NZ617746B2 - Compositions, uses and methods for treatment of metabolic disorders and diseases - Google Patents

Abstract

Disclosed is a peptide, comprising: a) an N-terminal region comprising at least seven amino acid residues, the N-terminal region having a first amino acid position and a last amino acid position, wherein the N-terminal region comprises DSSPL (SEQ ID NO: 121) or DASPH (SEQ ID NO: 122); and b) a C-terminal region having a first amino acid position and a last amino acid position, wherein the C-terminal region comprises: (i) a first C-terminal region sequence comprising WGDPIRLRHLYTSG (amino acids 16 to 29 of SEQ ID NO: 99 [FGF19]), wherein the W residue corresponds to the first amino acid position of the C-terminal region; and (ii) a second C-terminal region sequence comprising PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (amino acid residues 30 to 194 of SEQ ID NO: 99 [FGF19]) or a sequence comprising 1 to 5 amino acid substitutions, deletions or insertions thereof; wherein the peptide: (i) binds to fibroblast growth factor receptor 4 (FGFR4) with an affinity equal to or greater than FGF19 binding affinity for FGFR4; (ii) activates FGFR4 to an extent or amount equal to or greater than FGF19 activates FGFR4; (iii) has at least one of reduced hepatocellular carcinoma (HCC) formation; greater glucose lowering activity, less lipid increasing activity, less triglyceride activity, less cholesterol activity, less non-HDL activity or less HDL increasing activity, as compared to FGF19, or as compared to an FGF19 variant sequence having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence at amino acids 16-20 of FGF19 (SEQ ID NO: 99); and/or (iv) has less lean mass reducing activity as compared to FGF21. terminal region having a first amino acid position and a last amino acid position, wherein the C-terminal region comprises: (i) a first C-terminal region sequence comprising WGDPIRLRHLYTSG (amino acids 16 to 29 of SEQ ID NO: 99 [FGF19]), wherein the W residue corresponds to the first amino acid position of the C-terminal region; and (ii) a second C-terminal region sequence comprising PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (amino acid residues 30 to 194 of SEQ ID NO: 99 [FGF19]) or a sequence comprising 1 to 5 amino acid substitutions, deletions or insertions thereof; wherein the peptide: (i) binds to fibroblast growth factor receptor 4 (FGFR4) with an affinity equal to or greater than FGF19 binding affinity for FGFR4; (ii) activates FGFR4 to an extent or amount equal to or greater than FGF19 activates FGFR4; (iii) has at least one of reduced hepatocellular carcinoma (HCC) formation; greater glucose lowering activity, less lipid increasing activity, less triglyceride activity, less cholesterol activity, less non-HDL activity or less HDL increasing activity, as compared to FGF19, or as compared to an FGF19 variant sequence having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence at amino acids 16-20 of FGF19 (SEQ ID NO: 99); and/or (iv) has less lean mass reducing activity as compared to FGF21.

Description

Compositions, Uses and Methods for Treatment of Metabolic Disorders and Diseases Related Applications This application claims the benefit of priority of application serial no. 61/504,128, filed July 1, 2011, and application serial no. ,126, filed August 4, 2011, both ofwhich ations are expressly incorporated herein by reference in their entirety.

Field of the Invention The invention s to variants of fibroblast growth factor 19 (FGF19) ns and peptide sequences (and peptidomimetics) and fusions of fibroblast growth factor 19 (FGF19) and/or fibroblast growth factor 21 (FGF21) proteins and peptide sequences (and peptidomimetics), and variants of fusions of fibroblast growth factor 19 (FGF19) and/or ast growth factor 21 (FGF21) ns and peptide sequences (and peptidomimetics) having glucose ng activity, and methods for and uses in treatment of hyperglycemia and other disorders.

Introduction Diabetes mellitus is a debilitating metabolic disease caused by absent insulin production (type 1) or insulin resistance or insufficient insulin production (type 2) from pancreatic B—cells . B-cells are specialized endocrine cells that manufacture and store insulin for release following a meal. Insulin is a hormone that facilitates the transfer of glucose from the blood into tissues where it is needed. Patients with es must frequently monitor blood glucose levels and many require multiple daily insulin injections to survive. However, such patients rarely attain ideal glucose levels by insulin injection (Turner, R.C. et al. JAMA 281 :2005(1999)). Furthermore, prolonged elevation of insulin levels can result in detrimental side s such as hypoglycemic shock and desensitization of the body’s response to insulin.

Consequently, diabetic patients still develop long—term complications, such as vascular diseases, kidney e, blindness, nerve damage and wound healing ers (UK ctive Diabetes Study (UKPDS) Group, Lancet 3522837 (1998)).

Bariatric surgery has been proposed as a potential treatment for diabetes. It has been postulated that changes in gut hormone secretion after the surgery are responsible for the resolution of diabetic conditions. The underlying molecular ism has yet to be elucidated, although glucagon—like peptide 1 (GLP—l) has been speculated as a possible candidate (Rubino, F.

Diabetes Care 32 Suppl 2:S368(2009)). FGF19 is highly expressed in the distal small intestine and transgenic over-expression ofFGF19 improves glucose tasis (Tomlinson, E.

Endocrinology 143(5): 1741-7(2002)). Serum levels of FGF19 in humans are elevated following gastric bypass surgery. ted expression and secretion of FGF19 could at least partially explain the diabetes remission experienced following surgery.

Accordingly, there is a need for alternative treatments of hyperglycemic conditions such as es, prediabetes, insulin ance, hyperinsulinemia, glucose intolerance 0r metabolic syndrome, and other disorders and es associated with elevated glucose levels, in humans. The invention satisfies this need and provides related advantages.

Summary The invention is based, in part, on variants of fibroblast growth factor 19 (FGF 19) peptide sequences, s of fibroblast growth factor 19 (FGF19) and/or fibroblast growth factor 21 (FGF21) peptide sequences and variants of s (chimeras) of fibroblast growth factor 19 (FGF19) and/or fibroblast growth factor 21 (FGF21) peptide sequences having one or more activities, such as e lowering ty. Such variants and fusions (chimeras) of FGF 19 and/or FGF21 peptide sequences include sequences that do not increase or induce hepatocellular carcinoma (HCC) formation or HCC tumorigenesis. Such variants and fusions (chimeras) of FGF19 and/or FGF21 peptide sequences also include sequences that do not induce a substantial elevation or increase in lipid profile.

In one embodiment, a chimeric peptide sequence es or consists of: an N— terminal region having at least seven amino acid residues, the N—terminal region having a first amino acid position and a last amino acid on, where the N—terminal region has a DSSPL or DASPH sequence; and a C-terminal region having a portion ofFGF19, where the C-terminal region has a first amino acid position and a last amino acid position, where the C—terminal region includes amino acid residues 16-29 of FGF19 (WGDPIRLRHLYTSG), and where the W residue corresponds to the first amino acid position of the C-terminal region.

In another embodiment, a chimeric e sequence includes or consists of: an N- terminal region having a portion of FGF21, where the N—terminal region has a first amino acid position and a last amino acid position, where the N—terminal region has a GQV sequence, and where the V residue corresponds to the last amino acid position of the N-terminal region; and C—terminal region including a portion 9, the C—terminal region having a first amino acid position and a last amino acid position, where the C-terminal region includes amino acid residues 21-29 of FGF19 YTSG), and where the R residue ponds to the first position of the C—terminal region.

In a further embodiment, a chimeric peptide sequence includes or consists of any of: an N-terminal region comprising a portion of SEQ ID NO:1OO [FGF21], the N—terminal region having a first amino acid position and a last amino acid position, wherein the N-terminal region comprises at least 5 (or more) uous amino acids of SEQ ID NO:100 [FGF21] including the amino acid residues GQV, and wherein the V residue corresponds to the last amino acid position ofthe N-terminal region; and a C—terminal region comprising a n of SEQ ID NO:99 [FGF19], the C-terminal region having a first amino acid position and a last amino acid position, wherein the inal region comprises amino acid residues 21-29 of SEQ ID NO:99 [FGF19], RLRHLYTSG, and wherein the R residue ponds to the first position of the C—terminal region. In particular aspects, the N—terminal region comprises at least 6 contiguous amino acids (or more, e.g., 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30—40, 40-50, 50— 75, 75-100 contiguous amino acids) of SEQ ID NO: 100 [FGF21] including the amino acid residues GQV.

In an additional embodiment, a peptide sequence includes or consists of any of: a ast growth factor 19 (FGF19) sequence variant having one or more amino acid substitutions, insertions or ons compared to a reference or wild type FGF19; a ast growth factor 21 (FGF21) sequence variant having one or more amino acid substitutions, insertions or deletions compared to a reference or wild type FGF21; a portion of an FGF19 sequence fused to a portion of an FGF21 sequence; or a portion of an FGF19 sequence fused to a portion of an FGF21 sequence, wherein the FGF 19 and/or FGF21 sequence portion(s) have one or more amino acid substitutions, ions or deletions compared to a reference or wild type FGF19 and/or FGF21.

In still further embodiments, a peptide sequence or a chimeric peptide sequence includes or consists of amino-terminal amino acids 1-16 of SEQ ID NO:100 [FGF21] fused to carboxy-terminal amino acids 21-194 of SEQ ID NO:99 [FGF19], or the peptide sequence has amino-terminal amino acids 1-147 of SEQ ID NO:99 [FGF19] fused to carboxy-terminal amino acids 147-181 of SEQ ID NO:100 [FGF21] (M41), or the peptide sequence has terminal amino acids 1-20 of SEQ ID NO:99 [FGF19] fused to carboxy—terminal amino acids 17-181 of SEQ ID NO:100 [FGF21] (M44), or the peptide sequence has terminal amino acids 1—146 of SEQ ID NO:100 [FGF21] fused to carboxy-terminal amino acids 148-194 of SEQ ID NO:99 [FGF19] (M45), or the peptide sequence has amino-terminal amino acids 1-20 of SEQ ID NO:99 ] fused to al amino acidsl7-146 of SEQ ID NO:100 [FGF21] fused to y— terminal amino acids 148—194 of SEQ ID NO:99 [FGF19] (M46).

In yet additional embodiments, a peptide sequence or a chimeric peptide sequence has a WGDPI sequence motif corresponding to the WGDPI sequence of amino acids 16-20 of SEQ ID NO:99 [FGF 19], or has a substituted, mutated or absent WGDPI sequence motif ponding to FGF19 WGDPI sequence of amino acids 16—20 of FGF19, or the WGDPI sequence motif has one or more amino acids substituted, mutated or absent; or is distinct from an FGF l9 variant sequence having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the FGF19 WGDPI sequence at amino acids 16-20.

In yet fiirther embodiments, a peptide sequence or a chimeric peptide sequence has an N—terminal region that es or consists of amino acid residues VHYG, where the N—terminal region comprises amino acid residues DASPHVHYG, or where the N—terminal region comprises amino acid residues DSSPLVHYG, or where the N—terminal region comprises amino acid residues DSSPLLQ, or where the N—terminal region comprises amino acid residues DSSPLLQFGGQV. In particular aspects, the G ponds to the last position of the N—terminal region, or the Q residue is the last amino acid position of the N-terminal region, or the V residue corresponds to the last position of the N—terrninal region.

In still additional embodiments, a peptide ce or a chimeric peptide sequence has an N—terminal region that includes or consists of RHPIP, where R is the first amino acid position of the inal region; or HPIP (e.g., where HPIP are the first 4 amino acid residues of the N—terminal region), where H is the first amino acid position of the N—terminal region; or RPLAF, where R is the first amino acid position of the N—terminal region; or PLAF, where P is the first amino acid position of the N—terminal region; or R, where R is‘the first amino acid. position of the N-terminal region, or has at the N—terminal region any one of the following sequences: MDSSPL, MSDSSPL, SDSSPL, MSSPL or SSPL.

In other ments, a e sequence or a chimeric e sequence has, at the first position of the N—terminal region, an “M” residue, an “R” residue, a “S” e, a “H” residue, a “P” residue, a “L” residue or an “D” residue. In alternative ments, a peptide sequence or a chimeric peptide sequence does not have a “M” residue or an “R” e at the first amino acid position of the N—terminal region.

In still other ments, a peptide sequence or a chimeric peptide sequence has at the first and second positions of the inal region an MR sequence, or at the first and second positions of the N—terminal region an RM ce, or at the first and second positions of the N— al region an RD sequence, or at the first and second positions of the N-terminal region an DS sequence, or at the first and second ons of the N—terminal region an MD sequence, or at the first and second positions of the N—terminal region an MS sequence, or at the first through third positions of the N—terminal region an MDS sequence, or at the first through third positions of the N—terminal region an RDS sequence, or at the first through third positions of the N—terminal region an MSD sequence, or at the first through third positions of the N—terminal region an M88 sequence, or at the first through third positions of the N—terminal region an DSS sequence, or at the first through fourth positions of the N—terminal region an RDSS sequence, or at the first through fourth positions of the N—terminal region an MDSS sequence, or at the first through fifth positions of the N—terminal region an MRDSS sequence, or at the first through fifth positions of the N—terminal region an MSSPL sequence, or at the first through sixth positions of the N— terminal region an MDSSPL sequence, or at the first through h positions of the N—terminal region an MSDSSPL ce.

In still other embodiments, a peptide sequence or a chimeric peptide sequence an addition of amino acid residues 30—194 of SEQ ID NO:99 [FGF19] at the C-terminus, resulting in a chimeric polypeptide having at the last position of the C-terminal region that ponds to about residue 194 of SEQ ID NO:99 ]. In further other embodiments, a ic peptide sequence or peptide sequence comprises all or a portion of an FGF19 sequence (e.g., SEQ ID NO:99), oned at the inus of the peptide, or where the amino terminal “R” residue is deleted from the peptide. I In more particular embodiments, a chimeric e sequence or peptide sequence includes or consists Ofany ofMl—M98 variantpeptide sequences, or a subsequence or fragment of any ofthe Ml—M98 variant peptide sequences.

In additional particular embodiments, a chimeric peptide ce or e sequence has an N—terminal or a C-terminal region from about 20 to about 200 amino acid residues in length. In further particular ments, a chimeric e sequence or peptide sequence has at least one amino acid deletion. In still further particular embodiments, a chimeric peptide sequence or peptide sequence, or a subsequence or fragment thereof, has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid ons from the amino terminus, the carboxy-terminus or internally. In a particular non-limiting aspect, the amino acid substitution, or deletion is at any of amino acid positions 8-20 of FGF19 (AGPHVHYGWGDPI).

In more particular embodiments, a chimeric peptide sequence or peptide sequence includes or consists of an amino acid sequence of about 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more amino acids. In more particular embodiments, a chimeric peptide sequence or peptide sequence includes or consists of an amino acid sequence ofabout 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more amino acids of FGF19 or FGF21.

In further ular embodiments, chimeric peptide sequences and peptide sequences have particular functions or activities. In one aspect, a chimeric peptide sequence or peptide sequence ins or increases an FGFR4 mediated ty. In additional aspects, a chimeric peptide sequence or peptide sequence binds to fibroblast growth factor receptor 4 (FGFR4) or activates FGFR4, or does not detectably bind to ast growth factor receptor 4 (FGFR4) or te FGFR4, or binds to FGFR4 with an affinity less than, comparable to or greater than FGF19 binding affinity for FGFR4, or activates FGFR4 to an extent or amount less than, comparable to or greater than FGF19 activates FGFR4. In further aspects, a chimeric peptide sequence or peptide sequence has reduced hepatocellular carcinoma (HCC) formation ed to FGF19, or an FGF 19 variant sequence having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI tuted for the WGDPI ce at amino acids 16-20 of FGF19, and/or has greater glucose lowering activity compared to FGF19, or an FGF 19 variant sequence having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence at amino acids 16-20 of FGF19, and/or has less lipid increasing activity compared to FGF19, or an FGF 19 t sequence having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence at amino acids 16-20 of FGF19, and/or has less triglyceride, cholesterol, non-HDL or HDL increasing activity compared to FGF19, or an FGF 19 variant sequence having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence at amino acids 16-20 of FGF19, and/or has less lean mass reducing activity compared to FGF21.

Such functions and activities can be ascertained in vitro or in vivo, for e, in a db/db mouse. [0021a] In a particular embodiment the subject of the present invention, there is provided a e comprising: a) an inal region comprising at least seven amino acid residues, the N-terminal region having a first amino acid position and a last amino acid position, wherein the N- terminal region comprises DSSPL (SEQ ID NO:121) or DASPH (SEQ ID NO:122); and b) a C-terminal region having a first amino acid position and a last amino acid position, wherein the C-terminal region comprises i. a first C-terminal region sequence comprising WGDPIRLRHLYTSG (amino acids 16 to 29 of SEQ ID NO:99 [FGF19]), n the W residue ponds to the first amino acid position of the C-terminal region; and ii. a second C-terminal region sequence comprising PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSV RYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVS LSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPL ETDSMDPFGLVTGLEAVRSPSFEK (amino acid residues 30 to 194 of SEQ ID NO:99 [FGF19]) or a ce comprising 1 to 5 amino acid substitutions, deletions or insertions thereof; wherein the e i. binds to fibroblast growth factor receptor 4 (FGFR4) with an affinity equal to or greater than FGF19 binding affinity for FGFR4; ii. activates FGFR4 to an extent or amount equal to or greater than FGF19 activates FGFR4; iii. has at least one of reduced hepatocellular carcinoma (HCC) formation; greater e lowering activity, less lipid increasing activity, less triglyceride activity, less cholesterol activity, less non-HDL activity or less HDL increasing activity, as compared to FGF19, or as compared to an FGF19 variant sequence having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI tuted for the WGDPI ce at amino acids 16-20 of FGF19 (SEQ ID NO:99); and/or iv. has less lean mass reducing activity as compared to FGF21.

In still additional embodiments, chimeric peptide sequences and peptide sequences isolated or purified, and/or ic peptide sequences and peptide sequences can be included in compositions. In one embodiment, a chimeric peptide sequence or peptide ce is included in a pharmaceutical composition. Such compositions include combinations of inactive or other active ingredients. In one embodiment, a compositions, such as a pharmaceutical composition includes chimeric peptide sequence or peptide sequence and a glucose lowering agent.

In yet r ments, c acid les encoding the chimeric peptide sequence or peptide sequence are provided. Such molecules can further include an expression control element in operable linkage that confers expression of the nucleic acid molecule encoding the peptide in vitro, in a cell or in vivo, or a vector comprising the nucleic acid molecule (e.g., a viral vector). Transformed and host cells that express the chimeric e sequences and peptide sequences are also provided.

(FOLLOWED BY 7A) Uses and methods of treatment that include stration or delivery of any ic peptide sequence or peptide sequence are also provided. In particular embodiments, a use or method of ent of a subject includes administering an invention chimeric peptide or peptide sequence to a subject, such as a subject , or at risk of having, a disease or disorder treatable by an invention peptide sequence, in an amount effective for treating the disease or er. In particular embodiments the invention es for the use of the es described herein in the manufacture of a medicament for use in a method as described herein. In a further ment, a method includes administering an invention chimeric peptide or peptide sequence to a t, such as 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.

In particular aspects of the methods and uses, a chimeric peptide sequence or peptide ce is administered to a subject in an amount effective to improve glucose metabolism in the subject. In more ular aspects, a subject has a fasting plasma glucose level greater than 100 mg/dl or has a hemoglobin A1c (HbA1c) level above 6%, prior to administration.

In further embodiments, a use or method of treatment of a t is intended to or results in reduced glucose levels, increased insulin sensitivity, reduced insulin resistance, reduced glucagon, an improvement in glucose tolerance, or glucose metabolism or homeostasis, improved pancreatic function, or reduced triglyceride, cholesterol, IDL, LDL or VLDL levels, or a decrease in blood pressure, a decrease in intimal ning of the blood vessel, or a decrease in body mass or weight gain.

Methods of analyzing and/or fying a chimeric peptide sequence or peptide sequence are also provided, such as chimeric peptide sequences and peptide ces that have glucose lowering activity without substantial hepatocellular carcinoma (HCC) activity. In one embodiment, a method includes: a) providing a candidate chimeric peptide sequence or peptide ce; b) administering the candidate peptide ce to a test animal (e.g., a db/db mouse); c) measuring glucose levels of the animal after administration of the candidate peptide sequence, to ine if the candidate peptide sequence reduces glucose levels. In a particular aspect, the chimeric peptide sequence or peptide sequence is also analyzed for induction of HCC in the animal (e.g., assessing a hepatic tissue sample from the test animal), or sion of a marker correlating with HCC activity, wherein a candidate peptide having glucose lowering activity and not substantial HCC activity. Such methods identify the ate as having glucose lowering activity, optionally also without substantial hepatocellular carcinoma (HCC) activity.

(FOLLOWED BY 7B) Description of Drawings shows FGF19 and FGF21 protein sequences, and representative variant sequences, namely variant M5, variant M1, t M2, variant M69, variant M3, variant M48, variant M49, variant M50, t M51, variant M52, variant M53 and variant M70 peptide (FOLLOWED BY 8) WO 06486 sequences. 3 additional allelic (polymorphic) forms of FGF21, namely M71, M72 and M73 are also shown. shows representative domain exchanges n FGF21 (no shading) and FGF19 (grey shading) protein sequences, and the resultant fusion (chimeric) sequences. The amino acid regions from each ofFGF21 and FGF19 present in the fusion (chimera) are indicated by the s. e ng and lipid elevation are shown for each of the chimeric sequences. —3I show glucose lowering and body weight data. A) variant M5 ; B) variant M1; C) t M2 and variant M69; D) variant M3; E) variant M48 and variant M49; F) variant M51 and variant M50; G) variant M52 peptide; H) variant M53 e; and I) variant M70 peptide sequences all have glucose lowering (i.e., anti-diabetic) activity in db/db mice. Mice were injected with AAV vector expressing FGF19, FGF21, the selected variants, and saline and GFP are negative controls. —4I show serum lipid profile (triglyceride, total cholesterol, HDL and non- HDL) of db/db mice injected with AAV vector expressing FGF19, FGF21 or A) variant M5; B) variant Ml; C) variant M2 and variant M69; D) variant M3; E) variant M48 and t M49; F) variant M51 and variant M50; G) variant M52 peptide; H) variant M53 peptide; and I) t M70 e sequences. Variant M5 peptide sequence did not increase or elevate lipids, in contrast to FGF19, M1, M2 and M69 which increases and elevates lipids. Serum levels of all variants were comparable. Saline and GFP are negative controls.

