chimeric fibroblast growth factors with altered receptor specificity

专利摘要:
CHEMICAL FIBROBLAST GROWTH FACTORS WITH CHANGED RECEPTOR SPECIFICITY.The present invention relates to new polymptides with chimeric fibroblast growth factor (FGF), new DNA encoders of chimeric FGF polypeptides, and the re-combining production of chimeric FGF polypeptides for the therapeutic treatment of related metabolic disorders and other conditions, and to the preparation of pharmaceutically active compositions including chimeric FGF polypeptides, with compositions containing therapeutic and pharmacological properties including those associated with the treatment of related metabolic disorders and other conditions.
公开号:BR112012008907A2
申请号:R112012008907-2
申请日:2010-10-15
公开日:2020-11-24
发明作者:Junichiro SONODA
申请人:Genentech, Inc；
IPC主号:

专利说明:

[0001] [0001] This patent application claims priority benefit to provisional U.S. patent application serial number 61 / 252,074, filed on October 15, 2009, in which the whole is attached here for reference. FIELD OF THE INVENTION
[0002] [0002] The present invention generally relates to new polypeptides designated herein as chimeric fibroblast growth factor (FGF) polypeptides, to new DNAs encoding chimeric FGF polypeptides, and to the recombinant production of chimeric FGF polypeptides, and methods, compositions, and assays that use chimeric FGF polypeptides for the therapeutic treatment of related metabolic disorders and other conditions, and for the production of pharmaceutically active compositions, including chimeric FGF polypeptides, with compositions containing therapeutic and pharmacological properties including those associated with the treatment of related metabolic disorders and other conditions. BACKGROUND OF THE INVENTION
[0003] [0003] The family of fibroblast growth factors (FGF) includes the FGF19 subfamily, which consists of human FGF21, FGF23 and FGF19 and mouse FGF15. Unlike other members of the FGF family, which typically act on their original tissue in a paracrine manner, members of the FGF19 subfamily act on specific distal tissues in an endocrine manner. The effects of the FGF family members are the result of their heparin-dependent binding to one or more members of the FGF tyrosine kinase receptor (FGFR) family. This family of receptors includes four members, each containing a tyrosine kinase domain, FGFR1, FGFR2, FGFR3 and FGFRA4, as well as two splice variants each of FGFR1, FGFR2 and FGFR3. These splice variants, which occur in exon 3 of FGFR1, FGFR2 and FGFR3, are called "b" and "Cc" variants (ie, FGFR1b, FGFR2b, FGFR3c, FGFR1Ic, FGFR2c and FGFR3c, which are also known such as FGFR1 (III) b, FGFR2 (II) b, FGFR3 (Ill) c, FGFR1 (Ill) c, FGFR2 (Ill) and FGFR3 (Ill) c, respectively).
[0004] [0004] Members of the FGF19 subfamily have interfered with the regulation of numerous tissue-specific metabolic processes in mammals. Of particular interest is the FGF19 which has been shown to reach the target and which has effects on both adipocytes and hepatocytes. For example, mice treated with human recombinant FGF19 (rhFGF19), despite being on a high-calorie diet, have increased metabolic rates, increased lipid oxidation, a lower respiratory quotient and weight loss. In addition, these mice had low levels of leptin serum, insulin, cholesterol and triglycerides, and normal blood glucose levels despite a high fat diet, and with no reduced appetite. Obese mice with leptin deficiency, but which included an FGF19 transgen, showed weight loss and a reduction in cholesterol and triglycerides, and did not develop diabetes. Obese, diabetic mice lacking leptin, when injected with rhFGF19, showed a reversal of their metabolic characteristics in the form of weight loss and reduced blood glucose. (Fu, L. et al., Endocrinology 145 (6), 2594-2603 (2004); Tomlinson, E. et al,
[0005] [0005] Another member of the FGF19 subfamily, FGF21, is expressed primarily by the liver and has metabolic effects similar to those of FGF19, such as an increase in metabolism via its effects on adipose tissue, weight loss, low blood glucose levels, and resistance to obesity and diabetes. (Kharitonenkov, A. et al., J Clin Invest 115 (6), 1627-1635 (2005)). FGF21 transgenic mice were also resistant to diet-induced obesity. In addition, in models of diabetic rodents, the administration of FGF21 reduced blood glucose levels and triglyceride levels.
[0006] [0006] FGF21 has also been shown to play a role in regulating the growth hormone (GH) pathway. The anabolic effects of GH are mediated by insulin-like growth factor 1 (IGF-1) which is mainly produced by the liver. GH induces the transcription of IGF-1, thus increasing its circulation levels, through the activation of Janus kinase 2 (JAK2) by the GH receptor. Phosphorylated JAK2 members activated from the family of signal transducers and transcription activators (STAT), when phosphorylated, undergo nuclear translocation and bind to regulatory elements of target genes, including those of IGF-1. In particular, STAT5, in its phosphorylated form, has been shown to play a prominent role in this response.
[0007] [0007] The effects of GH on IGF-1 levels appear to be counterbalanced by hunger or fasting conditions that result in low levels of IGF-1 transcription and IGF-1 circulation. (Esta sen, J.P. et al., Endocr. Rev. 15, 80—101 (1994)). These effects on IGF-1 may be due to reduced levels of phosphorylated STATS. In particular, fasting rats injected with GH have lower levels of phosphorylated liver STATS than non-fasting rats. (Beauloye, V. et al., Endocrinology 143, 792-800 (2002)). FGF21, which is induced in the liver under conditions of starvation or fasting, can mediate this effect. FGF21 transgenic mice have been shown to have lower levels of IGF-1 and phosphorylated STATS. (Inagaki, T. et al., Cell Metabolism 8, 77-83 (2008)).
[0008] [0008] The metabolic effects of FGF19 and FGF21 are affected via their binding to FGFR1c, FGFR2c and FGFR3c receptors, among which the FGFR1c and FGFR2c bonds are the most significant. In addition, binding of FGF19 and FGF21 to these receptors requires the Klotho-beta co-receptor. Despite the predominance of these FGFR receptors, the metabolic effects of FGF19 and FGF21 are produced specifically for adipocytes, due to this requirement for the klotho-beta co-receptor, which has a specific location in the tissue.
[0009] [0009] FGF19 has also been shown to have effects that are distinct from those of FGF21. For example, FGF19 has been shown to regulate the production of bile by the liver via its specific effects on the liver. In response to postprandial bile production, FGF19 downregulates bile production by suppressing the transcription of the cholesterol 7-alpha-hydroxylase (CYP7A1) gene, at a limit enzyme rate in the synthesis of bile acids, and by stimulating filling of the bile vesicle In addition, FGF19 appears to have mitogenic effects of the liver that are not seen with regard to FGF21. For example, FGF19 transgenic mice develop — hepatic adenocarcinoma due to increased proliferation and dysplasia of hepatocytes, and mice treated with rhFGF19 exhibit hepatocyte proliferation of hepatocytes. (Nicholes, K. et al., Am J Pathol 160, 2295-2307 (2000).)
[0010] [0010] These additional FGF19 activities appear to be mediated via their link to FGFR4. FGF19 can bind FGFR4 in a way that is both Klotho-beta dependent and independent of
[0011] [0011] There is a need to develop new therapies for the treatment of related metabolic disorders, such as diabetes, obesity, high blood sugar, and other related disorders. There is also a need to develop new therapies for such related metabolic disorders in which the unwanted growth or potential proliferation (eg, tumorigenic potential) of such a therapy is eliminated or reduced. There is also a need to develop new therapies for such related metabolic disorders in which the potential for growth hormone resistance to such therapy is eliminated or reduced. BRIEF SUMMARY OF THE INVENTION
[0012] [0012] In a first aspect, the present invention provides a chimeric human fibroblast polypeptide 19 growth factor (hFGF19). In some embodiments of the present invention, the chimeric polypeptide sequence includes a C terminal portion that includes a C terminal portion of the hFGF19 polypeptide sequence and an N terminal portion that includes an N terminal portion of the hFGF21 polypeptide sequence. In certain embodiments, the C terminal portion of the hFGF19 polypeptide sequence has about 45 to about 185 residues in length and the N terminal portion of the hFGF21 polypeptide sequence has about 7 to about 140 residues in length.
[0013] In another embodiment of the present invention, a chimeric hFGF19 polypeptide is provided, in which the polypeptide sequence includes a C terminal portion that includes a C terminal portion of the hFGF21 polypeptide sequence and an N terminal portion that includes an N-terminal portion of the hFGF19 polypeptide sequence. In some embodiments, the C-terminal portion of the hNhFGF21 polypeptide sequence is approximately 8 to approximately 145 residues in length, and the N-terminal portion of the hFGF19 polypeptide sequence is approximately 45 to approximately 175 residues in length.
[0014] [0014] In another embodiment of the present invention, a chimeric hFGF19 polypeptide is provided in which the chimeric polypeptide sequence includes a first sequence of polypeptides containing at least a certain sequence identity with the sequence of the hFGF19 polypeptide, and wherein a portion of the first polypeptide sequence is replaced by a portion of a second polypeptide sequence, the second polypeptide sequence containing at least a certain sequence identity with respect to the hFGF21 polypeptide sequence, such that the substituted portion of the first polypeptide sequence is approximately 3 to approximately 185 residues in length.
[0015] [0015] In some embodiments of the present invention, a chimeric hFGF19 polypeptide is provided in which the sequence of the chimeric polypeptides includes the first polypeptide sequence containing at least a certain sequence identity with the sequence of the hFGF19 polypeptide, and in which a a portion of the first polypeptide sequence is replaced with more than a portion of a second polypeptide sequence, the second polypeptide sequence containing at least a certain sequence identity with the hFGF21 polypeptide sequence. In some embodiments, the chimeric hFGF19 polypeptide comprises a replacement of the B1-B2 loop of the first polypeptide, a replacement of the B10-B12 segment of the first polypeptide, and / or a replacement of the five WGDPI residues of the first polypeptide with the loop B1-B2 of the second polypeptide, segment B10-B12 of the second polypeptide, and / or the corresponding GQV sequence of the second polypeptide.
[0016] [0016] In certain embodiments of the present invention, the polypeptide - chimeric - hFGF19 “comprises the sequence: HPIPDSSPLLOFGGQAVRARYLYTSGPHGLSSCFLRIRADGVVDCARG QSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDC AFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLP
[0017] [0017] In certain embodiments, the chimeric hFGF19 polypeptide of the present invention is fused to a second polypeptide, the second polypeptide is the Fc portion of an immunoglobulin, an analog of the Fc portion of an immunoglobulin and one or more fragments of the Fc portion of an immunoglobulin. In certain embodiments, immunoglobulin is selected from the group consisting of: IgG-1, IgG-2, IgG-3, I9gG-4, IgA-1, I9gA-2, IgE, 1gD and IgM. In some modes of execution, the Fc portion is human or humanized. In some embodiments, the C-terminus of the chimeric hFGF19 polypeptide is fused to the N-terminus of the second polypeptide. In some embodiments, the C terminus of the chimeric hFGF19 polypeptide is fused to the N termination of the second polypeptide via a linker, the linker is selected from the group consisting of: a [Gly] n linker, a [Gly3Ser] linker m is a linker [Gly4Ser] m, where n is an integer 1-30 and m is an integer 1-6.
[0018] [0018] The present invention includes chimeric hFGF19 polypeptides that have a physiological lifespan that is at least or approximately the same as that of native hFGF19. The present invention includes chimeric hFGF19 polypeptides that have a physiological lifespan that is at least or approximately the same as the native hFGF21.
[0019] [0019] In certain embodiments, the chimeric hFGF19 polypeptide does not substantially activate FGFRA4, either in an independent Klotho-beta or dependent Klotho-beta. In certain embodiments, the chimeric hFGF19 polypeptide activates FGFR1IC in a Klotho-beta dependent manner
[0020] [0020] In certain embodiments, the chimeric hFGF19 polypeptide, when administered to an individual, does not reduce the individual's lean mass. In certain embodiments, the chimeric hFGF19 polypeptide, when administered to an individual, does not substantially reduce the individual's lean mass. In certain embodiments, the chimeric hFGF19 polypeptide, when administered to an individual, does not reduce the individual's bone density. In certain embodiments, the chimeric hFGF19 polypeptide, when administered to an individual, does not substantially reduce the individual's bone density. In certain embodiments, the chimeric hFGF19 polypeptide, when administered to an individual, does not reduce the individual's cardiac capacity. In certain embodiments, the chimeric hFGF19 polypeptide when administered to an individual does not substantially reduce the individual's cardiac capacity.
[0021] [0021] In certain embodiments, the chimeric hFGF19 polypeptide does not substantially reduce or reduce the amount of phosphorylated STATB5 polypeptide in vivo. In certain embodiments, the chimeric hFGF1I9 polypeptide, when administered to an individual, does not substantially reduce or reduce the amount of phosphorylated STAT5 polypeptide in the individual. In certain embodiments, when the chimeric hFGF19 polypeptide is administered to an individual, the amount of phosphorylated STAT5 polypeptide is reduced in the individual, but this amount of phosphorylated STAT5 polypeptide is greater than the amount of phosphorylated STATS polypeptide with administration of hFGF21 native to the individual. In certain embodiments, when the chimeric hFGF19 polypeptide is administered to an individual, the amount of phosphorylated STATS polypeptide is any: from 100% to 5%, from 100% to 10%, from 100% to 20%, from 100% up to 30%, 100% up to 40%, 100% up to 50%, 100% up to 60%, 100% up to 70%, 100% up to 80%, 100% up to 90% or 100% up to 95% of an amount of phosphorylated STATS polypeptide in the subject without such administration. In certain embodiments, when the chimeric hFGF19 polypeptide is administered to an individual, the reduction in the amount of phosphorylated STAT5 polypeptide is less than the reduction in an amount of phosphorylated STAT5 polypeptide with administration of native hFGF21. For example, the reduction of the phosphorylated STATS polypeptide, when the chimeric hFGF19 polypeptide is administered to an individual, is any one: from 0% to 5%, from 0% to 10%, from 0% to 20%, from 0% to 30%, 0% to 40%, 0% to 50%, 0% to 60%, 0% to 70%, 0% to 80%, 0% to 90% or 0% to 95 %, of a reduction in the amount of phosphorylated STATS polypeptide with the administration of native hFGF21.
[0022] [0022] In certain embodiments, the chimeric hFGF19 polypeptide does not substantially reduce or reduce the circulating amount of insulin-like growth factor 1 (IGF-1) in vivo. In certain embodiments, the chimeric hFGF19 polypeptide, when administered to an individual, does not substantially reduce or reduce the amount of IGF-1 circulating in the individual. In certain embodiments, when the chimeric hFGF19 polypeptide is administered to an individual, the amount of circulating IGF-1 is reduced, but this amount of circulating IGF-1 is greater than the amount of circulating IGF-1 with administration of hFGF21 native to the individual. In certain embodiments, when the chimeric hFGF19 polypeptide is administered to an individual, the amount of circulating IGF-1 is any: from 100% to 5%, from 100% to 10%, from 100% to 20%, from 100% to 30%, 100% to 40%, 100% to 50%, 100% to 60%, 100% to 70%, 100% to 80%, 100% to 90% or 100 % to 95% of the amount of IGF-1 circulating in the individual without such administration. In certain embodiments, when the chimeric hFGF19 polypeptide is administered to an individual, the reduction in the amount of circulating IGF-1 is less than a reduction in the amount of circulating IGF-1 under administration of native hFGF21. For example, the reduction in circulating IGF-1, when the chimeric hFGF19 polypeptide is administered to an individual, is any of: from 0% to 5%, from 0% to 10%, from 0% to 20%, from 0 % to 30%, 0% to 40%, 0% to 50%, 0% to 60%, 0% to 70%, 0% to 80%, 0% to 90% or 0% up to 95%, a reduction in the amount of circulating IGF-1 with administration of native hFGF21.
[0023] [0023] In another aspect, the present invention provides a pharmaceutical composition containing a therapeutically effective amount of a chimeric hFGF19 polypeptide of the present invention; and a pharmaceutically acceptable carrier.
[0024] [0024] In another aspect, the present invention provides methods of treating an individual who exhibits one or more of the factors: obesity, type 1 diabetes, type 2 diabetes, high blood glucose, atherosclerosis metabolic syndrome, hypercholesterolemia , stroke, osteoporosis, osteoarthritis, degenerative joint disease, muscle atrophy, sarcopenia, reduced lean body mass, baldness, wrinkles, increased fatigue, reduced stamina, reduced heart function, immune system dysfunction, cancer,
[0025] [0025] In another aspect, the present invention provides a method of reducing the blood glucose of an individual in need of such treatment, the method comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition of the present invention.
[0026] [0026] In another aspect, the present invention provides an isolated nucleic acid molecule comprising DNA and containing at least 80%, at least 90%, at least 95% or at least 99% sequence identity of a DNA molecule which encodes a polypeptide containing amino acid residues of approximately 1 to approximately 190 of SEQ ID NO: 5, or its complement.
[0027] [0027] In another aspect, the present invention provides an isolated nucleic acid molecule comprising the sequence:
[0028] [0028] In certain embodiments, an isolated nucleic acid of the present invention further comprises a sequence that encodes the amino acid residues that correspond to an Fc portion of an immunoglobulin.
[0029] [0029] In another aspect, the present invention provides an expression system that comprises the nucleic acid molecule of the present invention. In another aspect, the present invention provides a host cell that comprises an expression system or nucleic acid of the present invention.
[0030] [0030] In another aspect, the present invention provides an isolated polypeptide encoded by a nucleic acid molecule of the present invention.
[0031] [0031] In another aspect, the present invention provides a process for the preparation of an isolated polypeptide that comprises culturing a host cell of the present invention under conditions suitable for expression of the encoded polypeptide and recovery of the encoded polypeptide from the cell culture. In another aspect, the present invention provides an isolated polypeptide prepared by a process of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] [0032] FIG. 1 shows the amino acid sequence of human FGF19 polypeptides (SEQ ID NO: 1) and human pre-FGF19 polypeptides (SEQ ID NO: 3);
[0033] [0033] FIG. 2 shows the amino acid sequence of human FGF21 polypeptides (SEQ ID NO: 2) and human pre-FGF21 polypeptides (SEQ ID NO: 4);
[0034] [0034] FIG. 3 shows results of the examples of a receptor binding assay using chimeric FGF19 polypeptides of the present invention;
[0035] [0035] FIG. 4 shows results of examples of a receptor binding assay using chimeric FGF19 polypeptides of the present invention;
[0036] [0036] FIG. 5 shows results of the examples of a liver specific gene expression assay using chimeric FGF19 polypeptides of the present invention;
[0037] [0037] FIG. 6 shows results from the examples of an adipocyte-specific generic expression assay using chimeric FGF19 polypeptides of the present invention;
[0038] [0038] FIG. 7 shows results of the examples of a blood glucose reduction assay using chimeric FGF19 polypeptides of the present invention;
[0039] [0039] FIG. 8 shows results of the examples of a glucose tolerance test assay using chimeric FGF19 polypeptides of the present invention;
[0040] [0040] FIG. 9 shows results of the examples of an activity assay using Fc immunoglobulin fusions of chimeric FGF19 polypeptides of the present invention; and
[0041] [0041] FIG. 10-14 shows results from examples of a specific receptor assay using chimeric FGF19 polypeptides of the present invention.
[0042] [0042] FIG. 15 shows results of the examples of the use of a chimeric FGF19 polypeptide of the present invention at the levels of the phosphorylated Stat5 protein.
[0043] [0043] FIG. 16 shows results of the examples of the use of a chimeric FGF19 polypeptide of the present invention in the total metabolic activity of cells.
[0044] [0044] Figure 17 shows that FGFRA4 was requested for regulation of bile acid ("BA") but not for the improvement of glucose tolerance by FGF19. Figure 17A shows the glucose level of wild type ("WT") mice treated with FGF19 or PBS and the death of mice (Knockout) ("KO") with FGFR4 in a glucose tolerance test. * p <0.05. ** p <0.01. The p-value of the area under the curve (AUC) was p <0.02 (WT) and p <0.005 (KO). N = 6-8. Figure 17B shows various metabolic parameters (body weight (g), liver / BW ratio (body weight) (%), serum insulin (ng / mL), serum beta-hydroxybutyrate ("BHB")) (mg / L) , serum lactate (mg / dl), and serum triglyceride (mg / dl)) from mice treated with FGF19 or PBS WT and FGFR4 KO euthanized on day 7. The mice were euthanized and the serum was separated after 3 hours fasting. N = 6-8. Figure 17C shows the analysis of the composition of BA serum in WT mice treated with FGF1I19 or PBS and FGFR4 KO mice. Only the largest BA species are shown. CA: cholic acid, DCA: deoxycholic acid, MCA: muricolic acid, T-: conjugated taurine. Figure 17D shows relative expression of various liver genes (Egr-1, c-Fos, AFP, Cyp7a1, Cyp8b, Cyp27a1, Cyp7b, and GK) in WT and FGFR4 KO mice treated by FGF19 or PBS determined by real time qPCR. N = 6-8. The p-values for figures 17B-17D are: <0.05, ** <0.005 (PBS vs FGF19), t <0.05, tHt <0.005 (WT vs FGFR4KO).
[0045] [0045] Figure 18 shows the identification of FGF19 variants with reduced FGFR4 activity. Figure 18A shows the relative firefly luciferase activity normalized by the renilla luciferase activity (shown as the relative luciferase unit ("RLU")) of a GAL-EIk1 luciferase assay using L6 mouse cells transfected with KLB and FGFR1c or FGFRA4 and incubated with medium containing increasing concentrations of FGF19 (O) or FGF21 (A). Figure 18B shows drawings (in scale) of FGF19 (top), FGF21 (bottom), and various chimeric proteins with amino acid composition on the left. Based on the results of the GAL-EIlk1 assays shown in Fig. 18C, each chimera was classified in class (1), (II) or (Ill) as indicated on the right. Chimeras that did not exhibit FGFR1c activity equivalent to FGF21 or FGF19 when the conditioned medium was used are not shown here. Figure 18C shows the activation of FGFR1c or FGFR4 in a GAL-EIlk-1 assay using L6 cells cotransfected with KLB and / or FGFR (FGFR1c or FGFR4) and incubated with conditioned medium from 293 cells transiently transfected with various FGF constructs (see Figure 18B for amino acid compositions used in FGF constructs). The results are shown as a single fold induction on the conditioned control medium with transfected simulation cells. Figure 18D shows the fold induction for FGFR activation in a GAL-Elk1 luciferase assay using L6 mouse cells transfected with FGFR1c, FGFR4 + KLB, or FGFR4 and incubated with a medium containing increasing concentrations of FGF19 (O) purified or FGF19v (V) (the ft4 construct in Figs. 18B and 18C). Figure 18E shows results of solid phase binding assays for FGF19 and FGF19v to FGFRA4 fused to the Fc fragment. The schematic diagram of the experiments is shown on the right. Figure 18F shows that the anti-FGF19 antibody used in Figure 18E recognized FGF19 and FGF19v with indistinguishable affinity (ELISA control experiment). The schematic diagram for the experiments is shown on the right.
[0046] [0046] Figure 19 shows the RLU unit in a GAL-EIk1 luciferase assay in L6 mouse cells transfected with KLB and FGFR2c or FGFR3c and incubated with medium containing increasing concentrations of FGF19 (O) or FGF21 (A). L6 cells were cotransfected with expression vectors for KLB and the FGFR indicated together with GAL-EIk1, SV40-Renilla Luciferase, and reporter luciferase with response to Gal. Transfected cells were incubated with medium containing increasing concentrations of FGF19 or FGF21 for 6 hours before the luciferase assays. Transcriptional activation was performed by the relative luciferase activity normalized by the renilla luciferase activity and expressed as the relative luciferase unit (RLU). This figure shows that FGF21 and FGF19 activated FGFR2c and FGFR3c in the presence of KLB.
[0047] [0047] Figure 20 shows the activation of FGFR1c or FGFR4 in a GAL-Elk-1 assay using L6 cells cotransfected with KLB and / or FGFR (FGFR1c or FGFR4) and incubated with conditioned medium from 293 cells transiently transfected with various FGF constructs shown on the X-axis (see Figure 18B for amino acid compositions of FGF constructs used). The results are shown as fold induction by control means conditioned with simulated transfected cells. The numbering indicated on the X axis corresponds to the numbering of the construct shown in Fig. 18B.
