U.S. patent application number 15/040874 was filed with the patent office on 2016-09-15 for methods for increasing adiponectin.
The applicant listed for this patent is Acceleron Pharma Inc.. Invention is credited to Alan Koncarevic, Ravindra Kumar, Jennifer Lachey, Jasbir Seehra.
Application Number | 20160264681 15/040874 |
Document ID | / |
Family ID | 42337132 |
Filed Date | 2016-09-15 |
United States Patent
Application |
20160264681 |
Kind Code |
A1 |
Seehra; Jasbir ; et
al. |
September 15, 2016 |
METHODS FOR INCREASING ADIPONECTIN
Abstract
In certain aspects, the present invention provides compositions
and methods for increasing adiponectin in a patient in need thereof
by administering an antagonist of an ActRIIB signaling pathway.
Examples of such antagonists include ActRIIB polypeptides,
anti-ActRIIB antibodies, anti-activin A and/or B antibodies,
anti-myostatin antibodies, anti-GDF3 antibodies, and anti-BMP7
antibodies. Also provided are methods for ameliorating one or more
undesired effects of anti-androgen therapy, including muscle loss,
bone loss, increased adiposity, and/or increased insulin
resistance. A variety of disorders may be treated by causing an
increase in circulating adiponectin concentrations.
Inventors: |
Seehra; Jasbir; (Lexington,
MA) ; Kumar; Ravindra; (Acton, MA) ; Lachey;
Jennifer; (Arlington, MA) ; Koncarevic; Alan;
(Chicago, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Acceleron Pharma Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
42337132 |
Appl. No.: |
15/040874 |
Filed: |
February 10, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14319890 |
Jun 30, 2014 |
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15040874 |
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13404593 |
Feb 24, 2012 |
8765663 |
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14319890 |
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12657146 |
Jan 13, 2010 |
8138142 |
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13404593 |
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61204946 |
Jan 13, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 3/04 20180101; C07K
2317/24 20130101; C07K 2317/622 20130101; C07K 16/40 20130101; A61K
31/7088 20130101; A61P 3/10 20180101; A61K 31/00 20130101; A61P
5/50 20180101; A61P 9/10 20180101; C07K 14/705 20130101; A61K
38/179 20130101; C07K 14/71 20130101; A61K 2039/505 20130101; A61P
5/00 20180101; A61P 3/06 20180101; C07K 16/22 20130101; C07K
2317/54 20130101; C07K 2319/30 20130101; C07K 2317/55 20130101;
C07K 2317/76 20130101; C07K 2317/31 20130101; A61P 19/08 20180101;
A61P 3/08 20180101 |
International
Class: |
C07K 16/40 20060101
C07K016/40 |
Claims
1. A method for increasing adiponectin in a patient in need
thereof, the method comprising administering an effective amount of
an antibody, or antigen-binding fragment thereof, that binds to
ActRIIB.
2. The method of claim 1, wherein the antibody, or antigen-binding
fragment thereof, binds to a soluble ActRIIB peptide.
3. The method of claim 1, wherein the antibody, or antigen-binding
fragment thereof, is monoclonal.
4. The method of claim 1, wherein the antibody, of antigen-binding
fragment thereof, is chimeric.
5. The method claim 1, wherein the antibody, or antigen-binding
fragment thereof, is humanized.
6. The method of claim 1, wherein the antibody, or antigen binding
fragment thereof, is selected from: a single chain antibody, a
bispecific antibody, a F(ab).sub.2 fragment, and a Fab'
fragment.
7. The method of claim 1, wherein the patient has adiponectin
deficiency or insufficiency.
8. The method of claim 1, wherein the patient has low circulating
concentrations of adiponectin.
9. The method of claim 1, wherein administration of the antibody,
or antigen-binding fragment thereof, increase adiponectin
expression in adipocytes of the treated patient.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/657,146, filed Jan. 13, 2010, which claims the benefit of
U.S. Provisional Application No. 61/204,946, filed Jan. 13, 2009.
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 Feb. 10,
2012, is named PHPH-044-102seq.txt, and is 47,969 bytes in
size.
BACKGROUND OF THE INVENTION
[0003] Once thought to be merely an inert storage depot for excess
energy, adipose tissue is now recognized as an active endocrine and
paracrine organ secreting multiple mediators, known as adipokines,
that participate in diverse metabolic processes. The polypeptide
adipokine adiponectin is the most abundant known factor secreted by
adipocytes and accounts for approximately 0.01% of plasma protein.
Whereas levels of other adipokines increase with fat mass,
adiponectin levels vary inversely with fat mass/obesity. Decreased
adiponectin levels are also observed in type 2 diabetes and
cardiovascular disease. The strong correlation between low levels
of circulating adiponectin, or hypoadiponectinemia, and risk
factors for these major diseases may derive partly from
adiponectin's anti-inflammatory properties, which contrast with the
proinflammatory character of other adipokines (Szmitko et al.,
2007, Am J Physiol Heart Circ Physiol 292:H1655-H1663). Thus,
adiponectin appears to function as the protective adipokine,
counterbalancing the potentially detrimental actions of these other
adipokines.
[0004] Considerable evidence has emerged linking
hypoadiponectinemia with cardiovascular disease (Szmitko et al.,
supra). Adiponectin levels in patients with coronary heart disease
or cerebrovascular disease are lower than in healthy controls
(Hotta et al., 2000, Arterioscler Thromb Vasc Biol 20:1595-1599;
Kumada et al., 2003, Arterioscler Thromb Vasc Biol 23:85-89;
Pischon et al., 2004, JAMA 291:1730-1737) and vary inversely with
the severity of disease. Hypoadiponectinemia is associated with
increased risk of cardiovascular disease even in nonobese
individuals (Im et al., 2006, Metabolism 55:1546-1550).
Significantly, adiponectin inhibits development of atherosclerosis
in animal models (Okamoto et al., 2002, Circulation 106:2767-2770),
providing evidence for a causal relationship between low
adiponectin levels and cardiovascular disease. Therefore, there is
a need for ActRIIB-derived agents and other inhibitors of ActRIIB
signaling that can be used to treat or prevent
hypoadiponectinemia.
SUMMARY OF THE INVENTION
[0005] In certain aspects, the present disclosure provides methods
for increasing adiponectin levels in patients in need thereof by
using antagonists of the ActRIIB signaling pathway. Patients in
need of such therapy will typically exhibit low adiponectin,
particularly in the serum. Such patients are considered to have a
condition that is termed hypoadiponectinemia, Antagonists of the
ActRIIB signaling pathway may be, for example, soluble ActRIIB
proteins (e.g., ActRIIB-Fc fusion proteins), antagonists that bind
to ActRIIB or inhibit ActRIIB expression, and antagonists that bind
to or inhibit the expression of ligands that signal through ActRIIB
and regulate adiponectin expression and/or secretion. Such ligands
may include myostatin, GDF3, activins (particularly activin A,
activin B or activin AB), BMP7, BMP2 and BMP4. As demonstrated
herein, ActRIIB-Fc fusion proteins can be used to increase
adiponectin gene expression and increase circulating adiponectin
levels in diverse mouse models.
[0006] In certain aspects, the disclosure provides methods for
increasing adiponectin, or treating hypoadiponectinemia, by
administering to a patient in need thereof an effective amount of
an ActRIIB-related polypeptide. An ActRIIB-related polypeptide may
be an ActRIIB polypeptide (e.g., an ActRIIB extracellular domain or
portion thereof) that binds to an ActRIIB ligand such as GDF3,
BMP2, BMP4, BMP7, GDF8, GDF11, activin A, activin B, activin AB or
nodal. Optionally, the ActRIIB polypeptide binds to an ActRIIB
ligand with a Kd less than 10 micromolar or less than 1 micromolar,
100, 10 or 1 nanomolar. A variety of suitable ActRIIB polypeptides
have been described in the following published PCT patent
applications, all of which are incorporated by reference herein: WO
00/43781, WO 04/039948, WO 06/012627, WO 07/053775, WO 08/097541,
and WO 08/109167. Optionally, the ActRIIB polypeptide inhibits
ActRIIB signaling, such as intracellular signal transduction events
triggered by an ActRIIB ligand. A soluble ActRIIB polypeptide for
use in such a preparation may be any of those disclosed herein,
such as a polypeptide having an amino acid sequence selected from
SEQ ID NOs: 1, 2, 5, 12, 23 and 26 or having an amino acid sequence
that is at least 80%, 85%, 90%, 95%, 97% or 99% identical to an
amino acid sequence selected from SEQ ID NOs: 1, 2, 5, 12, 23 and
26. A soluble ActRIIB polypeptide may include a functional fragment
of a natural ActRIIB polypeptide, such as one comprising at least
10, 20 or 30 amino acids of a sequence selected from SEQ ID NOs: 1,
2, 5, 12, 23 and 26 or a sequence of SEQ ID NO: 1, lacking the
C-terminal 1, 2, 3, 4, 5 or 10 to 15 amino acids and lacking 1, 2,
3, 4 or 5 amino acids at the N-terminus. Optionally, polypeptides
will comprise a truncation relative to SEQ ID NO:1 of between 2 and
5 amino acids at the N-terminus and no more than 3 amino acids at
the C-terminus. Another polypeptide is that presented as SEQ ID
NO:12. A soluble ActRIIB polypeptide 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. A soluble ActRIIB polypeptide may be
a fusion protein that has, as one domain, an ActRIIB polypeptide
(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. A soluble ActRIIB fusion
protein may include an immunoglobulin constant domain, such as an
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 or non-repeating sequences of
threonine/serine and/or glycines (e.g., TG.sub.4 (SEQ ID NO: 6),
TG.sub.3 (SEQ ID NO: 27), SG.sub.4 (SEQ ID NO: 28), SG.sub.3 (SEQ
ID NO: 29), G.sub.4 (SEQ ID NO: 30), G.sub.3, G.sub.2, G). A fusion
protein may include a purification subsequence, such as an epitope
tag, a FLAG tag, a polyhistidine sequence, and a GST fusion.
Optionally, a soluble 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. In general, it is preferable that an
ActRIIB protein 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. Human and CHO cell lines have been used successfully, and
it is expected that other common mammalian expression vectors will
be useful.
[0007] In certain aspects, a compound disclosed herein may be
formulated as a pharmaceutical preparation for increasing
adiponectin in a patient in need thereof (e.g., the treatment of
hypoadiponectinemia). A pharmaceutical preparation may also include
one or more additional compounds such as a compound that is used to
treat an ActRIIB-associated disorder. Preferably, a pharmaceutical
preparation is substantially pyrogen free.
[0008] In certain aspects, the disclosure provides nucleic acids
encoding a soluble ActRIIB polypeptide, which do not encode a
complete ActRIIB polypeptide. An isolated polynucleotide may
comprise a coding sequence for a soluble ActRIIB polypeptide, such
as described above. For example, an isolated nucleic acid may
include a sequence coding for an extracellular domain (e.g.,
ligand-binding domain) of an ActRIIB polypeptide and a sequence
that would code for part or all of the transmembrane domain and/or
the cytoplasmic domain of an ActRIIB, but for a stop codon
positioned within the transmembrane domain or the cytoplasmic
domain, or positioned between the extracellular domain and the
transmembrane domain or cytoplasmic domain. For example, an
isolated polynucleotide may comprise a full-length ActRIIB
polynucleotide sequence such as SEQ ID NO: 4, or a partially
truncated version, said isolated polynucleotide further comprising
a transcription termination codon at least six hundred nucleotides
before the 3'-terminus or otherwise positioned such that
translation of the polynucleotide gives rise to an extracellular
domain optionally fused to a truncated portion of a full-length
ActRIIB Other suitable nucleic acids that encode ActRIIB
polypeptides are shown as SEQ ID NO: 3, 4, 10 or 24. Nucleic acids
disclosed herein may be operably linked to a promoter for
expression, and the disclosure provides cells transformed with such
recombinant polynucleotides. Preferably the cell is a mammalian
cell such as a CHO cell.
[0009] In certain aspects, the disclosure provides methods for
making a soluble ActRIIB polypeptide. Such a method may include
expressing any of the nucleic acids (e.g., SEQ ID NO: 3, 4, 10 or
27) 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 soluble ActRIIB
polypeptide, wherein said cell is transformed with a soluble
ActRIIB expression construct; and b) recovering the soluble ActRIIB
polypeptide so expressed. Soluble 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.
[0010] In certain aspects, a compound described herein may be used
in the management of a variety of forms of hypoadiponectinemia,
including patients having low adiponectin and an associated
condition (e.g., atherosclerosis, ischemic stroke, impaired glucose
tolerance, insulin resistance, diabetes type 2, hyperlipidemia,
hypertriglyceridemia, obesity). As shown herein, ActRIIB
polypeptides may be used to increase adiponectin gene expression
and/or circulating adiponectin levels while also having positive
effects on body composition, specifically on muscle, bone, and
adipose tissue.
[0011] In certain aspects, the disclosure provides uses of a
soluble ActRIIB polypeptide for making a medicament for the
treatment of a disorder or condition as described herein.
[0012] In certain aspects, the disclosure provides methods for
increasing adiponectin in a patient in need thereof (e.g., treating
hypoadiponectinemia), and such method may comprise administering an
effective amount of a compound selected from the group consisting
of: a polypeptide comprising an amino acid sequence that is at
least 90%, 93%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
5, 23 or 26 and a polypeptide encoded by a nucleic acid that
hybridizes under stringent hybridization conditions to a nucleic
acid of SEQ ID NO: 3 or 24. The polypeptide may be a fusion protein
comprising a heterologous portion. The polypeptide may be a dimer.
