U.S. patent application number 15/124877 was filed with the patent office on 2017-03-09 for methods of treating metabolic disorders associated with lipodystrophies and defects in insulin production or signaling.
This patent application is currently assigned to NOVARTIS AG. The applicant listed for this patent is John Louis DIENER, Jiaping GAO, Rick Jerome SCHIEBINGER. Invention is credited to John Louis DIENER, Jiaping GAO, Rick Jerome SCHIEBINGER.
Application Number | 20170065678 15/124877 |
Document ID | / |
Family ID | 52682963 |
Filed Date | 2017-03-09 |
United States Patent
Application |
20170065678 |
Kind Code |
A1 |
DIENER; John Louis ; et
al. |
March 9, 2017 |
METHODS OF TREATING METABOLIC DISORDERS ASSOCIATED WITH
LIPODYSTROPHIES AND DEFECTS IN INSULIN PRODUCTION OR SIGNALING
Abstract
The invention relates to the identification of new therapeutic
methods for the FGF21 polypeptide or protein, or mutants, variants,
and fusions thereof, for instance, in treating metabolic diseases
associated defects in insulin signaling (e.g. insulin receptor
mutation disorders (INSR disorders) and/or autoimmune insulin
receptor disorders (Type B insulin Resistance)), defects in insulin
production such as type 1 diabetes mellitus, mixed dyslipidemia,
nonalcoholic fatty liver disease (NAFLD), and other metabolic
disorders, and various lipodystrophies such as HIV-HAART induced
partial-lipodystrophy, and in reducing the mortality and morbidity
of critically ill patients.
Inventors: |
DIENER; John Louis;
(Cambridge, MA) ; GAO; Jiaping; (Ashland, MA)
; SCHIEBINGER; Rick Jerome; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DIENER; John Louis
GAO; Jiaping
SCHIEBINGER; Rick Jerome |
Cambridge
Ashland
Cambridge |
MA
MA
MA |
US
US
US |
|
|
Assignee: |
NOVARTIS AG
Basel
CH
|
Family ID: |
52682963 |
Appl. No.: |
15/124877 |
Filed: |
March 9, 2015 |
PCT Filed: |
March 9, 2015 |
PCT NO: |
PCT/US2015/019362 |
371 Date: |
September 9, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61950960 |
Mar 11, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 14/50 20130101; C07K 2319/30 20130101; A61K 38/1825 20130101;
A61P 1/06 20180101; A61P 3/06 20180101; A61P 3/00 20180101; A61P
3/10 20180101; A61P 3/08 20180101; A61P 1/16 20180101 |
International
Class: |
A61K 38/18 20060101
A61K038/18; C07K 14/50 20060101 C07K014/50 |
Claims
1. A method of treating an insulin receptor disorder (INSR
disorder) comprising administering to a subject in need thereof a
therapeutically effective amount of (a) an isolated human FGF21
polypeptide; or (b) an FGF21 protein variant.
2. The method of claim 1, wherein the INSR disorder is Type 1
diabetes mellitus.
3. The method of claim 1, wherein the INSR disorder is
dyslipidemia.
4. The method of claim 1, wherein the INSR disorder is
hyperglycemia.
5. The method of claim 1, wherein the INSR disorder is
hypoglycemia.
6. The method of claim 1, wherein the INSR disorder is glucose
intolerance.
7. The method of claim 1, wherein the INSR disorder is HIV-HAART
Induced Partial Lipodystrophy.
8. The method of claim 1, wherein the INSR disorder is metabolic
syndrome.
9. The method of claim 1, wherein the INSR disorder is nonalcoholic
fatty liver disease (NAFLD).
10. The method of claim 1, wherein the FGF21 protein variant is
Variant 76.
11. The method of claim 1, wherein the FGF21 protein variant is an
Fc fusion protein.
12. The method of claim 11, wherein the Fc fusion protein is
selected from Table 1.
13. The method of claim 11, wherein the Fc fusion protein is
Variant 101.
14. A method of treating an insulin receptor disorder (INSR
disorder) comprising administering to a subject in need thereof a
therapeutically effective amount of an FGF21 protein variant,
wherein the FGF21 protein variant is administered in the form of a
pharmaceutical composition comprising a therapeutically effective
amount of an FGF21 protein variant in admixture with a
pharmaceutically or physiologically acceptable formulation agent
selected for suitability with the mode of administration.
15. A method of treating an insulin receptor disorder (INSR
disorder) comprising administering to a subject in need thereof a
therapeutically effective amount of an FGF21 protein variant
covalently-modified with one or more PEG subunits.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods of treating
metabolic diseases associated with insulin resistance, specifically
insulin receptor disorders (INSR disorders, or IR disorders)
including those resulting from mutations in the insulin receptor
(e.g., Type A insulin resistance, Rabson Mendenhall and Donohue
Syndromes) and autoimmune insulin resistance (aka Type B insulin
resistance); insulin resistance and associated mixed dyslipidemias
caused by familial partial lipodystrophy, acquired partial
lipodystrophy, and HIV/HAART-induced partial lipodystrophy
disorders, and in reducing the mortality and morbidity of these
patients. The present invention also relates to methods of treating
patients with defects in insulin production, such as those
suffering from type 1 diabetes mellitus.
BACKGROUND OF THE INVENTION
[0002] The fibroblast growth factor (FGF) family is characterized
by 22 genetically distinct, homologous ligands, which are grouped
into seven subfamilies. According to the published literature, the
FGF family now consists of at least twenty-three members, FGF-1 to
FGF-23 (Reuss et al. (2003) Cell Tissue Res. 313:139-157).
[0003] FGF-21 was isolated from mouse embryos and is closest to
FGF-19 and FGF-23. This FGF subfamily regulates diverse
physiological processes uncommon to classical FGFs, namely energy
and bile acid homeostasis, glucose and lipid metabolism and
phosphate as well as vitamin D homeostasis. Moreover, unlike
classical FGFs, this subfamily acts in an endocrine fashion.
(Moore, D. D. (2007) Science 316, 1436-8). Fibroblast growth factor
21 (FGF21) has been reported to be preferentially expressed in the
liver (Nishimura et al., Biochimica et Biophysica Acta,
1492:203-206, (2000); patent publication WO01/36640; and patent
publication WO01/18172) and described as a treatment for ischemic
vascular disease, wound healing, and diseases associated with loss
of pulmonary, bronchia or alveolar cell function and numerous other
disorders.
[0004] FGF21 has been identified as a potent metabolic regulator.
Systemic administration of FGF21 to rodents and rhesus monkeys with
diet-induced or genetic obesity and diabetes exerts strong
anti-hyperglycemic and triglyceride-lowering effects, and reduction
of body weight. (Coskun, T, et al. (2008) Endocrinology
149:6018-6027; Kharitonenkov, A, et al. (2005) Journal of Clinical
Investigation 115:1627-1635; Kharitonenkov, A, et al. (2007)
Endocrinology 148:774-781; Xu, J, et al. (2009) Diabetes
58:250-259). FGF21 is a 209 amino acid polypeptide containing a 28
amino acid leader sequence. Human FGF21 has about 79% amino acid
identity to mouse FGF21 and about 80% amino acid identity to rat
FGF21.
[0005] Although FGF-21 activates FGF receptors and downstream
signaling molecules, including FRS2a and ERK, direct interaction of
FGFRs alone and FGF-21 has not been detected. Furthermore, many
cell types do not respond to FGF-21, even though they express
multiple FGFR isoforms. All of these data suggest that a cofactor
must mediate FGF-21 signaling through FGFRs. Recent studies have
identified .beta.-klotho, which is highly expressed in liver,
adipocytes and in pancreas, as a determinant of the cellular
response to FGF-21 (Kurosu, H. et al. (2007) J Biol Chem 282,
26687-95). .beta.-klotho preferentially binds to FGFR1c, FGFr2c,
FGFR3c, and FGFR4. The .beta.-klotho-FGFR complex, but not FGFR
alone, binds to FGF-21 in vitro (Kharitonenkov, A. et al. (2008) J
Cell Physiol 215, 1-7). A similar mechanism has been identified in
the FGF-23-klotho-FGFR system (Urakawa, I. et al. (2006) Nature
444, 770-4).
[0006] The bioactivity of FGF-21 was first identified in a mouse
3T3-L1 adipocyte glucose uptake assay (Kharitonenkov, A. et al.
(2005) J Clin Invest 115, 1627-35). Subsequently, FGF-21 was shown
to induce insulin-independent glucose uptake and GLUT1 expression.
FGF-21 has also been shown to ameliorate hyperglycemia in a range
of diabetic rodent models. In addition, transgenic mice
over-expressing FGF-21 were found to be resistant to diet-induced
metabolic abnormalities, including decreased body weight and fat
mass, and enhancements in insulin sensitivity (Badman, M. K. et al.
(2007) Cell Metab 5, 426-37). Administration of FGF-21 to diabetic
non-human primates caused a decline in fasting plasma glucose,
triglycerides, insulin and glucagon levels, and led to significant
improvements in lipoprotein profiles including a nearly 80%
increase in HDL cholesterol (Kharitonenkov, A. et al. (2007)
Endocrinology 148, 774-81). Importantly, hypoglycemia was not
observed at any point during this NHP study. Moreover, recent
studies identified FGF-21 as an important endocrine hormone that
helps to control adaptation to the fasting state. This provides a
previously missing link, downstream of PPAR.alpha., by which the
liver communicates with the rest of the body in regulating the
biology of energy homeostasis.
[0007] The FGF21 polypeptide and protein, as well as variants and
mutations thereof, have been evaluated for their metabolic effects
on obesity and type 2 diabetes (T2D). The effect of FGF21 on
insulin receptor mutation disorders (INSR disorders) and various
metabolic conditions associated with lipodystrophy, however, has
not been demonstrated. In this application, the effect of FGF21 on
T1D, insulin receptor mutation or autoimmune disorders (INSR
disorders) and HIV/HAART (Highly Active Anti-Retroviral Therapy
(used to treat patients with HIV infection)) induced partial
lipodystrophy is described, and the methods of the present
invention are useful for the treatment of said metabolic diseases
in addition to the known diseases associated with T2DM and insulin
resistance.
SUMMARY OF THE INVENTION
[0008] The invention relates to the identification of new
therapeutic functions for fibroblast growth factor 21 (FGF21)
proteins and polypeptides, including variants and mutations
thereof, and pharmaceutical compositions comprising the same, i.e.,
as agents to treat metabolic diseases associated with insulin
resistance, including metabolic conditions associated with insulin
receptor (INSR) mutations.
[0009] In some embodiments, the methods of the invention comprise
the wild type FGF21 protein, e.g., having NCBI reference sequence
number NP_061986.1, and encoded by the polynucleotide sequence
which has NCBI reference sequence number NM_019113.2, and found in
such issued patents as, e.g., U.S. Pat. No. 6,716,626B1, assigned
to Chiron Corporation.
[0010] In some embodiments, the methods of the invention comprise
variants of the FGF21 protein sequence, e.g., biologically active
FGF21 variants, and can include truncated versions of the FGF21
protein (in which residues from the C- and/or N-terminal regions
have been eliminated, thereby shortening/truncating the protein),
as well as variants with one or more point substitutions and/or
site-specific incorporation of amino acids at positions of interest
(e.g., with conservative amino acid residues, with non-conservative
residues, or with non-natural amino acid residues such as
pyrrolysine).
[0011] Representative examples of said variants are described,
e.g., in PCT Publication WO2012/066075 (filed 24 May 2012 by
Novartis A G). A preferred embodiment is described as "Variant 76,"
"FGF21 V76," "V76," or the like in PCT publication WO2012/066075
and herein, which is a genetically engineered FGF21 variant with
177 amino acid residues, including 9 point mutations. Another
preferred embodiment is described as "Variant 101," "FGF21 (v101),"
"V101," or the like in PCT publication WO2013/049247 and herein,
which is an Fc fusion protein linked by a two amino acid liker to a
genetically engineered FGF21 variant with 9 point mutations.
Preferred embodiments can be found in the following table:
TABLE-US-00001 TABLE 1 FGF21 Variant Fc fusion proteins SEQ ID NO:
Sequence Name* 1 DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT Full
Length N-term CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY Fc-Fusion
with 2 AA RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK Linker (GS)
and WT GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE FGF21 WESNGQPENN
YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKGSD
SSPLLQFGGQ VRQRYLYTDD AQQTEAHLEI REDGTVGGAA DQSPESLLQL KALKPGVIQI
LGVKTSRFLC QRPDGALYGS LHFDPEACSF RELLLEDGYN VYQSEAHGLP LHLPGNKSPH
RDPAPRGPAR FLPLPGLPPA LPEPPGILAP QPPDVGSSDP LSMVGPSQGR SPSYAS 2
DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT Full Length N-term
CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY Fc-Fusion with 15 AA
RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK Linker (GGGGS .times.
3) GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE between Fc and WT
WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG FGF21 NVFSCSVMHE
ALHNHYTQKS LSLSPGKGGG GSGGGGSGGG GSDSSPLLQF GGQVRQRYLY TDDAQQTEAH
LEIREDGTVG GAADQSPESL LQLKALKPGV IQILGVKTSR FLCQRPDGAL YGSLHFDPEA
CSFRELLLED GYNVYQSEAH GLPLHLPGNK SPHRDPAPRG PARFLPLPGL PPALPEPPGI
LAPQPPDVGS SDPLSMVGPS QGRSPSYAS 3 DSSPLLQFGG QVRQRYLYTD DAQETEAHLE
IREDGTVGGA Variant #76 = Protein AHQSPESLLE LKALKPGVIQ ILGVKTSRFL
CQKPDGALYG with 9 total SLHFDPEACS FRELLLEDGY NVYQSEAHGL PLHLPGNRSP
mutations relative to HCDPAPQGPA RFLPLPGLPP ALPEPPGILA PQPPDVGSSD
wild-type FGF21 (as PLAMVGPSQG RSPSYAS in WO01/018172) 4 DKTHTCPPCP
APEAAGGPSV FLFPPKPKDT LMISRTPEVT Variant #101 = N-term CVVVDVSHED
PEVKFNWYVD GVEVHNAKTK PREEQYNSTY Fc Fusion with the 2 AA RVVSVLTVLH
QDWLNGKEYK CKVSNKALPA PIEKTISKAK linker (GS) between Fc GQPREPQVYT
LPPSREEMTK NQVSLTCLVK GFYPSDIAVE and FGF21 = (Q55C, WESNGQPENN
YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG A109T, G148C, K150R, NVFSCSVMHE
ALHNHYTQKS LSLSPGKGSD SSPLLQFGGQ P158S, S195A, P199G, VRQRYLYTDD
ACQTEAHLEI REDGTVGGAA DQSPESLLQL G202A) KALKPGVIQI LGVKTSRFLC
QRPDGTLYGS LHFDPEACSF RELLLEDGYN VYQSEAHGLP LHLPCNRSPH RDPASRGPAR
FLPLPGLPPA LPEPPGILAP QPPDVGSSDP LAMVGGSQAR SPSYAS 5 DKTHTCPPCP
APEAAGGPSV FLFPPKPKDT LMISRTPEVT Variant #103 = N-term CVVVDVSHED
PEVKFNWYVD GVEVHNAKTK PREEQYNSTY Fc Fusion with the 2 AA RVVSVLTVLH
QDWLNGKEYK CKVSNKALPA PIEKTISKAK linker (GS) between Fc GQPREPQVYT
LPPSREEMTK NQVSLTCLVK GFYPSDIAVE and FGF21 = (Q55C, WESNGQPENN
YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG A109T, G148C, K150R, NVFSCSVMHE
ALHNHYTQKS LSLSPGKGSD SSPLLQFGGQ P158S, S195A, P199G, VRQRYLYTDD
ACQTEAHLEI REDGTVGGAA DQSPESLLQL G202A) KALKPGVIQI LGVKTSRFLC
QKPDGALYGS LHFDPEACSF RELLLEDGYN VYQSEAHGLP LHLPCNRSPH RDPASRGPAR
FLPLPGLPPA LPEPPGILAP QPPDVGSSDP LAMVGGSQAR SPSYAS 6 DKTHTCPPCP
APEAAGGPSV FLFPPKPKDT LMISRTPEVT Variant #104 = N-term CVVVDVSHED
PEVKFNWYVD GVEVHNAKTK PREEQYNSTY Fc Fusion with the 2 AA RVVSVLTVLH
QDWLNGKEYK CKVSNKALPA PIEKTISKAK linker (GS) = (Q55C, GQPREPQVYT
LPPSREEMTK NQVSLTCLVK GFYPSDIAVE D74H, Q82E, R105K, WESNGQPENN
YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG G148C, K150R, R159Q, NVFSCSVMHE
ALHNHYTQKS LSLSPGKGSD SSPLLQFGGQ P174L, S195A) VRQRYLYTDD
ACQTEAHLEI REDGTVGGAA HQSPESLLEL KALKPGVIQI LGVKTSRFLC QKPDGALYGS
LHFDPEACSF RELLLEDGYN VYQSEAHGLP LHLPCNRSPH RDPAPQGPAR FLPLPGLPPA
LPEPPGILAP QPPDVGSSDP LAMVGPSQGR SPSYAS 7 DKTHTCPPCP APEAAGGPSV
FLFPPKPKDT LMISRTPEVT Variant #183 = V104 CVVVDVSHED PEVKFNWYVD
GVEVHNAKTK PREEQYNSTY with 15 AA Linker RVVSVLTVLH QDWLNGKEYK
CKVSNKALPA PIEKTISKAK (GGGGS .times. 3) between Fc GQPREPQVYT
LPPSREEMTK NQVSLTCLVK GFYPSDIAVE and FGF21 = (Q55C, WESNGQPENN
YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG D74H, Q82E, R105K, NVFSCSVMHE
ALHNHYTQKS LSLSPGKGGG GSGGGGSGGG G148C, K150R, R159Q, GSDSSPLLQF
GGQVRQRYLY TDDACQTEAH LEIREDGTVG P174L, S195A) GAAHQSPESL
LELKALKPGV IQILGVKTSR FLCQKPDGAL YGSLHFDPEA CSFRELLLED GYNVYQSEAH
GLPLHLPCNR SPHRDPAPQG PARFLPLPGL PPALPEPPGI LAPQPPDVGS SDPLAMVGPS
QGRSPSYAS 8 DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT Variant
#188 = V103 CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY with 15 AA
Linker RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK (GGGGS .times.
3) between Fc GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE and FGF21
= (Q55C, WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG R105K, G148C,
K150R, NVFSCSVMHE ALHNHYTQKS LSLSPGKGGG GSGGGGSGGG P158S, S195A,
P199G, GSDSSPLLQF GGQVRQRYLY TDDACQTEAH LEIREDGTVG G202A)
GAADQSPESL LQLKALKPGV IQILGVKTSR FLCQKPDGAL YGSLHFDPEA CSFRELLLED
GYNVYQSEAH GLPLHLPCNR SPHRDPASRG PARFLPLPGL PPALPEPPGI LAPQPPDVGS
SDPLAMVGGS QARSPSYAS 9 DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT
Variant #204 = V101 CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
with 15 AA Linker RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK
(GGGGS .times. 3) between Fc GQPREPQVYT LPPSREEMTK NQVSLTCLVK
GFYPSDIAVE and FGF21 = (Q55C, WESNGQPENN YKTTPPVLDS DGSFFLYSKL
TVDKSRWQQG A109T, G148C, K150R, NVFSCSVMHE ALHNHYTQKS LSLSPGKGGG
GSGGGGSGGG P158S, S195A, P199G, GSDSSPLLQF GGQVRQRYLY TDDACQTEAH
LEIREDGTVG G202A) GAADQSPESL LQLKALKPGV IQILGVKTSR FLCQRPDGTL
YGSLHFDPEA CSFRELLLED GYNVYQSEAH GLPLHLPCNR SPHRDPASRG PARFLPLPGL
PPALPEPPGI LAPQPPDVGS SDPLAMVGGS QARSPSYAS *Note that the FGF21
wild-type sequence in this table refers to NCBI reference sequence
number NP_061986.1 unless otherwise specified.
