WO2016048995A2 - Troncatures et mutants fgf19 et utilisations de ceux-ci - Google Patents

Troncatures et mutants fgf19 et utilisations de ceux-ci Download PDF

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WO2016048995A2
WO2016048995A2 PCT/US2015/051402 US2015051402W WO2016048995A2 WO 2016048995 A2 WO2016048995 A2 WO 2016048995A2 US 2015051402 W US2015051402 W US 2015051402W WO 2016048995 A2 WO2016048995 A2 WO 2016048995A2
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protein
fgf19
seq
mutated
amino acids
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WO2016048995A3 (fr
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Ronald M. Evans
Michael Downes
Annette Atkins
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Salk Institute For Biological Studies
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]

Definitions

  • This application provides mutated FGF19 proteins, including FGF19 truncations, nucleic acid molecules encoding such proteins, and methods of their use, for example to treat a bile acid disease or a metabolic disease.
  • Bile acids regulate cholesterol, triglyceride, glucose and energy homeostasis, and facilitate digestion and absorption of lipids in the small intestine. Emulsification of lipids and fat- soluble vitamins in the intestine allows the formation of micelles that are transported via the lacteal system. Other functions of bile acids include driving the flow of bile to eliminate catabolites from the liver and aiding in the reduction of the bacteria flora found in the small intestine and biliary tract. Bile acids are also involved in the regulation of their own synthesis and enterohepatic circulation.
  • the primary bile acids (cholic acid and chenodeoxycholic acid) are synthesized in the liver), while the secondary bile acids (deoxycholic acid and lithocholic acid) are made by bacteria.
  • the synthesis of bile acids in the liver is negatively regulated by the hormone fibroblast growth factor (FGF) 19.
  • FGF19 is secreted from the intestine and signals to the liver to repress Cyp7al.
  • Intestinal FXR activation due to transintestinal bile acid flux after a meal induces FGF19 expression, which is released by small intestine epithelial cells and circulates to bind to hepatocyte FGF receptor 4 (FGFR4) receptors; the FGFR4 receptors signal a reduction in bile acid synthesis via c-Jun NF -terminal kinase (JNK) pathway activation.
  • JNK c-Jun NF -terminal kinase pathway activation.
  • Repression of CYP7A1 results in decreased synthesis of bile acids from intrahepatic cholesterol in response to the daily feeding-fasting cycle.
  • Abnormal bile acid homeostasis can cause, or exacerbate, several diseases, such as cholestasis, portosystemic shunt, Crohn's disease, and hepatic microvascular dysplasia.
  • bile acids play a role in modulating metabolic syndrome, a cluster of cardiovascular disease risk factors that include visceral obesity, insulin resistance, dyslipidemia, increased blood pressure, and hypercoagulability.
  • Bile acid synthesis is also associated with type 2 diabetes. Thus, modulation of bile acid synthesis (e.g., decreasing) can be used in the treatment of diabetes and pre-diabetes.
  • Type 2 diabetes and obesity are leading causes of mortality and are associated with the Western lifestyle, which is characterized by excessive nutritional intake and lack of exercise.
  • a central player in the pathophysiology of these diseases is the nuclear hormone receptor (NHR) PPARy, a lipid sensor and master regulator of adipogenesis.
  • PPARy is also the molecular target for the thiazolidinedione (TZD)-class of insulin sensitizers, which command a large share of the current oral anti-diabetic drug market.
  • TZDs TZDs
  • side effects associated with the use of TZDs such as weight gain, liver toxicity, upper respiratory tract infection, headache, back pain, hyperglycemia, fatigue, sinusitis, diarrhea, hypoglycemia, mild to moderate edema, and anemia.
  • the identification of new insulin sensitizers is needed.
  • mutants of fibroblast growth factor (FGF) 19 that can be used to reduce blood glucose in a mammal, treat a metabolic disease, and/or treat a bile acid related disease.
  • FGF fibroblast growth factor
  • methods of reducing fed and fasting blood glucose, improving insulin sensitivity and glucose tolerance, reducing systemic chronic inflammation, ameliorating hepatic steatosis in a mammal, reducing serum bile acids, or combinations thereof using the FGF 19 mutant proteins (or nucleic acids encoding such) are provided herein.
  • use of the disclosed methods result in one or more of: reduction in triglycerides, decrease in insulin resistance, reduction of hyperinsulinemia, increase in glucose tolerance, reduction of hyperglycemia, decrease serum bile acids, or combination thereof, in a mammal.
  • FGF19 mutants can have an N-terminal truncation, point mutation(s), or combinations thereof, and in some examples are part of a chimera, such as an FGF21/FGF19 chimeric protein.
  • These disclosed proteins, and the encoding nucleic acid sequences can have reduced or eliminated mitogenic activity relative to the native (e.g., wild type) FGF19 protein.
  • mutant FGF 19 proteins and chimeras of mutant FGF 19 and FGF21 peptide sequences can in some examples include sequences that do not increase or induce hepatocellular carcinoma (HCC) formation or HCC tumorigenesis, do not induce a substantial elevation or increase in lipid profile, or both.
  • HCC hepatocellular carcinoma
  • mutant FGF19 proteins and chimeras of mutant FGF19 and FGF21 peptide sequences can in some examples include sequences that have reduced HCC formation compared to native FGF19, have greater glucose lowering activity compared to native FGF19, have less lipid increasing activity compared to native FGF 19, have less triglyceride, cholesterol, non-HDL or HDL increasing activity compared to native FGF19, have less lean mass reducing activity compared to native FGF21, or combinations thereof.
  • Such FGF 19 mutants and chimeras can be used alone or in combination with other agents, such as other glucose reducing agents, such as thiazolidinedione.
  • methods of reducing blood glucose, treating a metabolic disease, and/or treating a bile acid disease in a mammal which include administering a therapeutically effective amount of a mutated mature FGF 19 protein to the mammal, or a nucleic acid molecule encoding the mutated mature FGF19 protein or a vector comprising the nucleic acid molecule, thereby reducing the blood glucose, treating the metabolic disease, and/or treating the bile acid disease.
  • Exemplary metabolic diseases that can be treated with the disclosed methods include but are not limited to: type 2 diabetes, non-type 2 diabetes, type 1 diabetes, polycystic ovary syndrome (PCOS), metabolic syndrome (MetS), obesity, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperlipidemia, hypertension, latent autoimmune diabetes (LAD), or maturity onset diabetes of the young (MODY).
  • PCOS polycystic ovary syndrome
  • MetS metabolic syndrome
  • NASH non-alcoholic steatohepatitis
  • NAFLD non-alcoholic fatty liver disease
  • LAD latent autoimmune diabetes
  • MODY maturity onset diabetes of the young
  • Exemplary bile acid diseases that can be treated with the disclosed methods include but are not limited to: a metabolic syndrome; a lipid or glucose disorder; cholesterol or triglyceride metabolism; type 2 diabetes; cholestasis, intrahepatic cholestasis, primary biliary cirrhosis (PBC), primary familial intrahepatic cholestasis (PFIC), progressive PFIC, primary sclerosing choangitis (PSC), pregnancy intrahepatic cholestasis (PIC), neonatal cholestasis, and drug induced cholestasis, diseases of extrahepatic cholestasis, bile duct compression from tumor, bile duct blockade by gall stones, bile acid malabsorption and other disorders involving the distal small intestine, ileal resection, inflammatory bowel diseases, Crohn's disease, ulcerative colitis, idiopathic disorders impairing absorption of bile acids, diarrhea, bile
  • the mutated mature FGF19 protein comprises a deletion of at least 5, at least 6, at least 7, at least, 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous N-terminal amino acids, and in some examples the mutated FGF19 protein has reduced mitogenic activity as compared to native mature FGF19 protein.
  • deletion of N-terminal amino acids further includes replacing the deleted N-terminal amino acids with at least 5 contiguous amino acids from FGF21, such as a mammalian FGF21 (e.g., SEQ ID NO: 15), such as at least 5 contiguous amino acids of HPIPDSSPLLQFGGQV (amino acids 1 to 16 of SEQ ID NO: 12).
  • FGF21 such as a mammalian FGF21 (e.g., SEQ ID NO: 15)
  • HPIPDSSPLLQFGGQV amino acids 1 to 16 of SEQ ID NO: 12
  • At least one point mutation includes a mutation at one or more of R23, Y26, R50, 160, 61, E102, Y109, Nl 10, P145, and L147, wherein the numbering refers to the sequence shown SEQ ID NO: 3, and wherein the mutated FGF19 protein in some examples has reduced mitogenic activity as compared to native mature FGF19 protein.
  • Specific exemplary point mutations are shown in Table 1.
  • the mutated FGF19 protein includes a combination of deletions and point mutations at its N-terminal end, such as an M70 mutation, wherein amino acids 2-6 of SEQ ID NO: 3 are deleted, and the following point mutations are made to the N-terminal end: M added to N terminus, A8S, G9S and HI 1L (see for example Zhou ei a/. Cancer Res. 74:3306- 16, 2014).
  • the resulting mutated FGF 19 has the N-terminal sequence of MRDSSPL (amino acids 1-7 of SEQ ID NO: 8).
  • the mutated mature FGF19 protein comprises at least 80%, at least
  • the mutated mature FGF19 protein comprises or consists of any of SEQ ID NOS: 6, 7, 8, 9, or 10.
  • the FGF 19 mutant is part of a chimeric protein, such as one that includes at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 10, at least 20, at least 30, at least 40, or at least 50 contiguous amino acids from FGF21.
  • chimeric proteins include a linker between the FGF 19 mutant and the FGF21 sequence.
  • the FGF21/FGF19 chimeric protein comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11 or 12.
  • the FGF21/FGF19 chimeric protein comprises or consists of the sequence shown in SEQ ID NO: 11 or 12.
  • mutated FGF19 proteins which can include deletion of an N- terminal portion of FGF19, point mutations (such as amino acid substitutions, deletions, additions, or combinations thereof), or combinations of N-terminal deletions and point mutations, and methods of their use to lower glucose, treat a metabolic disease, and/or treat a bile acid related disease (for example reduce fed and fasting blood glucose, improve insulin sensitivity and glucose tolerance, reduce systemic chronic inflammation, ameliorate hepatic steatosis in a mammal, reduce serum bile acids, or combinations thereof).
  • point mutations such as amino acid substitutions, deletions, additions, or combinations thereof
  • N-terminal deletions and point mutations and methods of their use to lower glucose, treat a metabolic disease, and/or treat a bile acid related disease (for example reduce fed and fasting blood glucose, improve insulin sensitivity and glucose tolerance, reduce systemic chronic inflammation, ameliorate hepatic steatosis in a mammal, reduce serum bile acids, or combinations thereof).
  • such mutations reduce the mitogenicity of mature FGF19 (e.g., SEQ ID NO: 3), such as a reduction of at least 20%, at least 50%, at least 75% or at least 90% .
  • the mutant FGF19 protein is a truncated version of the mature protein (e.g., SEQ ID NO: 3), which can include for example deletion of at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous N-terminal amino acids of mature FGF19.
  • the mutant FGF19 protein is a mutated version of the mature protein (e.g., SEQ ID NO: 3), such as one containing at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acid substitutions (such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions), such as one or more of those shown in Table 1.
  • the mutant FGF19 protein includes both an N-terminal truncation and point mutations.
  • the mutant FGF19 protein includes at least 30, at least 40, at least 50, at least 60 or at least 70 consecutive amino acids from amino acids 21- 153 of mature FGF19 (e.g., of SEQ ID NO: 3), (which in some examples can include 1-20 point mutations, such as substitutions, deletions, or additions).
  • the mutated mature FGF19 protein comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, or 10.
  • the mutated mature FGF19 protein comprises or consists of any of SEQ ID NOS: 6, 7, 8, 9, or 10.
  • the FGF19 mutants provided herein are used to generate a chimeric protein, such as an FGF21 FGF19 chimeric protein.
  • a chimeric protein such as an FGF21 FGF19 chimeric protein.
  • the C-terminal end or the N- terminal end of the disclosed FGF19 mutants can be joined directly or indirectly to a fragment of FGF21, such as at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous amino acids of a mammalian FGF21 (such as a fragment of SEQ ID NO: 15).
  • FGF21 that can be used in the FGF21/FGF19 chimeric protein is amino acids 1-16 of SEQ ID NO: 12 (HPIPDSSPLLQFGGQV).
  • Specific exemplary FGF19 mutant proteins are shown in SEQ ID NOS: 6, 7, 8, 9, and 10, which can be used to generate any of the chimeras provided herein.
  • Specific exemplary FGF21/FGF19 chimeras are shown in SEQ ID NOS: 11 and 12.
  • Such chimeric proteins can be used in the methods provided herein.
  • nucleic acid molecules encoding the disclosed mutant FGF19 proteins and chimeras.
  • Vectors and cells that include such nucleic acid molecules are also provided.
  • FIG. 1 shows an alignment of FGF1 (SEQ ID NO: 13), FGF2 (SEQ ID NO: 14), FGF19 (amino acids 26-175 of SEQ ID NO: 2), and FGF21 (amino acids 28-172 of SEQ ID NO: 15), with amino acids that form beta strands in bold, and other relevant residues highlighted and their interaction noted.
