U.S. patent application number 16/918088 was filed with the patent office on 2020-10-15 for method of treating or ameliorating metabolic disorders using growth differentiation factor 15 (gdf-15).
This patent application is currently assigned to AMGEN INC.. The applicant listed for this patent is AMGEN INC.. Invention is credited to Yang LI, Bei SHAN, Jackie Zeqi SHENG, Yumei XIONG, Wen-Chen YEH.
Application Number | 20200325200 16/918088 |
Document ID | / |
Family ID | 1000004929497 |
Filed Date | 2020-10-15 |
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United States Patent
Application |
20200325200 |
Kind Code |
A1 |
XIONG; Yumei ; et
al. |
October 15, 2020 |
METHOD OF TREATING OR AMELIORATING METABOLIC DISORDERS USING GROWTH
DIFFERENTIATION FACTOR 15 (GDF-15)
Abstract
Methods of treating metabolic diseases and disorders using a
GDF15 polypeptide are provided. In various embodiments the
metabolic disease or disorder is type 2 diabetes mellitus, obesity,
dyslipidemia, elevated glucose levels, elevated insulin levels and
diabetic nephropathy.
Inventors: |
XIONG; Yumei; (Palo Alto,
CA) ; LI; Yang; (Mountain View, CA) ; YEH;
Wen-Chen; (Belmont, CA) ; SHAN; Bei; (Redwood
City, CA) ; SHENG; Jackie Zeqi; (Thousand Oaks,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AMGEN INC. |
Thousand Oaks |
CA |
US |
|
|
Assignee: |
AMGEN INC.
Thousand Oaks
CA
|
Family ID: |
1000004929497 |
Appl. No.: |
16/918088 |
Filed: |
July 1, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14009790 |
Nov 6, 2013 |
10752664 |
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PCT/US12/32415 |
Apr 5, 2012 |
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16918088 |
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61473583 |
Apr 8, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/495 20130101;
A61K 38/18 20130101; A61K 38/00 20130101 |
International
Class: |
C07K 14/495 20060101
C07K014/495; A61K 38/18 20060101 A61K038/18 |
Claims
1. A method of improving glucose tolerance in a subject comprising
administering to a subject in need thereof a therapeutically
effective amount of an isolated human GDF15 polypeptide comprising
an amino acid sequence of SEQ ID NO: 10, and wherein administration
of the polypeptide improves glucose tolerance in the subject.
2. The method of claim 1, wherein the subject has type 2
diabetes.
3. The method of claim 1, wherein the subject has dyslipidemia.
4. The method of claim 1, wherein the subject is obese.
5. The method of claim 1, wherein the subject has diabetic
nephropathy.
6. The method of claim 1, wherein the subject has a fasting blood
glucose level of greater than or equal to 100 mg/dL.
7. The method of claim 1, wherein the subject is a mammal.
8. The method of claim 7, wherein the mammal is a human.
9. The method of claim 1, wherein the GDF15 protein comprises SEQ
ID NO: 2 or 6.
10. The method of claim 1, wherein the GDF15 polypeptide is encoded
by SEQ ID NO: 9.
11. The method of claim 1, wherein the GDF15 polypeptide is
administered in the form of a pharmaceutical composition comprising
the GDF15 polypeptide in admixture with a
pharmaceutically-acceptable carrier.
12. The method of claim 1, further comprising the step of
determining the subject's blood glucose level at a timepoint
subsequent to the administration.
13. The method of claim 1, further comprising the step of
determining the subject's serum insulin level at a timepoint
subsequent to the administration.
14. The method of claim 9, wherein the GDF15 polypeptide is encoded
by SEQ ID NO: 1 or 5, respectively.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/473,583 filed Apr. 8, 2011, which is
incorporated in its entirety by reference herein.
FIELD OF THE INVENTION
[0002] The disclosed invention relates to the treatment or
amelioration of a metabolic disorder, such as Type 2 diabetes,
elevated glucose levels, elevated insulin levels, dyslipidemia,
obesity or diabetic nephropathy, by administering a therapeutically
effective amount of GDF15 to a subject in need thereof.
BACKGROUND OF THE INVENTION
[0003] Growth differentiation factor 15 (GDF15) is a divergent
member of the TGF.beta. superfamily. It is also called macrophage
inhibitory cytokine 1 (MIC1) (Bootcov M R, 1997, Proc Natl Acad Sci
94:11514-9.), placental bone morphogenetic factor (PLAB) (Hromas R
1997, Biochim Biophys Acta. 1354:40-4.), placental transforming
growth factor beta (PTGFB) (Lawton L N 1997, Gene. 203:17-26),
prostate derived factor (PDF) (Paralkar V M 1998, J Biol Chem.
273:13760-7), and nonsteroidal anti-inflammatory drug-activated
gene (NAG-1) (Baek S J 2001, J Biol Chem. 276: 33384-92).
[0004] Human GDF15 gene is located on chromosome 19p13.2-13.1; rat
GDF15 gene is located on chromosome 16; and mouse GDF15 gene is
located on chromosome 8. The GDF15 open reading frames span two
exons (Bottner M 1999, Gene. 237:105-11 and NCBI). The mature GDF15
peptide shares low homology with other family members (Katoh M
2006, Int J Mol Med. 17:951-5.).
[0005] GDF15 is synthesized as a large precursor protein that is
cleaved at the dibasic cleavage site to release the carboxyterminal
mature peptide. The mouse and rat GDF15 prepro-peptides both
contain 303 amino acids. Human full-length precursor contains 308
amino acids. The rodent mature peptides contain 115 amino acids
after processing at the RGRR (SEQ ID NO:13) cleavage site. The
human mature peptide contains 112 amino acids after processing at
the RGRRRAR (SEQ ID NO:14) cleavage site. Human mature GDF15
peptide shared 66.1% and 68.1% sequence similarity with rat and
mouse mature GDF15 peptides (Bottner M 1999, Gene. 237:105-11;
Bauskin A R 2000, EMBO J. 19:2212-20; NCBI). There is no
glycosylation site in the mature GDF15 peptide.
[0006] The mature GDF15 peptide contains the seven conserved
cysteine residues required for the formation of the cysteine knot
motif and the single interchain disulfide bond that are typical for
TGF.beta. superfamily members. The mature peptide further contains
two additional cysteine residues that form a fourth intrachain
disulfide bond. Biologically active GDF15 is a 25 kD homodimer of
the mature peptide covalently linked by one interchain disulfide
bond.
[0007] GDF15 circulating levels have been reported to be elevated
in multiple pathological and physiological conditions, most notably
pregnancy (Moore A G 2000. J Clin Endocrinol Metab 85: 4781-4788),
.beta.-thalassemia (Tanno T 2007, Nat Med 13:1096-101) (Zimmermann
M B, 2008 Am J Clin Nutr 88:1026-31), congenital dyserythropoietic
anemia (Tamary H 2008, Blood. 112:5241-4). GDF15 has also been
linked to multiple biological activities in literature reports.
Studies of GDF15 knockout and transgenic mice suggested that GDF15
may be protective against ischemic/reperfusion- or overload-induced
heart injury (Kempf T, 2006, Circ Res. 98:351-60) (Xu J, 2006, Circ
Res. 98:342-50), protective against aging-associated motor neuron
and sensory neuron loss (Strelau J, 2009, J Neurosci. 29:13640-8.),
mildly protective against metabolic acidosis in kidney, and may
cause cachexia in cancer patients (Johnen H 2007 Nat Med.
11:1333-40). Many groups also studied the role of GDF15 in cell
apoptosis and proliferation and reported controversial results
using different cell culture and xenograft models. Studies on
transgenic mice showed that GDF15 is protective against carcinogen
or Apc mutation induced neoplasia in intestine and lung (Baek S J
2006, Gastroenterology. 131:1553-60) (Cekanova M 2009, Cancer Prev
Res 2:450-8.).
SUMMARY OF THE INVENTION
[0008] A method of treating a metabolic disorder is provided. In
one embodiment the method comprises administering to a subject in
need thereof a therapeutically effective amount of an isolated
human GDF15 polypeptide. In various embodiments, the metabolic
disorder is type 2 diabetes, dyslipidemia, obesity, or diabetic
nephropathy. In other embodiments, the metabolic disorder comprises
a condition in which the subject has a fasting blood glucose level
of greater than or equal to 100 mg/dL. The subject on which the
method is performed can be a mammal, for example a human. In
specific embodiments the GDF15 protein comprises one of SEQ ID
NOS:2, 6 and 10 and/or is encoded by the nucleic acid sequence of
SEQ ID NO:9. In some embodiments the GDF15 polypeptide is
administered in the form of a pharmaceutical composition comprising
the GDF15 polypeptide in admixture with a
pharmaceutically-acceptable carrier. In yet other embodiments the
provided method further comprises the step of determining the
subject's blood glucose level at a timepoint subsequent to the
administration. In still other embodiments the method further
comprises the step of determining the subject's serum insulin level
at a timepoint subsequent to the administration.
[0009] Also provided is another method of treating a metabolic
disorder. In one embodiment the method comprises administering to a
subject in need thereof a therapeutically effective amount of an
isolated human GDF15 polypeptide comprising an amino acid sequence
that has at least 90% sequence identity with one of SEQ ID NOS:2, 6
and 10. In various embodiments, the metabolic disorder is type 2
diabetes, dyslipidemia, obesity, or diabetic nephropathy. In other
embodiments, the metabolic disorder comprises a condition in which
the subject has a fasting blood glucose level of greater than or
equal to 100 mg/dL. The subject on which the method is performed
can be a mammal, for example a human. In specific embodiments the
GDF15 protein comprises one of SEQ ID NOS:2, 6 and 10 and/or is
encoded by one SEQ ID NOS:1, 5 and 9. In some embodiments the GDF15
polypeptide is administered in the form of a pharmaceutical
composition comprising the GDF15 polypeptide in admixture with a
pharmaceutically-acceptable carrier. In yet other embodiments the
provided method further comprises the step of determining the
subject's blood glucose level at a timepoint subsequent to the
administration. In still other embodiments the method further
comprises the step of determining the subject's serum insulin level
at a timepoint subsequent to the administration.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a series of two bar graphs showing the regulation
of GDF15 expression in murine liver (FIG. 1A) and murine fat (FIG.
1B) by nutritional states.
[0011] FIG. 2 is a series of two bar graphs showing the
upregulation of GDF15 expression in murine liver (FIG. 2A) and
murine 3T3-L1 adipocytes (FIG. 2B) by PPAR agonists.
[0012] FIG. 3 is a series of plots and bar graphs showing the
improvement in the metabolic profile of leptin-deficient ob/ob mice
following AAV-mediated treatment with murine GDF15; the effect of
AAV murine GDF15 injection on plasma glucose levels (FIG. 3A) and
body weight (FIG. 3C) were measured for two months. Plasma insulin
levels were measured two weeks after AAV injection (FIG. 3B) and
average daily food intake was measured three weeks after AAV
injection (FIG. 3D). Total cholesterol (FIG. 3E), NEFA (FIG. 3F),
triglyceride (FIG. 3G) and insulin levels (FIG. 3H) were measured
two months after AAV injection.
[0013] FIG. 4 is a series of two plots and two bar graphs showing
the glucose lowering activity of AAV-mediated treatment of ob/ob
mice with murine GDF15 and demonstrating that the glucose lowering
effect is independent of reduced body weight. The pair-feeding
study includes 3 groups of animals; one group injected with control
AVV and had free access to food, a second group injected with AAV
GDF15 and had free access to food, and a third group injected with
control AVV was fed the same amount of food that was consumed by
the group of animals injected with GDF15 AAV on the previous day.
The ratio of food intake to body weight over time in mice injected
with GDF15 or control AAV is shown in FIG. 4A;
[0014] FIG. 4B shows body weight over time in mice fed ad libitum
treated with control or GDF15 AAV and in mice pair fed injected
with control AAV; FIG. 4C shows plasma glucose levels at the end of
the pair-feeding study; and FIG. 4D shows body weight at the end of
the pair-feeding study.
[0015] FIG. 5 is a series of four plots and two bar graphs showing
the effects of murine GDF15 AAV on plasma glucose levels, body
weight and food intake, respectively in mice fed a high fat diet
(FIGS. 5A-5C) and the same thing in mice fed a normal chow diet
(FIGS. 5D-5F).
[0016] FIG. 6 is a series of four plots showing the effect of
AAV-mediated treatment with murine GDF15 on insulin sensitivity and
glucose tolerance in mice fed a high fat diet;
[0017] FIGS. 6A and 6B show the plasma glucose and plasma insulin
levels, respectively, measured during OGTT three weeks post AAV
injection, and FIGS. 6C and 6D show plasma glucose and plasma
glucose/basal glucose levels measured during ITT two weeks post AAV
injection.
[0018] FIG. 7 is a series of a plot and four bar graphs showing the
effect of AAV-mediated human GDF15 treatment of DIO mice on glucose
levels (FIG. 7A); food intake (FIG. 7B); body weight (FIG. 7C); and
the amount of human GDF15 expressed in DIO mice (FIG. 7D).
[0019] FIG. 8 is a plot and two bar graphs showing the effect of
AAV-mediated human GDF15 on the progression of glucose intolerance
in KKAy mice; FIG. 8A shows plasma glucose levels during an OGTT;
FIG. 8B body weight 3 weeks and 6 weeks after AAV injection; and
FIG. 8C insulin levels 3 weeks and 6 weeks after AAV injection.
[0020] FIG. 9 is a series of nine bar graphs showing the effect of
AAV-mediated human GDF15 on glucosuria in KKAy mice over a 3-4 week
period; FIG. 9A shows urine glucose levels; FIG. 9B urine volume;
FIG. 9C glucose excretion; FIG. 9D urine albumin; FIG. 9E albumin
excretion; FIG. 9F water intake; FIG. 9G insulin levels; FIG. 9H
plasma glucose levels; FIG. 9I human GDF15 levels; FIG. 9J shows
body weight and FIG. 9K food intake.
[0021] FIG. 10 is a series of four bar graphs showing the effect of
AAV-mediated murine GDF15 on the total body mass (FIG. 10A); fat
mass (FIG. 10B); fat mass/total body mass (FIG. 10C); and non-fat
mass/total body mass (FIG. 10D) in DIO mice.
[0022] FIG. 11 is a series of two plots and six bar graphs showing
the effect of AAV-mediated human GDF15 on DIO mice; FIG. 11A shows
body weight; FIG. 11B the amount of human GDF15 expressed; FIG. 11C
total body mass; FIG. 11D fat mass; FIG. 11E non-fat mass; FIG. 11F
bone mineral density; FIG. 11G percent of fat mass/body mass; and
FIG. 11H percent of non-fat mass/total body mass.
[0023] FIG. 12 is a series of three bar graphs showing the effect
of recombinant murine GDF15 on glucose and food intake in ob/ob
mice; FIG. 12A shows plasma glucose levels; FIG. 12B body weight;
and FIG. 12C food intake at day 1 and 2 after the injection of
vehicle or murine GDF15.
[0024] FIG. 13 is a series of three bar graphs showing the effect
of recombinant human GDF15 on plasma glucose levels, food intake
and body weight in ob/ob mice; FIG. 13A shows plasma glucose
levels; FIG. 13B food intake; and FIG. 13C body weight.
[0025] FIG. 14 is a series of a plot and two bar graphs showing the
effect of recombinant human GDF15 in DIO mice; FIG. 14A shows
plasma glucose levels measured during OGTT 3 days after protein
injection; FIG. 14B food intake; and FIG. 14C body weight.
[0026] FIG. 15 is a plot and a bar graph showing the effect of
recombinant human GDF15 on lipid metabolism in B6D2F1 male mice
following an oral lipid challenge; FIG. 15A shows plasma
triglyceride levels during the lipid tolerance test; and FIG. 15B
shows plasma exposure of recombinant human GDF15.