[0012] Said modifications of FGF21 used in the methods of the
present invention are designed to enhance the biological properties
of the variants relative to the wild-type FGF21 protein, as well
as, in some cases, serving as points of attachment for, e.g.,
labels and protein half-life extension agents, and for purposes of
affixing said variants to the surface of a solid support.
[0013] In various embodiments, said polypeptide and protein
variants disclosed herein can comprise (a) an amino-terminal
truncation of no more than 8 amino acid residues, wherein the
polypeptide is capable of lowering blood glucose in a mammal; (b) a
carboxyl-terminal truncation of no more than 12 amino acid
residues, wherein the polypeptide is capable of lowering blood
glucose in a mammal; or (c) an amino-terminal truncation of no more
than 8 amino acid residues and a carboxyl-terminal truncation of no
more than 12 amino acid residues, wherein the polypeptide is
capable of lowering blood glucose in a mammal.
[0014] In still other embodiments, the methods of the invention
comprise variants of the FGF21 protein sequence, such as those
described, e.g., in the following:
[0015] US issued U.S. Pat. No. 7,491,697; U.S. Pat. No. 8,541,369;
U.S. Pat. No. 8,012,931; U.S. Pat. No. 8,383,365;
[0016] US and PCT published patent applications US2011/0195895;
US2012/0052069; US2010/0216715; US2009/0118190; US2012/0264683;
WO10/084169; WO10/142665; US2008/0176790; WO11/089203; WO11/020319;
WO12/062078; US2010/0285131; WO10/042747; and US2011/0135657;
[0017] as well as any related applications, issued patents, and
family members of the above, both in the US and in the rest of the
world.
[0018] In still other embodiments, the methods of the invention
comprise variants of the FGF21 protein sequence in which disulfide
bonds have been engineered, e.g., by the addition of cysteine
residues. Said variants can be found, e.g., in PCT Publication
WO12/066075, as well as in any of the variants with engineered
disulfide bonds listed in the positions above.
[0019] In some embodiments, the methods of the invention comprise
PEGylated or otherwise half-life extended FGF21 polypeptides or
proteins, e.g., wild type FGF21, or mutants or variants
thereof.
[0020] In some embodiments, the methods of the invention comprise
polypeptide and protein variants covalently linked to one or more
polymers, such as polyethylene glycol (PEG) or polysialic acid,
whether at the position of site-specific amino acid modifications
made relative to the wild-type FGF21, or at the position of amino
acids commonly shared with the wild-type FGF21.
[0021] In some embodiments, the methods of the invention comprise
FGF21 fusion proteins, such as Fc fusions. Said fusions can
comprise wild type FGF21 or mutants or variants thereof. In some
embodiments, the methods of the present invention comprise
polypeptides which can be fused to a heterologous amino acid
sequence, optionally via a linker, such as GS or GGGGSGGGGSGGGGS
(SEQ ID NO:10). The heterologous amino acid sequence can be an IgG
constant domain or fragment thereof (e.g., the Fc region), Human
Serum Albumin (HSA), or albumin-binding polypeptides. Such methods
can comprise multimers of said fusion polypeptides. In some
embodiments, the methods of the present invention comprise fusion
proteins in which the heterologous amino acid sequence (e.g., HSA,
Fc, etc.) is fused to the amino-terminal of the FGF21 protein or
mutants or variants as described; in other embodiments, the fusion
occurs at the carboxyl-terminal of the FGF21 protein or mutants or
variants.
[0022] The invention also provides methods of treatment which
comprise pharmaceutical compositions comprising the polypeptide and
protein variants disclosed herein and a pharmaceutically acceptable
formulation agent. Such pharmaceutical compositions can be used in
a method for treating a metabolic disorder, and the method
comprises administering to a human patient in need thereof a
pharmaceutical composition of the invention. Non-limiting examples
of metabolic disorders that can be treated include type 1 diabetes
mellitus and INSR mutation disorders.
[0023] These and other aspects of the invention will be elucidated
in the following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIGS. 1A and 1B are graphical representations showing a
change in glucose levels when STZ mice are administered wild type
FGF21 (FIG. 1A) and Variant 76 (Cmpd-A) (FIG. 1B). [STZ is the beta
cell toxin streptozotocin.] Wild type FGF21 (3 mg/kg, daily dose)
decreased glucose in STZ mice from day 5 (D5). Variant 76 (referred
to as Cmpd-A)(1 or 3 mg/kg, 2 doses/week) decreased glucose in STZ
mice from day 2 (D2) with 3 mg/kg dose and day 11 (D11) with 1
mg/kg dose.
[0025] FIG. 2 is a graphical representation showing a change in
HbA1c levels. Variant 76 (referred to as Cmpd-A) was administered
at both 1 and 3 mg/kg doses, showing reduced HbA1c levels.
[0026] FIGS. 3A and 3B are graphical representations showing a
change in respiratory exchange ratio (RER) levels when STZ mice are
administered wild type FGF21 (FIG. 3A) and Variant 76 (FIG. 3B).
Wild type FGF21 (3 mg/kg, daily dose) increased RER in STZ mice,
and Variant 76 (referred to as Cmpd-A)(1 or 3 mg/kg, 2 doses/week)
increased glucose utilization in STZ mice.
[0027] FIGS. 4A and 4B are graphical representations showing a
change in energy expenditure levels when STZ mice are administered
wild type FGF21 (FIG. 4A) and Variant 76 (FIG. 4B). Wild type FGF21
(3 mg/kg, daily dose) increased energy expenditure in STZ mice, and
Variant 76 (referred to as Cmpd-A)(1 or 3 mg/kg, 2 doses/week)
increased energy expenditure in STZ mice.
[0028] FIGS. 5A and 5B are graphical representations showing a
change in food intake when STZ mice are administered wild type
FGF21 (FIG. 5A) and Variant 76 (Cmpd-A) (FIG. 5B). Both wild type
FGF21 (3 mg/kg, daily dose) and Variant 76 (1 or 3 mg/kg, 2
doses/week) decreased cumulative food intake in STZ mice.
[0029] FIGS. 6A and 6B are graphical representations showing a
change in ketogenesis when STZ mice are administered wild type
FGF21 (FIG. 6A) and Variant 76 (referred to as Cmpd-A)(FIG. 6B).
Both wild type FGF21 (3 mg/kg, daily dose) and Variant 76 (1 or 3
mg/kg, 2 doses/week) decreased ketogenesis in STZ mice.
[0030] FIGS. 7A and 7B are graphical representations showing a
change in epididymal fat weight when STZ mice are administered wild
type FGF21 (FIG. 7A) and Variant 76 (referred to as Cmpd-A)(FIG.
7B). Wild type FGF21 (3 mg/kg, daily dose) reduced loss of fat
tissue in STZ mice, and Variant 76 (1 or 3 mg/kg, 2 doses/week)
reduced loss of fat tissue ketogenesis in STZ mice.
[0031] FIGS. 8A and 8B show that blockade of Insulin Receptor
(INSR) via administration of inhibitory monoclonal antibody against
the insulin receptor completely blocks insulin dependent glucose
uptake by differentiated human adipocytes in the presence of excess
insulin (100 nM) (FIG. 8A); however 100 nM FGF21 is still able to
promote glucose uptake in the presence of up to 10 nM anti-INSR Ab
(FIG. 8b), demonstrating that an FGF21 therapy may be beneficial in
Type-B insulin resistance (autoimmune insulin resistance), and by
extension may also promote insulin independent glucose uptake in
the presence of a generally defective (i.e., mutated) insulin
receptor.
[0032] FIGS. 9A-C demonstrates the effect of HIV protease inhibitor
Ritonavir treatment on body weight gain, body fat gain, and plasma
triglycerides, respectively, in mice.
[0033] FIGS. 10A-E demonstrates the effect of Ritonavir with or
without FGF21 v76 according to the following readouts: body weight
(FIG. 10A); white adipose tissue (WAT) weight (FIG. 10B); plasma
glucose (during an oral glucose tolerance test (OGTT))(FIG. 10C);
Homeostatic Model Assessment of Insulin Resistance (HOMA-IR)(FIG.
10D); and liver lipid content (FIG. 10E), in all cases in mice
already treated for 51 days with Ritonavir to induce disease state,
and then treated for 23 days with both FGF21 and Ritonavir to treat
the same, compared to chow fed PBS treated control mice.
[0034] FIGS. 11A and B profile the effect of 0.6% t10, c12
conjugated linoleic acid (t10, c12 CLA) in the diet of mice on fat
pad weight (which is thought to induce lipodystrophy, as explained
in greater detail below) vs chow diet as a function of time. FIG.
11B shows said effect at the end of the study.
[0035] FIGS. 12A-D show that FGF21 (v101), administered in either
"prevention" or "treatment" modes (terms described herein), and
leptin administered in treatment mode, all reverse the increase in
liver weight (FIG. 12A), increase in liver fat content (FIG. 12B),
hyperglycemia (FIG. 12C), and hyperinsulinemia (FIG. 12D),
respectively, as induced by a 0.6% t10, c12 conjugated linoleic
acid (t10, c12 CLA) in the diet of mice. FIG. 12E shows that under
the same conditions, FGF21 (v101), administered in either
prevention or treatment mode, but not leptin in treatment mode, can
reverse said induced glucose intolerance, as measured using an
OGTT.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The methods of the present invention are based on the
discovery of an improved therapeutic treatment for subjects
afflicted with Type 1 diabetes and metabolic diseases associated
with insulin resistance, e.g., disorders associated with insulin
receptor (IR) mutations or lipodystrophies.
[0037] Multiple inactivating mutations of the INSR have been
described with varying phenotypes. Patients typically present with
severe resistance to the action of insulin which advances to
hyperglycemia at the time of puberty. The current standard of care
is treatment with very high doses of insulin when subjects become
hyperglycemic, which typically is inadequate in controlling
hyperglycemia. Treatments to date have been met with limited
success.
[0038] FGF21 polypeptides and proteins, as well as variants,
fusions, and mutations thereof, have long been recognized for their
metabolic effects on obesity and type 2 diabetes (T2D). The effect
of FGF21 on type 1 diabetes (T1D), insulin receptor mutation
disorders, and lipodystrophies, however, has not been demonstrated
until the present methods of the invention. In this application,
the effect of FGF21 on T1D and insulin receptor mutation disorders
and lipodystrophies is described, and the claimed methods of the
present invention can be employed for the treatment of said
metabolic diseases associated with insulin resistance, e.g., Type 1
DM, insulin receptor disorders (INSR disorders) including Type B
insulin resistance and HIV/HAART-induced partial lipodystrophy.
[0039] As described further herein, high dose streptozotocin
(STZ)-treated mice are commonly used as a rodent model of type 1
diabetes (T1D), which exhibit hyperglycemia, insulinopenia,
excessive lipid utilization and ketogenesis, as well as
hyperphagia. [STZ is a beta cell toxin, administered to destroy
beta cells, e.g., in the creation of mouse models with T1D-like
symptoms.] Treatment with either daily dose of wild type FGF21 or
two doses a week of half-life-extended FGF21 was efficacious on
improving overall disease condition in STZ mice, leading to a
reduction in hyperglycemia, excessive food consumption and
accumulation of ketone bodies, and an increase in utilization of
carbohydrate and energy expenditure.
[0040] Treatment with Variant 76 did not restore insulin secretion
in the STZ treated animals. Despite the early observation that
FGF21 could induce glucose uptake in adipocytes in the absence of
insulin, we are the first to propose and these STZ data are the
first to establish this effect in vivo and to demonstrate that
FGF21 treatment can work in the setting of T1D. Therefore, the
administration of wild type FGF21 polypeptide and protein, or of
variants, fusions, or mutants, thereof, can provide therapeutic
value in treating T1D.
[0041] Because of the demonstrated effect of FGF21 in the STZ model
in the absence of any increase in insulin secretion we propose that
an FGF21 analog could be used to treat diseases of impaired insulin
signaling caused by dysfunction of INSR itself (either by mutation
in INSR or by neutralizing autoantibodies to INSR). However, since,
INSR KO mice are not viable and die postnatally, an equivalent
animal model to the human condition does not currently exist.
Furthermore tissue specific KO of insulin receptor in mice fails to
fully recapitulate the various disease states seen in humans with
either mutation in INSR or autoantibodies to INSR. Therefore we
used an anti-human INSR antibody to demonstrate that under
conditions where glucose uptake by insulin was completely blocked
by the Ab (10 nM Ab), FGF21 could still promote significant glucose
uptake in these cells. These data support the novel use of an FGF21
therapy in patients with defects in INSR caused either by mutation
or by anti-insulin receptor antibodies.
[0042] It has been long established that an FGF21 analog can be
used to treat hyperglycemia, insulin resistance, obesity, and
dyslipidemia in diabetic leptin deficient ob/ob mice
(Kharitonenkov, A, et al. (2005) Journal of Clinical Investigation
115). A common property of these mice is significant accumulation
of liver fat. Liver fat in these animals is rapidly and dose
dependently reduced with FGF21 treatment. Significant increase in
liver fat is also associated with the types of insulin resistance
and associated mixed dyslipidemias caused by congenital generalized
or familial partial lipodystrophy, acquired generalized or acquired
partial lipodystrophy, HIV/HAART-induced partial lipodystrophy
and/or lipohypertrophy, and other lipid metabolism dysfunctions
caused by HAART. Here we demonstrate the novel method of treatment
of two chemically induced lipodystrophies (c10-t12 linoleic acid
and HIV protease inhibitor Ritanovir) using an FGF21 analog. In
these examples the exact mechanism of induction of lipodystrophy is
different and therefore these data should extend to the treatment
of any lipodystrophy associated with loss of peripheral fat and
accumulation of liver fat that leads to insulin resistance and
dyslipidemia using an FGF21 analog.
[0043] The methods of the present invention comprise wild type
FGF21 polypeptide and proteins, fusions, and variants and mutants
thereof. The FGF21 wild-type sequence has NCBI reference sequence
number NP_061986.1, and is encoded by the polynucleotide sequence
which has NCBI reference sequence number NM_019113.2, and can be
found in such issued patents as, e.g., U.S. Pat. No. 6,716,626B1,
assigned to Chiron Corporation.
[0044] The mature FGF21 sequence lacks a leader sequence and may
also include other modifications of a polypeptide such as
proteolytic processing of the amino terminus (with or without a
leader sequence) and/or the carboxyl terminus, cleavage of a
smaller polypeptide from a larger precursor, N-linked and/or
O-linked glycosylation, and other post-translational modifications
understood by those with skill in the art.
[0045] One skilled in the art of expression of proteins will
recognize that methionine or methionine-arginine sequence can be
introduced at the N-terminus of any of the FGF21 protein variants,
for expression in E. coli, and are contemplated within the context
of the methods of this invention.
[0046] The terms "FGF21 protein variant," "human FGF21 variant,"
"FGF21 polypeptide or protein variant," "variant," "FGF21 mutant,"
or any like terms, are defined as comprising human FGF21 in which a
naturally occurring (i.e., wild-type) FGF21 amino acid sequence has
been modified, e.g., in which at least one amino acid of the
wild-type protein has been substituted by another amino acid,
and/or removed. Additionally, the variants may include N- and/or
C-terminal truncations relative to the wild-type FGF21 protein.
Generally speaking, a variant possesses some modified property,
structural or functional, of the wild-type protein. For example,
the variant may have enhanced or improved physical stability in
concentrated solutions (e.g., less hydrophobic mediated
aggregation), enhanced or improved plasma stability when incubated
with blood plasma or enhanced or improved bioactivity while
maintaining a favorable bioactivity profile.
[0047] Acceptable amino acid substitutions and modifications which
constitute differences between the FGF21 polypeptide and protein
variants and mutants of the methods of the invention and wild-type
FGF21 include, but are not limited to, one or more amino acid
substitutions, including substitutions with non-naturally occurring
amino acid analogs, and truncations. Thus, FGF21 protein variants
include, but are not limited to, site-directed FGF21 mutants,
truncated FGF21 polypeptides, proteolysis-resistant FGF21 mutants,
aggregation-reducing FGF21 mutants, FGF21 combination mutants, and
FGF21 fusion proteins, as described herein.
[0048] The variant may possess increased compatibility with
pharmaceutical preservatives (e.g., m-cresol, phenol, benzyl
alcohol), thus enabling the preparation of a preserved
pharmaceutical formulation that maintains the physiochemical
properties and biological activity of the protein during storage.
Accordingly, variants with enhanced pharmaceutical stability
relative to wild-type FGF21, have improved physical stability in
concentrated solutions under both physiological and preserved
pharmaceutical formulation conditions, while maintaining biological
potency. By way of non-limiting example, the variants of the
invention may be more resistant to proteolysis and enzymatic
degradation; may have improved stability; and may be less likely to
aggregate, than their wild-type counterparts. As used herein, these
terms are not mutually exclusive or limiting, it being entirely
possible that a given variant has one or more modified properties
of the wild-type protein.
DEFINITIONS
[0049] Various definitions are used throughout this document. Most
words have the meaning that would be attributed to those words by
one skilled in the art. Words specifically defined either below or
elsewhere in this document have the meaning provided in the context
of the present invention as a whole and as are typically understood
by those skilled in the art.
[0050] As used herein, the term "FGF21" refers to a member of the
fibroblast growth factor (FGF) protein family and has the GenBank
Accession No. NP_061986.1 (the corresponding polynucleotide
sequence of which has NCBI reference sequence number NM_019113.2),
and can be found in such issued patents as, e.g., U.S. Pat. No.
6,716,626B1, assigned to Chiron Corporation.
[0051] As used herein, the term "FGF21 receptor" refers to a
receptor for FGF21 (Kharitonenkov, A, et al. (2008) Journal of
Cellular Physiology 215:1-7; Kurosu, H, et al. (2007) JBC
282:26687-26695; Ogawa, Y, et al. (2007) PNAS 104:7432-7437).
[0052] The term "FGF21 polypeptide" refers to a naturally-occurring
polypeptide expressed in humans. For purposes of this disclosure,
the term "FGF21 polypeptide" can be used interchangeably to refer
to any full-length FGF21 polypeptide, which consists of 209 amino
acid residues; any mature form of the polypeptide, which consists
of 181 amino acid residues, and in which the 28 amino acid residues
at the amino-terminal end of the full-length FGF21 polypeptide
(i.e., which constitute the signal peptide) have been removed; and
variants thereof.
[0053] The term "isolated nucleic acid molecule" refers to a
nucleic acid molecule of the present invention that (1) has been
separated from at least about 50 percent of proteins, lipids,
carbohydrates, or other materials with which it is naturally found
when total nucleic acid is isolated from the source cells, (2) is
not linked to all or a portion of a polynucleotide to which the
"isolated nucleic acid molecule" is linked in nature, (3) is
operably linked to a polynucleotide which it is not linked to in
nature, or (4) does not occur in nature as part of a larger
polynucleotide sequence. Preferably, the isolated nucleic acid
molecule of the present invention is substantially free from any
other contaminating nucleic acid molecules or other contaminants
that are found in its natural environment that would interfere with
its use in polypeptide production or its therapeutic, diagnostic,
prophylactic or research use.
[0054] The term "isolated polypeptide" refers to a polypeptide
(e.g., a FGF21 polypeptide or variant FGF21 polypeptide provided
herein) that has been separated from at least about 50 percent of
polypeptides, peptides, lipids, carbohydrates, polynucleotides, or
other materials with which the polypeptide is naturally found when
isolated from a source cell. Preferably, the isolated polypeptide
is substantially free from any other contaminating polypeptides or
other contaminants that are found in its natural environment that
would interfere with its therapeutic, diagnostic, prophylactic or
research use.
[0055] The term "vector" is used to refer to any molecule (e.g.,
nucleic acid, plasmid, or virus) used to transfer coding
information to a host cell.
[0056] The term "expression vector" refers to a vector that is
suitable for transformation of a host cell and contains nucleic
acid sequences that direct and/or control the expression of
inserted heterologous nucleic acid sequences. Expression includes,
but is not limited to, processes such as transcription,
translation, and RNA splicing, if introns are present.