FIG. SA-SI show hepatocellular carcinoma (HCC) — related data for A) variant M5; B) variant Ml; C) variant M2 and variant M69; D) variant M3; E) variant M48 and variant M49; F) variant M51 and variantMSO; G) variant M52; H) variant M53 peptide; and I) variant M70 peptide sequences. All variants did not cantly increase or induce hepatocellular carcinoma (HCC) formation or HCC tumorigenesis, in contrast to FGFl9. HCC score is recorded as the number ofHCC nodules on the surface of the entire liver from variants-injected mice divided by the number ofHCC s from wild type FGFl9—injected mice. —6I show lean mass or fat mass data for A) variant M5 ; B) variant Ml; C) variant M2 and variant M69; D) variant M3; E) variant M48 and variant M49; F) variant M51 and variant M50; G) variant M52; H) variant M53 peptide; and I) variant M70 peptide sequences.

Except for M2, M5 and M69, the variant peptide ces reduce lean mass or fat mass, in contrast to FGF21. —7B show graphical data demonstrating that injection of the recombinant A) variant M5; and B) variant M69 polypeptides reduce blood glucose in ob/ob mice. shows data indicating that liver expression of aldo-keto reductase family 1, member C18 (AkrlC18) and solute carrier family 1, member 2 (slcla2) s to correlate with HCC activity.

Detailed Description The invention provides ic and peptide sequences that are able to lower or reduce levels of glucose. In one embodiment, a chimeric peptide sequence includes or consists of an N-terminal region having at least seven amino acid residues and the N—terminal region having a first amino acid position and a last amino acid position, where the N—terminal region has a DSSPL or DASPH sequence; and a C-terminal region having a n ofFGF 19 and the C- terminal region having a first amino acid position and a last amino acid position, where the C— terminal region includes amino acid residues 16-29 of FGF19 RLRHLYTSG) and the W residue corresponds to the first amino acid position ofthe C—terminal region.

In another embodiment, a chimeric peptide sequence includes or consists of an N— terminal region having a portion ofFGF21 and the N—terminal region having a first amino acid position and a last amino acid position, where the N—terminal region has a GQV sequence and the V residue corresponds to the last amino acid position of the N—terminal region; and a C-terminal region having a portion of FGF19 and the inal region having a first amino acid position and a last amino acid position where the C-terminal region includes amino acid residues 21-29 of FGF19 (RLRHLYTSG) and the R residue corresponds to the first position of the C—terminal region.

In further embodiments, a peptide sequence includes or consists of a fibroblast growth factor 19 ) sequence variant having one or more amino acid substitutions, insertions or ons compared to a reference or wild type FGF19. In additional ments, a peptide ce includes or consists of a fibroblast growth factor 21 (FGF21) sequence variant having one or more amino acid substitutions, insertions or deletions ed to a reference or wild type FGF21. In yet additional embodiments, a peptide sequence includes or consists of a portion of an FGF19 sequence fused to a portion of an FGF21 ce. In still additional embodiments, a peptide sequence includes or consists of a portion of an FGF19 sequence fiised to a portion of an FGF21 sequence, where the FGF19 and/or FGF21 sequence portion(s) have one or more amino acid substitutions, insertions or deletions compared to a nce or wild type FGF 19 and/or FGF21.

The invention also provides methods and uses of ng a subject having or at risk of having a lic disorder treatable using variants and fusions of fibroblast growth factor 19 (FGF19) and/0r fibroblast growth factor 21 (FGF21) e sequences. In one embodiment, a WO 06486 2012/045087 method includes contacting or administering to a subject one or more variant or fusion fibroblast growth factor 19 (FGF19) and/0r fibroblast grth factor 21 (FGF21) peptide sequences in an amount effective for treating the disorder. In another embodiment, a method includes contacting or administering to a t one or more nucleic acid molecules ng a variant or fusion ast growth factor 19 (FGF19) and/or fibroblast growth factor 21 (FGF21) peptide ce (for example, an expression control element in operable linkage with the nucleic acid encoding the peptide ce, optionally including a vector), in an amount effective for treating the disorder.

Although an understanding of the underlying mechanism of action of the invention peptides is not required in order to practice the invention, without being bound to any particular theory or esis, it is believed that invention peptides mimic, at least in part, the effect that bariatric surgery has on, for example, glucose homeostasis and weight loss. Changes in gastrointestinal hormone secretion (e. g., glucagon—like peptide 1 (GLP-l)) after bariatric surgery are believed responsible for the resolution of, for example, diabetic conditions. FGF19 is highly sed in the distal small intestine, and enic over-expression of FGF19 improves glucose homeostasis. Because levels ofFGF19 in humans are also elevated following gastric bypass surgery, the elevated FGF19 might be involved with the remission of diabetes observed following bariatric y.

A representative reference or wild type FGF19 sequence is set forth as: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHR LPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPF GLVTGLEAVRSPSFEK (SEQ ID NO:99).

A representative reference or wild type FGF21 sequence is set forth as: HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKP GVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPG DPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (SEQ ID NO:100). FGF21 allelic variants are illustrated in Figure 1 (e.g., M70, M71 and M72).

The terms “peptide,” “protein,” and eptide” sequence are used interchangeably herein to refer to two or more amino acids, or “residues,” including chemical modifications and derivatives of amino acids, covalently linked by an amide bond or equivalent. The amino acids forming all or a part of a peptide may be from among the known 21 naturally occurring amino acids, which are referred to by both their single letter abbreviation or common three-letter abbreviation. In the peptide sequences of the invention, conventional amino acid residues have 2012/045087 their Conventional meaning. Thus, “Leu” is leucine, “Ile” is isoleucine, “Nle” is norleucine, and so on.

Exemplified herein are peptide sequences, distinct from reference FGF19 and FGF21 ptides set forth herein, that reduce or lower glucose, in viva (Tables 1-8 and Figure l). miting particular examples are a e sequence with amino-terminal amino acids 1—16 ofFGF21 fused to carboxy-terminal amino acids 21-194 of FGF19; a peptide sequence with amino—terminal amino acids 1—147 of FGF19 fused to carboxy—terminal amino acids 147-181 of FGF21; a peptide sequence with amino—terminal amino acids 1-20 of FGF19 fused to carboxy- terminal amino acids 17-181 of FGF21 ; a peptide sequence with amino-terminal amino acids 1- 146 ofFGF21 fused to carboxy—terminal amino acids 4’of FGF19; and a peptide sequence with amino-terminal amino acids 1-20 of FGF19 fused to internal amino acids 17—146 ofFGF21 fused to carboxy-terminal amino acids 148—194 of FGF19.

Additional particular peptides sequences have a WGDPI sequence motif corresponding to the WGDPI sequence of amino acids 16-20 of FGF19, lack a WGDPI sequence motif corresponding to the WGDPI sequence of amino acids 16—20 9, or have a substituted (i.e., mutated) WGDPI sequence motif corresponding to FGF19 WGDPI ce of amino acids 16—20 ofFGF19.

Particular e sequences ofthe ion also include ces distinct from FGF19 and FGF21 (e.g., as set forth herein), and FGF l9 variant ces having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for FGF19 WGDPI sequence at amino acids 16-20. Accordingly, the wild-type FGF19 and FGF21 (e.g., as set forth herein as SEQ ID NOS:99 and 100, respectively) may be excluded sequences, and FGF19 having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence at amino acids 16-20 of FGF19 may also be excluded. This exclusion, however, does not apply to where a sequence has, for example, 3 FGF21 residues fused to FGF19 having, for example, any of GQV, GQV, GDI, or GPI, or 2 FGF21 residues fused to any of WGPI, WGDI, GDPI, WDPI, WGDI, or WGDP.

Particular non-limiting examples of peptide sequences include or consist of all or a part of a sequence variant specified herein as Ml-M98 (SEQ ID NOs: 1-98). More particular non- limiting examples of peptide sequences include or consist of all or a part of a sequence set forth HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVAL RTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLS SAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGL EAVRSPSFEK (FGF21 sequences can also include an “R” residue at the amino terminus), or a subsequence or fragment thereof; or QFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTV AIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEELRPDGYNVYRSEKHRLPVSLSSAK QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAV K, or a subsequence or nt thereof; or RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHR LPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPF GLVTGLEAVRSPSFEK, or a subsequence or nt thereof; or RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK AVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRL PVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFG LVTGLEAVRSPSFEK, or a subsequence or fragment thereof; or DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSS AKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE AVRSPSFEK, or a subsequence or fragment f; or RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVAL RTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLS SAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGL EAVRSPSFEK (M69), or a subsequence or fragment thereof; or RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSA KQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA VRSPSFEK (M52), or a subsequence or nt thereof; or HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVAL RTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLS SAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGL EAVRSPSFEK (M5), or a subsequence or fragment thereof; HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKP GVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHSLPLPEPG NKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M71), or a subsequence or fragment thereof; or WO 06486 HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKP GVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPG NKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M72), or a subsequence or fragment thereof; or ' HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKP GVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPG NKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVVQDELQGVGGEG CHMHPENCKTLLTDIDRTHTEKPVWDGITGE (M73), or a subsequence or fragment thereof; RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHR LPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPF GLVTGLEAVRSPSFEK (M1), or a subsequence or fragment f; or RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRL PVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFG LVTGLEAVRSPSFEK (M2), or a subsequence or fragment thereof; or RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHR LPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPF GLVTGLEAVRSPSFEK (M3), or a subsequence or fragment thereof; or RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEH<AVALRT VAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSA KQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA VRSPSFEK (M48), or a subsequence or fragment thereof; or RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLP VSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGL VTGLEAVRSPSFEK (M49), or a subsequence or fragment thereof; or RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVA LRTVAIKGVHSVRYLCMGADGKMQGLLQYSBEDCAFEEEILEDGYNVYRSEKHRLPVSL SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTG LEAVRSPSFEK (M50), or a subsequence or fragment thereof; or RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVA LRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSL SSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTG LEAVRSPSFEK (M51), or a subsequence or fragment thereof; or lVHDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRT HSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSA KQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA VRSPSFEK (M53), or a subsequence or fragment thereof; and LVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAV ALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPV SLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLV TGLEAVRSPSFEK (M70), or a subsequence or fragment thereof, or for any of the foregoing e sequences the R terminal residue may be deleted. V Further particular non—limiting examples of peptide sequences include or consist of: HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVAL RTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLS SAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETIDSMDPFGLVTGL EAVRSPSFEK, or a subsequence or fragment thereof; or DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTV AIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAK QRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAV RSPSFEK, or a uence or fragment thereof; RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEI KAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHR LPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPF GLVTGLEAVRSPSFEK, or a subsequence or fragment f; DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIK AVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRL PVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFG AVRSPSFEK, or a subsequence or fragment thereof; DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALR TVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSS AKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE AVRSPSFEK, or a uence or fragment thereof.

Additional particular non-limiting examples of peptide sequences, having at the N— terminus, a peptide sequence including or consisting of all or a part of any of: HPIPDSSPLLQFGGQVRLRHLYTSG (M5); DSSPLLQFGGQVRLRHLYTSG (M6); RPLAFSDSSPLLQFGGQVRLRHLYTSG (M7); HPIPDSSPLLQWGDPIRLRHLYTSG (M8); HPIPDSSPLLQFGWGDPIRLRHLYTSG (M9); HPIPDSSPHVHYGWGDPI RLRHLYTSG (M10); RPLAFSDAGPLLQWGDPIRLRHLYTSG (M1 1); RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12); RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13); HPIPDSSPHVHYGGQVRLRHLYTSG (M14); RPLAFSDAGPHVHWGDPIRLRHLYTSG (M15); RPLAFSDAGPHVHWGDPI RLRHLYTSG (M16); RPLAFSDAGPHVGWGDPI RLRHLYTSG (M17); RPLAFSDAGPHYGWGDPIRLRHLYTSG (M18); RPLAFSDAGPVYGWGDPIRLRHLYTSG (M19); RPLAFSDAGPVHGWGDPI RLRHLYTSG (M20); RPLAFSDAGPVHYWGDPIRLRHLYTSG (M21); RPLAFSDAGPHVHGWGDPIRLRHLYTSG (M22); RPLAFSDAGPHHGWGDPIRLRHLYTSG (M23); DAGPHHYWGDPIRLRHLYTSG (M24); RPLAFSDAGPHVYWGDPIRLRHLYTSG (M25); RPLAFSDSSPLVHWGDPIRLRHLYTSG (M26); RPLAFSDSSPHVHWGDPIRLRHLYTSG (M22); RPLAFSDAGPHVWGDPIRLRHLYTSG (M28); RPLAFSDAGPHVHYWGDPIV TSG (M29); RPLAFSDAGPHVHYAWGDPIRLRHLYTSG (M30); RHPIPDSSPLLQFGAQVRLRHLYTSG (M3 1); RHPIPDSSPLLQFGDQVRLRHLYTSG (M32); RHPIPDSSPLLQFGPQVRLRHLYTSG (M33); RHPIPDSSPLLQFGGAVRLRHLYTSG (M34); RHPIPDSSPLLQFGGEVRLRHLYTSG (M35); RHPIPDSSPLLQFGGNVRLRHLYTSG (M36); SSPLLQFGGQARLRHLYTSG (M37); RHPIPDSSPLLQFGGQI RLRHLYTSG (M38); RHPIPDSSPLLQFGGQTRLRHLYTSG (M39); RHPIPDSSPLLQFGWGQPVRLRHLYTSG (M40); DAGPHVHYGWGDPIRLRHLYTSG (M74); DPIRLRHLYTSG (M75); RLRHLYTSG (M77); RHPIPDSSPLLQFGWGDPIRLRHLYTSG; RHPIPDSSPLLQWGDPIRLRHLYTSG; RPLAFSDAGPLLQFGWGDPI RLRHLYTSG; RHPIPDSSPHVHYGWGDPIRLRHLYTSG; RPLAFSDAGPLLQFGGQVRLRHLYTSG; RHPIPDSSPHVHYGGQVRLRHLYTSG; RPLAFSDAGPHVHYGGDIRLRHLYTSG; RDSSPLLQFGGQVRLRHLYTSG; RPLAFSDSSPLLQFGGQVRLRHLYTSG; RHPIPDSSPLLQFGAQVRLRHLYTSG; RHPIPDSSPLLQFGDQVRLRHLYTSG; RHPIPDSSPLLQFGPQVRLRHLYTSG; RHPIPDSSPLLQFGGAVRLRHLYTSG; RHPIPDSSPLLQFGGEVRLRHLYTSG; RHPIPDSSPLLQFGGNVRLRHLYTSG; RHPIPDSSPLLQFGGQARLRHLYTSG; RHPIPDSSPLLQFGGQIRLRHLYTSG; SSPLLQFGGQTRLRI—ILYTSG; RHPIPDSSPLLQFGWGQPVRLRHLYTSG; and for any of the foregoing peptide sequences the amino terminal R e may be deleted.

Peptide ces of the invention additionally include those with reduced or absent induction or formation of cellular carcinoma (HCC) ed to FGF19, or an FGF 19 variant sequence having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI tuted for the WGDPI sequence at amino acids 16-20 of FGF19. Peptide sequences of the ion also include those with greater glucose lowering activity compared to FGF19, or an FGF l9 variant sequence having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence at amino acids 16-20 of FGF19. Peptide sequences of the invention moreover include those with less lipid (e. g., triglyceride, cholesterol, non—HDL or HDL) increasing ty compared to FGF19, or an FGF 19 variant sequence having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence at amino acids 16-20 of FGF19.

Typically, the number of amino acids or residues in an invention peptide sequence will total less than about 250 (e.g.,amino acids or mimetics thereof). In various particular embodiments, the number of residues comprise from about 20 up to about 200 residues (e.g., amino acids or mimetics thereof). In additional embodiments, the number of residues comprise from about 50 up to about 200 residues (e.g., amino acids or mimetics thereof). In further embodiments, the number of residues comprise from about 100 up to about 195 residues (e.g., amino acids or mimetics thereof) in length.

Amino acids or residues can be linked by amide or by non—natural and non-amide chemical bonds including, for e, those formed with glutaraldehyde, N- hydroxysuccinimide esters, tional maleimides, or N, N’-dicyclohexylcarbodiimide (DCC).

Non-amide bonds include, for example, ketomethylene, aminomethylene, olefin, ether, thioether and the like (see, e.g, Spatola in Chem and Biochemistryflnino Acids, es and m, Vol. 7, pp 267-357 (1983), “Peptide and Backbone Modifications,” Marcel Decker, NY). Thus, when a peptide of the invention includes a portion of an FGF19 sequence and a portion of an FG21 sequence, the two portions need not be joined to each other by an amide bond, but can be joined by any other al moiety or conjugated together via a linker moiety.

The invention also includes subsequences, variants and modified forms of the exemplified peptide sequences (including the FGF19 and FGF21 variants and subsequences listed in Tables 1—8 and Figure l, and the FGF19/FGF21 fusions and chimeras listed in Tables 1—8 and Figure 1), so long as the foregoing retains at least a detectable or measureable activity or function. For e, certain exemplified t peptides have FGF19 C-terminal PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKM QGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPML PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK at the C-terminal portion, e. g., following the “TSG” amino acid residues of the variant.

Also, certain exemplified variant peptides, for example, those having all or a portion ofFGF21 sequence at the amino—terminus, have an “R” e positioned at the N—terminus, which can be d. Similarly, certain exemplified variant peptides, include an “M” residue positioned at the N-terminus, which can be appended to or further substituted for an d residue, such as an “R” residue. More particularly, in various embodiments peptide sequences at the N—terminus include any of: RDSS, DSS, MDSS or MRDSS. Furthermore, in cells when a “M” residue is nt to a “S” residue, the “M” residue may be cleaved suCh that the “M” e is deleted from the peptide sequence, whereas when the “M” residue is adjacent to a “D” residue, the “M” residue may not be cleaved. Thus, by way of example, in various embodiments peptide sequences include those with the following residues at the N—tenninus: MDSSPL, MSDSSPL (cleaved to SDSSPL) and MSSPL (cleaved to SSPL). [.0055] Accordingly, the “peptide,” eptide,” and “protein” sequences of the invention include subsequences, variants and modified forms of the FGF19 and FGF21 variants and uences listed in Tables 1-8 and Figure 1, and the FGF19/FGF21 fusions and chimeras listed in Tables 1-8 and Figure 1, so long as the subsequence, variant or modified form (e.g., fusion or chimera) s at least a detectable ty or function.

As used herein, the term “modify” and grammatical variations thereof, means that the composition deviates ve to a reference composition, such as a peptide sequence. Such modified peptide sequences, nucleic acids and other compositions may have r or less activity or function, or have a distinct function or ty compared with a reference unmodified peptide sequence, nucleic acid, or other composition, or may have a property desirable in a n formulated for therapy (e. g. serum half—life), to elicit dy for use in a detection assay, and/or for protein purification. For example, a peptide sequence of the invention can be modified to increase serum half—life, to increase in vitro and/or in vivo stability ofthe protein, etc.

Particular examples of such subsequences, variants and modified forms of the peptide sequences exemplified herein (e. g., a peptide ce listed in Tables 1—8 and Figure 1) include substitutions, deletions and/or insertions/additions of one or more amino acids, to or from the amino terminus, the carboxy-terminus or internally. One example is a substitution of an amino acid residue for another amino acid residue within the peptide ce. Another is a deletion of one or more amino acid residues from the peptide sequence, or an insertion or addition of one more amino acid residues into the peptide sequence.

The number of residues tuted, deleted or inserted/added are one or more amino acids (e.g., 1-3, 3—5, 5-10, 10-20, 20-30, 30-40, 40—50, 50-60, 60-70, 70-80, 80-90, 90-100, 100- 110, 110—120, 120—130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190—200, 200— 225, 225-250, or more) of a peptide sequence. Thus, an FGF19 or FGF21 sequence can have few or many amino acids tuted, deleted or inserted/added (e.g., 1-3, 3-5, 5-10, 10-20, 20—30, 30- 40, 40-50, 50-60, 60—70, 70-80, 80—90, 90—100, 100—110, 110-120, 120-130, 130—140, 140—150, 150-160, 160—170, 170—180, 180-190, 190-200, 200-225, 225—250, or more). In addition, an FGF19 amino acid sequence can include or t of an amino acid sequence of about 1—3, 3-5, —10, 10—20, 20-30, 30—40, 40-50, 50-60, 60-70, 70-80, 80-90, 90—100, 100-110, 110-120, 120— 130, 130—140, 140-150, 150—160, 160—170, 170-180, 0, 0, 200-225, 225-250, or more amino acids from FGF21; or an FGF21 amino acid or sequence can include or consist of an amino acid sequence of about 1-3, 3-5, 5-10, 10—20, 20—30, 30-40, 40—50, 50-60, 60—70, 70-80, 80—90, 90—100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 0, 170—180, 180- 190, 190-200, 200—225, 225-250, or more amino acids from FGF19.

Specific examples of tutions include substituting a D residue for an L-residue.

Accordingly, although residues are listed in the L-isomer configuration D—amino acids at any particular or all positions ofthe peptide sequences of the invention are included, unless a D- isomer leads to a sequence that has no able or measurable function.

Additional specific examples are non-conservative and conservative substitutions. A “conservative tution” is a replacement of one amino acid by a biologically, chemically or structurally similar residue. Biologically similar means that the substitution is compatible with a biological activity, e. g., glucose lowering activity. urally similar means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or having similar size, or the structure of a first, second or additional peptide sequence is maintained. Chemical similarity means that the residues have the same charge or are both hydrophilic and hydrophobic.

