KR20020059591A - Fibroblast Growth Factor-19 (FGF-19) Nucleic acid and Polypeptides and Methods of Use for the Treatment of Obesity - Google Patents

Abstract

The present invention relates to novel polypeptides belonging to the fibroblast growth factor family and nucleic acid molecules encoding these polypeptides. Also provided are vectors and host cells comprising these nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptide of the invention fused with heterologous polypeptide sequences, antibodies binding to the polypeptide of the invention, and methods of making the polypeptide of the invention. do. Also provided are methods of treating obesity.

Description

Fibroblast Growth Factor-19 (FGF-19) Nucleic acid and Polypeptides and Methods of Use for the Treatment of Obesity

Obesity is a chronic disease that is very widespread in modern societies, and not only social stigma, but also reduced lifespan, and psychological retrodevelopment, reproductive diseases such as polycystic ovarian disease, infections, skin diseases such as varicose veins, melanoma and eczema, and exercise tolerance , Diabetes, insulin resistance, hypertension, hypercholesterolemia, cholelithiasis, osteoarthritis, orthopedic injury, thromboembolic diseases, cancer and coronary heart disease [Rissanen et al., British Medical Journal, 301: 835-837 (1990).

Conventional obesity treatments include standard diet and exercise, low calorie diets, behavioral therapies, appetite suppressants, pyrogenic drugs, medical devices such as food absorption inhibitors, chin sutures, waist cords and instruments, And surgery [Jung and Chong, Clinical Endocrinology, 35: 11-20 (1991); Bray, Am. J. Clin. Nutr., 55: 538S-544S (1992). Modified fasting of protein retention has been reported to be effective in weight loss in adolescents [Lee et al., Clin. Pediatr., 31: 234-236 (4 April 1992). Calorie restriction as a treatment for obesity leads to catabolism of the protein store in the body and leads to negative nitrogen balance. Thus, protein supplemented diets have gained popularity as a means of reducing nitrogen loss during calorie restriction. Since the diet only results in moderate nitrogen retention, there is a need for more effective methods to preserve thin body weight and protein stores. In addition, if this regimen resulted in an increased loss of body fat, treatment of obesity would be improved. Various methods for this treatment are described in Weintraub and Bray, Med. Clinics N. Amer., 73: 237 (1989); Bray, Nutrition Reviews, 49:33 (1991).

Given the vastness of obesity in our society and the serious consequences associated with it as discussed above, any therapeutic drug that is potentially useful for reducing the weight of obese patients may have a very beneficial effect on their health. There is a need in the art for drugs that will reduce the weight of obese patients to their ideal weight without significant side effects and will help obese patients maintain reduced weight.

Thus, it would be desirable to provide a therapeutic regimen useful for restoring the body weight of obese patients to their normal, ideal weight.

Furthermore, it is also desirable to provide obesity treatments that result in long-term loss of weight maintenance.

In addition, it is desirable to prevent obesity and, once treatment is initiated, to prevent the onset of, or to prevent the evolution of, obesity-derived diseases such as atherosclerosis and polycystic ovary disease, or of obesity.

Such methods of treatment and related compositions are provided herein. Also provided are methods of screening novel proteins and nucleic acids, and modulators thereof. Other methods, treatments, and compositions presented herein will be apparent to those skilled in the art.

The present invention generally relates to the identification and isolation of novel DNA, the recombinant production of novel polypeptides referred to herein as fibroblast growth factor-19 (FGF-19) polypeptides, and therapeutic and pharmacology involving obesity treatment, and obesity treatment. It relates to methods, compositions and assays for using such polypeptides to produce pharmaceutically active substances having specific properties.

1 shows the nucleotide sequence (SEQ ID NO: 1) of a cDNA containing the nucleotide sequence (nucleotides 464-1111) encoding native sequence FGF-19, wherein the nucleotide sequence (SEQ ID NO: 1) is herein Clones labeled "DNA49435-1219". Also, the underlined bold portions are the starting and ending codon positions, respectively.

2 is the amino acid sequence of the native sequence FGF-19 polypeptide derived from the coding sequence of SEQ ID NO: 1 (SEQ ID NO: 2). In addition, approximate positions of various other important polypeptide domains are indicated.

3A and 3B show that MLC-FGF-19 transgenic mice weigh less than untransgenic litters (FIG. 3A) and have lower blood leptin levels (FIG. 3B). FIG. 3A shows 6-week-old FGF-19 transgenic mice (continuous bars) and non-transformation during libitum feeding (leftmost), after 6 and 24 hours fasting, and 24 hours after the end of 24 hours fasting (far right). Wild type) Shows the weight of litter (dotted bar). FIG. 3B shows the serum of the same group of mice shown in FIG. 3A in a leptin assay (vertical bar).

4A-4D are bar graphs showing that FGF-19 transgenic mice exhibit increased food intake and urine production, but have normal hematocrit. Mice from any group were monitored for food intake (FIG. 4A), water intake (FIG. 4B), urinary excretion (FIG. 4C) and hematocrit (FIG. 4D) during libitum feeding and 24 hours after the end of 24 hours fasting (each The results for the FGF-19 transgenic mice in the graph are shown as continuous bars and the results for wild type are shown as dotted bars).

5 is a bar graph showing that FGF-19 transgenic mice have increased oxygen consumption rate. Oxygen consumption is indicated for FGF-19 transgenic mice (continuous bars) and wild-type (dotted bars) at 24 hours after a 24 hour fast and at the end of a 24 hour fast during the light cycle (cancer or light).

6A and 6B are bar graphs showing that FGF-19 transgenic mice (continuous bars) have reduced triglycerides (FIG. 6A) and free fatty acids (FIG. 6B) compared to wild type mice (dotted bars).

7A and 7B show increased food intake (FIG. 7A) and oxygen compared to non-transgenic mice injected with FGF-19 (continuous bars) compared to mice injected with vehicle lacking FGF-19 (dotted bars). Bar graph indicating an increase in consumption (FIG. 7B), where “n” means night and “d” means low.

8A and 8B are bar graphs showing that FGF-19 increases leptin release from adipocytes (Fig. 8A) and decreases glucose uptake by adipocytes (Fig. 8B).

9 is a bar graph showing the fat pad weight of FGF-19 transgenic mice (hatched bars) or wild type mice (continuous bars) over time in a high fat diet (HFD) (bars horizontally from left) Results for weekly epididymis (HFD Ep), peripapillary and posterior peritoneum (HFD RP / PR), and epididymis, peripapillary and retroperitoneum at week 10 are shown)

FIG. 10 is a bar graph showing glucose tolerance of FGF-19 transgenic mice (hatched bars) or wild type mice (continuous bars) over time (both high fat diets for 10 weeks).

Summary of the Invention

Identifies cDNA clones (denoted herein as DNA49435-1219) encoding novel polypeptides that have some sequence similarity with members of the fibroblast growth factor family designated herein as "fibroblast growth factor-19 (FGF-19)". It was.

In one embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FGF-19 polypeptide.

In one aspect, an isolated nucleic acid molecule is (a) a DNA molecule encoding a PEACH polypeptide having an amino acid residue sequence of about 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2), or (b) (a At least about 80% nucleic acid sequence identity, or at least about 81% nucleic acid sequence identity, or at least about 82% nucleic acid sequence identity, or at least about 83% nucleic acid sequence identity, or at least about 84% nucleic acid sequence identity with the complement of the DNA molecule Or at least about 85% nucleic acid sequence identity, or at least about 86% nucleic acid sequence identity, or at least about 87% nucleic acid sequence identity, or at least about 88% nucleic acid sequence identity, or at least about 89% nucleic acid sequence identity, or about 90% At least about nucleic acid sequence identity, or at least about 91% nucleic acid sequence identity, or at least about 92% nucleic acid sequence identity, or at least about 93% nucleic acid sequence identity, or at least about 94% nucleic acid sequence identity, or about 95% In comprises the nucleic acid sequence identity, or at least about 96% nucleic acid sequence identity, or about 97% or more nucleic acid sequence identity, or a nucleotide sequence having at least about 98% nucleic acid sequence identity, or at least about 99% nucleic acid sequence identity.

In another aspect, an isolated nucleic acid molecule comprises (a) a nucleotide sequence encoding a FGF-19 polypeptide having an amino acid residue sequence of about 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2), or (b ) includes the complement of the nucleotide sequence of (a).

In another aspect, an isolated nucleic acid molecule is (a) the DNA molecule having a nucleotide sequence of about 464 or about 530 to about 1111 of FIG. 1 (SEQ ID NO: 1), or (b) the complement of the DNA molecule of (a) At least about 80% nucleic acid sequence identity, or at least about 81% nucleic acid sequence identity, or at least about 82% nucleic acid sequence identity, or at least about 83% nucleic acid sequence identity, or at least about 84% nucleic acid sequence identity, or at least about 85% Nucleic acid sequence identity, or at least about 86% nucleic acid sequence identity, or at least about 87% nucleic acid sequence identity, or at least about 88% nucleic acid sequence identity, or at least about 89% nucleic acid sequence identity, or at least about 90% nucleic acid sequence identity, or At least about 91% nucleic acid sequence identity, or at least about 92% nucleic acid sequence identity, or at least about 93% nucleic acid sequence identity, or at least about 94% nucleic acid sequence identity, or at least about 95% nucleic acid sequence identity, It comprises a nucleotide sequence having at least about 96% nucleic acid sequence identity, or about 97% or more nucleic acid sequence identity, or about 98% or more nucleic acid sequence identity, or at least about 99% nucleic acid sequence identity.

In another aspect, an isolated nucleic acid molecule comprises (a) the nucleotide sequence of about 464 or about 530 to about 1111 of FIG. 1 (SEQ ID NO: 1), or (b) the complement of the nucleotide sequence of (a) .

In a further aspect, the present invention provides a DNA molecule encoding the same mature polypeptide as that encoded by the human protein cDNA (DNA49435-1219) deposited at ATCC on November 21, 1997 under ATCC Accession No. 209480, or ( b) at least about 80% nucleic acid sequence identity, or at least about 81% nucleic acid sequence identity, or at least about 82% nucleic acid sequence identity, or at least about 83% nucleic acid sequence identity, or about 84% to the complement of the DNA molecule of (a) At least nucleic acid sequence identity, or at least about 85% nucleic acid sequence identity, or at least about 86% nucleic acid sequence identity, or at least about 87% nucleic acid sequence identity, or at least about 88% nucleic acid sequence identity, or at least about 89% nucleic acid sequence identity, Or at least about 90% nucleic acid sequence identity, or at least about 91% nucleic acid sequence identity, or at least about 92% nucleic acid sequence identity, or at least about 93% nucleic acid sequence identity, or at least about 94% nucleus Having acid sequence identity, or at least about 95% nucleic acid sequence identity, or at least about 96% nucleic acid sequence identity, or at least about 97% nucleic acid sequence identity, or at least about 98% nucleic acid sequence identity, or at least about 99% nucleic acid sequence identity It relates to an isolated nucleic acid molecule comprising a nucleotide sequence. In a preferred embodiment, the isolated nucleic acid molecule is (a) a nucleotide sequence encoding the same mature polypeptide as that encoded by the human protein cDNA (DNA49435-1219) deposited at ATCC on November 21, 1997 under ATCC accession number 209480, Or (b) the complement of the nucleotide sequence of (a).

In another aspect, the invention provides an antibody comprising (a) the full-length polypeptide coding sequence of a human protein cDNA (DNA49435-1219) deposited with ATCC as of November 21, 1997 under ATCC Accession No. 209480, or (b) of (a) At least about 80% nucleic acid sequence identity, or at least about 81% nucleic acid sequence identity, or at least about 82% nucleic acid sequence identity, or at least about 83% nucleic acid sequence identity, or at least about 84% nucleic acid sequence identity with the complement of the nucleotide sequence, or At least about 85% nucleic acid sequence identity, or at least about 86% nucleic acid sequence identity, or at least about 87% nucleic acid sequence identity, or at least about 88% nucleic acid sequence identity, or at least about 89% nucleic acid sequence identity, or at least about 90% nucleic acid Sequence identity, or at least about 91% nucleic acid sequence identity, or at least about 92% nucleic acid sequence identity, or at least about 93% nucleic acid sequence identity, or at least about 94% nucleic acid sequence identity, and Comprises a nucleotide sequence having at least about 95% nucleic acid sequence identity, or at least about 96% nucleic acid sequence identity, or at least about 97% nucleic acid sequence identity, or at least about 98% nucleic acid sequence identity, or at least about 99% nucleic acid sequence identity. To an isolated nucleic acid molecule. In a preferred embodiment, the isolated nucleic acid molecule is selected from (a) the full-length polypeptide coding sequence of DNA (DNA49435-1219) deposited at ATCC on November 21, 1997 under ATCC accession number 209480, or (b) of (a). Complement of the nucleotide sequence.

In another aspect, the present invention includes an active FGF as defined below comprising a nucleotide sequence that hybridizes with the complement of a nucleic acid sequence encoding one or about 23 to about 216 amino acids of FIG. 2 (SEQ ID NO: 2). -19 relates to an isolated nucleic acid molecule encoding a polypeptide. Preferably, hybridization occurs under stringent hybridization and washing conditions.

In another aspect, the present invention provides an active FGF-19 polypeptide as defined below comprising a nucleotide sequence that hybridizes with the complement of the nucleic acid sequence of 464 or about 530 to about 1111 of FIG. 1 (SEQ ID NO: 1). It relates to an isolated nucleic acid molecule encoding. Preferably, hybridization occurs under stringent hybridization and washing conditions.

In another aspect, the present invention provides, under stringent conditions, a test DNA molecule comprising (a) a DNA encoding a FGF-19 polypeptide having an amino acid residue sequence from about 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2). Molecule, or (b) hybrid with the complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80% nucleic acid sequence identity, or at least about 81% nucleic acid sequence identity, or about 82 with (a) or (b) % Or more nucleic acid sequence identity, or about 83% or more nucleic acid sequence identity, or about 84% or more nucleic acid sequence identity, or about 85% or more nucleic acid sequence identity, or about 86% or more nucleic acid sequence identity, or about 87% or more nucleic acid sequence identity Or at least about 88% nucleic acid sequence identity, or at least about 89% nucleic acid sequence identity, or at least about 90% nucleic acid sequence identity, or at least about 91% nucleic acid sequence identity, or at least about 92% nucleic acid sequence identity, or about 93% More than Nucleic acid sequence identity, or at least about 94% nucleic acid sequence identity, or at least about 95% nucleic acid sequence identity, or at least about 96% nucleic acid sequence identity, or at least about 97% nucleic acid sequence identity, or at least about 98% nucleic acid sequence identity, or If it has at least about 99% nucleic acid sequence identity, it relates to an isolated nucleic acid molecule having at least about 22 nucleotides, generated by isolating a test DNA molecule.

In another aspect, the invention provides an antibody comprising (a) at least about 80% positive, or at least about 81% positive, as compared to the amino acid residue sequence of about 1 or about 23 to 216 of FIG. 2 (SEQ ID NO: 2), or At least about 82% positive, or at least about 83% positive, or at least about 84% positive, or at least about 85% positive, or at least about 86% positive, or at least about 87% positive, or at least about 88% positive, or about At least 89% positive, or at least about 90% positive, or at least about 91% positive, or at least about 92% positive, or at least about 93% positive, or at least about 94% positive, or at least about 95% positive, or about 96 A nucleic acid sequence encoding a polypeptide having at least% positive, or at least about 97% positive, or at least about 98% positive, or at least about 99% positive, or (b) the nucleo of (a) It relates to an isolated nucleic acid molecule comprising the complement of SEQ ID NO de.

In certain aspects, the invention provides an isolated nucleic acid molecule comprising DNA encoding an FGF-19 polypeptide free of N-terminal signal sequence and / or initiating methionine, or complementary to said coding nucleic acid molecule. The signal peptide was found to temporarily extend from about 1 amino acid to about 22 amino acids in the sequence of FIG. 2 (SEQ ID NO: 2). However, note that while the C-terminal boundary of the signal peptide may vary, it will be only about 5 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C of the signal peptide Terminal boundaries are commonly used in the art for identifying types of amino acid sequence factors (eg, Nielsen et al., Prot. Eng. 10: 1-6 (1997) and von Heinje et al., Nucl. Acids. Res. 14 4683-4690 (1986). In addition, it has been found that in some cases, cleavage of signal sequences from secreted polypeptides is not completely uniform, resulting in one or more secretory species. These polypeptides, and polynucleotides encoding them, are contemplated in the present invention. As such, for purposes herein, the signal peptide of the FGF-19 polypeptide of FIG. 2 (SEQ ID NO: 2) extends from amino acid 1 of FIG. 2 (SEQ ID NO: 2) to X, where X is shown in FIG. Any amino acid of 17 to 27 of SEQ ID NO: 2). Thus, the mature forms of the FGF-19 polypeptides encompassed by the present invention include the amino acids X-216 of Figure 2 (SEQ ID NO: 2) and variants thereof as described below, wherein X is the Figure 2 (SEQ ID). NO: 2) is any amino acid of 17 to 27. Also contemplated are isolated nucleic acid molecules encoding these polypeptides.

Another embodiment may encode a fragment comprising, for example, a fragment of an FGF-19 polypeptide sequence comprising a coding sequence that may be useful as a hybridization probe, or optionally a binding site for an anti-FGF-19 antibody. A coding fragment of the FGF-19 polypeptide. The nucleic acid fragment is usually at least about 20 nucleotides, or at least about 30 nucleotides, or at least about 40 nucleotides, or at least about 50 nucleotides, or at least about 60 nucleotides, or at least about 70 nucleotides, or about 80 Or more nucleotides, or about 90 or more nucleotides, or about 100 or more nucleotides, or about 110 or more nucleotides, or about 120 or more nucleotides, or about 130 or more nucleotides, or about 140 or more nucleotides, or about 150 or more Nucleotides, or about 160 or more nucleotides, or about 170 or more nucleotides, or about 180 or more nucleotides, or about 190 or more nucleotides, or about 200 or more nucleotides, or about 250 or more nucleotides, or about 300 or more Nucleotides, or about 350 or more nucleotides, or about 400 or more nucleotides, or about 450 or more nucleotides, or about 500 or more nucleotides, or about 600 or more nucleotides, or about 700 or more nucleotides, or about 800 or more nucleotides , Or at least about 900 nucleotides, or at least about 1000 nucleotides, wherein the term “about” means ± 10% of the nucleotide sequence length mentioned. In a preferred embodiment, the nucleotide sequence fragment is derived from any coding region in the nucleotide sequence of FIG. 1 (SEQ ID NO: 1). New fragments of FGF-19 polypeptide-encoding nucleotide sequences can be used to arrange FGF-19 polypeptide-coding nucleotide sequences along with other known nucleotide sequences using any of a number of well-known sequence alignment programs, and which FGF-19 polypeptide-coding sequences. It can be determined in a conventional manner by determining if the nucleotide sequence fragment (s) is novel. All of the above FGF-19 polypeptide-encoding nucleotide sequences are contemplated herein and can be determined without undue experimentation. Also contemplated are FGF-19 polypeptide fragments encoded by these nucleotide molecule fragments, preferably FGF-19 polypeptide fragments comprising a binding site for an anti-FGF-19 antibody.

In another embodiment, the present invention provides a vector comprising a nucleotide sequence encoding FGF-19 or a variant thereof. The vector may comprise any of the above isolated nucleic acid molecules.

Also provided are host cells comprising the vectors. By way of example, the host cell can be a CHO cell, E. coli, baculovirus infected insect cell or yeast. Further provided are methods of making the FGF-19 polypeptide, comprising culturing the host cell under conditions suitable for expression of FGF-19 and recovering FGF-19 from the cell culture.

In another embodiment, the present invention provides an isolated FGF-19 polypeptide encoded by any of the above isolated nucleic acid sequences.

In certain aspects, the invention provides an isolated native sequence FGF-19 polypeptide comprising, in some embodiments, an amino acid sequence comprising residues of about 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2). do.

In another aspect, the invention provides about 80% or more amino acid sequence identity, or about 81% or more amino acid sequence identity, or about about 1 or about 23 to about 216 amino acid residue sequences of FIG. 2 (SEQ ID NO: 2) At least 82% amino acid sequence identity, or at least about 83% amino acid sequence identity, or at least about 84% amino acid sequence identity, or at least about 85% amino acid sequence identity, or at least about 86% amino acid sequence identity, or at least about 87% amino acid sequence Identity, or at least about 88% amino acid sequence identity, or at least about 89% amino acid sequence identity, or at least about 90% amino acid sequence identity, or at least about 91% amino acid sequence identity, or at least about 92% amino acid sequence identity, or about 93 At least% amino acid sequence identity, or at least about 94% amino acid sequence identity, or at least about 95% amino acid An isolated FGF-19 comprising an amino acid sequence having column identity, or at least about 96% amino acid sequence identity, or at least about 97% amino acid sequence identity, or at least about 98% amino acid sequence identity, or at least about 99% amino acid sequence identity. It relates to a polypeptide.

In an additional aspect, the present invention provides at least about 80% amino acid sequence identity, or about 81%, with an amino acid sequence encoded by the human protein cDNA deposited on ATCC as of November 21, 1997, under ATCC Accession No. 209480 (DNA49435-1219). At least amino acid sequence identity, or at least about 82% amino acid sequence identity, or at least about 83% amino acid sequence identity, or at least about 84% amino acid sequence identity, or at least about 85% amino acid sequence identity, or at least about 86% amino acid sequence identity, Or at least about 87% amino acid sequence identity, or at least about 88% amino acid sequence identity, or at least about 89% amino acid sequence identity, or at least about 90% amino acid sequence identity, or at least about 91% amino acid sequence identity, or at least about 92% Amino acid sequence identity, or at least about 93% amino acid sequence identity, or at least about 94% amino Amino acids having sequence identity, or at least about 95% amino acid sequence identity, or at least about 96% amino acid sequence identity, or at least about 97% amino acid sequence identity, or at least about 98% amino acid sequence identity, or at least about 99% amino acid sequence identity It relates to an isolated FGF-19 polypeptide comprising a sequence. In a preferred embodiment, the isolated FGF-19 polypeptide comprises an amino acid sequence encoded by the human protein cDNA (DNA49435-1219) deposited at ATCC on November 21, 1997 under ATCC Accession No. 209480.

In an additional aspect, the present invention provides about 80% or more positive, or about 81% or more positive, or about 82 when compared to the amino acid residue sequence of about 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2). At least% positive, or at least about 83% positive, or at least about 84% positive, or at least about 85% positive, or at least about 86% positive, or at least about 87% positive, or at least about 88% positive, or about 89% At least positive, or at least about 90% positive, or at least about 91% positive, or at least about 92% positive, or at least about 93% positive, or at least about 94% positive, or at least about 95% positive, or at least about 96% To an isolated FGF-19 polypeptide comprising an amino acid sequence having a positive, or at least about 97% positive, or at least about 98% positive, or at least about 99% positive will be.

