U.S. patent application number 17/358540 was filed with the patent office on 2022-06-02 for truncated actriib-fc fusion proteins.
The applicant listed for this patent is Acceleron Pharma Inc.. Invention is credited to Ravindra Kumar, Jasbir Seehra.
Application Number | 20220169996 17/358540 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-02 |
United States Patent
Application |
20220169996 |
Kind Code |
A1 |
Seehra; Jasbir ; et
al. |
June 2, 2022 |
TRUNCATED ACTRIIB-FC FUSION PROTEINS
Abstract
In certain aspects, the present invention provides compositions
and methods for modulating (promoting or inhibiting) growth of a
tissue, such as bone, cartilage, muscle, fat, brown fat and/or
neuronal tissue and for treating metabolic disorders such as
diabetes and obesity, as well as disorders associated with any of
the foregoing tissue.
Inventors: |
Seehra; Jasbir; (Cambridge,
MA) ; Kumar; Ravindra; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Acceleron Pharma Inc. |
Cambridge |
MA |
US |
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|
Appl. No.: |
17/358540 |
Filed: |
June 25, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16434841 |
Jun 7, 2019 |
11066654 |
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17358540 |
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15664172 |
Jul 31, 2017 |
10358633 |
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16434841 |
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14814040 |
Jul 30, 2015 |
9745559 |
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15664172 |
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13657649 |
Oct 22, 2012 |
9181533 |
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14814040 |
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12796307 |
Jun 8, 2010 |
8293881 |
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13657649 |
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61280543 |
Nov 3, 2009 |
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61268420 |
Jun 12, 2009 |
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International
Class: |
C12N 9/12 20060101
C12N009/12; C07K 14/475 20060101 C07K014/475; C07K 14/71 20060101
C07K014/71 |
Claims
1-29. (canceled)
30. A method for treating a metabolic disorder in a subject,
comprising administering to a subject an effective amount of a
polypeptide selected from the group consisting of: a. a polypeptide
that consists of the amino acid sequence of SEQ ID NO: 8; b. a
polypeptide produced by expressing in a mammalian cell the nucleic
acid of SEQ ID NO: 4; and c. a polypeptide produced by expressing
in a mammalian cell the nucleic acid of SEQ ID NO: 6.
31. The method of claim 30, wherein the subject has one or more of
the following characteristics: a. elevated serum triglyceride
levels; b. elevated free fatty acid levels; or c. elevated serum
insulin levels.
32. The method of claim 30, wherein the metabolic disorder is
selected from the group consisting of: type 2 diabetes, metabolic
syndrome, insulin resistance and obesity.
33. The method of claim 30, wherein the polypeptide is a
polypeptide of part (a).
34. The method of claim 30, wherein the polypeptide is a
polypeptide of part (b).
35. The method of claim 30, wherein the polypeptide is a
polypeptide of part (c).
36. The method of claim 30, wherein the polypeptide is capable of
causing a statistically significant decrease in serum triglyceride
levels in a mouse fed a high fat diet after four weeks of treatment
twice per week at a dose level of 10 mg/kg.
37. The method of claim 36, wherein the mean decrease is at least
50 mg/dl triglycerides.
38. The method of claim 30, wherein the polypeptide is capable of
causing a statistically significant decrease in serum free fatty
acid levels in a mouse fed a high fat diet after four weeks of
treatment twice per week at a dose level of 10 mg/kg.
39. The method of claim 38, wherein the mean decrease is at least
500 micromoles/dl free fatty acids.
40. The method of claim 30, wherein the polypeptide is covalently
associated with a second polypeptide to form a homodimer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/434,841, filed Jun. 7, 2019 (now allowed), which is a
continuation of U.S. application Ser. No. 15/664,172, filed Jul.
31, 2017 (now U.S. Pat. No. 10,358,633), which is a continuation of
U.S. application Ser. No. 14/814,040, filed Jul. 30, 2015 (now U.S.
Pat. No. 9,745,559), which is a continuation of U.S. Application
No. 13,657,649, filed Oct. 22, 2012 (now U.S. Pat. No. 9,181,533),
which is a divisional of U.S. patent application Ser. No.
12/796,307, filed Jun. 8, 2010 (now U.S. Pat. No. 8,293,881), which
claims the benefit of U.S. Provisional Application Nos. 61/280,543,
filed Nov. 3, 2009 (now expired), and 61/268,420, filed Jun. 12,
2009 (now expired). The specifications of each of the foregoing
applications are incorporated herein by reference in their
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Jun. 17,
2021, is named 1848179-0002-045-106_Seq.txt, and is 27,650 bytes in
size.
BACKGROUND OF THE INVENTION
[0003] The transforming growth factor-beta (TGF-beta) superfamily
contains a variety of growth factors that share common sequence
elements and structural motifs. These proteins are known to exert
biological effects on a large variety of cell types in both
vertebrates and invertebrates. Members of the superfamily perform
important functions during embryonic development in pattern
formation and tissue specification and can influence a variety of
differentiation processes, including adipogenesis, myogenesis,
chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and
epithelial cell differentiation. The family is represented by
proteins named, variously, the activins and inhibins, TGF-beta,
Growth and Differentiation Factors (GDFs) and Bone Morphogenetic
Factors (BMPs). Other members of the family are also known, such as
Nodal and Lefty. By manipulating the activity of a member of the
TGF-beta family, it is often possible to cause significant
physiological changes in an organism. For example, the Piedmontese
and Belgian Blue cattle breeds carry a loss-of-function mutation in
the GDF8 (also called myostatin) gene that causes a marked increase
in muscle mass. Grobet et al., Nat Genet. 1997, 17(1):71-4.
Furthermore, in humans, inactive alleles of GDF8 are associated
with increased muscle mass and, reportedly, exceptional strength.
Schuelke et al., N Engl J Med 2004, 350:2682-8.
[0004] Changes in muscle, bone, fat, cartilage and other tissues
may be achieved by agonizing or antagonizing signaling that is
mediated by an appropriate TGF-beta family member. Thus, there is a
need for agents that function as potent regulators of signaling by
members of the TGF-beta superfamily.
SUMMARY OF THE INVENTION
[0005] In certain aspects, the present disclosure provides novel
ActRIIB polypeptides, particularly amino- and carboxy-terminal
truncations and sequence alterations. In one embodiment,
polypeptides including amino acids 25-131 of human ActRIIB (SEQ ID
NO:1) or variants thereof, are described. Such polypeptides are
demonstrated to have surprising efficacy in the treatment of a
variety of disorders, but particularly disorders associated with
obesity, insulin resistance and other metabolic disorders. ActRIIB
polypeptides disclosed herein can be used to have a variety of
desirable effects in patients, including, for example, increasing
lean body mass, decreasing white fat mass, increasing brown fat
mass, decreasing serum triglycerides, decreasing serum insulin
levels or decreasing serum free fatty acid levels. ActRIIB
polypeptides disclosed herein may be used for the treatment of a
variety of disorders or conditions, including muscle and
neuromuscular disorders (e.g., muscular dystrophy, amyotrophic
lateral sclerosis (ALS), and muscle atrophy), adipose tissue
disorders (e.g., obesity, fatty liver disease), metabolic disorders
(e.g., type 2 diabetes, insulin resistance, metabolic syndrome),
neurodegenerative disorders, and muscle wasting associated with old
age (sarcopenia), prostate cancer therapy (e.g., androgen
deprivation therapy), and cachexia associated with a variety of
cancers. Examples of ActRIIB polypeptides include a human
ActRIIB-Fc fusion protein set forth in SEQ ID NO:8 and described
herein as ActRIIB (25-131)-hFc.
[0006] In certain aspects, the disclosure provides novel
polypeptides that are derived from ActRIIB (referred to as ActRIIB
polypeptides). In some embodiments, a polypeptide may be selected
from the group consisting of: a polypeptide comprising an amino
acid sequence wherein the amino acid sequence consists of the
sequence of SEQ ID NO:8 or an amino acid sequence that differs from
SEQ ID NO:8 at no more than one, two, three, four or five amino
acid positions; a polypeptide produced by the expression in a
mammalian cell of the nucleic acid of SEQ ID NO: 4 or a nucleic
acid that hybridizes under stringent condition to the complement
thereof a polypeptide produced by the expression in a mammalian
cell of the nucleic acid of SEQ ID NO:6 or a nucleic acid that
hybridizes under stringent conditions to the complement thereof. A
polypeptide disclosed herein may comprise a portion derived from
ActRIIB and one or more heterologous portions, wherein the portion
derived from ActRIIB may comprise an amino acid sequence consisting
of the sequence of amino acids 25-131 of SEQ ID NO:1 or an amino
acid sequence that differs the sequence of amino acids 25-131 of
SEQ ID NO:1 at no more than one, two, three, four or five amino
acid positions. The heterologous portion may comprise a constant
domain of an immunoglobulin, an Fc domain of an immunoglobulin or,
particularly, an Fc domain of a human IgG1 (the term "human IgG1
shall be understood to include variants of such Fc that are
compatible with use in humans). ActRIIB polypeptides may include a
portion derived from ActRIIB that comprises an amino acid sequence
consisting of the sequence of amino acids 25-131 of SEQ ID NO:1. An
ActRIIB polypeptide disclosed herein may be such that the amino
terminus has the sequence ETR. An ActRIIB polypeptide disclosed
herein may cause a statistically significant increase in lean body
mass in a mouse after four weeks of treatment twice per week at a
dose level of 10 mg/kg. The mean increase of lean tissue mass may
be at least 1, 2, 3, 4 or 5 or more grams. An ActRIIB polypeptide
disclosed herein may cause a statistically significant decrease in
fat mass in a mouse fed a high fat diet after four weeks of
treatment twice per week at a dose level of 10 mg/kg. The mean
decrease in fat mass may be 5, 7, 10, 15 or more grams. An ActRIIB
polypeptide disclosed herein may cause a statistically significant
decrease in serum triglyceride levels in a mouse fed a high fat
diet after four weeks of treatment twice per week at a dose level
of 10 mg/kg. The mean decrease in serum triglycerides may be at
least 50, 75, 100, 125 or 150 or more mg/dl. An ActRIIB polypeptide
disclosed herein may cause a statistically significant decrease in
serum free fatty acid levels in a mouse fed a high fat diet after
four weeks of treatment twice per week at a dose level of 10 mg/kg.
The mean decrease in free fatty acids may be at least 500, 750,
1000 or more micromoles/dl free fatty acids. An ActRIIB polypeptide
disclosed herein may cause a statistically significant decrease in
serum insulin levels in a mouse fed a high fat diet after four
weeks of treatment twice per week at a dose level of 10 mg/kg. The
mean decrease in serum insulin may be at least 0.5, 1, 1.5, 2 or
more ng/ml insulin. As used herein, the term "statistically
significant" generally refers to a p value or >0.05, but other
measures of significance may be recognized for different types of
statistical tests, and in such cases, the term "statistically
significant" should use the most widely used formula for assessing
the significance of the data. ActRIIB polypeptides may comprise at
least one N-linked sugar, and may include two, three or more
N-linked sugars. Such polypeptides may also comprise O-linked
sugars. ActRIIB polypeptides may be produced in a variety of cell
lines that glycosylate the protein in a manner that is suitable for
patient use, including engineered insect or yeast cells, and
mammalian cells such as COS cells, CHO cells, HEK cells and NSO
cells. ActRIIB polypeptides may form covalent or non-covalent
dimers, including homodimers. Generally, Fc fusion proteins tend to
form homodimers that are covalently linked. Any of the foregoing
polypeptides may be incorporated into a pharmaceutical
preparation.
[0007] In certain aspects, the ActRIIB polypeptides disclosed
herein bind to an ActRIIB ligand such as GDF8, GDF11, activin,
BMP7, GDF3 or nodal. Optionally, an ActRIIB polypeptide binds to an
ActRIIB ligand with a Kd less than 10 micromolar or less than 1
micromolar, 100, 10, 1 or 0.1 nanomolar. An ActRIIB polypeptide
disclosed herein may include one, two, three, four, five or more
alterations in the amino acid sequence (e.g., in the ligand-binding
domain) relative to a naturally occurring ActRIIB polypeptide. The
alteration in the amino acid sequence may, for example, alter
glycosylation of the polypeptide when produced in a mammalian,
insect or other eukaryotic cell or alter proteolytic cleavage of
the polypeptide relative to the naturally occurring ActRIIB
polypeptide. An ActRIIB polypeptide may be a fusion protein that
has, as one domain, an amino acid sequence derived from ActRIIB
(e.g., a ligand-binding domain of an ActRIIB or a variant thereof)
and one or more additional domains that provide a desirable
property, such as improved pharmacokinetics, easier purification,
targeting to particular tissues, etc. For example, a domain of a
fusion protein may enhance one or more of in vivo stability, in
vivo half life, uptake/administration, tissue localization or
distribution, formation of protein complexes, multimerization of
the fusion protein, and/or purification. An ActRIIB fusion protein
may include an immunoglobulin Fc domain (wild-type or mutant) or a
serum albumin. In certain embodiments, an ActRIIB-Fc fusion
comprises a relatively unstructured linker positioned between the
Fc domain and the extracellular ActRIIB domain. This unstructured
linker may correspond to the roughly 15 amino acid unstructured
region at the C-terminal end of the extracellular domain of ActRIIB
(the "tail"), or it may be an artificial sequence of between 5 and
15, 20, 30, 50 or more amino acids that are relatively free of
secondary structure. A linker may be rich in glycine and proline
residues and may, for example, contain repeating sequences of
threonine/serine and glycines (e.g., TG4 or SG4 repeats). In the
context of a polypeptide of SEQ ID NO:8, it appears to be
advantageous to use a short, flexible linker, such as one, two,
three, four or five glycine residues, optionally with one or more
small residues such as alanine, threonine or serine. A fusion
protein may include a purification subsequence, such as an epitope
tag, a FLAG tag, a polyhistidine sequence, and a GST fusion.
Optionally, an ActRIIB polypeptide includes one or more modified
amino acid residues selected from: a glycosylated amino acid, a
PEGylated amino acid, a farnesylated amino acid, an acetylated
amino acid, a biotinylated amino acid, an amino acid conjugated to
a lipid moiety, and an amino acid conjugated to an organic
derivatizing agent.
[0008] In certain aspects, an ActRIIB polypeptide may be formulated
as a pharmaceutical preparation. A pharmaceutical preparation will
preferably be pyrogen free (meaning pyrogen free to the extent
required by regulations governing the quality of products for
therapeutic use). A pharmaceutical preparation may also include one
or more additional compounds such as a compound that is used to
treat an ActRIIB-associated disorder.
In certain aspects, the disclosure provides nucleic acids encoding
an ActRIIB polypeptide. Such a nucleic acid may comprises a nucleic
acid sequence of 73-396 of SEQ ID NO:4 or one that hybridizes under
stringent conditions to the complement of nucleotides 73-396 of SEQ
ID NO:4. A nucleic acid may one that comprises the sequence of SEQ
ID NO:4. Such a nucleic acid may comprises a nucleic acid sequence
of 73-396 of SEQ ID NO:6 or one that hybridizes under stringent
conditions to the complement of nucleotides 73-396 of SEQ ID NO:6.
A nucleic acid may one that comprises the sequence of SEQ ID NO:6.
In certain aspects, an ActRIIB protein may be expressed in a
mammalian cell line that mediates suitably natural glycosylation of
the ActRIIB protein so as to diminish the likelihood of an
unfavorable immune response in a patient (including the possibility
of veterinary patients). Human and CHO cell lines have been used
successfully, and it is expected that other common mammalian
expression vectors will be useful. Thus the disclosure provides
cultured cells comprising any of the nucleic acids disclosed
herein. Such cells may be mammalian cells, including CHO cells, NSO
cells, HEK cells and COS cells. Other cells may be chosen depending
on the species of the intended patient. Other cells are disclosed
herein. Cultured cells are understood to mean cells maintained in
laboratory or other man-made conditions (e.g., frozen, or in media)
and not part of a living organism.
[0009] In certain aspects, the disclosure provides methods for
making a ActRIIB polypeptide. Such a method may include expressing
any of the nucleic acids (e.g., SEQ ID NO: 4 or 6, and nucleic
acids that hybridize thereto under stringent conditions) disclosed
herein in a suitable cell, such as a Chinese hamster ovary (CHO)
cell. Such a method may comprise: a) culturing a cell under
conditions suitable for expression of the ActRIIB polypeptide,
wherein said cell is transformed with an ActRIIB expression
construct; and b) recovering the ActRIIB polypeptide so expressed.
ActRIIB polypeptides may be recovered as crude, partially purified
or highly purified fractions using any of the well known techniques
for obtaining protein from cell cultures as well as techniques
described herein.
[0010] In certain aspects the disclosure provides methods for
treating a subject having a disorder associated with muscle loss or
insufficient muscle growth. Such a method may comprise
administering to the subject an effective amount of any of the
foregoing ActRIIB polypeptides or pharmaceutical preparations
thereof.
[0011] In certain aspects the disclosure provides methods for
increasing the lean mass or reducing the rate of loss of lean mass
in a subject in need thereof. Such a method may comprise
administering to the subject an effective amount of any of the
foregoing ActRIIB polypeptides or pharmaceutical preparations
thereof.
[0012] In certain aspects, the disclosure provides methods for
decreasing the body fat content or reducing the rate of increase in
body fat content in a subject. Such a method may comprise
administering to the subject an effective amount of any of the
foregoing ActRIIB polypeptides or pharmaceutical preparations
thereof.
[0013] In certain aspects, the disclosure provides methods for
treating a disorder associated with undesirable body weight gain in
a subject. Such a method may comprise administering to the subject
an effective amount of any of the foregoing ActRIIB polypeptides or
pharmaceutical preparations thereof.
[0014] In certain aspects, the disclosure provides methods for
treating a metabolic disorder in a subject. Such a method may
comprise administering to the subject an effective amount of any of
the foregoing ActRIIB polypeptides or pharmaceutical preparations
thereof. A patient eligible for treatment may have one or more of
the following characteristics: elevated serum triglyceride levels;
elevated free fatty acid levels; or elevated serum insulin levels.
Examples of metabolic disorders include type 2 diabetes, metabolic
syndrome, insulin resistance and obesity.
[0015] In certain aspects, an ActRIIB polypeptide disclosed herein
may be used in a method for treating a subject having a disorder
associated with muscle loss or insufficient muscle growth. Such
disorders include muscle atrophy, muscular dystrophy, amyotrophic
lateral sclerosis (ALS), and a muscle wasting disorder (e.g.,
cachexia, anorexia, DMD syndrome, BMD syndrome, AIDS wasting
syndrome, muscular dystrophies, neuromuscular diseases, motor
neuron diseases, diseases of the neuromuscular junction, and
inflammatory myopathies). A method may comprise administering to a
subject in need thereof an effective amount of an ActRIIB
polypeptide.
[0016] In certain aspects, an ActRIIB polypeptide disclosed herein
may be used in a method for decreasing the body fat content or
reducing the rate of increase in body fat content, and for treating
a disorder associated with undesirable body weight gain, such as
obesity, non-insulin dependent diabetes mellitus (NIDDM),
cardiovascular disease, cancer, hypertension, osteoarthritis,
stroke, respiratory problems, and gall bladder disease. These
methods may comprise administering to a subject in need thereof an
effective amount of an ActRIIB polypeptide.
[0017] In certain specific aspects, an ActRIIB polypeptide
disclosed herein may be used in a method for treating a disorder
associated with abnormal activity of GDF8. Such disorders include
metabolic disorders such as type 2 diabetes, impaired glucose
tolerance, metabolic syndrome (e.g., syndrome X), and insulin
resistance induced by trauma (e.g., burns or nitrogen imbalance);
adipose tissue disorders (e.g., obesity); muscular dystrophy
(including Duchenne muscular dystrophy); amyotrophic lateral
sclerosis (ALS); muscle atrophy; organ atrophy; frailty; carpal
tunnel syndrome; congestive obstructive pulmonary disease;
sarcopenia, cachexia and other muscle wasting syndromes;
osteoporosis; glucocorticoid-induced osteoporosis; osteopenia;
osteoarthritis; osteoporosis-related fractures; low bone mass due
to chronic glucocorticoid therapy, premature gonadal failure,
androgen suppression, vitamin D deficiency, secondary
hyperparathyroidism, nutritional deficiencies, and anorexia
nervosa. The method may comprise administering to a subject in need
thereof an effective amount of an ActRIIB polypeptide.