Particular es include the substitution of one hydrophobic e, such as isoleucine, , e or methionine for another, or the substitution of one polar residue for another, such as the tution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, serine for threonine, etc. Routine assays can be used to determine whether a subsequence, variant or modified form has activity, e. g., glucose ng activity.

Particular examples of subsequences, variants and modified forms of the peptide sequences exemplified herein (e.g., a peptide sequence listed in Tables 1-8 and Figure 1) have 50%—60%, 60%-70%, 70%—75%, %, 80%-85%, 85%—90%, 90%—95%, or 96%, 97%, 98%, or 99% identity to a reference peptide ce (for example, a peptide sequence in any of Tables 1—8 and Figure 1). The term “identity” and “homology” and grammatical variations thereof mean that two or more referenced entities are the same. Thus, where two amino acid sequences are identical, they have the identical amino acid sequence. “Areas, regions or domains of identity” mean that a portion of two or more referenced entities are the same. Thus, where two amino acid sequences are identical or homologous over one or more sequence regions, they share identity in these s.

The extent of identity between two sequences can be ascertained using a computer program and mathematical algorithm known in the art. Such algorithms that calculate percent sequence identity (homology) generally account for sequence gaps and mismatches over the comparison region. For example, a BLAST (e. g., BLAST 2.0) search thm (see, e.g., Altschul et al., J. Mol. Biol. 2152403 , ly available through NCBI) has exemplary search parameters as follows: Mismatch -2; gap open 5; gap extension 2. For e sequence comparisons, a BLASTP algorithm is typically used in combination with a scoring matrix, such as PAMlOO, PAM 250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and H ce comparison programs are also used to quantitate the extent of identity on et al., Proc. Natl. Acad. Sci. USA 8522444 (1988); Pearson, Methods Mol Biol. 132: 1 85 (2000); and Smith et al., J. Mol. Biol. 1472195 (1981)). Programs for quantitating protein structural similarity using Delaunay—based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 3041320 (2003)).

In the invention peptide sequences, including subsequences, variants and modified forms of the peptide ces exemplified herein (e.g., ces listed in Tables 1—8 and Figure 1) an “amino acid” or “residue” includes conventional alpha-amino acids as well as beta-amino acids, alpha, alpha disubstituted amino acids and N—substituted amino acids n at least one side chain is an amino acid side chain moiety as defined herein. An “amino acid” fiirther includes l alpha—amino acids, n the N—terminus amino group has a C1 to C6 linear or branched alkyl substituent. The term “amino acid” ore includes stereoisomers and ations of naturally occurring protein amino acids, non-protein amino acids, post- ationally modified amino acids (e.g., by glycosylation, phosphorylation, ester or amide cleavage, etc.), enzymatically modified or synthesized amino acids, derivatized amino acids, constructs or structures designed to mimic amino acids, amino acids with a side chain moiety modified, derivatized from naturally occurring moieties, or synthetic, or not naturally occurring, etc. Modified and unusual amino acids are included in the peptide sequences of the invention (see, for example, in Synthetic Pepfles: A User’s Guide; Hruby et al., Biochem. J. 268:249 (1990); and Toniolo C., Int. J. Peptide Protein Res. 35:287 ).

In addition, protecting and modifying groups of amino acids are included. The term “amino acid side chain moiety” as used herein includes any side chain of any amino acid, as the WO 06486 term “amino acid” is defined herein. This therefore includes the side chain moiety in naturally occurring amino acids. It further includes side chain es in modified naturally ing amino acids as set forth herein and known to one of skill in the art, such as side chain moieties in stereoisomers and modifications of naturally occurring protein amino acids, otein amino acids, post-translationally modified amino acids, enzymatically modified or synthesized amino acids, derivatized amino acids, constructs or structures designed to mimic amino acids, etc. For example, the side chain moiety of any amino acid disclosed herein or known to one of skill in the art is included within the definition.

A “derivative of an amino acid side chain moiety” is included within the definition of an amino acid side chain moiety. Non-limiting examples of derivatized amino acid side chain es include, for example: (a) adding one or more saturated or rated carbon atoms to an existing alkyl, aryl, or aralkyl chain; (b) substituting a carbon in the side chain with another atom, preferably oxygen or nitrogen; (c) adding a terminal group to a carbon atom of the side chain, including methyl (--CH3), methoxy (--OCH3), nitro (--N02), hydroxyl (——OH), or cyano ); (d) for side chain moieties including a hydroxy, thiol or amino groups, adding a suitable hydroxy, thiol or amino ting group; or (e) for side chain moieties including a ring structure, adding one or more ring substituents, including hydroxyl, halogen, alkyl, or aryl groups attached directly or through an ether linkage. For amino , suitable protecting groups are known to the skilled artisan. Provided such derivatization provides a d activity in the final peptide ce (e.g., glucose ng, improved glucose or lipid metabolism, anti-diabetic activity, absence of substantial HCC formation or tumorigenesis, absence of substantial modulation of lean or fat mass, etc.).

An “amino acid side chain moiety” includes all such derivatization, and particular non-limiting examples include: gamma-amino butyric acid, 12—amino dodecanoic acid, alpha- aminoisobutyric acid, 6-amino hexanoic acid, 4-(aminomethyl)—cyclohexane carboxylic acid, 8- amino octanoic acid, biphenylalanine, Boc--t-butoxycarbonyl, benzyl, l, line, diaminobutyric acid, pyrrollysine, diaminopropionic acid, 3,3-diphenylalanine, orthonine, citrulline, hydro—2H-isoindolecarboxylic acid, ethyl, uorenylmethoxycarbonyl, heptanoyl (CH3-—(CH2).sub.5--C(=O)--), hexanoyl (CH3—-(CH2)4--C(=O)—-), homoarginine, homocysteine, homolysine, homophenylalanine, homoserine, methyl, methionine sulfoxide, methionine e, norvaline (NVA), phenylglycine, propyl, isopropyl, sarcosine (SAR), tert- butylalanine, and benzyloxycarbonyl.

A single amino acid, including stereoisomers and modifications of naturally occurring protein amino acids, non—protein amino acids, post—translationally modified amino acids, enzymatically synthesized amino acids, non-naturally occurring amino acids including derivatized amino acids, an alpha, alpha disubstituted amino acid derived from any of the foregoing (i.e., an alpha, alpha disubstituted amino acid, wherein at least one side chain is the same as that of the residue from which it is derived), a beta-amino acid derived from any of the foregoing (i.e., a beta—amino acid which other than for the presence of a beta—carbon is otherwise the same as the residue from which it is derived) etc., including all of the foregoing can be referred to herein as a “residue.” Suitable substituents, in on to the side chain moiety of the alpha-amino acid, include C1 to C6 linear or branched alkyl. Aib is an example of an alpha, alpha disubstituted amino acid. While alpha, alpha disubstituted amino acids can be referred to using conventional L— and eric references, it is to be tood that such references are for convenience, and that where the substituents at the alpha-position are different, such amino acid can interchangeably be referred to as an alpha, alpha disubstituted amino acid derived from the L- or D-isomer, as appropriate, of a residue with the designated amino acid side chain moiety.

Thus (S)-2—Aminomethyl—hexanoic acid can be ed to as either an alpha, alpha disubstituted amino acid derived from L-Nle (norleucine) or as an alpha, alpha disubstituted amino acid derived from D-Ala. Similarly, Aib can be referred to as an alpha, alpha disubstituted amino acid derived from Ala. Whenever an alpha, alpha disubstituted amino acid is provided, it is to be understood as including all (R) and (S) configurations thereof.

An “N—substituted amino acid” includes any amino acid wherein an amino acid side chain moiety is covalently bonded to the backbone amino group, optionally where there are no substituents other than H in the alpha-carbon position. Sarcosine is an example of an N- substituted amino acid. By way of example, sarcosine can be referred to as an N—substituted amino acid derivative of Ala, in that the amino acid side chain moiety of ine and Ala is the same, i.e., methyl.

Covalent modifications of the invention peptide sequences, including subsequences, variants and modified forms of the e sequences ified herein (e. g., sequences listed in Tables 1—8 and Figure 1), are included in the invention. One type of covalent modification includes reacting targeted amino acid residues with an c derivatizing agent that is e of reacting with selected side chains or the N— or C—terminal residues of the peptide.

Derivatization with bifunctional agents is useful, for instance, for cross linking peptide to a water— insoluble support matrix or e for use in the method for ing anti-peptide antibodies, and vice-versa. Commonly used cross linking agents e, e.g., l,1-bis(diazoacetyl)—2— phenylethane, glutaraldehyde, N—hydroxysuccinimide esters, for e, esters with 4- azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidylpropionate), bifiinctional maleimides such as bis-N-maleimido-l,8-octane and agents such as methyl—3-[(p—azidophenyl)dithio]propioimidate.

WO 06486 2012/045087 Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, orylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha—amino groups of lysine, ne, and histidine side chains (T, E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 ), acetylation of the N—terminal amine, amidation of any C-terminal carboxyl group, etc.

Exemplified peptide sequences, and subsequences, variants and modified forms of the peptide sequences exemplified herein (e. g., ces listed in Tables 1-8 and Figure 1), can also include alterations of the backbone for stability, derivatives, and peptidomimetics. The term “peptidomimetic” includes a molecule that is a mimic of a residue (referred to as a “mimetic”), including but not limited to piperazine core molecules, keto-piperazine core molecules and diazepine core molecules. Unless otherwise specified, an amino acid mimetic of an invention peptide sequence includes both a carboxyl group and amino group, and a group ponding to an amino acid side chain, or in the case of a mimetic of Glycine, no side chain other than hydrogen.

By way of example, these would include compounds that mimic the sterics, surface charge distribution, polarity, etc. of a naturally occurring amino acid, but need not be an amino acid, which would impart stability in the biological . For e, Proline may be substituted by other lactams or lactones of suitable size and substitution; Leucine may be substituted by an alkyl ketone, N-substituted amide, as well as ions in amino acid side chain length using alkyl, alkenyl or other substituents, others may be apparent to the skilled n.

The essential element of making such substitutions is to provide a le of roughly the same size and charge and configuration as the e used to design the molecule. Refinement of these modifications will be made by analyzing the compounds in a functional (e.g., glucose lowering) or other assay, and comparing the structure activity relationship. Such methods are within the scope of the skilled artisan working in medicinal chemistry and drug development.

Another type of modification of the invention e sequences, including subsequences, sequence variants and modified forms of the exemplified peptide ces (including the peptides listed in Tables 1—8 and Figure l), is glycosylation. As used herein, “glycosylation” broadly refers to the presence, addition or attachment of one or more sugar (e.g., carbohydrate) moieties to proteins, lipids or other organic molecules. The use of the term “deglycosylation” herein is generally intended to mean the removal or deletion, of one or more sugar (e. g., ydrate) moieties. In addition, the phrase includes ative changes in the glycosylation of the native proteins involving a change in the type and proportions (amount) of the various sugar (e. g., carbohydrate) moieties present. 2012/045087 Glycosylation can be achieved by modification of an amino acid residue, or by adding one or more glycosylation sites that may or may not be present in the native sequence. For example, a typically non-glycosylated residue can be substituted for a residue that may be glycosylated. on of glycosylation sites can be accomplished by altering the amino acid sequence. The alteration to the peptide sequence may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues (for O-linked glycosylation sites) or asparaglne residues (for N—linked ylation sites). The structures ofN—linked and O-linked oligosaccharides and the sugar residues found in each type may be different. One type of sugar that is commonly found on both is N—acetylneuraminic acid (hereafter referred to as sialic acid).

Sialic acid is usually the terminal residue of both N—linked and ed oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the rotein.

Peptide sequences of the invention may optionally be altered through changes at the nucleotide (e.g., DNA) level, particularly by mutating the DNA encoding the peptide at preselected bases such that codons are generated that will translate into the desired amino acids. r means of increasing the number of carbohydrate moieties on the peptide is by chemical or enzymatic ng of glycosides to the ptide (see, for example, in WO 87/05330). De- glycosylation can be lished by removing the underlying glycosylation site, by deleting the glycosylation by chemical and/or enzymatic means, or by substitution of codons encoding amino acid residues that are glycosylated. Chemical deglycosylation techniques are known, and enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases.

Various cell lines can be used to produce proteins that are glycosylated. One non- limiting example is Dihydrofolate reductase (DHFR) — deficient Chinese Hamster Ovary (CHO) cells, which are a commonly used host cell for the production of recombinant glycoproteins.

These cells do not express the enzyme beta-galactoside 2,6—sialyltransferase and therefore do not add sialic acid in the alpha—2,6 e to N—linked oligosaccharides of glycoproteins produced in these cells. r type of ation is to conjugate (e.g., link) one or more additional components or molecules at the N— and/or C—tenninus of an invention e sequence, such as another protein (e. g., a protein having an amino acid sequence heterologous to the subject protein), or a carrier molecule. Thus, an exemplary e sequence can be a conjugate with another component or molecule.

In certain embodiments, the amino- or carboxy— terminus of an invention peptide sequence can be fused with an globulin Fc region (e.g., human PC) to form a fusion _ conjugate (or fiJsion molecule). Fc fusion conjugates can increase the systemic half-life of WO 06486 biopharmaceuticals, and thus the biopharmaceutical product may have prolonged activity or require less frequent administration. Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells that line the blood vessels, and, upon binding, the Fc fusion molecule is protected from degradation and re-released into the ation, keeping the le in circulation longer. This PC binding is believed to be the mechanism by which endogenous IgG s its long plasma half—life. Well—known and validated Fc—fusion drugs consist oftwo copies of a biopharmaceutical linked to the Fc region of an antibody to improve pharmacokinetics, solubility, and production efficiency. More recent Fc-fusion technology links a single copy of a biopharmaceutical to Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical as compared to traditional Fc-fusion conjugates.

A conjugate modification can be used to produce a peptide sequence that retains activity with an additional or complementary on or activity of the second molecule. For example, a peptide sequence may be conjugated to a molecule, e. g., to facilitate solubility, storage, in vivo or shelf half—life or stability, reduction in genicity, delayed or controlled release in vivo, etc. Other functions or activities include a ate that reduces toxicity relative to an unconjugated peptide ce, a ate that s a type of cell or organ more efficiently than an unconjugated peptide sequence, or a drug to further counter the causes or effects associated with a disorder or disease as set forth herein (e.g., diabetes).

Clinical effectiveness of protein therapeutics may be limited by short plasma half-life and tibility to degradation. Studies of various therapeutic proteins have shown that various modifications, including conjugating or linking the peptide sequence to any of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes (see, for example, typically via a linking moiety covalently bound to both the protein and the nonproteinaceous polymer (e.g., a PEG) can prolong half-life. Such PEG- ated biomolecules have been shown to possess clinically useful properties, including better physical and thermal stability, protection against susceptibility to enzymatic degradation, increased solubility, longer in vivo circulating ife and decreased clearance, reduced genicity and nicity, and reduced toxicity.

PEGs suitable for conjugation to an invention peptide sequence is generally soluble in water at room temperature, and have the general formula R(O-CH2-CH2)nO-R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. When R is a protective group, it generally has from 1 to 8 carbons. The PEG conjugated to the peptide sequence can be linear or branched. Branched PEG tives, “star— PEGs” and armed PEGs are included in the invention. ‘A molecular weight of the PEG used in the invention is not cted to any particular range, but certain embodiments have a molecular weight n 500 and 20,000 while other embodiments have a molecular weight between 4,000 and 10,000.

The invention includes compositions of conjugates n the PEGs have different “n” values and thus the various different PEGs are present in specific ratios. For example, some compositions comprise a mixture of conjugates where n=1, 2, 3 and 4. In some itions, the percentage of conjugates where n=l is 18-25%, the percentage of conjugates where n=2 is 50— 66%, the percentage of conjugates where n=3 is , and the percentage of conjugates where n=4 is up to 5%. Such compositions can be produced by reaction conditions and purification methods know in the art.

PEG may directly or indirectly (e. g., through an intermediate) bind to the peptide sequences of the invention. For example, in one embodiment, PEG binds via a terminal ve group (a “spacer”). The spacer, is, for example, a terminal reactive group which mediates a bond between the free amino or carboxyl groups of one or more of the peptide sequences and polyethylene glycol. The PEG having the spacer which may be bound to the free amino group includes N—hydroxysuccinylimide polyethylene glycol which may beprepared by activating succinic acid ester of polyethylene glycol with N—hydroxysuccinylimide. Another activated polyethylene glycol which may be bound to free amino group is 2,4-bis(O- methoxypolyethyleneglycol)—6-chloro-s-triazine which may be prepared by reacting polyethylene glycol monomethyl ether with cyanuric de. The activated polyethylene glycol which is bound to the free carboxyl group es polyoxyethylenediamine.

Conjugation of one or more of invention peptide sequences to PEG having a spacer may be carried‘out by various conventional methods. For example, the conjugation on can be carried out in solution at a pH of from 5 to 10, at temperature from 4°C to room temperature, for 30 minutes to 20 hours, utilizing a molar ratio of reagent to protein of from 4:1 to 30:1.

Reaction conditions may be selected to direct the reaction towards ing predominantly a - desired degree of substitution. In general, low temperature, low pH (e. g., pH=5), and short reaction time tend to decrease the number of PEGs attached, s high temperature, neutral to high pH (e.g., pHZ7), and longer reaction time tend to increase the number ofPEGs attached.

Various methods known in the art may be used to terminate the reaction. In some ments the reaction is terminated by acidifying the reaction mixture and freezing at, e. g., -20°C.

Invention peptide sequences including subsequences, sequence variants and modified forms of the exemplified peptide ces (including the peptides listed in Tables 1-8 and Figure 1), further include conjugation to large, slowly metabolized macromolecules such as proteins; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads; ric amino acids such as polygliitamic acid, sine; amino acid copolymers; inactivated Virus particles; inactivated ial toxins such as toxoid from diphtheria, tetanus, cholera, leukotoxin molecules; vated bacteria; and dendritic cells. Such conjugated forms, if desired, can be used to produce antibodies against peptide sequences of the invention.

Additional suitable components and molecules for conjugation include, for example, thyroglobulin; albumins such as human serum n (HSA); s toxoid; Diphtheria toxoid; polyamino acids such as poly(D—lysine:D—glutamic acid); VP6 polypeptides of rotaviruses; influenza virus hemaglutinin, influenza virus nucleoprotein; Keyhole Limpet Hemocyanin (KLH); and hepatitis B Virus core protein and surface antigen; or any combination of the foregoing.

Fusion of albumin to an invention peptide sequence can, for example, be achieved by c manipulation, such that the DNA coding for HSA (human serum albumin), or a fragment thereof, is joined to the DNA coding for a peptide sequence. Thereafter, a le host can be transformed or transfected with the fused nucleotide sequence in the form of, for example, a suitable plasmid, so as to s a fusion polypeptide. The sion may be effected in vitro from, for example, prokaryotic or eukaryotic cells, or in vivo from, for example, a transgenic organism. In some embodiments ofthe invention, the expression of the fusion protein is performed in mammalian cell lines, for example, CHO cell lines.

Further means for genetically fusing target proteins or peptides to albumin include a technology known as Albufuse® (Novozymes Biopharma A/S; Denmark), and the conjugated therapeutic peptide sequences ntly become much more effective with better uptake in the body. The logy has been utilized commercially to produce Albuferon® (Human Genome Sciences), a combination of albumin and interferon a—2B used to treat hepatitis C infection.

Another embodiment entails the use of one or more human domain antibodies (dAb). dAbs are the smallest fianctional binding units of human antibodies (IgGs) and have favorable stability and solubility teristics. The technology entails a dAb(s) conjugated to HSA (thereby forming a “AlbudAb”; see, e. g., 921B, W02005/118642 and W02006/051288) and a molecule of interest (e.g., a peptide sequence of the invention). AlbudAbs are often smaller and easier to manufacture in microbial expression systems, such as bacteria or yeast, than current technologies used for extending the serum half-life of es. As HSA has a half-life of about three weeks, the ing conjugated molecule improves the half—life. Use of the dAb logy may also enhance the efficacy of the molecule of st.

Additional suitable components and molecules for conjugation include those suitable for isolation or purification. Particular non-limiting examples e binding molecules, such as biotin (biotin-avidin specific binding pair), an antibody, a receptor, a ligand, a lectin, or molecules that comprise a solid support, including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test strips, and membranes.

Purification s such as cation exchange chromatography may be used to separate conjugates by charge ence, which effectively tes conjugates into their various molecular weights. For example, the cation exchange column can be loaded and then washed with ~20 mM sodium acetate, pH ~4, and then eluted with a linear (OM to 0.5M) NaCl gradient buffered at a pH from 3 to 5.5, preferably at pH ~4.5. The t of the fractions obtained by cation ge tography may be identified by molecular weight using conventional methods, for example, mass spectroscopy, SDS-PAGE, or other known methods for separating molecular entities by molecular weight. A fraction is then accordingly identified which contains the conjugate having the desired number of PEGs attached, d free from unmodified protein sequences and from conjugates having other numbers of PEGs attached.

In still other embodiments, an invention peptide ce is linked to a chemical agent (e. g., an immunotoxin or chemotherapeutic , including, but are not limited to, a cytotoxic agent, including taxol, cytochalasin B, gramicidin D, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, and analogs or homologs thereof.

Other chemical agents include, for example, antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6— thioguanine, cytarabine, S-fluorouracil decarbazine); alkylating agents (e. g., mechlorethamine, carmustine and lomustine, hosphamide, busulfan, omannitol, streptozotocin, mitomycin C, and cisplatin); otics (e.g., bleomycin); and anti-mitotic agents (e. g., vincristine and vinblastine). Cytotoxins can be conjugated to a peptide ofthe invention using linker technology known in the art and described herein.

Further suitable components and molecules for conjugation include those suitable for detection in an assay. Particular non-limiting examples include detectable labels, such as a sotope (e. g., 1251; 3 5 S, 32F; 33P), an enzyme which tes a detectable product (e.g., luciferase, B—galactosidase, horse radish peroxidase and alkaline phosphatase), a fluorescent protein, a chromogenic protein, dye (e. g., fluorescein isothiocyanate); fluorescence emitting metals (e.g., 152Eu); chemiluminescent nds (e. g., luminol and acridinium salts); bioluminescent compounds (e. g., luciferin); and fluorescent proteins. Indirect labels include labeled or detectable antibodies that bind to a peptide sequence, where the antibody may be detected.

