U.S. patent application number 15/891599 was filed with the patent office on 2018-10-25 for bmp-alk3 antagonists and uses for promoting bone growth.
This patent application is currently assigned to Acceleron Pharma Inc.. The applicant listed for this patent is Acceleron Pharma Inc.. Invention is credited to Jasbir Seehra.
Application Number | 20180305438 15/891599 |
Document ID | / |
Family ID | 42828671 |
Filed Date | 2018-10-25 |
United States Patent
Application |
20180305438 |
Kind Code |
A1 |
Seehra; Jasbir |
October 25, 2018 |
BMP-ALK3 ANTAGONISTS AND USES FOR PROMOTING BONE GROWTH
Abstract
In certain aspects, the present invention provides compositions
and methods for promoting bone growth and increasing bone density
and strength. In certain embodiments, the present invention
provides ALK3 polypeptides, including ALK3-Fc fusion proteins.
Inventors: |
Seehra; Jasbir; (Lexington,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Acceleron Pharma Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
Acceleron Pharma Inc.
Cambridge
MA
|
Family ID: |
42828671 |
Appl. No.: |
15/891599 |
Filed: |
February 8, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14611670 |
Feb 2, 2015 |
9914762 |
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15891599 |
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13724546 |
Dec 21, 2012 |
8945877 |
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14611670 |
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12750604 |
Mar 30, 2010 |
8338377 |
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13724546 |
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61314556 |
Mar 16, 2010 |
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61306331 |
Feb 19, 2010 |
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61211557 |
Mar 30, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/505 20130101;
C12Y 207/1103 20130101; A61P 19/10 20180101; A61P 19/08 20180101;
C07K 16/2863 20130101; A61P 19/00 20180101; A61P 35/04 20180101;
A61P 43/00 20180101; C07K 14/51 20130101; C07K 14/71 20130101; C07K
2319/30 20130101; A61P 1/02 20180101; C07K 16/22 20130101; C12N
9/12 20130101; A61P 35/00 20180101; A61K 38/00 20130101; A61K 39/00
20130101 |
International
Class: |
C07K 14/71 20060101
C07K014/71; C12N 9/12 20060101 C12N009/12; C07K 14/51 20060101
C07K014/51; C07K 16/22 20060101 C07K016/22 |
Claims
1-7. (canceled)
8. A method for promoting bone growth, increasing bone density or
increasing bone strength in a subject, the method comprising
administering to the subject an effective amount of an antibody or
fragment thereof that binds to ALK3 and inhibits ALK3-mediated
signaling.
9. The method of claim 8, wherein the antibody or fragment thereof
is a monoclonal antibody.
10. The method of claim 8, wherein the antibody or fragment thereof
is an antibody fragment.
11. The method of claim 10, wherein the antibody fragment is a
Fab.
12. The method of claim 8, wherein the antibody or fragment thereof
is humanized.
13. The method of claim 8, wherein the antibody or fragment thereof
is chimeric.
14. The method of claim 8, wherein the subject has
osteoporosis.
15. The method of claim 8, wherein the subject has a bone-related
disorder selected from the group consisting of: osteopetrosis,
osteoporosis, fibrous dysplasia, renal osteodystrophy,
post-menopausal osteoporosis, osteogenesis imperfecta,
hypophosphatemia, tumor-induced bone loss, cancer therapy induced
bone loss, bony metastases, multiple myeloma and Paget's
disease.
16. The method of claim 8, wherein the method further comprises
administering a second bone-active agent, wherein the second
bone-active agent is selected from the group consisting of: a
bisphosphonate, an estrogen, a selective estrogen receptor
modulator, a parathyroid hormone, a calcitonin, a calcium
supplement and a vitamin D supplement.
17. A method for promoting bone growth, increasing bone density or
increasing bone strength in a subject, the method comprising
administering to the subject an effective amount of a nucleic acid,
wherein the nucleic acid inhibits ALK3 expression.
18. A method for promoting bone growth, increasing bone density or
increasing bone strength in a subject, the method comprising
administering to the subject an effective amount of an antibody or
fragment thereof that binds BMP and disrupts binding of BMP to
ALK3.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 12/750,604, filed Mar. 30, 2010, which claims the benefit of
U.S. Provisional Application No. 61/211,557, filed on Mar. 30,
2009, 61/306,331, filed on Feb. 19, 2010, and 61/314,556, filed on
Mar. 16, 2010. The specifications of each of the foregoing
applications are incorporated herein by reference in their
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Dec. 21,
2012, is named PHPHO47102_Seq.txt, and is 99,757 bytes in size.
BACKGROUND OF THE INVENTION
[0003] Disorders of the bone, ranging from osteoporosis to
fractures, represent a set of pathological states for which there
are few effective pharmaceutical agents. Treatment instead focuses
on physical and behavioral interventions, including immobilization,
exercise and changes in diet. It would be beneficial to have
therapeutic agents that promote bone growth and increase bone
density for the purpose of treating a variety of bone
disorders.
[0004] Bone growth and mineralization are dependent on the
activities of two cell types, osteoclasts and osteoblasts, although
chondrocytes and cells of the vasculature also participate in
critical aspects of these processes. Developmentally, bone
formation occurs through two mechanisms, endochondral ossification
and intramembranous ossification, with the former responsible for
longitudinal bone formation and the later responsible for the
formation of topologically flat bones, such as the bones of the
skull. Endochondral ossification requires the sequential formation
and degradation of cartilaginous structures in the growth plates
that serve as templates for the formation of osteoblasts,
osteoclasts, the vasculature and subsequent mineralization. During
intramembranous ossification, bone is formed directly in the
connective tissues. Both processes require the infiltration of
osteoblasts and subsequent matrix deposition.
[0005] Fractures and other structural disruptions of bone are
healed through a process that, at least superficially, resembles
the sequence of developmental events of osteogenesis, including the
formation of cartilaginous tissue and subsequent mineralization.
The process of fracture healing can occur in two ways. Direct or
primary bone healing occurs without callus formation.
[0006] Indirect or secondary bone healing occurs with a callus
precursor stage. Primary healing of fractures involves the
reformation of mechanical continuity across a closely-set
disruption.
[0007] Under suitable conditions, bone-resorbing cells surrounding
the disruption show a tunnelling resorptive response and establish
pathways for the penetration of blood vessels and subsequent
healing. Secondary healing of bones follows a process of
inflammation, soft callus formation, callus mineralisation and
callus remodelling. In the inflammation stage, haematoma and
haemorrhage formation results from the disruption of periosteal and
endosteal blood vessels at the site of injury. Inflammatory cells
invade the area. In soft callus formation stage, the cells produce
new vessels, fibroblasts, intracellular material and supporting
cells, forming granulation tissue in the space between the fracture
fragments. Clinical union across the disruption is established by
fibrous or cartilaginous tissue (soft callus). Osteoblasts are
formed and mediate the mineralization of soft callus, which is then
replaced by lamellar bone and subjected to the normal remodeling
processes.
[0008] In addition to fractures and other physical disruptions of
bone structure, loss of bone mineral content and bone mass can be
caused by a wide variety of conditions and may result in
significant medical problems. Changes to bone mass occur in a
relatively predictable way over the life of an individual. Up to
about age 30, bones of both men and women grow to maximal mass
through linear growth of the endochondral growth plates and radial
growth. After about age 30 (for trabecular bone, e.g., flat bones
such as the vertebrae and pelvis) and age 40 (for cortical bone,
e.g., long bones found in the limbs), slow bone loss occurs in both
men and women. In women, a final phase of substantial bone loss
also occurs, probably due to postmenopausal estrogen deficiencies.
During this phase, women may lose an additional 10% of bone mass
from the cortical bone and 25% from the trabecular compartment.
Whether progressive bone loss results in a pathological condition
such as osteoporosis depends largely on the initial bone mass of
the individual and whether there are exacerbating conditions.
[0009] Bone loss is sometimes characterized as an imbalance in the
normal bone remodeling process. Healthy bone is constantly subject
to remodeling. Remodeling begins with resorption of bone by
osteoclasts. The resorbed bone is then replaced by new bone tissue,
which is characterized by collagen formation by osteoblasts, and
subsequent calcification. In healthy individuals the rates of
resorption and formation are balanced. Osteoporosis is a chronic,
progressive condition, marked by a shift towards resorption,
resulting in an overall decrease in bone mass and bone
mineralization. Osteoporosis in humans is preceded by clinical
osteopenia (bone mineral density that is greater than one standard
deviation but less than 2.5 standard deviations below the mean
value for young adult bone). Worldwide, approximately 75 million
people are at risk for osteoporosis.
[0010] Thus, methods for controlling the balance between osteoclast
and osteoblast activity can be useful for promoting the healing of
fractures and other damage to bone as well as the treatment of
disorders, such as osteoporosis, associated with loss of bone mass
and bone mineralization.
[0011] With respect to osteoporosis, estrogen, calcitonin,
osteocalcin with vitamin K, or high doses of dietary calcium are
all used as therapeutic interventions. Other therapeutic approaches
to osteoporosis include bisphosphonates, parathyroid hormone,
calcimimetics, statins, anabolic steroids, lanthanum and strontium
salts, and sodium fluoride. Such therapeutics, however, are often
associated with undesirable side effects.
[0012] Bone loss is also a significant complication of many
cancers, and may be caused by tumor metastases to bone, the
activation of osteoclasts or the effects of chemotherapeutic
treatment. In particular, anti-estrogen therapies that are used
widely in the treatment of breast cancer can cause significant bone
loss.
[0013] Other bone disorders, such as osteogenesis imperfecta, may
result from genetic, developmental, nutritional of other
pathologies and deficiencies.
[0014] Thus, it is an object of the present disclosure to provide
compositions and methods for promoting bone growth and
mineralization.
SUMMARY OF THE INVENTION
[0015] In part, the disclosure demonstrates that molecules having
ALK3 or BMP antagonist activity ("ALK3 antagonists" and "BMP
antagonists") can be used to increase bone density, promote bone
growth, and/or increase bone strength. This observation is
particularly surprising, given the large body of literature and
clinical experience indicating that many BMPs, and particularly
BMP2, BMP4 and BMP7, are potent stimulators of bone formation. The
disclosure demonstrates that a soluble form of ALK3 acts as an
inhibitor of BMP-ALK3 signaling and promotes increased bone
density, bone growth, and bone strength in vivo. While not wishing
to be bound to any particular mechanism, it appears that the
soluble form of ALK3 achieves this effect by inhibiting BMP2 and/or
BMP4, and perhaps other ligands which signal through ALK3. Thus,
the disclosure establishes that antagonists of the BMP-ALK3
signaling pathway may be used to increase bone density and promote
bone growth. While soluble ALK3 may affect bone through a mechanism
other than, or in addition to, BMP antagonism, the disclosure
nonetheless demonstrates that desirable therapeutic agents may be
selected on the basis of BMP-ALK3 antagonist activity. Therefore,
in certain embodiments, the disclosure provides methods for using
BMP-ALK3 antagonists, including, for example, BMP-binding ALK3
polypeptides, anti-BMP antibodies, anti-ALK3 antibodies, BMP- or
ALK3-targeted small molecules and aptamers, and nucleic acids that
decrease expression of BMP and ALK3, to treat disorders associated
with low bone density or low bone strength, such as osteoporosis,
or to promote bone growth in patients in need thereof, such as in
patients having a bone fracture. In additional embodiments, the
disclosure identifies truncated forms of ALK3 polypeptides (e.g.,
ALK3-Fc polypeptides) that have advantageous properties and retain
appropriate BMP2 or BMP4 binding.
[0016] In certain aspects, the disclosure provides polypeptides
comprising a soluble ALK3 polypeptide that binds to BMP2 and/or
BMP4. The soluble ALK3 polypeptide may bind to additional ligands
also. ALK3 polypeptides may be formulated as a pharmaceutical
preparation comprising the BMP-binding ALK3 polypeptide and a
pharmaceutically acceptable carrier. Preferably, the BMP-binding
ALK3 polypeptide binds to BMP2 and/or BMP4 with a K.sub.D less than
1 micromolar or less than 100, 10 or 1 nanomolar. Preferably the
composition is at least 95% pure, with respect to other polypeptide
components, as assessed by size exclusion chromatography, and more
preferably, the composition is at least 98% pure. A BMP-binding
ALK3 polypeptide for use in such a preparation may be any of those
disclosed herein, such as a polypeptide having an amino acid
sequence selected from SEQ ID NOs: 3, 7, 11, 14, 20, 22, 23, 25,
26, 28, 29, 30, 31, 33, 34, 35, 36, 38, 39, 40, or 41, or having an
amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%
or 99% identical to an amino acid sequence selected from SEQ ID
NOs: 3, 7, 11, 14, 20, 22, 23, 25, 26, 28, 29, 30, 31, 33, 34, 35,
36, 38, 39, 40, or 41, including N- and/or C-terminal truncations
of no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids of SEQ ID
NO:3, and optionally fused to an Fc fusion protein, with or without
a linker. In particular, the disclosure provides ALK3 polypeptides
with a truncation of 0 to 7 amino acids at the N-terminus of the
ALK3 ECD portion and 0 to 12 amino acids at the C-terminus of the
ALK3 ECD portion, thus describing a function portion corresponding
to amino acids 8 to 117 of SEQ ID NO:3 and polypeptides comprising
a protein that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99%
identical to the amino acid sequence of 8 to 117 of SEQ ID NO:3.
Notably, human ALK3 and murine ALK3 have 97 to 98% identity at the
amino acid sequence level in the extracellular domain, and proteins
comprising such domains from the human or mouse protein are shown
herein to exhibit similar activity in vitro and in vivo. A
BMP-binding ALK3 polypeptide may include a functional fragment of a
natural ALK3 polypeptide, such as one comprising at least 10, 20 or
30 amino acids of a sequence selected from SEQ ID NOs: 1 or 3.
Surprisingly, as demonstrated herein, ALK3 proteins that include a
deletion of amino acids at the C-terminal region of the ALK3
extracellular domain retain activity against BMP2 and BMP4 while
diminishing activity against other ligands (e.g., BMP6 and BMP7)
thus providing an improvement in ligand selectivity, which is
generally desirable to diminish unanticipated off-target effects in
clinical development or commercialization. Such variations may
include a deletion of no more than 6 or 7, no more than 12 or no
more than 24 amino acids from the C-terminus of SEQ ID NO:3.
Optionally, a form truncated at the C-terminus may also be
truncated by no more than 1, 2, 3, 4, 5, 6 or 7 amino acids at the
N-terminus. The aforementioned variations of ALK3 proteins may be
included in an ALK3-Fc fusion protein, which may comprise any
linker disclosed herein (or no linker at all), including a linker
having the sequence GGG or TGGG or SGGG, and an Fc portion derived
from a human IgG1, IgG2, IgG3 or IgG4 or other mammalian
immunoglobulin.
[0017] A soluble, BMP-binding ALK3 polypeptide may include one,
two, five or more alterations in the amino acid sequence (e.g., in
the ligand-binding domain) relative to a naturally occurring ALK3
polypeptide. The alteration in the amino acid sequence may, for
example, alter glycosylation of the polypeptide when produced in a
mammalian, insect or other eukaryotic cell or alter proteolytic
cleavage of the polypeptide relative to the naturally occurring
ALK3 polypeptide.
[0018] A BMP-binding ALK3 polypeptide may be a fusion protein that
has, as one domain, an ALK3 polypeptide (e.g., a ligand-binding
portion of ALK3) 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
BMP-binding ALK3 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 ALK3-Fc fusion comprises a
relatively unstructured linker positioned between the Fc domain and
the extracellular ALK3 domain. This unstructured linker may
correspond to the C-terminal end of the extracellular domain of
ALK3, or it 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/or glycines
(e.g., GGG, GGGG, TG.sub.4, SG.sub.4, TG.sub.3, or SG.sub.3
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 ALK3 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. A pharmaceutical
preparation may also include one or more additional compounds such
as a compound that is used to treat a bone disorder. Preferably, a
pharmaceutical preparation is substantially pyrogen free. In
general, it is preferable that an ALK3 protein be expressed in a
mammalian cell line that mediates suitably natural glycosylation of
the ALK3 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.
[0019] In certain aspects, the disclosure provides nucleic acids
encoding a soluble BMP-binding ALK3 polypeptide. An isolated
polynucleotide may comprise a coding sequence for a soluble,
BMP-binding ALK3 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 ALK3 and a
sequence that would code for part or all of the transmembrane
domain and/or the cytoplasmic domain of ALK3, 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 ALK3
polynucleotide sequence such as SEQ ID NO: 2 or 4, or a partially
truncated version, said isolated polynucleotide further comprising
a transcription termination codon at least six hundred nucleotides
before the 3'-terminus or otherwise positioned such that
translation of the polynucleotide gives rise to an extracellular
domain optionally fused to a truncated portion of a full-length
ALK3. Preferred nucleic acid sequences are SEQ ID NO: 12, 13, 15,
16, 19, 21, 24, 27, 32 or 37 and nucleic acids that hybridize to
such nucleic acids or the complements thereof under stringent
hybridization conditions. 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.
[0020] In certain aspects, the disclosure provides methods for
making a soluble, BMP-binding ALK3 polypeptide. Such a method may
include expressing any of the nucleic acids (e.g., SEQ ID NO: 2, 4,
12, 13, 15, 16, 19, 21, 24, 27, 32 or 37) disclosed herein in a
suitable cell, such as a Chinese hamster ovary (CHO) cell. Such a
method may comprise: a) culturing a cell under conditions suitable
for expression of the soluble ALK3 polypeptide, wherein said cell
is transformed with a soluble ALK3 expression construct; and b)
recovering the soluble ALK3 polypeptide so expressed. Soluble ALK3
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.
[0021] In certain aspects, a BMP-ALK3 antagonist disclosed herein,
such as a soluble, BMP-binding ALK3 polypeptide, may be used in a
method for promoting bone growth or increasing bone density in a
subject. In certain embodiments, the disclosure provides methods
for treating a disorder associated with low bone density, or to
promote bone growth, in patients in need thereof. A method may
comprise administering to a subject in need thereof an effective
amount of BMP-ALK3 antagonist. In certain aspects, the disclosure
provides uses of BMP-ALK3 antagonist for making a medicament for
the treatment of a disorder or condition as described herein.
[0022] In certain aspects, the disclosure provides a method for
identifying an agent that stimulates growth of, or increased
mineralization of, bone. The method comprises: a) identifying a
test agent that binds to BMPs or a ligand-binding domain of an ALK3
polypeptide; and b) evaluating the effect of the agent on growth
of, or mineralization of, bone.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The patent or application file contains at least one drawing
executed in color. Copies of this patent application publication
with color drawing(s) will be provided by the Office upon request
and payment of the necessary fee.
[0024] FIG. 1 shows the native amino acid sequence of human ALK3
precursor (SEQ ID NO: 1). The ALK3 extracellular domain (residues
24-152) is underlined.
[0025] FIG. 2 shows the native nucleotide sequence encoding human
ALK3 precursor (SEQ ID NO: 2). The sequence encoding the ALK3
extracellular domain (nucleotides 70-456) is underlined.
[0026] FIG. 3 shows the native amino acid sequence of the
extracellular domain of human ALK3 (SEQ ID NO: 3).
[0027] FIG. 4 shows the native nucleotide sequence encoding the
extracellular domain of human ALK3 (SEQ ID NO: 4).
[0028] FIG. 5 shows the native amino acid sequence of human IgG1 Fc
domain (SEQ ID NO: 5).
[0029] FIG. 6 shows the native nucleotide sequence encoding human
IgG1 Fc domain (SEQ ID NO: 6).
[0030] FIG. 7 shows the amino acid sequence of leaderless
hALK3(24-152)-hFc (SEQ ID NO: 7). The human ALK3 extracellular
domain (SEQ ID NO: 3) is underlined, and the TGGG linker sequence
is in bold.
[0031] FIG. 8 shows the full amino acid sequence of
hALK3(24-152)-hFc with TPA leader (SEQ ID NO: 11). The human ALK3
extracellular domain (SEQ ID NO: 3) is underlined, and the TGGG
linker sequence is in bold.
[0032] FIGS. 9A-9B show a nucleotide sequence encoding
hALK3(24-152)-hFc with TPA leader. SEQ ID NO: 12 corresponds to the
coding strand, and SEQ ID NO: 13 corresponds to the anti-coding
strand. The sequence encoding the human ALK3 extracellular domain
(SEQ ID NO: 4) is underlined.
[0033] FIG. 10 shows the full amino acid sequence of
hALK3(24-152)-mFc with TPA leader (SEQ ID NO: 14). The human ALK3
extracellular domain (SEQ ID NO: 3) is underlined, and the TGGG
linker sequence is in bold.
[0034] FIGS. 11A-11B show a nucleotide sequence encoding
hALK3(24-152)-mFc with TPA leader. SEQ ID NO: 15 corresponds to the
coding strand, and SEQ ID NO: 16 corresponds to the anti-coding
strand. The sequence encoding the human ALK3 extracellular domain
(SEQ ID NO: 4) is underlined.
[0035] FIG. 12 shows the effect of hALK3(24-152)-mFc treatment on
whole-body bone mineral density in female mice. Measurements were
made by dual energy x-ray absorptiometry (DEXA). Data are means
(n=8 per group).+-.SEM. *, P<0.05 vs. vehicle by unpaired
t-test. hALK3(24-152)-mFc increased whole-body bone density
significantly after 31 and 42 days of treatment.
[0036] FIG. 13 shows the effect of hALK3(24-152)-mFc treatment on
vertebral bone mineral density in female mice. Measurements of a
region containing the fourth and fifth lumbar vertebrae (L4, L5)
were made by DEXA. Data are means (n=8 per group).+-.SEM. **,
P<0.005 vs. vehicle by unpaired t-test. hALK3(24-152)-mFc
increased vertebral bone density significantly after 31 and 42 days
of treatment.
[0037] FIG. 14 shows the effect of hALK3(24-152)-mFc treatment on
cortical bone thickness in female mice. Measurements of the right
proximal tibia were made by micro-computed tomography (micro-CT).
Data are means (n=8 per group), and error bars represent.+-.two
times SEM. **, P<0.005 vs. vehicle by unpaired t-test.
hALK3(24-152)-mFc increased the thickness of cortical bone
significantly after 6 weeks of treatment.
[0038] FIG. 15 shows the effect of hALK3(24-152)-mFc treatment on
trabecular bone volume in female mouse. Measurements of the right
proximal tibia were made by micro-CT. Data are means (n=8 per
group), and error bars represent .+-.two times SEM. ***, P<0.001
vs. pretreatment baseline or vehicle by unpaired t-test.
hALK3(24-152)-mFc more than doubled the proportion of trabecular
bone after 4 weeks of treatment.
[0039] FIG. 16 shows the effect of hALK3(24-152)-mFc treatment on
mean trabecular thickness in female mice. Measurements of the right
proximal tibia were made by micro-CT. Data are group means (n=8 per
group), and error bars represent.+-.two times SEM. ***, P<0.001
vs. pretreatment baseline or vehicle by unpaired t-test.
hALK3(24-152)-mFc significantly increased trabecular thickness
after 4 weeks of treatment.
[0040] FIG. 17 shows the effect of hALK3(24-152)-mFc treatment for
4 weeks on trabecular bone microarchitecture in female mice.
Representative three-dimensional images of trabecular bone in the
proximal tibia were generated by micro-CT. Scale bars=300
.mu.m.
[0041] FIG. 18 shows examples of three approaches disclosed herein
to interfere with signaling by the BMP-ALK3 signaling axis for the
purpose of stimulating bone formation. A: ALK3-Fc. B: Antibody
against selected BMP ligand(s). C. Antibody against the ligand
binding region of ALK3 extracellular domain. "BMP2" is used to
illustrate that the BMP may be BMP2, BMP4 or another high affinity
ligand of ALK3.