[0018] In certain aspects, the disclosure provides a method for
identifying an agent that stimulates growth of a tissue such as
bone, cartilage, muscle and fat. The method comprises: a)
identifying a test agent that binds to a ligand-binding domain of
an ActRIIB polypeptide competitively with an ActRIIB polypeptide;
and b) evaluating the effect of the agent on growth of the
tissue.
[0019] In certain aspects, the disclosure provides methods for
antagonizing activity of an ActRIIB polypeptide or an ActRIIB
ligand (e.g., GDF8, GDF11, activin, GDF3, BMP7, and Nodal) in a
cell. The methods comprise contacting the cell with an ActRIIB
polypeptide. Optionally, the activity of the ActRIIB polypeptide or
the ActRIIB ligand is monitored by a signaling transduction
mediated by the ActRIIB/ActRIIB ligand complex, for example, by
monitoring cell proliferation. The cells of the methods include an
osteoblast, a chondrocyte, a myocyte, an adipocyte and a muscle
cell.
[0020] In certain aspects, the disclosure provides uses of an
ActRIIB polypeptide for making a medicament for the treatment of a
disorder or condition as described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Patent
Office upon request and payment of the necessary fee.
[0022] FIG. 1 shows the full, unprocessed amino acid sequence for
ActRIIB(25-131)-hFc (SEQ ID NO:3). The TPA leader (residues 1-22)
and double-truncated ActRIIB extracellular domain (residues 24-131,
using numbering based on the native sequence in SEQ ID NO:1) are
each underlined. Highlighted is the glutamate revealed by
sequencing to be the N-terminal amino acid of the mature fusion
protein, which is at position 25 relative to SEQ ID NO:1.
[0023] FIGS. 2A and 2B show a nucleotide sequence encoding
ActRIIB(25-131)-hFc (the coding strand is shown at top, SEQ ID
NO:4, and the complement shown at bottom 3'-5', SEQ ID NO:5).
Sequences encoding the TPA leader (nucleotides 1-66) and ActRIIB
extracellular domain (nucleotides 73-396) are underlined. The
corresponding amino acid sequence for ActRIIB(25-131) is also
shown.
[0024] FIGS. 3A and 3B show an alternative nucleotide sequence
encoding ActRIIB(25-131)-hFc (the coding strand is shown at top,
SEQ ID NO:6, and the complement shown at bottom 3'-5', SEQ ID
NO:7). This sequence confers a greater level of protein expression
in initial transformants, making cell line development a more rapid
process. Sequences encoding the TPA leader (nucleotides 1-66) and
ActRIIB extracellular domain (nucleotides 73-396) are underlined,
and substitutions in the wild type nucleotide sequence of the ECD
(see FIGS. 2A and 2B) are highlighted. The corresponding amino acid
sequence for ActRIIB(25-131) is also shown.
[0025] FIG. 4 shows the effect of four weeks treatment with
ActRIIB(25-131)-hFc on lean tissue mass in mouse. Vehicle was
Tris-buffered saline (TBS). Data are means (n=10 per group)
.+-.SEM. **, P<0.01 vs. TBS by unpaired t-test.
ActRIIB(25-131)-hFc treatment increased lean tissue mass in a clear
dose-dependent manner.
[0026] FIG. 5 shows the effect of four weeks treatment with
ActRIIB(25-131)-hFc on pectoralis muscle mass in mouse. Vehicle was
Tris-buffered saline (TBS). Data are means (n=10 per group)
.+-.SEM. *, P<0.05; **, P<0.01 vs. TBS by unpaired t-test.
ActRIIB(25-131)-hFc treatment increased pectoralis muscle mass in a
clear dose-dependent manner.
[0027] FIG. 6 shows the effect of ActRIIB(25-131)-hFc treatment on
grip strength in mouse. Vehicle was Tris-buffered saline (TBS).
Data are means (n=10 per group). **, P<0.01 vs. TBS by unpaired
t-test. ActRIIB(25-131)-hFc treatment increased grip strength in a
dose-dependent manner.
[0028] FIG. 7 shows the effect of four weeks treatment with
ActRIIB(25-131)-hFc on lean tissue mass in a mouse model of
androgen deprivation. Vehicle was Tris-buffered saline (TBS). Data
for orchidectomized (ORX) or sham-operated mice are means (n=10 per
group) .+-.SD. ***, P<0.001 vs. TBS control. ActRIIB(25-131)-hFc
increased lean tissue mass as effectively as did its full-length
counterpart ActRIIB(20-134)-mFc.
[0029] FIG. 8 shows the effect of ActRIIB(25-131)-hFc on lean
tissue mass in a mouse model of diet-induced obesity. Vehicle was
Tris-buffered saline (TBS). Data are means (n=9-10 per group). ***,
P<0.001 vs. TBS control. ActRIIB(25-131)-hFc increased lean
tissue mass effectively in mice fed a high fat diet.
[0030] FIG. 9 shows the effect of ActRIIB(25-131)-hFc on fat mass
in a mouse model of diet-induced obesity. Vehicle was Tris-buffered
saline (TBS). Data are means (n=9-10 per group) .+-.SD. *,
P<0.05; ***, P<0.001 vs. TBS control. Compared to vehicle,
ActRIIB(25-131)-hFc treatment for 12 weeks reduced fat mass by
approximately half in mice fed a high fat diet.
[0031] FIG. 10 depicts serum triglyceride concentrations in mice as
a function of diet and ActRIIB(25-131)-hFc treatment for 60 days.
Data are means.+-.SEM. ***, P<0.001. In mice fed a high-fat
diet, ActRIIB(25-131)-hFc reduced triglyceride concentrations by
more than 50%, thereby normalizing triglycerides to levels observed
in standard-diet controls.
[0032] FIG. 11 depicts serum free fatty acid (FFA) concentrations
in mice as a function of diet and ActRIIB(25-131)-hFc treatment for
60 days. Data are means.+-.SEM. ***, P<0.001. In mice fed a
high-fat diet, ActRIIB(25-131)-hFc reduced FFA concentrations by
nearly 55%, thereby normalizing FFA to levels observed in
standard-diet controls.
[0033] FIG. 12 depicts serum high-density lipoprotein (HDL)
concentrations in mice as a function of diet and
ActRIIB(25-131)-hFc treatment for 60 days. Data are means.+-.SEM.
***, P<0.001. In mice fed a high-fat diet, ActRIIB(25-131)-hFc
reduced HDL concentrations by nearly 50%, thereby normalizing HDL
to levels observed in standard-diet controls.
[0034] FIG. 13 depicts serum low-density lipoprotein (LDL)
concentrations in mice as a function of diet and
ActRIIB(25-131)-hFc treatment for 60 days. Data are means.+-.SEM.
*, P<0.05. In mice fed a high-fat diet, ActRIIB(25-131)-hFc
reduced LDL concentrations by more than 40%.
[0035] FIG. 14 depicts serum insulin concentrations in mice as a
function of diet and ActRIIB(25-131)-hFc treatment for 60 days.
Data are means.+-.SEM. **, P<0.01. In mice fed a high-fat diet,
ActRIIB(25-131)-hFc reduced insulin concentrations by more than
60%, thereby normalizing insulin to levels observed in
standard-diet controls.
[0036] FIG. 15 depicts serum adiponectin concentrations in mice as
a function of diet and ActRIIB(25-131)-hFc treatment for 60 days.
ELISA measurements detect all main oligomeric isoforms (total
adiponectin), and data are means.+-.SEM. **, P<0.01; ***,
P<0.001. In mice fed a high-fat diet, ActRIIB(25-131)-hFc
increased adiponectin concentrations by more than 75% and even
boosted adiponectin significantly above the levels observed in
standard-diet controls.
[0037] FIG. 16 shows thermogenic histological changes induced
within epididymal white adipose tissue by ActRIIB(25-131)-hFc
treatment for 60 days in a mouse model of diet-induced obesity. All
microscopic images shown at the same magnification. Hematoxylin and
eosin (H&E) staining indicates the ability of
ActRIIB(25-131)-hFc to reduce lipid droplet size and induce
clusters of multilocular adipocytes (arrows) characteristic of
brown fat. Immunostaining of non-adjacent sections reveals
widespread cytoplasmic induction of UCP1 (green fluorescence) in
both multilocular and unilocular adipocytes.
[0038] FIG. 17 shows the effect of ActRIIB(25-131)-hFc treatment
for 60 days on UCP1 mRNA levels in epididymal white fat in a mouse
model of diet-induced obesity. Data obtained by reverse
transcriptase polymerase chain reaction (RT-PCR), in relative units
(RU), are means.+-.SEM; n=6-7 per group; *, p<0.05.
ActRIIB(25-131)-hFc caused a 60-fold increase in mRNA encoding this
selective marker for brown fat, thus indicating upregulation of
thermogenic capability within this white fat depot.
[0039] FIG. 18 shows levels of adiponectin mRNA in epididymal white
fat of mice as a function of diet and ActRIIB(25-131)-hFc treatment
for 60 days. RT-PCR data, in relative units (RU), are means.+-.SEM;
n=7 per group; *, p<0.05. In mice fed a high-fat diet,
ActRIIB(25-131)-hFc increased adiponectin mRNA levels by more than
60%, thus contributing to elevated concentrations of circulating
adiponectin in these mice.
[0040] FIG. 19 shows the effect of ActRIIB(25-131)-hFc treatment
for 60 days on fatty liver deposits (hepatic steatosis) in a mouse
model of diet-induced obesity. Liver sections (all shown at the
same magnification) stained with Oil Red 0 reveal pronounced lipid
deposition under high-fat dietary conditions but not control
conditions. Arrows indicate several of many densely packed lipid
droplets, which are stained bright red but difficult to discern in
black-and-white images. ActRIIB(25-131)-hFc inhibited formation of
such lipid droplets and largely restored the appearance of liver
tissue to that of mice fed the standard diet.
[0041] FIG. 20 shows the effect of ActRIIB(25-131)-mFc treatment
for 35 days on the distribution of abdominal fat in a mouse model
of diet-induced obesity. Visceral and subcutaneous fat depots were
detected and differentiated in vivo by micro-computed tomography
(microCT) encompassing spinal cord segments T13-L5. N=4 per group;
scale bar=5 mm. Compared to controls fed a high-fat diet,
ActRIIB(25-131)-mFc treatment reduced the volume of both visceral
and subcutaneous depots of abdominal fat.
[0042] FIG. 21 shows the effect of ActRIIB(25-131)-mFc treatment
for 60 days on the volume of visceral fat as determined by microCT
in a mouse model of diet-induced obesity. Data are means.+-.SEM;
n=4 per group; ***, P<0.001. In mice fed a high-fat diet,
ActRIIB(25-131)-mFc reduced the volume of visceral fat by more than
60% compared to vehicle.
[0043] FIG. 22 shows the effect of ActRIIB(25-131)-mFc treatment
for 60 days on the volume of abdominal subcutaneous fat as
determined by microCT in a mouse model of diet-induced obesity.
Data are means.+-.SEM; n=4 per group; ***, P<0.001. In mice fed
a high-fat diet, ActRIIB(25-131)-mFc reduced the volume of
subcutaneous fat by nearly 60% compared to vehicle.
[0044] FIG. 23 shows photographs of bilateral pairs of
interscapular brown fat depots as a function of diet and
ActRIIB(25-131)-mFc treatment for 60 days in a mouse model of
diet-induced obesity. High-fat diet increased the size and
lightened the color of the depots, whereas ActRIIB(25-131)-mFc
largely reversed these changes.
[0045] FIG. 24 depicts the effect of ActRIIB(25-131)-mFc treatment
for 60 days on the mass of interscapular brown fat in a mouse model
of diet-induced obesity. Data are means.+-.SEM for combined left
and right depots; ***, p<0.001. ActRIIB(25-131)-mFc reversed the
effect of high-fat diet on the mass of this brown fat depot.
[0046] FIG. 25 depicts the effect of ActRIIB(25-131)-mFc treatment
for 60 days on the density of interscapular brown fat as determined
by microCT in a mouse model of diet-induced obesity. Data
(means.+-.SEM) are expressed in standardized units based on a
positive value for the bone mineral hydroxyapatite (HA) and a value
of zero for water; therefore, fat values are negative, with values
for white fat typically close to -120. **, p<0.01.
ActRIIB(25-131)-mFc completely reversed the effect of high-fat diet
on the density of this brown fat depot.
[0047] FIG. 26 depicts the effect of ActRIIB(25-131)-mFc treatment
on lean tissue mass as determined in a mouse model of aging by
nuclear magnetic resonance (NMR) analysis at multiple time points.
Data are means of 10-15 mice per group per time point; ***,
P<0.001 vs. vehicle at same time point. After 7 weeks of dosing,
lean tissue mass in aged mice treated with ActRIIB(25-131)-mFc
increased nearly 20% from baseline, in contrast to essentially
unchanged values in vehicle-treated controls.
[0048] FIG. 27 depicts the effect of ActRIIB(25-131)-mFc treatment
on forelimb grip strength as determined at multiple time points in
a mouse model of aging. Data are means of 13-15 mice per group per
time point; **, P<0.01 vs. vehicle at same time point. Mice
treated with ActRIIB(25-131)-mFc displayed an overall trend of
increasing grip strength across the study, in contrast to the
decline in grip strength observed in vehicle controls over the same
interval.
[0049] FIG. 28 depicts the effect of ActRIIB(25-131)-mFc treatment
for 8 weeks on bone mineral density as determined in a mouse model
of aging by dual energy x-ray absorptiometry (DEXA). Data are
means.+-.SEM; *, P<0.05. Bone mineral density in aged mice
treated with ActRIIB(25-131)-mFc (n=10) increased significantly
compared to vehicle-treated controls (n=14).
[0050] FIG. 29 depicts the effect of ActRIIB(25-131)-mFc treatment
on whole-body fat mass as determined in a mouse model of aging by
NMR analysis at multiple time points. Data are means of 10-15 mice
per group per time point. ***, P<0.001 vs. vehicle at same time
point. After 7 weeks of dosing, fat mass in aged mice treated with
ActRIIB(25-131)-mFc exhibited a percent decrease from baseline more
than twice the magnitude of that in vehicle-treated controls.
[0051] FIG. 30 depicts the effect of ActRIIB(25-131)-mFc treatment
for 8 weeks on serum insulin concentrations in a mouse model of
aging. Data are means.+-.SEM; *, P<0.05. Insulin concentrations
in aged mice treated with ActRIIB(25-131)-mFc (n=10) were reduced
by more than 40% compared to vehicle-treated controls (n=14).
[0052] FIG. 31 depicts the effect of ActRIIB(25-131)-mFc treatment
for 8 weeks on circulating glycated hemoglobin (A1C)
concentrations. Data are means.+-.SEM; n=5-6 per group; **,
P<0.01. ActRIIB(25-131)-mFc significantly reduced concentrations
of glycated hemoglobin, a widely accepted indicator of average
blood glucose concentrations over an extended period.
[0053] FIG. 32 depicts the effect of ActRIIB(25-131)-hFc treatment
for 5 weeks on lean tissue mass as determined by NMR analysis in a
mouse model of cancer cachexia. Data are means.+-.SEM; ***,
P<0.001. In tumor-implanted mice, vehicle treatment (n=7) was
associated with a 7% loss in lean tissue mass, whereas
ActRIIB(25-131)-hFc treatment (n=12) caused a 27% gain in lean
tissue mass from baseline.
DETAILED DESCRIPTION
1. Overview
[0054] In certain aspects, the present disclosure relates to
ActRIIB polypeptides. As used herein, the term "ActRIIB" refers to
a family of activin receptor type IIB (ActRIIB) proteins and
ActRIIB-related proteins, derived from any species. Members of the
ActRIIB family are generally all transmembrane proteins, composed
of a ligand-binding extracellular domain with cysteine-rich region,
a transmembrane domain, and a cytoplasmic domain with predicted
serine/threonine kinase specificity.
[0055] The term "ActRIIB polypeptide" is used to refer to
polypeptides comprising any naturally occurring polypeptide of an
ActRIIB family member as well as any variants thereof (including
mutants, fragments, fusions, and peptidomimetic forms) that retain
a useful activity. For example, ActRIIB polypeptides include
polypeptides derived from the sequence of any known ActRIIB having
a sequence at least about 80% identical to the sequence of an
ActRIIB polypeptide, and preferably at least 85%, 90%, 95%, 97%,
99% or greater identity.
[0056] The human ActRIIB precursor has the following amino acid
sequence, with the signal peptide underlined, the extracellular
domain indicated in bold, and the potential N-linked glycosylation
sites boxed (SEQ ID NO: 1) (NM 001106, 512 aa).
TABLE-US-00001 ##STR00001## ##STR00002##
AGGPEVTYEPPPTAPTLLTVLAYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPG
PPPPSPLVGLKPLQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFST
PGMKHENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMS
RGLSYLHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGD
THGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRCKAADGPVDEYMLP
FEEEIGQHPSLEELQEVVVHKKMRPTIKDHWLKHPGLAQLCVTIEECWDHDAEARLSAG
CVEERVSLIRRSVNGTTSDCLVSLVTSVTNVDLPPKESSI
[0057] ActRIIB polypeptides may include any naturally occurring
extracellular domain of an ActRIIB protein as well as any variants
thereof (including mutants, fragments and peptidomimetic forms)
that retain a useful activity. For example, the extracellular
domain of an ActRIIB protein binds to a ligand and is generally
soluble. The signal sequence can be a native signal sequence of an
ActRIIB, or a signal sequence from another protein, such as a
tissue plasminogen activator (TPA) signal sequence or a honey bee
melatin (HBM) signal sequence.
[0058] In part the disclosure provides a novel ActRIIB polypeptide
that is truncated, such that the portion derived from ActRIIB is
from amino acids 25-131 of SEQ ID NO:1. As shown herein,
polypeptides of this type when administered as an Fc construct,
ActRIIB(25-131)-hFc, promote the formation of lean body mass
(primarily muscle) and the loss of fat mass, while also having
marked desirable effects on metabolic parameters such as serum
triglycerides, serum free fatty acids and serum insulin levels.
Remarkably, ActRIIB(25-131)-hFc has a much greater effect on these
metabolic parameters than does a related protein, ActRIIB(20-134).
These data are presented in the Examples below.
[0059] TGF-.beta. signals are mediated by heteromeric complexes of
type I and type II serine/threonine kinase receptors, which
phosphorylate and activate downstream Smad proteins upon ligand
stimulation (Massague, 2000, Nat. Rev. Mol. Cell Biol. 1:169-178).
These type I and type II receptors are all transmembrane proteins,
composed of a ligand-binding extracellular domain with
cysteine-rich region, a transmembrane domain, and a cytoplasmic
domain with predicted serine/threonine specificity. Type I
receptors are essential for signaling; and type II receptors are
required for binding ligands and for expression of type I
receptors. Type I and II activin receptors form a stable complex
after ligand binding, resulting in phosphorylation of type I
receptors by type II receptors.
[0060] Two related type II receptors, ActRIIA and ActRIIB, have
been identified as the type II receptors for activins (Mathews and
Vale, 1991, Cell 65:973-982; Attisano et al., 1992, Cell 68:
97-108). Besides activins, ActRIIA and ActRIIB can biochemically
interact with several other TGF-.beta. family proteins, including
BMP7, Nodal, GDF8, and GDF11 (Yamashita et al., 1995, J. Cell Biol.
130:217-226; Lee and McPherron, 2001, Proc. Natl. Acad. Sci.