The polypeptide may be fused to a constant domain of an
immunoglobulin. The polypeptide may be fused to an Fc portion of an
immunoglobulin, such as an IgG1, IgG2, IgG3 or IgG4. The
polypeptide may comprise an amino acid sequence that is at least
80%, 90%, 93%, 95%, 97%, 98%, 99% or 100% identical to the sequence
of amino acids 29-109, 29-128, 29-131, 29-134, 25-109, 25-128,
25-131, 25-134 or 20-134 of SEQ ID NO:2. The polypeptide may
comprise an amino acid sequence that is at least 80%, 90%, 93%,
95%, 97%, 98%, 99% or 100% identical to the sequence of amino acids
of SEQ ID NO: 1, 2, 5, 12, 23 or 26. A patient to be treated with
such a compound may be one having a disorder described herein,
including, for example, hypoadiponectinemia and associated
conditions (e.g., atherosclerosis, ischemic stroke, impaired
glucose tolerance, insulin resistance, diabetes type 2,
hyperlipidemia, hypertriglyceridemia, or obesity).
[0013] In certain aspects, the disclosure provides methods for
increasing adiponectin in a patient in need thereof (e.g., treating
hypoadiponectinemia), the method comprising administering an
effective amount of a compound that inhibits the ActRIIB signaling
pathway, either by targeting ActRIIB or a ligand that signals
through ActRIIB Examples of such compounds include antagonists of
ActRIIB; antagonists of myostatin; antagonists of activin A;
antagonists of activin B; antagonists of BMP2; antagonists of BMP4
and antagonists of GDF3. Antagonists of each of the foregoing may
be an antibody or other protein that specifically binds to and
inhibits such target (e.g., an antibody such as a monoclonal
antibody, or a propeptide in the case of myostatin and GDF3).
Antagonists of the foregoing may also be a compound, such as a
nucleic acid based compound (e.g., an antisense or RNAi nucleic
acid) that inhibits the expression of ActRIIB or the ligand. A
patient to be treated with such a compound may be one having a
disorder described herein, including, for example, low adiponectin
level (hypoadiponectinemia), atherosclerosis, ischemic stroke,
impaired glucose tolerance, insulin resistance, diabetes type 2,
hyperlipidemia, hypertriglyceridemia, or obesity, and particularly
any of the foregoing wherein the patient additionally exhibits low
adiponectin levels.
[0014] In certain aspects, the disclosure provides methods for
concurrently increasing muscle, increasing bone, and increasing fat
in a patient in need thereof, the method comprising administering
to the patient an effective amount of an ActRIIB fusion protein,
wherein the ActRIIB fusion protein comprises an amino acid sequence
that is at least 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ
ID NO: 1, 2, 5, 23 or 26.
[0015] In certain aspects, the disclosure provides methods for
ameliorating one or more undesired effects of anti-androgen therapy
in a patient in need thereof, the method comprising administering
to the patient an effective amount of an ActRIIB fusion protein,
wherein the ActRIIB fusion protein comprises an amino acid sequence
that is at least 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ
ID NO: 2, 5, 23 or 26. The undesired effect of anti-androgen
therapy may be, for example, muscle loss, bone loss, increased
adiposity, or increased insulin resistance, or combinations of the
foregoing. In an exemplary embodiment, the undesired effect of
anti-androgen therapy is a combination of three or more of muscle
loss, bone loss, increased adiposity and insulin resistance.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] 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 Office
upon request and payment of the necessary fee.
[0017] FIG. 1 shows a human ActRIIB soluble (extracellular)
polypeptide sequence (SEQ ID NO: 1). The C-terminal "tail" is
underlined.
[0018] FIG. 2 shows human ActRIIB precursor protein sequence (SEQ
ID NO: 2). The signal peptide is underlined; the extracellular
domain is in bold (also referred to as SEQ ID NO: 1); and the
potential N-linked glycosylation sites are boxed.
[0019] FIG. 3 shows a nucleic acid sequence encoding a human
ActRIIB soluble (extracellular) polypeptide, designated as SEQ ID
NO: 3.
[0020] FIG. 4 shows a nucleic acid sequence encoding human ActRIIB
precursor protein, designated as SEQ ID NO: 4.
[0021] FIG. 5 shows an alignment of human ActRIIA (SEQ ID NO: 14)
and ActRIIB (SEQ ID NO: 33) with the residues that are deduced
herein, based on composite analysis of multiple ActRIIB and ActRIIA
crystal structures to directly contact ligand (the ligand binding
pocket) indicated with boxes.
[0022] FIG. 6 shows a multiple sequence alignment of various
vertebrate ActRIIB proteins and human ActRIIA.
[0023] FIG. 7 shows the full amino acid sequence of
ActRIIB(25-131)-hFc (SEQ ID NO: 23). The TPA leader (residues 1-22)
and truncated ActRIIB extracellular domain (native residues 25-131)
are each underlined. Highlighted is the glutamate revealed by
sequencing to be the N-terminal amino acid of the mature fusion
protein. FIG. 7 discloses SEQ ID NO: 24.
[0024] FIG. 8 shows a nucleotide sequence encoding
ActRIIB(25-131)-hFc (SEQ ID NO: 24) (the coding strand is shown at
top (SEQ ID NO: 25) and the complement shown at bottom 3'-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
(protein sequence is disclosed as residues 25-131 of SEQ ID NO:
24).
[0025] FIG. 9 shows body weight vs. time in mice as a function of
ORX and ActRIIB(R64 20-134)-mFc treatment. Vehicle was
Tris-buffered saline (TBS). Data shown are means (n=10 per group),
and Day 71 means that differ significantly (P<0.05, two-tailed
unpaired t-test) are designated by different letters. ActRIIB(R64
20-134)-mFc increased overall body weight gain under both ORX and
gonad-intact conditions.
[0026] FIG. 10 shows lean body mass vs. time in mice as a function
of ORX and ActRIIB(R64 20-134)-mFc treatment. Lean body mass (total
nonfat mass) was determined by NMR. Data shown are means (n=10 per
group), and Day 71 means that differ significantly (P<0.05,
two-tailed unpaired t-test) are designated by different letters.
Unlike ORX controls, ORX mice treated with ActRIIB(R64 20-134)-mFc
gained lean body mass over the course of the experiment, finishing
with values approximately 25% higher than in the former group. A
similar increase in lean body mass was also observed under
gonad-intact conditions for ActRIIB(R64 20-134)-mFc compared to
vehicle.
[0027] FIG. 11 shows skeletal muscle mass in mice as a function of
ORX and ActRIIB(R64 20-134)-mFc treatment for 71 days. Pectoralis,
rectus femoris, and gastrocnemius muscles were surgically removed
and weighed at study completion. Data shown are means.+-.SEM (n=10
per group), and those that differ significantly (P<0.05,
two-tailed unpaired t-test) are designated by different letters.
ActRIIB(R64 20-134)-mFc increased the mass of all three muscles
significantly under both ORX and gonad-intact conditions.
[0028] FIG. 12 shows whole-body bone area vs. time in mice as a
function of ORX and ActRIIB(R64 20-134)-mFc treatment. Measurements
were made by dual energy X-ray absorptiometry (DEXA). Data shown
are means (n=10 per group), and Day 47 means that differ
significantly (P<0.05, two-tailed unpaired t-test) are
designated by different letters. ActRIIB(R64 20-134)-mFc prevented
the progressive decrease in bone area observed under ORX conditions
and led to significantly increased bone area under gonad-intact
conditions.
[0029] FIG. 13 shows whole-body bone mineral content vs. time in
mice as a function of ORX and ActRIIB(R64 20-134)-mFc treatment.
Measurements were made by dual energy X-ray absorptiometry (DEXA)
analysis. Data shown are means (n=10 per group), and Day 47 means
that differ significantly (P<0.05, two-tailed unpaired t-test)
are designated by different letters. As with bone area, ActRIIB(R64
20-134)-mFc prevented the progressive decrease in bone mineral
content observed under ORX conditions and led to significantly
increased bone mineral content under gonad-intact conditions.
[0030] FIG. 14 shows whole-body bone mineral density vs. time in
mice as a function of ORX and ActRIIB(R64 20-134)-mFc treatment.
Measurements were made by dual energy X-ray absorptiometry (DEXA)
analysis. Data shown are means (n=10 per group), and Day 47 means
that differ significantly (P<0.05, two-tailed unpaired t-test)
are designated by different letters. ActRIIB(R64 20-134)-mFc
increased bone mineral density under ORX conditions but not
gonad-intact conditions.
[0031] FIG. 15 shows bone volume fraction in murine tibia as a
function of ORX and ActRIIB(R64 20-134)-mFc treatment for 71 days.
Measurements were made by micro-computed tomography (micro-CT).
Data shown are means.+-.SEM (n=7 per group), and those that differ
significantly (P<0.05, two-tailed unpaired t-test) are
designated by different letters. In ORX mice, ActRIIB(R64
20-134)-mFc increased bone volume fraction markedly compared to
vehicle, restoring this endpoint to levels typical in gonad-intact
mice treated with vehicle. ActRIIB(R64 20-134)-mFc increased this
endpoint in gonad-intact mice by a similar magnitude.
[0032] FIG. 16 shows trabecular number in murine tibia as a
function of ORX and ActRIIB(R64 20-134)-mFc treatment for 71 days.
Measurements were made by micro-CT and expressed as the mean number
of trabeculae per mm (of randomly positioned line segments through
the tissue). Data shown are means.+-.SEM (n=7 per group), and those
that differ significantly (P<0.05, two-tailed unpaired t-test)
are designated by different letters. In ORX mice, ActRIIB(R64
20-134)-mFc doubled the trabecular number observed with vehicle,
restoring this endpoint to levels typical in gonad-intact mice
treated with vehicle. ActRIIB(R64 20-134)-mFc increased this
endpoint in gonad-intact mice by a similar magnitude.
[0033] FIG. 17 shows trabecular thickness in murine tibia as a
function of ORX and ActRIIB(R64 20-134)-mFc treatment for 71 days.
Measurements were made by micro-CT. Data shown are means.+-.SEM
(n=7 per group), and those that differ significantly (P<0.05,
two-tailed unpaired t-test) are designated by different letters. In
ORX mice, ActRIIB(R64 20-134)-mFc increased trabecular thickness as
compared with vehicle, restoring this endpoint to levels typical in
gonad-intact mice treated with vehicle. ActRIIB(R64 20-134)-mFc
increased this endpoint in gonad-intact mice by a similar
percentage.
[0034] FIG. 18 shows trabecular separation in murine tibia as a
function of ORX and ActRIIB(R64 20-134)-mFc treatment for 71 days.
Measurements were made by micro-CT. Data shown are means.+-.SEM
(n=7 per group), and those that differ significantly (P<0.05,
two-tailed unpaired t-test) are designated by different letters. In
ORX mice, ActRIIB(R64 20-134)-mFc decreased trabecular separation
as compared with vehicle, restoring this endpoint to levels typical
in gonad-intact mice treated with vehicle. ActRIIB(R64 20-134)-mFc
decreased this endpoint in gonad-intact mice by a similar
percentage.
[0035] FIG. 19 shows tibial morphology in mice as a function of ORX
and ActRIIB(R64 20-134)-mFc treatment for 71 days. Images of
trabecular bone in the proximal tibia were obtained by micro-CT.
Scale bar=100 .mu.m. Tibial morphology in ORX mice treated with
ActRIIB(R64 20-134)-mFc closely resembled that in vehicle-treated
gonad-intact mice.
[0036] FIG. 20 shows fat tissue mass vs. time in mice as a function
of ORX and ActRIIB(R64 20-134)-mFc treatment. Measurements were
made by NMR. Data shown are means (n=10 per group), and Day 71
means that differ significantly (P<0.05, two-tailed unpaired
t-test) are designated by different letters. Fat mass in
vehicle-treated ORX mice tripled over the course of the study, and
ActRIIB(R64 20-134)-mFc treatment in ORX mice cut this increase by
more than 60%, restoring this endpoint to levels observed in
gonad-intact controls. ActRIIB(R64 20-134)-mFc decreased this
endpoint in gonad-intact mice by a similar percentage.
[0037] FIG. 21 shows adipocyte histology in ORX mice treated with
vehicle (TBS) or ActRIIB(R64 20-134)-mFc for 71 days. Sections were
stained with hematoxylin and eosin. Magnification=10.times..
ActRIIB-mFc reduced adipocyte size noticeably in subcutaneous and
epididymal fat depots but not in interscapular brown fat.
[0038] FIG. 22 shows serum adiponectin concentrations in mice as a
function of ORX and ActRIIB(R64 20-134)-mFc treatment for 71 days.
ELISA measurements detect all main oligomeric isoforms (total
adiponectin). Data shown are means.+-.SEM (n=10 per group), and
those that differ significantly (P<0.05, two-tailed unpaired
t-test) are designated by different letters. In both ORX and
gonad-intact mice, ActRIIB(R64 20-134)-mFc increased circulating
adiponectin concentrations significantly compared to their
vehicle-treated counterparts.
[0039] FIG. 23 shows serum leptin concentrations in mice as a
function of ORX and ActRIIB(R64 20-134)-mFc treatment for 71 days.
Data shown are means.+-.SEM (n=10 per group), and those that differ
significantly (P<0.05, two-tailed unpaired t-test) are
designated by different letters. In both ORX and gonad-intact mice,
ActRIIB(R64 20-134)-mFc reduced circulating leptin concentrations
significantly compared to their vehicle-treated counterparts.
[0040] FIG. 24 shows serum levels of adiponectin in mice as a
function of diet and ActRIIB-hFc treatment for 60 days. ELISA
measurements detect all main oligomeric isoforms (total
adiponectin), and data are means.+-.SEM; n=7-10 per group; **,
p<0.01; ***, p<0.001. In mice fed a high-fat diet,
ActRIIB(20-134)-hFc increased circulating adiponectin
concentrations by more than 50% to match those in standard-diet
controls, while ActRIIB(25-131)-hFc increased circulating
adiponectin concentrations by more than 75% to significantly exceed
those in standard-diet controls.
[0041] FIG. 25 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.
