. As shown in Figures 7A-B, administration of the recombinant human FGF-19 causes an increase in the intake of food when compared to mice infused with the carrier alone. In addition, the infusion of FGF-19 leads to an increase in metabolic rate as measured by oxygen consumption.

EXAMPLE 13 FGF-19 Reduces Glucose Absorption and Increases Peptide Release of Adipocytes To further investigate the mechanism by which FGF-19 alters metabolism, recombinant human FGF-19 was added to adipocyte cultures of primary rat and the absorption of glucose and the release of leptin by the cells was measured. As shown in Figures 8A-B, FGF-19 increases the release of leptin from, and reduces the absorption of glucose in the primary rat adipocytes.

EXAMPLE 14 Investigation of Glucose Tolerance and Weights of Fat Pads on Transgenic FGF-19 Mice Fed with High Fat Diets In general, mice (and humans) with a high fat diet will increase weight and adiposity and will become either diabetic or glucose intolerant.

To examine whether exposure to FGF-19 will have an impact on the adiposity and glucose tolerance groups of transgenic mice and their non-transgenic littermates (of corresponding age and sex), littermates were provided a diet high in fat as described by Rubeffe-Scrive et al Metabolism Vol. 42, No. 11 '1993 pp.1405-1409 and Surwit et al Metabolism, Vol. 44, No. 5 1995 pp. 645-651 with the modification that the sodium content was normalized with respect to the normal food (diets prepared by Research Diets Inc. Catalog No. D12330N). After ten weeks of either normal or high-fat diet, the mice (transgenic females and their non-transgenic littermates) were subjected to a glucose tolerance test. Therefore each mouse was injected intraperitoneally with 1.0 mg of glucose per kg of body weight and the concentration of glucose present in the blood was measured at intervals following the injection. The graph in Figure 10 shows the glucose levels in the mice and shows that 8/9 of the non-transgenic female mice that have been fed the high-fat diet could be defined as diabetic (glucose levels at 2 hours higher that 200 mg / dl; (World Book of Diabetes in Practice. Vo] 3; Ed. Krall, L.P .; Elsevier)) while 0/5 of the transgenic mice fed a comparable diet could be considered diabetic. The male mice that were fed the high-fat diet were sacrificed after being in the diet for either 6 or 10 weeks and the adiposity was determined by measuring the weights of the specific fat deposits. As shown in Figure 9, the transgenic mice that have been fed a high-fat diet were significantly less fat than the non-transgenic littermates.

Material Deposit The following materials have been deposited in the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209, USA (ATCC): ATCC Material Dep. No. Date of Deposit DNA49435-1219 209480 November 21, 1997 This deposit was made under the conditions of Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations under it (Budapest Treaty). This ensures the maintenance of a viable crop of the deposit for 30 years from the date of deposit. The deposit will be made available by the ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc., and the ATCC, which ensures the permanent and unrestricted availability of the progeny of the deposit culture to the public during the issuance of the US patent relevant and during the open presentation to the public of any U.S. patent application. or foreign, whichever is first, and ensures the availability of the progeny to a determined one by the United States Patent and Trademark Commissioner who will be authorized with respect to it in accordance with 35 USC §122 and the rules of the Commissioner with respect thereto (including 37 CFR §1.14 with particular reference to 886 OG 638). The assignee of the present application agrees that if a culture of the materials on the deposit must die or be lost or destroyed when cultivated under the right conditions, the materials will be quickly replaced during notification with others of the same type. The availability of the deposited material will not be considered as a license for the practice of the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws. The above written specification is considered to be sufficient to enable a person skilled in the art to practice the invention. The present invention will not be limited in scope by the deposited construction, since the deposited mode is proposed as a single illustration of certain aspects of the invention and any constructions that are functionally equivalent are within the scope of this invention. The deposit of the material here does not constitute an admission that the written description contained herein is inadequate to practice any aspect of the invention, including the best mode thereof, nor shall it be construed as limiting the scope of the claims for the illustrations. specific that it represents. Actually, various modifications of the invention in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (77)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. An isolated nucleic acid molecule, characterized in that it comprises the DNA having at least about 80% identity of the sequence with respect to (a) ) a DNA molecule encoding a FGF-19 polypeptide comprising the sequence of amino acid residues from about 1 or about 23 to about 216 of Figure 2 (SEQ ID NO: 2), or (b) the complement of the DNA molecule of (a).
