U.S. patent application number 16/840046 was filed with the patent office on 2020-08-06 for treatments of metabolic disorders using fgf binding protein 3.
This patent application is currently assigned to Georgetown University. The applicant listed for this patent is Georgetown University. Invention is credited to Anton Wellstein.
Application Number | 20200246425 16/840046 |
Document ID | / |
Family ID | 1000004782510 |
Filed Date | 2020-08-06 |
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United States Patent
Application |
20200246425 |
Kind Code |
A1 |
Wellstein; Anton |
August 6, 2020 |
Treatments of Metabolic Disorders Using FGF Binding Protein 3
Abstract
The invention relates to methods of treating a metabolic
disorder in a subject, the method comprising administering
fibroblast growth factor binding protein 3 (FGFBP3) or a variant
thereof to a subject in need of treatment of a metabolic
disorder.
Inventors: |
Wellstein; Anton;
(Washington, DC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Georgetown University |
Washington |
DC |
US |
|
|
Assignee: |
Georgetown University
Washington
DC
|
Family ID: |
1000004782510 |
Appl. No.: |
16/840046 |
Filed: |
April 3, 2020 |
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Application
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15784730 |
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16840046 |
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14853482 |
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61782382 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/1709
20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0001] Part of the work performed during development of this
invention utilized U.S. Government funds through National
Institutes of Health Grant Nos. RO1 CA71508 and PO1 HL068686. The
U.S. Government has certain rights in this invention.
Claims
1-20. (canceled)
21. A method of treating a metabolic disorder in a subject in need
thereof, the method comprising administering fibroblast growth
factor binding protein 3 (FGFBP3) to the subject; wherein the
metabolic disorder is selected from fatty liver, fatty liver
disease, or a combination thereof.
22. The method of claim 21, wherein the metabolic disorder is fatty
liver.
23. The method of claim 21, wherein the metabolic disorder is fatty
liver disease.
24. The method of claim 21, wherein the subject has metabolic
syndrome.
25. The method of claim 21, wherein the FGFBP3 is administered as a
complex with fibroblast growth factor 19 (FGF19).
Description
SEQUENCE LISTING INFORMATION
[0002] A computer readable text file, entitled
"SequenceListing.txt," created on or about 16 Oct. 2017 with a file
size of about 9 kb contains the sequence listing for this
application and is hereby incorporated by reference in its
entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The invention relates to methods of treating a metabolic
disorder in a subject, the method comprising administering
fibroblast growth factor binding protein 3 (FGFBP3) to a subject in
need of treatment of a metabolic disorder.
[0004] The invention also relates to methods of treating a
metabolic disorder in a subject, the method comprising
administering a complex of fibroblast growth factor 19 (FGF19),
fibroblast growth factor 21 (FGF21) or fibroblast growth factor 23
(FGF23), plus fibroblast growth factor binding protein 3 (FGFBP3)
to a subject in need of treatment of a metabolic disorder.
Background of the Invention
[0005] Fibroblast growth factor 19 (FGF19) and other members of the
FGF19 family (i.e. FGF21 and FGF23, the so-called "endocrine FGFs")
are involved in the regulation of metabolism. FGF19 and FGF21 have
also been recently described as a sensitizer to insulin. In
addition, some members of the FGF19 family interact with the
co-receptor klotho to affect metabolism.
[0006] Previous studies indicate that FGF-binding proteins (FGFBP)
can enhance the effects of FGF by mobilizing FGFs from their
storage depots found in extracellular glycosaminoglycans or
heparansulfate proteoglycans. In contrast to most other FGFs,
members of the FGF19 family of proteins, i.e., FGF19, FGF21 and
FGF23, show only very little binding to glycosaminoglycans or
heparansulfates in the extracellular matrix. The most recently
discovered member of the FGFBP3 family also appears to mobilize
FGFs from such storage depots and bind to FGFs including FGF19
family members.
SUMMARY OF THE INVENTION
[0007] The invention relates to methods of treating a metabolic
disorder in a subject, the method comprising administering
fibroblast growth factor binding protein 3 (FGFBP3) to a subject in
need of treatment of a metabolic disorder.
[0008] The invention also relates to methods of lowering blood
glucose levels in a subject, the method comprising administering
FGFBP3 to a subject in need of lowering of blood glucose
levels.
[0009] The invention also relates to methods of lowering a
subject's body weight, the method comprising administering FGFBP3
to a subject that is in need of lowering its body weight.
[0010] The invention also relates to methods of lowering a
subject's atherogenic serum lipids, the method comprising
administering FGFBP3 to a subject that is in need of lowering
atherogenic lipids.
[0011] The invention also relates to methods of treating a
metabolic disorder in a subject, the method comprising
administering a complex of fibroblast growth factor 19 (FGF19),
fibroblast growth factor 21 (FGF21) or fibroblast growth factor 23
(FGF23), plus fibroblast growth factor binding protein 3 (FGFBP3)
to a subject in need of treatment of a metabolic disorder.
[0012] The invention also relates to methods of lowering blood
glucose levels in a subject, the method comprising administering a
complex of FGF19, FGF21 or FGF23 and FGFBP3 to a subject in need of
lowering of blood glucose levels.
[0013] The invention also relates to methods of lowering a
subject's body weight, the method comprising administering a
complex of FGF19, FGF21 or FGF23 and FGFBP3 to a subject that is in
need of lowering its body weight.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 depicts the effects of a single dose of FGFBP3 alone
or FGF19+FGFBP3 treatments on glucose metabolism in fed ob/ob mice
that are diabetic. The Left Panel, shows that upon intraperitoneal
injection of FGFBP3 alone, glucose levels fell from a diabetic
level to roughly normal levels (100 to 150 mg/dl) beginning within
2 hours after the first treatment. Levels stayed close to normal
range (compared to controls) for 24 hours following injection. A
comparison with an injection of FGF19+FGFBP3 (both panels), shows
that FGFBP3 alone had the same absolute effect as the combination
with FGF19. BP3 showed a greater relative effect (Right Panel) on
lowering glucose levels when normalized to account for different
baseline glucose levels.
[0015] FIG. 2 depicts the effects of BP3 on glycemia in fed ob/ob
mice. Blood glucose levels after treatment with a single
intraperitoneal injection of BP3 or control protein
(=MBP).+-.pretreatment with an anti FGF15 antibody. Mean.+-.SEM;
n.gtoreq.5 mice/group. ns, non significant; ***, P<0.0001 vs.
control (=MBP). B. Blood glucose levels in fed ob/ob mice 24 hours
after a single intraperitoneal administration of increasing doses
of BP3, FGF19 or control (=MBP). The values are calculated relative
to baseline levels of blood glucose (mg/dl). Mean.+-.SEM;
n.gtoreq.5 mice/group. ***, P<0.0001 vs. control (=MBP). C.
Blood glucose of short-term starved ob/ob mice 2 hours after
treatment with a single intraperitoneal injection of BP3 (0.8
mg/kg), FGF19 (1 mg/kg), BP3+FGF19, and control (=MBP; 1 mg/kg).
Mean.+-.SEM; n=4 mice/group. **, P<0.001.
[0016] FIG. 3 depicts the ability of BP3 to inhibit gluconeogenesis
through IRS2/AKT and IL6/STAT3-dependent downregulation of G6PC by
modulating endogenous FGF15 activity. A. Changes in hepatic gene
expression determined by cDNA array (left) versus qRT-PCR (right).
Values are calculated as fold of control treatment levels.
Mean.+-.SEM, n=3/group. ns, non significant; *, P<0.05; **,
P<0.001; ***, P<0.0001. B. AKT and STAT3 phosphorylation in
ob/ob liver lysates from each experimental group were assayed by
immunoprecipitation and western blot with phosphospecific
antibodies. The expression of AKT and STAT3 were also determined by
immunoblotting with specific antibodies. The numbers below the
blots indicate the fold-change, corrected for the total protein
expression. The blots are representative of three independent
experiments. C. Schematic summary of the FGFR4-FGF19-BP3 regulatory
pathways in liver tissue. Sensitization of FGFR4/FGF19 pathway by
BP3 results in a downstream activation of IRS2/AKT and IL6/STAT3
signaling pathways, leading to the inhibition of gluconeogenesis,
through G6PC downregulation. Likewise, activation of STAT3 results
in an inhibition of PPARGC1B and SREBF1, leading to the inhibition
of lipogenesis. Activation of FGFR4/FGF19 by BP3 also results in a
suppression of bile acid biosynthesis through the downregulation of
CYP7A1 gene. D. Hierarchical cluster analysis of gene expression in
livers of fed ob/ob mice treated with recombinant human BP3, FGF19,
anti FGF15, anti FGF15+BP3 or MBP control for 4 hours. The cluster
analysis shows a separation of BP3 treatment from all other
conditions (n=3 independent samples per group).
[0017] FIG. 4 depicts the driver pathways of BP3 effects. Ingenuity
pathway analysis of the differentially expressed liver genes in
ob/ob mice treated with BP3, anti FGF15, BP3 +anti FGF15 (striped
bar) or FGF19 and normalized to the control group (=MBP). Upper
panel: Z-score predicting the activation of signaling pathways
based on Ingenuity upstream regulator analysis. Lower panel:
Z-score predicting the activation of metabolic functions identified
by Ingenuity global function analysis. Z-scores smaller (inhibited)
or greater (activated) than 2 were considered biologically
significant and are represented by dashed lines. The respective
P-values for the Z-scores are stated on the right.
[0018] FIG. 5 depicts the ability of BP3 to reduce blood glucose
levels in fed, healthy, non-diabetic C57BL mice. A. Blood glucose
levels of fed C57BL mice treated with a single intraperitoneal
injection of BP3 or MBP control (0.8 mg/kg). Blood glucose was
measured at 2 and 4 hours after administration. Mean.+-.SEM; n=3.
*, P<0.05; **, P<0.001; ***, P<0.0001. B. Changes in
hepatic gene expression determined by qRT-PCR (right) versus cDNA
array (Illumina) (left). Values are calculated as fold of MBP
control levels. Mean.+-.SEM, n=2. *, P<0.05; **, P<0.001;
***, P<0.0001. C. C57BL mice were administered MBP or BP3 (0.8
mg/kg) by intraperitoneal injection for four hours. AKT and STAT3
phosphorylation in liver lysates were assayed by
immunoprecipitation and western blot with specific antibodies. The
expression of total AKT and STAT3 were also determined by
immunoblotting with specific antibodies. The numbers below the
blots indicate the fold-change, corrected for the total protein
expression, relative to the control group. The blots are
representative of three independent experiments. D. Ingenuity
pathway analysis of the differentially expressed genes in C57BL
mouse livers treated with BP3 and normalized to the MBP control
group. Z-score predicting the activation of signaling pathways
based on Ingenuity upstream regulator analysis. Z-scores smaller
(inhibited) or greater (activated) than 2 were considered
biologically significant and are represented by dashed lines.
[0019] FIG. 6 depicts the ability of BP3 to selectively bind to
FGFR4. A. Binding of BP3 or MBP (ctrl) to immobilized FGFRs was
measured by direct ELISA with an anti MBP antibody. Mean.+-.SEM of
one of three independent experiments done in duplicate. ***,
P<0.0001 BP3 (black bars) vs. MBP ctrl (white bars). B: SPR
sensorgrams illustrating the binding kinetics of BP3 to immobilized
FGFR4 and FGFR1. The concentration of the BP3 analyte was varied
from 4 to 1 nM. RU, response units. C: Binding of increasing
concentrations of BP3 or MBP to immobilized FGFR4 measured by
direct ELISA with an anti MBP antibody. Mean.+-.SEM of one of three
independent experiments done in duplicate
[0020] FIG. 7 depicts that the C-terminal 66-amino acid long
FGF-binding domain of BP3 ("C66") is sufficient to reduce
hyperglycemia in diabetic mice and to stabilize FGFR4/FGF19 complex
formation. A. Coomassie blue staining of MBP-tagged C66 fusion
protein purified by amylose affinity chromatography. The arrow
indicates a band of an apparent molecular mass of 52 kDa. B. SPR
sensorgrams illustrating the binding of C66 to immobilized FGF19,
FGF2 and FGFR4. RU, response units. C. Schematic representation of
human BP3. The numbers correspond to the human BP3 amino acid
sequence (upper panel). Equilibrium binding of FGF19, C66 or their
combination to immobilized FGFR1 or FGFR4 was analyzed by SPR
(lower panel). Mean.+-.SEM of three independent experiments. Data
are represented as percent difference to FGF19 binding to FGFR1 or
FGFR4. ***, P<0.0001, FGF19+C66 vs. FGF19. D. Effect of C66 on
glycemia in ob/ob mice. Blood glucose levels of fed ob/ob mice
treated with a single intraperitoneal injection of C66, BP3 or MBP
control (0.8 mg/kg). Blood glucose was measured at 2 and 4 hours
after administration. Mean.+-.SEM; n=3-11 mice/group. *, P<0.05;
**, P<0.001; ***, P<0.0001. top asterisks: BP3 vs. MBP;
bottom asterisks: C66 vs. MBP. E. Changes in hepatic gene
expression determined by qRT-PCR in fed ob/ob mice treated with MBP
and C66 for 4 hours. Values are calculated as fold of MBP control
levels. Mean.+-.SEM, n=3/group. *, P<0.05; **, P<0.001.