DETAILED DESCRIPTION
1. Overview
[0042] In certain aspects, the present invention relates to
adiponectin (also known as Acrp30, AdipoQ, apM1, and GBP28), a
polypeptide hormone (247 amino acids) released from adipocytes in
multimeric form. Adiponectin acts through two receptors: AdipoR1,
which is expressed in skeletal muscle, vascular endothelial cells,
cardiomyocytes, and pancreatic .beta. cells, and AdipoR2, which is
expressed in liver and endothelial cells. Whereas other prominent
adipokines (adipocyte-derived hormones) such as leptin and resistin
are considered proinflammatory, adiponectin exerts
anti-inflammatory effects that seem to serve a counterbalancing
role (Szmitko et al., 2007, Am J Physiol Heart Circ Physiol
292:H1655-H1663). Circulating levels of adiponectin vary inversely
with adipose mass, and thus low adiponectin levels
(hypoadiponectinemia) may partially mediate the increased risk of
cardiovascular disease and type 2 diabetes associated with obesity.
However, hypoadiponectinemia is associated with increased risk of
cardiovascular disease and diabetes even in nonobese individuals
(Pellme et al., 2003, Diabetes 52:1182-1186; Im et al., 2006,
Metabolism 55:1546-1550). Thus the state having abnormally low
adiponectin levels is understood to represent an independent
dysfunctional state, and may also identify subset of patients
afflicted with another condition (e.g., type II diabetes, obesity
or cardiovascular disease) that are particularly amenable to
treatment with an agent described herein.
[0043] Evidence suggests a causal protective role for adiponectin
in the development of cardiovascular disease. Adiponectin levels in
patients with coronary heart disease or cerebrovascular disease are
lower than in healthy controls (Hotta et al., 2000, Arterioscler
Thromb Vasc Biol 20:1595-1599; Kumada et al., 2003, Arterioscler
Thromb Vasc Biol 23:85-89; Pischon et al., 2004, JAMA
291:1730-1737) and vary inversely with the severity of disease.
Moreover, administration of adiponectin inhibits development of
atherosclerosis in animal models (Okamoto et al., 2002, Circulation
106:2767-2770), providing evidence for a causal relationship
between adiponectin levels and cardiovascular disease. As described
in the Examples, ActRIIB-Fc fusion proteins can be used to increase
circulating adiponectin levels in diverse mouse models. Therefore,
ActRIIB-derived agents and other compounds that inhibit ActRIIB
signaling can be used to treat or prevent hypoadiponectinemia and
to treat a subset of patients having a condition such as
cardiovascular disease, diabetes, and obesity coupled with low
adiponectin.
[0044] Low adiponectin, or hypoadiponectinemia, may be understood
as the set of patients in the lowest quintile of adiponectin levels
(below about 10.5 mg/L per Pischon et al. JAMA 2004; 291:
1730-1737), and preferably below 4.0 mg/L or below 2.5 mg/L (see
also Im et al. Metabolism 2006; 55:1546-1550; Kumada et al.
Arterioscler Thromb Vasc Biol 2003; 23:85-89; Ryo et al. Circ J
2004; 68:975-981; Tsukinoki et al. Lipids Health Dis 2005; 4:27).
Values may be slightly higher in women than in men.
[0045] In certain aspects, the present invention 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. The amino acid sequence of
human ActRIIB precursor protein, including the native leader, is
illustrated in FIG. 2 (SEQ ID NO: 2) and is used throughout this
disclosure as the base sequence for numbering the amino acids of
any of the various truncations, mature forms, and variants of
ActRIIB
[0046] 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.
[0047] In a specific embodiment, the invention relates to soluble
ActRIIB polypeptides. As described herein, the term "soluble
ActRIIB polypeptide" generally refers to polypeptides comprising an
extracellular domain of an ActRIIB protein. The term "soluble
ActRIIB polypeptide," as used herein, includes 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. Examples of soluble ActRIIB polypeptides include
ActRIIB soluble polypeptides illustrated in FIG. 1 (SEQ ID NO: 1)
as well as SEQ ID Nos. 5 and 23. Other examples of soluble ActRIIB
polypeptides comprise a signal sequence in addition to the
extracellular domain of an ActRIIB protein, see Example 1. 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.
[0048] 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.
[0049] 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.
[0050] 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., a soluble
ActRIIB 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, GDF8, GDF11, and BMP7.
[0051] 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 (.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.
[0052] 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.
[0053] 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).
[0054] 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).
[0055] Bone morphogenetic protein (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. For example, BMP7 may be the
osteoinductive factor responsible for the phenomenon of epithelial
osteogenesis. It is also found that BMP7 plays a role in adipocyte
differentiation and brown fat formation. Like activin, BMP7 binds
to type II receptors, ActRIIA and IIB. 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).
[0056] In certain aspects, the present invention relates to the use
of certain ActRIIB polypeptides (e.g., soluble 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 activins, Nodal,
GDF8, GDF11, and BMP7, and may therefore be useful in the treatment
of additional disorders.
[0057] 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.
[0058] "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.
[0059] 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.
[0060] 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.
[0061] "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.
[0062] 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.
[0063] 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
[0064] In certain aspects, the invention relates to ActRIIB variant
polypeptides (e.g., soluble ActRIIB polypeptides). 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. Examples of ActRIIB
polypeptides include human ActRIIB precursor polypeptide (SEQ ID
NO: 2), and soluble human ActRIIB polypeptides (e.g., SEQ ID NOs:
1, 2, 5, 12, 23 and 26).
[0065] 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
April 15; 83(8):2163-70), which has an Alanine at the position
corresponding to amino acid 64 of SEQ ID NO: 2 (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.
[0066] Attisano et al. (Cell. 1992 January 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. Data presented here shows that an ActRIIB-Fc
fusion protein containing amino acids 20-119 of SEQ ID NO:2,
"ActRIIB(20-119)-Fc" has reduced binding to GDF-11 and activin
relative to an ActRIIB(20-134)-Fc, which includes the proline knot
region and the complete juxtamembrane domain. However, an
ActRIIB(20-129)-Fc protein retains similar but somewhat reduced
activity relative to the wild type, even though the proline knot
region is disrupted. Thus, ActRIIB extracellular domains that stop
at amino acid 134, 133, 132, 131, 130 and 129 are all expected to
be active, but constructs stopping at 134 or 133 may be most
active. Similarly, mutations at any of residues 129-134 are not
expected to alter ligand binding affinity by large margins. In
support of this, mutations of P129 and P130 do not substantially
decrease ligand binding. Therefore, an ActRIIB-Fc fusion protein
may end as early as amino acid 109 (the final cysteine), however,
forms ending at or between 109 and 119 are expected to have reduced
ligand binding. Amino acid 119 is poorly conserved and so is
readily altered or truncated. Forms ending at 128 or later retain
ligand binding activity. Forms ending at or between 119 and 127
will have an intermediate binding ability. Any of these forms may
be desirable to use, depending on the clinical or experimental
setting.
[0067] At the N-terminus of ActRIIB, it is expected that a protein
beginning at amino acid 29 or before will retain ligand binding
activity. Amino acid 29 represents the initial cysteine. An alanine
to asparagine mutation at position 24 introduces an N-linked
glycosylation sequence without substantially affecting ligand
binding. This confirms that mutations in the region between the
signal cleavage peptide and the cysteine cross-linked region,
corresponding to amino acids 20-29 are well tolerated. In
particular, constructs beginning at position 20, 21, 22, 23 and 24
will retain activity, and constructs beginning at positions 25, 26,
27, 28 and 29 are also expected to retain activity.
[0068] Taken together, an active portion of ActRIIB comprises amino
acids 29-109 of SEQ ID NO:2, presented here as SEQ ID NO: 26:
CIYYNANWELERTQSGLERCEGEQDKRLHCYASWRSSGTIELVKKGCWLDDFNCYDRQECVA
TEENPQVYFCCCEGNFC Constructs may, for example, begin at a residue
corresponding to amino acids 20-29 and end at a position
corresponding to amino acids 109-134 of SEQ ID NO: 2. Other
examples include constructs that begin at a position from 20-29 or
21-29 and end at a position from 119-134, 119-133 or 129-134,
129-133. Other examples include constructs that begin at a position
from 20-24 (or 21-24, or 22-25) and end at a position from 109-134
(or 109-133), 119-134 (or 119-133) or 129-134 (or 129-133).
Variants within these ranges are also contemplated, particularly
those having at least 80%, 85%, 90%, 95% or 99% identity to the
corresponding portion of SEQ ID NO:4.
[0069] The disclosure includes the results of an analysis of
composite ActRIIB structures, shown in FIG. 5, demonstrating that
the 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, a general formula for an active ActRIIB
variant protein is one that comprises amino acids 29-109, but
optionally beginning at a position ranging from 20-24 or 22-25 and
ending at a position ranging from 129-134, 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 29-109
of SEQ ID NO:4. 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.
[0070] 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 conserved. Accordingly, comparisons of ActRIIB sequences
from various vertebrate organisms provide insights into residues
that may be altered. Therefore, an active, human ActRIIB variant
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 Yin
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.
[0071] Further N-linked glycosylation sites (N-X-S/T) may be
introduced into the ActRIIb sequence. By introducing an asparagine
at position 24 (A24N construct), an NXT sequence is created. Other
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 defined in FIG. 12.
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.
[0072] The variations described may be combined in various ways.
Additionally, the results of mutagenesis program described
previously in WO 2006/012627 and WO 2008/097541 indicate that 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). Thus the disclosure provides a framework of amino acids
that may be conserved. 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).
[0073] In certain embodiments, isolated fragments of the ActRIIB
polypeptides can be obtained by screening polypeptides
recombinantly produced from the corresponding fragment of the
nucleic acid encoding an ActRIIB polypeptide (e.g., SEQ ID NOs: 3
and 4). In addition, fragments can be chemically synthesized using
techniques known in the art such as conventional Merrifield solid
phase f-Moc or t-Boc chemistry. The fragments can be produced
(recombinantly or by chemical synthesis) and tested to identify
those peptidyl fragments that can function, for example, as
antagonists (inhibitors) or agonists (activators) of an ActRIIB
protein or an ActRIIB ligand.
[0074] In certain embodiments, a functional variant of the ActRIIB
polypeptides has an amino acid sequence that is at least 75%
identical to an amino acid sequence selected from SEQ ID NOs: 1, 2,
5, 12, 23 and 26. In certain cases, the functional variant has an
amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or
100% identical to an amino acid sequence selected from SEQ ID NOs:
1, 2, 5, 12, 23 and 26.
[0075] 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.
[0076] 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.
[0077] For example, the disclosure provides variant ActRIIB
polypeptides that preferentially bind to GDF8/GDF11 relative to
activins. The disclosure further establishes the desirability of
such polypeptides for reducing off-target effects, although such
selective variants may be less desirable for the treatment of
severe diseases where very large gains in muscle mass may be needed
for therapeutic effect and where some level of off-target effect is
acceptable. For example, amino acid residues of the ActRIIB
protein, such as E39, K55, Y60, K74, W78, D80, and F101, are in the
ligand-binding pocket and mediate binding to its ligands such as
activin and GDF8. Thus, the present invention provides an altered
ligand-binding domain (e.g., GDF8-binding domain) of an ActRIIB
receptor, which comprises one or more mutations at those amino acid
residues. Optionally, the altered ligand-binding domain can have
increased selectivity for a ligand such as GDF8 relative to a
wild-type ligand-binding domain of an ActRIIB receptor. To
illustrate, these mutations increase the selectivity of the altered
ligand-binding domain for GDF8 over activin. Optionally, the
altered ligand-binding domain has a ratio of K.sub.d for activin
binding to K.sub.d for GDF8 binding that is at least 2, 5, 10, or
even 100 fold greater relative to the ratio for the wild-type
ligand-binding domain. Optionally, the altered ligand-binding
domain has a ratio of IC.sub.50 for inhibiting activin to IC.sub.50
for inhibiting GDF8 that is at least 2, 5, 10, or even 100 fold
greater relative to the wild-type ligand-binding domain.
Optionally, the altered ligand-binding domain inhibits GDF8 with an
IC.sub.50 at least 2, 5, 10, or even 100 times less than the
IC.sub.50 for inhibiting activin.
[0078] As a specific example, the positively-charged amino acid
residue Asp (D80) of the ligand-binding domain of ActRIIB can be
mutated to a different amino acid residue such that the variant
ActRIIB polypeptide preferentially binds to GDF8, but not activin.
Preferably, the D80 residue is changed to an amino acid residue
selected from the group consisting of: a uncharged amino acid
residue, a negative amino acid residue, and a hydrophobic amino
acid residue. As a further specific example, the hydrophobic
residue, L79, can be altered to the acidic amino acids aspartic
acid or glutamic acid to greatly reduce activin binding while
retaining GDF11 binding. As will be recognized by one of skill in
the art, most of the described mutations, variants or modifications
may be made at the nucleic acid level or, in some cases, by post
translational modification or chemical synthesis. Such techniques
are well known in the art.
[0079] In certain embodiments, the present invention contemplates
specific mutations of the ActRIIB polypeptides so as to alter the
glycosylation of the polypeptide. Exemplary glycosylation sites in
ActRIIB polypeptides are illustrated in FIG. 2. 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.
[0080] 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.
[0081] 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.
[0082] 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 an 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.
[0083] 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 (SEQ ID NO: 31)) 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).
[0084] The following is a specific example of Fc domains that may
be used (e.g., SEQ ID NO: 13).
TABLE-US-00001 THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*
[0085] The Fc domain may have 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.
[0086] 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.
[0087] In the case of fusion proteins, an ActRIIB polypeptide may
be 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 polyethylene glycol.
[0088] 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.
[0089] 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
[0090] In certain aspects, the invention provides isolated and/or
recombinant nucleic acids encoding any of the ActRIIB polypeptides
(e.g., soluble ActRIIB polypeptides), including any of the variants
disclosed herein. For example, SEQ ID NO: 4 encodes a naturally
occurring ActRIIB precursor polypeptide, while SEQ ID NO: 3 encodes
a soluble ActRIIB polypeptide. 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 or as direct therapeutic
agents (e.g., in a gene therapy approach).
[0091] In certain aspects, the subject nucleic acids encoding
ActRIIB polypeptides are further understood to include nucleic
acids that are variants of SEQ ID NO: 3. 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.
[0092] 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: 3, 10 and 24.
One of ordinary skill in the art will appreciate that nucleic acid
sequences complementary to SEQ ID NO: 3, and variants of SEQ ID NO:
3 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.
