U.S. patent application number 12/459202 was filed with the patent office on 2010-01-21 for methods for dosing an activin-actriia antagonist and monitoring of treated patients.
This patent application is currently assigned to Acceleron Pharma Inc.. Invention is credited to Niels Borgstein, Matthew L. Sherman.
Application Number | 20100015144 12/459202 |
Document ID | / |
Family ID | 41445149 |
Filed Date | 2010-01-21 |
United States Patent
Application |
20100015144 |
Kind Code |
A1 |
Sherman; Matthew L. ; et
al. |
January 21, 2010 |
Methods for dosing an activin-actriia antagonist and monitoring of
treated patients
Abstract
In certain aspects, the present invention provides methods for
dosing a patient with an activin-ActRIIa antagonist and methods for
managing patients treated with an activin-ActRIIa anatagonist. In
certain aspects, the methods involve measuring one or more
hematologic parameters in a patient.
Inventors: |
Sherman; Matthew L.;
(Newton, MA) ; Borgstein; Niels; (Medfield,
MA) |
Correspondence
Address: |
ROPES & GRAY LLP
PATENT DOCKETING 39/41, ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Assignee: |
Acceleron Pharma Inc.
Cambridge
MA
|
Family ID: |
41445149 |
Appl. No.: |
12/459202 |
Filed: |
June 26, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61133354 |
Jun 26, 2008 |
|
|
|
Current U.S.
Class: |
424/134.1 ;
424/158.1; 435/29; 436/66; 436/84; 436/86; 514/1.1; 600/485 |
Current CPC
Class: |
C07K 14/475 20130101;
G01N 2800/52 20130101; A61K 38/22 20130101; A61P 19/00 20180101;
C07K 14/72 20130101; G01N 2333/79 20130101; C07K 2319/30 20130101;
A61K 38/1796 20130101; A61P 7/06 20180101; G01N 33/721 20130101;
G01N 2333/47 20130101; G01N 33/80 20130101; G01N 33/90
20130101 |
Class at
Publication: |
424/134.1 ;
435/29; 436/84; 514/12; 436/66; 436/86; 424/158.1; 600/485 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12Q 1/02 20060101 C12Q001/02; G01N 33/20 20060101
G01N033/20; A61K 38/16 20060101 A61K038/16; G01N 33/72 20060101
G01N033/72; G01N 33/68 20060101 G01N033/68; A61P 19/08 20060101
A61P019/08; A61P 35/00 20060101 A61P035/00; A61B 5/021 20060101
A61B005/021 |
Claims
1. A method for managing a patient that has been treated with, or
is a candidate to be treated with, an activin-ActRIIa antagonist,
the method comprising monitoring in the patient one or more
hematologic parameters that correlate with increased red blood cell
levels.
2. The method of claim 1, wherein the hematologic parameters are
one or more of the following: red blood cell levels, blood
pressure, or iron stores.
3. The method of claim 1, the method further comprising
administering to the patient the activin-ActRIIa antagonist in an
amount that is appropriate to the observed level of said
hematologic parameter in the patient.
4. The method of claim 1, wherein, if the patient has blood
pressure elevated above baseline or is hypertensive, dosing with
the activin-ActRIIa antagonist is reduced, delayed or
terminated.
5. The method of claim 1, wherein, if the patient has blood
pressure elevated above baseline or is hypertensive, the patient is
treated with a blood pressure lowering agent prior to
administration of the activin-ActRIIa antagonist.
6. The method of claim 1, wherein if the patient has uncontrolled
hypertension, dosing with the activin-ActRIIa antagonist is
reduced, delayed or terminated.
7. The method of claim 1, wherein if the patient has a red blood
cell level greater than the normal range for patients of similar
age and sex, dosing with the activin-ActRIIa antagonist is reduced,
delayed or terminated.
8. The method of claim 1, wherein if the patient has a hemoglobin
level of greater than 15 g/dl, dosing with the activin-ActRIIa
antagonist is reduced, delayed or terminated.
9. The method of claim 1, wherein if the patient has a hemoglobin
level greater than 10, 11 or 12 g/dl, dosing with the
activin-ActRIIa antagonist is reduced, delayed or terminated.
10. The method of claim 1, wherein if the patient has iron stores
that are lower than the normal range for patients of similar age
and sex, dosing with the activin-ActRIIa antagonist is reduced,
delayed or terminated.
11. The method of claim 1, wherein if the patient has a transferrin
saturation of less than 20%, dosing with the activin-ActRIIa
antagonist is reduced, delayed or terminated.
12. The method of claim 1, wherein if the patient has a ferritin
level of less than 100 ng/ml, dosing with the activin-ActRIIa
antagonist is reduced, delayed or terminated.
13. The method of claim 1, wherein if the patient has iron stores
that are lower than the normal range for patients of similar age
and sex, the patient is treated with an iron supplement prior to
dosing with the activin-ActRIIa antagonist.
14. The method of claim 1, wherein if the patient has a transferrin
saturation of less than 20%, the patient is treated with an iron
supplement prior to dosing with the activin-ActRIIa antagonist.
15. The method of claim 1, wherein if the patient has a ferritin
level of less than 100 ng/ml, the patient is treated with an iron
supplement prior to dosing with the activin-ActRIIa antagonist.
16. The method of claim 1, wherein monitoring red blood cell levels
comprises monitoring one or more of the following: hemoglobin
levels and hematocrit levels.
17. The method of claim 1, wherein monitoring blood pressure
comprises monitoring one or more of the following: systolic blood
pressure, diastolic blood pressure or mean arterial blood
pressure.
18. The method of claim 1, wherein monitoring iron stores comprises
monitoring one or more of the following: transferrin saturation,
ferritin levels or total iron binding capacity.
19. A method for dosing a patient with an activin-ActRIIa
antagonist, the method comprising dosing the patient in amounts and
at intervals selected so as to reduce the risk of causing a rise in
hemoglobin levels greater than 1 g/dl in two weeks.
20. The method of claim 1, wherein the patient is suffering from
anemia.
21. The method of claim 1, wherein the patient is in need of bone
growth, increased bone density or increased bone strength.
22. The method of claim 1, wherein the patient is suffering from a
bone-related disorder.
23. The method of claim 1, wherein the patient has cancer.
24. The method of claim 23, wherein the patient has breast
cancer.
25. The method of claim 1, wherein the activin-ActRIIa antagonist
is an antibody that binds to a target protein selected from the
group consisting of: an activin and ActRIIA.
26. The method of claim 1, wherein the activin-ActRIIa antagonist
is inhibin or a conservative variant of inhibin.
27. The method of claim 1, wherein the activin-ActRIIa antagonist
is a protein comprising a follistatin domain that binds to and
antagonizes activin.
28. The method of claim 1, wherein the activin-ActRIIa antagonist
is a protein selected from the group consisting of: follistatin,
follastatin-related gene (FLRG) and a conservative variant of the
forgoing.
29. The method of claim 1, wherein the activin-ActRIIa antagonist
is an ActRIIa polypeptide selected from the group consisting of: a)
a polypeptide comprising an amino acid sequence at least 90%
identical to SEQ ID NO: 2; b) a polypeptide comprising an amino
acid sequence at least 90% identical to SEQ ID NO: 3; and c) a
polypeptide comprising at least 50 consecutive amino acids selected
from SEQ ID NO: 2. d) a polypeptide comprising an amino acid
sequence at least 90% identical to SEQ ID NO: 7; e) a polypeptide
comprising an amino acid sequence at least 90% identical to SEQ ID
NO: 12; and f) a polypeptide comprising at least 50 consecutive
amino acids selected from SEQ ID NO: 7.
30. The method of claim 29, wherein the polypeptide has one or more
of the following characteristics: i) binds to an ActRIIa ligand
with a KD of at least 10.sup.-7 M; and ii) inhibits ActRIIa
signaling in a cell.
31. The method of claim 29, wherein said polypeptide is a fusion
protein including, in addition to an ActRIIa polypeptide domain,
one or more polypeptide portions that enhance one or more of in
vivo stability, in vivo half life, uptake/administration, tissue
localization or distribution, formation of protein complexes,
and/or purification.
32. The method of claim 29, wherein said fusion protein includes a
polypeptide portion selected from the group consisting of: an
immunoglobulin Fc domain and a serum albumin.
33. The method of claim 29, wherein said 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.
34. The method of claim 29, wherein the activin-ActRIIa antagonist
is an ActRIIa-Fc fusion protein comprising an amino acid sequence
selected from the group consisting of: a) the amino acid sequence
of SEQ ID NO: 3, b) the amino acid sequence of SEQ ID NO: 2, c) the
amino acid sequence of SEQ ID NO: 7, d) an amino acid sequence that
is at least 90% identical to the amino acid sequence of SEQ ID NO:
7, e) an amino acid sequence that is at least 95% identical to the
amino acid sequence of SEQ ID NO: 7, f) the amino acid sequence of
SEQ ID NO: 12, and g) the amino acid sequence of SEQ ID NO: 13.
35. A method for administering an ActRIIa-Fc fusion protein to a
patient, the method comprising administering the ActRIIa-Fc fusion
protein no more frequently than once per 60 days.
36. The method of claim 35, wherein the activin antagonist is
administered to the patient no more frequently than once per 90
days.
37. The method of claim 35, wherein the activin antagonist is
administered to the patient no more frequently than once per 120
days.
38. A method for administering an ActRIIa-Fc fusion protein to a
patient in need thereof, the method comprising administering the
ActRIIa-Fc fusion protein to a patient having a hemoglobin level of
less than 10 g/dL.
39. The method of claim 38, wherein the patient has a hemoglobin
level of less than 11 g/dL.
40. The method of claim 38, wherein the patient has a hemoglobin
level of less than 12 g/dL.
41. The method of claim 38, wherein the patient is in need of bone
growth, increased bone density or increased bone strength.
42. The method of claim 38, wherein the patient is suffering from a
bone-related disorder.
43. The method of claim 38, wherein the patient has cancer.
44. The method of claim 38, wherein the patient has breast cancer.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/133,354, filed on Jun. 26, 2008, the
specification of which is incorporated by reference herein in its
entirety.
BACKGROUND OF THE INVENTION
[0002] The transforming growth factor-beta (TGF-beta) superfamily
contains a variety of growth factors that share common sequence
elements and structural motifs. These proteins are known to exert
biological effects on a large variety of cell types in both
vertebrates and invertebrates. Members of the superfamily perform
important functions during embryonic development in pattern
formation and tissue specification and can influence a variety of
differentiation processes, including adipogenesis, myogenesis,
chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and
epithelial cell differentiation. The family is divided into two
general branches: the BMP/GDF and the TGF-beta/Activin/BMP10
branches, whose members have diverse, often complementary effects.
By manipulating the activity of a member of the TGF-beta family, it
is often possible to cause significant physiological changes in an
organism. For example, the Piedmontese and Belgian Blue cattle
breeds carry a loss-of-function mutation in the GDF8 (also called
myostatin) gene that causes a marked increase in muscle mass.
Grobet et al., Nat Genet. 1997, 17(1):71-4. Furthermore, in humans,
inactive alleles of GDF8 are associated with increased muscle mass
and, reportedly, exceptional strength. Schuelke et al., N Engl J
Med 2004, 350:2682-8.
[0003] Changes in muscle, bone, cartilage and other tissues may be
achieved by agonizing or antagonizing signaling that is mediated by
an appropriate TGF-beta family member. It is an object of the
present disclosure to provide alternative methods for administering
modulators of the TGF-beta superfamily to patients.
SUMMARY OF THE INVENTION
[0004] In part, the disclosure relates to methods for administering
activin antagonists, as well as ActRIIa antagonists (collectively,
"activin-ActRIIa antagonists"), to patients in a manner that is
appropriate given the effects that such antagonists can have on a
variety of tissues, including red blood cells. In part, the
disclosure demonstrates that activin-ActRIIa antagonists can
increase red blood cell and hemoglobin levels and also increase
bone density. This dual effect has particular advantages in
patients that have both anemia and bone loss, such as many cancer
patients (where anemia and bone loss can be a consequence of the
tumor or a consequence of irradiation or chemotherapy), patients
with osteoporosis and patients with renal failure. In particular,
the disclosure demonstrates that a soluble form of ActRIIa acts as
an inhibitor of activin and, when administered in vivo, increases
red blood cell levels. While soluble ActRIIa may affect red blood
cell levels through a mechanism other than activin antagonism, the
disclosure nonetheless demonstrates that desirable therapeutic
agents may be selected on the basis of activin antagonism or
ActRIIa antagonism or both. Such agents are referred to
collectively as activin-ActRIIa antagonists. As described herein,
and in published patent applications WO/2009/038745,
WO/2008/100384, WO/2008/094708, WO/2008/076437, WO/2007/062188 and
WO/2006/012627, activin-ActRIIa antagonists also have a variety of
other therapeutic uses including, for example, promoting bone
growth, decreasing FSH levels, treating multiple myeloma and
treating breast cancer. In certain instances, when administering an
activin-ActRIIa antagonists for promoting bone growth or treating
breast cancer, it may be desirable to monitor the effects on red
blood cells during administration of an activin-ActRIIa
antagonists, or to determine or adjust the dosing of an
activin-ActRIIa antagonists, in order to reduce undesired effects
on red blood cells. For example, excessive increases in red blood
cell levels, hemoglobin levels, or hematocrit levels may cause
increases in blood pressure or other undesirable side effects. It
may also be desirable to restrict dosing of activin-ActRIIa
antagonists to patients who have appropriate hematologic
parameters. For example, it may be desirable to limit dosing to
only those patients who have a hemoglobin level below normal (e.g.,
below 12 g/dL, below 11 g/dL, below 10 g/dL or below 9 g/dL or
lower).
[0005] Therefore, in certain embodiments, the disclosure provides
methods for managing a patient that has been treated with, or is a
candidate to be treated with, an activin-ActRIIa antagonist,
including, for example, activin-binding ActRIIa polypeptides,
anti-activin antibodies, anti-ActRIIa antibodies, activin- or
ActRIIa-targeted small molecules and aptamers, and nucleic acids
that decrease expression of activin or ActRIIa, by monitoring in
the patient one or more hematologic parameters that correlate with
an increase in red blood cell levels, such as, for example, red
blood cell levels, blood pressure, or iron stores.
[0006] In certain aspects, the disclosure provides polypeptides
comprising a soluble, activin-binding ActRIIa polypeptide that
binds to activin. ActRIIa polypeptides may be formulated as a
pharmaceutical preparation comprising the activin-binding ActRIIa
polypeptide and a pharmaceutically acceptable carrier. The
activin-binding ActRIIa polypeptide may bind to activin with a KD
less than 1 micromolar or less than 100, 10 or 1 nanomolar.
Optionally, the activin-binding ActRIIa polypeptide selectively
binds activin versus GDF11 and/or GDF8, and optionally with a KD
that is at least 10-fold, 20-fold or 50-fold lower with respect to
activin than with respect to GDF11 and/or GDF8. While not wishing
to be bound to a particular mechanism of action, it is expected
that this degree of selectivity for activin inhibition over
GDF11/GDF8 inhibition in ActRIIa-Fc accounts for effects on bone or
erythropoiesis without a consistently measurable effect on muscle.
In many embodiments, an ActRIIa polypeptide will be selected for
causing less than 15%, less than 10% or less than 5% increase in
muscle at doses that achieve desirable effects on red blood cell
levels. In other embodiments, the effect on muscle is acceptable
and need not be selected against. The composition may be at least
95% pure, with respect to other polypeptide components, as assessed
by size exclusion chromatography, and optionally, the composition
is at least 98% pure. An activin-binding ActRIIa polypeptide for
use in such a preparation may be any of those disclosed herein,
such as a polypeptide having (i.e. comprising) an amino acid
sequence selected from SEQ ID NOs: 2, 3, 7, 12 or 13, or having
(i.e. comprising) 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: 2, 3, 7, 12 or 13. An activin-binding ActRIIa
polypeptide may include a functional fragment of a natural ActRIIa
polypeptide, such as one comprising at least 10, 20 or 30 amino
acids of a sequence selected from SEQ ID NOs: 1-3 or a sequence of
SEQ ID NO: 2, lacking the C-terminal 10 to 15 amino acids (the
"tail").
[0007] A soluble, activin-binding ActRIIa polypeptide may include
one or more alterations in the amino acid sequence (e.g., in the
ligand-binding domain) relative to a naturally occurring ActRIIa
polypeptide. Examples of altered ActRIIa polypeptides are provided
in WO 2006/012627, pp. 59-60 and pp. 55-58, respectively, which is
incorporated by reference herein, and throughout U.S. patent
application Ser. No. 12/012,652, incorporated by reference herein.
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 ActRIIa
polypeptide.