2. 2. The isolated nucleic acid molecule according to claim 1, characterized in that it comprises the sequence of the positions of the nucleotides from about 464 or about 530 to about llll of Figure 1 (SEQ ID NO: 1).
3. 3. The isolated nucleic acid molecule according to claim 1, characterized in that it comprises the nucleotide sequence of Figure 1 (SEQ ID NO: 1).
4. 4. The isolated nucleic acid molecule according to claim 1, characterized in that it comprises a nucleotide sequence that encodes the sequence of the amino acid residues from about 1 or about 23 to about 216 of Figure 2 (SEQ ID NO: 2) ).
5. 5. An isolated nucleic acid molecule, characterized in that it comprises DNA comprising at least about 80% sequence identity with respect to (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA deposited with the ATCC on November 21, 1997 under ATCC Deposit No. 209480 (DNA49435-1219), or (b) the complement of the DNA molecule of (a).
6. The isolated nucleic acid molecule according to claim 5, characterized in that it comprises the DNA encoding the same mature polypeptide encoded by the cDNA of the human protein deposited with the ATCC on November 21, 1997 under the ATCC Deposit No 209480 (DNA49435-1219).
7. 7. An isolated nucleic acid molecule, characterized in that it comprises the DNA comprising at least about 80% sequence identity with respect to (a) the coding sequence of the full length polypeptide of the human protein cDNA deposited in the ATCC on November 21, 1997 under ATCC Deposit No. 209480 (DNA49435-1219), or (b) the complement of the coding sequence of (a).
8. 8. The isolated nucleic acid molecule according to claim 7, characterized in that it comprises the coding sequence of the full length polypeptide of the human protein cDNA deposited with the ATCC on November 21, 1997 under the ATCC Deposit No. 209480 (DNA49435-1219).
9. 9. An isolated nucleic acid molecule, characterized in that it encodes a FGF-19 polypeptide comprising a DNA that hybridizes to the complement of the nucleic acid sequence encoding amino acids 1 or about 23 to about 216 of Figure 2 (SEQ. ID NO: 2).
10. 10. The isolated nucleic acid molecule according to claim 9, characterized in that the nucleic acid encoding amino acids 1 or about 23 to about 216 of Figure 2 (SEQ ID NO: 2), comprises nucleotides 464 or about 530 to about llll of Figure 1 (SEQ ID NO: 1).
11. 11. The isolated nucleic acid molecule according to claim 9, characterized in that the hybridization occurs under severe washing and hybridization conditions.
12. 12. An isolated nucleic acid molecule, characterized in that it comprises (a) the DNA encoding a polypeptide having a value of at least 80% positive when compared to the sequence of amino acid residues from 1 or about 23 to about 216 Figure 2 (SEQ ID NO: 2), or (b) the DNA complement of (a).
13. 13. An isolated nucleic acid molecule, characterized in that it comprises at least about 22 nucleotides and which is produced by the hybridization of a test DNA molecule under stringent hybridization conditions with (a) a DNA molecule which encodes a FGF-19 polypeptide comprising a sequence of amino acid residues from 1 or about 23 to about 216 of Figure 2 (SEQ ID NO: 2), or (b) the complement of the DNA sequence of (a), and isolating the DNA from the test DNA.
14. 14. The isolated nucleic acid molecule according to claim 13, characterized in that it has at least about 80% identity of the sequence with (a) or (b).
15. 15. A vector, characterized in that it comprises the nucleic acid molecule according to any of claims 1 to 14.
16. 16. The vector according to claim 15, characterized in that the nucleic acid molecule is operatively linked to the control sequences recognized by a host cell transformed with the vector.
17. 17. A nucleic acid molecule, characterized in that it is as deposited in the ATCC under accession number 209480 (DNA49435-1219).
18. 18. A host cell, characterized in that it comprises the vector according to claim 15.
19. 19. The host cell according to claim 18, characterized in that the cell is a CHO cell.
20. 20. The host cell according to claim 18, characterized in that the cell is one of E. coli.
21. 21. The host cell according to claim 18, characterized in that the cell is a yeast cell.