In certain aspects, the invention provides an isolated FGF-19 polypeptide that does not have an N-terminal signal sequence and / or an initiating methionine, and is encoded by a nucleotide sequence encoding an amino acid sequence as described above. Also described herein is a method of making this comprising culturing a host cell comprising a vector comprising a suitable coding nucleic acid molecule under conditions suitable for expression of the FGF-19 polypeptide and recovering the FGF-19 polypeptide from the cell culture. do.

In another aspect, the invention provides an isolated FGF-19 polypeptide comprising a amino acid residue sequence of about 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2), or a biologically active or anti-FGF- 19 to a fragment thereof sufficient to provide a binding site for an antibody, wherein the identification of FGF-19 polypeptide fragments having a biological activity or providing a binding site for an anti-FGF-19 antibody is well known in the art. Can be achieved in a conventional manner. Preferably, the FGF-19 fragment retains the qualitative biological activity of the native FGF-19 polypeptide, including its ability to treat obesity.

In an additional aspect, the present invention provides an antibody comprising (i) a FGF-19 polypeptide having an amino acid residue sequence of about 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2) under stringent conditions. Or (b) hybridizes with the complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80% nucleic acid sequence identity, or at least about 81% nucleic acid sequence identity with (a) or (b) Or at least about 82% nucleic acid sequence identity, or at least about 83% nucleic acid sequence identity, or at least about 84% nucleic acid sequence identity, or at least about 85% nucleic acid sequence identity, or at least about 86% nucleic acid sequence identity, or about 87% At least nucleic acid sequence identity, or at least about 88% nucleic acid sequence identity, or at least about 89% nucleic acid sequence identity, or at least about 90% nucleic acid sequence identity, or at least about 91% nucleic acid sequence identity, or at least about 92% nucleic acid sequence identity, Or about 93% Nucleic acid sequence identity, or at least about 94% nucleic acid sequence identity, or at least about 95% nucleic acid sequence identity, or at least about 96% nucleic acid sequence identity, or at least about 97% nucleic acid sequence identity, or at least about 98% nucleic acid sequence identity, Or, if it has at least about 99% nucleic acid sequence identity, (ii) culturing the 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 a chimeric molecule comprising a FGF-19 polypeptide fused with a heterologous polypeptide or amino acid sequence, which may comprise any FGF-19 polypeptide, variant or fragment thereof as described above. to provide. Examples of such chimeric molecules include FGF-19 polypeptides fused with an epitope tag sequence or an Fc region of an immunoglobulin.

In another embodiment, the present invention provides an antibody as defined below, which specifically binds to a FGF-19 polypeptide as described above. Optionally, the antibody is a monoclonal antibody, antibody fragment or single chain antibody.

In another embodiment, the present invention relates to agonists and antagonists of native FGF-19 polypeptides as defined below. In certain embodiments, the agent or antagonist is an anti-FGF-19 antibody or small molecule.

In an additional embodiment, the invention identifies an agent or antagonist for an FGF-19 polypeptide, comprising contacting the FGF-19 polypeptide with a candidate molecule and monitoring the biological activity mediated by the FGF-19 polypeptide. It is about a method. Preferably, the FGF-19 polypeptide is a native FGF-19 polypeptide.

In an additional embodiment, the present invention relates to a composition mixed with a carrier comprising an FGF-19 polypeptide, an agonist or antagonist of an FGF-19 polypeptide as described herein, or an anti-FGF-19 antibody. Optionally, the carrier is a pharmaceutically acceptable carrier.

Another embodiment of the invention is an FGF-19 polypeptide, an agonist or antagonist thereof as described herein, or an FGF-19 polypeptide, an agonist or antagonist thereof, or an anti-FGF-19 antibody of an anti-FGF-19 antibody. It relates to a use for the manufacture of a medicament useful for the treatment of a condition reactive to.

In one embodiment, a method for screening a biologically active agent capable of binding FGF-19 is provided. In one aspect, the method includes adding a biologically active candidate agent to the FGF-19 sample and determining binding of the candidate agent to the FGF-19, wherein the binding may bind to FGF-19. Biologically active agents.

In addition, a method of screening a biologically active agent capable of modulating the activity of FGF-19 is provided. In one embodiment, a method is provided that includes adding a biologically active candidate agent to a FGF-19 sample and determining a change in the biological activity of FGF-19, wherein the change can modulate the activity of FGF-19. Biologically active agents. In one embodiment, the FGF-19 activity is reduced uptake of glucose in the cell. In another embodiment, the FGF-19 activity is increased leptin free from the cells. In a preferred embodiment, the FGF-19 activity is reduced uptake of glucose and increased leptin free from the cells. Preferably, the cell is an adipocyte. In another embodiment, the FGF-19 activity is increased oxidation of lipids and carbohydrates. Preferably, the cells are liver or muscle cells.

In another embodiment, the present invention provides a method for identifying a receptor for FGF-19. In a preferred embodiment, the method binds FGF-19 with a composition comprising a cell membrane material, wherein FGF-19 complexes with a receptor on the cell membrane material and identifies the receptor as an FGF-19 receptor. Include. In one embodiment, the method comprises crosslinking the FGF-19 with a receptor. The cell membrane may be derived from the original cell or cell membrane extract.

In a further aspect of the invention, a method of inducing leptin release from a cell, preferably an adipocyte, is provided. In one embodiment, the method comprises administering FGF-19 to the cells in an amount effective to induce leptin release.

In the methods provided herein, FGF-19 may be administered in the form of a nucleic acid or protein expressing FGF-19. As described further below, FGF-19 may be administered as an infusion or sustained release formulation. Preferably, FGF-19 can be administered to a subject with a pharmaceutically acceptable carrier.

Also provided are methods for inducing a reduction in glucose uptake in cells, preferably adipocytes. In one embodiment, the method comprises administering FGF-19 to the cells in an amount effective to induce a decrease in glucose uptake.

In another aspect of the invention, a method of treating obesity in an individual is provided. In one embodiment, the method comprises administering to the subject a composition comprising FGF-19 in an amount effective to treat obesity. In this way, conditions associated with obesity, such as cardiovascular disease, can also be treated.

Also provided is a method of reducing the total body weight of an individual, comprising administering to the individual an effective amount of FGF-19. In a preferred embodiment, obesity (fat) in the subject is reduced.

Also provided are methods for reducing one or more levels of triglycerides and free fatty acids in a subject, comprising administering to the subject an effective amount of FGF-19. Also provided is a method of increasing the metabolic rate of an individual comprising administering to the individual an effective amount of FGF-19.

In addition, animal models are provided for determining the effects of FGF-19 and its modulators under various conditions and conditions. In one embodiment there is provided an animal, preferably a rodent, comprising a genome comprising a transforming gene encoding FGF-19.

<Detailed Description of the Preferred Embodiments>

1. Definition

As used herein, the terms "FGF-19 polypeptide", "FGF-19 protein" and "FGF-19" encompass native sequence FGF-19 and FGF-19 polypeptide variants (as further defined herein). FGF-19 polypeptides may be isolated from a variety of sources, such as human tissue types or another source, or may be prepared by recombinant and / or synthetic methods.

"Native sequence FGF-19" includes polypeptides having the same amino acid sequence as native FGF-19. The native sequence FGF-19 can be isolated naturally or can be prepared by recombinant and / or synthetic methods. The term “natural sequence FGF-19” specifically encompasses naturally truncated or secreted forms (eg, extracellular domain sequences), native variant forms (eg, alternatively sliced forms) and natural allelic variants of FGF-19. do. In one embodiment of the invention, native sequence FGF-19 is mature or full-length native sequence FGF-19 comprising the amino acids 1-216 of FIG. 2 (SEQ ID NO: 2). In addition, the FGF-19 polypeptide of FIG. 2 (SEQ ID NO: 2) is shown as beginning with a methionine residue designated herein as amino acid 1, but upstream or downstream of amino acid 1 of FIG. 2 (SEQ ID NO: 2). Another methionine residue located at may be used as the starting amino acid residue for the FGF-19 polypeptide.

“FGF-19 variant polypeptide” refers to (a) 1 or about 23 to 216 of the FGF-19 polypeptide of FIG. 2 (SEQ ID NO: 2), (b) the FGF-19 polypeptide of FIG. 2 (SEQ ID NO: 2) X to 216, wherein X is an amino acid residue from 17 to 27 of FIG. 2 (SEQ ID NO: 2), or (c) another specifically derived from the amino acid sequence of FIG. 2 (SEQ ID NO: 2) By active FGF-19 polypeptide as defined below, having at least about 80% amino acid sequence identity with the amino acid sequence of the fragment. Such FGF-19 variant polypeptides may have, for example, added or deleted one or more amino acid residues within the N- and / or C-terminus of the sequence of FIG. 2 (SEQ ID NO: 2), and one or more internal domains. FGF-19 polypeptides. Usually, the FGF-19 variant polypeptide comprises (a) 1 or about 23 to 216 of the FGF-19 polypeptide of FIG. 2 (SEQ ID NO: 2), and (b) the FGF-19 polypeptide of FIG. 2 (SEQ ID NO: 2). X to 216, wherein X is an amino acid residue from 17 to 27 of FIG. 2 (SEQ ID NO: 2), or (c) another specifically derived from the amino acid sequence of FIG. 2 (SEQ ID NO: 2) At least about 80% amino acid sequence identity, or at least about 81% amino acid sequence identity, or at least about 82% amino acid sequence identity, or at least about 83% amino acid sequence identity, or at least about 84% amino acid sequence identity, or about 85 At least% amino acid sequence identity, or at least about 86% amino acid sequence identity, or at least about 87% amino acid sequence identity, or at least about 88% amino acid sequence identity, or at least about 89% amino acid sequence identity, or at least about 90% amino acid sequence identity. , Or about 91% or more Amino acid sequence identity, or at least about 92% amino acid sequence identity, or at least about 93% amino acid sequence identity, or at least about 94% amino acid sequence identity, or at least about 95% amino acid sequence identity, or at least about 96% amino acid sequence identity, or At least about 97% amino acid sequence identity, or at least about 98% amino acid sequence identity, or at least about 99% amino acid sequence identity. FGF-19 variant polypeptides do not include native FGF-19 polypeptides. Usually, the FGF-19 variant polypeptide comprises at least about 10 amino acids, or at least about 20 amino acids, or at least about 30 amino acids, or at least about 40 amino acids, or at least about 50 amino acids, or at least about 60 amino acids, or About 70 or more amino acids, or about 80 or more amino acids, or about 90 or more amino acids, or about 100 or more amino acids, or about 150 or more amino acids, or about 200 or more amino acids, or about 300 or more amino acids, or More than amino acids.

“Percent amino acid sequence identity (%)” in relation to the FGF-19 polypeptide sequence identified herein refers to an amino acid residue of the FGF-19 sequence after aligning the sequence and introducing a gap as necessary to achieve the maximum sequence identity It is defined as a percentage of amino acid residues in the candidate sequence that are identical to and does not consider any conservative substitutions as part of sequence identity. Arrangements for determining the percentage of amino acid sequence identity may be used within the art using, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megaalign (DNASTAR) software. Can be achieved in a variety of ways. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve the maximum alignment for the full-length sequences to be compared. However, for purposes herein,% amino acid sequence identity values are obtained by using the sequence comparison computer program ALIGN-2, whose complete source code is set forth in Table 1, as described below. The authority of the ALIGN-2 sequence comparison computer program is from Genentech, Inc., and the source code of Table 1 is available from Washington, D., USA. Seed. It was filed with the user dissemination at the U.S. Copyright Office of 20559 and registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc. of South San Francisco, Calif., Or may be compiled from the source code shown in Table 1. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

For purposes herein, the percent amino acid sequence identity of the amino acid sequence A to the amino acid sequence B (or may be represented by the amino acid sequence A having or including a certain percent amino acid sequence identity to the amino acid sequence B) is It is calculated as follows:

100 X fraction X / Y

(Where X is the number of amino acid residues calculated to be the same when arranging A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues of B). If the length of amino acid sequence A is not equal to the length of amino acid sequence B, it will be appreciated that the% amino acid sequence identity of A to B will not be identical to the% amino acid sequence identity of B to A. As an example of% amino acid sequence identity calculations, Tables 2 and 3 illustrate a method of calculating% amino acid sequence identity of an amino acid sequence labeled "Comparative Protein" to an amino acid sequence labeled "PRO."

Unless specifically stated otherwise, all% amino acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program. However,% amino acid sequence identity can also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program can be downloaded from http://www.ncbi.nim.nih.gov or obtained from the National Institute of Sanitation, Bethesda, Maryland. NCBI-BLAST2 uses several irradiation parameters, all of which are, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.

When NCBI-BLAST2 is used for amino acid sequence comparison, to the amino acid sequence A having or including% amino acid sequence identity of the amino acid sequence A to the amino acid sequence B (or a certain% amino acid sequence identity to the amino acid sequence B) Can be expressed as:

100 X fraction X / Y

Where X is the number of amino acid residues calculated to be the same when arranging A and B by the sequence alignment program NCBI-BLAST2, and Y is the total amino acid residues of B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, it will be appreciated that the% amino acid sequence identity of A to B will not be identical to the% amino acid sequence identity of B to A.

“FGF-19 variant polynucleotide” or “FGF-19 variant nucleic acid sequence” refers to (a) a nucleic acid sequence encoding residue 1 or about 23-216 of the FGF-19 polynucleotide of FIG. 2 (SEQ ID NO: 2), (b) a nucleic acid sequence encoding amino acids X-216 of the FGF-19 polypeptide of FIG. 2 (SEQ ID NO: 2), wherein X is the amino acid residue of 17-27 of FIG. 2 (SEQ ID NO: 2) Or (c) an active FGF-19 as defined below having at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding another specifically derived fragment of the amino acid sequence of FIG. 2 (SEQ ID NO: 2). By nucleic acid molecules encoding polypeptides. Usually, the FGF-19 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 23-216 of the FGF-19 polynucleotide of FIG. 2 (SEQ ID NO: 2), (b) FIG. 2 (SEQ ID NO The nucleic acid sequence encoding amino acids X-216 of the FGF-19 polypeptide of: 2, wherein X is the amino acid residue of 17-27 of FIG. 2 (SEQ ID NO: 2), or (c) FIG. 2 (SEQ At least about 80% nucleic acid sequence identity, or at least about 81% nucleic acid sequence identity, or at least about 82% nucleic acid sequence identity, or about a nucleic acid sequence encoding another specifically derived fragment of the amino acid sequence of ID NO: 2) At least 83% nucleic acid sequence identity, or at least about 84% nucleic acid sequence identity, or at least about 85% nucleic acid sequence identity, or at least about 86% nucleic acid sequence identity, or at least about 87% nucleic acid sequence identity, or at least about 88% nucleic acid sequence Identity, or at least about 89% nucleic acid sequence identity, or about 9 0% or more nucleic acid sequence identity, or about 91% or more nucleic acid sequence identity, or about 92% or more nucleic acid sequence identity, or about 93% or more nucleic acid sequence identity, or about 94% or more nucleic acid sequence identity, or about 95% or more nucleic acid sequence Identity, or at least about 96% nucleic acid sequence identity, or at least about 97% nucleic acid sequence identity, or at least about 98% nucleic acid sequence identity, or at least about 99% nucleic acid sequence identity. FGF-19 polynucleotide variants do not comprise native FGF-19 nucleotide sequences.

Usually, the FGF-19 variant polynucleotides comprise at least about 30 nucleotides, or at least about 60 nucleotides, or at least about 90 nucleotides, or at least about 120 nucleotides, or at least about 150 nucleotides, or at least about 180 nucleotides, Or about 210 or more nucleotides, or about 240 or more nucleotides, or about 270 or more nucleotides, or about 300 or more nucleotides, or about 450 or more nucleotides, or about 600 or more nucleotides, or about 900 or more nucleotides, or It is more than that nucleotide.

With respect to the FGF-19 polypeptide encoding nucleic acid sequence identified herein, "% nucleic acid sequence identity" refers to the FGF-19 polypeptide- after aligning the sequence and introducing a gap if necessary to achieve the maximum sequence identity. It is defined as the percentage of nucleotides in the candidate sequence that are identical to the nucleotides of the coding nucleic acid sequence. Arrangements for determining the percentage of nucleic acid sequence identity can be varied within the art using, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megastar (DNASTAR) software. It can be achieved in a way. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve the maximum alignment for the total sequence to be compared. However, for purposes herein,% nucleic acid sequence identity values are obtained by using the sequence comparison computer program ALIGN-2, whose complete source code is set forth in Table 1, as described below. The authority of the ALIGN-2 sequence comparison computer program is Genentech, Inc. The source code of Table 1 is Washington, D., USA. Seed. It was filed with the user dissemination at the U.S. Copyright Office of 20559 and registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc. of South San Francisco, Calif., Or may be compiled from the source code shown in Table 1. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

For purposes herein, the% nucleic acid sequence identity of the nucleic acid sequence C to the nucleic acid sequence D (or can be represented by the nucleic acid sequence C having or including a certain percent nucleic acid sequence identity to the nucleic acid sequence D) is It is calculated as follows:

100 X Fraction W / Z

Where W is the number of nucleotides calculated to be the same when arranging C and D by the sequence alignment program ALIGN-2, and Z is the total number of nucleotides of D. It will be appreciated that if the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, then% nucleic acid sequence identity of C to D will not be identical to% nucleic acid sequence identity of D to C. As an example of calculating% nucleic acid sequence identity, Tables 4 and 5 illustrate methods for calculating% nucleic acid sequence identity of nucleic acid sequences labeled "Comparative DNA" to nucleic acid sequences labeled "PRO-DNA."

Unless specifically stated otherwise, all% nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program. However,% nucleic acid sequence identity can also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program can be downloaded from http://www.ncbi.nim.nih.gov or obtained from the National Institute of Sanitation, Bethesda, Maryland. NCBI-BLAST2 uses several irradiation parameters, all of which are, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoringmatrix = BLOSUM62.

When NCBI-BLAST2 is used for sequence comparison, it is expressed as the nucleic acid sequence C having or including% nucleic acid sequence identity of the nucleic acid sequence C to the nucleic acid sequence D (or having a certain percent nucleic acid sequence identity to the nucleic acid sequence D). Can be calculated as follows:

100 X Fraction W / Z

Where W is the number of nucleotides calculated to be the same when arranging C and D by the sequence alignment program NCBI-BLAST2, and Z is the total number of nucleotides of D. It will be appreciated that if the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, then the% amino acid sequence identity of C to D will not be the same as the% amino acid sequence identity of D to C.

In another embodiment, the FGF-19 variant polynucleotide is capable of hybridizing with the nucleotide sequence encoding the full-length FGF-19 polypeptide of FIG. 2 (SEQ ID NO: 2), preferably under stringent hybridization and wash conditions. Nucleic acid molecules encoding an active FGF-19 polypeptide. The FGF-19 variant polypeptide may be encoded by the FGF-19 variant polynucleotide.

With respect to amino acid sequence identity comparisons performed as described above, the term “positive” includes amino acid residues in the sequences being compared that are identical as well as have similar properties. An amino acid residue having a positive value for the amino acid residue of interest is the same as the amino acid residue of interest or a preferred substitution of the amino acid residue of interest (as defined in Table 6 below).

For purposes herein, the% positive value of the amino acid sequence A relative to the amino acid sequence B (or which may be represented by the amino acid sequence A having or comprising a certain% positive relative to the amino acid sequence B) is as follows: Is calculated:

100 X fraction X / Y

Where X is the number of amino acid residues having a positive value as defined above when arranging A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues of B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the% positive of A for B will not be the same as the% positive of B for A.

“Isolated,” as used to describe the various polypeptides disclosed herein, means polypeptides that have been identified, separated (and isolated) from components of the natural environment. Preferably, an isolated polypeptide is independent of all naturally related components. Contaminant components of the natural environment are generally substances that interfere with diagnostic or therapeutic uses of the polypeptide and include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the polypeptide is sufficient to obtain (1) at least 15 residues in the N-terminal or internal amino acid sequence by using a spinning cup sequencing, or (2) Coomassie blue or Preferably it will be purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using silver dyeing. Isolated polypeptides include polypeptides in situ in recombinant cells since at least one component of the FGF-19 natural environment will not be present. Usually, however, isolated polypeptide will be prepared by one or more purification steps.

An “isolated” nucleic acid molecule encoding an FGF-19 polypeptide is a nucleic acid molecule that has been identified and isolated from one or more contaminating nucleic acid molecules commonly associated within the natural source of FGF-19-encoding nucleic acid. Preferably the isolated nucleic acid molecule is independent of all naturally related components. Isolated FGF-19-encoding nucleic acid molecules are other than those found in nature. Thus, isolated nucleic acid molecules are distinguished from FGF-19-encoding nucleic acid molecules present in natural cells. However, an isolated nucleic acid molecule encoding a FGF-19 polypeptide can be used to refer to an FGF-19-encoding nucleic acid molecule, usually contained in cells expressing FGF-19, for example, where the nucleic acid molecule is at a chromosomal location different from that of natural cells. Include.

The term "control sequence" refers to a DNA sequence necessary for the expression of a coding sequence operably linked in a particular host organism. Suitable control sequences for prokaryotes include, for example, promoters, optionally effector sequences, and ribosomal binding sites. Eukaryotes are known to utilize promoters, polyadenylation signals, and amplifiers.

A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, the DNA of the prosequence or secretory leader sequence is operably linked to the DNA of the polypeptide when it is expressed as a proprotein that participates in the secretion of the polypeptide; A promoter or amplifier is operably linked to a coding sequence if it affects the transcription of the sequence; Or the ribosome binding site is operably linked with a coding sequence when it is located to promote transcription. In general, “operably linked” means that the linked DNA sequences are contiguous, and in the case of secretion leader sequences, are contiguous and in a read state. However, the amplifiers do not have to be contiguous. Linking is accomplished by linking at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide conjugates or linkers are typically used.

The term "antibody" is used in its broadest sense, and in particular, for example, a single anti-FGF-19 monoclonal antibody (including agonists, antagonists and neutralizing antibodies), anti-FGF-19 with multiepitope specificity Antibody compositions, single-chain anti-FGF-19 antibodies, and fragments of anti-FGF-19 antibodies (see below). The term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies (ie, identical except for the possibility of natural mutations in which small amounts of the individual antibodies that make up the population are present).

The “stringency” of the hybridization reaction can be readily determined by one skilled in the art and is generally an empirical calculation that depends on probe length, wash temperature and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. When complementary strands are present in an environment below their melting temperature, hybridization generally depends on the ability of the denatured DNA to reanneal. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, the higher the relative temperature, the more stringent the reaction conditions, and the lower the temperature tends to be less stringent. For further details and description of stringency of the hybridization reaction, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, 1995.