[0042] FIG. 19 shows the effect of hALK3(24-152)-mFc treatment for
6 weeks on maximum bone load in female mice. Unilateral analysis of
the femur was conducted ex vivo with an Instron mechanical testing
device. Data in newtons (N) are means (n=8 per group).+-.SEM. **,
P<0.01 vs. vehicle. hALK3(24-152)-mFc increased maximum bone
load by 30%.
[0043] FIG. 20 shows the effect of hALK3(24-152)-mFc treatment for
6 weeks on bone stiffness in female mice. Unilateral analysis of
the femur was conducted ex vivo with an Instron mechanical testing
device. Data in newtons (N) per mm are means (n=8 per
group).+-.SEM. * P<0.05 vs. vehicle. hALK3(24-152)-mFc increased
bone stiffness by 14%.
[0044] FIG. 21 shows the effect of hALK3(24-152)-mFc treatment for
6 weeks on energy to bone failure in female mice. Unilateral
analysis of the femur was conducted ex vivo with an Instron
mechanical testing device. Data in millijoules (mJ) are means (n=8
per group).+-.SEM. *, P<0.05 vs. vehicle. hALK3(24-152)-mFc
increased energy to failure by 32%.
[0045] FIG. 22 shows the effect of mALK3(24-152)-mFc treatment on
trabecular bone volume in an OVX mouse model of established
osteopenia. Measurements of the proximal tibia were made by
micro-CT. Data are means (n=7-8 per group), and error bars
represent .+-.2 SEM. *, P<0.05 vs. OVX+vehicle. Prior to dosing,
OVX mice had reduced trabecular bone volume compared to
sham-operated mice. Compared to OVX controls, mALK3(24-152)-mFc
increased bone volume significantly at 28 and 56 days of
treatment.
[0046] FIG. 23 shows the effect of mALK3(24-152)-mFc treatment on
cortical bone thickness in an OVX mouse model of osteopenia.
Measurements of cortical bone were made by micro-CT. Data are means
(n=7-8 per group), and error bars represent .+-.2 SEM. *, P<0.05
vs. OVX+vehicle. Compared to OVX controls, mALK3(24-152)-mFc
increased cortical thickness significantly at 56 days of
treatment.
[0047] FIG. 24 shows the effect of mALK3(24-152)-mFc treatment on
endosteal circumference in an OVX mouse model of osteopenia.
Measurements of the tibial shaft were made by micro-CT. Data are
means (n=7-8 per group), and error bars represent .+-.2 SEM. *,
P<0.05 vs. OVX+vehicle. Compared to OVX controls,
mALK3(24-152)-mFc reduced endosteal circumference significantly at
56 days of treatment, thus providing additional evidence of
cortical bone growth.
[0048] FIG. 25 shows the effect of mALK3(24-152)-mFc treatment on
whole-body bone mineral density in an OVX mouse model of osteopenia
as determined by DEXA. Data are means (n=7-8 per group).+-.SEM. *,
P<0.05 vs. OVX+vehicle. Compared to OVX controls,
mALK3(24-152)-mFc increased whole-body bone density significantly
at 14, 28, 42, and 56 days of treatment.
[0049] FIG. 26 shows the effect of mALK3(24-152)-mFc treatment on
vertebral bone mineral density in an OVX mouse model of osteopenia.
Analysis of the lumbar spine (vertebrae L1-L6) was conducted by
DEXA. Data are means (n=7-8 per group).+-.SEM. *, P<0.05 vs.
OVX+vehicle. Compared to OVX controls, mALK3(24-152)-mFc increased
vertebral bone density significantly at 14, 28, 42, and 56 days of
treatment.
[0050] FIG. 27 shows the effect of mALK3-mFc treatment on bone
mineral density of the femur-tibia in an OVX mouse model of
osteopenia as determined by DEXA. Analysis of the entire femur and
proximal tibia was conducted by DEXA. Data are means (n=7-8 per
group).+-.SEM. *, P<0.05 vs. OVX+vehicle. Compared to OVX
controls, mALK3(24-152)-mFc increased femoral-tibial bone density
significantly at 28, 42, and 56 days of treatment.
[0051] FIG. 28 shows the effect of mALK3(24-152)-mFc for 56 days on
vertebral bone microarchitecture in an OVX mouse model of
osteopenia. Representative three-dimensional images of trabecular
bone in lumbar vertebrae (L5) were generated ex vivo by micro-CT.
Scale bar=300 .mu.m.
[0052] FIG. 29 shows the effect of mALK3(24-152)-mFc on bone volume
in female mice as assessed in the distal femur by histomophometry.
Data are means.+-.SEM; n=6 per group per time point. **, P<0.01
vs. vehicle at corresponding time points. Compared to vehicle,
mALK3(24-152)-mFc increased bone volume significantly at all time
points.
[0053] FIG. 30 shows the effect of mALK3(24-152)-mFc on bone
formation rate in female mice as assessed in the distal femur by
histomophometry. Data are means.+-.SEM; n=6 per group per time
point. ***, P<0.001 vs. vehicle at corresponding time point.
Compared to vehicle, mALK3(24-152)-mFc increased bone formation
rate significantly at 28 days of treatment, thus providing evidence
of anabolic bone formation.
[0054] FIG. 31 shows the effect of mALK3(24-152)-mFc on bone
mineralizing surface in female mice as assessed in the distal femur
by histomophometry. Data are means.+-.SEM; n=6 per group per time
point. **, P<0.01; *, P<0.05 vs. vehicle at corresponding
time points. Compared to vehicle, mALK3(24-152)-mFc increased
mineralizing surface significantly at 14 and 28 days of treatment,
thus providing additional evidence of anabolic bone formation.
[0055] FIG. 32 shows the effect of mALK3(24-152)-mFc on osteoclast
surface in female mice as assessed in the distal femur by
histomophometry. Data are means.+-.SEM; n=6 per group per time
point. **, P<0.01 vs. vehicle at corresponding time point.
Compared to vehicle, mALK3(24-152)-mFc reduced osteoclast surface
significantly at 28 days of treatment, thus providing evidence of
antiresorptive bone formation.
[0056] FIG. 33 shows the effect of mALK3(24-152)-mFc on serum
levels of RANKL (receptor activator for nuclear factor KB ligand)
in female mice as determined by Luminex xMAP.RTM. assay. Data are
means.+-.SEM; n=6 per group per time point. **, P<0.01; *,
P<0.05 vs. vehicle at corresponding time points. Compared to
vehicle, mALK3(24-152)-mFc reduced circulating RANKL levels
significantly at all time points.
[0057] FIG. 34 shows the effect of mALK3(24-152)-mFc on serum
osteoprotegerin (OPG) levels in female mice as determined by
Luminex xMAP.RTM. assay. Data are means.+-.SEM; n=6 per group per
time point. **, P<0.01; *, P<0.05 vs. vehicle at
corresponding time points. Compared to vehicle, mALK3(24-152)-mFc
increased circulating OPG levels significantly at 28 and 42 days of
treatment.
[0058] FIG. 35 shows the effect of mALK3(24-152)-mFc on sclerostin
mRNA levels in the femur and tibia of female mice as assessed by
real-time polymerase chain reaction (RT-PCR). Data are
means.+-.SEM. ***, P<0.001; *, P<0.05 vs. vehicle at
corresponding time points. Compared to vehicle, mALK3(24-152)-mFc
reduced sclerostin mRNA levels significantly at 2, 7, and 28 days
of treatment.
[0059] FIG. 36 shows the effect of hALK3(24-152)-hFc on bone volume
in female mice. Bone volume was assessed in the proximal tibia by
micro-CT at Day 0 (baseline) and again at Day 42 (ex vivo). Data
are means.+-.SEM; n=6 per group. ***, P<0.001 vs. vehicle. Over
the course of the experiment, bone volume decreased by nearly 20%
in vehicle-treated controls but increased by more than 80% with
hALK3(24-152)-hFc treatment.
DETAILED DESCRIPTION OF THE INVENTION
1. Overview
[0060] In part, the present disclosure demonstrates the surprising
result that inhibitors of the BMP-ALK3 signaling pathway, such as
an ALK3-Fc protein, promote bone formation in animals. ALK3 is a
receptor for members of the transforming growth factor-beta
(TGF-beta)/bone morphogenetic protein (BMP) superfamily. The
TGF-beta/BMP 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.
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.
[0061] 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.
[0062] Activin receptor-like kinase-3 (ALK3) is a type I receptor
mediating effects of multiple ligands in the BMP family and is also
known as bone morphogenetic protein receptor, type IA (BMPR1A), or
activin A receptor, type II-like kinase (ACVRLK). Unlike several
type I receptors with ubiquitous tissue expression, ALK3 displays a
restricted pattern of expression consistent with more specialized
functionality (ten Dijke, 1993, Oncogene 8:2879-2887). ALK3 is
generally recognized as a high affinity receptor for BMP2, BMP4,
BMP7 and other members of the BMP family. BMP2 and BMP7 are potent
stimulators of osteoblastic differentiation, and are now used
clinically to induce bone formation in spine fusions and certain
non-union fractures. ALK3 is regarded as a key receptor in
mediating BMP2 and BMP4 signaling in osteoblasts (Lavery et al.,
2008, J. Biol. Chem. 283:20948-20958). A homozygous ALK3 knockout
mouse dies early in embryogenesis (day 9.5), however, adult mice
carrying a conditional disruption of ALK3 in osteoblasts have been
recently reported to exhibit increased bone mass, although the
newly formed bone showed evidence of disorganization (Kamiya, 2008,
J. Bone Miner. Res. 23:2007-2017; Kamiya, 2008, Development
135:3801-3811). This finding is in startling contrast to the
effectiveness of BMP2 and BMP7 (ligands for ALK3) as bone building
agents in clinical use.
[0063] As demonstrated herein, a soluble ALK3 polypeptide
(ALK3-Fc), which shows substantial preference in binding to BMP2
and BMP4 is effective to promote bone growth and increase bone
density in vivo. While not wishing to be bound to any particular
mechanism, it is expected that the effect of ALK3 is caused
primarily by a BMP antagonist effect, given the very strong BMP2
and BMP4 binding (picomolar dissociation constant) exhibited by the
particular soluble ALK3 construct used in these studies. Regardless
of mechanism, it is apparent from the data presented herein that
BMP-ALK3 antagonists do increase bone density in normal mice.
Surprisingly, the bone generated by treatment with ALK3-Fc shows no
evidence of the type of disorganization observed in the ALK3
conditional knockout mice. It should be noted that bone is a
dynamic tissue, with growth or shrinkage and increased or decreased
density depending on a balance of factors that produce bone and
stimulate mineralization (primarily osteoblasts) and factors that
destroy and demineralize bone (primarily osteoclasts). Bone growth
and mineralization may be increased by increasing the productive
factors, by decreasing the destructive factors, or both. The terms
"promote bone growth" and "increase bone mineralization" refer to
the observable physical changes in bone and are intended to be
neutral as to the mechanism by which changes in bone occur.
[0064] The mouse model for bone growth/density that was used in the
studies described herein is considered to be highly predictive of
efficacy in humans, and therefore, this disclosure provides methods
for using ALK3 polypeptides and other BMP-ALK3 antagonists to
promote bone growth and increase bone density in humans. BMP-ALK3
antagonists include, for example, BMP-binding soluble ALK3
polypeptides, antibodies that bind to BMP and disrupt ALK3 binding,
antibodies that bind to ALK3 and disrupt BMP binding, non-antibody
proteins selected for BMP or ALK3 binding (see e.g.,
WO/2002/088171, WO/2006/055689, WO/2002/032925, WO/2005/037989, US
2003/0133939, and US 2005/0238646 for examples of such proteins and
methods for design and selection of same), randomized peptides
selected for BMP or ALK3 binding, often affixed to an Fc domain.
Two different proteins (or other moieties) with BMP or ALK3 binding
activity, especially BMP 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
BMP-ALK3 signaling axis are also contemplated. Additionally,
nucleic acids, such as antisense molecules, siRNAs or ribozymes
that inhibit BMPs, or, particularly, ALK3 expression, can be used
as BMP-ALK3 antagonists.
[0065] 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.
[0066] "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.
[0067] 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.
[0068] 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.
[0069] "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.
[0070] 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.
[0071] 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. ALK3 Polypeptides
[0072] In certain aspects, the present invention relates to ALK3
polypeptides. As used herein, the term "ALK3" refers to a family of
activin receptor-like kinase-3 (ALK3) [also referred to as bone
morphogenetic protein receptor, type IA (BMPR1A), or activin A
receptor, type II-like kinase (ACVRLK)] proteins from any species
and variants derived from such ALK3 proteins by mutagenesis or
other modification. Reference to ALK3 herein is understood to be a
reference to any one of the currently identified forms. Members of
the ALK3 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.
[0073] The term "ALK3 polypeptide" includes polypeptides comprising
any naturally occurring polypeptide of an ALK3 family member as
well as any variants thereof (including mutants, fragments,
fusions, and peptidomimetic forms) that retain a useful activity.
For example, ALK3 polypeptides include polypeptides derived from
the sequence of any known ALK3 having a sequence at least about 80%
identical to the sequence of an ALK3 polypeptide, and preferably at
least 85%, 90%, 95%, 97%, 99% or greater identity. For example, an
ALK3 polypeptide of the invention may bind to and inhibit the
function of an ALK3 protein and/or BMPs. Preferably, an ALK3
polypeptide promotes bone growth and bone mineralization. Examples
of ALK3 polypeptides include human ALK3 precursor polypeptide (SEQ
ID NO: 1) and soluble human ALK3 polypeptides (e.g., SEQ ID NOs: 3,
7, 11, 14, 20, 22, 23, 25, 26, 28, 29, 30, 31, 33, 34, 35, 36, 38,
39, 40, or 41).
[0074] The human ALK3 precursor protein sequence (SEQ ID NO: 1) is
shown in FIG. 1, and the nucleic acid sequence encoding human ALK3
precursor protein (SEQ ID NO: 2; nucleotides 549-2144 of Genbank
entry NM_004329) is shown in FIG. 2. The human ALK3 soluble
(extracellular), processed polypeptide sequence (SEQ ID NO: 3) is
shown in FIG. 3, and the nucleic acid sequence encoding the human
ALK3 extracellular domain (SEQ ID NO: 4; nucleotides 618-1004 of
Genbank entry NM_004329) is shown in FIG. 4.
[0075] In a specific embodiment, the invention relates to soluble
ALK3 polypeptides. As described herein, the term "soluble ALK3
polypeptide" generally refers to polypeptides comprising an
extracellular domain of an ALK3 protein. The term "soluble ALK3
polypeptide," as used herein, includes any naturally occurring
extracellular domain of an ALK3 protein as well as any variants
thereof (including mutants, fragments and peptidomimetic forms). A
BMP-binding ALK3 polypeptide is one that retains the ability to
bind to BMPs, particularly BMP2 and BMP4. Preferably, a BMP-binding
ALK3 polypeptide will bind to BMP with a dissociation constant of 1
nM or less. The amino acid sequence of human ALK3 precursor protein
is provided in FIG. 1. The extracellular domain of an ALK3 protein
binds to BMP and is generally soluble, and thus can be termed a
soluble, BMP-binding ALK3 polypeptide. Examples of soluble,
BMP-binding ALK3 polypeptides include the soluble polypeptide
illustrated in SEQ ID NOs: 3, 7, 11, 14, 20, 22, 23, 25, 26, 28,
29, 30, 31, 33, 34, 35, 36, 38, 39, 40, or 41. SEQ ID NO:7 is
referred to as ALK3(24-152)-hFc, and is described further in the
Examples. Other examples of soluble, BMP-binding ALK3 polypeptides
comprise a signal sequence in addition to the extracellular domain
of an ALK3 protein, for example, the native ALK3 leader sequence
(SEQ ID NO: 8), the tissue plaminogen activator (TPA) leader (SEQ
ID NO: 9) or the honey bee melittin leader (SEQ ID NO: 10). The
ALK3-hFc polypeptide illustrated in SEQ ID NO: 11 uses a TPA
leader.
[0076] Functionally active fragments of ALK3 polypeptides can be
obtained by screening polypeptides recombinantly produced from the
corresponding fragment of the nucleic acid encoding an ALK3
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 ALK3 protein or signaling mediated by BMPs.
[0077] Functionally active variants of ALK3 polypeptides can be
obtained by screening libraries of modified polypeptides
recombinantly produced from the corresponding mutagenized nucleic
acids encoding an ALK3 polypeptide. The variants can be produced
and tested to identify those that can function as antagonists
(inhibitors) of ALK3 protein or signaling mediated by BMPs. In
certain embodiments, a functional variant of the ALK3 polypeptides
comprises an amino acid sequence that is at least 75% identical to
an amino acid sequence selected from SEQ ID NO: 3, 7, 11, 14, 20,
22, 23, 25, 26, 28, 29, 30, 31, 33, 34, 35, 36, 38, 39, 40, or 41.
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 NO: 3, 7, 11, 14, 20, 22,
23, 25, 26, 28, 29, 30, 31, 33, 34, 35, 36, 38, 39, 40, or 41.
[0078] Functional variants may be generated by modifying the
structure of an ALK3 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 ALK3
polypeptides, when selected to retain BMP binding, are considered
functional equivalents of the naturally-occurring ALK3
polypeptides. Modified ALK3 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 ALK3 polypeptide results in a
functional homolog can be readily determined by assessing the
ability of the variant ALK3 polypeptide to produce a response in
cells in a fashion similar to the wild-type ALK3 polypeptide.
[0079] In certain embodiments, the present invention contemplates
specific mutations of the ALK3 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 asparagines-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 ALK3
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 ALK3
polypeptide is by chemical or enzymatic coupling of glycosides to
the ALK3 polypeptide. Depending on the coupling mode used, the
sugar(s) may be attached to (a) arginine and histidine; (b) free
carboxyl groups; (c) free sulfhydryl groups such as those of
cysteine; (d) free hydroxyl groups such as those of serine,
threonine, or hydroxyproline; (e) aromatic residues such as those
of phenylalanine, tyrosine, or tryptophan; or (f) the amide group
of glutamine. These methods are described in WO 87/05330 published
Sep. 11, 1987, and in Aplin and Wriston (1981) CRC Crit. Rev.
Biochem., pp. 259-306, incorporated by reference herein. Removal of
one or more carbohydrate moieties present on an ALK3 polypeptide
may be accomplished chemically and/or enzymatically. Chemical
deglycosylation may involve, for example, exposure of the ALK3
polypeptide to the compound trifluoromethanesulfonic acid, or an
equivalent compound. This treatment results in the cleavage of most
or all sugars except the linking sugar (N-acetylglucosamine or
N-acetylgalactosamine), while leaving the amino acid sequence
intact. Chemical deglycosylation is further described by Hakimuddin
et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge et al.
(1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate
moieties on ALK3 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 ALK3
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,
ALK3 proteins for use in humans will be expressed in a mammalian
cell line that provides proper glycosylation, such as HEK293 or CHO
cell lines, although other mammalian expression cell lines, yeast
cell lines with engineered glycosylation enzymes and insect cells
are expected to be useful as well.
[0080] This disclosure further contemplates a method of generating
mutants, particularly sets of combinatorial mutants of an ALK3
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, ALK3 polypeptide variants which
can act as either agonists or antagonist, or alternatively, which
possess novel activities altogether. A variety of screening assays
are provided below, and such assays may be used to evaluate
variants. For example, an ALK3 polypeptide variant may be screened
for ability to bind to an ALK3 ligand, to prevent binding of an
ALK3 ligand to an ALK3 polypeptide or to interfere with signaling
caused by an ALK3 ligand.
[0081] The activity of an ALK3 polypeptide or its variants may also
be tested in a cell-based or in vivo assay. For example, the effect
of an ALK3 polypeptide variant on the expression of genes involved
in bone production or bone destruction may be assessed. This may,
as needed, be performed in the presence of one or more recombinant
ALK3 ligand proteins (e.g., BMP2 or BMP4), and cells may be
transfected so as to produce an ALK3 polypeptide and/or variants
thereof, and optionally, an ALK3 ligand. Likewise, an ALK3
polypeptide may be administered to a mouse or other animal, and one
or more bone properties, such as density or volume may be assessed.
The healing rate for bone fractures may also be evaluated.
Dual-energy x-ray absorptiometry (DEXA) is a well-established,
non-invasive, quantitative technique for assessing bone density in
an animal. In humans, central DEXA systems may be used to evaluate
bone density in the spine and pelvis. These are the best predictors
of overall bone density. Peripheral DEXA systems may be used to
evaluate bone density in peripheral bones, including, for example,
the bones of the hand, wrist, ankle and foot. Traditional x-ray
imaging systems, including CAT scans, may be used to evaluate bone
growth and fracture healing. The mechanical strength of bone may
also be evaluated.
[0082] Combinatorially-derived variants can be generated which have
a selective or generally increased potency relative to a naturally
occurring ALK3 polypeptide. Likewise, mutagenesis can give rise to
variants which have intracellular half-lives dramatically different
than the corresponding a wild-type ALK3 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
ALK3 polypeptide. Such variants, and the genes which encode them,
can be utilized to alter ALK3 polypeptide levels by modulating the
half-life of the ALK3 polypeptides. For instance, a short half-life
can give rise to more transient biological effects and can allow
tighter control of recombinant ALK3 polypeptide levels within the
patient. 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.
[0083] 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 ALK3 polypeptide
sequences. For instance, a mixture of synthetic oligonucleotides
can be enzymatically ligated into gene sequences such that the
degenerate set of potential ALK3 polypeptide nucleotide sequences
are expressible as individual polypeptides, or alternatively, as a
set of larger fusion proteins (e.g., for phage display).
[0084] 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 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. AG 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).
[0085] Alternatively, other forms of mutagenesis can be utilized to
generate a combinatorial library. For example, ALK3 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 (Leung et 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 ALK3
polypeptides.
[0086] 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 ALK3
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 BMP
binding assays and BMP-mediated cell signaling assays.
[0087] In certain embodiments, the ALK3 polypeptides of the
invention may further comprise post-translational modifications in
addition to any that are naturally present in the ALK3
polypeptides. Such modifications include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. As a result, the modified ALK3
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 ALK3 polypeptide may be tested as described
herein for other ALK3 polypeptide variants. When an ALK3
polypeptide is produced in cells by cleaving a nascent form of the
ALK3 polypeptide, post-translational processing may also be
important for correct folding and/or function of the protein.
Different cells (such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or
HEK293) have specific cellular machinery and characteristic
mechanisms for such post-translational activities and may be chosen
to ensure the correct modification and processing of the ALK3
polypeptides.
[0088] In certain aspects, functional variants or modified forms of
the ALK3 polypeptides include fusion proteins having at least a
portion of the ALK3 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.TM. system (Qiagen)
useful with (HIS.sub.6) fusion partners. As another example, a
fusion domain may be selected so as to facilitate detection of the
ALK3 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 ALK3 polypeptide is fused with a domain
that stabilizes the ALK3 polypeptide in vivo (a "stabilizer"
domain). By "stabilizing" is meant anything that increases serum
half life, regardless of whether this is because of decreased
destruction, decreased clearance by the kidney, or other
pharmacokinetic effect. Fusions with the Fc portion of an
immunoglobulin are known to confer desirable pharmacokinetic
properties on a wide range of proteins. Likewise, fusions to human
serum albumin can confer desirable properties. Other types of
fusion domains that may be selected include multimerizing (e.g.,
dimerizing, tetramerizing) domains and functional domains (that
confer an additional biological function, such as further
stimulation of bone growth or muscle growth, as desired).
[0089] As a specific example, the present invention provides a
fusion protein comprising a soluble extracellular domain of ALK3
fused to an Fc domain (e.g., SEQ ID NO: 5 in FIG. 5). Examples of
Fc domains are shown below:
TABLE-US-00001 THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*
[0090] 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.