98:9306-9311; Yeo and Whitman, 2001, Mol. Cell 7: 949-957; Oh et
al., 2002, Genes Dev. 16:2749-54).
[0061] In certain embodiments, the present invention relates to
antagonizing a ligand of ActRIIB receptors (also referred to as an
ActRIIB ligand) with a subject ActRIIB polypeptide (e.g., an
ActRIIB-Fc polypeptide). Thus, compositions and methods of the
present invention are useful for treating disorders associated with
abnormal activity of one or more ligands of ActRIIB receptors.
Exemplary ligands of ActRIIB receptors include some TGF-.beta.
family members, such as activin, Nodal, GDF3, GDF8, GDF11, and
BMP7.
[0062] Activins are dimeric polypeptide growth factors and belong
to the TGF-beta superfamily. There are three activins (A, B, and
AB) that are homo/heterodimers of two closely related .beta.
subunits (PAPA, .beta..sub.A.beta..sub.A, .beta..sub.B.beta..sub.B,
and .beta..sub.A.beta..sub.B). In the TGF-beta superfamily,
activins are unique and multifunctional factors that can stimulate
hormone production in ovarian and placental cells, support neuronal
cell survival, influence cell-cycle progress positively or
negatively depending on cell type, and induce mesodermal
differentiation at least in amphibian embryos (DePaolo et al.,
1991, Proc SocEp Biol Med. 198:500-512; Dyson et al., 1997, Curr
Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol. 55:953-963).
Moreover, erythroid differentiation factor (EDF) isolated from the
stimulated human monocytic leukemic cells was found to be identical
to activin A (Murata et al., 1988, PNAS, 85:2434). It was suggested
that activin A acts as a natural regulator of erythropoiesis in the
bone marrow. In several tissues, activin signaling is antagonized
by its related heterodimer, inhibin. For example, during the
release of follicle-stimulating hormone (FSH) from the pituitary,
activin promotes FSH secretion and synthesis, while inhibin
prevents FSH secretion and synthesis. Other proteins that may
regulate activin bioactivity and/or bind to activin include
follistatin (FS), follistatin-related protein (FSRP),
.alpha..sub.2-macroglobulin, Cerberus, and endoglin, which are
described below.
[0063] Bone morphogenetic protein 7 (BMP7), also called osteogenic
protein-1 (OP-1), is well known to induce cartilage and bone
formation. In addition, BMP7 regulates a wide array of
physiological processes. Notably, BMP7 has recently been identified
as a key promoter of brown adipocyte differentiation (Tseng et al.,
2008, Nature 454:1000-1004). In this study, genetic ablation of
BMP7 led to scarcity of brown fat and nearly complete absence of
UCP1 in murine embryos. Moreover, upregulation of BMP7 expression
in mice by adenovirus administration increased brown fat mass and
energy expenditure. Like activin, BMP7 binds to type II receptors,
ActRIIA and ActRIIB. However, BMP7 and activin recruit distinct
type I receptors into heteromeric receptor complexes. The major
BMP7 type I receptor observed was ALK2, while activin bound
exclusively to ALK4 (ActRIIB). BMP7 and activin elicited distinct
biological responses and activated different Smad pathways
(Macias-Silva et al., 1998, J Biol Chem. 273:25628-36).
[0064] Growth-and-Differentiation Factor-3 (GDF3), also known as
Vg1-related 2, plays an important role in embryonic development and
has also been implicated in adipogenesis during adulthood. In
brief, expression of GDF3 in white adipose tissue is correlated
with body mass or obesity (Weisberg et al., 2003, J Clin Invest
112:1796-1808), and adenovirus-mediated overexpression of GDF3
exaggerates the increase in adiposity observed under high-fat
dietary conditions in wildtype mice (Wang et al., 2004, Biochem
Biophys Res Commun 321:1024-1031). Importantly, mice with genetic
ablation of GDF3 are healthy and essentially normal when maintained
on a standard diet but are protected from obesity, and display an
increased basal metabolic rate, when maintained on a high-fat diet
(Shen et al., 2009, Mol Endocrinol 23:113-123). Taken together,
these findings implicate GDF3 specifically in diet-induced obesity
and more generally in the regulation of adiposity.
[0065] Nodal proteins have functions in mesoderm and endoderm
induction and formation, as well as subsequent organization of
axial structures such as heart and stomach in early embryogenesis.
It has been demonstrated that dorsal tissue in a developing
vertebrate embryo contributes predominantly to the axial structures
of the notochord and pre-chordal plate while it recruits
surrounding cells to form non-axial embryonic structures. Nodal
appears to signal through both type I and type II receptors and
intracellular effectors known as Smad proteins. Recent studies
support the idea that ActRIIA and ActRIIB serve as type II
receptors for Nodal (Sakuma et al., Genes Cells. 2002, 7:401-12).
It is suggested that Nodal ligands interact with their co-factors
(e.g., cripto) to activate activin type I and type II receptors,
which phosphorylate Smad2. Nodal proteins are implicated in many
events critical to the early vertebrate embryo, including mesoderm
formation, anterior patterning, and left-right axis specification.
Experimental evidence has demonstrated that Nodal signaling
activates pAR3-Lux, a luciferase reporter previously shown to
respond specifically to activin and TGF-beta. However, Nodal is
unable to induce pTlx2-Lux, a reporter specifically responsive to
bone morphogenetic proteins. Recent results provide direct
biochemical evidence that Nodal signaling is mediated by both
activin-TGF-beta pathway Smads, Smad2 and Smad3. Further evidence
has shown that the extracellular cripto protein is required for
Nodal signaling, making it distinct from activin or TGF-beta
signaling.
[0066] Growth and Differentiation Factor-8 (GDF8) is also known as
myostatin. GDF8 is a negative regulator of skeletal muscle mass.
GDF8 is highly expressed in the developing and adult skeletal
muscle. The GDF8 null mutation in transgenic mice is characterized
by a marked hypertrophy and hyperplasia of the skeletal muscle
(McPherron et al., Nature, 1997, 387:83-90). Similar increases in
skeletal muscle mass are evident in naturally occurring mutations
of GDF8 in cattle (Ashmore et al., 1974, Growth, 38:501-507;
Swatland and Kieffer, J. Anim. Sci., 1994, 38:752-757; McPherron
and Lee, Proc. Natl. Acad. Sci. USA, 1997, 94:12457-12461; and
Kambadur et al., Genome Res., 1997, 7:910-915) and, strikingly, in
humans (Schuelke et al., N Engl J Med 2004; 350:2682-8). Studies
have also shown that muscle wasting associated with HIV-infection
in humans is accompanied by increases in GDF8 protein expression
(Gonzalez-Cadavid et al., PNAS, 1998, 95:14938-43). In addition,
GDF8 can modulate the production of muscle-specific enzymes (e.g.,
creatine kinase) and modulate myoblast cell proliferation (WO
00/43781). The GDF8 propeptide can noncovalently bind to the mature
GDF8 domain dimer, inactivating its biological activity (Miyazono
et al. (1988) J. Biol. Chem., 263: 6407-6415; Wakefield et al.
(1988) J. Biol. Chem., 263; 7646-7654; and Brown et al. (1990)
Growth Factors, 3: 35-43). Other proteins which bind to GDF8 or
structurally related proteins and inhibit their biological activity
include follistatin, and potentially, follistatin-related proteins
(Gamer et al. (1999) Dev. Biol., 208: 222-232).
[0067] Growth and Differentiation Factor-11 (GDF11), also known as
BMP11, is a secreted protein (McPherron et al., 1999, Nat. Genet.
22: 260-264). GDF11 is expressed in the tail bud, limb bud,
maxillary and mandibular arches, and dorsal root ganglia during
mouse development (Nakashima et al., 1999, Mech. Dev. 80: 185-189).
GDF11 plays a unique role in patterning both mesodermal and neural
tissues (Gamer et al., 1999, Dev Biol., 208:222-32). GDF11 was
shown to be a negative regulator of chondrogenesis and myogenesis
in developing chick limb (Gamer et al., 2001, Dev Biol.
229:407-20). The expression of GDF11 in muscle also suggests its
role in regulating muscle growth in a similar way to GDF8. In
addition, the expression of GDF11 in brain suggests that GDF11 may
also possess activities that relate to the function of the nervous
system. Interestingly, GDF11 was found to inhibit neurogenesis in
the olfactory epithelium (Wu et al., 2003, Neuron. 37:197-207).
Hence, GDF11 may have in vitro and in vivo applications in the
treatment of diseases such as muscle diseases and neurodegenerative
diseases (e.g., amyotrophic lateral sclerosis).
[0068] In certain aspects, the present invention relates to the use
of certain ActRIIB polypeptides to antagonize the signaling of
ActRIIB ligands generally, in any process associated with ActRIIB
activity. Optionally, ActRIIB polypeptides of the invention may
antagonize one or more ligands of ActRIIB receptors, such as
activin, Nodal, GDF8, GDF11, and BMP7, and may therefore be useful
in the treatment of additional disorders.
[0069] Therefore, the present invention contemplates using ActRIIB
polypeptides in treating or preventing diseases or conditions that
are associated with abnormal activity of an ActRIIB or an ActRIIB
ligand. ActRIIB or ActRIIB ligands are involved in the regulation
of many critical biological processes. Due to their key functions
in these processes, they may be desirable targets for therapeutic
intervention. For example, ActRIIB polypeptides (e.g., ActRIIB-Fc
polypeptides) may be used to treat human or animal disorders or
conditions. Example of such disorders or conditions include, but
are not limited to, metabolic disorders such as type 2 diabetes,
impaired glucose tolerance, metabolic syndrome (e.g., syndrome X),
and insulin resistance induced by trauma (e.g., burns or nitrogen
imbalance); adipose tissue disorders (e.g., obesity); muscle and
neuromuscular disorders such as muscular dystrophy (including
Duchenne muscular dystrophy); amyotrophic lateral sclerosis (ALS);
muscle atrophy; organ atrophy; frailty; carpal tunnel syndrome;
congestive obstructive pulmonary disease; and sarcopenia, cachexia
and other muscle wasting syndromes. Other examples include
osteoporosis, especially in the elderly and/or postmenopausal
women; glucocorticoid-induced osteoporosis; osteopenia;
osteoarthritis; and osteoporosis-related fractures. Yet further
examples include low bone mass due to chronic glucocorticoid
therapy, premature gonadal failure, androgen suppression, vitamin D
deficiency, secondary hyperparathyroidism, nutritional
deficiencies, and anorexia nervosa. These disorders and conditions
are discussed below under "Exemplary Therapeutic Uses." As noted,
the truncated ActRIIB polypeptides disclosed herein appear to have
particularly beneficial effects on metabolic parameters.
[0070] The terms used in this specification generally have their
ordinary meanings in the art, within the context of this invention
and in the specific context where each term is used. Certain terms
are discussed below or elsewhere in the specification, to provide
additional guidance to the practitioner in describing the
compositions and methods of the invention and how to make and use
them. The scope or meaning of any use of a term will be apparent
from the specific context in which the term is used.
[0071] "About" and "approximately" shall generally mean an
acceptable degree of error for the quantity measured given the
nature or precision of the measurements. Typically, exemplary
degrees of error are within 20 percent (%), preferably within 10%,
and more preferably within 5% of a given value or range of
values.
[0072] Alternatively, and particularly in biological systems, the
terms "about" and "approximately" may mean values that are within
an order of magnitude, preferably within 5-fold and more preferably
within 2-fold of a given value. Numerical quantities given herein
are approximate unless stated otherwise, meaning that the term
"about" or "approximately" can be inferred when not expressly
stated.
[0073] The methods of the invention may include steps of comparing
sequences to each other, including wild-type sequence to one or
more mutants (sequence variants). Such comparisons typically
comprise alignments of polymer sequences, e.g., using sequence
alignment programs and/or algorithms that are well known in the art
(for example, BLAST, FASTA and MEGALIGN, to name a few). The
skilled artisan can readily appreciate that, in such alignments,
where a mutation contains a residue insertion or deletion, the
sequence alignment will introduce a "gap" (typically represented by
a dash, or "A") in the polymer sequence not containing the inserted
or deleted residue.
[0074] "Homologous," in all its grammatical forms and spelling
variations, refers to the relationship between two proteins that
possess a "common evolutionary origin," including proteins from
superfamilies in the same species of organism, as well as
homologous proteins from different species of organism. Such
proteins (and their encoding nucleic acids) have sequence homology,
as reflected by their sequence similarity, whether in terms of
percent identity or by the presence of specific residues or motifs
and conserved positions.
[0075] The term "sequence similarity," in all its grammatical
forms, refers to the degree of identity or correspondence between
nucleic acid or amino acid sequences that may or may not share a
common evolutionary origin.
[0076] However, in common usage and in the instant application, the
term "homologous," when modified with an adverb such as "highly,"
may refer to sequence similarity and may or may not relate to a
common evolutionary origin.
2. ActRIIB Polypeptides
[0077] In certain aspects, the invention relates to ActRIIB
polypeptides (e.g., ActRIIB-Fc polypeptides), and particularly
truncated forms exemplified by polypeptides comprising amino acids
25-131 of SEQ ID NO:1, and variants thereof. Optionally, the
fragments, functional variants, and modified forms have similar or
the same biological activities of their corresponding wild-type
ActRIIB polypeptides. For example, an ActRIIB variant of the
invention may bind to and inhibit function of an ActRIIB ligand
(e.g., activin A, activin AB, activin B, Nodal, GDF8, GDF11 or
BMP7). Optionally, an ActRIIB polypeptide modulates growth of
tissues such as bone, cartilage, muscle or fat or metabolic
parameters such as triglycerides, free fatty acids or insulin.
Examples of ActRIIB polypeptides include human ActRIIB precursor
polypeptide (SEQ ID NO: 1), and Fc fusion proteins, e.g., SEQ ID
Nos. 3 and 8. Variations on these polypeptides may be prepared
according to the following guidance. The numbering of amino acids
in the ActRIIB polypeptides is based on the sequence of SEQ ID
NO:1, regardless of whether the native leader sequence is used.
[0078] The disclosure identifies functionally active portions and
variants of ActRIIB. Applicants have ascertained that an Fc fusion
protein having the sequence disclosed by Hilden et al. (Blood. 1994
Apr. 15; 83(8):2163-70), which has an Alanine at the position
corresponding to amino acid 64 of SEQ ID NO: 1 (A64), has a
relatively low affinity for activin and GDF-11. By contrast, the
same Fc fusion protein with an Arginine at position 64 (R64) has an
affinity for activin and GDF-11 in the low nanomolar to high
picomolar range. Therefore, a sequence with an R64 is used as the
wild-type reference sequence for human ActRIIB in this
disclosure.
[0079] Attisano et al. (Cell. 1992 Jan. 10; 68(1):97-108) showed
that a deletion of the proline knot at the C-terminus of the
extracellular domain of ActRIIB reduced the affinity of the
receptor for activin. Mutations of P129 and P130 do not
substantially decrease ligand binding.
[0080] The ActRIIB ligand binding pocket is defined by residues
Y31, N33, N35, L38 through T41, E47, E50, Q53 through K55, L57,
H58, Y60, S62, K74, W78 through N83, Y85, R87, A92, and E94 through
F101. At these positions, it is expected that conservative
mutations will be tolerated, although a K74A mutation is
well-tolerated, as are R40A, K55A, F82A and mutations at position
L79. R40 is a K in Xenopus, indicating that basic amino acids at
this position will be tolerated. Q53 is R in bovine ActRIIB and K
in Xenopus ActRIIB, and therefore amino acids including R, K, Q, N
and H will be tolerated at this position. Thus, an ActRIIB protein
may be one that comprises amino acids 25-131 and comprising no more
than 1, 2, 5, 10 or 15 conservative amino acid changes in the
ligand binding pocket, and zero, one or more non-conservative
alterations at positions 40, 53, 55, 74, 79 and/or 82 in the ligand
binding pocket. Such a protein may retain greater than 80%, 90%,
95% or 99% sequence identity to the sequence of amino acids 25-131
of SEQ ID NO:1. Sites outside the binding pocket, at which
variability may be particularly well tolerated, include the amino
and carboxy termini of the extracellular domain (as noted above),
and positions 42-46 and 65-73. An asparagine to alanine alteration
at position 65 (N65A) actually improves ligand binding in the A64
background, and is thus expected to have no detrimental effect on
ligand binding in the R64 background. This change probably
eliminates glycosylation at N65 in the A64 background, thus
demonstrating that a significant change in this region is likely to
be tolerated. While an R64A change is poorly tolerated, R64K is
well-tolerated, and thus another basic residue, such as H may be
tolerated at position 64.
[0081] ActRIIB is well-conserved across nearly all vertebrates,
with large stretches of the extracellular domain conserved
completely. Many of the ligands that bind to ActRIIB are also
highly conserverd. Accordingly, comparisons of ActRIIB sequences
from various vertebrate organisms provide insights into residues
that may be altered. Therefore, an active, human ActRIIB may
include one or more amino acids at corresponding positions from the
sequence of another vertebrate ActRIIB, or may include a residue
that is similar to that in the human or other vertebrate sequence.
The following examples illustrate this approach to defining an
active ActRIIB variant. L46 is a valine in Xenopus ActRIIB, and so
this position may be altered, and optionally may be altered to
another hydrophobic residue, such as V, I or F, or a non-polar
residue such as A. E52 is a K in Xenopus, indicating that this site
may be tolerant of a wide variety of changes, including polar
residues, such as E, D, K, R, H, S, T, P, G, Y and probably A. T93
is a K in Xenopus, indicating that a wide structural variation is
tolerated at this position, with polar residues favored, such as S,
K, R, E, D, H, G, P, G and Y. F108 is a Y in Xenopus, and therefore
Y or other hydrophobic group, such as I, V or L should be
tolerated. E111 is K in Xenopus, indicating that charged residues
will be tolerated at this position, including D, R, K and H, as
well as Q and N. R112 is K in Xenopus, indicating that basic
residues are tolerated at this position, including R and H. A at
position 119 is relatively poorly conserved, and appears as P in
rodents and V in Xenopus, thus essentially any amino acid should be
tolerated at this position.
[0082] Further N-linked glycosylation sites (N--X--S/T) may be
added to an ActRIIB polypeptide, and may increase the serum
half-life of an ActRIIB-Fc fusion protein, relative to the
ActRIIB(R64)-Fc form. Examples of NX(T/S) sequences are found at
42-44 (NQS) and 65-67 (NSS), although the latter may not be
efficiently glycosylated with the R at position 64. N--X--S/T
sequences may be generally introduced at positions outside the
ligand binding pocket. Particularly suitable sites for the
introduction of non-endogenous N--X--S/T sequences include amino
acids 20-29, 20-24, 22-25, 109-134, 120-134 or 129-134. N--X--S/T
sequences may also be introduced into the linker between the
ActRIIB sequence and the Fc or other fusion component. Such a site
may be introduced with minimal effort by introducing an N in the
correct position with respect to a pre-existing S or T, or by
introducing an S or T at a position corresponding to a pre-existing
N. Thus, desirable alterations that would create an N-linked
glycosylation site are: A24N, R64N, S67N (possibly combined with an
N65A alteration), E106N, R112N, G120N, E123N, P129N, A132N, R112S
and R112T. Any S that is predicted to be glycosylated may be
altered to a T without creating an immunogenic site, because of the
protection afforded by the glycosylation. Likewise, any T that is
predicted to be glycosylated may be altered to an S. Thus the
alterations S67T and S44T are contemplated. Likewise, in an A24N
variant, an S26T alteration may be used. Accordingly, an ActRIIB
variant may include one or more additional, non-endogenous N-linked
glycosylation consensus sequences.
[0083] The variations described may be combined in various ways.
Additionally, there are amino acid positions in ActRIIB that are
often beneficial to conserve. These include position 64 (basic
amino acid), position 80 (acidic or hydrophobic amino acid),
position 78 (hydrophobic, and particularly tryptophan), position 37
(acidic, and particularly aspartic or glutamic acid), position 56
(basic amino acid), position 60 (hydrophobic amino acid,
particularly phenylalanine or tyrosine). Other positions that may
be desirable to conserve are as follows: position 52 (acidic amino
acid), position 55 (basic amino acid), position 81 (acidic), 98
(polar or charged, particularly E, D, R or K).