[0093] 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: 3,
10 or 24, complement sequence of SEQ ID NO: 3, or fragments
thereof. 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.
[0094] Isolated nucleic acids which differ from the nucleic acids
as set forth in SEQ ID NO: 3 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.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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 .beta.-gal containing pBlueBac III).
[0099] 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.
[0100] This invention also pertains to a host cell transfected with
a recombinant gene including a coding sequence (e.g., SEQ ID NO: 3,
4, 10 or 24) 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.
[0101] 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.
[0102] 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).
[0103] 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).
4. Antibodies
[0104] Another aspect of the invention pertains to antibodies. An
antibody that is specifically reactive with an ActRIIB polypeptide
(e.g., a soluble ActRIIB polypeptide) and which binds competitively
with the ActRIIB polypeptide may be used as an antagonist of
ActRIIB polypeptide activities. For example, by using immunogens
derived from an ActRIIB polypeptide, anti-protein/anti-peptide
antisera or monoclonal antibodies can be made by standard protocols
(see, for example, Antibodies: A Laboratory Manual ed. by Harlow
and Lane (Cold Spring Harbor Press: 1988)). A mammal, such as a
mouse, a hamster or rabbit can be immunized with an immunogenic
form of the ActRIIB polypeptide, an antigenic fragment which is
capable of eliciting an antibody response, or a fusion protein.
Techniques for conferring immunogenicity on a protein or peptide
include conjugation to carriers or other techniques well known in
the art. An immunogenic portion of an ActRIIB polypeptide can be
administered in the presence of adjuvant. The progress of
immunization can be monitored by detection of antibody titers in
plasma or serum. Standard ELISA or other immunoassays can be used
with the immunogen as antigen to assess the levels of
antibodies.
[0105] Following immunization of an animal with an antigenic
preparation of an ActRIIB polypeptide, antisera can be obtained
and, if desired, polyclonal antibodies can be isolated from the
serum. To produce monoclonal antibodies, antibody-producing cells
(lymphocytes) can be harvested from an immunized animal and fused
by standard somatic cell fusion procedures with immortalizing cells
such as myeloma cells to yield hybridoma cells. Such techniques are
well known in the art, and include, for example, the hybridoma
technique (originally developed by Kohler and Milstein, (1975)
Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar
et al., (1983) Immunology Today, 4: 72), and the EBV-hybridoma
technique to produce human monoclonal antibodies (Cole et al.,
(1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.
pp. 77-96). Hybridoma cells can be screened immunochemically for
production of antibodies specifically reactive with an ActRIIB
polypeptide and monoclonal antibodies isolated from a culture
comprising such hybridoma cells.
[0106] The term "antibody" as used herein is intended to include
fragments thereof which are also specifically reactive with a
subject ActRIIB polypeptide. Antibodies can be fragmented using
conventional techniques and the fragments screened for utility in
the same manner as described above for whole antibodies. For
example, F(ab).sub.2 fragments can be generated by treating
antibody with pepsin. The resulting F(ab).sub.2 fragment can be
treated to reduce disulfide bridges to produce Fab fragments. The
antibody of the present invention is further intended to include
bispecific, single-chain, and chimeric and humanized molecules
having affinity for an ActRIIB polypeptide conferred by at least
one CDR region of the antibody. In preferred embodiments, the
antibody further comprises a label attached thereto and able to be
detected (e.g., the label can be a radioisotope, fluorescent
compound, enzyme or enzyme co-factor).
[0107] In certain preferred embodiments, an antibody of the
invention is a monoclonal antibody, and in certain embodiments, the
invention makes available methods for generating novel antibodies.
For example, a method for generating a monoclonal antibody that
binds specifically to an ActRIIB polypeptide may comprise
administering to a mouse an amount of an immunogenic composition
comprising the ActRIIB polypeptide effective to stimulate a
detectable immune response, obtaining antibody-producing cells
(e.g., cells from the spleen) from the mouse and fusing the
antibody-producing cells with myeloma cells to obtain
antibody-producing hybridomas, and testing the antibody-producing
hybridomas to identify a hybridoma that produces a monoclonal
antibody that binds specifically to the ActRIIB polypeptide. Once
obtained, a hybridoma can be propagated in a cell culture,
optionally in culture conditions where the hybridoma-derived cells
produce the monoclonal antibody that binds specifically to the
ActRIIB polypeptide. The monoclonal antibody may be purified from
the cell culture.
[0108] The adjective "specifically reactive with" as used in
reference to an antibody is intended to mean, as is generally
understood in the art, that the antibody is sufficiently selective
between the antigen of interest (e.g., an ActRIIB polypeptide) and
other antigens that are not of interest that the antibody is useful
for, at minimum, detecting the presence of the antigen of interest
in a particular type of biological sample. In certain methods
employing the antibody, such as therapeutic applications, a higher
degree of specificity in binding may be desirable. Monoclonal
antibodies generally have a greater tendency (as compared to
polyclonal antibodies) to discriminate effectively between the
desired antigens and cross-reacting polypeptides. One
characteristic that influences the specificity of an
antibody:antigen interaction is the affinity of the antibody for
the antigen. Although the desired specificity may be reached with a
range of different affinities, generally preferred antibodies will
have an affinity (a dissociation constant) of about 10.sup.-6,
10.sup.-7, 10.sup.-8, 10.sup.-9 or less.
[0109] In addition, the techniques used to screen antibodies in
order to identify a desirable antibody may influence the properties
of the antibody obtained. For example, if an antibody is to be used
for binding an antigen in solution, it may be desirable to test
solution binding. A variety of different techniques are available
for testing interaction between antibodies and antigens to identify
particularly desirable antibodies. Such techniques include ELISAs,
surface plasmon resonance binding assays (e.g., the Biacore binding
assay, Bia-core AB, Uppsala, Sweden), sandwich assays (e.g., the
paramagnetic bead system of IGEN International, Inc., Gaithersburg,
Md.), western blots, immunoprecipitation assays, and
immunohistochemistry.
[0110] In certain aspects, the disclosure provides antibodies that
bind to a soluble ActRIIB polypeptide. Such antibodies may be
generated much as described above, using a soluble ActRIIB
polypeptide or fragment thereof as an antigen. Antibodies of this
type can be used, e.g., to detect ActRIIB polypeptides in
biological samples and/or to monitor soluble ActRIIB polypeptide
levels in an individual. In certain cases, an antibody that
specifically binds to a soluble ActRIIB polypeptide can be used to
modulate activity of an ActRIIB polypeptide and/or an ActRIIB
ligand, thereby regulating (promoting or inhibiting) growth of
tissues, such as bone, cartilage, muscle, fat, and neurons.
5. Screening Assays
[0111] In certain aspects, the present invention relates to the use
of the subject ActRIIB polypeptides (e.g., soluble ActRIIB
polypeptides) to identify compounds (agents) which are agonist or
antagonists of the ActRIIB polypeptides. Compounds identified
through this screening can be tested in tissues such as bone,
cartilage, muscle, fat, and/or neurons, to assess their ability to
modulate tissue growth in vitro. Optionally, these compounds can
further be tested in animal models to assess their ability to
modulate tissue growth in vivo.
[0112] There are numerous approaches to screening for therapeutic
agents for modulating tissue growth by targeting the ActRIIB
polypeptides. In certain embodiments, high-throughput screening of
compounds can be carried out to identify agents that perturb
ActRIIB-mediated effects on growth of bone, cartilage, muscle, fat,
and/or neurons. In certain embodiments, the assay is carried out to
screen and identify compounds that specifically inhibit or reduce
binding of an ActRIIB polypeptide to its binding partner, such as
an ActRIIB ligand (e.g., activin, Nodal, GDF8, GDF11 or BMP7).
Alternatively, the assay can be used to identify compounds that
enhance binding of an ActRIIB polypeptide to its binding protein
such as an ActRIIB ligand. In a further embodiment, the compounds
can be identified by their ability to interact with an ActRIIB
polypeptide.
[0113] A variety of assay formats will suffice and, in light of the
present disclosure, those not expressly described herein will
nevertheless be comprehended by one of ordinary skill in the art.
As described herein, the test compounds (agents) of the invention
may be created by any combinatorial chemical method. Alternatively,
the subject compounds may be naturally occurring biomolecules
synthesized in vivo or in vitro. Compounds (agents) to be tested
for their ability to act as modulators of tissue growth can be
produced, for example, by bacteria, yeast, plants or other
organisms (e.g., natural products), produced chemically (e.g.,
small molecules, including peptidomimetics), or produced
recombinantly. Test compounds contemplated by the present invention
include non-peptidyl organic molecules, peptides, polypeptides,
peptidomimetics, sugars, hormones, and nucleic acid molecules. In a
specific embodiment, the test agent is a small organic molecule
having a molecular weight of less than about 2,000 daltons.
[0114] The test compounds of the invention can be provided as
single, discrete entities, or provided in libraries of greater
complexity, such as made by combinatorial chemistry. These
libraries can comprise, for example, alcohols, alkyl halides,
amines, amides, esters, aldehydes, ethers and other classes of
organic compounds. Presentation of test compounds to the test
system can be in either an isolated form or as mixtures of
compounds, especially in initial screening steps. Optionally, the
compounds may be optionally derivatized with other compounds and
have derivatizing groups that facilitate isolation of the
compounds. Non-limiting examples of derivatizing groups include
biotin, fluorescein, digoxygenin, green fluorescent protein,
isotopes, polyhistidine, magnetic beads, glutathione S transferase
(GST), photoactivatible crosslinkers or any combinations
thereof.
[0115] In many drug screening programs which test libraries of
compounds and natural extracts, high throughput assays are
desirable in order to maximize the number of compounds surveyed in
a given period of time. Assays which are performed in cell-free
systems, such as may be derived with purified or semi-purified
proteins, are often preferred as "primary" screens in that they can
be generated to permit rapid development and relatively easy
detection of an alteration in a molecular target which is mediated
by a test compound. Moreover, the effects of cellular toxicity or
bioavailability of the test compound can be generally ignored in
the in vitro system, the assay instead being focused primarily on
the effect of the drug on the molecular target as may be manifest
in an alteration of binding affinity between an ActRIIB polypeptide
and its binding protein (e.g., an ActRIIB ligand).
[0116] Merely to illustrate, in an exemplary screening assay of the
present invention, the compound of interest is contacted with an
isolated and purified ActRIIB polypeptide which is ordinarily
capable of binding to an ActRIIB ligand, as appropriate for the
intention of the assay. To the mixture of the compound and ActRIIB
polypeptide is then added a composition containing an ActRIIB
ligand. Detection and quantification of ActRIIB/ActRIIB ligand
complexes provides a means for determining the compound's efficacy
at inhibiting (or potentiating) complex formation between the
ActRIIB polypeptide and its binding protein. The efficacy of the
compound can be assessed by generating dose response curves from
data obtained using various concentrations of the test compound.
Moreover, a control assay can also be performed to provide a
baseline for comparison. For example, in a control assay, isolated
and purified ActRIIB ligand is added to a composition containing
the ActRIIB polypeptide, and the formation of ActRIIB/ActRIIB
ligand complex is quantitated in the absence of the test compound.
It will be understood that, in general, the order in which the
reactants may be admixed can be varied, and can be admixed
simultaneously. Moreover, in place of purified proteins, cellular
extracts and lysates may be used to render a suitable cell-free
assay system.
[0117] Complex formation between the ActRIIB polypeptide and its
binding protein may be detected by a variety of techniques. For
instance, modulation of the formation of complexes can be
quantitated using, for example, detectably labeled proteins such as
radiolabeled (e.g., .sup.32P, .sup.35S, .sup.14C or .sup.3H),
fluorescently labeled (e.g., FITC), or enzymatically labeled
ActRIIB polypeptide or its binding protein, by immunoassay, or by
chromatographic detection.
[0118] In certain embodiments, the present invention contemplates
the use of fluorescence polarization assays and fluorescence
resonance energy transfer (FRET) assays in measuring, either
directly or indirectly, the degree of interaction between an
ActRIIB polypeptide and its binding protein. Further, other modes
of detection, such as those based on optical waveguides (PCT
Publication WO 96/26432 and U.S. Pat. No. 5,677,196), surface
plasmon resonance (SPR), surface charge sensors, and surface force
sensors, are compatible with many embodiments of the invention.
[0119] Moreover, the present invention contemplates the use of an
interaction trap assay, also known as the "two hybrid assay," for
identifying agents that disrupt or potentiate interaction between
an ActRIIB polypeptide and its binding protein. See for example,
U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;
Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al.
(1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993)
Oncogene 8:1693-1696). In a specific embodiment, the present
invention contemplates the use of reverse two hybrid systems to
identify compounds (e.g., small molecules or peptides) that
dissociate interactions between an ActRIIB polypeptide and its
binding protein. See for example, Vidal and Legrain, (1999) Nucleic
Acids Res 27:919-29; Vidal and Legrain, (1999) Trends Biotechnol
17:374-81; and U.S. Pat. Nos. 5,525,490; 5,955,280; and
5,965,368.
[0120] In certain embodiments, the subject compounds are identified
by their ability to interact with an ActRIIB polypeptide of the
invention. The interaction between the compound and the ActRIIB
polypeptide may be covalent or non-covalent. For example, such
interaction can be identified at the protein level using in vitro
biochemical methods, including photo-crosslinking, radiolabeled
ligand binding, and affinity chromatography (Jakoby W B et al.,
1974, Methods in Enzymology 46: 1). In certain cases, the compounds
may be screened in a mechanism based assay, such as an assay to
detect compounds which bind to an ActRIIB polypeptide. This may
include a solid phase or fluid phase binding event. Alternatively,
the gene encoding an ActRIIB polypeptide can be transfected with a
reporter system (e.g., .beta.-galactosidase, luciferase, or green
fluorescent protein) into a cell and screened against the library
preferably by a high throughput screening or with individual
members of the library. Other mechanism based binding assays may be
used, for example, binding assays which detect changes in free
energy. Binding assays can be performed with the target fixed to a
well, bead or chip or captured by an immobilized antibody or
resolved by capillary electrophoresis. The bound compounds may be
detected usually using colorimetric or fluorescence or surface
plasmon resonance.