[0021] FIG. 8 depicts the effects of multiple FGF19 or FGF19+BP3
treatments on glucose blood levels in ob/ob mice after a bolus
injection of glucose (=glucose tolerance test). A, Five treatments
with FGF19 reduced blood glucose levels at 180 minutes after the
beginning of the glucose tolerance test, and the curve returned to
the baseline 9 days after receiving no treatment. Filled Squares:
Baseline; Open Squares: FGF19 (one dose per day for 5 days); Open
Circles: FGF19 (one dose per day for 5 days+9 days of no
treatment). B, Five treatments with FGF19+BP3 improved the glucose
tolerance at 60, 120, and 180 minutes with a sustained effect for 9
days. Filled Squares: Baseline; Open Squares: FGF19+BP3 (one dose
per day for 5 days); Open Circles: FGF 19+BP3 (one dose per day for
5 days+9 days of no treatment). C, The area under the curve of the
glucose tolerance test (AUC) was improved after 5 treatments in
both groups. After another 9 days without any treatment, only the
FGF19+BP3 group exhibited an improved glucose tolerance test. Solid
Bars: Baseline, Open Bars: FGF19 or FGF19+BP3 (one dose per day for
5 days); Hatched Bars, FGF19 or FGF19+BP3 (one dose per day for 5
days+9 days of no treatment). * denotes P<0.01.
[0022] FIG. 9 depicts the effects of a single dose of FGF19 or
FGF19+BP3 treatment on glucose blood levels in ob/ob mice after a
bolus injection of glucose (=glucose tolerance test). A, The
glucose levels were not changed 2 days after a single dose of FGF19
alone. Filled Squares: Baseline, Open Squares: single dose
treatment. B, In the FGF19+BP3 group, the glucose levels were
significantly reduced at 15, 30 and 60 minutes post-injection of
glucose. C, The area under the curve of the glucose tolerance test
(AUC) was reduced 2 days after a single treatment in FGF19+BP3
group. Filled Squares: Baseline, Open Squares: Single treatment. *
denotes P<0.05, ** P<0.01.
[0023] FIG. 10 depicts the effects of acute single doses of BP3,
FGF19, and the complex of BP3+FGF19 in ob/ob mice in response to a
glucose tolerance test. FIGS. 3A, 3B show the effect when the test
was conducted two hours after treatment. FIG. 3C shows the effect
when the test was performed 24 hours after treatment. Even 7 days
of dosing of BP3 alone with a single daily dose of BP3 lacked an
effect on the baseline blood glucose or on the IPGTT test (FIG.
3D).
[0024] FIG. 11 depicts the body weight changes in ob/ob mice
following single or multiple treatments with FGF19 or FGF19+BP3. A,
The changes in body weight were significantly different between the
FGF19 group and the combination group immediately after 5
treatments (2.2.+-.0.8 g vs. -1.0.+-.0.7 g), and a clear trend was
also observed 2 days after a single treatment (p=0.055). Filled
Squares: single treatment, Open Squares: 5 treatments at one
dose/day. B, The percentage changes in body weight is also
different between the two groups immediately after 5 daily
treatments (6.04.+-.2.33% vs. -3.40.+-.2.50%). Filled Squares:
single treatment; Open Squares: 5 treatments (one dose/day). *
denotes P<0.05.
[0025] FIG. 12 depicts the presence of BP3 enhancing the ability of
FGF19 to induce phosphorylation of Erk1/2 in HepG2 cells. HepG2
cells were treated with FGF19.+-.BP3 or the negative control
protein MBP. pERK1/2 was measured in cell lysates at different
times after treatment.
[0026] FIG. 13 depicts a significant reduction of non-esterified
fatty acids (NEFA) in the serum (p=0.025) of transgenic animals
expressing mouse FGFBP3. No significant changes in the other lipids
were noted. In addition, the mice remained hyperinsulinemic, which
is a known phenotype for ob/ob mice. FGFBP3 alone did not change
glucagon or insulin levels significantly. The reduction of NEFA
after BP3 expression indicates an improved metabolic disease state
of the animals.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The invention relates to methods of treating a metabolic
disorder in a subject, the methods comprising administering a
fibroblast growth factor binding protein 3 (FGFBP3) or an
appropriate variant thereof to a subject in need of treatment of a
metabolic disorder. The invention also relates to methods of
treating a metabolic disorder in a subject, the methods comprising
administering a complex of fibroblast growth factor 19 (FGF19),
fibroblast growth factor 21 (FGF21) or fibroblast growth factor 23
(FGF23), plus fibroblast growth factor binding protein 3 (FGFBP3)
or variant thereof to a subject in need of treatment of a metabolic
disorder. As used herein, the term "subject" is used
interchangeably with the term "patient" and is also used to include
an animal, in particular a mammal, and even more particularly a
non-human or human primate or dog or cat to give examples.
[0028] The fibroblast growth factor binding protein 3 (herein
referred to interchangeably as BP3 or FGFBP3) is a secreted protein
that binds to human FGF19, FGF21 and FGF23. Fibroblast growth
factor 19 (FGF19) is the signature member of the FGF19 family of
proteins that is involved in nutrient metabolism. FGF19 is a
protein of 216 amino acids, with the signal peptide being amino
acids 1-24. As used herein, "fibroblast growth factor 19" or
"FGF19" can mean the full length FGF19 with or without the
N-terminus signal sequence. The mouse ortholog to human FGF19 is
known as FGF15 and is 218 amino acids in length, including the
25-amino acid signal sequence at the N-terminus. As used herein,
"fibroblast growth factor 15" or "FGF15" or "mFGF15" is used to
indicate any ortholog to hFGF19 and can include the full length
amino acid sequence, with or without the N-terminus signal
sequence. It is understood that a reference to "FGF19" or "hFGF19"
herein will also include a reference to its art-accepted orthologs,
such as mouse FGF15. In general the concentration of FGF19 (or
mFGF15) is upregulated after feeding and binds preferentially to
FGF Receptor 4 (FGFR4). Specifically, FGF19 is synthesized in the
distal small intestine in response to uptake of bile acids via the
nuclear bile receptor and controlled by food intake. FGF21
expression in the liver and fat tissues is also regulated by the
feeding or starving status and function in a temporal cascade with
insulin, glucagon and other hormones to regulate responses to
nutrition (Potthoff, et al., Genes and Development 2012).
[0029] FGFBP3 is believed to act as a co-receptor with FGFR4. The
full length amino acid sequence of human FGFBP3 is shown below as
SEQ ID NO:1. The full length amino acid sequence of human FGFBP3,
without the 26 amino-acid signal sequence, is shown below as SEQ ID
NO:2. The C-terminus of FGFBP3 is shown below as SEQ ID NO:3.
[0030] As used herein, "FGFBP3" means a peptide that comprises the
amino acid sequence of SEQ ID NO:3 or a variant thereof that still
retains activity similar to the wild-type FGFBP3. Thus, the amino
acid sequence of SEQ ID NOs:1 and 2 are just two embodiments of the
term FGFBP3 as it is used herein. "Variants" of FGFBP3 are
discussed below.
TABLE-US-00001 (SEQ ID NO: 1) MTPPKLRASL SPSLLLLLSG CLLAAARREK
GAASNVAEPV PGPTGGSSGR FLSPEQHACS 60 WQLLLPAPEA AAGSELALRC
QSPDGARHQC AYRGHPERCA AYAARRAHFW KQVLGGLRKK 120 RRPCHDPAPL
QARLCAGKKG HGAELRLVPR ASPPARPTVA GFAGESKPRA RNRGRTRERA 180
SGPAAGTPPP QSAPPKENPS ERKTNEGKRK AALVPNEERP MGTGPDPDGL DGNAELTETY
240 CAEKWHSLCN FFVNFWNG 258 (SEQ ID NO: 2) RREK GAASNVAEPV
PGPTGGSSGR FLSPEQHACS WQLLLPAPEA AAGSELALRC QSPDGARHQC 64
AYRGHPERCA AYAARRAHFW KQVLGGLRKK RRPCHDPAPL QARLCAGKKG HGAELRLVPR
124 ASPPARPTVA GFAGESKPRA RNRGRTRERA SGPAAGTPPP QSAPPKENPS
ERKTNEGKRK 184 AALVPNEERP MGTGPDPDGL DGNAELTETY CAEKWHSLCN FFVNFWNG
232 (SEQ ID NO: 3) LDGNAELTET YCAEKWHSLC NFFVNFWNG 29 (SEQ ID NO:
4) APPKENPSER KTNEGKRKAA LVPNEERPMG TGPDPDGLDG NAELTETYCA
EKWHSLCNFF 60 VNFWNG 66
[0031] The present invention is directed to methods that include
administration of FGFBP3. The FGFBP3 can, but need not, be
specifically interacting with FGF19, i.e., specifically binding to
one another. Other functions of FGFBP3 include but are not limited
to the ability to interact with other members of the family of
FGF19 proteins such as FGF21 and FGF23. FGFBP3 may exert its effect
by interacting with FGF21 and/or FGF23.
[0032] The present invention is also directed to methods that
include administration of a complex of FGF19 and FGFBP3. As used
herein, the term "complex" as it relates to FGF19 and FGFBP3 means
the presence of both FGFBP3 and FGF19. The FGF19 and FGFBP3 can,
but need not, specifically interact, i.e., specifically bind to one
another. In one embodiment, the FGF19 and FGFBP3 within the complex
are specifically bound to one another. In another embodiment, the
FGF19 and FGFBP3 within the complex are not necessarily
specifically binding to one another.
[0033] Accordingly, in some embodiment of the methods of the
present invention, full length FGFBP3 (a peptide amino acid
sequence of SEQ ID NO:1) is administered. In select of these
embodiments, the FGFBP3 comprises a peptide with an amino acid
sequence that is 100% identical to the amino acid sequence of SEQ
ID NO:1. In additional embodiments, the FGFBP3 consists of a
peptide with an amino acid sequence that is 100% identical to the
amino acid sequence of SEQ ID NO:1.
[0034] In other embodiments, full length FGFBP3 (a peptide amino
acid sequence of SEQ ID NO:1) is complexed with FGF19. In select of
these embodiments, the FGFBP3 in the complex comprises a peptide
with an amino acid sequence that is 100% identical to the amino
acid sequence of SEQ ID NO:1. In additional embodiments, the FGFBP3
in the complex consists of a peptide with an amino acid sequence
that is 100% identical to the amino acid sequence of SEQ ID
NO:1.
[0035] In additional embodiments of the methods of the present
invention, full length FGFBP3 without the signal sequence (a
peptide amino acid sequence of SEQ ID NO:2) is administered. In
select of these embodiments, the FGFBP3 peptide comprises an amino
acid sequence that is 100% identical to the amino acid sequence of
SEQ ID NO:2. In additional embodiments, the FGFBP3 peptide consists
of an amino acid sequence that is 100% identical to the amino acid
sequence of SEQ ID NO:2.
[0036] In other embodiments, full length FGFBP3 without the signal
sequence (a peptide amino acid sequence of SEQ ID NO:2) is
complexed with FGF19. In select of these embodiments, the FGFBP3 in
the complex comprises a peptide with an amino acid sequence that is
100% identical to the amino acid sequence of SEQ ID NO:2. In
additional embodiments, the FGFBP3 in the complex consists of a
peptide with an amino acid sequence that is 100% identical to the
amino acid sequence of SEQ ID NO:2.
[0037] In still additional embodiment of the methods of the present
invention, the C-terminal FGFBP3 (a peptide amino acid sequence of
SEQ ID NO:3) is administered. In select of these embodiments, the
FGFBP3 peptide comprises an amino acid sequence that is 100%
identical to the amino acid sequence of SEQ ID NO:3. In additional
embodiments, the FGFBP3 peptide consists of an amino acid sequence
that is 100% identical to the amino acid sequence of SEQ ID
NO:3.