[0084] In certain embodiments, the present invention contemplates
making functional variants by modifying the structure of an ActRIIB
polypeptide for such purposes as enhancing therapeutic efficacy, or
stability (e.g., ex vivo shelf life and resistance to proteolytic
degradation in vivo). Modified ActRIIB polypeptides can also be
produced, for instance, by amino acid substitution, deletion, or
addition. For instance, it is reasonable to expect that an isolated
replacement of a leucine with an isoleucine or valine, an aspartate
with a glutamate, a threonine with a serine, or a similar
replacement of an amino acid with a structurally related amino acid
(e.g., conservative mutations) will not have a major effect on the
biological activity of the resulting molecule. Conservative
replacements are those that take place within a family of amino
acids that are related in their side chains. Whether a change in
the amino acid sequence of an ActRIIB polypeptide results in a
functional homolog can be readily determined by assessing the
ability of the variant ActRIIB polypeptide to produce a response in
cells in a fashion similar to the wild-type ActRIIB polypeptide, or
to bind to one or more ligands, such as activin, GDF-11 or
myostatin in a fashion similar to wild type.
[0085] In certain specific embodiments, the present invention
contemplates making mutations in the extracellular domain (also
referred to as ligand-binding domain) of an ActRIIB polypeptide
such that the variant (or mutant) ActRIIB polypeptide has altered
ligand-binding activities (e.g., binding affinity or binding
specificity). In certain cases, such variant ActRIIB polypeptides
have altered (elevated or reduced) binding affinity for a specific
ligand. In other cases, the variant ActRIIB polypeptides have
altered binding specificity for their ligands.
[0086] In certain embodiments, the present invention contemplates
specific mutations of the ActRIIB polypeptides so as to alter the
glycosylation of the polypeptide. Such mutations may be selected so
as to introduce or eliminate one or more glycosylation sites, such
as O-linked or N-linked glycosylation sites. Asparagine-linked
glycosylation recognition sites generally comprise a tripeptide
sequence, asparagine-X-threonine (where "X" is any amino acid)
which is specifically recognized by appropriate cellular
glycosylation enzymes. The alteration may also be made by the
addition of, or substitution by, one or more serine or threonine
residues to the sequence of the wild-type ActRIIB polypeptide (for
O-linked glycosylation sites). A variety of amino acid
substitutions or deletions at one or both of the first or third
amino acid positions of a glycosylation recognition site (and/or
amino acid deletion at the second position) results in
non-glycosylation at the modified tripeptide sequence. Another
means of increasing the number of carbohydrate moieties on an
ActRIIB polypeptide is by chemical or enzymatic coupling of
glycosides to the ActRIIB polypeptide. Depending on the coupling
mode used, the sugar(s) may be attached to (a) arginine and
histidine; (b) free carboxyl groups; (c) free sulfhydryl groups
such as those of cysteine; (d) free hydroxyl groups such as those
of serine, threonine, or hydroxyproline; (e) aromatic residues such
as those of phenylalanine, tyrosine, or tryptophan; or (f) the
amide group of glutamine. These methods are described in WO
87/05330 published Sep. 11, 1987, and in Aplin and Wriston (1981)
CRC Crit. Rev. Biochem., pp. 259-306, incorporated by reference
herein. Removal of one or more carbohydrate moieties present on an
ActRIIB polypeptide may be accomplished chemically and/or
enzymatically. Chemical deglycosylation may involve, for example,
exposure of the ActRIIB polypeptide to the compound
trifluoromethanesulfonic acid, or an equivalent compound. This
treatment results in the cleavage of most or all sugars except the
linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while
leaving the amino acid sequence intact. Chemical deglycosylation is
further described by Hakimuddin et al. (1987) Arch. Biochem.
Biophys. 259:52 and by Edge et al. (1981) Anal. Biochem. 118:131.
Enzymatic cleavage of carbohydrate moieties on ActRIIB polypeptides
can be achieved by the use of a variety of endo- and
exo-glycosidases as described by Thotakura et al. (1987) Meth.
Enzymol. 138:350. The sequence of an ActRIIB polypeptide may be
adjusted, as appropriate, depending on the type of expression
system used, as mammalian, yeast, insect and plant cells may all
introduce differing glycosylation patterns that can be affected by
the amino acid sequence of the peptide. In general, ActRIIB
proteins for use in humans will be expressed in a mammalian cell
line that provides proper glycosylation, such as HEK293 or CHO cell
lines, although other mammalian expression cell lines are expected
to be useful as well.
[0087] This disclosure further contemplates a method of generating
variants, particularly sets of combinatorial variants of an ActRIIB
polypeptide, including, optionally, truncation variants; pools of
combinatorial mutants are especially useful for identifying
functional variant sequences. The purpose of screening such
combinatorial libraries may be to generate, for example, ActRIIB
polypeptide variants which have altered properties, such as altered
pharmacokinetics, or altered ligand binding. A variety of screening
assays are provided below, and such assays may be used to evaluate
variants. For example, an ActRIIB polypeptide variant may be
screened for ability to bind to an ActRIIB polypeptide, to prevent
binding of an ActRIIB ligand to an ActRIIB polypeptide.
[0088] The activity of an ActRIIB polypeptide or its variants may
also be tested in a cell-based or in vivo assay. For example, the
effect of an ActRIIB polypeptide variant on the expression of genes
involved in bone production in an osteoblast or precursor may be
assessed. This may, as needed, be performed in the presence of one
or more recombinant ActRIIB ligand protein (e.g., BMP7), and cells
may be transfected so as to produce an ActRIIB polypeptide and/or
variants thereof, and optionally, an ActRIIB ligand. Likewise, an
ActRIIB polypeptide may be administered to a mouse or other animal,
and one or more bone properties, such as density or volume may be
assessed. The healing rate for bone fractures may also be
evaluated. Similarly, the activity of an ActRIIB polypeptide or its
variants may be tested in muscle cells, adipocytes, and neuronal
cells for any effect on growth of these cells, for example, by the
assays as described below. Such assays are well known and routine
in the art. A SMAD-responsive reporter gene may be used in such
cell lines to monitor effects on downstream signaling.
[0089] Combinatorially-derived variants can be generated which have
a selective potency relative to a naturally occurring ActRIIB
polypeptide. Such variant proteins, when expressed from recombinant
DNA constructs, can be used in gene therapy protocols. Likewise,
mutagenesis can give rise to variants which have intracellular
half-lives dramatically different than the corresponding a
wild-type ActRIIB polypeptide. For example, the altered protein can
be rendered either more stable or less stable to proteolytic
degradation or other processes which result in destruction of, or
otherwise inactivation of a native ActRIIB polypeptide. Such
variants, and the genes which encode them, can be utilized to alter
ActRIIB polypeptide levels by modulating the half-life of the
ActRIIB polypeptides. For instance, a short half-life can give rise
to more transient biological effects and, when part of an inducible
expression system, can allow tighter control of recombinant ActRIIB
polypeptide levels within the cell.
[0090] In certain embodiments, the ActRIIB polypeptides of the
invention may further comprise post-translational modifications in
addition to any that are naturally present in the ActRIIB
polypeptides. Such modifications include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. As a result, the modified ActRIIB
polypeptides may contain non-amino acid elements, such as
polyethylene glycols, lipids, poly- or mono-saccharide, and
phosphates. Effects of such non-amino acid elements on the
functionality of a ActRIIB polypeptide may be tested as described
herein for other ActRIIB polypeptide variants. When an ActRIIB
polypeptide is produced in cells by cleaving a nascent form of the
ActRIIB polypeptide, post-translational processing may also be
important for correct folding and/or function of the protein.
Different cells (such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or
HEK293) have specific cellular machinery and characteristic
mechanisms for such post-translational activities and may be chosen
to ensure the correct modification and processing of the ActRIIB
polypeptides.
[0091] In certain aspects, functional variants or modified forms of
the ActRIIB polypeptides include fusion proteins having at least a
portion of the ActRIIB polypeptides and one or more fusion domains.
Well known examples of such fusion domains include, but are not
limited to, polyhistidine, Glu-Glu, glutathione S transferase
(GST), thioredoxin, protein A, protein G, an immunoglobulin heavy
chain constant region (e.g., an Fc), maltose binding protein (MBP),
or human serum albumin. A fusion domain may be selected so as to
confer a desired property. For example, some fusion domains are
particularly useful for isolation of the fusion proteins by
affinity chromatography. For the purpose of affinity purification,
relevant matrices for affinity chromatography, such as
glutathione-, amylase-, and nickel- or cobalt-conjugated resins are
used. Many of such matrices are available in "kit" form, such as
the Pharmacia GST purification system and the QIAexpress.TM. system
(Qiagen) useful with (HIS.sub.6) fusion partners. As another
example, a fusion domain may be selected so as to facilitate
detection of the ActRIIB polypeptides. Examples of such detection
domains include the various fluorescent proteins (e.g., GFP) as
well as "epitope tags," which are usually short peptide sequences
for which a specific antibody is available. Well known epitope tags
for which specific monoclonal antibodies are readily available
include FLAG, influenza virus haemagglutinin (HA), and c-myc tags.
In some cases, the fusion domains have a protease cleavage site,
such as for Factor Xa or Thrombin, which allows the relevant
protease to partially digest the fusion proteins and thereby
liberate the recombinant proteins therefrom. The liberated proteins
can then be isolated from the fusion domain by subsequent
chromatographic separation. In certain preferred embodiments, an
ActRIIB polypeptide is fused with a domain that stabilizes the
ActRIIB polypeptide in vivo (a "stabilizer" domain). By
"stabilizing" is meant anything that increases serum half life,
regardless of whether this is because of decreased destruction,
decreased clearance by the kidney, or other pharmacokinetic effect.
Fusions with the Fc portion of an immunoglobulin are known to
confer desirable pharmacokinetic properties on a wide range of
proteins. Likewise, fusions to human serum albumin can confer
desirable properties. Other types of fusion domains that may be
selected include multimerizing (e.g., dimerizing, tetramerizing)
domains and functional domains (that confer an additional
biological function, such as further stimulation of muscle
growth).
[0092] As a specific example, the present invention provides a
fusion protein as a GDF8 antagonist which comprises an
extracellular (e.g., GDF8-binding) domain fused to an Fc domain
(e.g., SEQ ID NO: 9).
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD (A) VSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK (A) VSNKALPVPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTT PPVLDSDG
PFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN (A) HYTQKSLSLSPGK*
[0093] Optionally, the Fc domain has one or more mutations at
residues such as Asp-265, lysine 322, and Asn-434. In certain
cases, the mutant Fc domain having one or more of these mutations
(e.g., Asp-265 mutation) has reduced ability of binding to the
Fc.gamma. receptor relative to a wildtype Fc domain. In other
cases, the mutant Fc domain having one or more of these mutations
(e.g., Asn-434 mutation) has increased ability of binding to the
MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fc
domain.
[0094] It is understood that different elements of the fusion
proteins may be arranged in any manner that is consistent with the
desired functionality. For example, an ActRIIB polypeptide may be
placed C-terminal to a heterologous domain, or, alternatively, a
heterologous domain may be placed C-terminal to an ActRIIB
polypeptide. The ActRIIB polypeptide domain and the heterologous
domain need not be adjacent in a fusion protein, and additional
domains or amino acid sequences may be included C- or N-terminal to
either domain or between the domains.
[0095] In certain embodiments, the ActRIIB polypeptides of the
present invention contain one or more modifications that are
capable of stabilizing the ActRIIB polypeptides. For example, such
modifications enhance the in vitro half life of the ActRIIB
polypeptides, enhance circulatory half life of the ActRIIB
polypeptides or reducing proteolytic degradation of the ActRIIB
polypeptides. Such stabilizing modifications include, but are not
limited to, fusion proteins (including, for example, fusion
proteins comprising an ActRIIB polypeptide and a stabilizer
domain), modifications of a glycosylation site (including, for
example, addition of a glycosylation site to an ActRIIB
polypeptide), and modifications of carbohydrate moiety (including,
for example, removal of carbohydrate moieties from an ActRIIB
polypeptide). In the case of fusion proteins, an ActRIIB
polypeptide is fused to a stabilizer domain such as an IgG molecule
(e.g., an Fc domain). As used herein, the term "stabilizer domain"
not only refers to a fusion domain (e.g., Fc) as in the case of
fusion proteins, but also includes nonproteinaceous modifications
such as a carbohydrate moiety, or nonproteinaceous polymer, such as
polyethylene glycol.
[0096] In certain embodiments, the present invention makes
available isolated and/or purified forms of the ActRIIB
polypeptides, which are isolated from, or otherwise substantially
free of, other proteins.
[0097] In certain embodiments, ActRIIB polypeptides (unmodified or
modified) of the invention can be produced by a variety of
art-known techniques. For example, such ActRIIB polypeptides can be
synthesized using standard protein chemistry techniques such as
those described in Bodansky, M. Principles of Peptide Synthesis,
Springer Verlag, Berlin (1993) and Grant G. A. (ed.), Synthetic
Peptides: A User's Guide, W. H. Freeman and Company, New York
(1992). In addition, automated peptide synthesizers are
commercially available (e.g., Advanced ChemTech Model 396;
Milligen/Biosearch 9600). Alternatively, the ActRIIB polypeptides,
fragments or variants thereof may be recombinantly produced using
various expression systems (e.g., E. coli, Chinese Hamster Ovary
cells, COS cells, baculovirus) as is well known in the art (also
see below). In a further embodiment, the modified or unmodified
ActRIIB polypeptides may be produced by digestion of naturally
occurring or recombinantly produced full-length ActRIIB
polypeptides by using, for example, a protease, e.g., trypsin,
thermolysin, chymotrypsin, pepsin, or paired basic amino acid
converting enzyme (PACE). Computer analysis (using a commercially
available software, e.g., MacVector, Omega, PCGene, Molecular
Simulation, Inc.) can be used to identify proteolytic cleavage
sites. Alternatively, such ActRIIB polypeptides may be produced
from naturally occurring or recombinantly produced full-length
ActRIIB polypeptides such as standard techniques known in the art,
such as by chemical cleavage (e.g., cyanogen bromide,
hydroxylamine).
3. Nucleic Acids Encoding ActRIIB Polypeptides
[0098] In certain aspects, the invention provides isolated and/or
recombinant nucleic acids encoding any of the ActRIIB polypeptides
disclosed herein. For example, SEQ ID NO: 4 encodes an
ActRIIB(25-131)-hFc precursor polypeptide, while SEQ ID NO: 6
encodes a the same protein but with an alternative sequence, and
nucleotides 73-396 of each of SEQ ID Nos. 4 and 6 encode the
ActRIIB-derived portion of the encoded proteins. The subject
nucleic acids may be single-stranded or double stranded. Such
nucleic acids may be DNA or RNA molecules. These nucleic acids are
may be used, for example, in methods for making ActRIIB
polypeptides.
[0099] For example, the following sequence encodes a naturally
occurring human ActRIIB precursor polypeptide (SEQ ID NO: 2)
(nucleotides 5-1543 of NM_001106, 1539 bp):
TABLE-US-00002 atgacggcgccctgggtggccctcgccctcctctggggatcgctgtggcc
cggctctgggcgtggggaggctgagacacgggagtgcatctactacaacg
ccaactgggagctggagcgcaccaaccagagcggcctggagcgctgcgaa
ggcgagcaggacaagcggctgcactgctacgcctcctggcgcaacagctc
tggcaccatcgagctcgtgaagaagggctgctggctagatgacttcaact
gctacgataggcaggagtgtgtggccactgaggagaacccccaggtgtac
ttctgctgctgtgaaggcaacttctgcaacgagcgcttcactcatttgcc
agaggctgggggcccggaagtcacgtacgagccacccccgacagccccca
ccctgctcacggtgctggcctactcactgctgcccatcgggggcctttcc
ctcatcgtcctgctggccttttggatgtaccggcatcgcaagccccccta
cggtcatgtggacatccatgaggaccctgggcctccaccaccatcccctc
tggtgggcctgaagccactgcagctgctggagatcaaggctcgggggcgc
tttggctgtgtctggaaggcccagctcatgaatgactttgtagctgtcaa
gatcttcccactccaggacaagcagtcgtggcagagtgaacgggagatct
tcagcacacctggcatgaagcacgagaacctgctacagttcattgctgcc
gagaagcgaggctccaacctcgaagtagagctgtggctcatcacggcctt
ccatgacaagggctccctcacggattacctcaaggggaacatcatcacat
ggaacgaactgtgtcatgtagcagagacgatgtcacgaggcctctcatac
ctgcatgaggatgtgccctggtgccgtggcgagggccacaagccgtctat
tgcccacagggactttaaaagtaagaatgtattgctgaagagcgacctca
cagccgtgctggctgactttggcttggctgttcgatttgagccagggaaa
cctccaggggacacccacggacaggtaggcacgagacggtacatggctcc
tgaggtgctcgagggagccatcaacttccagagagatgccttcctgcgca
ttgacatgtatgccatggggttggtgctgtgggagcttgtgtctcgctgc
aaggctgcagacggacccgtggatgagtacatgctgccctttgaggaaga
gattggccagcacccttcgttggaggagctgcaggaggtggtggtgcaca
agaagatgaggcccaccattaaagatcactggttgaaacacccgggcctg
gcccagctttgtgtgaccatcgaggagtgctgggaccatgatgcagaggc
tcgcttgtccgcgggctgtgtggaggagcgggtgtccctgattcggaggt
cggtcaacggcactacctcggactgtctcgtttccctggtgacctctgtc
accaatgtggacctgccccctaaagagtcaagcatctaa
[0100] The following sequence encodes a human soluble
(extracellular) ActRIIB polypeptide (SEQ ID NO: 10) (348 bp).
TABLE-US-00003 tctgggcgtggggaggctgagacacgggagtgcatctactacaacgccaa
ctgggagctggagcgcaccaaccagagcggcctggagcgctgcgaaggcg
agcaggacaagcggctgcactgctacgcctcctggcgcaacagctctggc
accatcgagctcgtgaagaagggctgctggctagatgacttcaactgcta
cgataggcaggagtgtgtggccactgaggagaacccccaggtgtacttct
gctgctgtgaaggcaacttctgcaacgagcgcttcactcatttgccagag
gctgggggcccggaagtcacgtacgagccacccccgacagcccccacc
[0101] In certain aspects, the subject nucleic acids encoding
ActRIIB polypeptides are further understood to include nucleic
acids that are variants of SEQ ID NO: 4 or 6. Variant nucleotide
sequences include sequences that differ by one or more nucleotide
substitutions, additions or deletions, such as allelic variants;
and will, therefore, include coding sequences that differ from the
nucleotide sequence of the coding sequence designated in SEQ ID NO:
4 or 6.
[0102] In certain embodiments, the invention provides isolated or
recombinant nucleic acid sequences that are at least 80%, 85%, 90%,
95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4 or 6, and
particularly those portions thereof that are derived from ActRIIB
(nucleotides 73-396). One of ordinary skill in the art will
appreciate that nucleic acid sequences complementary to SEQ ID NO:
4 or 6, and variants of SEQ ID NO: 4 are also within the scope of
this invention. In further embodiments, the nucleic acid sequences
of the invention can be isolated, recombinant, and/or fused with a
heterologous nucleotide sequence, or in a DNA library.