[0008] An activin-binding ActRIIa polypeptide may be a fusion
protein that has, as one domain, an ActRIIa polypeptide, (e.g., a
ligand-binding portion of an ActRIIa) and one or more additional
domains that provide a desirable property, such as improved
pharmacokinetics, easier purification, targeting to particular
tissues, etc. For example, a domain of a fusion protein may enhance
one or more of in vivo stability, in vivo half life,
uptake/administration, tissue localization or distribution,
formation of protein complexes, multimerization of the fusion
protein, and/or purification. An activin-binding ActRIIa fusion
protein may include an immunoglobulin Fc domain (wild-type or
mutant) or a serum albumin or other polypeptide portion that
provides desirable properties such as improved pharmacokinetics,
improved solubility or improved stability. In a preferred
embodiment, an ActRIIa-Fc fusion comprises a relatively
unstructured linker positioned between the Fc domain and the
extracellular ActRIIa domain. This unstructured linker may be an
artificial sequence of 1, 2, 3, 4 or 5 amino acids or a length of
between 5 and 15, 20, 30, 50 or more amino acids that are
relatively free of secondary structure, or a mixture of both. A
linker may be rich in glycine and proline residues and may, for
example, contain a single sequence of threonine/serine and glycines
or repeating sequences of threonine/serine and glycines (e.g.,
TG.sub.4 (SEQ ID NO: 15) or SG.sub.4 (SEQ ID NO: 16) singlets or
repeats). 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 ActRIIa polypeptide includes one
or more modified amino acid residues selected from: a glycosylated
amino acid, a PEGylated amino acid, a famesylated 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. A pharmaceutical preparation may also
include one or more additional compounds such as a compound that is
used to treat a bone disorder or a compound that is used to treat
anemia. Preferably, a pharmaceutical preparation is substantially
pyrogen free. In general, it is preferable that an ActRIIa protein
be expressed in a mammalian cell line that mediates suitably
natural glycosylation of the ActRIIa 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 systems will be useful.
[0009] As described herein, ActRIIa proteins designated ActRIIa-Fc
have desirable properties, including selective binding to activin
versus GDF8 and/or GDF11, high affinity ligand binding and serum
half life greater than two weeks in animal models and in human
patients. In certain embodiments the invention provides ActRIIa-Fc
polypeptides and pharmaceutical preparations comprising such
polypeptides and a pharmaceutically acceptable excipient.
[0010] In certain aspects, the disclosure provides nucleic acids
encoding a soluble activin-binding ActRIIa polypeptide. An isolated
polynucleotide may comprise a coding sequence for a soluble,
activin-binding ActRIIa 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 ActRIIa
and a sequence that would code for part or all of the transmembrane
domain and/or the cytoplasmic domain of an ActRIIa, 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 ActRIIa
polynucleotide sequence such as SEQ ID NO: 4 or a partially
truncated version of ActRIIa, such as a nucleic acid comprising the
nucleic acid sequence of SEQ ID NO:5, which corresponds to the
extracellular domain of ActRIIa. An isolated polynucleotide may
further comprise 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 ActRIIa. A preferred nucleic acid sequence for ActRIIa
is SEQ ID NO:14. 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.
[0011] In certain aspects, the disclosure provides methods for
making a soluble, activin-binding ActRIIa polypeptide. Such a
method may include expressing any of the nucleic acids (e.g., SEQ
ID NO: 4, 5 or 14) disclosed herein in a suitable cell, such as a
Chinese hamster ovary (CHO) cell or human cell. Such a method may
comprise: a) culturing a cell under conditions suitable for
expression of the soluble ActRIIa polypeptide, wherein said cell is
transformed with a soluble ActRIIa expression construct; and b)
recovering the soluble ActRIIa polypeptide so expressed. Soluble
ActRIIa polypeptides may be recovered as crude, partially purified
or highly purified fractions. Purification may be achieved by a
series of purification steps, including, for example, one, two or
three or more of the following, in any order: protein A
chromatography, anion exchange chromatography (e.g., Q sepharose),
hydrophobic interaction chromatography (e.g., phenylsepharose),
size exclusion chromatography, and cation exchange chromatography.
Soluble ActRIIa polypeptides may be formulated in liquid or solid
(e.g., lyophilized) forms.
[0012] In certain aspects, the disclosure provides a method for
dosing a patient with an activin-ActRIIa antagonist, comprising
dosing the patient in amounts and at intervals selected so as to
reduce the risk of causing a rise in hemoglobin levels greater than
0.5 g/dL, 1 g/dl or 1.5 g/dL in two weeks.
[0013] In certain aspects, the disclosure provides a method for
administering an ActRIIa-Fc fusion protein to a patient, comprising
administering the ActRIIa fusion protein no more frequently than
once per 60 days, once per 90 days, or once per 120 days. In
certain embodiments, the patient may be a patient in need of bone
growth or a patient suffering from or at risk for developing breast
cancer or multiple myeloma, or a patient in need of having
decreased FSH.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows the purification of ActRIIa-hFc expressed in
CHO cells. The protein purifies as a single, well-defined peak as
visualized by sizing column (left panel) and Coomassie stained
SDS-PAGE (right panel) (left lane: molecular weight standards;
right lane: ActRIIa-hFc).
[0015] FIG. 2 shows the binding of ActRIIa-hFc to activin and
GDF-11, as measured by BiaCore.TM. assay.
[0016] FIG. 3 shows the effects of ActRIIa-hFc on red blood cell
counts in female non-human primates. Female cynomolgus monkeys
(four groups of five monkeys each) were treated with placebo or 1
mg/kg, 10 mg/kg or 30 mg/kg of ActRIIa-hFc on day 0, day 7, day 14
and day 21. FIG. 3A shows red blood cell (RBC) counts. FIG. 3B
shows hemoglobin levels. Statistical significance is relative to
baseline for each treatment group. At day 57, two monkeys remained
in each group.
[0017] FIG. 4 shows the effects of ActRIIa-hFc on red blood cell
counts in male non-human primates. Male cynomolgus monkeys (four
groups of five monkeys each) were treated with placebo or 1 mg/kg,
10 mg/kg or 30 mg/kg of ActRIIa-hFc on day 0, day 7, day 14 and day
21. FIG. 4A shows red blood cell (RBC) counts. FIG. 4B shows
hemoglobin levels. Statistical significance is relative to baseline
for each treatment group. At day 57, two monkeys remained in each
group.
[0018] FIG. 5 shows the effects of ActRIIa-hFc on reticulocyte
counts in female non-human primates. Cynomolgus monkeys (four
groups of five monkeys each) were treated with placebo or 1 mg/kg,
10 mg/kg or 30 mg/kg of ActRIIa-hFc on day 0, day 7, day 14 and day
21. FIG. 5A shows absolute reticulocyte counts. FIG. 5B shows the
percentage of reticulocytes relative to RBCs. Statistical
significance is relative to baseline for each group. At day 57, two
monkeys remained in each group.
[0019] FIG. 6 shows the effects of ActRIIa-hFc on reticulocyte
counts in female non-human primates. Cynomolgus monkeys (four
groups of five monkeys each) were treated with placebo or 1 mg/kg,
10 mg/kg or 30 mg/kg of ActRIIa-hFc on day 0, day 7, day 14 and day
21. FIG. 6A shows absolute reticulocyte counts. FIG. 6B shows the
percentage of reticulocytes relative to RBCs. Statistical
significance is relative to baseline for each group. At day 57, two
monkeys remained in each group.
[0020] FIG. 7 shows results from the human clinical trial described
in Example 5, where the area-under-curve (AUC) and administered
dose of ActRIIa-hFc have a linear correlation, regardless of
whether ActRIIa-hFc was administered intravenously (IV) or
subcutaneously (SC).
[0021] FIG. 8 shows a comparison of serum levels of ActRIIa-hFc in
patients administered IV or SC.
[0022] FIG. 9 shows bone alkaline phosphatase (BAP) levels in
response to different dose levels of ActRIIa-hFc. BAP is a marker
for anabolic bone growth.
[0023] FIG. 10 depicts the median change from baseline of
hematocrit levels from the human clinical trial described in
Example 5. ActRIIa-hFc was administered intravenously (IV) at the
indicated dosage.
[0024] FIG. 11 depicts the median change from baseline of
hemoglobin levels from the human clinical trial described in
Example 5. ActRIIa-hFc was administered intravenously (IV) at the
indicated dosage.
[0025] FIG. 12 depicts the median change from baseline of RBC (red
blood cell) count from the human clinical trial described in
Example 5. ActRIIa-hFc was administered intravenously (IV) at the
indicated dosage.
[0026] FIG. 13 depicts the median change from baseline of
reticulocyte count from the human clinical trial described in
Example 5. ActRIIa-hFc was administered intravenously (IV) at the
indicated dosage.
[0027] FIG. 14 shows an alignment of human ActRIIA and ActRIIB 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.
[0028] FIG. 15 shows the effect of ActRIIA-mFc on hematocrit in a
mouse model of chemotherapy-induced anemia. Data are means.+-.SEM.
*, P<0.05 vs. vehicle at same time point. A single dose of
ActRIIA-mFc before chemotherapy prevented the decline in hematocrit
level otherwise observed after administration of the
chemotherapeutic paclitaxel.
[0029] FIG. 16 shows the dose-dependent effect of ActRIIA-mFc on
hematocrit in a mouse model of chemotherapy-induced anemia. Data
are means.+-.SEM. **, P<0.01; ***, P<0.001 vs. vehicle at
same time point. Two weeks after paclitaxel administration,
ActRIIA-mFc treatment increased hematocrit level as a function of
dose number.
[0030] FIG. 17 shows the effect of ActRIIA-mFc on hematocrit in a
partially nephrectomized (NEPHX) mouse model of chronic kidney
disease. Data are means.+-.SEM. *, P<0.05 vs. vehicle at same
time point. ActRIIA-mFc treatment prevented the decline in
hematocrit level otherwise observed at 4 weeks and produced a
beneficial trend in hematocrit at 8 weeks.
DETAILED DESCRIPTION OF THE INVENTION
1. Overview
[0031] The transforming growth factor-beta (TGF-beta) superfamily
contains a variety of growth factors that share common sequence
elements and structural motifs. These proteins are known to exert
biological effects on a large variety of cell types in both
vertebrates and invertebrates. Members of the superfamily perform
important functions during embryonic development in pattern
formation and tissue specification and can influence a variety of
differentiation processes, including adipogenesis, myogenesis,
chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and
epithelial cell differentiation. The family is divided into two
general branches: the BMP/GDF and the TGF-beta/Activin/BMP10
branches, whose members have diverse, often complementary effects.
By manipulating the activity of a member of the TGF-beta family, it
is often possible to cause significant physiological changes in an
organism. For example, the Piedmontese and Belgian Blue cattle
breeds carry a loss-of-function mutation in the GDF8 (also called
myostatin) gene that causes a marked increase in muscle mass.
Grobet et al., Nat Genet. 1997, 17(1):71-4. Furthermore, in humans,
inactive alleles of GDF8 are associated with increased muscle mass
and, reportedly, exceptional strength. Schuelke et al., N Engl J
Med 2004, 350:2682-8.
[0032] Activins are dimeric polypeptide growth factors that belong
to the TGF-beta superfamily. There are three principal activin
forms (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,
respectively). The human genome also encodes an activin C and an
activin E, which are primarily expressed in the liver, and
heterodimeric forms containing .beta..sub.C or .beta..sub.E are
also known. 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 Soc Ep 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 has been suggested that
activin A promotes 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) and
.alpha..sub.2-macroglobulin.
[0033] 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 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.
[0034] Two related type II receptors (ActRII), 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). ALK4 is the primary type I
receptor for activins, particularly for activin A, and ALK-7 may
serve as a receptor for activins as well, particularly for activin
B.
[0035] As demonstrated herein, a soluble ActRIIa polypeptide
(sActRIIa), which shows substantial preference in binding to
activin A as opposed to other TGF-beta family members, such as GDF8
or GDF11, is effective to increase red blood cell levels in vivo.
While not wishing to be bound to any particular mechanism, it is
expected that the effect of sActRIIa is caused primarily by an
activin antagonist effect, given the very strong activin binding
(picomolar dissociation constant) exhibited by the particular
sActRIIa construct used in these studies. Regardless of mechanism,
it is apparent from this disclosure that ActRIIa-activin
antagonists increase red blood cell levels in rodents, monkeys and
humans. It should be noted that hematopoiesis is a complex process,
regulated by a variety of factors, including erythropoietin, G-CSF
and iron homeostasis. The terms "increase red blood cell levels"
and "promote red blood cell formation" refer to clinically
observable metrics, such as hematocrit, red blood cell counts and
hemoglobin measurements, and are intended to be neutral as to the
mechanism by which such changes occur.
[0036] The data reported herein with respect to non-human primates
are reproducible in mice, rats and humans as well, and therefore,
this disclosure provides methods for using ActRIIa polypeptides and
other activin-ActRIIa antagonists to promote red blood cell
production and increase red blood cell levels in mammals ranging
from rodents to humans.
[0037] In addition to stimulating red blood cell levels,
activin-ActRIIa antagonists are useful for a variety of therapeutic
applications, including, for example, promoting bone growth (see
PCT Publication No. WO2007/062188, which is hereby incorporated by
reference in its entirety), and treating breast cancer (see PCT
Application No. PCT/US2008/001429, which is hereby incorporated by
reference in its entirety). In certain instances, when
administering an activin-ActRIIa antagonists for the purpose of
increasing bone or treating breast cancer, it may be desirable to
reduce or minimize or otherwise monitor effects on red blood cells.
In some instances, a dual effect on blood cells and bone or other
tissue will be desirable, but it is generally recognized that
pharmaceutically promoted increases in red blood cells, even up to
a level that is typically considered normal, can have adverse
effects on patients, and thus are often monitored or managed with
care. By monitoring various hematologic parameters in patients
being treated with, or who are candidates for treatment with, an
activin-ActRIIa antagonist, appropriate dosing (including amounts
and frequency of administration) may be determined based on an
individual patient's needs, baseline hematologic parameters, and
purpose for treatment. Furthermore, therapeutic progress and
effects on one or more hematologic parameters over time may be
useful in managing patients being dosed with an activin-ActRIIa
antagonist by facilitating patient care, determining appropriate
maintenance dosing (both amounts and frequency), etc.
[0038] Activin-ActRIIa antagonists include, for example,
activin-binding soluble ActRIIa polypeptides, antibodies that bind
to activin (particularly the activin A or B subunits, also referred
to as .beta..sub.A or .beta..sub.B) and disrupt ActRIIa binding,
antibodies that bind to ActRIIa and disrupt activin binding,
non-antibody proteins selected for activin or ActRIIa binding (see
e.g., WO/2002/088171, WO/2006/055689, and WO/2002/032925 for
examples of such proteins and methods for design and selection of
same), randomized peptides selected for activin or ActRIIa binding,
often affixed to an Fc domain. Two different proteins (or other
moieties) with activin or ActRIIa binding activity, especially
activin binders that block the type I (e.g., a soluble type I
activin receptor) and type II (e.g., a soluble type II activin
receptor) binding sites, respectively, may be linked together to
create a bifunctional binding molecule. Nucleic acid aptamers,
small molecules and other agents that inhibit the activin-ActRIIa
signaling axis are included as activin-ActRIIa antagonists. Various
proteins have activin-ActRIIa antagonist activity, including
inhibin (i.e., inhibin alpha subunit), although inhibin does not
universally antagonize activin in all tissues, follistatin (e.g.,
follistatin-288 and follistatin-315), FSRP, FLRG, activin C,
alpha(2)-macroglobulin, and an M108A (methionine to alanine change
at position 108) mutant activin A. Generally, alternative forms of
activin, particularly those with alterations in the type I receptor
binding domain can bind to type II receptors and fail to form an
active ternary complex, thus acting as antagonists. Additionally,
nucleic acids, such as antisense molecules, siRNAs or ribozymes
that inhibit activin A, B, C or E, or, particularly, ActRIIa
expression, can be used as activin-ActRIIa antagonists. The
activin-ActRIIa antagonist to be used may exhibit selectivity for
inhibiting activin-mediated signaling versus other members of the
TGF-beta family, and particularly with respect to GDF8 and
GDF11.
[0039] 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.
[0040] "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.
[0041] 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.
[0042] 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.
[0043] "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.
[0044] 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.
[0045] 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. ActRIIa Polypeptides
[0046] In certain aspects, the present invention relates to ActRIIa
polypeptides. As used herein, the term "ActRIIa" refers to a family
of activin receptor type IIa (ActRIIa) proteins from any species
and variants derived from such ActRIIa proteins by mutagenesis or
other modification. Reference to ActRIIa herein is understood to be
a reference to any one of the currently identified forms. Members
of the ActRIIa family are generally transmembrane proteins,
composed of a ligand-binding extracellular domain with a
cysteine-rich region, a transmembrane domain, and a cytoplasmic
domain with predicted serine/threonine kinase activity.
[0047] The term "ActRIIa polypeptide" includes polypeptides
comprising any naturally occurring polypeptide of an ActRIIa family
member as well as any variants thereof (including mutants,
fragments, fusions, and peptidomimetic forms) that retain a useful
activity. See, for example, WO/2006/012627. For example, ActRIIa
polypeptides include polypeptides derived from the sequence of any
known ActRIIa having a sequence at least about 80% identical to the
sequence of an ActRIIa polypeptide, and optionally at least 85%,
90%, 95%, 97%, 99% or greater identity. For example, an
ActRIIapolypeptide of the invention may bind to and inhibit the
function of an ActRIIa protein and/or activin. An ActRIIa
polypeptide may be selected for activity in promoting red blood
cell formation in vivo. Examples of ActRIIa polypeptides include
human ActRIIa precursor polypeptide (SEQ ID NO: 1) and soluble
human ActRIIa polypeptides (e.g., SEQ ID NOs: 2, 3, 7 and 12).