[0121] In certain aspects, the present invention provides methods
and agents for stimulating muscle growth and increasing muscle
mass, for example, by antagonizing functions of an ActRIIB
polypeptide and/or an ActRIIB ligand. Therefore, any compound
identified can be tested in whole cells or tissues, in vitro or in
vivo, to confirm their ability to modulate muscle growth. Various
methods known in the art can be utilized for this purpose. For
example, methods of the invention are performed such that the
signal transduction through an ActRIIB protein activated by binding
to an ActRIIB ligand (e.g., GDF8) has been reduced or inhibited. It
will be recognized that the growth of muscle tissue in the organism
would result in an increased muscle mass in the organism as
compared to the muscle mass of a corresponding organism (or
population of organisms) in which the signal transduction through
an ActRIIB protein had not been so effected.
[0122] For example, the effect of the ActRIIB polypeptides or test
compounds on muscle cell growth/proliferation can be determined by
measuring gene expression of Pax-3 and Myf-5 which are associated
with proliferation of myogenic cells, and gene expression of MyoD
which is associated with muscle differentiation (e.g., Amthor et
al., Dev Biol. 2002, 251:241-57). It is known that GDF8
down-regulates gene expression of Pax-3 and Myf-5, and prevents
gene expression of MyoD. The ActRIIB polypeptides or test compounds
are expected to antagonize this activity of GDF8. Another example
of cell-based assays includes measuring the proliferation of
myoblasts such as C(2)C(12) myoblasts in the presence of the
ActRIIB polypeptides or test compounds (e.g., Thomas et al., J Biol
Chem. 2000, 275:40235-43).
[0123] The present invention also contemplates in vivo assays to
measure muscle mass and strength. For example, Whittemore et al.
(Biochem Biophys Res Commun. 2003, 300:965-71) discloses a method
of measuring increased skeletal muscle mass and increased grip
strength in mice. Optionally, this method can be used to determine
therapeutic effects of test compounds (e.g., ActRIIB polypeptides)
on muscle diseases or conditions, for example those diseases for
which muscle mass is limiting.
[0124] In certain aspects, the present invention provides methods
and agents for modulating (stimulating or inhibiting) bone
formation and increasing bone mass. Therefore, any compound
identified can be tested in whole cells or tissues, in vitro or in
vivo, to confirm their ability to modulate bone or cartilage
growth. Various methods known in the art can be utilized for this
purpose.
[0125] For example, the effect of the ActRIIB polypeptides or test
compounds on bone or cartilage growth can be determined by
measuring induction of Msx2 or differentiation of osteoprogenitor
cells into osteoblasts in cell based assays (see, e.g., Daluiski et
al., Nat Genet. 2001, 27(1):84-8; Hino et al., Front Biosci. 2004,
9:1520-9). Another example of cell-based assays includes analyzing
the osteogenic activity of the subject ActRIIB polypeptides and
test compounds in mesenchymal progenitor and osteoblastic cells. To
illustrate, recombinant adenoviruses expressing an ActRIIB
polypeptide were constructed to infect pluripotent mesenchymal
progenitor C3H10T1/2 cells, preosteoblastic C2C12 cells, and
osteoblastic TE-85 cells. Osteogenic activity is then determined by
measuring the induction of alkaline phosphatase, osteocalcin, and
matrix mineralization (see, e.g., Cheng et al., J bone Joint Surg
Am. 2003, 85-A(8):1544-52).
[0126] The present invention also contemplates in vivo assays to
measure bone or cartilage growth. For example, Namkung-Matthai et
al., Bone, 28:80-86 (2001) discloses a rat osteoporotic model in
which bone repair during the early period after fracture is
studied. Kubo et al., Steroid Biochemistry & Molecular Biology,
68:197-202 (1999) also discloses a rat osteoporotic model in which
bone repair during the late period after fracture is studied. These
references are incorporated by reference herein in their entirety
for their disclosure of rat model for study on osteoporotic bone
fracture. In certain aspects, the present invention makes use of
fracture healing assays that are known in the art. These assays
include fracture technique, histological analysis, and
biomechanical analysis, which are described in, for example, U.S.
Pat. No. 6,521,750, which is incorporated by reference in its
entirety for its disclosure of experimental protocols for causing
as well as measuring the extent of fractures, and the repair
process.
[0127] In certain aspects, the present invention provides methods
and agents for controlling weight gain and obesity. At the cellular
level, adipocyte proliferation and differentiation is critical in
the development of obesity, which leads to the generation of
additional fat cells (adipocytes). Therefore, any compound
identified can be tested in whole cells or tissues, in vitro or in
vivo, to confirm their ability to modulate adipogenesis by
measuring adipocyte proliferation or differentiation. Various
methods known in the art can be utilized for this purpose. For
example, the effect of an ActRIIB polypeptide (e.g., a soluble
ActRIIB polypeptide) or test compounds on adipogenesis can be
determined by measuring differentiation of 3T3-L1 preadipocytes to
mature adipocytes in cell based assays, such as, by observing the
accumulation of triacylglycerol in Oil Red O staining vesicles and
by the appearance of certain adipocyte markers such as FABP
(aP2/422) and PPAR.gamma.2. See, for example, Reusch et al., 2000,
Mol Cell Biol. 20:1008-20; Deng et al., 2000, Endocrinology.
141:2370-6; Bell et al., 2000, Obes Res. 8:249-54. Another example
of cell-based assays includes analyzing the role of ActRIIB
polypeptides and test compounds in proliferation of adipocytes or
adipocyte precursor cells (e.g., 3T3-L1 cells), such as, by
monitoring bromodeoxyuridine (BrdU)-positive cells. See, for
example, Pico et al., 1998, Mol Cell Biochem. 189:1-7; Masuno et
al., 2003, Toxicol Sci. 75:314-20.
[0128] It is understood that the screening assays of the present
invention apply to not only the subject ActRIIB polypeptides and
variants of the ActRIIB polypeptides, but also any test compounds
including agonists and antagonist of the ActRIIB polypeptides.
Further, these screening assays are useful for drug target
verification and quality control purposes.
6. Exemplary Therapeutic Uses
[0129] In certain embodiments, compositions (e.g., ActRIIB
polypeptides) of the present invention can be used for treating or
preventing hypoadiponectinemia and interrelated 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.
[0130] 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.
[0131] As demonstrated herein, ActRIIB-Fc administration in vivo
promotes expression of adiponectin in white adipose tissue and
increases circulating adiponectin levels in diverse mouse models.
Accordingly, compositions disclosed herein may be used to treat or
prevent hypoadiponectinemia and associated disorders, including to
subset of patients with atherosclerosis, ischemic stroke, impaired
glucose tolerance, insulin resistance, diabetes type 2,
hyperlipidemia, hypertriglyceridemia, or obesity that also exhibit
low circulating adiponectin.
[0132] In other related embodiments, soluble ActRIIB polypeptides
and other compositions of the invention can be used as part of
treatment or prevention of 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.
[0133] Soluble ActRIIB polypeptides can also be used for treatment
or prevention of 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.
[0134] In related embodiments, soluble ActRIIB polypeptides and
other compositions of the invention can be used as part of
treatment or prevention of 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.
[0135] The subject ActRIIB polypeptides may further be used as a
therapeutic agent for slowing or preventing the development of
obesity. This approach is confirmed and supported by the data shown
herein, whereby an ActRIIB-Fc protein was shown to improve
metabolic status in mice on a high-fat diet.
[0136] 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.
[0137] In one specific embodiment, the present invention relates to
methods and compounds for reducing body weight and/or reducing
weight gain 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.
[0138] As demonstrated in WO 2006/012627 and WO 2008/097541,
compounds disclosed herein stimulate muscle growth. Accordingly,
these compounds may be particularly useful in diseases or
conditions with overlapping muscle and metabolic dysfunction.
[0139] In certain embodiments, compositions (e.g., soluble ActRIIB
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.
Moreover, declining muscle mass and diminishing physical activity
contribute to an imbalance between caloric intake and energy
expenditure, leading to unhealthy storage of excess energy as white
adipose tissue. 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).
[0140] 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.
[0141] 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.
[0142] 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.
[0143] ActRIIB polypeptide-induced increased muscle mass might also
benefit those suffering from muscle wasting diseases.
Gonzalez-Cadavid et al. (1998, PNAS 95:14938-43) reported 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.
[0144] Sarcopenia, the loss of muscle with aging is also often
associated with metabolic syndrome, diabetes, arteriosclerosis,
dyslipidemia, and other age-related metabolic conditions. ActRIIB
polypeptide-induced muscle mass might also benefit those suffering
from sarcopenia.
[0145] In particular, the present disclosure demonstrates that in
certain conditions, such as androgen deprivation, agents disclosed
herein can be used to promote muscle and bone formation while
decreasing adiposity, and therefore, the disclosure provides
methods for treating patients exhibiting low bone and muscle
content and elevated adiposity may be advantageously treated with
soluble ActRIIB polypeptides and other agents disclosed herein.
This may be particularly beneficial in patients receiving androgen
or estrogen antagonist therapy, elderly patients (e.g., combined
sarcopenia, osteoporosis and obesity) and patients with a muscle
wasting condition that are also receiving corticosteroid
therapy.
[0146] 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.
[0147] 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.
7 Pharmaceutical Compositions
[0148] 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.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] 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 nonaqueous 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.
[0153] 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.
[0154] 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.
[0155] 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.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] 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.
[0160] 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.
[0161] 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. In the case of muscle disorders, factors may include,
but are not limited to, amount of muscle mass desired to be formed,
the muscles most affected by disease, the condition of the
deteriorated muscle, the patient's age, sex, and diet, time of
administration, and other clinical factors. The addition of other
known growth factors to the final composition, may also affect the
dosage. Progress can be monitored by periodic assessment of muscle
growth and/or repair, for example, by strength testing, Mill
assessment of muscle size and analysis of muscle biopsies.
[0162] In certain embodiments of the invention, one or more ActRIIB
polypeptides can be administered, together (simultaneously) or at
different times (sequentially or overlapping). In addition, ActRIIB
polypeptides can be administered with another type of therapeutic
agents, for example, a cartilage-inducing agent, a bone-inducing
agent, a muscle-inducing agent, a fat-reducing, or a
neuron-inducing agent. The two types of compounds may be
administered simultaneously or at different times. It is expected
that the ActRIIB polypeptides of the invention may act in concert
with or perhaps synergistically with another therapeutic agent.
[0163] In a specific example, a variety of osteogenic,
cartilage-inducing and bone-inducing factors have been described,
particularly bisphosphonates. See e.g., European Patent Application
Nos. 148,155 and 169,016. For example, other factors that can be
combined with the subject ActRIIB polypeptides include various
growth factors such as epidermal growth factor (EGF), platelet
derived growth factor (PDGF), transforming growth factors
(TGF-.alpha. and TGF-.beta.), and insulin-like growth factor
(IGF).
[0164] In certain embodiments, the present invention also provides
gene therapy for the in vivo production of ActRIIB polypeptides.
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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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
[0169] 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 an ActRIIb-Fc Fusion Protein
[0170] Applicants constructed a soluble ActRIIb fusion protein that
has the extracellular domain of human ActRIIb fused to a human or
mouse Fc domain with a minimal linker (three glycine amino acids)
in between. The constructs are referred to as ActRIIb-hFc and
ActRIIb-mFc, respectively.
[0171] ActRIIb-hFc is shown below as purified from CHO cell lines
(SEQ ID NO: 5)
TABLE-US-00002 GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGT
IELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA
GGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLEPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0172] The ActRIIb-hFc and ActRIIb-mFc proteins were expressed in
CHO cell lines. Three different leader sequences were
considered:
(i) Honey bee mellitin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO:
7)
(ii) Tissue Plasminogen Activator (TPA): MDAMKRGLCCVLLLCGAVFVSP
(SEQ ID NO: 8)
[0173] (iii) Native: MGAAAKLAFAVFLISCSSGA (SEQ ID NO: 9).
[0174] The selected form employs the TPA leader and has the
following unprocessed amino acid sequence:
TABLE-US-00003 (SEQ ID NO: 32)
MDAMKRGLCCVLLLCGAVFVSPGASGRGEAETRECIYYNANWELERTNQS
GLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATE
ENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPC
PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
VPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGK.
[0175] This polypeptide is encoded by the following nucleic acid
sequence (SEQ ID NO:10):
TABLE-US-00004 A TGGATGCAAT GAAGAGAGGG CTCTGCTGTG TGCTGCTGCT
GTGTGGAGCA GTCTTCGTTT CGCCCGGCGC CTCTGGGCGT GGGGAGGCTG AGACACGGGA
GTGCATCTAC TACAACGCCA ACTGGGAGCT GGAGCGCACC AACCAGAGCG GCCTGGAGCG
CTGCGAAGGC GAGCAGGACA AGCGGCTGCA CTGCTACGCC TCCTGGCGCA ACAGCTCTGG
CACCATCGAG CTCGTGAAGA AGGGCTGCTG GCTAGATGAC TTCAACTGCT ACGATAGGCA
GGAGTGTGTG GCCACTGAGG AGAACCCCCA GGTGTACTTC TGCTGCTGTG AAGGCAACTT
CTGCAACGAG CGCTTCACTC ATTTGCCAGA GGCTGGGGGC CCGGAAGTCA CGTACGAGCC
ACCCCCGACA GCCCCCACCG GTGGTGGAAC TCACACATGC CCACCGTGCC CAGCACCTGA
ACTCCTGGGG GGACCGTCAG TCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGAT
CTCCCGGACC CCTGAGGTCA CATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT
CAAGTTCAAC TGGTACGTGG ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA
GGAGCAGTAC AACAGCACGT ACCGTGTGGT CAGCGTCCTC ACCGTCCTGC ACCAGGACTG
GCTGAATGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA GCCCTCCCAG TCCCCATCGA
GAAAACCATC TCCAAAGCCA AAGGGCAGCC CCGAGAACCA CAGGTGTACA CCCTGCCCCC
ATCCCGGGAG GAGATGACCA AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA
TCCCAGCGAC ATCGCCGTGG AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC
CACGCCTCCC GTGCTGGACT CCGACGGCTC CTTCTTCCTC TATAGCAAGC TCACCGTGGA
CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC GTGATGCATG AGGCTCTGCA
CAACCACTAC ACGCAGAAGA GCCTCTCCCT GTCTCCGGGT AAATGA
[0176] N-terminal sequencing of the CHO-cell produced material
revealed a major sequence of -GRGEAE (SEQ ID NO: 11). Notably,
other constructs reported in the literature begin with an -SGR . .