[0103] In other embodiments, nucleic acids of the invention also
include nucleotide sequences that hybridize under highly stringent
conditions to the nucleotide sequence designated in SEQ ID NO: 4 or
6, complement sequence of SEQ ID NO: 4 or 6, or fragments thereof
(e.g., nucleotides 73-396). As discussed above, one of ordinary
skill in the art will understand readily that appropriate
stringency conditions which promote DNA hybridization can be
varied. One of ordinary skill in the art will understand readily
that appropriate stringency conditions which promote DNA
hybridization can be varied. For example, one could perform the
hybridization at 6.0.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by a wash of 2.0.times.SSC at
50.degree. C. For example, the salt concentration in the wash step
can be selected from a low stringency of about 2.0.times.SSC at
50.degree. C. to a high stringency of about 0.2.times.SSC at
50.degree. C. In addition, the temperature in the wash step can be
increased from low stringency conditions at room temperature, about
22.degree. C., to high stringency conditions at about 65.degree. C.
Both temperature and salt may be varied, or temperature or salt
concentration may be held constant while the other variable is
changed. In one embodiment, the invention provides nucleic acids
which hybridize under low stringency conditions of 6.times.SSC at
room temperature followed by a wash at 2.times.SSC at room
temperature.
[0104] Isolated nucleic acids which differ from the nucleic acids
as set forth in SEQ ID NO: 4 or 6 due to degeneracy in the genetic
code are also within the scope of the invention. For example, a
number of amino acids are designated by more than one triplet.
Codons that specify the same amino acid, or synonyms (for example,
CAU and CAC are synonyms for histidine) may result in "silent"
mutations which do not affect the amino acid sequence of the
protein. However, it is expected that DNA sequence polymorphisms
that do lead to changes in the amino acid sequences of the subject
proteins will exist among mammalian cells. One skilled in the art
will appreciate that these variations in one or more nucleotides
(up to about 3-5% of the nucleotides) of the nucleic acids encoding
a particular protein may exist among individuals of a given species
due to natural allelic variation. Any and all such nucleotide
variations and resulting amino acid polymorphisms are within the
scope of this invention.
[0105] In certain embodiments, the recombinant nucleic acids of the
invention may be operably linked to one or more regulatory
nucleotide sequences in an expression construct. Regulatory
nucleotide sequences will generally be appropriate to the host cell
used for expression. Numerous types of appropriate expression
vectors and suitable regulatory sequences are known in the art for
a variety of host cells. Typically, said one or more regulatory
nucleotide sequences may include, but are not limited to, promoter
sequences, leader or signal sequences, ribosomal binding sites,
transcriptional start and termination sequences, translational
start and termination sequences, and enhancer or activator
sequences. Constitutive or inducible promoters as known in the art
are contemplated by the invention. The promoters may be either
naturally occurring promoters, or hybrid promoters that combine
elements of more than one promoter. An expression construct may be
present in a cell on an episome, such as a plasmid, or the
expression construct may be inserted in a chromosome. In a
preferred embodiment, the expression vector contains a selectable
marker gene to allow the selection of transformed host cells.
Selectable marker genes are well known in the art and will vary
with the host cell used.
[0106] In certain aspects of the invention, the subject nucleic
acid is provided in an expression vector comprising a nucleotide
sequence encoding an ActRIIB polypeptide and operably linked to at
least one regulatory sequence. Regulatory sequences are
art-recognized and are selected to direct expression of the ActRIIB
polypeptide. Accordingly, the term regulatory sequence includes
promoters, enhancers, and other expression control elements.
Exemplary regulatory sequences are described in Goeddel; Gene
Expression Technology: Methods in Enzymology, Academic Press, San
Diego, Calif. (1990). For instance, any of a wide variety of
expression control sequences that control the expression of a DNA
sequence when operatively linked to it may be used in these vectors
to express DNA sequences encoding an ActRIIB polypeptide. Such
useful expression control sequences, include, for example, the
early and late promoters of SV40, tet promoter, adenovirus or
cytomegalovirus immediate early promoter, RSV promoters, the lac
system, the trp system, the TAC or TRC system, T7 promoter whose
expression is directed by T7 RNA polymerase, the major operator and
promoter regions of phage lambda, the control regions for fd coat
protein, the promoter for 3-phosphoglycerate kinase or other
glycolytic enzymes, the promoters of acid phosphatase, e.g., PhoS,
the promoters of the yeast .alpha.-mating factors, the polyhedron
promoter of the baculovirus system and other sequences known to
control the expression of genes of prokaryotic or eukaryotic cells
or their viruses, and various combinations thereof. It should be
understood that the design of the expression vector may depend on
such factors as the choice of the host cell to be transformed
and/or the type of protein desired to be expressed. Moreover, the
vector's copy number, the ability to control that copy number and
the expression of any other protein encoded by the vector, such as
antibiotic markers, should also be considered.
[0107] A recombinant nucleic acid of the invention can be produced
by ligating the cloned gene, or a portion thereof, into a vector
suitable for expression in either prokaryotic cells, eukaryotic
cells (yeast, avian, insect or mammalian), or both. Expression
vehicles for production of a recombinant ActRIIB polypeptide
include plasmids and other vectors. For instance, suitable vectors
include plasmids of the types: pBR322-derived plasmids,
pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived
plasmids and pUC-derived plasmids for expression in prokaryotic
cells, such as E. coli.
[0108] Some mammalian expression vectors contain both prokaryotic
sequences to facilitate the propagation of the vector in bacteria,
and one or more eukaryotic transcription units that are expressed
in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt,
pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg
derived vectors are examples of mammalian expression vectors
suitable for transfection of eukaryotic cells. Some of these
vectors are modified with sequences from bacterial plasmids, such
as pBR322, to facilitate replication and drug resistance selection
in both prokaryotic and eukaryotic cells. Alternatively,
derivatives of viruses such as the bovine papilloma virus (BPV-1),
or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used
for transient expression of proteins in eukaryotic cells. Examples
of other viral (including retroviral) expression systems can be
found below in the description of gene therapy delivery systems.
The various methods employed in the preparation of the plasmids and
in transformation of host organisms are well known in the art. For
other suitable expression systems for both prokaryotic and
eukaryotic cells, as well as general recombinant procedures, see
Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook,
Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989)
Chapters 16 and 17. In some instances, it may be desirable to
express the recombinant polypeptides by the use of a baculovirus
expression system. Examples of such baculovirus expression systems
include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),
pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived
vectors (such as the -gal containing pBlueBac III).
[0109] In a preferred embodiment, a vector will be designed for
production of the subject ActRIIB polypeptides in CHO cells, such
as a Pcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4
vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo vectors
(Promega, Madison, Wis.). As will be apparent, the subject gene
constructs can be used to cause expression of the subject ActRIIB
polypeptides in cells propagated in culture, e.g., to produce
proteins, including fusion proteins or variant proteins, for
purification.
[0110] This invention also pertains to a host cell transfected with
a recombinant gene including a coding sequence (e.g., SEQ ID NO: 4
or 6) for one or more of the subject ActRIIB polypeptide. The host
cell may be any prokaryotic or eukaryotic cell. For example, an
ActRIIB polypeptide of the invention may be expressed in bacterial
cells such as E. coli, insect cells (e.g., using a baculovirus
expression system), yeast, or mammalian cells. Other suitable host
cells are known to those skilled in the art.
[0111] Accordingly, the present invention further pertains to
methods of producing the subject ActRIIB polypeptides. For example,
a host cell transfected with an expression vector encoding an
ActRIIB polypeptide can be cultured under appropriate conditions to
allow expression of the ActRIIB polypeptide to occur. The ActRIIB
polypeptide may be secreted and isolated from a mixture of cells
and medium containing the ActRIIB polypeptide. Alternatively, the
ActRIIB polypeptide may be retained cytoplasmically or in a
membrane fraction and the cells harvested, lysed and the protein
isolated. A cell culture includes host cells, media and other
byproducts. Suitable media for cell culture are well known in the
art. The subject ActRIIB polypeptides can be isolated from cell
culture medium, host cells, or both, using techniques known in the
art for purifying proteins, including ion-exchange chromatography,
gel filtration chromatography, ultrafiltration, electrophoresis,
and immunoaffinity purification with antibodies specific for
particular epitopes of the ActRIIB polypeptides. In a preferred
embodiment, the ActRIIB polypeptide is a fusion protein containing
a domain which facilitates its purification.
[0112] In another embodiment, a fusion gene coding for a
purification leader sequence, such as a poly-(His)/enterokinase
cleavage site sequence at the N-terminus of the desired portion of
the recombinant ActRIIB polypeptide, can allow purification of the
expressed fusion protein by affinity chromatography using a
Ni.sup.2+ metal resin. The purification leader sequence can then be
subsequently removed by treatment with enterokinase to provide the
purified ActRIIB polypeptide (e.g., see Hochuli et al., (1987) J.
Chromatography 411:177; and Janknecht et al., PNAS USA
88:8972).
[0113] Techniques for making fusion genes are well known.
Essentially, the joining of various DNA fragments coding for
different polypeptide sequences is performed in accordance with
conventional techniques, employing blunt-ended or stagger-ended
termini for ligation, restriction enzyme digestion to provide for
appropriate termini, filling-in of cohesive ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic ligation. In another embodiment, the fusion gene can be
synthesized by conventional techniques including automated DNA
synthesizers. Alternatively, PCR amplification of gene fragments
can be carried out using anchor primers which give rise to
complementary overhangs between two consecutive gene fragments
which can subsequently be annealed to generate a chimeric gene
sequence (see, for example, Current Protocols in Molecular Biology,
eds. Ausubel et al., John Wiley & Sons: 1992).
5. Exemplary Therapeutic Uses
[0114] In certain embodiments, compositions (e.g., ActRIIB
polypeptides) of the present invention can be used for treating or
preventing a disease or condition that is associated with abnormal
activity of an ActRIIB polypeptide and/or an ActRIIB ligand (e.g.,
GDF8). These diseases, disorders or conditions are generally
referred to herein as "ActRIIB-associated conditions." In certain
embodiments, the present invention provides methods of treating or
preventing an individual in need thereof through administering to
the individual a therapeutically effective amount of an ActRIIB
polypeptide as described above. These methods are particularly
aimed at therapeutic and prophylactic treatments of animals, and
more particularly, humans.
[0115] As used herein, a therapeutic that "prevents" a disorder or
condition refers to a compound that, in a statistical sample,
reduces the occurrence of the disorder or condition in the treated
sample relative to an untreated control sample, or delays the onset
or reduces the severity of one or more symptoms of the disorder or
condition relative to the untreated control sample. The term
"treating" as used herein includes prophylaxis of the named
condition or amelioration or elimination of the condition once it
has been established.
[0116] ActRIIB/ActRIIB ligand complexes play essential roles in
tissue growth as well as early developmental processes such as the
correct formation of various structures or in one or more
post-developmental capacities including sexual development,
pituitary hormone production, and creation of bone and cartilage.
Thus, ActRIIB-associated conditions include abnormal tissue growth
and developmental defects. In addition, ActRIIB-associated
conditions include, but are not limited to, disorders of cell
growth and differentiation such as inflammation, allergy,
autoimmune diseases, infectious diseases, and tumors.
[0117] Exemplary conditions for treatment include neuromuscular
disorders (e.g., muscular dystrophy and muscle atrophy), congestive
obstructive pulmonary disease (and muscle wasting associated with
COPD), muscle wasting syndrome, sarcopenia, cachexia, adipose
tissue disorders (e.g., obesity), type 2 diabetes, and bone
degenerative disease (e.g., osteoporosis). Other exemplary
conditions include musculodegenerative and neuromuscular disorders,
tissue repair (e.g., wound healing), neurodegenerative diseases
(e.g., amyotrophic lateral sclerosis), immunologic disorders (e.g.,
disorders related to abnormal proliferation or function of
lymphocytes), and obesity or disorders related to abnormal
proliferation of adipocytes.
[0118] In certain embodiments, compositions (e.g., ActRIIB-Fc
polypeptides) of the invention are used as part of a treatment for
a muscular dystrophy. The term "muscular dystrophy" refers to a
group of degenerative muscle diseases characterized by gradual
weakening and deterioration of skeletal muscles and sometimes the
heart and respiratory muscles. Muscular dystrophies are genetic
disorders characterized by progressive muscle wasting and weakness
that begin with microscopic changes in the muscle. As muscles
degenerate over time, the person's muscle strength declines.
Exemplary muscular dystrophies that can be treated with a regimen
including the subject ActRIIB polypeptides include: Duchenne
Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD),
Emery-Dreifuss Muscular Dystrophy (EDMD), Limb-Girdle Muscular
Dystrophy (LGMD), Facioscapulohumeral Muscular Dystrophy (FSH or
FSHD) (also known as Landouzy-Dejerine), Myotonic Dystrophy (MMD)
(also known as Steinert's Disease), Oculopharyngeal Muscular
Dystrophy (OPMD), Distal Muscular Dystrophy (DD), Congenital
Muscular Dystrophy (CMD).
[0119] Duchenne Muscular Dystrophy (DMD) was first described by the
French neurologist Guillaume Benjamin Amand Duchenne in the 1860s.
Becker Muscular Dystrophy (BMD) is named after the German doctor
Peter Emil Becker, who first described this variant of DMD in the
1950s. DMD is one of the most frequent inherited diseases in males,
affecting one in 3,500 boys. DMD occurs when the dystrophin gene,
located on the short arm of the X chromosome, is broken. Since
males only carry one copy of the X chromosome, they only have one
copy of the dystrophin gene. Without the dystrophin protein, muscle
is easily damaged during cycles of contraction and relaxation.
While early in the disease muscle compensates by regeneration,
later on muscle progenitor cells cannot keep up with the ongoing
damage and healthy muscle is replaced by non-functional fibro-fatty
tissue.
[0120] BMD results from different mutations in the dystrophin gene.
BMD patients have some dystrophin, but it is either insufficient in
quantity or poor in quality. Having some dystrophin protects the
muscles of those with BMD from degenerating as badly or as quickly
as those of people with DMD.
[0121] For example, recent researches demonstrate that blocking or
eliminating function of GDF8 (an ActRIIB ligand) in vivo can
effectively treat at least certain symptoms in DMD and BMD
patients. Thus, the subject ActRIIB polypeptides may act as GDF8
inhibitors (antagonists), and constitute an alternative means of
blocking the functions of GDF8 and/or ActRIIB in vivo in DMD and
BMD patients. This approach is confirmed and supported by the data
shown herein, whereby an ActRIIB-Fc protein was shown to increase
muscle mass in a mouse model of muscular dystrophy.
[0122] Similarly, the subject ActRIIB polypeptides provide an
effective means to increase muscle mass in other disease conditions
that are in need of muscle growth. For example, ALS, also called
Lou Gehrig's disease (motor neuron disease) is a chronic,
incurable, and unstoppable CNS disorder that attacks the motor
neurons, components of the CNS that connect the brain to the
skeletal muscles. In ALS, the motor neurons deteriorate and
eventually die, and though a person's brain normally remains fully
functioning and alert, the command to move never reaches the
muscles. Most people who get ALS are between 40 and 70 years old.
The first motor neurons that weaken are those leading to the arms
or legs. Those with ALS may have trouble walking, they may drop
things, fall, slur their speech, and laugh or cry uncontrollably.
Eventually the muscles in the limbs begin to atrophy from disuse.
This muscle weakness will become debilitating and a person will
need a wheel chair or become unable to function out of bed. Most
ALS patients die from respiratory failure or from complications of
ventilator assistance like pneumonia, 3-5 years from disease onset.
This approach is confirmed and supported by the data shown herein,
whereby an ActRIIB-Fc protein was shown to improve the appearance,
muscle mass and lifespan of a mouse model of ALS.
[0123] ActRIIB polypeptide-induced increased muscle mass might also
benefit those suffering from muscle wasting diseases.
Gonzalez-Cadavid et al. (supra) reported that that GDF8 expression
correlates inversely with fat-free mass in humans and that
increased expression of the GDF8 gene is associated with weight
loss in men with AIDS wasting syndrome. By inhibiting the function
of GDF8 in AIDS patients, at least certain symptoms of AIDS may be
alleviated, if not completely eliminated, thus significantly
improving quality of life in AIDS patients.
[0124] Since loss of GDF8 (an ActRIIB ligand) function is also
associated with fat loss without diminution of nutrient intake
(Zimmers et al., supra; McPherron and Lee, supra), the subject
ActRIIB polypeptides may further be used as a therapeutic agent for
slowing or preventing the development of obesity and type II
diabetes. This approach is confirmed and supported by the data
shown herein, whereby an ActRIIB-Fc protein was shown to improve
metabolic status in obese mice.
[0125] In certain embodiments, compositions (e.g., ActRIIB
polypeptides) of the invention are used as part of a treatment for
metabolic syndrome (also known as syndrome X and insulin resistance
syndrome), which is a combination of disorders and risk factors
that increase the risk of developing cardiovascular disease and
diabetes mellitus type II. Most patients are older, obese,
sedentary, and have some degree of insulin resistance. Central
(abdominal or visceral) adiposity is a significant feature of the
syndrome.
[0126] In related embodiments, ActRIIB polypeptides and other
compositions of the invention can be used as part of a treatment
for diabetes mellitus type II (also known as non-insulin-dependent
diabetes mellitus or adult-onset diabetes), which is characterized
by elevated blood glucose in the context of insulin resistance and
relative insulin deficiency. Complex and multifactorial metabolic
changes in diabetes often lead to damage and functional impairment
of many organs, most importantly the cardiovascular system.
Diabetes mellitus type II is often associated with obesity
(abdominal or visceral adiposity), hypertension, elevated
cholesterol, and metabolic syndrome. Important risk factors for
diabetes mellitus type II include aging, high-fat diets, and a
sedentary lifestyle.
[0127] In other related embodiments, ActRIIB polypeptides and other
compositions of the invention can be used as part of a treatment
for atherosclerosis, a chronic inflammatory condition in which
artery walls thicken due to the accumulation of fatty deposits,
often referred to as plaques. Risk factors for atherosclerosis
include aging, diabetes mellitus, dyslipoproteinemia, obesity
(abdominal or visceral adiposity), and a sedentary lifestyle.
[0128] ActRIIB polypeptides can also be used for lipodystrophic
disorders, which tend to be associated with metabolic syndrome.
Severe insulin resistance can result from both genetic and acquired
forms of lipodystrophy, including in the latter case human
immunodeficiency virus (HIV)-related lipodystrophy in patients
treated with antiretroviral therapy.
[0129] The cancer anorexia-cachexia syndrome is among the most
debilitating and life-threatening aspects of cancer. Progressive
weight loss in cancer anorexia-cachexia syndrome is a common
feature of many types of cancer and is responsible not only for a
poor quality of life and poor response to chemotherapy, but also a
shorter survival time than is found in patients with comparable
tumors without weight loss. Associated with anorexia, fat and
muscle tissue wasting, psychological distress, and a lower quality
of life, cachexia arises from a complex interaction between the
cancer and the host. It is one of the most common causes of death
among cancer patients and is present in 80% at death. It is a
complex example of metabolic chaos effecting protein, carbohydrate,
and fat metabolism. Tumors produce both direct and indirect
abnormalities, resulting in anorexia and weight loss. Currently,
there is no treatment to control or reverse the process. Cancer
anorexia-cachexia syndrome affects cytokine production, release of
lipid-mobilizing and proteolysis-inducing factors, and alterations
in intermediary metabolism. Although anorexia is common, a
decreased food intake alone is unable to account for the changes in
body composition seen in cancer patients, and increasing nutrient
intake is unable to reverse the wasting syndrome. Cachexia should
be suspected in patients with cancer if an involuntary weight loss
of greater than five percent of premorbid weight occurs within a
six-month period.
[0130] Since systemic overexpression of GDF8 in adult mice was
found to induce profound muscle and fat loss analogous to that seen
in human cachexia syndromes (Zimmers et al., supra), the subject
ActRIIB polypeptides as pharmaceutical compositions can be
beneficially used to prevent, treat, or alleviate the symptoms of
the cachexia syndrome, where muscle growth is desired.
[0131] In other embodiments, the present invention provides methods
of inducing bone and/or cartilage formation, preventing bone loss,
increasing bone mineralization or preventing the demineralization
of bone. For example, the subject ActRIIB polypeptides and
compounds identified in the present invention have application in
treating osteoporosis and the healing of bone fractures and
cartilage defects in humans and other animals. ActRIIB polypeptides
may be useful in patients that are diagnosed with subclinical low
bone density, as a protective measure against the development of
osteoporosis.