[0027] FIG. 16 is a series of four bar graphs showing the blood
chemistry of B6D21F mice fed a high-fat diet for three weeks after
AAV-mediated murine GDF15 administration; FIG. 16A shows plasma
insulin levels; FIG. 16B non-esterified fatty acid (NEFA) levels;
FIG. 16C total cholesterol levels; and FIG. 16D triglyceride
levels.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The instant disclosure provides a method of treating a
metabolic disorder, such as Type 2 diabetes mellitus (referred to
interchangeably herein as "type 2 diabetes"), elevated glucose
levels, elevated insulin levels, dyslipidemia or obesity, by
administering to a subject in need thereof a therapeutically
effective amount of an isolated human GDF15 polypeptide. Methods of
administration and delivery are also provided.
[0029] Recombinant polypeptide and nucleic acid methods used
herein, included in the Examples, are generally those set forth in
Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor Laboratory Press, 1989) and subsequent editions or
Current Protocols in Molecular Biology (Ausubel et al., eds., Green
Publishers Inc. and Wiley and Sons 1994) and subsequent editions,
both of which are incorporated herein by reference for any
purpose.
I. General Definitions
[0030] Following convention, as used herein "a" and "an" mean "one
or more" unless specifically indicated otherwise.
[0031] As used herein, the terms "amino acid" and "residue" are
interchangeable and, when used in the context of a peptide or
polypeptide, refer to both naturally occurring and synthetic amino
acids, as well as amino acid analogs, amino acid mimetics and
non-naturally occurring amino acids that are chemically similar to
the naturally occurring amino acids.
[0032] A "naturally occurring amino acid" is an amino acid that is
encoded by the genetic code, as well as those amino acids that are
encoded by the genetic code that are modified after synthesis,
e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. An amino acid analog is a compound that has the
same basic chemical structure as a naturally occurring amino acid,
i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an
amino group, and an R group, e.g., homoserine, norleucine,
methionine sulfoxide, methionine methyl sulfonium. Such analogs can
have modified R groups (e.g., norleucine) or modified peptide
backbones, but will retain the same basic chemical structure as a
naturally occurring amino acid.
[0033] An "amino acid mimetic" is a chemical compound that has a
structure that is different from the general chemical structure of
an amino acid, but that functions in a manner similar to a
naturally occurring amino acid. Examples include a methacryloyl or
acryloyl derivative of an amide, .beta.-, .gamma.-, .delta.-imino
acids (such as piperidine-4-carboxylic acid) and the like.
[0034] A "non-naturally occurring amino acid" is a compound that
has the same basic chemical structure as a naturally occurring
amino acid, but is not incorporated into a growing polypeptide
chain by the translation complex. "Non-naturally occurring amino
acid" also includes, but is not limited to, amino acids that occur
by modification (e.g., posttranslational modifications) of a
naturally encoded amino acid (including but not limited to, the 20
common amino acids) but are not themselves naturally incorporated
into a growing polypeptide chain by the translation complex. A
non-limiting lists of examples of non-naturally occurring amino
acids that can be inserted into a polypeptide sequence or
substituted for a wild-type residue in polypeptide sequence include
.beta.-amino acids, homoamino acids, cyclic amino acids and amino
acids with derivatized side chains.
[0035] Examples include (in the L-form or D-form; abbreviated as in
parentheses): citrulline (Cit), homocitrulline (hCit),
N.alpha.-methylcitrulline (NMeCit), N.alpha.-methylhomocitrulline
(N.alpha.-MeHoCit), ornithine (Orn), N.alpha.-Methylomithine
(N.alpha.-MeOrn or NMeOm), sarcosine (Sar), homolysine (hLys or
hK), homoarginine (hArg or hR), homoglutamine (hQ),
N.alpha.-methylarginine (NMeR), N.alpha.-methylleucine
(N.alpha.-MeL or NMeL), N-methylhomolysine (NMeHoK),
N.alpha.-methylglutamine (NMeQ), norleucine (Nle), norvaline (Nva),
1,2,3,4-tetrahydroisoquinoline (Tic), Octahydroindole-2-carboxylic
acid (Oic), 3-(1-naphthyl)alanine (1-Nal), 3-(2-naphthyl)alanine
(2-Nal), 1,2,3,4-tetrahydroisoquinoline (Tic), 2-indanylglycine
(IgI), para-iodophenylalanine (pI-Phe), para-aminophenylalanine
(4AmP or 4-Amino-Phe), 4-guanidino phenylalanine (Guf),
glycyllysine (abbreviated "K(N.epsilon.-glycyl)" or "K(glycyl)" or
"K(gly)"), nitrophenylalanine (nitrophe), aminophenylalanine
(aminophe or Amino-Phe), benzylphenylalanine (benzylphe),
.gamma.-carboxyglutamic acid (.gamma.-carboxyglu), hydroxyproline
(hydroxypro), p-carboxyl-phenylalanine (Cpa), .alpha.-aminoadipic
acid (Aad), N.alpha.-methyl valine (NMeVal), N-.alpha.-methyl
leucine (NMeLeu), N.alpha.-methylnorleucine (NMeNle),
cyclopentylglycine (Cpg), cyclohexylglycine (Chg), acetylarginine
(acetylarg), .alpha., .beta.-diaminopropionoic acid (Dpr), .alpha.,
.gamma.-diaminobutyric acid (Dab), diaminopropionic acid (Dap),
cyclohexylalanine (Cha), 4-methyl-phenylalanine (MePhe), .beta.,
.beta.-diphenyl-alanine (BiPhA), aminobutyric acid (Abu),
4-phenyl-phenylalanine (or biphenylalanine; 4Bip),
.alpha.-amino-isobutyric acid (Aib), beta-alanine,
beta-aminopropionic acid, piperidinic acid, aminocaprioic acid,
aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic
acid, N-ethylglycine, N-ethylaspargine, hydroxylysine,
allo-hydroxylysine, isodesmosine, allo-isoleucine, N-methylglycine,
N-methylisoleucine, N-methylvaline, 4-hydroxyproline (Hyp),
.gamma.-carboxyglutamate, .epsilon.-N,N,N-trimethyllysine,
.epsilon.-N-acetyllysine, O-phosphoserine, N-acetylserine,
N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,
.omega.-methylarginine, 4-Amino-O-Phthalic Acid (4APA), and other
similar amino acids, and derivatized forms of any of those
specifically listed.
[0036] The term "isolated nucleic acid molecule" refers to a single
or double-stranded polymer of deoxyribonucleotide or ribonucleotide
bases read from the 5' to the 3' end (e.g., a GDF15 nucleic acid
sequence provided herein), or an analog thereof, that has been
separated from at least about 50 percent of polypeptides, peptides,
lipids, carbohydrates, polynucleotides or other materials with
which the nucleic acid is naturally found when total nucleic acid
is isolated from the source cells. Preferably, an isolated nucleic
acid molecule is substantially free from any other contaminating
nucleic acid molecules or other molecules that are found in the
natural environment of the nucleic acid that would interfere with
its use in polypeptide production or its therapeutic, diagnostic,
prophylactic or research use.
[0037] The terms "isolated polypeptide" and "isolated protein" are
used interchangeably and refer to a polypeptide (e.g., a GDF15
polypeptide provided herein) that has been separated from at least
about 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99 percent of the
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.
[0038] The term "encoding" refers to a polynucleotide sequence
encoding one or more amino acids. The term does not require a start
or stop codon. An amino acid sequence can be encoded in any one of
six different reading frames provided by a polynucleotide
sequence.
[0039] The terms "identical" and percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same. "Percent
identity" means the percent of identical residues between the amino
acids or nucleotides in the compared molecules and is calculated
based on the size of the smallest of the molecules being compared.
For these calculations, gaps in alignments (if any) can be
addressed by a particular mathematical model or computer program
(i.e., an "algorithm"). Methods that can be used to calculate the
identity of the aligned nucleic acids or polypeptides include those
described in, e.g., Computational Molecular Biology, (Lesk, A. M.,
ed.), (1988) New York: Oxford University Press; Biocomputing
Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New
York: Academic Press; Computer Analysis of Sequence Data, Part I,
(Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey:
Humana Press; von Heinje, G., (1987) Sequence Analysis in Molecular
Biology, New York: Academic Press; Sequence Analysis Primer,
(Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton
Press; and Carillo et al., (1988) SIAM J. Applied Math.
48:1073.
[0040] In calculating percent identity, the sequences being
compared are aligned in a way that gives the largest match between
the sequences. The computer program used to determine percent
identity is the GCG program package, which includes GAP (Devereux
et al., (1984) Nucl. Acid Res. 12:387; Genetics Computer Group,
University of Wisconsin, Madison, Wis.). The computer algorithm GAP
is used to align the two polypeptides or polynucleotides for which
the percent sequence identity is to be determined. The sequences
are aligned for optimal matching of their respective amino acid or
nucleotide (the "matched span", as determined by the algorithm). A
gap opening penalty (which is calculated as 3.times. the average
diagonal, wherein the "average diagonal" is the average of the
diagonal of the comparison matrix being used; the "diagonal" is the
score or number assigned to each perfect amino acid match by the
particular comparison matrix) and a gap extension penalty (which is
usually 1/10 times the gap opening penalty), as well as a
comparison matrix such as PAM 250 or BLOSUM 62 are used in
conjunction with the algorithm. In certain embodiments, a standard
comparison matrix (see, Dayhoff et al., (1978) Atlas of Protein
Sequence and Structure 5:345-352 for the PAM 250 comparison matrix;
Henikoff et al., (1992) Proc. Natl. Acad. Sci. U.S.A.
89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by
the algorithm.
[0041] Recommended parameters for determining percent identity for
polypeptides or nucleotide sequences using the GAP program are the
following:
[0042] Algorithm: Needleman et al., 1970, J Mol. Biol.
48:443-453;
[0043] Comparison matrix: BLOSUM 62 from Henikoff et al., 1992,
supra;
[0044] Gap Penalty: 12 (but with no penalty for end gaps)
[0045] Gap Length Penalty: 4
[0046] Threshold of Similarity: 0
[0047] Certain alignment schemes for aligning two amino acid
sequences can result in matching of only a short region of the two
sequences, and this small aligned region can have very high
sequence identity even though there is no significant relationship
between the two full-length sequences. Accordingly, the selected
alignment method (e.g., the GAP program) can be adjusted if so
desired to result in an alignment that spans at least 50 contiguous
amino acids of the target polypeptide.
[0048] The terms "GDF15 polypeptide" and "GDF15 protein" are used
interchangeably and mean a naturally-occurring wild-type
polypeptide expressed in a mammal, such as a human or a mouse. For
purposes of this disclosure, the term "GDF15 polypeptide" can be
used interchangeably to refer to any full-length GDF15 polypeptide,
e.g., SEQ ID NO:2, which consist of 308 amino acid residues and
which is encoded by the nucleotide sequence SEQ ID NOs:1 (which,
when expressed recombinantly, may but need not comprise a stop
codon); any form comprising the active and prodomains of the
polypeptide, e.g., SEQ ID NO:6, which consist of 279 amino acid
residues and which is encoded by the nucleotide sequence SEQ ID
NO:5 (which, when expressed recombinantly, may but need not
comprise a stop codon), and in which the 29 amino acid residues at
the amino-terminal end of the full-length GDF15 polypeptide (i.e.,
which constitute the signal peptide) have been removed; and any
form of the polypeptide comprising the active domain from which the
prodomain and signal sequence have been removed, e.g., SEQ ID
NO:10, which consists of 112 amino acid residues and which is
encoded by the nucleotide sequence SEQ ID NO:9 (which, when
expressed recombinantly, may but need not comprise a stop codon),
in which the signal sequence and the pro domain have been removed.
GDF15 polypeptides can but need not comprise an amino-terminal
methionine, which may be introduced by engineering or as a result
of a bacterial expression process.
[0049] The term "GDF15 polypeptide" also encompasses a GDF15
polypeptide in which a naturally occurring GDF15 polypeptide
sequence (e.g., SEQ ID NOs:2, 6 or 10) has been modified. Such
modifications include, but are not limited to, one or more amino
acid substitutions, including substitutions with non-naturally
occurring amino acids non-naturally-occurring amino acid analogs
and amino acid mimetics.
[0050] In various embodiments, a GDF15 polypeptide comprises an
amino acid sequence that is at least about 85 percent identical to
a naturally-occurring GDF15 polypeptide (e.g., SEQ ID NOs:2, 6 or
10). In other embodiments, a GDF15 polypeptide comprises an amino
acid sequence that is at least about 90 percent, or about 95, 96,
97, 98, or 99 percent identical to a naturally-occurring GDF15
polypeptide amino acid sequence (e.g., SEQ ID NOs:2, 6 or 10). Such
GDF15 polypeptides preferably, but need not, possess at least one
activity of a wild-type GDF15 polypeptide, such as the ability to
lower blood glucose, insulin, triglyceride, or cholesterol levels;
the ability to reduce body weight; or the ability to improve
glucose tolerance, energy expenditure, or insulin sensitivity. The
present invention also encompasses nucleic acid molecules encoding
such GDF15 polypeptide sequences.
[0051] As stated herein, a GDF15 polypeptide can comprise a signal
sequence (residues 1-29 of SEQ ID NO:2) or it can have the signal
sequence removed (providing SEQ ID NO:6). In other embodiments, a
human GDF15 polypeptide can have the signal sequence removed and
can also be cleaved at residue 198, separating the primary sequence
of the prodomain (residues 30-198 of SEQ ID NO:2) from the primary
sequence of the active domain. The naturally-occurring biologically
active form of the GDF15 polypeptide is a homodimer of the
processed mature peptide (residues 199-308 of SEQ ID NO:2). In some
instances, a GDF15 polypeptide can be used to treat or ameliorate a
metabolic disorder in a subject is a mature form of GDF15
polypeptide that is derived from the same species as the
subject.
[0052] A GDF15 polypeptide is preferably biologically active. In
various respective embodiments, a GDF15 polypeptide has a
biological activity that is equivalent to, greater to or less than
that of the naturally occurring form of the mature GDF15 protein
from which the signal peptide has been removed from the N-terminus
of the full length GDF15 sequence and in which the prodomain has
been cleaved (but not necessarily removed from) the active domain.
Examples of biological activities include the ability to lower
blood glucose, insulin, triglyceride, or cholesterol levels; the
ability to reduce body weight; or the ability to improve glucose
tolerance, lipid tolerance, or insulin sensitivity; the ability to
lower urine glucose and protein excretion.
[0053] The terms "therapeutically effective dose" and
"therapeutically effective amount," as used herein, means an amount
of GDF15 polypeptide that elicits a biological or medicinal
response in a tissue system, animal, or human being sought by a
researcher, physician, or other clinician, which includes
alleviation or amelioration of the symptoms of the disease or
disorder being treated, i.e., an amount of a GDF15 polypeptide that
supports an observable level of one or more desired biological or
medicinal response, for example lowering blood glucose, insulin,
triglyceride, or cholesterol levels; reducing body weight; or
improving glucose tolerance, energy expenditure, or insulin
sensitivity.
II. GDF15 Polypeptides and Nucleic Acids
[0054] As disclosed herein, a GDF15 polypeptide described by the
instant disclosure can be engineered and/or produced using standard
molecular biology methodology. In various examples, a nucleic acid
sequence encoding a GDF15, which can comprise all or a portion of
SEQ ID NOs:2, 6 or 10 can be isolated and/or amplified from genomic
DNA, or cDNA using appropriate oligonucleotide primers. Primers can
be designed based on the nucleic and amino acid sequences provided
herein according to standard (RT)-PCR amplification techniques. The
amplified GDF15 nucleic acid can then be cloned into a suitable
vector and characterized by DNA sequence analysis.
[0055] Oligonucleotides for use as probes in isolating or
amplifying all or a portion of the GDF15 sequences provided herein
can be designed and generated using standard synthetic techniques,
e.g., automated DNA synthesis apparatus, or can be isolated from a
longer sequence of DNA.