In certain embodiments, a peptide sequence ofthe invention is conjugated to a radioactive isotope to generate a cytotoxic radiopharrnaceutical (radioimmunoconj ugates) usefiil as a stic or therapeutic agent. Examples of such radioactive isotopes e, but are not d to, iodine 13 indium 1 1', yttrium 9° and lutetium 177. Methods for preparing radioimmunoconjugates are known to the skilled artisan. Examples of radioimmunoconjugates that are commercially available include ibritumomab,-tiuxetan, and tositumomab.

Other means and methods included in the ion for prolonging the ation half-life, increasing stability, reducing clearance, or altering immunogenicity or allergenicity of a peptide sequence ofthe invention involves modification of the peptide sequence by hesylation, which utilizes hydroxyethyl starch tives linked to other molecules in order to modify the molecule’s teristics. Various aspects of hesylation are described in, for example, US.

Patent Appln. Nos. 2007/0134197 and 2006/0258607 Any of the foregoing components and molecules used to modify peptide sequences of the invention may optionally be conjugated via a linker. Suitable linkers include “flexible linkers” which are generally of sufficient length to permit some movement between the modified peptide sequences and the linked components and molecules. The linker molecules are generally about 6-50 atoms long. The linker molecules may also be, for example, aryl acetylene, ethylene glycol ers containing 2—10 monomer units, diamines, diacids, amino acids, or ations thereof. Suitable linkers can be readily selected and can be of any suitable length, such as l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30—50 amino acids (e.g., Gly).

Exemplary flexible s include e polymers (G)n, glycine-serine polymers (for example, (GS)n, GSGGSn and GGGSn, where n is an integer of at least one), e-alanine polymers, alanine—serine polymers, and other e linkers. Glycine and glycine-serine polymers are relatively unstructured, and therefore may serve as a neutral tether between components. Exemplary flexible linkers include, but are not limited to GGSG, GGSGG, GSGSG, GSGGG, GGGSG, and GSSSG. e sequences of the invention, including the FGF19 and FGF21 variants and subsequences and the FGF19/FGF21 fusions and chimeras listed in Tables 1—8 and Figure 1, as well as subsequences, sequence ts and modified forms of the sequences listed in Tables 1-8 and Figure 1 have one or more activities as set forth herein. One example of an activity is glucose lowering activity. Another example of an activity is reduced stimulation or formation of hepatocellular carcinoma (HCC), for e, as compared to FGF19. An additional example of an activity is lower or reduced lipid (e.g., triglyceride, cholesterol, non—HDL) or HDL sing activity, for example, as ed to FGF21. A further example of an ty is a lower or reduced lean muscle mass reducing activity, for e, as compared to FGF21. Yet another example of an activity is g to fibroblast growth factor receptor—4 (FGFR4), or activating FGFR4, for example, e sequences that bind to FGFR4 with an affinity comparable to or greater than FGF19 binding affinity for FGFR4; and peptide sequences that activate FGFR4 to an extent or amount comparable to or greater than FGF19 activates FGFR4. Still further examples 2012/045087 of activities include down—regulation or reduction of aldo—keto reductase gene expression, for example, compared to FGF 19; up-regulation or increased Slc1a2 gene sion compared to FGF21.

More particularly, peptide sequences of the invention, including the FGF19 and FGF21 variants and subsequences and the FGF19/FGF21 fusions and chimeras listed in Tables 1- 8 and Figure 1, as well as uences, variants and modified forms of the sequences listed in Tables 1-8 and Figure 1 include those with the following ties: peptide ces having reduced hepatocellular carcinoma (HCC) formation compared to FGF19, or an FGF 19 variant sequence having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence at amino acids 16-20 of FGF19; peptide sequences having greater glucose lowering activity compared to FGF19, or FGF 19 variant sequence having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence at amino acids 16-20 of FGF19; e sequences having less lipid increasing activity (e.g., less triglyceride, cholesterol, non-HDL) or more HDL increasing activity compared to FGF19, or an FGF 19 variant sequence having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence at amino acids 16-20 9; and peptide sequences having less lean mass reducing activity as compared to FGF21.

More particularly, peptide sequences of the invention, including the FGF19 and FGF21 ts and subsequences and the FGF19/FGF21 fusions and chimeras listed in Tables 1- 8 and Figure 1, as well as subsequences, variants and modified forms of the ces listed in Tables 1-8 and Figure 1 include those with the following activities: peptide sequences that bind to fibroblast growth factor receptor-4 ), or activate FGFR4, such as e sequences that bind to FGFR4 with an y comparable to or r than FGF19 binding y for FGFR4; peptide sequences that activate FGFR4 to an extent or amount comparable to or greater than FGF19 activates FGFR4; peptide sequences that down-regulate or reduce aldo-keto reductase gene expression, for example, compared to FGF19; and peptide sequences that up—regulate or increase solute carrier family 1, member 2 (Slc1a2) gene expression as compared to FGF21.

Activities such as, for example, hepatocellular carcinoma (HCC) ion or tumorigenesis, glucose lowering activity, lipid increasing activity, or lean mass reducing activity can be ascertained in an animal, such as a db/db mouse. Measurement of binding to FGFR4 or activation of FGFR4 can be ascertained by assays disclosed herein (see, for example, Example 1) or known to the skilled artisan.

The term “bind,” or “binding,” when used in reference to a peptide ce, means that the peptide sequence interacts at the molecular level. Thus, a peptide sequence that binds to FGFR4 binds to all or a part of the FGFR4 sequence. Specific and selective binding can be distinguished from non—specific binding using assays known in the art (e. g., competition binding, immunoprecipitation, ELISA, flow cytometry, Western blotting).

Peptides and peptidomimetics can be produced and isolated using methods known in the art. Peptides can be synthesized, in whole or in part, using chemical s (see, e.g., Caruthers (1980). Nucleic Acids Res. Symp. Ser. 215; Horn (1980); and Banga, A.K., Therapeutic Peptides and Proteins, Formulation, Processing and ry Systems (1995) Technomic Publishing Co., Lancaster, PA). e synthesis can be performed using various solid-phase techniques (see, e.g., Roberge Science 2692202 (1995); Merrifield, Methods l. 289:3 (1997)) and ted synthesis may be achieved, e. g., using the AB1431A Peptide Synthesizer (Perkin Elmer) in accordance with the cturer’s instructions. Peptides and peptide mimetics can also be synthesized using combinatorial methodologies. Synthetic residues and polypeptides incorporating mimetics can be sized using a variety of procedures and methodologies known in the art (see, e.g., Organic Syntheses Collective Volumes, Gilman, ez‘ al.

(Eds) John Wiley & Sons, Inc., NY). Modified peptides can be produced by chemical modification methods (see, for example, Belousov, Nucleic Acids Res. 2523440 (1997); Frenkel, Free Radic. Biol. Med. 19:373 (1995); and Blommers, mistry 33:7886 (1994)). Peptide sequence variations, derivatives, substitutions and modifications can also be made using methods such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR based nesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res, 1324331 (1986); Zoller et al., Nucl. Acids Res. 10:6487 (1987)), cassette nesis (Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA 3172415 (1986)) and other techniques can be performed on cloned DNA to produce invention peptide sequences, variants, fusions and chimeras, and variations, tives, substitutions and modifications thereof.

A “synthesized” or “manufactured” peptide sequence is a e made by method involving manipulation by the hand of man. Such methods include but are not limited to the aforementioned, such as al synthesis, recombinant DNA technology, biochemical or tic fragmentation of larger molecules, and combinations of the foregoing.

Peptide sequences of the invention including subsequences, sequence variants and modified forms of the exemplified peptide sequences (e. g., sequences listed in Tables 1-8 and Figure 1), can also be modified to form a chimeric molecule. In ance with the invention, there are provided peptide ces that include a heterologous . Such domains can be 2012/045087 ,‘ added to the amino-terminus or at the carboxyl-terminus of the peptide sequence. Heterologous domains can also be positioned within the peptide sequence, and/or alternatively flanked by FGF 19 and/or FGF2l derived amino acid sequences.

The term “peptide” also includes dimers or multimers mers) of peptides. In accordance with the invention, there are also ed dimers or multimers (oligomers) of the exemplified e sequences as well as subsequences, variants and modified forms of the exemplified peptide sequences (e.g., sequences listed in Tables 1-8 and Figure l).

The invention further provides nucleic acid molecules encoding peptide sequences of the invention, including subsequences, sequence variants and modified forms of the sequences listed in Tables 1-8 and Figure l, and vectors that include nucleic acid that s the peptide.

Accordingly, “nucleic acids” include those that encode the exemplified peptide sequences disclosed , as well as those encoding onal subsequences, ce variants and modified forms ofthe ified peptide sequences, so long as the foregoing retain at least detectable or measureable activity or function. For example, a subsequence, a t or modified form of an exemplified peptide sequence disclosed herein (e.g., a sequence listed in Tables 1-8 and Figure 1) that retains some ability to lower or reduce glucose, e normal glucose homeostasis, or reduce the histopathological conditions associated with chronic or acute hyperglycemia in viva, etc. c acid, which can also be referred to herein as a gene, polynucleotide, nucleotide sequence, primer, oligonucleotide or probe refers to natural or modified purine- and dine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides and oc-anomeric forms thereof. The two or more purine— and pyrimidine—containing polymers are typically linked by a phosphoester bond or analog thereof. The terms can be used interchangeably to refer to all forms of nucleic acid, ing deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The nucleic acids can be single strand, double, or triplex, linear or circular. Nucleic acids include genomic DNA and cDNA. RNA nucleic acid can be spliced or unspliced mRNA, rRNA, tRNA or antisense. Nucleic acids include naturally occurring, synthetic, as well as nucleotide analogues and derivatives.

As a result ofthe degeneracy of the genetic code, nucleic acid molecules include ' sequences degenerate with respect to nucleic acid molecules encoding the peptide ces of the invention. Thus, degenerate nucleic acid sequences encoding peptide sequences, including uences, ts and modified forms of the peptide sequences exemplified herein (e. g., sequences listed in Tables 1-8 and Figure l), are provided. The term “complementary,” when used in reference to a nucleic acid sequence, means the referenced regions are 100% complementary, i.e., t 100% base pairing with no mismatches.

Nucleic acid can be produced using any of a variety of known standard cloning and chemical synthesis methods, and can be altered intentionally by irected mutagenesis or other recombinant techniques known to one d in the art. Purity of polynucleotides can be determined through sequencing, gel electrophoresis, UV spectrometry.

Nucleic acids may be inserted into a nucleic acid construct in which expression of the nucleic acid is influenced or regulated by an “expression l t,” ed to herein as an “expression cassette.” The term “expression control element” refers to one or more nucleic acid sequence elements that regulate or influence sion of a nucleic acid sequence to which it is operatively . An expression control element can include, as riate, promoters, enhancers, transcription terminators, gene silencers, a start codon (e.g., ATG) in front of a protein-encoding gene, etc.

An expression l element operatively linked to a nucleic acid sequence controls transcription and, as appropriate, translation of the nucleic acid sequence. The term “operatively linked” refers to a juxtaposition wherein the referenced components are in a onship permitting them to function in their intended . Typically, expression control elements are juxtaposed at the 5’ or the 3’ ends of the genes but can also be intronic.

Expression control elements include elements that activate transcription constitutively, that are inducible (i.e., require an external signal or stimuli for activation), or derepressible (i.e., require a signal to turn transcription off; when the signal is no longer present, transcription is activated or “derepressed”). Also included in the expression cassettes of the invention are control elements sufficient to render gene expression controllable for specific cell- types or tissues (i.e., tissue-specific control elements). Typically, such elements are d upstream or downstream (i.e., 5’ and 3’) of the coding sequence. Promoters are generally oned 5’ of the coding sequence. Promoters, produced by recombinant DNA or synthetic techniques, can be used to provide for transcription of the polynucleotides of the invention. A “promoter” typically means a minimal ce element sufficient to direct transcription.

Nucleic acids may be inserted into a plasmid for transformationinto a host cell and for subsequent expression and/or c manipulation. A plasmid is a nucleic acid that can be stably propagated in a host cell; plasmids may optionally contain expression control ts in order to drive expression ofthe nucleic acid. For purposes of this invention, a vector is synonymous with a plasmid. ds and vectors generally contain at least an origin of ation for propagation in a cell and a promoter. Plasmids and vectors may also include an expression control element for expression in a host cell, and are therefore useful for expression and/or genetic manipulation of c acids encoding peptide sequences, expressing peptide sequences in host cells and organisms (e.g., a subject in need of treatment), or producing peptide sequences, for example.

As used herein, the term “transgene” means a polynucleotide that has been introduced into a cell or sm by e. For example, a cell having a transgene, the transgene has been introduced by c manipulation or “transformation” ofthe cell. A cell or progeny thereof into which the transgene has been uced is referred to as a “transformed cell” or “transformant.” Typically, the transgene is included in y of the transformant or becomes a part of the organism that develops from the cell. Transgenes may be inserted into the chromosomal DNA or maintained as a self-replicating plasmid, YAC, minichromosome, or the like.

Bacterial system promoters include T7 and inducible promoters such as pL of bacteriophage 2t, plac, ptrp, ptac (ptrp-lac hybrid promoter) and tetracycline responsive promoters. Insect cell system promoters include constitutive or inducible promoters (e.g., ecdysone). Mammalian cell constitutive promoters include SV40, RSV, bovine papilloma virus (BPV) and other Virus promoters, or inducible promoters derived from the genome of mammalian cells (e. g., metallothionein IIA er; heat shock promoter) or from mammalian Viruses (e. the adenovirus late promoter; the ble mouse mammary tumor Virus long terminal repeat).

Alternatively, a retroviral genome can be genetically modified for introducing and directing expression of a peptide sequence in riate host cells.

As s and uses of the invention include in vivo delivery, expression s further include vectors designed for in vivo use. Particular non-limiting examples include adenoviral vectors (U.S. Patent Nos. 5,700,470 and 5,731,172), adeno-associated vectors (U.S.

Patent No. 5,604,090), herpes simplex Virus vectors (U.S. Patent No. 5,501,979), retroviral s (U.S. Patent Nos. 5,624,820, 5,693,508 and 5,674,703), BPV vectors (U.S. Patent No. ,719,054), CMV vectors (U.S. Patent No. 5,561,063) and parvovirus, rus, Norwalk Virus and lentiviral vectors (see, e.g, U.S. Patent No. 6,013,516). Vectors e those that deliver genes to cells of the intestinal tract, including the stem cells (Croyle et al., Gene Ther. 5:645 (1998); SJ. Henning, Adv. Drug Deliv. Rev. 17:341 (1997), U.S. Patent Nos. 5,821,235 and 456). Many of these vectors have been approved for human studies.

Yeast vectors include constitutive and inducible promoters (see, e. g., Ausubel et al., In: Current ols in Molecular Biology, Vol. 2, Ch. 13, ed., Greene Publish. Assoc. & Wiley Interscience, 1988; Grant et al. Methods in Enzymology, 153:516 (1987), eds. Wu & Grossman; Bitter Methods in Enzymology, 152:673 (1987), eds. Berger & Kimmel, Acad. Press, N.Y.; and, Strathern eta1., The Molecular Biology ofthe Yeast Saccharomyces (1982) eds. Cold Spring Harbor Press, Vols. I and II). A constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (R. Rothstein In: DNA Cloning, A Practical Approach, Vol.11, Ch. 3, ed. D.M. Glover, IRL Press, Wash., DC, 1986). Vectors that facilitate integration of foreign c acid ces into a yeast chromosome, via homologous recombination for example, are known in the art. Yeast artificial chromosomes (YAC) are typically used when the inserted cleotides are too large for more conventional vectors (e. g., greater than about 12 Kb).

Expression s also can contain a selectable marker conferring resistance to a ive pressure or identifiable marker (e. g., beta-galactosidase), y allowing cells having the vector to be selected for, grown and expanded. Alternatively, a selectable marker can be on a second vector that is co—transfected into a host cell with a first vector containing a c acid encoding a peptide sequence. Selection s include but are not limited to herpes simplex virus thymidine kinase gene (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase gene (Szybalska et al., Proc. Natl. Acad. Sci. USA 48:2026 (1962)), and adenine oribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes that can be employed in tk—, hgprt— or aprt— cells, respectively. Additionally, antimetabolite resistance can be used as the basis of selection for dhfi, which confers resistance to methotrexate (O’Hare et al., Prac. Natl. Acad. Sci. USA 78: 1527 (1981)); the gpt gene, which confers resistance to mycophenolic acid (Mulligan et al., Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neomycz'n gene, which confers resistance to aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol. 150: 1(1981)); puromycin; and hygromycin gene, which confers resistance to hygromycin (Santerre et al., Gene 302147 (1984)). Additional selectable genes include trpB, which allows cells to utilize indolein place oftryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman eta1., Proc. Natl. Acad. Sci. USA 85:8047 (1988)); and ODC (ornithine decarboxylase), which confers resistance to the ornithine decarboxylase inhibitor, 2- (difluoromethyl)—DL—ornithine, DFMO (McConlogue (1987) In: t Communications in lar Biology, Cold Spring Harbor Laboratory).

In accordance with the invention, there are provided transformed cell(s) (in vitro, ex vivo and in vivo) and host cells that produce a t or fiasion of FGF19 and/or FGF21 as set forth herein, where expression of the variant or fusion of FGF 19 and/or FGF21 is red by a c acid encoding the variant or fusion of FGF19 and/or FGF21. Transformed and host cells that express invention peptide ces typically include a nucleic acid that encodes the invention peptide sequence. In one embodiment, a transformed or host cell is a yotic cell.

In another embodiment, a transformed or host cell is a eukaryotic cell. In various aspects, the eukaryotic cell is a yeast or mammalian (e. g., human, primate, etc.) cell.

As used herein, a “transformed” or “host” cell is a cell into which a nucleic acid is introduced that can be propagated and/0r transcribed for expression of an encoded peptide sequence. The term also es any y or subclones of the host cell.

Transformed and host cells include but are not limited to microorganisms such as bacteria and yeast; and plant, insect and mammalian cells. For example, bacteria transformed with inant bacteriophage nucleic acid, plasmid c acid or cosmid nucleic acid expression s; yeast ormed with recombinant yeast expression vectors; plant cell systems infected with recombinant virus expression vectors (e. g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant d expression vectors (e.g., Ti plasmid); insect cell s ed with recombinant virus expression vectors (e. g., baculovirus); and animal cell systems infected with inant virus expression vectors (e. g., retroviruses, adenovirus, vaccinia virus), or transformed animal cell systems engineered for transient or stable propagation or expression.

For gene therapy uses and methods, a transformed cell can be in a subject. A cell in a subject can be transformed with a nucleic acid that s an invention peptide sequence as set forth herein in vivo. Alternatively, a cell can be transformed in vitro with a transgene or polynucleotide, and then lanted into a tissue of subject in order to effect ent.

Alternatively, a primary cell isolate or an established cell line can be transformed with a transgene or polynucleotide that encodes a variant of FGF19 and/or FGFZl or a fusion/chimeric sequence (or variant) thereof, such as a chimeric peptide sequence including all or a portion of FGF19, or including all or a portion ofFGF21, and then optionally transplanted into a tissue of a subject.

Non-limiting target cells for expresSion of peptide sequences, particularly for expression in vivo, include pancreas cells (islet cells), muscle cells, mucosal cells and endocrine cells. Such endocrine cells can provide inducible production (secretion) of a variant ofFGF19 and/or FGF21, or a fusion/chimeric sequence (or variant) thereof, such as a ic peptide sequence including all or a portion of FGF19, or including all or a portion of FGF21. Additional cells to transform include stem cells or other multipotent or pluripotent cells, for example, progenitor cells that differentiate into the various pancreas cells (islet cells), muscle cells, mucosal cells and endocrine cells. Targeting stem cells provides longer term expression of e sequences of the invention.

As used herein, the term “cultured,” when used in reference to a cell, means that the cell is grown in vitro. A particular example of such a cell is a cell isolated from a subject, and grown or adapted for growth in tissue culture. Another example is a cell cally manipulated in vitro, and transplanted back into the same or a different subject.

The term “isolated,” when used in reference to a cell, means a cell that is separated from its naturally occurring in viva environment. “Cultured” and “isolated” cells may be manipulated by the hand of man, such as genetically transformed. These terms include any progeny of the cells, including progeny cells that may not be cal to the parental cell due to mutations that occur during cell on. The terms do not include an entire human being.

Nucleic acids encoding invention peptide ces can be introduced for stable expression into cells of a whole organism. Such organisms including non-human transgenic animals are USCfiJl for studying the effect of peptide expression in a whole animal and therapeutic benefit. For example, as disclosed herein, tion of a variant ofFGF19 and/or FGFZI or a fusion/chimeric ce (or variant) f, such as a chimeric peptide sequence including all or a portion of FGF19, or including all or a portion ofFGF21 as set forth herein, in mice lowered glucose and is anti—diabetic.

Mice strains that develop or are susceptible to developing a particular disease (e.g., es, degenerative disorders, cancer, etc.) are also useful for introducing therapeutic proteins as described herein in order to study the effect of therapeutic n expression in the disease susceptible mouse. Transgenic and genetic animal models that are susceptible to particular disease or physiological ions, such as streptozotocin (STZ)-induced diabetic (STZ) mice, are appropriate targets for expressing variants of FGF19 and/or FGF21, fusions/chimeric ces (or variant) thereof, such as a chimeric peptide sequence including all or a portion of FGF 19, or including all or a portion of FGF21, as set forth herein. Thus, in accordance with the invention, there are provided non-human transgenic animals that e a variant of FGF19 and/or FGF21, or a fusion/chimeric sequence (or variant) thereof, such as a chimeric peptide sequence including all or a portion of FGF19, or including all or a portion of FGF21, the production of which is not naturally occurring in the animal which is conferred by a transgene present in c or germ cells of the animal.