[0057] The term "operably linked" is used herein to refer to an
arrangement of flanking sequences wherein the flanking sequences so
described are configured or assembled so as to perform their usual
function. Thus, a flanking sequence operably linked to a coding
sequence may be capable of effecting the replication, transcription
and/or translation of the coding sequence. For example, a coding
sequence is operably linked to a promoter when the promoter is
capable of directing transcription of that coding sequence. A
flanking sequence need not be contiguous with the coding sequence,
so long as it functions correctly. Thus, for example, intervening
untranslated yet transcribed sequences can be present between a
promoter sequence and the coding sequence and the promoter sequence
can still be considered "operably linked" to the coding
sequence.
[0058] The term "host cell" is used to refer to a cell which has
been transformed, or is capable of being transformed with a nucleic
acid sequence and then of expressing a selected gene of interest.
The term includes the progeny of the parent cell, whether or not
the progeny is identical in morphology or in genetic make-up to the
original parent, so long as the selected gene is present.
[0059] The term "amino acid," as used herein, refers to naturally
occurring amino acids, unnatural amino acids, amino acid analogues
and amino acid mimetics that function in a manner similar to the
naturally occurring amino acids, all in their D and L stereoisomers
if their structure allows such stereoisomeric forms. Amino acids
are referred to herein by either their name, their commonly known
three letter symbols or by the one-letter symbols recommended by
the IUPAC-IUB Biochemical Nomenclature Commission.
[0060] The term "naturally occurring" when used in connection with
biological materials such as nucleic acid molecules, polypeptides,
host cells, and the like, refers to materials which are found in
nature and are not manipulated by man. Similarly, "non-naturally
occurring" as used herein refers to a material that is not found in
nature or that has been structurally modified or synthesized by
man. When used in connection with nucleotides, the term "naturally
occurring" refers to the bases adenine (A), cytosine (C), guanine
(G), thymine (T), and uracil (U). When used in connection with
amino acids, the term "naturally occurring" refers to the 20
conventional amino acids (i.e., alanine (A), cysteine (C), aspartic
acid (D), glutamic acid (E), phenylalanine (F), glycine (G),
histidine (H), isoleucine (I), lysine (K), leucine (L), methionine
(M), asparagine (N), proline (P), glutamine (Q), arginine (R),
serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine
(Y)), as well as selenocysteine, pyrrolysine (PYL), and
pyrroline-carboxy-lysine (PCL).
[0061] Pyrrolysine (PYL) is an amino acid naturally found within
methylamine methyltransferases of methanogenic archaea of the
family Methanosarcina. Pyrrolysine is a lysine analogue
co-translationally incorporated at in-frame UAG codons in the
respective mRNA, and it is considered the 22nd natural amino
acid.
[0062] As described at least in PCT patent publication WO2010/48582
(applicant IRM, LLC), attempts to biosynthesize pyrrolysine (PYL)
in E. coli resulted in the formation of a "demethylated
pyrrolysine," referred to herein as pyrroline-carboxy-lysine, or
PCL. "PCL," as used herein, refers to either PCL-A or PCL-B.
[0063] The terms "non-natural amino acid" and "unnatural amino
acid," as used herein, are interchangeably intended to represent
amino acid structures that cannot be generated biosynthetically in
any organism using unmodified or modified genes from any organism,
whether the same or different. The terms refer to an amino acid
residue that is not present in the naturally occurring (wild-type)
FGF21 protein sequence or the sequences of the FGF21 variants of
the present invention. These include, but are not limited to,
modified amino acids and/or amino acid analogues that are not one
of the 20 naturally occurring amino acids, selenocysteine,
pyrrolysine (PYL), or pyrroline-carboxy-lysine (PCL). Such
non-natural amino acid residues can be introduced by substitution
of naturally occurring amino acids, and/or by insertion of
non-natural amino acids into the naturally occurring (wild-type)
FGF21 protein sequence or the sequences of the FGF21 variants of
the invention. The non-natural amino acid residue also can be
incorporated such that a desired functionality is imparted to the
FGF21 molecule, for example, the ability to link a functional
moiety (e.g., PEG).
[0064] In addition, it is understood that such "unnatural amino
acids" require a modified tRNA and a modified tRNA synthetase (RS)
for incorporation into a protein. These "selected" orthogonal
tRNA/RS pairs are generated by a selection process as developed by
Schultz et al. or by random or targeted mutation. As way of
example, pyrroline-carboxy-lysine is a "natural amino acid" as it
is generated biosynthetically by genes transferred from one
organism into the host cells and as it is incorporated into
proteins by using natural tRNA and tRNA synthetase genes, while
p-aminophenylalanine (See, Generation of a bacterium with a 21
amino acid genetic code, Mehl R A, Anderson J C, Santoro S W, Wang
L, Martin A B, King D S, Horn D M, Schultz P G. J Am Chem Soc. 2003
Jan. 29; 125(4):935-9) is an "unnatural amino acid" because,
although generated biosynthetically, it is incorporated into
proteins by a "selected" orthogonal tRNA/tRNA synthetase pair.
[0065] Modified encoded amino acids include, but are not limited
to, hydroxyproline, .gamma.-carboxyglutamate, O-phosphoserine,
azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid,
beta-alanine, aminopropionic acid, 2-aminobutyric acid,
4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid,
2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic
acid, tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine,
2,2'-diaminopimelic acid, 2,3-diaminoproprionic acid,
N-ethylglycine, N-methylglycine, N-ethylasparagine, homoproline,
hydroxylysine, allo-hydroxylysine, 3-hydroxyproline,
4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine,
N-methylglycine, N-methylisoleucine, N-methylpentylglycine,
N-methylvaline, naphthalanine, norvaline, norleucine, ornithine,
pentylglycine, pipecolic acid and thioproline. The term "amino
acid" also includes naturally occurring amino acids that are
metabolites in certain organisms but are not encoded by the genetic
code for incorporation into proteins. Such amino acids include, but
are not limited to, ornithine, D-ornithine, and D-arginine.
[0066] The term "amino acid analogue," as used herein, refers to
compounds that have the same basic chemical structure as a
naturally occurring amino acid, by way of example only, an a-carbon
that is bound to a hydrogen, a carboxyl group, an amino group, and
an R group. Amino acid analogues include the natural and unnatural
amino acids which are chemically blocked, reversibly or
irreversibly, or their C-terminal carboxy group, their N-terminal
amino group and/or their side-chain functional groups are
chemically modified. Such analogues include, but are not limited
to, methionine sulfoxide, methionine sulfone,
S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide,
S-(carboxymethyl)-cysteine sulfone, aspartic acid-(beta-methyl
ester), N-ethylglycine, alanine carboxamide, homoserine,
norleucine, and methionine methyl sulfonium.
[0067] The term "amino acid mimetics," as used herein, refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but functions in a
manner similar to a naturally occurring amino acid.
[0068] The term "biologically active FGF21 variant" refers to any
FGF21 polypeptide variant described herein that possesses an
activity of the wild-type FGF21 polypeptide, such as the ability to
lower blood glucose, insulin, triglyceride, or cholesterol; reduce
body weight; and to improve glucose tolerance, energy expenditure,
or insulin sensitivity, regardless of the type or number of
modifications that have been introduced into the FGF21 polypeptide
variant. FGF21 polypeptide variants possessing a somewhat decreased
level of FGF21 activity relative to the wild-type FGF21 polypeptide
can nonetheless be considered to be biologically active FGF21
polypeptide variants.
[0069] The terms "effective amount" and "therapeutically effective
amount" each refer to the amount of an FGF21 protein variant used
to support an observable level of one or more biological activities
of the wild-type FGF21 polypeptide, such as the ability to lower
blood glucose, insulin, triglyceride or cholesterol levels; reduce
liver triglyceride or lipid levels; reduce body weight; or improve
glucose tolerance, energy expenditure, or insulin sensitivity. For
example, a "therapeutically-effective amount" administered to a
patient exhibiting, suffering, or prone to suffer from metabolic
diseases associated with insulin resistance (such as type 1 or type
2 diabetes mellitus, obesity, or metabolic syndrome), is such an
amount which induces, ameliorates or otherwise causes an
improvement in the pathological symptoms, disease progression,
physiological conditions associated with or resistance to
succumbing to the afore mentioned disorders. For the purposes of
the present invention a "subject" or "patient" is preferably a
human, but can also be an animal, more specifically, a companion
animal (e.g., dogs, cats, and the like), farm animals (e.g., cows,
sheep, pigs, horses, and the like) and laboratory animals (e.g.,
rats, mice, guinea pigs, and the like).
[0070] The term "pharmaceutically acceptable carrier" or
"physiologically acceptable carrier" as used herein refers to one
or more formulation materials suitable for accomplishing or
enhancing the delivery of an FGF21 protein, fusion, or variant.
[0071] The term "antigen" refers to a molecule or a portion of a
molecule that is capable of being bound by an antibody, and
additionally that is capable of being used in an animal to produce
antibodies that are capable of binding to an epitope of that
antigen. An antigen may have one or more epitopes.
[0072] The term "native Fc" refers to molecule or sequence
comprising the sequence of a non-antigen-binding fragment resulting
from digestion of whole antibody or produced by other means,
whether in monomeric or multimeric form, and can contain the hinge
region. The original immunoglobulin source of the native Fc is
preferably of human origin and can be any of the immunoglobulins,
although IgG1 and IgG2 are preferred. Native Fc molecules are made
up of monomeric polypeptides that can be linked into dimeric or
multimeric forms by covalent (i.e., disulfide bonds) and
non-covalent association. The number of intermolecular disulfide
bonds between monomeric subunits of native Fc molecules ranges from
1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass
(e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of a native
Fc is a disulfide-bonded dimer resulting from papain digestion of
an IgG (see Ellison et al., 1982, Nucleic Acids Res. 10: 4071-9).
The term "native Fc" as used herein is generic to the monomeric,
dimeric, and multimeric forms. The term "Fc variant" refers to a
molecule or sequence that is modified from a native Fc but still
comprises a binding site for the salvage receptor, FcRn (neonatal
Fc receptor). International Publication Nos. WO 97/34631 and WO
96/32478 describe exemplary Fc variants, as well as interaction
with the salvage receptor, and are hereby incorporated by
reference. Thus, the term "Fc variant" can comprise a molecule or
sequence that is humanized from a non-human native Fc. Furthermore,
a native Fc comprises regions that can be removed because they
provide structural features or biological activity that are not
required for the fusion molecules of the FGF21 mutants of the
present invention. Thus, the term "Fc variant" comprises a molecule
or sequence that lacks one or more native Fc sites or residues, or
in which one or more Fc sites or residues has be modified, that
affect or are involved in: (1) disulfide bond formation, (2)
incompatibility with a selected host cell, (3) N-terminal
heterogeneity upon expression in a selected host cell, (4)
glycosylation, (5) interaction with complement, (6) binding to an
Fc receptor other than a salvage receptor, or (7)
antibody-dependent cellular cytotoxicity (ADCC). Fc variants are
described in further detail hereinafter.
[0073] The term "Fc domain" encompasses native Fc and Fc variants
and sequences as defined above. As with Fc variants and native Fc
molecules, the term "Fc domain" includes molecules in monomeric or
multimeric form, whether digested from whole antibody or produced
by other means. In some embodiments of the present invention, an Fc
domain can be fused to FGF21 or a FGF21 mutant (including a
truncated form of FGF21 or a FGF21 mutant) via, for example, a
covalent bond between the Fc domain and the FGF21 sequence. Such
fusion proteins can form multimers via the association of the Fc
domains and both these fusion proteins and their multimers are an
aspect of the present invention.
[0074] The term "polyethylene glycol" or "PEG" refers to a
polyalkylene glycol compound or a derivative thereof, with or
without coupling agents or derviatization with coupling or
activating moieties.
[0075] The term "metabolic diseases associated with insulin
resistance," and terms similarly used herein, includes but is not
limited to type 1 diabetes mellitus, insulin receptor mutation
disorders (INSR disorders), mixed dyslipidemia, nonalcoholic fatty
liver disease (NAFLD), insulin resistance, and lipodystrophy.
[0076] The terms "insulin receptor disorders (INSR disorders),"
"disorders associated with severe inactivating mutations in the
insulin receptor," "metabolic diseases associated with insulin
resistance," and terms similarly used herein, describe conditions
in subjects afflicted with mutations in the insulin receptor (or
possibly, proteins directly downstream from it, e.g., IRS1 &
IRS2) which cause severe insulin resistance but are often seen
without the obesity common in Type 2 diabetes mellitus. Subjects
thereby afflicted fall into several categories of roughly
increasing severity, including: Type A Insulin Resistance, Type C
Insulin Resistance (also known as HAIR-AN Syndrome),
Rabson-Mendenhall Syndrome, and finally, Donohue's Syndrome, or
Leprechaunism. Type B Insulin Resistance is also included in the
general term "Insulin Receptor Disorder" and has a similar
phenotype to the genetic forms of insulin resistance outlined
herein, except that it is instead caused by neutralizing
auto-antibodies to the insulin receptor, as opposed to inactivating
mutations.
[0077] These disorders are associated with very high endogenous
insulin levels, and very often, hyperglycemia. Subjects thereby
afflicted also present with various clinical features associated
with "insulin toxicity," including hyperandrogenism, polycystic
ovary syndrome (PCOS, which is characterized by an increased
incidence of insulin receptor mutations), hirsutism, and acanthosis
nigricans (excessive growth and pigmentation) in the folds of the
skin.
[0078] "Type 2 diabetes mellitus" is a condition characterized by
excess glucose production in spite of the availability of insulin,
and circulating glucose levels remain excessively high as a result
of inadequate glucose clearance (insulin action).
[0079] "Type 1 diabetes mellitus" is a condition characterized by
high blood glucose levels caused by total lack of insulin. This
occurs when the body's immune system attacks the insulin-producing
beta cells in the pancreas and destroys them. The pancreas then
produces little or no insulin. Pancreatic removal or disease may
also lead to loss of insulin-producing beta cells.
[0080] "Dyslipidemia" is a disorder of lipoprotein metabolism,
including lipoprotein overproduction or deficiency. Dyslipidemias
may be manifested by elevation of the total cholesterol,
low-density lipoprotein (LDL) cholesterol and triglyceride
concentrations, and a decrease in high-density lipoprotein (HDL)
cholesterol concentration in the blood.
[0081] "Glucose intolerance," or Impaired Glucose Tolerance (IGT)
is a pre-diabetic state of dysglycemia that is associated with
increased risk of cardiovascular pathology. The pre-diabetic
condition prevents a subject from moving glucose into cells
efficiently and utilizing it as an efficient fuel source, leading
to elevated glucose levels in blood and some degree of insulin
resistance.
[0082] "HAART" Highly Active Anti-Retroviral Therapy used to treat
patients with HIV infection.
[0083] "HIV-HAART Induced Partial Lipodystrophy" Adverse effects,
including metabolic dysregulation and changes in body fat
deposition characterized by insulin resistance, dyslipidemia,
lipodystrophy, and increased visceral adiposity, which contribute
to an increased risk of cardiovascular disease among HIV patients
treated with HAART.
[0084] "Hyperglycemia" is defined as an excess of sugar (glucose)
in the blood.
[0085] "Hypoglycemia", also called low blood sugar, occurs when
blood glucose levels drop too low to provide enough energy to
maintain normal body function.
[0086] "Hyperinsulinemia" is defined as a higher-than-normal level
of insulin in the blood.
[0087] "Insulin resistance" is defined as a state in which a normal
amount of insulin produces a subnormal biologic response.
[0088] "Obesity," in terms of the human adult subject, can be
defined as a Body Mass Index (BMI) exceeding 30 kg/m.sup.2.
[0089] "Metabolic syndrome" can be defined as a cluster of at least
three of the following signs: abdominal fat--men, a greater than
40-inch waist and women, greater than 35-inch waist; high blood
sugar--at least 100 milligrams per deciliter (mg/dL) after fasting;
high triglycerides--at least 150 mg/dL in the bloodstream; low
HDL--less than 40 mg/dL for males and less than 50 mg/dL for
females; and, blood pressure of 130/85 mmHg or higher.
[0090] "Hypertension" or high blood pressure that is a transitory
or sustained elevation of systemic arterial blood pressure to a
level likely to induce cardiovascular damage or other adverse
consequences. Hypertension has been arbitrarily defined as a
systolic blood pressure above 140 mmHg or a diastolic blood
pressure above 90 mmHg.
[0091] "Cardiovascular diseases" are diseases related to the heart
or blood vessels.
[0092] "Atherosclerosis" is a vascular disease characterized by
irregularly distributed lipid deposits called plaque in the intima
of large and medium-sized arteries that may cause narrowing of
arterial lumens and proceed to fibrosis and calcification. Lesions
are usually focal and progress slowly and intermittently.
Occasionally plaque rupture occurs leading to obstruction of blood
flow resulting in tissue death distal to the obstruction.
Limitation of blood flow accounts for most clinical manifestations,
which vary with the distribution and severity of the
obstruction.
[0093] "Stroke" is any acute clinical event, related to impairment
of cerebral circulation, that lasts longer than 24 hours. A stroke
involves irreversible brain damage, the type and severity of
symptoms depending on the location and extent of brain tissue whose
circulation has been compromised.
[0094] "Heart failure", also called congestive heart failure, is a
condition in which the heart can no longer pump enough blood to the
rest of the body to meet demand.
[0095] "Coronary heart disease", also called coronary artery
disease, refers to atherosclerotic lesions or plaque in coronary
arteries which may cause narrowing of the small blood vessels that
supply blood and oxygen to the heart.
[0096] "Kidney disease" or nephropathy is any disease of the
kidney. Diabetic nephropathy is a major cause of morbidity and
mortality in people with type 1 or type 2 diabetes mellitus.
[0097] "Diabetic complications" are problems, caused by high blood
glucose levels, with other body functions such as kidneys, nerves
(neuropathies), feet (foot ulcers and poor circulation) and eyes
(e.g. retinopathies). Diabetes also increases the risk for heart
disease and bone and joint disorders. Other long-term complications
of diabetes include skin problems, digestive problems, sexual
dysfuntion and problems with teeth and gums.
[0098] "Neuroapathies" are any diseases involving the cranial
nerves or the peripheral or autonomic nervous system.
[0099] "Gastroparesis" is weakness of gastric peristalsis, which
results in delayed gastric emptying.
[0100] As used herein, the singular forms "a," "an" and "the"
include plural references unless the content clearly dictates
otherwise. Thus, for example, reference to "an antibody" includes a
mixture of two or more such antibodies.
[0101] As used herein, the term "about" refers to +/-20%, +/-10%,
or +/-5% of a value.
[0102] The terms "polypeptide" and "protein", are used
interchangeably and refer to a polymeric form of amino acids of any
length, which can include coded and non-coded amino acids,
chemically or biochemically modified or derivatized amino acids,
and polypeptides having modified peptide backbones. The term
includes fusion proteins, including, but not limited to, fusion
proteins with a heterologous amino acid sequence, fusions with
heterologous and homologous leader sequences, with or without
N-terminal methionine residues; immunologically tagged proteins;
and the like.
[0103] The terms "individual," "subject," "host," and "patient" are
used interchangeably and refer to any subject for whom diagnosis,
treatment, or therapy is desired, particularly humans. Other
subjects may include cattle, dogs, cats, guinea pigs, rabbits,
rats, mice, horses, and the like. In some preferred embodiments the
subject is a human.
[0104] As used herein, the term "sample" refers to biological
material from a patient. The sample assayed by the present
invention is not limited to any particular type. Samples include,
as non-limiting examples, single cells, multiple cells, tissues,
tumors, biological fluids, biological molecules, or supernatants or
extracts of any of the foregoing. Examples include tissue removed
for biopsy, tissue removed during resection, blood, urine, lymph
tissue, lymph fluid, cerebrospinal fluid, mucous, and stool
samples. The sample used will vary based on the assay format, the
detection method and the nature of the tumors, tissues, cells or
extracts to be assayed. Methods for preparing samples are well
known in the art and can be readily adapted in order to obtain a
sample that is compatible with the method utilized.