[0048] The human ActRIIa precursor protein sequence is as
follows:
TABLE-US-00001 (SEQ ID NO: 1)
MGAAAKLAFAVFLISCSSGAILGRSETQECLFFNANWEKDRTNQTGVEPC
YGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEV
YFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPYYNILLYSLVPLMLI
AGIVICAFWVYRHHKMAYPPVLVPTQDPGPPPPSPLLGLKPLQLLEVKAR
GRFGCVWKAQLLNEYVAVKIFPIQDKQSWQNEYEVYSLPGMKHENILQFI
GAEKRGTSVDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAETMARGL
AYLHEDIPGLKDGHKPAISHRDIKSKNVLLKNNLTACIADFGLALKFEAG
KSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELASR
CTAADGPVDEYMLPFEEEIGQHPSLEDMQEVVVHKKKRPVLRDYWQKHAG
MAMLCETIEECWDHDAEARLSAGCVGERITQMQRLTNIITTEDIVTVVTM
VTNVDFPPKESSL
[0049] The signal peptide is single underlined; the extracellular
domain is in bold and the potential N-linked glycosylation sites
are double underlined.
[0050] The human ActRIIa soluble (extracellular), processed
polypeptide sequence is as follows:
TABLE-US-00002 (SEQ ID NO: 2)
ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGS
IEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM
EVTQPTSNPVTPKPP
[0051] The C-terminal "tail" of the extracellular domain is
underlined. The sequence with the "tail" deleted (a .DELTA.15
sequence) is as follows:
TABLE-US-00003 (SEQ ID NO: 3)
ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGS
IEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM
[0052] The nucleic acid sequence encoding human ActRIIa precursor
protein is as follows (nucleotides 164-1705 of Genbank entry
NM.sub.--001616):
TABLE-US-00004 (SEQ ID NO: 4)
ATGGGAGCTGCTGCAAAGTTGGCGTTTGCCGTCTTTCTTATCTCCTGTTC
TTCAGGTGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTA
ATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGT
TATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATAT
TTCTGGTTCCATTGAAATAGTGAAACAAGGTTGTTGGCTGGATGATATCA
ACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTA
TATTTTTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTT
TCCAGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGC
CACCCTATTACAACATCCTGCTCTATTCCTTGGTGCCACTTATGTTAATT
GCGGGGATTGTCATTTGTGCATTTTGGGTGTACAGGCATCACAAGATGGC
CTACCCTCCTGTACTTGTTCCAACTCAAGACCCAGGACCACCCCCACCTT
CTCCATTACTAGGGTTGAAACCACTGCAGTTATTAGAAGTGAAAGCAAGG
GGAAGATTTGGTTGTGTCTGGAAAGCCCAGTTGCTTAACGAATATGTGGC
TGTCAAAATATTTCCAATACAGGACAAACAGTCATGGCAAAATGAATACG
AAGTCTACAGTTTGCCTGGAATGAAGCATGAGAACATATTACAGTTCATT
GGTGCAGAAAAACGAGGCACCAGTGTTGATGTGGATCTTTGGCTGATCAC
AGCATTTCATGAAAAGGGTTCACTATCAGACTTTCTTAAGGCTAATGTGG
TCTCTTGGAATGAACTGTGTCATATTGCAGAAACCATGGCTAGAGGATTG
GCATATTTACATGAGGATATACCTGGCCTAAAAGATGGCCACAAACCTGC
CATATCTCACAGGGACATCAAAAGTAAAAATGTGCTGTTGAAAAACAACC
TGACAGCTTGCATTGCTGACTTTGGGTTGGCCTTAAAATTTGAGGCTGGC
AAGTCTGCAGGCGATACCCATGGACAGGTTGGTACCCGGAGGTACATGGC
TCCAGAGGTATTAGAGGGTGCTATAAACTTCCAAAGGGATGCATTTTTGA
GGATAGATATGTATGCCATGGGATTAGTCCTATGGGAACTGGCTTCTCGC
TGTACTGCTGCAGATGGACCTGTAGATGAATACATGTTGCCATTTGAGGA
GGAAATTGGCCAGCATCCATCTCTTGAAGACATGCAGGAAGTTGTTGTGC
ATAAAAAAAAGAGGCCTGTTTTAAGAGATTATTGGCAGAAACATGCTGGA
ATGGCAATGCTCTGTGAAACCATTGAAGAATGTTGGGATCACGACGCAGA
AGCCAGGTTATCAGCTGGATGTGTAGGTGAAAGAATTACCCAGATGCAGA
GACTAACAAATATTATTACCACAGAGGACATTGTAACAGTGGTCACAATG
GTGACAAATGTTGACTTTCCTCCCAAAGAATCTAGTCTATGA
[0053] The nucleic acid sequence encoding a human ActRIIa soluble
(extracellular) polypeptide is as follows:
TABLE-US-00005 (SEQ ID NO: 5)
ATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATGCTAATTG
GGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACA
AAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCC
ATTGAAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGA
CAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTT
GCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATG
GAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCC
[0054] In a specific embodiment, the invention relates to soluble
ActRIIa polypeptides. As described herein, the term "soluble
ActRIIa polypeptide" generally refers to polypeptides comprising an
extracellular domain of an ActRIIa protein. The term "soluble
ActRIIa polypeptide," as used herein, includes any naturally
occurring extracellular domain of an ActRIIa protein as well as any
variants thereof (including mutants, fragments and peptidomimetic
forms). An activin-binding ActRIIa polypeptide is one that retains
the ability to bind to activin, including, for example, activin AA,
AB, BB, or forms that include a C or E subunit. Optionally, an
activin-binding ActRIIa polypeptide will bind to activin AA with a
dissociation constant of 1 nM or less. The extracellular domain of
an ActRIIa protein binds to activin and is generally soluble in
physiological conditions, and thus can be termed a soluble,
activin-binding ActRIIa polypeptide. Examples of soluble,
activin-binding ActRIIa polypeptides include the soluble
polypeptides illustrated in SEQ ID NOs: 2, 3, 7, 12 and 13. SEQ ID
NO:7 is referred to as ActRIIa-hFc, and is described further in the
Examples. Other examples of soluble, activin-binding ActRIIa
polypeptides comprise a signal sequence in addition to the
extracellular domain of an ActRIIa protein, for example, the honey
bee mellitin leader sequence (SEQ ID NO: 8), the tissue plaminogen
activator (TPA) leader (SEQ ID NO: 9) or the native ActRIIa leader
(SEQ ID NO: 10). The ActRIIa-hFc polypeptide illustrated in SEQ ID
NO:13 uses a TPA leader.
[0055] A general formula for an active ActRIIa variant protein is
one that comprises amino acids 12-82 of SEQ ID NO: 2, respectively,
but optionally beginning at a position ranging from 1-5 or 3-5 and
ending at a position ranging from 110-116 or 110-115, respectively,
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 comprise an
amino acid sequence that retains greater than 80%, 90%, 95% or 99%
sequence identity to the sequence of amino acids 29-109 of SEQ ID
NO: 2.
[0056] Functionally active fragments of ActRIIa polypeptides can be
obtained by screening polypeptides recombinantly produced from the
corresponding fragment of the nucleic acid encoding an ActRIIa
polypeptide. 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 as antagonists
(inhibitors) of ActRIIa protein or signaling mediated by
activin.
[0057] Functionally active variants of ActRIIa polypeptides can be
obtained by screening libraries of modified polypeptides
recombinantly produced from the corresponding mutagenized nucleic
acids encoding an ActRIIa polypeptide. The variants can be produced
and tested to identify those that can function as antagonists
(inhibitors) of ActRIIa protein or signaling mediated by activin.
In certain embodiments, a functional variant of the ActRIIa
polypeptides comprises an amino acid sequence that is at least 75%
identical to an amino acid sequence selected from SEQ ID NOs: 2 or
3. 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: 2 or
3.
[0058] Functional variants may be generated by modifying the
structure of an ActRIIa polypeptide for such purposes as enhancing
therapeutic efficacy, or stability (e.g., ex vivo shelf life and
resistance to proteolytic degradation in vivo). Such modified
ActRIIa polypeptides when selected to retain activin binding, are
considered functional equivalents of the naturally-occurring
ActRIIa polypeptides. Modified ActRIIa 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 ActRIIa polypeptide results in a
functional homolog can be readily determined by assessing the
ability of the variant ActRIIa polypeptide to produce a response in
cells in a fashion similar to the wild-type ActRIIa
polypeptide.
[0059] In certain embodiments, the present invention contemplates
specific mutations of the ActRIIa polypeptides so as to alter the
glycosylation of the polypeptide. Such mutations may be selected so
as to introduce or eliminate one or more glycosylation sites, such
as O-linked or N-linked glycosylation sites. Asparagine-linked
glycosylation recognition sites generally comprise a tripeptide
sequence, asparagine-X-threonine or asparagine-X-serine (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 ActRIIa
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
ActRIIa polypeptide is by chemical or enzymatic coupling of
glycosides to the ActRIIa 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. Removal of one or more carbohydrate
moieties present on an ActRIIa polypeptide may be accomplished
chemically and/or enzymatically. Chemical deglycosylation may
involve, for example, exposure of the ActRIIa 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. Enzymatic cleavage of
carbohydrate moieties on ActRIIa 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
ActRIIa 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, ActRIIa proteins for use in humans may 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. Other non-mammalian cell
lines may be used (e.g., yeast, E. coli, insect cells), and in some
cases, such cell lines may be engineered to include enzymes that
confer mammalian-type glycosylation patterns on the expressed
proteins.
[0060] This disclosure further contemplates a method of generating
mutants, particularly sets of combinatorial mutants of an ActRIIa
polypeptide, as well as truncation mutants; 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, ActRIIa polypeptide variants which
bind to activin or other ligands. A variety of screening assays are
provided below, and such assays may be used to evaluate variants.
For example, an ActRIIa polypeptide variant may be screened for
ability to bind to an ActRIIa ligand, to prevent binding of an
ActRIIa ligand to an ActRIIa polypeptide or to interfere with
signaling caused by an ActRIIa ligand.
[0061] The activity of an ActRIIa polypeptide or its variants may
also be tested in a cell-based or in vivo assay. For example, the
effect of an ActRIIa polypeptide variant on the expression of genes
involved in hematopoiesis may be assessed. This may, as needed, be
performed in the presence of one or more recombinant ActRIIa ligand
proteins (e.g., activin), and cells may be transfected so as to
produce an ActRIIa polypeptide and/or variants thereof, and
optionally, an ActRIIa ligand. Likewise, an ActRIIa polypeptide may
be administered to a mouse or other animal, and one or more blood
measurements, such as an RBC count, hemoglobin, or reticulocyte
count may be assessed.
[0062] Combinatorially-derived variants can be generated which have
a selective or generally increased potency relative to a naturally
occurring ActRIIa polypeptide. Likewise, mutagenesis can give rise
to variants which have intracellular half-lives dramatically
different than the corresponding a wild-type ActRIIa polypeptide.
For example, the altered protein can be rendered either more stable
or less stable to proteolytic degradation or other cellular
processes which result in destruction of, or otherwise inactivation
of a native ActRIIa polypeptide. Such variants, and the genes which
encode them, can be utilized to alter ActRIIa polypeptide levels by
modulating the half-life of the ActRIIa 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 ActRIIa polypeptide levels within
the cell. In an Fc fusion protein, mutations may be made in the
linker (if any) and/or the Fc portion to alter the half-life of the
protein.
[0063] A combinatorial library may be produced by way of a
degenerate library of genes encoding a library of polypeptides
which each include at least a portion of potential ActRIIa
polypeptide sequences. For instance, a mixture of synthetic
oligonucleotides can be enzymatically ligated into gene sequences
such that the degenerate set of potential ActRIIa polypeptide
nucleotide sequences are expressible as individual polypeptides, or
alternatively, as a set of larger fusion proteins (e.g., for phage
display).
[0064] There are many ways by which the library of potential
homologs can be generated from a degenerate oligonucleotide
sequence. Chemical synthesis of a degenerate gene sequence can be
carried out in an automatic DNA synthesizer, and the synthetic
genes can then be ligated into an appropriate vector for
expression. The synthesis of degenerate oligonucleotides is well
known in the art (see for example, Narang, S A (1983) Tetrahedron
39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland
Sympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevier pp
273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura
et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid
Res. 11:477). Such techniques have been employed in the directed
evolution of other proteins (see, for example, Scott et al., (1990)
Science 249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433;
Devlin et al., (1990) Science 249: 404-406; Cwirla et al., (1990)
PNAS USA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,
5,198,346, and 5,096,815).
[0065] Alternatively, other forms of mutagenesis can be utilized to
generate a combinatorial library. For example, ActRIIa polypeptide
variants can be generated and isolated from a library by screening
using, for example, alanine scanning mutagenesis and the like (Ruf
et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J.
Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118;
Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et
al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al., (1991)
Biochemistry 30:10832-10838; and Cunningham et al., (1989) Science
244:1081-1085), by linker scanning mutagenesis (Gustin et al.,
(1993) Virology 193:653-660; Brown et al., (1992) Mol. Cell Biol.
12:2644-2652; McKnight et al., (1982) Science 232:316); by
saturation mutagenesis (Meyers et al., (1986) Science 232:613); by
PCR mutagenesis (Leunget al., (1989) Method Cell Mol Biol 1:11-19);
or by random mutagenesis, including chemical mutagenesis, etc.
(Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL
Press, Cold Spring Harbor, N.Y.; and Greener et al., (1994)
Strategies in Mol Biol 7:32-34). Linker scanning mutagenesis,
particularly in a combinatorial setting, is an attractive method
for identifying truncated (bioactive) forms of ActRIIa
polypeptides.
[0066] A wide range of techniques are known in the art for
screening gene products of combinatorial libraries made by point
mutations and truncations, and, for that matter, for screening cDNA
libraries for gene products having a certain property. Such
techniques will be generally adaptable for rapid screening of the
gene libraries generated by the combinatorial mutagenesis of
ActRIIa polypeptides. The most widely used techniques for screening
large gene libraries typically comprises cloning the gene library
into replicable expression vectors, transforming appropriate cells
with the resulting library of vectors, and expressing the
combinatorial genes under conditions in which detection of a
desired activity facilitates relatively easy isolation of the
vector encoding the gene whose product was detected. Preferred
assays include activin binding assays and activin-mediated cell
signaling assays.
[0067] In certain embodiments, the ActRIIa polypeptides of the
invention may further comprise post-translational modifications in
addition to any that are naturally present in the ActRIIa
polypeptides. Such modifications include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. As a result, the modified ActRIIa
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 ActRIIa polypeptide may be tested as described
herein for other ActRIIa polypeptide variants. When an ActRIIa
polypeptide is produced in cells by cleaving a nascent form of the
ActRIIa 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, W138, 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 ActRIIa
polypeptides.
[0068] In certain aspects, functional variants or modified forms of
the ActRIIa polypeptides include fusion proteins having at least a
portion of the ActRIIa 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 (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 system (Qiagen) useful
with (HIS.sub.6) fusion partners. As another example, a fusion
domain may be selected so as to facilitate detection of the ActRIIa
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 ActRIIa polypeptide is fused with a
domain that stabilizes the ActRIIa 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. Constant domains from an
immunoglobulin, particularly an IgG heavy chain, may also be used
as stabilizing domains. 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 bone
growth).
[0069] As a specific example, the present invention provides a
fusion protein comprising a soluble extracellular domain of ActRIIa
fused to an Fc domain. An example of an IgG1 Fc domain is shown
below (SEQ ID NO: 6).
TABLE-US-00006 THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*
[0070] Optionally, the Fc domain has one or more mutations at
residues such as Asp-265, lysine 322, and Asn-434. In certain
cases, the mutant Fc domain having one or more of these mutations
(e.g., Asp-265 mutation) has reduced ability of binding to the
Fc.gamma. receptor relative to a wildtype Fc domain. In other
cases, the mutant Fc domain having one or more of these mutations
(e.g., Asn-434 mutation) has increased ability of binding to the
MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fc
domain. Fc domains from IgG2, IgG3 and IgG4 may also be used.
[0071] 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 ActRIIa polypeptide may be
placed C-terminal to a heterologous domain, or, alternatively, a
heterologous domain may be placed C-terminal to an ActRIIA
polypeptide. The ActRIIa 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.
[0072] In certain embodiments, the ActRIIaa polypeptides of the
present invention contain one or more modifications that are
capable of stabilizing the ActRIIa polypeptides. For example, such
modifications enhance the in vitro half life of the ActRIIa
polypeptides, enhance circulatory half life of the ActRIIa
polypeptides or reducing proteolytic degradation of the ActRIIa
polypeptides. Such stabilizing modifications include, but are not
limited to, fusion proteins (including, for example, fusion
proteins comprising an ActRIIa polypeptide and a stabilizer
domain), modifications of a glycosylation site (including, for
example, addition of a glycosylation site to an ActRIIa
polypeptide), and modifications of carbohydrate moiety (including,
for example, removal of carbohydrate moieties from an ActRIIa
polypeptide). As used herein, the term "stabilizer domain" not only
refers to a fusion domain (e.g., Fc) as in the case of fusion
proteins, but also includes nonproteinaceous modifications such as
a carbohydrate moiety, or nonproteinaceous moiety, such as
polyethylene glycol.
[0073] In certain embodiments, the present invention makes
available isolated and/or purified forms of the ActRIIa
polypeptides, which are isolated from, or otherwise substantially
free of, other proteins. ActRIIa polypeptides will generally be
produced by expression from recombinant nucleic acids.
3. Nucleic Acids Encoding ActRIIa Polypeptides
[0074] In certain aspects, the invention provides isolated and/or
recombinant nucleic acids encoding any of the ActRIIa polypeptides
(e.g., soluble ActRIIa polypeptides), including fragments,
functional variants and fusion proteins disclosed herein. For
example, SEQ ID NO: 4 encodes the naturally occurring human ActRIIa
precursor polypeptide, while SEQ ID NO: 5 encodes the processed
extracellular domain of ActRIIa. The subject nucleic acids may be
single-stranded or double stranded. Such nucleic acids may be DNA
or RNA molecules. These nucleic acids may be used, for example, in
methods for making ActRIIa polypeptides or as direct therapeutic
agents (e.g., in a gene therapy approach).