"Strict conditions" or "highly stringent conditions" refer to (1) use of low ionic strength and high temperature for washing, for example, use of 0.015 M sodium chloride / 0.015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50 ° C. ; (2) the use of denaturing agents such as formamide during hybridization, for example 0.1% bovine serum albumin / 0.1% picol / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5), 750 at 42 ° C. use of 50 v / v% formamide with mM sodium chloride, 75 mM sodium citrate; Or (3) 50% formamide at 42 ° C., 5 × SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt solution, Using sonicated salmon sperm DNA (50 μl / ml), 0.1% SDS and 10% dextran sulfate, washing in 0.2 × SSC (sodium chloride / sodium citrate) and 50% formamide at 42 ° C., followed by 55 ° C. Can be equated with a high stringency wash consisting of 0.1 x SSC containing EDTA.

“Severely stringent conditions” may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and less stringent washing solutions and hybridizations than those described above. The use of conditions (eg, temperature, ionic strength and% SDS). Examples of mildly stringent conditions include 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfide at 37 ° C. Incubate overnight in a solution containing pate and 20 mg / ml denatured salmon sperm DNA, and then wash the filter with 1 × SSC at about 37-50 ° C. Those skilled in the art will know how to adjust the temperature, ionic strength and the like if necessary to adapt factors such as probe length and the like.

The term “epitope tagged” refers to a chimeric polypeptide comprising a FGF-19 polypeptide fused with a “tag polypeptide”. The tag polypeptide has enough residues to provide an epitope from which the antibody can be made, but short enough to not interfere with the activity of the fused polypeptide. In addition, the tag polypeptide is preferably very unique so that the antibody does not cross react substantially with other epitopes. Suitable tag polypeptides generally have 6 or more amino acid residues, usually about 8 to 50 amino acid residues (preferably about 10 to 20 amino acid residues).

As used herein, the term “immunoadhesin” refers to a molecule such as an antibody that combines the binding specificity of a heterologous protein (“adhesin”) with the effector function of an immunoglobulin constant region. Structurally, an immunoadhesin comprises fusion of an antigen recognition and amino acid sequence with the desired binding specificity that is not (ie heterologous) the binding site of the antibody, and an immunoglobulin constant region sequence. The attachment site of an immunoadhesion molecule is generally a contiguous amino acid sequence comprising at least the binding site of a receptor or ligand. Immunoglobulin constant region sequences within an immunoadhesin may be any such as IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM From immunoglobulins.

For the purposes herein, "active" or "active" refers to the form (s) of FGF-19 that retain the biological and / or immunological activity of natural or natural FGF-19, wherein "biological" activity is natural Refers to the biological function (inhibition or stimulation) induced by native FGF-19, in addition to its ability to induce the production of antibodies against antigenic epitopes of FGF-19, and "immunological" activity refers to that of native FGF-19 Refers to the ability to induce the production of antibodies against antigenic epitopes. Preferred biological activities include one or more of the following activities: increased metabolism (metabolism) of the subject, reduced body weight of the subject, reduced fatness of the subject, decreased absorption of glucose into adipocytes, increased leptin release from adipocytes, Reduction of triglycerides in the subject and reduction of free fatty acids in the subject. Some of the activity of FGF-19 is directly induced by FGF-19, some indirectly, but each is a consequence of the presence of FGF-19, and in the absence of FGF-19 will not have such a result.

The term “antagonist” is used in its broadest sense and includes any molecule that partially, completely blocks, inhibits or neutralizes the biological activity of the native FGF-19 polypeptides described herein. Likewise, the term “agent” is used in its broadest sense and includes any molecule that mimics the biological activity of the native FGF-19 polypeptides described herein. Suitable agonist or antagonist molecules include, in particular, agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native FGF-19 polypeptides, peptides, small organic molecules and the like. Methods of identifying an agonist or antagonist of an FGF-19 polypeptide include contacting the FGF-19 polypeptide with a candidate agonist or antagonist molecule and determining the detectable change in one or more biological activities normally associated with the FGF-19 polypeptide.

"Treatment" refers to therapeutic and prophylactic treatment whose purpose is to prevent or alleviate (reduce) the target pathological condition or disease. Patients in need of treatment include those already afflicted with the disease, as well as patients susceptible to disease or those who need to prevent the disease.

"Chronic" administration, as opposed to acute mode, refers to the administration of the drug (s) in a continuous manner to maintain the long-term initial therapeutic effect. "Intermittent" administration is not done continuously without interference, but rather is a periodic treatment.

For purposes of treatment, "mammal" is any person classified as a mammal, including humans, livestock, and zoos, sports or pets, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, and the like. Say animal. Preferably, the mammal is a human.

An “individual” is any sample, preferably a mammal, more preferably a human.

“Obesity” refers to a state in which a mammal has a body weight index (BMI; calculated in weight (kg) / t ² ) of at least 25.9. Typically, a normal weight person has a BMI of less than 19.9 to 25.9. Obesity herein may be due to any cause, whether genetic or environmental. Examples of diseases that can cause or cause obesity include overeating, bulimia, polycystic ovarian disease, craniocephaloma, Prader-Willi syndrome, Frohlich syndrome, type II diabetes, GH-deficient samples, general Variant monocytes, Turner syndrome, and children with other pathological conditions, such as acute lymphoblastic leukemia, which indicate a decrease in metabolic activity or a decrease in resting energy consumption, which is a percentage of total fat-free mass.

"Obe-related conditions" include skin diseases such as infections, varicose veins, melanoma and eczema, exercise tolerance, diabetes, insulin resistance, hypertension, hypercholesterolemia, gallstones, osteoarthritis, orthopedic injury, thromboembolic diseases, It refers to a condition that is exacerbated by obesity or is a result of obesity, such as but not limited to cancer and coronary (or cardiovascular) heart disease, particularly cardiovascular diseases associated with high triglycerides and free fatty acids in an individual.

Administration “in conjunction with” one or more additional therapeutic agents includes simultaneous and continuous administration in any order.

As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient or stabilizer that is nontoxic to a cell or mammal exposed to it at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffer solution. Examples of physiologically acceptable carriers include buffers such as phosphoric acid, citric acid and other organic acids; Antioxidants including ascorbic acid; Low molecular weight (less than about 10 residues) polypeptides; Proteins such as 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 alcohols such as mannitol or sorbitol; Salt-forming counterions such as sodium; And / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG) and PLURONICS®.

The antibody fragment comprises a portion of the original antibody, preferably the antigen binding or variable region of the original antibody. Examples of antibody fragments include Fab, Fab ', F (ab') ₂ and Fv fragments; Diabodies; Linear antibodies (Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995)); Single chain antibody molecules; And multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen binding fragments, each with a single antigen binding site, called "Fab" fragments, and the remaining "Fc" fragments, which reflect the ability to easily crystallize. Pepsin treatment produces an F (ab ') ₂ fragment having two antigen binding sites and capable of cross linking antigens.

"Fv" is the minimum antibody fragment which contains a complete antigen recognition- and binding site. This region consists of a dimer of one heavy chain and one light chain variable region tightly non-covalently linked. In this arrangement, three CDRs of each variable region interact to define an antigen binding site on the surface of the VH-LH dimer. In total, six CDRs confer antigen binding specificity to the antibody. However, even a single variable region (or half of the Fv containing only three CDRs specific for the antigen) has a lower affinity than the total binding site, but has the ability to recognize and bind antigen.

The Fab fragment also contains the constant region of the light chain and the first constant region of the heavy chain (CHI). Fab fragments are distinguished from Fab 'fragments by the addition of several residues from the antibody hinge region to the carboxy terminus of the heavy chain CH1 region comprising one or more cysteines. Fab'-SH refers herein to Fab 'wherein the cysteine residue (s) of the constant region contain a free thiol group. F (ab ') ₂ antibody fragments are originally produced as pairs of Fab' fragments with hinge cysteines between them. In addition, other chemical couplings of antibody fragments are known.

The “light chain” of an antibody (immunoglobulin) from any vertebrate species can be classified into two distinct types, kappa and lambda, based on the amino acid sequence of its constant region.

Depending on the amino acid sequence of the constant region of their heavy chains, immunoglobulins can be classified into different groups. There are five main groups of immunoglobulins (IgA, IgD, IgE, IgG and IgM), some of which are further subgroups (isotypes) (e.g. IgG1, IgG2, IgG3, IgG4, IgA and IgA2). Can be classified.

"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL regions of an antibody, which are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL regions such that the sFv can form the structure required 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 bovine antibody fragments having two antigen binding sites, comprising a heavy chain variable region (VH) linked to a light chain variable region (VL) within the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between two regions on the same chain, the region has to pair with the complementary region of another chain to create two antigen binding sites. Diabodies are described, 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 that has been identified, isolated (and isolated) or recovered from components of its natural environment. Contaminant components of the natural environment are substances that interfere with the diagnostic or therapeutic use of antibodies and include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antibody comprises (1) up to 95% by weight or more of the antibody, as measured by the Lowry method, and (2) obtaining at least 15 residues in the N-terminal or internal amino acid sequence by using a spinning cup sequencer. To a degree sufficient, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie Blue, or preferably silver dyeing. Isolated antibody includes the polypeptide in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by one or more purification steps.

The term "label" refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody to produce a "labeled" antibody. The label may be detectable alone (eg, radioisotope labels or fluorescent labels), or, at the enzymatic level, may catalyze chemical changes in the detectable substrate compound or composition.

"Solid phase" means a non-aqueous matrix to which an antibody of the invention may attach. Examples of solid phases included herein include those formed partially or entirely of glass (eg, controlled pore glass), polysaccharides (eg, agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and silicone. In some embodiments, depending on the situation, the solid phase may comprise a well of an assay plate, where in the rest it is a purification column (eg, an affinity chromatography column). The term also includes discontinuous solid phases of discrete particles, such as those described in US Pat. No. 4,275,149.

A "liposome" is a vesicle composed of various types of lipids, phospholipids, and / or surfactants useful for the delivery of a drug (eg, an FGF-19 polypeptide or antibody thereof) to a mammal. The components of the liposomes are usually arranged in a double worm form, similar to the lipid configuration of the biological membrane.

"Small molecule" is defined herein as having a molecular weight of about 500 Daltons or less.

PRO XXXXXXXXXXXXXXX (length = 15 amino acids) Comparison protein XXXXXYYYYYYY (length = 12 amino acids) % Amino acid sequence identity = (number of identical amino acid residues between two polypeptide sequences as measured by ALIGN-2) / (total number of amino acid residues of PRO polypeptide) = 5/15 = 33.3%

PRO XXXXXXXXXX (length = 10 amino acids) Comparison protein XXXXXYYYYYYZZYZ (length = 15 amino acids) % Amino acid sequence identity = (number of identical amino acid residues between two polypeptide sequences as measured by ALIGN-2) / (total number of amino acid residues of PRO polypeptide) = 5/10 = 50%

PRO-DNA NNNNNNNNNNNNNN (length = 14 nucleotides) Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides) % Nucleic acid sequence identity = (number of identical nucleotides between two nucleic acid sequences, as measured by ALIGN-2) / (total number of nucleotides in PRO-DNA nucleic acid sequence) = 6/14 = 42.9%

PRO-DNA NNNNNNNNNNNN (length = 12 nucleotides) Comparison DNA NNNNLLLVV (length = 9 nucleotides) % Nucleic acid sequence identity = (number of identical nucleotides between two nucleic acid sequences as measured by ALIGN-2) / (total number of nucleotides in nucleic acid sequence of PRO-DNA) = 4/12 = 33.3%

II. Compositions and Methods of the Invention

A. Full-length FGF-19 Polypeptide

The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to herein as FGF-19 (or UNQ334). In particular, cDNA encoding the FGF-19 polypeptide was identified and isolated, as described in more detail in the Examples below. Note that proteins produced in separate rounds of expression may be assigned different PRO numbers, but UNQ numbers are unique for any given DNA and encoded protein and will not be altered. However, for the sake of simplicity, in this specification, the proteins encoded by DNA49435-1219, and additional natural homologs and variants included in the definition of FGF-19 (sometimes referred to as PRO533), also depend on their origin or mode of manufacture. It will be referred to as "FGF-19" regardless.

As described in the Examples below, cDNA clones designated here as DNA49435-1219 were deposited with the ATCC. The actual nucleotide sequence of the clone can be readily determined by one skilled in the art by sequencing the deposited clone using conventional methods in the art. Prospective amino acid sequences can be determined from nucleotide sequences using conventional techniques. For the FGF-19 polypeptides and coding nucleic acids described herein, Applicants have identified what is considered a reading frame that can best be identified with the sequence information available at the time.

When using the above-mentioned ALIGN-2 sequence array computer program, a full-length native sequence FGF-19 (Fig. 2 and SEQ ID NO: shown as 2) AF007268 _- it has a first and a part of amino acid sequence identity has been found . Thus, it is presently believed that the FGF-19 polypeptides described herein are newly identified members of the fibroblast growth factor protein family and may have one or more typical biological and / or immunological activities of that protein family.

B. FGF-19 Variants

In addition to the full-length native sequence FGF-19 polypeptides described herein, it is contemplated that FGF-19 variants may be prepared. FGF-19 variants can be prepared by introducing (or introducing) appropriate nucleotide changes into FGF-19 DNA and by synthesis of the desired FGF-19 polypeptide. Those skilled in the art will appreciate that amino acid changes can alter the post-transcriptional process of FGF-19, such as changing the number or location of glycosylation sites or changing membrane anchoring properties.

Changes in the natural full-length sequence FGF-19 or various regions of the FGF-19 described herein are described, for example, in any technique and for the conservative and non-conservative mutations set forth in, eg, US Pat. No. 5,364,934. Can be achieved using guidelines. The change can be a substitution, deletion or insertion of one or more codons encoding FGF-19 which results in a change in the amino acid sequence of FGF-19 compared to native sequence FGF-19. Optionally, the change is by replacing one or more amino acids with any other amino acid in one or more regions of FGF-19. Determining which amino acid residues can be inserted, substituted or deleted without adversely affecting the desired activity compares the sequence of FGF-19 with that of known homologous proteins and changes in amino acid sequence made in regions of high homology. This can be referred to by minimizing the number of. Amino acid substitutions may be the result of substituting leucine for serine, ie, replacing one amino acid with another amino acid having similar structural and / or chemical properties, such as conservative amino acid substitutions. Insertions or deletions may optionally range from about 1 to 5 amino acids. Acceptable changes can be determined by systematically inserting, deleting, or replacing amino acids in the sequence and testing the resulting variants for activity expressed by full-length or mature native sequences.

Provided herein are FGF-19 polypeptide fragments. Such fragments may be truncated at the N-terminus or C-terminus, or lack internal residues, for example when compared to a full-length native sequence. Some fragments lack amino acid residues that are not essential for the desired biological activity of the FGF-19 polypeptide.

FGF-19 fragments can be prepared by any of a number of conventional methods. Desired peptide fragments can be synthesized chemically. Alternatively, by enzymatic digestion, for example, by treating the protein with an enzyme known to cleave the protein at a site defined by a particular amino acid residue, or by digesting the DNA with an appropriate restriction enzyme and isolating the desired fragment. Generating the FGF-19 fragment. Another suitable method includes isolating DNA fragments encoding the desired polypeptide fragments and amplifying by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are used at the 5 'and 3' primers in PCR. Preferably, the FGF-19 polypeptide fragment shares one or more biological and / or immunological activities with the native FGF-19 polypeptide of FIG. 2 (SEQ ID NO: 2).

In certain embodiments, important conservative substitutions are shown in Table 6 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, the more substantial substitutions, indicated as exemplary substitutions in Table 6, or described further below with respect to the amino acid group, may be introduced and the product screened.

Original residue Exemplary Substitution Preferred Substitution 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) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Try (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu

Substantial alterations in the function or immunological properties of the FGF-19 polypeptide may include (a) the structure of the polypeptide backbone in the substitution region, such as, for example, a sheet or screw arrangement, (b) the charge or hydrophobicity of the molecules present at the target site Or (c) by choosing greatly different substitutions in their effect on the maintenance of the volume of the side chain. Natural residues are classified into the following groups based on common side chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilicity: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues affecting chain orientation: gly, pro; And

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will require exchanging a member of one of these groups for another group. Such substituted moieties will also be introduced into conservative substitution sites, or more preferably into the remaining (non-satisfactory) sites.

The change can be accomplished using methods known in the art, such as oligonucleotide mediated (site directed) mutagenesis, alanine injection and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res., 13: 4331 (1986); Zoller et al., Nucl. Acids Res., 10: 6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34: 315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317: 415 (1986)] or other known methods can be performed on cloned DNA to generate FGF-19 variant DNA.

Injection amino acid analysis can also be used to identify one or more amino acids along a contiguous sequence. Among the preferred injectable amino acids are relatively small and neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Alanine is generally the preferred injectable amino acid in this group because it will lessen the side chains of beta-carbon or more, thus changing the main chain form of the variant (Cunningham and Wells, Science, 244: 1081-1085 (1989)). Alanine is also the most common amino acid and is therefore generally preferred. Furthermore, it is frequently found in both hidden and exposed positions [Creighton, The Proteins (W. H. Freeman & Co., N. Y.); Chothia, J. Mol. Biol., 150: 1 (1976). If alanine substitutions fail to produce an adequate amount of variant, an equal density of amino acids can be used.

C. Changes in FGF-19

Covalent alterations of FGF-19 are included within the scope of the present invention. One type of covalent alteration involves reacting a target amino acid residue of the FGF-19 polypeptide with an organic derivatizing agent that can react with a selected side chain or N- or C-terminal residue of FGF-19. Derivatization with bifunctional agents is useful, for example, to crosslink or reverse FGF-19 to a water-insoluble support matrix or surface for use in methods of purifying anti-FGF-19 antibodies. Commonly used crosslinkers include, for example, disuccinimidyl esters such as 3,3-dithiobis (succinimidylpropionate), difunctional maleis such as bis-N-maleimido-1,8-octane. 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-, including agents such as mid and methyl-3-[(p-azidophenyl) dithio] propioimidate Hydroxysuccinimide esters such as esters with 4-azidosalicylic acid, homofunctional functional imidoesters.

Other alterations include deamidation of the glutamine and asparaginyl residues to the corresponding glutamyl and aspartyl, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, lysine, arginine And methylation of the α-amino group of the histidine side chain [T. E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of N-terminal amines and amidation of any C-terminal carboxyl groups.

Another type of covalent alteration of FGF-19 polypeptides within the scope of the present invention includes altering the natural glycosylation pattern of the polypeptide. For the purposes herein, "altering a natural glycosylation pattern" means that one or more carbohydrate moieties found in the native sequence FGF-19 are deleted (removing potential glycosylation sites or glycosylating by chemical and / or enzymatic means). By deleting (or deleting) one or more glycosylation sites that are not present in native sequence FGF-19. The phrase also includes qualitative changes in glycosylation of natural proteins, including changes in the nature and proportions of the various carbohydrate moieties present.

Adding glycosylation sites to the FGF-19 polypeptide can be accomplished by altering the amino acid sequence. The alteration can be accomplished, for example, by addition or substitution of one or more serine or threonine residues with the native sequence FGF-19 (for O-linked glycosylation sites). The FGF-19 amino acid sequence can be optionally altered through changes in the DNA level, in particular by mutating the DNA encoding the FGF-19 polypeptide at a preselected base to produce a codon to be transcribed into the desired amino acid.

Another means to increase the number of carbohydrate moieties on the FGF-19 polypeptide is to chemically or enzymatically couple glycosides to the polypeptide. Such methods are known in the art, eg WO 87/05330 (published Sept. 11, 1987) and in Aplin and Wriston, CRC Crit. Rev. Biochem., Pp. 259-306 (1981).

Removal of the carbohydrate moiety present on the FGF-19 polypeptide can be accomplished chemically or enzymatically, or by mutagenic substitution of codons encoding amino acid residues that serve as targets for glycosylation. Chemical deglycosylation methods are known in the art and are described, for example, in Hakimuddin et al., Arch. Biochem. Biophys., 259: 52 (1987) and Edge et al., Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides is described by Thotakura et al., Meth. Enzymol., 138: 350 (1987), can be accomplished by the use of various endo- and exo-glycosidases.

Another type of covalent alteration of FGF-19 is US Pat. No. 4,640,835; No. 4,496,689; 4,301,144; 4,670,417; 4,670,417; No. 4,791,192; Or by the method set forth in US Pat. No. 4,179,337, combining the FGF-19 polypeptide with one of a variety of nonproteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene.

The FGF-19 of the present invention may also be altered in such a way as to form chimeric molecules comprising FGF-19 fused to another heterologous polypeptide or amino acid sequence.

In one embodiment, the chimeric molecule comprises a fusion of FGF-19 with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. Epitope tags are generally located at the amino- or carboxy-terminus of FGF-19. The presence of such epitope-tagged forms of FGF-19 can be detected using antibodies to tag polypeptides. In addition, due to the provision of epitope tags, FGF-19 can be easily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to an epitope tag. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; flu HA tagged polypeptide and antibody 12CA5 [Field et al., Mol. Cell. Biol., 8: 2159-2165 (1988); c-myc tag and its 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies (Evan et al., Molecular and Cellular Biology, 5: 3610-3616 (1985)); And herpes simplex virus glycoprotein D (gD) tag and its antibodies (Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)). Other tag polypeptides include plaque-peptides (Hopp et al., Bio Technology, 6: 1204-1210 (1988)); KT3 epitope peptides (Martin et al., Science, 255: 192-194 (1992)); α-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266: 15163-15166 (1991); And T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA. 87: 6393-6397 (1990).

In an alternative embodiment, the chimeric molecule comprises fusion of FGF-19 with an immunoglobulin or a specific region of immunoglobulin. In the bivalent form of a chimeric molecule (also called an "immunoadhesin"), the fusion may be to the Fc region of an IgG molecule. Ig fusion preferably comprises substitution of the soluble (transmembrane region is deleted or inactivated) form of the FGF-19 polypeptide instead of one or more variable regions in the Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusions comprise hinges, CH2 and CH3, or hinges, CH1, CH2 and CH3 of the IgG1 molecule. For the generation of immunoglobulin fusions, see US Pat. No. 5,428,130 (June 27, 1995).

D. Preparation of FGF-19

The following technique relates primarily 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 FGF-19 may be prepared using alternative methods well known in the art. For example, FGF-19 sequences or portions thereof may be prepared by direct peptide synthesis using solid phase methods (eg, Stewart et al., Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, CA (1969). ); Merrifield, J. Am. Chem. Soc., 85: 2149-2154 (1963). In vitro protein synthesis can be performed using manual methods or by automation. Automated synthesis can be performed using, for example, an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) According to the manufacturer's instructions. Various 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 DNA Encoding FGF-19

DNA encoding FGF-19 can be obtained from a cDNA library prepared from tissues that have FGF-19 mRNA and are thought to express it at detectable levels. Thus, human FGF-19 DNA can be conveniently obtained from a cDNA library prepared from human tissue, as described in the Examples. FGF-19-encoding genes can also be obtained by genomic libraries or known synthetic processes (eg, automated nucleic acid synthesis).