[0038] In still additional embodiment of the methods of the present
invention, the C-terminal FGFBP3 (a peptide amino acid sequence of
SEQ ID NO:3) is complexed with FGF19. In select of these
embodiments, the FGFBP3 in the complex comprises a peptide with an
amino acid sequence that is 100% identical to the amino acid
sequence of SEQ ID NO:3. In additional embodiments, the FGFBP3 in
the complex consists of a peptide with an amino acid sequence that
is 100% identical to the amino acid sequence of SEQ ID NO:3.
[0039] In still additional embodiment of the methods of the present
invention, the C-terminal FGFBP3 "C66" peptide (a peptide amino
acid sequence of SEQ ID NO:4) is administered. In select of these
embodiments, the FGFBP3 peptide comprises an amino acid sequence
that is 100% identical to the amino acid sequence of SEQ ID NO:4.
In additional embodiments, the FGFBP3 peptide consists of an amino
acid sequence that is 100% identical to the amino acid sequence of
SEQ ID NO:4.
[0040] In still additional embodiment of the methods of the present
invention, the C-terminal FGFBP3 "C66" peptide (a peptide amino
acid sequence of SEQ ID NO:4) is complexed with FGF19. In select of
these embodiments, the FGFBP3 in the complex comprises a peptide
with an amino acid sequence that is 100% identical to the amino
acid sequence of SEQ ID NO:4. In additional embodiments, the FGFBP3
in the complex consists of a peptide with an amino acid sequence
that is 100% identical to the amino acid sequence of SEQ ID
NO:4.
[0041] The terms "peptide," "polypeptide" and "protein" are used
interchangeably herein. As used herein, an "isolated polypeptide"
is intended to mean a polypeptide that has been completely or
partially removed from its native environment. For example,
polypeptides that have been removed or purified from cells are
considered isolated. In addition, recombinantly produced
polypeptides molecules contained in host cells are considered
isolated for the purposes of the present invention. Moreover, a
peptide that is found in a cell, tissue or matrix in which it is
not normally expressed or found is also considered as "isolated"
for the purposes of the present invention. Similarly, polypeptides
that have been synthesized are considered to be isolated
polypeptides. "Purified," on the other hand is well understood in
the art and generally means that the peptides are substantially
free of cellular material, cellular components, chemical precursors
or other chemicals beyond, perhaps, buffer or solvent.
"Substantially free" is not intended to mean that other components
beyond the novel peptides are undetectable.
[0042] The invention also relates to the use of variants of FGFBP3
that still retain their ability to specifically interact, at least
partially, with FGF19. In one embodiment, FGFBP3 variants comprise
an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the amino acid sequences of SEQ ID NO: 3. In another
embodiment, the FGFBP3 variant consists of a peptide with an amino
acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the
amino acid sequences of SEQ ID NO: 3.
[0043] In one embodiment, FGFBP3 variants comprise an amino acid
sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the
amino acid sequences of SEQ ID NO: 4. In another embodiment, the
FGFBP3 variant consists of a peptide with an amino acid sequence
that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid
sequences of SEQ ID NO: 4.
[0044] In one embodiment, FGFBP3 variants comprise an amino acid
sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the
amino acid sequences of SEQ ID NO: 1. In another embodiment, the
FGFBP3 variant consists of a peptide with an amino acid sequence
that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid
sequences of SEQ ID NO: 1.
[0045] In one embodiment, FGFBP3 variants comprise an amino acid
sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the
amino acid sequences of SEQ ID NO: 2. In another embodiment, the
FGFBP3 variant consists of a peptide with an amino acid sequence
that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid
sequences of SEQ ID NO: 2.
[0046] As used herein, a metabolic disorder can be any disorder
associated with metabolism, and examples include but are not
limited to, obesity, central obesity, insulin resistance, glucose
intolerance, abnormal glycogen metabolism, type 2 diabetes,
hyperlipidemia, hypoalbuminemia, hypertriglyceridemia, metabolic
syndrome, syndrome X, a fatty liver, fatty liver disease,
polycystic ovarian syndrome, and acanthosis nigricans. In one
embodiment, the methods are directed towards treating at least one
component of postprandial metabolism, such as, but not limited to
hepatic glycogen synthesis, protein synthesis and clearance of
plasma glucose.
[0047] The terms "trait" and "phenotype" are used interchangeably
herein and refer to any visible, detectable or otherwise measurable
property of an organism such as symptoms of or susceptibility to a
disorder. Typically the terms "trait" or "phenotype" are used
herein to refer to symptoms of a metabolic disorder, or a
susceptibility to an metabolic disorder. Examples of traits of
metabolic disorders include but are not limited to high total
cholesterol, low high-density lipoprotein (HDL) cholesterol,
impaired fasting glucose levels, insulin resistance,
hyperproinsulinemia, central obesity, elevated triglyceride levels,
postprandial glucose levels, elevated uric acid levels, thyroid
dysfunction, increased body-mass index (BMI), hypertension,
impaired glucose tolerance, alterations in hormone and peptide
levels (e.g., leptin, ghrelin, obstatin, adiponectin, perilipin,
omentin), interactions with substances involved in insulin
signaling, lipid, amino acid and glucose metabolism, life
expectancy, increased systemic inflammatory state (e.g., as
reflected in levels of C-reactive protein, interleukin-6, and
TNF-alpha), depression, and sleep disordered breathing.
[0048] In additional embodiments, the peptide variants described
herein are functional and capable of altering a subject's response
in a glucose tolerance test when administered alone or in complex
with FGF19. In some embodiments, the FGFBP3 variants of the present
invention, alone or in complex with FGF19, have enhanced ability to
alter a subject's response in a glucose tolerance test compared to
wild-type FGFBP3. In some embodiments, the FGFBP3 variants of the
present invention also have enhanced stability compared to the
wild-type FGFBP3 regardless of their association with FGF19.
[0049] A polypeptide having an amino acid sequence at least, for
example, about 95% "identical" to a reference an amino acid
sequence, e.g., SEQ ID NO: 1, is understood to mean that the amino
acid sequence of the polypeptide is identical to the reference
sequence except that the amino acid sequence may include up to
about five modifications per each 100 amino acids of the reference
amino acid sequence. In other words, to obtain a peptide having an
amino acid sequence at least about 95% identical to a reference
amino acid sequence, up to about 5% of the amino acid residues of
the reference sequence may be deleted or substituted with another
amino acid or a number of amino acids up to about 5% of the total
amino acids in the reference sequence may be inserted into the
reference sequence. These modifications of the reference sequence
may occur at the N-terminus or C-terminus positions of the
reference amino acid sequence or anywhere between those terminal
positions, interspersed either individually among amino acids in
the reference sequence or in one or more contiguous groups within
the reference sequence.
[0050] As used herein, "identity" is a measure of the identity of
nucleotide sequences or amino acid sequences compared to a
reference nucleotide or amino acid sequence. In general, the
sequences are aligned so that the highest order match is obtained.
"Identity" per se has an art-recognized meaning and can be
calculated using well known techniques. While there are several
methods to measure identity between two polynucleotide or
polypeptide sequences, the term "identity" is well known to skilled
artisans (Carillo (1988) J. Applied Math. 48, 1073). Examples of
computer program methods to determine identity and similarity
between two sequences include, but are not limited to, GCG program
package (Devereux (1984) Nucleic Acids Research 12, 387), BLASTP,
ExPASy, BLASTN, FASTA (Atschul (1990) J. Mol. Biol. 215, 403) and
FASTDB. Examples of methods to determine identity and similarity
are discussed in Michaels (2011) Current Protocols in Protein
Science, Vol. 1, John Wiley & Sons.
[0051] In one embodiment of the present invention, the algorithm
used to determine identity between two or more polypeptides is
BLASTP. In another embodiment of the present invention, the
algorithm used to determine identity between two or more
polypeptides is FASTDB, which is based upon the algorithm of
Brutlag (1990) Comp. App. Biosci. 6, 237-245). In a FASTDB sequence
alignment, the query and reference sequences are amino sequences.
The result of sequence alignment is in percent identity. In one
embodiment, parameters that may be used in a FASTDB alignment of
amino acid sequences to calculate percent identity include, but are
not limited to: Matrix=PAM, k-tuple=2, Mismatch Penalty=1, Joining
Penalty=20, Randomization Group Length=0, Cutoff Score=1, Gap
Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of
the subject amino sequence, whichever is shorter.
[0052] If the reference sequence is shorter or longer than the
query sequence because of N-terminus or C-terminus additions or
deletions, but not because of internal additions or deletions, a
manual correction can be made, because the FASTDB program does not
account for N-terminus and C-terminus truncations or additions of
the reference sequence when calculating percent identity. For query
sequences truncated at the N- or C-termini, relative to the
reference sequence, the percent identity is corrected by
calculating the number of residues of the query sequence that are
N- and C-terminus to the reference sequence that are not
matched/aligned, as a percent of the total bases of the query
sequence. The results of the FASTDB sequence alignment determine
matching/alignment. The alignment percentage is then subtracted
from the percent identity, calculated by the above FASTDB program
using the specified parameters, to arrive at a final percent
identity score. This corrected score can be used for the purposes
of determining how alignments "correspond" to each other, as well
as percentage identity. Residues of the reference sequence that
extend past the N- or C-termini of the query sequence may be
considered for the purposes of manually adjusting the percent
identity score. That is, residues that are not matched/aligned with
the N- or C-termini of the comparison sequence may be counted when
manually adjusting the percent identity score or alignment
numbering.
[0053] For example, a 90 amino acid residue query sequence is
aligned with a 100 residue reference sequence to determine percent
identity. The deletion occurs at the N-terminus of the query
sequence and therefore, the FASTDB alignment does not show a
match/alignment of the first 10 residues at the N-terminus. The 10
unpaired residues represent 10% of the reference sequence (number
of residues at the N- and C-termini not matched/total number of
residues in the reference sequence) so 10% is subtracted from the
percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched (100% alignment) the
final percent identity would be 90% (100% alignment-10% unmatched
overhang). In another example, a 90 residue query sequence is
compared with a 100 reference sequence, except that the deletions
are internal deletions. In this case the percent identity
calculated by FASTDB is not manually corrected, since there are no
residues at the N- or C-termini of the subject sequence that are
not matched/aligned with the query. In still another example, a 110
amino acid query sequence is aligned with a 100 residue reference
sequence to determine percent identity. The addition in the query
occurs at the N-terminus of the query sequence and therefore, the
FASTDB alignment may not show a match/alignment of the first 10
residues at the N-terminus. If the remaining 100 amino acid
residues of the query sequence have 95% identity to the entire
length of the reference sequence, the N-terminal addition of the
query would be ignored and the percent identity of the query to the
reference sequence would be 95%.
[0054] As used herein, the terms "correspond(s) to" and
"corresponding to," as they relate to sequence alignment, are
intended to mean enumerated positions within the reference protein,
e.g., wild-type FGFBP3, and those positions in the variant or
ortholog of FGFBP3 that align with the positions with the reference
protein. Thus, when the amino acid sequence of a subject FGFBP3 is
aligned with the amino acid sequence of a reference FGFBP3, e.g.,
SEQ ID NO: 2, the amino acids in the subject sequence that
"correspond to" certain enumerated positions of the reference
sequence are those that align with these positions of the reference
sequence, e.g., SEQ ID NO: 2, but are not necessarily in these
exact numerical positions of the reference sequence. Methods for
aligning sequences for determining corresponding amino acids
between sequences are described herein.
[0055] The invention further embraces other species, preferably
mammalian, homologs with amino acid sequences that correspond to
FGFBP3. Species homologs, sometimes referred to as "orthologs," in
general, share at least 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with
the human version of the full length binding proteins or the full
length binding proteins without the signal sequence. Such
corresponding sequences account for FGFBP3 from across a variety of
species, such as canine, feline, mouse, rat, rabbit, monkey,
etc.
[0056] FGFBP3 with an additional methionine residue at position -1
(Met.sup.-1-peptide) are contemplated, as are variants with
additional methionine and lysine residues at positions -2 and -1
(Met.sup.-2-Lys.sup.-1-peptide). Variants of FGFBP3 with additional
Met, Met-Lys, or Lys residues (or one or more basic residues in
general) are particularly useful for enhanced recombinant protein
production in bacterial host cells.
[0057] Variants resulting from insertion of the polynucleotide
encoding FGFBP3 into an expression vector system are also
contemplated. For example, variants (usually insertions) may arise
from when the amino terminus and/or the carboxy terminus of FGFBP3
is/are fused to another polypeptide.
[0058] In another aspect, the invention provides deletion variants
wherein one or more amino acid residues in FGFBP3 are removed.
Deletions can be effected at one or both termini of the FGFBP3, or
with removal of one or more non-terminal amino acid residues of the
FGFBP3. Deletion variants, therefore, include all fragments of the
FGFBP3.