[0105] As used herein, the term "biological molecule" includes, but
is not limited to, polypeptides, nucleic acids, and
saccharides.
[0106] As used herein, the term "modulating" refers to a change in
the quality or quantity of a gene, protein, or any molecule that is
inside, outside, or on the surface of a cell. The change can be an
increase or decrease in expression or level of the molecule. The
term "modulates" also includes changing the quality or quantity of
a biological function/activity including, without limitation, the
ability to lower blood glucose, insulin, triglyceride, or
cholesterol levels; to reduce liver lipid or liver triglyceride
levels; to reduce body weight; and to improve glucose tolerance,
energy expenditure, or insulin sensitivity.
[0107] As used herein, the term "modulator" refers to a composition
that modulates one or more physiological or biochemical events
associated with a metabolic diseases associated with insulin
resistance, such as type 1 diabetes mellitus or a metabolic
condition like obesity. Said events include but are not limited to
the ability to lower blood glucose, insulin, triglyceride, or
cholesterol levels; to reduce liver lipid or liver triglyceride
levels; to reduce body weight; and to improve glucose tolerance,
energy expenditure, or insulin sensitivity.
[0108] A "gene product" is a biopolymeric product that is expressed
or produced by a gene. A gene product may be, for example, an
unspliced RNA, an mRNA, a splice variant mRNA, a polypeptide, a
post-translationally modified polypeptide, a splice variant
polypeptide etc. Also encompassed by this term are biopolymeric
products that are made using an RNA gene product as a template
(i.e. cDNA of the RNA). A gene product may be made enzymatically,
recombinantly, chemically, or within a cell to which the gene is
native. In some embodiments, if the gene product is proteinaceous,
it exhibits a biological activity. In some embodiments, if the gene
product is a nucleic acid, it can be translated into a
proteinaceous gene product that exhibits a biological activity.
[0109] "Modulation of FGF21 activity," as used herein, refers to an
increase or decrease in FGF21 activity that can be a result of, for
example, interaction of an agent with an FGF21 polynucleotide or
polypeptide, inhibition of FGF21 transcription and/or translation
(e.g., through antisense or siRNA interaction with the FGF21 gene
or FGF21 transcript, through modulation of transcription factors
that facilitate FGF21 expression), and the like. For example,
modulation of a biological activity refers to an increase or a
decrease in a biological activity. FGF21 activity can be assessed
by means including, without limitation, assaying blood glucose,
insulin, triglyceride, or cholesterol levels in a subject,
assessing FGF21 polypeptide levels, or by assessing FGF21
transcription levels. Comparisons of FGF21 activity can also be
accomplished by, e.g., measuring levels of an FGF21 downstream
biomarker, and measuring increases in FGF21 signaling. FGF21
activity can also be assessed by measuring: cell signaling; kinase
activity; glucose uptake into adipocytes; blood insulin,
triglyceride, or cholesterol level fluctuations; liver lipid or
liver triglyceride level changes; interactions between FGF21 and an
FGF21 receptor; or phosphorylation of an FGF21 receptor. In some
embodiments phosphorylation of an FGF21 receptor can be tyrosine
phosphorylation. In some embodiments modulation of FGF21 activity
can cause modulation of an FGF21-related phenotype.
[0110] A "FGF21 downstream biomarker," as used herein, is a gene or
gene product, or measurable indicia of a gene or gene product. In
some embodiments, a gene or activity that is a downstream marker of
FGF21 exhibits an altered level of expression, or in a vascular
tissue. In some embodiments, an activity of the downstream marker
is altered in the presence of an FGF21 modulator. In some
embodiments, the downstream markers exhibit altered levels of
expression when FGF21 is perturbed with an FGF21 modulator of the
present invention. FGF21 downstream markers include, without
limitation, glucose or 2-deoxy-glucose uptake, pERK and other
phosphorylated or acetylated proteins or NAD levels.
[0111] As used herein, the term "up-regulates" refers to an
increase, activation or stimulation of an activity or quantity. For
example, in the context of the present invention, FGF21 modulators
may increase the activity of an FGF21 receptor. In one embodiment,
one or more of FGFR-1c, FGFR-2c, FGFR-3c, or B-klotho may be
upregulated in response to an FGF21 modulator. Upregulation can
also refer to an FGF21-related activity, such as e.g., the ability
to lower blood glucose, insulin, triglyceride, or cholesterol
levels; to reduce liver lipid or triglyceride levels; to reduce
body weight; to improve glucose tolerance, energy expenditure, or
insulin sensitivity; or to cause phosphorylation of an FGF21
receptor; or to increase an FGF21 downstream marker. The FGFR21
receptor can be one or more of FGFR-1c, FGFR-2c, FGFR-3c, or
B-klotho. Up-regulation may be at least 25%, at least 50%, at least
75%, at least 100%, at least 150%, at least 200%, at least 250%, at
least 400%, or at least 500% as compared to a control.
[0112] As used herein, the term "N-terminus" refers to at least the
first 10 amino acids of a protein.
[0113] As used herein, the terms "N-terminal domain" and
"N-terminal region" are used interchangeably and refer to a
fragment of a protein that begins at the first amino acid of the
protein and ends at any amino acid in the N-terminal half of the
protein. For example, the N-terminal domain of FGF21 is from amino
acid 1 of wild type FGF21 to any amino acid between about amino
acids 10 and 105 of wild type FGF21.
[0114] As used herein, the term "C-terminus" refers to at least the
last 10 amino acids of a protein.
[0115] As used herein, the terms "C-terminal domain" and
"C-terminal region" are used interchangeably and refer to a
fragment of a protein that begins at any amino acid in the
C-terminal half of the protein and ends at the last amino acid of
the protein. For example, the C-terminal domain of FGF21 begins at
any amino acid from amino acid 105 to about amino acid 200 of wild
type FGF21 and ends at amino acid 209 of wild type FGF21.
[0116] The term "domain" as used herein refers to a structural part
of a biomolecule that contributes to a known or suspected function
of the biomolecule. Domains may be co-extensive with regions or
portions thereof and may also incorporate a portion of a
biomolecule that is distinct from a particular region, in addition
to all or part of that region.
[0117] As used herein, the term "signal domain" (also called
"signal sequence" or "signal peptide") refers to a peptide domain
that resides in a continuous stretch of amino acid sequence at the
N-terminal region of a precursor protein (often a membrane-bound or
secreted protein) and is involved in post-translational protein
transport. In many cases the signal domain is removed from the
full-length protein by specialized signal peptidases after the
sorting process has been completed. Each signal domain specifies a
particular destination in the cell for the precursor protein. The
signal domain of FGF21 is amino acids 1-28.
[0118] As used herein, the term "receptor binding domain" refers to
any portion or region of a protein that contacts a membrane-bound
receptor protein, resulting in a cellular response, such as a
signaling event.
[0119] As used herein, the term "ligand binding domain" refers to
any portion or region of a protein retaining at least one
qualitative binding activity of a corresponding native sequence of
FGF21.
[0120] The term "region" refers to a physically contiguous portion
of the primary structure of a biomolecule. In the case of proteins,
a region is defined by a contiguous portion of the amino acid
sequence of that protein. In some embodiments a "region" is
associated with a function of the biomolecule.
[0121] The term "fragment" as used herein refers to a physically
contiguous portion of the primary structure of a biomolecule. In
the case of proteins, a portion is defined by a contiguous portion
of the amino acid sequence of that protein and refers to at least
3-5 amino acids, at least 8-10 amino acids, at least 11-15 amino
acids, at least 17-24 amino acids, at least 25-30 amino acids, and
at least 30-45 amino acids. In the case of oligonucleotides, a
portion is defined by a contiguous portion of the nucleic acid
sequence of that oligonucleotide and refers to at least 9-15
nucleotides, at least 18-30 nucleotides, at least 33-45
nucleotides, at least 48-72 nucleotides, at least 75-90
nucleotides, and at least 90-130 nucleotides. In some embodiments,
portions of biomolecules have a biological activity.
[0122] A "native sequence" polypeptide is one that has the same
amino acid sequence as a polypeptide derived from nature. Such
native sequence polypeptides can be isolated from nature or can be
produced by recombinant or synthetic means. Thus, a native sequence
polypeptide can have the amino acid sequence of naturally occurring
human polypeptide, murine polypeptide, or polypeptide from any
other mammalian species.
[0123] As used herein, the term "mixing" refers to the process of
combining one or more compounds, cells, molecules, and the like
together in the same area. This may be performed, for example, in a
test tube, petri dish, or any container that allows the one or more
compounds, cells, or molecules, to be mixed.
[0124] As used herein, the term "substantially purified" refers to
a compound (e.g., either a polynucleotide or a polypeptide or an
antibody) that is removed from its natural environment and is at
least 60% free, at least 75% free, and at least 90% free from other
components with which it is naturally associated.
[0125] The term "pharmaceutically acceptable carrier" refers to a
carrier for administration of a therapeutic agent, such as
antibodies or a polypeptide, genes, and other therapeutic agents.
The term refers to any pharmaceutical carrier that does not itself
induce the production of antibodies harmful to the individual
receiving the composition, and which can be administered without
undue toxicity. Suitable carriers can be large, slowly metabolized
macromolecules such as proteins, polysaccharides, polylactic acids,
polyglycolic acids, polymeric amino acids, amino acid copolymers,
lipid aggregates and inactive virus particles. Such carriers are
well known to those of ordinary skill in the art. Pharmaceutically
acceptable carriers in therapeutic compositions can include liquids
such as water, saline, glycerol and ethanol. Auxiliary substances,
such as wetting or emulsifying agents, pH buffering substances, and
the like, can also be present in such vehicles.
[0126] Naturally occurring disulfide bonds, as provided by cysteine
residues, generally increase thermodynamic stability of proteins.
Successful examples of increased thermodynamic stability, as
measured in increase of the melting temperature, are multiple
disulfide-bonded mutants of the enzymes T4 lysozyme (Matsumura, et
al., PNAS 86:6562-6566 (1989)) and barnase (Johnson et al., J. Mol.
Biol. 268:198-208 (1997)). An aspect of the present invention is an
enhancement of the physical stability of FGF21 in the presence of a
preservative, achieved by the presence of disulfide bonds within
the variants, which constrain the flexibility of wild type FGF21
and thereby limit access of the preservative to the hydrophobic
core of the protein.
[0127] The present invention provides methods of treatment that
comprise variants or mutants of wild type human FGF21, or a
biologically active peptide thereof, with enhanced pharmaceutical
stability engendered by the incorporation of additional disulfide
bonds, e.g., via incorporating or substituting cysteine residues
into the wild-type FGF21 protein or the polypeptide and protein
variants of the invention. One skilled in the art will recognize
that the native cysteines, cysteine 103 and cysteine 121, could be
utilized as loci to introduce a novel disulfide bond that may
impart improved properties, in addition to the suggested
embodiments described herein and in the literature.
[0128] The methods of the present invention comprise pharmaceutical
compositions that may be administered by any means that achieve the
generally intended purpose: to treat metabolic diseases associated
with insulin resistance, such as type 2 diabetes mellitus, insulin
receptor mutation disorders (INSR disorders), nonalcoholic fatty
liver disease (NAFLD) and various forms of partial lipodystrophy
including familial partial lipodystrophy and HIV HAART induced
partial lipodystrophy as well as diseases associated with insulin
production (i.e., type 1 diabetes mellitus). The term "parenteral"
as used herein refers to modes of administration that include
intravenous, intramuscular, intraperitoneal, intrasternal,
subcutaneous, and intraarticular injection and infusion. The dosage
administered will be dependent upon the age, health, and weight of
the recipient, kind of concurrent treatment, if any, frequency of
treatment, and the nature of the effect desired. Included are all
compositions wherein an FGF21 polypeptide or protein, or fusion,
mutant, or variant thereof, is present in an amount that is
effective to achieve the desired medical effect for treatment of
metabolic diseases associated with insulin resistance. While
individual needs may vary from one patient to another, the
determination of the optimal ranges of effective amounts of all of
the components is within the ability of the clinician of ordinary
skill.
[0129] The variants of FGF21 comprising the methods of the present
invention can be formulated according to known methods to prepare
pharmaceutically useful compositions. A desired formulation would
be one that is a stable lyophilized product that is reconstituted
with an appropriate diluent or an aqueous solution of high purity
with optional pharmaceutically acceptable carriers, preservatives,
excipients or stabilizers [Remington's Pharmaceutical Sciences 16th
edition (1980)]. The variants of the present invention may be
combined with a pharmaceutically acceptable buffer, and the pH
adjusted to provide acceptable stability, and a pH acceptable for
administration.
[0130] For parenteral administration, in one embodiment, FGF21
variants are formulated generally by mixing one or more of them at
the desired degree of purity, in a unit dosage injectable form
(solution, suspension, or emulsion), with a pharmaceutically
acceptable carrier, i.e., one that is non-toxic to recipients at
the dosages and concentrations employed and is compatible with
other ingredients of the formulation. Preferably, one or more
pharmaceutically acceptable anti-microbial agents may be added.
Phenol, m-cresol, and benzyl alcohol are preferred pharmaceutically
acceptable anti-microbial agents.
[0131] Optionally, one or more pharmaceutically acceptable salts
may be added to adjust the ionic strength or tonicity. One or more
excipients may be added to further adjust the isotonicity of the
formulation. Glycerin, sodium chloride, and mannitol are examples
of an isotonicity adjusting excipient.
[0132] Those skilled in the art can readily optimize
pharmaceutically effective dosages and administration regimens for
therapeutic compositions comprising an FGF21 variant, as determined
by good medical practice and the clinical condition of the
individual patient. A typical dose range for the FGF21 variants of
the present invention will range from about 0.01 mg per day to
about 1000 mg per day (or about 0.07 mg per week to about 7000 mg
per week administered once per week) for an adult. Preferably, the
dosage ranges from about 0.1 mg per day to about 100 mg per day (or
about 0.7 mg per week to about 700 mg per week administered once
per week), more preferably from about 1.0 mg/day to about 10 mg/day
(or about 7 mg per week to about 70 mg per week administered once
per week). Most preferably, the dosage is about 1-5 mg/day (or
about 7 mg per week to about 35 mg per week administered once per
week). The appropriate dose of an FGF21 variant administered
according to the claimed methods of the invention will improve
metabolic profiles, e.g., will lower blood glucose levels, increase
energy expenditure, and/or promote more efficient glucose
utilization, and thus is useful for treating metabolic diseases
associated with insulin resistance, such as type 2 diabetes
mellitus, insulin receptor mutation disorders (INSR disorders),
nonalcoholic fatty liver disease (NAFLD) and various forms of
partial lipodystrophy including familial partial lipodystrophy and
HIV-HAART induced partial lipodystrophy as well as diseases
associated with insulin production (type 1 diabetes mellitus).
[0133] In addition, because hyperglycemia and insulin resistance
are common in critically ill patients given nutritional support,
some ICUs administer insulin to treat excessive hyperglycemia in
fed critically ill patients. In fact, recent studies document the
use of exogenous insulin to maintain blood glucose at a level no
higher than 110 mg per deciliter reduced morbidity and mortality
among critically ill patients in the surgical intensive care unit,
regardless of whether they had a history of diabetes (Van den
Berghe, et al. N Engl J Med., 345(19):1359, (2001)). Thus, methods
of the present invention are uniquely suited to help restore
metabolic stability in metabolically unstable critically ill
patients.
[0134] In another aspect of the present invention, variants of
FGF21 for use as a medicament for the treatment of metabolic
diseases associated with insulin resistance, such as type 2
diabetes mellitus, insulin receptor mutation disorders (INSR
disorders), nonalcoholic fatty liver disease (NAFLD), and various
forms of partial lipodystrophy including familial partial
lipodystrophy and HIV-HAART induced partial lipodystrophy as well
as diseases associated with insulin production (type 1 diabetes
mellitus), and in reducing the mortality and morbidity of
critically ill patients.
[0135] Having now described the present invention in detail, the
same will be more clearly understood by reference to the following
examples, which are included herewith for purposes of illustration
only and are not intended to be limiting of the invention.
[0136] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry,
biochemistry, molecular biology, immunology and pharmacology,
within the skill of the art. Such techniques are explained fully in
the literature. See, e.g., Remington's Pharmaceutical Sciences,
18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Methods
In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press,
Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M.
Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific
Publications); and Sambrook et al., Molecular Cloning: A Laboratory
Manual (2nd Edition, 1989).
Site-Specific FGF21 Mutants
[0137] The term "site-specific FGF21 mutant" or "substituted FGF21
mutant" refers to a FGF21 mutant polypeptide having an amino acid
sequence that differs from the amino acid sequence of a naturally
occurring FGF21 polypeptide sequence, e.g., having NCBI reference
sequence number NP_061986.1, and variants thereof. Site-specific
FGF21 mutants can be generated by introducing amino acid
substitutions, either conservative or non-conservative and using
naturally or non-naturally occurring amino acids, at particular
positions of the FGF21 polypeptide.
[0138] "Conservative amino acid substitution" can involve a
substitution of a native amino acid residue (i.e., a residue found
in a given position of the wild-type FGF21 polypeptide sequence)
with a nonnative residue (i.e., a residue that is not found in a
given position of the wild-type FGF21 polypeptide sequence) such
that there is little or no effect on the polarity or charge of the
amino acid residue at that position. Conservative amino acid
substitutions also encompass non-naturally occurring amino acid
residues that are typically incorporated by chemical peptide
synthesis rather than by synthesis in biological systems. These
include peptidomimetics, and other reversed or inverted forms of
amino acid moieties.
[0139] Naturally occurring residues can be divided into classes
based on common side chain properties:
[0140] (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
[0141] (2) neutral hydrophilic: Cys, Ser, Thr;
[0142] (3) acidic: Asp, Glu;
[0143] (4) basic: Asn, Gln, His, Lys, Arg;
[0144] (5) residues that influence chain orientation: Gly, Pro;
[0145] (6) aromatic: Trp, Tyr, Phe; and
[0146] (7) selenocysteine, pyrrolysine (PYL), and
pyrroline-carboxy-lysine (PCL).
[0147] Conservative substitutions can involve the exchange of a
member of one of these classes for another member of the same
class. Non-conservative substitutions can involve the exchange of a
member of one of these classes for a member from another class.
[0148] Desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired.
Truncated FGF21 Polypeptides
[0149] One embodiment of the present invention is directed to
methods of treatment comprising truncated forms of the mature FGF21
polypeptide. This embodiment of the present invention arose from an
effort to identify truncated FGF21 polypeptides that are capable of
providing an activity that is similar, and in some instances
superior, to untruncated forms of the mature FGF21 polypeptide.
[0150] As used herein, the term "truncated FGF21 polypeptide"
refers to an FGF21 polypeptide in which amino acid residues have
been removed from the amino-terminal (or N-terminal) end of the
FGF21 polypeptide, amino acid residues have been removed from the
carboxyl-terminal (or C-terminal) end of the FGF21 polypeptide, or
amino acid residues have been removed from both the amino-terminal
and carboxyl-terminal ends of the FGF21 polypeptide. The various
truncations disclosed herein were prepared as described herein.
[0151] The activity of N-terminally truncated FGF21 polypeptides
and C-terminally truncated FGF21 polypeptides can be assayed using
an in vitro phospho-ERK assay. Specific details of the in vitro
assays that can be used to examine the activity of truncated FGF21
polypeptides can be found in the examples.
[0152] The activity of the truncated FGF21 polypeptides of the
present invention can also be assessed in an in vivo assay, such as
ob/ob mice. Generally, to assess the in vivo activity of a
truncated FGF21 polypeptide, the truncated FGF21 polypeptide can be
administered to a test animal intraperitoneally. After a desired
incubation period (e.g., one hour or more), a blood sample can be
drawn, and blood glucose levels can be measured.