The library can be screened with probes (eg, antibodies against FGF-19 or oligonucleotides of about 20-80 or more bases) designed to identify the gene of interest or the protein encoded thereby. Screening cDNA or genomic libraries with selected probes can be performed using standard methods, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). . An alternative means of isolating genes encoding FGF-19 is to use PCR methodology [Sambrook et al., Supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995).

The following example describes a method of screening a cDNA library. The oligonucleotide sequence chosen as a probe should be long enough and clear enough to minimize the positive. Oligonucleotides are preferably labeled and can be detected upon hybridization with DNA in the library to be screened. Labeling methods are well known in the art and include the use of radiolabels, biotinylation or enzyme labels, such as ³² P-labeled ATP. Hybridization conditions are provided in Sambrook et al., Supra, including mild stringency and high stringency.

The sequences identified in the library screening method can be compared and arranged with other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at the amino acid or nucleic acid level) within defined regions or across full-length sequences of a molecule can be determined as described herein using methods known in the art.

Nucleic acids with protein coding sequences can be obtained by first screening selected cDNAs or genomic libraries using the putative amino acid sequences described herein, and, if necessary, the conventional primer extension described in Sambrook et al., Supra. The method is used to detect precursors and treatment intermediates of mRNA that would not be reverse transcribed into cDNA.

2. Selection and Transformation of Host Cells

For the production of FGF-19, the host cell is suitably transfected or transformed with the expression or cloning vector described herein, inducing a promoter, selecting a transformant, or amplifying a gene encoding a desired sequence. Cultured in altered conventional nutrient media. Culture conditions such as medium, temperature, pH and the like can be selected by those skilled in the art without undue experimentation. In general, principles, protocols and application techniques for maximizing the productivity of cell cultures are described in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., Supra.

Eukaryotic cell transfection and prokaryotic transformation methods, such as CaCl ₂ , CaPO ₄ , liposome-mediated and electroporation, are known to those skilled in the art. Depending on the host cell used, transformation is performed using standard methods appropriate for the cell. Calcium treatment or electroporation using calcium chloride as described in Sambrook et al., Supra, is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is described in some plant cells, as described in Shaw et al., Gene, 23: 315 (1983) and WO 89/05859 (June 29, 1989). Used for transformation of For mammalian cells without the cell wall, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52: 456-457 (1978) can be used. General aspects of mammalian cell host system transfection are described in US Pat. No. 4,399,216. Transformation with yeast is generally described by Van Solingen 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 may also be used, such as nuclear microinjection, electroporation, fusion of original cells with bacterial protoplasts, or politics (eg polybrene, polyornithine). For various methods for transforming 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 cloning or expressing the DNA in the vectors herein include prokaryote, yeast or higher eukaryotic cells. Suitable prokaryotes include, but are not limited to, Eubacterium such as Gram-negative or Gram-positive organisms, for example enterobacterial steel such as Escherichia coli. Various E. coli strains, for example E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strains W3110 (ATCC 27,325) and K5 772 (ATCC 53,635) are available publicly. Other suitable prokaryotic host cells include Escherichia (eg E. coli), Enterobacter, Erbinia, Klebsiella, Proteus, Salmonella (eg Salmonella typhimurium), Serratia (eg Serratia marcescans) and Shigella As well as enterobacterial steels, such as. B. subtilis and b. Vasily, p. Such as rickeniformis (B. licheniformis 41P as disclosed in DD 266,710, published April 12, 1989). Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative and not restrictive. Strain W3110 is a particularly preferred host or parent host since it is a common host strain for recombinant DNA product fermentation. Preferably, the host cell secretes minimal amounts of protease. For example, strain W3110 can be altered to achieve genetic mutations in genes encoding proteins that are endogenous to the host, and examples of such hosts include E. coli W3110 strain 1A2 with complete genotype tonA; E. coli W3110 strain 9E4 with complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244) with complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT kan; E. coli W3110 strain 37D6 with complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degPOmpT rbs7 ilvG kan; E. coli W3110 strain 40B4, which is strain 37D6 with kanamycin non-resistant degP deletion mutation; And E. coli strains having mutant periplasmic proteases described in US Pat. No. 4,946,783 (patent dated Aug. 7, 1990). Alternatively, in vitro cloning methods (eg PCR) or other nucleic acid polymerase reactions are suitable.

In addition to prokaryotic cells, eukaryotic microorganisms such as linear fungi or yeasts are also suitable cloning or expression hosts for the FGF-19-encoding vector. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. The rest are Schizosaccharomyces pombe (Beach and Nurse, nature, 290: 140 (1981); EP 139,383, published May 2, 1985); Kluyveromyces hosts (US Pat. No. 4,943,529; Fleer et al., Bio / Technology, 9: 968-975 (1991)), eg, K. K. lactis (MW98-8C, CBS683, CBS4574; Leuvencourt et al., J. Bacteriol., 154 (2): 737-742 (1983)), K. K. fragilis (ATCC 12,424), K. B. bulgalicus (ATCC 16,045), K. K. wicheramii (ATCC 24,178), K. K. waltii (ATCC 56,500), K. K. drosophilarum (ATCC 36,906; Van den Berg et al., Bio / Technology, 8: 135 (1990)), K. K. thermotolerans and K. K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28: 265-278 (1988)); candida; trichoderma lesia ( Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76: 5259-5263 (1979)); Schwanniomyces, eg For example, Schwanniomyces occidentalis (published as EP 394,538, Oct. 31, 1990); and fungi on board, such as Neurospora, Penicillium, Tolypocladium (WO 91/00357, published January 10, 1991), and Aspergilus 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., Proc, Natl. Acad. Sci. USA, 81: 1470-1474 (1984)) and A. niger (Kelly Hynes, EMBO J., 4: 475-479 (1985)) Methylstimulatory yeasts are suitable herein and include Hansenula, Candida, Kloechera, Pichia, Saccharomyces, Yeasts that can grow on methanol, selected from the genus consisting of Torulopsis and Rhodotorula, include, but are not limited to. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of 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 Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CV1 transformed with SV40 (COS-7, ATCC CRL 1651); Human-born cell lines (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977); Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23: 243-251 (1980)); human lung cells (W138, ATCC CCL 75); human Liver cells (Hep G2, HB 8065) and mouse carcinoma (MMT 060562, ATCC CCL51) Selection of suitable host cells will be considered within the skill of the art.

3. Selection and use of replicable vectors

Nucleic acids encoding FGF-19 (eg, cDNA or genomic DNA) can be inserted into a replicable vector for cloning (amplification of DNA) or for expression. Various vectors are available publicly. The vector may be, for example, in the form of a plasmid, cosmid, viral particles or phage. Appropriate nucleic acid sequences can be inserted into the vector by a variety of methods. In general, DNA is inserted into a suitable restriction endonuclease site (s) using techniques known in the art. Vector components generally include, but are not limited to, one or more signal sequences, replication origins, one or more label genes, amplifier factors, promoters and transcription termination sequences. Construction of suitable vectors containing one or more of these components uses standard linking techniques known to those skilled in the art.

FGF-19 may be prepared recombinantly as well as directly or as a fusion with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site present at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector or may be part of an FGF-19-encoding DNA inserted into the vector. The signal sequence can be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp or heat-stable enterotoxin II leader sequence. In yeast secretion, the signal sequence may be, for example, a yeast invertase leader sequence, an alpha factor leader sequence (including Saccharomyces, and the Kluyveromyces alpha factor leader sequence described in US Pat. No. 5,010,182), an acidic Phosphatase lead sequence, C. C. albicans glucoamylase leader sequence (published EP 362,179, 1990, 4, 4 characters), or the signal described in WO 90/13646 published November 15, 1990. In mammalian cell expression, mammalian signal sequences can be used for direct secretion of proteins, as well as viral secretion leader sequences, as well as signal sequences from secreted polypeptides of the same or related species.

Both expression and cloning vectors contain nucleic acid sequences that allow the vector to replicate in one or more secreted host cells. Such sequences are well known for various bacteria, yeasts and viruses. The replication origin from plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 μplasmid initiator is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are used to clone vectors in mammalian cells. useful.

Expression and cloning vectors will generally contain a selection gene, also known as a selectable label. Typical selection genes (e.g., genes encoding D-alanine racemase in vasilli) may (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, or (b ) Encodes a protein that compensates for nutrient deficiency, or (c) supplies important nutrients that are not available from the complex medium.

Examples of selectable labels suitable for mammalian cells are those that allow identification of cells capable of absorbing FGF-19-encoding nucleic acids, such as DHFR or thymidine kinase. When wild type DHFR is used, suitable host cells are described in Urlaub et al., Proc. Natl. Acad. Sci. USA, 77: 4216 (1980), which is a CHO cell line deficient in DHFR activity, prepared and expanded as described. Suitable selection genes for use in yeast are the trp1 gene present in yeast plasmid YRp7 [Stinchcomb et al., Nature, 282: 39 (1979); Kingsman et al., Gene, 7: 141 (1979); Tschemper et al., Gene, 10: 157 (1980)]. The trp1 gene provides a selection marker for mature strains of yeast that cannot grow on tryptophan (eg ATCC No. 44076 or PEP4-1) (Jones, Genetics, 85:12 (1977)).

Expression and cloning vectors usually contain a promoter operably linked to the FGF-19-encoding nucleic acid sequence to induce mRNA synthesis. Promoters recognized by various potential host cells are well known. Suitable promoters for use in 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, tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8: 4057 (1980); EP36,776, 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 facilitating sequences suitable for use in yeast hosts are described in 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255: 2073 (1980)] or other glycolysis enzymes [Hess et al., J. Adv. Enzyme Reg., 7: 149 (1968); Holland, Biochemistry, 17: 4900 (1978)], for example, enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6 -Promoters for phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triophosphate isomerase, phosphoglucose isomerase and glucokinase.

Other yeast promoters that are inducible promoters with the added benefit of transcription controlled by growth conditions include alcohol dehydrogenase 2, isocytochrome C, acidic phosphatase, degrading enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde Promoter region for -3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose use. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

FGF-19 transcription from a vector in a mammalian host cell can be determined by, for example, polyoma viruses, avianpoxviruses (UK 2,211,504 published July 5, 1989), adenoviruses if these promoters are compatible with the host cell system. Eg, genomes of viruses such as adenovirus 2), bovine papilloma virus, avian granulomas virus, cytomegalovirus, retrovirus, hepatitis-B virus and monkey virus 40 (SV40), heterologous mammalian promoters such as the actin promoter Or a promoter obtained from an immunoglobulin promoter, and a heat-shock promoter.

Transcription of the DNA encoding FGF-19 by higher eukaryotes can be increased by inserting an amplifier sequence into the vector. Amplifiers are cis-acting factors of DNA, usually about 10 to 300 bp, which act on the promoter and increase its transcription. Many amplifier sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). In general, however, amplifiers from eukaryotic cell viruses will be used. Examples include SV40 amplifiers (bp 100-270) on the back of the replication origin, cytomegalovirus early promoter amplifiers, polyoma amplifiers on the back of the replication origin and adenovirus amplifiers. The amplifier can be conjugated to the vector at the 5 'or 3' position relative to the FGF-19-coding sequence, but is preferably located at the 5 'position from the promoter.

Expression vectors used in eukaryotic host cells (nucleated cells from yeasts, fungi, insects, plants, animals, humans or other multicellular organisms) will also contain sequences necessary for termination of transcription and stabilization of mRNA. Such sequences are generally available from the 5 ', and sometimes 3', non-toxic regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide regions transcribed as polyadenylation fragments in the non-toxin portion of the mRNA encoding FGF-19.

Other methods, vectors and host cells suitable for alteration of FGF-19 in the synthesis of recombinant vertebrate cell cultures are described in Gothing 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 Amplification / Expression

Gene amplification and / or expression may be performed using, for example, conventional Southern blotting, Northern blotting to quantify transcription of mRNA, dot blotting (DNA analysis) using appropriately labeled probes based on the sequences provided herein Or directly in the sample by in situ hybridization. Alternatively, antibodies can be used that can recognize a particular pair of strands, including DNA pairs, RNA pairs, DNA-RNA pairs or DNA-protein pairs. In addition, an assay can be performed that can label the antibody and detect the presence of the antibody bound to the twin strand when the pair is bound to the surface to form the twin strand on the surface.

Alternatively, gene expression may be measured by immunological methods such as immunohistochemical staining of cell or tissue portions and cell culture or humoral assays to directly quantify expression of the gene product. Antibodies useful for immunohistochemical staining and / or sample bodily fluid assays can be monoclonal or polyclonal, and can be prepared in any mammal. For convenience, antibodies can be prepared against native sequence FGF-19 polypeptides, synthetic peptides based on the DNA sequences provided herein, or exogenous sequences fused with FGF-19 DNA and encoding specific antibody epitopes.

5. Purification of Polypeptides

Forms of FGF-19 can be recovered from medium or host cell lysate. If membrane-bound, this may be liberated from the membrane using a suitable surfactant solution (eg Triton-X 100) or by enzymatic cleavage. Cells used for expression of FGF-19 can be disrupted by various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption or cytolytic agents.

It may be desirable to purify FGF-19 from recombinant cell proteins or polypeptides. The following methods are examples of suitable purification methods: fractionation on an ion exchange column; Ethanol precipitation method; Reverse phase HPLC; Chromatography on silica, or cation exchange resins such as DEAE; Chromatofocusing; SDS-PAGE; Ammonium sulfate participation; Gel filtration using, for example, Sephadex G-75; Protein A Sepharose column to remove contaminants such as IgG; And a metal chelate column that binds to FGF-19 in epitope-tag form. Various protein purification methods can be used and such methods are 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 purification step (s) selected will depend, for example, on the nature of the preparation method used and the particular FGF-19 produced.

E. Use of FGF-19

Nucleotide sequences encoding FGFs (or their complements) have a variety of applications in the field of molecular biology, including hybridization probes, chromosomes and gene mapping and the generation of antisense RNAs and DNA. In addition, FGF-19 nucleic acids may be useful for preparing FGF-19 polypeptides by the recombinant techniques described herein.

Full length FGF-19 gene native sequence (SEQ ID NO: 1), or portions thereof, isolates full length FGF-19 cDNA having the desired sequence identity with the FGF-19 sequence described in FIG. 1 (SEQ ID NO: 1). Or as a hybridization probe to a cDNA library for isolating other cDNAs (eg, those encoding FGF-19 or native FGF-19 variants from other species). Optionally, the probe will be about 20 to about 50 bases in length. Hybridization probes will be derived from at least partially novel regions of the nucleotide sequences of SEQ ID NO: 1, which regions may be genome sequences comprising promoters, enhancer elements and introns of the native sequence of FGF-19 or without undue experimentation. It can be determined from. As an example, the screening method will include isolating the coding region of the FGF-19 gene using known DNA sequences to synthesize selected probes of about 40 bases. Hybridization probes can be labeled with a variety of labels, including ribonucleotides such as ³² P or ³⁵ S, or enzymatic labels such as alkaline phosphatase coupled to the probe by an avidin / biotin coupling system. Labeled probes having sequences complementary to the sequences of the FGF-19 genes of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of these libraries hybridize. Hybridization techniques will be described in more detail in the Examples below.

All EST sequences disclosed in the present application can be similarly used as probes using the methods disclosed herein.

Other useful fragments of FGF-19 nucleic acids include antisense or sense oligonucleotides, including single stranded nucleic acid sequences (RNA or DNA) capable of binding to target FGF-19 mRNA (sense) or FGF-19 DNA (antisense). . Antisense or sense oligonucleotides according to the present invention comprise fragments of coding regions of FGF-19 DNA. Such fragments generally comprise at least about 14 nucleotides, preferably about 14 to 30 nucleotides. The ability to induce antisense or sense oligonucleotides based on cDNA sequences encoding a given protein is described, for example, in Stein and Cohen, Cancer Res. 48: 2659, 1988 and van der Krol et al. BioTechniques 6: 958, 1988.

Binding of an antisense or sense oligonucleotide to a target nucleic acid sequence may result in the duplex that prevents the transcription or translation of the target sequence by one of several methods, including increased degradation of the duplex, early termination of transcription or translation, or other methods. Causes formation. Thus, antisense oligonucleotides can be used to block the expression of FGF-19 protein. In addition, antisense or sense oligonucleotides include oligonucleotides with modified glycophosphate diester backbones, wherein the sugar bonds are resistant to endogenous nucleases. Oligonucleotides having such resistant sugar linkages are stable in vivo (ie, able to withstand enzymatic degradation) but retain sequence specificity capable of binding to the target nucleotide sequence.

Other examples of sense or antisense oligonucleotides are oligonucleotides covalently linked to organic components, such as those described in WO 90/10048, and other components that increase the affinity of the oligonucleotide for the target nucleic acid sequence, for example. poly- (L-lysine). In addition, intercaltaing agents such as ellipsin and alkylating agents or metal complexes may be attached to the sense or antisense oligonucleotides to modify the binding specificity of the antisense or sense oligonucleotides to the target nucleotide sequence.

Antisense or sense oligonucleotides are introduced into cells containing target nucleic acid sequences using, for example, CaPO ₄ -mediated DNA transfection, any gene transfer method including electroinvasion, or gene transfer vectors such as Epstein-Barr virus. can do. In a preferred method, antisense or sense oligonucleotides are inserted into a suitable retroviral vector. Cells containing the target nucleic acid sequence are contacted with the recombinant retroviral vector in vivo or ex vivo. Suitable retroviruses include murine M-MuLV, N2 (retrovirus derived from M-MuLV), or duplex vectors labeled with DCT5A, DCT5B and DCT5C (see WO 90/13641), It is not limited to this.

In addition, sense or antisense oligonucleotides can be introduced into cells containing the target nucleotide sequence by forming conjugates with ligand binding molecules such as those described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, the conjugation of the ligand binding molecule substantially does not interfere with the ability of the ligand binding molecule to bind its corresponding molecule or receptor, or substantially prevents the sense or antisense oligonucleotide or its conjugated version from entering the cell. Do not.

Alternatively, the sense or antisense oligonucleotides can be introduced into cells containing the target nucleic acid sequence by oligonucleotide-lipid complex formation as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complexes are preferably degraded in cells by endogenous lipases.

In addition, probes can be used in PCR techniques to generate sequence pools for identifying significantly similar FGF-19 coding sequences.

The nucleotis sequence encoding FGF-19 can also be used to prepare hybridization probes for the mapping of genes encoding FGF-19 and for genetic analysis of individuals with genetic diseases. Nucleotide sequences provided herein can be mapped to specific regions of chromosomes and chromosomes using known techniques, such as in situ hybridization, association to known chromosomal markers, and hybridization screening of libraries.

When the coding sequence of FGF-19 encodes a protein that binds to another protein (eg, when FGF-19 is a receptor), FGF-19 is assayed to identify other proteins or molecules involved in binding interactions. Can be used in By this method, inhibitors of receptor / ligand binding interactions can be identified. In addition, proteins involved in such binding interactions can be used to screen peptides or small molecule inhibitors or agonists of binding interactions. In addition, receptor FGF-19 can be used to isolate correlated ligand (s). Screening assays can be designed to find inducible compounds that mimic the biological activity of native FGF-19 or receptors of FGF-19. Such screening assays include assays capable of high yield screening of compound libraries and make them particularly suitable for identifying small molecule drug candidates. Designed small molecules include synthetic organic or inorganic compounds. Assays can be performed in a variety of forms, including well characterized protein binding assays, biochemical screening assays, immunoassays and cell based assays in the art.

In addition, nucleic acids encoding FGF-19 or its modified forms can be used to generate transgenic or “knock-out” animals, which are also useful for developing and screening therapeutically useful reagents. . A transgenic animal (eg a mouse or rat) is an animal with cells containing the transgene, which is introduced before birth, for example in the embryonic stage, in the animal or parental zone of the animal. The transgene is DNA that is integrated into the genome of the cell from which the transgenic animal develops. In one embodiment, a cDNA encoding FGF-19 can be used to clone genomic DNA encoding FGF-19 according to established techniques, and a trait containing cells to express the DNA encoding FGF-19. Genomic sequences can be used to generate transgenic animals. Methods for generating transgenic animals, especially animals such as mice or rats, are conventional in the art and are described, for example, in US Pat. Nos. 4,736,866 and 4,870,009. In general, certain cells will be targeted for the introduction of the FGF-19 transgene along with tissue specific enhancers. Transgenic animals comprising a copy of a transgene encoding a FGF-19 introduced into the gonad of the animal at the embryonic stage may be used to investigate the effect of increased expression of DNA encoding the FGF-19. Such animals can be used, for example, as test animals for reagents thought to be able to protect against pathological symptoms associated with overexpression. According to this aspect of the invention, the animal is treated with a reagent and the reduced incidence of pathological symptoms as compared to untreated animals with the transgene will indicate potential therapeutic intervention for the pathological symptoms.

Alternatively, non-human homologous FGF-19 is a missing or modified gene as a result of homologous recombination between an endogenous gene to tote FGF-19 and a modified genomic DNA encoding FGF-19 introduced into animal embryonic liver cells. Can be used to prepare FGF-19 "knock out" animals. For example, cDNA encoding FGF-19 can be used to clone genomic DNA encoding FGF-19 according to established techniques. Portions of genomic DNA encoding FGF-19 can be deleted or replaced with other genes, such as genes encoding selection markers that can be used to monitor integration. In general, a number kb of unmodified flanking DNA can be included in the vector (Thomas and Capecchi, Cell, 51: 503 (1987) for a description of homologous recombination vectors, see also). This vector is introduced into an embryonic liver cell line (e.g., by electroinvasion) and cells into which the introduced DNA has undergone homologous recombination with endogenous DNA are selected [Li et al., Cell, 69: 915 (1992). , Reference]. The selected cells are then injected into the blastocysts of animals (eg mice or rats) to form aggregate chimeras [Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. The chimeric embryos are then transplanted into a suitable surrogate female parent animal and the embryos produce "knock out" animals. Progeny with homologous recombinant DNA in germ cells can be identified by standard techniques, and all cells of the animal can be used to breed animals containing homologous recombinant DNA. Knockout animals can be characterized, for example, by the ability to resist certain pathological symptoms and the expression of pathological symptoms due to the absence of the FGF-19 polypeptide. In addition, nucleic acids encoding FGF-19 can be used in gene therapy. In gene therapy applications, genes are introduced into cells to achieve synthesis of therapeutically effective genetic products in vivo, for example for replacement of missing genes. "Gene therapy" includes both conventional gene therapy in which a sustained effect is achieved by single treatment and in which the administration of the gene therapy involves a single or repeated administration of a therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can be used as therapeutic agents to interfere with the expression of certain genes in vivo. It has already been found that short antisense oligonucleotides can be transported into cells that can act as inhibitors despite their low intracellular concentration due to their limited uptake by the cell membrane (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83; 4143-4146 [1986]). Oligonucleotides can be modified with uncharged groups, for example by replacing their negatively charged diester phosphate groups to increase their uptake.