[0091] 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 ALK3 polypeptide may be
placed C-terminal to a heterologous domain, or, alternatively, a
heterologous domain may be placed C-terminal to an ALK3
polypeptide. The ALK3 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.
[0092] In certain embodiments, the ALK3 polypeptides of the present
invention contain one or more modifications that are capable of
stabilizing the ALK3 polypeptides. For example, such modifications
enhance the in vitro half life of the ALK3 polypeptides, enhance
circulatory half life of the ALK3 polypeptides or reduce
proteolytic degradation of the ALK3 polypeptides. Such stabilizing
modifications include, but are not limited to, fusion proteins
(including, for example, fusion proteins comprising an ALK3
polypeptide and a stabilizer domain), modifications of a
glycosylation site (including, for example, addition of a
glycosylation site to an ALK3 polypeptide), and modifications of
carbohydrate moiety (including, for example, removal of
carbohydrate moieties from an ALK3 polypeptide). In the case of
fusion proteins, an ALK3 polypeptide is fused to a stabilizer
domain such as an IgG molecule (e.g., an Fc domain). As used
herein, the term "stabilizer domain" not only refers to a fusion
domain (e.g., Fc) as in the case of fusion proteins, but also
includes nonproteinaceous modifications such as a carbohydrate
moiety, or nonproteinaceous polymer, such as polyethylene
glycol.
[0093] In certain embodiments, the present invention makes
available isolated and/or purified forms of the ALK3 polypeptides,
which are isolated from, or otherwise substantially free of, other
proteins. ALK3 polypeptides will generally be produced by
expression from recombinant nucleic acids.
3. Nucleic Acids Encoding ALK3 Polypeptides
[0094] In certain aspects, the invention provides isolated and/or
recombinant nucleic acids encoding any of the ALK3 polypeptides
(e.g., soluble ALK3 polypeptides), including fragments, functional
variants and fusion proteins disclosed herein. For example, SEQ ID
NO: 2 encodes the naturally occurring human ALK3 precursor
polypeptide, while SEQ ID NO: 4 encodes the processed extracellular
domain of ALK3. 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
ALK3 polypeptides or as direct therapeutic agents (e.g., in a gene
therapy approach).
[0095] In certain aspects, the subject nucleic acids encoding ALK3
polypeptides are further understood to include nucleic acids that
are variants of SEQ ID NO: 2 or 4. Variant nucleotide sequences
include sequences that differ by one or more nucleotide
substitutions, additions or deletions, such as allelic
variants.
[0096] In certain embodiments, the invention provides isolated or
recombinant nucleic acid sequences that are at least 80%, 85%, 90%,
95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2, 4, 12, 13,
15, 16, 19, 21, 24, 27, 32 or 37. One of ordinary skill in the art
will appreciate that nucleic acid sequences complementary to SEQ ID
NO: 2, 4, 12, 13, 15, 16, 19, 21, 24, 27, 32 or 37 and variants of
SEQ ID NO: 2, 4, 12, 13, 15, 16, 19, 21, 24, 27, 32 or 37 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.
[0097] In other embodiments, nucleic acids of the invention also
include nucleotide sequences that hybridize under highly stringent
conditions to the nucleotide sequence designated in SEQ ID NO: 2,
4, 12, 13, 15, 16, 19, 21, 24, 27, 32 or 37, complement sequence of
SEQ ID NO: 2, 4, 12, 13, 15, 16, 19, 21, 24, 27, 32 or 37, 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.
[0098] Isolated nucleic acids which differ from the nucleic acids
as set forth in SEQ ID NOs: 2, 4, 12, 13, 15, 16, 19, 21, 24, 27,
32 or 37 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.
[0099] 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.
[0100] In certain aspects of the invention, the subject nucleic
acid is provided in an expression vector comprising a nucleotide
sequence encoding an ALK3 polypeptide and operably linked to at
least one regulatory sequence. Regulatory sequences are
art-recognized and are selected to direct expression of the ALK3
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 ALK3 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.
[0101] 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 ALK3 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.
[0102] 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 pAcUW1), and pBlueBac-derived vectors (such as the
8-gal containing pBlueBac III).
[0103] In a preferred embodiment, a vector will be designed for
production of the subject ALK3 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 ALK3 polypeptides in
cells propagated in culture, e.g., to produce proteins, including
fusion proteins or variant proteins, for purification.
[0104] This disclosure also pertains to a host cell transfected
with a recombinant gene including a coding sequence (e.g., SEQ ID
NO: 2, 4, 12, 13, 15, 16, 19, 21, 24, 27, 32 or 37) for one or more
of the subject ALK3 polypeptides. The host cell may be any
prokaryotic or eukaryotic cell. For example, an ALK3 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.
[0105] Accordingly, the present invention further pertains to
methods of producing the subject ALK3 polypeptides. For example, a
host cell transfected with an expression vector encoding an ALK3
polypeptide can be cultured under appropriate conditions to allow
expression of the ALK3 polypeptide to occur. The ALK3 polypeptide
may be secreted and isolated from a mixture of cells and medium
containing the ALK3 polypeptide. Alternatively, the ALK3
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 ALK3 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 ALK3 polypeptides and affinity purification with an
agent that binds to a domain fused to the ALK3 polypeptide (e.g., a
protein A column may be used to purify an ALK3-Fc fusion). In a
preferred embodiment, the ALK3 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, ALK3-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 effects on bone in mice and an
acceptable safety profile in mice, rats and non-human primates.
[0106] 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 ALK3 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 ALK3 polypeptide (e.g., see Hochuli et al., (1987) J.
Chromatography 411:177; and Janknecht et al., PNAS USA
88:8972).
[0107] 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 BMP and ALK3 Antagonists
[0108] The data presented herein demonstrates that antagonists of
BMP-ALK3 signaling can be used to promote bone growth and bone
mineralization. Although soluble ALK3 polypeptides, and
particularly ALK3-Fc, are preferred antagonists, and although such
antagonists may affect bone through a mechanism other than BMP
antagonism (e.g., BMP 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 bone), other types of BMP-ALK3 antagonists are expected
to be useful, including anti-BMP (e.g., BMP2 or BMP4) antibodies,
anti-ALK3 antibodies, antisense, RNAi or ribozyme nucleic acids
that inhibit the production of ALK3, BMP2 or BMP4 and other
inhibitors of BMP or ALK3, particularly those that disrupt BMP-ALK3
binding.
[0109] An antibody that is specifically reactive with an ALK3
polypeptide (e.g., a soluble ALK3 polypeptide) and which either
binds competitively to ligand with the ALK3 polypeptide or
otherwise inhibits ALK3-mediated signaling may be used as an
antagonist of ALK3 polypeptide activities. Likewise, an antibody
that is specifically reactive with an BMP polypeptide and which
disrupts ALK3 binding may be used as an antagonist.
[0110] By using immunogens derived from an ALK3 polypeptide or a
BMP 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 ALK3
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 ALK3 or BMP 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.
[0111] Following immunization of an animal with an antigenic
preparation of an ALK3 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 ALK3
polypeptide and monoclonal antibodies isolated from a culture
comprising such hybridoma cells.
[0112] The term "antibody" as used herein is intended to include
fragments thereof which are also specifically reactive with a
subject polypeptide. Antibodies can be fragmented using
conventional techniques and the fragments screened for utility in
the same manner as described above for whole antibodies. For
example, F(ab).sub.2 fragments can be generated by treating
antibody with pepsin. The resulting F(ab).sub.2 fragment can be
treated to reduce disulfide bridges to produce Fab fragments. The
antibody of the present invention is further intended to include
bispecific, single-chain, chimeric, humanized and fully human
molecules having affinity for an ALK3 or BMP polypeptide conferred
by at least one CDR region of the antibody. An antibody may further
comprise a label attached thereto and able to be detected (e.g.,
the label can be a radioisotope, fluorescent compound, enzyme or
enzyme co-factor).
[0113] In certain embodiments, the antibody is a recombinant
antibody, which term encompasses any antibody generated in part by
techniques of molecular biology, including CDR-grafted or chimeric
antibodies, human or other antibodies assembled from
library-selected antibody domains, single chain antibodies and
single domain antibodies (e.g., human V.sub.H proteins or camelid
V.sub.HH proteins). 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 ALK3 polypeptide or BMP 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.
[0114] 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 ALK3 polypeptide) and
other antigens that are not of interest that the antibody is useful
for, at minimum, detecting the presence of the antigen of interest
in a particular type of biological sample. In certain methods
employing the antibody, such as therapeutic applications, a higher
degree of specificity in binding may be desirable. Monoclonal
antibodies generally have a greater tendency (as compared to
polyclonal antibodies) to discriminate effectively between the
desired antigens and cross-reacting polypeptides. One
characteristic that influences the specificity of an
antibody:antigen interaction is the affinity of the antibody for
the antigen. Although the desired specificity may be reached with a
range of different affinities, generally preferred antibodies will
have an affinity (a dissociation constant) of about 10.sup.-6,
10.sup.-7, 10.sup.-8, 10.sup.-9 or less. Given the extraordinarily
tight binding between BMPs and ALK3, it is expected that a
neutralizing anti-BMP or anti-ALK3 antibody would generally have a
dissociation constant of 10.sup.-9 or less.
[0115] 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.
[0116] Examples of categories of nucleic acid compounds that are
BMP or ALK3 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, 30, 25, 22, 20 or 18 nucleotides of the
full-length ALK3 nucleic acid sequence or BMP nucleic acid
sequence. The region of complementarity will preferably be at least
8 nucleotides, and optionally at least 10 or at least 15
nucleotides, and optionally between 15 and 25 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 internucleotide
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 preferably 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, bone growth and
mineralization.
5. Screening Assays
[0117] In certain aspects, the present invention relates to the use
of ALK3 polypeptides (e.g., soluble ALK3 polypeptides) and BMP
polypeptides to identify compounds (agents) which are agonist or
antagonists of the BMP-ALK3 signaling pathway. Compounds identified
through this screening can be tested to assess their ability to
modulate bone growth or mineralization in vitro. Optionally, these
compounds can further be tested in animal models to assess their
ability to modulate tissue growth in vivo.
[0118] There are numerous approaches to screening for therapeutic
agents for modulating tissue growth by targeting BMPs and ALK3
polypeptides. In certain embodiments, high-throughput screening of
compounds can be carried out to identify agents that perturb BMPs
or ALK3-mediated effects on bone. In certain embodiments, the assay
is carried out to screen and identify compounds that specifically
inhibit or reduce binding of an ALK3 polypeptide to BMPs.
Alternatively, the assay can be used to identify compounds that
enhance binding of an ALK3 polypeptide to BMPs. In a further
embodiment, the compounds can be identified by their ability to
interact with a BMP or ALK3 polypeptide.
[0119] A variety of assay formats will suffice and, in light of the
present disclosure, those not expressly described herein will
nevertheless be comprehended by one of ordinary skill in the art.
As described herein, the test compounds (agents) of the invention
may be created by any combinatorial chemical method. Alternatively,
the subject compounds may be naturally occurring biomolecules
synthesized in vivo or in vitro. Compounds (agents) to be tested
for their ability to act as modulators of tissue growth can be
produced, for example, by bacteria, yeast, plants or other
organisms (e.g., natural products), produced chemically (e.g.,
small molecules, including peptidomimetics), or produced
recombinantly. Test compounds contemplated by the present invention
include non-peptidyl organic molecules, peptides, polypeptides,
peptidomimetics, sugars, hormones, and nucleic acid molecules. In a
specific embodiment, the test agent is a small organic molecule
having a molecular weight of less than about 2,000 daltons.
[0120] The test compounds of the invention can be provided as
single, discrete entities, or provided in libraries of greater
complexity, such as made by combinatorial chemistry. These
libraries can comprise, for example, alcohols, alkyl halides,
amines, amides, esters, aldehydes, ethers and other classes of
organic compounds. Presentation of test compounds to the test
system can be in either an isolated form or as mixtures of
compounds, especially in initial screening steps. Optionally, the
compounds may be optionally derivatized with other compounds and
have derivatizing groups that facilitate isolation of the
compounds. Non-limiting examples of derivatizing groups include
biotin, fluorescein, digoxygenin, green fluorescent protein,
isotopes, polyhistidine, magnetic beads, glutathione S transferase
(GST), photoactivatible crosslinkers or any combinations
thereof.
[0121] In many drug screening programs which test libraries of
compounds and natural extracts, high throughput assays are
desirable in order to maximize the number of compounds surveyed in
a given period of time. Assays which are performed in cell-free
systems, such as may be derived with purified or semi-purified
proteins, are often preferred as "primary" screens in that they can
be generated to permit rapid development and relatively easy
detection of an alteration in a molecular target which is mediated
by a test compound. Moreover, the effects of cellular toxicity or
bioavailability of the test compound can be generally ignored in
the in vitro system, the assay instead being focused primarily on
the effect of the drug on the molecular target as may be manifest
in an alteration of binding affinity between an ALK3 polypeptide
and BMPs.
[0122] Merely to illustrate, in an exemplary screening assay of the
present invention, the compound of interest is contacted with an
isolated and purified ALK3 polypeptide which is ordinarily capable
of binding to BMPs. To the mixture of the compound and ALK3
polypeptide is then added a composition containing an ALK3 ligand.
Detection and quantification of ALK3/BMP complexes provides a means
for determining the compound's efficacy at inhibiting (or
potentiating) complex formation between the ALK3 polypeptide and
BMPs. The efficacy of the compound can be assessed by generating
dose response curves from data obtained using various
concentrations of the test compound. Moreover, a control assay can
also be performed to provide a baseline for comparison. For
example, in a control assay, isolated and a purified BMP is added
to a composition containing the ALK3 polypeptide, and the formation
of ALK3/BMP complex is quantitated in the absence of the test
compound. It will be understood that, in general, the order in
which the reactants may be admixed can be varied, and can be
admixed simultaneously. Moreover, in place of purified proteins,
cellular extracts and lysates may be used to render a suitable
cell-free assay system.
[0123] Complex formation between the ALK3 polypeptide and BMPs may
be detected by a variety of techniques. For instance, modulation of
the formation of complexes can be quantitated using, for example,
detectably labeled proteins such as radiolabeled (e.g., .sup.32,
.sup.35S, .sup.14C or .sup.3H), fluorescently labeled (e.g., FITC),
or enzymatically labeled ALK3 polypeptide or BMPs, by immunoassay,
or by chromatographic detection.
[0124] In certain embodiments, the present invention contemplates
the use of fluorescence polarization assays and fluorescence
resonance energy transfer (FRET) assays in measuring, either
directly or indirectly, the degree of interaction between an ALK3
polypeptide and its binding protein. Further, other modes of
detection, such as those based on optical waveguides (PCT
Publication WO 96/26432 and U.S. Pat. No. 5,677,196), surface
plasmon resonance (SPR), surface charge sensors, and surface force
sensors, are compatible with many embodiments of the invention.
[0125] Moreover, the present invention contemplates the use of an
interaction trap assay, also known as the "two hybrid assay," for
identifying agents that disrupt or potentiate interaction between
an ALK3 polypeptide and its binding protein. See for example, U.S.
Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et
al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993)
Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene
8:1693-1696). In a specific embodiment, the present invention
contemplates the use of reverse two hybrid systems to identify
compounds (e.g., small molecules or peptides) that dissociate
interactions between an ALK3 polypeptide and its binding protein.
See for example, Vidal and Legrain, (1999) Nucleic Acids Res
27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81;
and U.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.
[0126] In certain embodiments, the subject compounds are identified
by their ability to interact with an ALK3 or BMP polypeptide of the
invention. The interaction between the compound and the ALK3 or BMP
polypeptide may be covalent or non-covalent. For example, such
interaction can be identified at the protein level using in vitro
biochemical methods, including photo-crosslinking, radiolabeled
ligand binding, and affinity chromatography (Jakoby W B et al.,
1974, Methods in Enzymology 46: 1). In certain cases, the compounds
may be screened in a mechanism based assay, such as an assay to
detect compounds which bind to a BMP or ALK3 polypeptide. This may
include a solid phase or fluid phase binding event. Alternatively,
the gene encoding a BMP or ALK3 polypeptide can be transfected with
a reporter system (e.g., 3-galactosidase, luciferase, or green
fluorescent protein) into a cell and screened against the library
preferably by a high throughput screening or with individual
members of the library. Other mechanism based binding assays may be
used, for example, binding assays which detect changes in free
energy. Binding assays can be performed with the target fixed to a
well, bead or chip or captured by an immobilized antibody or
resolved by capillary electrophoresis. The bound compounds may be
detected usually using colorimetric or fluorescence or surface
plasmon resonance.
[0127] In certain aspects, the present invention provides methods
and agents for modulating (stimulating or inhibiting) bone
formation and increasing bone mass. Therefore, any compound
identified can be tested in whole cells or tissues, in vitro or in
vivo, to confirm their ability to modulate bone growth or
mineralization. Various methods known in the art can be utilized
for this purpose.
[0128] For example, the effect of the ALK3 or BMP polypeptides or
test compounds on bone or cartilage growth can be determined by
measuring induction of Msx2 or differentiation of osteoprogenitor
cells into osteoblasts in cell based assays (see, e.g., Daluiski et
al., Nat Genet. 2001, 27(1):84-8; Hino et al., Front Biosci. 2004,
9:1520-9). Another example of cell-based assays includes analyzing
the osteogenic activity of the subject ALK3 or BMP polypeptides and
test compounds in mesenchymal progenitor and osteoblastic cells. To
illustrate, recombinant adenoviruses expressing a BMP or ALK3
polypeptide can be constructed to infect pluripotent mesenchymal
progenitor C3H10T1/2 cells, preosteoblastic C2C12 cells, and
osteoblastic TE-85 cells. Osteogenic activity is then determined by
measuring the induction of alkaline phosphatase, osteocalcin, and
matrix mineralization (see, e.g., Cheng et al., J bone Joint Surg
Am. 2003, 85-A(8):1544-52).
[0129] The present invention also contemplates in vivo assays to
measure bone or cartilage growth. For example, Namkung-Matthai et
al., Bone, 28:80-86 (2001) discloses a rat osteoporotic model in
which bone repair during the early period after fracture is
studied. Kubo et al., Steroid Biochemistry & Molecular Biology,
68:197-202 (1999) also discloses a rat osteoporotic model in which
bone repair during the late period after fracture is studied.
Andersson et al., J. Endocrinol. 170:529-537 describe a mouse
osteoporosis model in which mice are ovariectomized, which causes
the mice to lose substantial bone mineral content and bone mineral
density, with the trabecular bone losing roughly 50% of bone
mineral density. Bone density could be increased in the
ovariectomized mice by administration of factors such as
parathyroid hormone. In certain aspects, the present invention
makes use of fracture healing assays that are known in the art.
These assays include fracture technique, histological analysis, and
biomechanical analysis, which are described in, for example, U.S.
Pat. No. 6,521,750, which is incorporated by reference in its
entirety for its disclosure of experimental protocols for causing
as well as measuring the extent of fractures, and the repair
process.
6. Exemplary Therapeutic Uses
[0130] In certain embodiments, BMP-ALK3 antagonists (e.g., ALK3
polypeptides) of the present invention can be used for treating or
preventing a disease or condition that is associated with bone
damage, whether, e.g., through breakage, loss or demineralization.
In certain embodiments, the present invention provides methods of
treating or preventing bone damage in an individual in need thereof
through administering to the individual a therapeutically effective
amount of an BMP-ALK3 antagonist, particularly an ALK3 polypeptide.
Given the potential for a dual effect on bone resorption and
formation, such compounds may be useful in a wide range of diseases
that are currently treated with anabolic (e.g., parathyroid hormone
and derivatives thereof) or anti-resorptive agents (e.g.,
bisphosphonates). In certain embodiments, the present invention
provides methods of promoting bone growth or mineralization in an
individual in need thereof through administering to the individual
a therapeutically effective amount of a BMP-ALK3 antagonist,
particularly an ALK3 polypeptide. These methods are optionally
aimed at therapeutic and prophylactic treatments of animals, and
more preferably, humans. In certain embodiments, the disclosure
provides for the use of BMP-ALK3 antagonists (particularly soluble
ALK3 polypeptides and neutralizing antibodies targeted to BMPs or
ALK3) for the treatment of disorders associated with low bone
density or decreased bone strength.
[0131] 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 and the
intended result of administration of the therapeutic agent.
[0132] The disclosure provides methods of inducing bone and/or
cartilage formation, preventing bone loss, increasing bone
mineralization or preventing the demineralization of bone. For
example, the subject BMP-ALK3 antagonists have application in
treating bone loss disorders, such as osteoporosis and the healing
of bone fractures and cartilage defects or other bone defects,
injuries and disorders in humans and other animals. ALK3 or BMP
polypeptides may be useful in patients that are diagnosed with
subclinical low bone density, as a protective measure against the
development of osteoporosis.
[0133] In one specific embodiment, methods and compositions of the
present invention may find medical utility in the healing of bone
fractures and cartilage defects in humans and other animals. The
subject methods and compositions may also have prophylactic use in
closed as well as open fracture reduction and also in the improved
fixation of artificial joints. De novo bone formation induced by an
osteogenic agent contributes to the repair of congenital,
trauma-induced, or oncologic resection induced craniofacial
defects, and also is useful in cosmetic plastic surgery. In certain
cases, the subject BMP-ALK3 antagonists may provide an environment
to attract bone-forming cells, stimulate growth of bone-forming
cells or induce differentiation of progenitors of bone-forming
cells. BMP-ALK3 antagonists of the invention may also be useful in
the treatment of osteoporosis.
[0134] Rosen et al. (ed) Primer on the Metabolic Bone Diseases and
Disorders of Mineral Metabolism, 7.sup.th ed. American Society for
Bone and Mineral Research, Washington D.C. (incorporated herein by
reference) provides an extensive discussion of bone disorders that
may be subject to treatment with ALK3-BMP antagonists. A partial
listing is provided herein. Methods and compositions of the
invention can be applied to conditions characterized by or causing
bone loss, such as osteoporosis (including secondary osteoporosis),
hyperparathyroidism, chronic kidney disease mineral bone disorder,
sex hormone deprivation or ablation (e.g. androgen and/or
estrogen), glucocorticoid treatment, rheumatoid arthritis, severe
burns, hyperparathyroidism, hypercalcemia, hypocalcemia,
hypophosphatemia, osteomalacia (including tumor-induced
osteomalacia), hyperphosphatemia, vitamin D deficiency,
hyperparathyroidism (including familial hyperparathyroidism) and
pseudohypoparathyroidism, tumor metastases to bone, bone loss as a
consequence of a tumor or chemotherapy, tumors of the bone and bone
marrow (e.g., multiple myeloma), ischemic bone disorders,
periodontal disease and oral bone loss, Cushing's disease, Paget's
disease, thyrotoxicosis, chronic diarrheal state or malabsorption,
renal tubular acidosis, or anorexia nervosa. Methods and
compositions of the invention may also be applied to conditions
characterized by a failure of bone formation or healing, including
non-union fractures, fractures that are otherwise slow to heal,
fetal and neonatal bone dysplasias (e.g., hypocalcemia,
hypercalcemia, calcium receptor defects and vitamin D deficiency),
osteonecrosis (including osteonecrosis of the jaw) and osteogenesis
imperfecta. Additionally, the anabolic effects will cause such
antagonists to diminish bone pain associated with bone damage or
erosion. As a consequence of the anti-resorptive effects, such
antagonists may be useful to treat disorders of abnormal bone
formation, such as osteoblastic tumor metastases (e.g., associated
with primary prostate or breast cancer), osteogenic osteosarcoma,
osteopetrosis, progressive diaphyseal dysplasia, endosteal
hyperostosis, osteopoikilosis, and melorheostosis. Other disorders
that may be treated include fibrous dysplasia and
chondrodysplasias.