IIA. Naturally-Occurring and Variant GDF15 Polypeptide and
Polynucleotide Sequences
[0056] In vivo, GDF15 is expressed as a contiguous amino acid
sequence comprising a signal sequence, a pro domain and an active
domain.
The 308 amino acid sequence of full length human GDF15 is (shown
with an optional N-terminal methionine codon in parentheses):
TABLE-US-00001 (SEQ ID NO: 2)
(M)PGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGP
SELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRI
LTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTASR
SWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARP
QLELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDL
GWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDT
VPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI
and is encoded by the DNA sequence (shown with an optional
N-terminal methionine codon in parentheses, and optional stop
codon):
TABLE-US-00002 (SEQ ID NO: 1)
(ATG)CCCGGGCAAGAACTCAGGACGGTGAATGGCTCTCAGATGC
TCCTGGTGTTGCTGGTGCTCTCGTGGCTGCCGCATGGGGGCGCCC
TGTCTCTGGCCGAGGCGAGCCGCGCAAGTTTCCCGGGACCCTCAG
AGTTGCACTCCGAAGACTCCAGATTCCGAGAGTTGCGGAAACGCT
ACGAGGACCTGCTAACCAGGCTGCGGGCCAACCAGAGCTGGGAAG
ATTCGAACACCGACCTCGTCCCGGCCCCTGCAGTCCGGATACTCA
CGCCAGAAGTGCGGCTGGGATCCGGCGGCCACCTGCACCTGCGTA
TCTCTCGGGCCGCCCTTCCCGAGGGGCTCCCCGAGGCCTCCCGCC
TTCACCGGGCTCTGTTCCGGCTGTCCCCGACGGCGTCAAGGTCGT
GGGACGTGACACGACCGCTGCGGCGTCAGCTCAGCCTTGCAAGAC
CCCAGGCGCCCGCGCTGCACCTGCGACTGTCGCCGCCGCCGTCGC
AGTCGGACCAACTGCTGGCAGAATCTTCGTCCGCACGGCCCCAGC
TGGAGTTGCACTTGCGGCCGCAAGCCGCCAGGGGGCGCCGCAGAG
CGCGTGCGCGCAACGGGGACCACTGTCCGCTCGGGCCCGGGCGTT
GCTGCCGTCTGCACACGGTCCGCGCGTCGCTGGAAGACCTGGGCT
GGGCCGATTGGGTGCTGTCGCCACGGGAGGTGCAAGTGACCATGT
GCATCGGCGCGTGCCCGAGCCAGTTCCGGGCGGCAAACATGCACG
CGCAGATCAAGACGAGCCTGCACCGCCTGAAGCCCGACACGGTGC
CAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCA
TTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACT
TGTTAGCCAAAGACTGCCACTGCATATGA.
The 303 amino acid sequence of full length murine GDF15 is (shown
with an optional N-terminal methionine codon in parentheses):
TABLE-US-00003 (SEQ ID NO: 4)
(M)APPALQAQPPGGSQLRFLLFLLLLLLLLSWPSQGDALAMPEQ
RPSGPESQLNADELRGRFQDLLSRLHANQSREDSNSEPSPDPAVR
ILSPEVRLGSHGQLLLRVNRASLSQGLPEAYRVHRALLLLTPTAR
PWDITRPLKRALSLRGPRAPALRLRLTPPPDLAMLPSGGTQLELR
LRVAAGRGRRSAHAHPRDSCPLGPGRCCHLETVQATLEDLGWSDW
VLSPRQLQLSMCVGECPHLYRSANTHAQIKARLHGLQPDKVPAPC
CVPSSYTPVVLMHRTDSGVSLQTYDDLVARGCHCA
and is encoded by the DNA sequence (shown with an optional
N-terminal methionine codon in parentheses, and optional stop
codon):
TABLE-US-00004 (SEQ ID NO: 3)
(ATG)GCCCCGCCCGCGCTCCAGGCCCAGCCTCCAGGCGGCTCTC
AACTGAGGTTCCTGCTGTTCCTGCTGCTGTTGCTGCTGCTGCTGT
CATGGCCATCGCAGGGGGACGCCCTGGCAATGCCTGAACAGCGAC
CCTCCGGCCCTGAGTCCCAACTCAACGCCGACGAGCTACGGGGTC
GCTTCCAGGACCTGCTGAGCCGGCTGCATGCCAACCAGAGCCGAG
AGGACTCGAACTCAGAACCAAGTCCTGACCCAGCTGTCCGGATAC
TCAGTCCAGAGGTGAGATTGGGGTCCCACGGCCAGCTGCTACTCC
GCGTCAACCGGGCGTCGCTGAGTCAGGGTCTCCCCGAAGCCTACC
GCGTGCACCGAGCGCTGCTCCTGCTGACGCCGACGGCCCGCCCCT
GGGACATCACTAGGCCCCTGAAGCGTGCGCTCAGCCTCCGGGGAC
CCCGTGCTCCCGCATTACGCCTGCGCCTGACGCCGCCTCCGGACC
TGGCTATGCTGCCCTCTGGCGGCACGCAGCTGGAACTGCGCTTAC
GGGTAGCCGCCGGCAGGGGGCGCCGAAGCGCGCATGCGCACCCAA
GAGACTCGTGCCCACTGGGTCCGGGGCGCTGCTGTCACTTGGAGA
CTGTGCAGGCAACTCTTGAAGACTTGGGCTGGAGCGACTGGGTGC
TGTCCCCGCGCCAGCTGCAGCTGAGCATGTGCGTGGGCGAGTGTC
CCCACCTGTATCGCTCCGCGAACACGCATGCGCAGATCAAAGCAC
GCCTGCATGGCCTGCAGCCTGACAAGGTGCCTGCCCCGTGCTGTG
TCCCCTCCAGCTACACCCCGGTGGTTCTTATGCACAGGACAGACA
GTGGTGTGTCACTGCAGACTTATGATGACCTGGTGGCCCGGGGCT GCCACTGCGCTTGA.
The amino acid sequence of human GDF15 following cleavage of the 29
residue signal sequence is (shown with an optional N-terminal
methionine codon in parentheses):
TABLE-US-00005 (SEQ ID NO: 6)
(M)LSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQ
SWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPE
ASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPALHLRLSP
PPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNGDHCPLG
PGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAA
NMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQT YDDLLAKDCHCI
and is encoded by the DNA sequence (shown with an optional
N-terminal methionine codon in parentheses, and optional stop
codon):
TABLE-US-00006 (SEQ ID NO: 5)
(ATG)CTGTCTCTGGCCGAGGCGAGCCGCGCAAGTTTCCCGGGAC
CCTCAGAGTTGCACTCCGAAGACTCCAGATTCCGAGAGTTGCGGA
AACGCTACGAGGACCTGCTAACCAGGCTGCGGGCCAACCAGAGCT
GGGAAGATTCGAACACCGACCTCGTCCCGGCCCCTGCAGTCCGGA
TACTCACGCCAGAAGTGCGGCTGGGATCCGGCGGCCACCTGCACC
TGCGTATCTCTCGGGCCGCCCTTCCCGAGGGGCTCCCCGAGGCCT
CCCGCCTTCACCGGGCTCTGTTCCGGCTGTCCCCGACGGCGTCAA
GGTCGTGGGACGTGACACGACCGCTGCGGCGTCAGCTCAGCCTTG
CAAGACCCCAGGCGCCCGCGCTGCACCTGCGACTGTCGCCGCCGC
CGTCGCAGTCGGACCAACTGCTGGCAGAATCTTCGTCCGCACGGC
CCCAGCTGGAGTTGCACTTGCGGCCGCAAGCCGCCAGGGGGCGCC
GCAGAGCGCGTGCGCGCAACGGGGACCACTGTCCGCTCGGGCCCG
GGCGTTGCTGCCGTCTGCACACGGTCCGCGCGTCGCTGGAAGACC
TGGGCTGGGCCGATTGGGTGCTGTCGCCACGGGAGGTGCAAGTGA
CCATGTGCATCGGCGCGTGCCCGAGCCAGTTCCGGGCGGCAAACA
TGCACGCGCAGATCAAGACGAGCCTGCACCGCCTGAAGCCCGACA
CGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGG
TGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATG
ATGACTTGTTAGCCAAAGACTGCCACTGCATATGA.
The amino acid sequence of murine GDF 15 following cleavage of the
32 residue signal sequence is (shown with an optional N-terminal
methionine codon in parentheses):
TABLE-US-00007 (SEQ ID NO: 8)
(M)SQGDALAMPEQRPSGPESQLNADELRGRFQDLLSRLHANQSR
EDSNSEPSPDPAVRILSPEVRLGSHGQLLLRVNRASLSQGLPEAY
RVHRALLLLTPTARPWDITRPLKRALSLRGPRAPALRLRLTPPPD
LAMLPSGGTQLELRLRVAAGRGRRSAHAHPRDSCPLGPGRCCHLE
TVQATLEDLGWSDWVLSPRQLQLSMCVGECPHLYRSANTHAQIKA
RLHGLQPDKVPAPCCVPSSYTPVVLMHRTDSGVSLQTYDDLVARG CHCA
and is encoded by the DNA sequence (shown with an optional
N-terminal methionine codon in parentheses, and optional stop
codon):
TABLE-US-00008 (SEQ ID NO: 7)
(ATG)TCGCAGGGGGACGCCCTGGCAATGCCTGAACAGCGACCCT
CCGGCCCTGAGTCCCAACTCAACGCCGACGAGCTACGGGGTCGCT
TCCAGGACCTGCTGAGCCGGCTGCATGCCAACCAGAGCCGAGAGG
ACTCGAACTCAGAACCAAGTCCTGACCCAGCTGTCCGGATACTCA
GTCCAGAGGTGAGATTGGGGTCCCACGGCCAGCTGCTACTCCGCG
TCAACCGGGCGTCGCTGAGTCAGGGTCTCCCCGAAGCCTACCGCG
TGCACCGAGCGCTGCTCCTGCTGACGCCGACGGCCCGCCCCTGGG
ACATCACTAGGCCCCTGAAGCGTGCGCTCAGCCTCCGGGGACCCC
GTGCTCCCGCATTACGCCTGCGCCTGACGCCGCCTCCGGACCTGG
CTATGCTGCCCTCTGGCGGCACGCAGCTGGAACTGCGCTTACGGG
TAGCCGCCGGCAGGGGGCGCCGAAGCGCGCATGCGCACCCAAGAG
ACTCGTGCCCACTGGGTCCGGGGCGCTGCTGTCACTTGGAGACTG
TGCAGGCAACTCTTGAAGACTTGGGCTGGAGCGACTGGGTGCTGT
CCCCGCGCCAGCTGCAGCTGAGCATGTGCGTGGGCGAGTGTCCCC
ACCTGTATCGCTCCGCGAACACGCATGCGCAGATCAAAGCACGCC
TGCATGGCCTGCAGCCTGACAAGGTGCCTGCCCCGTGCTGTGTCC
CCTCCAGCTACACCCCGGTGGTTCTTATGCACAGGACAGACAGTG
GTGTGTCACTGCAGACTTATGATGACCTGGTGGCCCGGGGCTGCC ACTGCGCTTGA.
[0057] The amino acid sequence of the recombinant active form of
the human GDF15, which comprises a homodimer comprising nine
intermolecular disulfide bonds (shown with an optional N-terminal
methionine residue in parentheses), is:
TABLE-US-00009 (SEQ ID NO: 10)
(M)ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVT
MCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMV
LIQKTDTGVSLQTYDDLLAKDCHCI
and is encoded by the DNA sequence (shown with an optional
N-terminal methionine codon in parentheses, and optional stop
codon):
TABLE-US-00010 (SEQ ID NO: 9)
(ATG)GCGCGCAACGGGGACCACTGTCCGCTCGGGCCCGGGCGTT
GCTGCCGTCTGCACACGGTCCGCGCGTCGCTGGAAGACCTGGGCT
GGGCCGATTGGGTGCTGTCGCCACGGGAGGTGCAAGTGACCATGT
GCATCGGCGCGTGCCCGAGCCAGTTCCGGGCGGCAAACATGCACG
CGCAGATCAAGACGAGCCTGCACCGCCTGAAGCCCGACACGGTGC
CAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCA
TTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACT
TGTTAGCCAAAGACTGCCACTGCATATAA.
[0058] The amino acid sequence of the recombinant active form of
the murine GDF15, which comprises a homodimer comprising nine
intermolecular disulfide bonds (shown with an optional N-terminal
methionine codon in parentheses), is:
TABLE-US-00011 (SEQ ID NO: 12)
(M)SAHAHPRDSCPLGPGRCCHLETVQATLEDLGWSDWVLSPRQL
QLSMCVGECPHLYRSANTHAQIKARLHGLQPDKVPAPCCVPSSYT
PVVLMHRTDSGVSLQTYDDLVARGCHCA
and is encoded by the DNA sequence (shown with an optional
N-terminal methionine codon in parentheses, and optional stop
codon):
TABLE-US-00012 (SEQ ID NO: 11)
(ATG)AGCGCGCATGCGCACCCAAGAGACTCGTGCCCACTGGGTC
CGGGGCGCTGCTGTCACCTGGAGACTGTGCAGGCAACTCTTGAAG
ACTTGGGCTGGAGCGACTGGGTGTTGTCCCCGCGCCAGCTGCAGC
TGAGCATGTGCGTGGGCGAGTGTCCCCACCTGTATCGCTCCGCGA
ACACGCATGCGCAGATCAAAGCACGCCTGCATGGCCTGCAGCCTG
ACAAGGTGCCTGCCCCGTGCTGTGTCCCCTCCAGCTACACCCCGG
TGGTTCTTATGCACAGGACAGACAGTGGTGTGTCACTGCAGACTT
ATGATGACCTGGTGGCCCGGGGCTGCCACTGCGCTTGA.
[0059] As stated herein, the term "GDF15 polypeptide" refers to a
GDF polypeptide comprising the human amino acid sequences SEQ ID
NOs:2, 6 and 10. The term "GDF15 polypeptide," however, also
encompasses polypeptides comprising an amino acid sequence that
differs from the amino acid sequence of a naturally occurring GDF
polypeptide sequence, e.g., SEQ ID NOs:2, 6 and 10, by one or more
amino acids, such that the sequence is at least 85% identical to
SEQ ID NOs:2, 6 and 10. GDF polypeptides can be generated by
introducing one or more amino acid substitutions, either
conservative or non-conservative and using naturally or
non-naturally occurring amino acids, at particular positions of the
GDF15 polypeptide.
[0060] A "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 non-native residue (i.e., a residue that is not found in
that same 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.
[0061] Naturally occurring residues can be divided into classes
based on common side chain properties:
[0062] (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
[0063] (2) neutral hydrophilic: Cys, Ser, Thr;
[0064] (3) acidic: Asp, Glu;
[0065] (4) basic: Asn, Gln, His, Lys, Arg;
[0066] (5) residues that influence chain orientation: Gly, Pro;
and
[0067] (6) aromatic: Trp, Tyr, Phe.
[0068] Additional groups of amino acids can also be formulated
using the principles described in, e.g., Creighton (1984) PROTEINS:
STRUCTURE AND MOLECULAR PROPERTIES (2d Ed. 1993), W.H. Freeman and
Company. In some instances it can be useful to further characterize
substitutions based on two or more of such features (e.g.,
substitution with a "small polar" residue, such as a Thr residue,
can represent a highly conservative substitution in an appropriate
context).
[0069] 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.
[0070] Synthetic, rare, or modified amino acid residues having
known similar physiochemical properties to those of an
above-described grouping can be used as a "conservative" substitute
for a particular amino acid residue in a sequence. For example, a
D-Arg residue may serve as a substitute for a typical L-Arg
residue. It also can be the case that a particular substitution can
be described in terms of two or more of the above described classes
(e.g., a substitution with a small and hydrophobic residue means
substituting one amino acid with a residue(s) that is found in both
of the above-described classes or other synthetic, rare, or
modified residues that are known in the art to have similar
physiochemical properties to such residues meeting both
definitions).