There are a variety of techniques that can be used to introduce nucleic acids into live cells. This technique depends on whether the nucleic acid can be delivered in culture in cells or in vivo in cells of a given host. Techniques suitable for delivery of nucleic acid into mammalian cells in vitro include the use of liposomes, electroinvasion, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation methods, and the like. Currently preferred in vivo gene transfer techniques include transfection by viral (generally retroviral) vectors and viral envelope protein-liposomal mediated transfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]. ]). In some cases, it is desirable to provide a nucleic acid source with reagents that target the target cell, such as cell surface membrane proteins or antibodies specific for the target cell, ligands for receptors on the target cell, and the like. If liposomes are used, proteins that bind to cell surface membrane proteins associated with endocytosis can be used for targeting (or) to facilitate uptake. For example, capsid proteins or fragments thereof specific to a particular cell type, antibodies to proteins undergoing internalization during the cycle, proteins that target intracellular placement and enhance intracellular half-life can be used. Techniques for receptor mediated endocytosis are described, for example, in Wu et al., J. Biol. Chem. 262, 4429-4432 (1987); And Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). For gene labeling and gene therapy protocols, see Anderson et al., Science 256, 808-813 (1992).

In addition, the FGF-19 polypeptides described herein can be used as molecular weight labels for protein electrophoresis purposes.

Nucleic acid molecules encoding the FGF-19 polypeptides or fragments thereof described herein are useful for chromosome identification. In this regard, there is a continuing need to identify new chromosomal labels because of the relatively small number of chromosomal labeling reagents based on the actual sequence data currently available. Each FGF-19 nucleic acid molecule of the invention can be used as a chromosome marker.

In addition, the FGF-19 polypeptides and nucleic acid molecules of the present invention can be used for histological testing, wherein the FGF-19 polypeptides of the present invention can be expressed differently in one tissue compared to other tissues. The FGF-19 nucleic acid molecule will find use as a production probe for PCR, Northern blot, Southern analysis and Western analysis.

In addition, the FGF-19 polypeptides thereof and modulators thereof may be used as therapeutic agents. FGF-19 polypeptides and modulators thereof of the present invention may be formulated according to known methods to prepare pharmaceutically useful compositions, whereby their FGF-19 products are mixed with a pharmaceutically acceptable carrier vehicle. . Therapeutic formulations are prepared for storage in lyophilized formulations or aqueous solutions, mixed with active ingredients of the desired degree of purity and optionally with physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Science 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients or stabilizers are not toxic to the recipient at the dosages and concentrations employed, and are buffered solutions such as phosphates, citrates and other organic acids, antioxidants including ascorbic acid, low molecular weight (about 10 residues) Polypeptides, proteins such as serum albumin, gelatin or immunoglobin, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disaccharides and glucose, mannose, or dextrins Other carbohydrates, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt forming counter ions such as sodium, and / or nonionic surfactants such as TWEEN ™, PLURONICS ™ or PEG.

The formulations used for in vivo administration must be sterile. This can be easily accomplished by filtration through sterile filtration membranes, prior to lyophilization and reconstitution.

Therapeutic compositions herein are generally placed in vials having a stopper that can be pierced with a sterile access port, such as an injection solution bag or a hypodermic needle.

The route of administration is in accordance with known methods such as intravenous, intraperitoneal, intracranial, intramuscular, intraocular, intraarterial or intralesional routes, topical administration, or injection, or infusion by a sustained release system.

Dosages and desired drug concentrations of the pharmaceutical compositions of the invention may vary depending upon the particular use contemplated. Appropriate doses or routes of administration are determined within the ordinary physician's skill. Animal experiments provide reliable guidance of effective dosages for phosphoric acid treatment. Species measures of effective dosages are described in 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, pp 42 Can be carried out according to the principles set out in [96].

When in vivo administration of an FGF-19 polypeptide or an agonist or antagonist thereof is adopted, the normal dosage ranges from about 10 ng / kg (mammalian body weight) to 100 mg / kg (mammalian body weight), preferably about 1 depending on the route of administration. May vary from μg / kg / day to 10 mg / kg / day. Detailed instructions on specific dosages and methods of delivery are provided in the literature. For example, US Pat. Nos. 4,657,760, 5,206,344 or 5,225,212 are described. It is expected that different agents will be effective for different therapeutic compounds and different diseases, and targeting administration to one organ or tissue will need to be delivered in a different manner than, for example, administration to another organ or tissue.

If the sustained release administration of the polypeptide or modulator of FGF-19 is desired in an agent with release properties suitable for the treatment of any disease or condition that requires administration of the FGF-19 polypeptide or modulator, microencapsulation is contemplated. Microencapsulation of recombinant proteins for sustained release formulations has been successfully performed with human growth hormone (rhGH), interferon- (rhIFN-)-, interleukin-2, and MN rgp120 [Johnson et al., Nat. Med., 2: 795-799 (1996); Yasuda, Biomed. Ther., 27: 1221-1223 (1993); Hora et al., Bio / Technology, 8: 755-758 (1990); Cleland, "Design and Production of Single Immunization Vaccines Using Polyvalent Polypeptide Polyglycolide Microsphere Systems." in Vaccine Deisign: 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. Patent No. 5,654,010].

Sustained release preparations of these proteins have been developed using these polymers due to the biocompatibility and the broad range of biodegradable properties of polylactic-coglycolic acid (PLGA) polymers. The degradation products, lactic acid and glycolic acid of PLGA can be quickly removed from the living body. Moreover, the resolution of this polymer can be adjusted from months to years depending on its molecular weight and composition. Lewis, "Controlled release of bioactive agents from lactide / glycolide polymer," in M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41.

Therapeutic agents and compositions comprising FGF-19 provided herein can be used in a number of applications. Applications include treating individuals with obesity or with symptoms associated with obesity. In one embodiment, FGF-19 is administered to a subject in need thereof in an amount effective to treat the condition. Preferably, the condition is one of one or more of the following symptoms to be treated: increased metabolism, reduced body weight, reduced body fat, reduced triglycerides, reduced free fatty acids, increased glucose outflow from adipocytes and / or fat Increase in leptin release from cells. Each of these parameters can be measured by standard methods such as measuring oxygen consumption to measure metabolic rate, using a scale to measure body weight, and measuring size to measure fat. In addition, the presence and amount of triglycerides, free fatty acids, glucose and leptin can be measured by standard methods. Each of these parameters is illustrated in the specific examples below.

Compositions comprising FGF-19 and FGF-19 are preferably used in vivo. However, as discussed below, administration can be in vitro as in the methods for screening FGF-19 modulators described below. However, modulators of FGF-19 can also be identified using animal models and patient samples.

The present invention includes methods for screening compounds to identify or mimic (or agonist) the FGF-19 polypeptide or to interfere with or inhibit the effect of the FGF-19 polypeptide (antagonist). Agonists and antagonists are referred to herein as modulators. Screening assays for antagonist drug candidates were used to identify compounds that bind to or complex the FGF-19 polypeptide encoded by the genes identified herein, or interfere with the interaction of the encoded polypeptide with other cellular proteins. Can not be done. Such screening assays will allow for high efficiency screening of compound libraries and will include assays that make them particularly suitable for identifying small molecule drug candidates.

Assays can be performed in a variety of formats, including well-characterized protein-protein binding assays, biochemical screening assays, immunoassays, and cell based assays.

All assays for antagonists are common in that they require contact of the drug candidate with the FGF-19 polylipeptides encoded by the nucleic acids identified herein under conditions and time sufficient for both components to interact.

In binding assays, the interactions are complexes that are formed and formed that can be isolated or detected in the reaction mixture. In specific embodiments, an FGF-19 polypeptide or drug candidate encoded by a gene identified herein is immobilized with a covalent or non-covalent attachment to a solid phase, such as a micro titer. Non-covalent attachment is generally accomplished by coding and drying the solid surface with FGF-19 polypeptide solution. Alternatively, an immobilized antibody, such as a monoclonal antibody, specific for the FGF-19 polypeptide to be immobilized can be used as an immobilization device for a solid surface. The analysis is performed by adding an unfixed component, which can be labeled on a coated surface containing an immobilized component, for example an immobilized component, with a detectable label. When the reaction is complete, unreacted components are removed by removal, for example by washing, and a complex immobilized on the solid surface is detected. When the original unimmobilized component has a label that can be detected, detection of the label immobilized on the surface indicates that the complex has occurred. When the present unimmobilized component does not have a label, the complex can be detected using, for example, a labeled antibody that specifically binds the immobilized complex.

If a candidate compound interacts with, but does not bind to, a particular FGF-19 polypeptide encoded by a gene identified herein, the interaction with that polypeptide can be analyzed by well known methods for detecting protein-protein interactions. Such assays include traditional approaches such as cross linking, co-immunoprecipitation, and co-purification via concentration gradients or chromatography columns. In addition, protein-protein interactions are described by Fields and co-workers as disclosed by Chevray and Nathans (Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991)). Can be monitored using a yeast based system described by Fields and Song, Nature (London), 340: 245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA, 88: 9578-9582 (1991). Many transcriptional activators, such as yeast GAL4, consist of two physically separate domains, one acting as a DNA-binding domain and the other acting as a transcriptional activation domain. The yeast expression system described in this publication (generally referred to as "two-hybrid system") has the advantage of this property, two hybrid proteins, one targeting protein GAL-4 The fusion to the DNA-binding domain of the other, the candidate activating protein is used to be fused to the activation domain. Expression of the GAL4- lac Z reporter gene under the control of the GAL4-activating promoter relies on reconstitution of GAL4 activity through protein-protein interactions. Colonies containing interacting polypeptides are detected as chromogenic substrates for β-galactosidase. A full kit (MATCHMAKER ™) for confirming protein-protein interactions between two specific proteins using two hybrid techniques is commercially available from Clontech. The system can also be used to map protein domains involved in specific protein interactions and amino acid residues in the correct positions critical for these interactions. Compounds that interfere with the interaction of genes encoding the FGF-19 polypeptides identified herein with other intracellular or extracellular components can be tested as follows: Conditions that generally allow for interaction and binding of the two products And a reaction mixture containing the gene product and intracellular or extracellular components under time. To test the ability of the candidate compound to inhibit binding, the reaction is performed in the presence and absence of the test compound. The binding (complex formation) between the test compound and the intracellular or extracellular component in the mixture is monitored as described above. Complex formation in the control reactant (s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound with its reaction partner.

To analyze antagonists, FGF-19 polypeptides can be added in the cell with the compound to be screened for specific activity, and the ability of the compound to inhibit target activity in the presence of the FGF-19 polypeptide is such that Indicates that it is an antagonist. Alternatively, the antagonist can be detected by combining the membrane bound FGF-19 polypeptide receptor or recombinant receptor with FGF-19 polypeptide and potential antagonist under conditions appropriate for competitive inhibition assays. FGF-19 polypeptides can be labeled, for example, radioactive, such that a number of FGF-19 polypeptide molecules bound to the receptor can be used to measure the potency of a potential antagonist. Genes encoding receptors can be identified by a number of methods known to those of ordinary skill in the art, for example ligand panning and FACS classification [Coligan et al., Current Protocols in Immun., 1 (2): Chapter 5 (1991)]. Preferably, polyadenylated RNA is prepared from cells reactive to the FGF-19 polypeptide, and cDNA libraries generated from this RNA are divided into pools and transduced COS cells or other cells that do not respond to the FGF-19 polypeptide. Expression cloning is used when used to produce. Transfected cells grown on glass slides are exposed to labeled FGF-19 polypeptides. FGF-19 polypeptides can be labeled in a variety of ways, including iodide and inclusion of recognition sites for site specific protein kinases. After fixation and incubation, the slides are analyzed by autoradiography. Reaction positive pools are identified, subpools are prepared, and the relevant subpools are retransfected with the use and rescreening method, resulting in a single clone encoding the putative receptor.

As another approach for receptor identification, the labeled FGF-19 polypeptide may be photoaffinity linked with a cell membrane or extract preparation that expresses the receptor molecule. Crosslinked material is analyzed by PAGE and exposed to X-ray film. The labeled complex containing the receptor is cut off, digested into peptide fragments and protein micro-sequenced. The amino acid sequences obtained from micro-sequencing will be used to devise a set of overlapping oligonucleotides for screening cDNA libraries to identify genes encoding putative receptors.

In other assays for antagonists, mammalian cell or membrane preparations expressing the receptor will be incubated with labeled FGF-19 polypeptide in the presence of the candidate compound. At this time, the ability of the compound to increase or block this interaction can be measured.

More specific examples of potential antagonists include oligonucleotides, in particular poly- and monoclonal antibodies and antibody fragments, single-stranded antibodies, antiioid antibodies and such antibodies and fragments that bind to fusions of FGF-19 polypeptides with immunoglobulins. Including but not limited to antibodies comprising a chimeric or humanized version of human antibodies and antibody fragments. Alternatively, the potential antagonist may be a mutated form of the FGF-19 polypeptide that competitively inhibits the action of the FGF-19 polypeptide by recognizing but without affecting a significantly related protein, such as a receptor.

In one embodiment herein, when a competitive binding assay is performed, an antibody against FGF receptor 4 or FGF-19 is used as a competition.

Another potential FGF-19 polypeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology, for example, the antisense RNA or DNA molecule acts to directly block mRNA translation by hybridizing to the target mRNA and interfering with protein translation. do. Antisense techniques can be used to regulate gene expression via triple helix formation or antisense DNA or RNA, both methods based on binding of polynucleotides to DNA or DNA. For example, the 5 'coding portion of a polynucleotide sequence encoding a mature FGF-19 polypeptide herein is used to design antisense RNA oligonucleotides of about 10 to 40 bases in length. DNA oligonucleotides are designed to be complementary to gene regions involved in transcription (triple helix-Lee et al., Nucl. Acids Res., 6: 3073 (1979); Cooney et al., Science, 241: 456 (1988) Dervan et al., Science, 251: 1360 (1991)), thereby interfering with the transcription and production of FGF-19 polypeptides. Antisense RNA oligonucleotides hybridize to mRNA in vivo and block the translation of mRNA molecules to FGF-19 polypeptides (Antisense-Ocano, Neurochem., 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC) Press: Boca Raton, FL, 1988) The oligonucleotides described above can also be delivered into cells to express antisense RNA or DNA in vivo to inhibit the production of FGF-19 polypeptides. When is used, oligodioxyribonucleotides derived from the translation initiation site, eg, about −10 to +10 positions of the target gene nucleotide sequence, are preferred.

Potential antagonists include small molecules that bind to the active site, receptor binding site, or growth factor or other related binding site of the FGF-19 polypeptide, thereby blocking the normal biological activity of the FGF-19 polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide like molecules, preferably soluble peptides, and synthetic non-peptide organic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act as endonuclease cleavage after sequence specific hybridization to the complementary target RNA. Specific ribozyme cleavage sites within potential RNA targets can be identified by known techniques. See Rossi, Current Biology, 4: 469-471 (1994), and PCT Publication WO 97/3351 (published Sep. 18, 1997).

In triple helix formation used for transcription inhibition, the nucleic acid molecule should be single stranded and composed of deoxynucleotides. The base composition of these oligonucleotides is designed to promote triple helix formation according to Hogsteen base pair law, which generally permits significant elongation of purine or pyrimidine on one strand of the double helix. See PCT Publication WO 97/33551 for details.

These small molecules can be identified using one or more of the screening assays described above and / or other screening techniques well known to those of ordinary skill in the art.

It is recognized that all assays provided herein are used to screen a wide range of candidate bioactive agents. As used herein, the term "candidate bioactive agent," "candidate agent" or "drug candidate" or grammatical equivalents thereof, refers directly to one of the cellular active phenotypes or expressions of the FGF-19 sequence, including both nucleic acid and protein sequences. Or any small molecule tested for bioactive agents that can be indirectly modified, such as proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, purine analogs, and the like.

Candidate agents encompass a broad group of compounds, but in general they are organic molecules, preferably small organic compounds having a molecular weight of at least 100 and up to about 2,500 Daltons (d). Small molecules are further defined herein as having molecular weights from 50 d to 2,000 d. In other embodiments, the small molecule has a molecular weight of 1,500 or less, or 1,200 or less, or 1,000 or less, or 750 or less, or 500 d or less. In one embodiment, small molecules as used herein have a molecular weight of about 100 to 200 d. Candidate agents include functional groups necessary for structural interaction with the protein, in particular hydrogen bonding, and generally comprise one or more amine, carbonyl, hydroxyl or carboxyl groups, preferably two or more chemical functional groups. Candidate agents often include cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among peptides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.

Candidate agents come from a wide variety of sources, including libraries of synthetic or natural compounds. For example, many methods can be used in the randomized and designated synthesis of a wide range of organic compounds and biomolecules, including the expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily prepared. In addition, libraries and compounds prepared by natural or synthetic methods are readily modified by conventional chemical, physical and biochemical methods. Known agents may be subject to designated or randomized chemical modifications, such as acylation, alkylation, esterification, amidation, to prepare structural analogs.

In a preferred embodiment, the candidate bioactive agent is a protein. By "protein" is meant herein an amino acid linked by two or more covalent bonds, which includes proteins, polypeptides, oligopeptides and peptides. Proteins may consist of naturally occurring amino acids and peptide bonds, or synthetic peptide mimetic structures. Thus, as used herein, "amino acid," or "peptide moiety" refers to both natural and synthetic amino acids. For example, homo-phenylalanine, citrulline and norleucine are considered amino acids for the purposes of the present invention. "Amino acids" also include imino acid residues such as proline and hydroxyproline. The side chains can be in either the (R) or (S) configuration. In a preferred embodiment, the amino acids are (S) or (L) -configuration. If a non-natural side chain is used, unsubstituted amino acid substituents can be used, for example, to prevent or delay degradation in vivo.

In a preferred embodiment, the candidate bioactive agent is a native protein or fragment of a native protein. Thus, for example, randomized or directed degradation of protein-containing intracellular extracts or proteinaceous intracellular extracts can be used. In this way libraries of prokaryotic and eukaryotic proteins can be used for screening in the methods of the invention. Especially preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, the latter being preferred, and human proteins being particularly preferred.

In a preferred embodiment, the candidate bioactive agent is a peptide of about 5 to about 30 amino acids, with about 5 to about 20 amino acids being preferred, and about 7 to about 15 amino acids being particularly preferred. The peptide can be a digest of a native protein, a randomized peptide, or a “biased” randomized peptide as outlined above. As used herein, “randomized” or grammatical equivalents mean that each nucleic acid and peptide consists essentially of randomized nucleotides and amino acids, respectively. In general, since these random peptides (or nucleic acids, described below) are chemically synthesized, they can introduce any nucleotide or amino acid at any position. Synthetic methods can be designed to generate randomized proteins or nucleic acids and allow the formation of almost any possible combination over the entire length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous materials.

In one embodiment, the library is completely randomized and there are no sequence preferences or restrictions at any location. In a preferred embodiment, the library is biased. That is, any position in the sequence is limited or selected within a limited range of possibilities. For example, in a preferred embodiment, the nucleotides or amino acid residues are formed in a limited class, for example hydrophobic amino acids, hydrophilic residues, stericly biased (small or large) residues, for the generation of nucleic acid binding domains, the formation of, crossover of cysteines. Randomized in binding, proline for the SH-3 domain, serine for the phosphorylation site, threonine, tyrosine or histidine and the like or purine and the like.

In a preferred embodiment, the candidate bioactive material is a nucleic acid. As used herein, "nucleic acid" or "oligonucleotide" or grammatical equivalent thereof means that two or more nucleotides are covalently linked to each other. Nucleic acids of the present invention will generally comprise phosphodiester bonds, but in some cases, for example, as described below, for example, phosphateamide (Beaucage et al., Tetrahedron 49 (10): 1925 (1993) and the above references. Ref., Letsinger, J. Org. Chem. 35: 3800 (1970); Sprinzl et al., Eur. J. Biochem. 81: 579 (1977); Letsinger et al., Nucl.Acids Res. 14: 3487 (1986); Sawai et al., Cehm. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110: 4470 (1988); and Pauwels et al., Chemica Scripta 26: 141 ( 1986)), thiothiophosphate (Mag et al., Nucleic Acids Res. 19: 1437 (1991); and US Pat. No. 5,644,048), dithiophosphate (Briu et al., J. Am. Chem. Soc. 111: 2321 (1989)), O-methylphosphoroamidite binding (see Eckstein, Oligonucelotides and Analogues: A Practical Approach, Oxford University Press) and peptide nucleic acid backbones and binding (Egholm, J. Am. Chem. Soc. 114 1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31: 1008 (1992) Nucleic acid analogs that may have other backbones, including Nielsen, Nature, 365: 566 (1993); Carlsson et al., Nature 380: 207 (1996)), all of which are incorporated by reference. Other analogue nucleic acids are described in US Pat. Nos. 5,235,033 and 5,034,506 and in Chapters 6 and 7, ASC Symphosium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y.S. Positive skeleton (Denpcy et al., Proc. Natl. Acad. Sci. USA 92: 6097 (1995)), including those described in Sanghui and P. Dan Cook; Nonionic backbones (US Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. Englisgh 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 Symphosium Series 580, "Carbohydrate Modifications in Antisense Research", Ed.YS Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem Lett. 4: 395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37: 743 (1996)) and non-lives backbones. Several nucleic acid analogs are described in Rawls, C & E News June 2,1997 page 35. All of these references are expressly incorporated herein by reference. These modifications of the ribose-phosphate backbone can be performed to facilitate the addition of additional ingredients such as labels or to increase the stability and half-life of such molecules in the physiological environment. In addition, mixtures of naturally occurring nucleic acids and analogs can be prepared. Alternatively, mixtures of different nucleic acid analogs and mixtures of native nucleic acids and analogs can be prepared. The nucleic acid may be single stranded or double stranded as specified, or may include both double stranded or single stranded sequence portions. The nucleic acid may be DNA, genome and cDNA, RNA or hybrids, wherein the nucleic acid is any combination of deoxyribose and ribo-nucleotides, and uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocyto May be any combination of bases including cin, isoguanine and the like.

As described above generally for proteins, the nucleic acid candidate bioactive material may be a naturally occurring nucleic acid, a randomized nucleic acid, or a 'biased' randomized nucleic acid. It can be used as outlined for the protein.

In a preferred embodiment, the candidate bioactive material is an organic chemical component, a wide range of which is described in the literature.