[0075] In certain aspects, the subject nucleic acids encoding
ActRIIa polypeptides are further understood to include nucleic
acids that are variants of SEQ ID NO: 4 or 5.
[0076] 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 NOs: 4 or 5. One of
ordinary skill in the art will appreciate that nucleic acid
sequences complementary to SEQ ID NOs: 4 or 5, and variants of SEQ
ID NOs: 4 or 5 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.
[0077] In other embodiments, nucleic acids of the invention also
include nucleotide sequences, and the ActRIIa polypeptides encoded
by such nucleic acids, that hybridize under highly stringent
conditions to the nucleotide sequence designated in SEQ ID NOs: 4
or 5, complement sequence of SEQ ID NOs: 4 or 5 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.
[0078] Isolated nucleic acids which differ from the nucleic acids
as set forth in SEQ ID NOs: 4 or 5 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.
[0079] 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.
[0080] In certain aspects of the invention, the subject nucleic
acid is provided in an expression vector comprising a nucleotide
sequence encoding an ActRIIa polypeptide and operably linked to at
least one regulatory sequence. Regulatory sequences are
art-recognized and are selected to direct expression of the ActRIIa
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 ActRIIa 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., Pho5,
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.
[0081] 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 ActRIIa 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.
[0082] 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, 3rd Ed., ed. by Sambrook,
Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 2001).
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 pAcUWI), and pBlueBac-derived vectors (such as the
.beta.-gal containing pBlueBac III).
[0083] In a preferred embodiment, a vector will be designed for
production of the subject ActRIIa 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 ActRIIa
polypeptides in cells propagated in culture, e.g., to produce
proteins, including fusion proteins or variant proteins, for
purification.
[0084] This disclosure also pertains to a host cell transfected
with a recombinant gene including a coding sequence (e.g., SEQ ID
NO: 4 or 5) for one or more of the subject ActRIIa polypeptides.
The host cell may be any prokaryotic or eukaryotic cell. For
example, an ActRIIa 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.
[0085] Accordingly, the present invention further pertains to
methods of producing the subject ActRIIa polypeptides. For example,
a host cell transfected with an expression vector encoding an
ActRIIa polypeptide can be cultured under appropriate conditions to
allow expression of the ActRIIa polypeptide to occur. The ActRIIa
polypeptide may be secreted and isolated from a mixture of cells
and medium containing the ActRIIa polypeptide. Alternatively, the
ActRIIa 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 ActRIIa 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,
immunoaffinity purification with antibodies specific for particular
epitopes of the ActRIIa polypeptides and affinity purification with
an agent that binds to a domain fused to the ActRIIa polypeptide
(e.g., a protein A column may be used to purify an ActRIIa-Fc
fusion). In a preferred embodiment, the ActRIIa polypeptide is a
fusion protein containing a domain which facilitates its
purification. In a preferred embodiment, purification is 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. As demonstrated herein, ActRIIa-hFc
protein was purified to a purity of >98% as determined by size
exclusion chromatography and >95% as determined by SDS PAGE.
This level of purity was sufficient to achieve desirable results in
mice, rats, non-human primates and humans.
[0086] 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 ActRIIa 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 ActRIIa polypeptide (e.g., see Hochuli et al., (1987) J.
Chromatography 411:177; and Janknecht et al., PNAS USA
88:8972).
[0087] 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. Alternative Activin and ActRIIa Antagonists
[0088] The data presented herein demonstrates that antagonists of
activin-ActRIIa signaling can be used to increase red blood cell or
hemoglobin levels. Although soluble ActRIIa polypeptides, and
particularly ActRIIa-Fc, are preferred antagonists, and although
such antagonists may affect red blood cell levels through a
mechanism other than activin antagonism (e.g., activin inhibition
may be an indicator of the tendency of an agent to inhibit the
activities of a spectrum of molecules, including, perhaps, other
members of the TGF-beta superfamily, and such collective inhibition
may lead to the desired effect on hematopoiesis), other types of
activin-ActRIIa antagonists are expected to be useful, including
anti-activin (e.g., activin .beta..sub.A, .beta..sub.B,
.beta..sub.C and .beta..sub.E) antibodies, anti-ActRIIa antibodies,
antisense, RNAi or ribozyme nucleic acids that inhibit the
production of ActRIIa, and other inhibitors of activin or ActRIIa,
particularly those that disrupt activin-ActRIIa binding.
[0089] An antibody that is specifically reactive with an ActRIIa
polypeptide (e.g., a soluble ActRIIa polypeptide) and which either
binds competitively to ligand with the ActRIIa polypeptide or
otherwise inhibits ActRIIa-mediated signaling may be used as an
antagonist of ActRIIa polypeptide activities. Likewise, an antibody
that is specifically reactive with an activin .beta..sub.A,
.beta..sub.B, .beta..sub.C or .beta..sub.E polypeptide, or any
heterodimer thereof, and which disrupts ActRIIa binding may be used
as an antagonist.
[0090] By using immunogens derived from an ActRIIa polypeptide or
an activin 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
activin or ActRIIa 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 ActRIIa or activin
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.
[0091] Following immunization of an animal with an antigenic
preparation of an activin or ActRIIa 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 activin or ActRIIa polypeptide and monoclonal
antibodies isolated from a culture comprising such hybridoma
cells.
[0092] The term "antibody" as used herein is intended to include
whole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc),
and includes fragments or domains of immunoglobulins which are
reactive with a selected antigen. Antibodies can be fragmented
using conventional techniques and the fragments screened for
utility and/or interaction with a specific epitope of interest.
Thus, the term includes segments of proteolytically-cleaved or
recombinantly-prepared portions of an antibody molecule that are
capable of selectively reacting with a certain protein.
Non-limiting examples of such proteolytic and/or recombinant
fragments include Fab, F(ab')2, Fab', Fv, and single chain
antibodies (scFv) containing a V[L] and/or V[H] domain joined by a
peptide linker. The scFv's may be covalently or non-covalently
linked to form antibodies having two or more binding sites. The
term antibody also includes polyclonal, monoclonal, or other
purified preparations of antibodies and recombinant antibodies. The
term "recombinant antibody", means an antibody, or antigen binding
domain of an immunoglobulin, expressed from a nucleic acid that has
been constructed using the techniques of molecular biology, such as
a humanized antibody or a fully human antibody developed from a
single chain antibody. Single domain and single chain antibodies
are also included within the term "recombinant antibody".
[0093] In certain 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 ActRIIa polypeptide or activin polypeptide may
comprise administering to a mouse an amount of an immunogenic
composition comprising the antigen 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 monocolonal antibody that binds specifically to the antigen. 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
antigen. The monoclonal antibody may be purified from the cell
culture.
[0094] 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 activin or ActRIIa
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 M or less.
[0095] 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.TM.
binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g.,
the paramagnetic bead system of IGEN International, Inc.,
Gaithersburg, Md.), western blots, immunoprecipitation assays, and
immunohistochemistry.
[0096] Examples of categories of nucleic acid compounds that are
activin or ActRIIa antagonists include antisense nucleic acids,
RNAi constructs and catalytic nucleic acid constructs. A nucleic
acid compound may be single or double stranded. A double stranded
compound may also include regions of overhang or
non-complementarity, where one or the other of the strands is
single stranded. A single stranded compound may include regions of
self-complementarity, meaning that the compound forms a so-called
"hairpin" or "stem-loop" structure, with a region of double helical
structure. A nucleic acid compound may comprise a nucleotide
sequence that is complementary to a region consisting of no more
than 1000, no more than 500, no more than 250, no more than 100, or
no more than 50, 35, 25, 22, 20, 18 or 15 nucleotides of the
full-length ActRIIa nucleic acid sequence or activin .beta..sub.A,
.beta..sub.B, .beta..sub.C, or .beta..sub.E nucleic acid sequence.
The region of complementarity will preferably be at least 8
nucleotides, and optionally about 18 to 35 nucleotides. A region of
complementarity may fall within an intron, a coding sequence or a
noncoding sequence of the target transcript, such as the coding
sequence portion. Generally, a nucleic acid compound will have a
length of about 8 to about 500 nucleotides or base pairs in length,
and optionally the length will be about 14 to about 50 nucleotides.
A nucleic acid may be a DNA (particularly for use as an antisense),
RNA or RNA:DNA hybrid. Any one strand may include a mixture of DNA
and RNA, as well as modified forms that cannot readily be
classified as either DNA or RNA. Likewise, a double stranded
compound may be DNA:DNA, DNA:RNA or RNA:RNA, and any one strand may
also include a mixture of DNA and RNA, as well as modified forms
that cannot readily be classified as either DNA or RNA. A nucleic
acid compound may include any of a variety of modifications,
including one or modifications to the backbone (the sugar-phosphate
portion in a natural nucleic acid, including intemucleotide
linkages) or the base portion (the purine or pyrimidine portion of
a natural nucleic acid). An antisense nucleic acid compound will
preferably have a length of about 15 to about 30 nucleotides and
will often contain one or more modifications to improve
characteristics such as stability in the serum, in a cell or in a
place where the compound is likely to be delivered, such as the
stomach in the case of orally delivered compounds and the lung for
inhaled compounds. In the case of an RNAi construct, the strand
complementary to the target transcript will generally be RNA or
modifications thereof. The other strand may be RNA, DNA or any
other variation. The duplex portion of double stranded or single
stranded "hairpin" RNAi construct will generally have a length of
18 to 40 nucleotides in length and optionally about 21 to 23
nucleotides in length, so long as it serves as a Dicer substrate.
Catalytic or enzymatic nucleic acids may be ribozymes or DNA
enzymes and may also contain modified forms. Nucleic acid compounds
may inhibit expression of the target by about 50%, 75%, 90% or more
when contacted with cells under physiological conditions and at a
concentration where a nonsense or sense control has little or no
effect. Preferred concentrations for testing the effect of nucleic
acid compounds are 1, 5 and 10 micromolar. Nucleic acid compounds
may also be tested for effects on, for example, red blood cell
levels.
[0097] In certain embodiments, an activin-ActRIIa antagonist may be
a follistatin polypeptide that antagonizes activin bioactivity
and/or binds to activin. The term "follistatin polypeptide"
includes polypeptides comprising any naturally occurring
polypeptide of follistatin as well as any variants thereof
(including mutants, fragments, fusions, and peptidomimetic forms)
that retain a useful activity, and further includes any functional
monomer or multimer of follistatin. Variants of follistatin
polypeptides that retain activin binding properties can be
identified based on previous studies involving follistatin and
activin interactions. For example, WO2008/030367 discloses specific
follistatin domains ("FSDs") that are shown to be important for
activin binding. As shown below in SEQ ID NOs: 19-21, the
N-terminus follistatin domain ("FSND" SEQ ID NO: 19), FSD2 (SEQ ID
NO: 20), and to a lesser extent FSD1 (SEQ ID NO: 21) represent
exemplary domains within follistatin important for activin binding.
In addition, methods for making and testing libraries of
polypeptides are described above in the context of ActRIIa
polypeptides and such methods also pertain to making and testing
variants of follistatin. Follistatin polypeptides include
polypeptides derived from the sequence of any known follistatin
having a sequence at least about 80% identical to the sequence of a
follistatin polypeptide, and optionally at least 85%, 90%, 95%,
97%, 99% or greater identity. Examples of follistatin polypeptides
include the mature follistatin polypeptide or shorter isoforms or
other variants of the human follistatin precursor polypeptide (SEQ
ID NO: 17) as described, for example, in WO2005/025601.
[0098] The human follistatin precursor polypeptide isoform FST344
is as follows:
TABLE-US-00007 MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTEL
SKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDC
GPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARC
KEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPA
SSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQC
TGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEA
ACSSGVLLEVKHGSCNSISEDTEEEEEDEDQDYSFPISSILEW (SEQ ID NO: 17;
NP_037541.1 FOLLISTATIN ISOFORM FST344)
The signal peptide is single underlined; the last 27 residues in
bold represent additional amino acids as compared to a shorter
follistatin isoform FST317 (NP.sub.--006341) below.
[0099] The human follistatin precursor polypeptide isoform FST317
is as follows:
TABLE-US-00008 (SEQ ID NO: 18)
MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTEL
SKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDC
GPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARC
KEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPA
SSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQC
TGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEA
ACSSGVLLEVKHSGSCN
The signal peptide is single underlined.
[0100] N-terminus follistatin domain (FSND) sequence is as
follows:
TABLE-US-00009 GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWM
IFNGGAPNCIPCK (SEQ ID NO: 19; FSND)
[0101] The FSD1 and FSD2 sequences are as follows:
TABLE-US-00010 ETCENVDCGPGKKCRMNKKNKPRCV (SEQ ID NO: 20; FSD1)
KTCRDVFCPGSSTCVVDQTNNAYCVT (SEQ ID NO: 21; FSD2)
[0102] In other embodiments, an activin-ActRIIa antagonist may be a
follistatin-like related gene (FLRG) that antagonizes activin
bioactivity and/or binds to activin. The term "FLRG polypeptide"
includes polypeptides comprising any naturally occurring
polypeptide of FLRG as well as any variants thereof (including
mutants, fragments, fusions, and peptidomimetic forms) that retain
a useful activity. Variants of FLRG polypeptides that retain
activin binding properties can be identified using routine methods
to assay FLRG and activin interactions. See, for example, U.S. Pat.
No. 6,537,966. In addition, methods for making and testing
libraries of polypeptides are described above in the context of
ActRIIa polypeptides and such methods also pertain to making and
testing variants of FLRG. FLRG polypeptides include polypeptides
derived from the sequence of any known FLRG having a sequence at
least about 80% identical to the sequence of an FLRG polypeptide,
and optionally at least 85%, 90%, 95%, 97%, 99% or greater
identity.
[0103] The human FLRG precursor polypeptide is as follows:
TABLE-US-00011 MRPGAPGPLWPLPWGALAWAVGFVSSMGSGNPAPGGVCWLQQGQEATCSL
VLQTDVTRAECCASGNIDTAWSNLTHPGNKINLLGFLGLVHCLPCKDSCD
GVECGPGKACRMLGGRPRCECAPDCSGLPARLQVCGSDGATYRDECELPA
ARCRGHPDLSVMYRGRCRKSCEHVVCPRPQSCVVDQTGSAHCVVCRAAPC
VPSSPGQELCGNNNVTYISSCHMRQATCFLGRSIGVRHAGSCAGTPEEPP GGESAEEEENFV
(SEQ ID NO: 22; NP_005851)
The signal peptide is single underlined.
[0104] In certain embodiments, functional variants or modified
forms of the follistatin polypeptides and FLRG polypeptides include
fusion protein having at least a portion of the follistatin
polypeptides or FLRG polypeptides and one or more fusion domains,
such as, for example, domains that facilitate isolation, detection,
stabilization or multimerization of the polypeptide. Suitable
fusion domains are discussed in detail above with reference to the
ActRIIa polypeptides. In one embodiment, an activin-ActRIIa
antagonist is a fusion protein comprising an activin binding
portion of a follistaton polypeptide fused to an Fc domain. In
another embodiment, an activin-ActRIIa antagonist is a fusion
protein comprising an activin binding portion of an FLRG
polypeptide fused to an Fc domain. Follistatin and FLRG have been
shown in the literature, and by the applicants with respect to
FLRG, to have affinities for Activin A in the picomolar range,
indicating that these agents will inhibit activin A signaling to a
similar degree as ActRIIa-Fc.
5. Exemplary Therapeutic Methods
[0105] In certain embodiments, the present invention provides
methods for managing a patient that has been treated with, or is a
candidate to be treated with, an activin-ActRIIa antagonist by
measuring one or more hematologic parameters in the patient. The
hematologic parameters may be used to evaluate appropriate dosing
for a patient who is a candidate to be treated with an
activin-ActRIIa anatagonist, to monitor the hematologic parameters
during treatment with an activin-ActRIIa antagonist, to evaluate
whether to adjust the dosage during treatment with an
activin-ActRIIa antagonist, and/or to evaluate an appropriate
maintenance dose of an activin-ActRIIa antagonist. If one or more
of the hematologic parameters are outside the normal level, dosing
with the activin-ActRIIa antagonist may be reduced, delayed or
terminated.
[0106] Hematologic parameters that may be measured in accordance
with the methods provided herein include, for example, red blood
cell levels, blood pressure, iron stores, and other agents found in
bodily fluids that correlate with increased red blood cell levels,
using art recognized methods. Such parameters may be determined
using a blood sample from a patient. Increases in red blood cell
levels, hemoglobin levels, and/or hematocrit levels may cause
increases in blood pressure.
[0107] Red blood cell levels may be determined, for example, by
determining a red blood cell count, by measuring hemoglobin levels
or by measuring hematocrit levels. A red blood cell count may be
determined using a commercially available Coulter Counter. The
normal range for a red blood cell count is between 4.2-5.9 million
cells/cm, although individual variations should be taken into
account. Hemoglobin levels may be determined by lysing the red
blood cells, converting the hemoglobin into cyanomethemoglobin and
measuring the amount of hemoglobin with a calorimeter. The normal
ranges for hemoglobin are 14-18 gm/dl for adult males and 12-16
gm-dl for adult females, although individual variations should be
taken into account. Hematocrit (Hct) or packed cell volume (PCV)
refers to the ratio of the volume of red blood cells to the volume
of whole blood. Hematocrit may be determined, for example, by
centrifugation of a blood sample followed by analysis of the layers
produced. Normal ranges for hematocrit are approximately 41-51% for
men and 35-45% for women, although individual variations should be
taken into account.