[0071] Nucleic acid sequences encoding a GDF15 polypeptide provided
herein, including those degenerate to SEQ ID NOs:1, 5 and 9, and
those encoding polypeptide variants of SEQ ID NOs:2, 6 and 10 form
other aspects of the instant disclosure.
IIB. GDF15 Vectors
[0072] In order to express the GDF15 nucleic acid sequences
provided herein, the appropriate coding sequences, e.g., SEQ ID
NOs:1, 5 or 9, can be cloned into a suitable vector and after
introduction in a suitable host, the sequence can be expressed to
produce the encoded polypeptide according to standard cloning and
expression techniques, which are known in the art (e.g., as
described in Sambrook, J., Fritsh, E. F., and Maniatis, T.
Molecular Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989). The invention also relates to such vectors
comprising a nucleic acid sequence according to the invention.
[0073] A "vector" refers to a delivery vehicle that (a) promotes
the expression of a polypeptide-encoding nucleic acid sequence; (b)
promotes the production of the polypeptide therefrom; (c) promotes
the transfection/transformation of target cells therewith; (d)
promotes the replication of the nucleic acid sequence; (e) promotes
stability of the nucleic acid; (f) promotes detection of the
nucleic acid and/or transformed/transfected cells; and/or (g)
otherwise imparts advantageous biological and/or physiochemical
function to the polypeptide-encoding nucleic acid. A vector can be
any suitable vector, including chromosomal, non-chromosomal, and
synthetic nucleic acid vectors (a nucleic acid sequence comprising
a suitable set of expression control elements). Examples of such
vectors include derivatives of SV40, bacterial plasmids, phage DNA,
baculovirus, yeast plasmids, vectors derived from combinations of
plasmids and phage DNA, and viral nucleic acid (RNA or DNA)
vectors.
[0074] A recombinant expression vector can be designed for
expression of a GDF15 protein in prokaryotic (e.g., E. coli) or
eukaryotic cells (e.g., insect cells, using baculovirus expression
vectors, yeast cells, or mammalian cells). Representative host
cells include those hosts typically used for cloning and
expression, including Escherichia coli strains TOP10F', TOP10,
DH10B, DH5a, HB101, W3110, BL21(DE3) and BL21 (DE3)pLysS,
BLUESCRIPT (Stratagene), mammalian cell lines CHO, CHO-KI, HEK293,
293-EBNA pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264:
5503-5509 (1989); pET vectors (Novagen, Madison Wis.).
Alternatively, the recombinant expression vector can be transcribed
and translated in vitro, for example using T7 promoter regulatory
sequences and T7 polymerase and an in vitro translation system.
Preferably, the vector contains a promoter upstream of the cloning
site containing the nucleic acid sequence encoding the polypeptide.
Examples of promoters, which can be switched on and off, include
the lac promoter, the T7 promoter, the trc promoter, the tac
promoter and the trp promoter.
[0075] Thus, provided herein are vectors comprising a nucleic acid
sequence encoding GDF15 that facilitate the expression of
recombinant GDF15. In various embodiments, the vectors comprise an
operably linked nucleotide sequence which regulates the expression
of GDF15. A vector can comprise or be associated with any suitable
promoter, enhancer, and other expression-facilitating elements.
Examples of such elements include strong expression promoters
(e.g., a human CMV IE promoter/enhancer, an RSV promoter, SV40
promoter, SL3-3 promoter, MMTV promoter, or HIV LTR promoter,
EF1alpha promoter, CAG promoter), effective poly (A) termination
sequences, an origin of replication for plasmid product in E. coli,
an antibiotic resistance gene as a selectable marker, and/or a
convenient cloning site (e.g., a polylinker). Vectors also can
comprise an inducible promoter as opposed to a constitutive
promoter such as CMV IE. In one aspect, a nucleic acid comprising a
sequence encoding a GDF15 polypeptide which is operatively linked
to a tissue specific promoter which promotes expression of the
sequence in a metabolically-relevant tissue, such as liver or
pancreatic tissue is provided.
IIC. Host Cells
[0076] In another aspect of the instant disclosure, host cells
comprising the GDF15 nucleic acids and vectors disclosed herein are
provided. In various embodiments, the vector or nucleic acid is
integrated into the host cell genome, which in other embodiments
the vector or nucleic acid is extra-chromosomal.
[0077] Recombinant cells, such as yeast, bacterial (e.g., E. coli),
and mammalian cells (e.g., immortalized mammalian cells) comprising
such a nucleic acid, vector, or combinations of either or both
thereof are provided. In various embodiments cells comprising a
non-integrated nucleic acid, such as a plasmid, cosmid, phagemid,
or linear expression element, which comprises a sequence coding for
expression of a GDF15 polypeptide, are provided.
[0078] A vector comprising a nucleic acid sequence encoding a GDF15
polypeptide provided herein can be introduced into a host cell by
transformation or by transfection. Methods of transforming a cell
with an expression vector are well known.
[0079] A GDF15-encoding nucleic acid can be positioned in and/or
delivered to a host cell or host animal via a viral vector. Any
suitable viral vector can be used in this capacity. A viral vector
can comprise any number of viral polynucleotides, alone or in
combination with one or more viral proteins, which facilitate
delivery, replication, and/or expression of the nucleic acid of the
invention in a desired host cell. The viral vector can be a
polynucleotide comprising all or part of a viral genome, a viral
protein/nucleic acid conjugate, a virus-like particle (VLP), or an
intact virus particle comprising viral nucleic acids and a GDF15
polypeptide-encoding nucleic acid. A viral particle viral vector
can comprise a wild-type viral particle or a modified viral
particle. The viral vector can be a vector which requires the
presence of another vector or wild-type virus for replication
and/or expression (e.g., a viral vector can be a helper-dependent
virus), such as an adenoviral vector amplicon. Typically, such
viral vectors consist of a wild-type viral particle, or a viral
particle modified in its protein and/or nucleic acid content to
increase transgene capacity or aid in transfection and/or
expression of the nucleic acid (examples of such vectors include
the herpes virus/AAV amplicons). Typically, a viral vector is
similar to and/or derived from a virus that normally infects
humans. Suitable viral vector particles in this respect, include,
for example, adenoviral vector particles (including any virus of or
derived from a virus of the adenoviridae), adeno-associated viral
vector particles (AAV vector particles) or other parvoviruses and
parvoviral vector particles, papillomaviral vector particles,
flaviviral vectors, alphaviral vectors, herpes viral vectors, pox
virus vectors, retroviral vectors, including lentiviral
vectors.
IID. Isolation of a GDF15 Polypeptide
[0080] A GDF15 polypeptide expressed as described herein can be
isolated using standard protein purification methods. A GDF15
polypeptide can be isolated from a cell in which is it naturally
expressed or it can be isolated from a cell that has been
engineered to express GDF15, for example a cell that does not
naturally express GDF15.
[0081] Protein purification methods that can be employed to isolate
a GDF15 polypeptide, as well as associated materials and reagents,
are known in the art. Exemplary methods of purifying a GDF15
polypeptide are provided in the Examples herein below. Additional
purification methods that may be useful for isolating a GDF15
polypeptide can be found in references such as Bootcov M R, 1997,
Proc. Nal. Acad. Sci. USA 94:11514-9, Fairlie W D, 2000, Gene 254:
67-76.
III. Pharmaceutical Compositions Comprising a GDF15 Polypeptide
[0082] Pharmaceutical compositions comprising a GDF15 polypeptide
are provided. Such GDF15 polypeptide pharmaceutical compositions
can comprise a therapeutically effective amount of a GDF15
polypeptide in admixture with a pharmaceutically or physiologically
acceptable formulation agent selected for suitability with the mode
of administration. The term "pharmaceutically acceptable carrier"
or "physiologically acceptable carrier" as used herein refers to
one or more formulation agents suitable for accomplishing or
enhancing the delivery of a GDF15 polypeptide into the body of a
human or non-human subject. The term includes any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like that are
physiologically compatible. Examples of pharmaceutically acceptable
carriers include one or more of water, saline, phosphate buffered
saline, dextrose, glycerol, ethanol and the like, as well as
combinations thereof. In some cases it will be preferable to
include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in a pharmaceutical
composition. Pharmaceutically acceptable substances such as wetting
or minor amounts of auxiliary substances such as wetting or
emulsifying agents, preservatives or buffers, which enhance the
shelf life or effectiveness of the GDF15 polypeptide can also act
as, or form a component of, a carrier. Acceptable pharmaceutically
acceptable carriers are preferably nontoxic to recipients at the
dosages and concentrations employed.
[0083] A pharmaceutical composition can contain formulation
agent(s) 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 agents
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: THE SCIENCE AND
PRACTICE OF PHARMACY, 19th edition, (1995); Berge et al., J. Pharm.
Sci., 6661), 1-19 (1977). Additional relevant principles, methods,
and agents are described in, e.g., Lieberman et al., PHARMACEUTICAL
DOSAGE FORMS-DISPERSE SYSTEMS (2nd ed., vol. 3, 1998); Ansel et
al., PHARMACEUTICAL DOSAGE FORMS & DRUG DELIVERY SYSTEMS (7th
ed. 2000); Martindale, THE EXTRA PHARMACOPEIA (31st edition),
Remington's PHARMACEUTICAL SCIENCES (16th-20.sup.th and subsequent
editions); The Pharmacological Basis Of Therapeutics, Goodman and
Gilman, Eds. (9th ed.--1996); Wilson and Gisvolds' TEXTBOOK OF
ORGANIC MEDICINAL AND PHARMACEUTICAL CHEMISTRY, Delgado and Remers,
Eds. (10th ed., 1998). Principles of formulating pharmaceutically
acceptable compositions also are described in, e.g., Aulton,
PHARMACEUTICS: THE SCIENCE OF DOSAGE FORM DESIGN, Churchill
Livingstone (New York) (1988), EXTEMPORANEOUS ORAL LIQUID DOSAGE
PREPARATIONS, CSHP (1998), incorporated herein by reference for any
purpose).
[0084] 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 a GDF15 polypeptide.
[0085] 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 polypeptide mutant
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. Furthermore,
the GDF15 polypeptide product can be formulated as a lyophilizate
using appropriate excipients such as sucrose.
[0086] The GDF15 polypeptide 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.
[0087] 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.
[0088] 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 GDF15 polypeptide in a pharmaceutically
acceptable vehicle. A particularly suitable vehicle for parenteral
injection is sterile distilled water in which a GDF15 polypeptide
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.
[0089] In one embodiment, a pharmaceutical composition can be
formulated for inhalation. For example, a GDF15 polypeptide can be
formulated as a dry powder for inhalation. GDF15 polypeptide
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.
[0090] It is also contemplated that certain formulations can be
administered orally. In one embodiment of the present invention,
GDF15 polypeptides 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 GDF15 polypeptide. Diluents, flavorings, low
melting point waxes, vegetable oils, lubricants, suspending agents,
tablet disintegrating agents, and binders can also be employed.
[0091] Another pharmaceutical composition can involve an effective
quantity of a GDF15 polypeptide 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.
[0092] Additional GDF15 polypeptide pharmaceutical compositions
will be evident to those skilled in the art, including formulations
involving GDF15 polypeptides 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). As described
herein, a hydrogel is an example of a sustained- or
controlled-delivery formulation.
[0093] 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.
[0094] A GDF15 polypeptide pharmaceutical composition to be used
for in vivo administration typically should 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.
[0095] 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.
[0096] 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).
[0097] The effective amount of a GDF15 polypeptide 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 a GDF15 polypeptide 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 g/kg up to about 100
mg/kg; or 5 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 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.
[0098] The frequency of dosing will depend upon the pharmacokinetic
parameters of the GDF15 polypeptide 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.
[0099] 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.
[0100] 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.
[0101] In order to deliver drug, e.g., a GDF15 polypeptide, at a
predetermined rate such that the drug concentration can be
maintained at a desired therapeutically effective level over an
extended period, a variety of different approaches can be employed.
In one example, a hydrogel comprising a polymer such as a gelatin
(e.g., bovine gelatin, human gelatin, or gelatin from another
source) or a naturally-occurring or a synthetically generated
polymer can be employed. Any percentage of polymer (e.g., gelatin)
can be employed in a hydrogel, such as 5, 10, 15 or 20%. The
selection of an appropriate concentration can depend on a variety
of factors, such as the therapeutic profile desired and the
pharmacokinetic profile of the therapeutic molecule.
[0102] Examples of polymers that can be incorporated into a
hydrogel include polyethylene glycol ("PEG"), polyethylene oxide,
polyethylene oxide-co-polypropylene oxide, co-polyethylene oxide
block or random copolymers, polyvinyl alcohol, poly(vinyl
pyrrolidinone), poly(amino acids), dextran, heparin,
polysaccharides, polyethers and the like.
[0103] Another factor that can be considered when generating a
hydrogel formulation is the degree of crosslinking in the hydrogel
and the crosslinking agent. In one embodiment, cross-linking can be
achieved via a methacrylation reaction involving methacrylic
anhydride. In some situations, a high degree of cross-linking may
be desirable while in other situations a lower degree of
crosslinking is preferred. In some cases a higher degree of
cross-linking provides a longer sustained release. A higher degree
of crosslinking may provide a firmer hydrogel and a longer period
over which drug is delivered.
[0104] Any ratio of polymer to crosslinking agent (e.g.,
methacrylic anhydride) can be employed to generate a hydrogel with
desired properties. For example, the ratio of polymer to
crosslinker can be, e.g., 8:1, 16:1, 24:1, or 32:1. For example,
when the hydrogel polymer is gelatin and the crosslinker is
methacrylate, ratios of 8:1, 16:1, 24:1, or 32:1 methyacrylic
anhydride:gelatin can be employed.
V. Therapeutic Uses of GDF15 Proteins and Nucleic Acids
[0105] GDF15 polypeptides can be used to treat, diagnose or
ameliorate, a metabolic condition or disorder. In one embodiment,
the metabolic disorder to be treated is diabetes, e.g., type 2
diabetes mellitus. In another embodiment, the metabolic condition
or disorder is obesity. In other embodiments the metabolic
condition or disorder is dyslipidemia, elevated glucose levels,
elevated insulin levels or diabetic nephropathy. For example, a
metabolic condition or disorder that can be treated or ameliorated
using a GDF15 polypeptide includes a state in which a human subject
has a fasting blood glucose level of 100 mg/dL or greater, for
example 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,
185, 190, 195, 200 or greater than 200 mg/dL. Blood glucose levels
can be determined in the fed or fasted state, or at random. The
metabolic condition or disorder can also comprise a condition in
which a subject is at increased risk of developing a metabolic
condition. For a human subject, such conditions include a fasting
blood glucose level of 100 mg/dL. Conditions that can be treated
using a pharmaceutical composition comprising a GDF15 polypeptide
can also be found in the relevant literature, e.g., American
Diabetes Association Standards of Medical Care in Diabetes
Care-2011, American Diabetes Association, Diabetes Care Vol. 34,
No. Supplement 1, S11-S61, 2010, incorporated herein by
reference.