[0135] In addition to the foregoing discussion, persons having any
of the following profiles may be candidates for treatment with an
ALK3 antagonist: a post-menopausal woman and not taking estrogen or
other hormone replacement therapy; a person with a personal or
maternal history of hip fracture or smoking; a post-menopausal
woman who is tall (over 5 feet 7 inches) or thin (less than 125
pounds); a man with clinical conditions associated with bone loss;
a person using medications that are known to cause bone loss,
including corticosteroids such as Prednisone.TM., various
anti-seizure medications such as Dilantin.TM. and certain
barbiturates, or high-dose thyroid replacement drugs; a person
having type 1 diabetes, liver disease, kidney disease, a family
history of osteoporosis; a person having high bone turnover (e.g.,
excessive collagen in urine samples); a person with a thyroid
condition, such as hyperthyroidism; a person who has experienced a
fracture after only mild trauma; a person who has had x-ray
evidence of vertebral fracture or other signs of osteoporosis.
[0136] Osteoporosis (meaning, generally speaking, a state of low
bone density or strength) may be caused by, or associated with,
various factors. Being female, particularly a post-menopausal
female, having a low body weight, and leading a sedentary lifestyle
are all risk factors for osteoporosis (loss of bone mineral
density, leading to fracture risk).
[0137] Osteoporosis can also result as a condition associated with
another disorder or from the use of certain medications.
Osteoporosis resulting from drugs or another medical condition is
known as secondary osteoporosis. In a condition known as Cushing's
disease, the excess amount of cortisol produced by the body results
in osteoporosis and fractures. The most common medications
associated with secondary osteoporosis are the corticosteroids, a
class of drugs that act like cortisol, a hormone produced naturally
by the adrenal glands. Although adequate levels of thyroid hormones
(which are produced by the thyroid gland) are needed for the
development of the skeleton, excess thyroid hormone can decrease
bone mass over time. Antacids that contain aluminum can lead to
bone loss when taken in high doses by people with kidney problems,
particularly those undergoing dialysis. Other medications that can
cause secondary osteoporosis include phenytoin (Dilantin) and
barbiturates that are used to prevent seizures; methotrexate
(Rheumatrex, Immunex, Folex PFS), a drug for some forms of
arthritis, cancer, and immune disorders; cyclosporine (Sandimmune,
Neoral), a drug used to treat some autoimmune diseases and to
suppress the immune system in organ transplant patients;
luteinizing hormone-releasing hormone agonists (Lupron, Zoladex),
used to treat prostate cancer and endometriosis; heparin
(Calciparine, Liquaemin), an anticlotting medication; and
cholestyramine (Questran) and colestipol (Colestid), used to treat
high cholesterol. Bone loss resulting from cancer therapy is widely
recognized and termed cancer therapy induced bone loss (CTIBL).
Bone metastases can create cavities in the bone that may be
corrected by treatment with BMP-ALK3 antagonists.
[0138] Optionally, BMP-ALK3 antagonists, particularly a soluble
ALK3, disclosed herein may be used in cancer patients. Patients
having certain tumors (e.g. prostate, breast, multiple myeloma or
any tumor causing hyperparathyroidism) are at high risk for bone
loss due to tumor-induced bone loss as well as bone metastases and
therapeutic agents. Such patients may be treated with BMP-ALK3
antagonists even in the absence of evidence of bone loss or bone
metastases. Patients may also be monitored for evidence of bone
loss or bone metastases, and may be treated with BMP-ALK3
antagonists in the event that indicators suggest an increased risk.
Generally, DEXA scans are employed to assess changes in bone
density, while indicators of bone remodeling may be used to assess
the likelihood of bone metastases. Serum markers may be monitored.
Bone specific alkaline phosphatase (BSAP) is an enzyme that is
present in osteoblasts. Blood levels of BSAP are increased in
patients with bone metastasis and other conditions that result in
increased bone remodeling. Osteocalcin and procollagen peptides are
also associated with bone formation and bone metastases. Increases
in BSAP have been detected in patients with bone metastasis caused
by prostate cancer, and to a lesser degree, in bone metastases from
breast cancer. Bone Morphogenetic Protein-7 (BMP-7) levels are high
in prostate cancer that has metastasized to bone, but not in bone
metastases due to bladder, skin, liver, or lung cancer. Type I
Carboxy-terminal telopeptide (ICTP) is a crosslink found in
collagen that is formed during the resorption of bone. Since bone
is constantly being broken down and reformed, ICTP will be found
throughout the body. However, at the site of bone metastasis, the
level will be significantly higher than in an area of normal bone.
ICTP has been found in high levels in bone metastasis due to
prostate, lung, and breast cancer. Another collagen crosslink, Type
I N-terminal telopeptide (NTx), is produced along with ICTP during
bone turnover. The amount of NTx is increased in bone metastasis
caused by many different types of cancer including lung, prostate,
and breast cancer. Also, the levels of NTx increase with the
progression of the bone metastasis. Therefore, this marker can be
used to both detect metastasis as well as measure the extent of the
disease. Other markers of resorption include pyridinoline and
deoxypyridinoline. Any increase in resorption markers or markers of
bone metastases indicate the need for BMP-ALK3 antagonist therapy
in a patient.
[0139] In another embodiment, BMP-ALK3 antagonists may be used in
patients with chronic kidney disease mineral bone disorder
(CKD-MBD), a broad syndrome of interrelated skeletal,
cardiovascular, and mineral-metabolic disorders arising from kidney
disease. CKD-MBD encompasses various skeletal pathologies often
referred to as renal osteodystrophy (ROD), which is a preferred
embodiment for treatment with BMP-ALK3 antagonists. Depending on
the relative contribution of different pathogenic factors, ROD is
manifested as diverse pathologic patterns of bone remodeling
(Hruska et al., 2008, Chronic kidney disease mineral bone disorder
(CKD-MBD); in Rosen et al. (ed) Primer on the Metabolic Bone
Diseases and Disorders of Mineral Metabolism, 7.sup.th ed. American
Society for Bone and Mineral Research, Washington D.C., pp
343-349). At one end of the spectrum is ROD with uremic
osteodystrophy and low bone turnover, characterized by a low number
of active remodeling sites, profoundly suppressed bone formation,
and low bone resorption. At the other extreme is ROD with
hyperparathyroidism, high bone turnover, and osteitis fibrosa.
Given that BMP-ALK3 antagonists exert both anabolic and
antiresorptive effects, these agents may be useful in patients
across the ROD pathology spectrum.
[0140] BMP-ALK3 antagonists may be conjointly administered with
other pharmaceutical agents. Conjoint administration may be
accomplished by administration of a single co-formulation, by
simultaneous administration or by administration at separate times.
BMP-ALK3 antagonists may be particularly advantageous if
administered with other bone-active agents. A patient may benefit
from conjointly receiving BMP-ALK3 antagonist and taking calcium
supplements, vitamin D, appropriate exercise and/or, in some cases,
other medication. Examples of other medications include,
bisphosphonates (alendronate, ibandronate and risedronate),
calcitonin, estrogens, parathyroid hormone and raloxifene. The
bisphosphonates (alendronate, ibandronate and risedronate),
calcitonin, estrogens and raloxifene affect the bone remodeling
cycle and are classified as anti-resorptive medications. Bone
remodeling consists of two distinct stages: bone resorption and
bone formation. Anti-resorptive medications slow or stop the
bone-resorbing portion of the bone-remodeling cycle but do not slow
the bone-forming portion of the cycle. As a result, new formation
continues at a greater rate than bone resorption, and bone density
may increase over time. Teriparatide, a form of parathyroid
hormone, increases the rate of bone formation in the bone
remodeling cycle. Alendronate is approved for both the prevention
(5 mg per day or 35 mg once a week) and treatment (10 mg per day or
70 mg once a week) of postmenopausal osteoporosis. Alendronate
reduces bone loss, increases bone density and reduces the risk of
spine, wrist and hip fractures. Alendronate also is approved for
treatment of glucocorticoid-induced osteoporosis in men and women
as a result of long-term use of these medications (i.e., prednisone
and cortisone) and for the treatment of osteoporosis in men.
Alendronate plus vitamin D is approved for the treatment of
osteoporosis in postmenopausal women (70 mg once a week plus
vitamin D), and for treatment to improve bone mass in men with
osteoporosis. Ibandronate is approved for the prevention and
treatment of postmenopausal osteoporosis. Taken as a once-a-month
pill (150 mg), ibandronate should be taken on the same day each
month. Ibandronate reduces bone loss, increases bone density and
reduces the risk of spine fractures. Risedronate is approved for
the prevention and treatment of postmenopausal osteoporosis. Taken
daily (5 mg dose) or weekly (35 mg dose or 35 mg dose with
calcium), risedronate slows bone loss, increases bone density and
reduces the risk of spine and non-spine fractures. Risedronate also
is approved for use by men and women to prevent and/or treat
glucocorticoid-induced osteoporosis that results from long-term use
of these medications (i.e., prednisone or cortisone). Calcitonin is
a naturally occurring hormone involved in calcium regulation and
bone metabolism. In women who are more than 5 years beyond
menopause, calcitonin slows bone loss, increases spinal bone
density, and may relieve the pain associated with bone fractures.
Calcitonin reduces the risk of spinal fractures. Calcitonin is
available as an injection (50-100 IU daily) or nasal spray (200 IU
daily). Estrogen therapy (ET)/Hormone therapy (HT) is approved for
the prevention of osteoporosis. ET has been shown to reduce bone
loss, increase bone density in both the spine and hip, and reduce
the risk of hip and spinal fractures in postmenopausal women. ET is
administered most commonly in the form of a pill or skin patch that
delivers a low dose of approximately 0.3 mg daily or a standard
dose of approximately 0.625 mg daily and is effective even when
started after age 70. When estrogen is taken alone, it can increase
a woman's risk of developing cancer of the uterine lining
(endometrial cancer). To eliminate this risk, healthcare providers
prescribe the hormone progestin in combination with estrogen
(hormone replacement therapy or HT) for those women who have an
intact uterus. ET/HT relieves menopause symptoms and has been shown
to have a beneficial effect on bone health. Side effects may
include vaginal bleeding, breast tenderness, mood disturbances and
gallbladder disease. Raloxifene, 60 mg a day, is approved for the
prevention and treatment of postmenopausal osteoporosis. It is from
a class of drugs called Selective Estrogen Receptor Modulators
(SERMs) that have been developed to provide the beneficial effects
of estrogens without their potential disadvantages. Raloxifene
increases bone mass and reduces the risk of spine fractures. Data
are not yet available to demonstrate that raloxifene can reduce the
risk of hip and other non-spine fractures. Teriparatide, a form of
parathyroid hormone, is approved for the treatment of osteoporosis
in postmenopausal women and men who are at high risk for a
fracture. This medication stimulates new bone formation and
significantly increases bone mineral density. In postmenopausal
women, fracture reduction was noted in the spine, hip, foot, ribs
and wrist. In men, fracture reduction was noted in the spine, but
there were insufficient data to evaluate fracture reduction at
other sites. Teriparatide is self-administered as a daily injection
for up to 24 months.
7. Pharmaceutical Compositions
[0141] In certain embodiments, BMP-ALK3 antagonists (e.g., ALK3
polypeptides) of the present invention are formulated with a
pharmaceutically acceptable carrier. For example, an ALK3
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.
[0142] 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 ALK3 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., ALK3 polypeptides) in the methods of the
invention.
[0143] Typically, ALK3 antagonists will be administered parentally.
Pharmaceutical compositions suitable for parenteral administration
may comprise one or more ALK3 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.
[0144] Further, the composition may be encapsulated or injected in
a form for delivery to a target tissue site (e.g., bone). In
certain embodiments, compositions of the present invention may
include a matrix capable of delivering one or more therapeutic
compounds (e.g., ALK3 polypeptides) to a target tissue site (e.g.,
bone), 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 ALK3 polypeptides. Such
matrices may be formed of materials presently in use for other
implanted medical applications.
[0145] 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.
[0146] In certain embodiments, methods of the invention can be
administered for orally, e.g., in the form of capsules, cachets,
pills, tablets, lozenges (using a flavored basis, usually sucrose
and acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or nonaqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of an agent as an
active ingredient. An agent may also be administered as a bolus,
electuary or paste.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] 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., ALK3
polypeptides). The various factors include, but are not limited to,
amount of bone weight desired to be formed, the degree of bone
density loss, the site of bone damage, the condition of the damaged
bone, the patient's age, sex, and diet, the severity of any disease
that may be contributing to bone loss, time of administration, and
other clinical factors. Optionally, the dosage may vary with the
type of matrix used in the reconstitution and the types of
compounds in the composition. The addition of other known growth
factors to the final composition, may also affect the dosage.
Progress can be monitored by periodic assessment of bone growth
and/or repair, for example, X-rays (including DEXA),
histomorphometric determinations, and tetracycline labeling.
[0152] In certain embodiments, the present invention also provides
gene therapy for the in vivo production of ALK3 polypeptides. Such
therapy would achieve its therapeutic effect by introduction of the
ALK3 polynucleotide sequences into cells or tissues having the
disorders as listed above. Delivery of ALK3 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 ALK3 polynucleotide sequences
is the use of targeted liposomes. Various viral vectors which can
be utilized for gene therapy as taught herein include adenovirus,
herpes virus, vaccinia, or, preferably, an RNA virus such as a
retrovirus. Preferably, the retroviral vector is a derivative of a
murine or avian retrovirus. Examples of retroviral vectors in which
a single foreign gene can be inserted include, but are not limited
to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma
virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous
Sarcoma Virus (RSV). A number of additional retroviral vectors can
incorporate multiple genes. All of these vectors can transfer or
incorporate a gene for a selectable marker so that transduced cells
can be identified and generated. Retroviral vectors can be made
target-specific by attaching, for example, a sugar, a glycolipid,
or a protein. Preferred targeting is accomplished by using an
antibody. Those of skill in the art will recognize that specific
polynucleotide sequences can be inserted into the retroviral genome
or attached to a viral envelope to allow target specific delivery
of the retroviral vector containing the ALK3 polynucleotide. In a
preferred embodiment, the vector is targeted to bone or
cartilage.
[0153] Alternatively, tissue culture cells can be directly
transfected with plasmids encoding the retroviral structural genes
gag, pol and env, by conventional calcium phosphate transfection.
These cells are then transfected with the vector plasmid containing
the genes of interest. The resulting cells release the retroviral
vector into the culture medium.
[0154] Another targeted delivery system for ALK3 polynucleotides is
a colloidal dispersion system. Colloidal dispersion systems include
macromolecule complexes, nanocapsules, microspheres, beads, and
lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, and liposomes. The preferred colloidal system of
this invention is a liposome. Liposomes are artificial membrane
vesicles which are useful as delivery vehicles in vitro and in
vivo. RNA, DNA and intact virions can be encapsulated within the
aqueous interior and be delivered to cells in a biologically active
form (see e.g., Fraley, et al., Trends Biochem. Sci., 6:77, 1981).
Methods for efficient gene transfer using a liposome vehicle, are
known in the art, see e.g., Mannino, et al., Biotechniques, 6:682,
1988. The composition of the liposome is usually a combination of
phospholipids, usually in combination with steroids, especially
cholesterol. Other phospholipids or other lipids may also be used.
The physical characteristics of liposomes depend on pH, ionic
strength, and the presence of divalent cations.
[0155] Examples of lipids useful in liposome production include
phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, and gangliosides. Illustrative
phospholipids include egg phosphatidylcholine,
dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.
The targeting of liposomes is also possible based on, for example,
organ-specificity, cell-specificity, and organelle-specificity and
is known in the art.
EXEMPLIFICATION
[0156] The invention now being generally described, it will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain
embodiments of the present invention, and are not intended to limit
the invention.
Example 1. Generation of ALK3-Fc Fusion Proteins
[0157] The amino acid sequence and corresponding nucleotide
sequence for native human ALK3 are shown in FIGS. 1, 2. Applicants
designed an ALK3-hFc fusion protein in which the extracellular
domain (native residues 24-152) of human ALK3 (FIGS. 3, 4) is fused
C-terminally with a human Fc domain (FIGS. 5, 6) via a minimal
linker (comprised of amino acid residues TGGG) to yield the protein
shown in FIG. 7. The following three leader sequences were
considered:
TABLE-US-00002 (i) Native: (SEQ ID NO: 8) MPQLYIYIRLLGAYLFIISRVQG
(ii) Tissue plasminogen activator (TPA): (SEQ ID NO: 9)
MDAMKRGLCCVLLLCGAVFVSP (iii) Honey bee melittin (HBML): (SEQ ID NO:
10) MKFLVNVALVFMVVYISYIYA
The selected form of hALK3(24-152)-hFc (SEQ ID NO: 11) employs the
TPA leader and has the unprocessed amino-acid sequence shown in
FIG. 8. A sense nucleotide sequence encoding this fusion protein
and the corresponding antisense sequence are indicated in FIG. 9.
Shown below is an alternative sense nucleotide sequence encoding
hALK3(24-152)-hFc, which incorporates a C.fwdarw.T substitution at
position 1137 (underlined) that does not alter the amino acid
sequence.
TABLE-US-00003 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC 41
TGTGTGGAGC AGTCTTCGTT TCGCCCGGCG CCCAGAATCT 81 GGATAGTATG
CTTCATGGCA CTGGGATGAA ATCAGACTCC 121 GACCAGAAAA AGTCAGAAAA
TGGAGTAACC TTAGCACCAG 161 AGGATACCTT GCCTTTTTTA AAGTGCTATT
GCTCAGGGCA 201 CTGTCCAGAT GATGCTATTA ATAACACATG CATAACTAAT 241
GGACATTGCT TTGCCATCAT AGAAGAAGAT GACCAGGGAG 281 AAACCACATT
AGCTTCAGGG TGTATGAAAT ATGAAGGATC 321 TGATTTTCAG TGCAAAGATT
CTCCAAAAGC CCAGCTACGC 361 CGGACAATAG AATGTTGTCG GACCAATTTA
TGTAACCAGT 401 ATTTGCAACC CACACTGCCC CCTGTTGTCA TAGGTCCGTT 441
TTTTGATGGC AGCATTCGAA CCGGTGGTGG AACTCACACA 481 TGCCCACCGT
GCCCAGCACC TGAACTCCTG GGGGGACCGT 521 CAGTCTTCCT CTTCCCCCCA
AAACCCAAGG ACACCCTCAT 561 GATCTCCCGG ACCCCTGAGG TCACATGCGT
GGTGGTGGAC 601 GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG 641
TGGACGGCGT GGAGGTGCAT AATGCCAAGA CAAAGCCGCG 681 GGAGGAGCAG
TACAACAGCA CGTACCGTGT GGTCAGCGTC 721 CTCACCGTCC TGCACCAGGA
CTGGCTGAAT GGCAAGGAGT 761 ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC
CAGCCCCCAT 801 CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA 841
CCACAGGTGT ACACCCTGCC CCCATCCCGG GAGGAGATGA 881 CCAAGAACCA
GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT 921 CTATCCCAGC GACATCGCCG
TGGAGTGGGA GAGCAATGGG 961 CAGCCGGAGA ACAACTACAA GACCACGCCT
CCCGTGCTGG 1001 ACTCCGACGG CTCCTTCTTC CTCTATAGCA AGCTCACCGT 1041
GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC 1081 TCCGTGATGC
ATGAGGCTCT GCACAACCAC TACACGCAGA 1121 AGAGCCTCTC CCTGTCTCCG
GGTAAATGA
[0158] A variant of hALK3(24-152)-Fc with the TPA leader and with
murine Fc substituted for human Fc is shown in FIG. 10. A sense
nucleotide sequence encoding this variant and its corresponding
antisense sequence are indicated in FIG. 11. Applicants constructed
a form of hALK3(24-152)-mFc having an asparagine at position 71
(position 70 in the native ALK3 ECD sequence). The protein was
expressed in CHO cell lines, and N-terminal sequencing revealed a
primary species with an N-terminal block, indicating a start at the
native glutamine (Q) residue, consistent with the protein of SEQ ID
NO:7, and a single minor sequence of GAQNLDSMLHGTGMK (SEQ ID NO:
17). Applicants additionally constructed a hALK3(24-152)-hFc
protein having the native ALK3 sequence. Another ALK3-Fc variant
comprising the murine ALK3 extracellular domain (native residues
24-152 in the murine precursor) and murine Fc domain was generated
by similar methods. The amino acid sequence of this variant,
mALK3(24-152)-mFc, is shown below with the ALK3 domain
underlined:
TABLE-US-00004 (SEQ ID NO: 18) 1 MDAMKRGLCC VLLLCGAVFV SPGAQNLDSM
LHGTGMKSDL DQKKPENGVT 51 LAPEDTLPFL KCYCSGHCPD DAINNTCITN
GHCFAIIEED DQGETTLTSG 101 CMKYEGSDFQ CKDSPKAQLR RTIECCRTNL
CNQYLQPTLP PVVIGPFFDG 151 SIRTGGGEPR VPITQNPCPP LKECPPCAAP
DLLGGPSVFI FPPKIKDVLM 201 ISLSPMVTCV VVDVSEDDPD VQISWFVNNV
EVHTAQTQTH REDYNSTLRV 251 VSALPIQHQD WMSGKEFKCK VNNRALPSPI
EKTISKPRGP VRAPQVYVLP 301 PPAEEMTKKE FSLTCMITGF LPAEIAVDWT
SNGRTEQNYK NTATVLDSDG 351 SYFMYSKLRV QKSTWERGSL FACSVVHEGL
HNHLTTKTIS RSLGK
Example 2. Ligand Binding to ALK3-Fc
[0159] Biacore.TM. methodology was used to determine the binding
affinity of ALK3-Fc fusion proteins for more than 15 members of the
BMP/GDF family. mALK3-mFc derived from HEK 293 cells displayed
high-affinity binding to hBMP2 and hBMP4
(K.sub.D=2.43.times.10.sup.-9 and 9.47.times.10.sup.-10,
respectively), as well as moderate-affinity binding to several
other ligands, including hBMP6 and hBMP7. hALK3(24-152)-hFc
displayed a similar binding profile. Specifically,
hALK3(24-152)-hFc derived from HEK 293 cells bound to hBMP2 and
hBMP4 with K.sub.DS of 6.53.times.10.sup.-10 and
1.02.times.10.sup.-9, respectively, while hALK3(24-152)-hFc derived
from CHO cells bound to hBMP2 and hBMP4 with K.sub.DS of
4.53.times.10.sup.-10 and 7.03.times.10.sup.-10, respectively. Like
mALK3(24-152)-mFc, hALK3(24-152)-hFc derived from both cell types
exhibited moderate affinity binding to hBMP6 and hBMP7, among other
ligands.
[0160] The overall selectivity of ALK3-Fc for BMP2 and BMP4 is
notable. While not wishing to be bound to any particular mechanism,
Applicants hypothesize, based on these results, that ALK3-Fc exerts
its effects in vivo primarily by binding BMP2 and BMP4 and thereby
inhibiting signaling by these ligands. Accordingly, it is predicted
that antibodies against BMP2 and/or BMP4 would also stimulate bone
formation. Alternatively, an antibody against the ALK3
ligand-binding domain would be expected to inhibit ALK3-mediated
signaling more broadly. FIG. 18 shows diagrammatically examples of
three approaches proposed here to interfere with signaling by BMP2,
BMP4, and potentially additional ligands for the purpose of
promoting bone formation.
[0161] A series of ALK3-Fc proteins incorporating truncated
variants of the human ALK3 extracellular domain (ECD) were
generated and compared with hALK3(24-152)-hFc for their ligand
binding affinities. ALK3 ECD variants with N-terminal deletions of
6, 12, 27, or 31 amino acids, C-terminal deletions of 6 or 12 amino
acids, and a double-truncation were expressed in HEK 293 cells and
purified by Mab chromatography (Protein A column). Biacore.TM.
methodology was used to screen members of the BMP/GDF/TGF.beta.
ligand superfamily for binding to these variants.