[0108] Blood pressure, including systolic blood pressure, diastolic
blood pressure, or mean arterial blood pressure, may be determined
using art recognized methods. Blood pressure is most commonly
measured using a sphygmomanometer. Typical values for a resting,
healthy adult human are approximately 120 mmHg systolic and 80 mmHg
diastolic, although individual variations should be taken into
account. Individuals suffering from hypertension typically have a
blood pressure .gtoreq.140 mmHg systolic and .gtoreq.90 diastolic
blood pressure. Individuals having a level above normal but less
than 140/90 mmHg are generally referred to as prehypertensive.
Additional methods for measuring blood pressure may be found in
Pickering et al., Hypertension 45: 142-161 (2005).
[0109] Iron stores may be measured using a variety of art
recognized techniques including, for example, by determining levels
of one or more of the following: serum ferritin (SF), transferrin
saturation (TSAT), total iron binding capacity, hemoglobin
concentration, zinc protoporphyrin, mean cell volume (MCV), or
transferrin receptor in serum. Serum ferritin levels may be
determined, for example, using an immunoassay such as an
enzyme-linked immunosorbent assay (ELISA) or immunoturbidometry. In
normal patients, serum ferritin levels range from 13 to 220 ng/mL,
although individual variations should be taken into account.
Transferrin saturation levels represent the proportion of
transferrin bound to iron and may be determined by dividing serum
iron by total iron biding capacity (TIBC). In normal patients,
transferrin saturation levels range from 20% to 40%, although
individual variations should be taken into account. Serum iron may
be determiend using colorimetry and is expressed as ug/dl or
umol/l. Total iron binding capacity reflects the total capacity of
circulating transferrin bound to iron and may be determined using a
colorimetric assay to determine the amount of iron that can be
bound to unsaturated transferrin in vitro. The normal range of
total iron binding capacity is about 250-450 ug/dl, although
individual variations should be taken into account. Additional
information about measuring iron stores may be found in the World
Health Organization report entitled Assessing the Iron Status of
Populations dated April 2004 and in Yamanishi et al., Clinical
Chemistry 48: 1565-1570 (2002).
[0110] In one embodiment, if one or more hematologic parameters are
outside the normal range, or on the high side of normal, in a
patient who is a candidate to be treated with an activin-ActRIIa
antagonist then onset of administration of the activin-ActRIIa
antagonist may be delayed until the hematologic parameters have
returned to a normal or acceptable level either naturally or via
therapeutic intervention. For example, if a candidate patient is
hypertensive or prehypertensive, then the patient may be treated
with a blood pressure lowering agent in order to reduce the
patient's blood pressure. Any blood pressure lowering agent
appropriate for the individual patient's condition may be used
including, for example, diuretics, adrenergic inhibitors (including
alpha blockers and beta blockers), vasodilators, calcium channel
blockers, angiotensin-converting enzyme (ACE) inhibitors, or
angiotensin II receptor blockers. Blood pressure may alternatively
be treated using a diet and exercise regimen. Similarly, if a
candidate patient has iron stores that are lower than normal, or on
the low side of normal, then the patient may be treated with an
appropriate regimen of diet and/or iron supplements until the
patient's iron stores have returned to a normal or acceptable
level. For patients having higher than normal red blood cell levels
and/or hemoglobin levels, then administration of the
activin-ActRIIa antagonist may be delayed until the levels have
returned to a normal or acceptable level.
[0111] In certain embodiments, if one or more hematologic
parameters are outside the normal range, or on the high side of
normal, in a patient who is a candidate to be treated with an
activin-ActRIIa antagonist then the onset of administration may be
not be delayed. However, the dosage amount or frequency of dosing
of the activin-ActRIIa antagonist may be set at an amount that
would reduce the risk of an unacceptable increase in the
hematologic parameters arising upon administration of the
activin-ActRIIa antagonist. Alternatively, a therapeutic regimen
may be developed for the patient that combines an activin-ActRIIa
antagonist with a therapeutic agent that addresses the undesirable
level of the hematologic parameter. For example, if the patient has
elevated blood pressure, then a therapeutic regimen involving
administration of an activin-ActRIIa antagonist and a blood
pressure lowering agent may be designed. For a patient having lower
than desired iron stores, a therapeutic regimen of an
activin-ActRIIa antagonist and iron supplementation may be
developed.
[0112] In one embodiment, baseline parameter(s) for one or more
hematologic parameters may be established for a patient who is a
candidate to be treated with an activin-ActRIIa antagonists and an
appropriate dosing regimen establish for that patient based on the
baseline value(s). Alternatively, established baseline parameters
based on a patient's medical history could be used to inform an
appropriate activin-ActRIIa antagonist dosing regimen for a
patient. For example, if a healthy patient has an established
baseline blood pressure reading that is above the defined normal
range it may not be necessary to bring the patient's blood pressure
into the range that is considered normal for the general population
prior to treatment with the activin-ActRIIa antagonist. A patient's
baseline values for one or more hematologic parameters prior to
treatment with an activin-ActRIIa antagonist may also be used as
the relevant comparative values for monitoring any changes to the
hematologic parameters during treatment with the activin-ActRIIa
antagonist.
[0113] In certain embodiments, one or more hematologic parameters
are measured in patients who are being treated with an
activin-ActRIIa antagonist. The hematologic parameters may be used
to monitor the patient during treatment and permit adjustment or
termination of the dosing with the activin-ActRIIa antagonist or
additional dosing with another therapeutic agent. For example, if
administration of an activin-ActRIIa antagonist results in an
increase in blood pressure, red blood cell level, or hemoglobin
level, or a reduction in iron stores, then the dose of the
activin-ActRIIa antagonists may be reduced in amount or frequency
in order to decrease the effects of the activin-ActRIIa antagonist
on the one or more hematologic parameters. If administration or an
activin-ActRIIa antagonist results in a change in one or more
hematologic parameters that is adverse to the patient, then the
dosing of the activin-ActRIIa antagonist may be terminated either
temporarily, until the hematologic parameter(s) return to an
acceptable level, or permanently. Similarly, if one or more
hematologic parameters are not brought within an acceptable range
after reducing the dose or frequency of administration of the
activin-ActRIIa antagonist then the dosing may be terminated. As an
alternative, or in addition to, reducing or terminating the dosing
with the activin-ActRIIa antagonist, the patient may be dosed with
an additional therapeutic agent that addresses the undesirable
level in the hematologic parameter(s), such as, for example, a
blood pressure lowering agent or an iron supplement. For example,
if a patient being treated with an activin-ActRIIa antagonist has
elevated blood pressure, then dosing with the activin-ActRIIa
antagonist may continue at the same level and a blood pressure
lowering agent is added to the treatment regimen, dosing with the
activin-ActRIIa antagonist may be reduce (e.g., in amount and/or
frequency) and a blood pressure lowering agent is added to the
treatment regimen, or dosing with the activin-ActRIIa antagonist
may be terminated and the patient may be treated with a blood
pressure lowering agent.
[0114] In certain embodiments, if a patient being treated with an
activin-ActRIIa antagonist or a patient who is a candidate for
treatment with an activin-ActRIIa antagonist has one or more of the
following: a hemoglobin level greater than 12 g/dl, a hemoglobin
level greater than 15 g/dl, a blood pressure .gtoreq.120/80 mmHg, a
blood pressure .gtoreq.140/90 mmHg, a transferrin saturation level
less than 20%, and/or a ferritin level less than 100 ng/ml, then
dosing with the activin-ActRIIa antagonist is reduced, delayed or
terminated. As an alternative, or in addition to, reducing,
delaying or terminating dosing with activin-ActRIIa antagonist, a
therapeutic agent that addresses the undesired level of one or more
hematologic parameters (such as a blood pressure lowering agent or
an iron supplement) may be administered to the patient.
[0115] In one embodiment, the present invention provides a method
for dosing a patient with an activin-ActRIIa antagonist by
administering to the patient an activin-ActRIIa antagonist in an
amount and at a frequency which reduces the risk of causing a rise
in hemoglobin levels greater than 1 g/dl over a two week period.
The methods may comprise measuring one or more hematologic
parameters either before beginning administration of the
activin-ActRIIa antagonist and/or during administration of the
activin-ActRIIa antagonist. The initial dose of the activin-ActRIIa
antagonist may be set so as to reduce the risk of causing a rise in
hemoglobin levels greater than 1 g/dl over a two week period. In
addition, the dose may be adjusted over time to in order to
maintain a reduced risk of causing a rise in hemoglobin levels
greater than 1 g/dl in two weeks.
[0116] In certain embodiments, the present invention provides a
method for administering an ActRIIa-Fc fusion protein to a patient
by administering the ActRIIa fusion protein no more frequently than
once per 60 days, once per 90 days, or once per 120 days, or 1-6
times per year, 2-6 times per year, 1-5 times per year, 2-5 times
per year, 1-4 times per year, 2-4 times per year, 1-3 times per
year, or 2-3 times per year. As demonstrated herein, increases in
red blood cell levels arising from administration peak around 60
days after administration. At about 90 days after administration, a
significant reduction in red blood cell levels is seen and after
about 120 days red blood cell levels return to the baseline level.
Accordingly, for patients in which the activin-ActRIIa antagonist
is being administered for purposes other than increasing red blood
cell levels, it may be desirable to administer subsequent doses of
the activin-ActRIIa antagonist after the peak increase in red blood
cell levels from the previous dose, or even after red blood cell
levels have returned to normal.
[0117] In certain embodiments, the invention provides methods for
determining dosing and monitoring therapeutic progress with
activin-ActRIIa antagonist treatment in patients in which the
activin-ActRIIa antagonist is being administered to increase red
blood cell levels. The methods involve determining one or more
hematologic parameters either prior to beginning dosing with the
activin-ActRIIa antagonist and/or during treatment with the
activin-ActRIIa antagonist. For example, one or more hematologic
parameters may be determined in a patient who is a candidate for
administration of an activin-ActRIIa antagonist for increasing
blood cell levels to facilitate determination of dosage amount and
frequency. One or more hematologic parameters may also be
determined in a patient being treated with an activin-ActRIIa
antagonist for purposes of increasing red blood cell levels in
order to monitor progress of the treatment, facilitate dosing
adjustments, and to determine maintenance dosing levels, etc.
[0118] In accordance with the methods of the invention, one or more
hematologic parameters may be measured at various time points and
at varying frequencies as needed for an individual patient based on
various factors such as a patient's baseline levels, responsiveness
to treatment with an activin-ActRIIa antagonist, general health,
age, sex, weight, etc. Measuring of one or more hematologic
parameters may be carried out before and/or during treatment with
an activin-ActRIIa antagonist. If conducting multiple measurements
of hematologic parameters at various time points, the same set of
hematologic parameter(s) need not be measured at each time point.
Similarly, the same test for an individual parameter need not be
used at each time point. Appropriate hematologic parameters and
tests for such parameters may be chosen for an individual taking
into account factors specific to the given individual. Testing of
hematologic parameters may occur as frequently as needed for a
given individual, such as, for example, once per day, once per
week, once per every two weeks, once per month, once per each 2
month period, once per each 3 month period, once per each 6 month
period, or once per year. In addition, the frequency of testing may
vary over time. For example, upon initial dosing of an individual
it may be desirable to test for one or more hematologic parameters
more frequently, e.g., once per day, once per week, once per every
two weeks, or once per month, and then decrease the frequency of
testing over time, e.g., after one month, two months, six months, 1
year, two years, or longer, of treatment, the frequency of testing
may reduced to, for example, once per month, once per every two
months, once per every three months, once per every six months, or
once per year. Similarly, it may be desirable to test more
frequently when adjusting a patient's dose of an activin-ActRIIa
antagonist (e.g., either amount or frequency of administration) and
then decrease the frequency of testing over time, for example, once
the patient's response to the activin-ActRIIa antagonist has been
established.
[0119] In various embodiments, patients being treated with an
activin-ActRIIa antagonist, or candidate patients for treatment
with an activin-ActRIIa antagonist, may be mammals such as rodents
and primates, and particularly human patients.
[0120] In certain embodiments, patients being treated with an
activin-ActRIIa antagonist, or candidate patients to be treated
with an activin-ActRIIa antagonist, are patients in need of bone
and/or cartilage formation, prevention of bone loss, increased bone
mineralization or prevention of bone demineralization, such as
patients with low bone density, decreased bone strength, or bone
damage due to breakage, loss or demineralization. In exemplary
embodiments, the patients or candidate patients may be patients
suffering from, or at risk for developing, osteoporosis (including
secondary osteoporosis), hyperparathyroidism, Cushing's disease,
Paget's disease, thyrotoxicosis, chronic diarrheal state or
malabsorption, renal tubular acidosis, or anorexia nervosa.
Osteoporosis resulting from drugs or another medical condition is
known as secondary osteoporosis. Medications that can cause
secondary osteoporosis include, for example, corticosteroids,
methotrexate (Rheumatrex, Immunex, Folex PFS), cyclosporine
(Sandimmune, Neoral), luteinizing hormone-releasing hormone
agonists (Lupron, Zoladex), and heparin (Calciparine, Liquaemin).
Bone loss resulting from cancer therapy is widely recognized and
termed cancer therapy induced bone loss (CTIBL).
[0121] In certain embodiments, patients being treated with an
activin-ActRIIa antagonist, or candidate patients to be treated
with an activin-ActRIIa antagonist, are patients suffering from or
at high risk for developing breast cancer. As every woman is at
risk for developing breast cancer, a woman with a high risk for
developing breast cancer is a woman whose risk factors confer a
greater probability of developing the disease compared to the
general population or the population of women within a certain age
group. Exemplary risk factors include age, family history or
genetic makeup, lifestyle habits such as exercise and diet,
exposure to radiation or other cancer-causing agents, age at the
time the first child was born, genetic changes, and weight gain
after menopause. Exemplary patients include, for example, patients
that have mutations in the BRCA1/2 genes or other genes shown to
predispose women to breast and ovarian cancer are also included.
Patients also include individuals with solid tumors, metastatic
cancer, precancerous lesions of the breast, benign lesions of the
breast, or with any abnormal proliferative lesions including
typical hyperplasia, atypical hyperplasia, and noninvasive or in
situ carcinoma. Patients also include those with both
hormone-dependent or hormone-responsive cancers (e.g., estrogen
receptor positive cancers) and hormone-independent cancers (e.g.,
estrogen receptor negative or estrogen receptor mutant cancers).
Patients suffering from cancers in which growth factors or
oncogenes are activated (e.g., breast cancers in which c-erbB-2
(also known as HER-2/Neu) tyrosine kinase is expressed) are also
contemplated.
[0122] In certain embodiments, patients being treated with an
activin-ActRIIa antagonist, or candidate patients to be treated
with an activin-ActRIIa antagonist, are patients with undesirably
low red blood cell or hemoglobin levels, such as patients having an
anemia, and those that are at risk for developing undesirably low
red blood cell or hemoglobin levels, such as those patients that
are about to undergo major surgery or other procedures that may
result in substantial blood loss, such as having blood drawn and
stored for a later transfusion. Patients and candidate patients may
also include those patients in need of an increase in red blood
cells and/or hemoglobin levels that do not respond well to Epo.
When observing hemoglobin levels in humans, a level of less than
normal for the appropriate age and gender category may be
indicative of anemia, although individual variations are taken into
account. Potential causes of anemia include blood-loss, nutritional
deficits, medication reaction, various problems with the bone
marrow and many diseases. More particularly, anemia has been
associated with a variety of disorders that include, for example,
chronic renal failure, myelodysplastic syndrome, rheumatoid
arthritis, bone marrow transplantation. Anemia may also be
associated with the following conditions: solid tumors (e.g. breast
cancer, lung cancer, colon cancer); tumors of the lymphatic system
(e.g. chronic lymphocyte leukemia, non-Hodgkins and Hodgkins
lymphomas); tumors of the hematopoietic system (eg. leukemia,
myelodysplastic syndrome, multiple myeloma); radiation therapy;
chemotherapy (e.g. platinum containing regimens); inflammatory and
autoimmune diseases, including, but not limited to, rheumatoid
arthritis, other inflammatory arthritides, systemic lupus
erythematosis (SLE), acute or chronic skin diseases (e.g.
psoriasis), inflammatory bowel disease (e.g. Crohn's disease and
ulcerative colitis); acute or chronic renal disease or failure
including idiopathic or congenital conditions; acute or chronic
liver disease; acute or chronic bleeding; situations where
transfusion of red blood cells is not possible due to patient allo-
or auto-antibodies and/or for religious reasons (e.g. some
Jehovah's Witnesses); infections (e.g. malaria, osteomyelitis);
hemoglobinopathies, including, for example, sickle cell disease,
thalassemias; drug use or abuse, e.g. alcohol misuse; pediatric
patients with anemia from any cause to avoid transfusion; and
elderly patients or patients with underlying cardiopulmonary
disease with anemia who cannot receive transfusions due to concerns
about circulatory overload.
[0123] 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. In either case, prevention or treatment may
be discerned in the diagnosis provided by a physician or other
health care provider and the intended result of administration of
the therapeutic agent.