[0153] a. N-Terminal Truncations
[0154] Some embodiments of the methods of the present invention
comprise N-terminal truncations with 1, 2, 3, 4, 5, 6, 7, or 8
amino acid residues from the N-terminal end of the mature FGF21
polypeptide. Truncated FGF21 polypeptides having N-terminal
truncations of fewer than 9 amino acid residues retain the ability
of the mature FGF21 polypeptide to lower blood glucose in an
individual. Accordingly, in particular embodiments, the present
invention encompasses truncated forms of the mature FGF21
polypeptide or FGF21 protein variants having N-terminal truncations
of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues.
[0155] b. C-Terminal Truncations
[0156] Some embodiments of the methods of the present invention
comprise C-terminal truncations with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, or 12 amino acid residues from the C-terminal end of the mature
FGF21 polypeptide. Truncated FGF21 polypeptides having C-terminal
truncations of fewer than 13 amino acid residues exhibited an
efficacy of at least 50% of the efficacy of wild-type FGF21 in an
in vitro ELK-luciferase assay (Yie J. et al. FEBS Letts 583:19-24
(2009)), indicating that these FGF21 mutants retain the ability of
the mature FGF21 polypeptide to lower blood glucose in an
individual. Accordingly, in particular embodiments, the present
invention encompasses truncated forms of the mature FGF21
polypeptide or FGF21 protein variants having C-terminal truncations
of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid
residues.
[0157] c. N-Terminal and C-Terminal Truncations
[0158] Some embodiments of the methods of the present invention
comprise truncated FGF21 polypeptides with a combination of
N-terminal and C-terminal truncations. Truncated FGF21 polypeptides
having a combination of N-terminal and C-terminal truncations share
the activity of corresponding truncated FGF21 polypeptides having
either the N-terminal or C-terminal truncations alone. In other
words, truncated FGF21 polypeptides having both N-terminal
truncations of fewer than 9 amino acid residues and C-terminal
truncations of fewer than 13 amino acid residues possess similar or
greater blood glucose-lowering activity as truncated FGF21
polypeptides having N-terminal truncations of fewer than 9 amino
acid residues or truncated FGF21 polypeptides having C-terminal
truncations of fewer than 13 amino acid residues. Accordingly, in
particular embodiments, the present invention encompasses truncated
forms of the mature FGF21 polypeptide or FGF21 protein variants
having both N-terminal truncations of 1, 2, 3, 4, 5, 6, 7, or 8
amino acid residues and C-terminal truncations of 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, or 12 amino acid residues.
[0159] As with all FGF21 variants of the methods of the present
invention, truncated FGF21 polypeptides can optionally comprise an
amino-terminal methionine residue, which can be introduced by
directed mutation or as a result of a bacterial expression
process.
[0160] The truncated FGF21 polypeptides comprising the methods of
the present invention can be prepared as described in the examples
described herein. Those of ordinary skill in the art, familiar with
standard molecular biology techniques, can employ that knowledge,
coupled with the instant disclosure, to make and use the truncated
FGF21 polypeptides of the present invention. Standard techniques
can be used for recombinant DNA, oligonucleotide synthesis, tissue
culture, and transformation (e.g., electroporation, lipofection).
See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,
supra, which is incorporated herein by reference for any purpose.
Enzymatic reactions and purification techniques can be performed
according to manufacturer's specifications, as commonly
accomplished in the art, or as described herein. Unless specific
definitions are provided, the nomenclatures utilized in connection
with, and the laboratory procedures and techniques of, analytical
chemistry, synthetic organic chemistry, and medicinal and
pharmaceutical chemistry described herein are those well known and
commonly used in the art. Standard techniques can be used for
chemical syntheses; chemical analyses; pharmaceutical preparation,
formulation, and delivery; and treatment of patients.
[0161] The truncated FGF21 polypeptides of the methods of the
present invention can also be fused to another entity, which can
impart additional properties to the truncated FGF21 polypeptide. In
one embodiment of the present invention, a truncated FGF21
polypeptide can be fused to an IgG constant domain or fragment
thereof (e.g., the Fc region), Human Serum Albumin (HSA), or
albumin-binding polypeptides. Such fusion can be accomplished using
known molecular biological methods and/or the guidance provided
herein. The benefits of such fusion polypeptides, as well as
methods for making such fusion polypeptides, are discussed in more
detail herein.
FGF21 Fusion Proteins
[0162] As used herein, the term "FGF21 fusion polypeptide" or
"FGF21 fusion protein" refers to a fusion of one or more amino acid
residues (such as a heterologous protein or peptide) at the
N-terminus or C-terminus of any FGF21 protein variant described
herein.
[0163] Heterologous peptides and polypeptides include, but are not
limited to, an epitope to allow for the detection and/or isolation
of an FGF21 protein variant; a transmembrane receptor protein or a
portion thereof, such as an extracellular domain or a transmembrane
and intracellular domain; a ligand or a portion thereof which binds
to a transmembrane receptor protein; an enzyme or portion thereof
which is catalytically active; a polypeptide or peptide which
promotes oligomerization, such as a leucine zipper domain; a
polypeptide or peptide which increases stability, such as an
immunoglobulin constant region; a functional or non-functional
antibody, or a heavy or light chain thereof; and a polypeptide
which has an activity, such as a therapeutic activity, different
from the FGF21 protein variants of the present invention. Also
encompassed by the present invention are FGF21 mutants fused to
human serum albumin (HSA).
[0164] FGF21 fusion proteins can be made by fusing heterologous
sequences at either the N-terminus or at the C-terminus of an FGF21
protein variant. As described herein, a heterologous sequence can
be an amino acid sequence or a non-amino acid-containing polymer.
Heterologous sequences can be fused either directly to the FGF21
protein variant or via a linker or adapter molecule. A linker or
adapter molecule can be one or more amino acid residues (or -mers),
e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 residues (or -mers), preferably
from 10 to 50 amino acid residues (or -mers), e.g., 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 residues (or
-mers), and more preferably from 15 to 35 amino acid residues (or
-mers). A linker or adapter molecule can also be designed with a
cleavage site for a DNA restriction endonuclease or for a protease
to allow for the separation of the fused moieties.
[0165] a. Fc Fusions
[0166] In one embodiment of the present invention, an FGF21 protein
variant is fused to one or more domains of an Fc region of human
IgG. Antibodies comprise two functionally independent parts, a
variable domain known as "Fab," that binds an antigen, and a
constant domain known as "Fc," that is involved in effector
functions such as complement activation and attack by phagocytic
cells. An Fc has a long serum half-life, whereas a Fab is
short-lived (Capon et al., 1989, Nature 337: 525-31). When joined
together with a therapeutic protein, an Fc domain can provide
longer half-life or incorporate such functions as Fc receptor
binding, protein A binding, complement fixation, and perhaps even
placental transfer (Capon et al., 1989).
[0167] In vivo pharmacokinetic analysis indicated that human FGF21
has a short half-life of about 0.5 to 1 hours in mice due to rapid
clearance and in vivo degradation. Therefore, to extend the
half-life of FGF21 an Fc sequence was fused to the N- or C-terminal
end of the FGF21 polypeptide. The fusion of an Fc region to
wild-type FGF21, in particularly Fc fused to the N-terminus of
wild-type FGF21, did not extend the half-life as expected, however,
which led to an investigation of the proteolytic degradation of
FGF21 in vivo and the identification of FGF21 mutants that were
resistant to such degradation.
[0168] Throughout the disclosure, Fc-FGF21 refers to a fusion
protein in which the Fc sequence is fused to the N-terminus of
FGF21. Similarly, throughout the disclosure, FGF21-Fc refers to a
fusion protein in which the Fc sequence is fused to the C-terminus
of FGF21.
[0169] The resulting FGF21 fusion protein can be purified, for
example, by the use of a Protein A affinity column. Peptides and
proteins fused to an Fc region have been found to exhibit a
substantially greater half-life in vivo than the unfused
counterpart. Also, a fusion to an Fc region allows for
dimerization/multimerization of the fusion polypeptide. The Fc
region can be a naturally occurring Fc region, or can be altered to
improve certain qualities, such as therapeutic qualities,
circulation time, or reduced aggregation.
[0170] Useful modifications of protein therapeutic agents by fusion
with the "Fc" domain of an antibody are discussed in detail in
International Publication No. WO 00/024782, which is hereby
incorporated by reference in its entirety. This document discusses
linkage to a "vehicle" such as polyethylene glycol (PEG), dextran,
or an Fc region.
[0171] b. Fusion Protein Linkers
[0172] When forming the fusion proteins of the present invention, a
linker can, but need not, be employed. When present, the linker's
chemical structure may not critical, since it serves primarily as a
spacer. The linker can be made up of amino acids linked together by
peptide bonds. In some embodiments of the present invention, the
linker is made up of from 1 to 20 amino acids linked by peptide
bonds, wherein the amino acids are selected from the 20 naturally
occurring amino acids. In various embodiments, the 1 to 20 amino
acids are selected from the amino acids glycine, serine, alanine,
proline, asparagine, glutamine, and lysine. In some embodiments, a
linker is made up of a majority of amino acids that are sterically
unhindered, such as glycine and alanine. In some embodiments,
linkers are polyglycines, polyalanines, combinations of glycine and
alanine (such as poly(Gly-Ala)), or combinations of glycine and
serine (such as poly(Gly-Ser)). While a linker of 15 amino acid
residues has been found to work particularly well for FGF21 fusion
proteins, the present invention contemplates linkers of any length
or composition.
[0173] The linkers described herein are exemplary, and linkers that
are much longer and which include other residues are contemplated
by the present invention. Non-peptide linkers are also contemplated
by the present invention. For example, alkyl linkers such as can be
used. These alkyl linkers can further be substituted by any
non-sterically hindering group, including, but not limited to, a
lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g., Cl, Br), CN,
NH2, or phenyl. An exemplary non-peptide linker is a polyethylene
glycol linker, wherein the linker has a molecular weight of 100 to
5000 kD, for example, 100 to 500 kD.
Chemically-Modified FGF21 Mutants
[0174] Chemically modified forms of the FGF21 protein variants
described herein, including the truncated forms of FGF21 described
herein, can be prepared by one skilled in the art, given the
disclosures described herein. Such chemically modified FGF21
mutants are altered such that the chemically modified FGF21 mutant
is different from the unmodified FGF21 mutant, either in the type
or location of the molecules naturally attached to the FGF21
mutant. Chemically modified FGF21 mutants can include molecules
formed by the deletion of one or more naturally-attached chemical
groups.
[0175] In one embodiment, FGF21 protein variants of the present
invention can be modified by the covalent attachment of one or more
polymers. For example, the polymer selected is typically
water-soluble so that the protein to which it is attached does not
precipitate in an aqueous environment, such as a physiological
environment. Included within the scope of suitable polymers is a
mixture of polymers. Preferably, for therapeutic use of the
end-product preparation, the polymer will be pharmaceutically
acceptable. Non-water soluble polymers conjugated to FGF21 protein
variants of the present invention also form an aspect of the
invention.
[0176] Exemplary polymers each can be of any molecular weight and
can be branched or unbranched. The polymers each typically have an
average molecular weight of between about 2 kDa to about 100 kDa
(the term "about" indicating that in preparations of a
water-soluble polymer, some molecules will weigh more and some less
than the stated molecular weight). The average molecular weight of
each polymer is preferably between about 5 kDa and about 50 kDa,
more preferably between about 12 kDa and about 40 kDa, and most
preferably between about 20 kDa and about 35 kDa.
[0177] Suitable water-soluble polymers or mixtures thereof include,
but are not limited to, N-linked or O-linked carbohydrates, sugars,
phosphates, polyethylene glycol (PEG) (including the forms of PEG
that have been used to derivatize proteins, including
mono-(C1-C10), alkoxy-, or aryloxy-polyethylene glycol),
monomethoxy-polyethylene glycol, dextran (such as low molecular
weight dextran of, for example, about 6 kD), cellulose, or other
carbohydrate based polymers, poly-(N-vinyl pyrrolidone)
polyethylene glycol, propylene glycol homopolymers, polypropylene
oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g.,
glycerol), and polyvinyl alcohol. Also encompassed by the present
invention are bifunctional crosslinking molecules that can be used
to prepare covalently attached FGF21 protein variant multimers.
Also encompassed by the present invention are FGF21 mutants
covalently attached to polysialic acid.
[0178] In some embodiments of the present invention, an FGF21
mutant is covalently, or chemically, modified to include one or
more water-soluble polymers, including, but not limited to,
polyethylene glycol (PEG), polyoxyethylene glycol, or polypropylene
glycol. See, e.g., U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192; and 4,179,337. In some embodiments of the
present invention, an FGF21 mutant comprises one or more polymers,
including, but not limited to, monomethoxy-polyethylene glycol,
dextran, cellulose, another carbohydrate-based polymer,
poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol
homopolymers, a polypropylene oxide/ethylene oxide co-polymer,
polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, or
mixtures of such polymers.
[0179] In some embodiments of the present invention, an FGF21
mutant is covalently-modified with PEG subunits. In some
embodiments, one or more water-soluble polymers are bonded at one
or more specific positions (for example, at the N-terminus) of the
FGF21 mutant. In some embodiments, one or more water-soluble
polymers are randomly attached to one or more side chains of an
FGF21 mutant. In some embodiments, PEG is used to improve the
therapeutic capacity of an FGF21 mutant. Certain such methods are
discussed, for example, in U.S. Pat. No. 6,133,426, which is hereby
incorporated by reference for any purpose.
[0180] In embodiments of the present invention wherein the polymer
is PEG, the PEG group can be of any convenient molecular weight,
and can be linear or branched. The average molecular weight of the
PEG group will preferably range from about 2 kD to about 100 kDa,
and more preferably from about 5 kDa to about 50 kDa, e.g., 10, 20,
30, 40, or 50 kDa. The PEG groups will generally be attached to the
FGF21 mutant via acylation or reductive alkylation through a
reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol,
or ester group) to a reactive group on the FGF21 mutant (e.g., an
aldehyde, amino, or ester group).
[0181] Branched PEG derivatives, also known as "Y-shaped" PEG
derivatives, contain two linear methoxy PEG chain attached to a
central core. The sterically bulky structure of these "Y-shaped"
PEG derivatives will facilitate the single point attachment of the
modified molecules. By way of example, three kinds of "Y-shaped"
PEG derivatives are Y-NHS-40K (useful for amine PEGylation);
Y-MAL-40K (useful for thiol PEGylation); and Y-ALD-40K (e.g.,
Y-AALD-40K and Y-PALD-40K)(useful for N-terminal PEGylation). For
amine PEGylation, the "Y-shape" NHS ester will react with the amino
group of lysine(s) or the N-terminal amine in biological active
molecules to produce a stable amide linkage(s). This NHS ester will
couple with the targeted molecules at pH 7-8. For thiol PEGylation,
the "Y-shape" maleimide will react with the thiol groups in
biological active molecules to generates a stable
3-thiosuccinimidyl ether linkage. This maleimide will couple with
the targeted molecules at pH 5.0-6.5 in the presence of other
functional groups. For N-terminal PEGylation, The "Y-shape"
aldehyde will preferably react with the N-terminal amine in
biological active molecules to produce a stable amine linkage in
the presence of a reducing reagent such as sodium cyanoborohydride.
This aldehyde will couple with the N-terminal amine of the targeted
molecules at pH 5-8. Reagents for performing branched PEGylation
are available through, e.g., JenKem Technology.
[0182] The PEGylation of a polypeptide, including the FGF21 mutants
of the present invention, can be specifically carried out using any
of the PEGylation reactions known in the art. Such reactions are
described, for example, in the following references: Francis et
al., 1992, Focus on Growth Factors 3: 4-10; European Patent Nos. 0
154 316 and 0 401 384; and U.S. Pat. No. 4,179,337. For example,
PEGylation can be carried out via an acylation reaction or an
alkylation reaction with a reactive polyethylene glycol molecule
(or an analogous reactive water-soluble polymer) as described
herein. For the acylation reactions, a selected polymer should have
a single reactive ester group. For reductive alkylation, a selected
polymer should have a single reactive aldehyde group. A reactive
aldehyde is, for example, polyethylene glycol propionaldehyde,
which is water stable, or mono C1-C10 alkoxy or aryloxy derivatives
thereof (see, e.g., U.S. Pat. No. 5,252,714).
[0183] In some embodiments of the present invention, a useful
strategy for the attachment of the PEG group to a polypeptide
involves combining, through the formation of a conjugate linkage in
solution, a peptide and a PEG moiety, each bearing a special
functionality that is mutually reactive toward the other. The
peptides can be easily prepared with conventional solid phase
synthesis. The peptides are "preactivated" with an appropriate
functional group at a specific site. The precursors are purified
and fully characterized prior to reacting with the PEG moiety.
Ligation of the peptide with PEG usually takes place in aqueous
phase and can be easily monitored by reverse phase analytical HPLC.
The PEGylated peptides can be easily purified by preparative HPLC
and characterized by analytical HPLC, amino acid analysis and laser
desorption mass spectrometry.
[0184] Polysaccharide polymers are another type of water-soluble
polymer that can be used for protein modification. Therefore, the
FGF21 mutants of the present invention fused to a polysaccharide
polymer form embodiments of the present invention. Dextrans are
polysaccharide polymers comprised of individual subunits of glucose
predominantly linked by alpha 1-6 linkages. The dextran itself is
available in many molecular weight ranges, and is readily available
in molecular weights from about 1 kD to about 70 kD. Dextran is a
suitable water-soluble polymer for use as a vehicle by itself or in
combination with another vehicle (e.g., Fc). See, e.g.,
International Publication No. WO 96/11953. The use of dextran
conjugated to therapeutic or diagnostic immunoglobulins has been
reported. See, e.g., European Patent Publication No. 0 315 456,
which is hereby incorporated by reference. The present invention
also encompasses the use of dextran of about 1 kD to about 20
kD.
[0185] In general, chemical modification can be performed under any
suitable condition used to react a protein with an activated
polymer molecule. Methods for preparing chemically modified
polypeptides will generally comprise the steps of: (a) reacting the
polypeptide with the activated polymer molecule (such as a reactive
ester or aldehyde derivative of the polymer molecule) under
conditions whereby a FGF21 protein variant becomes attached to one
or more polymer molecules, and (b) obtaining the reaction products.
The optimal reaction conditions will be determined based on known
parameters and the desired result. For example, the larger the
ratio of polymer molecules to protein, the greater the percentage
of attached polymer molecule. In one embodiment of the present
invention, chemically modified FGF21 mutants can have a single
polymer molecule moiety at the amino-terminus (see, e.g., U.S. Pat.
No. 5,234,784)
[0186] In another embodiment of the present invention, FGF21
protein variants can be chemically coupled to biotin. The
biotin/FGF21 protein variants are then allowed to bind to avidin,
resulting in tetravalent avidin/biotin/FGF21 protein variants.
FGF21 protein variants can also be covalently coupled to
dinitrophenol (DNP) or trinitrophenol (TNP) and the resulting
conjugates precipitated with anti-DNP or anti-TNP-IgM to form
decameric conjugates with a valency of 10.
[0187] Generally, conditions that can be alleviated or modulated by
the administration of the present chemically modified FGF21 mutants
include those described herein for FGF21 protein variants. However,
the chemically modified FGF21 mutants disclosed herein can have
additional activities, enhanced or reduced biological activity, or
other characteristics, such as increased or decreased half-life, as
compared to unmodified FGF21 mutants.
[0188] Therapeutic Compositions of FGF21 Mutants and Administration
Thereof
[0189] Therapeutic compositions comprising FGF21 mutants are within
the scope of the methods of the present invention, and are
specifically contemplated in light of the identification of several
mutant FGF21 sequences exhibiting enhanced properties. Such FGF21
mutant pharmaceutical compositions can comprise a therapeutically
effective amount of an FGF21 protein variant in admixture with a
pharmaceutically or physiologically acceptable formulation agent
selected for suitability with the mode of administration.
[0190] Acceptable formulation materials preferably are nontoxic to
recipients at the dosages and concentrations employed.