In a preferred embodiment, as outlined above, screens can be generated on individual genes and gene products (proteins). In a preferred embodiment, the gene or protein is associated with a particular tissue as described in the Examples below and thus is identified as a differently expressed gene associated with a disease associated with that tissue. Thus, in one embodiment, the screen is first designed to find candidate agents capable of binding to FGF-19, which can then be used in the assay to assess the ability of the candidate agents to modulate FGF-19 activity. . Thus, many different analyzes can be performed, as recognized by those of ordinary skill in the art.

In addition, screening of drugs that modulate FGF-19 activity can be performed. In a preferred embodiment, the method for screening bioactive agents capable of modulating the activity of FGF-19 comprises adding a candidate bioactive agent to a FGF-19 sample and measuring the change in biological activity of FGF-19. do. "Modulating activity of FGF-19" includes increasing activity, decreasing activity, or changing the type or type of current activity. Thus, in this embodiment, the candidate agent must be able to bind (but not necessarily) to FGF-19 and alter its biological or biochemical activity as defined herein. This method includes both in vitro screening methods such as those generally outlined above for alterations in the presence, expression, distribution, activity or amount of FGF-19 and in vivo screening of cells.

Thus, in this embodiment, the method comprises combining the sample and the candidate bioactive agent and assessing the effect on FGF-19 activity. As used herein, "FGF-19 protein activity" or grammatical equivalents refers to the biological activity of one or more FGF-19 proteins 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. Thus, 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 sequence are used in drug screening assays by evaluating the effect of drug candidates on FGF-19. Cell types include normal cells, tumor cells and fat cells.

Methods for evaluating FGF-19 activity, such as changes in glucose uptake, leptin release, metabolism, triglyceride and free fatty acid levels, body weight and body fat, are known in the examples below and known in the art.

In a preferred embodiment, the method comprises adding a candidate bioactive agent as defined above to the cells comprising FGF-19. Preferred cell types include most all cells. The cell comprises a nucleic acid encoding FGF-19, preferably a recombinant. In a preferred embodiment, the library of candidate agents is tested on multiple cells.

In one aspect, the assay is pharmacological, including physiological signals such as hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, chemotherapy drugs, radioactivity, carcinogens or other cells (ie, cell-cell contact). Evaluated in the presence or absence of the substance or in the prior or post exposure to these substances. In other embodiments, the measurements are measured at different stages of cell cycle progression.

In addition, the FGF-19 sequences provided herein can be used in diagnostic methods. Overexpression of FGF-19 will result in abnormally high metabolic rate and underexpression will show obesity tendency. In addition, samples of patients can be analyzed for mutated or dysfunctional FGF-19. In general, such methods include comparing a sample of a patient with a sample of a control for FGF-19 expression.

F. Anti-FGF-19 Antibodies

In addition, the present invention provides anti-FGF-19 antibodies. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific and heterozygous antibodies.

1. Polyclonal Antibodies

Anti-FGF-19 antibodies may comprise polyclonal antibodies. Methods of making polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be produced in mammals, for example by one or more injections of an immunizing agent and, if desired, an adjuvant. In general, the immunizing agent and / or adjuvant will be injected into the mammal by a number of subcutaneous or intraperitoneal injections. Immunizing agents will include FGF-19 polypeptides or fusion proteins thereof. It would be useful to conjugate the immunizing agent to a protein known to be immunized in the immunized mammal. Examples of such immunogenic proteins include, but are not limited to, keyholelimpethemocyanin, serum albumin, bovine tyroglobin, and soybean trypsin inhibitors. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicholinomycolate). Immunization protocols can be selected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

Alternatively, the anti-FGF-19 antibody may be a monoclonal antibody. Monoclonal antibodies can be prepared using hybridoma methods such as those described in Kohler and Milstein, Nature, 256: 495 (1975). In the hybridoma method, mice, hamsters, or other suitable host animals are generally immunized with an immunizing agent to produce lymphocytes that may or may produce antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

Immunizing agents generally comprise an FGF-19 polypeptide or a fusion protein thereof. Generally, peripheral blood lymphocytes (“PBLs”) are used when cells of human origin are required and spleen cells or lymph node cells are used when non-human mammalian origin is required. Lymphocytes are then fused with the immobilized cell line using a suitable fusing agent such as polyethylene glycol to form hybridoma cells [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103. Immobilized cell lines are generally transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. In general, rat or mouse myeloma cell lines are used. Hybridoma cells may preferably be cultured in a suitable culture medium containing one or more substances that inhibit the growth or survival of the unfused, immobilized cells. For example, if the parental cells lack the enzyme hypoxanthin guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium of hybridomas is generally hypoxanthine, aminoterin, and thymine (“HAT” medium). And these substances interfere with the growth of HGPRT-deficient cells.

Preferred immobilized cell lines are those that fuse effectively, support stable high levels of antibody expression by selected antibody forming cells, and are sensitive to media such as HAT media. More preferred immobilized cell lines are murine myeloma cell lines, which can be obtained, for example, from the Salk Institute Cell Distribution Cnter (San Diego, Calif.) And American Type Culture Collection (Manassas, Va.). In addition, human myeloma and mouse-human heteromyeloma cell lines have 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) pp. 51-63.

The cell medium in which hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies against FGF-19. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is measured by in vitro binding assays such as immunoprecipitation or radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. Binding affinity of monoclonal antibodies is described, for example, in Scatchard analysis of Munson and Pollard, Anal. Biochem., 107: 220 (1980).

After the desired hybridoma cells were identified, clones were subcloned in a limiting dilution method and cultured in a standard way [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites cells in a mammal.

The monoclonal antibodies secreted by the subclones may be cultured by conventional immunoglobulin purification methods such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. It can be isolated or purified from ascites fluid.

In addition, monoclonal antibodies can be prepared by recombinant DNA methods such as those described in US Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated by conventional methods (e.g., using oligonucleotide probes that can specifically bind to the genes encoding the H and L chains of murine antibodies). May be sequenced. The hybridoma cells of the present invention are used as a preferred source of such DNA. After isolation, the DNA can be located in an expression vector, and then Simian COS cells, Chinese hamster ovary (CHO) cells, which otherwise do not produce immunoglobin proteins, to obtain the synthesis of monoclonal antibodies in recombinant host cells. Or transfected into host cells such as myeloma cells. In addition, DNA can be modified, for example, by substituting coding sequences for the constant domains of the H and L chains in place of homologous murine sequences, or by covalently binding to all or part of the immunoglobulin coding sequence for non-immunoglobine polypeptides. [US Pat. No. 4,816,567; Morrison et al., Supra. Such non-immunoglobine polypeptides can be substituted in the constant domains of the antibodies of the invention to produce chimeric divalent antibodies, or in the variable domains of one of the antigen binding sites of the antibodies of the invention. The antibody may be a monovalent antibody. Methods of making monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin L chains and modified H chains. The H chain is generally cleaved at any one point in the Fc region to prevent crosslinking of the H chain. Alternatively, related cysteine residues are substituted or deleted with other amino acid residues to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies. Antibody digestion for preparing these fragments, particularly Fab fragments, can be accomplished using conventional techniques known in the art.

3. Human and Humanized Antibodies

In addition, the anti-FGF-19 antibodies of the present invention include humanized antibodies or human antibodies. Humanized antibodies of non-human (eg murine) antibodies include chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab ', F () that contain a minimal sequence derived from non-human immunoglobulin. ab ') ₂ or another antibody binding subsequence of the antibody). Humanized antibodies include human immunoglobin (receptor antibodies), and the residues of the complementarity determining regions (CDRs) of the receptors are CDRs (donor) of non-human species such as mice, rats or rabbits with the desired specificity, affinity and capacity. Antibody). In some cases, the Fv framework residues of human immunoglobin are replaced with corresponding non-human residues. Humanized antibodies also include residues that are not found in the receptor antibody or in the imported CDR or framework sequences. In general, humanized antibodies will comprise substantially all one or more, generally two variable domains, all or substantially all CDR regions corresponding to those of non-human immunoglobin, and all or almost all FR regions are human immunoglobin. Consensus sequence. In addition, the humanized antibody will optionally comprise at least a portion of an immunoglobin constant region (Fc), generally of human immunoglobin [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 of humanizing nonhuman antibodies are well known in the art. Humanized antibodies generally have one or more amino acid residues derived from a non-human origin and introduced into the antibody. These nonhuman amino acid residues are often referred to as "import" residues, which are generally derived from "import" variable domains. Humanization can be performed essentially in accordance with the methods of Winter and its co-workers by replacing the corresponding sequences of human antibodies with rodent CDRs and CDR sequences. [Jones et al., Nature, 321: 522-525 ( 1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988). Thus, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567), wherein the substantially intact human variable domains are substituted with the corresponding sequences of non-human species. In practice, humanized antibodies are generally human antibodies in which some CDR residues and possibly some FR residues are substituted by residues of analogous sites in rodent antibodies.

Human antibodies can also be prepared using a variety of methods known in the art, including phage display libraries [Hoogenhoom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Bio., 222: 581 (1991). In addition, techniques such as Cole and Boerner are useful for the preparation of human monoclonal antibodies (Cole 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 partially or completely lack the human immunoglobin locus in transgenic animals, eg, endogenous immunoglobin genes. After immunization, human antibody production is observed, which is very similar to that observed in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. For example, US Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,6612,016 and the following scientific publications: Marks et al., Bio / Technology 10 779-783 (1992); Lonberg e tal., Nature 368, 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fihgwild 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 antibodies that have binding specificities for at least two different antigens, monoclonal, preferably human or humanized. In this case, one of the binding specificities is for FGF-19, the other is for any other antigen, and preferably for cell surface proteins or receptors or receptor subunits.

Methods of making bispecific antibodies are known in the art. Typically, recombinant production of bispecific antibodies is based on co-expression of two immunoglobin H chains / L chains, where the two H chains have different specificities [Milstein and Cuello, Nature, 305: 537-539 (1983 )]. Due to the random combination of immunoglobulin H and L chains, these hybridomas (quamers) produce possible mixtures of 10 different antibody molecules, where only one has the correct bispecific structure. Purification of the correct molecule is generally accomplished by affinity chromatography. Similar methods are disclosed in WO 93/08829 (published May 13, 1993) and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).

Antibody variable domains (antibody-antigen binding sites) with the desired binding specificities can be fused to immunoglobulin constant domain sequences. The fusion preferably has an immunoglobulin H chain constant domain and comprises at least some of the hinge, CH2, and CH3 regions. It is preferred to have the H chain constant region (CH1) required for L chain bond present in one or more fusions. The DNA encoding the immunoglobulin H chain fusion and, if desired, the immunoglobulin L chain are inserted into separate expression vectors and cotransfected in a suitable host organism. See Suresh et al., Methods in Enzymology, 121: 210 (1986) for details of bispecific antibody production.

According to another approach described in WO 96/27011, the preferred contact surface between a pair of antibody molecules can be engineered to maximize the proportion of heterodimers recovered from recombinant cell culture. Preferred contact surfaces comprise at least a portion of the CH3 region of an antibody constant domain. In this method, at least one small amino acid side chain at the contact surface of the first antibody molecule is replaced with a large side chain (eg tyrosine or tryptophan). Compensatory "cavities" of the same or similar size as the large side chain (s) are produced on the contact surface of the second antibody molecule by replacing large amino acid chains with small amino acids (eg alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other undesired end products such as homodimers.

Heterospecific antibodies can be prepared as full length antibodies or antibody fragments (eg, F (ab ') ₂ heterospecific antibodies). Techniques for generating heterospecific antibodies from antibody fragments are described in the literature. For example, heterologous antibodies can be prepared using chemical bonds. Brennan et al. (Science 229: 81 (1985)) disclose a method in which intact antibodies are cleaved by proteases to produce F (ab ') ₂ fragments. These fragments are reduced in the presence of the dithiol complex sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide bond formation. The Fab 'fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reduced to mercaptoethylamine and converted to Fab'-thiol and mixed with the same amount of other Fab'-TNB derivatives to form a heterospecific antibody. The resulting heterospecific antibody can be used as a substance for selective immobilization of enzymes.

Fab 'fragments can be recovered directly from E. coli and chemically coupled to form heterologous antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describe the production of fully humanized heterospecific antibody F (ab ') ₂ molecules. Each Fab 'fragment is secreted separately from Escherichia coli and grows the designated chemical coupling in vitro to form heterologous antibodies. The heterospecific antibody thus formed was able to bind Erb2 receptors and cells that overexpress normal human T cells, and also induce lytic activity of human cytotoxic lymphocytes against human breast cancer targets.

In addition, various techniques have been described for preparing and isolating heterospecific antibody fragments directly from recombinant cell culture. For example, heterologous antibodies were prepared using leucine zippers. Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992). Leucine zipper peptides derived from Fos and Jun proteins were bound to the Fab 'portion of two different antibodies by gene fusion. The antibody homodimer was reduced in the hinge region to form a monomer, which was then reoxidized to form an antibody heterodimer. This method can also be used for antibody homodimer preparation. Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provides a "diabody" technique that provides an alternative mechanism for preparing heterospecific antibody fragments. The fragment comprises an H chain variable domain (V _H ) linked to the L chain variable domain (V _L ) by a linker that is too short to allow pairing between two domains on the same chain. Therefore, V _H and V _L domains of one fragment are formed complementary to the V _H and V _L pairs and the other fragment, thereby forming two antigen-binding sites. Other methods for preparing heterospecific antibody fragments by the use of single stranded F _V (sF _V ) dimers have also been reported. Gruber et al., J. Immunol. See 152: 5368 (1994).

Bivalent or higher antibodies have been designed. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60 (1991).

Exemplary heterospecific antibodies may bind to two different epitopes on a FGF-19 polypeptide given herein. Alternatively, an anti-FGF-19 polypeptide arm may be used, such as a T cell receptor molecule (e.g., CD2, CD3, CD28 or B7) to focus on cellular defense mechanisms against cells expressing a particular FGF-19 polypeptide. Lymphocytes or cancers that bind to inducible molecules on the Fc receptor (Fc γR) for IgG such as Fc γRI (CD64), Fc γRII (CD32) and Fc γRIII (CD16). Heterospecific antibodies may also be used to limit cytotoxic agents to cells expressing certain FGF-19 polypeptides. These antibodies have FGF-19 binding cancers and cancers that bind to cytotoxic agents or radionuclide chelators such as EOTUBE, DPTA, DOTA, or TETA. Other desired heterospecific antibodies bind to the FGF-19 polypeptide and also bind to tissue factor (TF).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies consist of two covalently bound antibodies. Such antibodies include, for example, cells in which the target immune system is not desired [US Pat. No. 4,676,980] and the treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that antibodies can be prepared in vitro using methods known in the synthetic protein chemistry, including the use of crosslinkers. For example, immunotoxins can be prepared using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolates and methyl-4-mercaptobutylimidates and those as disclosed in US Pat. No. 4,676,980.

6. Effector Function Engineering

It is desirable to modify the antibodies of the invention with respect to effector function, for example, to enhance the efficacy of the antibody in the treatment of cancer. For example, cysteine residue (s) can be introduced into the Fc region, thereby allowing intrachain disulfide bond formation in this region. Homodimeric antibodies thus formed may have improved endocytosis and / or increased complement mediated cell killing and antibody dependent cytotoxicity (ADCC). Caron et al., J. Exp. Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). In addition, homodimeric antibodies with improved antitumor activity can be prepared using heterofunctional crosslinkers as described in Wolff et al. (Cancer Research, 53: 2560-2565 (1993)). Alternatively, the antibody may be engineered to have a double Fc region, thereby having improved complement lysis and ADCC ability. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

7. Immunoconjugates

The invention also relates to cytotoxic substances such as chemotherapeutic agents, toxins (eg, enzymatically active toxins from bacteria, fungi, plants or animals, or fragments thereof), or radioactive isotopes (ie, radioconjugates). It relates to an immunoconjugate comprising an antibody conjugated to).

Chemotherapeutic agents useful in the production of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chains, unbound active fragments of diphtheria toxins, exotoxin A chains (from Pseudomonas aeruginosa), lysine A chains, avirin A chains, modeline A chains, Alpha-sarsine, Alurites fordi protein, diatin protein, Phytoraca americana protein (PAPI, PAPII, and PAP-S), Momordica Charantia Inhibitor, Kursin, Cro Tin, sapaonaria opininalis inhibitors, gelonin, mivogelin, restritocin, phenomycin, enomycin and triotecene. Various radionuclides can be used for the production of radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re.

Conjugates of antibodies and cytotoxic substances include N-succinylimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), difunctional derivatives of imidoesters (e.g., Dimethyl adipimidate HCL), active esters (e.g. disuccinylimidyl suverate), aldehydes (e.g. glutareldihydr), bis-azido compounds (e.g. bis (p-a) Zidobenzoyl) hexanediamine), bis-diazonium derivatives (eg bis- (p-diazoniumbenzoyl) ethylenediamine), diisocyanates (eg tolene 2,6-diisocyanate), and bis-activity It is prepared using various difunctional protein coupling agents such as fluorine compounds (eg 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocinanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radionucleotides to antibodies. See WO 94/11026.

In another embodiment, the "ligand" conjugated to a cytotoxic substance (eg, radionucleotide) after the antibody-receptor conjugate has been administered to the patient, and the unbound conjugate is removed from circulation using a chelating agent. The antibody can be conjugated to a "receptor" (eg, streptavidin) for use in tumor pretargeting to which the antibody is administered.

8. Immunoliposomes

The antibodies disclosed herein may be formulated with immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art and described, for example, in Epstein et al., Proc. Natl. Acad. Sci, USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); And US Pat. Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in US Pat. No. 5,013,556.

Particularly useful liposomes can be produced by reverse phase evaporation methods with lipid compositions comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through filters of defined pore size to produce liposomes with the desired diameter. Fab ′ fragments of antibodies of the invention are described in Martin et al., J. Biol. Chem., 257: 286-288 (1982), can be conjugated to liposomes by disulfide exchange reactions. Chemotherapeutic agents (eg, Doxorubicin) are optionally contained in liposomes. See Gabizon et al., J. National Cancer Inst., 81 (19); 1484 (1989).

9. Pharmaceutical Compositions of Antibodies

Antibodies that specifically bind to FGF-19 as identified herein can be administered for the treatment of various diseases in the form of pharmaceutical compositions like other molecules identified in the screening assays disclosed herein.

If the FGF-19 polypeptide is in the cell and the whole antibody is used as an inhibitor, then the incoming antibody is preferred. However, lipofection or liposomes can also be used to deliver the antibody, or antibody fragment, into the cell. If antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable region sequence of an antibody, a peptide molecule is devised that possesses the ability to bind the target protein sequence. Such peptides can be chemically synthesized and / or prepared by recombinant DNA technology. Marasco et al., Proc. Natl. Acad. Sci. USA. See 90: 7889-7893 (1993). The formulations herein also include one or more active compounds required for the particular condition to be treated, especially those of complementary activity that do not adversely affect one another. Alternatively, or in addition, the composition may comprise agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

In addition, the active ingredient may be a coacervation technique or contact surface polymerization, e.g. For example, they can be captured in microcapsules prepared by hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively. Such techniques are described in Remington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

Sustained-release preparations may be prepared, suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of tangible articles, for example films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol), polylactide (US Pat. No. 3,773,919), L-glutamic acid and copolymers of γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT ™ (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate) And poly-D-(-)-3-hydroxybutyl acid While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules over 100 days, certain hydrogel-releasing proteins If the encapsulated antibody remains in the body for a long time, they denature or coagulate due to exposure to moisture at 37 ° C., and there is a lack of biological activity. And, depending on the mechanism involved, a rational strategy can be devised for stabilization, for example, if the aggregation mechanism is found to be intramolecular SS bond formation via thio-disulfide exchange, Stabilization can be achieved by modifying sulfhydryl residues, freeze drying from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

G. Uses of Anti-FGF-19 Antibodies

The anti-FGF-19 antibodies of the present invention have a variety of uses. For example, anti-FGF-19 antibodies can be used for diagnostic analysis of FGF-19, eg, for detecting its expression in specific cells, tissues, or serum. Various diagnostic assay techniques known in the art can be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays performed on heterologous or homologous [Zola, Monoclaonal Antibodies: A Manual of Techniques, CRC Press] , Inc. (1987) pp. 147-158. Antibodies used in diagnostic assays can be labeled with components that can be detected. Components that can be detected should be able to generate, directly or indirectly, a detectable signal. For example, a detectable moiety can be a radioisotope, fluorescent or chemiluminescent compound such as ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹²⁵ I, for example phosphor isocyanate, rhodamine, or Luciferin, or an enzyme such as alkaline phosphatase, beta-galactoxydase or horseradish peroxidase. Hunter et al., Nature, 144: 945 (1962); David et al., Biocehmistry, 13: 1014 (1974); Pain et al., J. Immunol. Meth., 40: 219 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407 (1982), can be used any method known for conjugating antibodies to detectable components.

In addition, anti-FGF-19 antibodies are useful for affinity purification of FGF-19 from recombinant cell culture or natural sources. In this method, the antibody against FGF-19 is immobilized on a suitable support such as Sephadex resin or filter paper using methods well known in the art. The immobilized antibody is then contacted with a sample containing FGF-19 to be purified, and then the support is washed with a suitable solvent that substantially removes all material in the sample except FGF-19 bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that releases FGF-19 from the antibody.

The following examples are given for illustrative purposes only and are not intended to limit the invention in any way.

All patents and references cited herein are hereby incorporated by reference in their entirety.

Commercially available reagents cited in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of these cells identified by the ATCC accession number throughout the following examples, and throughout the specification, is the American Type Culture Collection, Manassas, VA.

Example 1

Isolation of cDNA Clones Encoding Human FGF-19

EST Accession No. AF007268, Murine Fibroblast Growth Factor (FGF-19) was used to search various published EST databases (eg, GenBank, Dayhoff, etc.). The search was performed by using the computer programs BLAST or BLAST2 [Altschul et al., Methods in Enzymology, 266: 460-480 (1996)] to compare the ECD protein sequence with a six frame translation of the ESF sequence. The search was hit on GenBank EST AA220994, which was identified as STRATAGENE NT2 neuronal precursor 937230. The sequence of AA220994 was also referred to herein as DNA47412.

Based on the DNA47412 sequence, oligonucleotides were synthesized for the following purposes: 1) to identify the cDNA library containing the desired sequence by PCR, and 2) to isolate clones of the full-length coding sequence of FGF-19. For use as a probe. Forward and reverse PCR primers are generally 20-30 nucleotides and are often designed to produce PCR products about 100-1,000 bp in length. Probe sequences are generally 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kb. To screen several libraries for full length clones, DNA was screened by PCR amplification from PCR primer pairs according to Ausubel et al., Current Proteocols in Molecular Biology, supra. A positive library was then used to isolate clones encoding the genes of interest using one of the probe oligonucleotide and primer pairs.

PCR primers (forward and reverse) were synthesized:

Forward PCR primer 5'-ATCCGCCCAGATGGCTACAATGTGTA-3 '(SEQ ID NO: 3), and

Reverse PCR primer 5'-CCAGTCCGGTGACAAGCCCAAA-3 '(SEQ ID NO: 4).