TABLE-US-00005 Binding Affinity (K.sub.D, in pM) of Selected Human
Ligands for Human ALK3 ECD Variants Ligand Construct Expressed in
293 Cells hBMP2 hBMP4 hBMP6 hBMP7 Full Length hALK3(24-152)- 653
1020 17300 5990 hFc ALK3N.DELTA.6 hALK3(30-152)- 869 1610 12800 --
hFc ALK3N.DELTA.12 hALK3(36-152)- 1040 -- 5280 -- hFc
ALK3N.DELTA.27 hALK3(51-152)- 1570 -- 8040 4290 hFc ALK3N.DELTA.31
hALK3(55-152)- 663 -- 17000 3670 hFc ALK3C.DELTA.6 hALK3(24-146)-
532 396 -- -- hFc ALK3C.DELTA.12 hALK3(24-140)- 769 446 -- 5900 hFc
ALK3N.DELTA.6C.DELTA.6 hALK3(30-146)- 437 329 -- -- hFc -- no
detectable binding
[0162] As shown above, the C-terminal truncations that were
evaluated display similar or increased binding affinity for
BMP2/BMP4 compared to full-length ALK3 ECD, with generally reduced
binding to BMP6/BMP7, although ALK3C.DELTA.12 retains binding to
BMP7 at an affinity similar to the full-length ALK3 ECD. In
contrast, N-terminal truncations tend to reduce binding to BMP2,
abolish binding to BMP4, and display varying effects on binding to
BMP6/BMP7. Interestingly, the double-truncated variant
ALK3N.DELTA.6C.DELTA.6 displays increased affinity for BMP2/BMP4
compared to full-length ALK3 ECD, in combination with undetectable
binding to BMP6/BMP7. Molecules with greater selectivity for the
desired targets, BMP2 and BMP4 are useful because they will have
fewer "off target" effects in patients. N-terminal sequencing
demonstrated that the nucleic acid encoding a six amino acid
truncation at the N-terminus, when expressed in cell culture, gave
rise to a population of polypeptides having the six amino acid
truncation and a population of polypeptides having a seven amino
acid truncation. Taken together, these demonstrate that hALK3-hFc
polypeptides containing up to a seven amino acid truncation at the
N-terminus and up to a twelve amino acid truncation at the
C-terminus retain useful activity and demonstrate the desirable and
surprising reduction in binding to off-target ligands. Thus, an
ALK3 polypeptide comprising at least amino acids 8 to 117 of SEQ ID
NO:3 may be used for the purposes described herein.
[0163] Ligand binding properties were used to compare the quality
of hALK3(24-152)-hFc protein derived from CHO cells with that
derived from HEK 293 cells. As determined by Biacore.TM.
methodology, the affinity (Kd) of BMP2 for hALK3(24-152)-hFc did
not differ depending on the source of fusion protein; however, the
percentage of active protein generated by CHO cells was higher than
that of HEK 293 cells based on their respective Rmax values. Rmax
is a measure of protein quality equal to
(MW.sub.A/MW.sub.L).times.R.sub.L.times.S.sub.M, where MW.sub.A is
the molecular weight of analyte, MW.sub.L is the molecular weight
of ligand, R.sub.L in the immobilization level in response units,
and S.sub.M is the molar stoichiometry. Corresponding analysis of
BMP4 binding revealed that protein derived from CHO cells exhibited
higher affinity for BMP4 than did that from HEK 293 cells (K.sub.DS
of 314 pM vs. 1020 pM, respectively), and the Rmax value for
protein generated by CHO cells was three times that for protein
from HEK 293 cells, again indicating a higher percentage of active
protein. Therefore, unexpected benefits of CHO cells as the source
of hALK3 (24-152)-hFc protein include higher binding affinity of
protein to BMP4 and greater bioavailability predicted to result
from higher protein quality (Rmax value).
Example 3. hALK3-mFc Improves Bone Status in Mice
[0164] Applicants investigated the ability of a version of ALK3-mFc
to improve bone status in mice. Twelve-week-old female C57BL/6 mice
(n=8 per group) were treated with hALK3(24-152)-mFc, 10 mg/kg, or
vehicle (Tris-buffered saline) by intraperitoneal injection twice
per week for a total of six weeks. Compared to vehicle,
hALK3(24-152)-mFc significantly increased whole-body bone density,
as determined by dual energy x-ray absorptiometry (DEXA), by Day 31
and maintained this effect through study completion on Day 42 (FIG.
12). A similar effect of hALK3(24-152)-mFc treatment on bone
density was observed for localized analysis of lumbar vertebrae by
DEXA at these same time points (FIG. 13). In addition,
high-resolution measurements of the tibial shaft and proximal tibia
were conducted by micro-computed tomography (micro-CT) to determine
the effect of hALK3(24-152)-mFc on cortical bone and trabecular
bone, respectively. As compared to vehicle, hALK3(24-152)-mFc
treatment significantly increased: i) thickness of cortical bone by
Week 6 (FIG. 14), ii) volume of trabecular bone by Week 4 (FIG.
15), and iii) mean trabecular thickness by Week 4 (FIG. 16).
Representative three-dimensional reconstructions of
micro-CT-generated sections through the proximal tibia (FIG. 17)
underscore the robust stimulatory effect of hALK(24-152)-mFc
treatment (4 weeks) on trabecular bone microarchitecture.
Importantly, hALK(24-152)-mFc treatment did not cause significant
changes in lean tissue mass, fat mass, or red blood cell mass over
the course of the study.
[0165] Taken together, the foregoing data demonstrate that
hALK3(24-152)-mFc can be used in vivo to selectively improve bone
status through increased bone mineral density and increased net
formation of both cortical and trabecular bone.
Example 4. hALK3-mFc Increases Bone Strength in Mice
[0166] In the experiment described in Example 3, Applicants also
investigated the ability of hALK3(24-152)-mFc to increase bone
strength. After 6 weeks of dosing, femurs were collected and stored
frozen at -20.degree. C. Bones were later thawed to ambient
temperature, and destructive four-point bend tests were performed
on the left femur midshaft with an Instron mechanical testing
instrument (Instron 4465 retrofitted to 5500). Separation between
the fixed supports was 7 mm, and separation between the two points
of load application was 2.5 mm. Load was applied at a constant
displacement rate of 3 mm/min until bone breakage, and maximum
load, stiffness, and energy absorption data were calculated with
Bluehill v 2.5 software. Compared to vehicle, hALK3(24-152)-mFc
significantly increased maximum bone load by 30% (FIG. 19), bone
stiffness by 14% (FIG. 20), and energy to bone failure by 32% (FIG.
21). These findings demonstrate that increased bone strength
accompanies the improvement in bone composition observed with
hALK3(24-152)-mFc treatment (Example 3).
Example 5. Effects of mALK3-mFc on Bone in an OVX Mouse Model of
Osteopenia
[0167] Estrogen deficiency in postmenopausal women promotes bone
loss, particularly loss of trabecular bone. Therefore, Applicants
investigated the ability of mALK3(24-152)-mFc to improve bone
status in an ovariectomized (OVX) mouse model of osteopenia with
established bone loss. Eight-week-old female C57BL/6 mice underwent
bilateral OVX or sham surgery, then remained untreated for an
eight-week interval. At the end of eight weeks, baseline
measurements by micro-CT and DEXA confirmed significant bone loss
in the OVX mice compared to sham treatment. Most notable was a 43%
reduction in trabecular bone volume (FIG. 22, Day 0 time point), as
determined in the proximal tibia by micro-CT. Mice were then
treated with mALK3(24-152)-mFc, 10 mg/kg, or vehicle (Tris-buffered
saline), by ip injection twice per week for 8 weeks.
[0168] Treatment with mALK3(24-152)-mFc led to improvement in both
trabecular and cortical bone despite continuing estrogen
deficiency. By study completion on Day 56, trabecular bone volume
in the proximal tibia of OVX mice treated with mALK3(24-152)-mFc
was increased by nearly 250% compared to OVX controls and by more
than 80% compared to sham controls (FIG. 22). mALK3(24-152)-mFc
treatment also caused growth of cortical bone, as indicated by
increased cortical thickness (FIG. 23) and reduced endosteal
circumference (FIG. 24) in the tibial shaft compared to OVX
controls. These improvements were accompanied by increased bone
mineral density. Compared to OVX controls, mALK3(24-152)-mFc
treatment significantly increased whole-body bone mineral density
(as determined by DEXA) by Day 14 and maintained this improvement
through study completion (FIG. 25). Similar effects of
mALK3(24-152)-mFc treatment were observed on mineral density in the
lumbar spine (FIG. 26) and femur-tibia (FIG. 27). Three-dimensional
images of vertebral trabecular bone derived from micro-CT analysis
(FIG. 28) underscore the robust improvement in bone status
associated with mALK3(24-152)-mFc treatment despite ongoing
estrogen deficiency. These findings demonstrate that
mALK3(24-152)-mFc can reverse the deterioration of bone, including
trabecular bone, associated with estrogen withdrawal in a mouse
model of osteopenia. The ability of mALK3(24-152)-mFc to transform
bone from an osteopenic condition to one which surpasses the
quantity (FIGS. 22-24, 28) and matches the quality (FIGS. 25-27) of
bone in gonad-intact controls is evidence that this agent exerts
effects which are not only antiresorptive but anabolic.
Example 6. Effects of mALK3-mFc on Bone Histomorphometry and Serum
Biomarkers in Mice
[0169] In a separate study, Applicants investigated the ability of
mALK3-mFc to improve bone status in mice as assessed by
histomorphometry and serum biomarkers. Twelve-week-old female
C57BL/6 mice were treated with mALK3(24-152)-mFc, 10 mg/kg, or
vehicle (Tris-buffered saline) by intraperitoneal injection twice
per week. Cohorts of mice were necropsied after 14, 28, and 42 days
of treatment to permit collection of bone and serum. The
fluorescent compounds calcein (20 mg/kg) and demeclocycline (20
mg/kg) were administered intraperitoneally to mice 9 days and 2
days before necropsy, respectively, for dynamic histomorphometric
analysis.
[0170] Bone was prepared for histomorphometry as follows. At
necropsy, the right femur was detached, and the distal quarter of
the femur underwent histological preparation consisting of
dehydration, infiltration by methylmethacrylate, and embedding in
methylmethacrylate. A rotary microtome was used to obtain sets of
frontal sections at thicknesses of 4 and 8 .mu.m. The thinner
sections were stained with Goldner's trichrome and used for
analysis of static parameters, whereas the thicker sections were
mounted unstained and used for analysis of dynamic parameters.
Histomorphometry was performed in a treatment-blind manner with a
Nikon Eclipse E4000 light/epifluorescent microscope connected to a
video subsystem running OsteoMeasure image analysis software.
[0171] Histomorphometric analysis of the distal femur revealed both
anabolic and antiresorptive effects of ALK3-Fc. Compared to
vehicle, mALK3(24-152)-mFc significantly increased bone volume at
all three time points by up to 90% (FIG. 29). Importantly,
mALK3(24-152)-mFc increased bone formation rate by as much as 120%
(FIG. 30) and bone mineralizing surface by as much as 115% (FIG.
31). These latter parameters are considered to be indicative of
anabolic bone growth, although additional markers of anabolic
effects--osteoblast surface and osteoid surface--showed more modest
or negligible increases. Histomorphometric analysis also provided
evidence of temporal antiresorptive effects, as mALK3(24-152)-mFc
reduced osteoclast surface significantly at Day 28 only (FIG. 32),
and a similar effect on eroded surface was observed.
[0172] Effects of mALK3(24-152)-mFc treatment on serum biomarkers
of bone status were also investigated. RANKL (receptor activator of
nuclear factor-KB ligand) is produced by osteoblasts and is a key
activator of osteoclast differentiation, whereas osteoprotegerin
(OPG) is an endogenous inhibitor of RANKL signaling. Thus, the
RANKL/OPG ratio is an important determinant of osteoclastic
activity, bone mass, and bone quality (Boyce et al., 2008, Arch
Biochem Biophys 473:139-146). In the present experiment, serum
levels of RANKL and OPG were measured with Millipore products
(MBN2A-41K and MBN-41K-1OPG) incorporating Luminex xMAP.RTM.
technology. mALK3(24-152)-mFc treatment significantly reduced serum
RANKL levels at all three time points (FIG. 33) and significantly
increased serum OPG levels at 28 and 42 days (FIG. 34) compared to
vehicle. These results indicate that mALK3(24-152)-mFc treatment
stimulates bone formation in part through an antiresorptive
action.
Example 7. Effects of mALK3-mFc on Sclerostin Gene Expression in
Mice
[0173] Sclerostin protein is a key negative regulator of bone
formation, and interference with sclerostin signaling has been
reported to exert anabolic effects on bone in vivo (Li et al.,
2009, J Bone Miner Res 24:578-588). Applicants therefore
investigated whether mALK3(24-152)-mFc treatment in vivo alters
sclerostin gene expression in bone and thus whether a reduction in
sclerostin levels could potentially mediate some of the
bone-rebuilding effects of ALK3-Fc. Twelve-week-old female C57BL/6
mice were treated with mALK3(24-152)-mFc or vehicle (PBS) by
intraperitoneal injection twice per week. Cohorts of mice were
necropsied after 2, 7, 14, and 28 days of treatment to permit
bilateral collection of femurs and tibias, which were separated and
cleaned of any residual muscle or connective tissue.
[0174] Sclerostin gene expression was analyzed as follows. Bones
were trimmed to expose the interior marrow shaft, and marrow cells
were flushed out with sterile saline using a 21-gauge needle
attached to a 3-mL syringe. The femurs and tibias from each mouse
were pulverized together, and RNA was extracted from the resulting
powder with a Ribopure kit (Ambion) according to the manufacturer's
instructions. RNA integrity in bone samples was confirmed with RNA
Nano Chips (Agilent Technologies) run on an Agilent Technologies
2100 Bioanalyzer according to the manufacturer's instructions. RNA
was reverse-transcribed using TaqMan RT reagents (Applied
Biosystems), and real-time polymerase chain reaction (PCR) was
performed with sclerostin probe/primers and Eukaryotic 18S rRNA
Endogenous Control (both from Applied Biosystems). Amplifications
were performed with an Applied Biosystems 7300 System, and results
were analyzed using the 2.sup.-.DELTA..DELTA.ct method.
[0175] Compared to vehicle, treatment with mALK3(24-152)-mFc
reduced bone levels of sclerostin mRNA significantly at three of
the four time points investigated (FIG. 35). This finding indicates
that reduced expression of sclerostin may contribute to the
anabolic and/or antiresorptive effects of mALK3(24-152)-mFc on
bone.
Example 8. Effect of hALK3-hFc on Bone Status in Mice
[0176] Applicants investigated effects of the human construct
hALK3(24-152)-hFc on bone status in mice. Twelve-week-old female
C57BL/6 mice (n=6 per group) were treated with hALK3(24-152)-hFc,
10 mg/kg, or vehicle (Tris-buffered saline) by intraperitoneal
injection twice per week for a total of 6 weeks. Over the course of
the experiment, trabecular bone volume decreased nearly 20% in
vehicle-treated controls but increased more than 80% with
hALK3(24-152)-hFc treatment, as determined by micro-CT analysis of
the proximal tibia (FIG. 36). Significant increases from baseline
in trabecular number (34%) and trabecular thickness (20%) were also
observed with hALK3(24-152)-hFc, but not vehicle, by study
conclusion. Compared to vehicle, hALK3(24-152)-hFc significantly
increased whole-body bone mineral density, as determined by DEXA,
at study conclusion. Localized analysis of lumbar vertebrae (L1-L6)
by DEXA also revealed a significant stimulatory effect (21%
increase) of hALK3(24-152)-hFc on bone mineral density at study
conclusion compared to vehicle.
[0177] These results demonstrate that the human construct
hALK3(24-152)-hFc can improve bone status in mice, although it is
expected that the magnitude of its effects in rodents would be
blunted by an immune response. Collectively, the foregoing findings
demonstrate that ALK3-Fc constructs 1) promote bone formation in
both the axial skeleton and appendicular skeleton through both
antiresorptive and anabolic actions, 2) improve bone mechanical
strength, and 3) reverse bone loss induced by estrogen deficiency
in a mouse model of established osteopenia.
Example 9. Exemplary hALK3-hFc Nucleic Acids and Proteins
[0178] This example summarizes nucleic acid constructs used to
express ALK3 constructs in CHO cells, according to the methods
provided herein, and provides the mature proteins isolated from
cell culture.
A. The nucleic acid of SEQ ID NO:19 was expressed in CHO cells and
the following ALK3-Fc species were isolated:
[0179] (1) The hALK3(24-152)-hFc sequence shown in SEQ ID NO:7,
beginning with a glutamine (which tends to be blocked for
N-terminal sequencing by Edman degradation).
[0180] (2) The hALK3(GA,24-152)-hFc sequence shown below (SEQ ID
NO: 20), which retains an initial glycine-alanine from the leader
sequence.
TABLE-US-00006 (SEQ ID NO: 20) GAQNLDSM LHGTGMKSDS DQKKSENGVT
LAPEDTLPFL KCYCSGHCPD DAINNTCITN GHCFAIIEED DQGETTLASG CMKYEGSDFQ
CKDSPKAQLR RTIECCRTNL CNQYLQPTLP PVVIGPFFDG SIRTGGGTHT CPPCPAPELL
GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ
YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR
EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS
RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK*
B. A nucleic acid encoding hALK3(24-146)-hFc, shown below (SEQ ID
NO: 21) was expressed in CHO cells:
TABLE-US-00007 (SEQ ID NO: 21) AT GGATGCAATG AAGAGAGGGC TCTGCTGTGT
GCTGCTGCTG TGTGGAGCAG TCTTCGTTTC GCCCGGCGCC CAGAATCTGG ATAGTATGCT
TCATGGCACT GGGATGAAAT CAGACTCCGA CCAGAAAAAG TCAGAAAATG GAGTAACCTT
AGCACCAGAG GATACCTTGC CTTTTTTAAA GTGCTATTGC TCAGGGCACT GTCCAGATGA
TGCTATTAAT AACACATGCA TAACTAATGG ACATTGCTTT GCCATCATAG AAGAAGATGA
CCAGGGAGAA ACCACATTAG CTTCAGGGTG TATGAAATAT GAAGGATCTG ATTTTCAGTG
CAAAGATTCT CCAAAAGCCC AGCTACGCCG GACAATAGAA TGTTGTCGGA CCAATTTATG
TAACCAGTAT TTGCAACCCA CACTGCCCCC TGTTGTCATA GGTCCGTTTA CCGGTGGTGG
AACTCACACA TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT CAGTCTTCCT
CTTCCCCCCA AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT
GGTGGTGGAC GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT
GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA CGTACCGTGT
GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA
GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA
GCCCCGAGAA CCACAGGTGT ACACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA
GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA
GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG ACTCCGACGG
CTCCTTCTTC CTCTATAGCA AGCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT
CTTCTCATGC TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC
CCTGTCTCCG GGTAAATGA
The following protein species were isolated:
[0181] (1) The hALK3(24-146)-hFc shown below (SEQ ID NO:22),
beginning with a glutamine (which tends to be blocked for
N-terminal sequencing by Edman degradation).
TABLE-US-00008 (SEQ ID NO: 22) QNLDSMLHGT GMKSDSDQKK SENGVTLAPE
DTLPFLKCYC SGHCPDDAIN NTCITNGHCF AIIEEDDQGE TTLASGCMKY EGSDFQCKDS
PKAQLRRTIE CCRTNLCNQY LQPTLPPVVI GPFTGGGTHT CPPCPAPELL GGPSVFLFPP
KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV
LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL
TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC
SVMHEALHNH YTQKSLSLSP GK*
[0182] (2) The hALK3(GA,24-146)-hFc sequence shown below (SEQ ID
NO: 23), which retains an initial glycine-alanine from the leader
sequence.
TABLE-US-00009 (SEQ ID NO: 23) GA QNLDSMLHGT GMKSDSDQKK SENGVTLAPE
DTLPFLKCYC SGHCPDDAIN NTCITNGHCF AIIEEDDQGE TTLASGCMKY EGSDFQCKDS
PKAQLRRTIE CCRTNLCNQY LQPTLPPVVI GPFTGGGTHT CPPCPAPELL GGPSVFLFPP
KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV
LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL
TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC
SVMHEALHNH YTQKSLSLSP GK*
C. A nucleic acid encoding hALK3(24-140)-hFc, shown below (SEQ ID
NO: 24) was expressed in CHO cells:
TABLE-US-00010 (SEQ ID NO: 24) ATGG ATGCAATGAA GAGAGGGCTC
TGCTGTGTGC TGCTGCTGTG TGGAGCAGTC TTCGTTTCGC CCGGCGCCCA GAATCTGGAT
AGTATGCTTC ATGGCACTGG GATGAAATCA GACTCCGACC AGAAAAAGTC AGAAAATGGA
GTAACCTTAG CACCAGAGGA TACCTTGCCT TTTTTAAAGT GCTATTGCTC AGGGCACTGT
CCAGATGATG CTATTAATAA CACATGCATA ACTAATGGAC ATTGCTTTGC CATCATAGAA
GAAGATGACC AGGGAGAAAC CACATTAGCT TCAGGGTGTA TGAAATATGA AGGATCTGAT
TTTCAGTGCA AAGATTCTCC AAAAGCCCAG CTACGCCGGA CAATAGAATG TTGTCGGACC
AATTTATGTA ACCAGTATTT GCAACCCACA CTGCCCCCTA CCGGTGGTGG AACTCACACA
TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT CAGTCTTCCT CTTCCCCCCA
AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC
GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT GGAGGTGCAT
AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC
CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC
AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA
CCACAGGTGT ACACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA GGTCAGCCTG
ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG
CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG ACTCCGACGG CTCCTTCTTC
CTCTATAGCA AGCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC
TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG
GGTAAATGA
The following protein species were isolated:
[0183] (1) The hALK3(24-140)-hFc shown below (SEQ ID NO:25),
beginning with a glutamine (which tends to be blocked for
N-terminal sequencing by Edman degradation.
TABLE-US-00011 (SEQ ID NO: 25) QNLD SMLHGTGMKS DSDQKKSENG
VTLAPEDTLP FLKCYCSGHC PDDAINNTCI TNGHCFAIIE EDDQGETTLA SGCMKYEGSD
FQCKDSPKAQ LRRTIECCRT NLCNQYLQPT LPPTGGGTHT CPPCPAPELL GGPSVFLFPP
KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV
LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL
TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC
SVMHEALHNH YTQKSLSLSP GK*
[0184] (2) The hALK3(GA,24-140)-hFc sequence shown below (SEQ ID
NO: 26), which retains an initial glycine-alanine from the leader
sequence.