The term genic ” refers to an animal whose somatic or germ line cells bear genetic information ed, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by microinjection or infection with recombinant Virus. The term “transgenic” further includes cells or tissues (i.e., “transgenic Cell,” “transgenic tissue”) obtained from a transgenic animal genetically manipulated as described herein. In the present context, a “transgenic animal” does not ass animals ed by classical crossbreeding or in vitro fertilization, but rather denotes animals in which one or more cells receive a nucleic acid molecule. Invention transgenic animals can be either heterozygous or homozygous with respect to the transgene. Methods for producing transgenic animals, including mice, sheep, pigs and frogs, are well known in the art (see, e. g., U.S. Patent Nos. 5,721,367, 5,695,977, 5,650,298, and ,614,396) and, as such, are onally included.

Peptide sequences, nucleic acids encoding peptide sequences, vectors and transformed host cells expressing peptide sequences include isolated and purified forms. The term “isolated,” when used as a r of an invention composition, means that the ition is separated, substantially completely or at least in part, from one or more components in an environment. Generally, compositions that exist in nature, when isolated, are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate or cell membrane. The term ted” does not exclude alternative physical forms of the composition, such as variants, modifications or derivatized forms, fusions and chimeras, multimers/oligomers, etc., or forms expressed in host cells. The term “isolated” also does not exclude forms (e.g., ceutical compositions, ation compositions, etc.) in which there are combinations therein, any one of which is produced by the hand of man.

An “isolated” composition can also be “purified” when free of some,ra substantial number of, or most or all of one or more other materials, such as a contaminant or an undesired substance or material. Peptide sequences of the invention are generally not known or believed to exist in nature. r, for a composition that does exist in nature, an isolated composition will generally be free of some, a substantial number of, or most or all other als with which it typically associates with in nature. Thus, an isolated peptide sequence that also occurs in nature does not include polypeptides or polynucleotides present among millions of other sequences, such as proteins of a protein library or nucleic acids in a genomic or cDNA y, for example. A “purified” composition includes combinations with one or more other inactive or active molecules. For example, a peptide sequence of the invention combined with another drug or agent, such as a glucose ng drug or therapeutic agent, for e.

As used herein, the term “recombinant,” when used as a modifier of e sequences, nucleic acids encoding peptide ces, etc., means that the compositions have been manipulated (i. e., engineered) in a fashion that generally does not occur in nature (e.g., in vitro).

A ular example of a recombinant peptide would be where a peptide sequence of the invention is sed by a cell transfected with a nucleic acid encoding the peptide sequence. A particular example of a recombinant nucleic acid would be where a nucleic acid (e.g, genomic or cDNA) encoding a peptide sequence cloned into a plasmid, with or without 5’, 3’ or intron regions that the gene is normally uous with in the genome of the organism. Another example of a recombinant peptide or nucleic acid is a hybrid or fusion sequence, such as a chimeric peptide sequence comprising a n of FGF19 and a portion ofFGF21.

In accordance with the invention, there are provided compositions and mixtures of invention peptide ces, ing subsequences, variants and modified forms of the exemplified peptide sequences (including the FGF19 and FGFZl variants and subsequences listed in Tables 1-8 and Figure l, and the FGF19/FGF21 fusions and chimeras listed in Tables 1-8 and Figure 1). In one ment, a mixture includes one or more peptide sequences and a pharmaceutically acceptable carrier or excipient. In another embodiment, a e includes one or more peptide sequences and an adjunct drug or therapeutic agent, such as an anti—diabetic, or glucose lowering, drug or therapeutic agent. Examples of drugs and therapeutic agents are set forth hereafter. Combinations, such as one or more peptide sequences in a pharmaceutically able carrier or excipient, with one or more of an anti-diabetic, or glucose lowering drug or therapeutic agent are also provided. Such combinations of peptide sequence of the invention with another drug or agent, such as a glucose ng drug or therapeutic agent, for example are useful in accordance with the invention methods and uses, for example, for treatment of a t.

Combinations also include incorporation of e sequences or nucleic acids of the ion into particles or a polymeric substances, such as polyesters, carbohydrates, ine acids, hydrogel, polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose, carboxymethylcellulose, protamine e, or lactide/glycolide copolymers, polylactide/glycolide copolymers, or ethylenevinylacetate copolymers; entrapment in microcapsules prepared by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatin-microcapsules, or poly (methylmethacrolate) microcapsules, respectively; incorporation in colloid drug delivery and dispersion systems such as macromolecule complexes, nano-capsules, microspheres, beads, and lipid-based systems (e. g., N— ‘ fatty acyl groups such as oyl, N-oleoyl, fatty amines such as dodecyl amine, oleoyl amine, etc., see US Patent No. 6,638,513), including oil-in—water emulsions, micelles, mixed micelles, and liposomes, for example.

Invention es including subsequences, variants and modified forms of the exemplified peptide sequences (including the FGF19 and FGF21 variants and subsequences listed in Tables 1—8 and Figure l, and the FGF19/FGF21 fusions and chimeras listed in Tables 1—8 and Figure l) as set forth herein can be used to modulate e metabolism and facilitate transport of glucose from the blood to key metabolic organs such as muscle, liver and fat. Such peptide sequences can be produced in s sufficient or effective to restore glucose tolerance and/or to improve or provide normal glucose tasis.

As disclosed herein, administration of various FGF19 and/ FGF21 variants and fusion e sequences to mice successfully reduced glucose levels. Furthermore, in contrast to FGF19, certain peptide sequences did not stimulate or induce HCC formation or tumorigenesis in 2012/045087 mice. Thus, administration of invention es, including subsequences, variants and d forms of the exemplified peptide ces (including the FGF19 and FGF21 variants and subsequences listed in Tables 1-8 and Figure l, and the FGFl9/FGF21 fusions and chimeras listed in Tables 1—8 and Figure 1), into an , either by direct or indirect in vivo or by ex vivo methods (e.g., administering the variant or fusion peptide, a nucleic acid encoding the variant or fusion peptide, or a ormed cell or gene therapy vector expressing the variant or fusion peptide), can be used to treat various disorders.

Accordingly, the invention includes in vitro, ex vivo and in viva (e. g., on or in a subject) methods and uses. Such methods and uses can be practiced with any of the peptide ces of the invention set forth herein.

In accordance with the ion, there are provided methods of treating a subject having, or at risk of having, a disorder. In various embodiments, a method includes stering a peptide sequence, such as an FGF19 or FGF21 variant, fusion or chimera listed in Tables 1—8 and Figure l, or a subsequence, a variant or d form of an FGF19 0r FGF21 variant, fusion or chimera listed in Tables 1-8 and Figure l, to a subject in an amount effective for treating the disorder.

Exemplary disorders treatable, preventable, and the like with invention peptides, and methods and uses, include metabolic diseases and disorders. Non limiting examples of diseases and disorders e: 1. Glucose utilization ers and the sequelae associated therewith, including diabetes mellitus (Type I and Type-2), gestational diabetes, hyperglycemia, insulin resistance, abnormal glucose metabolism, iabetes” (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 s such as pancreatic B-cell ction. For treatment, invention peptide sequences can be administered to subjects having a fasting plasma glucose (FPG) level greater than about 100 mg/dl. Peptide ces of the invention may also be useful in other hyperglycemic-related disorders, including kidney damage (e.g., tubule damage or pathy), liver degeneration, eye damage (e.g., diabetic retinopathy or cataracts), and diabetic foot disorders; 2. Dyslipidemias and their ae 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 d to, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), and polycystic ovarian syndrome (PCOS)), and also include thromboses, hypercoagulable and prothrombotic states (arterial and venous), hypertension, cardiovascular disease, stroke and heart failure; 4.

Disorders or conditions in which inflammatory reactions are involved, including atherosclerosis, chronic inflammatory bowel diseases (e.g., Crohn's disease and tive colitis), , lupus erythematosus, arthritis, or other inflammatory rheumatic disorders; 5. Disorders of cell cycle or cell differentiation processes such as adipose cell tumors, lipomatous carcinomas ing, for example, liposarcomas, solid tumors, and neoplasms; 6. egenerative diseases and/or demyelinating disorders of the central and peripheral nervous systems and/or neurological diseases involving neuroinflammatory 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.

As used herein, the term “hyperglycemic” or “hyperglycemia,” when used in reference to a condition of a subject means a transient or chronic ally high level of glucose present in the blood of a subject. The ion can be caused by a delay in glucose metabolism or absorption such that the subject exhibits glucose intolerance or a state of elevated glucose not typically found in normal subjects (e. g., in e-intolerant pre-diabetic subjects at risk of developing diabetes, or in diabetic subjects). Fasting plasma e (FPG) levels for normoglycemia are less than about 100 mg/dl, for ed glucose metabolism, between about 100 and 126 mg/dl, and for diabetics greater than about 126 mg/dl.

As disclosed herein, the invention es methods of preventing (e.g., in subjects predisposed to having a particular disorder(s)), delaying, slowing or inhibiting progression of, the onset of, or treating (e. g., ameliorating) obesity or an undesirable body mass (e. g., a greater than normal body mass index, or “BMI” relative to an riate matched subject of comparable age, gender, race, etc.) Thus, in various embodiments, a method of the invention for, for example, treating obesity or an undesirable body mass (including the bid conditions of obesity, e.g., obstructive sleep apnea, arthritis, cancer (e.g., , endometrial, and colon), gallstones or hyperglycemia, es contacting or administering a peptide of the ion as set forth herein (e. g., a variant or fusion ofFGF19 and/or FGFZl as set forth in Tables 1-8 or Figure l, for example) in an amount effective to treat obesity or an undesirable body mass. In particular aspects, a subject has a body mass index greater than 25, for example, 25-30, 30-35, 35—40, or greater than 40.

Moreover, the invention includes methods of preventing (e. g., in subjects predisposed to having a particular disorder(s)), slowing or inhibiting the progression of, delaying the onset of, or treating undesirable levels or ally eleyated serum/plasma LDL, VLDL, triglycerides or cholesterol, all of which, alone or in combination, can lead to, for e, plaque formation, narrowing or blockage of blood vessels, and increased risk of hypertension, stroke and coronary artery disease. Such disorders can be due to, for example, genetic position or diet, for example.

The term “subject” refers to an animal. Typically, the animal is a mammal that would benefit from treatment with a peptide sequence of the invention. Particular examples include primates (e.g., humans), dogs, cats, horses, cows, pigs, and sheep. ts include those having a disorder, e. g., a hyperglycemic er, such as es, or subjects that do not have a disorder but may be at risk of developing the disorder, e.g., pre-diabetic subjects having FPG levels r than 100 mg/dl, for example, n about 100 and 126 mg/dl. Subjects at risk of developing a disorder e, for example, those whose diet may contribute to development of acute or chronic hyperglycemia (e.g., diabetes), undesirable body mass or obesity, as well as those which may have a family history or genetic predisposition towards development of acute or chronic lycemia, or undesirable body mass or obesity.

As disclosed herein, treatment methods include contacting or administering a peptide of the invention as set forth herein (e.g., a variant or fusion of FGF19 and or FGF21 as set forth in Tables 1-8 or Figure 1, for example) in an amount effective to achieve a desired outcome or result in a subject. A treatment that results in a desired outcome or result includes decreasing, reducing or preventing severity or frequency of one or more ms ofthe condition in the subject, e. an ement in the subj ect’s condition or a “beneficial effect” or peutic .” Therefore, treatment can decrease or reduce or prevent the severity or frequency of one or more symptoms of the disorder, stabilize or inhibit progression or worsening of the disorder, and in some instances, reverse the disorder, transiently (e.g., for 1-6, 6-12, or 12-24 hours), for medium term (e. g., 1—6, 6-12, 12-24 or 24-48 days) or long term (e.g., for 1-6, 6-12, 12—24, 24-48 weeks, or greater than 24—48 weeks). Thus, in the case of a hyperglycemic disorder, for example, treatment can lower or reduce blood glucose, improve glucose tolerance, improve glucose metabolism, provide normal glucose homeostasis, lower or reduce insulin resistance, lower or reduce insulin levels, or decrease, prevent, improve, or reverse metabolic syndrome, or a athological change associated with or that results from the hyperglycemic disorder, such as diabetes.

For example, a e sequence, method or use can lower or reduce glucose in one more ts having FPG levels greater than 100 mg/dl, for example, n about 100 and 125 mg/dl, or greater than 125 mg/dl, by 5-10%, 10-20%, 20-30%, or 30-50%, or more, or for example from greater than 200 mg/dl to less than 200 mg/dl, for greater than 150 mg/dl to less than 150 mg/dl, from greater than 125 mg/dl to less than 125 mg/dl, etc. In addition, a peptide sequence, method or use can lower or reduce glucose, for example, for pre-diabetes or for diabetes (e.g., Type 2) subjects with baseline HbAIc levels greater than about 5%, 6%, 7%, 8%, 9% or 10%, in particular 5%, 6%, or 7%.

Non—limiting es of an improvement of a athological change associated with a hyperglycemic condition include, for e, decreasing, inhibiting, reducing or arresting: the destruction or degeneration of as cells (e.g., B—cells), kidney damage such as tubule calcification or nephropathy, degeneration of liver, eye damage (e.g., diabetic retinopathy, cataracts), ic foot, ulcerations in mucosa such as mouth and gums, periodontitis, excess bleeding, slow or delayed healing of injuries or wounds (e. g., that lead to diabetic carbuncles), skin infections and other cutaneous disorders, cardiovascular and coronary heart disease, peripheral ar disease, stroke, dyslipidemia, hypertension, obesity, or the risk of developing any of the foregoing. Improvement in undesirable body mass or obesity can include, for example, a reduction of body mass (as reflected by BMI or the like) or an improvement in an associated disorder, such as a decrease in triglyceride, cholesterol, LDL or VLDL levels, a decrease in blood pressure, a decrease in intimal thickening of the blood vessel, a decreased or reduced risk of cardiovascular disease, or stroke, decrease in resting heart rate, etc.

An “effective amount” or a “sufficient amount” for use and/or for treating a subject refer to an amount that provides, in single or multiple doses, alone, or in combination with one or more other itions (therapeutic agents such as a drug or treatment for hyperglycemia), treatments, protocols, or therapeutic regimens agents, a detectable response of any duration of time (transient, medium or long term), a desired outcome in or an objective or subjective benefit to a t of any measurable or detectable degree or for any duration of time (e.g., for hours, days, months, years, or cured). such s typically are effective to ameliorate a disorder, or one, le or all adverse symptoms, uences or complications of the disorder, to a able extent, although reducing or inhibiting a progression or worsening of the disorder, is considered a satisfactory outcome.

As used herein, the term “ameliorate” means an improvement in the subject’s disorder, a reduction in the severity ofthe disorder, or an inhibition of progression or worsening of the disorder (e.g., stabilizing the disorder). In the case of a hyperglycemic disorder (e.g., diabetes, insulin resistance, glucose intolerance, metabolic syndrome, etc.), for example, an ement can be a lowering or a reduction in blood glucose, a reduction in insulin ance, a reduction in glucagon, an improvement in glucose tolerance, or glucose lism or homeostasis. An improvement in a hyperglycemic er also can e improved pancreatic function (e.g,, inhibit or prevent B-cell/islet destruction or enhance B -cell number and/or function), a decrease in a pathology ated with or ing from the disorder, such as an improvement in histopathology of an affected tissue or organ, as set forth herein. In the case of undesirable body mass or obesity, for example, an improvement can be a decrease in weight gain, a ion of body mass (as reflected in reduced BMI, for example) or an improvement in a condition ated with rable body mass obesity, for example, as set forth herein (e.g., a lowering or a reduction of blood glucose, triglyceride, terol, LDL or VLDL levels, a decrease in blood pressure, a decrease in intimal thickening of the blood vessel, etc.).

A therapeutic benefit or improvement therefore need not be complete ablation of any one, most or all symptoms, complications, consequences or underlying causes associated with the disorder or disease. Thus, a satisfactory endpoint is achieved when there is a transient, medium or long term, incremental improvement in a subject’s condition, or a l reduction in the ence, frequency, severity, ssion, or duration, or inhibition or reversal, of one or more associated adverse symptoms or complications or consequences or underlying causes, ing or ssion (e.g., stabilizing one or more ms or complications of the condition, disorder or disease), of the disorder or disease, over a duration of time , days, weeks, months, etc.). .[0151] Thus, in the case of a disorder treatable by a peptide sequence of the invention, the amount of peptide sufficient to ameliorate a disorder will depend on the type, severity and extent, or duration of the disorder, the therapeutic effect or outcome desired, and can be readily ascertained by the skilled artisan. Appropriate amounts will also depend upon the individual subject (e.g., the bioavailability within the subject, gender, age, etc.). For example, a transient, or partial, restoration of normal glucose tasis in a subject can reduce the dosage amount or frequency of insulin injection, even though complete freedom from insulin has not resulted.

An effective amount can be ascertained, for example, by measuring one or more relevant physiological effects. In a particular non-limiting example in the case of a hyperglycemic condition, a lowering or ion of blood e or an improvement in glucose tolerance test can be used to determine whether the amount of ion e sequence, including subsequences, sequence variants and d forms of the exemplified peptide sequences (e. g., sequences listed in Tables 1-8 and Figure l) is effective to treat a hyperglycemic condition. In another particular non-limiting example, an effective amount is an amount sufficient to reduce or decrease any level (e.g., a baseline level) of FPG, wherein, for example, an amount sufficient to reduce a FPG level greater than 200 mg/dl to less than 200 mg/dl, an amount sufficient to reduce a FPG level between 175 mg/dl and 200 mg/dl to less than the pre- administration level, an amount sufficient to reduce a FPG level between 150 mg/dl and 175 mg/dl to less than the pre—administration level, an amount sufficient to reduce a FPG level between 125 mg/dl and 150 mg/dl to less than the pre-administration level, and so on (e.g., reducing FPG levels to less than 125 mg/dl, to less than 120 mg/dl, to less than 115 mg/dl, to less than 110 mg/dl, etc.). In the case ofHbAIc , an effective amount includes an amount sufficient to reduce or decrease levels by more than about 10% to 9%, by more than about 9% to 8%, by more than about 8% to 7%, by more than about 7% to 6%, by more than about 6% to 5%, and so on. More particularly, a ion or decrease ofHbAIc levels by about 0.1%, 0.25%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, or more is an effective amount in accordance with the invention. In yet another particular non-limiting example in the case of undesirable body mass or y, an ive amount is an amount sufficient to decrease or reduce the body mass index (BMI) of a subject, a se or reduction of e, a decrease or reduction in serum/plasma levels of triglyceride, lipid, cholesterol, fatty acids, LDL and/or VLDL. In yet fiirther particular non-limiting examples, an amount is an amount sufficient to decrease or reduce any of the aforementioned parameters by, for example, about 0.1%, 0.25%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, %, 10%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, or more.

Methods and uses of the invention for ng a subject are applicable for prophylaxis to prevent a disorder in a t, such as a hyperglycemic er, or development of undesirable body mass or obesity. Alternatively, methods and uses can be practiced during or following treatment of a subject. For e, prior to, during or ing treatment of a subject to lower glucose using insulin or another glucose lowering drug or therapeutic agent, for example, a method or use of the invention can, for example, a peptide sequence of the invention can be administered to the subject. In addition, a composition such as a peptide sequence of the invention can be combined with another drug or agent, such as a glucose lowering drug or therapeutic agent, for example.

Accordingly, methods and uses of the invention for treating a subject can be practiced prior to, substantially contemporaneously with or following another treatment, and can be supplemented with other forms of therapy. Supplementary therapies include other glucose lowering treatments, such as insulin, an insulin sensitivity enhancer and other drug ents, a change in diet (low sugar, fats, etc.), weight loss surgery— (reducing stomach volume by gastric bypass, gastrectomy), gastric banding, gastric balloon, gastric sleeve, etc. For example, a method or use of the ion for treating a hyperglycemic or insulin resistance disorder can be used in combination with drugs or other pharmaceutical compositions that lower glucose or se insulin sensitivity in a subject. Drugs for treating diabetes include, for example, biguanides and sulphonylureas (e.g., tolbutamide, chlorpropamide, acetohexamide, tolazamide, clamide and ide), thiazolidinediones (rosiglitazone, pioglitazone), GLP-l analogues, Dipeptidyl peptidase-4 (DPP—4) inhibitors, bromocriptine formulations (e.g. and bile acid sequestrants (e. g., velam), and insulin (bolus and basal analogs), metformin (e. g., metformin hydrochloride) with or without a thiazolidinedione (TZD), and SGLT-2 inhibitors. Appetite suppression drugs are also well known and can be used in combination with the methods of the invention.

Supplementary therapies can be administered prior to, contemporaneously with or following invention methods and uses.

Peptide sequences of the invention including subsequences, sequence variants and modified forms of the exemplified peptide sequences (sequences listed in Tables 1—8 and Figure 1), may be formulated in a unit dose or unit dosage form. In a particular embodiment, a peptide sequence is in an amount effective to treat a subject in need of treatment, e.g., due to hyperglycemia. Exemplary unit doses range from about , 250-500, 500-1000, 1000-2500 or 2500—5000, 5000—25,000, 25,000-50,000 ng; from about 25-250, 250—500, 500-1000, 1000- 2500 or 2500—5000, 5000-25,000, 25,000-50,000 ug; and from about 25-250, 250-500, 00, 1000—2500 or 2500-5000, 5000-25,000, 25,000-50,000 mg.

Peptide sequences of the invention ing subsequences, sequence ts and modified forms ofthe exemplified peptide sequences (sequences listed in Tables 1—8 and Figure 1) can be stered to provide the intended effect as a single dose or le s, for example, in an effective or sufficient amount. ary doses range from about , 250- 500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000, —50,000’pg/kg; from about 50—500, 500—5000, 5000—25,000 or 25,000-50,000 ng/kg; and from about 25-250, 250-500, 500-1000, 1000-2500 or 2500—5000, 5000-25,000, 25,000-50,000 [Lg/kg. Single or multiple doses can be administered, for e, multiple times per day, on consecutive days, alternating days, weekly or intermittently (e.g., twice per week, once every 1, 2, 3, 4, 5, 6, 7 or 8 weeks, or once every 2, 3, 4, 5 or 6 months).