  • FIGS. 2A and 2B show an exemplary wild-type mature FGF19 sequence (SEQ ID NO: 3), point mutations that can be made to mature FGF19 (SEQ ID NO: 6), N-terminal deletions (in some examples with point mutations) that can be made to mature FGF19 (SEQ ID NOS: 7- 10), and chimeras with FGF21/FGF19 (SEQ ID NOS: 11- 12).
  • nucleic and amino acid sequences are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NOS: 1 and 2 provide an exemplary human FGF19 nucleic acid and protein sequences, respectively.
  • the signal peptide is amino acids 1-22 (nt 464-529), and the mature peptide ammo acids 23-216 (shown in SEQ ID NO: 3) (encoded by nt 530-11 1 1).
  • SEQ ID NO: 3 provides an exemplary mature form of a human FGF19 protein sequence
  • SEQ ID NOS: 4 and 5 provide an exemplary mouse FGF15 nucleic acid and protein sequences, respectively.
  • Signal peptide is nt 148-22, aa 1-25.
  • Mature peptide is nt 223-801 , aa 26-218.
  • SEQ ID NO: 6 provides an exemplary mature form of FGF19 with point mutations R23V and Nl 10V (wherein numbering refers to SEQ ID NO: 3) to reduce mitogenic activity.
  • SEQ ID NO: 7 provides an exemplary N-terminally truncated form of FGF19, with some of the amino acids deleted and some mutated, resulting the a N-terminal sequence of MRDSSPL (FGF19-M70 (23-216 ⁇ )) to reduce mitogenic activity.
  • SEQ ID NO: 8 provides an exemplary N-terminally truncated form of FGF19, with some of the amino acids deleted and some mutated, resulting in a N-terminal sequence of MRDSSPL, and point mutations, underlined (R23V, Nl lOV, wherein numbering refers to SEQ ID NO: 3) (FGF19-M70 (23-216 ⁇ )), to reduce mitogenic activity.
  • SEQ ID NO: 9 provides an exemplary N-terminally truncated form of FGF19
  • FGF19 ANT (43-216acc), which has the first 20 N-terminal amino acids deleted from the mature form (SEQ ID NO: 3).
  • SEQ ID NO: 10 provides an exemplary mature form of FGF19 with an N-terminal deletion (20 amino acids from the mature form) and two point mutations (R23V and Nl 10V, wherein numbering refers to SEQ ID NO: 3), FGF19 ANT (43-216aa) R23V, Nl lOV.
  • SEQ ID NO: 1 1 provides an exemplary FGF21/FGF19 chimera, wherein the 16 N- terminal amino acids (underlined) are from FGF21, and the 20 N-terminal amino acids of the mature form of FGF19 are deleted (FGF21/19 (34-216 ⁇ )).
  • SEQ ID NO: 12 provides an exemplary FGF21/FGF19 chimera with two point muations in bold (R23V, Nl lOV, wherein numbering refers to SEQ ID NO: 3), wherein the 16 N-terminal amino acids (underlined) are from FGF21, and the 20 N- terminal amino acids of the mature form of FGF19 are deleted (FGF21/19 (34-216 ⁇ ) R23V, N110V).
  • SEQ ID NO: 13 provides an exemplary mature form of FGF1 (140 aa, sometimes referred to in the art as FGF1 15-154).
  • SEQ ID NO: 14 provides an exemplary portion of an FGF2 protein sequence.
  • SEQ ID NO: 15 provides an exemplary exemplary human FGF21 protein sequence. Obtained from GenBank Accession No. AAQ89444.1. The mature form of FGF21 is about amino acids 21-208. DETAILED DESCRIPTION
  • exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
  • Bile acid related or associated disorders/diseases A transient or chronic abnormal level of one or more bile acids present in a subject (such as a mammalian or human subject), such as disrupted bile acid production, so that administration of mutant FGF19 treats the disease.
  • the disease can be caused by an increase or decrease or a delay in bile acid synthesis, metabolism or absorption such that the subject has a bile acid level not typically found in normal subjects.
  • Exemplary bile acid disorders that can be treated with the FGF19 mutants and chimeras provided herein include but are not limited to: metabolic syndrome; a lipid- or glucose- related disorder; cholesterol or triglyceride metabolism; type 2 diabetes; cholestasis, including, for example diseases of intrahepatic cholestasis (e.g., primary biliary cirrhosis (PBC), primary familial intrahepatic cholestasis (PFIC) (e.g., progressive PFIC), primary sclerosing choangitis (PSC), pregnancy intrahepatic cholestasis (PIC), neonatal cholestasis, and drug induced cholestasis (e.g., estrogen)), and diseases of extrahepatic cholestasis (e.g., bile duct compression from tumor, bile duct blockade by gall stones); bile acid malabsorption and other disorders involving the distal small intestine, including ile
  • bile acid diarrhea e.g., bile acid diarrhea (BAD)
  • BAD bile acid diarrhea
  • GI GI, liver, and/or biliary cancers (e.g., colon cancer and hepatocellular cancer); and/or bile acid synthesis abnormalities, such as those contributing to non-alcoholic steatohepatitis
  • NASH Newcastle disease virus
  • cirrhosis cirrhosis and portal hypertension.
  • Other specific examples are known in the art (e.g., see WO 2014/105939, herein incorporated by reference).
  • C-terminal portion A region of a protein sequence that includes a contiguous stretch of amino acids that begins at or near the C-terminal residue of the protein.
  • a C-terminal portion of the protein can be defined by a contiguous stretch of amino acids (e.g. , a number of amino acid residues).
  • Chimeric protein A protein that includes at least a portion of the sequence of a full- length first protein (e.g., FGF19) and at least a portion of the sequence of a full-length second protein (e.g., FGF21), where the first and second proteins are different.
  • a chimeric polypeptide also encompasses polypeptides that include two or more non-contiguous portions derived from the same polypeptide. The two different peptides can be joined directly or indirectly, for example using a linker.
  • Diabetes mellitus A group of metabolic diseases in which a subject has high blood sugar, either because the pancreas does not produce enough insulin, or because cells do not respond to the insulin that is produced. Type 1 diabetes results from the body's failure to produce insulin.
  • IDDM insulin-dependent diabetes mellitus
  • NIDDM non insulin-dependent diabetes mellitus
  • adult-onset diabetes The defective responsiveness of body tissues to insulin is believed to involve the insulin receptor.
  • Diabetes mellitus is characterized by recurrent or persistent hyperglycemia, and in some examples diagnosed by demonstrating any one of:
  • Effective amount or Therapeutically effective amount The amount of agent, such as a mutated FGF19 protein or chimera (or nucleic acid encoding such) disclosed herein, that is an amount sufficient to prevent, treat (including prophylaxis), reduce and/or ameliorate the symptoms and/or underlying causes of any of a disorder or disease.
  • an "effective amount" is sufficient to reduce or eliminate a symptom of a disease, such as a bile acid disorder, or a metabolic disorder such as diabetes (such as type II diabetes), for example by lowering serum bile acids and/or blood glucose.
  • Fibroblast Growth Factor 19 (e.g., OMEVI 603891). Includes FGF19 nucleic acid molecules and proteins (known as Fgf 15 in rodents). FGF19 is a hormone-like protein that regulates carbohydrate, lipid and bile acid metabolism. FGF 19 acts through receptor complex FGFR4 ⁇ -Klotho (KLB) to regulate bile acid metabolism. The murine ortholog of FGF19 is Fgf 15. FGF19 sequences are publically available, for example from the GenBank® sequence database (e.g., Accession Nos. NP_005108.1 , AAQ88669.1 , NP_032029.1 ,
  • NP_570109.1 and NP_032029.1 provide exemplary native FGF19 protein sequences, while Accession Nos. AY358302.1, NM_008003.2, and NM_005117.2 provide exemplary native FGF19 nucleic acid sequences). Specific examples are provided in SEQ ID NOS: 1-5.
  • FGF19 sequence is one that does not include a mutation that alters the normal activity of the protein (e.g., SEQ ID NO: 3 or amino acids 26-218 of SEQ ID NO: 5).
  • a mutated FGF19 is a variant of FGF19 with different biological activity, such as reduced mitogenicity, such as reduced effects on tumor growth (e.g., a variant of any of SEQ ID NOS: 1-5, such as one having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any of SEQ ID NOS: 1-5).
  • such a variant includes an N- terminal truncation, at least one point mutation, or combinations thereof, such as changes that decrease mitogenicity of FGF19.
  • Specific exemplary FGF19 mutant proteins are shown in SEQ ID NOS: 6, 7, 8, 9, and 10.
  • Mutated FGF19 proteins also include chimeric proteins that include a mutated FGF19 protein, for example chimeras that include a portion of FGF21 (such as a mammalian or human FGF21).
  • Specific exemplary FGF21/FGF19 chimeric proteins are shown in SEQ ID NOS: 11 and 12.
  • Fibroblast Growth Factor 21 (e.g., OMEVI 609436). Includes FGF21 nucleic acid molecules and proteins. FGF21 stimulates glucose uptake in adipocytes. FGF21 sequences are publically available, for example from the GenBank® sequence database (e.g., Accession Nos. AAQ89444.1 , NP_061986, and AAH49592.1 provide exemplary native FGF21 protein sequences, while Accession Nos. AY359086.1 and BC049592 provide exemplary native FGF21 nucleic acid sequences). One of ordinary skill in the art can identify additional FGF21 nucleic acid and protein sequences, including FGF21 variants. An exemplary FGF21 protein sequence is shown in SEQ ID NO: 15.
  • Host cells Cells in which a vector can be propagated and its DNA expressed.
  • the cell may be prokaryotic or eukaryotic.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used.
  • host cells can be transgenic, in that they include nucleic acid molecules that have been introduced into the cell, such as a nucleic acid molecule encoding a mutant FGF19 protein disclosed herein.
  • Isolated An "isolated" biological component (such as a mutated FGF19 protein or nucleic acid molecule) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA and RNA, and proteins.
  • Nucleic acids molecules and proteins which have been "isolated” thus include nucleic acids and proteins purified by standard purification methods.
  • the term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • a purified or isolated cell, protein, or nucleic acid molecule can be at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.
  • Linker A moiety or group of moieties that joins or connects two or more discrete separate peptide or proteins, such as monomer domains, for example to generate a chimeric protein (such as an FGF21/mutant FGF19 protein).
  • a linker is a substantially linear moiety.
  • Exemplary linkers that can be used to generate the chimeric proteins provided herein include but are not limited to: peptides, nucleic acid molecules, peptide nucleic acids, and optionally substituted alkylene moieties that have one or more oxygen atoms incorporated in the carbon backbone.
  • a linker can be a portion of a native sequence, a variant thereof, or a synthetic sequence.
  • Linkers can include naturally occurring amino acids, non-naturally occurring amino acids, or a combination of both.
  • a linker is composed of at least 5, at least 10, at least 15 or at least 20 amino acids, such as 5 to 10, 5 to 20, or 5 to 50 amino acids.
  • the linker is a poly alanine.
  • Mammal This term includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects (such as cats, dogs, cows, and pigs).
  • Metabolic disorder/disease A disease or disorder that results from the disruption of the normal mammalian process of metabolism. Includes metabolic syndrome.
  • glucose utilization disorders and the sequelae associated therewith including diabetes mellitus (Type I and Type-2), gestational diabetes, hyperglycemia, insulin resistance, abnormal glucose metabolism, "pre-diabetes” (Impaired Fasting Glucose (IFG) or Impaired Glucose Tolerance (IGT)), and other physiological disorders associated with, or that result from, the hyperglycemic condition, including, for example, histopathological changes such as pancreatic ⁇ -cell destruction; (2) dyslipidemias and their sequelae such as, for example, atherosclerosis, coronary artery disease, cerebrovascular disorders and the like; (3) other conditions which may be associated with the metabolic syndrome, such as obesity and elevated body mass (including the co-morbid conditions thereof such as, but not limited to, nonalcoholic fatty liver disease (NAFLD), nonalcoholic fatty liver disease (NAFLD), nonalcoholic fatty liver disease
  • NASH steatohepatitis
  • PCOS polycystic ovarian syndrome
  • inflammatory reactions are involved, including atherosclerosis, chronic inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis), asthma, lupus erythematosus, arthritis, or other inflammatory rheumatic disorders; (5) disorders of cell cycle or cell differentiation processes such as adipose cell tumors, lipomatous carcinomas including, for example, liposarcomas, solid tumors, and neoplasms; (6) neurodegenerative diseases and/or demyelinating disorders of the central and peripheral nervous systems and/or neurological diseases involving neuroinfiammatory processes and/or other peripheral neuropathies, including Alzheimer's disease, multiple sclerosis, Parkinson's disease, progressive multifocal leukoencephalopathy and Guillian-Barre syndrome; (7) skin and dermatological disorders and/or disorders of wound healing processes, including erythemato-squamous dermatoses; and (8) other disorders such as syndrome X, osteoarthritis, and acute respiratory distress syndrome.
  • Other examples are
  • N-terminal portion A region of a protein sequence that includes a contiguous stretch of amino acids that begins at or near the N-terminal residue of the protein.
  • An N-terminal portion of the protein can be defined by a contiguous stretch of amino acids (e.g. , a number of amino acid residues).
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence (such as a mutated FGF19 coding sequence).
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions e.g. , powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Promoter Ann array of nucleic acid control sequences which direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
  • a recombinant nucleic acid molecule is one that has a sequence that is not naturally occurring (e.g., a mutated FGF19 or chimeric protein) or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by routine methods, such as chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, such as by genetic engineering techniques.