. sequence.
[0177] Purification could be achieved by 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.
[0178] ActRIIb-Fc fusion proteins were also expressed in HEK293
cells and COS cells. Although material from all cell lines and
reasonable culture conditions provided protein with muscle-building
activity in vivo, variability in potency was observed perhaps
relating to cell line selection and/or culture conditions.
Example 2
Generation of ActRIIb-Fc Mutants
[0179] Applicants generated a series of mutations in the
extracellular domain of ActRIIB and produced these mutant proteins
as soluble fusion proteins between extracellular ActRIIB and an Fc
domain. The background ActRIIB-Fc fusion has the sequence (Fc
portion underlined)(SEQ ID NO:12):
TABLE-US-00005 SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSG
TIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPE
AGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLEPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0180] Various mutations, including N- and C-terminal truncations,
were introduced into the background ActRIIB-Fc protein. Based on
the data presented in Example 1, it is expected that these
constructs, if expressed with a TPA leader, will lack the
N-terminal serine. Mutations were generated in ActRIIB
extracellular domain by PCR mutagenesis. After PCR, fragments were
purified through a Qiagen column, digested with SfoI and AgeI and
gel purified. These fragments were ligated into expression vector
pAID4 (see WO2006/012627) such that upon ligation it created fusion
chimera with human IgG1. Upon transformation into E. coli DH5
alpha, colonies were picked and DNAs were isolated. For murine
constructs (mFc), a murine IgG2a was substituted for the human
IgG1. All mutants were sequence verified.
[0181] All of the mutants were produced in HEK293T cells by
transient transfection. In summary, in a 500 ml spinner, HEK293T
cells were set up at 6.times.10.sup.5 cells/ml in Freestyle
(Invitrogen) media in 250 ml volume and grown overnight. Next day,
these cells were treated with DNA:PEI (1:1) complex at 0.5 ug/ml
final DNA concentration. After 4 hrs, 250 ml media was added and
cells were grown for 7 days. Conditioned media was harvested by
spinning down the cells and concentrated.
[0182] Mutants were purified using a variety of techniques,
including, for example, protein A column and eluted with low pH
(3.0) glycine buffer. After neutralization, these were dialyzed
against PBS.
[0183] Mutants were also produced in CHO cells by similar
methodology.
[0184] Mutants were tested in binding assays and/or bioassays. In
some instances, assays were performed with conditioned medium
rather than purified proteins.
Example 3
Generation of Truncated Variant ActRIIB(25-131)-hFc
[0185] Applicants generated a truncated fusion protein,
ActRIIB(25-131)-hFc (FIGS. 7-8), which exhibits effects on muscle
that are similar to those observed with ActRIIB(20-134)-hFc (while
exhibiting superior effects on other tissues and parameters).
ActRIIB(25-131)-hFc was generated using the same leader and
methodology as described above with respect to ActRIIB(20-134)-hFc.
The mature ActRIIB(25-131)-hFc protein purified after expression in
CHO cells has the sequence shown below (SEQ ID NO: 23). Amino acids
1-107 (underlined) are derived from ActRIIB.
TABLE-US-00006 (SEQ ID NO: 23) FTRECIYYNA 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
[0186] 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 4
High-Affinity Ligand Binding by ActRIIB(25-131)-hFc
[0187] 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-00007 [0188] 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 5
Bioassay for GDF-11 and Activin-Mediated Signaling
[0189] An A-204 Reporter Gene Assay was used to evaluate the
effects of ActRIIB-Fc proteins on signaling by GDF-11 and Activin
A. Cell line: Human Rhabdomyosarcoma (derived from muscle).
Reporter vector: pGL3(CAGA)12 (Described in Dennler et al, 1998,
EMBO 17: 3091-3100.) See FIG. 5. The CAGA12 motif is present in
TGF-Beta responsive genes (PAI-1 gene), so this vector is of
general use for factors signaling through Smad2 and 3.
[0190] Day 1: Split A-204 cells into 48-well plate.
[0191] Day 2: A-204 cells transfected with 10 ug pGL3(CAGA)12 or
pGL3(CAGA)12 (10 ug)+pRLCMV (1 ug) and Fugene.
[0192] Day 3: Add factors (diluted into medium+0.1% BSA).
Inhibitors need to be preincubated with Factors for 1 hr before
adding to cells. 6 hrs later, cells rinsed with PBS, and lyse
cells.
[0193] This is followed by a Luciferase assay. In the absence of
any inhibitors, Activin A showed 10 fold stimulation of reporter
gene expression and an ED50.about.2 ng/ml. GDF-11: 16 fold
stimulation, ED50: .about.1.5 ng/ml.
[0194] ActRIIB(R64, 20-134) is a potent inhibitor of activin, GDF-8
and GDF-11 activity in this assay. Variants were tested in this
assay as well.
Example 6
GDF-11 Inhibition by N-Terminal and C-Terminal Truncations
[0195] Truncations at the N-terminus and C-terminus of the ActRIIB
portion ActRIIB-Fc (R64, 20-134) were generated and tested for
activity as inhibitors of GDF-11 and activin. The activities are
shown below (as measured in conditioned media):
C-Terminal ActRIIb-hFc Truncations:
TABLE-US-00008 [0196] IC50 (ng/mL) GDF-11 Activin ActRIIb-hFc (R64,
20-134) 45 2 ActRIIb-hFc (R64, 20-132) 87 32 ActRIIb-hFc (R64,
20-131) 120 44 ActRIIb-hFc (R64, 20-128) 130 158
[0197] As can be seen, truncations of three (ending with . . .
PPT), six (ending with . . . YEP) or more amino acids at the
C-terminus causes a threefold or greater decrease in the activity
of the molecule. The truncation of the final 15 amino acids of the
ActRIIB portion causes a greater loss of activity (see
WO2006/012627).
[0198] Amino terminal truncations were made in the background of an
ActRIIb-hFc (R64 20-131) protein. The activities are shown below
(as measured in conditioned media):
N-Terminal ActRIIb-hFc Truncations:
TABLE-US-00009 [0199] IC50 (ng/mL) GDF-11 Activin ActRIIb-hFc (R64,
20-131) 183 201 (GRG . . . ) ActRIIb-hFc (R64, 21-131) 121 325 (RGE
. . . ) ActRIIb-hFc (R64, 22-131) 71 100 (GEA . . . ) ActRIIb-hFc
(R64, 23-131) 60 43 (EAE . . . ) ActRIIb-hFc (R64, 24-131) 69 105
(AET . . . )
[0200] Accordingly, truncations of two, three or four amino acids
from the N-terminus lead to the production of a more active protein
than the versions with a full-length extracellular domain.
Additional experiments show that a truncation of five amino acids,
ActRIIb-hFc (R64, 25-131) has activity equivalent to the
untruncated form, and additional deletions at the N-terminus
continue to degrade the activity of the protein. Therefore, optimal
constructs will have a C-terminus ending between amino acid 133-134
of SEQ ID NO:4 and an N-terminus beginning at amino acids 22-24 of
SEQ ID NO:4. An N-terminus corresponding to amino acids 21 or 25
will give activity that is similar to the ActRIIb-hFc (R64, 20-134)
construct, although the protein designated as SEQ ID NO: 23 has
been characterized as having superior effects in some regards.
Example 7
ActRIIb-Fc Variants, Cell-Based Activity
[0201] Activity of ActRIIB-Fc proteins was tested in a cell based
assay, as described above. Results are summarized in Table 1,
below. Some variants were tested in different C-terminal truncation
constructs. As discussed above, truncations of five or fifteen
amino acids caused reduction in activity. Remarkably, the L79D and
L79E variants showed substantial loss of activin binding while
retaining almost wild-type inhibition of GDF-11.
Soluble ActRIIB-Fc Binding to GDF11 and Activin A:
TABLE-US-00010 [0202] Portion of ActRIIB (corresponds to amino
GDF11 Activin ActRIIB-Fc acids of SEQ ID Inhibition Inhibition
Variations NO: 4) Activity Activity 64R 20-134 +++ +++ (approx.
(approx. 10.sup.-8 M K.sub.1) 10.sup.-8 M K.sub.1) 64A 20-134 + +
(approx. (approx. 10.sup.-6 M K.sub.1) 10.sup.-6 M K.sub.1) 64R
20-129 +++ +++ 64R K74A 20-134 ++++ ++++ 64R A24N 20-134 +++ +++
64R A24N 20-119 ++ ++ 64R A24N K74A 20-119 + + R64 L79P 20-134 + +
R64 L79P K74A 20-134 + + R64 L79D 20-134 +++ + R64 L79E 20-134 +++
+ R64K 20-134 +++ +++ R64K 20-129 +++ +++ R64 P129S P130A 20-134
+++ +++ R64N 20-134 + + + Poor activity (roughly 1 .times.
10.sup.-6 K.sub.1) ++ Moderate activity (roughly 1 .times.
10.sup.-7 K.sub.1) +++ Good (wild-type) activity (roughly 1 .times.
10.sup.-8 K.sub.1) ++++ Greater than wild-type activity
Example 8
GDF-11 and Activin A Binding
[0203] Binding of certain ActRIIB-Fc proteins to ligands was tested
in a BiaCore.TM. assay.
[0204] The ActRIIB-Fc variants or wild-type protein were captured
onto the system using an anti-hFc antibody. Ligands were injected
and flowed over the captured receptor proteins. Results are
summarized in tables, below.
Ligand Binding Specificity IIB Variants.
TABLE-US-00011 [0205] GDF11 Protein Kon (1/Ms) Koff (1/s) KD (M)
ActRIIB-hFc (R64 20-134) 1.34e-6 1.13e-4 8.42e-11 ActRIIB-hFc (R64,
A24N 20- 1.21e-6 6.35e-5 5.19e-11 134) ActRIIB-hFc (R64, L79D 20-
6.7e-5 4.39e-4 6.55e-10 134) ActRIIB-hFc (R64, L79E 20- 3.8e-5
2.74e-4 7.16e-10 134) ActRIIB-hFc (R64K 20-134) 6.77e-5 2.41e-5
3.56e-11 GDF8 Protein Kon (1/Ms) Koff (1/s) KD (M) ActRIIB-hFc (R64
20-134) 3.69e-5 3.45e-5 9.35e-11 ActRIIB-hFc (R64, A24N 20- 134)
ActRIIB-hFc (R64, L79D 20- 3.85e-5 8.3e-4 2.15e-9 134) ActRIIB-hFc
(R64, L79E 20- 3.74e-5 9e-4 2.41e-9 134) ActRIIB-hFc (R64K 20-134)
2.25e-5 4.71e-5 2.1e-10 ActRIIB-hFc (R64K 20-129) 9.74e-4 2.09e-4
2.15e-9 ActRIIB-hFc (R64, P129S, 1.08e-5 1.8e-4 1.67e-9 P130R
20-134) ActRIIB-hFc (R64, K74A 20- 2.8e-5 2.03e-5 7.18e-11 134)
ActivinA Protein Kon (1/Ms) Koff (1/s) KD (M) ActRIIB-hFc (R64
20-134) 5.94e6 1.59e-4 2.68e-11 ActRIIB-hFc (R64, A24N 20- 3.34e6
3.46e-4 1.04e-10 134) ActRIIB-hFc (R64, L79D 20- Low binding 134)
ActRIIB-hFc (R64, L79E 20- Low binding 134) ActRIIB-hFc (R64K
20-134) 6.82e6 3.25e-4 4.76e-11 ActRIIB-hFc (R64K 20-129) 7.46e6
6.28e-4 8.41e-11 ActRIIB-hFc (R64, P129S, 5.02e6 4.17e-4 8.31e-11
P130R 20-134)
[0206] Other variants have been generated and tested, as reported
in WO2006/012627, using ligands coupled to the device and flowing
receptor over the coupled ligands. A table of data with respect to
these variants is reproduced below:
Soluble ActRIIB-Fc Variants Binding to GDF11 and Activin A (BiaCore
Assay)
TABLE-US-00012 [0207] ActRIIB ActA GDF11 WT (64A) KD = 1.8e-7M KD =
2.6e-7M (+) (+) WT (64R) na KD = 8.6e-8M (+++) +15tail KD~2.6 e-8M
KD = 1.9e-8M (+++) (++++) E37A * * R40A - - - D54A - * K55A ++ *
R56A * * K74A KD = 4.35e-9M KD = 5 .3 e-9M +++++ +++++ K74Y * --
K74F * -- K74I * -- W78A * * L79A + * D80K * * D80R * * D80A * *
D80F * * D80G * * D80M * * D80N * * D80I * -- F82A ++ - * No
observed binding -- <1/5 WT binding - ~1/2 WT binding + WT ++
<2x increased binding +++ ~5x increased binding ++++ ~10x
increased binding +++++ ~40x increased binding
Example 9
Effect of ActRIIB-Fc on Bone Loss and Adiposity Caused by
Orchidectomy
[0208] Androgen-deprivation therapy, most prominently used in the
treatment of prostate cancer, can cause pathological loss of muscle
and bone, as well as enlargement of adipose tissue. Applicants
investigated effects of ActRIIB-Fc in the orchidectomized (ORX)
mouse, an animal model which mimics many of the changes associated
with androgen deprivation. Nine-week-old C57BL/6 mice were ORX or
sham-operated, and ten days later treatment was initiated with
ActRIIB(R64 20-134)-mFc or Tris-buffered-saline (TBS) vehicle (n=10
per group) twice per week at 10 mg/kg, i.p., for a period of 10
weeks (71 days).