Peptide sequences of the invention including uences, variants and modified forms of the exemplified peptide sequences (sequences listed in Tables 1-8 and Figure 1) can be stered and methods may be practiced via systemic, regional or local administration, by route. For example, a peptide sequence can be stered parenterally (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally), orally (e. g., ingestion, buccal, or sublingual), inhalation, intradermally, intracavity, intracranially, transdermally (topical), transmucosally or rectally. Peptide sequences ofthe invention ing subsequences, variants and modified forms of the exemplified peptide sequences (sequences listed in Tables 1-8 and Figure 1) and methods of the invention including pharmaceutical compositions can be administered via a (micr0)encapsulated delivery system or packaged into an implant for administration.

The invention fithher es “pharmaceutical compositions,” which include a peptide sequence (or sequences) of the invention, including uences, variants and modified forms of the ified peptide sequences (sequences listed in Tables 1-8 and Figure 1), and one or more pharmaceutically acceptable or physiologically acceptable diluent, carrier or excipient. In particular embodiments, a peptide sequence or sequences are present in a therapeutically acceptable amount. The pharmaceutical compositions may be used in accordance with the invention methods and uses. Thus, for example, the pharmaceutical compositions can be administered ex vivo or in vivo to a subject in order to practice treatment methods and uses of the invention.

Pharmaceutical compositions of the invention can be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are set forth herein. In addition, the pharmaceutical compositions may further comprise other therapeutically active agents or compounds disclosed herein (e.g., glucose lowering agents) or known to the skilled artisan which can be used in the treatment or prevention of various diseases and ers as set forth herein. ceutical compositions typically comprise a therapeutically effective amount of at least one of the peptide sequences of the invention, including subsequences, ts and d forms of the exemplified peptide sequences (sequences listed in Tables 1-8 and Figure l) and one or more pharmaceutically and physiologically acceptable formulation agents. Suitable pharmaceutically able or physiologically acceptable diluents, carriers or excipients include, but are not limited to, idants (e. g., ascorbic acid and sodium ate), preservatives (e. benzyl alcohol, methyl parabens, ethyl or n—propyl, p-hydroxybenzoate), emulsifying agents, suspending agents, dispersing agents, solvents, fillers, g agents, buffers, vehicles, diluents, and/or nts. For example, a suitable vehicle may be physiological saline solution or citrate buffered saline, possibly supplemented with other materials common in pharmaceutical compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary es. Those skilled in the art will readily recognize a variety of buffers that could be used in the pharmaceutical compositions and dosage forms used in the invention. Typical buffers include, but are not limited to pharmaceutically acceptable weak acids, weak bases, or mixtures f. Buffer components also include water e materials such as phosphoric acid, tartaric acids, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, ic acid, ic acid, and salts thereof.

A primary solvent in a vehicle may be either aqueous or ueous in . In addition, the vehicle may contain other pharmaceutically able excipients for modifying or maintaining the pH, osmolarity, viscosity, sterility or stability of the pharmaceutical composition.

In certain embodiments, the pharmaceutically acceptable e is an aqueous buffer. In other embodiments, a vehicle comprises, for example, sodium chloride and/or sodium citrate.

Pharmaceutical compositions of the invention may contain still other pharmaceutically-acceptable formulation agents for modifying or ining the rate of release 2012/045087 of an invention e. Such formulation agents include those substances known to artisans skilled in preparing sustained release formulations. For further reference pertaining to pharmaceutically and physiologically acceptable formulation agents, see, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., , Pa. 18042) pages 1435-1712, The Merck Index, 12th Ed. (1996, Merck Publishing Group, Whitehouse, NJ); and Pharmaceutical Principles of Solid Dosage Forms (1993, Technonic Publishing Co., Inc., Lancaster, Pa.). Additional pharmaceutical compositions riate for administration are known in the art and are applicable in the methods and compositions of the invention.

A pharmaceutical composition may be stored in a e vial as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such compositions may be stored either in a ready to use form, a lyophilized form requiring reconstitution prior to use, a liquid form requiring dilution prior to use, or other able form. In some embodiments, a pharmaceutical composition is provided in a single—use container (e. g., a single-use vial, ampoule, syringe, or autoinjector (similar to, e. g., an EpiPen®)), whereas a multi-use container (e.g., a multi—use vial) is provided in other embodiments. Any drug delivery apparatus may be used to r invention peptides, including implants (e. g., implantable pumps) and catheter systems, both of which are known to the skilled n. Depot injections, which are generally administered subcutaneously or intramuscularly, may also be,utilized to release invention peptides over a defined period of time. Depot ions are usually either solid- or oil-based and generally comprise at least one of the ation components set forth herein. The skilled artisan is familiar with possible formulations and uses of depot ions.

A pharmaceutical composition can be formulated to be compatible with its intended route of administration. Thus, pharmaceutical compositions include carriers, diluents, or ents suitable for administration by routes including parenteral (e.g., subcutaneous (s.c.), intravenous, intramuscular, or intraperitoneal), intradermal, oral (e. g., ingestion), inhalation, intracavity, intracranial, and transdermal (topical).

Pharmaceutical compositions may be in the form of a e inj ectable aqueous or oleagenous suspension. This suspension may be formulated using le dispersing or wetting agents and suspending agents sed herein or known to the skilled artisan. The sterile inj ectable preparation may also be a sterile inj ectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a on in 1,3-butane diol.

Acceptable diluents, solvents and dispersion media that may be employed include water, Ringer’s solution, isotonic sodium chloride solution, hor ELTM (BASF, Parsippany, NJ) or phosphate ed saline (PBS), ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. Moreover, fatty— acids such as oleic acid find use in the preparation of ables. Prolonged tion of particular injectable formulations can be achieved by including an agent that delays absorption (e.g., aluminum earate or gelatin).

Pharmaceutical itions may be in a form suitable for oral use, for example, as tablets, capsules, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups, solutions, microbeads or elixirs. Pharmaceutical compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions. Such itions may contain one or more agents such as sweetening agents, flavoring agents, ng agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets containing an invention e may be in admixture with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients e, for example, diluents, such as calcium ate, sodium ate, lactose, calcium phosphate or sodium phosphate; ating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or , and lubricating , for e magnesium stearate, stearic acid or talc.

Tablets, capsules and the like suitable for oral administration may be uncoated or they may be coated by known techniques to delay egration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by techniques known in the art to form osmotic therapeutic tablets for controlled release. Additional agents include biodegradable or biocompatible particles or a polymeric substance such as polyesters, polyamine acids, hydrogel, nyl pyrrolidone, polyanhydrides, polyglycolic acid, ethylene-vinylacetate, cellulose, carboxymethylcellulose, protamine sulfate, or lactide/glycolide copolymers, polylactide/glycolide copolymers, or ethylenevinylacetate copolymers in order to control delivery of an administered composition. For example, the oral agent can be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization, by the use of hydroxymethylcellulose or gelatin-microcapsules or poly (methylmethacrolate) microcapsules, respectively, or in a colloid drug delivery system. - dal dispersion systems include macromolecule complexes, nano-capsules, pheres, microbeads, and lipid—based systems, including oil-in—water emulsions, micelles, mixed micelles, and liposomes. Methods for ation of such formulations are known to those skilled in the art and are commercially available.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, m phosphate, kaolin or rystalline ose, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions n the active materials in ure with excipients suitable for the cture thereof. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum ; dispersing or wetting agents may be a naturally—occurring phosphatide, for example lecithin, or sation ts of an alkylene oxide with fatty acids, for e polyoxy—ethylene stearate, or sation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ne oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or t oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.

Dispersible powders and granules suitable for preparation of an aqueous suspension by addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. le dispersing or wetting agents and suspending agents are exemplified herein. .

Pharmaceutical compositions ofthe invention may also be in the form of oil—in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example, liquid paraffin, or mixtures of these. Suitable emulsifying agents may be naturally—occurring gums, for example, gum acacia or gum tragacanth; naturally-occurring phosphatides, for e, soy bean, lecithin, and esters or partial esters derived from fatty acids; hexitol anhydrides, for example, sorbitan eate; and condensation products of partial esters with ethylene oxide, for example, polyoxyethylene an eate.

Pharmaceutical compositions can also include carriers to protect the composition against rapid degradation or elimination from the body, such as a controlled release formulation, including implants, liposomes, hydrogels, prodrugs and microencapsulated delivery systems. For example, a time delay material such as glyceryl monostearate or glyceryl stearate alone, or in combination with a wax, may be employed. Prolonged absorption of inj ectable pharmaceutical compositions can be achieved by including an agent that delays absorption, for example, aluminum monostearate or gelatin. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

The invention also includes invention peptides in the form of suppositories for rectal administration. The itories can be ed by mixing an invention peptide with a le non—irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to e the drug. Such als include, but are not limited to, cocoa butter and polyethylene glycols.

In accordance with the invention, there are provided methods of identifying a peptide (or a subsequence, variant or modified form as set forth herein) having glucose lowering activity without substantial hepatocellular carcinoma (HCC) activity. In one embodiment, a method includes: screening (e.g., assaying or measuring) a peptide sequence (or a subsequence, variant or ed form as set forth herein) for e lowering activity; and screening (e.g., ng or measuring) a e ce (or a subsequence, variant or modified form as set forth herein) for HCC activity, or expression of a marker correlating with HCC activity. A peptide having glucose lowering activity and reduced or absent HCC activity thereby identifies the peptide. In particular aspects, the marker correlating with HCC activity comprises lipid profile- a peptide that has less lipid increasing activity compared to FGF19 indicates the peptide has reduced or absent HCC activity; or the marker correlating with HCC activity ses aldo-keto reductase gene sion— a e that down—regulates or decreases aldo-keto reductase gene expression compared to FGF19 indicates that the peptide has reduced or absent HCC activity; or the marker indicative ofHCC activity comprises Slcla2 gene expression— a peptide that up—regulates or ses Slc1a2 gene expression compared to FGF19 indicates that the e has reduced or absent HCC activity.

The terms “assaying” and “measuring” and grammatical variations thereof are used interchangeably herein and refer to either qualitative or quantitative determinations, or both qualitative and quantitative determinations. When the terms are used in reference to detection, any means of ing the relative amount is contemplated, including the various methods set forth herein and known in the art. For e, gene sion can be assayed or measured by a Northern blot, Western blot, immunoprecipitation assay, or by measuring activity, function or amount of the expressed protein (e.g., aldo-keto reductase or Slcla2). 2012/045087 Risk factors for HCC, the most common type of liver cancer, include type 2 diabetes (probably exacerbated by obesity). The risk ofHCC in type 2 diabetics is greater (from ~25 to ~7 times the non-diabetic risk) depending on the duration of diabetes and treatment protocol.

Various methodologies can be used in the screening and diagnosis ofHCC and are well known to the skilled n. Indicators for HCC include ion of a tumor maker such as elevated alpha-fetoprotein (AFP) or des—gamma carboxyprothrombin (DCP) levels. A number of different seaming and imaging techniques are also helpful, including ultrasound, CT scans and MRI. In relation to the invention, evaluation of r a peptide (e.g., a candidate peptide) exhibits ce of inducing HCC may be determined in vivo by, for example, fying HCC nodule formation in an animal model, such as db/db mice, administered a peptide, compared to HCC nodule formation by wild type FGFl9. Macroscopically, liver cancer may be nodular, where the tumor s (which are round-to-oval, grey or green, well circumscribed but not encapsulated) appear as either one large mass or multiple smaller masses. Alternatively, HCC may be t as an infiltrative tumor which is diffuse and poorly circumscribed and frequently infiltrates the portal veins.

Pathological assessment of hepatic tissue samples is generally performed after the results of one or more of the aforementioned techniques te the likely presence of HCC.

Thus, methods of the invention may r include assessing a c tissue sample from an in vivo animal model (e.g., a db/db mouse) useful in HCC studies in order to determine whether a peptide sequence exhibits evidence of inducing HCC. By microscopic assessment, a pathologist can determine whether one of the four general architectural and cytological types rns) of HCC are present (i.e., fibrolamellar, pseudoglandular (adenoid), pleomorphic (giant cell) and clear cell).

The invention also includes the generation and use of antibodies, and fragments thereof, that bind the peptide sequences of the invention, including subsequences, sequence variants and modified forms of the exemplified peptide sequences (including the peptides listed in Tables 1—8 and Figure l).

As used herein, the terms “antibodies” (Abs) and “immunoglobulins” (Igs) refer to glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to an antigen, immunoglobulins include both antibodies and other antibody-like molecules which may lack antigen specificity.

The term “antibody” includes intact monoclonal antibodies, onal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody binding fragments including Fab and 2, provided that they exhibit the desired ical activity. The basic antibody structural unit comprises a tetramer, and each tetramer is composed oftwo identical pairs of polypeptide chains, each pair having one “light” chain (about kDa) and one “heavy” chain (about 50—70 kDa). The terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen ition. In contrast, the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light , s human heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the dy's isotype as IgM, IgD, IgA, and IgE, respectively. Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain antibodies.

Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain ofthe heavy chain. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. The antibody chains all exhibit the same general structure of relatively conserved ork s (FR) joined by three hyper-variable regions, also called complementarity-determining s or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C- terminal, both light and heavy chains comprise the s FRl, CDRl, FR2, CDRZ, FR3, CDR3 and FR4.

An intact antibody has two binding sites and, except in bifunctional or ific antibodies, the two binding sites are the same. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. ific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' nts.

As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of ntially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which include different antibodies directed t different determinants (epitopes), each monoclonal antibody is ed against a single determinant on the antigen.

A “neutralizing dy” is an antibody molecule that is able to eliminate or significantly reduce an effector function of a target antigen to which it binds.

Antibody binding fragments may be produced by enzymatic or chemical cleavage of intact antibodies. Digestion of antibodies with the enzyme papain results in two identical antigen-binding fragments, also known as “Fab” fragments, and an “F0” fragment which has no n—binding activity. Digestion of dies with the enzyme pepsin results in a F(ab')2 fragment in which the two arms of the antibody molecule remain linked and comprise two- antigen binding sites. The 2 fragment has the ability to crosslink antigen.

The term “Fab” refers to a nt of an antibody that comprises the nt domain of the light chain and the CH1 domain of the heavy chain. The term “Fv” when used herein refers to the minimum fragment of an antibody that retains both antigen-recognition and antigen-binding sites. In a ain Fv species, this region consists of a dimer of one heavy- chain and one light-chain variable domain in non—covalent association. In a single-chain Fv species, one heavy-chain and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two—chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH—VL dimer.

While the six CDRS, collectively, confer antigen-binding specificity to the antibody, even a single variable domain (or half of an Fv comprising only three CDRs specific for an n) has the ability to recognize and bind antigen.

The term “complementarity determining regions” or “CDRs” refers to parts of immunological receptors that make contact with a specific ligand and determine its city.

The term “hypervariable region” refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” and/or those residues from a “hypervariable loop”.

As used herein, the term “epitope” refers to binding sites for antibodies on protein ns. ic inants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, as well as c three dimensional structural and charge characteristics. An antibody is said to bind an antigen when the dissociation constant is $1 uM, preferably S 100 nM, and most ably 5 10 nM. An increased equilibrium constant (“KD”) means that there is less affinity n the epitope and the antibody, whereas a decreased equilibrium constant means that there is a higher affinity between the epitope and the antibody. An antibody with a KD of “no more than” a certain amount means that the antibody will bind to the epitope with the given KB or more strongly. Whereas KD describes the binding characteristics of an epitope and an antibody, “potency” describes the effectiveness of the antibody itself for a function of the dy. There is not necessarily a ation between an equilibrium constant and potency; thus, for example, a relatively low KD does not automatically mean a high potency.

The term “selectively binds” in reference to an antibody does not mean that the antibody only binds to a single substance, but rather that the KB of the antibody to a first substance is less than the KB of the antibody to a second substance. An dy that exclusively binds to an epitope only binds to that single epitope.

When administered to humans, antibodies that contain rodent (murine or rat) variable and/or constant regions are mes associated with, for example, rapid clearance from the body or the generation of an immune response by the body against the antibody. In order to avoid the utilization of rodent-derived antibodies, fully human antibodies can be generated through the uction of human antibody function into a rodent so that the rodent produces fully human antibodies. Unless specifically identified , “human” and “fully human” antibodies can be used interchangeably herein. The term “fully human” can be useful when guishing antibodies that are only partially human from those that are completely, or fully human. The skilled artisan is aware of various methods of generating fiJlly human antibodies.

In order to address possible human anti—mouse antibody responses, chimeric or otherwise humanized antibodies can be utilized. Chimeric antibodies have a human constant region and a murine variable region, and, as such, human anti-chimeric antibody responses may be ed in some patients. Therefore, it is advantageous to provide fully human antibodies against multimeric enzymes in order to avoid possible human anti-mouse antibody or human anti- chimeric antibody responses.

Fully human monoclonal antibodies can be prepared, for example, by the generation ofhybridoma cell lines by techniques known to the skilled artisan. Other preparation methods involve the use of sequences encoding particular antibodies for ormation of a suitable mammalian host cell, such as a CHO cell. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example, packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection ures known in the art. s for introducing logous polynucleotides into mammalian cells are well known in the art and e dextran—mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast filSlOIl, electroporation, ulation of the cleotide(s) in liposomes, and direct microinjection of the DNA into . Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to CHO cells, HeLa cells, and human hepatocellular carcinoma cells.

Antibodies can be used diagnostically and/or therapeutically. For example, the dies can be used as a diagnostic by detecting the level of one or more peptides of the invention in a subject, and either comparing the detected level to standard control level or to a baseline level in a subject determined previously (e. g., prior to any illness). The antibodies can be used as a therapeutic to modulate the activity of one or more peptides ofthe inventidn, y having an effect on a condition or disorder.

The invention provides kits including, but not limited to, peptide sequences of the invention, optionally in combination with one or more therapeutic agents, compositions and pharmaceutical itions thereof, packaged into suitable packaging al. A kit ally includes a label or packaging insert including a ption of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein. Exemplary instructions include instructions for reducing or lowering blood glucose, treatment of hyperglycemia, treatment of diabetes, etc.

A kit can contain a collection of such components, e.g., two or more peptide sequences alone, or a combination of a peptide sequence with another therapeutically useful composition (e. g., an anti—diabetic drug, such as a gastrin compound).

The term “packaging material” refers to a physical structure housing the components of the kit. The packaging material can maintain the components sterilely, and can be made of material commonly used for such es (e.g, paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.).

Kits of the invention can include labels or s. Labels or inserts include “printed matter,” e. g., paper or cardboard, separate or affixed to a component, a kit or packing material (e. g., a box), or ed to, for example, an , tube or vial containing a kit component.

Labels or inserts can additionally include a computer readable medium, such as a disk (e. g., hard disk, card, memory disk), optical disk such as CD— or DVD—ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or s of these such as ic/optical e media, FLASH media or memory type cards.

Labels or inserts can include identifying information of one or more ents therein, dose amounts, clinical pharmacology of the active ingredient(s) ing mechanism of , pharmacokinetics and pharmacodynamics. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location and date.

Labels or inserts can include information on a condition, disorder, disease or symptom for which a kit component may be used. Labels or inserts can include instructions for the clinician or for a subject for using one or more of the kit components in a , ent protocol or therapeutic regimen. Instructions can include dosage amounts, frequency or duration, and instructions for practicing any of the methods, ent protocols or therapeutic regimes set forth herein. ary instructions include instructions for treatment or use of a peptide sequence as set forth herein. Kits ofthe invention therefore can additionally include labels or instructions for practicing any of the methods and uses of the invention described herein including treatment methods and uses.

Labels or inserts can include information on any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information on potential adverse side effects, such as warnings to the subject or clinician regarding situations where it would not be appropriate to use a particular composition. Adverse side effects could also occur when the subject has, will be or is currently taking one or more other medications that may be incompatible with the composition, or the subject has, will be or is tly undergoing another ent protocol or therapeutic regimen which would be incompatible with the composition and, ore, instructions could include information ing such incompatibilities.

Invention kits can additionally include other components. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package. Invention kits can be designed for cold storage. Invention kits can further be designed to contain peptide sequences of the invention, or that contain nucleic acids encoding peptide sequences. The cells in the kit can be maintained under appropriate storage conditions until ready to use.

Unless ise defined, all technical and scientific terms used herein have the same meaning as commonly tood by one of ordinary skill in the art to which this invention belongs. Although methods and materials r or lent to those bed herein can be used in the practice or testing of the invention, le methods and materials are described herein.

All applications, publications, s and other references, GenBank citations and ATCC ons cited herein are incorporated by reference in their entirety. In case of conflict, the specification, including definitions, will control. As used herein, the singular forms “a”, “and,” and “the” include plural referents unless the t clearly indicates otherwise. Thus, for example, reference to “a peptide sequence” or a “treatment,” includes a plurality of such sequences, treatments, and so forth.

As used herein, numerical values are often presented in a range format throughout this document. The use of a range format is merely for convenience and brevity and should not be 2012/045087 construed as an ble limitation on the scope of the invention unless the t clearly indicates otherwise. Accordingly, the use of a range expressly includes all possible subranges, all individual numerical values within that range, and all numerical values or numerical ranges including integers within such ranges and ons of the values or the integers within ranges unless the context clearly indicates otherwise. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document. Thus, for example, reference to a range of 90—100% includes 91—99%, 92—98%, 93-95%, 91-98%, 91-97%, 91-96%, 91-95%, 91- 94%, 91-93%, and so forth. Reference to a range of 90-100% also es 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth.