[0048] [0048] Figure 21 shows the effects of FGF19v in rats fed standard chow (fed mice). Figure 21A shows the relative expression of several genes (c-Fos, Egr-1, GK, SHP, and Cyp7a1) in FVB mice injected (via the tail vein) with 1mg / kg of FGF21, FGF19, or FGF19v or PBS of control. The p values: * <0.05, ** <0.01, *** <0.001 (vs PBS). 4 hours after injection, liver MRNA was prepared from each mouse and subjected to real-time qQPCR analysis for the indicated genes. Figure 21B shows the relative expression of several genes (c-Fos, Egr-1, GK, SHP, and Cyp7a1i) in WT or FGFR4 KO (KO) mice (N = 5-7) injected via i.p. with 1Img / kg of FGF21 or FGF19 or control PBS. Mice that fasted overnight were injected via i.p.com FGF protein or control PBS. 4 hours after injection, liver MRNA was prepared from each mouse and subjected to real-time qPCR analysis for the indicated genes. P-values: * <0.05, ** <0.01, *** <0.001. Figure 21C shows the proliferation of HepG2 cells treated with FGF21, FGF19, or FGF19v in various concentrations (10 ng / mL, 60 ng / mL, and 200 ng / mL) measured by fluorescent intensity (x 10º) in a growth assay anchoring independent cell. The proliferation of HepG2 cells on soft agar was estimated based on the conversion of resazurin (Alamer Blue), a non-fluorescent indicator dye, to resorufin. Figure 21D shows the number of Brdu + hepatocytes fold change in FVB mice infused with FGF19 or FGF19v. N = 6, * p <0.01, *** p <5E-5 (vs PBS), ttHp <0.0002 (vs FGF19). An osmotic pump was implanted in FVB mice to continuously infuse the indicated FGF protein at 1ng / hour (-0.8mg / kg / day) (day O). The mouse also received a daily injection of 1 mg / kg / day of FGF protein (qd) and 30 mg / kg / day of BrdU (bid) starting on day 1. On the seventh day, livers were dissected and subjected to dyeing with anti- BrdU. The results are shown as a fold induction in animals treated by simulating the number of positive BrdU hepatocytes per analyzed area. Figure 21E shows representative images for the study shown in figure 21C. Figure 21F shows the relative expression of several genes (c-Fos, Egr-1, AFP, GK, Cyp7ai and Cyp8b) in mice used in Figures 21D and 21E. N = 6. * p <0.05, ** p <0.005, *** p <0.001 (vs PBS), ttp <0.05, tHttp <0.005 (vs FGF19).
[0049] [0049] Figure 22 shows that FGF19v and FGF21 had similar metabolic effects and improved hyperglycemia in ob / ob mice. 11-week old ob / ob mice underwent subcutaneous implantation with an osmotic pump to infuse 1ng / hour of FGF protein (0.4mg / Kkg / day) or control PBS (N = 7). Figure 22A shows body weight (g) and blood glucose (mg / dl) in ob / ob mice infused with an osmotic pump of 1ng / hour of FGF21 or FGF19v (0, Amg / kg / day) or PBS control (N = 7). The osmotic pump was implanted on day 0. Figure 22B shows blood glucose levels (mg / dl) in ob / ob mice infused with FGF21, FGF19v or control PBS in a random feeding condition and after fasting overnight . Figure 22C shows levels of non-esterified fatty acid ("NEFA") serum in ob / ob mice infused with control FGF21, FGF19v or PBS on day 8. Figure 22D shows results of a glucose tolerance test conducted on ob / ob mice infused with FGF21, FGF19v or PBS control on the day
[0050] [0050] The present invention provides new chimeric FGF19 polypeptides. In some embodiments of the present invention, a chimeric FGF19 polypeptide sequence includes a portion of a FGF1I9 polypeptide sequence and a portion of a FGF21 polypeptide sequence. In certain preferred embodiments, the FGF19 polypeptide is a processed human FGF19 (hFGF19) polypeptide in which the sequence is defined in SEQ ID NO: 1. In certain preferred embodiments, the FGF21 polypeptide is human processed FGF21 (hFGF21) polypeptide in which the sequence is defined in SEQ ID NO: 2. In another aspect, the present invention provides new chimeric FGF19 polypeptides that are further fused to an immunoglobulin domain, such as the Fc domain.
[0051] [0051] In another aspect, the present invention provides new chimeric FGF19 polypeptides that have altered receptor specificity. In certain preferred embodiments, a chimeric FGF19 polypeptide of the present invention does not substantially activate FGFR4 in a Klotho-beta-dependent or Klotho-beta-independent manner. In certain embodiments, a chimeric FGF19 polypeptide of the present invention activates at least one of FGFR1c, FGFR2c or FGFR3c in a Klotho-beta-dependent manner.
[0052] [0052] In some embodiments, a chimeric FGF19 polypeptide of the present invention may have one or more of the following advantageous characteristics: the polypeptide does not substantially induce hepatocyte proliferation in an individual with its administration, the polypeptide does not substantially induce resistance to growth hormone in an individual with its administration, the polypeptide does not include a residue that is polymorphic in the population, the polypeptide has a physiological lifespan in vivo that is at least or about the same as the native FGF19 polypeptide (such as native hFGF19 polypeptide ), the polypeptide has an in vivo physiological lifespan that is at least or approximately the same as that of the native FGF21 polypeptide (such as native hrFGF21 polypeptide), the polypeptide does not substantially reduce the lean mass in an individual with its administration, the polypeptide does not substantially reduce bone density in an individual with its administration, and the polypeptide it does not substantially reduce cardiac capacity in an individual with its administration.
[0053] [0053] Other advantageous characteristics of a chimeric FGF19 polypeptide of the present invention may include one or more of the following: the polypeptide reduces blood glucose in an individual, in an individual in need of such treatment; the polypeptide activates at least one of FGFR1c, FGFR2c or FGFR3c in a Klotho-beta-dependent manner; the polypeptide does not substantially activate FGFR4 in a Klotho-beta-dependent manner; the polypeptide does not substantially activate FGFR4 in a Klotho-beta-independent manner; the polypeptide does not substantially reduce or reduce the amount of phosphorylated STATS polypeptide in an individual with its administration; the polypeptide when administered to an individual, the amount of phosphorylated STATS polypeptide in the individual is reduced but this amount of phosphorylated STATS polypeptide is greater than the amount of phosphorylated STATS polypeptide with administration of the individual's native hFGF21; the polypeptide when administered to an individual the reduction in the amount of phosphorylated STATS polypeptide in the individual is less than the reduction in the amount of phosphorylated STATS polypeptide with administration of native hFGF21 in the individual; the polypeptide does not substantially reduce or reduce the amount of circulating IGF-1 polypeptides in an individual with its administration; the polypeptide when administered to an individual the amount of IGF-1 circulating in the individual is reduced but this amount of IGF-1 circulation is greater than the amount of IGF-1 circulating with the administration of hFGF21 native to the individual; the polypeptide when administered to an individual the reduction in the amount of circulating IGF-1 in the individual is less than a reduction in the amount of circulating IGF-1 under the administration of native hFGF21 in the individual; and the polypeptide does not substantially reduce or reduce the amount of IGF-1 polypeptide circulating in an individual containing a normal or supranormal amount of GH.
[0054] [0054] In certain embodiments, a chimeric FGF19 polypeptide of the present invention has advantageous characteristics of activating at least one FGFR1c, FGFR2c or FGFR3c in a Klotho-beta-dependent manner, and does not substantially activate FGFR4 in a Klotho-beta manner -dependent or Klotho-beta “independent. In certain embodiments, a chimeric FGF19 polypeptide of the present invention has the advantageous characteristics of activating at least one of FGFR1c, FGFR2c or FGFR3c in a Klotho-beta-dependent manner, does not substantially activate FGFR4 in a Klotho-beta-dependent manner or Klotho-beta -independent, and reduces the amount of phosphorylated STATS polypeptide in an individual but this amount of phosphorylated STATS polypeptide is greater than the amount of phosphorylated STATS polypeptide with administration of native hFGF21 to the individual. In certain embodiments, a chimeric FGF19 polypeptide of the present invention has the advantageous characteristics of activating at least one FGFR1c, FGFR2c or FGFR3c in a Klotho-beta-dependent manner, and does not substantially activate FGFR4 in a Klotho-beta-dependent manner or Klotho-beta -independent, and does not include a residue that is polymorphic in the population. In certain embodiments, a chimeric FGF19 polypeptide of the present invention has the advantageous characteristics that it activates for one of the FGFR1c, FGFR2c or FGFR3c in a Klotho-beta-dependent manner, does not substantially activate FGFR4 in a Klotho-beta-dependent or Klotho manner -beta “independent, reduces the amount of phosphorylated STATS polypeptides in an individual but this amount of phosphorylated STATS polypeptides is greater than the amount of phosphorylated STATS polypeptides with administration of hFGF21 native to the individual and does not include a residue that is polymorphic in the population.
[0055] [0055] In another aspect, the present invention provides new chimeric FGF19 polypeptides that have altered receptor specificity. In certain preferred embodiments, the chimeric FGF19 polypeptide of the present invention does not substantially activate FGFR4 in a Klotho-beta-dependent or Klotho-beta-independent manner. In certain embodiments, the chimeric FGF19 polypeptide of the present invention activates at least one of FGFR1Ic, FGFR2c or FGFR3c in a Klotho-beta-dependent manner.
[0056] [0056] In another aspect, the chimeric polypeptide FGF19 does not effect an activity of resistance to growth hormone in an individual compared to the resistance to growth hormone made by native FGF21. In another aspect, the chimeric polypeptide FGF19 does not perform a substantial resistance activity to growth hormone in an individual when compared to the resistance to growth hormone made by native FGF21. In certain embodiments, the chimeric FGF19 polypeptide does not substantially reduce or reduce the amount of phosphorylated STAT5 polypeptide in an individual. In certain embodiments when the chimeric hFGF19 polypeptide is administered to an individual, the amount of phosphorylated STATS polypeptide is reduced in the individual but this amount of phosphorylated STAT5 polypeptide is greater than the amount of phosphorylated STATS5 polypeptide with administration of native hFGF21 polypeptide. . In certain embodiments when the chimeric hFGF19 polypeptide is administered to an individual, the reduction in the amount of phosphorylated STATS polypeptide is less than the reduction in the amount of phosphorylated STAT5 polypeptide under administration of native hFGF21. In certain embodiments, the FGFI9 chimeric polypeptide does not substantially reduce or reduce the amount of circulating insulin-like growth factor 1 (IGF-1). In certain embodiments when the chimeric hFGF19 polypeptide is administered to an individual, the amount of circulating IGF-1 is reduced but this amount of circulating IGF-1 is greater than the amount of circulating IGF-1 with administration of native hFGF21 for the individual. In certain embodiments when the chimeric hFGF19 polypeptide is administered to an individual, the reduction in the amount of circulating IGF-1 is less than a reduction in the amount of circulating IGF-1 under administration of native hFGF21.
[0057] [0057] In another aspect, the present invention provides new isolated nucleic acid molecules containing a sequence encoding a chimeric FGF19 polypeptide of the present invention, new expression systems that include a nucleic acid molecule of the present invention, and host cells that include a nucleic acid molecule of the invention or an expression system of the invention.
[0058] [0058] In another aspect, the present invention includes antibodies that can specifically bind to a chimeric FGF19 polypeptide of the present invention.
[0059] [0059] In another aspect, the present invention provides pharmaceutical compositions that include a chimeric FGF19 polypeptide of the present invention and a pharmaceutically acceptable carrier.
[0060] [0060] In another aspect, the present invention provides methods of treating an individual against a related metabolic disorder by administering a chimeric FGF19 polypeptide of the present invention, or an appropriate pharmaceutical formulation thereof. In another aspect, the present invention provides methods for effecting at least one or more of the following effects on an individual: decreased blood glucose, reduced obesity, increased metabolic rate, increased lipid oxidation, reduced weight, reduced of serum glucose levels, leptin, insulin, cholesterol! and / or triglycerides, treatment of diabetes, and other metabolic effects, such effects being due to the administration to the individual of a therapeutic amount of a chimeric FGF19 polypeptide of the invention or a pharmaceutical formulation thereof.
[0061] [0061] In one aspect of the present invention, a chimeric FGF19 polypeptide sequence includes a C-terminal portion and an N-terminal portion. The N-terminal portion of the FGF19 chimeric polypeptide sequence includes an N-terminal portion of a FGF21 polypeptide sequence and a C-terminal portion of the FGF19 chimeric polypeptide sequence includes a C-terminal portion of a FGF19 polypeptide sequence. In some preceding embodiments, the C-terminal portion of the FGF19 polypeptide sequence and the N-terminal portion of the chimeric FGF21 polypeptide sequence are contiguously linked. In some of the foregoing embodiments, a C-terminal portion of the FGF19 polypeptide sequence and the N-terminal portion of the chimeric FGF21 polypeptide sequence are contiguously linked by overlapping 1, 2, 3 or more residues in common between the two portions. In some alternative embodiments, the C terminal portion of the FGF19 polypeptide sequence and the N terminal portion of the chimeric FGF21 polypeptide sequence have an intervening spacer of 1, 2, 3, 4, 5 or more amino acid residues.
[0062] [0062] In certain preferred embodiments, the FGF19 polypeptide is a human FGF19 polypeptide (hFGF19) in which the sequence is defined in SEQ ID NO: 1 (Fig. 1). In some embodiments, the terminal C portion of the hFGF19 polypeptide sequence is approximately 45 to approximately 185 residues in length, expressly including the sequence lengths of the terminal C portions of hFGF19 shown in Table 1. In some embodiments, the terminal C portion of the chimeric polypeptide sequence FGF19 includes a terminal C portion of the polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity in the hFGF19 polypeptide sequence. In some embodiments, the FGF19 polypeptide is a human FGF19 preprocessed polypeptide (hFGF19), which includes its signal sequence and in which the sequence is defined in SEQ ID NO: 3 (Fig. 1).
[0063] [0063] In certain preferred embodiments, the FGF21 polypeptide is human FGF21 polypeptide (hFGF21) in which the sequence is defined in SEQ ID NO: 2 (Fig. 2). In some embodiments, the N terminal portion of the hFGF21 polypeptide sequence is approximately 7 to approximately 140 residues in length, expressly including the sequence lengths of the N terminal hFGF19 portion shown in table 2. In some embodiments, the portion N-terminus of the chimeric polypeptide sequence FGF19 includes an N-terminal portion of a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% of amino acid sequence identity of the hFGF21 polypeptide sequence. In some embodiments, the FGF21 polypeptide is a preprocessed human FGF21 (hFGF21) polypeptide,
[0064] [0064] As used herein, a C terminal portion, an N terminal portion, a substituted portion or a portion that replaces a polypeptide sequence, such as those hFGF19 or hFGF21 polypeptide sequences, has a first position and an end position. These positions correspond to positions in the polypeptide sequence from which the portion is referenced. Therefore, the sequence of a defined portion is the contiguous sequence of amino acids that start at or around the position in the polypeptide sequence that corresponds to the first position, and end at or around the position in the polypeptide sequence that corresponds to the final position. In some embodiments, the final position of a C-terminal portion of a polypeptide corresponds to or approximately the final residue of the polypeptide. In some embodiments, the first position of an N-terminal portion of a polypeptide corresponds to or approximately the first residue of the polypeptide.
[0065] [0065] Examples of C terminal portions of the hFGF19 polypeptide sequence referred to in the present invention include, without limitation, those having a first position that corresponds to or approximately any of the positions 10, 11, 25, 26, 27, 28, 30, 33, 35, 37, 40, 41, 42, 43, 44, 45, 52, 53, 54, 56, 57, 58, 59, 72, 73, 74, 79, 80, 81, 143, 144, 145 or 146 of SEQ ID NO: 1. Each terminal C portion of the examples also has a final position that corresponds to or approximately position 194 of SEQ ID NO: 1. Table 1 shows the sequences of hFGF19 C terminal polypeptide portions of the examples. Other analogous portions of hFGF19 are also considered here.
[0066] Examples of N-terminal portions of the hFGF21 polypeptide sequence referred to in the present invention include, without limitation, those having a final position that corresponds to or approximately any of positions 7, 8, 20, 21, 22, 23, 25 , 27, 29, 31, 34, 35, 36, 37, 38, 39, 46, 47, 48, 50, 51, 52, 53, 66, 67, 68, 73, 74, 75, 135, 136, 137 and 138 of SEQ ID NO: 2. Each N terminal portion of the examples also has a first position that corresponds to or approximately position 1 SEQ ID NO: 2. Table 2 shows a list of hFGF21 N terminal portions of the examples. Other analogous portions of hFGF21 are also contemplated here.
[0067] [0067] It is intended that a C terminal, N terminal portion or any other portion of a polypeptide sequence defined herein may include, independently and optionally, from one to five or more additional residues or less in the defined starting or ending position. For example, a C terminal portion of a polypeptide containing a first position on the residue or close to residue 100 may independently (i) optionally include 1, 2, 3, 4, 5 or more additional N terminal residues to the residue at position 100 , (ii) optionally include 1, 2, 3, 4, 5 or more additional C terminal residues to the final residue, (li) optionally start at a position 1, 2, 3, 4, 5 or more C terminal residues to the residue in position 100 or (iv) optionally terminate in a position 1, 2, 3, 4, 5 or more N terminal waste to the final waste. If present, one or more of the additional residues may or may not be the same (s) as the residues in the corresponding position in the polypeptide.
[0068] [0068] In some embodiments of a chimeric FGF19 polypeptide of the present invention, the N-terminal portion of this sequence includes a sequence that is selected from the N-terminal portion of the hFGF21 polypeptide sequence listed in table 2, and the C-terminal portion of its sequence includes the sequence that is selected from the C-terminal portions of the hFGF19 polypeptide sequence listed in table 1. In some embodiments, the selected N-terminal portion hFGF21 and the selected C-terminal portion hFGF19 are selected independently of each other . In some embodiments, the N-terminal hPFGF21 sequence portion and the C-terminal hFGF19 sequence portion are selected such that the C terminus of the N terminal sequence portion and the N terminal end of the C terminal sequence portion have at least 1, at least 2 or at least 3 or more residues in common. In some embodiments, the FGF19 chimeric polypeptide sequence comprises the sequence in which the C terminal portion of the FGF19 polypeptide sequence and the N terminal portion of the FGF21 chimeric polypeptide sequence are contiguous without intervening amino acids between them. In some preceding embodiments, the FGF19 chimeric polypeptide sequence comprises a sequence in which the C terminal portion of the FGF19 polypeptide sequence and the N terminal portion of the FGF21 chimeric polypeptide sequence are contiguously joined by overlapping 1, 2, 3 or more waste in common between the two portions. In some alternative embodiments, the FGF19 chimeric polypeptide sequence comprises a sequence which includes the C terminal portion of the FGF1I9 polypeptide sequence and the N terminal portion of the FGF21 chimeric polypeptide sequence, and further includes an intervening spacer between them. 1,2,3,4, or more amino residues.
[0069] [0069] Chimeric FGF19 polypeptide sequences of the examples of the present invention are shown in table 3, where its N-terminal portion includes an N-terminal portion of a hFGF21 polypeptide sequence and its C-terminal portion includes a C-terminal portion of a sequence of hFGF19 polypeptides.
[0070] In a second aspect of the present invention, a sequence of chimeric FGF19 polypeptides includes a C terminal portion and an N terminal portion. The N-terminal portion of the chimeric FGF19 polypeptide sequence includes an N-terminal portion of a FGF19 polypeptide sequence and a C-terminal portion of the FGF19 chimeric polypeptide sequence includes a C-terminal portion of a FGF21 polypeptide sequence. In some preceding embodiments, the C-terminal portion of the FGF21 polypeptide sequence and the N-terminal portion of the chimeric FGF19 polypeptide sequence are contiguously linked. In some preceding embodiments, the C-terminal portion of a FGF21 polypeptide sequence and the N-terminal portion of the FGF19 chimeric polypeptide sequence are contiguously linked by overlapping 1, 2, 3 or more residues in common between the two portions. In some alternative embodiments, a C terminal portion of a FGF21 polypeptide sequence and the N terminal portion of the FGF19 chimeric polypeptide sequence have an intervening spacer of 1, 2, 3, 4, 5 or more amino residues.
[0071] [0071] In certain preferred embodiments, the FGF19 polypeptide is human FGF19 polypeptide (hFGF19) in which the sequence is defined in SEQ ID NO: 1. In some embodiments, the N-terminal portion of the hFGF1I9 polypeptide sequence is approximately 45 to approximately 175 residues in length, expressly including the lengths of the N-terminal sequence hFGF19 shown in table 4. In some embodiments, the N-terminus of the chimeric polypeptide sequence FGF19 includes an N-terminal portion of a polypeptide sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with the hFGF19 polypeptide sequence.
[0072] [0072] In certain preferred embodiments, the FGF21 polypeptide is human FGF21 polypeptide (hFGF21) in which the sequence is defined in SEQ ID NO: 2. In some embodiments, the terminal C portion of the hFGF21 polypeptide sequence is approximately 8 to approximately 145 residues in length, expressly including the sequence lengths of the terminal C portions of hDFGF19 shown in table 5. In some embodiments, the C-terminal portion of the chimeric polypeptide sequence FGF21 includes a C-terminal portion of a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% of identity of the amino acid sequence with the hFGF21 polypeptide sequence.
[0073] [0073] Examples of N-terminal portions of the hFGF19 polypeptide sequence referred to in the present invention include, without limitation, those having a final position that corresponds to or approximately any of the 9, 10, 24, 25, 26, 27 positions, 39, 40, 41, 42, 43, 44, 51, 52, 53, 55, 56, 57, 58, 71, 72, 73, 78, 79, 80, 142, 143, 144 and 145 of SEQ ID NO: 1. Each N terminal portion of the example also has a final position that corresponds to or approximately position 1 of SEQ ID NO: 1. Table 4 shows the polypeptide sequence of N-terminal portions of hFGF19 of the examples. Other analogous portions are also contemplated here.
[0074] Examples of terminal C portions of the hFGF21 polypeptide sequence referred to in the present invention include, without limitation, those having a final position that corresponds to or approximately any of positions 8, 9, 21, 22, 23, 24, 35 , 36, 37, 38, 39, 40, 47, 48, 49, 51, 52, 53, 54, 67, 68, 69, 146, 147, 148 and 149 of SEQ ID NO: 2. Each terminal C portion of the example also has a final position that corresponds to or approximately position 181 of SEQ ID NO: 2. Table 5 shows a list of hFGF21 C terminal portions of the examples. Other analogous portions of hFGF21 are also considered here.
[0075] [0075] In some embodiments of a chimeric FGF19 polypeptide of the present invention, the N-terminal portion of its sequence includes a sequence that is selected from the hFGF19 polypeptide sequence portions listed in table 4, and a C-terminal portion of its sequence includes the sequence that is selected from the hFGF21 polypeptide sequence portions listed in table 5. In some embodiments, the selected N-terminal hFGF19 portion and the selected C-terminal hFGF21 portion are selected independently of each other. In some embodiments, the N-terminal portion of the hFGF19 sequence and the C-terminal portion of hFGF21 are selected such that the C-terminus of the N-terminal sequence and the end of the N-termination of the C-terminal portion have at least at least 1, at least 2 or at least 3 or more residues in common. In some embodiments, the FGF19 chimeric polypeptide sequence comprises the sequence in which the N-terminal portion of a FGF19 polypeptide sequence and a C-terminal portion of the FGF21 chimeric polypeptide sequence are contiguous without the intervening of amino acids between them. In some embodiments, the FGF19 chimeric polypeptide sequence comprises the sequence in which the N-terminal portion of a FGF19 polypeptide sequence and a C-terminal portion of the chimeric FGF21 polypeptide sequence are contiguously joined by overlapping 1, 2, 3 or more waste in common between the two portions. In some alternative embodiments, the chimeric FGF19 polypeptide sequence comprises a sequence that includes the N-terminal portion of a FGF1I9 polypeptide sequence and the C-terminal portion of the chimeric FGF21 polypeptide sequence, and further includes an intervening spacer between them. 1, 2, 3, 4, 5 or more amino residues.
[0076] [0076] Sequences of the FGF19 chimeric polypeptide examples of the present invention are shown in table 6, where its N-terminal portion includes an N-terminal portion of a hFGF19 polypeptide sequence and its C-terminal portion includes a C-terminal portion of a sequence of hFGF21 polypeptide.
[0077] [0077] In a third aspect of the present invention, a chimeric FGF19 polypeptide sequence includes a first polypeptide sequence in which a portion of a first polypeptide sequence is replaced with a portion of a second polypeptide sequence. In preferred embodiments, the first polypeptide is human FGF19 (hFGF19) polypeptide in which the sequence is defined in SEQ ID NO: 1. In some embodiments, the first polypeptide sequence is a sequence of polypeptides that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity amino acid sequence with the hFGF19 polypeptide sequence.
[0078] [0078] In certain preferred embodiments, the second polypeptide is a human FGF21 (hFGF21) polypeptide in which the sequence is defined in SEQ ID NO: 2. In some embodiments, the FGF21 polypeptide sequence is a polypeptide sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity amino acid sequence for the hFGF21 polypeptide sequence.