[0106] In application, a metabolic disorder or condition, such as
Type 2 diabetes, elevated glucose levels, elevated insulin levels,
dyslipidemia, obesity or diabetic nephropathy, can be treated by
administering a therapeutically effective dose of a GDF15
polypeptide, e.g., a human GDF15 polypeptide such as SEQ ID NOs:2,
6 or 10, to a patient in need thereof. The administration can be
performed as described herein, such as by IV injection,
intraperitoneal (IP) injection, subcutaneous injection,
intramuscular injection, or orally in the form of a tablet or
liquid formation. In some situations, a therapeutically effective
or preferred dose of a GDF15 polypeptide can be determined by a
clinician. A therapeutically effective dose of GDF15 polypeptide
will depend, inter alia, upon the administration schedule, the unit
dose of agent administered, whether the GDF15 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 an amount
of GDF15 polypeptide that elicits a biological or medicinal
response in a tissue system, animal, or human being sought by a
researcher, medical doctor, or other clinician, which includes
alleviation or amelioration of the symptoms of the disease or
disorder being treated, i.e., an amount of a GDF15 polypeptide that
supports an observable level of one or more desired biological or
medicinal response, for example lowering blood glucose, insulin,
triglyceride, or cholesterol levels; reducing body weight; or
improving glucose tolerance, energy expenditure, or insulin
sensitivity.
[0107] It is noted that a therapeutically effective dose of a GDF15
polypeptide can also vary with the desired result. Thus, for
example, in situations in which a lower level of blood glucose is
indicated a dose of GDF15 will be correspondingly higher than a
dose in which a comparatively lower level of blood glucose is
desired. Conversely, in situations in which a higher level of blood
glucose is indicated a dose of GDF15 will be correspondingly lower
than a dose in which a comparatively higher level of blood glucose
is desired.
[0108] In various embodiments, a subject is a human having a blood
glucose level of 100 mg/dL or greater can be treated with a GDF15
polypeptide.
[0109] In one embodiment, a method of the instant disclosure
comprises first measuring a baseline level of one or more
metabolically-relevant compounds such as glucose, insulin,
cholesterol, lipid in a subject. A pharmaceutical composition
comprising a GDF15 polypeptide is then administered to the subject.
After a desired period of time, the level of the one or more
metabolically-relevant compounds (e.g., blood glucose, insulin,
cholesterol, lipid) in the subject is again measured. The two
levels can then be compared in order to determine the relative
change in the metabolically-relevant compound in the subject.
Depending on the outcome of that comparison another dose of the
pharmaceutical composition comprising a GDF15 molecule can be
administered to achieve a desired level of one or more
metabolically-relevant compound.
[0110] It is noted that a pharmaceutical composition comprising a
GDF15 polypeptide can be co-administered with another compound. The
identity and properties of compound co-administered with the GDF15
polypeptide will depend on the nature of the condition to be
treated or ameliorated. A non-limiting list of examples of
compounds that can be administered in combination with a
pharmaceutical composition comprising a GDF15 polypeptide include
rosiglitizone, pioglitizone, repaglinide, nateglitinide, metformin,
exenatide, stiagliptin, pramlintide, glipizide,
glimeprirideacarbose, and miglitol.
VI. Kits
[0111] Also provided are kits for practicing the disclosed methods.
Such kits can comprise a pharmaceutical composition such as those
described herein, including nucleic acids encoding the peptides or
proteins provided herein, vectors and cells comprising such nucleic
acids, and pharmaceutical compositions comprising such nucleic
acid-containing compounds, which can be provided in a sterile
container. Optionally, instructions on how to employ the provided
pharmaceutical composition in the treatment of a metabolic disorder
can also be included or be made available to a patient or a medical
service provider.
[0112] In one aspect, a kit comprises (a) a pharmaceutical
composition comprising a therapeutically effective amount of a
GDF15 polypeptide; and (b) one or more containers for the
pharmaceutical composition. Such a kit can also comprise
instructions for the use thereof; the instructions can be tailored
to the precise metabolic disorder being treated. The instructions
can describe the use and nature of the materials provided in the
kit. In certain embodiments, kits include instructions for a
patient to carry out administration to treat a metabolic disorder,
such as elevated glucose levels, elevated insulin levels, obesity,
type 2 diabetes, dyslipidemia or diabetic nephropathy.
[0113] Instructions can be printed on a substrate, such as paper or
plastic, etc, and can be present in the kits as a package insert,
in the labeling of the container of the kit or components thereof
(e.g., associated with the packaging), etc. In other embodiments,
the instructions are present as an electronic storage data file
present on a suitable computer readable storage medium, e.g.
CD-ROM, diskette, etc. In yet other embodiments, the actual
instructions are not present in the kit, but means for obtaining
the instructions from a remote source, such as over the internet,
are provided. An example of this embodiment is a kit that includes
a web address where the instructions can be viewed and/or from
which the instructions can be downloaded.
[0114] Often it will be desirable that some or all components of a
kit are packaged in suitable packaging to maintain sterility. The
components of a kit can be packaged in a kit containment element to
make a single, easily handled unit, where the kit containment
element, e.g., box or analogous structure, may or may not be an
airtight container, e.g., to further preserve the sterility of some
or all of the components of the kit.
EXAMPLES
[0115] The following examples, including the experiments conducted
and results achieved, are provided for illustrative purposes only
and are not to be construed as limiting the present invention.
Example 1
Preparation of GDF15 Polypeptides
[0116] E. coli that were transformed with a GDF 15 expression
vector constructed with an affinity tag were grown to an optical
density of 9 at 600 nm and then induced and harvested at an optical
density of 63 by centrifugation 6 hours later. Frozen cell paste
was thawed and re-suspended into buffer at 15% (wt./vol.) with a
low shear homogenizer until the slurry was homogeneous. The cells
were then subjected to high shear homogenization to break open and
release product-containing inclusion bodies. The resulting
homogenate was then centrifuged at 5,000.times.g for an hour at 5 C
to harvest the inclusion bodies as a pellet, leaving the
cytoplasmic contaminants in the discarded supernatant. The residual
cytoplasm is washed from the inclusion bodies by homogeneously
re-suspending the pellet to the original homogenate volume using
chilled water and a low shear homogenizer followed by
centrifugation as before. The resulting pellet, washed inclusion
bodies (WIBS), is then frozen at -80 C.
[0117] A sufficient amount of WIBS and guanidine hydrochloride
(GnHCl) was used at pH 8.5 in a reducing-solubilization to result
in approximately 25 mg/ml reduced product and 6 M GnHCl final
concentrations. The solubilization was then rapidly diluted 25-fold
with stirring into a refolding buffer containing redox reagents,
chaotrope and co-solvents at alkaline pH. The refold solution was
allowed to gently stir and air oxidize at 6 C for 72 hours or until
the solution was negative to Ellman's reagent. The refold solution
at 5 C was then clarified by depth filtration to allow for a
10-fold ultra-filtration concentration and subsequent diafiltration
into a buffer containing 50 mM sodium phosphate and low chaotrope
concentration at pH 8.5. The subsequent retentate was warmed to 25
C and then the pH lowered into the acidic range to cause
precipitation of contaminants. The precipitate was removed by
centrifugation at 5,000.times.g for 30 min at 25 C and the
resulting supernatant further clarified by 0.45 um filtration. The
filtrate (AP) was then adjusted to pH 8.5, and low salt
concentration to permit the first step of purification involving
immobilized metal affinity chromatography (IMAC).
[0118] Following protein folding and AP, the GDF 15 was purified
using a two-step chromatography train. The adjusted AP was applied
to an IMAC column that is equilibrated with buffered chaotrope
containing a low salt concentration at pH 8.5. The column was next
washed with equilibration buffer until a baseline ultraviolet (UV)
level is obtained. Product and contaminants are eluted by step-wise
increases in displacer concentration and the elutions were
collected and subsequently assayed by Coomasie-stained SDS-PAGE
(sodium dodecyl sulfate polyacrylamide gel electrophoresis) to
identify which eluate fractions contained a polypeptide that
migrates at the predicted molecular weight of GDF 15. After the
IMAC was completed, the pooled fraction containing product is
adjusted to pH 7.2 and 5 mM EDTA at 25 C. The product was converted
into the mature length GDF 15 by adding a low concentration of an
enzyme to cleave off the affinity tag at 25 C for several hours.
The cleavage reaction mixture was adjusted with an organic modifier
and acidic pH by the addition of acetic acid and organic solvent.
This allowed for the final chromatography step consisting of a
linear gradient elution of product from a reverse phase column
conducted at 25 C. The elution from the chromatography was
collected as fractions and then assayed by SDS-PAGE to determine
the appropriate fractions to pool for homogeneous product. The
resulting pool was buffer exchanged by diafiltration into a weakly
acidic buffer, concentrated by ultra-filtration, sterile filtered,
and stored at 5 C or frozen.
Example 2
Regulation of Murine GDF15 in Liver and Epidydimal Tissue
[0119] Liver and fat tissues are major metabolic organs in
mammalians. To identify potential novel therapeutic targets for
treatment of metabolic disorders, a microarray study was conducted
to compare gene expression patterns in liver and fat tissues of fed
or fasted wildtype or obese ob/ob mice. Liver or epidydimal fat
tissues were harvested for RNA extraction from age-matched C57B16
or ob/ob male mice (Jackson Labs) that had free access to food
("fed") or that were fasted for 24 hr ("fast"). cRNA samples were
hybridized to custom made micro array chips (Agilent). Data was
analyzed to compare gene expression patterns between wildtype and
ob/ob mice and between fed and fasted mice. Murine GDF15 (SEQ ID
NO:4; NCBI Accession Number BC067248.1) was identified as a target
gene regulated by feeding/fasting in liver and fat tissues as well
as differentially expressed in wildtype and ob/ob mice.
[0120] FIGS. 1A and 1B shows the change of signal intensity of
GDF15 in liver and fat tissues, respectively. It is noted that
GDF15 expression levels are significantly higher in liver tissues
from ob/ob mice than C57Bl/6 mice. GDF15 expression levels were
observed to be downregulated by fasting in liver in both C57Bl/6
mice and ob/ob mice. GDF15 expression levels were also
significantly higher in fat tissues from ob/ob mice than C57Bl/6
mice. Fasting increased GDF15 gene expression levels in both
C57Bl/6 mice and ob/ob mice. However, the fold induction was less
robust in ob/ob mice. These data suggest that GDF15 may be a novel
metabolic regulator.
Example 3
Induction of GDF15 by PPAR Agonists
[0121] PPAR.alpha. is nuclear receptor regulating metabolism in
liver and a major therapeutic target for metabolic disorders.
PPAR.alpha. is reported to be the master regulator mediating
fasting-induced FGF21 upregulation in liver (Inagaki T 2007 Cell
Metab 5:415-25). Male C57B16 mice (Jackson) were treated with a
PPAR.alpha. agonist clofibrate (500 mg/kg), and liver tissues were
harvested 1 day after treatment for RNA extraction. cRNA samples
were hybridized to mouse 10K micro array (Motorola). Data was
analyzed to identify PPAR target genes in mouse liver. FIG. 2A
shows that GDF15 expression was largely induced by clofibrate
treatment in mouse liver and demonstrates that GDF15 is a
downstream target gene of PPAR.alpha. in liver.
[0122] PPAR.gamma. is a master regulator of gene expression in the
fat tissue and PPAR.gamma. agonists are clinically approved or
being developed for diabetes treatment. Some PPAR.gamma. target
genes, such as adiponectin, an adipokine and a PPAR.gamma. target
gene in the fat tissue (Maeda N 2001 Diabetes 50:2094-9), are also
considered as therapeutic targets for treatment of type 2 diabetes.
Gene expression patterns in 3T3-L1 adipocytes treated with vehicle
or PPAR.gamma. agonist BRL49653 were compared, and murine GDF15 was
identified as a target gene inducible PPAR.gamma. agonist treatment
in adipocytes. Differentiated 3T3-L1 adipocytes were treated with
10 uM BRL49653 for 24 hours. RNA samples were isolated and cRNA
samples were hybridized to mouse 10K micro array (Motorola). Data
was analyzed to identify PPAR.gamma. genes in 3T3-L1 adipocytes.
FIG. 2B shows that GDF15 expression was largely induced by BRL49653
treatment in 3T3-L1 mouse adipocytes and demonstrates that GDF15 is
a downstream target gene of PPAR.gamma. in adipocytes, suggesting
that GDF15 has the potential to be a therapeutic target for
diabetes treatment.
Example 4
Murine GDF15 Reduces Food Intake, Body Weight Gain, Blood Insulin
Levels, Blood Glucose Levels and Blood Lipid Levels in Ob/Ob
Mice
[0123] Since GDF15 was robustly regulated by metabolic changes or
pharmacological treatments that activate major pathways regulating
metabolism, we examined if overexpression of GDF15 in vivo would
cause metabolic phenotypes in obese and diabetic ob/ob mice
(Coleman D L 1973 Diabetologia 9:287-93). Adeno-associated virus
(AAV) was used to achieve in vivo overexpression for two major
advantages. First, unlike transgene, AAV can be applied to adult
animals and does not interfere with fetal development.
[0124] Secondly, unlike other types of virus used for in vivo gene
overexpression, AAV produced with helper-free system is
replication-defective and is not pathogenic (Matsushita T 1998 Gene
Therapy 5: 938-45). muGDF15 full-length cDNA (SEQ ID NO:3) was
cloned into AAV vector with EF1a promoter and bGH polyA. AAVs were
produced with helper-free system and purified by chromatography and
gradient centrifugation. Seven-week-old male ob/ob mice (Jackson
Labs) were injected with 8.times.10.sup.12 genomic copy/animal
AAV-muGDF15 or control virus through the tail vein.
[0125] Glucose levels and body weight were examined on days 10, 17,
24, and 63 (FIGS. 3A and 3C, respectively). Food intake was
measured weekly from day 3 to day 24 (FIG. 3D). Blood insulin was
also measured on day 17 (FIG. 3B). Total cholesterol (FIG. 3E),
free fatty acids (FIG. 3F), triglyceride (FIG. 3G), and insulin
levels (FIG. 3H) were measured on day 63.
[0126] The lowered body weight, food intake, blood glucose,
insulin, triglyceride and cholesterol levels in AAV-muGDF15 group
compared to control virus treated group demonstrated that AAV
mediated in vivo overexpression of muGDF15 largely corrected
metabolic abnormalities in ob/ob mice, including hyperphagia,
obesity, hyperglycemia, hyperinsulinemia and dyslipidemia. This
data confirmed our hypothesis that GDF15 regulates body metabolism
and can be a potential therapeutic target for the treatment of a
metabolic disorder, such as obesity, diabetes and dyslipidemia.
Example 5
Murine GDF15 Improves Hyperglycemia in Ob/Ob Mice, Independent of
Reduction of Food Intake and without Body Weight Gain
[0127] GDF15 significantly reduced excessive food intake and body
weight gain in ob/ob mice, raising the question whether the
improvement of hyperglycemia is secondary to the lowered food
intake and reduced body weight gain. A pair-feeding study was
performed to determine whether GDF15 could improve hyperglycemia
independently from reduced food intake and without body weight
gain. Seven-week old male ob/ob mice (Jackson Labs) were injected
with 8.times.10.sup.12 genomic copy/animal AAV-muGDF15 or a control
virus through the tail vein as described in Example 4.
[0128] One group of control virus-injected mice (pair-feeding
group) had limited food access. The amount of food given to the
pair-feeding group (grams food intake/grams body weight) was
calculated to be equal to the amount of food consumed by
AAV-muGDF15 injected mice the day before (grams food intake/grams
body weight), after normalized by body weight. Body weight and food
intake were monitored daily, and the effect of GDF15 on these
parameters is shown in FIGS. 4A and 4B, respectively. Glucose
levels and body weight were measured at the end of the study and
the effect of GDF15 on these parameters is shown in FIGS. 4C and
4D, respectively.
[0129] Through the course of the 17-day pair-feeding study, the
GDF15 group had reduced food intake and body weight gain compared
to control virus group, and the pair-fed group maintained similar
body weight to the GDF15 group (FIGS. 4A and 4B). However, GDF15
group had significantly lower glucose levels than both the control
group and the pair-fed group, suggesting that GDF15 can improve
glucose management in hyperglycemic ob/ob mice independently of
food intake or body weight.