[0059] Within the confines of the disclosed percent identity, the
invention also relates to substitution variants of disclosed
polypeptides of the invention. Substitution variants include those
polypeptides wherein one or more amino acid residues of FGFBP3 are
removed and replaced with alternative residues. For example two
variants of SEQ ID NOs: 1 or 2 are known to exist and the invention
contemplates the use of these known variants in the methods
described herein. Specifically, a variant of FGFBP3 wherein Alanine
at position 107 of SEQ ID NO:1 is replaced with Threonine (A107T)
is included in the methods of the present invention. Another
variant of FGFBP3 wherein Glutamate at position 206 of SEQ ID NO:1
is replaced with Valine (E206V) is included in the methods of the
present invention. Of course, positions 107 and 206 of SEQ ID NO:1
correspond to positions 81 and 180 of SEQ ID NO:2, and position 206
of SEQ ID NO:1 also corresponds to position 14 of SEQ ID NO:4. In
one aspect, the substitutions are conservative in nature; however,
the invention embraces substitutions that are also
non-conservative. Conservative substitutions for this purpose may
be defined as set out in the tables below. Amino acids can be
classified according to physical properties and contribution to
secondary and tertiary protein structure. A conservative
substitution is recognized in the art as a substitution of one
amino acid for another amino acid that has similar properties.
Exemplary conservative substitutions are set out in below.
TABLE-US-00002 TABLE I Conservative Substitutions Side Chain
Characteristic Amino Acid Aliphatic Non-polar Gly, Ala, Pro, Iso,
Leu, Val Polar-uncharged Cys, Ser, Thr, Met, Asn, Gln Polar-charged
Asp, Glu, Lys, Arg Aromatic His, Phe, Trp, Tyr Other Asn, Gln, Asp,
Glu
[0060] Alternatively, conservative amino acids can be grouped as
described in Lehninger (1975) Biochemistry, Second Edition; Worth
Publishers, pp. 71-77, as set forth below.
TABLE-US-00003 TABLE II Conservative Substitutions Side Chain
Characteristic Amino Acid Non-polar (hydrophobic) Aliphatic: Ala,
Leu, Iso, Val, Pro Aromatic: Phe, Trp Sulfur-containing: Met
Borderline: Gly Uncharged-polar Hydroxyl: Ser, Thr, Tyr Amides:
Asn, Gln Sulfhydryl: Cys Borderline: Gly Positively Charged
(Basic): Lys, Arg, His Negatively Charged (Acidic) Asp, Glu
[0061] And still other alternative, exemplary conservative
substitutions are set out below.
TABLE-US-00004 TABLE III Conservative Substitutions Original
Residue Exemplary Substitution Ala (A) Val, Leu, Ile Arg (R) Lys,
Gln, Asn Asn (N) Gln, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q)
Asn Glu (E) Asp His (H) Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met,
Ala, Phe Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met
(M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro (P) Gly Ser (S)
Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp, Phe, Thr, Ser Val (V) Ile,
Leu, Met, Phe, Ala
[0062] It should be understood that the definition of peptides or
polypeptides of the invention is intended to include polypeptides
bearing modifications other than insertion, deletion, or
substitution of amino acid residues. By way of example, the
modifications may be covalent in nature, and include for example,
chemical bonding with polymers, lipids, other organic and inorganic
moieties. Such derivatives may be prepared to increase circulating
half-life of a polypeptide, or may be designed to improve the
targeting capacity of the polypeptide for desired cells, tissues or
organs. Similarly, the invention further embraces FGFBP3 or
variants thereof that have been covalently modified to include one
or more water-soluble polymer attachments such as polyethylene
glycol, polyoxyethylene glycol or polypropylene glycol.
[0063] Compositions in which the FGFBP3 or variants thereof is
linked to a polymer are included within the scope of the present
invention. The polymer may be water soluble to prevent
precipitation of the protein in an aqueous environment, such as a
physiological environment. Suitable water-soluble polymers may be
selected from the group consisting of, for example, polyethylene
glycol (PEG), monomethoxypolyethylene glycol, dextran, cellulose,
or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone)
polyethylene glycol, polypropylene glycol homopolymers, a
polypropylene oxide/ethylene oxide copolymer polyoxyethylated
polyols (e.g., glycerol) and polyvinyl alcohol. The selected
polymer is usually modified to have a single reactive group, such
as an active ester for acylation or an aldehyde for alkylation, so
that the degree of polymerization may be controlled. Polymers may
be of any molecular weight, and may be branched or unbranched, and
mixtures of such polymers may also be used. When the chemically
modified NgR polymer is destined for therapeutic use,
pharmaceutically acceptable polymers will be selected for use.
[0064] Pegylation of FGFBP3 or variants thereof may be carried out
by any of the pegylation reactions known in the art. In one method,
the pegylation is carried out via an acylation reaction or an
alkylation reaction with a reactive polyethylene glycol molecule
(or an analogous reactive water-soluble polymer). A preferred
water-soluble polymer for pegylation of polypeptides is
polyethylene glycol (PEG), including, but not limited to
bi-functional PEGs. As used herein, "polyethylene glycol" is meant
to encompass any of the forms of PEG that have been used to
derivatize other proteins, such as mono (Cl--ClO) alkoxy- or
aryloxy-polyethylene glycol.
[0065] Chemical derivatization of FGFBP3 or variants thereof may be
performed under any suitable conditions used to react a
biologically active substance with an activated polymer molecule.
Methods for preparing pegylated FGFBP3 or variants thereof will
generally comprise the steps of (a) reacting the polypeptide with
polyethylene glycol, such as a reactive ester or aldehyde
derivative of PEG, under conditions whereby FGFBP3 or variants
thereof becomes attached to one or more PEG groups, and (b)
obtaining the reaction products. It will be apparent to one of
ordinary skill in the art to select the optimal reaction conditions
or the acylation reactions based on known parameters and the
desired result.
[0066] Pegylated and other polymer-modified FGFBP3 or variants
thereof may generally be used in the methods of the current
invention. The chemically-derivatized polymer-modified FGFBP3 or
variants thereof disclosed herein may have additional activities,
enhanced or reduced biological activity, or other characteristics,
such as increased or decreased half-life, as compared to the
nonderivatized molecules. The modified FGFBP3 or variants thereof,
alone or in complex, may be employed alone, together, or in
combination with other pharmaceutical compositions. For example,
cytokines, growth factors, antibiotics, anti-inflammatories and/or
chemotherapeutic agents may be co-administered as is appropriate
for the indication being treated.
[0067] The present invention provides compositions comprising
purified polypeptides, alone or in complex, of the invention.
Examples of compositions include but are not limited to a
pharmaceutically acceptable, i.e., sterile and non-toxic, liquid,
semisolid, or solid diluent that serves as a pharmaceutical
vehicle, excipient or medium. Any diluent known in the art may be
used. Exemplary diluents include, but are not limited to, water,
saline solutions, polyoxyethylene sorbitan monolaurate, magnesium
stearate, methyl- and propylhydroxybenzoate, talc, alginates,
starches, lactose, sucrose, dextrose, sorbitol, mannitol, glycerol,
calcium phosphate, mineral oil and cocoa butter.
[0068] In one embodiment, the invention provides fusion proteins
comprising at least a first and a second fusion peptide. The fusion
partners are, generally speaking, covalently bonded to one another
via a typical amine bond between the fusion peptides, thus creating
one contiguous amino acid chain. Types of fusion proteins provided
by the present invention include but are not limited to, fusions
with secretion signals and other heterologous functional regions.
Thus, for instance, a region of additional amino acids,
particularly charged amino acids, may be added to the N-terminus of
the FGFBP3 or variant thereof to improve stability and persistence
in the host cell, during purification or during subsequent handling
and storage.
[0069] Additional fusion proteins include fusions for enhancing
translocation of the protein across cell membranes. For example,
Tat is an 86-amino acid protein involved in the replication of
human immunodeficiency virus type 1 (HIV-1). The HIV-1 Tat
transactivation protein is efficiently taken up by cells, and it
has been demonstrated that low concentrations (nM) are sufficient
to transactivate a reporter gene expressed from the HIV-1 promoter.
Exogenous Tat protein is able to translocate through the plasma
membrane and reach the nucleus to transactivate the viral genome.
Tat peptide-mediated cellular uptake and nuclear translocation have
been demonstrated in several systems. Chemically coupling a
Tat-derived peptide (residues 37-72 of Tat) to several proteins
results in their internalization in several cell lines or tissues
(Fawell (1994) Proc. Natl. Acad. Sci. USA 91, 664-668.
[0070] It is well-known that a region of the Tat protein centered
on a cluster of basic amino acids is responsible for this
translocation activity. A synthetic peptide consisting of the Tat
basic amino acids 48-60 with a cysteine residue at the C-terminus
coupled to fluorescein maleimide translocates to the cell nucleus
as determined by fluorescence microscopy. In addition, a fusion
protein (Tat-NLS-.beta.-Gal) consisting of Tat amino acids 48-59
fused by their amino-terminus to .beta.-galactosidase amino acids
9-1023 translocates to the cell nucleus in an ATP-dependent,
cytosolic factor-independent manner. Accordingly, the fusion
proteins of the present invention may comprise all or a portion of
HIV-Tat, such as any sequential residues of the Tat protein basic
peptide motif 37-72 (37-CFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ-72
(SEQ ID NO: 5). The minimum number of amino acid residues can be in
the range of from about three to about six. In one embodiment, the
Tat portion of the fusion protein is from about three to about five
contiguous amino acids in length. In another embodiment, the Tat
portion of the fusion protein is about four amino acids in length,
i.e., the minimal requirement for one alpha helical turn. In
another embodiment, the Tat portion of the fusion protein comprises
Tat protein residues 48-57 (GRKKRRQRRR) (SEQ ID NO: 6).
[0071] In additional embodiments of fusion proteins, a region may
be added to facilitate purification. For example, "histidine tags"
("his tags") or "lysine tags" may be appended to the first fusion
peptide. Examples of histidine tags include, but are not limited to
hexaH, heptaH and hexaHN. Examples of lysine tags include, but are
not limited to pentaL, heptaL and FLAG. Such regions may be removed
prior to final preparation of the FGFBP3 or variant thereof. Other
examples of a second fusion peptide include, but are not limited
to, glutathione S-transferase (GST) and alkaline phosphatase
(AP).
[0072] The addition of peptide moieties to proteins, whether to
engender secretion or excretion, to improve stability and to
facilitate purification or translocation, among others, is a
familiar and routine technique in the art and may include modifying
amino acids at the terminus to accommodate the tags. For example in
SEQ ID NOs: 1, 2, 3 or 4, the N-terminus amino acid may be modified
to, for example, arginine and/or serine to accommodate a tag. Of
course, the amino acid residues of the C-terminus may also be
modified to accommodate tags. One particularly useful fusion
protein comprises a heterologous region from immunoglobulin that
can be used solubilize proteins. For example, EP A0464 533
discloses fusion proteins comprising various portions of constant
region of immunoglobin molecules together with another human
protein or part thereof. In many cases, the Fc part in a fusion
protein is thoroughly advantageous for use in therapy and diagnosis
and thereby results, for example, in improved pharmacokinetic
properties (EP A0232 262). On the other hand, for some uses, it
would be desirable to be able to delete the Fc part after the
fusion protein has been expressed, detected and purified in the
advantageous manner described.
[0073] The fusion proteins of the current invention can be
recovered and purified from recombinant cell cultures by well-known
methods including, but not limited to, ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography, e.g.,
immobilized metal affinity chromatography (IMAC), hydroxylapatite
chromatography and lectin chromatography. High performance liquid
chromatography ("HPLC") may also be employed for purification.
Well-known techniques for refolding protein may be employed to
regenerate active conformation when the fusion protein is denatured
during isolation and/or purification.
[0074] Fusion proteins of the present invention include, but are
not limited to, products of chemical synthetic procedures and
products produced by recombinant techniques from a prokaryotic or
eukaryotic host, including, for example, bacterial, yeast, higher
plant, insect and mammalian cells. Depending upon the host employed
in a recombinant production procedure, the fusion proteins of the
present invention may be glycosylated or may be non-glycosylated.
In addition, fusion proteins of the invention may also include an
initial modified methionine residue, in some cases as a result of
host-mediated processes.
[0075] The FGFBP3 or variant, alone or in complex, thereof can be
prepared as a pharmaceutical composition. For example, one or more
cofactors may also be added to the FGFBP3 or variant thereof, or to
the complex of FGFBP3 or variant thereof and FGF19, to form a
composition. Cofactors that may be added include, but are not
limited to, heparin, hyaluronic acid, a fibronectin, an elastin, a
laminin, albumin, a proteoglycan, collagen, gelatin, a divalent
cation, calcium chloride, zinc sulfate, magnesium chloride, sodium
bicarbonate, sodium chloride, sodium acetate, or sodium phosphate.