  • a recombinant protein is one encoded for by a recombinant nucleic acid molecule.
  • a recombinant or transgenic cell is one that contains a recombinant nucleic acid molecule and expresses a recombinant protein.
  • Sequence identity The similarity between amino acid (or nucleotide) sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.
  • BLAST Basic Local Alignment Search Tool
  • NCBI Bethesda, MD
  • sequence analysis programs blastp, blastn, blastx, tblastn and tblastx A description of how to determine sequence identity using this program is available on the NCBI website on the internet.
  • Variants of the mutated FGF19 proteins and coding sequences disclosed herein are typically characterized by possession of at least about 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity counted over the full length alignment with the amino acid sequence using the NCBI Blast 2.0, gapped blastp set to default parameters.
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • sequence identity When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 95%, at least 98%, or at least 99% sequence identity.
  • homologs and variants When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or at least 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
  • a mutant FGF19 protein disclosed herein can have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 6, 7, 8, 9, or 10.
  • exemplary FGF21/mutant FGF19 chimeras in some examples have at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11 or 12.
  • Subject Any mammal, such as humans, non-human primates, pigs, sheep, cows, dogs, cats, rodents and the like which is to be the recipient of the particular treatment, such as treatment with a mutated FGF19 protein or chimeric protein (or corresponding nucleic acid molecule) provided herein.
  • a subject is a human subject or a murine subject.
  • the subject has a metabolic disease, such as type 2 diabetes, non-type 2 diabetes, type 1 diabetes, polycystic ovary syndrome (PCOS), metabolic syndrome (MetS), obesity, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperlipidemia, hypertension, latent autoimmune diabetes (LAD), and maturity onset diabetes of the young (MODY).
  • a metabolic disease such as type 2 diabetes, non-type 2 diabetes, type 1 diabetes, polycystic ovary syndrome (PCOS), metabolic syndrome (MetS), obesity, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperlipidemia, hypertension, latent autoimmune diabetes (LAD), and maturity onset diabetes of the young (MODY).
  • the subject has a bile acid disorder.
  • the subject has elevated blood glucose, serum bile acids, or both.
  • a virus or vector "transduces” a cell when it transfers nucleic acid into the cell.
  • a cell is “transformed” or “transfected” by a nucleic acid transduced into the cell when the DNA becomes stably replicated by the cell, either by incorporation of the nucleic acid into the cellular genome, or by episomal replication.
  • fusion e.g., liposomes
  • receptor- mediated endocytosis e.g., DNA-protein complexes, viral envelope/capsid-DNA complexes
  • viruses such as recombinant viruses ⁇ Wolff, J. A., ed, Gene Therapeutics, Birkhauser, Boston, USA ( 1994) ⁇ .
  • retroviruses the infecting retrovirus particles are absorbed by the target cells, resulting in reverse transcription of the retroviral RNA genome and integration of the resulting provirus into the cellular DNA.
  • Transgene An exogenous gene supplied by a vector.
  • a transgene includes a mutated FGF19 coding sequence.
  • a vector may include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication.
  • a vector may also include one or more mutated FGF19 coding sequences and/or selectable marker genes and other genetic elements known in the art.
  • a vector can transduce, transform or infect a cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell.
  • a vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating or the like.
  • Hepatocellular carcinoma one of the leading causes of cancer-related death, develops from premalignant lesions in chronically damaged livers. While it is known that fibroblast growth factor (FGF) 19 acts through receptor complex FGFR4- -Klotho (KLB) to regulate bile acid metabolism, FGF19 is also implicated in the development of HCC. In humans, FGF 19 is amplified in HCC and its expression is induced in the liver under cholestatic and cirrhotic conditions. In mice, ectopic overexpression of FGF19 drives HCC development in a process that requires FGFR4.
  • FGF fibroblast growth factor
  • mutants of FGF19 that can reduce or eliminate undesirable mitogenic activity and retain the therapeutic potential for FGF19, which includes the treatment of chronic liver diseases, as well as obesity and diabetes and other metabolic disorders.
  • methods of reducing blood glucose, treating a metabolic disease, and/or treating a bile acid disease in a mammal include administering a therapeutically effective amount of a mutated mature FGF19 protein to the mammal, or a nucleic acid molecule encoding the mutated mature FGF19 protein or a vector comprising the nucleic acid molecule, thereby reducing the blood glucose, treating the metabolic disease, and/or treating the bile acid disease.
  • Exemplary metabolic diseases that can be treated with the disclosed methods include but are not limited to: type 2 diabetes, non-type 2 diabetes, type 1 diabetes, polycystic ovary syndrome (PCOS), metabolic syndrome (MetS), obesity, nonalcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperlipidemia, hypertension, latent autoimmune diabetes (LAD), or maturity onset diabetes of the young (MODY).
  • PCOS polycystic ovary syndrome
  • MetS metabolic syndrome
  • NASH nonalcoholic steatohepatitis
  • NAFLD non-alcoholic fatty liver disease
  • LAD latent autoimmune diabetes
  • MODY maturity onset diabetes of the young
  • Exemplary bile acid diseases that can be treated with the disclosed methods include but are not limited to: a metabolic syndrome; a lipid or glucose disorder; cholesterol or triglyceride metabolism; type 2 diabetes; cholestasis, intrahepatic cholestasis, primary biliary cirrhosis (PBC), primary familial intrahepatic cholestasis (PFIC), progressive PFIC, primary sclerosing choangitis (PSC), pregnancy intrahepatic cholestasis (PIC), neonatal cholestasis, and drug induced cholestasis, diseases of extrahepatic cholestasis, bile duct compression from tumor, bile duct blockade by gall stones, bile acid malabsorption and other disorders involving the distal small intestine, ileal resection, inflammatory bowel diseases, Crohn's disease, ulcerative colitis, idiopathic disorders impairing absorption of bile acids, diarrhea, bile
  • Such methods can include administering a therapeutically effective amount of a mutated mature FGF19 protein to the mammal, or a nucleic acid molecule encoding the mutated FGF19 protein or a vector comprising the nucleic acid molecule, thereby reducing fed and fasting blood glucose, improving insulin sensitivity and glucose tolerance, reducing systemic chronic inflammation, ameliorating hepatic steatosis, reducing serum bile acids, or combinations thereof, in a mammal.
  • the fed and fasting blood glucose is reduced in the treated subject by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%), or at least 90% as compared to an absence of administration of mutant FGF19.
  • insulin sensitivity and glucose tolerance is increased in the treated subject by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 90% as compared to an absence of administration of mutant FGF19.
  • systemic chronic inflammation is reduced in the treated subject by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 90% as compared to an absence of administration of mutant FGF19.
  • hepatic steatosis is reduced in the treated subject by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 90% as compared to an absence of administration of mutant FGF19.
  • serum bile acids are reduced in the treated subject by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 90% as compared to an absence of administration of mutant FGF1 . In some examples, combinations of these reductions are achieved.
  • the mutated mature FGF19 protein used in the disclosed methods can include a deletion of at least six contiguous N-terminal amino acids, at least one point mutation, or combinations thereof. Specific examples of such proteins are provided herein.
  • the mutated mature FGF19 protein has reduced hepatocellular carcinoma (HCC) formation compared to native FGF19, has greater glucose lowering activity compared to native FGF19 (e.g. , SEQ ID NO: 3 or amino acids 26-218 of SEQ ID NO: 5), has less lipid increasing activity compared to native FGF19, has less triglyceride, cholesterol, non-HDL or HDL increasing activity compared to native FGF19, has less lean mass reducing activity compared to native FGF21, or combinations thereof.
  • HCC hepatocellular carcinoma
  • mitogenic activity such as HCC formation
  • mitogenic activity is reduced with the mutated FGF19 by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%-, or at least 90% as compared to native FGF19.
  • glucose lowering activity is increased with the mutated FGF19 by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 90% as compared to native FGF19.
  • lipid increasing activity is reduced with the mutated FGF19 by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 90% as compared to native FGF19.
  • triglyceride, cholesterol, non-HDL and/or or HDL increasing activity is reduced with the mutated FGF19 by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 90% as compared to native FGF19.
  • lean mass reducing activity is reduced with the mutated FGF19 by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 90% as compared to native FGF21.
  • the mutated mature FGF19 protein used in the disclosed methods has at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous N-terminal amino acids deleted from the mature native FGF19 protein, wherein the mutated FGF19 protein has reduced mitogenic activity as compared to native mature FGF19 protein.
  • the deleted N-terminal amino acids are replaced with other amino acids.
  • the mutated mature FGF19 protein used in the disclosed methods has at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous N-terminal amino acids deleted from the mature native FGF19 protein, wherein the mutated FGF19 protein has reduced mitogenic activity as compared to native mature FGF19 protein.
  • the deleted N-terminal amino acids are replaced with other amino acids.
  • the mutated mature FGF19 protein used in the disclosed methods has at least 9, at least 10, at
  • FGF19 protein used in the disclosed methods has at least one point mutation at one or more of R23, Y26, R50, 160, K61, E102, Y109, N110, P145, and L147, wherein the numbering refers to the sequence shown SEQ ID NO: 3, and wherein the mutated FGF19 protein has reduced mitogenic activity as compared to native mature FGF19 protein.
  • Exemplary point mutations are provided in Table 1.
  • the at least one point mutation includes a mutation at R23 and N110 (such as R23V and Nl lOV), wherein the numbering refers to the sequence shown SEQ ID NO: 3, and wherein the mutated FGF19 protein has reduced mitogenic activity as compared to wild-type mature FGF19 protein.
  • the mutated mature FGF19 protein used in the disclosed methods has a combination of N-terminal deletions and amino acid substitutions.
  • Specific exemplary mutated mature FGF19 proteins include those having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, or 10 (which in some examples include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the mutations shown in Table 1), and which have reduced mitogenic activity.
  • the mutated mature FGF19 protein includes or consists of any of SEQ ID NOS: 6, 7, 8, 9, or 10.
  • the mutated mature FGF19 protein is part of a chimeric protein further comprising at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous amino acids of a mammalian FGF21.
  • the FGF21/mutated FGF19 chimeric protein comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11 or 12 (which in some examples include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the mutations shown in Table 1).
  • the FGF21/mutated FGF19 chimeric protein comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11
  • FGF21/mutated FGF19 chimeric protein includes or consists of SEQ ID NO: 11 or 12. Any routine method of administration can be used, such as subcutaneous, intraperitoneal, intramuscular, or intravenous. In some examples, the therapeutically effective amount of the mutated mature FGF19 protein is at least 0.5 mg/kg.
  • Exemplary subjects that can be treated with the disclosed methods include mammals, such as human and veterinary subjects, such as a cat or dog or livestock.
  • the mammal, such as a human, cat or dog has diabetes.
  • the mammal, such as a human, cat or dog has a metabolic disease.
  • the mammal, such as a human, cat or dog has a bile acid disorder.
  • mutated FGF19 proteins that can include an N-terminal deletion, one or more point mutations (such as amino acid substitutions, deletions, additions, or combinations thereof), or combinations of N-terminal deletions and point mutations.
  • Such mutated FGF19 proteins can be part of a chimeric protein, such as one that includes a portion of FGF21 (e.g., SEQ ID NO: 15).
  • FGF21 e.g., SEQ ID NO: 15
  • such reference also includes reference to FGF21/ mutated FGF19 chimeras.
  • an isolated mutated mature FGF19 protein has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, or 10 (which in some examples include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the mutations shown in Table 1).
  • FGF19 mutant proteins or their nucleic acid coding sequences
  • mutations in FGF19 reduce the mitogenicity of mature FGF19 (e.g., SEQ ID NO: 3), such as a reduction of at least 20%, at least 50%, at least 75% or at least 90% relative to a native mature FGF19 (e.g., SEQ ID NO: 3 or amino acids 26-218 of SEQ ID NO: 5).
  • the mutant FGF19 protein is a truncated version of the mature protein (e.g., SEQ ID NO: 3 or amino acids 26-218 of SEQ ID NO: 5), which can include for example deletion of at least 5, at least 6, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive N- terminal amino acids, such as the N-terminal 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,1 5, 16, 17, 18, 19 or 20 amino acids of mature FGF19.
  • such an N-terminally deleted FGF19 protein has reduced mitogenic activity as compared to a native mature FGF19 protein.
  • the deleted N-terminal amino acids are replaced with contiguous amino acids from FGF21 (e.g., see SEQ ID NO: 15), to generate an FGF21/FGF19 chimera, such as at least 3, at least 4, at least 5, at least 10, at least 15, or at least 20 contiguous amino acids from FGF21 (such as from the N-terminal half or N-terminal quarter of FGF21), such as 5 to 20, 4-6, 4-9, 3- 10, 12- 16, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19 or 20 contiguous amino acids from FGF21.
  • Examples of an FGF19 mutated protein with an N- terminal deletion having 16 contiguous amino acids from FGF21 are shown in SEQ ID NOs: 11 and 12.
  • amino acids from other FGFs besides FGF21 can be used, including those having low affinity for FGFR4, including FGF3, FGF5, FGF7, FGF9 and FGF10.
  • mutations in FGF19 increase the thermostability of mature or truncated FGF19, such as an increase of at least 20%, at least 50%, at least 75% or at least 90%.