Example 6
The Efficacy of Murine GDF15 is More Robust in a High-Fat Diet
Induced Obesity (DIO) Model than in a Normal Chow-Fed Model
[0130] We next examined the efficacy of AAV mediated GDF15
overexpression in B6D2F1 mice on high fat diet, another rodent
model to examine efficacy of diabetic therapeutics (Karasawa H 2009
Metab Clin Exp 58:296-33). For comparison, mice fed normal chow
were also included in the study. Four-week-old male B6D2F1 mice
(Harlan Labs) were put on 60% high fat diet or normal chow for 3
weeks. They were subsequently injected with 8.times.10.sup.12
genomic copy/ms AAV-muGDF15 or a control virus through tail vein as
described in Example 4.
[0131] Glucose levels and body weight were measured on days 7, 13
and 28 by glucometer; the results are shown in FIGS. 5A and 5B,
respectively. Food intake was measured weekly for four weeks and
the results are shown in FIG. 5C. The results for the control
animals fed a normal chow diet are shown in FIGS. 5D-5F.
AAV-muGDF15 largely decreased blood glucose levels and body weight
in mice on high fat diet. On contrary, in mice on regular chow of
normal blood glucose levels, the effect was very mild.
[0132] These results indicate that GDF15 is a metabolic regulator
that takes effect selectively in the disease model, and will likely
not cause hypoglycemia, unlike some diabetes therapies.
Example 7
Murine GDF15 Improves Insulin Sensitivity and Glucose Tolerance in
DIO Mice
[0133] Diabetes is a metabolic disease of insulin resistance and
insulin insufficiency. To further understand the potential of GDF15
for diabetes treatment, we tested glucose tolerance and insulin
sensitivity in mice fed high fat diet treated that had been
administered with AAV-muGDF15 or control virus. Male B6D2F1 mice
(Harlan Labs) were fed a 60% high fat diet for three weeks and then
injected with 8.times.10.sup.2 genomic copy/animal AAV-muGDF15 or
control virus through the tail vein as described in Example 4.
[0134] A glucose tolerance test (GTT) was performed three weeks
after the AAV injection. The GTT was performed as follows: animals
were fasted for 4 hours. Following a measurement of body weight and
glucose levels (by glucometer) and bleeding for insulin
measurement, a 20% glucose solution in water was orally
administered at 10 ml/kg. Glucose levels at 15, 30, 60, 120 min
after glucose dosing were measured by glucometer. Blood samples
were collected at 15, 30, 60 min for measurement of serum insulin
levels. FIGS. 6A and 6B show the glucose curve and insulin curve
during the GTT, respectively. In the GTT study, the GDF15 group had
lower glucose levels at all time points compared to control group
(FIG. 6A), indicating that GDF15 treated animals have improved
glucose tolerance. The glucose-induced insulin secretion (GSIS) was
also lower at all time points (FIG. 6B), indicating that less
insulin was required for glucose disposal after the oral glucose
load, which suggests GDF15 treatment improved insulin sensitivity
in these mice.
[0135] To directly test insulin sensitivity in these mice, an
insulin sensitivity test (IST) was performed two weeks after AAV
injection on 4 hr fasted mice; i.p. dosing of 0.5 u/kg insulin was
used. The insulin sensitivity test (IST) was performed as follows:
animals were fasted for 4 hours. Following measurement of body
weight and glucose levels by glucometer, animals were i.p. dosed
with 10 ml/kg of 0.5 u/10 ml Novolin solution. Glucose levels at
15, 30, 60, 120 min after glucose dosing were measured by
glucometer. FIG. 6C shows the glucose curve during IST. The GDF15
treated group had lower glucose at all time points compared to
control group. FIG. 6D shows the glucose levels normalized to basal
glucose, and GDF15 treated group had lower glucose/basal glucose
ratio at 30, 60, 90 min compared to control group, strongly
indicating improved insulin sensitivity in GDF15 treated
animals.
Example 8
Human GDF15 Improves Glucose Tolerance in DIO Mice
[0136] Mouse GDF15 mature peptide and human GDF15 mature peptide
share 68.7% homology. To examine whether human GDF15 is functional
in mouse models, glucose tolerance was tested in B6D2F1 DIO mice
treated with AAV-huGDF15 or control virus. Male B6D2F1 mice (Harlan
Labs) were put on 60% high fat diet for five months, then injected
with 8.times.10.sup.12 genomic copy/animal AAV-huGDF15 or control
virus through tail vein as described in Example 4.
[0137] A glucose tolerance test was performed as described in
Example 7 two weeks after AAV injection with 4 hour fasted mice; a
2 g/kg oral glucose challenge was used. FIG. 7A depicts the results
of the GTT. Food intake was measured every three days for 12 days.
FIG. 7B shows the results of the food intake measurement over the
12 day period.
[0138] Body weight was measured before the glucose tolerance test
was performed, at the two week timepoint. FIG. 7C depicts the
results of the body weight measurements at the two week
timepoint.
[0139] Finally, plasma GDF15 levels at the two week time point were
measured by huGDF15 ELISA (R&D systems). FIG. 7D shows the
amount of huGDF15 detected. The circulating GDF15 levels in rodent
are not clear due to lack of detection method. In normal humans,
circulating GDF15 levels are reported to be several hundred pg/ml
(Moore A G, 2000 J Clin Endocrinol Metab 85: 4781-8). Our data
shows that AAV-hGDF15 treated group had several nanograms of
huGDF15 in circulation (FIG. 7D).
[0140] Collectively, this data demonstrates that similarly to mouse
GDF15, human GDF15 is efficacious in mouse models and the function
conserved well between the two homologs, even though they only
share 68.7% sequence homology.
Example 9
Human GDF15 Prevents Worsening of Insulin Sensitivity and Glucose
Tolerance in KK-Ay Mice
[0141] We further tested the efficacy of GDF15 in KK-Ay mice, an
obese-diabetic rodent model with different etiology and symptoms
from ob/ob and DIO mice (Iwatsuka H 1970 Endocrinol Jpn 17:23-35).
Seventeen-week-old male KK.Cg-Ay mice (Jackson Labs) were injected
with 8.times.10.sup.12 genomic copy/animal AAV-huGDF15 or control
virus through tail vein as described in Example 4.
[0142] A glucose tolerance test was performed on four hour fasted
mice at three and at six week timepoints after AAV injection; a 2
g/kg oral glucose challenge was used. The control group became more
glucose intolerant at 6 weeks as animals grew older and disease
progressed, while GDF15 group maintained similar glucose tolerance
6 weeks and 3 weeks post AAV injection (FIG. 8A), suggesting that
GDF15 treatment prevented disease progression in these animals. The
body weight and blood insulin levels of the mice were examined
before glucose challenge. The effect of the AAV injection on body
weight and blood insulin is shown in FIGS. 8B and 8C, respectively.
Both control group and GDF15 group were slightly hyperinsulinemic 3
weeks after injection (FIG. 8C). While the control group became
more hyperinsulinemic at 6 weeks, GDF15 group showed trend of
improved hyperinsulinemia (FIG. 8C), suggesting that similar to
what was observed in B6D2F1 high fat diet mice, GDF15 treatment
improved glucose tolerance in KK-Ay mice through enhanced insulin
sensitivity.
[0143] These data implies that GDF15 improves glucose tolerance in
all diabetic disease mouse models tested.
Example 10
Human GDF15 Improves Glucosuria and Proteinuria in KKAY Mice
[0144] A very well-documented diabetic phenotype in KK-Ay mice is
renal complications, including glucosuria and proteinuria (Reddi A
S 1988 Adv Exp Med Biol 246:7-15). We also examined the glucose and
albumin excretion in KK-Ay mice after GDF15 treatment.
Seventeen-week-old male KK.Cg-Ay mice (Jackson Labs) were injected
with 8.times.10.sup.12 genomic copy/animal AAV-huGDF15 or control
virus through tail vein as described in Example 4.
[0145] Three weeks after AAV injection, urine glucose levels, urine
albumin levels, urine volume, daily water intake, serum insulin
levels, blood glucose levels, serum huGDF15 levels, body weight,
and food intake were examined. The results are shown in FIGS. 9A-9K
(FIG. 9A shows urine glucose levels, 9B shows urine volume, 9C
glucose excretion, 9D urine albumin, 9E albumin excretion, 9F water
intake, 9G insulin levels, 9H glucose levels, 9I body weight, and
9J food intake, and 9K huGDF15 levels. GDF15 group had
significantly improved glucosuria compared with control group,
demonstrated with lowering of urine glucose levels (FIG. 9A), urine
volume (FIG. 9B) and total glucose excretion (FIG. 9C). Similarly,
they also had significantly improved proteinuria, as demonstrated
by lowered urine albumin levels (FIG. 9D), urine volume (FIG. 9B)
and total urine albumin excretion (FIG. 9E). GDF15 group also
reduced water intake to about 6 ml/d/animal, which is similar to
water intake of a normal animal, while water intake of control
group was about 19 ml/d/animal.
[0146] These results indicate that GDF15 significantly improved
glucose and albumin excretion in urine and may have additional
beneficial effect in diabetic nephropathy.
Example 11
Murine GDF15 Reduces Fat Mass and Fat Mass/Total Body Mass Ratio in
a DIO Model
[0147] Since GDF15 robustly reduced food intake and body weight
gain in ob/ob and B6D2F1 DIO mice, body mass and fat mass were
measured in B6D2F1 DIO mice after AAV-muGDF15 injection to
determine if GDF15 mainly lowers body fat mass and may be useful as
an obesity treatment or mainly lowers body lean mass, which would
be undesired. Four-week-old male B6D2F1 mice (Harlan) were put on
60% high fat diet or normal chow for 3 week, then injected with
8.times.10.sup.12 genomic copy/animal AAV-muGDF15 or control virus
through tail vein as described in Example 4.
[0148] Five months after AAV injection, total body mass and fat
mass were measured by DEXA scan (PIXImus II, GE). The results are
shown in FIGS. 10A (total body mass) and 10B (fat mass). The ratio
of fat mass/total body mass and the ratio of non-fat mass/total
mass were also calculated (FIGS. 10C and 10D, respectively). After
5 months, mice on high fat diet with no exposure to exogenous GDF15
(control group) gained much weight and were excessively obese while
GDF15 group maintained normal body mass similar to lean and young
animals (FIG. 10A). The GDF15 group also had much lower body fat
mass (FIG. 10B) and body fat mass/total mass ratio (FIG. 10C). In
contrast, non-fat mass/total mass ratio had increased in GDF15
group (FIG. 10D). This data suggests that GDF15 treatment mainly
lowers body fat mass and could be considered as a treatment for
obesity.
Example 12
Human GDF15 Reduces Fat Mass and Fat Mass/Total Body Mass Ratio in
DIO Model
[0149] For reasons similar to those outlined in Example 12, body
mass and fat mass were measured in B6D2F1 DIO mice after
AAV-huGDF15 injection. Male B6D2F1 mice (Harlan Labs) were put on
60% high fat diet for five months, then injected with
8.times.10.sup.12 genomic copy/animal AAV-huGDF15 or control virus
through tail vein as described in Example 4.
[0150] One year after AAV injection, the body weight of the
AAV-hGDF15 treated group was maintained at around 30 g (FIG. 11A),
and huGDF15 plasma level was maintained at around 5 ng/ml (FIG.
11B). Total body mass (FIG. 11C), fat mass (FIG. 11D), and bone
mineral density (FIG. 11E) were measured by DEXA scan (PIXImus II,
GE). The ratio of fat mass/total body mass and the ratio of non-fat
mass/total mass ratio were also calculated (FIGS. 11G and 11H,
respectively). A group of 12-week-old male B6D2F1 mice on normal
chow was included in the DEXA scan for comparison.
[0151] This experiment demonstrates that human GDF15 exhibits
anti-obesity properties by decreasing fat mass and increasing
non-fat mass/total body bass ratio.
Example 13
Recombinant Murine GDF15 Protein Improves Hyperglycemia and
Hyperphagia in Leptin-Deficient Ob/Ob Mice
[0152] We demonstrated strong metabolic efficacy of mouse and human
GDF15 in different mouse models through AAV mediated in vivo
expression. Next, we tested the efficacy of recombinant mouse GDF15
proteins in ob/ob mice. Six-week-old male ob/ob mice (Jackson Labs)
were dosed subcutaneously with 5 mg/kg rmGDF15 protein or vehicle
buffer twice per day. Briefly, ob/ob male mice were randomized by
food intake, body weight and glucose levels into vehicle and
treatment group. Animals were subcutaneously dosed twice daily with
5 mg/kg recombinant muGDF15 protein or vehicle buffer for 2
days.
[0153] Glucose, body weight, food intake before injection and on
days 1 and 2 were measured. For all timepoints, the glucose levels
are shown in FIG. 12A, body weight is shown in FIG. 12B and food
intake is shown in FIG. 12C.
[0154] These results demonstrate that exogenously administered
recombinant mouse GDF15 protein is efficacious in ob/ob mice,
similar to AAV-muGDF15.
Example 14
Recombinant Human GDF15 Protein Improves Hyperglycemia and
Hyperphagia in Leptin-Deficient Ob/Ob Mice
[0155] The efficacy of recombinant human GDF15 protein was also
tested in ob/ob mice. Seven-week-old male ob/ob mice (Jackson Labs)
were dosed subcutaneously with 5, 1.5, 0.5, 0.15 mg/kg rhGDF15
protein or vehicle buffer, by single injection. Animals were
randomized as described in Example 13.
[0156] Glucose, body weight, food intake were measured 16-17 hours
after treatment. Glucose levels are shown in FIG. 13A, food intake
is shown in FIG. 13B and body weight is shown in FIG. 13C.
[0157] The data indicates that exogenously administered recombinant
human GDF15 protein acutely improves hyperphagia and hyperglycemia
in ob/ob mice, and the efficacy was dose-dependent.
Example 15
Recombinant Human GDF15 Protein Improves Glucose Tolerance in DIO
Mice
[0158] We further tested the efficacy of recombinant human GDF15
protein in DIO model. Male B6D2F1 mice (Harlan Labs) on 60% high
fat diet for six months were dosed subcutaneously with 5 mg/kg
rhGDF15 protein or vehicle buffer by single dosing, animals were
randomized as described in Example 13.
[0159] A glucose tolerance test (GTT) was performed three days
after dosing on 4 hr fasted mice; a 1 g/kg oral glucose challenge
was used. The results of the GTT are shown in FIG. 14A. Food intake
was measured daily and is shown in FIG. 14B. Body weight was
measured before the GTT was performed and is shown in FIG. 14C.
[0160] Collectively these results indicate that recombinant human
GDF15 protein is efficacious in a DIO mouse model.
Example 16
Recombinant Human GDF15 Improves Lipid Tolerance
[0161] Another interesting metabolic activity of GDF15 we
discovered is that GDF15 acutely improves lipid tolerance in mice.
Male B6D2F1 mice (Harlan Labs) on 60% high fat diet for two months
were dosed subcutaneously with 5 mg/kg rhGDF15 protein or vehicle
buffer. Four hours later, mice were orally dosed with 20 ml/kg 20%
Intralipid.RTM.. Serum triglyceride levels were measured at 0, 60,
90, 120, 180 min after lipid challenge by a colorimetric assay
(Sigma). The measured serum triglyceride levels are presented in
FIG. 15A. Serum rhGDF15 levels at 180 min were measured by huGDF15
ELISA (R&D Systems) and are shown in FIG. 15B.
[0162] Serum triglyceride levels increased at 30, 60, 90 min after
oral intralipid challenge in both GDF15 and vehicle treated animals
(FIG. 15A). However, the triglyceride levels at 60 and 90 min were
significantly lower in treated group, indicating that GDF15 acutely
improved lipid tolerance in these animals (FIG. 15A). Dyslipidemia
including hypertriglycerdeamia is a major risk factor for
cardiovascular disease, the leading outcome that causes mortality
in diabetes patients (Hokanson J E 1996 J Cardiovasc. Risk
3:213-219). The acute improvement of lipid tolerance by GDF15
suggests that GDF15 can provide a beneficial effect in diabetic
dyslipidemia, particularly postprandial dyslipidemia.