[0191] The pharmaceutical composition can contain formulation
materials for modifying, maintaining, or preserving, for example,
the pH, osmolarity, viscosity, clarity, color, isotonicity, odor,
sterility, stability, rate of dissolution or release, adsorption,
or penetration of the composition. Suitable formulation materials
include, but are not limited to, amino acids (such as glycine,
glutamine, asparagine, arginine, or lysine), antimicrobials,
antioxidants (such as ascorbic acid, sodium sulfite, or sodium
hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl,
citrates, phosphates, or other organic acids), bulking agents (such
as mannitol or glycine), chelating agents (such as ethylenediamine
tetraacetic acid (EDTA)), complexing agents (such as caffeine,
polyvinylpyrrolidone, beta-cyclodextrin, or
hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,
disaccharides, and other carbohydrates (such as glucose, mannose,
or dextrins), proteins (such as serum albumin, gelatin, or
immunoglobulins), coloring, flavoring and diluting agents,
emulsifying agents, hydrophilic polymers (such as
polyvinylpyrrolidone), low molecular weight polypeptides,
salt-forming counterions (such as sodium), preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, chlorhexidine,
sorbic acid, or hydrogen peroxide), solvents (such as glycerin,
propylene glycol, or polyethylene glycol), sugar alcohols (such as
mannitol or sorbitol), suspending agents, surfactants or wetting
agents (such as pluronics; PEG; sorbitan esters; polysorbates such
as polysorbate 20 or polysorbate 80; triton; tromethamine;
lecithin; cholesterol or tyloxapal), stability enhancing agents
(such as sucrose or sorbitol), tonicity enhancing agents (such as
alkali metal halides; preferably sodium or potassium chloride; or
mannitol sorbitol), delivery vehicles, diluents, excipients and/or
pharmaceutical adjuvants (see, e.g., Remington's Pharmaceutical
Sciences (18th Ed., A. R. Gennaro, ed., Mack Publishing Company
1990), and subsequent editions of the same, incorporated herein by
reference for any purpose).
[0192] The optimal pharmaceutical composition will be determined by
a skilled artisan depending upon, for example, the intended route
of administration, delivery format, and desired dosage (see, e.g.,
Remington's Pharmaceutical Sciences, supra). Such compositions can
influence the physical state, stability, rate of in vivo release,
and rate of in vivo clearance of the FGF21 protein variant.
[0193] The primary vehicle or carrier in a pharmaceutical
composition can be either aqueous or non-aqueous in nature. For
example, a suitable vehicle or carrier for injection can be water,
physiological saline solution, or artificial cerebrospinal fluid,
possibly supplemented with other materials common in compositions
for parenteral administration. Neutral buffered saline or saline
mixed with serum albumin are further exemplary vehicles. Other
exemplary pharmaceutical compositions comprise Tris buffer of about
pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can
further include sorbitol or a suitable substitute. In one
embodiment of the present invention, FGF21 protein variant
compositions can be prepared for storage by mixing the selected
composition having the desired degree of purity with optional
formulation agents (Remington's Pharmaceutical Sciences, supra) in
the form of a lyophilized cake or an aqueous solution. Further, the
FGF21 protein variant product can be formulated as a lyophilizate
using appropriate excipients such as sucrose.
[0194] The FGF21 protein variant pharmaceutical compositions can be
selected for parenteral delivery. Alternatively, the compositions
can be selected for inhalation or for delivery through the
digestive tract, such as orally. The preparation of such
pharmaceutically acceptable compositions is within the skill of the
art.
[0195] The formulation components are present in concentrations
that are acceptable to the site of administration. For example,
buffers are used to maintain the composition at physiological pH or
at a slightly lower pH, typically within a pH range of from about 5
to about 8.
[0196] When parenteral administration is contemplated, the
therapeutic compositions for use in this invention can be in the
form of a pyrogen-free, parenterally acceptable, aqueous solution
comprising the desired FGF21 protein variant in a pharmaceutically
acceptable vehicle. A particularly suitable vehicle for parenteral
injection is sterile distilled water in which an FGF21 protein
variant is formulated as a sterile, isotonic solution, properly
preserved. Yet another preparation can involve the formulation of
the desired molecule with an agent, such as injectable
microspheres, bio-erodible particles, polymeric compounds (such as
polylactic acid or polyglycolic acid), beads, or liposomes, that
provides for the controlled or sustained release of the product
which can then be delivered via a depot injection. Hyaluronic acid
can also be used, and this can have the effect of promoting
sustained duration in the circulation. Other suitable means for the
introduction of the desired molecule include implantable drug
delivery devices.
[0197] In one embodiment, a pharmaceutical composition can be
formulated for inhalation. For example, an FGF21 protein variant
can be formulated as a dry powder for inhalation. FGF21 protein
variant inhalation solutions can also be formulated with a
propellant for aerosol delivery. In yet another embodiment,
solutions can be nebulized. Pulmonary administration is further
described in International Publication No. WO 94/20069, which
describes the pulmonary delivery of chemically modified
proteins.
[0198] It is also contemplated that certain formulations can be
administered orally. In one embodiment of the present invention,
FGF21 protein variants that are administered in this fashion can be
formulated with or without those carriers customarily used in the
compounding of solid dosage forms such as tablets and capsules. For
example, a capsule can be designed to release the active portion of
the formulation at the point in the gastrointestinal tract when
bioavailability is maximized and pre-systemic degradation is
minimized. Additional agents can be included to facilitate
absorption of the FGF21 protein variant. Diluents, flavorings, low
melting point waxes, vegetable oils, lubricants, suspending agents,
tablet disintegrating agents, and binders can also be employed.
[0199] Another pharmaceutical composition can involve an effective
quantity of FGF21 protein variants in a mixture with non-toxic
excipients that are suitable for the manufacture of tablets. By
dissolving the tablets in sterile water, or another appropriate
vehicle, solutions can be prepared in unit-dose form. Suitable
excipients include, but are not limited to, inert diluents, such as
calcium carbonate, sodium carbonate or bicarbonate, lactose, or
calcium phosphate; or binding agents, such as starch, gelatin, or
acacia; or lubricating agents such as magnesium stearate, stearic
acid, or talc.
[0200] Additional FGF21 protein variant pharmaceutical compositions
will be evident to those skilled in the art, including formulations
involving FGF21 protein variants in sustained- or
controlled-delivery formulations. Techniques for formulating a
variety of other sustained- or controlled-delivery means, such as
liposome carriers, bio-erodible microparticles or porous beads and
depot injections, are also known to those skilled in the art (see,
e.g., International Publication No. WO 93/15722, which describes
the controlled release of porous polymeric microparticles for the
delivery of pharmaceutical compositions, and Wischke &
Schwendeman, 2008, Int. J Pharm. 364: 298-327, and Freiberg &
Zhu, 2004, Int. J Pharm. 282: 1-18, which discuss
microsphere/microparticle preparation and use).
[0201] Additional examples of sustained-release preparations
include semipermeable polymer matrices in the form of shaped
articles, e.g. films, or microcapsules. Sustained release matrices
can include polyesters, hydrogels, polylactides (U.S. Pat. No.
3,773,919 and European Patent No. 0 058 481), copolymers of
L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983,
Biopolymers 22: 547-56), poly(2-hydroxyethyl-methacrylate) (Langer
et al., 1981, J. Biomed. Mater. Res. 15: 167-277 and Langer, 1982,
Chem. Tech. 12: 98-105), ethylene vinyl acetate (Langer et al.,
supra) or poly-D-3-hydroxybutyric acid (European Patent No. 0 133
988). Sustained-release compositions can also include liposomes,
which can be prepared by any of several methods known in the art.
See, e.g., Epstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:
3688-92; and European Patent Nos. 0 036 676, 0 088 046, and 0 143
949.
[0202] The FGF21 protein variant pharmaceutical composition to be
used for in vivo administration typically must be sterile. This can
be accomplished by filtration through sterile filtration membranes.
Where the composition is lyophilized, sterilization using this
method can be conducted either prior to, or following,
lyophilization and reconstitution. The composition for parenteral
administration can be stored in lyophilized form or in a solution.
In addition, parenteral compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0203] Once the pharmaceutical composition has been formulated, it
can be stored in sterile vials as a solution, suspension, gel,
emulsion, solid, or as a dehydrated or lyophilized powder. Such
formulations can be stored either in a ready-to-use form or in a
form (e.g., lyophilized) requiring reconstitution prior to
administration.
[0204] In a specific embodiment, the present invention is directed
to kits for producing a single-dose administration unit. The kits
can each contain both a first container having a dried protein and
a second container having an aqueous formulation. Also included
within the scope of this invention are kits containing single and
multi-chambered pre-filled syringes (e.g., liquid syringes and
lyosyringes).
[0205] The effective amount of an FGF21 protein variant
pharmaceutical composition to be employed therapeutically will
depend, for example, upon the therapeutic context and objectives.
One skilled in the art will appreciate that the appropriate dosage
levels for treatment will thus vary depending, in part, upon the
molecule delivered, the indication for which the FGF21 protein
variant is being used, the route of administration, and the size
(body weight, body surface, or organ size) and condition (the age
and general health) of the patient. Accordingly, the clinician can
titer the dosage and modify the route of administration to obtain
the optimal therapeutic effect. A typical dosage can range from
about 0.1 .mu.g/kg to up to about 100 mg/kg or more, depending on
the factors mentioned above. In other embodiments, the dosage can
range from 0.1 .mu.g/kg up to about 100 mg/kg; or 1 .mu.g/kg up to
about 100 mg/kg; or 5 .mu.g/kg, 10 .mu.g/kg, 15 .mu.g/kg, 20
.mu.g/kg, 25 .mu.g/kg, 30 .mu.g/kg, 35 .mu.g/kg, 40 .mu.g/kg, 45
.mu.g/kg, 50 .mu.g/kg, 55 .mu.g/kg, 60 .mu.g/kg, 65 .mu.g/kg, 70
.mu.g/kg, 75 .mu.g/kg, up to about 100 mg/kg. In yet other
embodiments, the dosage can be 50 .mu.g/kg, 100 .mu.g/kg, 150
.mu.g/kg, 200 .mu.g/kg, 250 .mu.g/kg, 300 .mu.g/kg, 350 .mu.g/kg,
400 .mu.g/kg, 450 .mu.g/kg, 500 .mu.g/kg, 550 .mu.g/kg, 600
.mu.g/kg, 650 .mu.g/kg, 700 .mu.g/kg, 750 .mu.g/kg, 800 .mu.g/kg,
850 .mu.g/kg, 900 .mu.g/kg, 950 .mu.g/kg, 100 .mu.g/kg, 200
.mu.g/kg, 300 .mu.g/kg, 400 .mu.g/kg, 500 .mu.g/kg, 600 .mu.g/kg,
700 .mu.g/kg, 800 .mu.g/kg, 900 .mu.g/kg, 1000 .mu.g/kg, 2000
.mu.g/kg, 3000 .mu.g/kg, 4000 .mu.g/kg, 5000 .mu.g/kg, 6000
.mu.g/kg, 7000 .mu.g/kg, 8000 .mu.g/kg, 9000 .mu.g/kg or 10
mg/kg.
[0206] The frequency of dosing will depend upon the pharmacokinetic
parameters of the FGF21 protein variant in the formulation being
used. Typically, a clinician will administer the composition until
a dosage is reached that achieves the desired effect. The
composition can therefore be administered as a single dose, as two
or more doses (which may or may not contain the same amount of the
desired molecule) over time, or as a continuous infusion via an
implantation device or catheter. Further refinement of the
appropriate dosage is routinely made by those of ordinary skill in
the art and is within the ambit of tasks routinely performed by
them. Appropriate dosages can be ascertained through use of
appropriate dose-response data.
[0207] The route of administration of the pharmaceutical
composition is in accord with known methods, e.g., orally; through
injection by intravenous, intraperitoneal, intracerebral
(intraparenchymal), intracerebroventricular, intramuscular,
intraocular, intraarterial, intraportal, or intralesional routes;
by sustained release systems (which may also be injected); or by
implantation devices. Where desired, the compositions can be
administered by bolus injection or continuously by infusion, or by
implantation device.
[0208] Alternatively or additionally, the composition can be
administered locally via implantation of a membrane, sponge, or
other appropriate material onto which the desired molecule has been
absorbed or encapsulated. Where an implantation device is used, the
device can be implanted into any suitable tissue or organ, and
delivery of the desired molecule can be via diffusion,
timed-release bolus, or continuous administration.
Therapeutic Uses of FGF21
[0209] FGF21 polypeptide or protein, or mutants, variants, and
fusions thereof, can be used to treat, diagnose, ameliorate, or
prevent a number of diseases, disorders, or conditions, including,
but not limited to treating metabolic diseases associated with
insulin resistance, such as type 2 diabetes mellitus, insulin
receptor mutation disorders (INSR disorders), nonalcoholic fatty
liver disease (NAFLD) and various forms of partial lipodystrophy
including familial partial lipodystrophy and HIV-HAART induced
partial lipodystrophy as well as diseases associated with insulin
production (type 1 diabetes mellitus), and in reducing the
mortality and morbidity of critically ill patients.
[0210] A disorder or condition such as type 1 diabetes mellitus or
insulin receptor mutation disorders can be treated by administering
an FGF21 polypeptide or protein, or mutant, variant, or fusion
thereof to a patient in need thereof in the amount of a
therapeutically effective dose. The administration can be performed
as described herein, such as by IV injection, intraperitoneal
injection, intramuscular injection, or orally in the form of a
tablet or liquid formation. In most situations, a desired dosage
can be determined by a clinician, as described herein, and can
represent a therapeutically effective dose of the FGF21. It will be
apparent to those of skill in the art that a therapeutically
effective dose of FGF21 will depend, inter alia, upon the
administration schedule, the unit dose of antigen administered,
whether the nucleic acid molecule or polypeptide is administered in
combination with other therapeutic agents, the immune status and
the health of the recipient. The term "therapeutically effective
dose," as used herein, means that amount of FGF21 mutant
polypeptide that elicits the biological or medicinal response in a
tissue system, animal, or human being sought by a researcher,
medical doctor, or other clinician, which includes alleviation of
the symptoms of the disease or disorder being treated.
Pharmaceutical Compositions
[0211] The present invention also provides methods comprising
pharmaceutical compositions comprising one or more of the FGF21
polypeptide or protein, or mutants, variants, and fusions thereof
described herein and a pharmaceutically acceptable carrier. In some
embodiments the pharmaceutical compositions are prepared as
injectables, either as liquid solutions or suspensions; solid forms
suitable for solution in, or suspension in, liquid vehicles prior
to injection can also be prepared. Liposomes are included within
the definition of a pharmaceutically acceptable carrier.
Pharmaceutically acceptable salts can also be present in the
pharmaceutical composition, e.g., mineral acid salts such as
hydrochlorides, hydrobromides, phosphates, sulfates, and the like;
and the salts of organic acids such as acetates, propionates,
malonates, benzoates, and the like. A thorough discussion of
pharmaceutically acceptable excipients is available in Remington:
The Science and Practice of Pharmacy (1995) Alfonso Gennaro,
Lippincott, Williams, & Wilkins.
Fusion Proteins and FGF21-Derived Peptidic Compounds
[0212] In another embodiment, the methods of the present invention
comprise FGF21 proteins, variants, or mutants which can be made
into a fusion protein or peptidic compound derived from the FGF21
amino acid sequences. Such fusion proteins and peptidic compounds
can be made using standard techniques known in the art. For
example, peptidic compounds can be made by chemical synthesis using
standard peptide synthesis techniques and then introduced into
cells by a variety of means known in the art for introducing
peptides into cells (e.g., liposome and the like).
[0213] The in vivo half-life of the fusion protein or peptidic
compounds of the invention can be improved by making peptide
modifications, such as the addition of N-linked glycosylation sites
into FGF21 proteins, variants, or mutants, or conjugating FGF21
proteins, variants, or mutants to poly(ethylene glycol)(PEG;
pegylation), e.g., via lysine-monopegylation or
cysteine-monopegylation. Such techniques have proven to be
beneficial in prolonging the half-life of therapeutic protein
drugs. It is expected that pegylation of the FGF21 proteins,
variants, or mutants comprising the methods of the invention may
result in similar pharmaceutical advantages.
[0214] In addition, pegylation can be achieved in any part of an
FGF21 proteins, variants, or mutants comprising the methods of the
invention by the introduction of a nonnatural amino acid. Certain
nonnatural amino acids can be introduced by the technology
described in Deiters et al., J Am Chem Soc 125:11782-11783, 2003;
Wang and Schultz, Science 301:964-967, 2003; Wang et al., Science
292:498-500, 2001; Zhang et al., Science 303:371-373, 2004 or in
U.S. Pat. No. 7,083,970. Briefly, some of these expression systems
involve site-directed mutagenesis to introduce a nonsense codon,
such as an amber TAG, into the open reading frame encoding a
polypeptide of the invention. Such expression vectors are then
introduced into a host that can utilize a tRNA specific for the
introduced nonsense codon and charged with the nonnatural amino
acid of choice. Particular nonnatural amino acids that are
beneficial for purpose of conjugating moieties to the polypeptides
of the invention include those with acetylene and azido side
chains. The FGF21 proteins, variants, or mutants comprising the
methods of the invention containing these novel amino acids can
then be pegylated at these chosen sites in the protein.
EXAMPLES
Example 1
In Vivo Administration of FGF21 in Type 1 Diabetes Mouse Models
[0215] Human type 1 diabetes (T1D) exhibits high plasma glucose and
low insulin levels due to the inability of pancreatic .beta.-cell
to produce and secrete insulin. As a consequence of a very low
insulin level in circulation, patients with T1D have reduced
utilization of carbohydrate and increased utilization of lipid,
which leads to loss of body fat storage and ketosis.
[0216] High dosages of the .beta.-cell toxin streptozocin (STZ)
induce severe insulin deficiency and T1D with ketosis and
hyperphagia. When high dose STZ is injected to adult animals,
.beta.-cell regeneration is diminished and animals remain in T1D
condition.
[0217] Diabetes was induced in twenty-two-week old male C57BL mice
via an intraperitoneal injection of STZ for 3 consecutive days at
70 mg/kg/day. When being fully diabetic (12-19 days from the first
STZ injection), in two separate studies, the mice received either
vehicle (PBS) or wild type FGF21 (3 mg/kg/day, subcutaneous
injection) for 12 days, or vehicle or Variant 76 two times a week
(1 mg or 3 mg/kg, 2.times./wk, subcutaneous injection) for 26 days.
One group of non-STZ mice was used as normal control for each
study. All the animals were acclimated in TSE system for at least
24 h before the RER and energy expenditure measurements.
[0218] Groups of mice in Study 1: Normal-vehicle (n=6); STZ-vehicle
(n=6); STZ-wild type FGF21 (n=6). Groups of mice in Study 2:
Normal-vehicle (n=8); STZ-vehicle (n=8); STZ-Cmpd A 1 mg/kg (n=8);
STZ-Cmpd A 3 mg/kg (n=8). The following measurements were taken:
glucose, insulin, HbA1c, respiratory exchange ratio (RER), energy
expenditure, food intake, body weight, epididymal fat mass, and
ketone body levels. As seen in Tables 1 and 2 below, STZ treated
mice became hyperglycemic and hypoinsulinemic.
[0219] In seen in Tables 2 and 3 and FIGS. 1-7, the following were
observed after administering wild type FGF21 and half-life-extended
FGF21 (Variant 76): a decrease of hyperglycemia in STZ-induced T1D
mice but without restoration of insulin secretion (FIG. 1 and
Tables 2 and 3). Variant 76 also decreased HbA1c level during the
study (FIG. 2). Both wild type FGF21 and Variant 76 increased
carbohydrate utilization (as seen in FIG. 3) and energy expenditure
(as seen in FIG. 4), reduced hyperphagia (as seen in FIG. 5),
ketogenesis (as seen in FIG. 6) and loss of fat mass (as seen in
FIG. 7).