6. Pharmaceutical Compositions
[0124] In certain embodiments, activin-ActRIIa antagonists (e.g.,
ActRIIa polypeptides) of the present invention are formulated with
a pharmaceutically acceptable carrier. For example, an ActRIIa
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.
[0125] In certain embodiments, the therapeutic method of the
invention includes administering the composition 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. Therapeutically
useful agents other than the activin-ActRIIa antagonists which may
also optionally be included in the composition as described above,
may be administered simultaneously or sequentially with the subject
compounds (e.g., ActRIIa polypeptides) in the methods of the
invention.
[0126] Typically, activin-ActRIaI antagonists will be administered
parenterally. Pharmaceutical compositions suitable for parenteral
administration may comprise one or more ActRIIa 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.
[0127] Further, the composition may be encapsulated or injected in
a form for delivery to a target tissue site (e.g., bone marrow). In
certain embodiments, compositions of the present invention may
include a matrix capable of delivering one or more therapeutic
compounds (e.g., ActRIIa polypeptides) to a target tissue site
(e.g., bone marrow), 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 ActRIIa
polypeptides. Such matrices may be formed of materials presently in
use for other implanted medical applications.
[0128] 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.
[0129] In certain embodiments, methods of the invention can be
administered for orally, e.g., in the form of capsules, cachets,
pills, tablets, lozenges (using a flavored basis, usually sucrose
and acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of an agent as an
active ingredient. An agent may also be administered as a bolus,
electuary or paste.
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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.
[0134] 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., ActRIIa
polypeptides). The various factors include, but are not limited to,
the patient's red blood cell count, hemoglobin level or other
diagnostic assessments, the desired target red blood cell count,
the patient's age, sex, and diet, the severity of any disease that
may be contributing to a depressed red blood cell level, 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 red
blood cell and hemoglobin levels, as well as assessments of
reticulocyte levels and other indicators of the hematopoietic
process.
[0135] Experiments with primates and humans have demonstrated that
effects of ActRIIa-Fc on red blood cell levels are detectable when
the compound is dosed at intervals and amounts sufficient to
achieve serum concentrations of about 100 ng/ml or greater, for a
period of at least about 20 to 30 days. Dosing to obtain serum
levels of 200 ng/ml, 500 ng/ml, 1000 ng/ml or greater for a period
of at least 20 to 30 days may also be used. Bone effects can be
observed at serum levels of about 200 ng/ml, with substantial
effects beginning at about 1000 ng/ml or higher, over a period of
at least about 20 to 30 days. Thus, if it is desirable to achieve
effects on red blood cells while having little effect on bone, a
dosing scheme may be designed to deliver a serum concentration of
between about 100 and 1000 ng/ml over a period of about 20 to 30
days. Alternatively, if it is desirable to achieve effects on bone,
breast cancer, etc., while having little effect on, or reducing
effects on red blood cell levels, a dosing scheme may be designed
to deliver a dosing scheme of between about 100 and 1000 ng/ml with
a dosing frequency that occurs less than once every 60 days, once
every 90 days, or once every 120 days. In humans, serum levels of
200 ng/ml may be achieved with a single dose of 0.1 mg/kg or
greater and serum levels of 1000 ng/ml may be achieved with a
single dose of 0.3 mg/kg or greater. The observed serum half-life
of the molecule is between about 20 and 30 days, substantially
longer than most Fc fusion proteins, and thus a sustained effective
serum level may be achieved, for example, by dosing with about 0.05
to 0.5 mg/kg on a weekly or biweekly basis, or higher doses may be
used with longer intervals between dosings. For example, doses of
0.1 to 1 mg/kg might be used on a monthly or bimonthly basis.
[0136] In certain embodiments, the present invention also provides
gene therapy for the in vivo production of ActRIIa polypeptides.
Such therapy would achieve its therapeutic effect by introduction
of the ActRIIa polynucleotide sequences into cells or tissues
having the disorders as listed above. Delivery of ActRIIa
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 ActRIIa
polynucleotide sequences is the use of targeted liposomes.
[0137] Various viral vectors which can be utilized for gene therapy
as taught herein include adenovirus, herpes virus, vaccinia, or an
RNA virus such as a retrovirus. The retroviral vector may be 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
ActRIIa polynucleotide.
[0138] 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.
[0139] Another targeted delivery system for ActRIIa 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.
[0140] 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.
Ememplification
[0141] 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
ActRIIa-Fc Fusion Proteins
[0142] Applicants constructed a soluble ActRIIa fusion protein that
has the extracellular domain of human ActRIIa fused to a human or
mouse Fc domain with a minimal linker in between. The constructs
are referred to as ActRIIa-hFc and ActRIIa-mFc, respectively.
[0143] ActRIIa-hFc is shown below as purified from CHO cell lines
(SEQ ID NO: 7):
TABLE-US-00012 ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGS
IEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM
EVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0144] The ActRIIa-hFc and ActRIIa-mFc proteins were expressed in
CHO cell lines. Three different leader sequences were
considered:
TABLE-US-00013 (i) Honey bee mellitin (HBML): MKFLVNVALVFMVVYISYLYA
(SEQ ID NO: 8) (ii) Tissue Plasminogen Activator (TPA):
MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 9) (iii) Native:
MGAAAKLAFAVFLISCSSGA. (SEQ ID NO: 10)
[0145] The selected form employs the TPA leader and has the
following unprocessed amino acid sequence:
TABLE-US-00014 (SEQ ID NO:13)
MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQT
GVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKK
DSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPP
CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK
[0146] This polypeptide is encoded by the following nucleic acid
sequence:
TABLE-US-00015 (SEQ ID NO: 14)
ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGC
AGTCTTCGTTTCGCCCGGCGCCGCTATACTTGGTAGATCAGAAACTCAGG
AGTGTCTTTTTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTG
GTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCT
ACCTGGAAGAATATTTCTGGTTCCATTGAATAGTGAAACAAGGTTGTTGG
CTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGA
CAGCCCTGAAGTATATTTCTGTTGCTGTGAGGGCAATATGTGTAATGAAA
AGTTTTCTTATTTTCCGGAGATGGAAGTCACACAGCCCACTTCAAATCCA
GTTACACCTAAGCCACCCACCGGTGGTGGAACTCACACATGCCCACCGTG
CCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAA
AACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTG
GTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT
GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGT
ACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC
TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCC
AGTCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC
CACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGT
GGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTG
GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA
TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG
GTAAATGAGAATTC
[0147] Both ActRIIa-hFc and ActRIIa-mFc were remarkably amenable to
recombinant expression. As shown in FIG. 1, the protein was
purified as a single, well-defined peak of protein. N-terminal
sequencing revealed a single sequence of -ILGRSTQE (SEQ ID NO: 11).
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. The ActRIIa-hFc protein
was purified to a purity of >98% as determined by size exclusion
chromatography and >95% as determined by SDS PAGE.
[0148] ActRIIa-hFc and ActRIIa-mFc showed a high affinity for
ligands, particularly activin A. GDF-11 or Activin A ("ActA") were
immobilized on a Biacore CM5 chip using standard amine coupling
procedure. ActRIIa-hFc and ActRIIa-mFc proteins were loaded onto
the system, and binding was measured. ActRIIa-hFc bound to activin
with a dissociation constant (KD) of 5.times.10.sup.-12, and the
protein bound to GDF11 with a KD of 9.96.times.10.sup.-9. See FIG.
2. ActRIIa-mFc behaved similarly.
[0149] The ActRIIa-hFc was very stable in pharmacokinetic studies.
Rats were dosed with 1 mg/kg, 3 mg/kg or 10 mg/kg of ActRIIa-hFc
protein and plasma levels of the protein were measured at 24, 48,
72, 144 and 168 hours. In a separate study, rats were dosed at 1
mg/kg, 10 mg/kg or 30 mg/kg. In rats, ActRIIa-hFc had an 11-14 day
serum half life and circulating levels of the drug were quite high
after two weeks (11 .mu.g/ml, 110 .mu.g/ml or 304 .mu.g/ml for
initial administrations of 1 mg/kg, 10 mg/kg or 30 mg/kg,
respectively.) In cynomolgus monkeys, the plasma half life was
substantially greater than 14 days and circulating levels of the
drug were 25 .mu.g/ml, 304 .mu.g/ml or 1440 .mu.g/ml for initial
administrations of 1 mg/kg, 10 mg/kg or 30 mg/kg, respectively.
Preliminary results in humans suggests that the serum half life is
between about 20 and 30 days.
Example 2
Characterization of an ActRIIa-hFc Protein
[0150] ActRIIa-hFc fusion protein was expressed in stably
transfected CHO-DUKX B11 cells from a pAID4 vector (SV40
ori/enhancer, CMV promoter), using a tissue plasminogen leader
sequence of SEQ ID NO:9. The protein, purified as described above
in Example 1, had a sequence of SEQ ID NO:7. The Fc portion is a
human IgG1 Fc sequence, as shown in SEQ ID NO:7. Sialic acid
analysis showed that the protein contained, on average, between
about 1.5 and 2.5 moles of sialic acid per molecule of ActRIIa-hFc
fusion protein.
[0151] This purified protein showed a remarkably long serum
half-life in all animals tested, including a half-life of 25-32
days in human patients (see Example 6, below). Additionally, the
CHO cell expressed material has a higher affinity for activin B
ligand than that reported for an ActRIIa-hFc fusion protein
expressed in human 293 cells (del Re et al., J Biol Chem. Dec. 17,
2004;279(51):53126-35.) Additionally, the use of the tPa leader
sequence provided greater production than other leader sequences
and, unlike ActRIIa-Fc expressed with a native leader, provided a
highly pure N-terminal sequence. Use of the native leader sequence
resulted in two major species of ActRIIa-Fc, each having a
different N-terminal sequence.
Example 3
ActRIIa-hFc Increases Red Blood Cell Levels in Non-Human
Primates
[0152] The study employed four groups of five male and five female
cynomolgus monkeys each, with three per sex per group scheduled for
termination on Day 29, and two per sex per group scheduled for
termination on Day 57. Each animal was administered the vehicle
(Group I) or ActRIIa-Fc at doses of 1, 10, or 30 mg/kg (Groups 2, 3
and 4, respectively) via intravenous (IV) injection on Days 1, 8,
15 and 22. The dose volume was maintained at 3 mL/kg. Various
measures of red blood cell levels were assessed two days prior to
the first administration and at days 15, 29 and 57 (for the
remaining two animals) after the first administration.
[0153] The ActRIIa-hFc caused statistically significant increases
in mean red blood cell parameters (red blood cell count [RBC],
hemoglobin [HGB], and hematocrit [HCT]) for males and females, at
all dose levels and time points throughout the study, with
accompanying elevations in absolute and relative reticulocyte
counts (ARTC; RTC). See FIGS. 3-6.
[0154] Statistical significance was calculated for each treatment
group relative to the mean for the treatment group at baseline.
[0155] Notably, the increases in red blood cell counts and
hemoglobin levels are roughly equivalent in magnitude to effects
reported with erythropoietin. The onset of these effects is more
rapid with ActRIIa-Fc than with erythropoietin.
[0156] Similar results were observed with rats and mice.
Example 4
ActRIIa-hFc Increases Red Blood Cell Levels and Markers of Bone
Formation in Human Patients
[0157] The ActRIIa-hFc fusion protein described in Example 1 was
administered to human patients in a randomized, double-blind,
placebo-controlled study that was conducted to evaluate, primarily,
the safety of the protein in healthy, postmenopausal women.
Forty-eight subjects were randomized in cohorts of 6 to receive
either a single dose of ActRIIa-hFc or placebo (5 active:1
placebo). Dose levels ranged from 0.01 to 3.0 mg/kg intravenously
(IV) and 0.03 to 0.1 mg/kg subcutaneously (SC). All subjects were
followed for 120 days. In addition to pharmacokinetic (PK)
analyses, the biologic activity of ActRIIa-hFc was also assessed by
measurement of biochemical markers of bone formation and
resorption, and FSH levels.
[0158] To look for potential changes, hemoglobin and RBC numbers
were examined in detail for all subjects over the course of the
study and compared to the baseline levels. Platelet counts were
compared over the same time as the control. There were no
clinically significant changes from the baseline values over time
for the platelet counts.
[0159] PK analysis of ActRIIa-hFc displayed a linear profile with
dose, and a mean half-life of approximately 25-32 days. The
area-under-curve (AUC) for ActRIIa-hFc was linearly related to
dose, and the absorption after SC dosing was essentially complete
(see FIGS. 7 and 8). These data indicate that SC is a desirable
approach to dosing because it provides equivalent bioavailability
and serum-half life for the drug while avoiding the spike in serum
concentrations of drug associated with the first few days of IV
dosing (see FIG. 8). ActRIIa-hFc caused a rapid, sustained
dose-dependent increase in serum levels of bone-specific alkaline
phosphatase (BAP), which is a marker for anabolic bone growth, and
a dose-dependent decrease in C-terminal type 1 collagen telopeptide
and tartrate-resistant acid phosphatase 5b levels, which are
markers for bone resorption. Other markers, such as P1NP showed
inconclusive results. BAP levels showed near saturating effects at
the highest dosage of drug, indicating that half-maximal effects on
this anabolic bone biomarker could be achieved at a dosage of 0.3
mg/kg, with increases ranging up to 3 mg/kg. Calculated as a
relationship of pharmacodynamic effect to AUC for drug, the EC50 is
51,465 (day*ng/ml). See FIG. 9. These bone biomarker changes were
sustained for approximately 120 days at the highest dose levels
tested. There was also a dose-dependent decrease in serum FSH
levels consistent with inhibition of activin.
[0160] Overall, there was a very small non-drug related reduction
in hemoglobin over the first week of the study probably related to
study phlebotomy in the 0.01 and 0.03 mg/kg groups whether given IV
or SC. The 0.1 mg/kg SC and IV hemoglobin results were stable or
showed modest increases by Day 8-15. At the 0.3 mg/kg IV dose level
there was a clear increase in HGB levels seen as early as Day 2 and
often peaking at Day 15-29 that was not seen in the placebo
subjects. At the 1.0 mg/kg IV dose and the 3.0 mg/kg IV dose, mean
increases in hemoglobin of greater than 1 g/dl were observed in
response to the single dose, with corresponding increases in RBC
counts and hematocrit. These hematologic parameters peaked at about
60 days after the dose and substantial decrease by day 120. This
indicates that dosing for the purpose of increasing red blood cell
levels may be more effective if done at intervals less than 120
days (i.e., prior to return to baseline), with dosing intervals of
90 days or less or 60 days or less may be desirable. For a summary
of hematological changes, see FIGS. 10-13.
[0161] Overall, ActRIIa-hFc showed a dose-dependent effect on red
blood cell counts and reticulocyte counts, and a dose-dependent
effect on markers of bone formation.
Example 5
Treatment of an Anemic Patient with ActRIIa-hFc
[0162] A clinical study was designed to treat patients with
multiple doses of ActRIIa-hFc, at dose levels of 0.1 mg/kg, 0.3
mg/kg and 1.0 mg/kg, with dosing every thirty days. Normal healthy
patients in the trial exhibited an increase in hemoglobin and
hematocrit that is consistent with the increases seen in the Phase
I clinical trial reported in Example 4, except that, in some
instances, the hemoglobin and hematocrit were elevated beyond the
normal range. An anemic patient with hemoglobin of approximately
7.5 also received two doses at the 1 mg/kg level, resulting in a
hemoglobin level of approximately 10.5 after two months. The
patient's anemia was a microcytic anemia, thought to be caused by
chronic iron deficiency.
Example 6
ActRIIa-mFc Increases Red Blood Cell Levels in Mice by Stimulation
of Splenic Red Blood Cell Release
[0163] In this study the effects of the in vivo administration of
ActRIIa-mFc on the frequency of hematopoietic progenitors in bone
marrow and spleen was analyzed. One group of mice was injected with
PBS as a control and a second group of mice administered two doses
of ActRIIa-mFc at 10 mg/kg and both groups sacrificed after 8 days.
Peripheral blood was used to perform complete blood counts and
femurs and spleens were used to perform in vitro clonogenic assays
to assess the lymphoid, erythroid and myeloid progenitor cell
content in each organ. In the peripheral blood a significant
increase in the red blood cell and hemoglobin content was seen in
compound treated mice. In the femurs there was no difference in the
nucleated cell numbers or progenitor content between the control
and treated groups. In the spleens, the compound treated group
experienced a statistically significant increase in the nucleated
cell number before red blood cell lysis and in the mature erythroid
progenitor (CFU-E) colony number per dish, frequency and total
progenitor number per spleen. In addition, and increase was seen in
the number of myeloid (CFU-GM), immature erythroid (BFU-E) and
total progenitor number per spleen.
[0164] Animals:
[0165] Sixteen BDF1 female mice 6-8 weeks of age were used in the
study. Eight mice were injected subcutaneously with test compound
ActRIIa-mFc at days 1 and 3 at a dose of 10 mg/kg and eight mice
were injected subcutaneously with vehicle control, phosphate
buffered saline (PBS), at a volume of 100 .mu.L per mouse. All mice
were sacrificed 8 days after first injection in accordance with the
relevant Animal Care Guidelines. Peripheral blood (PB) samples from
individual animals were collected by cardiac puncture and used for
complete blood counts and differential (CBC/Diff). Femurs and
spleens were harvested from each mouse.
[0166] Tests Performed:
[0167] CBC/Diff Counts
[0168] PB from each mouse was collected via cardiac puncture and
placed into the appropriate microtainer tubes. Samples were sent to
CLV for analysis on a CellDyn 3500 counter.