Example 17
Murine GDF15 Improves the Insulin and Lipid Profile in a DIO
Model
[0163] Since GDF15 acutely improves lipid tolerance, we also
examined if chronically, GDF15 improves blood lipid profiles.
B6D2F1 mice (Harlan Labs) were put on 60% high fat diet and
injected with 8.times.10.sup.12 genomic copy/animal AAV-muGDF15 or
control virus through tail vein as described in Example 4. Blood
insulin, total cholesterol, NEFA and triglyceride levels were
measured 3 weeks after AAV injection. The results are shown in FIG.
16; FIG. 16A shows insulin levels, FIG. 16B NEFA levels, FIG. 16C
total cholesterol levels, and FIG. 16D triglyceride levels. GDF15
group had lower cholesterol levels (FIG. 16C) and triglyceride
levels compared to control group, demonstrating that GDF15
chronically improves lipid profile. This data further indicates
that GDF15 treatment can provide a beneficial effect in diabetic
dyslipidemia.
Sequence CWU 1
1
121927DNAHomo sapiensCDS(1)..(927) 1atg ccc ggg caa gaa ctc agg acg
gtg aat ggc tct cag atg ctc ctg 48Met Pro Gly Gln Glu Leu Arg Thr
Val Asn Gly Ser Gln Met Leu Leu1 5 10 15gtg ttg ctg gtg ctc tcg tgg
ctg ccg cat ggg ggc gcc ctg tct ctg 96Val Leu Leu Val Leu Ser Trp
Leu Pro His Gly Gly Ala Leu Ser Leu 20 25 30gcc gag gcg agc cgc gca
agt ttc ccg gga ccc tca gag ttg cac tcc 144Ala Glu Ala Ser Arg Ala
Ser Phe Pro Gly Pro Ser Glu Leu His Ser 35 40 45gaa gac tcc aga ttc
cga gag ttg cgg aaa cgc tac gag gac ctg cta 192Glu Asp Ser Arg Phe
Arg Glu Leu Arg Lys Arg Tyr Glu Asp Leu Leu 50 55 60acc agg ctg cgg
gcc aac cag agc tgg gaa gat tcg aac acc gac ctc 240Thr Arg Leu Arg
Ala Asn Gln Ser Trp Glu Asp Ser Asn Thr Asp Leu65 70 75 80gtc ccg
gcc cct gca gtc cgg ata ctc acg cca gaa gtg cgg ctg gga 288Val Pro
Ala Pro Ala Val Arg Ile Leu Thr Pro Glu Val Arg Leu Gly 85 90 95tcc
ggc ggc cac ctg cac ctg cgt atc tct cgg gcc gcc ctt ccc gag 336Ser
Gly Gly His Leu His Leu Arg Ile Ser Arg Ala Ala Leu Pro Glu 100 105
110ggg ctc ccc gag gcc tcc cgc ctt cac cgg gct ctg ttc cgg ctg tcc
384Gly Leu Pro Glu Ala Ser Arg Leu His Arg Ala Leu Phe Arg Leu Ser
115 120 125ccg acg gcg tca agg tcg tgg gac gtg aca cga ccg ctg cgg
cgt cag 432Pro Thr Ala Ser Arg Ser Trp Asp Val Thr Arg Pro Leu Arg
Arg Gln 130 135 140ctc agc ctt gca aga ccc cag gcg ccc gcg ctg cac
ctg cga ctg tcg 480Leu Ser Leu Ala Arg Pro Gln Ala Pro Ala Leu His
Leu Arg Leu Ser145 150 155 160ccg ccg ccg tcg cag tcg gac caa ctg
ctg gca gaa tct tcg tcc gca 528Pro Pro Pro Ser Gln Ser Asp Gln Leu
Leu Ala Glu Ser Ser Ser Ala 165 170 175cgg ccc cag ctg gag ttg cac
ttg cgg ccg caa gcc gcc agg ggg cgc 576Arg Pro Gln Leu Glu Leu His
Leu Arg Pro Gln Ala Ala Arg Gly Arg 180 185 190cgc aga gcg cgt gcg
cgc aac ggg gac cac tgt ccg ctc ggg ccc ggg 624Arg Arg Ala Arg Ala
Arg Asn Gly Asp His Cys Pro Leu Gly Pro Gly 195 200 205cgt tgc tgc
cgt ctg cac acg gtc cgc gcg tcg ctg gaa gac ctg ggc 672Arg Cys Cys
Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly 210 215 220tgg
gcc gat tgg gtg ctg tcg cca cgg gag gtg caa gtg acc atg tgc 720Trp
Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys225 230
235 240atc ggc gcg tgc ccg agc cag ttc cgg gcg gca aac atg cac gcg
cag 768Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala
Gln 245 250 255atc aag acg agc ctg cac cgc ctg aag ccc gac acg gtg
cca gcg ccc 816Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp Thr Val
Pro Ala Pro 260 265 270tgc tgc gtg ccc gcc agc tac aat ccc atg gtg
ctc att caa aag acc 864Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val
Leu Ile Gln Lys Thr 275 280 285gac acc ggg gtg tcg ctc cag acc tat
gat gac ttg tta gcc aaa gac 912Asp Thr Gly Val Ser Leu Gln Thr Tyr
Asp Asp Leu Leu Ala Lys Asp 290 295 300tgc cac tgc ata tga 927Cys
His Cys Ile3052308PRTHomo sapiens 2Met Pro Gly Gln Glu Leu Arg Thr
Val Asn Gly Ser Gln Met Leu Leu1 5 10 15Val Leu Leu Val Leu Ser Trp
Leu Pro His Gly Gly Ala Leu Ser Leu 20 25 30Ala Glu Ala Ser Arg Ala
Ser Phe Pro Gly Pro Ser Glu Leu His Ser 35 40 45Glu Asp Ser Arg Phe
Arg Glu Leu Arg Lys Arg Tyr Glu Asp Leu Leu 50 55 60Thr Arg Leu Arg
Ala Asn Gln Ser Trp Glu Asp Ser Asn Thr Asp Leu65 70 75 80Val Pro
Ala Pro Ala Val Arg Ile Leu Thr Pro Glu Val Arg Leu Gly 85 90 95Ser
Gly Gly His Leu His Leu Arg Ile Ser Arg Ala Ala Leu Pro Glu 100 105
110Gly Leu Pro Glu Ala Ser Arg Leu His Arg Ala Leu Phe Arg Leu Ser
115 120 125Pro Thr Ala Ser Arg Ser Trp Asp Val Thr Arg Pro Leu Arg
Arg Gln 130 135 140Leu Ser Leu Ala Arg Pro Gln Ala Pro Ala Leu His
Leu Arg Leu Ser145 150 155 160Pro Pro Pro Ser Gln Ser Asp Gln Leu
Leu Ala Glu Ser Ser Ser Ala 165 170 175Arg Pro Gln Leu Glu Leu His
Leu Arg Pro Gln Ala Ala Arg Gly Arg 180 185 190Arg Arg Ala Arg Ala
Arg Asn Gly Asp His Cys Pro Leu Gly Pro Gly 195 200 205Arg Cys Cys
Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly 210 215 220Trp
Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys225 230
235 240Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala
Gln 245 250 255Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp Thr Val
Pro Ala Pro 260 265 270Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val
Leu Ile Gln Lys Thr 275 280 285Asp Thr Gly Val Ser Leu Gln Thr Tyr
Asp Asp Leu Leu Ala Lys Asp 290 295 300Cys His Cys Ile3053912DNAMus
musculusCDS(1)..(912) 3atg gcc ccg ccc gcg ctc cag gcc cag cct cca
ggc ggc tct caa ctg 48Met Ala Pro Pro Ala Leu Gln Ala Gln Pro Pro
Gly Gly Ser Gln Leu1 5 10 15agg ttc ctg ctg ttc ctg ctg ctg ttg ctg
ctg ctg ctg tca tgg cca 96Arg Phe Leu Leu Phe Leu Leu Leu Leu Leu
Leu Leu Leu Ser Trp Pro 20 25 30tcg cag ggg gac gcc ctg gca atg cct
gaa cag cga ccc tcc ggc cct 144Ser Gln Gly Asp Ala Leu Ala Met Pro
Glu Gln Arg Pro Ser Gly Pro 35 40 45gag tcc caa ctc aac gcc gac gag
cta cgg ggt cgc ttc cag gac ctg 192Glu Ser Gln Leu Asn Ala Asp Glu
Leu Arg Gly Arg Phe Gln Asp Leu 50 55 60ctg agc cgg ctg cat gcc aac
cag agc cga gag gac tcg aac tca gaa 240Leu Ser Arg Leu His Ala Asn
Gln Ser Arg Glu Asp Ser Asn Ser Glu65 70 75 80cca agt cct gac cca
gct gtc cgg ata ctc agt cca gag gtg aga ttg 288Pro Ser Pro Asp Pro
Ala Val Arg Ile Leu Ser Pro Glu Val Arg Leu 85 90 95ggg tcc cac ggc
cag ctg cta ctc cgc gtc aac cgg gcg tcg ctg agt 336Gly Ser His Gly
Gln Leu Leu Leu Arg Val Asn Arg Ala Ser Leu Ser 100 105 110cag ggt
ctc ccc gaa gcc tac cgc gtg cac cga gcg ctg ctc ctg ctg 384Gln Gly
Leu Pro Glu Ala Tyr Arg Val His Arg Ala Leu Leu Leu Leu 115 120
125acg ccg acg gcc cgc ccc tgg gac atc act agg ccc ctg aag cgt gcg
432Thr Pro Thr Ala Arg Pro Trp Asp Ile Thr Arg Pro Leu Lys Arg Ala
130 135 140ctc agc ctc cgg gga ccc cgt gct ccc gca tta cgc ctg cgc
ctg acg 480Leu Ser Leu Arg Gly Pro Arg Ala Pro Ala Leu Arg Leu Arg
Leu Thr145 150 155 160ccg cct ccg gac ctg gct atg ctg ccc tct ggc
ggc acg cag ctg gaa 528Pro Pro Pro Asp Leu Ala Met Leu Pro Ser Gly
Gly Thr Gln Leu Glu 165 170 175ctg cgc tta cgg gta gcc gcc ggc agg
ggg cgc cga agc gcg cat gcg 576Leu Arg Leu Arg Val Ala Ala Gly Arg
Gly Arg Arg Ser Ala His Ala 180 185 190cac cca aga gac tcg tgc cca
ctg ggt ccg ggg cgc tgc tgt cac ttg 624His Pro Arg Asp Ser Cys Pro
Leu Gly Pro Gly Arg Cys Cys His Leu 195 200 205gag act gtg cag gca
act ctt gaa gac ttg ggc tgg agc gac tgg gtg 672Glu Thr Val Gln Ala
Thr Leu Glu Asp Leu Gly Trp Ser Asp Trp Val 210 215 220ctg tcc ccg
cgc cag ctg cag ctg agc atg tgc gtg ggc gag tgt ccc 720Leu Ser Pro
Arg Gln Leu Gln Leu Ser Met Cys Val Gly Glu Cys Pro225 230 235
240cac ctg tat cgc tcc gcg aac acg cat gcg cag atc aaa gca cgc ctg
768His Leu Tyr Arg Ser Ala Asn Thr His Ala Gln Ile Lys Ala Arg Leu
245 250 255cat ggc ctg cag cct gac aag gtg cct gcc ccg tgc tgt gtc
ccc tcc 816His Gly Leu Gln Pro Asp Lys Val Pro Ala Pro Cys Cys Val
Pro Ser 260 265 270agc tac acc ccg gtg gtt ctt atg cac agg aca gac
agt ggt gtg tca 864Ser Tyr Thr Pro Val Val Leu Met His Arg Thr Asp
Ser Gly Val Ser 275 280 285ctg cag act tat gat gac ctg gtg gcc cgg
ggc tgc cac tgc gct tga 912Leu Gln Thr Tyr Asp Asp Leu Val Ala Arg
Gly Cys His Cys Ala 290 295 3004303PRTMus musculus 4Met Ala Pro Pro
Ala Leu Gln Ala Gln Pro Pro Gly Gly Ser Gln Leu1 5 10 15Arg Phe Leu
Leu Phe Leu Leu Leu Leu Leu Leu Leu Leu Ser Trp Pro 20 25 30Ser Gln
Gly Asp Ala Leu Ala Met Pro Glu Gln Arg Pro Ser Gly Pro 35 40 45Glu
Ser Gln Leu Asn Ala Asp Glu Leu Arg Gly Arg Phe Gln Asp Leu 50 55
60Leu Ser Arg Leu His Ala Asn Gln Ser Arg Glu Asp Ser Asn Ser Glu65
70 75 80Pro Ser Pro Asp Pro Ala Val Arg Ile Leu Ser Pro Glu Val Arg
Leu 85 90 95Gly Ser His Gly Gln Leu Leu Leu Arg Val Asn Arg Ala Ser
Leu Ser 100 105 110Gln Gly Leu Pro Glu Ala Tyr Arg Val His Arg Ala
Leu Leu Leu Leu 115 120 125Thr Pro Thr Ala Arg Pro Trp Asp Ile Thr
Arg Pro Leu Lys Arg Ala 130 135 140Leu Ser Leu Arg Gly Pro Arg Ala
Pro Ala Leu Arg Leu Arg Leu Thr145 150 155 160Pro Pro Pro Asp Leu
Ala Met Leu Pro Ser Gly Gly Thr Gln Leu Glu 165 170 175Leu Arg Leu
Arg Val Ala Ala Gly Arg Gly Arg Arg Ser Ala His Ala 180 185 190His
Pro Arg Asp Ser Cys Pro Leu Gly Pro Gly Arg Cys Cys His Leu 195 200
205Glu Thr Val Gln Ala Thr Leu Glu Asp Leu Gly Trp Ser Asp Trp Val
210 215 220Leu Ser Pro Arg Gln Leu Gln Leu Ser Met Cys Val Gly Glu
Cys Pro225 230 235 240His Leu Tyr Arg Ser Ala Asn Thr His Ala Gln
Ile Lys Ala Arg Leu 245 250 255His Gly Leu Gln Pro Asp Lys Val Pro
Ala Pro Cys Cys Val Pro Ser 260 265 270Ser Tyr Thr Pro Val Val Leu
Met His Arg Thr Asp Ser Gly Val Ser 275 280 285Leu Gln Thr Tyr Asp
Asp Leu Val Ala Arg Gly Cys His Cys Ala 290 295 3005840DNAHomo
sapiensCDS(1)..(840) 5ctg tct ctg gcc gag gcg agc cgc gca agt ttc
ccg gga ccc tca gag 48Leu Ser Leu Ala Glu Ala Ser Arg Ala Ser Phe
Pro Gly Pro Ser Glu1 5 10 15ttg cac tcc gaa gac tcc aga ttc cga gag
ttg cgg aaa cgc tac gag 96Leu His Ser Glu Asp Ser Arg Phe Arg Glu
Leu Arg Lys Arg Tyr Glu 20 25 30gac ctg cta acc agg ctg cgg gcc aac
cag agc tgg gaa gat tcg aac 144Asp Leu Leu Thr Arg Leu Arg Ala Asn
Gln Ser Trp Glu Asp Ser Asn 35 40 45acc gac ctc gtc ccg gcc cct gca
gtc cgg ata ctc acg cca gaa gtg 192Thr Asp Leu Val Pro Ala Pro Ala
Val Arg Ile Leu Thr Pro Glu Val 50 55 60cgg ctg gga tcc ggc ggc cac
ctg cac ctg cgt atc tct cgg gcc gcc 240Arg Leu Gly Ser Gly Gly His
Leu His Leu Arg Ile Ser Arg Ala Ala65 70 75 80ctt ccc gag ggg ctc