In addition, reference to a range of l-3, 3-5, 5-10, 10-20, 20-30, 30—40, 40-50, 50-60, 60—70, 70-80, 80—90, 90-100, 100-110, 110-120, 120-130, 130-140, 140—150, 150—160, 160-170, 170-180, 180-190, 0, 200-225, 225-250 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, , 16, 17, 18, 19, 20, etc. In a further example, reference to a range of25—250, 250—500, 500— 1000, 1000-2500 or 2500-5000, 5,000, 5000-50,000 includes any numerical value or range within or encompassing such values, e.g., 25, 26, 27, 28, 29.250, 251, 252, 253, 254. . ..500, 501, 502, 503, 504..., etc.

As also used herein a series of ranges are disclosed throughout this document. The use of a series of ranges e combinations of the upper and lower ranges to provide another range. This construction applies regardless of the breadth of the range and in all contexts throughout this patent nt. Thus, for example, reference to a series of ranges such as 5-10, —20, 20-30, 30-40, 40—50, 50-75, , 100-150, includes ranges such as 5-20, 5—30, 5-40, 5- 50, 5—75, 5-100, 5-150, and 10—30, 10-40, 10-50, 10-75, 10-100, 10-150, and 20-40, 20-50, 20—75, -100, 20-150, and so forth.

For the sake of conciseness, certain abbreviations are used herein. One example is the single letter abbreviation to represent amino acid residues. The amino acids and their ponding three letter and single letter abbreviations are as follows: alanine Ala (A) arginine Arg (R) asparagine Asn (N) ic acid Asp (D) cysteine Cys (C) glutamic acid Glu (E) glutamine Gln (Q) glycine Gly (G) histidine His (H) cine Ile (I) leucine Leu (L) lysine Lys (K) methionine Met (M) phenylalanine Phe (F) proline Pro (P) serine Ser (S) threonine Thr (T) tryptophan Trp (W) tyrosine Tyr (Y) valine Val (V) The invention is generally disclosed herein using affirmative language to describe the numerous embodiments. The invention also specifically includes ments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include, aspects that are not expressly included in the invention are nevertheless. disclosed herein.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate but not limit the scope of ion described in the claims.

Exa_mpl_es_ The following is a description of various methods and als used in the studies herein.

Animals. db/db mice were purchased from The Jackson Laboratory (Bar Habor, ME), Mice were kept in ance with welfare guidelines under controlled light (12 hr light and 12 hr dark cycle, dark 6:30 pm-6:30 am), temperature (22i4°C) and ty (50%i20%) conditions. They had free access to water (autoclaved distilled water) and were fed ad libitum on a commercial diet (Harlan Laboratories, apolis, IN, Irradiated 2018 Teklad Global 18% Protein Rodent Diet) containing 17 kcal% fat, 23 kcal% protein and 60 kcal% carbohydrate. For diet—induced obesity, C57BL6/J mice (Jackson tory) were ined on a high-fat diet (D12492, Research Diet, New Brunswick, NJ. USA) containing 60 kcal% fat, 20 kcal% protein and 20 kcal% carbohydrate for 16—20 weeks. All animal studies were approved by the NGM Institutional Animal Care and Use Committee.

DNA and amino acid sequences. cDNA of ORF encoding human FGF19 (Homo sapiens FGF19, GenBank Accession No. NM_005117.2) variants Protein sew encodfly the cDNA (GenBank Accession No. NP 005108.11 PCR. FGF19 ORF was amplified with polymerase chain reaction (PCR) using inant DNA (cDNA) prepared from human small intestinal tissue. PCR reagents kits with Phusion high— fidelity DNA polymerase were sed from New England BioLabs (F—530L, Ipswich, MA).

The following primers were used: forward PCR primer: 5’ CCGACTAGTCACCatgcggagcgggtgtgtgg and reverse PCR primer: 5’ ATAAGAATGCGGCCGCTTACTTCTCAAAGCTGGGACTCCTC. ed DNA fragment was digested with restriction enzymes Spe I and Not I (the restriction sites were included in the 5’ or 3’ PCR primers, respectively) and was then ligated with AAV transgene vectors that had been digested with the same restriction enzymes. The vector used for expression contained a selectable marker and an expression cassette composed of a strong eukaryotic promoter 5’ of a site for insertion of the cloned coding sequence, followed by a 3’ untranslated region and bovine growth hormone polyadenylation tail. The expression construct is also flanked by internal terminal repeats at the 5’ and 3’ ends. tion and purification of AAV. AAV293 cells (obtained from Agilent Technologies, Santa Clara, CA) were cultured in Dulbeco’s ation of Eagle’s Medium (DMEM, Mediatech, Inc. Manassas, VA) supplemented with 10% fetal bovine serum and 1x antibiotic-antimycotic solution (Mediatech, Inc. Manassas, VA). The cells were plated at 50% density on day 1 in 150 mm cell culture plates and transfected on day 2, using calcium phosphate precipitation method with the following 3 plasmids (20 te of each): AAV transgene plasmid, r ds (Agilent logies) and AAV2/9plasmid (Gao et al., J. Viral. 78:6381 ). 48 hours after ection, the cells were scraped off the plates, pelleted by centrifugation at 3000xg and resuspended in buffer containing 20 mM Tris pH 8.5, 100 mM NaCl and 1 mM MgC12. The suspension was frozen in an l dry ice bath and was then thawed in 37 OC water bath. The freeze and thaw cycles were repeated three times; Benzenase (Sigma- aldrich, St. Louis, MO) was added to 50 units/m1; holate was added to a final tration of 0.25%. After an incubation at 37°C for 30 min, cell debris was pelleted by centrifugation at 5000 X g for 20 min. Viral particles in the supernatant were purified using a discontinued iodixanal (Sigma-aldrich, St. Louis, MO) gradient as previously described (Zolotukhin S. et a1 (1999) Gene 77761”. 6:973). The viral stock was concentrated using Vivaspin (MW cutoff 100,000 Dalton, Sartorius Stedim h, Aubagne, France) and re—suspended in phosphate—buffered saline (PBS) with 10% glycerol and stored at -80°C. To determine the viral genome copy number, 2 [Ll of viral stock were incubated in 6 ul of solution containing 50 units/ml Benzonase, 50 mM Tris-HCl pH 7.5, 10 mM MgC12 and 10 mM CaClz at 37°C for 30 minutes.

Afterwards, 15 ul of the solution containing 2 mg/ml of Proteinase K, 0.5% SDS and mM EDTA were added and the mixture was incubated for onal 20 min at 55°C to release viral DNA. Viral DNA was cleaned with mini DNeasy Kit (Qiagen, ia, CA) and eluted with 40 ul of water. Viral genome copy (GC) was determined by using quantitative PCR.

Viral stock was diluted with PBS to ble GC/ml. Viral working solution (200 pl) was delivered into mice via tail vein injection.

Blood glucose assay. Blood glucose in mouse tail snip was measured using EK Active test strips read by ACCU—CHEK Active meter (Roche Diagnostics, Indianapolis, IN) following cturer’s ction.

Lipid profile assay. Whole blood from mouse tail snips was collected into plain capillary tubes (BD Clay Adams SurePrep, Becton son and Co. Sparks, MD). Serum and blood cells were separated by spinning the tubes in an Autocrit Ultra 3 (Becton Dickinson and Co. Sparks, MD). Serum samples were assayed for lipid profile (triglyceride, total cholesterol, HDL, and non—HDL) using Integra 400 Clinical Analyzer'(Roche Diagnostics, Indianapolis, IN) following the manufacturer’s instructions.

Serum FGF19/FGF21/variants exposure level assay. Whole blood (about 50 til/mouse) from mouse tail snips was ted into plain capillary tubes (BD Clay Adams SurePrep, Becton Dickenson and Co. Sparks, MD). Serum and blood cells were separated by spinning the tubes in an Autocrit Ultra 3 n Dickinson and Co. Sparks, MD). FGFl9, FGF21, and variant exposure levels in serum were determined using EIA kits (Biovendor) by following the manufacturer’s instructions.

Hepatocellular carcinoma (HCC) assay. Liver specimen was harvested from db/db mice 6 months after AAV injection. HCC score is recorded as the number ofHCC nodules on the surface of the entire liver from variants—injected mice d by the number ofHCC s from wildtype FGFl9—inj ected mice.

Liver gene expression assay. Liver specimen was harvested and homogenized in Trizol reagent (Invitrogen). Total RNA was extracted following manufacturer’s instruction. RNA was treated with DNase (Ambion) followed by quantitative RT-PCR is using Taqman primers and reagents from Applied Biosystems. Relative mRNA levels of aldo-keto reductase and slc1a2 in the liver was calculated using AACt method.

FGFR4 binding and activity assays. Solid phase ELISA (binding) and ERK phosphorylation assay were performed using purified recombinant ns. FGFR binding assay was conduCted using solid phase ELISA. Briefly, 96well plate was coated with 2 ug/ml anti-hFc antibody and incubated withl ug/ml FGFRl-hFc or FGFR4-hFc. Binding to FGF19 ts in the presence of 1 ug/ ml soluble b— klotho and 20 ug/ml heparin were detected by biotinylated anti- FGF19 antibodies (0.2ug/mL), followed by avidin- HRP incubation (lOOng/mL). For FGFR4 activation assay, Hep3B cells were stimulated with FGF19 variants for 10 minutes at 37C, then immediately lysed and assayed for ERK phosphorylation using a commercially available kit from Cis-Bio.

Example 2 The following is a description of studies showing the glucose ng activity of various sequence variants of FGF19 and FGF21, and FGF21 fusion constructs.

Figure 2 illustrates exemplary FGF19/FGF21 filSlOI’l constructs, and the segments from each of FGF19 and FGF21 present in the fusion peptides. These peptides were analyzed for glucose lowering activity and statistically significant lipid elevating Or increasing activity (Tables 1—8 and Figure l).

Mice ) were injected with Viral vector expressing FGF19, FGF21 or variants, and analyzed after injection. Glucose-lowering activity of each sequence is represented by a “+” symbol (a “-” symbol means no glucose lowering activity, a “+/-“ symbol means variants retain minimal glucose-lowering ty); lipid ing activity is represented by a “+”symbol (a “-” symbol means no lipid ing ty, a “+/-“ symbol means ts retain minimal lipid- elevating activity, Figure 2).

Two fusions ofFGF21 and FGF19, denoted variant M5 and variant 45 (M45), ted glucose lowering activity and an absence of statistically significant lipid elevating or increasing activity. Variants denoted M1, M2 and M69, respectively (Figure 1, also ted glucose ng activity (Figures 3B and 3C, Table 5). Data comparing M5, M1, M2 and M69 glucose lowering activity and lipid elevating or increasing activity to FGF19 and FGF21 are illustrated in Figures 3A—3C and 4A-4C.

Example 3 The following is a description of studies showing that variants M5, M1, M2 and M69 are not tumorigenic, as determined by hepatocellular carcinoma (HCC) formation, and that variants M5, M2 and M69 also do not reduce lean muscle and fat mass.

Animals (db/db) were ed with AAV vectors expressing FGF19, FGF21, M5, M1, M2, or M69, or injected with saline, and analyzed 6 months after injection. The data indicate that variants M5, M1, M2, and M69 did not induce (HCC) formation significantly es SA-SC). s (db/db mice) were also injected with viral vector expressing FGF19, FGF21, M5, M1, M2 or M69, or injected with saline, and analyzed 6 months after injection for the effect of on lean mass and fat mass. The data indicate that M5, M2 and M69 peptides did not cause a statistically cant reduction in lean mass or fat mass, in contrast to FGF21, and that M1 e reduces lean mass (Figures 6A—6C).

Example 4 The following is a data y of 25 additional variant peptides analyzed for lipid ing activity and tumorigenesis. The data clearly show a positive correlation between lipid elevation and tumorigenesis, as determined by hepatocellular carcinoma (HCC) formation in db/db mice.

Tables 1 to 3 summarize data for 26 different variant peptides. Such exemplified variant peptides have FGF19 C-terminal sequence: PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKM QGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPML PMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK at the C-terminal portion, e.g, ing the “TSG” amino acid residues. Notably, t peptides (7 total, including M5) that did not cause a statistically significant elevation of lipids did not induce hepatocellular carcinoma (HCC) ion. In contrast, all variant peptides (17 total) that caused a statistically significant elevation of lipids also caused hepatocellular carcinoma (HCC) formation in mice. This data indicates that there is a strong positive correlation between lipid elevating activity and hepatocellular carcinoma (HCC) formation. Accordingly, lipid elevating activity can be used as an indicator and/or predictor of hepatocellular carcinoma (HCC) formation in animals.

Table 1: Elevated Triglyceride and Cholesterol in (lb/db Mice Appears to Positively ate With HCC Formation.

N—terminal Domain Core Lipid HCC Elevation A Formation FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG + + FGF21 HPIPDSSPLLQ--FGGQV RQRYLYTDD - — M5 R—HPIPDSSPLLQ--FGGQV RLRHLYTSG - M74 R------------------DAGPHVHYGWGDPI RLRHLYTSG + + M75 R--------------------------- VHYGWGDPI RLRHLYTSG - M76 R---'-----------------------------------GDPI RLRHLYTSG - M77 R---------------------------------------------- RLRHLYTSG - - M78 --------------AGPHVHYGWGDPI RLRHLYTSG + + M79 R--------------------- GPHVHYGWGDPI RLRHLYTSG + + M80 R----------------------- WGDPI RLRHLYTSG - - M81 --------------------HVHYGVKHWI RLRHLYTSG — — Table 2: Elevated Triglyceride and Cholesterol in db/db Mice Appears to Positively Correlate with HCC Formation N—terminal Domain Core Lipid HCC A Elevation Formation FGF ]9 RPLAFSDAGPHVHYGWGDPI TSG + + FGFZl SPLLQ--FGGQV RQRYLYTDD — - M82 RPLAFSAAGPHVHYGWGDPI RLRHLYTSG + + M83 RPLAFSDAAPHVHYGWGDPI RLRHLYTSG +/- --/ M84 RPLAFSDAGAHVHYGWGDPI RLRHLYTSG +/- --/ M85 RPLAFSDAGPHVHYGAGDPI RLRHLYTSG - - M86 RPLAFSDAGPHVHYGWGAPI RLRHLYTSG + + M87 RPLAFSDAGPHVHYGWGDAI RLRHLYTSG + + Table 3: Elevated Triglyceride and terol in db/db Mice s to Positively Correlate with HCC Formation Lipid HCC Elevatlon N—terminal Domain Formation l—~—L——i FGF19 DAGPHVHYGWGDPI RLRéE§¥EG + + FGFZl HPIPDSSPLLQ——FGGQV RQRYLYTDD - H3lA/Sl4lA(M88) FGF19 + + H31A/H142A(M89) FGFl9 + + Kl27A/R129A(M90) FGFlg + + K127A/Sl4lA(M9l) FGF19 + + K127A/Hl42A(M92) FGF19 + + R129A/Sl4lA(M93) FGF19 + + Sl4lA/Hl42A(M94) FGF19 + + K127A/Hl42A(M95) FGF19 + + K127A/R129A/3141A(M97> FGF19 + + K127A/R129A/H142A(M98) FGF19 + + R129A/Sl4lA/Hl42A(M99) FGF19 + + Examgle 5 The following is a data summary of additional FGF 19 variant peptides analyzed for glucose lowering activity and lipid elevating activity.

Table 4 illustrates the e “core sequences” of 35 additional FGF19 variants, denoted M5 to M40. Such exemplified variant peptides have FGF19 C-terminal sequence, PHGLSSCFLRIRADGVVDCARGQSAHSLLEH<AVALRTVAIKGVHSVRYLCMGADGKM QGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPML PMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK at the C—terminal portion, e.g., following the “TSG” amino acid residues of the core sequence. The data y show that ts M6, M7, M8, mM38 and M39 have the desired characteristics of glucose lowering activity and not tically significant lipid elevating activity in db/db mice.

Table 4: Additional Variants and Fine Mapping of the N—terminal Domain inal Domain Core Glucose Lipid Lowering ion FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG + + FGF21 HP1PDSSPLLQ-—FGGQV RQRYLYTDD + - V15 R-HPIPDSSPLLQ—-FGGQV RLRHLYTSG + - M6 R-------DSSPLLQ--FGGQV RLRHLYTSG + - M7» RPLAFSDSSPLLQ--FGGQV RLRHLYTSG + - M8 R—HPIPDSSPLLQ--WGDPI RLRHLYTSG + — M9 R-HPIPDSSPLLQFGWGDPI RLRHLYTSG + + M10 R-HPIPDSSPHVHYGWGDPI RLRHLYTSG - + V111 RPLAFSDAGPLLQ-—WGDPI RLRHLYTSG N/D N/D M12 RPLAFSDAGPLLQFGWGDPI RLRHLYTSG - + M13 RPLAFSDAGPLLQ--FGGQV RLRHLYTSG - M14 R-HPIPDSSPHVHYG--GQV RLRHLYTSG - V115 RPLAFSDAGPHVHYG-—GQV RLRHLYTSG + + M16 RPLAFSDAGPHVH--WGDPI RLRHLYTSG N/D N/D M17 RPLAFSDAGPHV--GWGDP1 RLRHLYTSG N/D N/D V118 RPLAFSDAGPH——YGWGDP1 RLRHLYTSG N/D N/D V119 RPLAFSDAGP—V-YGWGDPI RLRHLYTSG N/D N/D M20 RPLAFSDAGP-VH-GWGDPI RLRHLYTSG N/D N/D M21 RPLAFSDAGP-VHY-WGDPI RLRHLYTSG N/D N/D M22 RPLAFSDAGPHVH—GWGDPI RLRHLYTSG N/D N/D V123 RPLAFSDAGPH-H-GWGDPI TSG N/D N/D V124 RPLAFSDAGPH-HY—WGDPI RLRHLYTSG N/D N/D M25 RPLAFSDAGPHV—Y-WGDPI RLRHLYTSG N/D N/D V126 RPLAFSDSSPLVH--WGDPI RLRHLYTSG N/D N/D M27 RPLAFSDSSPHVH-—WGDPI RLRHLYTSG N/D N/D M28 RPLAFSDAPHV---WGDPI RLRHLYTSG N/D N/D M29 RPLAFSDAGPHVHY-WGDPI RLRHLYTSG N/D N/D M30 RPLAFSDAGPHVHYAWGDPI RLRHLYTSG N/D N/D V131 DSSPLLQ——FGAQV RLRHLYTSG --/- - V132 R-HPIPDSSPLLQ--FGIYQV RLRHLYTSG - - M33 R-HPIPDSSPLLQ--FGGQV RLRHLYTSG - - M34 R—HPIPDSSPLLQ--FG7AV RLRHLYTSG --/- - M35 R—HPIPDSSPLLQ-—FGGEV TSG --/- +/ V136 R—HPIPDSSPLLQ--FGGQV RLRHLYTSG +/— - V137 R-HPIPDSSPLLQ--FGGUA RLRHLYTSG - M38 R-HPIPDSSPLLQ-—FGGQT RLRHLYTSG + - M39 R-HPIPDSSPLLQ--FGGQT TSG + — V140 R-HPIPDSSPLLQFGWGQPO RLRHLYTSG - + Table 4a: N-terminal Domain Lowerino W W ’———'—’—’—'—\ Core FGF19 RPLAFSDAGPHVHYGVVGDPI RLRHLYTSG + + + FGFZ 1 SPLLQuFGGQV RQRYLYTDD + — ..

M5 R—HPIPDSSPLLQ--FGGQV RLRHLYTSG + - - M9 R-HPIPDSSPLLQFG\VGDPI RLRHLYTSG + + “ N18 R—HPIPDSSPLLQ—-\VGDPI RLRHLYTSG + + u M12 RPLAFSDAGPLLQFGVVGDPI RLRHLYTSG — + + N110 R—HPIPDSSPHVHYGW’GDPI RLRHLYTSG - + + N11 3 RPLAFSDAGPLLQ--FGGQV RLRHLYTSG - + + M15 RPLAFSDAGPHVHYGnGQV RLRHLYTSG - - +,I’- N114 R-HPIPDSSPHVHYG——GQV RLRHLYTSG — - +/- N143 RPLAFSDAGPHVHYG-GD-I RLRHLYTSG - - +/- M6 R-----DSSPLLQ--FGGQV TSG + - M7 RPLAFSDSSPLLQnFGGQV RLRHLYTSG + - Table 4b: Glucose Lipid HCC N-terminal Domain Lowering Elevation F0mm tiox . \ Core FGF19 RPLAFSDAGPHVHYG‘WGDPI RLRHLYTSG + + + FGFL’ 1 HPIPDSSPLLQ--FGGQ_V RQRYLYTDD " N15 R-HPIPDSSPLLQ--FGGQV RLRHLYTSG “ M31 R-HPIPDSSPLLQuFGAQV RLRHLYTSG “ - + N132 R—HPIPDSSPLLQnFGDQV RLRHLYTSG + - N133 R—HPIPDSSPLLQ—-FGPQV TSG - - + M34 R—HPIPDSSPLLQ--FGGAV RLRHLYTSG — *- 1V135 R-HPIPDSSPLLQ--FGGEV RLRHLYTSG - -- N136 R—HPIPDSSPLLQuFGGNV RLRHLYTSG + - _,/_ M37 R—HPIPDSSPLLQ--FGGQA TSG - _ + MSS DSSPLLQnFGGQI RLRHLYTSG -— N139 R-HPIPDSSPLLQ--FGGQT RLRHLYTSG u M40 R-HPIPDSSPLLQFGWGQPV RLRHLYTSG - + + Table 4c: e Lipid HCC inal Domain Lowen'ug Elevation Formation I—'—‘%—l Core FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG + ‘1' + FGFZl HPIPDSSPLLQ--FGGQV RQRYLYTDD + MS R-HPIPDSSPLLQ--FGGQV RLRHLYTSG + M52 DSSPLLQ--WGDPI RLRHLYTSG + + - MS4 RPLAFSDAGPLLQ--WGDPI RLRHLYTSG + + MSS RPLAFSDAGPH--YGWGDPI RLRHLYTSG + + V156 RPLAFSDAGP—V—YGWGDPI TSG + + 157 RPLAl-‘SDAGP-VH-GWGDPI RLRHLYTSG *1 + \158 RPLAFSDAGP-VHY-WGDPI RLRHLYTSG + + ‘v159 RPLAFSDAGPH-H-GWGDPI RLRHLYTSG + + \460 DAGPH—HY-WGDPI RLRHLYTSG + + MGl RPLAFSDAGPHV--GWGDPI RLRHLYTSG + + MGZ RPLAFSDAGPHV-Y-WGDPI RLRHLYTSG - + + V163 RPLAFSDAGPHVH-—WGDP1 RLRHLYTSG + + + 164 RPLAFSDSSPLVHv—WGDPI RLRHLYTSG + + «1— M65 RPLAFSDSSPHVH--WGDPI RLRHLYTSG - + + N166 RPLAFSDAGPHLQ--WGDPI RLRHLYTSG — — + M67 RPLAFSDAGPHV—--WGDPI RLRHLYTSG - - +/'- MGS RPLAFSDAGPHVHY—WGDPI RLRHLYTSG - + - M4 RPLAFSDAGPHVHYAWGDPI RLRHLYTSG — + + \469 R—-———DSSPLVHYGWGDPI RLRHLYTSG + 4 \170 MR—---DSSPLVHYGWGDPI RLRHLYTSG + + MSS M-----DSSPLVHYGWGDPI RLRHLYTSG + + Table 5 illustrates the peptide sequences of 3 additional FGF19 variants, denoted M1, M2 and M69. The data clearly show that these three variants have the desired characteristics of glucose ng activity in db/db mice (Figures 3B and 3C). These three variants appear to elevate lipids in db/db mice Figures 4B and 4C).