[0132] In one specific embodiment, methods and compositions of the
present invention may find medical utility in the healing of bone
fractures and cartilage defects in humans and other animals. The
subject methods and compositions may also have prophylactic use in
closed as well as open fracture reduction and also in the improved
fixation of artificial joints. De novo bone formation induced by an
osteogenic agent contributes to the repair of congenital,
trauma-induced, or oncologic resection induced craniofacial
defects, and also is useful in cosmetic plastic surgery. Further,
methods and compositions of the invention may be used in the
treatment of periodontal disease, and in other tooth repair
processes. In certain cases, the subject ActRIIB polypeptides may
provide an environment to attract bone-forming cells, stimulate
growth of bone-forming cells or induce differentiation of
progenitors of bone-forming cells. ActRIIB polypeptides of the
invention may also be useful in the treatment of osteoporosis.
Further, ActRIIB polypeptides may be used in cartilage defect
repair and prevention/reversal of osteoarthritis.
[0133] In another specific embodiment, the invention provides a
therapeutic method and composition for repairing fractures and
other conditions related to cartilage and/or bone defects or
periodontal diseases. The invention further provides therapeutic
methods and compositions for wound healing and tissue repair. The
types of wounds include, but are not limited to, burns, incisions
and ulcers. See e.g., PCT Publication No. WO84/01106. Such
compositions comprise a therapeutically effective amount of at
least one of the ActRIIB polypeptides of the invention in admixture
with a pharmaceutically acceptable vehicle, carrier or matrix.
[0134] In another specific embodiment, methods and compositions of
the invention can be applied to conditions causing bone loss such
as osteoporosis, hyperparathyroidism, Cushing's disease,
thyrotoxicosis, chronic diarrheal state or malabsorption, renal
tubular acidosis, chronic renal failure or anorexia nervosa. Many
people know that being female, having a low body weight, and
leading a sedentary lifestyle are risk factors for osteoporosis
(loss of bone mineral density, leading to fracture risk). However,
osteoporosis can also result from the long-term use of certain
medications. Osteoporosis resulting from drugs or another medical
condition is known as secondary osteoporosis. In a condition known
as Cushing's disease, the excess amount of cortisol produced by the
body results in osteoporosis and fractures. The most common
medications associated with secondary osteoporosis are the
corticosteroids, a class of drugs that act like cortisol, a hormone
produced naturally by the adrenal glands. Although adequate levels
of thyroid hormones (which are produced by the thyroid gland) are
needed for the development of the skeleton, excess thyroid hormone
can decrease bone mass over time. Antacids that contain aluminum
can lead to bone loss when taken in high doses by people with
kidney problems, particularly those undergoing dialysis. Other
medications that can cause secondary osteoporosis include phenytoin
(Dilantin) and barbiturates that are used to prevent seizures;
methotrexate (Rheumatrex, Immunex, Folex PFS), a drug for some
forms of arthritis, cancer, and immune disorders; cyclosporine
(Sandimmune, Neoral), a drug used to treat some autoimmune diseases
and to suppress the immune system in organ transplant patients;
luteinizing hormone-releasing hormone agonists (Lupron, Zoladex),
used to treat prostate cancer and endometriosis; heparin
(Calciparine, Liquaemin), an anticlotting medication; and
cholestyramine (Questran) and colestipol (Colestid), used to treat
high cholesterol. Gum disease causes bone loss because these
harmful bacteria in our mouths force our bodies to defend against
them. The bacteria produce toxins and enzymes under the gum-line,
causing a chronic infection.
[0135] In other embodiments, the present invention provides
compositions and methods for regulating body fat content in an
animal and for treating or preventing conditions related thereto,
and particularly, health-compromising conditions related thereto.
According to the present invention, to regulate (control) body
weight can refer to reducing or increasing body weight, reducing or
increasing the rate of weight gain, or increasing or reducing the
rate of weight loss, and also includes actively maintaining, or not
significantly changing body weight (e.g., against external or
internal influences which may otherwise increase or decrease body
weight). One embodiment of the present invention relates to
regulating body weight by administering to an animal (e.g., a
human) in need thereof an ActRIIB polypeptide.
[0136] In one specific embodiment, the present invention relates to
methods and compounds for reducing fat mass and/or reducing gain of
fat mass in an animal, and more particularly, for treating or
ameliorating obesity in patients at risk for or suffering from
obesity. In another specific embodiment, the present invention is
directed to methods and compounds for treating an animal that is
unable to gain or retain weight (e.g., an animal with a wasting
syndrome). Such methods are effective to increase body weight
and/or mass, or to reduce weight and/or mass loss, or to improve
conditions associated with or caused by undesirably low (e.g.,
unhealthy) body weight and/or mass.
7. Pharmaceutical Compositions
[0137] In certain embodiments, compounds (e.g., ActRIIB
polypeptides) of the present invention are formulated with a
pharmaceutically acceptable carrier. For example, an ActRIIB
polypeptide can be administered alone or as a component of a
pharmaceutical formulation (therapeutic composition). The subject
compounds may be formulated for administration in any convenient
way for use in human or veterinary medicine.
[0138] In certain embodiments, the therapeutic method of the
invention includes administering the composition topically,
systemically, or locally as an implant or device. When
administered, the therapeutic composition for use in this invention
is, of course, in a pyrogen-free, physiologically acceptable form.
Further, the composition may desirably be encapsulated or injected
in a viscous form for delivery to a target tissue site (e.g., bone,
cartilage, muscle, fat or neurons), for example, a site having a
tissue damage. Topical administration may be suitable for wound
healing and tissue repair. Therapeutically useful agents other than
the ActRIIB polypeptides which may also optionally be included in
the composition as described above, may alternatively or
additionally, be administered simultaneously or sequentially with
the subject compounds (e.g., ActRIIB polypeptides) in the methods
of the invention.
[0139] In certain embodiments, compositions of the present
invention may include a matrix capable of delivering one or more
therapeutic compounds (e.g., ActRIIB polypeptides) to a target
tissue site, providing a structure for the developing tissue and
optimally capable of being resorbed into the body. For example, the
matrix may provide slow release of the ActRIIB polypeptides. Such
matrices may be formed of materials presently in use for other
implanted medical applications.
[0140] The choice of matrix material is based on biocompatibility,
biodegradability, mechanical properties, cosmetic appearance and
interface properties. The particular application of the subject
compositions will define the appropriate formulation. Potential
matrices for the compositions may be biodegradable and chemically
defined calcium sulfate, tricalciumphosphate, hydroxyapatite,
polylactic acid and polyanhydrides. Other potential materials are
biodegradable and biologically well defined, such as bone or dermal
collagen. Further matrices are comprised of pure proteins or
extracellular matrix components. Other potential matrices are
non-biodegradable and chemically defined, such as sintered
hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices
may be comprised of combinations of any of the above mentioned
types of material, such as polylactic acid and hydroxyapatite or
collagen and tricalciumphosphate. The bioceramics may be altered in
composition, such as in calcium-aluminate-phosphate and processing
to alter pore size, particle size, particle shape, and
biodegradability.
[0141] In certain embodiments, methods of the invention can be
administered for orally, e.g., in the form of capsules, cachets,
pills, tablets, lozenges (using a flavored basis, usually sucrose
and acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of an agent as an
active ingredient. An agent may also be administered as a bolus,
electuary or paste.
[0142] In solid dosage forms for oral administration (capsules,
tablets, pills, dragees, powders, granules, and the like), one or
more therapeutic compounds of the present invention may be mixed
with one or more pharmaceutically acceptable carriers, such as
sodium citrate or dicalcium phosphate, and/or any of the following:
(1) fillers or extenders, such as starches, lactose, sucrose,
glucose, mannitol, and/or silicic acid; (2) binders, such as, for
example, carboxymethylcellulose, alginates, gelatin, polyvinyl
pyrrolidone, sucrose, and/or acacia; (3) humectants, such as
glycerol; (4) disintegrating agents, such as agar-agar, calcium
carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate; (5) solution retarding agents,
such as paraffin; (6) absorption accelerators, such as quaternary
ammonium compounds; (7) wetting agents, such as, for example, cetyl
alcohol and glycerol monostearate; (8) absorbents, such as kaolin
and bentonite clay; (9) lubricants, such a talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof; and (10) coloring agents. In the
case of capsules, tablets and pills, the pharmaceutical
compositions may also comprise buffering agents. Solid compositions
of a similar type may also be employed as fillers in soft and
hard-filled gelatin capsules using such excipients as lactose or
milk sugars, as well as high molecular weight polyethylene glycols
and the like.
[0143] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups, and elixirs. In addition to the active
ingredient, the liquid dosage forms may contain inert diluents
commonly used in the art, such as water or other solvents,
solubilizing agents and emulsifiers, such as ethyl alcohol,
isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,
benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring, coloring, perfuming, and
preservative agents.
[0144] Suspensions, in addition to the active compounds, may
contain suspending agents such as ethoxylated isostearyl alcohols,
polyoxyethylene sorbitol, and sorbitan esters, microcrystalline
cellulose, aluminum metahydroxide, bentonite, agar-agar and
tragacanth, and mixtures thereof.
[0145] Certain compositions disclosed herein may be administered
topically, either to skin or to mucosal membranes. The topical
formulations may further include one or more of the wide variety of
agents known to be effective as skin or stratum corneum penetration
enhancers. Examples of these are 2-pyrrolidone,
N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide,
propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide,
and azone. Additional agents may further be included to make the
formulation cosmetically acceptable. Examples of these are fats,
waxes, oils, dyes, fragrances, preservatives, stabilizers, and
surface active agents. Keratolytic agents such as those known in
the art may also be included. Examples are salicylic acid and
sulfur.
[0146] Dosage forms for the topical or transdermal administration
include powders, sprays, ointments, pastes, creams, lotions, gels,
solutions, patches, and inhalants. The active compound may be mixed
under sterile conditions with a pharmaceutically acceptable
carrier, and with any preservatives, buffers, or propellants which
may be required. The ointments, pastes, creams and gels may
contain, in addition to a subject compound of the invention (e.g.,
an ActRIIB polypeptide), excipients, such as animal and vegetable
fats, oils, waxes, paraffins, starch, tragacanth, cellulose
derivatives, polyethylene glycols, silicones, bentonites, silicic
acid, talc and zinc oxide, or mixtures thereof.
[0147] Powders and sprays can contain, in addition to a subject
compound, excipients such as lactose, talc, silicic acid, aluminum
hydroxide, calcium silicates, and polyamide powder, or mixtures of
these substances. Sprays can additionally contain customary
propellants, such as chlorofluorohydrocarbons and volatile
unsubstituted hydrocarbons, such as butane and propane.
[0148] In certain embodiments, pharmaceutical compositions suitable
for parenteral administration may comprise one or more ActRIIB
polypeptides in combination with one or more pharmaceutically
acceptable sterile isotonic aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions, or sterile powders which may
be reconstituted into sterile injectable solutions or dispersions
just prior to use, which may contain antioxidants, buffers,
bacteriostats, solutes which render the formulation isotonic with
the blood of the intended recipient or suspending or thickening
agents. Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0149] The compositions of the invention may also contain
adjuvants, such as preservatives, wetting agents, emulsifying
agents and dispersing agents. Prevention of the action of
microorganisms may be ensured by the inclusion of various
antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption,
such as aluminum monostearate and gelatin.
[0150] It is understood that the dosage regimen will be determined
by the attending physician considering various factors which modify
the action of the subject compounds of the invention (e.g., ActRIIB
polypeptides). The various factors will depend upon the disease to
be treated.
[0151] In certain embodiments, the present invention also provides
gene therapy for the in vivo production of ActRIIB polypeptides or
other compounds disclosed herein. Such therapy would achieve its
therapeutic effect by introduction of the ActRIIB polynucleotide
sequences into cells or tissues having the disorders as listed
above. Delivery of ActRIIB polynucleotide sequences can be achieved
using a recombinant expression vector such as a chimeric virus or a
colloidal dispersion system. Preferred for therapeutic delivery of
ActRIIB polynucleotide sequences is the use of targeted
liposomes.
[0152] Various viral vectors which can be utilized for gene therapy
as taught herein include adenovirus, herpes virus, vaccinia, or,
preferably, an RNA virus such as a retrovirus. Preferably, the
retroviral vector is a derivative of a murine or avian retrovirus.
Examples of retroviral vectors in which a single foreign gene can
be inserted include, but are not limited to: Moloney murine
leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),
murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A
number of additional retroviral vectors can incorporate multiple
genes. All of these vectors can transfer or incorporate a gene for
a selectable marker so that transduced cells can be identified and
generated. Retroviral vectors can be made target-specific by
attaching, for example, a sugar, a glycolipid, or a protein.
Preferred targeting is accomplished by using an antibody. Those of
skill in the art will recognize that specific polynucleotide
sequences can be inserted into the retroviral genome or attached to
a viral envelope to allow target specific delivery of the
retroviral vector containing the ActRIIB polynucleotide. In one
preferred embodiment, the vector is targeted to bone, cartilage,
muscle or neuron cells/tissues.
[0153] Alternatively, tissue culture cells can be directly
transfected with plasmids encoding the retroviral structural genes
gag, pol and env, by conventional calcium phosphate transfection.
These cells are then transfected with the vector plasmid containing
the genes of interest. The resulting cells release the retroviral
vector into the culture medium.
[0154] Another targeted delivery system for ActRIIB polynucleotides
is a colloidal dispersion system. Colloidal dispersion systems
include macromolecule complexes, nanocapsules, microspheres, beads,
and lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, and liposomes. The preferred colloidal system of
this invention is a liposome. Liposomes are artificial membrane
vesicles which are useful as delivery vehicles in vitro and in
vivo. RNA, DNA and intact virions can be encapsulated within the
aqueous interior and be delivered to cells in a biologically active
form (see e.g., Fraley, et al., Trends Biochem. Sci., 6:77, 1981).
Methods for efficient gene transfer using a liposome vehicle, are
known in the art, see e.g., Mannino, et al., Biotechniques, 6:682,
1988. The composition of the liposome is usually a combination of
phospholipids, usually in combination with steroids, especially
cholesterol. Other phospholipids or other lipids may also be used.
The physical characteristics of liposomes depend on pH, ionic
strength, and the presence of divalent cations.
[0155] Examples of lipids useful in liposome production include
phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, and gangliosides. Illustrative
phospholipids include egg phosphatidylcholine,
dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.
The targeting of liposomes is also possible based on, for example,
organ-specificity, cell-specificity, and organelle-specificity and
is known in the art.
EXEMPLIFICATION
[0156] The invention now being generally described, it will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain
embodiments and embodiments of the present invention, and are not
intended to limit the invention.
Example 1. Generation of ActRIIB(25-131)-hFc with Alternative
Nucleotide Sequences
[0157] To generate ActRIIB(25-131)-hFc, the human ActRIIB
extracellular domain with N-terminal and C-terminal truncations
(residues 25-131 of the native protein) was fused N-terminally with
a TPA leader sequence substituted for the native ActRIIB leader and
C-terminally with a human Fc domain via a minimal linker (three
glycine residues) (FIG. 1). A nucleotide sequence encoding this
fusion protein is shown in FIG. 2. Applicants modified the codons
and found a variant nucleic acid encoding the ActRIIB(25-131)-hFc
protein that provided substantial improvement in the expression
levels of initial transformants (FIGS. 3A and 3B).
[0158] The mature protein has an amino acid sequence as follows
(N-terminus confirmed by N-terminal sequencing)(SEQ ID NO: 8):
TABLE-US-00004 ETRECIYYNA NWELERTNQS GLERCEGEQD KRLHCYASWR
NSSGTIELVK KGCWLDDFNC YDRQECVATE ENPQVYFCCC EGNFCNERFT HLPEAGGPEV
TYEPPPTGGG THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE
VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI
EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK
TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK
Amino acids 1-107 are derived from ActRIIB.
[0159] The expressed molecule was purified using a series of column
chromatography steps, including for example, three or more of the
following, in any order: Protein A chromatography, Q sepharose
chromatography, phenylsepharose chromatography, size exclusion
chromatography and cation exchange chromatography. The purification
could be completed with viral filtration and buffer exchange.
Example 2. High-Affinity Ligand Binding by ActRIIB(25-131)-hFc
[0160] Affinities of several ligands for ActRIIB(25-131)-hFc and
its full-length counterpart ActRIIB(20-134)-hFc were evaluated in
vitro with a Biacore.TM. instrument, and the results are summarized
in the table below. Kd values were obtained by steady-state
affinity fit due to very rapid association and dissociation of the
complex, which prevented accurate determination of k.sub.on and
k.sub.off. ActRIIB(25-131)-hFc bound activin A, activin B, and
GDF11 with high affinity. Intriguingly, ActRIIB(25-131)-hFc appears
to show a higher affinity for GDF3 than ActRIIB(20-134)-hFc (data
not shown).
Ligand Affinities of ActRIIB-hFc Forms:
TABLE-US-00005 [0161] Activin A Activin B GDF11 Fusion Construct
(e-11) (e-11) (e-11) ActRIIB(20-134)-hFc 1.6 1.2 3.6
ActRIIB(25-131)-hFc 1.8 1.2 3.1
Example 3. ActRIIB(25-131)-hFc Increases Muscle Mass and Strength
In Vivo
[0162] Applicants investigated the ability of ActRIIB(25-131)-hFc
to increase muscle mass and strength in the mouse. Male mice (n=10
per group) were treated subcutaneously twice per week with vehicle
(Tris-buffered saline) or one of five doses of ActRIIB(25-131)-hFc.
Four weeks of treatment with ActRIIB(25-131)-hFc produced a clear
dose-dependent increase in lean tissue mass (FIG. 4), as determined
by whole-body nuclear magnetic resonance (NMR) scanning. Increased
muscle mass was confirmed at study termination for specific
muscles, including the pectoralis (FIG. 5), rectus femoris, and
gastrocnemius. Importantly, increased muscle mass was accompanied
by increased strength, as assessed by grip strength, compared to
vehicle (FIG. 6). These results provide compelling evidence that
ActRIIB(25-131)-hFc increases both muscle mass and muscle strength
in vivo.
Example 4. ActRIIB(25-131)-hFc Prevents Muscle Loss in Mouse Model
of Androgen Deprivation
[0163] Applicants investigated the ability of ActRIIB(25-131)-hFc
to prevent muscle loss in a mouse model of androgen deprivation, a
standard therapeutic intervention for advanced prostate cancer in
men. Male mice (n=10 per group) were orchidectomized (ORX) or
sham-operated and treated subcutaneously twice per week with TBS
vehicle, ActRIIB(25-131)-hFc at 10 mg/kg, or its full-length murine
counterpart ActRIIB(20-134)-mFc at 10 mg/kg. Lean tissue mass was
determined by whole-body NMR scan. ORX mice treated for four weeks
with either of the ActRIIB-Fc forms displayed an increase in lean
tissue mass from baseline, which was highly significant compared to
the decrease observed in ORX controls over that period (FIG. 7). An
analogous, highly significant increase was observed under
gonad-intact conditions for both ActRIIB-Fc forms compared to sham
controls (FIG. 7). These results demonstrate that
ActRIIB(25-131)-hFc can increase lean tissue mass (prevent muscle
loss) as effectively as its full-length counterpart
ActRIIB(20-134)-mFc in this androgen deprivation model.