TABLE-US-00012 (SEQ ID NO: 26) GAQNLD SMLHGTGMKS DSDQKKSENG
VTLAPEDTLP FLKCYCSGHC PDDAINNTCI TNGHCFAIIE EDDQGETTLA SGCMKYEGSD
FQCKDSPKAQ LRRTIECCRT NLCNQYLQPT LPPTGGGTHT CPPCPAPELL GGPSVFLFPP
KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV
LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL
TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC
SVMHEALHNH YTQKSLSLSP GK*
D. A nucleic acid encoding hALK3(30-152)-hFc, shown below (SEQ ID
NO: 27) was expressed in CHO cells:
TABLE-US-00013 (SEQ ID NO: 27) AT GGATGCAATG AAGAGAGGGC TCTGCTGTGT
GCTGCTGCTG TGTGGAGCAG TCTTCGTTTC GCCCGGCGCC CTTCATGGCA CTGGGATGAA
ATCAGACTCC GACCAGAAAA AGTCAGAAAA TGGAGTAACC TTAGCACCAG AGGATACCTT
GCCTTTTTTA AAGTGCTATT GCTCAGGGCA CTGTCCAGAT GATGCTATTA ATAACACATG
CATAACTAAT GGACATTGCT TTGCCATCAT AGAAGAAGAT GACCAGGGAG AAACCACATT
AGCTTCAGGG TGTATGAAAT ATGAAGGATC TGATTTTCAG TGCAAAGATT CTCCAAAAGC
CCAGCTACGC CGGACAATAG AATGTTGTCG GACCAATTTA TGTAACCAGT ATTTGCAACC
CACACTGCCC CCTGTTGTCA TAGGTCCGTT TTTTGATGGC AGCATTCGAA CCGGTGGTGG
AACTCACACA TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT CAGTCTTCCT
CTTCCCCCCA AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT
GGTGGTGGAC GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT
GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA CGTACCGTGT
GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA
GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA
GCCCCGAGAA CCACAGGTGT ACACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA
GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA
GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG ACTCCGACGG
CTCCTTCTTC CTCTATAGCA AGCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT
CTTCTCATGC TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC
CCTGTCTCCG GGTAAATGA
The following protein species were isolated:
[0185] (1) The hALK3(GA,30-152)-hFc shown below (SEQ ID NO:28),
which retains an initial glycine-alanine from the leader
sequence.
TABLE-US-00014 (SEQ ID NO: 28) GA LHGTGMKSDS DQKKSENGVT LAPEDTLPFL
KCYCSGHCPD DAINNTCITN GHCFAIIEED DQGETTLASG CMKYEGSDFQ CKDSPKAQLR
RTIECCRTNL CNQYLQPTLP PVVIGPFFDG SIRTGGGTHT CPPCPAPELL GGPSVFLFPP
KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV
LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL
TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC
SVMHEALHNH YTQKSLSLSP GK*
[0186] (2) The hALK3(A,30-152)-hFc shown below (SEQ ID NO:29),
which retains an initial alanine from the leader sequence.
TABLE-US-00015 (SEQ ID NO: 29) A LHGTGMKSDS DQKKSENGVT LAPEDTLPFL
KCYCSGHCPD DAINNTCITN GHCFAIIEED DQGETTLASG CMKYEGSDFQ CKDSPKAQLR
RTIECCRTNL CNQYLQPTLP PVVIGPFFDG SIRTGGGTHT CPPCPAPELL GGPSVFLFPP
KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV
LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL
TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC
SVMHEALHNH YTQKSLSLSP GK*
[0187] (3) The hALK3(31-152)-hFc sequence shown below (SEQ ID NO:
30), in which the leader and the initial leucine are removed,
leaving an initial histidine (effectively N.DELTA.7).
TABLE-US-00016 (SEQ ID NO: 30) HGTGMKSDS DQKKSENGVT LAPEDTLPFL
KCYCSGHCPD DAINNTCITN GHCFAIIEED DQGETTLASG CMKYEGSDFQ CKDSPKAQLR
RTIECCRTNL CNQYLQPTLP PVVIGPFFDG SIRTGGGTHT CPPCPAPELL GGPSVFLFPP
KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV
LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL
TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC
SVMHEALHNH YTQKSLSLSP GK*
[0188] (4) An additional species, hALK3(30-152)-hFc, shown below
(SEQ ID NO:31) was expected but not identified by N-terminal
sequencing.
TABLE-US-00017 (SEQ ID NO: 31) LHGTGMKSDS DQKKSENGVT LAPEDTLPFL
KCYCSGHCPD DAINNTCITN GHCFAIIEED DQGETTLASG CMKYEGSDFQ CKDSPKAQLR
RTIECCRTNL CNQYLQPTLP PVVIGPFFDG SIRTGGGTHT CPPCPAPELL GGPSVFLFPP
KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV
LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL
TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC
SVMHEALHNH YTQKSLSLSP GK*
E. A nucleic acid encoding hALK3(30-146)-hFc, shown below (SEQ ID
NO: 32) was expressed in CHO cells:
TABLE-US-00018 (SEQ ID NO: 32) ATGG ATGCAATGAA GAGAGGGCTC
TGCTGTGTGC TGCTGCTGTG TGGAGCAGTC TTCGTTTCGC CCGGCGCCCT TCATGGCACT
GGGATGAAAT CAGACTCCGA CCAGAAAAAG TCAGAAAATG GAGTAACCTT AGCACCAGAG
GATACCTTGC CTTTTTTAAA GTGCTATTGC TCAGGGCACT GTCCAGATGA TGCTATTAAT
AACACATGCA TAACTAATGG ACATTGCTTT GCCATCATAG AAGAAGATGA CCAGGGAGAA
ACCACATTAG CTTCAGGGTG TATGAAATAT GAAGGATCTG ATTTTCAGTG CAAAGATTCT
CCAAAAGCCC AGCTACGCCG GACAATAGAA TGTTGTCGGA CCAATTTATG TAACCAGTAT
TTGCAACCCA CACTGCCCCC TGTTGTCATA GGTCCGTTTA CCGGTGGTGG AACTCACACA
TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT CAGTCTTCCT CTTCCCCCCA
AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC
GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT GGAGGTGCAT
AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC
CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC
AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA
CCACAGGTGT ACACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA GGTCAGCCTG
ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG
CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG ACTCCGACGG CTCCTTCTTC
CTCTATAGCA AGCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC
TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG
GGTAAATGA
The following protein species were isolated:
[0189] (1) The hALK3(GA,30-146)-hFc shown below (SEQ ID NO:33),
which retains an initial glycine-alanine from the leader
sequence.
TABLE-US-00019 (SEQ ID NO: 33) GALHGT GMKSDSDQKK SENGVTLAPE
DTLPFLKCYC SGHCPDDAIN NTCITNGHCF AIIEEDDQGE TTLASGCMKY EGSDFQCKDS
PKAQLRRTIE CCRTNLCNQY LQPTLPPVVI GPFTGGGTHT CPPCPAPELL GGPSVFLFPP
KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV
LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL
TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC
SVMHEALHNH YTQKSLSLSP GK*
[0190] (2) The hALK3(A,30-146)-hFc shown below (SEQ ID NO:34),
which retains an initial alanine from the leader sequence.
TABLE-US-00020 (SEQ ID NO: 34) ALHGT GMKSDSDQKK SENGVTLAPE
DTLPFLKCYC SGHCPDDAIN NTCITNGHCF AIIEEDDQGE TTLASGCMKY EGSDFQCKDS
PKAQLRRTIE CCRTNLCNQY LQPTLPPVVI GPFTGGGTHT CPPCPAPELL GGPSVFLFPP
KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV
LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL
TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC
SVMHEALHNH YTQKSLSLSP GK*
[0191] (3) The hALK3(31-146)-hFc sequence shown below (SEQ ID NO:
35), in which the leader and the initial leucine are removed,
leaving an initial histidine (effectively N.DELTA.7C.DELTA.6).
TABLE-US-00021 (SEQ ID NO: 35) HGT GMKSDSDQKK SENGVTLAPE DTLPFLKCYC
SGHCPDDAIN NTCITNGHCF AIIEEDDQGE TTLASGCMKY EGSDFQCKDS PKAQLRRTIE
CCRTNLCNQY LQPTLPPVVI GPFTGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR
TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN
GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS
DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH
YTQKSLSLSP GK*
[0192] (4) An additional species, hALK3(30-146)-hFc, shown below
(SEQ ID NO:36) was expected but not identified by N-terminal
sequencing.
TABLE-US-00022 (SEQ ID NO: 36) LHGT GMKSDSDQKK SENGVTLAPE
DTLPFLKCYC SGHCPDDAIN NTCITNGHCF AIIEEDDQGE TTLASGCMKY EGSDFQCKDS
PKAQLRRTIE CCRTNLCNQY LQPTLPPVVI GPFTGGGTHT CPPCPAPELL GGPSVFLFPP
KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV
LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL
TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC
SVMHEALHNH YTQKSLSLSP GK*
F. A nucleic acid encoding hALK3(30-140)-hFc, shown below (SEQ ID
NO: 37) may be expressed in CHO cells:
TABLE-US-00023 (SEQ ID NO: 37) ATGGAT GCAATGAAGA GAGGGCTCTG
CTGTGTGCTG CTGCTGTGTG GAGCAGTCTT CGTTTCGCCC GGCGCCCTTC ATGGCACTGG
GATGAAATCA GACTCCGACC AGAAAAAGTC AGAAAATGGA GTAACCTTAG CACCAGAGGA
TACCTTGCCT TTTTTAAAGT GCTATTGCTC AGGGCACTGT CCAGATGATG CTATTAATAA
CACATGCATA ACTAATGGAC ATTGCTTTGC CATCATAGAA GAAGATGACC AGGGAGAAAC
CACATTAGCT TCAGGGTGTA TGAAATATGA AGGATCTGAT TTTCAGTGCA AAGATTCTCC
AAAAGCCCAG CTACGCCGGA CAATAGAATG TTGTCGGACC AATTTATGTA ACCAGTATTT
GCAACCCACA CTGCCCCCTA CCGGTGGTGG AACTCACACA TGCCCACCGT GCCCAGCACC
TGAACTCCTG GGGGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG ACACCCTCAT
GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC GTGAGCCACG AAGACCCTGA
GGTCAAGTTC AACTGGTACG TGGACGGCGT GGAGGTGCAT AATGCCAAGA CAAAGCCGCG
GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC CTCACCGTCC TGCACCAGGA
CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT
CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT ACACCCTGCC
CCCATCCCGG GAGGAGATGA CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT
CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA
GACCACGCCT CCCGTGCTGG ACTCCGACGG CTCCTTCTTC CTCTATAGCA AGCTCACCGT
GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT
GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAATGA
The following protein species may be isolated:
[0193] (1) The hALK3(GA,30-140)-hFc shown below (SEQ ID NO:38),
which retains an initial glycine-alanine from the leader sequence.
[0194] GALHGTGMKS DSDQKKSENG VTLAPEDTLP FLKCYCSGHC
TABLE-US-00024 [0194] (SEQ ID NO: 38) PDDAINNTCI TNGHCFAIIE
EDDQGETTLA SGCMKYEGSD FQCKDSPKAQ LRRTIECCRT NLCNQYLQPT LPPTGGGTHT
CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH
NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE
PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF
LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK*
[0195] (2) The hALK3(A,30-140)-hFc shown below (SEQ ID NO:39),
which retains an initial alanine from the leader sequence.
TABLE-US-00025 (SEQ ID NO: 39) ALHGTGMKS DSDQKKSENG VTLAPEDTLP
FLKCYCSGHC PDDAINNTCI TNGHCFAIIE EDDQGETTLA SGCMKYEGSD FQCKDSPKAQ
LRRTIECCRT NLCNQYLQPT LPPTGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR
TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN
GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS
DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH
YTQKSLSLSP GK*
[0196] (3) The hALK3(31-140)-hFc sequence shown below (SEQ ID NO:
40), in which the leader and the initial leucine are removed,
leaving an initial histidine (effectively N.DELTA.7C.DELTA.12).
TABLE-US-00026 (SEQ ID NO: 40) HGTGMKS DSDQKKSENG VTLAPEDTLP
FLKCYCSGHC PDDAINNTCI TNGHCFAIIE EDDQGETTLA SGCMKYEGSD FQCKDSPKAQ
LRRTIECCRT NLCNQYLQPT LPPTGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR
TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN
GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS
DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH
YTQKSLSLSP GK*
[0197] (4) An additional species, hALK3(30-140)-hFc, shown below
(SEQ ID NO:41).
TABLE-US-00027 (SEQ ID NO: 41) LHGTGMKS DSDQKKSENG VTLAPEDTLP
FLKCYCSGHC PDDAINNTCI TNGHCFAIIE EDDQGETTLA SGCMKYEGSD FQCKDSPKAQ
LRRTIECCRT NLCNQYLQPT LPPTGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR
TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN
GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS
DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH
YTQKSLSLSP GK*
INCORPORATION BY REFERENCE
[0198] 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.
[0199] 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
481532PRTHomo sapiens 1Met Pro Gln Leu Tyr Ile Tyr Ile Arg Leu Leu
Gly Ala Tyr Leu Phe 1 5 10 15 Ile Ile Ser Arg Val Gln Gly Gln Asn
Leu Asp Ser Met Leu His Gly 20 25 30 Thr Gly Met Lys Ser Asp Ser
Asp Gln Lys Lys Ser Glu Asn Gly Val 35 40 45 Thr Leu Ala Pro Glu
Asp Thr Leu Pro Phe Leu Lys Cys Tyr Cys Ser 50 55 60 Gly His Cys
Pro Asp Asp Ala Ile Asn Asn Thr Cys Ile Thr Asn Gly 65 70 75 80 His
Cys Phe Ala Ile Ile Glu Glu Asp Asp Gln Gly Glu Thr Thr Leu 85 90
95 Ala Ser Gly Cys Met Lys Tyr Glu Gly Ser Asp Phe Gln Cys Lys Asp
100 105 110 Ser Pro Lys Ala Gln Leu Arg Arg Thr Ile Glu Cys Cys Arg
Thr Asn 115 120 125 Leu Cys Asn Gln Tyr Leu Gln Pro Thr Leu Pro Pro
Val Val Ile Gly 130 135 140 Pro Phe Phe Asp Gly Ser Ile Arg Trp Leu
Val Leu Leu Ile Ser Met 145 150 155 160 Ala Val Cys Ile Ile Ala Met
Ile Ile Phe Ser Ser Cys Phe Cys Tyr 165 170 175 Lys His Tyr Cys Lys
Ser Ile Ser Ser Arg Arg Arg Tyr Asn Arg Asp 180 185 190 Leu Glu Gln
Asp Glu Ala Phe Ile Pro Val Gly Glu Ser Leu Lys Asp 195 200 205 Leu
Ile Asp Gln Ser Gln Ser Ser Gly Ser Gly Ser Gly Leu Pro Leu 210 215
220 Leu Val Gln Arg Thr Ile Ala Lys Gln Ile Gln Met Val Arg Gln Val
225 230 235 240 Gly Lys Gly Arg Tyr Gly Glu Val Trp Met Gly Lys Trp
Arg Gly Glu 245 250 255 Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu
Glu Ala Ser Trp Phe 260 265 270 Arg Glu Thr Glu Ile Tyr Gln Thr Val
Leu Met Arg His Glu Asn Ile 275 280 285 Leu Gly Phe Ile Ala Ala Asp
Ile Lys Gly Thr Gly Ser Trp Thr Gln 290 295 300 Leu Tyr Leu Ile Thr
Asp Tyr His Glu Asn Gly Ser Leu Tyr Asp Phe 305 310 315 320 Leu Lys
Cys Ala Thr Leu Asp Thr Arg Ala Leu Leu Lys Leu Ala Tyr 325 330 335
Ser Ala Ala Cys Gly Leu Cys His Leu His Thr Glu Ile Tyr Gly Thr 340
345 350 Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser Lys Asn
Ile 355 360 365 Leu Ile Lys Lys Asn Gly Ser Cys Cys Ile Ala Asp Leu
Gly Leu Ala 370 375 380 Val Lys Phe Asn Ser Asp Thr Asn Glu Val Asp
Val Pro Leu Asn Thr 385 390 395 400 Arg Val Gly Thr Lys Arg Tyr Met
Ala Pro Glu Val Leu Asp Glu Ser 405 410 415 Leu Asn Lys Asn His Phe
Gln Pro Tyr Ile Met Ala Asp Ile Tyr Ser 420 425 430 Phe Gly Leu Ile
Ile Trp Glu Met Ala Arg Arg Cys Ile Thr Gly Gly 435 440 445 Ile Val
Glu Glu Tyr Gln Leu Pro Tyr Tyr Asn Met Val Pro Ser Asp 450 455 460
Pro Ser Tyr Glu Asp Met Arg Glu Val Val Cys Val Lys Arg Leu Arg 465
470 475 480 Pro Ile Val Ser Asn Arg Trp Asn Ser Asp Glu Cys Leu Arg
Ala Val 485 490 495 Leu Lys Leu Met Ser Glu Cys Trp Ala His Asn Pro
Ala Ser Arg Leu 500 505 510 Thr Ala Leu Arg Ile Lys Lys Thr Leu Ala
Lys Met Val Glu Ser Gln 515 520 525 Asp Val Lys Ile 530
21596DNAHomo sapiens 2atgcctcagc tatacattta catcagatta ttgggagcct
atttgttcat catttctcgt 60gttcaaggac agaatctgga tagtatgctt catggcactg
ggatgaaatc agactccgac 120cagaaaaagt cagaaaatgg agtaacctta
gcaccagagg ataccttgcc ttttttaaag 180tgctattgct cagggcactg
tccagatgat gctattaata acacatgcat aactaatgga 240cattgctttg
ccatcataga agaagatgac cagggagaaa ccacattagc ttcagggtgt
300atgaaatatg aaggatctga ttttcagtgc aaagattctc caaaagccca
gctacgccgg 360acaatagaat gttgtcggac caatttatgt aaccagtatt
tgcaacccac actgccccct 420gttgtcatag gtccgttttt tgatggcagc
attcgatggc tggttttgct catttctatg 480gctgtctgca taattgctat
gatcatcttc tccagctgct tttgttacaa acattattgc 540aagagcatct
caagcagacg tcgttacaat cgtgatttgg aacaggatga agcatttatt
600ccagttggag aatcactaaa agaccttatt gaccagtcac aaagttctgg
tagtgggtct 660ggactacctt tattggttca gcgaactatt gccaaacaga
ttcagatggt ccggcaagtt 720ggtaaaggcc gatatggaga agtatggatg
ggcaaatggc gtggcgaaaa agtggcggtg 780aaagtattct ttaccactga
agaagccagc tggtttcgag aaacagaaat ctaccaaact 840gtgctaatgc
gccatgaaaa catacttggt ttcatagcgg cagacattaa aggtacaggt
900tcctggactc agctctattt gattactgat taccatgaaa atggatctct
ctatgacttc 960ctgaaatgtg ctacactgga caccagagcc ctgcttaaat
tggcttattc agctgcctgt 1020ggtctgtgcc acctgcacac agaaatttat
ggcacccaag gaaagcccgc aattgctcat 1080cgagacctaa agagcaaaaa
catcctcatc aagaaaaatg ggagttgctg cattgctgac 1140ctgggccttg
ctgttaaatt caacagtgac acaaatgaag ttgatgtgcc cttgaatacc
1200agggtgggca ccaaacgcta catggctccc gaagtgctgg acgaaagcct
gaacaaaaac 1260cacttccagc cctacatcat ggctgacatc tacagcttcg
gcctaatcat ttgggagatg 1320gctcgtcgtt gtatcacagg agggatcgtg
gaagaatacc aattgccata ttacaacatg 1380gtaccgagtg atccgtcata
cgaagatatg cgtgaggttg tgtgtgtcaa acgtttgcgg 1440ccaattgtgt
ctaatcggtg gaacagtgat gaatgtctac gagcagtttt gaagctaatg
1500tcagaatgct gggcccacaa tccagcctcc agactcacag cattgagaat
taagaagacg 1560cttgccaaga tggttgaatc ccaagatgta aaaatc
15963129PRTHomo sapiens 3Gln Asn Leu Asp Ser Met Leu His Gly Thr
Gly Met Lys Ser Asp Ser 1 5 10 15 Asp Gln Lys Lys Ser Glu Asn Gly
Val Thr Leu Ala Pro Glu Asp Thr 20 25 30 Leu Pro Phe Leu Lys Cys
Tyr Cys Ser Gly His Cys Pro Asp Asp Ala 35 40 45 Ile Asn Asn Thr
Cys Ile Thr Asn Gly His Cys Phe Ala Ile Ile Glu 50 55 60 Glu Asp
Asp Gln Gly Glu Thr Thr Leu Ala Ser Gly Cys Met Lys Tyr 65 70 75 80
Glu Gly Ser Asp Phe Gln Cys Lys Asp Ser Pro Lys Ala Gln Leu Arg 85
90 95 Arg Thr Ile Glu Cys Cys Arg Thr Asn Leu Cys Asn Gln Tyr Leu
Gln 100 105 110 Pro Thr Leu Pro Pro Val Val Ile Gly Pro Phe Phe Asp
Gly Ser Ile 115 120 125 Arg 4387DNAHomo sapiens 4cagaatctgg
atagtatgct tcatggcact gggatgaaat cagactccga ccagaaaaag 60tcagaaaatg
gagtaacctt agcaccagag gataccttgc cttttttaaa gtgctattgc
120tcagggcact gtccagatga tgctattaat aacacatgca taactaatgg
acattgcttt 180gccatcatag aagaagatga ccagggagaa accacattag
cttcagggtg tatgaaatat 240gaaggatctg attttcagtg caaagattct
ccaaaagccc agctacgccg gacaatagaa 300tgttgtcgga ccaatttatg
taaccagtat ttgcaaccca cactgccccc tgttgtcata 360ggtccgtttt
ttgatggcag cattcga 3875225PRTHomo sapiens 5Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 1 5 10 15 Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 20 25 30 Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 35 40 45
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 50
55 60 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val 65 70 75 80 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu 85 90 95 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys 100 105 110 Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 115 120 125 Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr 130 135 140 Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 145 150 155 160 Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 165 170 175
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 180
185 190 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu 195 200 205 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly 210 215 220 Lys 225 6678DNAHomo sapiens 6actcacacat
gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 60ttccccccaa
aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg
120gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt
ggacggcgtg 180gaggtgcata atgccaagac aaagccgcgg gaggagcagt
acaacagcac gtaccgtgtg 240gtcagcgtcc tcaccgtcct gcaccaggac
tggctgaatg gcaaggagta caagtgcaag 300gtctccaaca aagccctccc
agcccccatc gagaaaacca tctccaaagc caaagggcag 360ccccgagaac
cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag
420gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt
ggagtgggag 480agcaatgggc agccggagaa caactacaag accacgcctc
ccgtgctgga ctccgacggc 540tccttcttcc tctatagcaa gctcaccgtg
gacaagagca ggtggcagca ggggaacgtc 600ttctcatgct ccgtgatgca
tgaggctctg cacaaccact acacgcagaa gagcctctcc 660ctgtccccgg gtaaatga
6787358PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Gln Asn Leu Asp Ser Met Leu His Gly Thr Gly
Met Lys Ser Asp Ser 1 5 10 15 Asp Gln Lys Lys Ser Glu Asn Gly Val
Thr Leu Ala Pro Glu Asp Thr 20 25 30 Leu Pro Phe Leu Lys Cys Tyr
Cys Ser Gly His Cys Pro Asp Asp Ala 35 40 45 Ile Asn Asn Thr Cys
Ile Thr Asn Gly His Cys Phe Ala Ile Ile Glu 50 55 60 Glu Asp Asp
Gln Gly Glu Thr Thr Leu Ala Ser Gly Cys Met Lys Tyr 65 70 75 80 Glu
Gly Ser Asp Phe Gln Cys Lys Asp Ser Pro Lys Ala Gln Leu Arg 85 90
95 Arg Thr Ile Glu Cys Cys Arg Thr Asn Leu Cys Asn Gln Tyr Leu Gln
100 105 110 Pro Thr Leu Pro Pro Val Val Ile Gly Pro Phe Phe Asp Gly
Ser Ile 115 120 125 Arg Thr Gly Gly Gly Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu 130 135 140 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp 145 150 155 160 Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 165 170 175 Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 180 185 190 Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 195 200 205 Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 210 215
220 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu 245 250 255 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn 260 265 270 Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile 275 280 285 Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr 290 295 300 Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 305 310 315 320 Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 340
345 350 Ser Leu Ser Pro Gly Lys 355 823PRTUnknownDescription of
Unknown Native leader sequence 8Met Pro Gln Leu Tyr Ile Tyr Ile Arg
Leu Leu Gly Ala Tyr Leu Phe 1 5 10 15 Ile Ile Ser Arg Val Gln Gly
20 922PRTUnknownDescription of Unknown Tissue plasminogen activator
leader sequence 9Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu
Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro 20 1021PRTApis
sp. 10Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr
Ile 1 5 10 15 Ser Tyr Ile Tyr Ala 20 11382PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
11Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1
5 10 15 Ala Val Phe Val Ser Pro Gly Ala Gln Asn Leu Asp Ser Met Leu
His 20 25 30 Gly Thr Gly Met Lys Ser Asp Ser Asp Gln Lys Lys Ser
Glu Asn Gly 35 40 45 Val Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe
Leu Lys Cys Tyr Cys 50 55 60 Ser Gly His Cys Pro Asp Asp Ala Ile
Asn Asn Thr Cys Ile Thr Asn 65 70 75 80 Gly His Cys Phe Ala Ile Ile
Glu Glu Asp Asp Gln Gly Glu Thr Thr 85 90 95 Leu Ala Ser Gly Cys
Met Lys Tyr Glu Gly Ser Asp Phe Gln Cys Lys 100 105 110 Asp Ser Pro
Lys Ala Gln Leu Arg Arg Thr Ile Glu Cys Cys Arg Thr 115 120 125 Asn
Leu Cys Asn Gln Tyr Leu Gln Pro Thr Leu Pro Pro Val Val Ile 130 135
140 Gly Pro Phe Phe Asp Gly Ser Ile Arg Thr Gly Gly Gly Thr His Thr
145 150 155 160 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe 165 170 175 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro 180 185 190 Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val 195 200 205 Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr 210 215 220 Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 225 230 235 240 Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 245 250 255
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 260
265 270 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro 275 280 285 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val 290 295 300 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly 305 310 315 320 Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp 325 330 335 Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 340 345 350 Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 355 360 365 Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380