[0079] [0079] In some embodiments, the sequence of the chimeric polypeptides = FGF19 includes a sequence of hFGF19 polypeptides in which the portion to be replaced is from a group that includes, without limitation, portions that have (i) a first position that corresponds to or approximately any of positions 1, 10, 11, 17, 18, 21, 22, 25, 26, 27, 28, 40, 41, 42, 43, 44, 45, 52, 53, 54, 56 , 57, 58, 59, 63, 72, 73, 74, 79, 80, 81, 143, 144, 145 and 146 in SEQ ID NO: 1, and (ii) a final position that corresponds to or approximately any one of positions 9, 10, 24, 25, 26, 27, 29, 31, 32, 34, 36, 39, 40, 41, 42, 43, 44, 51, 52, 53, 55, 56, 57, 58, 66, 71, 72, 73, 78, 79, 80, 142, 143, 144, 145 and 194 in SEQ ID NO: 1, such that the final position is C terminal for the first position and the positions are selected independently. The table
[0080] [0080] The substituted portion of the hFGF19 polypeptide sequence is replaced with a portion of the hFGF21 polypeptide sequence. Substitute portions of the hFGF21 polypeptide sequence of the examples include, without limitation, portions that have (i) a first position that corresponds to or approximately any of positions 1, 8, 9, 13, 14, 17, 18, 21, 22 , 23, 24, 35, 36, 37, 38, 39, 40, 47, 48, 49, 51, 52, 53, 54, 58, 67, 68, 69, 74, 75, 76, 136, 137, 138 and 139 in SEQ ID NO: 2, and (ii) a final position that corresponds to or approximately any of positions 7, 8, 20, 21, 22, 23, 24, 25, 27, 29, 31, 34 , 35, 36, 37, 38, 39, 46, 47, 48, 50, 51, 52, 53, 61, 66, 67, 68, 73, 74, 75, 135, 136, 137, 138 and 181 in SEQ ID NO: 2, such that the final position is C terminal for the first position and the positions are selected independently. Table 8 shows a list of portions of the hFGF21 polypeptide sequence from the examples that should replace a portion of the hFGF19 polypeptide sequence.
[0081] [0081] In some embodiments, a chimeric FGF19 polypeptide includes the hFGF19 polypeptide sequence with a portion of its sequence, such as a sequence selected from the portions of the hFGF19 polypeptide sequence listed in table 7, replaced with a portion of the sequence of hFGF21 polypeptides, such as the sequence portions listed in table 8. In some embodiments, the selected N terminal hFGF19 portion and the selected C terminal hFGF21 portion are selected independently of each other. In some embodiments, the hFGF19 portion to be replaced includes the N-terminal residue of the hFGF19 polypeptide. In some embodiments, the hFGF19 portion to be replaced includes the C-terminal residue of the hFGF19 polypeptide. In some embodiments, the substituted hFGF21 portion includes the N-terminal residue of the hFGF21 polypeptide. In some embodiments, the substituted hFGF21 portion includes the C terminal residue of the hFGF21 polypeptide. In some embodiments, the hFGF19 portion to be replaced includes the N terminal residue of the hFGF19 polypeptide, and the substitute hFGF21 portion also includes the N terminal residue of the hFGF21 polypeptide. In some embodiments, the hFGF19 portion to be replaced includes the C terminal residue of the hFGF19 polypeptide, and the substituted hFGF21 portion also includes the C terminal residue of the hFGF21 polypeptide.
[0082] [0082] In some embodiments, the hFGF19 sequence portion and the hPFGF21 sequence portion are selected such that at least one of their respective ends (e.g., the N terminal end of the hFGF19 portion and the N terminal end of the portion hFGF21, the C terminal end of the hFGF1I9 portion and the C terminal end of the hFGF21 portion, or both) have at least 1, at least 2 or at least 3 or more residues in common at the corresponding corresponding ends. In some embodiments, the FGF19 chimeric polypeptide sequence comprises the sequence in which the substituting portion of the hFGF21 polypeptide sequence is contiguous to the remaining hFGF19 polypeptide sequence by overlapping 1, 2, 3 or more residues in common between the two portions .
[0083] [0083] In some embodiments, the sequence of the chimeric FGF19 polypeptides comprises the sequence in which the portion of the hFGF21 polypeptide sequence is replaced in the hFGF19 polypeptide sequence such that the sequence of hFGF1I9 and hFGF21 polypeptides are contiguous and without additional amino acid interposition , intervening between them. In some alternative embodiments, the FGF19 chimeric polypeptide sequence comprises the sequence in which the portion of the hFGF21 polypeptide sequence is replaced in the hFGF19 polypeptide sequence such that the FGF1I9 polypeptide - chimeric sequence still includes an intervening spacer between them 1 , 2, 3, 4, 5 or more amino residues between the hFGF19 and hFGF21 sequences.
[0084] [0084] Sequences of chimeric FGF19 polypeptides from the examples of the present invention are shown in Table 9, in which the hFGF19 portion is replaced by a hFGF21 portion.
[0085] [0085] In some embodiments of any of the chimeric hFGF19, the chimeric hFGF19 polypeptide includes a first sequence of polypeptides containing at least a certain sequence identity with the sequence of the hFGF19 polypeptide, and in which a portion of the first polypeptide sequence is replaced by more than one portion of a second polypeptide sequence, the second polypeptide sequence containing at least a certain sequence identity with the sequence of the hFGF21 polypeptide. In some embodiments, the chimeric hFGF19 polypeptide further comprises a replacement of the B1-B2 loop of the first polypeptide, a replacement of the B10-B12 segment of the first polypeptide, and / or a replacement of the five WGDPI residues (SEQ ID NO: 287) of the first polypeptide with the loop (loop) B1-B2 of the second polypeptide, segment B10-B12 of the second polypeptide, and / or the corresponding GQV sequence of the second polypeptide. In some embodiments, the chimeric hFGF19 polypeptide further comprises a replacement of the FGF19 loop B1-B2 (50-57 amino acid residues (SGPHGLSS (SEQ ID NO: 288)) with the loop B1-B2 (amino acid residues 51-57 of FGF21 (DDAQQTE (SEQ ID NO: 289)) of FGF21. In some embodiments, the chimeric hFGF19 polypeptide still comprises a substitution of segment B10-B12 (amino acid residues 146-162 of FGF19 (SSAKORQLYKNRGFLPL (SEQ ID NO: 290)) of FGF19 with segment B10-B12 (amino acid residues 147-161 of FGF21 (PGNKSPHRDPAPRGP (SEQ ID NO: 291)) of FGF21. In some embodiments, chimeric hFGF19 polypeptides still comprise a substitution of residues amino acids 38-42 (WGDPI (SEQ ID NO: 287)) of FGF19 by amino acid residues 41-43 (GQV) of FGF21.
[0086] [0086] FGF19 chimeric polypeptides of the present invention, and particularly their pharmaceutically active compositions and methods that use said FGF19 chimeric polypeptides in the therapeutic treatment of one or more diseases, conditions, etc. listed or described herein or known in the art have certain advantages over the use of either native FGF19 (e.g., hDFGF19) or native FGF21 (e.g., hnFGF21).
[0087] [0087] In some embodiments, chimeric FGF19 polypeptides may be less immunogenic than one or both of their native parental FGFs. A native FGF19 and / or FGF21 (such as hFGF19 or hFGF21) can be present in the population in more than one allelic variation, where there is at least one amino acid residue that is different between allelic forms. For example, hFGF21 is known to have a polymorphism at position 146 in the mature form, where this residue can be leucine (as in Fig. 2 and SEQ ID NO: 2) or proline in different alleles. Such polymorphism may limit the usefulness of native hFGF21 as a therapeutic composition. For example, when administering a FGF1I9 polypeptide to an individual, in which the individual's endogenous FGF19 has a different sequence than the FGF19 administered, an individual's immune response to the hFGF21a to be administered may result. Therefore, in some embodiments, a chimeric FGF19 polypeptide of the present invention can include a portion of the hFGF21 polypeptide sequence and a portion of the hFGF19 polypeptide sequence, both portions of which include only portions of the respective polypeptide sequences that are non-polymorphic. . This can be achieved, for example, by replacing a portion of the polymorphic sequence of one FGF with the analogous, non-polymorphic portion of the other FGF polypeptide. For example, a chimeric FGF19 polypeptide of the present invention, such as cFGF21 / 19-2 (cf. table 3) that includes a portion of hFGF21 but does not include position 146, has no polymorphism at this position. In this way, chimeric FGF19 polypeptides of the present invention may advantageously be less immunogenic, and therefore may be advantageously more suitable for administration to a wide range of individuals.
[0088] [0088] In some embodiments, chimeric FGF19 polypeptides may be less tumorigenic than one or both of their corresponding native FGFs. In particular, chimeric FGF19 polypeptides may be less tumorigenic than native hFGF19. Native hFGF19, as discussed earlier, demonstrates potential tumorigenic activity via its binding to FGFRA4. This tumorigenic activity appears separable from the metabolic effects of hFGF19, which, like hFGF21, are carried out via Klotho-beta-dependent binding to FGFR1c, FGFR2c and / or FGFR3c. In some embodiments, the chimeric FGF19 polypeptides of the present invention include a portion of the hFGF19 polypeptide sequence that does not include the FGFRA effector motif, and are instead replaced with a corresponding hFGF21 sequence. In some embodiments, the chimeric FGF19 polypeptides of the present invention no longer bind to FGFR4 substantially and / or do not substantially activate FGFR4. In some embodiments, chimeric polypeptides of the present invention FGF19 no longer bind substantially to and / or do not substantially activate a receptor, such as FGFRA4, in a Klotho-beta-independent manner. In this way, the chimeric FGF19 polypeptides of the present invention can be advantageously less tumorigenic, and therefore can be advantageously more suitable for administration to a wide range of individuals.
[0089] [0089] In some embodiments, chimeric FGF19 polypeptides may not effect growth hormone (GH) resistance, or demonstrate substantially less GH resistance activity, than one or both of their corresponding native FGFs. In some embodiments, chimeric FGF19 polypeptides may have less GH resistance activity than native FGF21, such as native hFGF21. GH normally has growth and metabolic effects that are mediated by insulin-like growth factor 1 (IGF-1). The binding of GH to its receptor results in the activation of Janus kinase 2 (JAK2), which then phosphorylates the STAT5 protein. Phosphorylated STAT5 is translocated to the nucleus and binds to regulatory response elements in the genes that promote IGF-1 expression.
[0090] [0090] The effects of GH in individuals can be mitigated by raising FGF21 levels or by prolonged fasting or starvation, which also raises FGF21 levels. The effects of GH resistance in individuals include energy conservation, increased torpor, decreased body temperature, reduced physical activity, inhibited growth, loss of lean mass, and ketone induction in body synthesis. Native hFGF21 affects GH resistance, for example, by reducing the level of IGF-1 that is normally induced by GH. Without being limited to theory, this resistance, this GF resistance activity of hFGF21 can be mediated by its ability to reduce the amount of phosphorylated STAT5 polypeptides and, as a result, reduce the translocation of phosphorylated STATS to the nucleus and thereby , reduce the expression of IGF-1, thus resisting the effects of GH.
[0091] [0091] In some embodiments, chimeric FGF19 polypeptides of the present invention do not substantially reduce or reduce the amount of phosphorylated STATS polypeptides. In this way, chimeric FGF19 polypeptides of the present invention can demonstrate less or substantially no resistance activity against GH, and therefore are advantageously more suitable for administration to a range of individuals.
[0092] [0092] In some embodiments, chimeric FGF19 polypeptides do not substantially promote anchorage of independent cell growth. In some embodiments, polypeptides - chimeric FGF19 may not substantially increase metabolic activity and / or cell proliferation in an environment that requires anchorage-independent growth. In some embodiments, chimeric FGF1I9 polypeptides can promote the growth of anchorage-independent cells to a certain extent that is less than the corresponding growth of anchorage-independent cells of native FGF19. In some embodiments, chimeric FGF19 polypeptides can promote increased metabolic activity and / or cell proliferation in an environment that requires anchorage-independent growth to a limit that is less than the corresponding effect of native FGF19. As the independent anchoring factor is one of the defining characteristics of transformed cells, such chimeric FGF19 polypeptides of the present invention, which do not promote the growth of anchorage-independent cells, or do not substantially promote increased metabolic activity, and / or cell proliferation in an environment that requires growth independent of anchorage, they may be less able to differentially promote or increase the growth and / or metabolic activity of transformed cells, and therefore may be advantageously more suitable for administration to a wide range of individuals. WV. Definitions
[0093] [0093] The terms "FGF19 polypeptide", "FGF19 protein" and "FGF19" when used here encompass a polypeptide containing an amino acid sequence that is the same as a native sequence of a member of the fibroblast growth factor family 19. Members of such a family include the human amino acid sequence 194 FGF19 (hFGF19) as provided by SEQ ID NO: 1 and in FIG. 1. A FGF19 polypeptide can be isolated from nature or can be prepared by recombinant and / or synthetic means. A FGF19 polypeptide specifically covers naturally occurring truncated or secreted forms, naturally occurring variant forms (e.g. spliced forms) and naturally occurring allelic variants of FGF19. One - FGF1I9 polypeptide also specifically covers both processed and unprocessed forms, such as, for example, the 216 amino acid sequence of the pre-human FGF19 polypeptide, as provided by SEQ ID NO: 3 and in FIG.1.
[0094] [0094] The terms "FGF21 polypeptide", "FGF21 protein" and "FGF21" when used herein encompass a polypeptide containing an amino acid sequence that is the same as the native sequence of a member of the fibroblast growth factor 21 family. Members of such families include the amino acid sequence 181 of human FGF21 (hFGF21) as provided by SEQ ID NO: 2 and in FIG. 2. A FGF21 polypeptide can be isolated from nature or can be produced by recombinant and / or synthetic means. A FGF21 polypeptide specifically covers naturally occurring truncated or secreted forms, naturally occurring variant forms (for example, alternatively joined, spliced forms and naturally occurring allele variants of FGF21. A FGF21 polypeptide also specifically covers both processed forms, and unprocessed FGF21, such as for example the 209 amino acid sequence of the pre-human FGF21 polypeptide as provided by SEQ ID NO: 4 and in FIG.2.
[0095] [0095] The terms "FGF polypeptide" and "FGF protein" when used herein encompass a polypeptide containing the native sequence of a member of the FGF family, such as FGF1, FGF2, FGF3, FGFA, FGF5, FGF6, FGF7, FGF8, FGF9 , FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, humans and their mammalian counterparts. A native sequence of a given FGF polypeptide can be isolated from nature or produced by recombinant and / or synthetic means. The native sequence of a given FGF specifically encompasses naturally occurring truncated or secreted forms, naturally occurring variant forms (for example, alternatively intertwined forms, its naturally occurring allelic variants, and both processed and unprocessed forms of FGF.
[0096] [0096] The terms "chimeric polypeptides" and "chimeric protein" when used herein encompass a polypeptide containing a sequence that includes at least a portion of a full length sequence of the first polypeptide sequence and at least a portion of a length sequence total of a second polypeptide sequence, where the first and second polypeptides are different polypeptides. A chimeric polypeptide also encompasses polypeptides that include two or more non-contiguous portions derived from the same polypeptide. A chimeric polypeptide also encompasses polypeptides containing at least one substitution, the chimeric polypeptide including a first polypeptide sequence in which a portion of the first polypeptide sequence has been replaced by a portion of a second polypeptide sequence.
[0097] [0097] The term "portion," when used here with respect to a new polypeptide sequence, refers to a contiguous length of the given polypeptide sequence that is less than the full length sequence of the given polypeptide. A portion of a given polypeptide can be defined by its first position and its final position, in which the first position and the final position each correspond to a position in a sequence of the given polypeptides, the position of the sequence corresponding to the first position is located N-terminally to the position of the sequence that corresponds to the final position, and where the sequence of the portions is the contiguous sequence of amino acids in the polypeptide since it starts at the position of the sequence that corresponds to the first position, and ends at the position of the sequence that corresponds to the final position. A portion can also be defined by reference to the position in the given sequence of polypeptides and a length of residues relative to the referenced position, the sequence of the portion being a contiguous sequence of amino acids in the polypeptide since it has the defined length and is located in the polypeptide given with respect to the defined position
[0098] [0098] The term "N-terminal portion" of a given polypeptide sequence is a contiguous length of the given polypeptide sequence that begins at or near the N-terminal residue of the given polypeptide sequence. An N-terminal portion of the given polypeptide can be defined by a length. Similarly, the term "C-terminal portion" of a given polypeptide sequence is a contiguous length of the given polypeptide sequence that ends at or near the C-terminal residue of the given polypeptide sequence. A C-terminal portion of the given polypeptide can be defined by a length.
[0099] [0099] The terms "chimeric FGF polypeptides" and "chimeric FGF proteins" when used herein encompass a polypeptide containing a sequence that includes at least a portion of a first FGF polypeptide sequence and a portion of a second FGF polypeptide sequence, in which the first and second polypeptides also encompass polypeptides that include two or more non-contiguous portions derived from the same FGF polypeptide. A chimeric FGF polypeptide also encompasses polypeptides containing at least one substitution, wherein the chimeric FGF polypeptide includes a first FGF polypeptide sequence, in which a portion of the first FGF polypeptide sequence has been replaced by a portion of a second FGF polypeptide sequence.
[00100] [00100] The terms "chimeric FGF19 polypeptides" and "chimeric FGF19 proteins" when used herein encompass chimeric FGF polypeptides containing a sequence that includes at least a portion of a FGF19 polypeptide sequence and a portion of a second polypeptide sequence. For example, a chimeric FGF19 polypeptide comprises polypeptides in which the second sequence of polypeptides is a sequence of FGF21 polypeptides.
[00101] [00101] A chimeric FGF1I9 polypeptide also encompasses polypeptides that include two or more non-contiguous portions derived from a FGF19 polypeptide sequence. A FGF1I9 chimeric polypeptide also encompasses polypeptides containing at least one substitution, in which the chimeric FGF19 polypeptide sequence includes a FGF19 polypeptide sequence in which a portion of the FGF19 polypeptide sequence has been replaced by a portion of a second polypeptide sequence. In such cases, a chimeric FGF19 polypeptide expressly encompasses polypeptides in which the substitute portion is a portion of a sequence of FGF21 polypeptides.
[00102] [00102] A chimeric FGF19 polypeptide also encompasses a polypeptide in which the sequence consists only of derived portions or of a sequence of FGF19 polypeptides or a second sequence of polypeptides, such as a sequence of FGF21 polypeptides. Unless otherwise indicated, a chimeric FGF19 polypeptide is not limited to, nor does it imply, unless otherwise indicated, in the respective order or locations of the FGF19 polypeptide sequence with respect to any other portions of sequences within of the chimeric polypeptide sequence FGF19.
[00103] [00103] "Percentage (%) of amino acid sequence identity" with respect to a given polypeptide sequence identified here is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the reference sequence , after aligning the strings and inserting spaces, if necessary, to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. The alignment for the purposes of determining the sequence identity percentage of amino acids can be achieved in a number of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. can determine appropriate parameters for measurement, alignment, including any necessary algorithms to achieve maximum alignment over the total length of the compared sequences.
[00104] [00104] For the purposes mentioned here, the% amino acid sequence identity of a given amino acid sequence, for, with or against a given B amino acid sequence (which can alternatively be formulated as a given A amino acid sequence that has or comprises a certain% identity of a sequence of amino acids for, with or against a given sequence of amino acids B) is calculated as follows: 100 times the fraction XY, where X is the number of amino acid residues scored as identical, coincides with a given sequence alignment program, in which the alignment program of A and
[00105] [00105] The "percentage (%) nucleic acid sequence identity" with respect to a polypeptide-encoding nucleic acid sequence is defined here as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in the sequence nucleic acid encoding polypeptides, after aligning the sequences and introducing spaces, if necessary, to achieve the maximum percentage of sequence identity. Alignment for the purposes of determining the percentage of nucleic acid sequence identity can be achieved in a number of ways that are within the scope of the technique, for example, the use of publicly available computer software such as BLAST, BLAST- 2, ALIGN, ALIGN-2 or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment of the total length of the sequences being compared.
[00106] [00106] For the purposes set forth herein, the% nucleic acid sequence identity of a given C nucleic acid sequence for, with or against a given D nucleic acid sequence (which alternatively may be formulated as a given acid sequence nucleic C which has or comprises a certain% nucleic acid sequence identity for, with or against a given nucleic acid sequence D (which may alternatively be formulated as a given nucleic acid sequence C which has or comprises a certain% identity of nucleic acid sequence for, with, or against a given acid nucleic acid sequence D) is calculated as follows: 100 times the W / Z fraction where W is the number of nucleotides scored with identical results for a given alignment program of sequence in that CebD alignment, although Z is the total number of nucleotides in D. It will be appreciated that where the length of the C nucleic acid sequence is not equal to the length of the sequence ia D of nucleic acid, the% of the nucleic acid sequence identity from C to D will not be equal to the% of the nucleic acid sequence identity from D to C.
[00107] [00107] "Isolated," when used to describe the various polypeptides “disclosed here, means polypeptide that has been identified and separated and / or recovered from a component of its natural environment. Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated. Contaminating components of your natural environment are materials that would typically interfere with the diagnosis or therapeutic uses of the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of the N terminal or internal amino acid sequence through the use of a spinning cup sequenator, or (2) for the homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stains. Isolated polypeptides include polypeptides in situ within recombinant cells, since at least one component of the polypeptide's natural environment will not be present. Usually, however, isolated polypeptides will be prepared by at least one purification step.
[00108] [00108] An "isolated" nucleic acid molecule encoding a polypeptide is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in the natural source of nucleic acid that it encodes the polypeptide. Preferably, the isolated nucleic acid is free of association with all components with which it is naturally associated. An isolated nucleic acid molecule encoding the polypeptide is different in the form or fit in which it is found in nature. Isolated nucleic acid molecules are therefore distinct from the nucleic acid molecule that encodes the polypeptide as they exist in natural cells. However, an isolated nucleic acid molecule encoding a polypeptide includes nucleic acid molecules encoding polypeptides - contained in the cells of those polypeptides normally expressed in which, for example, the nucleic acid molecule is at a different chromosomal location than that of natural cells.
[00109] [00109] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are appropriate for prokaryotes, for example, include a promoter, optionally a sequence operator, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals and enhancers.
[00110] [00110] Nucleic acid is "operably linked to" when it is placed in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or a secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operationally linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operationally linked to" means that the DNA sequences that are linked are contiguous and, in the case of a secretory leader, contiguous and in the reading phase. However, intensifiers need not be contiguous. Bonding is achieved by bonding at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or ligands are used according to conventional practice.
[00111] [00111] The term "antibody" is used in the broadest sense and specifically covers, for example, single monoclonal antibodies (including agonists, antagonists, and neutralizing antibodies), antibody compositions with polyepitopic specificity, single chain antibodies, and fragments antibodies (see below). The term "monoclonal antibody", as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, eg, individual antibodies comprising the population are identical except for possible naturally occurring mutations, which may be present in smaller quantities.
[00112] [00112] Regarding the attachment of a polypeptide, antibody, oligopeptide or other organic molecule to a target molecule or cognate receptor, the term "specific binding" or "specifically binds to" or is "specific to" a polypeptide in particular, or an epitope on a particular target polypeptide or cognate receptor, means binding that is measurably different from a non-specific interaction. Specific connection can be measured,
[00113] [00113] The "stringency" of hybridization reactions is readily determinable by an individual with normal knowledge of the technique, and is generally an empirical calculation dependent on the length of the sample, the washing temperature and the salt concentration. In general, longer samples require higher temperatures for proper association, while shorter samples require lower temperatures. Hybridization generally depends on the ability to reassociate denatured DNA when complementary strands are present in an environment below its fusion temperature. The higher the degree of desired homology between the samples and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less stringent. For further details and explanation of the stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
[00114] [00114] "Stringent conditions" or "stringent conditions", as defined here, can be identified as those that: (1) employ low ionic resistance and high temperature for washing, for example 0.015 M sodium chloride / citrate of sodium 0.0015 M / 0.1% sodium dodecyl sulfate at 50 ° C; (2) use during denaturation a denaturing agent, such as formamide, for example 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C; or (3) use of 50% formamide, 5xSSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt solution, Sonicated salmon sperm DNA (50 µg / ml), 0.1% SDS, and 10% dextran sulfate at 42 ° C, with washes at 42 ° C in 0.2xSSC (sodium chloride / sodium citrate) and 50 % formamide at 55º C, followed by a high stringency wash consisting of EDTA containing 0.1xSSC at 55º C.