In some embodiments, a protein or a protein fragment may be added
as a cofactor to the FGFBP3 or variant thereof. In other
embodiments, a protein or a protein fragment may be added as a
cofactor to the complex of FGFBP3 or variant thereof and FGF19.
[0076] As used herein, "pharmaceutically acceptable carrier" or
"pharmaceutical carrier" is intended to include any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like,
compatible with pharmaceutical administration. The nature of the
pharmaceutical carrier or other ingredients will depend on the
specific route of administration and particular embodiment of the
invention to be administered. Examples of techniques and protocols
that are useful in this context are, inter alia, found in
Remington: The Science and Practice of Pharmacy (2010), Lippincott
Williams & Wilkins. Examples of such pharmaceutical carriers or
diluents include, but are not limited to, water, saline, Ringer's
solution, dextrose solution and 5% human serum albumin. Liposomes
and non-aqueous vehicles such as fixed oils may also be used. The
use of such media and agents for pharmaceutically active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active compound, use thereof in
the compositions is contemplated. Supplementary active compounds
can also be incorporated into the compositions.
[0077] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include oral and parenteral
(e.g., intravenous, intradermal, subcutaneous, inhalation,
transdermal (topical), transmucosal and rectal administration).
Solutions or suspensions used for parenteral, intradermal or
subcutaneous application can include, but are not limited to, a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents, antibacterial agents such as benzyl alcohol or
methyl parabens, antioxidants such as ascorbic acid or sodium
bisulfite, chelating agents such as ethylenediaminetetraacetic
acid, buffers such as acetates, citrates or phosphates, and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases, such as hydrochloric
acid or sodium hydroxide. The parenteral preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
[0078] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable pharmaceutical carriers include
physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF)
or phosphate buffered saline (PBS). In all cases, the compositions
must be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The pharmaceutical carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it may be
desirable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0079] Sterile injectable solutions can be prepared by
incorporating the active compound/composition in the required
amount in an appropriate solvent with one or a combination of
ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle that contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, methods of preparation
are vacuum drying and freeze-drying that yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0080] Oral compositions generally include an inert diluent or an
edible pharmaceutical carrier. They can be enclosed in gelatin
capsules or compressed into tablets. For the purpose of oral
therapeutic administration, the active compound can be incorporated
with excipients and used in the form of tablets, troches or
capsules. Pharmaceutically compatible binding agents, and/or
adjuvant materials can be included as part of the composition. The
tablets, pills, capsules, troches and the like may contain any of
the following ingredients, or compounds of a similar nature, such
as but not limited to a binder, such as microcrystalline cellulose,
gum tragacanth or gelatin, an excipient such as starch or lactose,
a disintegrating agent such as alginic acid, Primogel or corn
starch, a lubricant such as magnesium stearate or Sterotes, a
glidant such as colloidal silicon dioxide, a sweetening agent such
as sucrose or saccharin, or a flavoring agent such as peppermint,
methyl salicylate or flavoring.
[0081] In one embodiment, the active is prepared with
pharmaceutical carriers that will protect the active against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These compositions can be
prepared according to methods known to those skilled in the art,
for example, as described in U.S. Pat. No. 4,522,811. It is
especially advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of the
active calculated to produce the desired therapeutic effect in
association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on the unique characteristics of
the active compound and the particular therapeutic effect to be
achieved.
[0082] The pharmaceutical compositions can be included in a
container, pack or dispenser together with instructions for
administration.
[0083] The dosage of the FGFBP3 and/or the dosage of the
FGFBP3-FGF19 complex will depend on the disorder or condition to be
treated and other clinical factors such as weight and condition of
the human or animal and the route of administration of the
compound. For treating human or animals, the FGFBP3 or variant
thereof, or the complex, can be administered at a dose of between
about 0.005 mg/kg of body weight to 500 mg/kg of body weight.
Therapy is typically administered at lower dosages and is continued
until the desired therapeutic outcome is observed.
[0084] Methods of determining the dosages of composition to be
administered to a patient and modes of administering compositions
to an organism are disclosed in, for example, WO 96/22976. Those
skilled in the art will appreciate that such descriptions are
applicable to the present invention and can be easily adapted to
it.
[0085] The proper dosage depends on various factors such as the
type of disorder being treated, the particular composition being
used and the size and physiological condition of the patient.
Therapeutically effective doses for the compositions described
herein can be estimated initially from cell culture and animal
models. For example, a dose can be formulated in animal models to
achieve a circulating concentration range that initially takes into
account the IC.sub.50 as determined in cell culture assays. The
animal model data can be used to more accurately determine useful
doses in humans.
[0086] The invention also relates to methods of altering
intracellular signaling of a cell, comprising contacting cells with
FGFBP3 or a variant thereof, or comprising contacting cells with
the complex of FGFBP3 or a variant thereof plus FGF19, wherein the
cell possesses a receptor that specifically binds to or associates
with FGFBP3. In one embodiment, the receptor is the fibroblast
growth factor receptor 4 (FGFR4). The specific binding of the
FGFBP3 or the complex to a receptor will, in turn, initiate the
intracellular signaling cascade that is normally associated with
FGFBP3. For example, FIG. 12 demonstrates that administration of
the complex of FGFBP3 or a variant thereof and FGF19 results in
phosphorylation of Erk1/2. Accordingly, the present invention
provides for methods of stimulating phosphorylation of Erk1/2 in a
cell comprising contacting the cell(s) with a complex of FGF19 and
FGFBP3 or a variant thereof. Additional methods of the present
invention comprise assessing the levels of Erk1/2 phosphorylation,
both before and after contacting the cell(s) with the complexes of
the present invention and determining the increase or decrease of
Erk1/2 phosphorylation in response to the complexes of the present
invention.
[0087] Recently, it was shown that FGF19 alone was able to induce
phosphorylation of the p90 ribosomal S6 kinase (p90RSK), which is a
downstream target of phosphorylated ERK1/2. In turn, phosphorylated
p90RSK is known to phosphorylate both ribosomal protein S6 (rpS6)
and the eukaryotic translation initiation factor 4B (eIF4B). It was
also recently shown that FGF19 alone was able to induce
phosphorylation of both rpS6 and eIF4B. Thus one embodiment of the
present invention comprises methods of stimulating phosphorylation
of p90RSK, rpS6 and/or eIF4B in cells. These methods of
phosphorylating p90RSK, rpS6 and/or eIF4B comprise contacting the
cells with FGFBP3or a variant thereof, alone or in complex with
FGF19, in an amount sufficient to stimulate phosphorylation
thereof.
[0088] One target of a phosphorylated p90RSK is glycogen synthase
kinase 3.alpha. and 3.beta. (GSK3 kinases), which, when
phosphorylated, are responsible for inhibition of glycogen synthase
(GS). The GSK3 kinases are also inhibited or inactivated when they
themselves are phosphorylated. Specifically, phosphorylated p90RSK
inhibits or inactivates the GSK3 kinases which block the inhibition
of GS. Once the inhibition of GS is removed, GS is activated and,
in turn, can trigger production of glycogen. Thus one embodiment of
the present invention is directed to methods of increasing glycogen
production in a subject in need thereof, with the methods
comprising administering a FGFBP3 or a variant thereof, alone or in
complex with FGF19, in a subject in need thereof in an amount
sufficient to stimulate production of glycogen.
[0089] Phosphorylated p90RSK also stimulates protein synthesis at
least in the liver. Accordingly, one embodiment of the present
invention is directed towards increasing protein synthesis in a
subject in need thereof, with the methods comprising administering
FGFBP3 or a variant, alone or in complex with FGF19, thereof to the
subject in an amount sufficient to stimulate protein synthesis. One
example of a liver-synthesized protein is albumin. Accordingly, one
specific embodiment of the present invention is directed towards
increasing production of albumin in a subject in need thereof, with
the methods comprising administering FGFBP3 or a variant thereof,
alone or in complex with FGF19, to the subject in an amount
sufficient to stimulate production of albumin.
[0090] Likewise, the present invention provides methods of
stimulating promoter activity in a cell or population of cells,
where the promoter is responsive to activated Erk1/2 or p90RSK with
the methods comprising contacting the cell(s) with FGFBP3 or a
variant thereof, alone or in complex with FGF19. One of skill in
the art would be aware of promoters that respond to activated
Erk1/2 or p90RSK. The activity of a variant of FGFBP3, alone or in
complex with FGF19, with respect to stimulating Erk1/2-responsive
promoters or p90RSK-responsive promoters may or may not be altered
relative to the variant's ability to complex with FGF19. One of
skill in the art can readily determine if a promoter is more or
less activated over control groups using well known techniques such
as transcription of reporter genes, ELISA assays, etc. Additional
methods of the present invention comprise assessing the activity of
an Erk1/2-responsive promoter both before and after contacting the
cell(s) with the FGFBP3 or variant thereof, alone or in complex
with FGF19, of the present invention and determining the increase
or decrease of the promoter in response to the FGFBP3 or variant
thereof, alone or in complex with FGF19, of the present
invention.
[0091] The present invention also provides methods of altering the
activity or expression of cell signaling molecules in a cell or
population of cells in which there is a need to alter the
expression or activity thereof. For example, contacting the cell or
cells with FGFBP3 or a variant thereof, alone or in complex with
FGF19, causes a reduction in the activity and/or expression of the
CYP7A1 enzyme (Cholesterol 7.alpha.-hydrolase), a reduction in the
activity or expression of glucose-6-phosphatase (G6PC), and/or a
reduction in the activity or expression of peroxisome
proliferator-activated receptor-.gamma. coactivator-1.beta.
(PPARGC1B). In another example, contacting the cell or cells with
FGFBP3 or a variant thereof, alone or in complex with FGF19, causes
an increase in the activity and/or expression of interleukin-6
(IL-6), an increase in the activity and/or expression of insulin
receptor substrate (IRS2) and/or an increase in the activity and/or
expression of suppressor of cytokine signaling 3 (SOCS3). In other
embodiments, the methods comprise contacting a cell or cells with
FGFBP3 or a variant thereof, alone or in complex with FGF19, to a
cell or cell in need thereof to alter the phosphorylation state of
cell signaling molecules such as but not limited to AKT, STAT3 and
forkhead box O1 (FoxO1). Specifically, contacting the cells with
FGFBP3 or a variant thereof, alone or in complex with FGF19, will
cause an increase in levels of phosphorylated AKT, STAT3 and/or
FoxO1.
[0092] As used herein, "contacting," when used in connection with
the methods of the present invention means bringing the compounds
or compositions of the present invention in proximity to the target
cells such that a specific binding event or a biological effect is
possible. Thus, contacting can include adding the FGFBP3 in culture
medium and applying the culture medium to cells in culture. Of
course, contacting would also include administration of the FGFBP3,
or pharmaceutical compositions thereof, of the present invention to
cells in an intact organism. Compositions for administering the
FGFBP3 of the present invention have been described herein.
[0093] As used herein, "administering," and "administer" are used
to mean introducing FGFBP3 or variant thereof, alone or in complex
with FGF19, of the present invention into a subject. When
administration is for the purpose of treatment, the composition is
provided at, or after the onset of, a symptom or condition in need
of treatment. The therapeutic administration of this composition
serves to attenuate any symptom, or prevent additional symptoms
from arising. When administration is for the purposes of preventing
a condition from arising ("prophylactic administration"), the
composition is provided in advance of any visible or detectable
symptom. The prophylactic administration of the composition serves
to attenuate subsequently arising symptoms or prevent symptoms from
arising altogether. The route of administration of the composition
includes, but is not limited to, topical, transdermal, intranasal,
vaginal, rectal, oral, subcutaneous intravenous, intraarterial,
intramuscular, intraosseous, intraperitoneal, epidural and
intrathecal as previously disclosed herein.
[0094] Furthermore, the methods would also include coadministering
one or more substances in addition to the composition the present
invention. The term "coadminister" indicates that each of at least
two substances, with one of the substances being FGFBP3 or a
variant thereof, alone or in complex with FGF19, is administered
during a time frame wherein the respective periods of biological
activity or effects of each of the substances overlap. Thus the
term includes sequential as well as coextensive administration of
the FGFBP3 of the present invention with another substance. And
similar to administering the compositions of the present invention,
coadministration of more than one substance can be for therapeutic
and/or prophylactic purposes. If more than one substance is
coadministered, the routes of administration of the two or more
substances need not be the same.