22. 22. A process for the production of a FGF-19 polypeptide, characterized in that it comprises culturing the host cell according to claim 18 under conditions suitable for expression of the FGF-19 polypeptide and recovering the FGF-19 polypeptide from the culture. cell phone.
23. 23. An isolated FGF-19 polypeptide, characterized in that it comprises an amino acid sequence comprising at least 80% sequence identity with the sequence of amino acid residues from about 1 or about 23 to about 216 of Figure 2 ( SEQ ID NO: 2).
24. 24. The FGF-19 polypeptide isolated according to claim 23, characterized in that it comprises amino acid residues 1 or approximately 23 to approximately 216 of Figure 2 (SEQ ID NO: 2).
25. 25. An isolated FGF-19 polypeptide, characterized in that it has at least about 80% sequence identity to the polypeptide encoded by the vector's cDNA insert deposited with the ATCC on November 21, 1997 as the ATCC Repository No. 209480 (DNA49435-1219).
26. 26. The FGF-19 polypeptide isolated according to claim 25, characterized in that it is encoded by the cDNA insert of the vector deposited with the ATCC on November 21, 1997 as the ATCC Deposit No. 209480 (DNA49435-1219) .
27. 27. An isolated FGF-19 polypeptide having a value of at least 80% positive when compared to the sequence of amino acid residues from 1 or about 23 to about 216 of Figure 2 (SEQ ID NO: 2).
28. 28. An isolated FGF-19 polypeptide, characterized in that it comprises the sequence of the amino acid residues from 1 or about 23 to about 216 of Figure 2 (SEQ ID NO: 2), or a fragment thereof sufficient to provide a site of binding for an anti-FGF-19 antibody.
29. 29. An isolated polypeptide, characterized in that it is produced by (i) hybridizing a test DNA molecule under severe conditions with (a) a DNA molecule encoding a FGF-19 polypeptide comprising the sequence of amino acid residues from 1 or about 23 to about 216 of Figure 2 (SEQ ID NO: 2), or (b) the complement of the DNA molecule of (a), (ii) culturing a host cell comprising the test DNA molecule under suitable conditions for the expression of the polypeptide; and (iii) recovering the polypeptide from the cell culture.
30. 30. The isolated polypeptide according to claim 29, characterized in that the test DNA has at least about 80% identity of the sequence with respect to (a) or (b). .
31. 31. A chimeric molecule, characterized in that it comprises a FGF-19 polypeptide fused to a heterologous amino acid sequence.
32. 32. The chimeric molecule according to claim 31, characterized in that the heterologous amino acid sequence is a sequence of the epitope tag.
33. 33. The chimeric molecule according to claim 31, characterized in that the heterologous amino acid sequence is an Fc region of an immunoglobulin.
34. 34. An antibody, characterized in that it binds specifically to a FGF-19 polypeptide.
35. 35. The antibody according to claim 34, characterized in that the antibody is a monoclonal antibody.
36. 36. The antibody according to claim 34, characterized in that the antibody is a humanized antibody.
37. 37. The antibody according to claim 34, characterized in that the antibody is an antibody fragment.
38. 38. An agonist, characterized in that it is for a polypeptide of FGF-19.
39. 39. An antagonist, characterized in that it is for a polypeptide of FGF-19.
40. 40. A composition of material, characterized in that it comprises (a) a polypeptide of FGF-19, (b) an agonist for a polypeptide of FGF-19, (c) an antagonist for a polypeptide of FGF-19, or (d) an anti-FGF-19 antibody mixed with a pharmaceutically acceptable carrier.
41. 41. A method for the selection of a bioactive agent capable of binding to FGF-19, characterized in that it comprises: a) adding a candidate bioactive agent to a sample of FGF-19, and b) determining the binding of the candidate agent to FGF-19 , wherein the linkage indicates a bioactive agent capable of binding to FGF-19.
42. 42. A method for the selection of a bioactive agent capable of modulating the activity of FGF-19, the method is characterized in that it comprises the steps of: a) adding a candidate bioactive agent to a sample of FGF-19, and b) determining a alteration in the biological activity of FGF-19, where an alteration indicates a bioactive agent capable of modulating the activity of the FGF-19.
43. 43. A method according to claim 42, characterized in that the biological activity is the reduced absorption of glucose in the adipocytes.
44. 44. A method in accordance with the claim 42, characterized in that the biological activity is the increased release of leptin from the adipocytes.