[00115] [00115] "Moderately stringent conditions" can be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions
[00116] [00116] The term "tag epitope" when used here refers to a polypeptide fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, but it is short enough not to interfere with the activity of the polypeptide to which it is fused. The preferred tag polypeptide is also quite unique, so the antibody does not cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between approximately 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues)
[00117] [00117] As used herein, the term "immunoadhesin" designates antibody-like molecules that combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of the immunoglobulin constant domains. Structurally, immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity that is other than the recognition antigen and the binding site of an antibody (e.g., it is "heterologous"), and a sequence of constant immunoglobulin domain. The adhesin part of an immunoadhesin molecule is typically a contiguous sequence of amino acids comprising at least the binding site of a receptor or a linker. The immunoglobulin constant sequence in the immunoadhesin can be obtained from any immunoglobulin, such as subtypes I9G-1, I9G-2, I9G-3, or I9G-4, IgA (including IgA-1 and IgA-2) , IgE, IgD or IgM.
[00118] [00118] "Active" or "activity" for the purposes described herein refers to the chimeric FGF19 polypeptides, which retain at least one biological and / or immunological activity of the FGF19 polypeptides and / or of the naturally occurring or native FGF21 polypeptides, particularly the hFGF1I9 polypeptides and / or the native or naturally occurring hFGF21 polypeptides. "Biological" activity refers to a biological function (either inhibitory, stimulating or cooperative) caused by a native or naturally occurring FGF1I9 polypeptide and / or a FGF21 polypeptide, in addition to the ability to induce the production of an antibody against an epitope antigen possessed by a native or naturally occurring FGF19 polypeptide and / or FGF21 polypeptide "Biological" activity can also refer to a cellular or biochemical function of FGF19 polypeptides and / or naturally occurring or native FGF21 polypeptides, such as the ability to bind to one or more of their respective cognate receptors "Immunological" activity refers to the ability of a FGF1I9 polypeptide and / or a naturally occurring or FGF21 polypeptide to induce the production of an antibody against an antigenic epitope. Preferred biological activity includes any of the following activities in the examples: increased metabolism (or metabolic rate) in an individual, dim decrease in body weight of an individual, decrease in adiposity in an individual, decrease in glucose uptake in adipocytes, increase the release of adipocyte leptin, decrease triglycerides in an individual, decrease free fatty acids in an individual, through Klotho-beta-dependent binding to a cognate FGF receptor, and Klotho-beta-independent binding to a cognate FGF receptor. It is understood that some of the activities of the FGF19 and / or FGF21 polypeptides are induced - directly by the polypeptides and some are indirectly induced, however, each of them is the result of the presence of the FGF19 and / or FGF21 polypeptides and would otherwise not have the result, in the absence of polypeptides.
[00119] [00119] The term "antagonist" is used in the broadest sense and includes any molecule that partially or totally blocks, inhibits or neutralizes the biological activity of a native or chimeric polypeptide disclosed here. In a similar way, the term "agonist" is used in the broadest sense and includes any molecule that mimics the biological activity of a native or chimeric polypeptide disclosed here. Antagonistic antibodies or fragments of antibodies, fragments or variants of amino acid sequences of native polypeptides, peptides, small organic molecules, etc. Methods for identifying agonists or antagonists of a polypeptide may comprise contacting a polypeptide with a candied agonist or antagonist molecule, and measuring a detectable change in one or more biological activities associated with the polypeptide.
[00120] [00120] As used here, "treatment" ("treatment" or "treating") is an approach to obtain beneficial or desired results, including clinical results. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: decrease in one or more symptoms resulting from the disease, reduction in the extent of the disease, stabilization of the disease (for example, prevention or delayed worsening of the disease), delayed or delayed disease progression, improved disease status, decreased dose or other medications required to treat the disease, and / or increased quality of life.
[00121] [00121] As used here, "delay" in progression means to postpone, prevent, decrease, delay, stabilize and / or postpone the development of the disease. This delay can be of several time intervals, depending on the history of the disease and / or the individual being treated.
[00122] [00122] In some embodiments, the treatment methods described here improve (eg, reduce the incidence of, the duration of, reduce or decrease the severity of) one or more symptoms of the disease.
[00123] [00123] "A" symptom "is any phenomenon that is morbid or distant from normal in function, structure or sensation, experienced by the individual.
[00124] [00124] An "effective amount" of a polypeptide, antibody, its agonist or antagonist as disclosed here is an amount sufficient to perform a specifically mentioned purpose. An "effective amount" can be determined empirically in a routine manner, in relation to the mentioned purpose.
[00125] [00125] The term "therapeutically effective amount" refers to an amount of an antibody, polypeptide, or other drug effective to "treat" a disease or disorder in an individual or mammal. See the definition of "treat" here.
[00126] [00126] "Chronic" administration refers to the administration of the agent (s) in a continuous mode as opposed to an acute mode, in order to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is treatment that is not done consecutively without interruption, but instead is cyclical in nature.
[00127] [00127] "" Mammalian "for the purpose of treatment refers to any animal classified as a mammal, including humans, domestic animals and farm, zoo and sports animals, or pets, such as dogs, cats, cattle , horses, sheep, pigs, goats, rabbits, etc. Preferably the mammal is human.
[00128] [00128] "Individual" is any mammal, preferably a human.
[00129] [00129] "Obesity" refers to a condition in which a mammal has a body mass index (BMI), which is calculated as weight (kg) per height (meters) of at least 25.9. Conventionally, those with normal weight have a BMI of 19.9 to less than 25.9. Obesity here can be due to any cause, both genetic and environmental. Examples of disorders that can result in obesity or be the cause of obesity include overfeeding and bulimia, polycyclic ovarian disease, craniopharyngioma, Prader-Willi syndrome, Frohlich syndrome, type II diabetes, GH-deficient individuals, normal variant of short stature, Turner syndrome and other pathological conditions that show reduced metabolic activity or a decrease in resting energy expenditure as a percentage of the total fat-free mass, for example children with acute lymphoblastic leukemia.
[00130] [00130] "Conditions related to obesity" refer to conditions that are the result of or that are exasperated by obesity, such as, but not limited to, dermatological disorders such as infections, varicose veins, Acanthosis nigricans, and eczema, intolerance exercise, diabetes mellitus, insulin resistance,
[00131] [00131] Administration "in combination with" or "in conjunction with" one or more other therapeutic agents includes simultaneous, concurrent, consecutive and sequential administration, in any order.
[00132] [00132] "Transporters" as used herein include transporters - pharmaceutically acceptable, excipients, or stabilizers that are non-toxic to the cell or mammal being exposed to it at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers, such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidones; amino acids such as glycines, glutamines, asparagines, arginines or lysines; 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 non-ionic surfactants such as TWEEN'Y, polyethylene glycol (PEG), and PLURONICS'Y,
[00133] [00133] Papain's digestion of antibodies produces two identical antigen-binding fragments, the so-called "Fab" fragments, and a residual "Fc" fragment, a designation that reflects the ability to readily crystallize. The Fab fragment consists of an entire L chain along with the domain of the variable region of the H chain (Vs), and the first constant domain of a heavy chain (Cm). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a unique antigen binding site. Treatment of an antibody with pepsin produces a single large F (ab ') fragment, which roughly corresponds to two disulfites bound to Fab fragments containing divalent antigen binding activity and is also capable of cross-linking with an antigen. Fab 'fragments differ from Fab fragments in that they have some additional residues at the CH-carboxy terminus including one or more cysteines from the antibody hinge region. Fab'-SH is the designation here for Fab 'in which the cysteine residue (s) of the constant domains contain a free thiol group. Antibody fragments F (ab ') 2 were originally produced as pairs of Fab' fractions that have cysteine hinges between them. Other chemical bonds of antibody fragments are also known.
[00134] [00134] "Fv" is the minimum antibody fragment that contains an antigen recognition site and complete antigen binding site. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain in tight and non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv that comprises only three antigen-specific CDRs) has the ability to recognize and bind antigens, despite a lesser affinity than that of the entire binding site.
[00135] [00135] The "Fc" fragment comprises the carboxy terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which is also the part recognized by the Fc receptors (FCcR) found in certain types of cells.
[00136] [00136] The Fab fragment also contains the constant domain of the light chain and the first constant domain (Cx1) of the heavy chain. Fab fragments differ from Fab 'fragments by the addition of some residues at the carboxy terminus of the Cn heavy chain domain including one or more cysteines from the antibody hinge region.
[00137] [00137] The "light chains" of antibodies (immunoglobulins) of any species of vertebrates can be attributed to one or two clearly distinct types, called Kappa and lambda, based on the amino acid sequences of their constant domains.
[00138] [00138] Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), eg, IgG1, IgG2, 1g9G3, IgG4, IgA, and IgA2.
[00139] [00139] "Single chain Fv" antibody fragments or "sFv antibody fragments" comprise the VH and VL antibody domains, where these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the sFvs to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer publisher, New York, p. 269-315 (1994).
[00140] [00140] The term "diabody" refers to small antibody fragments with two antigen binding sites, whose fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same chain polypeptide (VH-VL). Using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen binding sites. Diabodies are described in more detail in, for example, EP Patent Publication 0404097; PCT International Patent Publication WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).
[00141] [00141] An "isolated" antibody is one that has been identified and separated, and / or recovered from a component of its natural environment. Contaminating components of its natural environment are materials that interfere with the diagnosis or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to more than 95% by weight of the antibody as determined by the Lowry method, and more preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of the terminal or internal N amino acid sequence by using a spinning cup sequenator, or (3) until homogeneity by SDS-PAGE under conditions of reduction or non-reduction using "Coomassie blue" or, preferably "silver stain". The isolated antibody includes the antibody in situ within the recombinant cells, since at least one component of the antibody's natural environment will not be present. It is common, however, for isolated antibodies to be prepared by at least one purification step.
[00142] [00142] The word "label" when used here refers to a detectable compound or composition that is conjugated directly or indirectly with the antibody in order to generate a "labeled" antibody. The tag can be detectable by itself (eg radioisotope tags or fluorescent tags) or, in the case of an enzyme tag, it can catalyze a chemical change of a compound or substrate composition that is detectable.
[00143] [00143] By "solid phase" is meant a non-aqueous matrix to which the antibody of the present invention can adhere. Examples of solid phases covered here include those formed partially or entirely of glass (e.g., glass with controlled pore), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the cavity of a test plate; in other words, the purification column (eg, an affinity chromatographic column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.
[00144] [00144] A "liposome" is a small vesicle made up of various types of lipids, phospholipids and / or surfactants, which is employable for the supply of a medication (such as a chimeric polymer FGF19 or its antibody) to a mammal. The components of the liposome are normally arranged in a two-layer formation, similar to the lipid arrangement of biological membranes.
[00145] [00145] Reference to "approximately" a value or parameter here includes (and describes) variations that are close to that value or parameter per se. For example, the description for "approximately X" includes the description of "X" '
[00146] [00146] As used here and in the appended claims, the singular forms "a," "or," and "os / as" include references in the plural, unless the context clearly explains otherwise. It is understood that aspects and variations of the invention described here include aspects and variations of "consisting" and / or "consisting essentially of". V. Chimeric variants FGF19
[00147] [00147] In addition to the chimeric FGF19 polypeptides described here, it turns out that chimeric FGF19 variant polypeptides (or "chimeric FGF19 variants") can be prepared. Chimeric FGF19 variants can be prepared by introducing appropriate nucleotide changes in a DNA encoding chimeric or native FGF19 or FGF21 polypeptides, and / or by synthesis of the desired chimeric FGF19 variant. Those skilled in the art will find that changes in amino acids can alter post-translational processes of the chimeric FG19 variant, such as a change in the number or position of glycosylation sites or a change in membrane anchoring characteristics.
[00148] [00148] Variations in the chimeric FGF19 polypeptides of the present invention or in its various domains can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for example, in U.S. Pat. No. 5,364,934. Variations can be a substitution, deletion or insertion of one or more codons encoding the chimeric FGF19 polypeptide which results in a change in the amino acid sequence of the chimeric FGF19 polypeptides. The variations, with respect to one or more codons, can encode the chimeric polypeptide FGF19 which is derived from the native polypeptide FGF19 or the polypeptide FGF21. Optionally, the variation is by substituting at least one amino acid with any other amino acid in one or more of the domains of the chimeric polypeptides FGF19. Guidance for determining which amino acid residue can be inserted, replaced or deleted without adversely affecting the desired activity can be found by comparing the sequence of the FGF19 chimeric polypeptides with that of the known homologues of the protein molecules and minimizing the number of changes in the sequence of amino acids made in regions of high homology. Amino acid substitutions can be the result of replacing an amino acid containing similar structural and / or chemical properties, such as replacing a leucine with a serine, that is, amino acid substitutions - conservative. Insertions or deletions can optionally be in the range of about 1 to 5 amino acids. The allowed variation can be determined by insertions, deletions, or substitutions of amino acids, made systematically, in the sequence and testing the resulting variants for the activity displayed by the total length or mature native sequence.
[00149] [00149] Fragments of chimeric FGF19 polypeptides ("or chimeric FGF19 fragments") are provided here. Such fragments can be truncated at the N-termination or at the C-termination, or they may lack internal residues, for example, when compared to the total length of a native protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity of the chimeric FGF19 polypeptides.
[00150] [00150] Chimeric FGF19 fragments can be prepared by any of a number of conventional techniques. Desired peptide fragments can be synthesized chemically. An alternative approach involves the generation of chimeric FGF19 fragments by enzymatic digestion, for example, by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting DNA with appropriate restriction enzymes and isolating the desired fragment. Yet another appropriate technique involves the isolation and amplification of a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired terms of the DNA fragment are used in the 5 'and 3 primers in the PCR. Preferably, chimeric FGF19 polypeptide fragments share at least one biological and / or immunological activity with a native FGF19 polypeptide, such as the hFGF19 polypeptides shown in FIG. 1 (SEQ ID NO: 1) or in the native FGF21 polypeptide, such as hFGF21 shown in FIG. 2 (SEQ ID NO: 2).
[00151] [00151] In particular modes of execution, conservative substitutions of interest are shown in table 10 under the heading preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, called substitutions of the examples in table 10, or as described below with reference to classes of amino acids, are introduced and the products are analyzed.
[00152] [00152] Substantial modifications in the immune function or immunological identity of the chimeric polypeptide FGF19 are achieved by selecting substitutions that differ significantly in their effect in maintaining (a) the structure of the polypeptide backbone in the substitution area, for example, as a sheet conformation or helical, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulge of the side chain. Naturally occurring residues are divided into groups based on the common properties of the side chains: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, Ilys, arg; (5) residues that influence the chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.
[00153] [00153] - Non-conservative substitutions will imply the exchange of a member of one of these classes for another class. Such substituted residues can also be introduced in conservative substitution sites or, more preferably, in the remaining (non-conserved) sites.
[00154] [00154] Variations can be made using methods known in the art, such as oligonucleotide-mediated (directed site) alanine-mediated mutagenesis and PCR mutagenesis. Mutagenesis (site-directed) [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 techniques can be performed on the cloned DNA to produce the DNA encoding the chimeric FGF 19 variant polypeptides.
[00155] [00155] Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the scanning amino acids, relatively small, neutral amino acids are preferred. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side chain in addition to beta-carbon and is less likely to alter the main chain conformation of the variant [Cunningham and Wells, Science, 244: 1081-1085 (1989) ]. Alanine is also typically preferred because it is the most common amino acid. In addition, it is often found in both buried and exposed positions [Creighton, "The Proteins", (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150: 1 (1976)]. If the substitution with alanine does not produce adequate amounts of variant, an isoteric amino acid can be used.
[00156] [00156] Covalent modifications of FGF19 chimeric polypeptides are included within the scope of this invention. One type of covalent modification includes the reaction of target amino acid residues of a chimeric FGF19 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N-terminal or C-terminal residues of the chimeric FGF19 polypeptides. Derivatization with bifunctional agents is useful, for example, for chimeric FGF19 polypeptides by cross-linking them with a water-insoluble support matrix or surface for use in the antibody purification process, and vice versa. Crosslinking agents commonly used include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3 , 3 "- dithiobis (succinimidylpropionate), bifunctional maleimides, such as bis-N-maleimido-1,8-octane / and agents such as methyl-3 - [(p-azidophenyl) dithio] propioimidate.
[00157] [00157] Other modifications include deamidation of glutaminyl and asparaginyl residues in the corresponding glutamyl and aspartyl residues, respectively, proline and lysine hydroxylation, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine , and histidine side chains [T. E. Creighton, Proteins: Molecular Structure and Properties, W.H. Freeman & Co., San Francisco, p. 79-86 (1983)], acetylation of the N-terminal amine and, amidation of any C-terminal carboxyl group.
[00158] [00158] Another type of covalent modification of the chimeric polypeptide FGF19 included within the scope of this invention comprises altering the glycosylation pattern of the polypeptide. By "altering the native glycosylation pattern" for purposes of use here the meaning of deleting one or more portions of carbohydrates found in the corresponding native FGF19 polypeptides and / or in the sequence of FGF21 polypeptides (either by removal of the underlying glycosylation site) or by deleting the glycosylation by chemical and / or enzymatic means), and / or adding one or more glycosylation sites that are not present in the native FGF19 polypeptide and / or in the FGF21 polypeptide sequence. In addition, the phrase includes qualitative changes in the glycosylation of native proteins, involving a change in nature and proportions of the various carbohydrate parcels present.
[00159] [00159] The addition of glycosylation sites to the chimeric polypeptide FGF19 can be achieved by changing the amino acid sequence. The change can be made, for example, by adding, or replacing with, one or more serine or threonine residues to the chimeric polypeptide FGF19 (for O-linked glycosylation sites). The amino acid sequence of the chimeric FGF19 polypeptides can optionally be altered by changes in the level of DNA, particularly by mutating the DNA encoding the chimeric polypeptide FGF19 on pre-selected bases, so that codons are generated that will be translated into the desired amino acids.
[00160] [00160] Another means of increasing the number of carbohydrate parcels in the chimeric polypeptide FGF19 is by chemically or enzymatically linking glycosides to the polypeptide. Such methods are described in the art, for example, in WO 87/05330 published on September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., P. 259-306 (1981).
[00161] [00161] The removal of carbohydrate portions present in the chimeric polypeptide FGF19 can be achieved chemically or enzymatically or by mutational replacement of codons that encode amino acid residues that serve as a target for glycosylation. Chemical deglycosylation techniques are known in the art and described, for example, by Hakimuddin, et al., Arch.
[00162] [00162] Another type of covalent modification of chimeric FGF19 polypeptides comprises linking the chimeric FGF19 polypeptide to one of a variety of non-proteinaceous polymers, for example polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylenes, in the manner indicated in U.S. Pat. No. 4,640,835;
[00163] [00163] The chimeric FGF1I9 polypeptide of the present invention can also be modified by fusing the chimeric FGF19 polypeptide fused to another heterologous polypeptide or amino acid sequence.
[00164] [00164] In one embodiment, such a chimeric molecule comprises a fusion of the chimeric polypeptide FGF19 with a polypeptide tag which provides an epitope to which an anti-tag antibody can selectively bind. The tag epitope is generally placed at the amino or carboxyl terminus of the chimeric FGF19 polypeptides. The presence of such forms of tag epitopes of the chimeric FGF19 polypeptides can be detected using an antibody against the tag polypeptide. Also, the provision of tag epitopes allows the chimeric FGF19 polypeptide to be readily purified through affinity purification using an anti-tag antibody or other type of affinity matrix that binds to the tag epitope. 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; polypeptide tags flu
[00165] [00165] In an alternative embodiment, a polypeptide of the present invention may comprise a fusion of a chimeric FGF19 polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be the Fc portion of an immunoglobulin, an analog of the Fc portion of an immunoglobulin and one or more fragments of the Fc portion of an immunoglobulin. In some embodiments, immunoglobulin is selected from the group consisting of: IgG-1, IgG-2, I19G-3, 19G-4, IgA-1, I9gA-2, IgE, 1gD and IgM. In some modes of execution, the Fc portion is human or humanized.
[00166] [00166] In some embodiments, the C-terminus of the chimeric polypeptide FGF19 and the N-terminus of the Fc portion are fused. In some embodiments, the N-terminus of the chimeric polypeptide FGF19 and the C-terminus of the Fc portion are fused. In some embodiments, immunoglobulin fusion includes the hinge, CH2 and CH3 regions, or the CH1, CH2 and CH3 hinge regions of an IgG1 molecule. For the production of immunoglobulin fusions, see also U.S. Pat. No. 5,428,130 issued June 27, 1995. In some embodiments, the C-terminus of the chimeric polypeptide FGF19 is fused to the N-terminus of the Fc portion via a linker, and the linker is selected from the group consisting of: a linker [Gly] n, a linker [GIly3Ser] m and a linker [Gly4Ser] m, where n is an integer 1-30 and m is an integer 1-6.
[00167] [00167] The chimeric polypeptides FGF19 and their modulators described here can also be used as therapeutic agents. The chimeric FGF19 polypeptides and their modulators of the present invention can be formulated according to known methods for the preparation of pharmaceutically useful compositions, whereby the chimeric FGF19 polypeptide in question is combined in admixture with a pharmaceutically acceptable carrier. Therapeutic formulations are prepared for storage by mixing the active ingredient with the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to containers at the dosages and concentrations employed and include buffers, such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone, amino acids, such as glycines, glutamines, asparagines, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA;
[00168] [00168] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, before or after lyophilization and reconstitution.
[00169] [00169] The therapeutic compositions described here are generally placed in a container containing a sterile access port, for example, a bag or vial for intravenous solution containing a stopper punctured by a hypodermic injection needle.
[00170] [00170] The route of administration is in accordance with known methods, for example injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or intralesional, topical administration, or by sustainable delivery systems.
[00171] [00171] The desired dosage and drug concentrations of the pharmaceutical compositions of the present invention may vary depending on the particular intended use. Determining the appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal experiments have provided a reliable guide for determining effective doses for human therapy. Scaling between species of effective doses can be performed following the principles provided by Mordenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press , New York 1989, p. 42-96.
[00172] [00172] When an in vivo administration of a chimeric FGF19 polypeptide or an agonist or antagonist thereof is used, normal dosage amounts can vary from approximately ng / kg to 100 mg / kg of the mammal's body weight or more per day. preferably approximately 1 µg / kg / day to 10 mg / kg / day, depending on the route of administration. A guide to specific dosages and methods of administration is provided in the literature; see, for example, U.S. Pat. No. 4,657,760; 5,206,344; or 5,225,212. It is anticipated that different formulations will be effective for different treatment compounds and different disorders, and that targeting an organ or tissue, for example, may require administration differently than to another organ or tissue.
[00173] [00173] Microencapsulation is contemplated where administration with sustained release of a chimeric FGF19 polypeptide or modulator is desired in a formulation with appropriate detachment characteristics for the treatment of any disease or disorder that requires administration of the chimeric FGF19 polypeptide or modulator. Microencapsulation of recombinant proteins for sustained release was 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 Polylactide Polyglycolide Microsphere Systems," in Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman, eds, (Plenum Press: New York, 1995), p. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat. No. 5,654,010.
[00174] [00174] Formulations with sustained release of these proteins were developed using poly-lactic-coglycolic acid polymer (PLGA) due to their biocompatibility and wide range of biodegradable properties. PLGA degradation products,
[00175] [00175] Therapeutic agents and compositions comprising chimeric FGF19 polypeptides provided here can be used in a number of applications. Applications include treating an individual with obesity or a condition associated with obesity. In one aspect, chimeric FGF19 polypeptide is administered to an individual in need of him in an amount effective to treat the condition. Preferably, the condition is one that requires at least one of the following to be treated: reduction in blood glucose, an increase in metabolism, a reduction in body weight, a reduction in body fat, a reduction in triglycerides, a reduction in fatty acids free, an increase in the release of glucose from adipocytes and / or an increase in the release of leptin from adipocytes. Each of these parameters can be measured by standard methods, for example, by measuring oxygen consumption to determine the metabolic rate, using scales to determine weight, and measuring size to determine fat. In addition, the presence and amount of triglycerides, free fatty acids, glucose and leptin can be determined by standard methods. Claims include the treatment of an individual with one or more type 1 diabetes, type 2 diabetes, high blood glucose, metabolic syndrome, atherosclerosis, hypercholesterolemia, stroke, osteoporosis, osteoarthritis, degenerative joint disease, muscle atrophy, sarcopenia, reduced lean body mass , bald, wrinkles, increased fatigue, reduced stamina, decreased cardiac function, immune system dysfunction, cancer, Parkinson's disease, senile dementia, Alzheimer's disease and reduced cognitive function.
[00176] [00176] FGFI9 chimeric polypeptides and compositions comprising FGF19 chimeric polypeptides are preferably used in vivo. However, as discussed below, administration can be in vitro as in the methods described below for scanning FGF19 chimeric polypeptide modulators. Although it is understood that modulators of chimeric polypeptides FGF19 can also be identified by the use of animal models and samples of individuals.