[0095] The invention also relates to methods of lowering blood
glucose levels in a subject, the method comprising administering
FGFBP3 to a subject in need of lowering of blood glucose levels. In
one embodiment, the subject is screened prior to administration of
the FGFBP3.
[0096] The invention also relates to methods of lowering a
subject's body weight, the method comprising administering FGFBP3
or a variant thereof to a subject that is in need of lowering its
body weight. In one embodiment, the subject is screened prior to
administration of the FGFBP3.
[0097] The invention also relates to methods of lowering blood
glucose levels in a subject, the method comprising administering a
complex of FGF19 and FGFBP3 to a subject in need of lowering of
blood glucose levels. In one embodiment, the subject is screened
prior to administration of the complex.
[0098] The invention also relates to methods of lowering a
subject's body weight, the method comprising administering a
complex of FGF19 and FGFBP3 to a subject that is in need of
lowering its body weight. In one embodiment, the subject is
screened prior to administration of the complex.
[0099] The following examples are illustrative and are not intended
to limit the scope of the invention described herein.
EXAMPLES
Materials and Methods
[0100] Human BP3 cDNA, (the amino acid sequence of SEQ ID NO:2 and
corresponding to amino acids 27-258 of SEQ ID NO:1), and the
C-terminal hBP3 region (SEQ ID NO:4 and corresponding to amino
acids 167-232 of SEQ ID NO:2), were subcloned into a pMAL-p2X
vector (New England BioLabs, Ipswich, Mass.) and MBP-tagged
recombinant proteins (hBP3 and C66, respectively) were generated as
has been previously described in Xie, B., et al., J. Biol. Chem.
281, 1137-1144 (2006). MBP and MBP-BP3 are referred to herein as
"control" and "BP3", respectively. Recombinant proteins were
purified by fast protein liquid chromatography (FPLC). Briefly,
bacterial cell lysates were loaded onto an MBPTrap.TM. HP columns
(Dextrin Sepharose) (GE Healthcare Life Sciences, Piscataway, N.J.)
and MBP-tagged proteins eluted with 20 ml of a gradient of 0-10 mM
Maltose in Column Buffer (20 mM Tris HCl pH 7.4, 200 mM NaCl, 1 mM
EDTA). Positive fractions were then loaded onto HiTrap.TM. Heparin
HP columns (GE Healthcare Life Sciences) and proteins eluted with
20 ml of a gradient of 0-1.5M NaCl in Column Buffer. Eluted
proteins were analyzed by immunoblotting with an anti hBP3 rabbit
polyclonal antibody (Abgent, San Diego, Calif.) or with an anti MBP
mouse monoclonal antibody (New England BioLabs). Eluted hBP3 was
resolved on a 4-12% Bis-Tris gel (Life Technologies, Carlsbad,
Calif.), visualized by Coomassie Blue staining and the bands
excised from the gel. Mass spectrometry analysis was conducted as
described previously in Zhang, W. et al., J. Biol. Chem.,
283:28329-28337 (2008).
[0101] MaxiSorp.TM. microtiter plates (Sigma Aldrich, St. Louis,
Mo.) were coated with 100 .mu.l/well of recombinant proteins [human
recombinant FGF2 (Life Technologies), human recombinant FGF19, or
human recombinant FGFR4 Fc Chimera (R&D Systems Minneapolis,
Minn.); 7.5 .mu.g/ml] and incubated overnight at 4.degree. C.
Plates were washed thrice between each incubation step with washing
buffer [1X Phosphate buffered saline (PBS) with 0.2% Tween 20, pH
7.4 (PBST)]. Blocking was carried out with 100 .mu.l/well of 5% dry
milk diluted in PBST for 1 hour at room temperature. Subsequently,
plates were incubated for 2 hours at room temperature with 100
.mu.l/well of an MBP-tagged recombinant protein (MBP control or
BP3) at a fixed concentration (1 .mu.g/ml) or in serial dilutions.
Detection was carried out with 100 .mu./well of an anti MBP mouse
monoclonal antibody (New England BioLabs) and with an
affinity-purified goat anti-mouse horseradish peroxidase
(HRP)-conjugated antibody (GE Healthcare Life Sciences) (1:1,000
dilution in PBS). The reactions were visualized with the aid of
1-Step Turbo TMB (Thermo Scientific, Pittsburgh, Pa.), according to
the manufacturer's protocol, and read with an Ultramark Microplate
Imaging System (Bio-Rad Laboratories, Hercules, Calif.) at 450 nm
absorbance.
[0102] MaxiSorp.TM. microtiter plates were coated with 0.75 mg of
recombinant FGFR4 and incubated overnight at 4.degree. C. plates
were washed thrice between each incubation step with PBS. Blocking
was carried out with 100 .mu./well of 5% dry milk diluted in PBS
for 1 hour at room temperature. Subsequently, plates were incubated
for 2 hours at room temperature with 100 .mu./well of FGF19 (2
.mu./ml).+-.BP3 or MBP control (1 .mu./ml). Bound proteins were
detected by western blot analysis with 1 .mu./ml of an anti FGFR4
(LD1; Genentech, South San Francisco, Calif.), anti MBP (New
England BioLabs) or anti FGF19 (Abnova, Walnut, Calif.) mouse
monoclonal antibodies.
[0103] Biacore T200 instrument (GE Healthcare) was used for surface
plasmon resonance measurements. Human recombinant FGFR4 or FGFR1 Fc
chimera in HBS-P buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05%
P-20) were immobilized on a flow cell of a CM-5 sensor chip (GE
Healthcare, Piscataway, N.J.) via amine coupling. A blank flow cell
was used as a negative control for non-specific binding to the
sensor surface. FGF19 (47.1 and 23.55 nM), C66 (245 nM) in HBS-P
buffer, alone or in combination, or BP3 (4, 2, or 1 nM) were
injected over the immobilized receptor with a flow rate of 10
.mu.L/min for 60 seconds and the resulting maximum responses were
obtained. Dissociation constants were calculated from the
association and dissociation rates of the proteins after washing.
The experiments were performed in triplicate.
[0104] Six to nine week-old female ob/ob or C57BL mice were
purchased from The Jackson Laboratory (Bar Harbor, Me.). Animals
were maintained in a normal light-cycle room and were provided with
rodent chow and water ad libitum. Mice were treated with a single
intraperitoneal injection of recombinant proteins and blood glucose
levels were determined with a portable glucose meter (Contour,
Bayer, Whippany, N.J.) at different times after treatment (0-48
hours). Animal experiments were reviewed and approved by the
Institutional Animal Care and Use Committee of Georgetown
University.
[0105] Immunoprecipitation and Western Blot analyses were performed
as described earlier in Tassi, E., et al., Am. J. Pathol.,
179:2220-2232 (2011). Briefly, livers from ob/ob or C57BL mice were
homogenized in 1 mL of lysis buffer with a MagNa lyser homogenizer
(Roche, Indianapolis, Ind.). 5 mg of total lysates were
immunoprecipitated with 10 .mu.l of sepharose-conjugated anti-AKT
or phospho STAT3 antibodies and immunoblotted with an anti-phospho
AKT or anti STAT3 rabbit polyclonal antibodies, respectively. Total
AKT and STAT3 in the liver lysates (50 .mu.g) were detected using
an anti AKT and STAT3 rabbit polyclonal antibodies, respectively.
ERK1/2 phosphorylation studies in HepG2 cells were performed by
immunoblotting for phospho ERK1/2 and anti ERK1/2 rabbit polyclonal
antibodies, as described in Tassi, E., et al., J. Biol. Chem.,
276:40247-40253 (2001). All antibodies were purchased from Cell
Signaling (Danvers, Mass.). Detection of MBP and MBP-BP3 in mouse
sera was carried out by immunoprecipitation and immunoblot with
anti MBP magnetic beads and anti MBP rabbit polyclonal antibody
(New England BioLabs), respectively.
[0106] Total RNA was isolated from ob/ob or C57BL mouse livers or
WAT using RNeasy Mini kit or RNeasy Lipid Tissue kit (Qiagen,
Valencia, Calif.), according to the manufacturer's
instructions.
[0107] Liver total RNA was reverse transcribed into complementary
RNA (cRNA), biotin-UTP labeled, and hybridized to the Illumina
mouseRef-8v2.0 Expression BeadChip (Illumina Inc., San Diego,
Calif.).
[0108] cDNA was synthesized from 1 .mu.g of total RNA using the
iScript.TM. cDNA Synthesis Kit, according to the manufacturer's
protocol (Bio-Rad Laboratories, Hercules, Calif.). Real-time PCR
was performed in a Realplex2 (Eppendorf, Hauppauge, N.Y.) using the
iQ SYBR Green Supermix (Bio-Rad Laboratories) under the following
conditions: 95.degree. C. for 3 minutes, followed by 40 cycles
(95.degree. C. for 20 seconds, 60.degree. C. for 30 seconds, and
72.degree. C. for 40 seconds). The following PCR primers were used:
mouse .beta.-actin sense 5'-GGCGCTTTTGACTCAGGATTTAA-3' (SEQ ID NO:
7), antisense 5'-CCTCAGCCACATTTGTAGAACTTT-3' (SEQ ID NO: 8); mouse
CYP7A1 sense 5'-CCCACAGTTAATGCACTTGGATCCTG-3' (SEQ ID NO: 9) and
antisense 5'-GGGCATGTAGAAATACTTCAGCTTGTTTCC-3' (SEQ ID NO: 10);
mouse SOCS3 sense 5'-TCTTTGTCGGAAGACTGTCAACGG-3' (SEQ ID NO: 11)
and antisense 5'-CATCATACTGATCCAGGAACTCCCGA-3' (SEQ ID NO: 12);
mouse IL6 sense 5'-GTCACTTTGAGATCTACTCGGCAAACC-3' (SEQ ID NO: 13)
and antisense 5'-TCTGACCACAGTGAGGAATGTCCA-3' (SEQ ID NO: 14); mouse
G6PC 5'-CGACTCGCTATCTCCAAGTGA-3' (SEQ ID NO: 15) and antisense
5'-GTTGAACCAGTCTCCGACCA (SEQ ID NO: 16); mouse PPARGC1B
5'-TCCTGTAAAAGCCCGGAGTAT-3' (SEQ ID NO: 17) and antisense
5'-GCTCTGGTAGGGGCAGTGA-3' (SEQ ID NO: 18); mouse IRS2
5'-ACCGACTTGGTCAGCGAAG-3' (SEQ ID NO: 19) and antisense
5'-CACGAGCCCGTAGTTGTCAT-3' (SEQ ID NO: 20).
[0109] The web-based Ingenuity Pathways Analysis (IPA) (Ingenuity
Systems.RTM., www.ingenuity.com) was used to identify functional
networks and pathways analyses. Activation z-score was calculated
as a measure of functional and translational activation in
Functions and Upstream regulators analysis. z-scores greater than 2
or smaller than -2 were considered significant.
Example 1
[0110] Six to nine week old female ob/ob mice, obtained from
Jackson Laboratories, were randomly assigned to treatment groups
with FGFBP3 alone, or FGF19 in combination with BP3. Ob/ob mice
show pathologically increased glucose levels and exhibit glucose
intolerance in traditional glucose tolerance tests. They are a
leptin-deficient diabetes model.
[0111] The substances were administered by single intraperitoneal
(i.p.) injection. To assess potential therapeutic effects on the
metabolic profile with FGFBP3 or FGF19+FGFBP3 treatments, glucose
levels were measured while the animals were fed ad libitum. Two
series of experiments were performed to investigate acute and
sustained effects with the different treatments.
[0112] Before starting the experiments, the animals were weighed to
determine the amount of proteins (FGFBP3 or others) to inject.
Contrary to alternative approaches that use glucose challenge after
prolonged fasting (=glucose tolerance test), the animals were not
fasted and were fed ad libitum. This was done to mimic the natural
setting. For the measurements of blood glucose levels, blood was
sampled from the tail tip at 0, 2, 3, 4 and 24 hours after protein
injection. Blood glucose levels were determined with a portable
glucose meter (Contour.RTM., Bayer HealthCare).
[0113] The results in FIG. 1 show that the effects of FGFBP3 alone
or FGF19+FGFBP3 treatments on glucose metabolism in ob/ob mice.
Left Panel, shows that upon intraperitoneal injection of either
FGFBP3 alone or FGF19+FGFBP3, glucose levels fell to roughly normal
levels (100 to 150 mg/dl) within 2 hours after the first treatment.
Levels stayed close to normal range (compared to controls) for 24
hours following injection. Right Panel, shows that FGFBP3 had a
greater effect on lowering glucose levels when normalized to
account for different baseline glucose levels.