45. 45. A method of identifying a receptor for FGF-19, the method is characterized in that it comprises combining FGF-19 with a composition comprising the material of the cell membrane wherein FGF-19 forms a complex with a receptor on the material of the cell membrane, and identify the receptor as a receptor for FGF-19.
46. 46. The method of compliance with the claim 45, characterized in that FGF-19 binds to the receptor, and the method further includes a cross-linking step of FGF-19 and the receptor.
47. 47. The method according to claim 45, characterized in that the composition is a cell.
48. 48. The method according to claim 45, characterized in that the composition is a preparation of the cell membrane extract.
49. 49. A method of inducing the release of leptin from adipocyte cells, the method is characterized in that it comprises administering FGF-19 to the cells in an amount effective to induce the release of leptin.
50. 50. The method according to claim 49, characterized in that FGF-19 is administered as a protein.
51. 51. The method according to claim 49, characterized in that the FGF-19 is administered as a nucleic acid.
52. 52. A method for inducing a reduction in glucose uptake in adipocyte cells, the method is characterized in that it comprises administering FGF-19 to the cells in an amount effective to induce a reduction in glucose uptake.
53. 53. The method according to claim 52, characterized in that FGF-19 is administered as a protein.
54. 54. The method according to claim 52, characterized in that the FGF-19 is administered as a nucleic acid.
55. 55. A method of treating obesity in an individual, the method is characterized in that it comprises administering to the individual a composition comprising FGF-19 in an amount effective to treat obesity.
56. 56. The method according to claim 55, characterized in that the treatment of obesity also leads to the treatment of a condition related to obesity.
57. 57. The method according to claim 55, characterized in that FGF-19 is administered as a protein.
58. 58. The method of compliance with the claim 55, characterized in that FGF-19 is administered as a nucleic acid.
59. 59. The method according to claim 55, characterized in that the composition further comprises a pharmaceutically acceptable carrier.
60. 60. The method according to claim 55, characterized in that the FGF-19 has at least about 85% identity of the amino acid sequence with respect to the amino acid sequence shown in Figure 2 (SEQ ID NO: 2).
61. 61. A method for reducing total body mass in an individual, the method is characterized in that it comprises administering to the individual an effective amount of FGF-19.
62. 62. The method according to claim 61, characterized in that FGF-19 is administered as a protein.
63. 63. The method according to the claim 61, characterized in that FGF-19 is administered as a nucleic acid.
64. 64. The method according to claim 61, characterized in that the FGF-19 is administered with a pharmaceutically acceptable carrier.
65. 65. The method according to claim 61, characterized in that the reduction in total body mass includes a reduction in the individual's fat.
66. 66. The method according to claim 61, characterized in that the FGF-19 has at least about 85% identity of the amino acid sequence with respect to the amino acid sequence shown in Figure 2 (SEQ ID NO: 2).
67. 67. A method of reducing the level of at least one of triglycerides and free fatty acids in an individual, the method is characterized in that it comprises administering to the individual an effective amount of FGF-19.
68. 68. The method of compliance with the claim 67, characterized in that FGF-19 is administered as a protein.
69. 69. The method according to claim 67, characterized in that the FGF-19 is administered as a nucleic acid.
70. 70. The method according to claim 67, characterized in that the FGF-19 is administered with a pharmaceutically acceptable carrier.
71. 71. The method according to claim 67, characterized in that the FGF-19 has at least about 85% identity of the amino acid sequence with the amino acid sequence shown in Figure 2 (SEQ ID NO: 2).
72. 72. A method for increasing metabolic rate in an individual, the method is characterized in that it comprises administering to the individual an effective amount of FGF-19.
73. 73. The method according to claim 72, characterized in that the FGF-19 is administered as a protein.
74. 74. The method according to claim 72, characterized in that the FGF-19 is administered as a nucleic acid.
75. 75. The method according to claim 72, characterized in that the FGF-19 is administered with a pharmaceutically acceptable carrier-
76. 76. The method according to the claim 72, characterized in that FGF-19 has at least about 85% identity of the amino acid sequence with the amino acid sequence shown in Figure 2 (SEQ ID NO: 2).
77. 77. A rodent, characterized in that it comprises a genome comprising a transgene encoding FGF-19.