[00177] [00177] The present invention also includes aspects in which a chimeric FGF19 polypeptide of the present invention or a pharmaceutical composition thereof is administered to an individual in combination with a second agent, wherein the second agent is preferably a pharmacological agent. In some embodiments, the chimeric FGF19 polypeptide of the present invention or a pharmaceutical composition thereof is administered in a therapeutically effective amount in combination with a therapeutically effective amount of the second agent. In some embodiments, the chimeric polypeptide FGF19 or its pharmaceutical composition is administered in conjunction with the second agent, eg, the respective administration periods are part of a single administrative regimen. In some embodiments, the chimeric polypeptide FGF19 or its pharmaceutical composition and the second agent are administered simultaneously, e.g., the respective administration periods overlap each other. In some embodiments, the chimeric polypeptide FGF19 or its pharmaceutical composition and the second agent are administered non-concurrently, eg, the respective administration periods do not overlap each other. In some embodiments, the chimeric polypeptide FGF19 or its pharmaceutical composition and the second agent are administered sequentially, e.g., The chimeric polypeptide FGF19 or its pharmaceutical composition is administered before and / or after administration of the second agent. In some embodiments, the chimeric polypeptide FGF19 or its pharmaceutical composition and the second agent are administered simultaneously as separate compositions. In some embodiments, the chimeric polypeptide FGF19 or its pharmaceutical composition and the second agent are administered simultaneously as part of the same composition.
[00178] [00178] In some embodiments, the second agent is a chimeric FGF19 polypeptide different from the present invention. In some embodiments, the second agent is an anti-inflammatory agent, an anti-diabetic agent, and / or a cholesterol-lowering drug of the "statin" class. In some embodiments, the second active agent is insulin. In some modes of execution, insulin is fast-acting, slow-acting, regular-acting, intermediate-acting, or long-acting insulin. In some embodiments, insulin is and / or comprises Humalog, Lispro, Novolog, Apidra, Humulin, Aspart, regular insulin, NPH, Lens, Ultralent, Lantus, Glargine, Levemir, or Detemir. In some embodiments, the second active agent is a statin. In some embodiments, the statin is and / or comprises Atorvastatin (eg, Lipitor or Torvast), Cerivastatin (eg, Lipobay or Baycol), Fluvastatin (eg, Lescol or Lescol), Lovastatin (eg, Mevacor, Altocor, or Altoprev) Mevastatin, Pitavastatin (eg, Livalo or Pitava), Pravastatin (eg, Pravachol, Selektine, or Lipostat) Rosuvastatin (eg, Crestor) , Simvastatin (eg, Zocor or Lipex), Vytorin, Advicor, Besylate Caduet or Simcor.
[00179] [00179] In another aspect of the present invention, a chimeric FGF19 polypeptide of the present invention or a pharmaceutical composition thereof is administered to an individual in combination with a second therapy performed on the individual, wherein the second therapy comprises surgery. In some embodiments, the chimeric polypeptide FGF19 of the present invention or a pharmaceutical composition thereof is administered in a therapeutically effective amount in combination with the second therapy. In some embodiments, the chimeric polypeptide FGF19 or pharmaceutical composition thereof is administered together with the second therapy, eg, administration and therapy are part of a single administrative regimen. In some embodiments, the chimeric polypeptide FGF19 or pharmaceutical composition thereof is administered simultaneously with the second therapy, e.g., the respective periods of administration and therapy overlap each other. In some embodiments, the chimeric polypeptide FGF19 or pharmaceutical composition thereof and the second agent is administered non-simultaneously with a second therapy, e.g., the respective periods of administration and therapy do not overlap each other. In some embodiments, the chimeric polypeptide FGF19 or pharmaceutical composition thereof and the second agent is administered sequentially with a second therapy, e.g., The chimeric polypeptide FGF19 or pharmaceutical composition thereof is administered before and / or after a second therapy. In some embodiments, the chimeric polypeptide FGF19 or pharmaceutical composition thereof and the second agent is administered simultaneously with the second therapy.
[00180] [00180] The chimeric FGF19 polypeptides described here can also be used as molecular weight markers for the purpose of protein electrophoresis.
[00181] [00181] The chimeric FGF19 polypeptides and nucleic acid molecules of the present invention can also be used for tissue types, in which the chimeric FGF19 polypeptides of the present invention can be expressed differently in one tissue compared to another. The chimeric polypeptides FGF19 of the nucleic acid molecules will find use to generate samples for PCR, Northern analysis, Southern analysis and Western analysis.
[00182] [00182] —Chimeric FGF19 polypeptides of the present invention that bind to another protein (example, one of the FGFRs), the chimeric FGF19 polypeptide can be used in assays to identify the other proteins or molecules involved in the binding interaction. By such methods, inhibitors of receptor / linker binding interaction can be identified. The proteins involved in such binding interactions can also be used to monitor peptides or small molecule inhibitors or agonists of binding interactions. Also, the chimeric polypeptide FGF19 can be used to isolate related ligand (s). Monitoring assays can be designed to find leading components that mimic the biological activity of a native FGF19, native FGF21, chimeric FGF19 polypeptides, or a FGF19 and / or FGF21 receptor. Such monitoring assays will include assays for monitoring high-yield chemical libraries, - making them - particularly - suitable for identifying small molecule drug candidates. Small molecules contained include synthetic organic or inorganic compounds. The assays can be performed in a number of formats, including protein-protein binding assays, biochemical monitoring assays, immunoassays and cell-based assays, which are well characterized in the art.
[00183] [00183] As an alternative approach to receptor identification, the chimeric FGF19 labeled polypeptide may be linked by photo-affinity to a cell membrane or extracts of preparations that express the receptor molecule. The cross-linked material is resolved by PAGE and exposed to an X-ray film. The labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from the micro-sequencing would be used to design a set of degenerate oligonucleoitide samples to monitor a cDNA library to identify the gene encoding the putative receptor.
[00184] [00184] In an embodiment here in which competitive binding assays are performed, FGFR1c, FGFR2c, FGFR3c and / or FGFR4 or an antibody to the chimeric polypeptide FGF19 is used as a competitor.
[00185] [00185] Antibodies to chimeric anti-FGF19 polypeptides may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be cultured in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and / or the adjuvant will be injected into the mammal by means of multiple subcutaneous or intraperitoneal injections. The immunizing agent can include the chimeric polypeptide FGF19 or a fusion protein therefrom. It may be useful to couple the immunizing agent to a protein known to be immunogenic in the mammal to be immunized. Examples of such immunogenic proteins include but are not limited to Californian keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and sodium trypsin inhibitor. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorinomycinate). The immunization protocol can be selected by a person skilled in the art without undoing the experiment.
[00186] [00186] Antibodies to chimeric anti-FGF19 polypeptides may alternatively be monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256: 495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to obtain lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. In preferred embodiments, the anti-chimeric polypeptide - FGF19 antibody specifically binds to the polypeptide of the present invention. In more preferred embodiments, the antibody that specifically binds does not bind to the native FGF19 polypeptide or the native FGF21 polypeptide.
[00187] [00187] The immunizing agent will typically include the chimeric polypeptide FGF19 or a fusion protein thereof. Generally, peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells if non-human mammalian sources are desired. Lymphocytes are then fused to an immortalized cell line using an appropriate fusion agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pages. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly rodent, bovine and human myeloma cells. Usually rat or mouse myeloma cell lines are employed. Hybridoma cells can be grown in an appropriate culture medium that preferably contains one or more substances that inhibit the growth or survival of the immortalized, unfused cells. For example, if parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas will typically include hypoxanthine, aminopterin and thymidine ("HAT medium"), whose substances prevent the growth of cells deficient in HGPRT .
[00188] [00188] Preferred immortalized cell lines are those that fuse effectively, support the high stable level of antibody expression by the selected antibody producing cells and are sensitive to a medium such as HAT medium. Most preferred immortalized cell lines are murine myeloma lines, which can be obtained for example from the Salk Institute Cell Distribution Center, San Diego, Calif. and in the American Type Culture Collection, Manassas, Va. Cell lines of human myeloma and mouse-human heteromyeloma have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) p. 51-63].
[00189] [00189] The culture medium in which the hybridoma cells are cultured can then be tested for the presence of monoclonal antibodies directed against chimeric FGF19 polypeptides. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody, for example, can be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107: 220 (1980).
[00190] [00190] After the desired hybridoma cells have been identified, the clones can be subcloned by limiting dilution procedures and cultured by standard procedures [Goding, supra]. A culture medium suitable for this purpose includes, for example, Dulbecco's Modified Eagles Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be cultured in vivo as ascites in a mammal.
[00191] [00191] Monoclonal antibodies secreted by the subclones can be isolated or purified from ascites culture medium or fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, chromatography with hydroxylapatite, gel electrophoresis, dialysis, or affinity chromatography.
[00192] [00192] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNAs encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (eg, using samples of oligonucleotides that are able to specifically bind to genes encoding murine heavy and light chains) ). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein. , to obtain the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by replacing the coding sequence for constant domains of heavy and light human chains in place of homologous murine sequences. [U.S. Pat. No. 4,816,567; Morrison et al., Supra] or covalently adding to the immunoglobulin coding sequence all or part of the polypeptide coding sequence - non-immunoglobulin. Such a non-immunoglobulin polypeptide can be replaced by a constant domain of an antibody of the invention, or it can be replaced by the variable domains of an antigen combining site with an antibody of the invention to create a bivalent chimeric antibody.
[00193] [00193] The antibodies can be monovalent antibodies. Methods of preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of the immunoglobulin light chain and modified heavy chain. The heavy chain is generally truncated at any point in the Fc region in order to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are replaced with another amino acid residue or are deleted to prevent cross-linking.
[00194] [00194] In vitro methods are also appropriate for preparing monovalent antibodies. Digestion of antibodies to produce their fragments, particularly Fab fragments, can be accomplished using routine techniques known in the art.
[00195] [00195] The antibodies to the chimeric anti-FGF19 polypeptides of the invention may still comprise humanized antibodies or human antibodies. Humanized forms of non-human antibodies (for example, murines) are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab,
[00196] [00196] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are commonly referred to as "imported" residues, which are typically taken from an "imported" variable domain. Humanization can be performed essentially following the method of Winter et al. [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988)], replacing sequences of rodent CDRs or CDRs with the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat No. 4,816,567) in which substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies.
[00197] [00197] Human antibodies can also be produced using various techniques known in the art, including phage representation libraries [Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol. 222: 581 (1991)]. Cole et al. and Boerner et al. are also available 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 can be produced by introducing human immunoglobulin loci into transgenic animals, for example, mice in which the endogenous immunoglobulin genes have been partially or totally deactivated. production of a human antibody that closely resembles that seen in humans in all aspects, including gene rearrangement, and antibody repertoire This approach is described, for example, in US Pat. Nos. 5,545,807; 5,545 .806; 5,569,825; 5,625,126; 5,633,425;
[00198] [00198] Bispecific antibodies are monoclonal antibodies, preferably human or humanized antibodies, which have specificities of binding to at least two different antigens. In the present case, one of the binding specificities is for FGF19, the other is for any other antigen, and preferably for a cell surface protein or receptor or receptor subunit.
[00199] [00199] Methods for preparing bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain / light chain pairs, in which the two heavy chains have different specificities [Milstein and Cuello, Nature, 305: 537-539 (1983)] . Due to the random variety of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published on May 13, 1993, and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).
[00200] [00200] Variable domains of antibodies with the desired binding specificities (combined antibody-antigen sites) can be fused to constant-domain immunoglobulin sequences. The preferred fusion is with a constant domain heavy chain immunoglobulin, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) with the site necessary for binding the light chains present to at least one of the fusions. The DNAs encoding the immunoglobulin heavy chain fusions, and if desired the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into an appropriate host organism. For more details on bispecific antibody generation, see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).
[00201] [00201] According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from a recombinant cell culture. The preferred interface comprises at least part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (eg tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain (s) are created at the interface of the second antibody molecule by replacing amino acid side chains with smaller ones (for example alanine or threonine) ). This provides a mechanism for increasing the production of heterodimers over other unwanted end products such as homodimers.
[00202] Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg bispecific antibodies F (ab ').). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical bonding. Brennan et al., Science
[00203] [00203] Fab 'fragments can be directly recovered from E. coli and chemically linked to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describes the production of a fully humanized bispecific F (ab ') antibody molecule. Each Fab 'fragment was secreted separately from E. coli and subjected to chemical ligation by chemical coupling directed in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells that overexpress the ErbB2 receptor and normal human T cells, as well as to trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
[00204] [00204] Various techniques for producing and isolating fragments of antibodies - bispecific - directly from the culture of recombinant cells have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelhy et al., J. Immunol. 148 (5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab 'portions of two different antibodies by gene fusion. The antibody homodimers were reduced in the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be used for the production of antibody homodimers. The diabody technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provided an alternative mechanism for producing bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thus forming two antigen binding sites. Another strategy for producing bispecific antibody fragments has also been reported using single-chain Fv (sFv) dimers. See, Gruber et al., J. Immunol. 152: 5368 (1994). Antibodies with more than two valences are considered. For example, specific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60 (1991).
[00205] [00205] - Bispecific antibodies of the examples can bind to two different epitopes on a given FGF19 polypeptide. Alternatively, an anti-polypeptide from the chimeric FGF19 polypeptide arm can be combined with an arm that binds to a triggering molecule in a leukocyte, such as a T cell receptor molecule (eg CD2, CD3, CD28, or B7) , or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FceyRII (CD32) and FcyRIIl (CD16) in order to focus on cellular defense mechanisms for the cell to express the particular FGF19 polypeptide. Bispecific antibodies can also be used to locate cytotoxic agents in cells that express a particular FGF19 polypeptide. These antibodies have an FGF1I9 binding arm and an arm that binds to a cytotoxic agent or chelating agent for radionuclides, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the FGF19 polypeptide and still binds to tissue factor (TF).
[00206] [00206] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies have, for example, been proposed to target cells of the immune system as unwanted cells [U.S. Pat. At the.
[00207] [00207] It may be desirable to modify the antibody of the invention with respect to the effector function, in order to increase, for example, the effectiveness of the antibody. For example, cysteine residue (s) can be introduced into the Fc region, thus allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent cell cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148:
[00208] [00208] The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent, such as a chemotherapeutic agent, toxin (eg, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (eg a radioconjugate).
[00209] [00209] Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments that can be employed include diphtheria A chain, active non-binding fragments of diphtheria toxin, A-chain exotoxin (from Pseudomonas aeruginosa), A-chain castor, A-chain abrin, A-chain modeccin, alpha-sarcina, aleurites fordii proteins, diantina proteins, American Phytolaca A chain (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcine, crotin, saponaria, officinalis inhibitor, gelonin, mitogelin, restrictocine, phenomycin, enomycin, and trichothecenes. A variety of radionuclides are available for the production of radioconjugate antibodies. Examples include 212Bi, 1311, 131In, 90Y, and 186Re.
[00210] [00210] Antibody and cytotoxic agent conjugates are prepared using a variety of bifunctional protein binding agents, such as N-succinimidyl-3- (2-pyridyldithiol) propionate
[00211] [00211] In another embodiment, the antibody can be conjugated to a "receptor" (such as streptavidin) for use in a pre-target tumor in which the antibody-receptor conjugate is administered to the individual, followed by removal of unbound conjugate circulation using a clearing agent and then administering a "linker" (eg, avidin) that is conjugated to a cytotoxic agent (eg, a radionucleotide).
[00212] [00212] The antibodies disclosed here can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. We.
[00213] [00213] Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamines (PEG-PE). Liposomes are extruded through defined pore size filters to produce liposomes with the desired diameter. Fab 'fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide exchange reaction.
[00214] [00214] Antibodies that specifically bind to a FGF19 polypeptide identified here, as well as other molecules identified by the monitoring assays disclosed earlier, can be administered for the treatment of various disorders in the form of pharmaceutical compositions.
[00215] [00215] If the FGF19 polypeptide is intracellular and whole antibodies are used as inhibitors, internalized antibodies are preferred. However, lipofections or liposomes can also be used to deliver the antibody, or an antibody fragment, to cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the target protein binding domain is preferred. For example, based on the variable region sequences of an antibody, peptide molecules can be designed that retain the ability to bind to the target protein sequence. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993). The formulation here can also contain more than one active compound as needed for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibiting agent. Such molecules are suitably present in combination, in amounts that are effective for the intended purpose.
[00216] [00216] The active ingredients can also be trapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example hydroxymethylcellulose or gelatin microcapsules and poly- (methyl methacrylate) microcapsules, respectively, in delivery systems colloidal drugs (eg, liposomes, albumin microspheres, microemulsions, - nano-particles, = and nanocapsules)) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.
[00217] [00217] The formulations to be used for in vivo administration must be sterile. These are readily obtained by filtration through sterile filtration membranes.
[00218] [00218] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, whose matrices are in the form of molded articles, for example, films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl methacrylate), or poly (vinyl) alcohol, polylactides (US Pat. No. 3,773,919), L-glutamic acid copolymers and Y-ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOTY (injectable microspheres composed of copolymer of lactic acid-glycolic acid and leuprolide acetate), and poly acid -D - (-) - 3-hydroxybutyric. While polymers, such as ethylene-vinyl acetate and lactic acid-glycolic acid allow molecules to detach for more than 100 days, certain hydrogels detach proteins for shorter periods of time. When encapsulated antibodies remain in the body for a long time, they can denature or aggregate as a result of exposure to moisture at 37º C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be designed for stabilization depending on the mechanism involved. For example, if it is discovered that the aggregation mechanism is intermolecular, the formation of the S - S bond exchanges through the thio-disulfide, stabilization can be obtained by modifying the sulfohydryl residues, lyophilizing from acidic solutions, controlling the moisture content, using appropriate additives and developing specific polymer matrix compositions. G. Uses of Antibodies
[00219] [00219] The antibodies to the chimeric anti-FGF19 polypeptide of the invention have several uses. For example, anti-FGF19 antibodies can be used in diagnostic assays for chimeric FGF19 polypeptides, eg, by detecting their expression in specific cells, tissues or serum. Various diagnostic testing techniques known in the art can be employed, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted either in heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987) pgs. 147-158]. The antibodies used in the diagnostic assays can be marked with a detectable portion. The detectable portion should be able to produce, both directly and indirectly, a detectable signal. For example, the detectable portion may be a radioisotope, such as 3H, 14C, 32P, 358, or 1251], a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase , beta-galactosidase or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety can be employed, including those methods described by Hunter et al., Nature, 144: 945 (1962); David et al., Biochemistry, 13: 1014 (1974); Pain et al., J. Imnmunol. Meth., 40: 219 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407 (1982).
[00220] [00220] - Chimeric FGF19 anti-polypeptide antibodies are also useful for the affinity purification of the chimeric FGF19 polypeptide from recombinant cell culture or natural sources. In this process, antibodies against chimeric FGF19 polypeptides are mobilized on an appropriate support, such as a Sephadex resin or paper filter, using methods well known in the art. The immobilized antibody is then contacted with a sample containing the chimeric polypeptide FGF1I9 to be purified, and then the support is washed with an appropriate solvent that will remove substantially all of the material in the sample except the chimeric FGF19 polypeptides, which are bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will detach the chimeric polypeptide FGF19 from the antibody.
[00221] [00221] The description below reports primarily on the production of a chimeric FGF1I9 polypeptide by means of culture cells transformed or transfected with a vector containing nucleic acid encoding chimeric FGF19 polypeptides. It is considered, of course, that alternative methods that are well known in the art can be employed to prepare chimeric FGF19 polypeptides. For example, chimeric FGF19 polypeptides, or portions thereof, can be produced by direct peptide synthesis using solid phase techniques [see, eg, Stewart et al., Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, Calif. (1969); Merrífield, J. Am. Chem. Soc., 85: 2149-2154 (1963)]. In vitro protein synthesis can be performed using manual techniques or by automation; Automated synthesis can be achieved, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) With instructions from the manufacturer. Various portions of the chimeric FGF19 polypeptides can be chemically synthesized separately and combined using chemical or enzymatic methods to produce full-length chimeric FGF19 polypeptides.
[00222] [00222] "CDNA fragments encoding the chimeric FGF polypeptides of the present invention can be generated using PCR methodology using cDNA encoding at least a portion of the native FGF19 polypeptides and at least a portion of native FGF21 polypeptides as a template. For example, in one example, a cDNA fragment encoding an N terminal portion of the FGF21 polypeptide and a cDNA fragment encoding a C terminal portion of the FGF19 polypeptide are separately amplified and purified by a standard procedure, such as using PCR followed by agarose gel electrophoresis. Primary sequences are designed such that there is an overlap of 18 nucleotides at the 3 'end of the FGF21 cDNA fragment and at the 5' end of the FGF19 cDNA fragment. A second amplification using PCR is conducted using a mixture of the two cDNA fragments as a template, resulting in a cDNA encoding the chimeric polypeptide of the FGF21 fragment and the FGF19 fragment. The resulting cDNA fragment is digested with an appropriate restriction of enzymes, purified by agarose gel electrophoresis, and cloned into a plasmid vector pRK5.sm (a plasmid vector based on pUC containing a CMV promoter for mammalian expression) using standard procedures. The sequence of the resulting plasmids was confirmed by Sanger's DNA sequencing method.
[00223] [00223] The DNA encoding the chimeric FGF19 polypeptide can be obtained from a cDNA library prepared from tissue believed to have the FGF19 and / or FGF21 nRNA and express it at a detectable level. Accordingly, human DNA FGF19 and / or FGF21 can be conveniently obtained from a cDNA library prepared from human tissue, as described in the examples. The gene encoding FGF19 and / or FGF21 can also be obtained from a genomic library or by known synthetic procedures (eg, automated nucleic acid synthesis).
[00224] [00224] Libraries can be monitored with samples (such as antibodies to FGF19 and / or FGF21 or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Monitoring of the CDNA or genomic library with the selected sample can be conducted using standard procedures, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means of isolating the gene encoding FGF19 is to use the PCR methodology [Sambrook et al., Supra; Dieffenbach et al., POR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
[00225] [00225] The examples below describe techniques for monitoring a cDNA library. The oligonucleotide sequences - selected as samples should be long enough and unambiguous enough for false positives to be minimized. The oligonucleotide is preferably marked (labeled) such that it can be detected with DNA hybridization in the monitored library. Labeling methods are well known in the art, and include the use of radiolabelers such as labeled ATP ºP, biotinylation or labeling enzyme. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., Supra.
[00226] [00226] The sequences identified in such library scanning methods can be compared and aligned to other deposited sequences and available in public databases, such as GenBank or other private database sequences. Sequence identity (either at the amino acid or nucleotide level) within defined regions of the molecule or through the full length sequence can be determined using methods known in the art and as described here.
[00227] [00227] Nucleic acid containing protein coding sequence can be obtained by scanning cDNA or genomic libraries using the deduced amino acid sequence discussed here for the first time, and, if necessary, using conventional primary extension procedures as described in Sambrook et al ., supra, to detect precursors and process mMRNA intermediates that may not have been reverse transcribed into cDNA.
[00228] [00228] Host cells are transfected or transformed by the expression or cloning vectors described here for the production of chimeric polypeptides FGF19 and cultured in conventional modified nutrient medium if appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences . Culture conditions, such as medium, temperature, pH and the like, can be selected by the person skilled in the art without undue experimentation. In general, principles, protocols and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., Supra.
[00229] [00229] Methods of transfecting eukaryotic cells and transforming prokaryotic cells are known to the common specialist, for example, CaCl2, CaPO4, mediated by liposome and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. Calcium treatment using calcium chloride, as described in Sambrook et al., Supra, or electroporation is generally used for prokaryotes. Infection with agrobacterium tumefaciens is used for the transformation of certain plant cells, as described by Shaw et al., Gene, 23: 315 (1983) and WO 89/05859 published on June 29, 1989. For mammalian cells without such cell walls, the Graham and van der Eb calcium phosphate precipitation method, Virology, 52: 456-457 (1978) can be employed. General aspects of transfections of the mammalian cell host system have been described in U.S. Pat. No. 4,399,216. Yeast transformations are typically performed according to the method of 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 can also be used, such as by nuclear microinjection, electroporation, fusion of bacterial protoplasts with intact cells, or polycations, for example, polybrene, polyiornin. For various techniques for transforming mammalian cells, see Keown et al, Methods in Enzymology, 185: 527-537 (1990) and Mansour et al., Nature,
[00230] [00230] Host cells suitable for cloning or expressing DNA in the vectors described herein include prokaryotic cells, yeasts, or larger eukaryotic cells. Suitable prokaryotes include but are not limited to eubacteria, such as organisms - gram-negative or gram-positive, for example, enterobacteria such as E. coli. Various strains of E. coli are publicly available, such as the E. coli K12 strain MM294 (ATCC
[00231] [00231] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeasts are suitable hosts for cloning or expression for vectors encoding FGF19. Saccharomyces cerevisiae is a eukaryotic host microorganism - low “normally - used. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140; EP 139,383 published on May 2, 1985); Kluyveromyces hosts (US Pat. No. 4,943,529; Fleer et al., Bio / Technology, 9: 968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154 (2): 737-742), K. fragilis (ATCC 12.424), K. bulgaricus (ATCC 16.045), K. wickeramii (ATCC
[00232] [00232] Host cells suitable for the expression of glycosylated chimeric FGF19 polypeptides 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 cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (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)); sertoli mouse 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 breast tumor (MMT 060562, ATCC CCL51). Selection of appropriate host cells is considered to be within the skill of the art.