TABLE-US-00002 TABLE 2 Body weight, glucose and insulin levels
before (day 1) and after treatment (day 12) with wild type FGF21 at
3 mg/kg with daily dosing in Study 1. Body weight (g) Glucose
(mg/dl) Insulin (ng/ml) Group day 1 day 12 day 1 day 12 day 1 day
12 Non-STZ 27.9 .+-. 0.8 28.6 .+-. 0.7 216.8 .+-. 13.6 125.2 .+-.
0.9 3.02 .+-. 0.20 2.03 .+-. 0.52 STZ 27.8 .+-. 1.5 27.1 .+-. 1.4
548.0 .+-. 24.4 521.2 .+-. 35.5 0.92 .+-. 0.20 0.48 .+-. 0.16 STZ-
26.7 .+-. 0.7 27.3 .+-. 0.9 540.3 .+-. 23.2 320.5 .+-. 69.3* 0.95
.+-. 0.34 0.52 .+-. 0.14 FGF21 Values are means .+-. SEM for n = 6
per group. Glucose and insulin were measured in a fed condition.
*indicates p < 0.05 vs. STZ group by one-way ANOVA.
TABLE-US-00003 TABLE 3 Body weight, glucose and insulin levels
before (day 1) and after treatment (day 26) with Variant 76 at 1 or
3 mg/kg with 2x/week dosing in Study 2. Body weight (g) Glucose
(mg/dl) Insulin (ng/ml) Group day 1 day 26 day 1 day 26 day 1 day
26 Non-STZ 32.5 .+-. 1.0 31.3 .+-. 0.9 164.8 .+-. 9.2 159.1 .+-.
5.8 1.96 .+-. 0.53 1.52 .+-. 0.30 STZ 28.6 .+-. 0.4 27.2 .+-. 0.5
452.3 .+-. 38.2 595.9 .+-. 69.7 0.49 .+-. 0.13 0.45 .+-. 0.06
STZ-Cmpd A-1 29.0 .+-. 0.3 27.2 .+-. 0.5 453.1 .+-. 40.0 306.3 .+-.
62.4* 0.44 .+-. 0.08 0.34 .+-. 0.05 STZ-Cmpd A-3 29.0 .+-. 0.5 26.8
.+-. 0.3 452.1 .+-. 32.8 218.3 .+-. 13.3* 0.66 .+-. 0.11 0.33 .+-.
0.04 Values are means .+-. SEM for n = 8 per group. Glucose and
insulin were measured in a fed condition. *indicates p < 0.05
vs. STZ group by one-way ANOVA.
Example 2
Type B Insulin Resistance: FGF21 Stimulates Glucose Uptake in Human
Adipocytes in the Presence of Anti-Insulin Receptor Monoclonal
Antibody
[0220] INSR knockout mice are not viable and typically die a few
days after birth. While tissue specific knockout of INSR in mice is
tolerated, the full spectrum of symptoms seen in patients with INSR
mutation is not replicated in these animals. Thus, to further
validate the clinical hypothesis that FGF21 can play a role in in
the context of INSR inactivation, we simply measured the ability of
FGF21 to promote glucose uptake in human adipocytes in the presence
of a neutralizing anti-INSR antibody under conditions where insulin
signaling is significantly impaired. This experiment effectively
replicates the phenotype of Type-B insulin resistance (autoimmune
insulin resistance), which is caused by development of neutralizing
auto-antibodies to INSR.
[0221] Measurement of glucose uptake in differentiated human
adipocytes is a widely used physiologically relevant readout. FGF21
exhibits glucose uptake in an insulin independent manner in human
adipocytes. To study an INSR dysfunctional state in vitro, we
tested the activity of insulin and FGF21 to stimulate glucose
uptake in the absence and presence of an anti-INSR monoclonal
antibody (Millipore Catalogue # MAB1137).
[0222] Primary human adipocytes were seeded in a 96-well collagen
coated plate at 15,000 cells/well, differentiated in adipocyte
differentiation media (Cell Application) for 12 days, and then
treated for 3 days with adipocyte maintenance media (Cell
Application). For glucose uptake measurement, adipocytes were
treated with 100 nM insulin for 30-min or 100 nM FGF21 for 24 h at
37.degree. C. Adipocytes were washed with Krebs ringer phosphate
(KRP) buffer and treated with KRP containing 0.05 .mu.Ci of
2-deoxy-D-[3H] glucose (2-DOG) for 1 hr at 37.degree. C. Cells were
lysed and glucose uptake measured using MicroBeta counter (Perkin
Elmer). To study the glucose uptake of insulin or FGF21 in the
presence of anti-INSR antibody, the adipocytes were pretreated with
0.1 nM, 10 nM, and 100 nM of antibody for 1 h at 37.degree. C.
prior to insulin or FGF21 treatment.
[0223] As shown in FIG. 8A, 100 nM insulin on its own stimulated a
robust glucose uptake response in human adipocytes. In the presence
of 0.1 nM anti-INSR antibody, insulin-stimulated glucose uptake was
unaltered. However, pre-treatment with 10 nM and 100 nM of
anti-INSR antibody abolished insulin stimulated glucose uptake.
However as shown in FIG. 8B, 100 nM FGF21 stimulated glucose uptake
in the absence of anti-INSR antibody. Pre-treatment with anti-INSR
antibody at 0.1 nM and 10 nM did not significantly reduce (10% and
12% respectively) FGF21 stimulate glucose uptake. FGF21 activity
was only abolished in the presence at high concentration of
anti-INSR antibody, administered at 100 nM.
[0224] These data support use of an FGF21 analog in patients with
Type B insulin resistance (autoimmune insulin resistance) as well
as any with other insulin receptor deficiencies (e.g., caused by
mutations in the insulin receptor, such as Type A insulin
resistance, Rabson Mendonhall Syndrome and Donohue Syndrome).
Example 3
HIV HAART Induced Partial Liposystrophy
[0225] To study the effect of FGF21 in a model of lipodystrophy
induced by HIV highly active antiretroviral therapy (HAART),
12-week-old C57BL mice (Taconic) were provided with PicoLab mouse
diet #5058 (10% fat content) formulated by Research Diets with 0.1
or 0.2% HIV protease inhibitor Ritonavir. After 50 days of
Ritonavir treatment, mice developed lipodystrophy and were divided
into two subgroups, receiving either FGF21 V76 (5 mpk, s.c.) or PBS
vehicle 2.times./wk for 4 wks. Body weight, % body fat mass, plasma
glucose, insulin and TG were measured during the study. Oral
glucose tolerance test (OGTT) was assessed on treatment day 23.
Liver lipid content was measured at the termination of the
study.
[0226] Prior to V76 treatment, mice on diet mixed with Ritonavir
developed a significant reduction in body weight gain (FIG. 9A) and
fat content (FIG. 9B), as well as increased plasma triglyceride
levels (FIG. 9C) relative to mice on control diet.
[0227] Body weight was significantly reduced with FGF21 v76
treatment (FIG. 10A). Fat mass was also significantly reduced with
FGF21 v76 treatment (FIG. 10B). Mice treated with FGF21 v76 had
significantly improved glucose excursion during OGTT (FIG. 10C).
Ritonavir treatment resulted in increased HOMA-IR and liver lipid
content. Treatment with FGF21 v76 completely normalized HOMA-IR
(FIG. 10D) and liver steatosis (FIG. 10E) in both 0.1 and 0.2%
Ritonavir groups.
[0228] In the HIV HAART-induced partial lipodystrophy mouse model,
treatment with FGF21 v76 for 25 days (5 mpk, 2.times./wk)
completely alleviated insulin resistance and liver steatosis
induced by 75 days of diet mixture with Ritonavir. The preclinical
data support a novel approach of FGF21 therapy in patients
developed lipodystrophy by HIV protease inhibitors.
Example 4
t10, c12 Conjugated Linoleic Acid Induced Lipodystrophy
[0229] To study the effects of FGF21 V101 in a second more severe
lipodystrophy model, mice were fed a diet containing 0.6% t10, c12
conjugated linoleic acid (t10, c12 CLA). Mice were treated with
FGF21 V101 starting either 1) one week after diet initiation
("Prevention" mode) or 2) three weeks after diet initiation
("Reversal" mode). The effects of recombinant murine leptin were
also tested as a positive control in the "Reversal" mode. Body
weight, liver fat, adipose mass, glucose, insulin and plasma lipids
were the primary read-outs.
[0230] Animals:
[0231] C57B6J male mice were housed individually and acclimated to
facility for 6 weeks on normal chow prior to starting special diets
at 18 weeks of age. The mice were grouped into categories listed in
Table 5, below.
[0232] Diets:
[0233] Purified, t10, c12 CLA was synthesized by Matreya, LLC and
delivered to Research Diets, Inc. Purified custom diets will be
prepared fresh weekly by Research Diets, Inc. The t10, c12 CLA diet
contained 0.6% t10, c12 CLA. Daily rations were bagged, purged with
Nitrogen, vacuum-sealed, stored at -20.degree. C. and served daily
to the mice. Diet ingredients are itemized in Table 4 below.
TABLE-US-00004 TABLE 4 t10, c12 CLA diet Control Diet gm kcal gm
kcal % Protein 18.5 19.2 18.5 19.2 Carbohydrate 67.3 70.9 67.3 70.9
Fat 4.3 10.0 4.3 10.0 Total 100.0 100.0 kcal/gm 3.85 3.85
Ingredient Casein 192 768 192 768 L-Cystine 3 12 3 12 Corn Starch
325 1300 325 1300 Maltodextrin 35 140 35 140 Sucrose 350 1400 350
1400 Cellulose 48.7 48.7 t10, c12 CLA 6 54 0 Sunflower Oil 39 351
45 405 Mineral Mix S10026 10 10 DiCalcium Phosphate 13 13 Calcium
Carbonate 6 6 Potassium Citrate, 1 H2O 17 17 Vitamin Mix V10001 10
40 10 40 Choline Bitartrate 2 2 Total 1056 4065 1056 4065
[0234] After 2 week acclimation to facility mice were switched to
0.6% t10, c12 CLA diet or the control diet. One group of chow-fed
mice (GROUP 11 (see Table 4 for the specifics of this and the other
groups)) was sacrificed at study start for day zero measurements of
liver fat and adipose mass. In addition, a group of mice on the
t10, c12 CLA diet (GROUP 9) and a group of mice on the control diet
(GROUP 10) were sacrificed after one week on diet and also after
three weeks of diet (GROUPS 7 and 8) without any additional
treatments. Data for groups 7-10 are used to establish liver fat
and adipose mass prior to FGF21 v101 or leptin treatment. Another
group (GROUP 6) were maintained on the control diet until study end
to serve as a normal control for study endpoints.
[0235] One group of mice (GROUP 1) started FGF21 v101 treatment
after one week on the t10, c12 CLA diet to access whether FGF21 can
"prevent" the lipodystrophic progression. Another group of mice
(GROUP 2) started FGF21 v101 treatment after three weeks on the
t10, c12 CLA diet to access whether FGF21 v101 could "reverse" the
disease. The control group for FGF21 v101 (GROUP 3) received PBS
injections starting after three weeks on the t10, c12 CLA diet.
Thereafter GROUPS 1-3 received subcutaneous injections of PBS or
FGF21 once per week. After three weeks on diet, GROUP 4 and 5 were
implanted subcutaneously with Alzet osmotic minipumps to
continuously deliver Leptin or saline, respectively. Thereafter,
body weight, blood glucose, plasma insulin and plasma lipids (tail
bleed) were measured weekly for GROUPS 1-5.
[0236] After five weeks on diet, GROUPS 1-6 received an oral
glucose tolerance test (OGTT) (2 g glucose per kg) with glucose
measured 0, 10, 20, 40 and 90 min after glucose challenge. After
six weeks on diet, body weight, blood glucose, plasma insulin and
plasma lipid (tail bleed) were measured for GROUPS 1-6 prior to
sacrifice. Liver, adipose depots (epidydimal, retroperitoneal, and
inguinal), muscle (soleus and TA) and blood for plasma (cadiac
stick) were collected at sacrifice. Liver and adipose depots were
weighed. Liver and muscle tissues was analyzed for fat content
using a Bruker minispectrophotometer. Total liver fat (g) is
calculated as (liver weight.times.(% liver fat/100)).
[0237] Dose Administration:
[0238] FGF21 V101 was provided as a solution in PBS. 1 mg/kg
administered subcutaneously once per week (using a 0.25 mg/ml
solution of FGF21 V101).
[0239] Murine Leptin from Sigma, Cat# L3772, Lot#081M1287V,
recombinant expressed in E. coli. lyophilized powder dissolved in
PBS, was administered at 1 mg/kg/d dosage via continuous
subcutaneous infusion via Alzet osmotic minipump.
TABLE-US-00005 TABLE 5 Study Mice GROUP 1 = FGF21 prevention (n =
10) GROUP 2 = FGF21 reversal (n = 10) GROUP 3 = Vehicle Injection
(n = 10) GROUP 4 = Leptin Pump (n = 10) GROUP 5 = Vehicle Pump (n =
10) GROUP 6 = Control diet for study end (n = 10) GROUP 7 = 3 week
CLA time zero (n = 7) GROUP 8 = 3 week Control time zero (n = 7)
GROUP 9 = 1 week CLA time zero (n = 7) GROUP 10 = 1 week Control
time zero (n = 7) GROUP 11 = chow fed mice (n = 5)
[0240] Adipose Depots
[0241] The time course of loss in adipose mass is depicted in FIG.
11A. The weight of adipose depots (FIG. 11B) was measured as the
sum of epidydimal, retroperitoneal and subcutaneous inguinal fat.
These three depots were the only substantial depots detected since
we used normal mice which were fed a diet with a low fat content
(10% kcal from fat). Mice fed t10, c12 CLA diet (and treated with
PBS) lost approximately 85% of their adipose mass during the six
week study as compared to mice on Control Diet (1.6 g for Control
diet versus 0.27 g for CLA diet).
[0242] In the "prevention" mode, mice treated with FGF21 v101 lost
an additional 0.15 g adipose mass (P<0.05 by one-way ANOVA). In
the "reversal" mode, mice treated with FGF21 v101 lost an
additional 0.1 g adipose mass (not statistically significant).
Similarly mice treated with leptin lost an additional 0.1 g adipose
mass (not statistically significant). As expected, neither FGF21
v101 nor leptin treatment restored peripheral fat mass.
[0243] Liver Fat
[0244] At time of sacrifice, the weight of the liver was measured
(FIG. 12A). Small pieces of liver (approximately 40 mg) were
sampled for measurement of percent liver fat using a Bruker
minispectrophomoter tissue composition analyzer. Total liver fat
(g) was calculated as (liver weight.times.(% liver fat/100)) (FIG.
12B). Mice fed t10, c12 CLA diet (and treated with PBS),
accumulated 12-fold more fat in their livers during the six week
study as compared to mice on Control Diet (0.06 g for Control diet
versus 0.75 g for CLA diet, P<0.05 by one-way ANOVA). Liver fat
accumulation was reduced to half in mice treated with FGF21 (0.75 g
for PBS treated group, 0.34 g for FGF21 v101 "prevention" group,
0.36 g for FGF21 v101 "reversal" group.) Similarly, liver fat
accumulation was reduced in mice treated with leptin. (0.75 g for
PBS treated group, 0.48 g for leptin group.) All reductions in
liver fat were statistically significant versus PBS-treated groups
(P<0.05 by one-way ANOVA). The reduction in FGF21-treated groups
was not significantly different from the reduction in the
leptin-treated groups.
[0245] Fed Blood Glucose and Plasma Insulin
[0246] At week 6 of the study, mice fed t10, c12 CLA diet and
treated with PBS, had a 40% increase in fed blood glucose
(P<0.05 by one-way ANOVA) and a 20-fold increase in fed plasma
insulin (p<0.05 by one-way ANOVA) consistent with significant
insulin resistance and impaired glucose tolerance induced by the
t10, c12 CLA diet and lipodistrophy.
[0247] However, blood glucose was not elevated in mice on the CLA
diet treated with FGF21 v101, in either the "treatment" or
"prevention" modes. Blood glucose was partially elevated in mice on
the CLA diet who were treated with leptin (230 mg/dL for
PBS-treated group on CLA diet, 165 mg/dL for FGF21 "prevention"
group on CLA diet, 163 mg/dL for FGF21 "reversal" group on CLA
diet, 203 mg/dL for leptin group on CLA diet, 167 mg/dL for mice on
Control diet) (FIG. 12C).
[0248] Similarly, the increase in plasma insulin was markedly
blunted in mice on the CLA diet who were treated with FGF21, in
both the "treatment" or "prevention" modes and in mice treated with
leptin (6.7-8.7 ng/mL for PBS-treated groups on CLA diet, 1.8 ng/ml
for FGF21 "prevention" group on CLA diet, 2.2 ng/ml for FGF21
"reversal" group on CLA diet, 2.4 ng/ml for leptin group on CLA
diet, 0.3 ng/ml for mice on Control diet)(FIG. 12D).
[0249] Taken together, the elevation in blood glucose and plasma
insulin that resulted from CLA diet, indicates that CLA promoted
insulin resistance. The reduction in blood glucose and plasma
insulin in mice on CLA diet which resulted from treatment with
FGF21 (in both the "treatment" or "prevention") and treatment with
leptin, indicates that these treatments prevented and/or reversed
the CLA-induced insulin resistance.
[0250] Oral Glucose Tolerance Test
[0251] At week 5 of the study, an OGTT was performed after a 5 h
fast. Mice were dosed orally with glucose (2 g/kg, 8 ml/kg). Blood
glucose was measured before the glucose dose and 10, 20, 40 and 90
min after the glucose dose and the blood glucose AUC 0-90 was
calculated (FIG. 12E).
[0252] The blood glucose AUC 0-90 was 30% higher in mice fed t10,
c12 CLA diet (treated with PBS) as compared to mice fed the Control
diet. Blood glucose AUC in control mice and mice on the CLA diet
who were treated with FGF21, in either the "treatment" or
"prevention" modes was reduced compared to the PBS treated group on
the CLA diet.
SUMMARY
[0253] FGF21 V101 (1 mg/kg/wk) prevented and reversed liver lipid
accumulation and insulin resistance in a lipodystrophy model using
mice fed 0.6% t10, c12 conjugated linoleic acid. These data suggest
that and FGF21 analog could be useful for treatment of insulin
resistance and/or hyperglycemia induced by significant fat loss
(lipodystrophy).
Example 5
Effect of FGF21 Fusion Protein V103 in a Lipodystrophy Mouse
Model
[0254] Methods
[0255] Animals
[0256] Twenty-week old male C57BL mice (Taconic, Germantown, N.Y.)
were housed four per cage in a normal light cycle room (light on
from 6:00 a.m. to 6:00 p.m.) and given access to food and water ad
libitum. Lipodystrophy was induced by feeding mice a specially
formulated diet containing ritonavir (0.2% w/w) in PicoLab Mouse
Diet 20#5058 (21.6% kcal from fat). The mice in a control group
were given Diet 20#5058 without ritonavir. When the mice on
ritonavir diet became lipodystrophic (defined by increased plasma
triglyceride level and weight loss at day 50 on the diet), they
received either PBS (phosphate buffered saline) vehicle or FGF21
fusion protein V103, as described herein, (1 mg/kg, subcutaneous
injection) for a total of 3 injections given on days 1, 8 and 17 of
the treatment. Mice not on the ritonavir diet were also injected
with PBS during the study. The total treatment length of the study
was 25 days.
[0257] Experiments were conducted under an approved Institutional
Animal Care and Use Committee protocol 12CVM043. All procedures in
this study were in compliance with the Animal Welfare Act
Regulations 9 CFR Parts 1, 2 and 3, and other guidelines.
[0258] Measurements
[0259] Body weight and food intake were measured throughout the
study. Plasma glucose, insulin, triglyceride (TG) and free fatty
acid (FFA) concentrations were measured in a fed or fasted
condition. An insulin facilitated glucose tolerance test (IFGTT,
with an intraperitoneal (i.p.) injection of a mixture of glucose at
1 g/kg and insulin at 0.5 U/kg) was given on day 17 after 5 hours
of fast. Plasma glucose levels were measured before and during the
IFGTT. An oral glucose tolerance test (OGTT, with an oral dose of
glucose at 1 g/kg) was given on day 24 after an overnight fast.