[0169] Clonogenic Assays
[0170] Clonogenic progenitors of the myeloid, erythroid and
lymphoid lineages were assessed using the in vitro
methylcellulose-based media systems described below.
[0171] Mature Erythroid Progenitors:
[0172] Clonogenic progenitors of the mature erythroid (CFU-E)
lineages were cultured in MethoCult.TM. 3334, a
methylcellulose-based medium containing recombinant human (rh)
Erythropoietin (3 U/mL).
[0173] Lymphoid Projenitors:
[0174] Clonogenic progenitors of the lymphoid (CFU-pre-B) lineage
were cultured in MethoCult.RTM. 3630, a methylcellulose-based
medium containing rh Interleukin 7 (10 ng/mL).
[0175] Myeloid and Immature Erythroid Progenitors:
[0176] Clonogenic progenitors of the granulocyte-monocyte (CFU-GM),
erythroid (BFU-E) and multipotential (CFU-GEMM) lineages were
cultured in MethoCult.TM. 3434, a methylcellulose-based medium
containing recombinant murine (rm) Stem Cell Factor (50 ng/mL), rh
Interleukin 6 (10 ng/mL), rm Interleukin 3 (10 ng/mL) and rh
Erythropoietin (3 U/mL).
[0177] Methods:
[0178] Mouse femurs and spleens were processed by standard
protocols. Briefly, bone marrow was obtained by flushing the
femoral cavity with Iscove's Modified Dulbecco's Media containing
2% fetal bovine serum (IMDM 2% FBS) using a 21 gauge needle and 1
cc syringe. Spleen cells were obtained by crushing spleens through
a 70 .mu.M filter and rinsing the filter with IMDM 2% FBS.
Nucleated cell counts in 3% glacial acetic acid were then performed
on the single cells suspensions using a Neubauer counting chamber
so that the total cells per organ could be calculated. To remove
contaminating red blood cells, total spleen cells were then diluted
with 3 times the volume of ammonium chloride lysis buffer and
incubated on ice 10 minutes. The cells were then washed and
resuspended in
[0179] IMDM 2% FBS and a second cell count were performed to
determine the cell concentration of cells after lysis.
[0180] Cell stocks were made and added to each
methylcellulose-based media formulation to obtain the optimal
plating concentrations for each tissue in each media formulation.
Bone marrow cells were plated at 1.times.10.sup.5 cells per dish in
MethoCult.TM. 3334 to assess mature erythroid progenitors,
2.times.10.sup.5 cells per dish in MethoCult.TM. 3630 to assess
lymphoid progenitors and 3.times.10.sup.4 cells per dish in
MethoCult.TM. 3434 to assess immature erythroid and myeloid
progenitors. Spleen cells were plated at 4.times.10.sup.5 cells per
dish in MethoCult.TM. 3334 to assess mature erythroid progenitors,
4.times.10.sup.5 cells per dish in MethoCult.TM. 3630 to assess
lymphoid progenitors and 2.times.10.sup.5 cells per dish in
MethoCult.TM. 3434 to assess immature erythroid and myeloid
progenitors. Cultures plated in triplicate dishes were incubated at
37.degree. C., 5% CO2 until colony enumeration and evaluation was
performed by trained personnel. Mature erythroid progenitors were
cultured for 2 days, lymphoid progenitors were cultured for 7 days
and mature erythroid and myeloid progenitors were cultured for 12
days.
[0181] Analysis:
[0182] The mean.+-.1 standard deviation was calculated for the
triplicate cultures of the clonogenic assays and for the control
and treatment groups for all data sets.
[0183] Frequency of colony forming cells (CFC) in each tissue was
calculated as follows:
[0184] Cells plated per dish
[0185] Mean CFC scored per dish
[0186] Total CFC per femur or spleen was calculated as follows:
[0187] Total CFC scored x nucleated cell count per femur or spleen
(following RBC lysis)
[0188] Number of nucleated cells cultured
[0189] Standard t-tests were performed to assess if there was a
differences in the mean number of cells or hematopoietic
progenitors between the PBS control mice and compound treated mice.
Due to the potential subjectivity of colony enumeration, a p value
of less than 0.01 is deemed significant. Mean values (.+-.SD) for
each group are shown in the tables below.
TABLE-US-00016 TABLE Hematologic Parameters White Blood Cells Red
Blood Cells Hemoglobin Hematocrit Treatment Group
(.times.10.sup.9/L) (.times.10.sup.9/L) (g/L) (L/L) PBS 6.37 +/-
2.83 10.9 +/- 0.7 154.5 +/- 5.9 0.506 +/- 0.029 (n = 8) ActRIIa-mFc
8.92 +/- 3.69 11.8 +/- 0.3* 168.3 +/- 4.3** 0.532 +/- 0.014 (n = 8)
*= p < 0.01 **= p < 0.0005
TABLE-US-00017 TABLE CFC From Femur and Spleen Total CFC per Total
CFC per Total CFU-E per Total CFU-E per Treatment Group Femur
Spleen Femur Spleen PBS 33437 +/- 7118 4212 +/- 1148 27185 +/-
12893 6743 +/- 1591 (n = 8) ActRIIa-mFc 31068 +/- 8024 6816 +/-
1516* 18118 +/- 6672 27313 +/- 11790 (n = 8) *= p < 0.005 **= p
< 0.0001
[0190] Treatment of mice with ActRIIa-mFc resulted in significant
increases in a number of hematopoietic parameters. In the
peripheral blood a significant increase in the red blood cell and
hemoglobin content was seen in compound treated mice. In the femurs
there was no difference in the nucleated cell numbers or progenitor
content between the control and treated groups. In the spleens, the
compound treated group experienced a statistically significant
increase in the nucleated cell number before red blood cell lysis
and in the mature erythroid progenitor (CFU-E) colony number per
dish, frequency and total progenitor number per spleen. In
addition, an increase was seen in the number of myeloid (CFU-GM),
immature erythroid (BFU-E) and total progenitor number per
spleen.
Example 7
Alternative ActRIIa-Fc Proteins
[0191] A variety of ActRIIa variants that may be used according to
the methods described herein are described in the International
Patent Application published as WO2006/012627 (see e.g., pp.
55-58), incorporated herein by reference in its entirety. An
alternative construct may have a deletion of the C-terminal tail
(the final 15 amino acids of the extracellular domain of ActRIIa.
The sequence for such a construct is presented below (Fc portion
underlined)(SEQ ID NO: 12):
TABLE-US-00018 ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGS
IEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM
TGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK
Example 8
Effect of ActRIIA-mFc on Chemotherapy-Induced Anemia in Mice
[0192] Applicants investigated the effect of ActRIIA-mFc on
chemotherapy-induced anemia in mice. In the first of two studies,
6-week-old female C57BL/6 mice were treated with a single dose of
ActRIIA-mFc (10 mg/kg, s.c.) or vehicle (phosphate-buffered saline)
3 days before a single dose of the chemotherapeutic paclitaxel (20
mg/kg, i.p.). Blood samples were collected before chemotherapy and
then 3, 7, and 14 days (n=6 per cohort per time point) after
paclitaxel. ActRIIA-mFc prevented the decline in hematocrit level
otherwise observed after paclitaxel (FIG. 15), and similar effects
were observed for hemoglobin concentration and RBC count. In a
second study, 6-week-old female C57BL/6 mice were given a varying
number of ActRIIA-mFc doses (10 mg/kg, s.c.), or vehicle (PBS),
beginning before paclitaxel (20 mg/kg single dose, i.p.) and
continuing at intervals of 3 or 4 days. Blood samples were
collected 3, 7, and 14 days (n=8 per cohort per time point) after
paclitaxel. At 14 days, ActRIIA-mFc treatment increased hematocrit
level progressively as a function of dose number (FIG. 16). Thus,
ActRIIA-mFc can stimulate erythropoiesis sufficiently to attenuate
or prevent chemotherapy-induced anemia.
Example 9
Effect of ActRIIA-mFc on Anemia in a Mouse Model of Chronic Kidney
Disease
[0193] Applicants investigated the effect of ActRIIA-mFc on
nephrectomy-induced anemia in mice as a model of chronic kidney
disease. In the first of two studies, female C57BL/6 mice underwent
a partial surgical nephrectomy, with removal of approximately
five-sixths of total kidney volume, to reduce production of
erythropoietin. Mice were given a 4-week recovery period with a
high-fat diet to further promote renal deficiency and were then
treated twice-weekly with ActRIIA-mFc (10 mg/kg, s.c.) or vehicle
(PBS) for a total of 8 weeks. Blood samples were collected before
the onset of dosing, after 4 weeks of treatment, and after 8 weeks
of treatment (n=8 per cohort per time point). Control mice
exhibited a decline in hematocrit level over the 8-week treatment
period, whereas ActRIIA-mFc treatment prevented the decline at 4
weeks and also produced a beneficial trend at 8 weeks (FIG. 17).
Similar benefits of ActRIIA-mFc treatment over control were
observed in a second study that differed mainly in the use of a
longer recovery period (2 months) and a standard diet. Thus,
ActRIIA-mFc can stimulate erythropoiesis sufficiently to prevent or
attenuate anemia in a model of chronic kidney disease.
[0194] Taken together, these findings indicate that soluble
ActRIIA-Fc fusion proteins can be used as antagonists of signaling
by TGF-family ligands to increase circulating levels of red blood
cells, and thereby, to treat hypoproliferative anemias resulting
from chronic diseases such as cancer and renal disease, and
potentially other inflammatory or infectious diseases as well. Note
that effects of ACE-011 on anemia in human patients are typically
robust compared to the more modest effects in rodents.
INCORPORATION BY REFERENCE
[0195] 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.
[0196] 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
251513PRTHomo sapiens 1Met Gly Ala Ala Ala Lys Leu Ala Phe Ala Val
Phe Leu Ile Ser Cys1 5 10 15Ser Ser Gly Ala Ile Leu Gly Arg Ser Glu
Thr Gln Glu Cys Leu Phe 20 25 30Phe Asn Ala Asn Trp Glu Lys Asp Arg
Thr Asn Gln Thr Gly Val Glu 35 40 45Pro Cys Tyr Gly Asp Lys Asp Lys
Arg Arg His Cys Phe Ala Thr Trp 50 55 60Lys Asn Ile Ser Gly Ser Ile
Glu Ile Val Lys Gln Gly Cys Trp Leu65 70 75 80Asp Asp Ile Asn Cys
Tyr Asp Arg Thr Asp Cys Val Glu Lys Lys Asp 85 90 95Ser Pro Glu Val
Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu 100 105 110Lys Phe
Ser Tyr Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn 115 120
125Pro Val Thr Pro Lys Pro Pro Tyr Tyr Asn Ile Leu Leu Tyr Ser Leu
130 135 140Val Pro Leu Met Leu Ile Ala Gly Ile Val Ile Cys Ala Phe
Trp Val145 150 155 160Tyr Arg His His Lys Met Ala Tyr Pro Pro Val
Leu Val Pro Thr Gln 165 170 175Asp Pro Gly Pro Pro Pro Pro Ser Pro
Leu Leu Gly Leu Lys Pro Leu 180 185 190Gln Leu Leu Glu Val Lys Ala
Arg Gly Arg Phe Gly Cys Val Trp Lys 195 200 205Ala Gln Leu Leu Asn
Glu Tyr Val Ala Val Lys Ile Phe Pro Ile Gln 210 215 220Asp Lys Gln
Ser Trp Gln Asn Glu Tyr Glu Val Tyr Ser Leu Pro Gly225 230 235
240Met Lys His Glu Asn Ile Leu Gln Phe Ile Gly Ala Glu Lys Arg Gly
245 250 255Thr Ser Val Asp Val Asp Leu Trp Leu Ile Thr Ala Phe His
Glu Lys 260 265 270Gly Ser Leu Ser Asp Phe Leu Lys Ala Asn Val Val
Ser Trp Asn Glu 275 280 285Leu Cys His Ile Ala Glu Thr Met Ala Arg
Gly Leu Ala Tyr Leu His 290 295 300Glu Asp Ile Pro Gly Leu Lys Asp
Gly His Lys Pro Ala Ile Ser His305 310 315 320Arg Asp Ile Lys Ser
Lys Asn Val Leu Leu Lys Asn Asn Leu Thr Ala 325 330 335Cys Ile Ala
Asp Phe Gly Leu Ala Leu Lys Phe Glu Ala Gly Lys Ser 340 345 350Ala
Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro 355 360
365Glu Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg
370 375 380Ile Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Ala
Ser Arg385 390 395 400Cys Thr Ala Ala Asp Gly Pro Val Asp Glu Tyr
Met Leu Pro Phe Glu 405 410 415Glu Glu Ile Gly Gln His Pro Ser Leu
Glu Asp Met Gln Glu Val Val 420 425 430Val His Lys Lys Lys Arg Pro
Val Leu Arg Asp Tyr Trp Gln Lys His 435 440 445Ala Gly Met Ala Met
Leu Cys Glu Thr Ile Glu Glu Cys Trp Asp His 450 455 460Asp Ala Glu
Ala Arg Leu Ser Ala Gly Cys Val Gly Glu Arg Ile Thr465 470 475
480Gln Met Gln Arg Leu Thr Asn Ile Ile Thr Thr Glu Asp Ile Val Thr
485 490 495Val Val Thr Met Val Thr Asn Val Asp Phe Pro Pro Lys Glu
Ser Ser 500 505 510Leu 2115PRTHomo sapiens 2Ile Leu Gly Arg Ser Glu
Thr Gln Glu Cys Leu Phe Phe Asn Ala Asn1 5 10 15Trp Glu Lys Asp Arg
Thr Asn Gln Thr Gly Val Glu Pro Cys Tyr Gly 20 25 30Asp Lys Asp Lys
Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile Ser 35 40 45Gly Ser Ile
Glu Ile Val Lys Gln Gly Cys Trp Leu Asp Asp Ile Asn 50 55 60Cys Tyr
Asp Arg Thr Asp Cys Val Glu Lys Lys Asp Ser Pro Glu Val65 70 75
80Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser Tyr
85 90 95Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn Pro Val Thr
Pro 100 105 110Lys Pro Pro 1153100PRTHomo sapiens 3Ile Leu Gly Arg
Ser Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala Asn1 5 10 15Trp Glu Lys
Asp Arg Thr Asn Gln Thr Gly Val Glu Pro Cys Tyr Gly 20 25 30Asp Lys
Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile Ser 35 40 45Gly
Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu Asp Asp Ile Asn 50 55
60Cys Tyr Asp Arg Thr Asp Cys Val Glu Lys Lys Asp Ser Pro Glu Val65
70 75 80Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser
Tyr 85 90 95Phe Pro Glu Met 10041542DNAHomo sapiens 4atgggagctg
ctgcaaagtt ggcgtttgcc gtctttctta tctcctgttc ttcaggtgct 60atacttggta
gatcagaaac tcaggagtgt cttttcttta atgctaattg ggaaaaagac
120agaaccaatc aaactggtgt tgaaccgtgt tatggtgaca aagataaacg
gcggcattgt 180tttgctacct ggaagaatat ttctggttcc attgaaatag
tgaaacaagg ttgttggctg 240gatgatatca actgctatga caggactgat
tgtgtagaaa aaaaagacag ccctgaagta 300tatttttgtt gctgtgaggg
caatatgtgt aatgaaaagt tttcttattt tccagagatg 360gaagtcacac
agcccacttc aaatccagtt acacctaagc caccctatta caacatcctg
420ctctattcct tggtgccact tatgttaatt gcggggattg tcatttgtgc
attttgggtg 480tacaggcatc acaagatggc ctaccctcct gtacttgttc
caactcaaga cccaggacca 540cccccacctt ctccattact agggttgaaa
ccactgcagt tattagaagt gaaagcaagg 600ggaagatttg gttgtgtctg
gaaagcccag ttgcttaacg aatatgtggc tgtcaaaata 660tttccaatac
aggacaaaca gtcatggcaa aatgaatacg aagtctacag tttgcctgga
720atgaagcatg agaacatatt acagttcatt ggtgcagaaa aacgaggcac
cagtgttgat 780gtggatcttt ggctgatcac agcatttcat gaaaagggtt
cactatcaga ctttcttaag 840gctaatgtgg tctcttggaa tgaactgtgt
catattgcag aaaccatggc tagaggattg 900gcatatttac atgaggatat
acctggccta aaagatggcc acaaacctgc catatctcac 960agggacatca
aaagtaaaaa tgtgctgttg aaaaacaacc tgacagcttg cattgctgac
1020tttgggttgg ccttaaaatt tgaggctggc aagtctgcag gcgataccca
tggacaggtt 1080ggtacccgga ggtacatggc tccagaggta ttagagggtg
ctataaactt ccaaagggat 1140gcatttttga ggatagatat gtatgccatg
ggattagtcc tatgggaact ggcttctcgc 1200tgtactgctg cagatggacc
tgtagatgaa tacatgttgc catttgagga ggaaattggc 1260cagcatccat
ctcttgaaga catgcaggaa gttgttgtgc ataaaaaaaa gaggcctgtt
1320ttaagagatt attggcagaa acatgctgga atggcaatgc tctgtgaaac
cattgaagaa 1380tgttgggatc acgacgcaga agccaggtta tcagctggat
gtgtaggtga aagaattacc 1440cagatgcaga gactaacaaa tattattacc