  • Exemplary mutations that can be used to increase the thermostability of mutated FGF19 include but are not limited to one or more of: R23V, Nl 10V, I60V, K61I, and E102V, wherein the numbering refers to SEQ ID NO: 3.
  • mutated FGF19 can be mutated to increase the thermostability of the protein compared to an FGF19 protein without the modification. Methods of measuring thermostability are known in the art.
  • the mutant FGF19 protein is a mutated version of the mature protein (e.g., SEQ ID NO: 3 or amino acids 26-218 of SEQ ID NO: 5), such as one containing at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24 or at least 25 amino acid substitutions, such as 1-20, 1-10, 2-4, 4-8, 5-25, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions.
  • the mutant FGF19 protein is a mutated version of the mature protein (e.g., SEQ ID NO: 3 or amino acids 26-218 of SEQ ID NO: 5), such as one containing at least 1, at least 2, at least 3, at least 4, at least 5, at least 6,
  • FGF19 protein includes deletion of one or more amino acids, such as deletion of 1- 10, 10-20, 4- 8, 5- 10, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.
  • the mutant FGF19 protein includes a combination of amino acid substitutions and deletions, such as at least 1 substitution and at least 1 deletion, such as 1 to 10 substitutions with 1 to 20 deletions.
  • Exemplary FGF19 mutations are shown in Table 1 below, with amino acids referenced to either SEQ ID NO: 2 or 3.
  • these mutations can be used singly, or in combination (such as 1-10, 1 -8, 1-2, 2-4, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of these amino acid substitutions).
  • mutant FGF19 proteins can be part of a chimeric protein, such as with a portion of FGF21.
  • Table 1 Exemplary FGF19 mutations
  • the mutant FGF19 protein includes mutations at one or more of the following positions, such as 1 , 2, 3 4, 5, 6 ,7, 8, 9 or 10 of these positions: R23, Y26, R50, 160, K61 , E102, Y109, N110, P145, or L147, (wherein the numbering refers to SEQ ID NO: 3), such as one or more of R23V, R23C, Y26F, Y26A, Y26V, R50E, R50V, I60V, K61L E102V, E102A, E102S, E102T, Y109V, Y109F, Y109A, N110V, N1 10A, N110S, N1 10T, P145S, P145A, L147S, and L147A (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of these mutations).
  • positions such as 1 , 2, 3 4, 5, 6 ,7, 8, 9 or 10 of these positions: R23, Y26, R50, 160,
  • mutant FGF 19 protein can include other changes, such as 1-20, 1-10, or 1-5 conservative amino acid substitutions that do not adversely affect the function of the mutated protein (such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions).
  • the mutant FGF19 protein includes mutations at R23 and Nl 10 (wherein the numbering refers to SEQ ID NO: 3), such as one of R23V, R23C and one of Nl 10V, Nl 10A, Nl 10S, and Nl 10T.
  • the mutant FGF19 protein includes at least 30, at least 40, at least 50, at least 60 or at least 70 consecutive amino acids from amino acids 21-153 of mature FGF19 (e.g., of SEQ ID NO: 3 or amino acids 26-218 of SEQ ID NO: 5), (which in some examples can include further deletion of 1 to 20 N-terminal amino acids and/or 1-5, 1-10 or 1-20 point mutations, such as substitutions, deletions, or additions).
  • the mutant FGF19 protein includes both an N-terminal truncation and point mutations, such as deletion of at least 5 N-terminal amino acids (such as deletion of 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19 or 20 contiguous N-terminal amino acids) and at least one point mutation (such as at least 2, at least 4, at least 5, at least 8, at least 10, at least 15, at least 20, or at least 30 point mutations, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 ,13, 14, 15, 16, 17, 18, 19 or 20 point mutations).
  • Specific exemplary FGF19 mutant proteins are shown in SEQ ID NOS: 6- 12.
  • the FGF19 mutant includes an N-terminal deletion, but retains a methionine at the N-terminal position.
  • the FGF19 mutant is 140- 300 or 140- 190 amino acids in length.
  • the FGF19 mutant protein is part of a chimeric protein.
  • one end of the mutant FGF19 mutant protein can be joined directly or indirectly to the end of FGF21, such as at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 contiguous amino acids of FGF21 (such as at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 contiguous amino acids contiguous amino acids of SEQ ID NO: 15, such as in the region of amino acids 21-75, 21-50 or 21- 100 of SEQ ID NO: 5).
  • the mutated FGF19 portion of the chimera is at the N-terminus of the chimera
  • the FGF21 portion is the C-terminus
  • the mutant FGF19 and FGF21 portion are linked indirectly through the use of a linker, such as one composed of at least 5, at least 10, at least 15 or at least 20 amino acids.
  • the linker is a poly alanine.
  • the FGF19 mutant protein includes at least 80% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, or 10.
  • the FGF19 mutant protein can have at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, or 10.
  • the FGF19 mutant protein includes or consists of any of SEQ ID NOS: 6, 7, 8, 9, or 10.
  • the disclosure encompasses variants of the disclosed FGF19 mutant proteins, such as any of SEQ ID NOS: 6, 7, 8, 9, or 10 having 1 to 8, 2 to 10, 1 to 5, 1 to 6, or 5 to 10 mutations, such as one or more of those in Table 1, for example in combination with conservative amino acid substitutions.
  • a FGF21/mutant FGF19 chimera protein includes at least 80% sequence identity to SEQ ID NO: 11 or 12.
  • the FGF21/mutant FGF19 chimeric protein can have at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 11 or 12.
  • the FGF21/mutant FGF19 protein includes or consists of SEQ ID NO: 1 1 or 12.
  • the disclosure encompasses variants of the disclosed FGF21/mutant FGF19 chimera proteins, such as SEQ ID NO: 11 or 12 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations, such as conservative amino acid substitutions.
  • nucleic acid molecules encoding the disclosed mutated FGF19 proteins and chimeras, such as a nucleic acid molecule encoding a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9 10, 11 or 12 (which in some examples include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the mutations shown in Table 1).
  • SEQ ID NOS: 6 which in some examples include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the mutations shown in Table 1.
  • SEQ ID NOS: 6 and 4 Based on the coding sequence of native FGF19 shown in SEQ ID NOS: 1 and 4, one skilled in the art can generate a coding sequence of any FGF19 mutant provided herein.
  • Vectors and cells that include such nucleic acid molecules are also provided.
  • nucleic acid molecules can be expressed in a host cell, such as a bacterium or yeast cell (e.g., E. coli), thereby permitting expression of the mutated FGF19 protein.
  • a host cell such as a bacterium or yeast cell (e.g., E. coli)
  • yeast cell e.g., E. coli
  • the resulting mutated FGF19 protein can be purified from the cell.
  • the mutated mature FGF19 protein can include a deletion of at least six contiguous N-terminal amino acids, at least one point mutation, or combinations thereof.
  • such methods include administering a therapeutically effective amount of a disclosed mutated FGF19 protein or chimeric protein including the mutant FGF19 mutant protein, (such as at least 0.01 , at least 0.1 mg kg, or at least 0.5 mg/ g) (or nucleic acid molecules encoding such) to reduce blood glucose and/or serum bile acids in a mammal, such as a decrease of at least 5%, at least 10%, at least 25% or at least 50%.
  • a therapeutically effective amount of a disclosed mutated FGF19 protein or chimeric protein including the mutant FGF19 mutant protein such as at least 0.01 , at least 0.1 mg kg, or at least 0.5 mg/ g
  • nucleic acid molecules encoding such to reduce blood glucose and/or serum bile acids in a mammal, such as a decrease of at least 5%, at least 10%, at least 25% or at least 50%.
  • use of the FGF19 mutants or chimeric proteins including a mutant FGF19 mutant protein, disclosed herein does not lead to (or significantly reduces, such as a reduction of at least 20%, at least 50%, at least 75%, or at least 90%) the adverse side effects observed with thiazolidinediones (TZDs) therapeutic insulin sensitizers, including weight gain, increased liver steatosis and bone fractures (e.g., reduced affects on bone mineral density, trabecular bone architecture and cortical bone thickness).
  • ZTDs thiazolidinediones
  • the present disclosure provides mutated FGF19 proteins that can include an N-terminal deletion, one or more point mutations (such as amino acid substitutions, deletions, additions, or combinations thereof), or combinations of N-terminal deletions and point mutations.
  • Such proteins and corresponding coding sequences can be used in the methods provided herein.
  • the disclosed FGF19 mutant proteins have reduced mitogenicity compared to mature native FGF19 (e.g., SEQ ID NO: 3 or amino acids 26-218 of SEQ ID NO: 5), such as a reduction of at least 20%, at least 50%, at least 75% or at least 90% .
  • Methods of measuring mitogenicity are known in the art.
  • the mutant FGF19 protein is a truncated version of the native mature protein (e.g., SEQ ID NO: 3 or amino acids 26-218 of SEQ ID NO: 5), which can include for example deletion of at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive N-terminal amino acids.
  • the mutant FGF19 protein is a truncated version of the mature protein (e.g., SEQ ID NO: 3 or amino acids 26-218 of SEQ ID NO: 5), such a deletion of the N-terminal 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
  • the FGF19 mutant includes an N-terminal deletion, but retains a methionine at the N-terminal position.
  • the mutated FGF19 protein includes a combination of deletions and point mutations at its N-terminal end, such as an M70 mutation, wherein amino acids 2-6 of SEQ ID NO: 3 are deleted, and the following point mutations are made to the N-terminal end: M added to N terminus, A8S, G9S and HI 1L (see for example Zhou ei a/. Cancer Res. 74:3306- 16, 2014).
  • MRDSSPL amino acids 1-7 of SEQ ID NO: 8
  • such an N-terminally deleted FGF19 protein has reduced mitogenic activity as compared to wild-type mature FGF19 protein.
  • the deleted N-terminal amino acids are replaced with a portion of an FGF21 sequence (e.g., see SEQ ID NO: 15 for a native FGF21 protein), resulting in an FGF21 sequence (e.g., see SEQ ID NO: 15 for a native FGF21 protein), resulting in an FGF21 sequence (e.g., see SEQ ID NO: 15 for a native FGF21 protein), resulting in an FGF21 sequence (e.g., see SEQ ID NO: 15 for a native FGF21 protein), resulting in an FGF21 sequence (e.g., see SEQ ID NO: 15 for a native FGF21 protein), resulting in an FGF21 sequence (e.g., see SEQ ID NO: 15 for a native FGF21 protein), resulting in an FGF21 sequence (e.g., see SEQ ID NO: 15 for a native FGF21 protein), resulting in an FGF21 sequence (e.g., see SEQ ID NO: 15 for a native FGF21 protein), resulting
  • FGF21/FGF19 chimera such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least
  • FGF21 contiguous amino acids from FGF21, such as 1 -5, 1-4, 2-4, 4-6, 4-9, 3- 10, 5 to
  • FGF21 contiguous amino acids from FGF21 (such as from the N-terminal half of FGF21 ).
  • FGF19 mutated protein with an N-terminal deletion having amino acids from FGF21 are shown in SEQ ID NOs: 1 1 and 12.
  • contiguous amino acids from another FGF having low affinity for FGFR4 including FGF3, FGF5, FGF7, FGF9 and FGF10 can be used to reduce mitogenicity of the resulting FGF19 mutant protein.
  • the mutant FGF19 protein includes at least 30, at least 40, at least 50, at least 60, or at least 70 consecutive amino acids from amino acids 21- 153 of mature FGF19 (e.g., of SEQ ID NO: 3), (which in some examples can include further deletion of 1-20 N-terminal amino acids and/or point mutations, such as substitutions, deletions, or additions).
  • the mutant FGF19 protein is a mutated version of the mature protein (e.g., SEQ ID NO: 3 or amino acids 26-218 of SEQ ID NO: 5), or a N-terminal truncation of the mature protein (e.g., SEQ ID NOS: 7- 1 1), such as one containing at least 1 , at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acid substitutions, such as 1-20, 1- 10, 4-8, 5- 12, 5-10, 5-25, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 amino acid substitutions.
  • point mutations can be introduced into an FGF19 sequence to decrease mitogenicity and/or increase stability, compared to the FGF19 protein without the modification. Specific exemplary point mutations that can be used are shown above in Table 1.
  • the mutant FGF19 protein includes mutations (such as a substitution or deletion) at one or more of the following positions, such as 1 , 2, 3 4, 5, 6, 7, 8, 9 or 10 of these positions: R23, Y26, R50, 160, 61, E102, Y109, N110, P145, or L147, (wherein the numbering refers to SEQ ID NO: 3), such as one or more of R23V, R23C, Y26F, Y26A, Y26V, R50E, R50V, I60V, K61I, E102V, E102A, E102S, E102T, Y109V, Y109F, Y109A, N110V, Nl lOA, Nl lOS, Nl lOT, P145S, P145A, L147S, and L147A (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of these mutations).
  • such an FGF19 protein with one or more point mutations has reduced mitogenic activity as compared to wild-type mature FGF19 protein.
  • FGF19 mutant proteins containing point mutations include but are not limited to the protein sequence shown in any of SEQ ID NOs: 6, 8, 10 and 12.
  • mutations in FGF19 increase the thermostability of mature or truncated FGF 19.
  • mutations can be made at one or more of the following positions.