Additionally, synthetic oligonucleotide hybridization probes were prepared from DNA47412 having the following nucleotide sequences:

Hybridization probe

5'-GCCTCCCGGTCTCCCTGAGCAGTGCCAAACAGCGGCAGTGTA-3 '(SEQ ID NO: 5)

RNA for cDNA library preparation was isolated from the human fetal retina. The cDNA library used to isolate the cDNA clones was prepared by standard methods using reagents such as those commercially available from Invitrogen (San Diego, Calif.). cDNA contains NotI site, primed with oligo dT blunted to SalI anti-phosphorylated adapter, cleaved with NotI, roughly sized in gel electrophoresis, unique XhoI and NotI sites In a suitable vector (e.g., pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain a SfiI site; see Holmes et al., Science, 253: 1278-1280 (1991)).

DNA sequencing of the isolated clones as described above was derived for the full-length DNA sequence for the full-length FGF-19 polypeptide (indicated herein as DNA49435-1219 [FIG. 1, SEQ ID NO: 1]) and for the FGF-19 polypeptide. Protein sequences were provided.

The full length clone identified above comprises a single open reading frame with a clear translation initiation site at nucleotides 464-466 and a termination signal at nucleotides 1112-1114 (FIG. 1, SEQ ID NO: 1). The estimated polypeptide precursor is 216 amino acids in length, with a calculated molecular weight of about 24,003 Daltons and an estimated pi of about 6.99. Analysis of the full length FGF-19 shown in FIG. 2 (SEQ ID NO: 2) demonstrates the presence of the various important polypeptide domains shown in FIG. 2, where the positions established for these important polypeptide domains are similar to those described above. Chromosomal mapping demonstrates that the FGF-19 coding nucleic acid maps to human chromosome 11q13.1, band q13.1. Clone DNA49435-1219 was deposited with ATCC on November 21, 1997 and was assigned ATCC accession number 209480.

Analysis of the Dayhoff database (version 35.45 SwissProt 35) using the ALIGN-2 sequence alignment of the full length sequence shown in FIG. 2 demonstrated the identity of the FGF-19 amino acid sequence with the following Dayhoff sequence: 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.

DNA comprising the coding sequence of full length or mature FGF-19 is used as a probe to screen homologous DNA (such as those encoding natural variants of FGF-19) in a human tissue cDNA library or human tissue genomic library.

Hybridization and washing of filters containing library DNA is performed under the following stringent hybridization conditions. Hybridization of the radiolabeled FGF-19 induced probe to the filter was performed with 50% formaldehyde, 5x SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, 2x Denhardt solution, and 10% dextran sulfate solution. Was carried out at 42 ° C. for 20 hours. Filter washes were performed at 42 ° C. in an aqueous solution of 0.1 × SSC and 0.1% SDS.

The DNA encoding the full-length native type FGF-19 and the DNA having the desired sequence identity can then be identified using standard techniques known in the art.

Example 3

Expression of FGF-19 in Escherichia Coli

This example illustrates the preparation of the unglycosulated form of FGF-29 by recombinant expression in E. coli.

DNA sequences encoding FGF-19 are amplified using the first selected PCR primers. The primer should include a restriction enzyme site corresponding to the restriction enzyme site on the selected expression vector. Various expression vectors can be used. An example of a suitable vector is pBR322 (derived from E. coli: Bolivar et al., Gene, 2:95 (1977)) comprising ampicillin and tetracycline resistance genes. This vector is cleaved with restriction enzymes and dephosphorylated. The PCR amplified sequence is then linked to the vector. The vector preferably contains an antibiotic resistance gene, a trp promoter, a poly-His leader (including the first six STII codons, a poly-his enterokinase cleavage site), an FGF-19 coding region, a lambda transcription terminator, and an argU gene. It includes the coding sequence.

The ligation mixture is then used to transform selected E. coli strains using the method described in Sambrook et al., Supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are selected. Plasmid DNA can be isolated and confirmed by restriction enzyme analysis and DNA sequencing.

Selected colonies can be grown overnight in liquid culture medium, such as LB culture supplemented with antibiotics. Overnight cultures are then used to inoculate large scale cultures. The cells are then grown to the desired absorbance during which the expression promoters are run.

After incubating the cells for several hours, the cells can be collected by centrifugation. Cell pellets obtained by centrifugation can be lysed using various reagents known in the art, and the lysed FGF-19 protein can then be purified using a metal chelating column under conditions that allow strong binding of the protein.

FGF-19 can be expressed in E. coli in the form of a poly-His tag using the following method. DNA encoding FGF-19 is amplified using the first selected PCR primers. The primers will include restriction enzyme sites corresponding to restriction enzyme sites on the selected expression vector and other useful sequences that provide efficient and reliable initiation of translation, rapid purification on metal chelate columns, and proteolytic removal by enterokinase. The PCR-amplified, poly-His tagged sequence is then linked in the expression vector, which is used to transform E. coli hosts based on strain 52 (W3110 fuhA (tonA) lon galE rpoHts (htpRts) clpP (lacIq). Transformants were first incubated in LB containing 50 mg / ml carbenicillin with stirring at 30 ° C. and reached an OD 600 of 3-5. The culture was then 50-100-fold in CRAP medium (3.57 in 500 ml water). g (NH ₄ ) ₂ SO ₄ , 0.71 g sodium citrate.2H ₂ 0, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF, and 110 mM MPOS, pH 7.3, 0.55% (w / v) Diluted with glucose and 7 mM MgSO ₄ ) and grown for about 20-30 hours with stirring at 30 ° C. Samples were removed to confirm expression by SDS-PAGE analysis and centrifuged in bulk culture cells. Pellets Cell pellets are frozen until purification and refolding.

E. coli paste (6-10 g pellets) in a 0.5-1 L fermentor was resuspended at 10 vol (w / v) in 7M guanidine, 20 mM Tris (pH 8) buffer. Solid sodium sulfite and sodium tetrathionate were added at final concentrations of 0.1 M and 0.02 M, respectively, and the solution was stirred overnight at 4 ° C. In this step all cysteine residues produce denatured proteins blocked by sulfite. The solution was centrifuged for 30 minutes at 40,000 rpm in a Beckman ultracentrifuge. The supernatant was diluted with 3-5 volumes of metal chelate column buffer (6M guanidine, 20 mM Tris, pH 7.4) and filtered through a 0.22 micron filter to clarify. The characterized extracts were loaded onto a Qiagen Ni-NTA metal chelate column equilibrated with metal chelate column buffer. The column was washed with additional buffer containing pH 7.4, 50 mM imidazole (Calbiochem, Ultrol grade). The protein was eluted with a buffer containing 250 mM imidazole. Fractions containing the desired protein were collected and stored at 4 ° C. The protein concentration was estimated by absorbance at 280 nm using the absorbance coefficient calculated based on the amino acid sequence.

The protein was refolded by slowly diluting the sample with freshly prepared refolding buffer consisting of 20 mM Tris, pH8.6, 0.3M NaCl, 2.5M urea, 5mM cysteine, 20mM glycine and 1mM EDTA. The refolding volume was chosen to give a final protein concentration of 50-100 mg / ml. The refolding solution was stirred gently at 4 ° C. for 12-36 hours. TFA was added to a final concentration of 0.4% (pH approximately 3) to stop the refolding reaction. Prior to further protein purification, the solution was filtered through a 0.22 micron filter and acetonitrile was added at 2-10% final concentration. The refolded protein was chromatographed by eluting with 10-80% concentration gradient acetonitrile on a Poros RI / H reversed phase column using 0.1% TFA mobile phase buffer. Fraction aliquots of A280 absorbance were analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein were collected. In general, most protein classes that are properly refolded elute at the lowest concentrations of acetonitrile because these classes are the most compact so that their hydrophobic interiors are shielded from interaction with reversed phase resins. Aggregated classes are generally eluted at the highest acetonitrile concentrations. In addition to separating the misfolded form of protein from the desired form, the reverse phase also removes endotoxins from the sample.

Fractions containing the desired folded FGF-19 polypeptide were collected and acetonitrile was removed by gently shooting the nitrogen stream towards the solution. Proteins were formulated with 20 mM Hepes, pH 6.8, 0.14 chloride and 4% mannitol by gel infiltration using dialysis or formulation buffer and sterile filtered G25 Superfine (Pharmacia) resin.

Example 4

Expression of FGF-19 in Mammalian Cells

This example illustrates the preparation of a potentially glycated form of FGF-19 by recombinant expression in mammalian cells.

The vector, pRK5 (EP 307,247, published March 15, 1989) is used as the expression vector. Optionally, FGF-19 DNA is linked to pRK5 with selected restriction enzymes that allow insertion of FGF-19 DNA using a linkage method such as described in Sambrook et al., Supra.

In one embodiment, the selected host cell can be 293 cells. Human 293 cells (ATCC CCL 1573) grow full on tissue culture plates in medium such as fetal calf serum and optionally DMEM supplemented with nutrients and / or antibiotics. About 10 μg of pRK5-FGF-19 DNA was mixed with about 1 μg of DNA encoding the VA RNA gene (Thimmappaya et al., Cell, 31: 543 (1982)) and 500 μl of 1 mM Tris-HCl, 0.1 dissolved in mM EDTA, 0.227 M CaCl ₂ . To this mixture, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO ₄ was added dropwise and a precipitate formed at 25 ° C. for about 10 minutes. The precipitate was suspended and added to 293 cells and allowed to settle at 37 ° C. for about 4 hours. Culture medium was aspirated and 2 ml of 20% glycol in PBS was added for 30 seconds. 293 cells were washed with serum free medium, fresh medium was added and the cells were incubated for about 5 days.

About 24 hours after transfection, the culture medium was removed and replaced with culture medium (alone) or with culture medium containing 200 μCi / ml ³⁵ S-cysteine and 200 μCi / ml ³⁵ S-methionine. After 12 hours of incubation, the adjusted medium was collected, concentrated on a spin filter and loaded on a 15% SDS gel. The treated gel can be dried and exposed to the film for a selected time to reveal the presence of the FGF-19 polypeptide. Cultures containing transfected cells can be further cultured (in serum free medium) and the medium is tested in selected bioassays.

In another technique, FGF-19 is described by Somparyrac et al., Proc. Natl. Acad. Sci. 12: 7575 (1981) can be introduced into 293 cells temporarily using the dextran sulfate method described. 293 cells are grown to maximum density in a rotating flask and 700 μg of pRK5-FGF-19 DNA is added. First, cells are concentrated by centrifugation from a rotating flask and washed with PBS. DNA-dextran precipitates are incubated on cell pellets for 4 hours. Cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium and reintroduced into a rotating flask containing tissue culture medium, 5 μg / ml bovine insulin and 0.1 μg / ml bovine transferrin. After 4 days, the conditioned medium is centrifuged and filtered to remove cells and debris.

The sample containing expressed FGF-19 can then be concentrated and purified by any method selected, such as dialysis and / or column chromatography.

In other embodiments, FGF-19 can be expressed in CHO cells. Known reagents such as CaPO ₄ or DEAE-dextran can be used to transfect pRK5-FGF-19 into CHO cells. As described above, the cell culture can be cultured and the medium can be exchanged with a culture medium (alone) or with a medium containing radiolabels such as ³⁵ S-methionine. After confirming the presence of the FGF-19 polypeptide, the culture medium can be exchanged for serum free medium. Preferably, after culturing the medium for about 6 days, the adjusted medium is collected. The medium containing the expressed FGF-19 can then be concentrated and purified by any method selected.

In addition, epitope-tagged FGF-19 can be expressed in host CHO cells. FGF-19 can be subcloned from the pRK5 vector. The subclonal insert can be PCR to be framed fusion into a baculovirus expression vector with a selected epitope tag such as a poly-his tag. The poly-his tagged FGF-19 insert can then be subcloned into an SV40 derived vector containing a selection marker such as DHFR to select stable clones. Finally, CHO cells can be transfected with SV40 derived vectors (as described above). Labeling can be performed to confirm expression as described above. The culture medium containing the expressed poly-His tagged FGF-19 can then be concentrated and purified by any method selected, such as Ni ²⁺ -chelate affinity chromatography.

In addition, FGF-19 can be expressed according to transient expression methods in CHO cells and / or COS cells, or according to other stable expression methods in CHO cells.

Stable expression in CHO cells is performed using the following method. The protein is expressed as an IgG construct (immunoadhesion), wherein the soluble form (eg, extracellular domain) of each protein is an IgG1 containing a hinge, CH2 and CH2 domain, and / or poly-His tagged form. Fused to the structure.

After PCR amplification, each DNA is subcloned in a CHO expression vector using standard techniques such as described in Ausubel et al., Current Proteocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression vectors are prepared with 5 'and 3' conformity restriction enzyme sites of the DNA of interest to allow for convenient shuffling of the cDNA. Vectors used in expression in CHO cells are described by Lucas et al., Nucl. Acids Res. 24: 9, 1774-1779 (1996), and use the SV40 early promoter / enhancer to induce expression of the desired cDNA and dihydrofolate reductase (DHFR). DHFR expression allows selection for stable maintenance of the plasmid after transfection.

Commercially available transfection reagent Superfect of 12 µg of the desired plasmid DNA (Quiagen), Dosper Or Fugene (Boehringer Mannheim) is used to introduce about 10 million CHO cells. The cells grow as described in Lucas et al., Supra. About 3 × 10 ⁻⁷ cells are frozen in ampoules for further growth and production as described below.

Ampoules containing plasmid DNA are transferred to a bath and melted and mixed by vortex. The contents were pipetted into centrifuge tubes containing 10 mL of medium and centrifuged at 1,000 rpm for 5 minutes. Aspirate supernatant and resuspend cells in selection medium (0.2 μm filtered PS20 with 5% 0.2 μm diafiltered fetal bovine serum). The cells were then aliquoted in 100 mL spinners containing 90 mL selection medium. After 1-2 days, cells were transferred to 250 mL spinners filled with 150 mL selective growth medium and incubated at 37 ° C. After an additional 2-3 days, 250 mL, 500 mL and 2,000 mL spinners were inoculated at 3 × 10 ⁵ cells / ml. Cell medium was centrifuged to exchange with fresh medium and resuspended in production medium. Any suitable CHO medium may be used, but the production medium described in US Pat. No. 5,122,469 (registered June 16, 1992) may actually be used. 3 L production spinners were inoculated at 1.2 × 10 ⁶ cells / ml. On day 0, the cell number pH was measured. On day 1, the spinner was sampled and sparging of the filtered air was started. On day 2, the spinner was sampled, the temperature was changed to 33 ° C., 30 mL of 500 g / L glucose and 0.6 mL of 10% antifoam (eg 35% polydimethylsiloxane emulsion, Dow Corning 365 medical grade emulsion) Was used. Throughout the production process, it was necessary to keep the pH around 7.2, adjusting it. After 10 days, or until viability was below 70%, cell cultures were collected by centrifugation and filtered through a 0.22 μm filter. The filtrate was quickly loaded into the column for storage or purification at 4 ° C.

For poly-His tagged constructs, proteins were purified using a Ni-NTA column (Qiagen). Prior to purification, imidazole was added at 5 mM concentration in the adjusted medium. The conditioned medium was pumped at 4 ° C. at a rate of 4-5 ml / min to a 6 ml Ni-NTA column equilibrated with 20 mM Hepes buffer, pH 7.4, containing 0.3 M NaCl and 5 mM imidazole. After loading, the column was washed with additional equilibration buffer and the protein was eluted with equilibration buffer containing 0.25M imidazole. Highly purified protein was desalted into a 25 ml G25 Superfine (Pharmacia) column in storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8 and stored at −80 ° C.

The immunoadhesin (Fc-containing) constructs were purified from media adjusted as follows. The conditioned medium was pumped onto a 5 ml Protein A column (Pharmacia) equilibrated with 20 mM Na phosphate buffer (pH 6.8). After loading, the column was washed thoroughly with equilibration buffer before eluting with 100 mM citric acid (pH 3.5). Eluted proteins were quickly neutralized by collecting 1 ml fractions into a tube containing 275 μl 1M Tris buffer (pH 9). Highly purified proteins were then desalted into storage buffer for poly-His tagged proteins as described above. Uniformity was assessed by N-terminal amino acid sequencing by SDS polyacrylamide gel and Edman degradation.

Example 5

Expression of FGF-19 in Yeast

The following method describes recombinant expression of FGF-19 in yeast.

First, yeast expression vectors were prepared for intracellular production or secretion of FGF-19 from the ADH2 / GAPDH promoter. DNAs and promoters encoding FGF-19 were inserted into suitable restriction enzyme sites in selected plasmids to cause intracellular expression of FGF-19. For secretion, the ADH2 / GAPDH promoter for FGF-19 expression, a native FGF-19 signal peptide or other mammalian signal peptide, such as a yeast alpha-factor or invertase secretion signal / leader sequence, and a linker sequence (if necessary) DNA encoding FGF-19 can be cloned into a selected plasmid along with DNA encoding a).

Yeast cells, such as yeast strain AB110, can be transformed with expression plasmids as described above and cultured in selected fermentation medium. Transformed yeast supernatants can be analyzed by gel staining with Coomassie Blue staining after precipitation with 10% trichloroacetic acid and separation by SDS-PAGE.

Recombinant FGF-19 can then be isolated and centrifuged to remove yeast cells from the fermentation medium and concentrated to purify the medium using selected cartridge filters. In addition, concentrates containing FGF-19 can be purified using selected column chromatography resins.

Example 6

Expression of FGF-19 in Baculovirus Infected Insect Cells

The following method describes recombinant expression of FGF-19 in baculovirus infected insect cells.

The sequence encoding FGF-19 is fused upstream of the epitope tag contained within the baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (Fc regions of IgG). Various plasmids can be used, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). In short, if the sequence encoding the FGF-19 or the protein is an extracellular protein, the desired portion of the coding sequence of FGF-19, such as the sequence encoding the extracellular domain of the transmembrane protein or the sequence encoding the mature protein, is 5 PCR amplification with primers complementary to the 'and 3' regions. The 5 'primer can insert flanking (selected) restriction enzyme sites. The product is then cleaved with the selected restriction enzymes and subcloned into the expression vector.

Recombinant baculovirus combines the plasmid and BaculoGold ™ viral DNA (Pharmingen) together with Lipofectin (commercially available from GIBCO-BRL) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711). Generate by transfection with. After 4-5 days of incubation at 28 ° C., the released virus was collected and then used for amplification. Viral infection and protein expression were performed as described in O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).

The expressed poly-his tagged FGF-19 can then be purified by Ni ²⁺ -chelate affinity chromatography, for example as follows. Extracts were prepared from recombinant virus infected Sf9 cells as described in Rupert et al., Nature, 362: 175-179 (1993). In brief, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl ₂ ; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl) and iced Sonication was performed twice in the stomach for 20 seconds. The sonicates were labeled by centrifugation and the supernatant diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. Ni ²⁺ -NTA agarose column (commercially available from Qiagen) was prepared in 5 mL fill volume, washed with 25 mL water and equilibrated with 25 mL loading buffer. The filtered cell extracts were loaded into the column at 0.5 mL / min. The column was washed up to baseline A ₂₈₀ with loading buffer and fraction collection started at this point. The column was then washed with secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH6.0), eluting nonspecifically bound protein. After reaching the A ₂₈₀ baseline, the column was developed with a gradient of 0-500 mM imidazole in secondary wash buffer. 1 mL fractions were collected, analyzed by SDS-PAGE analysis and western blot with Ni ²⁺ -NTA (Qiagen) conjugated to silver staining or alkaline phosphatase. Fractions containing eluted His ₁₀ -tagged FGF-19 were collected and dialyzed against loading buffer.

Alternatively, purification of IgG tagged (or Fc tagged) FGF-19 can be carried out using known chromatography techniques such as Protein A or Protein G column chromatography.

Example 7

Preparation of Antibody Binding to FGF-19

This example illustrates the preparation of monoclonal antibodies capable of binding FGF-19.

Techniques for preparing monoclonal antibodies are known in the art and are described, for example, in Goding, supra. Immunogens that can be used include purified FGF-19, fusion proteins comprising FGF-19, and cells expressing recombinant FGF-19 on the cell surface. One skilled in the art can select immunogens without undue experimentation.

Mice such as Balb / c were immunized with emulsified FGF-19 immunogen in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount of 1-100 μg. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Rivi Immunochemical Research, Hamilton, Montana) and injected into the heel of the animal. The immunized mice were then boosted after 10-12 days with additional immunogens emulsified in the selected adjuvant. Thereafter, mice were boosted by additional immunization for several weeks. Serum samples can be obtained periodically from mice by tracheal bleeding for testing in ELISA assays to detect anti-FGF-19 antibodies.

After determining the appropriate antibody titers, animals that are "positive" for the antibody can be injected by final intravenous injection of FGF-19. After 3-4 days, mice were killed and spleen cells were collected. ATCC, No. Spleen cells were fused to selected murine myeloma cell lines such as P3X63AgU.1, which is obtained with CRL 1597 (using 35% polyethylene glycol). Fusion is performed on hybridoma cells that can be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminoterin, and thymidine) medium to inhibit proliferation of unfused cells, myeloma hybrids and splenic cell hybrids. Generated.

Hybridoma cells will be screened for reactivity with FGF-19 in ELISA. Determination of "positive" hybridoma cells secreting monoclonal antibodies against FGF-19 of interest is within the skill of the art.

Positive hybridoma cells can be injected intraperitoneally into syngenic Balb / c mice to produce ascites containing anti-FGF-19 monoclonal antibodies. Alternatively, the hybridoma cells can be grown in culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using gel exclusion chromatography after ammonium sulfate precipitation. Alternatively, affinity chromatography based on antibody binding to Protein A or Protein G can be used.

Example 8

Purification of FGF-19 Polypeptides Using Specific Antibodies

Various standard techniques in the field of protein purification can purify natural or recombinant FGF-19 polypeptides. For example, pro-FGF-19 polypeptides, mature FGF-19 polypeptides, or pre-FGF-19 polypeptides are purified by immunoaffinity chromatography using antibodies specific for the desired FGF-19 polypeptide. In general, immunoaffinity columns are prepared by covalently coupling an anti-FGF-19 polypeptide antibody to an activated chromatography resin.

Polyclonal immunoglobin was prepared from immune sera by purification on protein A (Pharmacia LKB Biotechnology, Piscataway, NJ), either precipitated or immobilized with ammonium sulfate. Similarly, monoclonal antibodies were prepared from mouse ascites by precipitation with ammonium sulfate or purification on immobilized Protein A. Partially purified immunoglobin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE ™ (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked and the derivative resin is washed according to the manufacturer's instructions.