[0209] In this experiment, ActRIIB-mFc treatment increased body
weight as the net effect of beneficial changes in muscle mass, bone
mass, and fat mass. As shown in FIG. 9, ActRIIB-mFc increased the
rate of body weight gain, compared to controls, under ORX
conditions as well as gonad-intact conditions. This effect was due
to a pronounced increase in lean body mass. Whereas ORX controls
showed a slight decline in lean body mass over 10 weeks, ORX mice
treated with ActRIIB-mFc displayed a marked increase in lean body
mass, reaching a mean value 25% higher than controls at study
completion (FIG. 10). A similar increase was observed under
gonad-intact conditions for ActRIIB-mFc compared to vehicle (FIG.
10). Part of this increase in lean body mass was due to a
stimulatory effect of ActRIIB-mFc on muscle mass under both ORX
conditions and gonad-intact conditions, as exemplified by three
different skeletal muscles (FIG. 11).
[0210] ActRIIB-mFc exerted a series of beneficial effects on bone.
As determined by whole-body analysis with dual energy X-ray
absorptiometry (DEXA), ActRIIB-mFc prevented progressive decreases
in bone area and bone mineral content evident under ORX conditions
and led to significantly increased bone area and bone mineral
content under gonad-intact conditions (FIGS. 12, 13). ActRIIB-mFc
also increased whole-body bone mineral density under ORX conditions
(FIG. 14). Moreover, micro-CT analysis of trabecular bone in the
proximal tibia revealed that ActRIIB-mFc treatment restored several
bone parameters in ORX mice to levels observed in gonad-intact
controls. With respect to ORX controls, these changes included: 1)
a tripling of the bone volume fraction (FIG. 15), 2) a doubling of
trabecular number (FIG. 16), 3) increased trabecular thickness
(FIG. 17), and 4) reduced trabecular separation (FIG. 18). The
similarity of tibial morphology in ORX mice treated with
ActRIIB-mFc to that in gonad-intact controls is evident from images
shown in FIG. 19. For each of the foregoing tibia-based endpoints,
ActRIIB-mFc also produced changes in gonad-intact mice comparable
in direction and magnitude to those in ORX mice (FIGS. 15-18).
[0211] ActRIIB-mFc also exerted beneficial effects on fat mass. As
determined by NMR, total fat mass in ORX controls tripled over the
course of the study. ActRIIB-mFc treatment in ORX mice cut this
increase by more than 60%, restoring fat mass under ORX conditions
to levels observed in gonad-intact controls (FIG. 20). ActRIIB-mFc
also reduced the gain in fat mass observed in gonad-intact mice
during the study. Consistent with these results, a histologic
survey of fat depots indicated that ActRIIB-mFc reduced adipocyte
size in subcutaneous and epididymal depots but not appreciably in
interscapular brown fat (FIG. 21).
[0212] Finally, ActRIIB-mFc treatment altered circulating
concentrations of adiponectin and leptin, endocrine molecules
originating in adipose tissue (adipokines). There is general
agreement that 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. Moreover, low adiponectin levels are associated
with cardiovascular risk factors even in nonobese healthy
individuals (Im et al., 2006, Metabolism 55:1546-1550). Thus, it is
important that ActRIIB-mFc treatment increased serum adiponectin
concentrations significantly in both ORX and gonad-intact mice
compared to their vehicle-treated counterparts (FIG. 22). The
higher adiponectin concentrations in ORX mice compared to their
gonad-intact counterparts are consistent with the known inhibitory
effect of androgen on adiponectin (Nishizawa et al., 2002, Diabetes
51:2734-2741). ActRIIB-mFc also reduced serum concentrations of
leptin, another indicator of adipocyte status, in both ORX and
gonad-intact mice compared to vehicle (FIG. 23).
[0213] Taken together, these data indicate that soluble ActRIIB-Fc
chimeras can be used as antagonists of signaling by TGF-.beta.
family ligands in males to treat bone loss and increased adiposity
arising from androgen deprivation and potentially other conditions
as well.
Example 10
Effect of ActRIIB-Fc Variants on Adiponectin Levels in Mice Fed a
High-Fat Diet
[0214] Applicants investigated the effects of ActRIIB(20-134)-hFc
or ActRIIB(25-131)-hFc on circulating concentrations of adiponectin
in male mice fed a high-fat diet. Ten-week-old C57BL/6 mice were
weight-matched and treated subcutaneously with ActRIIB(20-134)-hFc
(10 mg/kg), ActRIIB(25-131)-hFc (10 mg/kg), or Tris-buffered-saline
(TBS) vehicle twice per week 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. An additional group of mice
maintained on the standard chow diet was also treated with TBS
vehicle and followed as a dietary control. By Day 60,
ActRIIB(20-134)-hFc treatment increased serum adiponectin
concentrations in mice fed the high-fat diet to approximately the
same levels seen in mice fed the standard diet, while
ActRIIB(25-131)-hFc treatment raised serum adiponectin
concentrations significantly beyond these control levels (FIG. 24).
Contributing to elevated adiponectin concentrations was an increase
in adiponectin gene expression in white fat. Analysis of white
adipose tissue by real-time polymerase chain reaction (RT-PCR)
revealed that ActRIIB(25-131)-hFc increased adiponectin mRNA levels
by more than 60% compared to high-fat diet controls (FIG. 25).
[0215] Taken together, these findings demonstrate that ActRIIB-Fc
proteins can be used in vivo to increase adiponectin gene
expression in white adipose tissue and to increase circulating
adioponectin levels under a variety of physiological
conditions.
INCORPORATION BY REFERENCE
[0216] 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.
[0217] 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
341116PRTHomo sapiens 1Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys
Ile Tyr Tyr Asn Ala 1 5 10 15 Asn Trp Glu Leu Glu Arg Thr Asn Gln
Ser Gly Leu Glu Arg Cys Glu 20 25 30 Gly Glu Gln Asp Lys Arg Leu
His Cys Tyr Ala Ser Trp Arg Asn Ser 35 40 45 Ser Gly Thr Ile Glu
Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe 50 55 60 Asn Cys Tyr
Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln 65 70 75 80 Val
Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr 85 90
95 His Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro
100 105 110 Thr Ala Pro Thr 115 2512PRTHomo sapiens 2Met Thr Ala
Pro Trp Val Ala Leu Ala Leu Leu Trp Gly Ser Leu Trp 1 5 10 15 Pro
Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr 20 25
30 Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg
35 40 45 Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser
Trp Arg 50 55 60 Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly
Cys Trp Leu Asp 65 70 75 80 Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys
Val Ala Thr Glu Glu Asn 85 90 95 Pro Gln Val Tyr Phe Cys Cys Cys
Glu Gly Asn Phe Cys Asn Glu Arg 100 105 110 Phe Thr His Leu Pro Glu
Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro 115 120 125 Pro Pro Thr Ala
Pro Thr Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu 130 135 140 Pro Ile
Gly Gly Leu Ser Leu Ile Val Leu Leu Ala Phe Trp Met Tyr 145 150 155
160 Arg His Arg Lys Pro Pro Tyr Gly His Val Asp Ile His Glu Asp Pro
165 170 175 Gly Pro Pro Pro Pro Ser Pro Leu Val Gly Leu Lys Pro Leu
Gln Leu 180 185 190 Leu Glu Ile Lys Ala Arg Gly Arg Phe Gly Cys Val
Trp Lys Ala Gln 195 200 205 Leu Met Asn Asp Phe Val Ala Val Lys Ile
Phe Pro Leu Gln Asp Lys 210 215 220 Gln Ser Trp Gln Ser Glu Arg Glu
Ile Phe Ser Thr Pro Gly Met Lys 225 230 235 240 His Glu Asn Leu Leu
Gln Phe Ile Ala Ala Glu Lys Arg Gly Ser Asn 245 250 255 Leu Glu Val
Glu Leu Trp Leu Ile Thr Ala Phe His Asp Lys Gly Ser 260 265 270 Leu
Thr Asp Tyr Leu Lys Gly Asn Ile Ile Thr Trp Asn Glu Leu Cys 275 280
285 His Val Ala Glu Thr Met Ser Arg Gly Leu Ser Tyr Leu His Glu Asp
290 295 300 Val Pro Trp Cys Arg Gly Glu Gly His Lys Pro Ser Ile Ala
His Arg 305 310 315 320 Asp Phe Lys Ser Lys Asn Val Leu Leu Lys Ser
Asp Leu Thr Ala Val 325 330 335 Leu Ala Asp Phe Gly Leu Ala Val Arg
Phe Glu Pro Gly Lys Pro Pro 340 345 350 Gly Asp Thr His Gly Gln Val
Gly Thr Arg Arg Tyr Met Ala Pro Glu 355 360 365 Val Leu Glu Gly Ala
Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg Ile 370 375 380 Asp Met Tyr
Ala Met Gly Leu Val Leu Trp Glu Leu Val Ser Arg Cys 385 390 395 400
Lys Ala Ala Asp Gly Pro Val Asp Glu Tyr Met Leu Pro Phe Glu Glu 405
410 415 Glu Ile Gly Gln His Pro Ser Leu Glu Glu Leu Gln Glu Val Val
Val 420 425 430 His Lys Lys Met Arg Pro Thr Ile Lys Asp His Trp Leu
Lys His Pro 435 440 445 Gly Leu Ala Gln Leu Cys Val Thr Ile Glu Glu
Cys Trp Asp His Asp 450 455 460 Ala Glu Ala Arg Leu Ser Ala Gly Cys
Val Glu Glu Arg Val Ser Leu 465 470 475 480 Ile Arg Arg Ser Val Asn
Gly Thr Thr Ser Asp Cys Leu Val Ser Leu 485 490 495 Val Thr Ser Val
Thr Asn Val Asp Leu Pro Pro Lys Glu Ser Ser Ile 500 505 510
3348DNAHomo sapiens 3tctgggcgtg 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
34841539DNAHomo sapiens 4atgacggcgc 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
15395343PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 5Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile
Tyr Tyr Asn Ala Asn 1 5 10 15 Trp Glu Leu Glu Arg Thr Asn Gln Ser
Gly Leu Glu Arg Cys Glu Gly 20 25 30 Glu Gln Asp Lys Arg Leu His
Cys Tyr Ala Ser Trp Arg Asn Ser Ser 35 40 45 Gly Thr Ile Glu Leu
Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn 50 55 60 Cys Tyr Asp
Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val 65 70 75 80 Tyr
Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His 85 90
95 Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr
100 105 110 Ala Pro Thr Gly Gly Gly Thr His Thr Cys Pro Pro Cys Pro
Ala Pro 115 120 125 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys 130 135 140 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val 145 150 155 160 Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp 165 170 175 Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 180 185 190 Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 195 200 205 Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 210 215
220 Pro Val Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
225 230 235 240 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys 245 250 255 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp 260 265 270 Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys 275 280 285 Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser 290 295 300 Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 305 310 315 320 Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 325 330 335
Leu Ser Leu Ser Pro Gly Lys 340 65PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 6Thr Gly Gly Gly Gly 1 5
721PRTApis sp. 7Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val
Val Tyr Ile 1 5 10 15 Ser Tyr Ile Tyr Ala 20
822PRTUnknownDescription of Unknown Tissue plasminogen activator
8Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1
5 10 15 Ala Val Phe Val Ser Pro 20 920PRTHomo sapiens 9Met Gly Ala
Ala Ala Lys Leu Ala Phe Ala Val Phe Leu Ile Ser Cys 1 5 10 15 Ser
Ser Gly Ala 20 101107DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 10atggatgcaa
tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60tcgcccggcg
cctctgggcg tggggaggct gagacacggg agtgcatcta ctacaacgcc
120aactgggagc tggagcgcac caaccagagc ggcctggagc gctgcgaagg
cgagcaggac 180aagcggctgc actgctacgc ctcctggcgc aacagctctg
gcaccatcga gctcgtgaag 240aagggctgct ggctagatga cttcaactgc
tacgataggc aggagtgtgt ggccactgag 300gagaaccccc aggtgtactt
ctgctgctgt gaaggcaact tctgcaacga gcgcttcact 360catttgccag
aggctggggg cccggaagtc acgtacgagc cacccccgac agcccccacc
420ggtggtggaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg
gggaccgtca 480gtcttcctct tccccccaaa acccaaggac accctcatga
tctcccggac ccctgaggtc 540acatgcgtgg tggtggacgt gagccacgaa
gaccctgagg tcaagttcaa ctggtacgtg 600gacggcgtgg aggtgcataa
tgccaagaca aagccgcggg aggagcagta caacagcacg 660taccgtgtgg
tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac
720aagtgcaagg tctccaacaa agccctccca gtccccatcg agaaaaccat
ctccaaagcc 780aaagggcagc cccgagaacc acaggtgtac accctgcccc
catcccggga ggagatgacc 840aagaaccagg tcagcctgac ctgcctggtc
aaaggcttct atcccagcga catcgccgtg 900gagtgggaga gcaatgggca
gccggagaac aactacaaga ccacgcctcc cgtgctggac 960tccgacggct
ccttcttcct ctatagcaag ctcaccgtgg acaagagcag gtggcagcag
1020gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta
cacgcagaag 1080agcctctccc tgtctccggg taaatga 1107116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Gly
Arg Gly Glu Ala Glu 1 5 12344PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 12Ser Gly Arg Gly Glu Ala
Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala 1 5 10 15 Asn Trp Glu Leu
Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu 20 25 30 Gly Glu
Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser 35 40 45
Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe 50
55 60 Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro
Gln 65 70 75 80 Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu
Arg Phe Thr 85 90 95 His Leu Pro Glu Ala Gly Gly Pro Glu Val Thr
Tyr Glu Pro Pro Pro 100 105 110 Thr Ala Pro Thr Gly Gly Gly Thr His
Thr Cys Pro Pro Cys Pro Ala 115 120 125 Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro 130 135 140 Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 145 150 155 160 Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 165 170 175
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 180
185 190 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln 195 200 205 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala 210 215 220 Leu Pro Val Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro 225 230 235 240 Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr 245 250 255 Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 260 265 270 Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 275 280 285 Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 290 295 300
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 305
310 315 320 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys 325 330 335 Ser Leu Ser Leu Ser Pro Gly Lys 340
13225PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 13Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro 1 5 10 15 Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 20 25 30 Arg Thr Pro Glu Val Thr Cys
Val Val Val Xaa Val Ser His Glu Asp 35 40 45 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 50 55 60 Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 65 70 75 80 Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 85 90
95 Tyr Lys Cys Xaa Val Ser Asn Lys Ala Leu Pro Val Pro Ile Glu Lys
100 105 110 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 115 120 125 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr 130 135 140 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 145 150 155 160 Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu 165 170 175 Asp Ser Asp Gly Pro
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 180 185 190 Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 195 200 205 Ala
Leu His Xaa His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 210 215
220 Lys 225 14116PRTHomo sapiens 14Ile Leu Gly Arg Ser Glu Thr Gln
Glu Cys Leu Phe Phe Asn Ala Asn 1 5 10 15 Trp Glu Lys Asp Arg Thr
Asn Gln Thr Gly Val Glu Pro Cys Tyr Gly 20 25 30 Asp Lys Asp Lys
Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile Ser 35 40 45 Gly Ser
Ile Glu Ile Val Lys Gln Gly Cys Trp Leu Asp Asp Ile Asn 50 55 60
Cys Tyr Asp Arg Thr Asp Cys Val Glu Lys Lys Asp Ser Pro Glu Val 65
70 75 80 Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu Lys Phe
Ser Tyr 85 90 95 Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn
Pro Val Thr Pro 100 105 110 Lys Pro Pro Thr 115 15150PRTRattus sp.