[0114] Treatment of the mice with an anti-FGF15 antibody and BP3
did not result in measurably different glucose levels compared to
mice treated with the control protein only. Moreover, the effect of
the antibody pretreatment did not significantly alter hyperglycemic
levels when co-injected with a control protein at all time points
(FIG. 2A).
[0115] A dose-response curve for BP3 was established and compared
that to exogenous FGF19. The administration of a single high dose
of FGF19 (1 mg/kg) did not impact hyperglycemia in the ob/ob mice,
whereas BP3 treatment resulted in a dose-dependent decrease of
blood glucose with an ED50 below 0.2 mg/kg (FIG. 2B). To evaluate
if the BP3 effect was impacted by the feeding status of the
animals, the effect of BP3 in starved animals, which have reduced
FGF15 levels, was tested. In this setting, BP3 effects on glucose
were absent and comparable to those of a single treatment of FGF19
or of the control protein. Normoglycemic levels were achieved when
starved animals were co-treated with a combination of BP3 and
exogenous FGF19 (FIG. 2C).
[0116] Gene expression patterns in livers of ob/ob mice using the
treatment cohort above were assayed. Organs were harvested after
four hours when the glucose lowering effect of BP3 had reached
normoglycemic levels and included the following treatment groups:
control protein (MBP), FGF19, BP3 alone, anti-FGF15 antibody alone,
or BP3 plus anti-FGF15. As a signature gene of the FGFR4/FGF19
signaling axis CYP7A1 (Cholesterol 7.alpha.-hydrolase) was first
probed for expression. CYP7A1 mRNA was reduced 32-fold after
administration of exogenous FGF19 suggesting that control of CYP7A1
transcription in the liver in response to the postprandial
secretion by ileal entherocytes of FGF15/19 activates FGFR4 in the
hepatocytes, thereby resulting in the repression of bile acid (BA)
biosynthesis is intact. Administration of exogenous FGF19
significantly reduced CYP7A1 transcription by 32 fold versus
control mice, thus indicating the efficacy of FGF19 treatment. BP3
treatment, however, resulted in a more potent downregulation of
CYP7A1, whose transcription was found reduced by 110 fold when
compared to the expression levels observed in control mice.
Moreover, the reduction of endogenous FGF15 levels by a specific
neutralizing antibody blunted BP3-induced CYP7A1 inhibition,
thereby indicating that BP3 can modulate FGF15/19-dependent gene
regulation (FIG. 3A).
[0117] Interestingly, one of the most prominently downregulated
genes in livers from BP3 treated mice was glucose-6-phosphatase
(G6PC), a key gluconeogenic enzyme, which was reduced about 130
fold when compared to control levels. In addition, there were
corresponding increases of G6PC upstream regulatory genes, such as
insulin receptor substrate 2 (IRS2). BP3 treatment also resulted in
a marked upregulation of both interleukin 6 (IL6) and its
downstream effector, suppressor of cytokine signaling 3 (SOCS3),
which in turn blocks gluconeogenesis through G6PC suppression.
Lastly, it is known that FGF19/15 can negatively modulate
lipogenesis in mouse livers by inhibiting peroxisome
proliferator-activated receptor-.gamma. coactivator-1.beta.
(PPARGC1B) expression. Here, BP3 treatment significantly enhanced
this effect (FIG. 3A). In these experimental settings, whilst a
single administration of FGF19 was not sufficient to alter the
expression of these gluconeogenic or lipogenic genes,
neutralization of endogenous FGF15 reverted BP3-induced gene
regulation to levels indistinguishable from those observed in
control livers. These microarray data were validated with qRT-PCR,
and it was observed that the results were highly consistent with
those obtained in the cDNA array, as shown in the right panel of
FIG. 3A.
[0118] BP3's ability to modulate FGF15/19-induced downstream
activity was examined. Specifically, the phosphorylation state of
candidate signaling molecules upstream of gluconeogenesis and
lipogenesis in the same ob/ob mouse livers was analyzed. Protein
kinase B (AKT) is a critical effector kinase downstream of IRS2
whose phosphorylation results in a negative regulation of
gluconeogenic genes, such as G6PC. Administration of BP3, but not
of FGF19, induced a two-fold increase of AKT phosphorylation over
the basal status in control levels, without affecting AKT
expression. In addition, activation of AKT by BP3 was reduced to
baseline intensities by an anti FGF15 antibody (FIG. 3B). The
addition of exogenous FGF19 to BP3 treatment evoked a comparable
AKT phosphorylation to that induced by BP3 only.
[0119] Several reports have described the contribution of
interleukin-6 (IL6) pathway activation in improving insulin
sensitivity through the activation of STAT3, with resulting G6PC
downregulation. The status of endogenous STAT3 phosphorylation was
examined in the same ob/ob mouse livers. Treatment with BP3, but
not with FGF19, resulted in a 3.9-fold increase of STAT3
phosphorylation over baseline levels, whereas total STAT3
expression remained unchanged. Moreover, neutralization of
endogenous FGF15 by a specific antibody blunted BP3-induced STAT3
phosphorylation (FIG. 3B). The graphic model depicted in FIG. 3C
summarizes the molecular pathways utilized by BP3 to modulate BA
biosynthesis, gluconeogenesis and lipogenesis in mouse livers.
[0120] To further compare the expression pattern of the hepatic
genes between different treatments, a sample dendrogram was
generated by hierarchical cluster analysis. The analysis revealed a
clear separation between the BP3 treated liver samples and control
groups, indicating that global gene expression in the former
experimental group was significantly altered (FIG. 3D). It is
noteworthy that the transcript pattern from livers co-treated with
BP3 and a neutralizing anti FGF15 antibody clustered together with
those of the control group, and they also co-clustered with
transcripts from FGF19 or anti FGF15 treated mouse livers.
[0121] The Ingenuity Pathway Analysis software was used to
integrate gene expression with key biochemical networks in response
to the different experimental treatments (FIG. 4). An overall
analysis of metabolic functions indicated that carbohydrate, fatty
acid and protein metabolism were significantly activated in
response of BP3 treatment, but not of FGF19 alone (FIG. 4, bottom).
Analysis of activated pathways revealed a significant activation of
FGF2 and FGF19-induced signaling after BP3 treatment. Moreover,
major biochemical effector molecules activated by the engagement of
FGF/FGFR axes, such as p38, ERK1/2 and JNK, were significantly
activated. Amongst all others, the IL6/STAT3 signaling pathway
showed the highest induction upon BP3 administration, with z-scores
of 7.98 and 6.15, respectively. Commensurate with an upregulation
of IL6, IL6 receptor (IL6R)-induced downstream signaling was also
enhanced.
[0122] Activated AKT phosphorylates the downstream forkhead box O1
(FoxO1) transcription factor, which results in the suppression of
gluconeogenic gene transcription, such as G6PC. Both AKT and FoxO1
pathways are concomitantly activated with BP3 treatment, whereas
treatment with FGF19 alone or an anti-FGF15 antibody maintains
these pathways in an inhibitory state. Commensurate with
BP3-mediated suppression of PPARGC1B transcription via STAT3
activation (FIG. 3A), BP3 treatment induced the inhibition of
sterol regulatory element-binding protein 1c (SREBF1) signaling
pathway, a PPARGC1B downstream effector molecule, thus indicating a
reduction of lipogenesis.
[0123] It has been reported that FGF19 can promote initiation of
protein translation in mouse livers by inducing eukaryotic
initiation factor 4E (EIF4E) phosphorylation. Here, the EIF4E
pathway was highly activated in response to BP3 administration,
whereas FGF19 alone failed to activate it.
[0124] It is also noteworthy that BP3 treatment in leptin deficient
ob/ob mice significantly restored leptin-induced signaling pathway,
thus suggesting that BP3 can trigger a downstream molecular
response that mimicks that of leptin. Neutralization of endogenous
FGF15 drastically reduced the induction of the aforementioned
pathways and metabolic functions upon BP3 treatment, thus
indicating that BP3 can sensitize and modulate FGF15/19-driven
downstream biochemical signaling. The integration of
BP3/FGF19/FGFR4-induced transcriptional regulation with activated
pathways analyzed in silico is depicted in the schematic model in
FIG. 3C.
Example 2
[0125] The impact of BP3 treatment on ad libitum fed normoglycemic
wild type C57BL mice was examined using a similar protocol as
above. Similar to the results seen in the ob/ob mice, a single
intraperitoneal dose of BP3, but not of a control protein, was
sufficient to significantly reduce plasma glucose levels (FIG. 5A).
Similarly to what observed in ob/ob mouse livers, BP3 treatment of
wild type mice induced a marked downregulation of hepatic CYP7A1,
CYP8B1, G6PC, and PPARGC1B, and an upregulation of IL6 and SOCS3
(FIG. 5B) and a prominent increase of endogenous AKT and STAT3
phosphorylation levels, without affecting basal expression (FIG.
5B). Conversely, CYP8B1 transcription was not suppressed by BP3
treatment in ob/ob mice. Lastly, in this experimental setting, in
silico analysis of BP3-activated pathways in C57BL mice (FIG. 5D)
was commensurate with the results obtained from the ob/ob model
(see FIG. 4).
Example 3
[0126] FGF19 exhibits a high affinity for FGFR4, and BP3 can
enhance FGFR4/FGF19 complex formation. The data herein shows that
BP3 binds to FGFR4, but not to other FGFR5 (FIG. 6A), and contains
a high-affinity binding site for FGFR4, as determined in a dose
response assay by surface plasmon resonance (SPR) and ELISA (FIG.
6B-C). A 66 amino acid-long BP3 C-terminal fragment has been
previously identified as the FGF2 binding domain. This binding
domain was purified, and an MBP-tagged C66 fusion protein (referred
as C66) (FIG. 7A) and used as a tool for binding studies. Whilst
C66 retained its ability to bind to FGF2, its binding to FGF19 was
even higher. The C66 fragment, however, did not bind to FGFR4,
indicating that the FGF-binding domain of BP3 is not sufficient to
elicit BP3 binding to FGFR4 (FIG. 7B). The contribution of C66 to
FGFR4/FGF19 complex formation was assessed in vitro. C66
significantly enhanced FGF19 binding to immobilized FGFR4, but
immobilized FGFR1 did not display any binding to FGF19 or C66,
either alone or in combination, which is similar to the binding
characteristics of full length BP3 (FIG. 7C). Moreover, when
administered to ad libitum fed ob/ob diabetic mice for four hours
in a single dose, C66 reverted hyperglycemia to normoglycemic
levels, indistinguishable from those resulted from the treatment
with full-length BP3, whereas mice treated with a control protein
remained diabetic (FIG. 7D). Analogous to full-length BP3
treatment, gene expression analysis of livers from C66-treated
ob/ob mice revealed a marked suppression of CYP7A1 and G6PC and
upregulation of IL6 (FIG. 7E). From these experiments, the
FGF-binding domain of BP3 is sufficient to increase FGFR4/FGF19
binding affinity and to reduce hyperglycemia in diabetic mice to
the same extent as full-length BP3. Moreover, binding of BP3 to
FGFR4 is not required for BP3 regulation of glucose
homeostasis.
Example 4
[0127] Five week old female ob/ob mice, obtained from Jackson
Laboratories, were randomly assigned to treatment groups with FGF19
alone, or FGF19 in combination with BP3. The substances were
administered in the morning by single intraperitoneal (i.p.)
injection. To assess potential therapeutic effects on the metabolic
profile with FGF19 or FGF19+BP3 treatments, glucose tolerance tests
were performed and changes in body weights were measured. Two
series of experiments were performed to investigate acute and
sustained effects with the different treatments.
TABLE-US-00005 Series I Series II 1. Baseline 1. Baseline
(non-treated) 2. FGF19 or FGF19 + BP3 treated 2. Post-treatment (1
dose/day for 5 days) (2 days following a single dose) 3.
Post-treatment (9 days after treatment)
[0128] For all experiments reported in FIGS. 8-10, mice were fasted
overnight (14 h) before they were subjected to a standard glucose
tolerance test (GTT). The GTT is used to evaluate the ability of an
organism to metabolize exogenous glucose. In clinical practice the
GTT is used to uncover patients with latent diabetes or patients at
risk for diabetes, e.g. during pregnancy and is performed after a
fasting period by oral administration of a glucose containing
drink. In the animal model oral GTT is performed by gavage of a
glucose solution, i.e. orally administration. Intraperitoneal
injection of a sterile glucose solution was chosen because this
allows for a tighter control of dosing of the glucose. Before
starting the experiments, the animals were weighed to determine the
amount of glucose to inject. The glucose tolerance test was
performed in a quiet room and handling was kept down to a minimum
to reduce stress during the procedure. A bolus of glucose (1 g/kg)
was injected into the intraperitoneal cavity (30%
D-glucose:H.sub.2O solution) and blood was sampled from the tail
tip at 0, 15, 30, 60, 120 and 180 minutes after glucose injection.