Plasma glucose and insulin levels were measured before and during
the OGTT. Body composition was determined with Echo MRI before and
after the ritonavir diet induction, and at the end of V103
treatment (on day 24). At the termination of the study, liver,
epididymal fat and inguinal fat were collected and weighed. Liver
and soleus muscle collected at the end of the study were analyzed
for tissue lipid content.
[0260] Glucose was measured using a glucose meter (Embrace, Omnis
Health, Natick, Mass.). Insulin was measured using an ultra
sensitive mouse insulin enzyme-linked immunosorbent assay (ELISA)
kit (Crystal Chem, Inc., Chicago, Ill., cat#90080). TG and FFA were
measured using a highly sensitive fluorescent assay based on the
horseradish peroxidase catalyzed oxidation of Amplex.RTM. Red by
hydrogen peroxide to resorufin. Body composition was measured using
EchoMRI-100 (Echo Medical Systems, Huston, Tex.). Liver and muscle
lipid content was measured using NMR Mq-60 tissue fat analyzer
(BRUKER Optics Inc., The Woodlands. Tex.).
[0261] Data Analysis
[0262] Statistical analysis was performed using GraphPad Prism 5.0
(GraphPad Software, San Diego, Calif.). Time course analysis was
performed by a two-way analysis of variance (ANOVA) followed by a
post-hoc test using Bonferroni's method for each time point.
Analysis among the three groups without time course was conducted
using a one-way ANOVA followed by Dunnett's test. Analysis between
the two ritonavir groups was also performed using a non-paired and
two-tailed Student t-test. Data are presented as mean.+-.standard
error of the mean (SEM). The level of statistical significance was
set at p<0.05.
[0263] Results
[0264] Effect of Ritonavir on body weight and percent fat mass
[0265] Mice treated with Ritonavir for 8 weeks show a significant
body weight loss and mild reduction in fat mass in. Due to a high
fat content in both control and ritonavir diets, fat mass was more
than doubled in both diet groups at the end of the diet
treatment.
[0266] Effect of Ritonavir on plasma lipids, glucose and
insulin
[0267] Following the Ritonavir diet, there was a significant
increase in both plasma triglycerides (TG) and free fatty acids
(FFA). Plasma glucose and insulin were not affected by Ritonavir
treatment; however, insulin levels in both groups were much higher
on day 50 compared to the values on day 1, suggesting that the high
fat content of the diets led to insulin resistance in both
groups.
[0268] Effect of V103 on Body Weight and Percent Fat Mass
[0269] Treatment with V103 significantly reduced body weight (BW)
and percent fat mass. Food intake was not affected by the treatment
(data not shown). Body weight was measured during the V103
treatment, and percent fat mass was measured at the end of the
treatment (day 24). V103 treatment led to a significant weight loss
as well as a reduction in fat mass. #p<0.05 by a two-way ANOVA
compared to the Control-Vehicle group; *p<0.05 by a two-way
ANOVA (BW) or a one-way ANOVA (fat mass) compared to the
Ritonavir-Vehicle group.
[0270] Effect of V103 on Plasma Lipids, Glucose and Insulin
[0271] Treatment with V103 led to a significant reduction in plasma
TG and FFA in Ritonavir-treated mice to the levels similar to those
seen in the control diet group. Both plasma glucose and insulin
were significantly reduced in mice treated with V103. The insulin
level in the control diet group was elevated during the study,
indicating insulin resistance as a result of a high diet fat
content. In mice feed a standard chow diet, plasma insulin
concentration is normally around 1 ng/ml (RD-2005-50000).
[0272] Data are mean.+-.SEM. N=7-8 per group. Plasma TG and FFA
were measured in a fed condition at the end of the treatment (day
25). V103 treatment significantly reduced plasma TG and FFA levels.
#p<0.05 by a one-way ANOVA compared to the Control-Vehicle
group; *p<0.05 by a one-way ANOVA compared to the
Ritonavir-Vehicle group.
[0273] Data are mean.+-.SEM. N=7-8 per group. Plasma glucose and
insulin were measured in a fed condition during the V103 treatment.
V103 treatment significantly reduced both plasma glucose and
insulin. #p<0.05 by a two-way ANOVA compared to the
Control-Vehicle group; *p<0.05 by a two-way ANOVA compared to
the Ritonavir-Vehicle group.
[0274] Effect of V103 on Insulin Sensitivity and Glucose
Utilization
[0275] Insulin sensitivity and glucose utilization were assessed
using an IFGTT on day 17 and an OGTT on day 24. V103 treatment led
to significant improvement in insulin sensitivity as indicated by a
much suppressed glucose excursion during the IFGTT, during which
exogenous insulin together with glucose were administered. Data are
mean.+-.SEM. N=7-8 per group. Plasma glucose was measured in a
fasted condition during the IFGTT. Glucose levels in V103 treated
group were significantly lower than the vehicle treated mice.
*p<0.05 by a two-way ANOVA compared to the Ritonavir-Vehicle
group.
[0276] Similarly, treatment with V103 significantly improved
glucose tolerance during an OGTT in both plasma glucose and
insulin. Data are mean.+-.SEM. N=7-8 per group. Plasma glucose and
insulin were measured in a fasted condition during the OGTT. Both
glucose and insulin levels in V103 treated group were significantly
lower than the vehicle treated mice. *p<0.05 by a two-way ANOVA
compared to the Ritonavir-Vehicle group.
[0277] In addition, HOMA-IR (homeostasis model of
assessment-insulin resistance, calculated based on fasting glucose
and insulin levels) was significantly improved in mice treated with
V103. Thus, V103 was highly efficacious on improving insulin
sensitivity in ritonavir treated mice. Data are mean.+-.SEM. N=7-8
per group. Fasting plasma glucose and insulin were measured on day
24 following an overnight fast. HOMA-IR in V103 treated group was
significantly lower than the vehicle treated mice. *p<0.05 by a
one-way ANOVA compared to the Ritonavir-Vehicle group.
[0278] Effect of V103 on Liver and Muscle Lipid Content and Tissue
Weight
[0279] V103 treatment resulted in a significantly reduction of
lipid content in both liver and muscle. Data are mean.+-.SEM. N=7-8
per group. Lipid content of liver and soleus were measured at the
end of the treatment (day 25). V103 treatment significantly reduced
lipid content in both liver and soleus muscle. Lipid content in
soleus muscle was also low in the Ritonavir-Vehicle group.
#p<0.05 by a one-way ANOVA compared to the Control-Vehicle
group; *p<0.05 by a one-way ANOVA (liver lipid content) or
Student t-test (soleus lipid content) compared to the
Ritonavir-Vehicle group.
[0280] Ritonavir treatment resulted in lipoatrophy with an
increased liver mass. Liver weight was significantly increased
while white adipose pad weight was decreased with Ritonavir
treatment. V103 treatment resulted in a normalization of liver
weight but a further reduction of adipose pad weight.
[0281] Data are mean.+-.SEM. N=7-8 per group. Tissue weight of
liver, epididymal fat and inguinal fat were measured at the end of
the treatment (day 25). As indicated, tissue weight was
significantly increased in the liver but decreased in epididymal
fat in the Ritonavir-Vehicle group, and V103 treatment
significantly reduced weight of all three tissues. #p<0.05 by a
one-way ANOVA compared to the Control-Vehicle group; *p<0.05 by
a one-way ANOVA compared to the Ritonavir-Vehicle group.
[0282] Plasma Levels of V103 During the Study
[0283] Plasma levels of V103 were measured acutely after the first
dose (4 and 24 hours post dose) and chronically after the second (9
days after the dose) and third (8 days after the dose) doses.
Plasma levels of V103 were well maintained during the study. Data
are mean.+-.SEM. N=8 per group. Plasma V103 levels were measured at
4 and 24 hours after the first dose, 9 days after the second dose
and 8 days after the third dose.
DISCUSSION
[0284] We evaluated the effect of V103 in an HIV proteinase
inhibitor-induced lipodystrophy mouse model. With a chronic daily
treatment of ritonavir provided in diet, mice developed
lipodystrophy conditions, such as increased plasma TG and FFA
levels, increased liver lipid content, and reduced fat mass and
body weight. Since lean mice on a standard chow diet maintain a low
fat mass and body weight, in order to provide a sufficient amount
of fat for redistributing from adipose tissue to the liver with
ritonavir treatment, Diet 20#5058 was used with or without (for the
control group) addition of ritonavir. The diet, which has been used
for mouse breeding purpose, contains double amount of fat compared
to a standard chow diet. A disadvantage of using this diet is that
it may cause insulin resistance due to its high fat content.
Indeed, in this study we observed an increase in plasma insulin
level and percent body fat mass in the control diet fed mice.
[0285] Treatment with V103 for just three doses in 25 days
effectively improved dyslipidemic condition with significantly
reduced plasma TG and FFA levels as well as lipid content in the
liver and muscle. In addition, V103 treatment significantly
enhanced insulin sensitivity and glucose utilization as illustrated
by a suppression of glucose excursion during both IFGTT and OGTT as
well as an improved HOMA-IR.
[0286] The results from this study demonstrated that V103 can
provide a therapeutic approach for treating patients with
lipodystrophy. The profound effect of V103 on weight loss and lipid
reduction suggests its valuable potential for treating obesity and
fatty liver diseases.
[0287] Unless defined otherwise, the technical and scientific terms
used herein have the same meaning as that usually understood by a
specialist familiar with the field to which the disclosure
belongs.
[0288] Unless indicated otherwise, all methods, steps, techniques
and manipulations that are not specifically described in detail can
be performed and have been performed in a manner known per se, as
will be clear to the skilled person. Reference is for example again
made to the standard handbooks and the general background art
mentioned herein and to the further references cited therein.
Unless indicated otherwise, each of the references cited herein is
incorporated in its entirety by reference.
[0289] Claims to the invention are non-limiting and are provided
below.
[0290] Although particular aspects and claims have been disclosed
herein in detail, this has been done by way of example for purposes
of illustration only, and is not intended to be limiting with
respect to the scope of the appended claims, or the scope of
subject matter of claims of any corresponding future application.
Other aspects, advantages, and modifications considered to be
within the scope of the following claims. Those skilled in the art
will recognize or be able to ascertain, using no more than routine
experimentation, many equivalents of the specific aspects of the
invention described herein. Such equivalents are intended to be
encompassed by the following claims. Redrafting of claim scope in
later filed corresponding applications may be due to limitations by
the patent laws of various countries and should not be interpreted
as giving up subject matter of the claims.
Sequence CWU 1
1
101406PRTHomo sapiens 1Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90
95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220 Pro Gly Lys Gly Ser Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln
225 230 235 240 Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln
Thr Glu Ala 245 250 255 His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly
Gly Ala Ala Asp Gln 260 265 270 Ser Pro Glu Ser Leu Leu Gln Leu Lys
Ala Leu Lys Pro Gly Val Ile 275 280 285 Gln Ile Leu Gly Val Lys Thr
Ser Arg Phe Leu Cys Gln Arg Pro Asp 290 295 300 Gly Ala Leu Tyr Gly
Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe 305 310 315 320 Arg Glu
Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala 325 330 335
His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp 340
345 350 Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu
Pro 355 360 365 Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln
Pro Pro Asp 370 375 380 Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly
Pro Ser Gln Gly Arg 385 390 395 400 Ser Pro Ser Tyr Ala Ser 405
2419PRTHomo sapiens 2Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90
95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220 Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240 Gly Ser Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln
Val Arg Gln 245 250 255 Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr
Glu Ala His Leu Glu 260 265 270 Ile Arg Glu Asp Gly Thr Val Gly Gly
Ala Ala Asp Gln Ser Pro Glu 275 280 285 Ser Leu Leu Gln Leu Lys Ala
Leu Lys Pro Gly Val Ile Gln Ile Leu 290 295 300 Gly Val Lys Thr Ser
Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu 305 310 315 320 Tyr Gly
Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu 325 330 335
Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu 340
345 350 Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala
Pro 355 360 365 Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro
Pro Ala Leu 370 375 380 Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro
Pro Asp Val Gly Ser 385 390 395 400 Ser Asp Pro Leu Ser Met Val Gly
Pro Ser Gln Gly Arg Ser Pro Ser 405 410 415 Tyr Ala Ser 3177PRTHomo
sapiens 3Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln
Arg Tyr 1 5 10 15 Leu Tyr Thr Asp Asp Ala Gln Glu Thr Glu Ala His
Leu Glu Ile Arg 20 25 30 Glu Asp Gly Thr Val Gly Gly Ala Ala His
Gln Ser Pro Glu Ser Leu 35 40 45 Leu Glu Leu Lys Ala Leu Lys Pro
Gly Val Ile Gln Ile Leu Gly Val 50 55 60 Lys Thr Ser Arg Phe Leu
Cys Gln Lys Pro Asp Gly Ala Leu Tyr Gly 65 70 75 80 Ser Leu His Phe
Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu 85 90 95 Glu Asp
Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu 100 105 110
His Leu Pro Gly Asn Arg Ser Pro His Cys Asp Pro Ala Pro Gln Gly 115
120 125 Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro
Glu 130 135 140 Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly
Ser Ser Asp 145 150 155 160 Pro Leu Ala Met Val Gly Pro Ser Gln Gly
Arg Ser Pro Ser Tyr Ala 165 170 175 Ser 4406PRTHomo sapiens 4Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10
15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145
150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys Gly Ser Asp
Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln 225 230 235 240 Val Arg Gln
Arg Tyr Leu Tyr Thr Asp Asp Ala Cys Gln Thr Glu Ala 245 250 255 His
Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln 260 265
270 Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile
275 280 285 Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg
Pro Asp 290 295 300 Gly Thr Leu Tyr Gly Ser Leu His Phe Asp Pro Glu
Ala Cys Ser Phe 305 310 315 320 Arg Glu Leu Leu Leu Glu Asp Gly Tyr
Asn Val Tyr Gln Ser Glu Ala 325 330 335 His Gly Leu Pro Leu His Leu
Pro Cys Asn Arg Ser Pro His Arg Asp 340 345 350 Pro Ala Ser Arg Gly
Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro 355 360 365 Pro Ala Leu
Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp 370 375 380 Val
Gly Ser Ser Asp Pro Leu Ala Met Val Gly Gly Ser Gln Ala Arg 385 390
395 400 Ser Pro Ser Tyr Ala Ser 405 5406PRTHomo sapiens 5Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20
25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150
155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys Gly Ser Asp Ser
Ser Pro Leu Leu Gln Phe Gly Gly Gln 225 230 235 240 Val Arg Gln Arg
Tyr Leu Tyr Thr Asp Asp Ala Cys Gln Thr Glu Ala 245 250 255 His Leu
Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln 260 265 270
Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile 275
280 285 Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Lys Pro
Asp 290 295 300 Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala
Cys Ser Phe 305 310 315 320 Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn
Val Tyr Gln Ser Glu Ala 325 330 335 His Gly Leu Pro Leu His Leu Pro
Cys Asn Arg Ser Pro His Arg Asp 340 345 350 Pro Ala Ser Arg Gly Pro
Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro 355 360 365 Pro Ala Leu Pro
Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp 370 375 380 Val Gly
Ser Ser Asp Pro Leu Ala Met Val Gly Gly Ser Gln Ala Arg 385 390 395
400 Ser Pro Ser Tyr Ala Ser 405 6406PRTHomo sapiens 6Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155
160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys Gly Ser Asp Ser Ser
Pro Leu Leu Gln Phe Gly Gly Gln 225 230 235 240 Val Arg Gln Arg Tyr
Leu Tyr Thr Asp Asp Ala Cys Gln Thr Glu Ala 245 250 255 His Leu Glu
Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala His Gln 260 265 270 Ser
Pro Glu Ser Leu Leu Glu Leu Lys Ala Leu Lys Pro Gly Val Ile 275 280
285 Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Lys Pro Asp
290 295 300 Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys
Ser Phe 305 310 315 320 Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val
Tyr Gln Ser Glu Ala 325 330 335 His Gly Leu Pro Leu His Leu Pro Cys
Asn Arg Ser Pro His Arg Asp 340 345 350 Pro Ala Pro Gln Gly Pro Ala
Arg Phe Leu Pro Leu Pro Gly Leu Pro 355 360 365 Pro Ala Leu Pro Glu
Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp 370 375 380 Val Gly Ser
Ser Asp Pro Leu Ala Met Val Gly Pro Ser Gln Gly Arg 385 390 395 400
Ser Pro Ser Tyr Ala Ser 405 7419PRTHomo sapiens 7Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35
40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 65
70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220 Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly 225 230 235 240 Gly Ser Asp Ser Ser Pro Leu Leu Gln
Phe Gly Gly Gln Val Arg Gln 245 250 255 Arg Tyr Leu Tyr Thr Asp Asp
Ala Cys Gln Thr Glu Ala His Leu Glu 260 265 270 Ile Arg Glu Asp Gly
Thr Val Gly Gly Ala Ala His Gln Ser Pro Glu 275 280 285 Ser Leu Leu
Glu Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu 290 295 300 Gly
Val Lys Thr Ser Arg Phe Leu Cys Gln Lys Pro Asp Gly Ala Leu 305 310
315 320 Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu
Leu 325 330 335 Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala
His Gly Leu 340 345 350 Pro Leu His Leu Pro Cys Asn Arg Ser Pro His
Arg Asp Pro Ala Pro 355 360 365 Gln Gly Pro Ala Arg Phe Leu Pro Leu
Pro Gly Leu Pro Pro Ala Leu 370 375 380 Pro Glu Pro Pro Gly Ile Leu
Ala Pro Gln Pro Pro Asp Val Gly Ser 385 390 395 400 Ser Asp Pro Leu
Ala Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser 405 410 415 Tyr Ala
Ser 8419PRTHomo sapiens 8Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85
90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210
215 220 Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly 225 230 235 240 Gly Ser Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly
Gln Val Arg Gln 245 250 255 Arg Tyr Leu Tyr Thr Asp Asp Ala Cys Gln
Thr Glu Ala His Leu Glu 260 265 270 Ile Arg Glu Asp Gly Thr Val Gly
Gly Ala Ala Asp Gln Ser Pro Glu 275 280 285 Ser Leu Leu Gln Leu Lys
Ala Leu Lys Pro Gly Val Ile Gln Ile Leu 290 295 300 Gly Val Lys Thr
Ser Arg Phe Leu Cys Gln Lys Pro Asp Gly Ala Leu 305 310 315 320 Tyr
Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu 325 330
335 Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu
340 345 350 Pro Leu His Leu Pro Cys Asn Arg Ser Pro His Arg Asp Pro
Ala Ser 355 360 365 Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu
Pro Pro Ala Leu 370 375 380 Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln
Pro Pro Asp Val Gly Ser 385 390 395 400 Ser Asp Pro Leu Ala Met Val
Gly Gly Ser Gln Ala Arg Ser Pro Ser 405 410 415 Tyr Ala Ser
9419PRTHomo sapiens 9Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90
95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220 Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240 Gly Ser Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln
Val Arg Gln 245 250 255 Arg Tyr Leu Tyr Thr Asp Asp Ala Cys Gln Thr
Glu Ala His Leu Glu 260 265 270 Ile Arg Glu Asp Gly Thr Val Gly Gly
Ala Ala Asp Gln Ser Pro Glu 275 280 285 Ser Leu Leu Gln Leu Lys Ala
Leu Lys Pro Gly Val Ile Gln Ile Leu 290 295 300 Gly Val Lys Thr Ser
Arg Phe Leu Cys Gln Arg Pro Asp Gly Thr Leu 305 310 315 320 Tyr Gly
Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu 325 330 335
Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu 340
345 350 Pro Leu His Leu Pro Cys Asn Arg Ser Pro His Arg Asp Pro Ala
Ser 355 360 365 Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro
Pro Ala Leu 370 375 380 Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro
Pro Asp Val Gly Ser 385 390 395 400 Ser Asp Pro Leu Ala Met Val Gly
Gly Ser Gln Ala Arg Ser Pro Ser 405 410 415 Tyr Ala Ser 1015PRTHomo
sapiens 10Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser 1 5 10 15
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