[00233] [00233] The nucleic acid (eg, cDNA or genomic DNA) encoding chimeric FGF19 polypeptides can be inserted into a replicable vector for cloning (amplification of DNA) or for expression. Several vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle or phage. The appropriate nucleic acid sequence can be inserted into the vector by a variety of procedures. In general, the DNA is inserted into an appropriate endonuclease restriction site (s) using techniques known in the art. The components of the vector generally include, but are not limited to, one or more signal sequences, an origin of replication, one or more gene markers, an enhancer element, a promoter and a transcription termination sequence. The construction of appropriate vectors containing one or more of these components employs standard ligation techniques that are known to those skilled in the art.
[00234] [00234] The chimeric polypeptide FGF19 can be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which can be a signal sequence or other polypeptide containing a specific cleavage site at the N-termination of the mature protein or polypeptide. In general, the signal sequence can be a component of the vector, or it can be a part of the DNA encoding the FGF19 chimeric polypeptides that is inserted into the vector. The signal sequence can be the original FGF19 or FGF21 signal sequence, such as hFGF19 or hFGF21. Therefore, in such embodiments, a chimeric FGF19 polypeptide of the present invention can include at least an N-terminal portion of pre-hFGF19, such as at least residues 1-22 of SEQ ID NO: 3. In such embodiments, a chimeric FGF19 polypeptide of the present invention can include at least an N-terminal portion of pre-hFGF1, such as at least residues 1-28 of SEQ ID NO: 4.
[00235] [00235] The signal sequence can be a prokaryotic signal sequence selected, for example, from the group of alkaline phosphatases, penicillinase,! Pp, or heat-stable enterotoxin | l leaders. For yeast secretion, the signal sequence may be, for example, the yeast invertase leader, alpha factor leader (including saccharomyces and Kluyveromyces factor a. Leaders, the latter described in US Pat. No. 5,010,182 ), or acid phosphatase leader, the glucoamylase leader of C. albicans (EP 362,179 published on April 4, 1990), or the signal described in WO 90/13646 published on November 15, 1990. In mammalian cell expression, sequences of mammalian signal can be used for direct secretion of the protein, such as signal sequences of the secreted polypeptides of the same species or related species, as well as viral secretory leaders.
[00236] [00236] Both expression and cloning vectors contain a nucleic acid sequence that allows the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeasts, and viruses. The origin of replication of plasmid pBR322 is appropriate for most gram-negative bacteria, the origin of plasmid 21 is suitable for yeast, and several viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
[00237] [00237] Expression and cloning vectors will typically contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, for example, ampicillin, neomycin, methotrexates, or tetracyclines, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for bacilli.
[00238] [00238] An example of selectable markers suitable for mammalian cells are those that allow the identification of cells that are competent to absorb the FGF19-encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell, when wild-type DHFR is employed, is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77: 4216 (1980). An appropriate selection gene for use in yeast is the trp1 gene present in the YRp7 yeast plasmid [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 a mutant strain of yeast unable to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
[00239] [00239] Expression and cloning vectors usually contain a promoter operably linked to the FGF19-encoding nucleic acid sequence to direct mMRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with hosts —procariotics include the B-lactamase and lactose promoter systems [Chang et al., Nature, 275: 615 (1978); Goedde! L et al., Nature, 281: 544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8: 4057 (1980); EP
[00240] [00240] “Examples of promoter sequences suitable for use with yeast hosts include 3-phosphoglycerate kinase promoters [Hitzeman et al., J. Biol. Chem., 255: 2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme
[00241] [00241] Other promoters of yeast, which are inducible promoters containing the additional advantage of transcription controlled by growing conditions, are the promoter regions of alcohol dehydrogenase 2, isocitocrome C, acid phosphatase, degradable enzymes associated with the metabolism of nitrogen, metallothionein, glyceraldehyde -3-phosphate-dehydrogenase, and enzymes responsible for the use of maltose and galactose. Vectors and promoters suitable for use in yeast expression are further described in EP 73,657.
[00242] [00242] Vector transcription in mammalian host cells is controlled, for example, by promoters obtained from virus genomes, such as the polyoma virus, fowlpox virus (UK 2,211,504 published on July 5, 1989), adenovirus ( such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and simian virus 40 (SV40), from heterologous mammalian promoters, eg the actin promoter or an immunoglobulin promoter, and heat shock promoters, provided such promoters are compatible with host cell systems.
[00243] [00243] The transcription of a DNA encoding the chimeric polypeptide FGF19 by higher eukaryotes can be increased by inserting an enhancer sequence in the vector. Enhancers are elements of DNA with cis action, usually about 10 to 300 bp, that act on a promoter to increase its transcription. Many promoter sequences of mammalian genes are now known (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, a eukaryotic cell virus promoter will be used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the late side polyoma enhancer on the replication origin and adenovirus enhancers . The enhancer can be divided into the vector at the 5 'or 3' position in the coding sequence for the chimeric polypeptide FGF19, but it is preferably located at a 5 'site of the promoter.
[00244] [00244] Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilization of MRNA. Such sequences are usually available from the 5 'and, occasionally, 3' regions, untranslated from eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mMRNA encoding the FGF19 chimeric polypeptides.
[00245] [00245] Still other methods, vectors and host cells appropriate for adaptation to the synthesis of chimeric polypeptides FGF19 in recombinant cell culture of vertebrates are described in Gething et al., Nature, 293: 620-625 (1981); Mantei et al., Nature, 281: 40-46 (1979); EP 117,060; and EP 117,058.
[00246] [00246] The amplification and / or expression of genes can be measured in a sample directly, by conventional Southern blotting, Northern blotting to quantify mMRNA transcription [Thomas, Proc. Natl. Acad. Sci. USA, 77: 5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled sample, based on the sequences provided here. Alternatively, antibodies can be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and hybrid DNA-RNA duplexes or DNA-protein duplexes. The antibodies in turn can be labeled and the assay can be performed in which the duplex is attached to a surface, so that with the formation of duplex on the surface, the presence of the antibody attached to the duplex can be detected.
[00247] [00247] The expression of the genes, alternatively, can be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to directly quantify the expression of the gene product. Antibodies useful for immunohistochemical staining and / or testing sample fluids can be either monoclonal or polyclonal, and can be prepared in any mammal. Conveniently, antibodies can be prepared against a chimeric FGF19 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to DNA encoding the chimeric polypeptide FGF19 and encoding a specific antibody epitope.
[00248] [00248] Forms of chimeric FGF19 polypeptides can be recovered from the culture medium or from host cell lysates. If attached to the membrane, they can be detached from the membrane using an appropriate detergent solution (eg Triton-X 100) or by enzymatic cleavage. Cells employed in the expression of a chimeric FGF19 polypeptide can be disrupted by various physical or chemical means, such as freeze / thaw cycle, sonication, mechanical interruption, or cell lysis agents.
[00249] [00249] It may be desired to purify the chimeric FGF1I9 polypeptides from recombinant cell proteins or polypeptides. The following procedures are examples of appropriate purification procedures: by fractionation in an ion exchange column; ethanol precipitation; Reverse phase HPLC; chromatography on silica or a cation exchange resin such as DEAE; chromatofocalization; SDS-PAGE; precipitation with ammonium sulfate; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as I | gG; and metal chelate columns to link epitope-tagged forms of the chimeric FGF19 polypeptides. Various methods of protein purification can be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer publisher, New York (1982). The purification step (s) selected will depend, for example, on the nature of the production process used and the particular chimeric FGF19 polypeptide produced.
[00250] [00250] The present invention includes in another aspect nucleotide sequences (or their complement) that encode the FGF19 chimeric polypeptides (or "FGF19 chimeric nucleic acids") of the present invention. Chimeric nucleic acids FGF19 of the present invention have several applications in the technique of molecular biology, including uses as hybridization samples, in the mapping of chromosomes and genes and in the generation of antisense RNA and DNA. Chimeric FGF19 nucleic acids will also be useful for the preparation of chimeric FGF19 polypeptides by the recombinant techniques described here.
[00251] [00251] In some embodiments, chimeric FGF19 polypeptides may include one or more epitopes tag (epitope tag). In some embodiments, a tag epitope is positioned at the N-terminus of the chimeric FGF19 polypeptides. In some embodiments, a label epitope is positioned at the C terminus of the chimeric FGF19 polypeptides. In some embodiments, a tag epitope is positioned at the N-terminus of the chimeric FGF19 polypeptides.
[00252] [00252] In some embodiments, chimeric FGF19 polypeptides can include one or more tag epitopes. In some embodiments, a tag epitope is positioned at the N-terminus of the chimeric FGF19 polypeptides. In some embodiments, a label epitope is positioned at the C terminus of the chimeric FGF19 polypeptides. In some embodiments, a tag epitope is positioned at the N-terminus of the chimeric FGF19 polypeptides. In some embodiments, the label epitope comprises the amino acid sequence DYKDDDDK (SEQ ID NO: 279).
[00253] [00253] In an exemplary embodiment, a chimeric FGF 19 nucleic acid of the present invention includes the sequence:
[00254] [00254] In another exemplary embodiment, a chimeric FGF 19 nucleic acid of the present invention includes the sequence: ATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGgC TGTGGGTTTCTGTGCTGGCTGGTCTTCTGCTGGGAGCCTGCCAG
[00255] [00255] In another exemplary embodiment, a chimeric FGF19 nucleic acid of the present invention includes the sequence:
[00256] [00256] In another exemplary embodiment, a chimeric FGF19 nucleic acid of the present invention includes the sequence: ATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGGC TGTGGGTTTCTGTGCTGGCTGGTCTTCTGCTGGGAGCCTGCCAG GCACACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCEGGGGGC CAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGAC AGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGCGGC GCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCCTT GAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTT CCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACT TTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGAC GGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCAC CTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGG ACCAGCTCGCTTCCTTCCACTCTCTCATTTCOTGCCCATGCOTGCC CATGGTCCCAGAGGAGCCTGAGGACCTCAGGGGCCACTTGGAAT CTGACATGTTCTCTTCGCCCCTGGAGACCGACAGCATGGACCCAT
[00257] [00257] The full-length native nucleic acid sequence of the hFGF19 gene (SEQ ID NO: 5), the full-length native nucleic acid sequence of the FGF21 gene (SEQ ID NO: 6), the native nucleic acid sequence of Total length of a chimeric FGF19 polypeptide of the present invention, or any of its previous portions, can be used as a hybridization sample for detecting or scanning FGFI19 polypeptide - chimeric polypeptides of the present invention. Optionally, the length of the samples will be about 20 to approximately 50 bases. As an example, a scanning method will comprise isolating the FGF19 gene coding region using the known DNA sequence to synthesize a selected sample of about 40 bases. Hybridization samples can be labeled by a variety of labels, including radionucleotides such as 32P or 358, or enzymatic labels such as alkaline phosphatase bound to the sample via avidin / biotin binding systems. Labeled samples containing a sequence complementary to that of the FGF19 gene of the present invention can be used for scanning human cDNA, DNA or genomic mMRNA libraries to determine which members of such libraries are hybridized to the sample. Hybridization techniques are described in more detail in the examples below.
[00258] [00258] “Any EST sequences disclosed in the present patent application can be used similarly as samples, using the methods disclosed here.
[00259] [00259] Other useful fragments of chimeric FGF19 nucleic acids include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to chimeric FGF19 mRNA (sense) or (antisense) chimeric target sequences FGF19 DNA. Antisense or sense oligonucleotides according to the present invention comprise a fragment of the FGF19 chimeric DNA coding region. Such a fragment generally comprises at least about 14 nucleotides, preferably approximately 14 to 30 nucleotides. The ability to derive an antisense or sense oligonucleotide, based on a cDNA sequence 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).
[00260] [00260] Binding of antisense or sense oligoncleotides to target nucleic acid sequences results in the formation of duplexes that block the transcription or translation of the target sequence by one of several means, including increased degradation of the duplexes, premature transcription termination or translation , or by other means. The antisense oligonucleotides therefore can be used to block the expression of the chimeric FGF19 polypeptides. Antisense or sense oligonucleotides further comprise oligonucleotides containing modified sugar-phosphodiester backbones (or other sugar bonds, such as those described in WO 91/06629) and in which such sugar bonds are resistant to endogenous nucleases. Such sugar-resistant oligonucleotides are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to the target nucleotide sequences.
[00261] [00261] Other examples of sense or antisense oligonucleotides - include those —oligonucleotides that are covalently linked to organic parcels, such as those described in WO 90/10048, and other parcels that increase the affinity of oligonucleotides to a target nucleic acid sequence , such as poly- (L-lysine). In addition, intercalating agents, such as ellipticin, and alkylating agents or metal complexes can be linked to antisense or antisense oligonucleotides to modify the specificities of antisense or antisense oligonucleotides to the target nucleotide sequence.
[00262] [00262] —Sense or antisense ligonucleotides can be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaPO-mediated DNA transfection; electroporation, or by the use of gene transfer vectors such as viruses
[00263] [00263] —O sense or antisense ligonucleotides can also be introduced into a cell containing a target nucleotide sequence by forming a conjugate with a linker binding molecule, as described in WO 91/04753. Suitable linker binding molecules include, but are not limited to, surface receptors, growth factors, other cytokines or other linkers that bind to cell surface receptors. Preferably, the conjugation of the linker binding molecule does not substantially interfere with the ability of the binding molecule to bind to its corresponding molecule or receptor, or to block the entry of the oligonucleotide in the sense or antisense or its conjugated version into the cell.
[00264] [00264] & Alternatively, a sense or antisense oligonucleotide can be introduced into a cell containing the target nucleic acid sequence by forming an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.
[00265] [00265] The samples can also be used in PCR techniques to generate a set of sequences for the identification of closely related FGF19 chimeric coding sequences.
[00266] [00266] Nucleic acid encoding the chimeric polypeptide FGF19 can also be used in gene therapy. In gene therapy applications, genes are introduced into cells to achieve in vivo synthesis of a therapeutically effective gene product, for example, to replace a defective gene. "Gene therapy" includes both conventional therapy genes in which a long-lasting effect is obtained by a single treatment, and the administration of therapeutic gene agents, which involves the single or repeated administration of a therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can be used as therapeutic agents to block the expression of certain genes in vivo. It has been shown that antisense oligonucleotides can be imported into cells in which they act as inhibitors, despite their low intracellular concentrations caused by their restricted absorption by the cell membrane. (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83: 4143-4146). Oligonucleotides can be modified to increase their absorption, for example by replacing their negatively charged phosphodiester groups with uncharged groups.
[00267] [00267] There are a variety of techniques available for introducing nucleic acids into viable cells. Techniques vary depending on whether the nucleic acid is transferred to cells cultured in vitro, or in vivo in the cells of the intended host. Appropriate techniques for the transfer of nucleic acid in mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. Currently preferred in vivo gene transfer techniques include transfection with viral vectors (typically retrovirus) and liposome-mediated transfection of coated viral protein (Dzau et al., Trends in Biotechnology 11, 205- 210). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell membrane protein or target cell, a linker for a receptor on the target cell, etc. Where liposomes are used, proteins that bind to a cell surface membrane protein associated with endocytosis can be used to target it and / or to facilitate absorption, eg capsid proteins or tropic fragments for one cell type in particular, antibodies to proteins that undergo internalization in cyclization, proteins that target the intracellular location of the target and increase the intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al ,, Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). For a review of gene labeling and gene therapy protocol see Anderson et al., Science 256, 808-813 (1992). X. Articles of Manufacture
[00268] [00268] In another embodiment of the invention, an article of manufacture is provided containing materials useful for the treatment of disorders related to metabolism, conditions or symptoms as described above. Preferably, the article of manufacture comprises: (a) a container comprising a composition with a chimeric FGF19 polypeptide described herein and a pharmaceutically acceptable carrier or diluent within the container; and (b) an information leaflet with instructions for administering the composition to an individual who suffers or exhibits disorders, conditions or symptoms related to metabolism.
[00269] [00269] In some modes of execution, the individual has disorders, conditions or symptoms related to metabolism. In some modes of execution, the individual is at risk of developing disorders, conditions or symptoms related to metabolism. In some modes of execution, the individual has one or more characteristics selected from the group consisting of (a) waist circumference of approximately 102 cm or more in men and approximately 88 cm or more in women, (b) fasting triglycerides of approximately 150 mg / dL or more, (c) a fasting glucose of approximately 95 mg / dL or greater, and (d) high levels of oxidized LDL. In some modes of execution, the individual still has inflammation associated with diabetes. In some modes of execution, the individual has a high glycemic level of about 95 mg / dL or greater after fasting overnight. In some embodiments, the individual has a high blood glucose level of approximately 126 mg / dL or more after an overnight fast. In some embodiments, the individual has a blood glucose level of approximately 140 mg / dL after a two-hour oral glucose tolerance test. In some embodiments, the individual has a blood glucose level of approximately 200 mg / dL after a two-hour oral glucose tolerance test. In some modes of execution, the individual has pre-diabetes. In some modes of execution, the individual has diabetes. In some embodiments, diabetes is selected from the group consisting of type I diabetes, type II diabetes, and gestational diabetes. In some embodiments, diabetes is type-II diabetes.
[00270] [00270] The manufactured article comprises a container and an information label or leaflet in or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers can be formed by a variety of materials such as glass or plastic. The container has or contains a composition that is effective for the treatment of multiple sclerosis and may have a sterile access port (for example, the container may be a pouch or an intravenous solution bottle containing a stopper punctured by a hypodermic injection needle). At least one active agent in the composition is the chimeric polypeptide FGF19. The label or leaflet indicates that the composition is used to treat disorders, conditions or symptoms related to metabolism in an individual suffering from them with a specific guide with regard to dosage amounts and ranges of antibody and any other medications provided. The manufactured article may further comprise a second container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. The article of manufacture may also include other materials desirable from a commercial and user point of view, including other buffer solutions, diluents, filters, needles and syringes.
[00271] [00271] - Optionally, the article of manufacture described herein further comprises a container comprising a second agent other than the polypeptide for treatment and further comprising instructions for treating the mammal with such an agent. In some embodiments, the second agent is an anti-inflammatory agent, an anti-diabetic agent, and / or a cholesterol-lowering drug of the "statin" class. In some embodiments, the second active agent is insulin. In some modes of execution, insulin is a fast-acting, short-acting, regular-acting, intermediate-acting, or long-acting insulin. In some embodiments, insulin is and / or comprises humalog, lispro, novolog, apidra, humulin, aspart, regular insulin, NPH, lens, ultralente, lantus, glargine, levemir, or detemir. In some embodiments, the second active agent is a statin. In some embodiments, the statin is and / or comprises atorvastatin (eg, lipitor or torvast), cerivastatin (eg,
[00272] [00272] A "leaflet" is used to refer to instructions normally included in commercial therapeutic product packages, which contain information about the indications, uses, dosages, administration, contraindications or other therapeutic products to be combined with the product of the package and / or notices concerning the use of such therapeutic products, etc.
[00273] [00273] The following examples are offered for illustrative purposes only, and are in no way intended to limit the scope of the present invention.
[00274] [00274] All references in the literature cited in the present patent application are hereby incorporated by reference in their entirety. EXAMPLES
[00275] [00275] Commercially available reagents referred to in the examples were used according to the manufacturer's instructions unless otherwise indicated. The source of the cells identified in the following examples and through the patent application by the ATCC accession numbers is the American Type Culture Collection, Manassas, VA. Cells were grown in Dulbecco's Modified Eagle Medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS) at 37 ºC under 5% CO2, unless otherwise indicated.
[00276] The in vitro binding activity to the FGF receptor of a chimeric FGF polypeptide of the present invention was measured using an enzyme-linked immunosorbent assay (ELISA). With reference to Fig. 3A (top), a schematic diagram of ELISA is represented for measuring the in vitro binding activity of the FGF receptor (FGFR) and its corresponding control.
[00277] [00277] Monoclonal antibodies specific for the human IgG-Fc fragment (Jackson ImmunoResearch, West Grove, PA, USA) were immobilized in the wells of the 96 wells of the MaxisorpTM flat-bottom plates (Nunc, Thermo Fisher Scientific, Rochester, New York) during overnight by incubation with 100 µl per well of 2 µg / ml of antibody solution. Each well was then incubated or with 1 FGFR4-Fc upgiml (a recombinant polypeptide comprising a human FGFRA4 extracellular domain fused to a human IgG1 Fc fragment; catalog no. 685-FR-050, R&D Systems, Inc., Minneapolis, Minnesota ) or 1 µg / ml of FGFR1c-Fc (a polypeptide recombinant “comprising a human FGFR1c extracellular domain fused to a human IgG1 Fc fragment; catalog no. 658-FR-050, R&D Systems).
[00278] [00278] FGFR4-Fc or FGFR1c-Fce polypeptides immobilized on the surface were incubated for 1 hour with native human FGF19-Flag polypeptides with a C terminal tag epitope (see FGF19-Flag in table 10, SEQ ID NO: 237) in concentrations of 1 ug / ml, 0.4 ug / ml, 0.16 ug / ml, 0.064 ug / ml, 0.0256 pg / ml, 0.004096 pg / ml or 0.0016384 upg / ml, each with 2 ug / ml heparin, to allow binding of the FGF19 polypeptide to the receptor domain. Similarly, FGFR4-Fc or FGFR1c-Fe immobilized surface polypeptides were incubated for 1 hour with a chimeric FGF19 polypeptide with a C terminal tag epitope (see cFEGF21 / 19-2 / Flag in table 10; SEQ ID NO: 242) a concentrations of 1 µg / mL, 0.4 µg / mL, 0.16 µg / mL, 0.064 upg / mL, 0.0256 µg / mL, 0.004096 µg / mL or 0.0016384 µg / mL, each with 2 µg / ml heparin, to allow binding of the chimeric polypeptide FGF19 to the receptor domain. After incubation,
[00279] [00279] With reference to Fig. 3A, the results of the in vitro FGFR binding assay for the FGFR4-Fc or FGFR1IcC-Fe polypeptides with immobilized surface showed that the native human FGF19 polypeptides bound to FGFR4-Fc in a concentration- dependent, Klotho-beta-independent, but did not bind to FGFR1Ic-Fc. Klotho-beta-independent binding of chimeric FGFR19 polypeptides (cFGF21 / 19-2 / Flag; SEQ ID NO: 242) to both FGFR4-Fc and FGFR1c-Fc was not detected.
[00280] [00280] With reference to Fig. 3B, a schematic diagram of an assay for FGFR activation is presented. In this assay, transiently transfected L6 cells express a FGF receptor, such as human FGFR1c or human FGFR4, on their cell surfaces. Effective binding of a ligand to the FGF receptor can result in the activation of an endogenous MAP kinase pathway, which can result in the phosphorylation of a chimeric transcriptional activator containing an Elk-1 activation domain and a binding domain
[00281] [00281] In this assay, rat L6 myoblasts in a 96-well plate were transiently transfected with an expression vector encoding a human FGFR4 polypeptide, an expression vector encoding a GAL4-Elk-1 transcriptional activator (in catalog 219005, pFA2 -Elk1, Stratagene, La Jolla, California), an expression vector encoding a firefly luciferase reporter gene under the control of the upstream yeast GALA4 activator (catalog number 219050, pFR-luc, Stratagene). A vector for the constitutive expression of Renilla luciferase (catalog number E2231, pRL-SVA40, Promega, Madison, Wisconsin) was also transfected into the cells. Transfections were performed using a FUGENE HD transfection reagent (catalog number 04 709 705 001, Roche Applied Science, Indianapolis, Indiana) according to the manufacturer's instructions.