  • Exemplary mutations that can be used to increase the thermostability of mutated FGF19 include but are not limited to one or more of: R23V, Nl 10V, 160 V, K61I, and El 02V, wherein the numbering refers to SEQ ID NO: 3.
  • the FGF19 mutant protein is part of a chimeric protein.
  • any mutant FGF19 protein provided herein can be joined directly or indirectly at its N- or C- terminal end to a portion of FGF21, such as contiguous amino acids from the N- terminal half or quarter of mature FGF21 (such as amino acids 21- 100 or 21-50 of SEQ ID NO: 15).
  • At least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, or at least 40 contiguous amino acids of mature FGF21 can be part of the chimera.
  • Examples of fragments of FGF21 that can be used are shown in SEQ ID NOS: 11 and 12.
  • the mutant FGF19 protein includes both an N-terminal truncation and point mutations. Specific exemplary FGF19 mutant proteins are shown in SEQ ID NOS: 6, 7, 8, 9 and 10.
  • the FGF19 mutant protein includes at least 80% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9 and 10 (which in some examples include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the mutations shown in Table 1).
  • the FGF19 mutant protein can have at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9 and 10.
  • the FGF19 mutant protein includes or consists of any of SEQ ID NOS: 6, 7, 8, 9 and 10.
  • the disclosure encompasses variants of the disclosed FGF19 mutant proteins, such as any of SEQ ID NOS: 6, 7, 8, 9 and 10 having 1 to 20, 1 to 15, 1 to 10, 1 to 8, 2 to 10, 1 to 5, 1 to 6, 2 to 12, 3 to 12, 5 to 12, or 5 to 10 mutations, such as conservative amino acid substitutions.
  • Such mutant FGF19 proteins can be used to generate an FGF19 mutant chimera.
  • the mutant FGF19 protein has at its N-terminus a methionine. In some examples, the mutant FGF19 protein is at least 120 amino acids in length, such as at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, or at least 175 amino acids in length, such as 140 to 300, 140 to 200, 140 to 190, 150 to 200 or 160 to 190 amino acids in length.
  • N-terminally truncated FGF19 sequences and FGF19 point mutations that can he used to generate an FGF19 mutant protein are shown in Table 1 (as well as those provided in any of SEQ ID NOS: 6, 7, 8, 9 and 10).
  • Table 1 Exemplary N-terminally truncated FGF19 sequences and FGF19 point mutations that can he used to generate an FGF19 mutant protein are shown in Table 1 (as well as those provided in any of SEQ ID NOS: 6, 7, 8, 9 and 10).
  • any N-terminal truncation provided herein can be combined with any FGF19 point mutation in
  • mutations can be made to the sequences shown in any of SEQ ID NOS: 6, 7, 8, 9 and 10, such as one or more of the mutations discussed herein (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, such as conservative amino acid substitutions, deletions, or additions).
  • Exemplary mutant FGF19 proteins are provided in SEQ ID NOS: 6, 7, 8, 9, and 10, and FGF21/mutant FGF19 chimeras in SEQ ID NOS: 1 1 and 12.
  • FGF21/mutant FGF19 chimeras in SEQ ID NOS: 1 1 and 12.
  • minor variations can be made to these sequences, without adversely affecting the function of the protein (such as its ability to reduce blood glucose, reduce serum bile acids, or combinations thereof).
  • variants of the mutant FGF19 proteins include those having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, and 10 (which in some examples include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the mutations shown in Table 1), but retain the ability to treat a metabolic disease, or decrease blood glucose in a mammal (such as a mammal with type II diabetes).
  • variants of any of SEQ ID NOS: 6, 7, 8, 9, and 10 retaining at least 90% or 95% sequence identity are of use in the disclosed methods.
  • Mature forms of FGF19 can be mutated to control ⁇ e.g., reduce) the mitogenicity of the protein and to provide glucose-lowering ability and/or serum bile acid level reduction activity to the protein. Mutations can also be introduced into a wild-type mature FGF19 sequence that affects the stability and receptor binding selectivity of the protein.
  • FGF19 includes SEQ ID NO: 2 or 5, but without the N-terminal methionine.
  • the mature/active form of FGF19 is one where a portion of the N- terminus is removed, such as the N-terminal 20, 21, 22, 23, 24, 25 or 26 amino acids from SEQ ID NO: 2 or 5.
  • the active form of FGF19 comprises or consists of amino acids 23-216 of SEQ ID NO: 2 ⁇ e.g., see SEQ ID NO: 3) or amino acids 26-218 of SEQ ID NO: 5.
  • the mature form of FGF19 that can be mutated includes SEQ ID NO: 3 with a methionine added to the N-terminus (wherein such a sequence can be mutated as discussed herein).
  • the mutated mature FGF19 protein can include an N-terminal truncation. Mutations can be introduced into a wild- type FGF19 (such as SEQ ID NO: 2, 3, or 5). In some examples, multiple types of mutations disclosed herein are made to the FGF19 protein. Although mutations below are noted by a particular amino acid for example in SEQ ID NO: 2, 3 or 5, one skilled in the art will appreciate that the corresponding amino acid can be mutated in any FGF19 sequence.
  • mutations are made to the N-terminal region of FGF19 (such as SEQ ID NO: 3), such as deletion of the first 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of SEQ ID NO: 3.
  • Mutations can be made to FGF19 (such as SEQ ID NO: 3) to reduce its mitogenic activity.
  • such mutations reduce mitogenic activity by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or even complete elimination of detectable mitogenic activity.
  • Methods of measuring mitogenic activity are known in the art, such as thymidine incorporation into DNA in serum-starved cells (e.g. , NIH 3T3 cells) stimulated with the mutated FGF19,
  • methylthiazoletetrazolium (MTT) assay for example by stimulating serum-starved cells with mutated FGF19 for 24 hr then measuring viable cells, cell number quantification or BrdU incorporation.
  • MTT methylthiazoletetrazolium
  • the assay provided by Fu et al, World J. Gastroenterol. 10:3590-6, 2004; Klingenberg et al, J. Biol. Chem. 274: 18081-6, 1999; Shen et al, Protein Expr Purif. 81: 119-25, 2011, or Zou et al, Chin. Med. J. 121:424-429, 2008 is used to measure mitogenic activity.
  • Cancer Res 74:3306- 16, 2014 is used to measure HCC tumor growth.
  • mutations include, but are not limited to those at R23 and Nl 10, such as R23V, Nl 10V (wherein the numbering refers to the sequence shown SEQ ID NO: 3).
  • a portion of contiguous N-terminal residues are removed, such as amino acids 1- 11 or 1-20 of SEQ ID NO: 3, to produce a non-mito genie form of FGF19. Examples are shown in SEQ ID NOS: 7, 8, 9, 10 and 11.
  • mitogenicity is reduced or eliminated by deleting the N-terminal region of FGF19 (such as the region that binds FGFR4) and replacing the amino acids deleted with the sequence MRDSSPL.
  • mutations are introduced to improve stability of FGF19.
  • Methods of measuring FGF19 stability are known in the art, such as measuring denaturation of FGF19 or mutants by fluorescence and circular dichroism in the presence of 1.5 M urea or isothermal equilibrium denaturation by guanidine hydrochloride.
  • the assay provided by Dubey et al, J. Mol. Biol. 371:256-268, 2007 is used to measure FGF19 stability.
  • mutations that can be used to increase stability of the protein include, but are not limited to, one or more of R23V, Nl 10V, 160 V, K61I, El 02V (wherein the numbering refers to the sequence shown SEQ ID NO: 3).
  • mutations are introduced to improve the thermostability of FGF19.
  • the cysteines in FGF19 form two intramolecular disulfide bridges, so in some examples these are not mutated.
  • FGF19 K61 (reference to SEQ ID NO: 3) is mutated to a hydrophobic residue, such as isoleucine.
  • mutations are introduced to increase protease resistance of FGF19 (e.g., see Kobielak et al., Protein Pept Lett. 21(5):434-43, 2014).
  • the mutant FGF19 protein or chimera is PEGylated at one or more positions, such as at Nl 10 (for example see methods of Niu et al. , /. Chromatog. 1327:66-72, 2014, herein incorporated by reference).
  • Pegylation consists of covalently linking a polyethylene glycol group to surface residues and/or the N-terminal amino group. N110 is involved in receptor binding, thus is on the surface of the folded protein. As mutations to surface exposed residues could potentially generate immunogenic sequences, pegylation is an alternative method to abrogate a specific interaction. Pegylation is an option for any surface exposed site implicated in the receptor binding and/or proteolytic degradation. Pegylation can "cover" functional amino acids, e.g. N110, as well as increase serum stability.
  • the mutant FGF19 protein or chimera includes an immunoglobin FC domain (for example see Czajkowsky et al., EMBO Mol. Med. 4: 1015-28, 2012, herein incorporated by reference).
  • the conserved FC fragment of an antibody can be incorporated either n-terminal or c-terminal of the mutant FGF19 protein or chimera, and can enhance stability of the protein and therefore serum half-life.
  • the FC domain can also be used as a means to purify the proteins on protein A or Protein G sepharose beads. This makes the FGF19 mutants having heparin binding mutations easier to purify.
  • variants of the sequences shown SEQ ID NOS: 6, 7, 8, 9, 10, 11, and 12 can contain one or more mutations, such as a single insertion, a single deletion, a single substitution.
  • the mutant FGF19 protein includes 1-20 insertions, 1-20 deletions, 1-20 substitutions, or any combination thereof (e.g., single insertion together with 1-19 substitutions).
  • the disclosure provides a variant of any disclosed mutant FGF19 protein having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
  • SEQ ID NO: 6, 7, 8, 9, 10, 11, or 12 includes and additional 1-8 insertions, 1-15 deletions, 1- 10 substitutions, or any combination thereof (e.g., 1- 15, 1-4, or 1-5 amino acid deletions together with 1-10, 1-5 or 1 -7 amino acid substitutions).
  • the disclosure provides a variant of SEQ ID NO: 6, 7, 8, 9, 10 ,11, or 12, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid changes.
  • such variant peptides are produced by manipulating the nucleotide sequence encoding a peptide using standard procedures such as site-directed mutagenesis or PCR. Such variants can also be chemically synthesized.
  • One type of modification or mutation includes the substitution of amino acids for amino acid residues having a similar biochemical property, that is, a conservative substitution (such as 1-4, 1-8, 1- 10, or 1-20 conservative substitutions).
  • conservative substitutions have little to no impact on the activity of a resulting peptide.
  • a conservative substitution is an amino acid substitution in any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, and 12, that does not substantially affect the ability of the peptide to decrease blood glucose and/or decrease serum bile acids in a mammal.
  • An alanine scan can be used to identify which amino acid residues in a mutant FGF19 protein, such as any of SEQ ID NOS: 6, 7, 8, 9, 10, 1 1 , and 12, can tolerate an amino acid substitution.
  • the blood glucose lowering activity and/or the serum bile acid lowering activity of FGF19, or any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, and 12, is not altered by more than 25%, for example not more than 20%, for example not more than 10%, when an alanine, or other conservative amino acid, is substituted for 1-4, 1-8, 1- 10, or 1-20 native amino acids.
  • amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions include: Ser for Ala; Lys for Arg; Gin or His for Asn; Glu for Asp; Ser for Cys; Asn for Gin; Asp for Glu; Pro for Gly; Asn or Gin for His; Leu or Val for He; He or Val for Leu; Arg or Gin for Lys; Leu or lie for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and He or Leu for Val.
  • substitutions that are less conservative e.g., selecting residues that differ more significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the polypeptide at the target site; or (c) the bulk of the side chain.
  • substitutions that in general are expected to produce the greatest changes in polypeptide function are those in which: (a) a hydrophilic residue, e.g., serine or threonine, is substituted for (or by) a hydrophobic residue, e.g., leucine, isoleucine,
  • phenylalanine, valine or alanine (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysine, arginine, or histidine, is substituted for (or by) an electronegative residue, e.g., glutamic acid or aspartic acid; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.
  • electropositive side chain e.g., lysine, arginine, or histidine
  • an electronegative residue e.g., glutamic acid or aspartic acid
  • a residue having a bulky side chain e.g., phenylalanine
  • the effects of these amino acid substitutions can be assessed by analyzing the function of the mutant FGF19 protein, such as any of SEQ ID NOS: 6, 7, 8, 9, 10, 1 1 or 12, by analyzing the ability of the variant protein to decrease blood glucose and/or decrease serum bile acids in a mammal.
  • mutated FGF19 proteins can be carried out by conventional means, such as preparative chromatography and immunological separations. Once expressed, mutated FGF19 proteins can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, Protein Purification, Springer- Verlag, N.Y., 1982). Substantially pure compositions of at least about 90 to 95% homogeneity are disclosed herein, and 98 to 99% or more homogeneity can be used for pharmaceutical purposes.
  • mutated FGF19 proteins disclosed herein can also be constructed in whole or in part using standard peptide synthesis.
  • mutated FGF19 proteins are synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end (such as by the use of the coupling reagent N, N'-dicylohexylcarbodimide) are well known in the art.
  • Nucleic acid molecules encoding a mutated FGF19 protein are encompassed by this disclosure. Based on the genetic code, nucleic acid sequences coding for any mutated FGF19 protein or chimera, such as those having at least 90% or at least 95% sequence identity to those shown in any of SEQ ID NOS: 6, 7, 8, 9, 10, 1 1 or 12 can be routinely generated. In some examples, such a sequence is optimized for expression in a host cell, such as a host cell used to express the mutant FGF19 protein.