acagaggaca ttgtaacagt ggtcacaatg 1500gtgacaaatg ttgactttcc
tcccaaagaa tctagtctat ga 15425345DNAHomo sapiens 5atacttggta
gatcagaaac tcaggagtgt cttttcttta atgctaattg ggaaaaagac 60agaaccaatc
aaactggtgt tgaaccgtgt tatggtgaca aagataaacg gcggcattgt
120tttgctacct ggaagaatat ttctggttcc attgaaatag tgaaacaagg
ttgttggctg 180gatgatatca actgctatga caggactgat tgtgtagaaa
aaaaagacag ccctgaagta 240tatttttgtt gctgtgaggg caatatgtgt
aatgaaaagt tttcttattt tccagagatg 300gaagtcacac agcccacttc
aaatccagtt acacctaagc caccc 3456225PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
6Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro1 5
10 15Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 20 25 30Arg Thr Pro Glu Val Thr Cys Val Val Val Xaa Val Ser His
Glu Asp 35 40 45Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn 50 55 60Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val65 70 75 80Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu 85 90 95Tyr Lys Cys Xaa Val Ser Asn Lys Ala
Leu Pro Val Pro Ile Glu Lys 100 105 110Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 115 120 125Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 130 135 140Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu145 150 155
160Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
165 170 175Asp Ser Asp Gly Pro Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 180 185 190Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 195 200 205Ala Leu His Xaa His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 210 215 220Lys2257344PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
7Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala Asn1 5
10 15Trp Glu Lys Asp Arg Thr Asn Gln Thr Gly Val Glu Pro Cys Tyr
Gly 20 25 30Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn
Ile Ser 35 40 45Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu Asp
Asp Ile Asn 50 55 60Cys Tyr Asp Arg Thr Asp Cys Val Glu Lys Lys Asp
Ser Pro Glu Val65 70 75 80Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys
Asn Glu Lys Phe Ser Tyr 85 90 95Phe Pro Glu Met Glu Val Thr Gln Pro
Thr Ser Asn Pro Val Thr Pro 100 105 110Lys Pro Pro Thr Gly Gly Gly
Thr His Thr Cys Pro Pro Cys Pro Ala 115 120 125Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 130 135 140Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val145 150 155
160Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
165 170 175Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln 180 185 190Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln 195 200 205Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala 210 215 220Leu Pro Val Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro225 230 235 240Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 245 250 255Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 260 265 270Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 275 280
285Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
290 295 300Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe305 310 315 320Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys 325 330 335Ser Leu Ser Leu Ser Pro Gly Lys
340821PRTApis sp. 8Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met
Val Val Tyr Ile1 5 10 15Ser Tyr Ile Tyr Ala
20922PRTUnknownDescription of Unknown Tissue plasminogen activator
leader sequence 9Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu
Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Pro
201020PRTUnknownDescription of Unknown Native leader sequence 10Met
Gly Ala Ala Ala Lys Leu Ala Phe Ala Val Phe Leu Ile Ser Cys1 5 10
15Ser Ser Gly Ala 20118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 11Ile Leu Gly Arg Ser Thr Gln
Glu1 512329PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 12Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu
Phe Phe Asn Ala Asn1 5 10 15Trp Glu Lys Asp Arg Thr Asn Gln Thr Gly
Val Glu Pro Cys Tyr Gly 20 25 30Asp Lys Asp Lys Arg Arg His Cys Phe
Ala Thr Trp Lys Asn Ile Ser 35 40 45Gly Ser Ile Glu Ile Val Lys Gln
Gly Cys Trp Leu Asp Asp Ile Asn 50 55 60Cys Tyr Asp Arg Thr Asp Cys
Val Glu Lys Lys Asp Ser Pro Glu Val65 70 75 80Tyr Phe Cys Cys Cys
Glu Gly Asn Met Cys Asn Glu Lys Phe Ser Tyr 85 90 95Phe Pro Glu Met
Thr Gly Gly Gly Thr His Thr Cys Pro Pro Cys Pro 100 105 110Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 115 120
125Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
130 135 140Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr145 150 155 160Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu 165 170 175Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His 180 185 190Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys 195 200 205Ala Leu Pro Val Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 210 215 220Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met225 230 235
240Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
245 250 255Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn 260 265 270Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu 275 280 285Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val 290 295 300Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln305 310 315 320Lys Ser Leu Ser Leu
Ser Pro Gly Lys 32513369PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 13Met Asp Ala Met Lys Arg
Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser
Pro Gly Ala Ala Ile Leu Gly Arg Ser Glu Thr 20 25 30Gln Glu Cys Leu
Phe Phe Asn Ala Asn Trp Glu Lys Asp Arg Thr Asn 35 40 45Gln Thr Gly
Val Glu Pro Cys Tyr Gly Asp Lys Asp Lys Arg Arg His 50 55 60Cys Phe
Ala Thr Trp Lys Asn Ile Ser Gly Ser Ile Glu Ile Val Lys65 70 75
80Gln Gly Cys Trp Leu Asp Asp Ile Asn Cys Tyr Asp Arg Thr Asp Cys
85 90 95Val Glu Lys Lys Asp Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu
Gly 100 105 110Asn Met Cys Asn Glu Lys Phe Ser Tyr Phe Pro Glu Met
Glu Val Thr 115 120 125Gln Pro Thr Ser Asn Pro Val Thr Pro Lys Pro
Pro Thr Gly Gly Gly 130 135 140Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro145 150 155 160Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 165 170 175Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 180 185 190Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 195 200
205Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
210 215 220Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu225 230 235 240Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Val Pro Ile Glu Lys 245 250 255Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 260 265 270Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr 275 280 285Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 290 295 300Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu305 310 315
320Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
325 330 335Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 340 345 350Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 355 360
365Lys 141114DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 14atggatgcaa tgaagagagg
gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60tcgcccggcg ccgctatact
tggtagatca gaaactcagg agtgtctttt tttaatgcta 120attgggaaaa
agacagaacc aatcaaactg gtgttgaacc gtgttatggt gacaaagata
180aacggcggca ttgttttgct acctggaaga atatttctgg ttccattgaa
tagtgaaaca 240aggttgttgg ctggatgata tcaactgcta tgacaggact
gattgtgtag aaaaaaaaga 300cagccctgaa gtatatttct gttgctgtga
gggcaatatg tgtaatgaaa agttttctta 360ttttccggag atggaagtca
cacagcccac ttcaaatcca gttacaccta agccacccac 420cggtggtgga
actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc
480agtcttcctc ttccccccaa aacccaagga caccctcatg atctcccgga
cccctgaggt 540cacatgcgtg gtggtggacg tgagccacga agaccctgag
gtcaagttca actggtacgt 600ggacggcgtg gaggtgcata atgccaagac
aaagccgcgg gaggagcagt acaacagcac 660gtaccgtgtg gtcagcgtcc
tcaccgtcct gcaccaggac tggctgaatg gcaaggagta 720caagtgcaag
gtctccaaca aagccctccc agtccccatc gagaaaacca tctccaaagc
780caaagggcag ccccgagaac cacaggtgta caccctgccc ccatcccggg
aggagatgac 840caagaaccag gtcagcctga cctgcctggt caaaggcttc
tatcccagcg acatcgccgt 900ggagtgggag agcaatgggc agccggagaa
caactacaag accacgcctc ccgtgctgga 960ctccgacggc tccttcttcc
tctatagcaa gctcaccgtg gacaagagca ggtggcagca 1020ggggaacgtc
ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa
1080gagcctctcc ctgtctccgg gtaaatgaga attc 1114155PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Thr
Gly Gly Gly Gly1 5165PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 16Ser Gly Gly Gly Gly1
517343PRTHomo sapiens 17Met Val Arg Ala Arg His Gln Pro Gly Gly Leu
Cys Leu Leu Leu Leu1 5 10 15Leu Leu Cys Gln Phe Met Glu Asp Arg Ser
Ala Gln Ala Gly Asn Cys 20 25 30Trp Leu Arg Gln Ala Lys Asn Gly Arg
Cys Gln Val Leu Tyr Lys Thr 35 40 45Glu Leu Ser Lys Glu Glu Cys Cys
Ser Thr Gly Arg Leu Ser Thr Ser 50 55 60Trp Thr Glu Glu Asp Val Asn
Asp Asn Thr Leu Phe Lys Trp Met Ile65 70 75 80Phe Asn Gly Gly Ala
Pro Asn Cys Ile Pro Cys Lys Glu Thr Cys Glu 85 90 95Asn Val Asp Cys
Gly Pro Gly Lys Lys Cys Arg Met Asn Lys Lys Asn 100 105 110Lys Pro
Arg Cys Val Cys Ala Pro Asp Cys Ser Asn Ile Thr Trp Lys 115 120
125Gly Pro Val Cys Gly Leu Asp Gly Lys Thr Tyr Arg Asn Glu Cys Ala
130 135 140Leu Leu Lys Ala Arg Cys Lys Glu Gln Pro Glu Leu Glu Val
Gln Tyr145 150 155 160Gln Gly Arg Cys Lys Lys Thr Cys Arg Asp Val
Phe Cys Pro Gly Ser 165 170 175Ser Thr Cys Val Val Asp Gln Thr Asn
Asn Ala Tyr Cys Val Thr Cys 180 185 190Asn Arg Ile Cys Pro Glu Pro
Ala Ser Ser Glu Gln Tyr Leu Cys Gly 195 200 205Asn Asp Gly Val Thr
Tyr Ser Ser Ala Cys His Leu Arg Lys Ala Thr 210 215 220Cys Leu Leu
Gly Arg Ser Ile Gly Leu Ala Tyr Glu Gly Lys Cys Ile225 230 235
240Lys Ala Lys Ser Cys Glu Asp Ile Gln Cys Thr Gly Gly Lys Lys Cys
245 250 255Leu Trp Asp Phe Lys Val Gly Arg Gly Arg Cys Ser Leu Cys
Asp Glu 260 265 270Leu Cys Pro Asp Ser Lys Ser Asp Glu Pro Val Cys
Ala Ser Asp Asn 275 280 285Ala Thr Tyr Ala Ser Glu Cys Ala Met Lys
Glu Ala Ala Cys Ser Ser 290 295 300Gly Val Leu Leu Glu Val Lys His
Gly Ser Cys Asn Ser Ile Ser Glu305 310 315 320Asp Thr Glu Glu Glu
Glu Glu Asp Glu Asp Gln Asp Tyr Ser Phe Pro 325 330 335Ile Ser Ser
Ile Leu Glu Trp 34018317PRTHomo sapiens 18Met Val Arg Ala Arg His
Gln Pro Gly Gly Leu Cys Leu Leu Leu Leu1 5 10 15Leu Leu Cys Gln Phe
Met Glu Asp Arg Ser Ala Gln Ala Gly Asn Cys 20 25 30Trp Leu Arg Gln
Ala Lys Asn Gly Arg Cys Gln Val Leu Tyr Lys Thr 35 40 45Glu Leu Ser
Lys Glu Glu Cys Cys Ser Thr Gly Arg Leu Ser Thr Ser 50 55 60Trp Thr
Glu Glu Asp Val Asn Asp Asn Thr Leu Phe Lys Trp Met Ile65 70 75
80Phe Asn Gly Gly Ala Pro Asn Cys Ile Pro Cys Lys Glu Thr Cys Glu
85 90 95Asn Val Asp Cys Gly Pro Gly Lys Lys Cys Arg Met Asn Lys Lys
Asn 100 105 110Lys Pro Arg Cys Val Cys Ala Pro Asp Cys Ser Asn Ile
Thr Trp Lys 115 120 125Gly Pro Val Cys Gly Leu Asp Gly Lys Thr Tyr
Arg Asn Glu Cys Ala 130 135 140Leu Leu Lys Ala Arg Cys Lys Glu Gln
Pro Glu Leu Glu Val Gln Tyr145 150 155 160Gln Gly Arg Cys Lys Lys
Thr Cys Arg Asp Val Phe Cys Pro Gly Ser 165 170 175Ser Thr Cys Val
Val Asp Gln Thr Asn Asn Ala Tyr Cys Val Thr Cys 180 185 190Asn Arg
Ile Cys Pro Glu Pro Ala Ser Ser Glu Gln Tyr Leu Cys Gly 195 200
205Asn Asp Gly Val Thr Tyr Ser Ser Ala Cys His Leu Arg Lys Ala Thr
210 215 220Cys Leu Leu Gly Arg Ser Ile Gly Leu Ala Tyr Glu Gly Lys
Cys Ile225 230 235 240Lys Ala Lys Ser Cys Glu Asp Ile Gln Cys Thr
Gly Gly Lys Lys Cys 245 250 255Leu Trp Asp Phe Lys Val Gly Arg Gly
Arg Cys Ser Leu Cys Asp Glu 260 265 270Leu Cys Pro Asp Ser Lys Ser
Asp Glu Pro Val Cys Ala Ser Asp Asn 275 280 285Ala Thr Tyr Ala Ser
Glu Cys Ala Met Lys Glu Ala Ala Cys Ser Ser 290 295 300Gly Val Leu
Leu Glu Val Lys His Ser Gly Ser Cys Asn305 310 3151963PRTHomo
sapiens 19Gly Asn Cys Trp Leu Arg Gln Ala Lys Asn Gly Arg Cys Gln
Val Leu1 5 10 15Tyr Lys Thr Glu Leu Ser Lys Glu Glu Cys Cys Ser Thr
Gly Arg Leu 20 25 30Ser Thr Ser Trp Thr Glu Glu Asp Val Asn Asp Asn
Thr Leu Phe Lys 35 40 45Trp Met Ile Phe Asn Gly Gly Ala Pro Asn Cys
Ile Pro Cys Lys 50 55 602025PRTHomo sapiens 20Glu Thr Cys Glu Asn
Val Asp Cys Gly Pro Gly Lys Lys Cys Arg Met1 5 10 15Asn Lys Lys Asn
Lys Pro Arg Cys Val 20 252126PRTHomo sapiens 21Lys Thr Cys Arg Asp
Val Phe Cys Pro Gly Ser Ser Thr Cys Val Val1 5 10 15Asp Gln Thr Asn
Asn Ala Tyr Cys Val Thr 20 2522262PRTHomo sapiens 22Met Arg Pro Gly
Ala Pro Gly Pro Leu Trp Pro Leu Pro Trp Gly Ala1 5 10 15Leu Ala Trp
Ala Val Gly Phe Val Ser Ser Met Gly Ser Gly Asn Pro 20 25 30Ala Pro
Gly Gly Val Cys Trp Leu Gln Gln Gly Gln Glu Ala Thr Cys 35 40 45Ser
Leu Val Leu Gln Thr Asp Val Thr Arg Ala Glu Cys Cys Ala Ser 50 55
60Gly Asn Ile Asp Thr Ala Trp Ser Asn Leu Thr His Pro Gly Asn Lys65
70 75 80Ile Asn Leu Leu Gly Phe Leu Gly Leu Val His Cys Leu Pro Cys
Lys 85 90 95Asp Ser Cys Asp Gly Val Glu Cys Gly Pro Gly Lys Ala Cys
Arg Met 100 105 110Leu Gly Gly Arg Pro Arg Cys Glu Cys Ala Pro Asp
Cys Ser Gly Leu 115 120 125Pro Ala Arg Leu Gln Val Cys Gly Ser Asp
Gly Ala Thr Tyr Arg Asp 130 135 140Glu Cys Glu Leu Arg Ala Ala Arg
Cys Arg Gly His Pro Asp Leu Ser145 150 155 160Val Met Tyr Arg Gly
Arg Cys Arg Lys Ser Cys Glu His Val Val Cys 165 170 175Pro Arg Pro
Gln Ser Cys Val Val Asp Gln Thr Gly Ser Ala His Cys 180 185 190Val
Val Cys Arg Ala Ala Pro Cys Val Pro Ser Ser Pro Gly Gln Glu 195 200
205Leu Cys Gly Asn Asn Asn Val Thr Tyr Ile Ser Ser Cys His Met Arg
210 215 220Gln Ala Thr Cys Phe Leu Gly Arg Ser Ile Gly Val Arg His
Ala Gly225 230 235 240Ser Cys Ala Gly Thr Pro Glu Glu Pro Pro Gly
Gly Glu Ser Ala Glu 245 250 255Glu Glu Glu Asn Phe Val
260236PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6xHis tag 23His His His His His His1 524116PRTHomo
sapiens 24Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe Phe Asn
Ala Asn1 5 10 15Trp Glu Lys Asp Arg Thr Asn Gln Thr Gly Val Glu Pro
Cys Tyr Gly 20 25 30Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp
Lys Asn Ile Ser 35 40 45Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp
Leu Asp Asp Ile Asn 50 55 60Cys Tyr Asp Arg Thr Asp Cys Val Glu Lys
Lys Asp Ser Pro Glu Val65 70 75 80Tyr Phe Cys Cys Cys Glu Gly Asn
Met Cys Asn Glu Lys Phe Ser Tyr 85 90 95Phe Pro Glu Met Glu Val Thr
Gln Pro Thr Ser Asn Pro Val Thr Pro 100 105 110Lys Pro Pro Thr
11525115PRTHomo sapiens 25Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys
Ile Tyr Tyr Asn Ala Asn1 5 10 15Trp Glu Leu Glu Arg Thr Asn Gln Ser
Gly Leu Glu Arg Cys Glu Gly 20 25 30Glu Gln Asp Lys Arg Leu His Cys
Tyr Ala Ser Trp Arg Asn Ser Ser 35 40 45Gly Thr Ile Glu Leu Val Lys
Lys Gly Cys Trp Leu Asp Asp Phe Asn 50 55 60Cys Tyr Asp Arg Gln Glu
Cys Val Ala Thr Glu Glu Asn Pro Gln Val65 70 75 80Tyr Phe Cys Cys
Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His 85 90 95Leu Pro Glu
Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr 100 105 110Ala
Pro Thr 115
* * * * *