Such immunoaffinity columns are used in the purification of FGF-19 by preparing fractions from cells containing the soluble form of the FGF-19 polypeptide. This preparation is induced by solubilization of whole cell or subcellular fractions obtained by differential centrifugation by addition of surfactants or by other methods well known in the art. Alternatively, soluble FGF-19 polypeptides, including signal sequences, may be secreted in useful amounts into the medium in which the cells are grown.

Soluble FGF-19 polypeptide-containing preparation was passed through an immunoaffinity column and the column was washed under conditions that allow for selective uptake of the FGF-19 polypeptide (eg, strong ionic strength buffer in the presence of a surfactant). The column is then subjected to conditions under conditions that disrupt antibody / FGF-19 polypeptide binding (e.g., low pH, such as pH about 2-3, or high concentrations of chaotrope, such as urea or thiocyanate ions). Eluted and FGF-19 polypeptide was collected.

Example 9

Drug screening

The present invention is particularly useful for screening compounds using FGF-19 polypeptides or combinations of these fragments, among other various drug screening techniques. The FGF-19 polypeptide or fragment used in such a test may be free in solution, attached to a solid support, contained on the cell surface, or located within the cell. One method of drug screening uses eukaryotic or prokaryotic host cells stably transformed with a recombinant nucleic acid expressing an FGF-19 polypeptide or fragment. Drugs are screened for such transformed cells in a competitive binding assay. Such cells may be live or in fixed form or used in standard binding assays. For example, one method can measure complex formation between a FGF-19 polypeptide or fragment and a drug being tested. Alternatively, one method can investigate the reduction of complex formation between the FGF-19 polypeptide and its target cell or target receptor due to the agent being tested.

Accordingly, the present invention provides a method for screening a drug or any other agent that may affect a FGF-19 polypeptide associated disease or disorder. These methods include contacting such agents with FGF-19 polypeptides or fragments thereof and methods well known in the art, such as (i) the presence of a complex between the drug and the FGF-19 polypeptide or fragment, or (ii) FGF-19 Analyzing the presence of the complex between the polypeptide or fragment and the cell. In such competitive binding assays, the FGF-19 polypeptide or fragment is generally labeled. After suitable incubation, the free FGF-19 polypeptide or fragment is isolated from what is in the bound form and the label that does not form a free or complex indicates that the specific agent binds to the FGF-19 polypeptide or FGF-19 polypeptide / cell. It is a measure of the ability to interfere with the complex.

Other drug screening techniques provide high yield screening for compounds with suitable binding affinity for polypeptides, which are described in WO 84/03564 (published Sep. 13, 1984). In short, many different small peptide test compounds are synthesized on a solid support, such as a plastic pin or other surface. For applications in FGF-19 polypeptides, peptide test compounds are reacted with and washed with FGF-19 polypeptides. Bound FGF-19 polypeptides are detected by methods well known in the art. In addition, the purified FGF-19 polypeptide may be coated directly onto the plate in the drug screening techniques mentioned above. In addition, non-neutralized antibodies can be used to capture peptides and immobilize them on solid supports.

The present invention also provides for the use of competitive drug screening assays in which neutralizing antibodies capable of specifically binding to FGF-19 polypeptides compete with test compounds for binding to FGF-19 polypeptides or fragments thereof. In this way, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with the FGF-19 polypeptide.

Example 10

Reasonable Drug Design

The goal of rational drug design is to produce structural analogs of biologically active target polypeptides (eg, FGF-19 polypeptides) or small molecules with which they interact, such as agonists, antagonists, or inhibitors. All these examples can be used to design drugs that may interfere in vivo with the function of the more active or stable forms of FGF-19 polypeptide or FGF-19 polypeptide (see Hodgson, Bio / Technology, 9). 19-21 (1991).

In one approach, the tertiary structure of the FGF-19 polypeptide, or FGF-19 polypeptide-inhibitor complex, is determined by x-ray crystallography, computer modeling, most commonly a combination of the two methods. The shape and charge of the FGF-19 polypeptide should be identified to elucidate the structure and determine the active site (s) of the molecule. Rarely, modeling based on the structure of homologous proteins provides useful information about the structure of the FGF-19 polypeptide. In both cases, relevant structural information is used to design similar FGF-19 polypeptide like molecules or to identify effective inhibitors. Useful examples of rational drug design include molecules or molecules with improved activity or stability, such as those shown in Braxton and Wells, Biochemistry, 31: 7796-7801 (1992). Athauda et al, J. Biocehm., 113: 742 -746 (1993)] will include molecules that act as inhibitors, agonists, or antagonists of natural peptides.

It is also possible to isolate selected target specific antibodies by functional analysis as described above and to reveal their crystal structure. This approach theoretically creates drug centers on which subsequent drug design can be based. It is possible to circumvent protein crystallography by generating anti-factor antibodies (anti-ids) against functional, pharmaceutically active antibodies. As a mirror image of the mirror image, the binding site of the anti-ids is expected to be an analog of the original receptor. The anti-id can then be used to identify and isolate peptides from chemically or biologically produced peptide banks. The isolated peptide will then act as drug center.

By the present invention, sufficient amounts of FGF-19 polypeptide can be prepared to perform such analytical studies such as X-ray crystallography. In addition, the FGF-19 polypeptide amino acid sequence provided herein provides instructions for using computer modeling in place of or in conjunction with x-ray conjunctivology.

Example 11

Investigation of body weight, leptin levels, town food intake, urine production, oxygen consumption, and triglyceride and free fatty acid levels in FGF-19 transgenic mice

As disclosed herein, FGF-19 is newly identified as a member of an emerging family of secreted proteins associated with fibroblast growth factor. FGF-19 has been characterized herein as interacting with FGF receptor 4 and has not been shown to act as a mitogen. To further investigate the function of this protein, transgenic mice expressing human FGF-19 were prepared.

In particular, cDNA encoding human FGF-19 was colonized into plasmids containing a promoter for myosin L chain. This promoter is sufficient for muscle specific transcription of the transgene. Also included are splice receptors and donors at 5 'of the FGF-19 cDNA to increase expression levels and splice donors at 3' of the FGF-19 cDNA to increase transcription levels and provide transcription termination sites. And receptor and poly A addition signals were included.

DNA (transgene) comprising MLC promoter, 5 'splice receptor and donor, FGF-19 cDNA and 3' splice receptor and donor and transcription termination site is released from bacterial vector sequence using appropriate restriction enzymes, Purified by size fractionation on agarose gel. Purified DNA was injected into one pronucleus of fertilized mouse eggs and transgenic mice were generated and identified as described above (Genetic Modification of Animals; Tim Stewart; In Exploring Genetic Mechanisms pp 565-598; 1997 Eds M Singer and P Berg; Univertisy Science Books; Sausalito, Calif). Six-week-old mice were used to measure water intake, food consumption, urine excretion and red blood cell volume as described below. Leptin, triglycerides and free fatty acids were measured in the same animals at 8 weeks of age.

As the results discussed below show, these mice showed increased food intake and increased metabolic rate, as evidenced by their oxygen consumption rate. Despite increased food intake, these mouse weights were significantly reduced compared to untransformed compatriots. This reduced body weight appears to be the result of a decrease in leptin in transgenic mice that is closely associated with adipose tissue weight in humans and rodents. As further supporting evidence, the transgenic mice have normal length growth, as assessed by measuring the length from the growth nose to the buttocks. They are normal in terms of body temperature, body size (bone length) and blood levels. With increased food intake, transgenic mice have increased urine excretion. Increased urine excretion will be due to increased food metabolism, as mice do not appear to drink more and are not dehydrated as measured by normal erythrocyte volume fraction. Since FGF-19 reduces fat accumulation without altering muscle weight or long bone formation, FGF-19 has been shown to be an effective therapeutic in the treatment of obesity and related diseases.

More specifically, MLC-FGF-19 transgenic mice were weighed at various time points under different fasting and feeding conditions. More specifically, the body weight of FGF-19 transgenic mice and their untransformed litters were measured at 6 weeks of age, 6 hours and 24 hours after fasting and 24 hours after 24 hours fasting termination. It was. As shown in FIG. 3A, under all conditions, the FGF-19 transgene mouse (solid bar) weighed less than its wild-type, untransformed equivalent (dot bar).

FIG. 3B shows the serum analyzed for leptin of the same group of mice shown in FIG. 3A. Reduced leptin in FGF-19 transgenic mice is consistent with low body weight (FIG. 3A) due to reduced fat accumulation.

Six week old transgenic mice were monitored for food intake (FIG. 4A), water intake (FIG. 4B), urine excretion (FIG. 4C) and erythrocyte volume fraction (FIG. 4D). As shown, FGF-19 transgenic mice (solid bar) consumed more food than wild-type litter, but did not drink more water. Although there was no change in water intake, the transgenic mice produced more urine (FIG. 4C). Despite the increase in urine production, transgenic mice were not dehydrated as evidenced by normal red blood cell volume ratio (FIG. 4D).

Weight loss (FIG. 3) along with increased food intake (FIG. 4) can be explained by increased metabolic rate. Metabolic rate was determined by measuring oxygen consumption. As shown in FIG. 5, FGF-19 transgenic mice show increased metabolic rate during the light cycle, 24 hours after 24 hours fasting and 24 hours after termination.

Obesity and elevated triglycerides and free fatty acids are risk factors for cardiovascular disease. Since FGF-19 reduced one of the risk factors for cardiovascular disease (obesity (FIG. 3)), it was investigated whether FGF-19 could also reduce other risk factors. As shown in FIG. 6, the levels of triglycerides and free fatty acids were also low in FGF-19 transgenic mice.

Example 12

FGF-19 injection causes increased food intake and increased oxygen consumption

To confirm the effects seen in FGF-19 transgenic mice induced by FGF-19 protein, osmotic pressure-induced transplantation of recombinant FGF-19 (1 mg / kg / day, intravenous) into a group of untransformed FvB mice was performed. Delivered by pump. As shown in FIGS. 7A-B, administration of recombinant human FGF-19 resulted in an increase in food intake compared to mice injected with carrier only. In addition, FGF-19 infusion resulted in an increase in metabolic rate as measured by oxygen consumption.

Example 13

FGF-19 reduces glucose uptake and increases the release of leptin from adipocytes

To further investigate the mechanism by which FGF-19 alters metabolism, recombinant human FGF-19 was added in primary rat adipocyte cultures and glucose uptake and leptin release by these cells were measured. As shown in Figures 8A-B, FGF-19 increased the release of leptin and decreased glucose uptake into primary rat adipocytes.

Example 14

Investigation of Glucose Tolerance and Fatty Layer Weight in High Fat Dieted FGF-19 Transgenic Mice

In general, mice (and humans) that have a high fat diet gain weight and fat accumulation and become glucose intolerant or diabetic. To investigate whether exposure to FGF-19 affects fat accumulation and glucose tolerance in the group of transgenic mice and their untransformed mice (adapted to age and gender), the litters were found to have a high sodium content relative to their normal diet. Standardized modifications were made to substantially reduce the resistance as described in Rebuffe-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. A high content diet was administered (the diet was prepared according to Research Diets Inc. Catalog no.D1233ON).

After 10 weeks with a normal mouse diet or a high fat diet, glucose tolerance tests were performed on mice (female transformants and their untransformed litters). Therefore, each mouse was injected intraperitoneally at 1.0 mg of glucose / kg body weight and blood glucose levels were measured at intervals after injection. The graph in FIG. 10 shows glucose levels in mice and 8/9 female untransformed mice fed a high fat diet were defined as diabetes (2 hours glucose levels greater than 200 mg / dl; (World Book of Diabetics in Practice.Vol 3; Ed Krall, LP; Elsevier)), demonstrating that 0/5 of transformed mice fed the corresponding diet are considered diabetic.

Male mice fed a high fat diet were killed after 6 or 10 week diets and fat accumulation was determined by measuring specific fat stocks. As shown in FIG. 9, the transgenic mice fed the high fat diet were significantly less fat than the untransformed litter.

Substance deposit

The following materials were deposited in the American Cell Type Culture Collection (10801 Blvd University, Manassas, Virginia 20110-2209; ATCC).

matter ATCC Deposit Number Deposit date

DNA49435-12192094801997. 11. 21

The deposit was made in accordance with the provisions of the Budapest Treaty (Budapest Treaty) under the international recognition of microbial deposits for patent purposes and treaty purposes. This ensures that the product of the deposit remains for 30 years from the date of deposit. Deposits may be obtained from the ATCC under the Budapest Regulations, and in consultation with Genentech Inc. and the ATCC, regardless of which one comes first, upon the registration of the relevant U.S. patent or disclosure of the U.S. or foreign patent application. To ensure the permanent and unrestricted availability of the progeny of the culture and to ensure that the offspring are registered by the US Patent and Trademark Commission in accordance with USC § 122 and related Commission regulations (see 37 CFR §1.14 specifically 886 OG 638). Guarantee to be determined

The assignee of the present application has agreed to promptly replace with another identical material, notifying that if the deposited material dies, is lost or destroyed when incubated under suitable conditions. The availability of deposited materials shall not be construed as an authorization to carry out the invention under any governmental authority, in violation of the rights granted under its patent law.

The specification set forth above is believed to be sufficient to enable one skilled in the art to practice the invention. Since the deposited material is intended to be one example of certain aspects of the invention, the invention is not to be limited in scope by the deposited structure, and all functionally equivalent structures are within the scope of the invention. Deposits of materials herein are not admitted to be inappropriate to enable the implementation of any aspect of the invention, including their best aspects, and the claims are limited to the specific examples shown herein. It is not to be interpreted. Indeed, various modifications of the invention from the above description in addition to those shown and described herein are apparent to those skilled in the art and fall within the appended claims.

Claims (77)

(a) a DNA molecule encoding a FGF-19 polypeptide comprising the amino acid residue sequence at position 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2), or (b) a DNA molecule of (a) An isolated nucleic acid molecule comprising DNA having at least about 80% sequence identity with the complement. The isolated nucleic acid molecule of claim 1, comprising the nucleotide sequence at position 464 or about 530 to about 1111 of FIG. 1 (SEQ ID NO: 1). The isolated nucleic acid molecule of claim 1, comprising the nucleotide sequence of FIG. 1 (SEQ ID NO: 1). The isolated nucleic acid molecule of claim 1 comprising a nucleotide sequence encoding the amino acid residue sequence at position 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2). (a) a DNA molecule encoding the same mature polypeptide as encoded by the human protein cDNA (DNA49435-1219) deposited at ATCC on November 21, 1997 under ATCC accession number 209480, or (b) the DNA molecule of (a) An isolated nucleic acid molecule comprising DNA having at least about 80% sequence identity with the complement of. The isolated nucleic acid molecule of claim 5 comprising DNA encoding the same mature polypeptide as encoded by the human protein cDNA (DNA49435-1219) deposited at ATCC on November 21, 1997 under ATCC Accession No. 209480. (a) about 80% of the full-length polypeptide coding sequence of the human protein cDNA (DNA49435-1219) deposited with ATCC on November 21, 1997 under ATCC Accession No. 209480, or (b) the complement of the coding sequence of (a) An isolated nucleic acid molecule comprising DNA having the above sequence identity. The isolated nucleic acid molecule of claim 7 comprising the full-length polypeptide coding sequence of the human protein cDNA (DNA49435-1219) deposited at ATCC on November 21, 1997 under ATCC Accession No. 209480. 2. An isolated nucleic acid molecule encoding a FGF-19 polypeptide comprising DNA hybridizing with the complement of a nucleic acid sequence encoding amino acid at position 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2). The nucleic acid of claim 9, wherein the nucleic acid encoding an amino acid at position 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2) is located at position 464 or about 530 to about 1111 of FIG. Isolated nucleic acid molecule comprising a nucleotide. The isolated nucleic acid molecule of claim 9, wherein the hybridization occurs under stringent hybridization and wash conditions. (a) DNA encoding a polypeptide having at least 80% positive, as compared to the amino acid residue sequence at position 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2), or (b) the DNA of (a) An isolated nucleic acid molecule comprising the complement of. Under stringent hybridization conditions, the test DNA may comprise (a) a DNA molecule encoding a FGF-19 polypeptide comprising the amino acid residue sequence at position 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2), or (b) An isolated nucleic acid molecule comprising about 22 or more nucleotides, generated by hybridizing with molecular complement of the DNA of (a). The isolated nucleic acid molecule of claim 13, having at least about 80% sequence identity with (a) or (b). A vector comprising the nucleic acid molecule of any one of claims 1-14. The vector of claim 15, wherein said nucleic acid molecule is operably linked with a control sequence recognized by a host cell transformed with the vector. Nucleic acid molecule deposited on ATCC under accession number 209480 (DNA49435-1219). A host cell comprising the vector of claim 15. 19. The host cell of claim 18, wherein said cell is a CHO cell. 19. The host cell of claim 18, wherein said cell is Escherichia coli. 19. The host cell of claim 18, wherein said cell is a yeast cell. A method for producing an FGF-19 polypeptide, comprising culturing the host cell of claim 18 under conditions suitable for expression of the FGF-19 polypeptide, and recovering the FGF-19 polypeptide from the cell culture. An isolated FGF-19 polypeptide comprising an amino acid sequence having at least about 80% sequence identity with an amino acid residue sequence at position 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2). The isolated FGF-19 polypeptide of claim 23 comprising an amino acid residue at position 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2). An isolated FGF-19 polypeptide having at least about 80% sequence identity with a polypeptide encoded by a cDNA insert (DNA49435-1219) of a vector deposited with ATCC as ATCC Accession No. 209480. The isolated FGF-19 polypeptide of claim 25, encoded by the cDNA insert (DNA49435-1219) of the vector deposited with the ATCC as ATCC Accession No. 209480. An isolated FGF-19 polypeptide having at least 80% positives, as compared to the amino acid residue sequence at position 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2). Isolated FGF-19 comprising the amino acid residue sequence at position 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2), or a fragment thereof sufficient to provide a binding site for an anti-FGF-19 antibody Polypeptide. (i) under stringent conditions, the test DNA molecule comprises (a) a DNA molecule encoding a FGF-19 polypeptide comprising the amino acid residue sequence at position 1 or about 23 to about 216 of FIG. 2 (SEQ ID NO: 2), or (b) hybridizing with the complement of the DNA molecule of (a), (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. An isolated polypeptide produced by. The isolated polypeptide of claim 29, wherein said test DNA has at least about 80% sequence identity with (a) or (b). A chimeric molecule comprising a FGF-19 polypeptide fused with a heterologous amino acid sequence. 32. The chimeric molecule of claim 31, wherein said heterologous amino acid sequence is an epitope tag sequence. 32. The chimeric molecule of claim 31, wherein said heterologous amino acid sequence is an Fc region of an immunoglobulin. An antibody that specifically binds to a FGF-19 polypeptide. The antibody of claim 34, which is a monoclonal antibody. The antibody of claim 34, which is a humanized antibody. The antibody of claim 34 which is an antibody fragment. Agents for FGF-19 Polypeptides. Antagonist against FGF-19 polypeptide. (a) an FGF-19 polypeptide, (b) an agent against an FGF-19 polypeptide, (c) an antagonist against an FGF-19 polypeptide, or (d) an anti-FGF-19 antibody with a pharmaceutically acceptable carrier Composition. a) adding a biologically active candidate agent to the FGF-19 sample, b) determining binding of the candidate agent to the FGF-19, wherein the binding indicates a biologically active agent capable of binding to FGF-19. Screening methods for biologically active agents. a) adding a biologically active candidate agent to the FGF-19 sample, b) determining the change in the biological activity of FGF-19, wherein the change indicates a biologically active agent capable of modulating the activity of FGF-19. A method of screening a biologically active agent. 43. The method of claim 42, wherein said biological activity is a reduction in glucose uptake in adipocytes. 43. The method of claim 42, wherein said biological activity is an increase in leptin release from adipocytes. A method of identifying a receptor for FGF-19 comprising binding FGF-19 with a composition comprising a cell membrane material in which FGF-19 complexes with a receptor on the cell membrane material and identifying the receptor as an FGF-19 receptor. 46. The method of claim 45, further comprising binding FGF-19 to said receptor and crosslinking said FGF-19 and receptor. 46. The method of claim 45, wherein said composition is a cell. 46. The method of claim 45, wherein said composition is a cell membrane extract. A method of inducing leptin release from adipocytes comprising administering FGF-19 to the adipocytes in an amount effective to induce leptin release. The method of claim 49, wherein said FGF-19 is administered as a protein. The method of claim 49, wherein said FGF-19 is administered as a nucleic acid. A method of inducing a decrease in glucose uptake from adipocytes, comprising administering FGF-19 to the adipocytes in an amount effective to induce a decrease in glucose uptake. The method of claim 52, wherein said FGF-19 is administered as a protein. The method of claim 52, wherein said FGF-19 is administered as a nucleic acid. A method of treating obesity in a subject, comprising administering to the subject a composition comprising FGF-19 in an amount effective to treat obesity. The method of claim 55, wherein said treating obesity further results in treating a disease associated with obesity. The method of claim 55, wherein said FGF-19 is administered as a protein. The method of claim 55, wherein said FGF-19 is administered as a nucleic acid. 56. The method of claim 55, wherein said composition further comprises a pharmaceutically acceptable carrier. The method of claim 55, wherein said FGF-19 has at least about 85% amino acid sequence identity with the amino acid sequence of FIG. 2 (SEQ ID NO: 2). A method of reducing the subject's total weight, comprising administering to the subject an effective amount of FGF-19. 62. The method of claim 61, wherein said FGF-19 is administered as a protein. 62. The method of claim 61, wherein said FGF-19 is administered as a nucleic acid. 62. The method of claim 61, wherein said FGF-19 is administered with a pharmaceutically acceptable carrier. 62. The method of claim 61, wherein the reduction in total weight comprises a decrease in fat of the subject. The method of claim 61, wherein the FGF-19 has at least about 85% amino acid sequence identity with the amino acid sequence of FIG. 2 (SEQ ID NO: 2). A method of reducing one or more levels of triglycerides and free fatty acids in a subject, comprising administering to the subject an effective amount of FGF-19. 68. The method of claim 67, wherein said FGF-19 is administered as a protein. 68. The method of claim 67, wherein said FGF-19 is administered as a nucleic acid. 68. The method of claim 67, wherein said FGF-19 is administered with a pharmaceutically acceptable carrier. The method of claim 67, wherein said FGF-19 has at least about 85% amino acid sequence identity with the amino acid sequence of FIG. 2 (SEQ ID NO: 2). A method of increasing the metabolic rate of an individual comprising administering to the individual an effective amount of FGF-19. The method of claim 72, wherein said FGF-19 is administered as a protein. The method of claim 72, wherein said FGF-19 is administered as a nucleic acid. 73. The method of claim 72, wherein said FGF-19 is administered with a pharmaceutically acceptable carrier. The method of claim 72, wherein said FGF-19 has at least about 85% amino acid sequence identity with the amino acid sequence of FIG. 2 (SEQ ID NO: 2). Rodents comprising a genome comprising a transforming gene encoding FGF-19.