  • a nucleic acid sequence coding for a mutant FGF19 protein has at least 80%, at least 90%, at least 92%, at least 95%, at lest 96%, at least 97%, at least 99% or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10,
  • nucleic acids 1 1 or 12 can readily be produced by one of skill in the art, using the amino acid sequences provided herein, and the genetic code.
  • one of skill can readily construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same mutant FGF19 protein sequence.
  • Nucleic acid molecules include DNA, cDNA and RNA sequences which encode a mutated FGF19 peptide. Silent mutations in the coding sequence result from the degeneracy (i.e., redundancy) of the genetic code, whereby more than one codon can encode the same amino acid residue.
  • leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; aspartic acid can be encoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; and isoleucine can be encoded by ATT, ATC, or ATA. Tables showing the standard genetic code can be found in various sources (see, for example, Stryer, 1988, Biochemistry, 3 rd Edition, W.H. 5 Freeman and Co., NY).
  • Codon preferences and codon usage tables for a particular species can be used to engineer isolated nucleic acid molecules encoding a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) that take advantage of the codon usage preferences of that particular species.
  • the mutated FGF19 proteins disclosed herein can be designed to have codons that are preferentially used by a particular organism of interest.
  • a nucleic acid encoding a mutant FGF19 protein (such as a protein generated using the mutations shown in Table 1 , for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) can be cloned or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self- sustained sequence replication system (3SR) and the ⁇ ) ⁇ replicase amplification system (QB).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription-based amplification system
  • 3SR self- sustained sequence replication system
  • QB replicase amplification system
  • nucleic acids encoding sequences encoding a mutant FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least
  • Nucleic acid sequences encoding a mutated FGF19 protein can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol.
  • a mutant FGF19 protein (such as a protein generated using the mutations shown in Table 1 , for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) is prepared by inserting the cDNA which encodes the mutant FGF19 protein into a vector. The insertion can be made so that the mutant FGF19 protein is read in frame so that the mutant FGF 19 protein is produced.
  • the mutated FGF 19 protein nucleic acid coding sequence (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, such as a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11 , or 12, which in some examples includes at least 1 of the mutations shown in Table 1) can be inserted into an expression vector including, but not limited to a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in either prokaryotes or eukaryotes.
  • an expression vector including, but not limited to a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in either prokaryotes or eukaryotes.
  • Hosts can include microbial, yeast, insect, plant and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art.
  • the vector can encode a selectable marker, such as a thymidine kinase gene.
  • Nucleic acid sequences encoding a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) can be operatively linked to expression control sequences.
  • An expression control sequence operatively linked to a mutated FGF19 protein coding sequence is ligated such that expression of the mutant FGF19 protein coding sequence is achieved under conditions compatible with the expression control sequences.
  • the expression control sequences include, but are not limited to appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a mutated FGF19 protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of rnRNA, and stop codons.
  • vectors are used for expression in yeast such as S. cerevisiae, P. pastoris, or Kluyveromyces lactis.
  • yeast expression systems such as the constitutive promoters plasma membrane H + -ATPase (PMA1), glyceraldehyde-3-phosphate dehydrogenase (GPD), phosphoglycerate kinase-1 (PGK1), alcohol dehydrogenase- 1 (ADH1), and pleiotropic drug-resistant pump (PDR5).
  • PMA1 plasma membrane H + -ATPase
  • GPD glyceraldehyde-3-phosphate dehydrogenase
  • PGK1 phosphoglycerate kinase-1
  • ADH1 alcohol dehydrogenase- 1
  • PDR5 pleiotropic drug-resistant pump
  • inducible promoters are of use, such as GALl-10 (induced by galactose), PH05 (induced by low extracellular inorganic phosphate), and tandem heat shock HSE elements (induced by temperature elevation to 37 °C).
  • Promoters that direct variable expression in response to a titratable inducer include the methionine-responsive MET3 and MET25 promoters and copper- dependent CUP1 promoters. Any of these promoters may be cloned into multicopy (2 ⁇ ) or single copy (CEN) plasmids to give an additional level of control in expression level.
  • the plasmids can include nutritional markers (such as URA3, ADE3, HIS1 , and others) for selection in yeast and antibiotic resistance (AMP) for propagation in bacteria. Plasmids for expression on K. lactis are known, such as p LACl. Thus, in one example, after amplification in bacteria, plasmids can be introduced into the corresponding yeast auxotrophs by methods similar to bacterial transformation.
  • nucleic acid molecules encoding a mutated FGF19 protein can also be designed to express in insect cells.
  • a mutated FGF19 protein such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12
  • a mutated FGF19 protein such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12
  • a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) can be expressed in a variety of yeast strains. For example, seven pleiotropic drug-resistant transporters, YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11, and PDR15, together with their activating transcription factors, PDR1 and PDR3, have been simultaneously deleted in yeast host cells, rendering the resultant strain sensitive to drugs.
  • Yeast strains with altered lipid composition of the plasma membrane can also be utilized. Proteins that are highly sensitive to proteolysis can be expressed in a yeast cell lacking the master vacuolar endopeptidase Pep4, which controls the activation of other vacuolar hydrolases. Heterologous expression in strains carrying
  • ts temperature- sensitive alleles of genes can be employed if the corresponding null mutant is inviable.
  • Viral vectors can also be prepared that encode a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N- terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12).
  • Exemplary viral vectors include polyoma, SV40, adenovirus, vaccinia virus, adeno- associated virus, herpes viruses including HSV and EBV, Sindbis viruses, alphaviruses and retroviruses of avian, murine, and human origin.
  • Baculovirus vectors are also known in the art, and may be obtained from commercial sources.
  • suitable vectors include retrovirus vectors, orthopox vectors, avipox vectors, fowlpox vectors, capripox vectors, suipox vectors, adenoviral vectors, herpes virus vectors, alpha virus vectors, baculovirus vectors, Sindbis virus vectors, vaccinia virus vectors and poliovirus vectors.
  • poxvirus vectors such as vaccinia virus, fowlpox virus and a highly attenuated vaccinia virus (MVA), adenovirus, baculovirus and the like.
  • Pox viruses of use include orthopox, suipox, avipox, and capripox virus.
  • Orthopox include vaccinia, ectromelia, and raccoon pox.
  • One example of an orthopox of use is vaccinia.
  • Avipox includes fowlpox, canary pox and pigeon pox.
  • Capripox include goatpox and sheeppox.
  • the suipox is swinepox.
  • Other viral vectors that can be used include other DNA viruses such as herpes virus and adenoviruses, and RNA viruses such as retroviruses and polio.
  • Viral vectors that encode a mutated truncated FGF19 protein can include at least one expression control element operationally linked to the nucleic acid sequence encoding the mutated FGF19 protein.
  • the expression control elements are inserted in the vector to control and regulate the expression of the nucleic acid sequence.
  • expression control elements of use in these vectors includes, but is not limited to, lac system, operator and promoter regions of phage lambda, yeast promoters and promoters derived from polyoma, adenovirus, retrovirus or SV40. Additional operational elements include, but are not limited to, leader sequence, termination codons, polyadenylation signals and any other sequences necessary for the appropriate transcription and subsequent translation of the nucleic acid sequence encoding the mutated FGF19 protein in the host system.
  • the expression vector can contain additional elements necessary for the transfer and subsequent replication of the expression vector containing the nucleic acid sequence in the host system. Examples of such elements include, but are not limited to, origins of replication and selectable markers.
  • Such techniques involve, for example, homologous recombination between the viral DNA sequences flanking the DNA sequence in a donor plasmid and homologous sequences present in the parental virus.
  • the vector can be constructed for example by steps known in the art, such as by using a unique restriction endonuclease site that is naturally present or artificially inserted in the parental viral vector to insert the heterologous DNA.
  • Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding an mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12), and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • an mutated FGF19 protein such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • a nucleic acid molecule encoding a mutated FGF19 protein disclosed herein can be used to transform cells and make transformed cells.
  • cells expressing a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) are disclosed.
  • Cells expressing a mutated FGF19 protein disclosed herein can be eukaryotic or prokaryotic. Examples of such cells include, but are not limited to bacteria, archea, plant, fungal, yeast, insect, and mammalian cells, such as Lactobacillus, Lactococcus,
  • Bacillus such as B. subtilis
  • Escherichia such as E. coli
  • Clostridium Saccharomyces or
  • Pichia such as S. cerevisiae or P. pastoris
  • Kluyveromyces lactis Kluyveromyces lactis
  • Salmonella typhimurium Salmonella typhimurium
  • SF9 cells C129 cells
  • 293 cells Neurospora
  • immortalized mammalian myeloid and lymphoid cell lines
  • Cells expressing a mutated FGF19 protein are transformed or recombinant cells.
  • Such cells can include at least one exogenous nucleic acid molecule that encodes a mutated FGF19 protein, for example a sequence encoding a mutant FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12). It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host cell, are known in the art.
  • Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known.
  • the host is prokaryotic, such as E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCh method using procedures well known in the art.
  • MgC or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.
  • Techniques for the propagation of mammalian cells in culture are well-known (see, Jakoby and Pastan (eds), 1979, Cell Culture. Methods in Enzymology, volume 58, Academic Press, Inc., Harcourt Brace Jovanovich, N.Y.). Examples of commonly used mammalian host cell lines are VERO and HeLa cells, CHO cells, and WI38, BHK, and COS cell lines, although cell lines may be used, such as cells designed to provide higher expression desirable glycosylation patterns, or other features. Techniques for the transformation of yeast cells, such as polyethylene glycol transformation, protoplast transformation and gene guns are also known in the art.
  • compositions that include
  • compositions that include a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1 alone or in combination with an N-terminal deletion, such as a protein having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) or a nucleic acid encoding these proteins, can be formulated with an appropriate pharmaceutically acceptable carrier, depending upon the particular mode of administration chosen.
  • a mutated FGF19 protein such as a protein generated using the mutations shown in Table 1 alone or in combination with an N-terminal deletion, such as a protein having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least
  • the pharmaceutical composition consists essentially of a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1 , for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) (or a nucleic acid encoding such a protein) and a pharmaceutically acceptable carrier.
  • additional therapeutically effective agents are not included in the compositions.
  • the pharmaceutical composition includes a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1 , for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) (or a nucleic acid encoding such a protein) and a pharmaceutically acceptable carrier.
  • Additional therapeutic agents such as agents for the treatment of diabetes or other metabolic disorder, or a bile acid disorder, can be included.
  • the pharmaceutical compositions can include a therapeutically effective amount of another agent.
  • agents include, without limitation, anti-apoptotic substances such as the Nemo-Binding Domain and compounds that induce proliferation such as cyclin dependent kinase (CDK)-6, CDK-4 and cyclin Dl .
  • active agents can be utilized, such as antidiabetic agents for example, metformin, sulphonylureas (e.g., glibenclamide, tolbutamide, glimepiride), nateglinide, repaglinide, thiazolidinediones (e.g., rosiglitazone, pioglitazone), peroxisome proliferator- activated receptor (PPAR)-gamma-agonists (such as C 1262570) and antagonists, PPAR-gamma/alpha modulators (such as KRP 297), alpha-glucosidase inhibitors (e.g., acarbose, voglibose), dipeptidyl peptidase
  • compositions containing a mutated FGF19 protein can further include a therapeutically effective amount of other FGFs, such as FGF21 , heparin, or combinations thereof.
  • the methods further include administration of therapeutic agents for bile acid disorders, such as modified bile acids ⁇ e.g., 6oc-ethyl chenodeoxycholic acid, 6-eCDCA).
  • parenteral formulations usually include injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like.
  • injectable fluids such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like.
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, pH buffering agents, or the like, for example sodium acetate or sorbitan monolaurate.
  • auxiliary substances such as wetting or emulsifying agents, preservatives, pH buffering agents, or the like, for example sodium acetate or sorbitan monolaurate.
  • Excipients that can be included are, for instance, other proteins, such as human serum albumin or plasma preparations.
  • a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1 , for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) is included in a controlled release formulation, for example, a microencapsulated formulation.
  • a controlled release formulation for example, a microencapsulated formulation.
  • a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1 , for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9,
  • Nanodispersion systems and methods for producing such nanodispersions are well known to one of skill in the art. See, e.g., U.S. Pat. No.
  • a nanodispersion system includes a biologically active agent and a dispersing agent (such as a polymer, copolymer, or low molecular weight surfactant).
  • a dispersing agent such as a polymer, copolymer, or low molecular weight surfactant.
  • Exemplary polymers or copolymers include
  • polyvinylpyrrolidone PVP
  • PVA poly(D,L-lactic acid)
  • PLGA poly(D,L-lactic-co-glycolic acid
  • exemplary low molecular weight surfactants include sodium dodecyl sulfate, hexadecyl pyridinium chloride, polysorbates, sorbitans, poly(oxyethylene) alkyl ethers, poly(oxyethylene) alkyl esters, and combinations thereof.
  • the nanodispersion system includes PVP and ODP or a variant thereof (such as 80/20 w/w).
  • the nanodispersion is prepared using the solvent evaporation method, see for example, Kanaze et al, Drug Dev. Indus. Pharm. 36:292-301, 2010; Kanaze et al, J. Appl. Polymer Sci. 102:460-471, 2006.