15Met Thr Ala Pro Trp Ala
Ala Leu Ala Leu Leu Trp Gly Ser Leu Cys 1 5 10 15 Ala Gly Ser Gly
Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr 20 25 30 Asn Ala
Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg 35 40 45
Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Pro 50
55 60 Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu
Asp 65 70 75 80 Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr
Glu Glu Asn 85 90 95 Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn
Phe Cys Asn Glu Arg 100 105 110 Phe Thr His Leu Pro Glu Pro Gly Gly
Pro Glu Val Thr Tyr Glu Pro 115 120 125 Pro Pro Thr Ala Pro Thr Leu
Leu Thr Val Leu Ala Tyr Ser Leu Leu 130 135 140 Pro Ile Gly Gly Leu
Ser 145 150 16150PRTSus sp. 16Met Thr Ala Pro Trp Ala Ala Leu Ala
Leu Leu Trp Gly Ser Leu Cys 1 5 10 15 Val Gly Ser Gly Arg Gly Glu
Ala Glu Thr Arg Glu Cys Ile Tyr Tyr 20 25 30 Asn Ala Asn Trp Glu
Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg 35 40 45 Cys Glu Gly
Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg 50 55 60 Asn
Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp 65 70
75 80 Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu
Asn 85 90 95 Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys
Asn Glu Arg 100 105 110 Phe Thr His Leu Pro Glu Ala Gly Gly Pro Glu
Val Thr Tyr Glu Pro 115 120 125 Pro Pro Thr Ala Pro Thr Leu Leu Thr
Val Leu Ala Tyr Ser Leu Leu 130 135 140 Pro Ile Gly Gly Leu Ser 145
150 17150PRTMus sp. 17Met Thr Ala Pro Trp Ala Ala Leu Ala Leu Leu
Trp Gly Ser Leu Cys 1 5 10 15 Ala Gly Ser Gly Arg Gly Glu Ala Glu
Thr Arg Glu Cys Ile Tyr Tyr 20 25 30 Asn Ala Asn Trp Glu Leu Glu
Arg Thr Asn Gln Ser Gly Leu Glu Arg 35 40 45 Cys Glu Gly Glu Gln
Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg 50 55 60 Asn Ser Ser
Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp 65 70 75 80 Asp
Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn 85 90
95 Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg
100 105 110 Phe Thr His Leu Pro Glu Pro Gly Gly Pro Glu Val Thr Tyr
Glu Pro 115 120 125 Pro Pro Thr Ala Pro Thr Leu Leu Thr Val Leu Ala
Tyr Ser Leu Leu 130 135 140 Pro Ile Gly Gly Leu Ser 145 150
18150PRTHomo sapiens 18Met Thr Ala Pro Trp Val Ala Leu Ala Leu Leu
Trp Gly Ser Leu Cys 1 5 10 15 Ala Gly Ser Gly Arg Gly Glu Ala Glu
Thr Arg Glu Cys Ile Tyr Tyr 20 25 30 Asn Ala Asn Trp Glu Leu Glu
Arg Thr Asn Gln Ser Gly Leu Glu Arg 35 40 45 Cys Glu Gly Glu Gln
Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg 50 55 60 Asn Ser Ser
Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp 65 70 75 80 Asp
Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn 85 90
95 Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg
100 105 110 Phe Thr His Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr
Glu Pro 115 120 125 Pro Pro Thr Ala Pro Thr Leu Leu Thr Val Leu Ala
Tyr Ser Leu Leu 130 135 140 Pro Ile Gly Gly Leu Ser 145 150
19150PRTUnknownDescription of Unknown Bovine IIb peptide 19Met Thr
Ala Pro Trp Ala Ala Leu Ala Leu Leu Trp Gly Ser Leu Cys 1 5 10 15
Ala Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr 20
25 30 Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu
Arg 35 40 45 Cys Glu Gly Glu Arg Asp Lys Arg Leu His Cys Tyr Ala
Ser Trp Arg 50 55 60 Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys
Gly Cys Trp Leu Asp 65 70 75 80 Asp Phe Asn Cys Tyr Asp Arg Gln Glu
Cys Val Ala Thr Glu Glu Asn 85 90 95 Pro Gln Val Tyr Phe Cys Cys
Cys Glu Gly Asn Phe Cys Asn Glu Arg 100 105 110 Phe Thr His Leu Pro
Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro 115 120 125 Pro Pro Thr
Ala Pro Thr Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu 130 135 140 Pro
Val Gly Gly Leu Ser 145 150 20150PRTXenopus sp. 20Met Gly Ala Ser
Val Ala Leu Thr Phe Leu Leu Leu Leu Ala Thr Phe 1 5 10 15 Arg Ala
Gly Ser Gly His Asp Glu Val Glu Thr Arg Glu Cys Ile Tyr 20 25 30
Tyr Asn Ala Asn Trp Glu Leu Glu Lys Thr Asn Gln Ser Gly Val Glu 35
40 45 Arg Leu Val Glu Gly Lys Lys Asp Lys Arg Leu His Cys Tyr Ala
Ser 50 55 60 Trp Arg Asn Asn Ser Gly Phe Ile Glu Leu Val Lys Lys
Gly Cys Trp 65 70 75 80 Leu Asp Asp Phe Asn Cys Tyr Asp Arg Gln Glu
Cys Ile Ala Lys Glu 85 90 95 Glu Asn Pro Gln Val Phe Phe Cys Cys
Cys Glu Gly Asn Tyr Cys Asn 100 105 110 Lys Lys Phe Thr His Leu Pro
Glu Val Glu Thr Phe Asp Pro Lys Pro 115 120 125 Gln Pro Ser Ala Ser
Val Leu Asn Ile Leu Ile Tyr Ser Leu Leu Pro 130 135 140 Ile Val Gly
Leu Ser Met 145 150 21150PRTHomo sapiens 21Met Gly Ala Ala Ala Lys
Leu Ala Phe Ala Val Phe Leu Ile Ser Cys 1 5 10 15 Ser Ser Gly Ala
Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe 20 25 30 Phe Asn
Ala Asn Trp Glu Lys Asp Arg Thr Asn Gln Thr Gly Val Glu 35 40 45
Pro Cys Tyr Gly Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp 50
55 60 Lys Asn Ile Ser Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp
Leu 65 70 75 80 Asp Asp Ile Asn Cys Tyr Asp Arg Thr Asp Cys Val Glu
Lys Lys Asp 85 90 95 Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu Gly
Asn Met Cys Asn Glu 100 105 110 Lys Phe Ser Tyr Phe Pro Glu Met Glu
Val Thr Gln Pro Thr Ser Asn 115 120 125 Pro Val Thr Pro Lys Pro Pro
Tyr Tyr Asn Ile Leu Leu Tyr Ser Leu 130 135 140 Val Pro Leu Met Leu
Ile 145 150 22154PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Consensus sequence 22Met Thr Ala Pro Trp Ala Ala
Xaa Leu Ala Leu Leu Trp Gly Ser Leu 1 5 10 15 Cys Ala Gly Ser Gly
Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr 20 25 30 Tyr Asn Ala
Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu 35 40 45 Arg
Leu Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser 50 55
60 Trp Arg Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp
65 70 75 80 Leu Asp Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala
Thr Glu 85 90 95 Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly
Asn Phe Cys Asn 100 105 110 Glu Arg Phe Thr His Leu Pro Glu Xaa Gly
Gly Pro Glu Val Thr Tyr 115 120 125 Glu Pro Lys Pro Pro Thr Ala Pro
Thr Leu Leu Thr Val Leu Ala Tyr 130 135 140 Ser Leu Leu Pro Ile Gly
Gly Leu Ser Met 145 150 23335PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 23Glu Thr Arg Glu Cys Ile
Tyr Tyr Asn Ala Asn Trp Glu Leu Glu Arg 1 5 10 15 Thr Asn Gln Ser
Gly Leu Glu Arg Cys Glu Gly Glu Gln Asp Lys Arg 20 25 30 Leu His
Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr Ile Glu Leu 35 40 45
Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn Cys Tyr Asp Arg Gln 50
55 60 Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe Cys Cys
Cys 65 70 75 80 Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu Pro
Glu Ala Gly 85 90 95 Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr
Gly Gly Gly Thr His 100 105 110 Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val 115 120 125 Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 130 135 140 Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu 145 150 155 160 Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 165 170 175
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 180
185 190 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys 195 200 205 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile 210 215 220 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro 225 230 235 240 Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu 245 250 255 Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 260 265 270 Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 275 280 285 Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 290 295 300
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 305
310 315 320 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 330 335 24360PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 24Met Asp Ala Met Lys Arg
Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val
Ser Pro Gly Ala Ala Glu Thr Arg Glu Cys Ile Tyr 20 25 30 Tyr Asn
Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu 35 40 45
Arg Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp 50
55 60 Arg Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp
Leu 65 70 75 80 Asp Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala
Thr Glu Glu 85 90 95 Asn Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly
Asn Phe Cys Asn Glu 100 105 110 Arg Phe Thr His Leu Pro Glu Ala Gly
Gly Pro Glu Val Thr Tyr Glu 115 120 125 Pro Pro Pro Thr Gly Gly Gly
Thr His Thr Cys Pro Pro Cys Pro Ala 130 135 140 Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 145 150 155 160 Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 165 170 175
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 180
185 190 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln 195 200 205 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln 210 215 220 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala 225 230 235 240 Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro 245 250 255 Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 260 265 270 Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 275 280 285 Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 290 295 300
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 305
310 315 320 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe 325 330 335 Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys 340 345 350 Ser Leu Ser Leu Ser Pro Gly Lys 355 360
251083DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 25atggatgcaa 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 10 gag 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 25 cag 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 Gly 30 35 40 45 acc 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 60 tac 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 75 ttc
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
90 cca 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
100 105 ggtggtggaa 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 10832681PRTHomo
sapiens 26Cys Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn
Gln Ser 1 5 10 15 Gly Leu Glu Arg Cys Glu Gly Glu
Gln Asp Lys Arg Leu His Cys Tyr 20 25 30 Ala Ser Trp Arg Asn Ser
Ser Gly Thr Ile Glu Leu Val Lys Lys Gly 35 40 45 Cys Trp Leu Asp
Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala 50 55 60 Thr Glu
Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe 65 70 75 80
Cys 274PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Thr Gly Gly Gly 1 285PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 28Ser
Gly Gly Gly Gly 1 5 294PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 29Ser Gly Gly Gly 1
304PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Gly Gly Gly Gly 1 316PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag
31His His His His His His 1 5 32368PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
32Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1
5 10 15 Ala Val Phe Val Ser Pro Gly Ala Ser Gly Arg Gly Glu Ala Glu
Thr 20 25 30 Arg Glu Cys Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu
Arg Thr Asn 35 40 45 Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu Gln
Asp Lys Arg Leu His 50 55 60 Cys Tyr Ala Ser Trp Arg Asn Ser Ser
Gly Thr Ile Glu Leu Val Lys 65 70 75 80 Lys Gly Cys Trp Leu Asp Asp
Phe Asn Cys Tyr Asp Arg Gln Glu Cys 85 90 95 Val Ala Thr Glu Glu
Asn Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly 100 105 110 Asn Phe Cys
Asn Glu Arg Phe Thr His Leu Pro Glu Ala Gly Gly Pro 115 120 125 Glu
Val Thr Tyr Glu Pro Pro Pro Thr Ala Pro Thr Gly Gly Gly Thr 130 135
140 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
145 150 155 160 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg 165 170 175 Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro 180 185 190 Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 195 200 205 Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val 210 215 220 Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 225 230 235 240 Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile Glu Lys Thr 245 250 255
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 260
265 270 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys 275 280 285 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser 290 295 300 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp 305 310 315 320 Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser 325 330 335 Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala 340 345 350 Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 355 360 365
33115PRTHomo sapiens 33Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile
Tyr Tyr Asn Ala Asn 1 5 10 15 Trp Glu Leu Glu Arg Thr Asn Gln Ser
Gly Leu Glu Arg Cys Glu Gly 20 25 30 Glu Gln Asp Lys Arg Leu His
Cys Tyr Ala Ser Trp Arg Asn Ser Ser 35 40 45 Gly Thr Ile Glu Leu
Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn 50 55 60 Cys Tyr Asp
Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val 65 70 75 80 Tyr
Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His 85 90
95 Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr
100 105 110 Ala Pro Thr 115 34108PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 34Ala Glu Thr Arg Glu
Cys Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu 1 5 10 15 Arg Thr Asn
Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu Gln Asp Lys 20 25 30 Arg
Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr Ile Glu 35 40
45 Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn Cys Tyr Asp Arg
50 55 60 Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe
Cys Cys 65 70 75 80 Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His
Leu Pro Glu Ala 85 90 95 Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro
Pro Thr 100 105
* * * * *