[00282] [00282] L6 transfected cells were cultured overnight in DMEM (prepared from Cellgro 50-013-PC, Mediatech, Inc., Manassas, Virginia) containing 10% FBS (catalog number F2442, Sigma-Aldrich, St. Louis, Missouri). The cells were then washed and cultured for an additional 6 hours in an enriched serum-free medium derived from the 50:50 F12 / DME mixture containing 25 mg / L of porcine heparin and a given concentration of the FGF19 polypeptide. The FGF19 polypeptides that were tested were FGF19-
[00283] [00283] With reference again to Fig. 3B, each normalized luciferase activity is shown as a mean and standard error of the mean of the three replicates. The results show that L6 cells that express FGFR4 but not Klotho-beta, when treated with native FGF19 polypeptides, show a dose-dependent activation of luciferase activity, whereas neither the native FGF21 polypeptide nor the chimeric FGF19 polypeptide showed such activity.
[00284] [00284] In this assay, rat L6 myoblasts in a 96-well plate were transiently transfected with an expression vector encoding either the human FGFR4 polypeptide (based on the NCBI reference sequence: NM 002011.3) or the human FGFR1Ic polypeptide (based on the sequence reference code NCBI: NM 015850.3), an expression vector encoding the Klotho-beta (KLB) polypeptide (based on the NCBI reference sequence: NM 175737.3 fused to an LEDYKDDDDK C terminal epitope sequence), an expression vector encoding a transcriptional activator GAL4-EIlk-1 (pFA2-EIlk1, Stratagene), and an expression vector encoding a firefly luciferase reporter gene under control of the yeast GAL4 upstream activator (pFR-luc, Stratagene). A vector for the constitutive expression of Renilla luciferase (pRL-SV40, Promega) was also transfected into the cells. Transfections were performed using a FUGENE HD transfection reagent (Roche Applied Science) according to the manufacturer's instructions.
[00285] [00285] The transfected L6 cells were cultured overnight in DMEM containing 10% FBS, as above. The cells were then washed and cultured for an additional 6 hours in a serum-free medium containing 25 mg / L of porcine heparin and a given concentration of a FGF polypeptide. The FGF polypeptides that were tested were human native FGF19-Flag polypeptide (see FGF19-Flag in table 10, SEQ ID NO: 237), human native FGF21-His polypeptide (see FGF21-His in table 10, SEQ ID NO: 238) and a chimeric FGF19-Flag polypeptide (see cFEGF21 / 19-2 / Flag in table 10; SEQ ID NO: 242). The cells were incubated with the FGF19 polypeptide at concentrations of 500 ng / mL, 83.3 ng / mL, 13.9 ng / mL, 2.3 ng / mL, 0.39 ng / mL, 0.064 ng / mL or 0.011 ng / ml. The cells were incubated with the chimeric polypeptide FGF19 at concentrations of 2667 ng / ml, 444.4 ng / ml, 74.1 ng / ml, 12.3 ng / ml, 2.06 ng / ml, 0.34 ng / ml or 0.057 ng / ml. The cells were then lysed with the PLB reagent (Promega) and the luciferase activity was determined in each well using Dual-Glo Luciferase Assay System (Promega) and EnVision Multilabel Reader (PerkinElmer) according to the respective manufacturer's instructions. Each firefly luciferase activity was normalized to co-expressed renilla luciferase activity, and each sample condition was performed in triplicate.
[00286] [00286] With reference to Fig. 4, each normalized luciferase activity is shown as a mean and standard error of the mean of the three replicates. The results show that native FGF19 polypeptide and chimeric FGF19 polypeptide show similar dose-dependent luciferase activation in the presence of KLB and FGFR1c, with a
[00287] [00287] In this example, FVB mice fasted overnight. A sample group (n = 5 or 6) of fasting mice was injected via the tail vein with a native human FGF19-Flag polypeptide (FGF19-Flag in table 10, native human FGF21-His polypeptide (FGF21-His in table 10 ), chimeric polypeptide FGF19-Flag (cFGF21 / 19-2 / Flag in table 10; SEQ ID NO: 242) or phosphate buffer solution (PBS) control vehicle. Polypeptides were supplied in PBS at a dose of 1 mg / kg In 4 hours post injection, the liver tissue of each mouse was harvested and snap-frozen (subjected to rapid freezing) in liquid nitrogen.The total RNA tissue was isolated from the liver tissue harvested using Qiazol (no. catalog 79306, Qiagen, Germantown, Maryland) and used as a model for cDNA synthesis (Quantitect Reverse Transcription Kit, catalog number 205311, Qiagen). Following standard protocols for quantitative real-time PCR, the cDNA was quantified using the SYBR Green dye (catalog number
[00288] [00288] The example was performed as described in example 3, with brown adipose tissue (BAT) and white adipose tissue (WAT) harvested 4 hours after injection and quickly frozen (snap-frozen) in liquid nitrogen. With reference to Fig. 6, the results show that such levels of Egr-1 mMRNA in WAT and UCP-3 mMRNA in BAT, none of which expresses a detectable FGFR4, were similarly regulated by the employed FGF polypeptides. Levels with values of p <0.05, <0.01 and <0.001 are indicated by "*", "**" and "***", respectively.
[00289] [00289] In this example, an 11-month-old ob / ob mouse (stockt & 000632, The Jackson Laboratory, Bar Harbor, Maine) was implanted subcutaneously with an osmotic pump (catalog number 2001, Alzet, Cupertino, California) containing 200 ul of human native FGF21 polypeptide (FGF21-FlagN in table 10; Img / mlL in PBS), chimeric FGF19 polypeptide (cFGF21 / 19-2 / Flag in table 10; SEQ ID NO: 242) (Img / ml in PBS) or vehicle control (PBS). Each sample group consisted of nine (9) mice. The osmotic pump was configured to deliver polypeptides at a rate of - 0.4 mg / kg / day.
[00290] [00290] With reference to Fig. 7A, the body weight and blood glucose level (glucose in the blood) randomly fed from each mouse was measured starting three (3) days before implantation of the pump up to five (5) days after implantation in time points indicated. Blood glucose was measured using the One Touch 2 Ultra Blood glucose monitoring system (LifeScan, Milpitas, California). Fig. 7A shows the average body weight and blood glucose level of each sample in the group (levels with p values <0.05, <0.001 and <5 x 107 are indicated with "*", "tm E Ne, respectively On day 5, the mice fasted overnight and blood glucose was measured the next morning.With reference to Fig. 7B, the blood glucose levels for each mouse on days 5 and 6 (which fasted overnight) are shown (levels with p values <0.002, <0.0005 and <5 x 10-10 are indicated by "*", "tm" E me, respectively). The results show that both native human FGF21 polypeptides and chimeric FGF19 polypeptides reduced blood glucose to similar levels in these mice.
[00291] [00291] The mice in example 5 were injected intraperitoneally with glucose bolus in PBS (19g / kg) following the overnight fast on day 6 to test glucose tolerance. The bolus injection occurred at the point corresponding to time time = O in Fig. 8A.
[00292] [00292] In this example, conditioned media containing a FGF-Fc fusion polypeptide is harvested from cells transfected with the corresponding expression vector. HEK293S cells were transiently transfected with an expression vector encoding native human FGF 19 polypeptides fused to the N-terminus of a human IgG1-Fc fragment via a linker with 21 amino acids GGGGSGGGGSDYKDDDDKGRAQVT (SEQ ID NO: 286) FGF-terminated human polypeptides. N of a human IgG1-Fc fragment via a 4-amino acid GGGG linker, or a chimeric human FGF19 polypeptide (cFGF21 / 19-2) fused to the N-terminus of the human IgG1-Fc fragment via a 4-amino acid GGGS linker. Simulated transfected cells were used as controls. The cells were grown overnight in DMEM containing 10% FBS, as above. The cells were then washed and cultured in an enriched serum-free medium derived from the 50:50 F12 / DME mixture for two (2) days. A conditioned medium was taken from each sample. Equal volumes (6.5 µl) of each conditioned medium from each sample were used for immunoblot analysis using antibodies specific for the human IgG-Fc fragment. The immunoblot results are shown in Fig. 9B, which shows the presence of a polypeptide containing an Fc fragment with the expected molecular weight in the conditioned media that were harvested from cells transformed with FGF19-Fc, FGF21-Fc and cFGF21 / 19- fusion. 2-Fc.
[00293] [00293] In this example, conditioned media were used to demonstrate the activity of the FGF-Fc fusion polypeptides. HEK293S cells in a 96-well plate were transiently transfected with an expression vector encoding the GAL4-Elk-1 transcriptional activator (pFA2-Elk1, Stratagene), and a vector expression encoding a firefly luciferase encoding reporter gene under control of the upstream activator of the yeast GAL4 (pFR-luc, Stratagene). In some experiments, cells were also transfected with an expression vector that encodes a Klotho-beta polypeptide (KLB). A vector for the constitutive expression of Renilla luciferase (pRL-SV40, Promega) was also transfected in the cells. Transfections were performed using a FuGENE HD Transfection Reagent (Roche Applied Science) according to the manufacturer's instructions.
[00294] [00294] The transfected cells were cultured overnight in DMEM containing 10% FBS, as above. The cells were then washed and cultured for an additional 6 hours in a medium prepared from 1 part of the conditioned medium diluted with 3 parts of serum from an enriched serum-free medium derived from the 50:50 F12 / DME mixture, the final medium containing 25 mg / L of porcine heparin. The cells were then lysed with a PLB reagent (catalog number E1941, Promega) and the luciferase activity in each well was determined using Dual-Glo Luciferase Assay System (Promega) and EnVision Multilabel Reader (PerkinElmer) according to the respective instructions manufacturer. Each firefly luciferase activity was normalized to co-expressed renilla luciferase activity, and each sample condition was performed in triplicate.
[00295] [00295] With reference to Fig. 9A, each normalized luciferase activity is shown as an average and the standard error of the mean of the three replicates. The results show that the luciferase activity of the transformed HEK293S cells, with the presence of KLB, can be activated by a native human FGF19-Fc polypeptide or by the chimeric FGF19-Fc polypeptides in a similar manner to their respective Fc non-fusion analogues. However, in contrast to the FC non-fusion analogs corresponding to native FGF21 polypeptides, FGF21-Fc fusion polypeptides showed substantially less activation of firefly luciferase even in the presence of KLB.
[00296] [00296] In this example, rat L6 myoblasts in a 48-well plate were transiently transfected with an expression vector encoding either human FGFR4 polypeptides, human FGFR1c polypeptides or a control vector. An expression vector encoding the Klotho-beta polypeptide (KLB), an expression vector encoding a transcriptional GAL4-Elk-1 activator (pFA2-EIk1, Stratagene), an expression vector encoding a firefly luciferase reporter gene under the control of the GALA4 yeast upstream activator (pFR-luc, Stratagene), and the vector for the constitutive expression of renilla luciferase (pRL-SV40, Promega). Transfections were performed using the transfection reagent
[00297] [00297] The transfected L6 cells were cultured overnight in DMEM containing 10% FBS, as above. The cells were then washed and cultured for an additional 6 hours in serum-free conditioned medium (each conditioned medium was produced and harvested according to Example 7, diluted for use with an equal volume of serum-free medium) containing 25 mg / L of porcine heparin. The conditioned medium contained both a control vector (group A in Fig. 10) (vector derived from pUC containing a CMV promoter :); human native FGF21-FlagC polypeptides (FGF21-FlagC in table 10; group B in Fig. 10); native human FGF19-Flag polypeptides (FGF19-Flag in table 10; group C in Fig. 10); a first chimeric FGF19 polypeptide containing an N-terminal sequence derived from native human FGF21 (group D in Fig. 10) (cFGF21 / 19-13 / Flag in table 10, HPIPDSSPLLQOFGGQAQVRARYLYTDDAQQTEAHLEIREDGT VGGAADOQSPESLLQLKALKPGVIQILGVKTSRFLCARPDGALYGSLH FDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGP
[00298] [00298] With reference to Fig. 10, each normalized luciferase activity is shown as a fold induction on the corresponding transfected cells in group A samples, which were incubated in conditioned control medium derived from cells transfected with the control vector . Each fold induction is shown as an average and a standard error of the average of the three replicates. The results show that the fold induction of normalized luciferase activity in L6 cells expressing FGFR1c was comparable between the non-control samples. However, fold induction in L6 cells expressing FGFR4 was significantly greater in cells incubated with native FGF19 than in those cells incubated with either native FGF21 polypeptides or chimeric FGF 19 polypeptides.
[00299] [00299] In this example, chimeric FGF19 polypeptides containing N-terminal domains derived from the native human FGF19 polypeptide were tested for activity. All of the tested polypeptides also contained a Flag terminal C label epitope. HEK293S cells were transiently transfected with an expression vector encoding the Klotho-beta polypeptide (KLB), an expression vector encoding a transcriptional GAL4-Elk-1 activator (pFA2- EIlk1, Stratagene), an expression vector encoding a firefly luciferase reporter gene under control of the upstream GALA yeast activator (pFR-luc, Stratagene), and the vector for the constitutive expression of renilla luciferase (pRL-SV40, Promega). Rat L6 myoblasts were transiently transfected with an expression vector that encodes both human FGFR4 polypeptide and human FGFR1c polypeptide, an expression vector that encodes Klotho-beta polypeptide (KLB), an expression vector that encodes a GAL4 transcriptional activator -Elk-1 / - (pFA2-EIk1, Stratagene), an expression vector encoding a luciferase reporter gene under the control of the upstream activator of the yeast GAL4 (pFR-luc, Stratagene), and the vector for the constitutive expression of Renilla luciferase (pRL-SV40, Promega). Transfections were performed using the FUGENE HD transfection reagent (Roche Applied Science) according to the manufacturer's instructions.
[00300] The transfected HEK293S and L6 cells were cultured overnight in DMEM containing 10% FBS, as above. The cells were then washed and cultured for an additional 6 hours in serum-free conditioned medium (each conditioned medium was produced and harvested according to Example 7, diluted for use with an equal volume of serum-free medium) containing 25 mg / L of porcine heparin. The conditioned media were harvested from cells transfected with human native FGF21-FlagC polypeptides (A in Fig. 11), chimeric FGF1I9 cFGF19 / 21-1 / Flag polypeptides (cFGF19 / 21-1 / Flag in table 10; B in Fig. 11 ), chimeric polypeptides FGF19 cFGF19 / 21-2 / Flag (cFGF19 / 21-2 / Flag in table 10; C in Fig. 11), chimeric polypeptides FGF19 cFGF19 / 21-3 / Flag (cFGF19 / 21-3 / Flag na table 10; D in Fig. 11), chimeric polypeptides FGF19 cFGF19 / 21-4 / Flag (cFGF19 / 21-4 / Flag in table 10; E in Fig. 11), chimeric polypeptides FGF19 cFGF19 / 21-5 / Flag ( cFGF19 / 21-5 / Flag in table 10; F in Fig. 11), chimeric polypeptides FGF19 cFGF19 / 21-6 / Flag (cFGF19 / 21-6 / Flag in table 10; G in Fig. 11), chimeric polypeptides FGF19 cFGF19 / 21-7 / Flag (cFGF19 / 21-7 / Flag in table 10; H in Fig. 11), FGF19 chimeric polypeptides cFGF19 / 21-8 / Flag (cFGF19 / 21-8 / Flag in table 10; | na Fig. 11), chimeric polypeptides FGF19 cFGF19 / 21-9 / Flag (cFGF19 / 21-9 / Flag in table 10; J in Fig. 11), chimeric polypeptides rich FGF19 cFGF19 / 21 / 19-29 / Flag (cFGF19 / 21 / 19-29 / Flag in table 10; K in Fig. 11), or FGF19-Flag native polypeptides (L in Fig. 11).
[00301] [00301] With reference to Fig. 11, the firefly luciferase activity for each sample was normalized to the co-expressed renilla luciferase activity, and each sample condition was performed in triplicate. The normalized luciferase activity was compared to that of the human native FGF19-Flag polypeptides, where "+" indicates activity substantially equivalent to that of the native human FGF19-Fc polypeptide, "+/-" indicates intermediate activity, and "-" indicates no detectable activity. Conditioned media that showed no detectable or intermediate activity in HEK293S cells were not tested in L6 cells.
[00302] [00302] In this example, chimeric FGF19 polypeptides containing N terminal domains derived from the native human FGF21 polypeptide were tested for activity. All tested polypeptides also contained a C flag terminal tag epitope. The assay was performed as described in example 9. The conditioned media were harvested from cells transfected with human native FGF19-Flag polypeptide (FGF19-Flag in table 10; A in Fig. 12), chimeric FGF19 cFGF21 / 19-1 / Flag polypeptide (cFGF21 / 19-1 / Flag in table 10; B in Fig. 12), chimeric polypeptide FGF19 cFGF21 / 19-2 / Flag (cFGF21 / 19-2 / Flag in table 10; C in Fig. 12), chimeric polypeptide FGF19 cFGF21 / 19-7 / Flag (cFGF21 / 19-7 / Flag in table 10; D in Fig. 12), chimeric polypeptide FGF19 cFGF21 / 19-8 / Flag (cFGF21 / 19- 8 / Flag in table 10; E in Fig. 12), chimeric polypeptide FGF19 cFGF21 / 19-9 / Flag (cFGF21 / 19-9 / Flag in table 10; F in Fig. 12), chimeric polypeptide - FGF19 cFGF21 / 19-10 / Flag (cFGF21 / 19 - 10 / Flag in table 10; G in Fig. 12), chimeric polypeptide FGF19 cFGF21 / 19-11 / Flag (cFGF21 / 19-11 / Flag in table 10; H in Fig. 12), chimeric polypeptide - FGF19 cFGF21 / 19-12 / Flag (cFGF21 / 19-12 / Flag in table 10; | in Fig. 12), chimeric polypeptide FGF19 cFGF21 / 19-13 / Flag (cFGF21 / 19-13 / Flag in table 10; J in Fig. 12), or native FGF21-FlagC polypeptide (FGF21-FlagC in table 10; K in Fig. 12).
[00303] [00303] With reference to Fig. 12, the firefly luciferase activity for each sample was normalized to the co-expressed Renilla luciferase activity, and each sample condition was performed in triplicate. The normalized luciferase activity was compared to the activity of the human native FGF19-Fc fusion polypeptide, where "+" indicates activity substantially equivalent to that of the human native FGF19-Fc fusion polypeptide, "+/-" indicates intermediate activity, and " - "indicates no detectable activity. Conditioned media that showed no detectable or intermediate activity in HEK293S cells were not tested in L6 cells.
[00304] [00304] In this example, chimeric FGF19 polypeptides containing N terminal domains derived from human native FGF21 polypeptides were tested for activity. All of the tested polypeptides also contained a Flag terminal C label epitope. The assay was performed as described in example 9, except that only the transfected HEK293S cells and the transfected L6 cells FGFRA4 were used in the test. The conditioned media were harvested from cells transfected with human native FGF21-FlagC polypeptides (FGF21-FlagC in table 10; A in Fig. 13), human native FGF19-Flag polypeptides (FGF19-Flag in table 10; B in Fig. 13) , chimeric polypeptides FGF19 cFGF21 / 19-1 / Flag (cFGF21 / 19-1 / Flag in table 10; C in Fig. 13), chimeric polypeptides FGF19 cFGF21 / 19-2 / Flag (cFGF21 / 19-2 / Flag in table 10; D in Fig. 13), chimeric polypeptides FGF19 cFGF21 / 19-3 / Flag (cFGF21 / 19-3 / Flag in table 10; E in Fig. 13), chimeric polypeptides FGF19 cFGF21 / 19-4 / Flag (cFGF21 / 19-4 / Flag in table 10; F in Fig. 13), chimeric polypeptides FGF19 cFGF21 / 19-5 / Flag (cFGF21 / 19-5 / Flag in table 10; G in Fig. 13), or chimeric FGF19 polypeptides cFGF21 / 19-6 / Flag (cFGF21 / 19-6 / Flag in table 10; G in Fig. 13).
[00305] [00305] With reference to Fig. 13, the firefly luciferase activity for each sample was normalized to the co-expressed renilla luciferase activity, and each sample condition was performed in triplicate. Normalized luciferase activity was compared to that of human native FGF19-Flag polypeptides, where "+" indicates activity substantially equivalent to that of native human FGF19-Flagn polypeptides, "+/-" indicates intermediate activity, and "-" indicates none detectable activity. Conditioned media that showed no detectable activity or showed intermediate activity in HEK293S cells were not tested in L6 cells.
[00306] [00306] Fig. 13 still shows a proposed alignment of the respective amino acid sequences of the N terminal portion of the tested polypeptides. Selected amino acid residues that correspond to the LYT and LxxIxxG motifs conserved in each polypeptide are indicated in the alignment by outlined boxes.
[00307] [00307] In this example, chimeric FGF19 polypeptides containing N-terminal and internal domains derived from human native FGF21 polypeptides were tested for activity. All of the tested polypeptides also contained a Flag terminal C label epitope. The assay was performed as described in example 9. The conditioned media were harvested from cells transfected with human native FGF21-FlagC polypeptides (as shown in Fig. 14), human native FGF19-Flag polypeptides (as shown in Fig. 14), polypeptides chimeric FGF19 cFGF21 / 19-2 / Flag (cFGF21 / 19-2 / Flag in table 10; A in Fig. 14), chimeric FGF19 cFGF21 / 19-3 / Flag polypeptides (cFGF21 / 19-3 / Flag in table 10; B in Fig. 14), chimeric polypeptides FGF19 cFGF21 / 19-4 / Flag (cFGF21 / 19-4 / Flag in table 10; C in Fig. 14), chimeric polypeptides FGF19 cFGF21 / 19-5 / Flag (cFGF21 / 19 -5 / Flag in table 10; D in Fig. 14), chimeric polypeptides FGF19 cFGF21 / 19-6 / Flag (cFGF21 / 19-6 / Flag in table 10; E in Fig. 14), chimeric polypeptides FGF19 cFGF19 / 21 / 19-1 / Flag (cFGF19 / 21 / 19-1 / Flag in table 10; F in Fig. 14), chimeric polypeptides FGF19 cFGF19 / 21 / 19-2 / Flag (cFGF19 / 21 / 19-2 / Flag na table 10; G in Fig.
[00308] [00308] With reference to Fig. 14, the firefly luciferase activity for each sample was normalized to the co-expressed renilla luciferase activity, and each sample condition was performed in triplicate. Normalized luciferase activity was compared to activity for native human FGF19-Flag polypeptides, where "+" indicates activity substantially equivalent to that of native human FGF19-Flag polypeptides, "+/—" indicates intermediate activity, and "-" indicates none detectable activity. Conditioned media that showed no detectable or intermediate activity in HEK293S cells were not tested in L6 cells.
[00309] [00309] Fig. 14 still shows a proposed alignment of the respective amino acid sequences of the N-terminal portions of the tested polypeptides. Selected amino acid residues that correspond to the conserved LYT and LxxIXxG motifs in each polypeptide are indicated in alignment by outlined boxes.
[00310] [00310] In this example, a chimeric FGF19 polypeptide of the present invention was tested for its effect on dephosphorylation in STAT5. Five-week-old male mice
[00311] [00311] With reference to Fig. 15, the results show that the Tyr694-phosphorylated Stat5 protein was not detectable in the mice that were injected with native human FGF21 polypeptides. However, mice that were injected with the chimeric polypeptide FGF19 showed significant levels of the phosphorylated Stat5 protein.
[00312] [00312] In this example, a chimeric FGF19 polypeptide of the present invention was tested for its effect on anchorage-dependent growth of human HepG2 hepatoma cells, which express KLB and FGFR4. A 96 well plate was filled with 50 µL per well of molten agar base (DMEM, 0.5% agarose and 10% FBS). After the base agar solidified, about 670 HepG2 cells suspended in 50 μL of melted top agar solution (DMEM, 0.35% agarose and 10% FBS) were added to the agar base in each well and allowed to solidify.
[00313] [00313] Following the solidification of the cell suspension, 20 µl of the culture medium (DMEM and 10% FBS) was added to each well on a day designated as zero day (0). For a given experimental sample, the culture medium also included both human native FGF19-Flag polypeptides, human native FGF21-His polypeptides (catalog number 2539-FG-025 / CF, R&D Systems, Inc., Minneapolis, Minnesota), chimeric polypeptides labeled FGF19 flag (cFGF21 / 19-2 / Flag; SEQ ID NO: 242), or no FGF polypeptide as a control. The concentration of polypeptide in the culture medium added on day zero was either 120 ng / ml or 1200 ng / ml, so that the final concentration in each well becomes 20 ng / ml or 200 ng / ml, respectively. On each subsequent day 2, 4, 6 and 8, another 20 μl of the culture medium was added to each well, where another culture medium added to a given well contained the same FGF polypeptide as in applications prior to that well, but with one-sixth of the FGF polypeptide concentration (ie 20 ng / ml or 200 ng / ml) than that of day zero. A subset of the sample wells was also treated with the G418 protein synthesis inhibitor to provide a background fluorescence signal.