Blood glucose levels were determined with a portable glucose meter
(Contour.RTM., Bayer HealthCare).
[0129] Ob/ob mice exhibit glucose intolerance and are generally
used as a leptin-deficient diabetes model and reflect the human
disease well. To examine the metabolic capacity of the animals for
glucose the intraperitoneal glucose tolerance test (IPGTT) was
performed in ob/ob mice and blood glucose levels were read at 0,
15, 30, 60, 120 and 180 minutes post-injection of glucose. The
baseline blood glucose levels were much higher than the normal
range (70-120 mg/dl) (FIGS. 8A, 8B, Baseline). After 5 days of
treatment (one dose/day) with FGF19, ob/ob mice (n=6) showed an
improved glucose tolerance as evidenced by reduced blood glucose
levels at 180 minutes post-injection of glucose (p<0.05). The
glucose tolerance test (IPGTT) was repeated 9 days after treatment
and glucose levels returned to the levels seen before the treatment
(FIG. 8A).
[0130] For the combination treatment group (FGF19+BP3, n=5), the
glucose tolerance test (IPGTT) was seen improved at 60, 120 and 180
minutes post-injection of glucose (p<0.05). The respective blood
glucose levels were significantly lower than those of the control
group. This effect was sustained even 9 days after the last of 5
doses of FGF19+BP3 (FIG. 8B). The results from the glucose
tolerance test, shown as the area under the curve of the blood
glucose levels after glucose injection (AUC), was reduced by 26%
compared with baseline in the FGF19 group and by 33% in the
FGF19+BP3 group. Most strikingly, the FGF19+BP3 group displayed an
improved AUC compared with baseline even 9 days after completed
treatment (p<0.01), whereas no such effect was observed in mice
receiving FGF19 alone (FIG. 8C). In addition, the combination
treatment (n=5) improved the AUC even 2 days after a single dose of
FGF19+BP3, while FGF19 alone (n=3) did not (p<0.01, FIG. 9).
These data suggest that BP3 dramatically enhances the FGF19 effect
in a standardized IPGTT glucose tolerance test in diabetic
mice.
[0131] The effects of acute single doses of BP3 or FGF19 alone, and
the combination of BP3+FGF19 were tested in ob/ob mice by IPGTT
described above (FIGS. 10A, B). Mice were starved overnight (14
hours) and then treated for two hours by i.p. injection of vehicle
(filled squares, FIGS. 10A, B) or FGF19 alone or BP3+FGF19 (open
squares FIGS. 10A, B). The IPGTT test was initiated two hours after
treatments. The combination of BP3+FGF19 induced a striking effect
and normalized the baseline blood glucose and the IPGTT blood
glucose curve (FIG. 10A). This effect was still present 24 hours
after the single dose of BP3+FGF19 (FIG. 10C). FGF19 alone showed
no significant effect in the test (FIG. 10B). Also, treatment of
animals with BP3 alone (without FGF19) showed no significant
effect, and even 7 days of dosing of BP3 with a single daily dose
of BP3 lacked an effect on the baseline blood glucose or on the
IPGTT test (FIG. 10D).
[0132] Two days after a single dose treatment with FGF19 (n=3) or
FGF19+BP3 (n=5), ob/ob mice were weighed and the changes in body
weight were compared between the two groups. No significant changes
in body weight were found (FIGS. 11A, 11B, 1 treatment) between the
groups. However, 5 days of treatment (one dose/day) with FGF19+BP3
lowered the body weight by 1.0.+-.0.7 g/animal, whereas 5 days of
treatment (one dose/day) with FGF19 alone was associated with an
increase in body weight (2.2.+-.0.8g/animal), which was similar to
vehicle treated animals. For the 5 days of treatment, the changes
in body weight were significantly different, in both grams and in
percent body weight (p<0.05, FIGS. 11A, 11B, 5 daily
treatments).
[0133] BP3 enhances and prolongs the effects of FGF19 on glucose
homeostasis, i.e., there is an improvement of the glucose
tolerance, from either single or multiple doses. The combination of
FGF19+BP3 also reduced the body weight per mouse after 1 dose/day
for 5 days, while the animals in FGF19 treatment group gained
weight. The combination of BP3 and FGF19 has an unexpectedly better
therapeutic effect on obesity than FGF19 alone.
[0134] Given the proposed mechanism of action, it was surprising
that a FGFBP3 enhanced the effects of FGF19, i.e., a molecule that
that will not be retained by the extracellular matrix and that
administration of a complex of FGF19 and FGFBP3 would affect
metabolism more quickly, (i.e. after a single dose), for a longer
period (i.e for up to two days after a single dose) and more
profoundly (i.e. better efficacy in normalizing glucose tolerance)
than FGF19 alone. These data support the concept that BP3+FGF19
treatment improves glucose metabolism in subjects with diabetes and
that a single daily dose may be sufficient to last beyond 24
hours.
[0135] To further explore a possible mechanism of action, HepG2
cells (hepatocellular carcinoma cells) were treated with FGF19 with
or without BP3. FIG. 12 shows that the presence of BP3 enhanced the
ability of FGF19 to induce phosphorylation of Erk1/2.
Example 5
[0136] To understand the longterm effect, FGFBP3 was expressed in
ob/ob, leptin deficient mice that develop metabolic disease. For
this experiment, mouse FGFBP3 was used to avoid immune rejection of
a human protein. Exogenously introduced FGFBP3 expression was
achieved by injecting mice twice weekly with a liposomally packaged
FGFBP3 expression plasmid to achieve uptake and expression of the
exogenously introduce FGFBP3 gene. After 2 1/2 weeks of treatments
the content of blood in mice was analyzed as shown in FIG. 13.
Sequence CWU 1
1
201258PRTArtificial SequenceSynthetic peptide - full length FGFBP3
1Met Thr Pro Pro Lys Leu Arg Ala Ser Leu Ser Pro Ser Leu Leu Leu1 5
10 15Leu Leu Ser Gly Cys Leu Leu Ala Ala Ala Arg Arg Glu Lys Gly
Ala 20 25 30Ala Ser Asn Val Ala Glu Pro Val Pro Gly Pro Thr Gly Gly
Ser Ser 35 40 45Gly Arg Phe Leu Ser Pro Glu Gln His Ala Cys Ser Trp
Gln Leu Leu 50 55 60Leu Pro Ala Pro Glu Ala Ala Ala Gly Ser Glu Leu
Ala Leu Arg Cys65 70 75 80Gln Ser Pro Asp Gly Ala Arg His Gln Cys
Ala Tyr Arg Gly His Pro 85 90 95Glu Arg Cys Ala Ala Tyr Ala Ala Arg
Arg Ala His Phe Trp Lys Gln 100 105 110Val Leu Gly Gly Leu Arg Lys
Lys Arg Arg Pro Cys His Asp Pro Ala 115 120 125Pro Leu Gln Ala Arg
Leu Cys Ala Gly Lys Lys Gly His Gly Ala Glu 130 135 140Leu Arg Leu
Val Pro Arg Ala Ser Pro Pro Ala Arg Pro Thr Val Ala145 150 155
160Gly Phe Ala Gly Glu Ser Lys Pro Arg Ala Arg Asn Arg Gly Arg Thr
165 170 175Arg Glu Arg Ala Ser Gly Pro Ala Ala Gly Thr Pro Pro Pro
Gln Ser 180 185 190Ala Pro Pro Lys Glu Asn Pro Ser Glu Arg Lys Thr
Asn Glu Gly Lys 195 200 205Arg Lys Ala Ala Leu Val Pro Asn Glu Glu
Arg Pro Met Gly Thr Gly 210 215 220Pro Asp Pro Asp Gly Leu Asp Gly
Asn Ala Glu Leu Thr Glu Thr Tyr225 230 235 240Cys Ala Glu Lys Trp
His Ser Leu Cys Asn Phe Phe Val Asn Phe Trp 245 250 255Asn
Gly2232PRTArtificial SequenceSynthetic peptide - full length FGFBP3
without the signal sequence 2Arg Arg Glu Lys Gly Ala Ala Ser Asn
Val Ala Glu Pro Val Pro Gly1 5 10 15Pro Thr Gly Gly Ser Ser Gly Arg
Phe Leu Ser Pro Glu Gln His Ala 20 25 30Cys Ser Trp Gln Leu Leu Leu
Pro Ala Pro Glu Ala Ala Ala Gly Ser 35 40 45Glu Leu Ala Leu Arg Cys
Gln Ser Pro Asp Gly Ala Arg His Gln Cys 50 55 60Ala Tyr Arg Gly His
Pro Glu Arg Cys Ala Ala Tyr Ala Ala Arg Arg65 70 75 80Ala His Phe
Trp Lys Gln Val Leu Gly Gly Leu Arg Lys Lys Arg Arg 85 90 95Pro Cys
His Asp Pro Ala Pro Leu Gln Ala Arg Leu Cys Ala Gly Lys 100 105
110Lys Gly His Gly Ala Glu Leu Arg Leu Val Pro Arg Ala Ser Pro Pro
115 120 125Ala Arg Pro Thr Val Ala Gly Phe Ala Gly Glu Ser Lys Pro
Arg Ala 130 135 140Arg Asn Arg Gly Arg Thr Arg Glu Arg Ala Ser Gly
Pro Ala Ala Gly145 150 155 160Thr Pro Pro Pro Gln Ser Ala Pro Pro
Lys Glu Asn Pro Ser Glu Arg 165 170 175Lys Thr Asn Glu Gly Lys Arg
Lys Ala Ala Leu Val Pro Asn Glu Glu 180 185 190Arg Pro Met Gly Thr
Gly Pro Asp Pro Asp Gly Leu Asp Gly Asn Ala 195 200 205Glu Leu Thr
Glu Thr Tyr Cys Ala Glu Lys Trp His Ser Leu Cys Asn 210 215 220Phe
Phe Val Asn Phe Trp Asn Gly225 230329PRTArtificial
SequenceSynthetic peptide - C-terminal FGFBP3 3Leu Asp Gly Asn Ala
Glu Leu Thr Glu Thr Tyr Cys Ala Glu Lys Trp1 5 10 15His Ser Leu Cys
Asn Phe Phe Val Asn Phe Trp Asn Gly 20 25466PRTArtificial
SequenceSynthetic peptide - C-terminal FGFBP3 "C66" peptide 4Ala
Pro Pro Lys Glu Asn Pro Ser Glu Arg Lys Thr Asn Glu Gly Lys1 5 10
15Arg Lys Ala Ala Leu Val Pro Asn Glu Glu Arg Pro Met Gly Thr Gly
20 25 30Pro Asp Pro Asp Gly Leu Asp Gly Asn Ala Glu Leu Thr Glu Thr
Tyr 35 40 45Cys Ala Glu Lys Trp His Ser Leu Cys Asn Phe Phe Val Asn
Phe Trp 50 55 60Asn Gly65536PRTArtificial SequenceSynthetic peptide
- Tat protein basic peptide motif 37-72 5Cys Phe Ile Thr Lys Ala
Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg1 5 10 15Arg Gln Arg Arg Arg
Pro Pro Gln Gly Ser Gln Thr His Gln Val Ser 20 25 30Leu Ser Lys Gln
35610PRTArtificial SequenceSynthetic peptide - Tat protein residues
48-57 6Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5
10723DNAArtificial SequencePCR Primer 7ggcgcttttg actcaggatt taa
23824DNAArtificial SequencePCR Primer 8cctcagccac atttgtagaa cttt
24926DNAArtificial SequencePCR Primer 9cccacagtta atgcacttgg atcctg
261030DNAArtificial SequencePCR Primer 10gggcatgtag aaatacttca
gcttgtttcc 301124DNAArtificial SequencePCR Primer 11tctttgtcgg
aagactgtca acgg 241226DNAArtificial SequencePCR Primer 12catcatactg
atccaggaac tcccga 261327DNAArtificial SequencePCR Primer
13gtcactttga gatctactcg gcaaacc 271424DNAArtificial SequencePCR
Primer 14tctgaccaca gtgaggaatg tcca 241521DNAArtificial SequencePCR
Primer 15cgactcgcta tctccaagtg a 211620DNAArtificial SequencePCR
Primer 16gttgaaccag tctccgacca 201721DNAArtificial SequencePCR
Primer 17tcctgtaaaa gcccggagta t 211819DNAArtificial SequencePCR
Primer 18gctctggtag gggcagtga 191919DNAArtificial SequencePCR
Primer 19accgacttgg tcagcgaag 192020DNAArtificial SequencePCR
Primer 20cacgagcccg tagttgtcat 20
* * * * *
References