Example 5. ActRIIB(25-131)-hFc Improves Body Composition in Mouse
Model of Diet-Induced Obesity
[0164] Applicants also investigated the ability of
ActRIIB(25-131)-hFc to increase muscle mass and reduce fat mass in
a mouse model of diet-induced obesity. Male mice (n=10 per group)
were fed either a standard chow diet or a high fat diet and treated
intraperitoneally twice per week with TBS vehicle or
ActRIIB(25-131)-hFc at 10 mg/kg. Lean tissue mass and fat mass were
determined by whole-body NMR scan. Treatment of mice on the high
fat diet with ActRIIB(25-131)-hFc for four weeks resulted in more
than a 25% increase in lean tissue mass as compared to a 2%
increase with vehicle treatment (FIG. 8). Similar results were
obtained in mice on the control diet with ActRIIB(25-131)-hFc as
compared to vehicle (FIG. 8). Moreover, continued treatment was
found to improve adiposity. Compared to vehicle,
ActRIIB(25-131)-hFc treatment for 12 weeks reduced fat mass by
approximately half in mice on the high fat diet as well as in those
on the control diet (FIG. 9).
[0165] Taken together, these data demonstrate that
ActRIIB(25-131)-hFc can be used to improve body composition in vivo
under a variety of conditions, including androgen deprivation and
high fat intake.
Example 6: ActRIIB(25-131)-hFc Normalizes Serum Lipids, Insulin,
and Adiponectin in Mouse Model of Diet-Induced Obesity
[0166] Applicants investigated the effects of ActRIIB(25-131)-hFc
on serum concentrations of clinically important lipids, insulin,
adiponectin, and on other metabolic endpoints in male mice fed a
high-fat diet. Ten-week-old C57BL/6 mice were weight-matched and
treated with ActRIIB(25-131)-hFc (n=10) or Tris-buffered-saline
(TBS) vehicle (n=7) twice per week at 10 mg/kg, s.c., for 60 days.
During this period, mice had unlimited access to a diet containing
58% fat instead of the standard chow containing 4.5% fat.
[0167] ActRIIB(25-131)-hFc treatment caused a constellation of
noteworthy metabolic effects. In mice fed a high-fat diet,
ActRIIB(25-131)-hFc reduced the pathologically elevated serum
concentrations of triglycerides, free fatty acids, high-density
lipoprotein (HDL), and low-density lipoprotein (LDL) (FIG. 10-13),
in most cases normalizing these parameters to levels observed in
mice fed a standard diet. Importantly, ActRIIB(25-131)-hFc
treatment also normalized insulin concentrations in high-fat-diet
mice (FIG. 14) and increased concentrations of adiponectin
significantly above even those in mice fed a standard diet (FIG.
15). Adiponectin is a key biomarker of body composition, as
circulating adiponectin levels are known to vary inversely with fat
mass/obesity, and adiponectin enhances insulin sensitivity in
target tissues. ActRIIB(25-131)-hFc also reduced serum
concentrations of leptin, another major indicator of adipocyte
status, by nearly 50% (P<0.05). Finally, the aforementioned
effects were accompanied by beneficial changes in body composition,
as determined by nuclear magnetic resonance (NMR) at baseline and
Day 48. Under high-fat dietary conditions, total fat mass in
vehicle-treated controls tripled during this 48-day period, and
ActRIIB(25-131)-hFc treatment cut this increase by nearly 40%. By
Day 48, total fat mass was 27% of body weight in ActRIIB-Fc-treated
mice vs. 39% in control mice, whereas lean tissue mass was 59% of
body weight in ActRIIB(25-131)-hFc-treated mice vs. 55% in control
mice. Thus, the net result was a healthier body composition under
conditions of high-fat diet.
[0168] For the foregoing serum parameters, ActRIIB(25-131)-hFc
consistently outperformed ActRIIB(20-134)-hFc, which was also
evaluated in this same study. Thus, ActRIIB(25-131)-hFc improved
triglyceride levels nearly 6 times as much, FFA levels nearly twice
as much, HDL levels nearly 4 times as much, insulin levels more
than twice as much, and adiponectin levels nearly 1.5 times as much
as ActRIIB(20-134)-hFc did at the same dose.
Example 7: ActRIIB(25-131)-hFc Induces Thermogenic Properties in
White Fat in Mouse Model of Diet-Induced Obesity
[0169] In the study described above (Example 6), Applicants also
investigated effects of ActRIIB(25-131)-hFc on thermogenic
properties of white adipose tissue. Under high-fat dietary
conditions, ActRIIB(25-131)-hFc treatment triggered histological
changes and a gene expression profile in white adipose tissue that
were consistent with thermogenic capability. As shown in FIG. 16,
histological examination of epididymal white fat indicated that
ActRIIB(25-131)-hFc reduced lipid droplet size and caused formation
of clusters of multilocular adipocytes that are a hallmark of brown
fat. Moreover, immunohistochemical analysis of this tissue revealed
widespread cytoplasmic induction of UCP1 in both multilocular and
unilocular adipocytes as a result of ActRIIB(25-131)-hFc treatment
(FIG. 16).
[0170] Accompanying these histological changes were significant
changes in the expression of key thermogenic and metabolic
regulatory genes in epididymal white fat, as determined by
quantitative RT-PCR (reverse transcription polymerase chain
reaction). In mice on the high-fat diet, ActRIIB(25-131)-hFc
treatment increased UCP1 mRNA levels more than 60-fold compared to
vehicle (FIG. 17), a particularly impressive change since this
strain of mouse displays severely blunted induction of UCP1 and
brown adipocytes within key white fat depots compared to other
mouse strains (Guerra et al., 1998, J Clin Invest 102:412-420; Xue
et al., 2007, J Lipid Res 48:41-51). In addition,
ActRIIB(25-131)-hFc treatment increased levels of mRNA encoding the
sirtuin SIRT-1 (silent information regulator two, homolog 1), an
energy-sensitive master regulator (deacetylase) that protects
against metabolic damage induced by a high-fat diet (Pfluger et
al., 2008, Proc Natl Acad Sci USA 105:9793-9798) and is implicated
as an important control of fatty acid mobilization (Rodgers et al.,
2008, FEBS Lett 582:46-53). Significantly, ActRIIB(25-131)-hFc
treatment also increased levels of mRNA encoding PGC-la (peroxisome
proliferator-activated receptor gamma coactivator-la), a
well-documented target of SIRT-1 that, in turn, controls expression
of many genes necessary for mitochondrial biogenesis and
thermogenic capability in brown adiopose tissue (Uldry et al.,
2006, Cell Metab, 3:333-341). Notably, forced expression of PGC-la
in white adipocytes has been shown to induce a thermogenic program
of gene expression, including UCP1, closely resembling that in
brown adipocytes (Hansen et al., 2006, Biochem J 398:153-168). In
the present study, ActRIIB(25-131)-hFc restored PGC-la gene
expression in white adipose tissue under high-fat dietary
conditions to levels indistinguishable from those in mice fed the
standard diet.
[0171] Additional changes associated with treatment constitute a
prominent link between the altered expression profile in white
adipose tissue and beneficial hormonal and metabolic effects. Thus,
in epididymal white fat, ActRIIB(25-131)-hFc increased levels of
mRNA encoding Foxo-1 (forkhead box-containing, protein 0
subfamily-1), a transcription factor that is both a target of
SIRT-1 and a key inducer of adiponectin expression (Qiao et al.,
2006, J Biol Chem 281:39915-39924). Consistent with Foxo-1 mRNA
induction, ActRIIB(25-131)-hFc treatment raised levels of
adiponectin mRNA in white fat (FIG. 18), which helps to account for
increased circulating levels of adiponectin (FIG. 15, Example 6),
enhanced insulin sensitivity in target tissues, and normalized
insulin concentrations (FIG. 14, Example 6) in these animals. In
summary, ActRIIB(25-131)-hFc treatment under high-fat dietary
conditions resulted in 1) histological changes and a gene
expression profile in white adipose tissue that were consistent
with thermogenic capability and 2) beneficial changes in a wide
range of hormonal and metabolic parameters.
Example 8: Effects of ActRIIB(25-131)-hFc on Liver and Muscle in
Mouse Model of Diet-Induced Obesity
[0172] Nonalcoholic fatty liver disease (NAFLD) is a spectrum of
increasingly common hepatic disorders widely considered to be the
hepatic manifestation of metabolic syndrome and characterized by
fat accumulation in the liver (steatosis), often with deleterious
effects. A subset of NAFLD patients develop an inflammatory
condition referred to as nonalcoholic steatohepatitis (NASH), which
can progress further to hepatic fibrosis, cirrhosis, and
hepatocellular carcinoma (Perlemuter et al., 2007, Nat Clin Pract
Endocrinol Metab 3:458-469). In the study described above (Examples
6-7), Applicants investigated whether ActRIIB(25-131)-hFc could
inhibit hepatic steatosis associated with a high-fat diet. At study
completion, hepatic tissue of mice fed the high-fat diet displayed
large numbers of densely packed lipid droplets, as assessed by
staining with Oil Red O, whereas mice fed the standard diet showed
no evidence of hepatic lipid deposits (FIG. 19). Treatment with
ActRIIB(25-131)-hFc almost completely reversed hepatic lipid
deposition and normalized the appearance of hepatic tissue despite
the high-fat diet. Thus, ActRIIB(25-131)-hFc was an effective
inhibitor of hepatic steatosis caused by high-fat diet.
[0173] ActRIIB(25-131)-hFc treatment also increased muscle mass in
this model of diet-induced obesity, consistent with findings in
other models (Examples 3-5). Specifically, ActRIIB(25-131)-hFc
increased pectoralis mass by more than 70% (P<0.001),
gastrocnemius mass by nearly 40% (P<0.001), and rectus femoris
mass by more than 25% (P<0.001) compared to high-fat diet
controls. These changes in muscle mass were accompanied by changes
in muscle gene expression, as determined in gastrocnemius tissue by
RT-PCR. Compared to high-fat diet controls, ActRIIB(25-131)-hFc
increased PGC-la mRNA levels and Foxo-1 mRNA levels by
approximately 50% each (P<0.05) in gastrocnemius.
Example 9: Effect of ActRIIB(25-131)-mFc on Visceral White Fat in
Mouse Model of Diet-Induced Obesity
[0174] Accumulation of visceral fat, as opposed to subcutaneous
fat, plays a critical role in the development of cardiovascular
disease and obesity-related disorders such as diabetes mellitus,
hyperlipidemia, hypertension, and metabolic syndrome (Matsuzawa et
al., 2006, FEBS Lett 580:2917-2921). Due to its location, visceral
(or intra-abdominal) fat has ready access to the liver via the
hepatic portal circulation, where it could influence metabolism,
promote insulin resistance, and cause steatosis. Therefore, in a
study similar to that described above (Examples 6-8), Applicants
investigated effects of the truncated variant ActRIIB(25-131)-mFc
on the quantities of visceral fat vs. abdominal subcutaneous fat
under high-fat dietary conditions. Nine-week-old C57BL/6 mice were
treated with ActRIIB(25-131)-mFc (n=20), at 10 mg/kg, s.c., or
Tris-buffered-saline (TBS) vehicle (n=10) twice per week for 60
days. Beginning 7 days before the start of dosing, mice had
unlimited access to a diet containing 58% fat instead of the
standard chow containing 4.5% fat. An additional group of mice
(n=10) maintained on the standard chow diet was also treated with
TBS vehicle and followed as a dietary control. Fat volumes were
determined by microCT for a subset of mice (n=4 per group) whose
percentages of total body fat, as determined by nuclear magnetic
resonance (NMR) analysis, were closest to group means (all mice
were subjected to NMR analysis).
[0175] Visceral fat and abdominal subcutaneous fat both varied
markedly in size with diet and ActRIIB(25-131)-mFc treatment.
Three-dimensional reconstruction of microCT images obtained partway
through the study (35 days) demonstrates that the depots of
visceral fat and subcutaneous fat both expanded as a result of the
high-fat diet and that ActRIIB(25-131)-mFc largely reversed those
increases (FIG. 20). When analyzed quantitatively at study
conclusion (60 days), the effect of ActRIIB(25-131)-mFc compared to
high-fat diet alone was highly significant for both visceral fat
(FIG. 21) and abdominal subcutaneous fat (FIG. 22).
Example 10: Effect of ActRIIB(25-131)-mFc on Brown Fat Properties
in Mouse Model of Diet-Induced Obesity
[0176] In the study described in Example 9, Applicants also
investigated effects of ActRIIB(25-131)-mFc on properties of
intrascapular brown fat depots under high-fat dietary conditions.
Compared to the standard diet, the high-fat diet produced several
changes in the interscapular depot of brown adipose tissue, and
ActRIIB(25-131)-mFc treatment either completely or largely reversed
each of these changes. Specifically, high-fat diet caused a
pronounced enlargement of the interscapular depot as well as
lightening of its color from red to pink (FIG. 23). This
diet-induced enlargement reflected a doubling of the mass (FIG. 24)
and a reduction in the density (FIG. 25) of brown fat depots. Depot
density was determined by micro-computed tomography (microCT) in
situ for a subset of mice (n=4 per group) whose percentages of
total body fat, as determined by nuclear magnetic resonance (NMR)
analysis, were closest to the group means (all mice were subjected
to NMR analysis). ActRIIB(25-131)-mFc treatment completely reversed
diet-induced changes in brown fat mass (FIG. 24) and density (FIG.
25), while largely reversing diet-induced changes in size and color
of the depot (FIG. 23). These results indicate that, under high-fat
dietary conditions, ActRIIB(25-131)-mFc largely or completely
restores properties likely to correlate with healthy brown fat
function and thus improves the quality of brown fat as it decreases
the overall size of brown fat depots.
Example 11: Effects of ActRIIB(25-131)-mFc on Muscle, Bone, Fat,
and Metabolic Hormones in Mouse Model of Aging
[0177] Body composition changes with aging in a predictable manner.
Normal age-dependent decline in muscle mass and strength, known as
sarcopenia, begins around age 30 and accelerates after age 60
(Stenholm et al, 2008, Curr Opin Clin Nutr Metab Care 11:693-700).
Bone mass and strength exhibit a similar decline with age, leading
to an increased risk of osteoporosis in the elderly. Whole-body fat
mass increases with age until around age 70, then declines in
absolute terms but remains a roughly constant proportion of total
body mass (Cartwright et al., 2007, Exp Gerontol 42:463-471). Based
on efficacy observed in other models and described herein,
Applicants investigated effects of ActRIIB(25-131)-mFc on muscle,
bone, fat, and insulin levels in a mouse model of aging.
Nineteen-month-old male C57BL/6 mice were given unlimited access to
a standard chow diet and treated with ActRIIB(25-131)-mFc (n=16),
at 10 mg/kg, s.c., or TBS vehicle (n=15) twice per week for 8
weeks. As a frame of reference, median life expectancy in this
mouse strain was previously found to be approximately 27 months
under standard dietary condtions (Turturro et al., 2002, J Gerontol
A Biol Sci Med Sci 57:B379-389).
[0178] ActRIIB(25-131)-mFc treatment generated a series of notable
changes in body composition and metabolic hormone effects in these
aged mice. As determined by whole-body NMR analysis, lean tissue
mass was essentially unchanged in control mice over the course of
the study, whereas in ActRIIB(24-131)-mFc-treated mice it increased
progressively to almost 20% above baseline by 7 weeks (FIG. 26).
Consistent with this whole-body effect, ActRIIB(25-131)-mFc also
significantly increased the mass of individual muscle groups,
including the pectoralis (increased 55%), rectus femoris (40%),
triceps (40%), and gastrocnemius (28%), compared to vehicle-treated
controls at 8 weeks. Importantly, ActRIIB(25-131)-mFc treatment
improved neuromuscular function, as determined by forelimb grip
strength testing according to an established protocol (FIG.
27).
[0179] Several bone-related parameters improved with
ActRIIB(25-131)-mFc treatment in aged mice. As determined by DEXA
analysis at baseline and 8-week time points, ActRIIB(25-131)-mFc
increased whole-body bone mineral density over the course of the
study, whereas controls were essentially unchanged (FIG. 28). In
addition, microCT analysis of the proximal tibia demonstrated that
ActRIIB(25-131)-mFc treatment for 8 weeks doubled the bone volume
fraction of the proximal tibia compared to controls
(P<0.01).
[0180] ActRIIB(25-131)-mFc exerted major effects on fat in aged
mice. As determined by NMR analysis at multiple time points, there
was a progressive decline in whole-body fat mass in vehicle-treated
controls over the course of the study (FIG. 29), consistent with
findings from humans in advanced old age. ActRIIB(25-131)-mFc
treatment accelerated this change, triggering a decrease of twice
the magnitude observed in controls (-44% vs.-19%, respectively)
(FIG. 29). By the terminal time point, ActRIIB(25-131)-mFc
significantly reduced the mass of the individual epididymal,
inguinal, and retroperitoneal depots of white fat by amounts
ranging from 48-54%. Interestingly, ActRIIB(25-131)-mFc treatment
also reduced the mass of the interscapular brown fat depots by
nearly 45% (P<0.05), similar to results obtained for this tissue
in the mouse model of dietary obesity (Example 10). Finally, as
determined by microCT analysis in a representative subset of mice
(n=4) from each group, ActRIIB(25-131)-mFc reduced the volume of
the visceral component of abdominal fat by 65% (P<0.01) and the
subcutaneous component of abdominal fat by 49% (P<0.01). Hence,
the critical visceral fat compartment was strongly targeted by
ActRIIB(25-131)-mFc in this model of aging.
[0181] ActRIIB(25-131)-mFc also produced beneficial changes in
important metabolic hormones in aged mice. Eight weeks of treatment
with ActRIIB(25-131)-mFc nearly doubled circulating adiponectin
concentrations (P<0.001) and reduced circulating insulin
concentrations by more than 40% (FIG. 30). An elevated fasting
insulin concentration (hyperinsulinemia) is a widely accepted
surrogate measure of insulin resistance (Weyer et al., 2000,
Diabetes 49:2094-2101), and increased adiponectin concentrations
are likely contributing to improved insulin sensitivity in the
present study. Glycated hemoglobin (A1C) concentrations were
significantly reduced by ActRIIB(25-131)-mFc in this study (FIG.
31), thereby providing additional evidence for improved glucose
regulation with ActRIIB(25-131)-mFc treatment in this model of
aging.
Example 12: Effect of ActRIIB(25-131)-hFc on Lean Tissue in Mouse
Model of Cancer Cachexia
[0182] Cachexia is undesired weight loss resulting from loss of
muscle and adipose tissue. Many tumors are associated with loss of
appetite and severe muscle loss, and patients exhibiting cachexia
have a poorer prognosis than non-cachectic patients. Since the
colon-cancer cell line CT26 induces profound cachexia in mice,
ActRIIB(25-131)-hFc was tested in this mouse model for potential
effects on xenograft-induced cachexia. Eight-week-old BALB/c mice
were injected subcutaneously with 10.sup.6 Colon-26 adenocarcinoma
(CT26) cells per mouse. Two weeks after tumor implantation,
treatment was initiated with ActRIIB(25-131)-hFc (n=15), at 10
mg/kg, s.c., or Tris-buffered-saline (TBS) vehicle (n=13) twice per
week. Additional groups of BALB/c mice did not receive CT26 cells
but were treated with ActRIIB(25-131)-hFc or vehicle as above.
Treatment with ActRIIB(25-131)-hFc resulted in a significant
increase in body weight that was maintained across the study. At 5
weeks post tumor implantation, vehicle-treated mice exhibited a 7%
loss of lean tissue mass from baseline, as determined by NMR
analysis, whereas mice treated with ActRIIB(25-131)-hFc exhibited a
27% increase in lean mass from baseline (FIG. 32). Fat mass did not
differ significantly between the groups. These results demonstrate
that ActRIIB(25-131)-hFc can alleviate cachexia in tumor-bearing
mice and could be an effective therapy for treating cachexia in
cancer patients.
[0183] Taken together, these data indicate that ActRIIB(25-131)-hFc
fusion protein can be used as an antagonist of signaling by
TGF-family ligands to reverse many pathological metabolic changes
associated with diet-induced obesity, and thereby, to treat
metabolic conditions exacerbated by high caloric intake. Moreover,
ActRIIB(25-131)-hFc can be used to treat pathologic metabolic
changes associated with aging or cancer cachexia.