[00314] [00314] On day 59.10 ul of AlamarBlue reagent (catalog number DAL1100, Invitrogen) was added to each sample well and the plate was further incubated for 5 hours to test the total metabolic activity in each well. The resulting fluorescent intensity was measured using an EnVision Multilabel Reader (PerkinElmer). Five (5) replicates of each sample were tested.
[00315] [00315] With reference to Fig. 16, the results are shown as fluorescent intensity above the background and represent the mean and standard deviation of the five (5) replicates. The results show that the total metabolic activity, as a proposed indicator of anchorage-independent cell growth, was promoted by the addition of human native FGF19 polypeptides, but such activity was reduced with the addition of human native FGF21 polypeptides or chimeric FGF19 polypeptides ( p values <0.05 compared to the treated samples simulated according to the Student's t test).
[00316] [00316] To investigate the requirements for FGFR4 in mediating FGF19 activity through the use of mice deficient in Fgfr4 as well as a protein variant of FGF19, which is specifically enhanced in its ability to activate FGFRA4.
[00317] [00317] Expression of recombinant FGF protein. Amino acid sequences of FGF19, FGF21, and chimeras have been constructed, the drawings of the chimera constructs made are shown in figure 18B). The constructs numbered 1-17 shown in the figure
[00318] [00318] Unless otherwise stated, human recombinant FGF21, FGF19 and variants produced in CHO cells transiently transfected and purified to homogeneity in PBS were used for experiments. For some experiments, FGF21 (2539-FG / CF, R&D systems) derived from E. coli were used. All purified proteins were tested by cell-based GAL-EIlk1i assays for their activity before application in another assay. For experiments in Fig 18B, 18C, and 20, FGF proteins were expressed in transiently transfected HEK293 cells and freshly conditioned (fresh) serum-free medium was used for assays without purification.
[00319] [00319] Assay with luciferase. All cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS) at 37ºC under 5% CO 2. Rat L6 myoblasts in a 96-well plate were transiently transfected with expression vectors encoding renilla luciferase (pRL-SV40, Promega), human KLB, appropriate human FGFR, GAL4-Elk-1 transcriptional activator (pFA2-EIk1, Stratagene) , and GAL4 binding sites carried by firefly luciferase reporter (pFR-luc, Stratagene), using FUGENE HD transfection reagent (Roche Applied Science). The following day, the transfected cells were cultured for an additional 6-8 hours in a serum-free medium containing 25 mg / L of porcine heparin (Sigma) and FGF protein at various concentrations. The cells were then lysed with a PLB reagent (Promega) and the luciferase activity in each well was determined using a Dual-Glo Luciferase Assay System (Promega) and EnVision Multilabel Reader (PerkinElmer) assay. The firefly luciferase activity was normalized to co-expressed renilla luciferase activity, and was shown as a mean and standard error of the mean of the three replicates.
[00320] [00320] Assay-independent cell proliferation assay. A 96-well plate was filled with 50 µl / well of 0.5% agarose melted in culture medium. After the agarose base had solidified, about 670 HepG2 cells suspended in 50 μl of molten agarose top solution (0.35% agarose in culture medium) were added to the agar base in each well, and allowed to solidify. Following solidification, 20 µl of a culture medium containing an appropriate amount of FGF protein was added to each well on the day designated day O. On each of the subsequent days 2, 4, 6 and 8, another culture medium of 20 µl with an appropriate amount of FGF protein was added to each well. A subset of the sample wells was also treated with a Geneticin protein synthesis inhibitor (invitrogen) to provide a background fluorescence signal. On day 9, 10 µl of AlamarBlue reagent (Invitrogen) was added to each sample well and the plate was further incubated for 5 hours. The resulting fluorescent intensity was measured using EnvVision Multilabel Reader (PerkinElmer) and used as an indication of the total metabolic activity in each well. Five replicates of each sample were tested.
[00321] [00321] FGFR / linker assay. FGFR binding activity of FGF19 and FGF19v was measured as described in Desnoyers et al, Oncogene 27 (1): 85-97 (2008) employing the biotinylated anti-FGF19 antibody (BAF969, R&D systems) in the presence of 2 µgml of heparin. ELSA control experiments were performed using the anti-FGF19 antibody (AF969, R&D systems) and the biotinylated anti-FGF19 antibody (BAF969, R&D systems) to confirm that the antibody reacts to FGF19 and FGF19v in an indistinguishable manner.
[00322] [00322] Mouse Studies. Mice were kept in a pathogen-free animal facility (dog kennel) at 21 ºC under a 12 h light / 12 h dark cycle pattern with access to feed (a standard rodent feed (Labdiet 5010, 12.7% calories) fat) or a high fat diet, rich in carbohydrates (Harlan Teklad TD.03584, 58.4% of calories from fat) and water ad libitum. Male mice were used for all experiments. FGFR4 KO mice on C57BL / background 6 were previously described by Weinstein et al., Development 1998 125 (18): 3615-23 (1998). C57BL / 6 mice, ob / ob mice in C57BL / 6 background and FVB / NJ mice were purchased from Jackson Laboratory. Continuous infusion of FGF protein, an osmotic pump (Alzet 2001) was implanted subcutaneously. For the glucose tolerance test, glucose levels were measured using a One Touch Ultra glucometer. Statistics were performed using the Student t test. present as mean +/- standard deviation. BrdU staining was performed as described as fNicholes, 2002 tH79) and positive BrdU hepatocytes were counted using the automated Ariol image analysis system. All animal studies were performed under approved protocols from Genentech's Institutional Animal Care and Use Committee.
[00323] [00323] Analysis of serum. Total cholesterol, triglycerides, B-hydroxybutylates (BHB), Thermo DMA lactates) and non-esterified fatty acid (Roche) were determined by the use of enzymatic reactions. Serum insulin levels were determined by ELISA (crystal chem). The BA composition was determined by analyzing liquid chromatography coupled with mass spectrometry as previously described by Stedman et al., J Biol Chem. 279 (12): 11336-43 (2004).
[00324] [00324] Analysis of gene expression. Tissue RNAs were isolated using the QIAzol reagent (Qiagen). cDNA was synthesized with The Quantitect Reverse Transcription Kit (Qiagen) transcription kit. For real-time qPCR, samples were made in triplicate on the ABI Prism 7900HT (Applied Biosystems) using a universal mix see SYBR (Invitrogen) or the universal mix Tagman (Roche) and normalized to levels of 36B4. Pre-designed primers Quantitect primers for GK, SHP, Cyp8b1, IGFBP2, and AFP were obtained from Qiagen and all other primers were designed using an express primer software (Applied Biosystems).
[00325] [00325] To determine which of the metabolic effects caused by FGF19 are mediated by FGFR4, WT mice fed with HFD or Fgfr4 KO were treated with recombinant FGF19 or control vehicle, and metabolic phenotypes and gene expression were studied. To obtain sustained exposure to FGF19,
[00326] [00326] To assess changes in the metabolism of BA (bile acid), the composition of BA serum was determined by mass spectrometry - liquid chromatography (Fig. 17C). Infusion of FGF19 reduced free cholic acid (CA) and conjugated taurine and CA-derived secondary deoxycholic acid in WT mice, while having a minimal effect on CDCA metabolites (CDCA). This finding is consistent with a shift in BA synthesis to the alternative (acidic) pathway, bypassing Cypral-suppressed FGF19 and proceeding to Cyprbl (Fig 17D).
[00327] [00327] To quantitatively evaluate the specific activation of FGFRs by FGF19, an FGF response assay to GAL-EIk1 luciferase reporter was introduced into rat L6 cells. In these assays, the effective binding of a ligand to FGFR results in the activation of an endogenous MAP kinase pathway, leading to the activation of a chimeric transcriptional activator comprising an Elk-1 activation domain and a GAL4 DNA binding domain.
[00328] [00328] A chimeric construct classified as a class Il molecule, consisting of amino acids 1-20 of FGF21 and 25-194 of FGF19 (> 90% identical to FGF19), was selected for large scale synthesis in CHO cells and this variant is referred to as "FGF19v". When compared to FGF19 using the luciferase reporter assay, the FGF19v protein exhibited dose-dependent activity similar to FGF19 in L6 cells cotransfected with KLB and FGFR1c (Fig 18D). However, FGF19v activity was significantly reduced in L6 cells cotransfected with or with FGFRA4 alone or a combination of FGFRA4 and KLB (Fig. 18D), FGF19 has previously been shown to bind directly to FGFR4 even in the absence of KLB. FGF19, but not FGF19v, exhibited dose-dependent FGFR4 binding activity (Fig. 18E and F).
[00329] [00329] FGF19v activity was further tested in vivo compared to FGF19 and FGF21 by intravenous injection in fasted FVB mice overnight. Livers were harvested 4 hours after injection and the expression of hepatic mMRNA was determined by QPCR. Genes that were intensely induced by FGF19 but not FGF21, such as Egr-1 and c-Fos, were not effectively induced by FGF19v, which is consistent with the reduced FGFR4 activity of FGF19v (Fig. 21A). FGF19v had an activity similar to FGF19 or FGF21 in genes that were co-
[00330] [00330] FGF1I9 increased anchorage-independent proliferation of HepG2 cells on soft agar, and this effect was much less apparent for FGF19v or FGF21 proteins (Fig. 21C). To see if FGF19v also exhibited reduced ability to induce hepatocyte proliferation in vivo, mice were administered infusion with FGF19, FGF19v (1 ng / h) or control vehicle via an osmotic minipump. In addition, intraperitoneally was injected 1 mg / kg / day of FGF protein daily for 7 days to the same mice to obtain high peak exposures. To capture intermittent proliferative events, a BrdU solution (30 mg / kg) was injected twice a day for a total of 13 times. Hepatocyte proliferation was determined by measuring BrdU positive hepatocytes in liver harvested on day 7. As previously reported, treatment with FGF19 resulted in a dramatic increase in BrdU uptake; however, this response was significantly masked for FGF19v (Fig. 21D and E). Hepatic MRNA for Egr-1, c-Fos, and the AFP hepatocyte proliferation marker were all dramatically induced by FGF19 and these inductions were largely absent for FGF19v, while the regulation of GK, Cyp7a1 and Cyp8b1 did not differ between FGF19 and
[00331] [00331] The in vitro and in vivo results described above raised the question of whether FGF19v, a variant of FGF19 with reduced FGFR4 activity and proliferative potential, could improve hyperglycemia in diabetic animals as does FGF21. FGF21, FGF19v (1ng / h) or control vehicle were continuously administered by subcutaneous infusion in ob / ob mice using osmotic minipumps. While the infusion did not significantly affect body weight (Fig. 22A), both FGF21 and FGF19v dramatically reduced blood glucose (glycemia) levels in both randomly fed and fasted mice. (Fig. 22A and B), reduced circulating free fatty acid levels (Fig. 22C), and improved glucose tolerance (Fig. 22D).
[00332] [00332] To visualize the proliferation of hepatocytes, BrdU animals were injected 4 hours before sacrifice on day 7. Neither FGF21 nor FGF19v increased the incorporation of hepatic BrdU (not shown), but the gross liver weight was significantly reduced ( Fig. 22E) and no significant change in hepatic AFP mRNA expression was observed (Fig. 22F). Taken together, FGF19v can improve the metabolic status of obese mice without inducing hepatocyte proliferation.
[00333] [00333] A number of genes have been identified and exhibited normally altered expression in ob / ob mice treated with FGF21 and FGF19v. In the liver, both proteins induced IGFBP2 (a recently demonstrated anti-diabetic protein), and suppressed stearoyl-Coenzyme A desaturase 1 (SCD-1; a lipogenic gene) and Cyp8b1 (the determinant of the balance between CA and CDCA production ). In addition, both induced a UCP-1
[00334] [00334] Examining the individual BA serum, it was shown that recombinant FGF19, acting through Fgfr4, suppressed Cyp7a1 causing the synthesis of bile acid to proceed to the alternative Cyp7a1-independent pathway (acid) that leads to the production of CDCA at the expense of HERE. The expression of Cyp8b1 increased the death (knockout) in Fgfr4 mice several times and that treatment with FGF19 suppresses Cyp8b1i, a mandatory enzymatic step for the synthesis of CA. FGFRA4 was a determinant of the proportion of CDCA for the production of CA, through a negative regulation of both Cyp7a1l and Cyp8b1. Activation of FGFR4 shifts BA production to CDCA, while its abrogation leads to the formation of CA. In addition, FGF19 increased the hepatic expression of AFP in a Fgafrd4-dependent manner. FGF19 improved glucose tolerance in FGFR4 KO mice fed HFD (Fig 17) and FGF19v, a protein specifically improved for FGFRA4 binding and activation, and improved hyperglycemia in ob / ob mice (Figs 18 and 21-22). In addition to the effects of insulin resistance and glucose metabolism, FGF19 increased serum BHB levels even in KO FGFRA4 mice (Fig. 17), such as FGF21. Both FGF19 and FGF21 can bind and activate FGFR1c, FGFR2c, and FGFR3c in the presence of KLB. Therefore FGFR1c, FGFR2c, or FGFR3c, in cooperation with KLB, can mediate the common metabolic effects of FGF19 and FGF21.

权利要求:
Claims (24)
[1]
1. Chimeric fibroblast growth factor 19 (FGF19) polypeptide, characterized by the fact that the polypeptide sequence comprises: a C-terminal portion comprising a C-terminal portion of the hFGF19 polypeptide sequence; and an N-terminal portion comprising an N-terminal portion of the hFGF21 sequence, wherein the C-terminal portion of the hFGF19 polypeptide sequence contains about 45 to about 185 residues in length, the C-terminal portion of the hFGF19 polypeptide sequence contains a first position and an end position, the N-terminal portion of the hFGF21 polypeptide sequence contains about 7 to about 140 residues in length, the C-terminal portion of the hFGF21 polypeptide sequence contains a first position and an end position.
[2]
2. Chimeric FGF19 polypeptide according to claim 1, characterized in that the first position of the C-terminal portion of the hFGF polypeptide sequence corresponds to a position of SEQ ID NO: 1, selected from the group consisting of: 10, 11 , 25, 26, 27, 28, 30, 33, 35, 37, 40, 41, 42, 43, 44, 45, 52, 53, 54, 56, 57, 58, 59, 72, 73, 74, 79 , 80, 81, 143, 144, 145 and 146; and the final position of the C-terminal portion of the hFGF polypeptide sequence approximately corresponds to position 194 of SEQ ID NO: 1, and / or the first position of the N-terminal portion of the hFGF21 polypeptide sequence corresponds approximately to the | of SEQ ID NO: 2, and the final position of the N-terminal portion of the hFGF21 polypeptide sequence corresponds to a position of SEQ ID NO: 2, selected from the group consisting of: 7, 8, 20, 21, 22, 23, 25, 27, 29, 31, 34, 35, 36, 37, 38, 39, 46, 47, 48, 50, 51, 52, 53, 66, 67, 68, 73, 74,
75, 135, 136, 137 and 138.
[3]
Chimeric polypeptide FGF1I19 according to claim 1 or 2, characterized by the fact that: the first position of the C-terminal portion of the hFGF19 polypeptide sequence corresponds to position 25 of SEQ ID NO: 1, and the final position of the C-terminal portion the hFGF polypeptide sequence corresponds approximately to position 194 of SEQ ID NO: 1 and / or the first position of the N-terminal portion of the hFGF21 polypeptide sequence corresponds approximately to position 1 of SEQ ID NO: 2; wherein the final position of the N-terminal portion of the hFGF21 polypeptide sequence corresponds to position 20 of SEQ ID NO: 2.
[4]
4. Chimeric polypeptide FGF19, characterized by the fact that the polypeptide sequence comprises: a first polypeptide sequence that contains at least 85% of the sequence identity SEQ ID NO: 1, with a portion of the first polypeptide sequence being replaced by a portion of a second polypeptide sequence, and the second polypeptide sequence contains at least 85% of the sequence identity SEQ ID NO: 2, and the substituted portion of the first polypeptide sequence has a first and a last position, the first position of the substituted portion corresponds to the position in SEQ ID NO: 1, selected from the group consisting of: 1, 10, 11, 17, 18, 21, 22, 25, 26, 27, 28, 40, 41, 42, 43, 44, 45, 52, 53, 54, 56, 57, 58, 59, 63, 72, 73, 74, 79, 80, 81, 143, 144, 145 and 146, and the last position of the replaced portion corresponds to the position in SEQ ID NO: 1, selected from the group consisting of: 9, 10, 24, 25, 26, 27, 29, 31, 32, 34, 36, 39, 40, 41, 42, 43, 44, 51 , 52, 53, 55, 56, 57, 58, 66, 71, 72, 73, 78, 79, 80, 142, 143, 144, 145 and 194, and the first position of the replaced portion and the last position of the portion substituted are selected in such a way that the substituted portion of the first polypeptide sequence is about 3 to about 185 residues in length.
[5]
Chimeric FGF19 polypeptide according to claim 1 or 4, characterized in that the chimeric hFGF19 polypeptide further comprises a substitution of the B1-B2 loop of the first polypeptide, a substitution of the B10-B12 segment of the first polypeptide, and / or a replacement of amino acid residues 38 to 42 of the first polypeptide with loop B1-B2 of the second polypeptide, segment B10-B12 of the second polypeptide, and / or the corresponding sequence of amino acid residues of the second polypeptide.
[6]
6. Chimeric polypeptide FGF19, characterized by the fact that it comprises the sequence SEQ ID NO: 5.
[7]
Chimeric FGF19 polypeptide according to any one of claims 1 to 6, characterized in that the chimeric hFGF19 polypeptide is fused to a second polypeptide, the second polypeptide is selected from the group consisting of: the Fc portion of an immunoglobulin, of an analog of the Fc portion of an immunoglobulin and one or more fragments of the Fc portion of an immunoglobulin.
[8]
8. Chimeric FGF19 polypeptide according to claim 7, characterized by the fact that: (i) the immunoglobulin is selected from the group consisting of: IgG-1, IgG-2, IgG-3, I9G-4, IgA-1 , IgA-2, IgE, IgD and IgM; and / or (ii) the Fc portion is human or humanized; and / or (ili) the C terminus of the chimeric hFGF19 polypeptide is fused to the N termination of the second polypeptide, where, optionally, the C terminus of the chimeric hFGF19 polypeptide is fused to the N termination of the second polypeptide via a linker, the linker is selected of the group consisting of: a [Gly] n linker, a [Gly3Ser] linker m and a [GIly4Ser] linker, where n is an integer from 1 to 30 and is an integer from 1 to 6.
[9]
Chimeric FGF19 polypeptide according to any one of claims 1 to 8, characterized in that the chimeric hFGF19 polypeptide: (i) does not substantially activate FGFRA4, both in an independent Klotho-beta and dependent Klotho-beta; (ii) activates FGFR1c in a Klotho-beta dependent manner; (iii) when administered to an individual, it does not reduce the level of phosphorylated STAT5 polypeptide of the individual, where, optionally, the individual is a human; (iv) when administered to an individual, it reduces the amount of STATS polypeptides in the individual, but this amount of phosphorylated STATS polypeptides is greater than the amount of phosphorylated STAT5 polypeptides under administration of natural hFGF21 in an individual, where, optionally, the individual is a human; (v) when administered to an individual, reduces the amount of phosphorylated STATS5 polypeptides to an amount between: 100% to 5%, 100% to 10%, 100% to 20%, 100% to 30%, 100 % up to 40%, 100% up to 50%, 100% up to 60%, 100% up to 70%, 100% up to 80%, 100% up to 90% or 100% up to 95%, of the amount of phosphorylated STAT5 polypeptides in the individual without administration, where, optionally, the individual is a human; (vi) when administered to an individual, the reduction in the amount of phosphorylated STAT5 polypeptide is less than the reduction in the amount of phosphorylated STATS polypeptide under administration of native hFGF21, where, optionally, said reduction is a percentage: 0% up to 5%, from 0% to 10%, from 0% to 20%, from 0% to 30%, from 0% to 40%, from 0% to 50%, from 0% to 60%, from 0% to 70%, from 0% to 80%, from 0% to 90% or from 0% to 95%, of the reduction in the amount of phosphorylated STATS5 polypeptide under the administration of native hFGF21, where, optionally, the individual is human; (vii) when administered to an individual, it does not induce resistance to growth hormone, where, optionally, the individual is human; (vili) has a physiological half-life in vivo that is at least or approximately equal to that of FGF19; or (ix) has a physiological half-life in vivo that is at least or approximately equal to that of FGF21.
[10]
10. Pharmaceutical composition, characterized in that it comprises: (a) a therapeutically effective amount of the chimeric polypeptide FGF19, as defined in any one of claims 1age (b) an acceptable pharmaceutical carrier.
[11]
11. Use of a therapeutically effective amount of the pharmaceutical composition, as defined in claim 10, characterized by the fact that it is for the preparation of a medicine for the treatment of an individual who exhibits one or more: obesity, type 1 diabetes, diabetes type 2, high blood glucose level, metabolic syndrome, atherosclerosis, hypercholesterolemia, stroke, osteoporosis, osteoarthritis, degenerative joint diseases, muscle atrophy, sarcopenia, decreased lean body mass, baldness, wrinkles, increased fatigue, reduced stamina, cardiac function reduced, immune system dysfunction, cancer, Parkinson's disease, senile dementia, Alzheimer's disease and impaired cognitive function, where, optionally, the individual is a human.
[12]
12. Use of a therapeutically effective amount of the pharmaceutical composition, as defined in claim 10, characterized by the fact that it is for the preparation of a drug to reduce blood glucose levels in an individual in need of this treatment, in which, optionally, the individual is a human.
[13]
13. Isolated nucleic acid molecule, characterized by the fact that it comprises DNA containing one of: at least 80%, at least 90%, at least 95% or at least 99% of a sequence identity to: (a) a DNA molecule encoding a polypeptide containing the amino acid sequence with SEQ ID NO: 5; or (b) the complement of the DNA molecule of (a).
[14]
An isolated nucleic acid molecule according to claim 13, characterized in that it comprises: (1) the nucleic acid sequence with SEQ ID NO: 7; (ii) a DNA molecule encoding a polypeptide that comprises the amino acid residue sequence 1 through 190 of SEQ ID NO: 5, or (b) the complement of the DNA molecule of (a); (iii) a DNA molecule encoding a polypeptide that contains at least 85% of the amino acid sequence to residues 1 through 190 of SEQ ID NO: 5, or (b) the complement of the DNA molecule of (a); or (iv) the nucleic acid molecule comprising the DNA having one of: at least 80%, at least 90%, at least 95% or at least 99% of the sequence identity with the sequence SEQ ID NO: 7, where the encoded polypeptide further comprises amino acid residues that correspond to the Fc portion of an immunoglobulin.
[15]
15. Expression system, characterized by the fact that it comprises the nucleic acid molecule, as defined in claim 13 or 14.
[16]
16. Host cell, characterized by the fact that it comprises the nucleic acid molecule, as defined in claim 13 or 14, or the expression system, as defined in claim 15.
[17]
17. Isolated polypeptide, characterized by the fact that it is encoded by the nucleic acid molecule, as defined in claim 13 or 14.
[18]
18. Process for the preparation of an isolated polypeptide, characterized by the fact that it comprises: (i) the culture of the host cell, as defined in claim 16, under appropriate conditions for the expression of the encoded polypeptides; and (ii) recovering the encoded polypeptide from cell culture.
[19]
19. Isolated polypeptide, characterized by the fact that it is prepared by the process, as defined in claim 18.
[20]
20. Antibody, characterized by the fact that it specifically binds to any of the chimeric FGF19 polypeptides, as defined in any one of claims 1 to 9.
[21]
21. Antibody according to claim 20, characterized by the fact that: (i) the antibody is monocional; and / or (ii) the antibody does not bind to FGF19 native polypeptides or FGF21 native polypeptides.
[22]
22. Vector, characterized by the fact that it comprises a nucleic acid sequence, as defined in claim 13, in addition to comprising at least one component, among: a signal sequence, an origin of replication, a gene marker, an enhancer element, a promoter and / or a transcription termination sequence.
[23]
23. Article of manufacture, characterized by the fact that it comprises: (a) a container comprising a composition with a chimeric polypeptide FGF19, as defined in any of claims 1 to 9, and a pharmaceutically acceptable carrier or diluent within the container; and (b) an information leaflet with instructions for administering the composition to an individual.
[24]
24. Invention, characterized by any of its embodiments or categories of claim encompassed by the material initially disclosed in the patent application or in its examples presented here.

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法律状态:
2020-12-01| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2020-12-08| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2021-03-16| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-04-06| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-07-20| B11B| Dismissal acc. art. 36, par 1 of ipl - no reply within 90 days to fullfil the necessary requirements|

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2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US25207409P| true| 2009-10-15|2009-10-15|

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US61/252,074|2009-10-15|

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PCT/US2010/052852|WO2011047267A1|2009-10-15|2010-10-15|Chimeric fibroblast growth factors with altered receptor specificity|

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