ccc gag gcc tcc cgc ctt cac cgg gct ctg ttc 288Leu Pro Glu Gly Leu
Pro Glu Ala Ser Arg Leu His Arg Ala Leu Phe 85 90 95cgg ctg tcc ccg
acg gcg tca agg tcg tgg gac gtg aca cga ccg ctg 336Arg Leu Ser Pro
Thr Ala Ser Arg Ser Trp Asp Val Thr Arg Pro Leu 100 105 110cgg cgt
cag ctc agc ctt gca aga ccc cag gcg ccc gcg ctg cac ctg 384Arg Arg
Gln Leu Ser Leu Ala Arg Pro Gln Ala Pro Ala Leu His Leu 115 120
125cga ctg tcg ccg ccg ccg tcg cag tcg gac caa ctg ctg gca gaa tct
432Arg Leu Ser Pro Pro Pro Ser Gln Ser Asp Gln Leu Leu Ala Glu Ser
130 135 140tcg tcc gca cgg ccc cag ctg gag ttg cac ttg cgg ccg caa
gcc gcc 480Ser Ser Ala Arg Pro Gln Leu Glu Leu His Leu Arg Pro Gln
Ala Ala145 150 155 160agg ggg cgc cgc aga gcg cgt gcg cgc aac ggg
gac cac tgt ccg ctc 528Arg Gly Arg Arg Arg Ala Arg Ala Arg Asn Gly
Asp His Cys Pro Leu 165 170 175ggg ccc ggg cgt tgc tgc cgt ctg cac
acg gtc cgc gcg tcg ctg gaa 576Gly Pro Gly Arg Cys Cys Arg Leu His
Thr Val Arg Ala Ser Leu Glu 180 185 190gac ctg ggc tgg gcc gat tgg
gtg ctg tcg cca cgg gag gtg caa gtg 624Asp Leu Gly Trp Ala Asp Trp
Val Leu Ser Pro Arg Glu Val Gln Val 195 200 205acc atg tgc atc ggc
gcg tgc ccg agc cag ttc cgg gcg gca aac atg 672Thr Met Cys Ile Gly
Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met 210 215 220cac gcg cag
atc aag acg agc ctg cac cgc ctg aag ccc gac acg gtg 720His Ala Gln
Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp Thr Val225 230 235
240cca gcg ccc tgc tgc gtg ccc gcc agc tac aat ccc atg gtg ctc att
768Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile
245 250 255caa aag acc gac acc ggg gtg tcg ctc cag acc tat gat gac
ttg tta 816Gln Lys Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp
Leu Leu 260 265 270gcc aaa gac tgc cac tgc ata tga 840Ala Lys Asp
Cys His Cys Ile 2756279PRTHomo sapiens 6Leu Ser Leu Ala Glu Ala Ser
Arg Ala Ser Phe Pro Gly Pro Ser Glu1 5 10 15Leu His Ser Glu Asp Ser
Arg Phe Arg Glu Leu Arg Lys Arg Tyr Glu 20 25 30Asp Leu Leu Thr Arg
Leu Arg Ala Asn Gln Ser Trp Glu Asp Ser Asn 35 40 45Thr Asp Leu Val
Pro Ala Pro Ala Val Arg Ile Leu Thr Pro Glu Val 50 55 60Arg Leu Gly
Ser Gly Gly His Leu His Leu Arg Ile Ser Arg Ala Ala65 70 75 80Leu
Pro Glu Gly Leu Pro Glu Ala Ser Arg Leu His Arg Ala Leu Phe 85 90
95Arg Leu Ser Pro Thr Ala Ser Arg Ser Trp Asp Val Thr Arg Pro Leu
100 105 110Arg Arg Gln Leu Ser Leu Ala Arg Pro Gln Ala Pro Ala Leu
His Leu 115 120 125Arg Leu Ser Pro Pro Pro Ser Gln Ser Asp Gln Leu
Leu Ala Glu Ser 130 135 140Ser Ser Ala Arg Pro Gln Leu Glu Leu His
Leu Arg Pro Gln Ala Ala145 150 155 160Arg Gly Arg Arg Arg Ala Arg
Ala Arg Asn Gly Asp His Cys Pro Leu 165 170 175Gly Pro Gly Arg Cys
Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu 180 185 190Asp Leu Gly
Trp Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val 195 200 205Thr
Met Cys Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met 210 215
220His Ala Gln Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp Thr
Val225 230 235 240Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn Pro
Met Val Leu Ile 245 250 255Gln Lys Thr Asp Thr Gly Val Ser Leu Gln
Thr Tyr Asp Asp Leu Leu 260 265 270Ala Lys Asp Cys His Cys Ile
2757816DNAMus musculusCDS(1)..(816) 7tcg cag ggg gac gcc ctg gca
atg cct gaa cag cga ccc tcc ggc cct 48Ser Gln Gly Asp Ala Leu Ala
Met Pro Glu Gln Arg Pro Ser Gly Pro1 5 10 15gag tcc caa ctc aac gcc
gac gag cta cgg ggt cgc ttc cag gac ctg 96Glu Ser Gln Leu Asn Ala
Asp Glu Leu Arg Gly Arg Phe Gln Asp Leu 20 25 30ctg agc cgg ctg cat
gcc aac cag agc cga gag gac tcg aac tca gaa 144Leu Ser Arg Leu His
Ala Asn Gln Ser Arg Glu Asp Ser Asn Ser Glu 35 40 45cca agt cct gac
cca gct gtc cgg ata ctc agt cca gag gtg aga ttg 192Pro Ser Pro Asp
Pro Ala Val Arg Ile Leu Ser Pro Glu Val Arg Leu 50 55 60ggg tcc cac
ggc cag ctg cta ctc cgc gtc aac cgg gcg tcg ctg agt 240Gly Ser His
Gly Gln Leu Leu Leu Arg Val Asn Arg Ala Ser Leu Ser65 70
75 80cag ggt ctc ccc gaa gcc tac cgc gtg cac cga gcg ctg ctc ctg
ctg 288Gln Gly Leu Pro Glu Ala Tyr Arg Val His Arg Ala Leu Leu Leu
Leu 85 90 95acg ccg acg gcc cgc ccc tgg gac atc act agg ccc ctg aag
cgt gcg 336Thr Pro Thr Ala Arg Pro Trp Asp Ile Thr Arg Pro Leu Lys
Arg Ala 100 105 110ctc agc ctc cgg gga ccc cgt gct ccc gca tta cgc
ctg cgc ctg acg 384Leu Ser Leu Arg Gly Pro Arg Ala Pro Ala Leu Arg
Leu Arg Leu Thr 115 120 125ccg cct ccg gac ctg gct atg ctg ccc tct
ggc ggc acg cag ctg gaa 432Pro Pro Pro Asp Leu Ala Met Leu Pro Ser
Gly Gly Thr Gln Leu Glu 130 135 140ctg cgc tta cgg gta gcc gcc ggc
agg ggg cgc cga agc gcg cat gcg 480Leu Arg Leu Arg Val Ala Ala Gly
Arg Gly Arg Arg Ser Ala His Ala145 150 155 160cac cca aga gac tcg
tgc cca ctg ggt ccg ggg cgc tgc tgt cac ttg 528His Pro Arg Asp Ser
Cys Pro Leu Gly Pro Gly Arg Cys Cys His Leu 165 170 175gag act gtg
cag gca act ctt gaa gac ttg ggc tgg agc gac tgg gtg 576Glu Thr Val
Gln Ala Thr Leu Glu Asp Leu Gly Trp Ser Asp Trp Val 180 185 190ctg
tcc ccg cgc cag ctg cag ctg agc atg tgc gtg ggc gag tgt ccc 624Leu
Ser Pro Arg Gln Leu Gln Leu Ser Met Cys Val Gly Glu Cys Pro 195 200
205cac ctg tat cgc tcc gcg aac acg cat gcg cag atc aaa gca cgc ctg
672His Leu Tyr Arg Ser Ala Asn Thr His Ala Gln Ile Lys Ala Arg Leu
210 215 220cat ggc ctg cag cct gac aag gtg cct gcc ccg tgc tgt gtc
ccc tcc 720His Gly Leu Gln Pro Asp Lys Val Pro Ala Pro Cys Cys Val
Pro Ser225 230 235 240agc tac acc ccg gtg gtt ctt atg cac agg aca
gac agt ggt gtg tca 768Ser Tyr Thr Pro Val Val Leu Met His Arg Thr
Asp Ser Gly Val Ser 245 250 255ctg cag act tat gat gac ctg gtg gcc
cgg ggc tgc cac tgc gct tga 816Leu Gln Thr Tyr Asp Asp Leu Val Ala
Arg Gly Cys His Cys Ala 260 265 2708271PRTMus musculus 8Ser Gln Gly
Asp Ala Leu Ala Met Pro Glu Gln Arg Pro Ser Gly Pro1 5 10 15Glu Ser
Gln Leu Asn Ala Asp Glu Leu Arg Gly Arg Phe Gln Asp Leu 20 25 30Leu
Ser Arg Leu His Ala Asn Gln Ser Arg Glu Asp Ser Asn Ser Glu 35 40
45Pro Ser Pro Asp Pro Ala Val Arg Ile Leu Ser Pro Glu Val Arg Leu
50 55 60Gly Ser His Gly Gln Leu Leu Leu Arg Val Asn Arg Ala Ser Leu
Ser65 70 75 80Gln Gly Leu Pro Glu Ala Tyr Arg Val His Arg Ala Leu
Leu Leu Leu 85 90 95Thr Pro Thr Ala Arg Pro Trp Asp Ile Thr Arg Pro
Leu Lys Arg Ala 100 105 110Leu Ser Leu Arg Gly Pro Arg Ala Pro Ala
Leu Arg Leu Arg Leu Thr 115 120 125Pro Pro Pro Asp Leu Ala Met Leu
Pro Ser Gly Gly Thr Gln Leu Glu 130 135 140Leu Arg Leu Arg Val Ala
Ala Gly Arg Gly Arg Arg Ser Ala His Ala145 150 155 160His Pro Arg
Asp Ser Cys Pro Leu Gly Pro Gly Arg Cys Cys His Leu 165 170 175Glu
Thr Val Gln Ala Thr Leu Glu Asp Leu Gly Trp Ser Asp Trp Val 180 185
190Leu Ser Pro Arg Gln Leu Gln Leu Ser Met Cys Val Gly Glu Cys Pro
195 200 205His Leu Tyr Arg Ser Ala Asn Thr His Ala Gln Ile Lys Ala
Arg Leu 210 215 220His Gly Leu Gln Pro Asp Lys Val Pro Ala Pro Cys
Cys Val Pro Ser225 230 235 240Ser Tyr Thr Pro Val Val Leu Met His
Arg Thr Asp Ser Gly Val Ser 245 250 255Leu Gln Thr Tyr Asp Asp Leu
Val Ala Arg Gly Cys His Cys Ala 260 265 2709339DNAHomo
sapiensCDS(1)..(339) 9gcg cgc aac ggg gac cac tgt ccg ctc ggg ccc
ggg cgt tgc tgc cgt 48Ala Arg Asn Gly Asp His Cys Pro Leu Gly Pro
Gly Arg Cys Cys Arg1 5 10 15ctg cac acg gtc cgc gcg tcg ctg gaa gac
ctg ggc tgg gcc gat tgg 96Leu His Thr Val Arg Ala Ser Leu Glu Asp
Leu Gly Trp Ala Asp Trp 20 25 30gtg ctg tcg cca cgg gag gtg caa gtg
acc atg tgc atc ggc gcg tgc 144Val Leu Ser Pro Arg Glu Val Gln Val
Thr Met Cys Ile Gly Ala Cys 35 40 45ccg agc cag ttc cgg gcg gca aac
atg cac gcg cag atc aag acg agc 192Pro Ser Gln Phe Arg Ala Ala Asn
Met His Ala Gln Ile Lys Thr Ser 50 55 60ctg cac cgc ctg aag ccc gac
acg gtg cca gcg ccc tgc tgc gtg ccc 240Leu His Arg Leu Lys Pro Asp
Thr Val Pro Ala Pro Cys Cys Val Pro65 70 75 80gcc agc tac aat ccc
atg gtg ctc att caa aag acc gac acc ggg gtg 288Ala Ser Tyr Asn Pro
Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val 85 90 95tcg ctc cag acc
tat gat gac ttg tta gcc aaa gac tgc cac tgc ata 336Ser Leu Gln Thr
Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile 100 105 110taa
33910112PRTHomo sapiens 10Ala Arg Asn Gly Asp His Cys Pro Leu Gly
Pro Gly Arg Cys Cys Arg1 5 10 15Leu His Thr Val Arg Ala Ser Leu Glu
Asp Leu Gly Trp Ala Asp Trp 20 25 30Val Leu Ser Pro Arg Glu Val Gln
Val Thr Met Cys Ile Gly Ala Cys 35 40 45Pro Ser Gln Phe Arg Ala Ala
Asn Met His Ala Gln Ile Lys Thr Ser 50 55 60Leu His Arg Leu Lys Pro
Asp Thr Val Pro Ala Pro Cys Cys Val Pro65 70 75 80Ala Ser Tyr Asn
Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val 85 90 95Ser Leu Gln
Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile 100 105
11011351DNAMus musculusCDS(1)..(351) 11atg agc gcg cat gcg cac cca
aga gac tcg tgc cca ctg ggt ccg ggg 48Met Ser Ala His Ala His Pro
Arg Asp Ser Cys Pro Leu Gly Pro Gly1 5 10 15cgc tgc tgt cac ctg gag
act gtg cag gca act ctt gaa gac ttg ggc 96Arg Cys Cys His Leu Glu
Thr Val Gln Ala Thr Leu Glu Asp Leu Gly 20 25 30tgg agc gac tgg gtg
ttg tcc ccg cgc cag ctg cag ctg agc atg tgc 144Trp Ser Asp Trp Val
Leu Ser Pro Arg Gln Leu Gln Leu Ser Met Cys 35 40 45gtg ggc gag tgt
ccc cac ctg tat cgc tcc gcg aac acg cat gcg cag 192Val Gly Glu Cys
Pro His Leu Tyr Arg Ser Ala Asn Thr His Ala Gln 50 55 60atc aaa gca
cgc ctg cat ggc ctg cag cct gac aag gtg cct gcc ccg 240Ile Lys Ala
Arg Leu His Gly Leu Gln Pro Asp Lys Val Pro Ala Pro65 70 75 80tgc
tgt gtc ccc tcc agc tac acc ccg gtg gtt ctt atg cac agg aca 288Cys
Cys Val Pro Ser Ser Tyr Thr Pro Val Val Leu Met His Arg Thr 85 90
95gac agt ggt gtg tca ctg cag act tat gat gac ctg gtg gcc cgg ggc
336Asp Ser Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Val Ala Arg Gly
100 105 110tgc cac tgc gct tga 351Cys His Cys Ala 11512116PRTMus
musculus 12Met Ser Ala His Ala His Pro Arg Asp Ser Cys Pro Leu Gly
Pro Gly1 5 10 15Arg Cys Cys His Leu Glu Thr Val Gln Ala Thr Leu Glu
Asp Leu Gly 20 25 30Trp Ser Asp Trp Val Leu Ser Pro Arg Gln Leu Gln
Leu Ser Met Cys 35 40 45Val Gly Glu Cys Pro His Leu Tyr Arg Ser Ala
Asn Thr His Ala Gln 50 55 60Ile Lys Ala Arg Leu His Gly Leu Gln Pro
Asp Lys Val Pro Ala Pro65 70 75 80Cys Cys Val Pro Ser Ser Tyr Thr
Pro Val Val Leu Met His Arg Thr 85 90 95Asp Ser Gly Val Ser Leu Gln
Thr Tyr Asp Asp Leu Val Ala Arg Gly 100 105 110Cys His Cys Ala
115
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