Table 5: Additional Variants M1:RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEK HRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMD PFGLVTGLEAVRSPSFEK M2:RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSL LEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEK LSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMD GLEAVRSPSFEK M69:RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKA VALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLP VSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGL VTGLEAVRSPSFEK Examgle 6 The following is a data summary showing that FGF19 reduces body weight in diet— induced obese mice and in ob/ob mice, and liver tumor formation activity and body weight in db/db mice.

Mice were injected with FGF19 or FGF21 in AAV vector. Body weight was recorded 4 weeks after ion.

Table 6: FGF19 reduces body weight in diet-induced obese mice and in oblob mice Body Weight— Body Weight— N—terminal Domain Lcwering in DIO Lowering in (Db/Ob ,—_;—_‘ Care FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG + + FGF21 H?IP£‘!SSPLLQ——FGGQV RQRYLYTDD + + Table 7: Correlation of body weight and liver tumor formation of FGF19, FGF21 and selected variants in (lb/db mice . Liver m N—terminal Domain Tumor M Nodule l’"“—"—‘._—"—t Core FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG + Increased FGF21 HPIPDSSPLLQ-—FGGQV RQRYLYTDD - Decreased M5 R—HPIPDSSPLLQ-—FGGQV RLRHLYTSG- - Increased M6 R—————DSSPLLQ--FGGQV RLRHLYTSG - Decreased M32 ,R-HPIPDSSPLLQ--FGDQV TSG - Decreased M52 R—————DSSPLLQ—-WGDPI RLRHLYTSG - Decreased M69 R—————DSSPLVHYGWGDPI RLRHLYTSG - Increased The ing is a study showing that variant M5 and variant M69 peptides reduce blood glucose.

Mice (ob/ob) were injected (subcutaneously) with M5 (0.1 and 1 mg/kg, s.c.) or FGF19 (1 mg/kg, s.c.), or variant M69 (0.1 and 1 mg/kg, s.c.) or FGF19 (1 mg/kg, s.c.). Plasma glucose levels were measured at 2, 4, 7, and 24 hours after injection, and the results are shown in Figure 7. M5 e 7A) and t M69 (Figure 7B) showed similar glucose ng effects as wild type FGF19.

Example 8 This example describes a study showing that liver expression of aldo—keto reductase family 1, member C18 (AkrlC18) and solute carrier family 1, member 2 (slcla2) appears to correlate with HCC activity.

Mice (db/db) were injected with viral vector expressing FGF19 (HCC+), FGFZl (HCC-), dN2 (HCC-) or MS (HCC-), or injected with GFP. Liver samples were harvested and ed by tative RT-PCR 2 weeks after injection. The data, shown in Figure 8, shows that liver expression of AkrlCl8 and slcla2 appears to correlate with HCC activity.

Table 8: Summary of FGF19 Variants in 3T3L1 Adipocyte Signaling Assay P-Erk assay in 3T3L1 adioc tes FGF19 FGF21 M5 M2 M63 M64 M1 M8 —----- -433 —-------- ———---Immm __------- 3-74 4-24 4-15 4.16 0-24 0-14

Claims (113)

What we claim is:

1. A peptide, comprising: a) an N-terminal region comprising at least seven amino acid residues, the N-terminal region having a first amino acid on and a last amino acid position, wherein the N-terminal region comprises DSSPL (SEQ ID NO:121) or DASPH (SEQ ID NO:122); and b) a inal region having a first amino acid position and a last amino acid position, wherein the C-terminal region comprises (i) a first C-terminal region sequence comprising WGDPIRLRHLYTSG (amino acids 16 to 29 of SEQ ID NO:99 ]), wherein the W residue corresponds to the first amino acid position of the C-terminal region; and (ii) a second C-terminal region sequence comprising CFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAI KGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGY NVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPM DLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS PSFEK (amino acid residues 30 to 194 of SEQ ID NO:99 [FGF19]) or a sequence comprising 1 to 5 amino acid tutions, deletions or insertions thereof; wherein the peptide (i) binds to fibroblast growth factor receptor 4 (FGFR4) with an affinity equal to or greater than FGF19 binding affinity for FGFR4; (ii) activates FGFR4 to an extent or amount equal to or r than FGF19 activates FGFR4; (iii) has at least one of reduced hepatocellular carcinoma (HCC) formation; greater glucose lowering activity, less lipid increasing activity, less triglyceride activity, less cholesterol activity, less non-HDL activity or less HDL increasing activity, as compared to FGF19, or as compared to an FGF19 variant sequence having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence at amino acids 16-20 of FGF19 (SEQ ID NO:99); and/or (iv) has less lean mass reducing activity as ed to FGF21.

2. The e of claim 1, wherein the second inal region sequence of the peptide comprises from 1 to 5 amino acid substitutions, deletions or insertions.

3. The peptide of claim 1, wherein the peptide is less than about 250 amino acids in length.

4. The peptide of claim 1, n the N-terminal region ses amino acid residues VHYG (SEQ ID NO:101), DASPHVHYG (SEQ ID NO:102), or DSSPLVHYG (SEQ ID NO:103).

5. The peptide of claim 4, wherein the G corresponds to the last position of the N- terminal .

6. The e of claim 1, wherein the N-terminal region comprises amino acid residues DSSPLLQ (SEQ ID NO:104), and wherein the Q residue is the last amino acid position of the N-terminal region.

7. The peptide of claim 5, wherein the N-terminal region further comprises: RHPIP (SEQ ID NO:106), wherein R is the first amino acid position of the N- terminal region; HPIP (SEQ ID NO:107), wherein H is the first amino acid position of the N- terminal region; RPLAF (SEQ ID NO:108), wherein R is the first amino acid position of the N- terminal region; PLAF (SEQ ID NO:109), wherein P is the first amino acid position of the N- terminal region; or R, wherein R is the first amino acid position of the N-terminal region.

8. The peptide of claim 6, wherein the N-terminal region further comprises: RHPIP (SEQ ID NO:106), n R is the first amino acid position of the N- terminal region; HPIP (SEQ ID NO:107), n H is the first amino acid position of the N- terminal region; RPLAF (SEQ ID ), wherein R is the first amino acid position of the N- terminal region; PLAF (SEQ ID NO:109), wherein P is the first amino acid position of the N- terminal region; or R, wherein R is the first amino acid position of the N-terminal region.

9. The peptide of claim 1, wherein the N-terminal region comprises amino acid es DSSPLLQFGGQV (SEQ ID NO:105), and wherein the V e corresponds to the last position of the N-terminal region.

10. The peptide of claim 1, wherein amino acid residues HPIP (SEQ ID NO:107) are the first 4 amino acid residues of the N-terminal region.

11. The peptide of claim 1, wherein the first position of the N-terminal region is a R or M residue; the first and second positions of the N-terminal region is a MR, RM, RD, DS, MD or MS ce; the first h third positions of the N-terminal region is a MDS, RDS, MSD, MSS, or DSS sequence; the first through fourth positions of the inal region is a RDSS (SEQ ID NO:115) or MDSS (SEQ ID NO:116) sequence; the first through fifth positions of the N-terminal region is an MRDSS (SEQ ID NO:117) sequence; the first through sixth positions of the N-terminal region is an MDSSPL (SEQ ID NO:119) sequence; or the first through seventh positions of the inal region is an MSDSSPL (SEQ ID NO:120) sequence.

12. The e of claim 1, wherein the peptide comprises an N-terminus region and a first C-terminal region having an amino acid sequence comprising or consisting of any of: RPLAFSDASPHVHYGWGDPIRLRHLYTSG (amino acids 1-29 of SEQ ID NO:1); PLAFSDASPHVHYGWGDPIRLRHLYTSG (amino acids 2-29 of SEQ ID NO:1); RPLAFSDSSPLVHYGWGDPIRLRHLYTSG (amino acids 1-29 of SEQ ID NO:2); PLAFSDSSPLVHYGWGDPIRLRHLYTSG (amino acids 2-29 of SEQ ID NO:2); RHPIPDSSPLLQWGDPIRLRHLYTSG (amino acids 1-26 of SEQ ID NO:8); RHPIPDSSPLLQFGWGDPIRLRHLYTSG (amino acids 1-28 of SEQ ID NO:9); RPLAFSDSSPLVHWGDPIRLRHLYTSG (amino acids 1-27 of SEQ ID NO:26); PLAFSDSSPLVHWGDPIRLRHLYTSG (amino acids 2-27 of SEQ ID NO:26); SPLLQWGDPIRLRHLYTSG (amino acids 1-25 of SEQ ID NO:47); RDSSPLLQWGDPIRLRHLYTSG (amino acids 1-22 of SEQ ID NO:52); DSSPLLQWGDPIRLRHLYTSG (amino acids 2-22 of SEQ ID NO:52); MDSSPLVHYGWGDPIRLRHLYTSG (amino acids 1-24 of SEQ ID NO:53); VHYGWGDPIRLRHLYTSG (amino acids 1-24 of SEQ ID NO:69); DSSPLVHYGWGDPIRLRHLYTSG (amino acids 2-24 of SEQ ID NO:69); MRDSSPLVHYGWGDPIRLRHLYTSG (amino acids 1-25 of SEQ ID NO:70); DSSPLVHYGWGDPIRLRHLYTSG (amino acids 1-23 of SEQ ID NO:141); SPLLQFGWGDPIRLRHLYTSG (amino acids 1-27 of SEQ ID ).

13. The peptide of claim 1, wherein the N-terminal region first amino acid position is a methionine (M), ne (R), serine (S), histidine (H), proline (P), e (L) or aspartic acid (D) residue.

14. The peptide of claim 1, wherein the N-terminal region does not have a methionine (M) or arginine (R) residue at the first amino acid position of the N-terminal region.

15. The peptide of claim 1, n the N-terminal region comprises any one of the following amino acid sequences: MDSSPL (SEQ ID NO:119), MSDSSPL (SEQ ID NO:120), or SDSSPL (SEQ ID ).

16. The peptide of claim 1, wherein the peptide has an amino acid sequence comprising or consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:26, SEQ ID NO:47, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:69, SEQ ID NO:70; SEQ ID NO:141 or SEQ ID NO:163.

17. The peptide of claim 16, wherein the peptide has an amino acid sequence comprising or consisting of SEQ ID NOs:1, 2, 8, 9, 26, 52 or 69, wherein the arginine (R) residue at the first amino acid position of the N-terminal region of the sequence is deleted.

18. The e of claim 16, wherein the peptide has an amino acid sequence comprising or consisting of SEQ ID NO:1.

19. The peptide of claim 16, n the peptide has an amino acid sequence comprising or consisting of SEQ ID NO:2.

20. The peptide of claim 16, wherein the peptide has an amino acid sequence comprising or consisting of SEQ ID NO:8.

21. The peptide of claim 16, wherein the peptide has an amino acid sequence comprising or consisting of SEQ ID NO:9.

22. The peptide of claim 16, wherein the peptide has an amino acid sequence comprising or ting of SEQ ID NO:26.

23. The peptide of claim 16, wherein the peptide has an amino acid sequence comprising or consisting of SEQ ID NO:47.

24. The peptide of claim 16, wherein the peptide has an amino acid sequence comprising or consisting of SEQ ID NO:52.

25. The peptide of claim 16, wherein the peptide has an amino acid sequence comprising or consisting of SEQ ID NO:53.

26. The peptide of claim 16, wherein the peptide has an amino acid ce comprising or consisting of SEQ ID .

27. The peptide of claim 16, wherein the peptide has an amino acid sequence sing or consisting of SEQ ID NO:163.

28. The peptide of claim 1 wherein said peptide is fused with an immunoglobulin Fc region.

29. The peptide of claim 1, n the peptide has at least one of reduced HCC formation; greater glucose ng activity, or less lipid increasing activity as ed to FGF19, or as compared to an FGF19 variant having any of GQV, GDI, WGPI, WGDPV, WGDI, GDPI, GPI, WGQPI, WGAPI, AGDPI, WADPI, WGDAI, WGDPA, WDPI, WGDI, WGDP or FGDPI substituted for the WGDPI sequence at amino acids 16-20 of FGF19 (SEQ ID NO:99).

30. The peptide of claim 29, wherein the HCC formation, glucose ng activity, or lipid increasing activity is ascertained in a db/db mouse.

31. The peptide of claim 29, wherein the peptide has less lean mass reducing activity as compared to the lean mass reducing activity of FGF21.

32. The peptide of claim 31, wherein the lean mass reducing activity is ascertained in a db/db mouse.

33. A pharmaceutical composition, comprising the e of any one of claims 1 to 32, and a pharmaceutically acceptable carrier.

34. A pharmaceutical composition, comprising the peptide of any one of claims 1 to 32, a glucose lowering agent, and a pharmaceutically acceptable carrier.

35. A nucleic acid molecule encoding the peptide of any one of claims 1 to 32.

36. The nucleic acid le of claim 35, further comprising an expression control element in operable linkage that confers expression of the nucleic acid molecule encoding the peptide in vitro, in a cell or in vivo.

37. A vector comprising the nucleic acid molecule of claim 35 or 36.

38. The vector of claim 37, n the vector comprises a viral vector.

39. An in vitro transformed or host cell that expresses the peptide of any one of claims 1 to 32.

40. A peptide having an amino acid sequence comprising or ting of MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHS LLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDG YNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGH LESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO:70).

41. The peptide of claim 40, wherein the peptide has an amino acid sequence comprising SEQ ID NO:70.

42. The e of claim 40, wherein the peptide has an amino acid sequence consisting of SEQ ID NO:70.

43. The peptide of claim 40, wherein said peptide is fused with an immunoglobulin Fc region.

44. A peptide having an amino acid ce comprising or consisting of RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLL EIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYN VYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLES DMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO:69).

45. The e of claim 44, wherein the peptide has an amino acid sequence comprising SEQ ID NO:69.

46. The peptide of claim 44, wherein the peptide has an amino acid ce consisting of SEQ ID NO:69.

47. The peptide of claim 44, wherein said peptide is fused with an immunoglobulin Fc region.

48. A pharmaceutical composition, comprising the e of any one of claims 40 to 47, and a ceutically acceptable carrier.

49. A pharmaceutical composition, sing the peptide of any one of claims 40 to 47, a glucose lowering agent, and a pharmaceutically acceptable carrier.

50. Use of the peptide of any one of claims 1 to 32 and 40 to 47 in the manufacture of a medicament for treating a t having, or at risk of having, a disease or disorder treatable by the peptide.

51. Use of the e of any one of claims 1 to 32 and 40 to 47 in the manufacture of a medicament for reducing glucose levels in a subject.

52. The use of claim 51, wherein the medicament is for administration to said subject in combination with a supplementary therapy.

53. The use of claim 52, n said supplemental therapy is a weight loss surgery, gastric bypass, gastrectomy, gastric banding, gastric balloon, or gastric sleeve.

54. The use of claim 52, wherein said supplemental therapy is a glucose lowering agent, insulin, GLP1 analogue, biguanide, sulphonylurea, thiazolidinedione, a dipeptidyl peptidase-4 (DPP-4) inhibitor, a bromocriptine formulation, a bile acid sequestrant, metformin, a thiazolidinedione (TZD), a SGLT-2 inhibitor, or any combination thereof.

55. The use of claim 54, wherein said biguanide or sulphonylurea is selected from the group consisting of tolbutamide, ropamide, acetohexamide, tolazamide, clamide, glipizide or any combination thereof.

56. The use of claim 54, n said thiazolidinedione is rosiglitazone or pioglitazone, or ation thereof.

57. The use of claim 54, wherein said bile acid sequestrant is colesevelam.

58. The use of claim 52, wherein the medicament is for administration prior to, contemporaneously with or following said supplemental therapy.

59. The use of claim 51, wherein the glucose levels are blood glucose levels.

60. The use of claim 51, n the subject has nonalcoholic fatty liver disease (NAFLD).

61. The use of claim 51, wherein the subject has nonalcoholic steatohepatitis (NASH).

62. The use of claim 51, wherein the t has a fasting plasma glucose (FPG) level of greater than 100 mg/dl.

63. The use of claim 51, wherein the subject has an FPG level of 125 mg/dl or above.

64. The use of claim 51, wherein the subject has an FPG level of between about 100 and 125 mg/dl.

65. The use of claim 51, wherein the glucose levels are d by at least 5%.

66. The use of claim 51, wherein the subject has a hemoglobin A1c (HbA1c) level above

67. Use of the peptide of any one of claims 1 to 32 and 40 to 47 in the manufacture of a medicament for the treatment of a hyperglycemic condition in a subject.

68. The use of claim 67, wherein the hyperglycemic condition is diabetes.

69. The use of claim 67, wherein the hyperglycemic condition is insulin-dependent (type I) diabetes.

70. The use of claim 67, wherein the hyperglycemic condition is type II diabetes.

71. The use of claim 67, wherein the hyperglycemic condition is gestational diabetes.

72. The use of claim 67, wherein the hyperglycemic condition is prediabetes.

73. Use of the peptide of any one of claims 1 to 32 and 40 to 47 in the manufacture of a medicament for the treatment of insulin resistance in a subject.

74. Use of the e of any one of claims 1 to 32 and 40 to 47 in the manufacture of a medicament for the treatment of hyperinsulinemia in a subject.

75. Use of the peptide of any one of claims 1 to 32 and 40 to 47 in the manufacture of a medicament for the treatment of e intolerance in a subject.

76. Use of the peptide of any one of claims 1 to 32 and 40 to 47 in the manufacture of a medicament for the treatment of a metabolic me in a subject.

77. Use of the peptide of any one of claims 1 to 32 and 40 to 47 in the manufacture of a medicament for the treatment of obesity in a subject.

78. Use of the peptide of any one of claims 1 to 32 and 40 to 47 in the manufacture of a medicament for reducing glucagon in a subject.

79. Use of the peptide of any one of claims 1 to 32 and 40 to 47 in the manufacture of a ment for increasing glucose metabolism or homeostasis in a subject.

80. Use of the peptide of any one of claims 1 to 32 and 40 to 47 in the manufacture of a medicament for ing pancreatic function in a subject.

81. Use of the peptide of any one of claims 1 to 32 and 40 to 47 in the manufacture of a medicament for reducing triglycerides in a subject.

82. Use of the peptide of any one of claims 1 to 32 and 40 to 47 in the manufacture of a medicament for ng terol in a subject.

83. Use of the peptide of any one of claims 1 to 32 and 40 to 47 in the manufacture of a medicament for ng IDL, LDL or VLDL levels in a subject.

84. Use of the peptide of any one of claims 1 to 32 and 40 to 47 in the manufacture of a medicament for decreasing blood pressure in a subject.

85. Use of the peptide of any one of claims 1 to 32 and 40 to 47 in the manufacture of a medicament for decreasing intimal thickening of the blood vessel in a subject.

86. Use of the peptide of any one of claims 1 to 32 and 40 to 47 in the manufacture of a medicament for decreasing body mass or weight gain in a subject.

87. A e according to claim 1, substantially as herein described or exemplified.

88. A pharmaceutical composition according to claim 33, ntially as herein described or exemplified.

89. A pharmaceutical composition according to claim 34, substantially as herein described or ified.

90. A nucleic acid according to claim 35, substantially as herein described or exemplified.

91. A vector according to claim 37, substantially as herein described or exemplified.

92. An in vitro transformed or host cell according to claim 39, substantially as herein described or exemplified.

93. A peptide according to claim 40, substantially as herein described or exemplified.

94. A peptide according to claim 44, substantially as herein described or exemplified.

95. A pharmaceutical composition according to claim 48 substantially as herein bed or exemplified.

96. A pharmaceutical composition according to claim 49 ntially as herein described or exemplified.

97. A use according to claim 50 ntially as herein described or exemplified.

98. A use according to claim 51 substantially as herein described or exemplified.

99. A use ing to claim 67 substantially as herein described or exemplified.

100. A use according to claim 73 substantially as herein described or exemplified.

101. A use according to claim 74 substantially as herein described or exemplified.

102. A use according to claim 75 substantially as herein described or exemplified.

103. A use according to claim 76 substantially as herein described or exemplified.

104. A use according to claim 77 substantially as herein described or exemplified.

105. A use according to claim 78 substantially as herein described or exemplified.

106. A use according to claim 79 substantially as herein described or exemplified.

107. A use according to claim 80 substantially as herein bed or exemplified.

108. A use according to claim 81 substantially as herein described or exemplified.

109. A use according to claim 82 ntially as herein described or ified.

110. A use according to claim 83 substantially as herein described or ified.

111. A use ing to claim 84 substantially as herein described or exemplified.

112. A use according to claim 85 substantially as herein described or exemplified.

113. A use according to claim 86 substantially as herein described or exemplified.