INCORPORATION BY REFERENCE
[0184] All publications and patents mentioned herein are hereby
incorporated by reference in their entirety as if each individual
publication or patent was specifically and individually indicated
to be incorporated by reference.
[0185] While specific embodiments of the subject matter have been
discussed, the above specification is illustrative and not
restrictive. Many variations will become apparent to those skilled
in the art upon review of this specification and the claims below.
The full scope of the invention should be determined by reference
to the claims, along with their full scope of equivalents, and the
specification, along with such variations.
Sequence CWU 1
1
151512PRTHomo sapiens 1Met Thr Ala Pro Trp Val Ala Leu Ala Leu Leu
Trp Gly Ser Leu Trp1 5 10 15Pro Gly Ser Gly Arg Gly Glu Ala Glu Thr
Arg Glu Cys Ile Tyr Tyr 20 25 30Asn Ala Asn Trp Glu Leu Glu Arg Thr
Asn Gln Ser Gly Leu Glu Arg 35 40 45Cys Glu Gly Glu Gln Asp Lys Arg
Leu His Cys Tyr Ala Ser Trp Arg 50 55 60Asn Ser Ser Gly Thr Ile Glu
Leu Val Lys Lys Gly Cys Trp Leu Asp65 70 75 80Asp Phe Asn Cys Tyr
Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn 85 90 95Pro Gln Val Tyr
Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg 100 105 110Phe Thr
His Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro 115 120
125Pro Pro Thr Ala Pro Thr Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu
130 135 140Pro Ile Gly Gly Leu Ser Leu Ile Val Leu Leu Ala Phe Trp
Met Tyr145 150 155 160Arg His Arg Lys Pro Pro Tyr Gly His Val Asp
Ile His Glu Asp Pro 165 170 175Gly Pro Pro Pro Pro Ser Pro Leu Val
Gly Leu Lys Pro Leu Gln Leu 180 185 190Leu Glu Ile Lys Ala Arg Gly
Arg Phe Gly Cys Val Trp Lys Ala Gln 195 200 205Leu Met Asn Asp Phe
Val Ala Val Lys Ile Phe Pro Leu Gln Asp Lys 210 215 220Gln Ser Trp
Gln Ser Glu Arg Glu Ile Phe Ser Thr Pro Gly Met Lys225 230 235
240His Glu Asn Leu Leu Gln Phe Ile Ala Ala Glu Lys Arg Gly Ser Asn
245 250 255Leu Glu Val Glu Leu Trp Leu Ile Thr Ala Phe His Asp Lys
Gly Ser 260 265 270Leu Thr Asp Tyr Leu Lys Gly Asn Ile Ile Thr Trp
Asn Glu Leu Cys 275 280 285His Val Ala Glu Thr Met Ser Arg Gly Leu
Ser Tyr Leu His Glu Asp 290 295 300Val Pro Trp Cys Arg Gly Glu Gly
His Lys Pro Ser Ile Ala His Arg305 310 315 320Asp Phe Lys Ser Lys
Asn Val Leu Leu Lys Ser Asp Leu Thr Ala Val 325 330 335Leu Ala Asp
Phe Gly Leu Ala Val Arg Phe Glu Pro Gly Lys Pro Pro 340 345 350Gly
Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro Glu 355 360
365Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg Ile
370 375 380Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Val Ser
Arg Cys385 390 395 400Lys Ala Ala Asp Gly Pro Val Asp Glu Tyr Met
Leu Pro Phe Glu Glu 405 410 415Glu Ile Gly Gln His Pro Ser Leu Glu
Glu Leu Gln Glu Val Val Val 420 425 430His Lys Lys Met Arg Pro Thr
Ile Lys Asp His Trp Leu Lys His Pro 435 440 445Gly Leu Ala Gln Leu
Cys Val Thr Ile Glu Glu Cys Trp Asp His Asp 450 455 460Ala Glu Ala
Arg Leu Ser Ala Gly Cys Val Glu Glu Arg Val Ser Leu465 470 475
480Ile Arg Arg Ser Val Asn Gly Thr Thr Ser Asp Cys Leu Val Ser Leu
485 490 495Val Thr Ser Val Thr Asn Val Asp Leu Pro Pro Lys Glu Ser
Ser Ile 500 505 51021539DNAHomo sapiens 2atgacggcgc cctgggtggc
cctcgccctc ctctggggat cgctgtggcc cggctctggg 60cgtggggagg ctgagacacg
ggagtgcatc tactacaacg ccaactggga gctggagcgc 120accaaccaga
gcggcctgga gcgctgcgaa ggcgagcagg acaagcggct gcactgctac
180gcctcctggc gcaacagctc tggcaccatc gagctcgtga agaagggctg
ctggctagat 240gacttcaact gctacgatag gcaggagtgt gtggccactg
aggagaaccc ccaggtgtac 300ttctgctgct gtgaaggcaa cttctgcaac
gagcgcttca ctcatttgcc agaggctggg 360ggcccggaag tcacgtacga
gccacccccg acagccccca ccctgctcac ggtgctggcc 420tactcactgc
tgcccatcgg gggcctttcc ctcatcgtcc tgctggcctt ttggatgtac
480cggcatcgca agccccccta cggtcatgtg gacatccatg aggaccctgg
gcctccacca 540ccatcccctc tggtgggcct gaagccactg cagctgctgg
agatcaaggc tcgggggcgc 600tttggctgtg tctggaaggc ccagctcatg
aatgactttg tagctgtcaa gatcttccca 660ctccaggaca agcagtcgtg
gcagagtgaa cgggagatct tcagcacacc tggcatgaag 720cacgagaacc
tgctacagtt cattgctgcc gagaagcgag gctccaacct cgaagtagag
780ctgtggctca tcacggcctt ccatgacaag ggctccctca cggattacct
caaggggaac 840atcatcacat ggaacgaact gtgtcatgta gcagagacga
tgtcacgagg cctctcatac 900ctgcatgagg atgtgccctg gtgccgtggc
gagggccaca agccgtctat tgcccacagg 960gactttaaaa gtaagaatgt
attgctgaag agcgacctca cagccgtgct ggctgacttt 1020ggcttggctg
ttcgatttga gccagggaaa cctccagggg acacccacgg acaggtaggc
1080acgagacggt acatggctcc tgaggtgctc gagggagcca tcaacttcca
gagagatgcc 1140ttcctgcgca ttgacatgta tgccatgggg ttggtgctgt
gggagcttgt gtctcgctgc 1200aaggctgcag acggacccgt ggatgagtac
atgctgccct ttgaggaaga gattggccag 1260cacccttcgt tggaggagct
gcaggaggtg gtggtgcaca agaagatgag gcccaccatt 1320aaagatcact
ggttgaaaca cccgggcctg gcccagcttt gtgtgaccat cgaggagtgc
1380tgggaccatg atgcagaggc tcgcttgtcc gcgggctgtg tggaggagcg
ggtgtccctg 1440attcggaggt cggtcaacgg cactacctcg gactgtctcg
tttccctggt gacctctgtc 1500accaatgtgg acctgccccc taaagagtca
agcatctaa 15393360PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 3Met Asp Ala Met Lys Arg Gly Leu Cys
Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Pro Gly Ala
Ala Glu Thr Arg Glu Cys Ile Tyr 20 25 30Tyr Asn Ala Asn Trp Glu Leu
Glu Arg Thr Asn Gln Ser Gly Leu Glu 35 40 45Arg Cys Glu Gly Glu Gln
Asp Lys Arg Leu His Cys Tyr Ala Ser Trp 50 55 60Arg Asn Ser Ser Gly
Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu65 70 75 80Asp Asp Phe
Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu 85 90 95Asn Pro
Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu 100 105
110Arg Phe Thr His Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu
115 120 125Pro Pro Pro Thr Gly Gly Gly Thr His Thr Cys Pro Pro Cys
Pro Ala 130 135 140Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro145 150 155 160Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val 165 170 175Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val 180 185 190Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 195 200 205Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 210 215 220Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala225 230
235 240Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro 245 250 255Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr 260 265 270Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser 275 280 285Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr 290 295 300Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr305 310 315 320Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 325 330 335Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 340 345
350Ser Leu Ser Leu Ser Pro Gly Lys 355 36041083DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
polynucleotideCDS(73)..(396) 4atggatgcaa tgaagagagg gctctgctgt
gtgctgctgc tgtgtggagc agtcttcgtt 60tcgcccggcg cc gct gag aca cgg
gag tgc atc tac tac aac gcc aac tgg 111 Ala Glu Thr Arg Glu Cys Ile
Tyr Tyr Asn Ala Asn Trp 1 5 10gag ctg gag cgc acc aac cag agc ggc
ctg gag cgc tgc gaa ggc gag 159Glu Leu Glu Arg Thr Asn Gln Ser Gly
Leu Glu Arg Cys Glu Gly Glu 15 20 25cag gac aag cgg ctg cac tgc tac
gcc tcc tgg cgc aac agc tct ggc 207Gln Asp Lys Arg Leu His Cys Tyr
Ala Ser Trp Arg Asn Ser Ser Gly30 35 40 45acc atc gag ctc gtg aag
aag ggc tgc tgg cta gat gac ttc aac tgc 255Thr Ile Glu Leu Val Lys
Lys Gly Cys Trp Leu Asp Asp Phe Asn Cys 50 55 60tac gat agg cag gag
tgt gtg gcc act gag gag aac ccc cag gtg tac 303Tyr Asp Arg Gln Glu
Cys Val Ala Thr Glu Glu Asn Pro Gln Val Tyr 65 70 75ttc tgc tgc tgt
gaa ggc aac ttc tgc aac gag cgc ttc act cat ttg 351Phe Cys Cys Cys
Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu 80 85 90cca gag gct
ggg ggc ccg gaa gtc acg tac gag cca ccc ccg aca 396Pro Glu Ala Gly
Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr 95 100 105ggtggtggaa
ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca
456gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac
ccctgaggtc 516acatgcgtgg tggtggacgt gagccacgaa gaccctgagg
tcaagttcaa ctggtacgtg 576gacggcgtgg aggtgcataa tgccaagaca
aagccgcggg aggagcagta caacagcacg 636taccgtgtgg tcagcgtcct
caccgtcctg caccaggact ggctgaatgg caaggagtac 696aagtgcaagg
tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc
756aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga
ggagatgacc 816aagaaccagg tcagcctgac ctgcctggtc aaaggcttct
atcccagcga catcgccgtg 876gagtgggaga gcaatgggca gccggagaac
aactacaaga ccacgcctcc cgtgctggac 936tccgacggct ccttcttcct
ctatagcaag ctcaccgtgg acaagagcag gtggcagcag 996gggaacgtct
tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag
1056agcctctccc tgtccccggg taaatga 108351083DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
5tcatttaccc ggggacaggg agaggctctt ctgcgtgtag tggttgtgca gagcctcatg
60catcacggag catgagaaga cgttcccctg ctgccacctg ctcttgtcca cggtgagctt
120gctatagagg aagaaggagc cgtcggagtc cagcacggga ggcgtggtct
tgtagttgtt 180ctccggctgc ccattgctct cccactccac ggcgatgtcg
ctgggataga agcctttgac 240caggcaggtc aggctgacct ggttcttggt
catctcctcc cgggatgggg gcagggtgta 300cacctgtggt tctcggggct
gccctttggc tttggagatg gttttctcga tgggggctgg 360gagggctttg
ttggagacct tgcacttgta ctccttgcca ttcagccagt cctggtgcag
420gacggtgagg acgctgacca cacggtacgt gctgttgtac tgctcctccc
gcggctttgt 480cttggcatta tgcacctcca cgccgtccac gtaccagttg
aacttgacct cagggtcttc 540gtggctcacg tccaccacca cgcatgtgac
ctcaggggtc cgggagatca tgagggtgtc 600cttgggtttt ggggggaaga
ggaagactga cggtcccccc aggagttcag gtgctgggca 660cggtgggcat
gtgtgagttc caccacctgt cgggggtggc tcgtacgtga cttccgggcc
720cccagcctct ggcaaatgag tgaagcgctc gttgcagaag ttgccttcac
agcagcagaa 780gtacacctgg gggttctcct cagtggccac acactcctgc
ctatcgtagc agttgaagtc 840atctagccag cagcccttct tcacgagctc
gatggtgcca gagctgttgc gccaggaggc 900gtagcagtgc agccgcttgt
cctgctcgcc ttcgcagcgc tccaggccgc tctggttggt 960gcgctccagc
tcccagttgg cgttgtagta gatgcactcc cgtgtctcag cggcgccggg
1020cgaaacgaag actgctccac acagcagcag cacacagcag agccctctct
tcattgcatc 1080cat 108361083DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotideCDS(73)..(396)
6atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt
60tcgcccggcg cc gcc gaa acc cgc gaa tgt att tat tac aat gct aat tgg
111 Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn Trp 1 5 10gaa
ctc gaa cgg acg aac caa tcc ggg ctc gaa cgg tgt gag ggg gaa 159Glu
Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu 15 20
25cag gat aaa cgc ctc cat tgc tat gcg tcg tgg agg aac tcc tcc ggg
207Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser
Gly30 35 40 45acg att gaa ctg gtc aag aaa ggg tgc tgg ctg gac gat
ttc aat tgt 255Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp Asp
Phe Asn Cys 50 55 60tat gac cgc cag gaa tgt gtc gcg acc gaa gag aat
ccg cag gtc tat 303Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn
Pro Gln Val Tyr 65 70 75ttc tgt tgt tgc gag ggg aat ttc tgt aat gaa
cgg ttt acc cac ctc 351Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu
Arg Phe Thr His Leu 80 85 90ccc gaa gcc ggc ggg ccc gag gtg acc tat
gaa ccc ccg ccc acc 396Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu
Pro Pro Pro Thr 95 100 105ggtggtggaa ctcacacatg cccaccgtgc
ccagcacctg aactcctggg gggaccgtca 456gtcttcctct tccccccaaa
acccaaggac accctcatga tctcccggac ccctgaggtc 516acatgcgtgg
tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg
576gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta
caacagcacg 636taccgtgtgg tcagcgtcct caccgtcctg caccaggact
ggctgaatgg caaggagtac 696aagtgcaagg tctccaacaa agccctccca
gcccccatcg agaaaaccat ctccaaagcc 756aaagggcagc cccgagaacc
acaggtgtac accctgcccc catcccggga ggagatgacc 816aagaaccagg
tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg
876gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc
cgtgctggac 936tccgacggct ccttcttcct ctatagcaag ctcaccgtgg
acaagagcag gtggcagcag 996gggaacgtct tctcatgctc cgtgatgcat
gaggctctgc acaaccacta cacgcagaag 1056agcctctccc tgtccccggg taaatga
108371083DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 7tcatttaccc ggggacaggg agaggctctt
ctgcgtgtag tggttgtgca gagcctcatg 60catcacggag catgagaaga cgttcccctg
ctgccacctg ctcttgtcca cggtgagctt 120gctatagagg aagaaggagc
cgtcggagtc cagcacggga ggcgtggtct tgtagttgtt 180ctccggctgc
ccattgctct cccactccac ggcgatgtcg ctgggataga agcctttgac
240caggcaggtc aggctgacct ggttcttggt catctcctcc cgggatgggg
gcagggtgta 300cacctgtggt tctcggggct gccctttggc tttggagatg
gttttctcga tgggggctgg 360gagggctttg ttggagacct tgcacttgta
ctccttgcca ttcagccagt cctggtgcag 420gacggtgagg acgctgacca
cacggtacgt gctgttgtac tgctcctccc gcggctttgt 480cttggcatta
tgcacctcca cgccgtccac gtaccagttg aacttgacct cagggtcttc
540gtggctcacg tccaccacca cgcatgtgac ctcaggggtc cgggagatca
tgagggtgtc 600cttgggtttt ggggggaaga ggaagactga cggtcccccc
aggagttcag gtgctgggca 660cggtgggcat gtgtgagttc caccaccggt
gggcgggggt tcataggtca cctcgggccc 720gccggcttcg gggaggtggg
taaaccgttc attacagaaa ttcccctcgc aacaacagaa 780atagacctgc
ggattctctt cggtcgcgac acattcctgg cggtcataac aattgaaatc
840gtccagccag caccctttct tgaccagttc aatcgtcccg gaggagttcc
tccacgacgc 900atagcaatgg aggcgtttat cctgttcccc ctcacaccgt
tcgagcccgg attggttcgt 960ccgttcgagt tcccaattag cattgtaata
aatacattcg cgggtttcgg cggcgccggg 1020cgaaacgaag actgctccac
acagcagcag cacacagcag agccctctct tcattgcatc 1080cat
10838335PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 8Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn
Trp Glu Leu Glu Arg1 5 10 15Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu
Gly Glu Gln Asp Lys Arg 20 25 30Leu His Cys Tyr Ala Ser Trp Arg Asn
Ser Ser Gly Thr Ile Glu Leu 35 40 45Val Lys Lys Gly Cys Trp Leu Asp
Asp Phe Asn Cys Tyr Asp Arg Gln 50 55 60Glu Cys Val Ala Thr Glu Glu
Asn Pro Gln Val Tyr Phe Cys Cys Cys65 70 75 80Glu Gly Asn Phe Cys
Asn Glu Arg Phe Thr His Leu Pro Glu Ala Gly 85 90 95Gly Pro Glu Val
Thr Tyr Glu Pro Pro Pro Thr Gly Gly Gly Thr His 100 105 110Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 115 120
125Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
130 135 140Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu145 150 155 160Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys 165 170 175Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser 180 185 190Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys 195 200 205Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 210 215 220Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro225 230 235
240Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
245 250 255Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 260 265 270Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 275 280
285Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
290 295 300Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu305 310 315 320His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 325 330 3359225PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMOD_RES(43)..(43)Asp or
AlaMOD_RES(100)..(100)Lys or AlaMOD_RES(212)..(212)Asn or Ala 9Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro1 5 10
15Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
20 25 30Arg Thr Pro Glu Val Thr Cys Val Val Val Xaa Val Ser His Glu
Asp 35 40 45Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn 50 55 60Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val65 70 75 80Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 85 90 95Tyr Lys Cys Xaa Val Ser Asn Lys Ala Leu
Pro Val Pro Ile Glu Lys 100 105 110Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr 115 120 125Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr 130 135 140Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu145 150 155 160Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 165 170
175Asp Ser Asp Gly Pro Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
180 185 190Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 195 200 205Ala Leu His Xaa His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 210 215 220Lys22510348DNAHomo sapiens 10tctgggcgtg
gggaggctga gacacgggag tgcatctact acaacgccaa ctgggagctg 60gagcgcacca
accagagcgg cctggagcgc tgcgaaggcg agcaggacaa gcggctgcac
120tgctacgcct cctggcgcaa cagctctggc accatcgagc tcgtgaagaa
gggctgctgg 180ctagatgact tcaactgcta cgataggcag gagtgtgtgg
ccactgagga gaacccccag 240gtgtacttct gctgctgtga aggcaacttc
tgcaacgagc gcttcactca tttgccagag 300gctgggggcc cggaagtcac
gtacgagcca cccccgacag cccccacc 348115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Thr
Gly Gly Gly Gly1 5125PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 12Ser Gly Gly Gly Gly1
5136PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6xHis tag 13His His His His His His1 514108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
14Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu1
5 10 15Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu Gln Asp
Lys 20 25 30Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr
Ile Glu 35 40 45Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn Cys
Tyr Asp Arg 50 55 60Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val
Tyr Phe Cys Cys65 70 75 80Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe
Thr His Leu Pro Glu Ala 85 90 95Gly Gly Pro Glu Val Thr Tyr Glu Pro
Pro Pro Thr 100 10515108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 15Ala Glu Thr Arg Glu Cys
Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu1 5 10 15Arg Thr Asn Gln Ser
Gly Leu Glu Arg Cys Glu Gly Glu Gln Asp Lys 20 25 30Arg Leu His Cys
Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr Ile Glu 35 40 45Leu Val Lys
Lys Gly Cys Trp Leu Asp Asp Phe Asn Cys Tyr Asp Arg 50 55 60Gln Glu
Cys Val Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe Cys Cys65 70 75
80Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu Pro Glu Ala
85 90 95Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr 100 105
* * * * *