  • one approach to administration of nucleic acids is direct treatment with plasmid DNA, such as with a mammalian expression plasmid.
  • nucleotide sequence encoding a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) can be placed under the control of a promoter to increase expression of the protein.
  • a mutated FGF19 protein such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12
  • release delivery systems are available and known. Examples include polymer based systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent No. 5,075, 109.
  • Delivery systems also include non-polymer systems, such as lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and triglycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and triglycerides
  • hydrogel release systems such as silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • mutated FGF19 protein such as a protein generated using the mutations shown in Table 1 , for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12), or polynucleotide encoding this protein, is contained in a form within a matrix such as those described in U.S. Patent Nos.
  • Long-term sustained release implant may be particularly suitable for treatment of chronic conditions, such as diabetes.
  • Long-term release means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 30 days, or at least 60 days.
  • Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above. These systems have been described for use with nucleic acids (see U.S. Patent No. 6,218,371).
  • nucleic acids and peptides are preferably relatively resistant to degradation (such as via endo- and exo-nucleases).
  • modifications of the disclosed mutated FGF19 proteins such as the inclusion of a C-terminal amide, can be used.
  • the dosage form of the pharmaceutical composition can be determined by the mode of administration chosen.
  • topical, inhalation, oral and suppository formulations can be employed.
  • Topical preparations can include eye drops, ointments, sprays, patches and the like.
  • Inhalation preparations can be liquid ⁇ e.g., solutions or suspensions) and include mists, sprays and the like.
  • Oral formulations can be liquid ⁇ e.g., syrups, solutions or suspensions), or solid ⁇ e.g., powders, pills, tablets, or capsules).
  • Suppository preparations can also be solid, gel, or in a suspension form.
  • conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, cellulose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.
  • compositions that include a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N- terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) can be formulated in unit dosage form, suitable for individual administration of precise dosages.
  • a mutated FGF19 protein such as a protein generated using the mutations shown in Table 1, for example in combination with an N- terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12
  • a unit dosage contains from about 1 mg to about 1 g of a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12), such as about 10 mg to about 100 mg, about 50 mg to about 500 mg, about
  • a mutated FGF19 protein such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12
  • a therapeutically effective amount of a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N- terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) is about 0.01 mg/kg to about 50 mg/kg, for example, about 0.5 mg kg to about 25 mg/kg or about 1 mg/kg to about 10 mg/kg.
  • a therapeutically effective amount of a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N- terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11 , or 12) is about 1 mg kg to about 5 mg/kg, for example about 2 mg/kg.
  • a mutated FGF19 protein such as a protein generated using the mutations shown in Table 1, for example in combination with an N- terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11 , or 12
  • a therapeutically effective amount of a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs:
  • NOS: 6, 7, 8, 9, 10, 11, or 12 includes about 1 mg/kg to about 10 mg/kg, such as about 2 mg/kg.
  • the disclosed mutated FGF19 proteins and chimeras (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%), at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12), or nucleic acids encoding such proteins, can be administered to a subject, for example to treat a metabolic disease, for example by reducing fed and fasting blood glucose, improving insulin sensitivity and glucose tolerance, reducing systemic chronic inflammation, ameliorating hepatic steatosis in a mammal, or combinations thereof.
  • a metabolic disease for example by reducing fed and fasting blood glucose, improving insulin sensitivity and glucose tolerance, reducing systemic chronic inflammation, ameliorating hepatic steatosis in a mammal, or combinations thereof.
  • the disclosed mutated FGF19 proteins and chimeras (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12), or nucleic acids encoding such proteins, can be administered to a subject, for example to reduce glucose levels, increase insulin sensitivity, reduce insulin resistance, reduce glucagon, improve glucose tolerance, or glucose metabolism or homeostasis, improve pancreatic function, reduce triglyceride, cholesterol, IDL, LDL or VLDL levels, decrease blood pressure, decrease intimal thickening of the blood vessel, decrease body mass or weight gain, or combinations thereof.
  • a subject for example to reduce glucose levels, increase insulin sensitivity, reduce insulin resistance, reduce glucagon, improve glucose tolerance, or glucose metabolism or
  • the disclosed mutated FGF19 proteins and chimeras can be administered to subjects having a fasting plasma glucose (FPG) level greater than about 100 mg/d and/or has a hemoglobin Ale (HbAlc) level above 6%.
  • FPG fasting plasma glucose
  • HbAlc hemoglobin Ale
  • the disclosed mutated FGF19 proteins and chimeras (such as a protein generated using the mutations shown in Table 1, for example in combination with an N- terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12), or nucleic acids encoding such proteins, can be administered to a subject, for example to treat a subject having a hyperglycemic condition ⁇ e.g., diabetes, such as insulin- dependent (type I) diabetes, type II diabetes, or gestational diabetes), insulin resistance, hyperinsulinemia, glucose intolerance or metabolic syndrome, or is obese or has an undesirable body mass.
  • a hyperglycemic condition ⁇ e.g., diabetes, such as insulin- dependent (type I) diabetes, type II diabetes, or gestational diabetes
  • insulin resistance hyperinsulinemia
  • the disclosed mutated FGF19 proteins and chimeras (such as a protein generated using the mutations shown in Table 1, for example in combination with an N- terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12), or nucleic acids encoding such proteins, can be administered to a subject, for example to treat other hyperglycemic -related disorders, including kidney damage (e.g., tubule damage or nephropathy), liver degeneration, eye damage (e.g., diabetic retinopathy or cataracts), and diabetic foot disorders; dyslipidemias and their sequelae such as, for example,
  • kidney damage e.g., tubule damage or nephropathy
  • liver degeneration e.g., eye damage (e.g., diabetic retinopathy or cataracts
  • the disclosed mutated FGF19 proteins and chimeras (such as a protein generated using the mutations shown in Table 1, for example in combination with an N- terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12), or nucleic acids encoding such proteins, can be administered to a subject, for example to treat a bile acid disease, for example by reducing serum bile acids.
  • a bile acid disease for example by reducing serum bile acids.
  • a subject having a bile acid related or associated disorder such as one or more of bile acid malabsorption, diarrhea, cholestasis (e.g., intrahepatic or extrahepatic cholestasis), primary billiary cirrhosis, primary sclerosing cholangitis, and PFIC (e.g., progressive PFIC) can be treated with the disclosed methods.
  • a bile acid related or associated disorder such as one or more of bile acid malabsorption, diarrhea, cholestasis (e.g., intrahepatic or extrahepatic cholestasis), primary billiary cirrhosis, primary sclerosing cholangitis, and PFIC (e.g., progressive PFIC)
  • cholestasis e.g., intrahepatic or extrahepatic cholestasis
  • primary billiary cirrhosis e.g., primary billiary cirrhosis
  • bile-acid related or associated disorders that can be treated with the disclosed methods include: metabolic syndrome; a lipid- or glucose-related disorder; cholesterol or triglyceride metabolism; type 2 diabetes; cholestasis, including, for example diseases of intrahepatic cholestasis (e.g., PBC, PFIC, PSC, PIC, neonatal cholestasis, and drug induced cholestasis (e.g., estrogen)), and diseases of extrahepatic cholestasis (e.g., bile duct compression from tumor, bile duct blockade by gall stones); bile acid malabsorption and other disorders involving the distal small intestine, including ileal resection, inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis), disorders impairing absorption of bile acids not otherwise characterized (idiopathic)) leading to diarrhea (e.g., BAD) and GI symptoms, and GI,
  • compositions of this disclosure that include a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N- terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) (or nucleic acids encoding these molecules) can be administered to humans or other animals by any means, including orally, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, intrathecally, subcutaneously, via inhalation or via suppository.
  • a mutated FGF19 protein such as a protein generated using the mutations shown in Table 1, for example in combination with an N- terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at
  • the composition is administered via injection.
  • site-specific administration of the composition can be used, for example by administering a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1 , for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11 , or 12) (or a nucleic acid encoding these molecules) to pancreas tissue (for example by using a pump, or by implantation of a slow release form at the site of the pancreas).
  • a mutated FGF19 protein such as a protein generated using the mutations shown in Table 1 , for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 9
  • Treatment can involve daily or multi-daily or less than daily (such as weekly or monthly etc.) doses over a period of a few days to months, or even years.
  • a therapeutically effective amount of a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) can be
  • treatment involves once daily dose or twice daily dose.
  • the amount of mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1 , for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) administered can be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and can be left to the judgment of the prescribing clinician. Within these bounds, the formulation to be administered will contain a quantity of the mutated FGF19 protein in amounts effective to achieve the desired effect in the subject being treated.
  • a therapeutically effective amount of mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%), at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) can be the amount of the mutant FGF19 protein, or a nucleic acid encoding these molecules that is necessary to treat diabetes, reduce blood glucose levels, and/or reduce serum bile acids (for example a reduction of at least 5%, at least 10%, at least 20%, or at least 50%).
  • mutated FGF19 protein such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%)
  • a viral vector When a viral vector is utilized for administration of an nucleic acid encoding a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12), the recipient can receive a dosage of each recombinant virus in the composition in the range of from about 10 s to about 10 10 plaque forming units/mg mammal, although a lower or higher dose can be administered.
  • a mutated FGF19 protein such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 9
  • compositions into mammals include, but are not limited to, exposure of cells to the recombinant virus ex vivo, or injection of the composition into the affected tissue or intravenous, subcutaneous, intradermal or intramuscular
  • the recombinant viral vector or combination of recombinant viral vectors may be administered locally by direct injection into the pancreases in a pharmaceutically acceptable carrier.
  • the quantity of recombinant viral vector, carrying the nucleic acid sequence of the mutated FGF19 protein to be administered is based on the titer of virus particles.
  • An exemplary range to be administered is 10 s to 10 10 virus particles per mammal, such as a human.
  • a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1 , for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11 , or 12), or a nucleic acid encoding the mutated FGF19 protein, is administered in combination (such as sequentially or simultaneously or contemporaneously) with one or more other agents, such as those useful in the treatment of diabetes, insulin resistance and/or a bile acid disorder.
  • one or more other agents such as those useful in the treatment of diabetes, insulin resistance and/or a bile acid disorder.
  • Anti-diabetic agents are generally categorized into six classes: biguanides;
  • the anti-diabetic agents include those agents disclosed in Diabetes Care, 22(4):623-634.
  • One class of anti-diabetic agents of use is the sulfonylureas, which are believed to increase secretion of insulin, decrease hepatic glucogenesis, and increase insulin receptor sensitivity.
  • Another class of anti-diabetic agents use the biguanide antihyperglycemics, which decrease hepatic glucose production and intestinal absorption, and increase peripheral glucose uptake and utilization, without inducing hyperinsulinemia.
  • the methods further include administration of therapeutic agents for bile acid disorders, such as modified bile acids (e.g., 6oc-ethyl chenodeoxycholic acid, 6-eCDCA).
  • modified bile acids e.g., 6oc-ethyl chenodeoxycholic acid, 6-eCDCA.
  • mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1 , for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9,
  • lipid lowering compounds such as statins or fibrates
  • agents that reduce serum bile acids such as modified bile acids, e.g., 6oc-ethyl chenodeoxycholic acid, 6-eCDCA.
  • administration in combination refers to both concurrent and sequential administration of the active agents.
  • Administration of mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1 , for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) or a nucleic acid encoding such a mutant FGF19 protein, may also be in combination with lifestyle modifications, such as increased physical activity, low fat diet, low sugar diet, and smoking cessation.
  • Additional agents of use include, without limitation, anti-apoptotic substances such as the Nemo-Binding Domain and compounds that induce proliferation such as cyclin dependent kinase (CDK)-6, CDK-4 and Cyclin D 1.
  • Other active agents can be utilized, such as antidiabetic agents for example, metformin, sulphonylureas (e.g., glibenclamide, tolbutamide, glimepiride), nateglinide, repaglinide, thiazolidinediones (e.g., rosiglitazone, pioglitazone), peroxisome proliferator- activated receptor (PPAR)-gamma-agonists (such as C1262570) and antagonists, PPAR- gamma/alpha modulators (such as KRP 297), alpha-glucosidase inhibitors (e.g., acarbose, voglibose), Dipeptidyl peptidas
  • the agent is an immunomodulatory factor such as anri-CD3 mAb, growth factors such as HGF, vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), lactogens, or parathyroid hormone related protein (PTHrP).
  • growth factors such as HGF, vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), lactogens, or parathyroid hormone related protein (PTHrP).
  • the mutated FGF19 protein is administered in combination with a therapeutically effective amount of another FGF, such as FGF21 , heparin, or combinations thereof.
  • methods are provided for treating diabetes or pre-diabetes in a subject by administering a therapeutically effective amount of a composition including a mutated FGF 19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12), or a nucleic acid encoding the mutated FGF19 protein, to the subject.
  • the subject can have diabetes type I or diabetes type II.
  • the subject can be any mammalian subject, including human subjects and veterinary subjects such as cats and dogs.
  • the subject can be a child or an adult.
  • the subject can also be administered insulin.
  • the method can include measuring blood glucose levels.
  • the method includes selecting a subject with diabetes, such as type I or type II diabetes, or a subject at risk for diabetes, such as a subject with pre-diabetes.
  • a subject with diabetes such as type I or type II diabetes
  • a subject at risk for diabetes such as a subject with pre-diabetes.
  • These subjects can be selected for treatment with the disclosed mutated FGF19 proteins (such as a protein generated using the mutations shown in Table 1, for example in combination with an N- terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) or nucleic acid molecules encoding such.
  • the disclosed mutated FGF19 proteins such as a protein generated using the mutations shown in Table 1, for example in combination with an N- terminal deletion, or a protein having at least 80%, at least
  • a subject with diabetes may be clinically diagnosed by a fasting plasma glucose (FPG) concentration of greater than or equal to 7.0 millimole per liter (mmol/L) (126 milligram per deciliter (mg/dL)), or a plasma glucose concentration of greater than or equal to 11.1 mmol/L (200 mg/dL) at about two hours after an oral glucose tolerance test (OGTT) with a 75 gram (g) load, or in a patient with classic symptoms of hyperglycemia or hyperglycemic crisis, a random plasma glucose concentration of greater than or equal to 11.1 mmol/L (200 mg/dL), or HbAlc levels of greater than or equal to 6.5%.
  • FPG fasting plasma glucose
  • a subject with pre-diabetes may be diagnosed by impaired glucose tolerance (IGT).
  • IGT impaired glucose tolerance
  • An OGTT two-hour plasma glucose of greater than or equal to 140 mg/dL and less than 200 mg/dL (7.8- 11.0 mM), or a fasting plasma glucose (FPG) concentration of greater than or equal to 100 mg/dL and less than 125 mg/dL (5.6-6.9 mmol/L), or HbAlc levels of greater than or equal to 5.7% and less than 6.4% (5.7-6.4%) is considered to be IGT, and indicates that a subject has pre-diabetes. Additional information can be found in Standards of Medical Care in Diabetes— 2010
  • the subject treated with the disclosed compositions and methods has
  • HbAlC of greater than 6.5% or greater than 7%.
  • treating diabetes includes one or more of increasing glucose tolerance, decreasing insulin resistance (for example, decreasing plasma glucose levels, decreasing plasma insulin levels, or a combination thereof), decreasing serum triglycerides, decreasing free fatty acid levels, and decreasing HbAlc levels in the subject.
  • the disclosed methods include measuring glucose tolerance, insulin resistance, plasma glucose levels, plasma insulin levels, serum triglycerides, free fatty acids, and/or HbAlc levels in a subject.
  • a mutated FGF19 protein such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12) or pre-diabetes, by decreasing of HbAlC, such as a reduction of at least 0.5%, at least 1%, or at least 1.5%, such as a decrease of 0.5% to 0.8%, 0.5% to 1%, 1 to 1.5% or 0.5% to 2%.
  • HbAlC such as a reduction of at least 0.5%, at least 1%, or at least 1.5%, such as a decrease of 0.5% to 0.8%, 0.5% to 1%, 1 to 1.5% or 0.5% to 2%.
  • the target for HbAlC is less than about 6.5%, such as about 4-6%, 4-6.4%, or 4-6.2%. In some examples, such target levels are achieved within about 26 weeks, within about 40 weeks, or within about 52 weeks.
  • Methods of measuring HbAlC are routine, and the disclosure is not limited to particular methods. Exemplary methods include HPLC, immunoassays, and boronate affinity chromatography.
  • a mutated FGF19 protein such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12), or nucleic acid molecule encoding such, treats diabetes or prediabetes by increasing glucose tolerance, for example, by decreasing blood glucose levels (such as two-hour plasma glucose in an OGTT or FPG) in a subject.
  • blood glucose levels such as two-hour plasma glucose in an OGTT or FPG
  • the method includes decreasing blood glucose by at least 5% (such as at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or more) as compared with a control (such as no administration of any of insulin, a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1 , for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12).
  • a control such as no administration of any of insulin, a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1 , for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least
  • a decrease in blood glucose level is determined relative to the starting blood glucose level of the subject (for example, prior to treatment with a mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12), or nucleic acid molecule encoding such).
  • a mutated FGF19 protein such as a protein generated using the mutations shown in Table 1, for example in combination with an N-terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12), or nucleic
  • decreasing blood glucose levels of a subject includes reduction of blood glucose from a starting point (for example greater than about 126 mg/dL FPG or greater than about 200 mg/dL OGTT two-hour plasma glucose) to a target level (for example, FPG of less than 126 mg/dL or OGTT two-hour plasma glucose of less than 200 mg/dL).
  • a target FPG may be less than 100 mg/dL.
  • a target OGTT two-hour plasma glucose may be less than 140 mg/dL.
  • the disclosed methods include comparing one or more indicator of diabetes (such as glucose tolerance, triglyceride levels, free fatty acid levels, or HbAlc levels) to a control (such as no administration of any of insulin, any mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N- terminal deletion, or a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12), or a nucleic acid molecule encoding such), wherein an increase or decrease in the particular indicator relative to the control (as discussed above) indicates effective treatment of diabetes.
  • a control such as no administration of any of insulin, any mutated FGF19 protein (such as a protein generated using the mutations shown in Table 1, for example in combination with an N- terminal deletion, or a protein having at least 80%
  • the control can be any suitable control against which to compare the indicator of diabetes in a subject.
  • the control is a sample obtained from a healthy subject (such as a subject without diabetes).
  • the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of subjects with diabetes, or group of samples from subjects that do not have diabetes).
  • the control is a reference value, such as a standard value obtained from a population of normal individuals that is used by those of skill in the art. Similar to a control population, the value of the sample from the subject can be compared to the mean reference value or to a range of reference values (such as the high and low values in the reference group or the 95% confidence interval).
  • the control is the subject (or group of subjects) treated with placebo compared to the same subject (or group of subjects) treated with the therapeutic compound in a cross-over study.
  • the control is the subject (or group of subjects) prior to treatment.
  • the subject treated with the disclosed compositions and methods has a serum bile acid that is greater than 11 pmol/L.
  • the disclosed methods include measuring serum bile acid levels in a subject.
  • Mutated FGF19 proteins can be made using known methods (e.g., see Xia et al. , PLoS One. 7(l l):e48210, 2012 and Kong and Guo, PLOS one, e85890, 2014). An example is provided below.
  • a nucleic acid sequence encoding an FGF19 mutant protein ⁇ e.g., any of SEQ ID NOs: 6, 7, 8, 9, 10, 11, or 12
  • a thioredoxin fusion protein e.g., see Kong and Guo, PLOS one, e85890, 2014.
  • the mutant FGF19 protein can be expressed from an E. coli host after induction with isopropyl-P-D-thio-galactoside.
  • the expressed protein can be purified utilizing sequential column chromatography on Ni- nitrilotriacetic acid (NTA) affinity resin followed by cation and anion exchange chromatography.
  • NTA Ni- nitrilotriacetic acid
  • the purified protein can be digested with tobacco etch virus (TEV) protease to remove the N-terminal (His)6 tag and thioredoxin fusion.
  • TSV tobacco etch virus
  • a subsequent second Ni-NTA chromatographic step can be utilized to remove the released N-terminal mutant FGF19 protein (along with any uncleaved fusion protein).
  • Final purification can be performed using HiLoad Superdex 75 size exclusion chromatography equilibrated to 50 mM Na 2 P0 4 , 100 mM NaCl, 10 mM (NH 4 )2S0 4 , 0.1 mM
  • ethylenediaminetetraacetic acid EDTA
  • 5 mM L-Methionine pH at 6.5
  • L- Methionine can be included in PBX buffer to limit oxidization of reactive thiols and other potential oxidative degradation.
  • the purified mutant FGF19 protein can be sterile filtered through a 0.22 micron filter, purged with N2, snap frozen in dry ice and stored at -80°C prior to use.
  • the purity of the mutant FGF19 protein can be assessed by both Coomassie Brilliant Blue and Silver Stain Plus (BIO-RAD Laboratories, Inc., Hercules CA) stained sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE).
  • Mutant FGF19 proteins can be prepared in the absence of heparin. Prior to IV bolus, PBS can be added to the protein.
  • This example describes methods that can be used to test any of the FGF19 mutants provided herein (e.g., any of SEQ ID NOS: 6-12) for their ability to lower blood glucose in vivo.
  • Animals e.g., any of SEQ ID NOS: 6-12
  • Mice are housed in a temperature-controlled environment with a 12-hour light/ 12- hour dark cycle and handled according to institutional guidelines complying with U.S.
  • mice Male ob/ob mice (B6.V-Lep ob /J, Jackson laboratories) and male C57BL/6J mice receive a standard or high fat diet (MI laboratory rodent diet 5001, Harlan Teklad; high fat (60%) diet F3282, Bio-Serv) and acidified water ad libitum. STZ-induced diabetic mice on the C57BL/6J background can be purchased from Jackson laboratories. 0.1 mg/ml solutions in PBS of mouse FGF15, human FGF19, or mutated FGF19 proteins can be injected.
  • MI laboratory rodent diet 5001, Harlan Teklad high fat (60%) diet F3282, Bio-Serv
  • Blood can be collected by tail bleeding either in the ad libitum fed state or following overnight fasting.
  • Free fatty acids (Wako), triglycerides (Thermo) and cholesterol (Thermo) can be measured using enzymatic colorimetric methods following the manufacturer' s instructions.
  • Serum insulin levels can be measured using an Ultra Sensitive Insulin ELISA kit (Crystal Chem).
  • Plasma adipokine and cytokine levels can be measured using MilliplexTM MAP and Bio-Plex ProTM kits (Millipore and Bio-Rad).
  • Glucose tolerance tests can be conducted after o/n fasting. Mice can be injected i.p. with 1 g of glucose per/kg bodyweight and blood glucose was monitored at 0, 15, 30, 60, and 120 min using a OneTouch Ultra glucometer (Lifescan Inc). Insulin tolerance tests (ITT) can be conducted after 3h fasting. Mice can be injected i.p. with 2U of insulin/kg bodyweight (Humulin R; Eli Lilly) and blood glucose monitored at 0, 15, 30, 60, and 90 min using a OneTouch Ultra glucometer (Lifescan Inc).

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Abstract

La présente invention concerne des protéines mutantes FGF19 telles que celles ayant une délétion ou des mutations ponctuelles N-terminales ou des combinaisons de celles-ci, pouvant réduire les acides biliaires sériques et/ou le glucose sanguin chez un mammifère. De telles protéines mutantes FGF19 peuvent faire partie d'une protéine chimère qui comprend une partie de FGF21. Dans certains exemples, les protéines mutantes FGF19 présentent une activité mitogénique réduite. L'invention concerne aussi des molécules d'acides nucléiques qui codent pour ces protéines, ainsi que des vecteurs et des cellules comprenant ces acides nucléiques. L'invention concerne également des procédés d'utilisation des molécules selon l'invention pour réduire les taux de glucose sanguin et réduire les acides biliaires sériques, par exemple pour traiter un trouble métabolique ou un trouble lié aux acides biliaires.
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US10369199B2 (en) 2013-10-28 2019-08-06 Ngm Biopharmaceuticals, Inc. Methods of using variants of FGF19 polypeptides for the treatment of cancer
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US10398758B2 (en) 2014-05-28 2019-09-03 Ngm Biopharmaceuticals, Inc. Compositions comprising variants of FGF19 polypeptides and uses thereof for the treatment of hyperglycemic conditions
US10413590B2 (en) 2011-07-01 2019-09-17 Ngm Biopharmaceuticals, Inc. Methods of using compositions comprising variants of FGF19 polypeptides for reducing body mass in a subject
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US10517929B2 (en) 2014-10-23 2019-12-31 Ngm Biopharmaceuticals, Inc. Pharmaceutical compositions comprising FGF19 variants
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US11066454B2 (en) 2012-11-28 2021-07-20 Ngm Biopharmaceuticals, Inc. Compositions comprising variants and fusions of FGF19 polypeptides
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US11065302B2 (en) 2011-07-01 2021-07-20 Ngm Biopharmaceuticals, Inc. Compositions comprising fusion variants of FGF19 polypeptides
US11066454B2 (en) 2012-11-28 2021-07-20 Ngm Biopharmaceuticals, Inc. Compositions comprising variants and fusions of FGF19 polypeptides
US10758590B2 (en) 2012-11-28 2020-09-01 Ngm Biopharmaceuticals, Inc. Methods of using compositions comprising variants and fusions of FGF 19 polypeptides for treating diabetes
US11564972B2 (en) 2012-12-27 2023-01-31 Ngm Biopharmaceuticals, Inc. Methods of using compositions comprising variants of FGF19 polypeptides for treating primary biliary cirrhosis in a subject
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US10398758B2 (en) 2014-05-28 2019-09-03 Ngm Biopharmaceuticals, Inc. Compositions comprising variants of FGF19 polypeptides and uses thereof for the treatment of hyperglycemic conditions
US11241481B2 (en) 2014-06-16 2022-02-08 Ngm Biopharmaceuticals, Inc. Methods and uses for modulating bile acid homeostasis and treatment of bile acid disorders and diseases
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US10517929B2 (en) 2014-10-23 2019-12-31 Ngm Biopharmaceuticals, Inc. Pharmaceutical compositions comprising FGF19 variants
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US10744185B2 (en) 2015-11-09 2020-08-18 Ngm Biopharmaceuticals, Inc. Methods of using variants of FGF19 polypeptides for the treatment of pruritus
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US11370841B2 (en) 2016-08-26 2022-06-28 Ngm Biopharmaceuticals, Inc. Methods of treating fibroblast growth factor 19-mediated cancers and tumors
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