121146DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 12atggatgcaa tgaagagagg gctctgctgt
gtgctgctgc tgtgtggagc agtcttcgtt 60tcgcccggcg cccagaatct ggatagtatg
cttcatggca ctgggatgaa atcagactcc 120gaccagaaaa agtcagaaaa
tggagtaacc ttagcaccag aggatacctt gcctttttta 180aagtgctatt
gctcagggca ctgtccagat gatgctatta ataacacatg cataactaat
240ggacattgct ttgccatcat agaagaagat gaccagggag aaaccacatt
agcttcaggg 300tgtatgaaat atgaaggatc tgattttcag tgcaaagatt
ctccaaaagc ccagctacgc 360cggacaatag aatgttgtcg gaccaattta
tgtaaccagt atttgcaacc cacactgccc 420cctgttgtca taggtccgtt
ttttgatggc agcattcgaa ccggtggggg tactcacaca 480tgcccaccgt
gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca
540aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt
ggtggtggac 600gtgagccacg aagaccctga ggtcaagttc aactggtacg
tggacggcgt ggaggtgcat 660aatgccaaga caaagccgcg ggaggagcag
tacaacagca cgtaccgtgt ggtcagcgtc 720ctcaccgtcc tgcaccagga
ctggctgaat ggcaaggagt
acaagtgcaa ggtctccaac 780aaagccctcc cagcccccat cgagaaaacc
atctccaaag ccaaagggca gccccgagaa 840ccacaggtgt acaccctgcc
cccatcccgg gaggagatga ccaagaacca ggtcagcctg 900acctgcctgg
tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg
960cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg
ctccttcttc 1020ctctatagca agctcaccgt ggacaagagc aggtggcagc
aggggaacgt cttctcatgc 1080tccgtgatgc atgaggctct gcacaaccac
tacacgcaga agagcctctc cctgtccccg 1140ggtaaa 1146131146DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
13tttacccggg gacagggaga ggctcttctg cgtgtagtgg ttgtgcagag cctcatgcat
60cacggagcat gagaagacgt tcccctgctg ccacctgctc ttgtccacgg tgagcttgct
120atagaggaag aaggagccgt cggagtccag cacgggaggc gtggtcttgt
agttgttctc 180cggctgccca ttgctctccc actccacggc gatgtcgctg
ggatagaagc ctttgaccag 240gcaggtcagg ctgacctggt tcttggtcat
ctcctcccgg gatgggggca gggtgtacac 300ctgtggttct cggggctgcc
ctttggcttt ggagatggtt ttctcgatgg gggctgggag 360ggctttgttg
gagaccttgc acttgtactc cttgccattc agccagtcct ggtgcaggac
420ggtgaggacg ctgaccacac ggtacgtgct gttgtactgc tcctcccgcg
gctttgtctt 480ggcattatgc acctccacgc cgtccacgta ccagttgaac
ttgacctcag ggtcttcgtg 540gctcacgtcc accaccacgc atgtgacctc
aggggtccgg gagatcatga gggtgtcctt 600gggttttggg gggaagagga
agactgacgg tccccccagg agttcaggtg ctgggcacgg 660tgggcatgtg
tgagtacccc caccggttcg aatgctgcca tcaaaaaacg gacctatgac
720aacagggggc agtgtgggtt gcaaatactg gttacataaa ttggtccgac
aacattctat 780tgtccggcgt agctgggctt ttggagaatc tttgcactga
aaatcagatc cttcatattt 840catacaccct gaagctaatg tggtttctcc
ctggtcatct tcttctatga tggcaaagca 900atgtccatta gttatgcatg
tgttattaat agcatcatct ggacagtgcc ctgagcaata 960gcactttaaa
aaaggcaagg tatcctctgg tgctaaggtt actccatttt ctgacttttt
1020ctggtcggag tctgatttca tcccagtgcc atgaagcata ctatccagat
tctgggcgcc 1080gggcgaaacg aagactgctc cacacagcag cagcacacag
cagagccctc tcttcattgc 1140atccat 114614395PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
14Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1
5 10 15 Ala Val Phe Val Ser Pro Gly Ala Gln Asn Leu Asp Ser Met Leu
His 20 25 30 Gly Thr Gly Met Lys Ser Asp Ser Asp Gln Lys Lys Ser
Glu Asn Gly 35 40 45 Val Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe
Leu Lys Cys Tyr Cys 50 55 60 Ser Gly His Cys Pro Asp Asp Ala Ile
Asn Asn Thr Cys Ile Thr Asn 65 70 75 80 Gly His Cys Phe Ala Ile Ile
Glu Glu Asp Asp Gln Gly Glu Thr Thr 85 90 95 Leu Ala Ser Gly Cys
Met Lys Tyr Glu Gly Ser Asp Phe Gln Cys Lys 100 105 110 Asp Ser Pro
Lys Ala Gln Leu Arg Arg Thr Ile Glu Cys Cys Arg Thr 115 120 125 Asn
Leu Cys Asn Gln Tyr Leu Gln Pro Thr Leu Pro Pro Val Val Ile 130 135
140 Gly Pro Phe Phe Asp Gly Ser Ile Arg Thr Gly Gly Gly Glu Pro Arg
145 150 155 160 Val Pro Ile Thr Gln Asn Pro Cys Pro Pro Leu Lys Glu
Cys Pro Pro 165 170 175 Cys Ala Ala Pro Asp Leu Leu Gly Gly Pro Ser
Val Phe Ile Phe Pro 180 185 190 Pro Lys Ile Lys Asp Val Leu Met Ile
Ser Leu Ser Pro Met Val Thr 195 200 205 Cys Val Val Val Asp Val Ser
Glu Asp Asp Pro Asp Val Gln Ile Ser 210 215 220 Trp Phe Val Asn Asn
Val Glu Val His Thr Ala Gln Thr Gln Thr His 225 230 235 240 Arg Glu
Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile 245 250 255
Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn 260
265 270 Asn Arg Ala Leu Pro Ser Pro Ile Glu Lys Thr Ile Ser Lys Pro
Arg 275 280 285 Gly Pro Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro
Pro Ala Glu 290 295 300 Glu Met Thr Lys Lys Glu Phe Ser Leu Thr Cys
Met Ile Thr Gly Phe 305 310 315 320 Leu Pro Ala Glu Ile Ala Val Asp
Trp Thr Ser Asn Gly Arg Thr Glu 325 330 335 Gln Asn Tyr Lys Asn Thr
Ala Thr Val Leu Asp Ser Asp Gly Ser Tyr 340 345 350 Phe Met Tyr Ser
Lys Leu Arg Val Gln Lys Ser Thr Trp Glu Arg Gly 355 360 365 Ser Leu
Phe Ala Cys Ser Val Val His Glu Gly Leu His Asn His Leu 370 375 380
Thr Thr Lys Thr Ile Ser Arg Ser Leu Gly Lys 385 390 395
151185DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 15atggatgcaa tgaagagagg gctctgctgt
gtgctgctgc tgtgtggagc agtcttcgtt 60tcgcccggcg cccagaatct ggatagtatg
cttcatggca ctgggatgaa atcagactcc 120gaccagaaaa agtcagaaaa
tggagtaacc ttagcaccag aggatacctt gcctttttta 180aagtgctatt
gctcagggca ctgtccagat gatgctatta ataacacatg cataactaat
240ggacattgct ttgccatcat agaagaagat gaccagggag aaaccacatt
agcttcaggg 300tgtatgaaat atgaaggatc tgattttcag tgcaaagatt
ctccaaaagc ccagctacgc 360cggacaatag aatgttgtcg gaccaattta
tgtaaccagt atttgcaacc cacactgccc 420cctgttgtca taggtccgtt
ttttgatggc agcattcgaa ccggtggggg tgagcccaga 480gtgcccataa
cacagaaccc ctgtcctcca ctcaaagagt gtcccccatg cgcagctcca
540gacctcttgg gtggaccatc cgtcttcatc ttccctccaa agatcaagga
tgtactcatg 600atctccctga gccccatggt cacatgtgtg gtggtggatg
tgagcgagga tgacccagac 660gtccagatca gctggtttgt gaacaacgtg
gaagtacaca cagctcagac acaaacccat 720agagaggatt acaacagtac
tctccgggtg gtcagtgccc tccccatcca gcaccaggac 780tggatgagtg
gcaaggagtt caaatgcaag gtcaacaaca gagccctccc atcccccatc
840gagaaaacca tctcaaaacc cagagggcca gtaagagctc cacaggtata
tgtcttgcct 900ccaccagcag aagagatgac taagaaagag ttcagtctga
cctgcatgat cacaggcttc 960ttacctgccg aaattgctgt ggactggacc
agcaatgggc gtacagagca aaactacaag 1020aacaccgcaa cagtcctgga
ctctgatggt tcttacttca tgtacagcaa gctcagagta 1080caaaagagca
cttgggaaag aggaagtctt ttcgcctgct cagtggtcca cgagggtctg
1140cacaatcacc ttacgactaa gaccatctcc cggtctctgg gtaaa
1185161185DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 16tttacccaga gaccgggaga tggtcttagt
cgtaaggtga ttgtgcagac cctcgtggac 60cactgagcag gcgaaaagac ttcctctttc
ccaagtgctc ttttgtactc tgagcttgct 120gtacatgaag taagaaccat
cagagtccag gactgttgcg gtgttcttgt agttttgctc 180tgtacgccca
ttgctggtcc agtccacagc aatttcggca ggtaagaagc ctgtgatcat
240gcaggtcaga ctgaactctt tcttagtcat ctcttctgct ggtggaggca
agacatatac 300ctgtggagct cttactggcc ctctgggttt tgagatggtt
ttctcgatgg gggatgggag 360ggctctgttg ttgaccttgc atttgaactc
cttgccactc atccagtcct ggtgctggat 420ggggagggca ctgaccaccc
ggagagtact gttgtaatcc tctctatggg tttgtgtctg 480agctgtgtgt
acttccacgt tgttcacaaa ccagctgatc tggacgtctg ggtcatcctc
540gctcacatcc accaccacac atgtgaccat ggggctcagg gagatcatga
gtacatcctt 600gatctttgga gggaagatga agacggatgg tccacccaag
aggtctggag ctgcgcatgg 660gggacactct ttgagtggag gacaggggtt
ctgtgttatg ggcactctgg gctcaccccc 720accggttcga atgctgccat
caaaaaacgg acctatgaca acagggggca gtgtgggttg 780caaatactgg
ttacataaat tggtccgaca acattctatt gtccggcgta gctgggcttt
840tggagaatct ttgcactgaa aatcagatcc ttcatatttc atacaccctg
aagctaatgt 900ggtttctccc tggtcatctt cttctatgat ggcaaagcaa
tgtccattag ttatgcatgt 960gttattaata gcatcatctg gacagtgccc
tgagcaatag cactttaaaa aaggcaaggt 1020atcctctggt gctaaggtta
ctccattttc tgactttttc tggtcggagt ctgatttcat 1080cccagtgcca
tgaagcatac tatccagatt ctgggcgccg ggcgaaacga agactgctcc
1140acacagcagc agcacacagc agagccctct cttcattgca tccat
11851715PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Gly Ala Gln Asn Leu Asp Ser Met Leu His Gly Thr
Gly Met Lys 1 5 10 15 18395PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 18Met Asp Ala Met Lys Arg
Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val
Ser Pro Gly Ala Gln Asn Leu Asp Ser Met Leu His 20 25 30 Gly Thr
Gly Met Lys Ser Asp Leu Asp Gln Lys Lys Pro Glu Asn Gly 35 40 45
Val Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys Cys Tyr Cys 50
55 60 Ser Gly His Cys Pro Asp Asp Ala Ile Asn Asn Thr Cys Ile Thr
Asn 65 70 75 80 Gly His Cys Phe Ala Ile Ile Glu Glu Asp Asp Gln Gly
Glu Thr Thr 85 90 95 Leu Thr Ser Gly Cys Met Lys Tyr Glu Gly Ser
Asp Phe Gln Cys Lys 100 105 110 Asp Ser Pro Lys Ala Gln Leu Arg Arg
Thr Ile Glu Cys Cys Arg Thr 115 120 125 Asn Leu Cys Asn Gln Tyr Leu
Gln Pro Thr Leu Pro Pro Val Val Ile 130 135 140 Gly Pro Phe Phe Asp
Gly Ser Ile Arg Thr Gly Gly Gly Glu Pro Arg 145 150 155 160 Val Pro
Ile Thr Gln Asn Pro Cys Pro Pro Leu Lys Glu Cys Pro Pro 165 170 175
Cys Ala Ala Pro Asp Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro 180
185 190 Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Met Val
Thr 195 200 205 Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val
Gln Ile Ser 210 215 220 Trp Phe Val Asn Asn Val Glu Val His Thr Ala
Gln Thr Gln Thr His 225 230 235 240 Arg Glu Asp Tyr Asn Ser Thr Leu
Arg Val Val Ser Ala Leu Pro Ile 245 250 255 Gln His Gln Asp Trp Met
Ser Gly Lys Glu Phe Lys Cys Lys Val Asn 260 265 270 Asn Arg Ala Leu
Pro Ser Pro Ile Glu Lys Thr Ile Ser Lys Pro Arg 275 280 285 Gly Pro
Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Ala Glu 290 295 300
Glu Met Thr Lys Lys Glu Phe Ser Leu Thr Cys Met Ile Thr Gly Phe 305
310 315 320 Leu Pro Ala Glu Ile Ala Val Asp Trp Thr Ser Asn Gly Arg
Thr Glu 325 330 335 Gln Asn Tyr Lys Asn Thr Ala Thr Val Leu Asp Ser
Asp Gly Ser Tyr 340 345 350 Phe Met Tyr Ser Lys Leu Arg Val Gln Lys
Ser Thr Trp Glu Arg Gly 355 360 365 Ser Leu Phe Ala Cys Ser Val Val
His Glu Gly Leu His Asn His Leu 370 375 380 Thr Thr Lys Thr Ile Ser
Arg Ser Leu Gly Lys 385 390 395 191149DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
19atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt
60tcgcccggcg cccagaatct ggatagtatg cttcatggca ctgggatgaa atcagactcc
120gaccagaaaa agtcagaaaa tggagtaacc ttagcaccag aggatacctt
gcctttttta 180aagtgctatt gctcagggca ctgtccagat gatgctatta
ataacacatg cataactaat 240ggacattgct ttgccatcat agaagaagat
gaccagggag aaaccacatt agcttcaggg 300tgtatgaaat atgaaggatc
tgattttcag tgcaaagatt ctccaaaagc ccagctacgc 360cggacaatag
aatgttgtcg gaccaattta tgtaaccagt atttgcaacc cacactgccc
420cctgttgtca taggtccgtt ttttgatggc agcattcgaa ccggtggtgg
aactcacaca 480tgcccaccgt gcccagcacc tgaactcctg gggggaccgt
cagtcttcct cttcccccca 540aaacccaagg acaccctcat gatctcccgg
acccctgagg tcacatgcgt ggtggtggac 600gtgagccacg aagaccctga
ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 660aatgccaaga
caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc
720ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa
ggtctccaac 780aaagccctcc cagcccccat cgagaaaacc atctccaaag
ccaaagggca gccccgagaa 840ccacaggtgt acaccctgcc cccatcccgg
gaggagatga ccaagaacca ggtcagcctg 900acctgcctgg tcaaaggctt
ctatcccagc gacatcgccg tggagtggga gagcaatggg 960cagccggaga
acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc
1020ctctatagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt
cttctcatgc 1080tccgtgatgc atgaggctct gcacaaccac tacacgcaga
agagcctctc cctgtctccg 1140ggtaaatga 114920360PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
20Gly Ala Gln Asn Leu Asp Ser Met Leu His Gly Thr Gly Met Lys Ser 1
5 10 15 Asp Ser Asp Gln Lys Lys Ser Glu Asn Gly Val Thr Leu Ala Pro
Glu 20 25 30 Asp Thr Leu Pro Phe Leu Lys Cys Tyr Cys Ser Gly His
Cys Pro Asp 35 40 45 Asp Ala Ile Asn Asn Thr Cys Ile Thr Asn Gly
His Cys Phe Ala Ile 50 55 60 Ile Glu Glu Asp Asp Gln Gly Glu Thr
Thr Leu Ala Ser Gly Cys Met 65 70 75 80 Lys Tyr Glu Gly Ser Asp Phe
Gln Cys Lys Asp Ser Pro Lys Ala Gln 85 90 95 Leu Arg Arg Thr Ile
Glu Cys Cys Arg Thr Asn Leu Cys Asn Gln Tyr 100 105 110 Leu Gln Pro
Thr Leu Pro Pro Val Val Ile Gly Pro Phe Phe Asp Gly 115 120 125 Ser
Ile Arg Thr Gly Gly Gly Thr His Thr Cys Pro Pro Cys Pro Ala 130 135
140 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
145 150 155 160 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val 165 170 175 Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val 180 185 190 Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln 195 200 205 Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln 210 215 220 Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 225 230 235 240 Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 245 250 255
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 260
265 270 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser 275 280 285 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr 290 295 300 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr 305 310 315 320 Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe 325 330 335 Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys 340 345 350 Ser Leu Ser Leu
Ser Pro Gly Lys 355 360 211113DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 21atggatgcaa
tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60tcgcccggcg
cccagaatct ggatagtatg cttcatggca ctgggatgaa atcagactcc
120gaccagaaaa agtcagaaaa tggagtaacc ttagcaccag aggatacctt
gcctttttta 180aagtgctatt gctcagggca ctgtccagat gatgctatta
ataacacatg cataactaat 240ggacattgct ttgccatcat agaagaagat
gaccagggag aaaccacatt agcttcaggg 300tgtatgaaat atgaaggatc
tgattttcag tgcaaagatt ctccaaaagc ccagctacgc 360cggacaatag
aatgttgtcg gaccaattta tgtaaccagt atttgcaacc cacactgccc
420cctaccggtg gtggaactca cacatgccca ccgtgcccag cacctgaact
cctgggggga 480ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc
tcatgatctc ccggacccct 540gaggtcacat gcgtggtggt ggacgtgagc
cacgaagacc ctgaggtcaa gttcaactgg 600tacgtggacg gcgtggaggt
gcataatgcc aagacaaagc cgcgggagga gcagtacaac 660agcacgtacc
gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag
720gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa
aaccatctcc 780aaagccaaag ggcagccccg agaaccacag gtgtacaccc
tgcccccatc ccgggaggag 840atgaccaaga accaggtcag cctgacctgc
ctggtcaaag gcttctatcc cagcgacatc 900gccgtggagt gggagagcaa
tgggcagccg gagaacaact acaagaccac gcctcccgtg 960ctggactccg
acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg
1020cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa
ccactacacg 1080cagaagagcc tctccctgtc tccgggtaaa tga
111322346PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 22Gln Asn Leu Asp Ser Met Leu His Gly Thr Gly
Met Lys Ser Asp Ser 1 5 10 15 Asp Gln Lys Lys Ser Glu Asn Gly Val
Thr Leu Ala Pro Glu Asp Thr 20 25 30 Leu Pro Phe Leu Lys Cys Tyr
Cys Ser Gly His Cys Pro Asp Asp
Ala 35 40 45 Ile Asn Asn Thr Cys Ile Thr Asn Gly His Cys Phe Ala
Ile Ile Glu 50 55 60 Glu Asp Asp Gln Gly Glu Thr Thr Leu Ala Ser
Gly Cys Met Lys Tyr 65 70 75 80 Glu Gly Ser Asp Phe Gln Cys Lys Asp
Ser Pro Lys Ala Gln Leu Arg 85 90 95 Arg Thr Ile Glu Cys Cys Arg
Thr Asn Leu Cys Asn Gln Tyr Leu Gln 100 105 110 Pro Thr Leu Pro Pro
Thr Gly Gly Gly Thr His Thr Cys Pro Pro Cys 115 120 125 Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 130 135 140 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 145 150
155 160 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp 165 170 175 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 180 185 190 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 195 200 205 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 210 215 220 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 225 230 235 240 Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 245 250 255 Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 260 265 270
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 275
280 285 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 290 295 300 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 305 310 315 320 Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr 325 330 335 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 340 345 23348PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 23Gly Ala Gln Asn Leu Asp
Ser Met Leu His Gly Thr Gly Met Lys Ser 1 5 10 15 Asp Ser Asp Gln
Lys Lys Ser Glu Asn Gly Val Thr Leu Ala Pro Glu 20 25 30 Asp Thr
Leu Pro Phe Leu Lys Cys Tyr Cys Ser Gly His Cys Pro Asp 35 40 45
Asp Ala Ile Asn Asn Thr Cys Ile Thr Asn Gly His Cys Phe Ala Ile 50
55 60 Ile Glu Glu Asp Asp Gln Gly Glu Thr Thr Leu Ala Ser Gly Cys
Met 65 70 75 80 Lys Tyr Glu Gly Ser Asp Phe Gln Cys Lys Asp Ser Pro
Lys Ala Gln 85 90 95 Leu Arg Arg Thr Ile Glu Cys Cys Arg Thr Asn
Leu Cys Asn Gln Tyr 100 105 110 Leu Gln Pro Thr Leu Pro Pro Thr Gly
Gly Gly Thr His Thr Cys Pro 115 120 125 Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe 130 135 140 Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 145 150 155 160 Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 165 170 175
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 180
185 190 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr 195 200 205 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val 210 215 220 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala 225 230 235 240 Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg 245 250 255 Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly 260 265 270 Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 275 280 285 Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 290 295 300
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 305
310 315 320 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His 325 330 335 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 241131DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 24atggatgcaa tgaagagagg
gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60tcgcccggcg cccttcatgg
cactgggatg aaatcagact ccgaccagaa aaagtcagaa 120aatggagtaa
ccttagcacc agaggatacc ttgccttttt taaagtgcta ttgctcaggg
180cactgtccag atgatgctat taataacaca tgcataacta atggacattg
ctttgccatc 240atagaagaag atgaccaggg agaaaccaca ttagcttcag
ggtgtatgaa atatgaagga 300tctgattttc agtgcaaaga ttctccaaaa
gcccagctac gccggacaat agaatgttgt 360cggaccaatt tatgtaacca
gtatttgcaa cccacactgc cccctgttgt cataggtccg 420ttttttgatg
gcagcattcg aaccggtggt ggaactcaca catgcccacc gtgcccagca
480cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa
ggacaccctc 540atgatctccc ggacccctga ggtcacatgc gtggtggtgg
acgtgagcca cgaagaccct 600gaggtcaagt tcaactggta cgtggacggc
gtggaggtgc ataatgccaa gacaaagccg 660cgggaggagc agtacaacag
cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 720gactggctga
atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc
780atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt
gtacaccctg 840cccccatccc gggaggagat gaccaagaac caggtcagcc
tgacctgcct ggtcaaaggc 900ttctatccca gcgacatcgc cgtggagtgg
gagagcaatg ggcagccgga gaacaactac 960aagaccacgc ctcccgtgct
ggactccgac ggctccttct tcctctatag caagctcacc 1020gtggacaaga
gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct
1080ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaatg a
113125346PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 25Gln Asn Leu Asp Ser Met Leu His Gly Thr Gly
Met Lys Ser Asp Ser 1 5 10 15 Asp Gln Lys Lys Ser Glu Asn Gly Val
Thr Leu Ala Pro Glu Asp Thr 20 25 30 Leu Pro Phe Leu Lys Cys Tyr
Cys Ser Gly His Cys Pro Asp Asp Ala 35 40 45 Ile Asn Asn Thr Cys
Ile Thr Asn Gly His Cys Phe Ala Ile Ile Glu 50 55 60 Glu Asp Asp
Gln Gly Glu Thr Thr Leu Ala Ser Gly Cys Met Lys Tyr 65 70 75 80 Glu
Gly Ser Asp Phe Gln Cys Lys Asp Ser Pro Lys Ala Gln Leu Arg 85 90
95 Arg Thr Ile Glu Cys Cys Arg Thr Asn Leu Cys Asn Gln Tyr Leu Gln
100 105 110 Pro Thr Leu Pro Pro Thr Gly Gly Gly Thr His Thr Cys Pro
Pro Cys 115 120 125 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 130 135 140 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys 145 150 155 160 Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp 165 170 175 Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 180 185 190 Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 195 200 205 His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 210 215
220 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
225 230 235 240 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu 245 250 255 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 260 265 270 Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 275 280 285 Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 290 295 300 Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 305 310 315 320 Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 325 330 335
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 26348PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
26Gly Ala Gln Asn Leu Asp Ser Met Leu His Gly Thr Gly Met Lys Ser 1
5 10 15 Asp Ser Asp Gln Lys Lys Ser Glu Asn Gly Val Thr Leu Ala Pro
Glu 20 25 30 Asp Thr Leu Pro Phe Leu Lys Cys Tyr Cys Ser Gly His
Cys Pro Asp 35 40 45 Asp Ala Ile Asn Asn Thr Cys Ile Thr Asn Gly
His Cys Phe Ala Ile 50 55 60 Ile Glu Glu Asp Asp Gln Gly Glu Thr
Thr Leu Ala Ser Gly Cys Met 65 70 75 80 Lys Tyr Glu Gly Ser Asp Phe
Gln Cys Lys Asp Ser Pro Lys Ala Gln 85 90 95 Leu Arg Arg Thr Ile
Glu Cys Cys Arg Thr Asn Leu Cys Asn Gln Tyr 100 105 110 Leu Gln Pro
Thr Leu Pro Pro Thr Gly Gly Gly Thr His Thr Cys Pro 115 120 125 Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 130 135
140 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
145 150 155 160 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe 165 170 175 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro 180 185 190 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr 195 200 205 Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val 210 215 220 Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 225 230 235 240 Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 245 250 255
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 260
265 270 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro 275 280 285 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser 290 295 300 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln 305 310 315 320 Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His 325 330 335 Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 340 345 271131DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
27atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt
60tcgcccggcg cccttcatgg cactgggatg aaatcagact ccgaccagaa aaagtcagaa
120aatggagtaa ccttagcacc agaggatacc ttgccttttt taaagtgcta
ttgctcaggg 180cactgtccag atgatgctat taataacaca tgcataacta
atggacattg ctttgccatc 240atagaagaag atgaccaggg agaaaccaca
ttagcttcag ggtgtatgaa atatgaagga 300tctgattttc agtgcaaaga
ttctccaaaa gcccagctac gccggacaat agaatgttgt 360cggaccaatt
tatgtaacca gtatttgcaa cccacactgc cccctgttgt cataggtccg
420ttttttgatg gcagcattcg aaccggtggt ggaactcaca catgcccacc
gtgcccagca 480cctgaactcc tggggggacc gtcagtcttc ctcttccccc
caaaacccaa ggacaccctc 540atgatctccc ggacccctga ggtcacatgc
gtggtggtgg acgtgagcca cgaagaccct 600gaggtcaagt tcaactggta
cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 660cgggaggagc
agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag
720gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct
cccagccccc 780atcgagaaaa ccatctccaa agccaaaggg cagccccgag
aaccacaggt gtacaccctg 840cccccatccc gggaggagat gaccaagaac
caggtcagcc tgacctgcct ggtcaaaggc 900ttctatccca gcgacatcgc
cgtggagtgg gagagcaatg ggcagccgga gaacaactac 960aagaccacgc
ctcccgtgct ggactccgac ggctccttct tcctctatag caagctcacc
1020gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat
gcatgaggct 1080ctgcacaacc actacacgca gaagagcctc tccctgtctc
cgggtaaatg a 113128354PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 28Gly Ala Leu His Gly Thr
Gly Met Lys Ser Asp Ser Asp Gln Lys Lys 1 5 10 15 Ser Glu Asn Gly
Val Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu 20 25 30 Lys Cys
Tyr Cys Ser Gly His Cys Pro Asp Asp Ala Ile Asn Asn Thr 35 40 45
Cys Ile Thr Asn Gly His Cys Phe Ala Ile Ile Glu Glu Asp Asp Gln 50
55 60 Gly Glu Thr Thr Leu Ala Ser Gly Cys Met Lys Tyr Glu Gly Ser
Asp 65 70 75 80 Phe Gln Cys Lys Asp Ser Pro Lys Ala Gln Leu Arg Arg
Thr Ile Glu 85 90 95 Cys Cys Arg Thr Asn Leu Cys Asn Gln Tyr Leu
Gln Pro Thr Leu Pro 100 105 110 Pro Val Val Ile Gly Pro Phe Phe Asp
Gly Ser Ile Arg Thr Gly Gly 115 120 125 Gly Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly 130 135 140 Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 145 150 155 160 Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 165 170 175
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 180
185 190 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg 195 200 205 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys 210 215 220 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu 225 230 235 240 Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr 245 250 255 Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu 260 265 270 Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 275 280 285 Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 290 295 300
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 305
310 315 320 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His 325 330 335 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 340 345 350 Gly Lys 29353PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
29Ala Leu His Gly Thr Gly Met Lys Ser Asp Ser Asp Gln Lys Lys Ser 1
5 10 15 Glu Asn Gly Val Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu
Lys 20 25 30 Cys Tyr Cys Ser Gly His Cys Pro Asp Asp Ala Ile Asn
Asn Thr Cys 35 40 45 Ile Thr Asn Gly His Cys Phe Ala Ile Ile Glu
Glu Asp Asp Gln Gly 50 55 60 Glu Thr Thr Leu Ala Ser Gly Cys Met
Lys Tyr Glu Gly Ser Asp Phe 65 70 75 80 Gln Cys Lys Asp Ser Pro Lys
Ala Gln Leu Arg Arg Thr Ile Glu Cys 85 90 95 Cys Arg Thr Asn Leu
Cys Asn Gln Tyr Leu Gln Pro Thr Leu Pro Pro 100 105 110 Val Val Ile
Gly Pro Phe Phe Asp Gly Ser Ile Arg Thr Gly Gly Gly 115 120 125 Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 130 135
140 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
145 150 155 160 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp
165 170 175 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn 180 185 190 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val 195 200 205 Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu 210 215 220 Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys 225 230 235 240 Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 245 250 255 Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 260 265 270 Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 275 280
285 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
290 295 300 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 305 310 315 320 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu 325 330 335 Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 340 345 350 Lys 30351PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
30His Gly Thr Gly Met Lys Ser Asp Ser Asp Gln Lys Lys Ser Glu Asn 1
5 10 15 Gly Val Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys Cys
Tyr 20 25 30 Cys Ser Gly His Cys Pro Asp Asp Ala Ile Asn Asn Thr
Cys Ile Thr 35 40 45 Asn Gly His Cys Phe Ala Ile Ile Glu Glu Asp
Asp Gln Gly Glu Thr 50 55 60 Thr Leu Ala Ser Gly Cys Met Lys Tyr
Glu Gly Ser Asp Phe Gln Cys 65 70 75 80 Lys Asp Ser Pro Lys Ala Gln
Leu Arg Arg Thr Ile Glu Cys Cys Arg 85 90 95 Thr Asn Leu Cys Asn
Gln Tyr Leu Gln Pro Thr Leu Pro Pro Val Val 100 105 110 Ile Gly Pro
Phe Phe Asp Gly Ser Ile Arg Thr Gly Gly Gly Thr His 115 120 125 Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 130 135
140 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
145 150 155 160 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu 165 170 175 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys 180 185 190 Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser 195 200 205 Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys 210 215 220 Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 225 230 235 240 Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 245 250 255
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 260
265 270 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn 275 280 285 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser 290 295 300 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg 305 310 315 320 Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu 325 330 335 His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 31352PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
31Leu His Gly Thr Gly Met Lys Ser Asp Ser Asp Gln Lys Lys Ser Glu 1
5 10 15 Asn Gly Val Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys
Cys 20 25 30 Tyr Cys Ser Gly His Cys Pro Asp Asp Ala Ile Asn Asn
Thr Cys Ile 35 40 45 Thr Asn Gly His Cys Phe Ala Ile Ile Glu Glu
Asp Asp Gln Gly Glu 50 55 60 Thr Thr Leu Ala Ser Gly Cys Met Lys
Tyr Glu Gly Ser Asp Phe Gln 65 70 75 80 Cys Lys Asp Ser Pro Lys Ala
Gln Leu Arg Arg Thr Ile Glu Cys Cys 85 90 95 Arg Thr Asn Leu Cys
Asn Gln Tyr Leu Gln Pro Thr Leu Pro Pro Val 100 105 110 Val Ile Gly
Pro Phe Phe Asp Gly Ser Ile Arg Thr Gly Gly Gly Thr 115 120 125 His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 130 135
140 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
145 150 155 160 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro 165 170 175 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala 180 185 190 Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val 195 200 205 Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr 210 215 220 Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 225 230 235 240 Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 245 250 255
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 260
265 270 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser 275 280 285 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp 290 295 300 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser 305 310 315 320 Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala 325 330 335 Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350
321113DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 32atggatgcaa tgaagagagg gctctgctgt
gtgctgctgc tgtgtggagc agtcttcgtt 60tcgcccggcg cccttcatgg cactgggatg
aaatcagact ccgaccagaa aaagtcagaa 120aatggagtaa ccttagcacc
agaggatacc ttgccttttt taaagtgcta ttgctcaggg 180cactgtccag
atgatgctat taataacaca tgcataacta atggacattg ctttgccatc
240atagaagaag atgaccaggg agaaaccaca ttagcttcag ggtgtatgaa
atatgaagga 300tctgattttc agtgcaaaga ttctccaaaa gcccagctac
gccggacaat agaatgttgt 360cggaccaatt tatgtaacca gtatttgcaa
cccacactgc cccctgttgt cataggtccg 420tttaccggtg gtggaactca
cacatgccca ccgtgcccag cacctgaact cctgggggga 480ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
540gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 600tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
cgcgggagga gcagtacaac 660agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc aggactggct gaatggcaag 720gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 780aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
840atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 900gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 960ctggactccg acggctcctt cttcctctat
agcaagctca ccgtggacaa gagcaggtgg 1020cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1080cagaagagcc
tctccctgtc tccgggtaaa tga 111333348PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
33Gly Ala Leu His Gly Thr Gly Met Lys Ser Asp Ser Asp Gln Lys Lys 1
5 10 15 Ser Glu Asn Gly Val Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe
Leu 20 25 30 Lys Cys Tyr Cys Ser Gly His Cys Pro Asp Asp Ala Ile
Asn Asn Thr 35 40 45 Cys Ile Thr Asn Gly His Cys Phe Ala Ile Ile
Glu Glu Asp Asp Gln 50 55 60 Gly Glu Thr Thr Leu Ala Ser Gly Cys
Met Lys Tyr Glu Gly Ser Asp 65 70 75 80 Phe Gln Cys Lys Asp Ser Pro
Lys Ala Gln Leu Arg Arg Thr Ile Glu 85 90 95 Cys Cys Arg Thr Asn
Leu Cys Asn Gln Tyr Leu Gln Pro Thr Leu Pro 100 105 110 Pro Val Val
Ile Gly Pro Phe Thr Gly Gly Gly Thr His Thr Cys Pro 115 120 125 Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 130 135
140 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
145 150 155 160 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe 165 170 175 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro 180 185 190 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr 195 200 205 Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val 210 215 220 Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 225 230 235 240 Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 245 250 255
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 260
265 270 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro 275 280 285 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser 290 295 300 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln 305 310 315 320 Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His 325 330 335 Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 340 345 34347PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
34Ala Leu His Gly Thr Gly Met Lys Ser Asp Ser Asp Gln Lys Lys Ser 1
5 10 15 Glu Asn Gly Val Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu
Lys 20 25 30 Cys Tyr Cys Ser Gly His Cys Pro Asp Asp Ala Ile Asn
Asn Thr Cys 35 40 45 Ile Thr Asn Gly His Cys Phe Ala Ile Ile Glu
Glu Asp Asp Gln Gly 50 55 60 Glu Thr Thr Leu Ala Ser Gly Cys Met
Lys Tyr Glu Gly Ser Asp Phe 65 70 75 80 Gln Cys Lys Asp Ser Pro Lys
Ala Gln Leu Arg Arg Thr Ile Glu Cys 85 90 95 Cys Arg Thr Asn Leu
Cys Asn Gln Tyr Leu Gln Pro Thr Leu Pro Pro 100 105 110 Val Val Ile
Gly Pro Phe Thr Gly Gly Gly Thr His Thr Cys Pro Pro 115 120 125 Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 130 135
140 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
145 150 155 160 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn 165 170 175 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg 180 185 190 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val 195 200 205 Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser 210 215 220 Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 225 230 235 240 Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 245 250 255
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 260
265 270 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu 275 280 285 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe 290 295 300 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly 305 310 315 320 Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr 325 330 335 Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 340 345 35345PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 35His Gly Thr Gly Met
Lys Ser Asp Ser Asp Gln Lys Lys Ser Glu Asn 1 5 10 15 Gly Val Thr
Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys Cys Tyr 20 25 30 Cys
Ser Gly His Cys Pro Asp Asp Ala Ile Asn Asn Thr Cys Ile Thr 35 40
45 Asn Gly His Cys Phe Ala Ile Ile Glu Glu Asp Asp Gln Gly Glu Thr
50 55 60 Thr Leu Ala Ser Gly Cys Met Lys Tyr Glu Gly Ser Asp Phe
Gln Cys 65 70 75 80 Lys Asp Ser Pro Lys Ala Gln Leu Arg Arg Thr Ile
Glu Cys Cys Arg 85 90 95 Thr Asn Leu Cys Asn Gln Tyr Leu Gln Pro
Thr Leu Pro Pro Val Val 100 105 110 Ile Gly Pro Phe Thr Gly Gly Gly
Thr His Thr Cys Pro Pro Cys Pro 115 120 125 Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 130 135 140 Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 145 150 155 160 Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 165 170
175 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
180 185 190 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His 195 200 205 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys 210 215 220 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln 225 230 235 240 Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met 245 250 255 Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 260 265 270 Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 275 280 285 Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 290 295
300 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
305 310 315 320 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln 325 330 335 Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345
36346PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 36Leu His Gly Thr Gly Met Lys Ser Asp Ser Asp
Gln Lys Lys Ser Glu 1 5 10 15 Asn Gly Val Thr Leu Ala Pro Glu Asp
Thr Leu Pro Phe Leu Lys Cys 20 25 30 Tyr Cys Ser Gly His Cys Pro
Asp Asp Ala Ile Asn Asn Thr Cys Ile 35 40 45 Thr Asn Gly His Cys
Phe Ala Ile Ile Glu Glu Asp Asp Gln Gly Glu 50 55 60 Thr Thr Leu
Ala Ser Gly Cys Met Lys Tyr Glu Gly Ser Asp Phe Gln 65 70 75 80 Cys
Lys Asp Ser Pro Lys Ala Gln Leu Arg Arg Thr Ile Glu Cys Cys 85 90
95 Arg Thr Asn Leu Cys Asn Gln Tyr Leu Gln Pro Thr Leu Pro Pro Val
100 105 110 Val Ile Gly Pro Phe Thr Gly Gly Gly Thr His Thr Cys Pro
Pro
Cys 115 120 125 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 130 135 140 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 145 150 155 160 Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp 165 170 175 Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 180 185 190 Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 195 200 205 His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 210 215 220
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 225
230 235 240 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu 245 250 255 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 260 265 270 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 275 280 285 Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 290 295 300 Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 305 310 315 320 Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 325 330 335 Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 371095DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
37atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt
60tcgcccggcg cccttcatgg cactgggatg aaatcagact ccgaccagaa aaagtcagaa
120aatggagtaa ccttagcacc agaggatacc ttgccttttt taaagtgcta
ttgctcaggg 180cactgtccag atgatgctat taataacaca tgcataacta
atggacattg ctttgccatc 240atagaagaag atgaccaggg agaaaccaca
ttagcttcag ggtgtatgaa atatgaagga 300tctgattttc agtgcaaaga
ttctccaaaa gcccagctac gccggacaat agaatgttgt 360cggaccaatt
tatgtaacca gtatttgcaa cccacactgc cccctaccgg tggtggaact
420cacacatgcc caccgtgccc agcacctgaa ctcctggggg gaccgtcagt
cttcctcttc 480cccccaaaac ccaaggacac cctcatgatc tcccggaccc
ctgaggtcac atgcgtggtg 540gtggacgtga gccacgaaga ccctgaggtc
aagttcaact ggtacgtgga cggcgtggag 600gtgcataatg ccaagacaaa
gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 660agcgtcctca
ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc
720tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa
agggcagccc 780cgagaaccac aggtgtacac cctgccccca tcccgggagg
agatgaccaa gaaccaggtc 840agcctgacct gcctggtcaa aggcttctat
cccagcgaca tcgccgtgga gtgggagagc 900aatgggcagc cggagaacaa
ctacaagacc acgcctcccg tgctggactc cgacggctcc 960ttcttcctct
atagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc
1020tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag
cctctccctg 1080tctccgggta aatga 109538342PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
38Gly Ala Leu His Gly Thr Gly Met Lys Ser Asp Ser Asp Gln Lys Lys 1
5 10 15 Ser Glu Asn Gly Val Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe
Leu 20 25 30 Lys Cys Tyr Cys Ser Gly His Cys Pro Asp Asp Ala Ile
Asn Asn Thr 35 40 45 Cys Ile Thr Asn Gly His Cys Phe Ala Ile Ile
Glu Glu Asp Asp Gln 50 55 60 Gly Glu Thr Thr Leu Ala Ser Gly Cys
Met Lys Tyr Glu Gly Ser Asp 65 70 75 80 Phe Gln Cys Lys Asp Ser Pro
Lys Ala Gln Leu Arg Arg Thr Ile Glu 85 90 95 Cys Cys Arg Thr Asn
Leu Cys Asn Gln Tyr Leu Gln Pro Thr Leu Pro 100 105 110 Pro Thr Gly
Gly Gly Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 115 120 125 Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 130 135
140 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
145 150 155 160 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly 165 170 175 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn 180 185 190 Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp 195 200 205 Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro 210 215 220 Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 225 230 235 240 Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 245 250 255
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 260
265 270 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr 275 280 285 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys 290 295 300 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys 305 310 315 320 Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu 325 330 335 Ser Leu Ser Pro Gly Lys
340 39341PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 39Ala Leu His Gly Thr Gly Met Lys Ser Asp Ser
Asp Gln Lys Lys Ser 1 5 10 15 Glu Asn Gly Val Thr Leu Ala Pro Glu
Asp Thr Leu Pro Phe Leu Lys 20 25 30 Cys Tyr Cys Ser Gly His Cys
Pro Asp Asp Ala Ile Asn Asn Thr Cys 35 40 45 Ile Thr Asn Gly His
Cys Phe Ala Ile Ile Glu Glu Asp Asp Gln Gly 50 55 60 Glu Thr Thr
Leu Ala Ser Gly Cys Met Lys Tyr Glu Gly Ser Asp Phe 65 70 75 80 Gln
Cys Lys Asp Ser Pro Lys Ala Gln Leu Arg Arg Thr Ile Glu Cys 85 90
95 Cys Arg Thr Asn Leu Cys Asn Gln Tyr Leu Gln Pro Thr Leu Pro Pro
100 105 110 Thr Gly Gly Gly Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu 115 120 125 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr 130 135 140 Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val 145 150 155 160 Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val 165 170 175 Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 180 185 190 Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 195 200 205 Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 210 215
220 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
225 230 235 240 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln 245 250 255 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala 260 265 270 Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr 275 280 285 Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu 290 295 300 Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 305 310 315 320 Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 325 330 335
Leu Ser Pro Gly Lys 340 40339PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 40His Gly Thr Gly Met Lys
Ser Asp Ser Asp Gln Lys Lys Ser Glu Asn 1 5 10 15 Gly Val Thr Leu
Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys Cys Tyr 20 25 30 Cys Ser
Gly His Cys Pro Asp Asp Ala Ile Asn Asn Thr Cys Ile Thr 35 40 45
Asn Gly His Cys Phe Ala Ile Ile Glu Glu Asp Asp Gln Gly Glu Thr 50
55 60 Thr Leu Ala Ser Gly Cys Met Lys Tyr Glu Gly Ser Asp Phe Gln
Cys 65 70 75 80 Lys Asp Ser Pro Lys Ala Gln Leu Arg Arg Thr Ile Glu
Cys Cys Arg 85 90 95 Thr Asn Leu Cys Asn Gln Tyr Leu Gln Pro Thr
Leu Pro Pro Thr Gly 100 105 110 Gly Gly Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly 115 120 125 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 130 135 140 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 145 150 155 160 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 165 170 175
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 180
185 190 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 195 200 205 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 210 215 220 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 225 230 235 240 Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser 245 250 255 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 260 265 270 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 275 280 285 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 290 295 300
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 305
310 315 320 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 325 330 335 Pro Gly Lys 41340PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
41Leu His Gly Thr Gly Met Lys Ser Asp Ser Asp Gln Lys Lys Ser Glu 1
5 10 15 Asn Gly Val Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys
Cys 20 25 30 Tyr Cys Ser Gly His Cys Pro Asp Asp Ala Ile Asn Asn
Thr Cys Ile 35 40 45 Thr Asn Gly His Cys Phe Ala Ile Ile Glu Glu
Asp Asp Gln Gly Glu 50 55 60 Thr Thr Leu Ala Ser Gly Cys Met Lys
Tyr Glu Gly Ser Asp Phe Gln 65 70 75 80 Cys Lys Asp Ser Pro Lys Ala
Gln Leu Arg Arg Thr Ile Glu Cys Cys 85 90 95 Arg Thr Asn Leu Cys
Asn Gln Tyr Leu Gln Pro Thr Leu Pro Pro Thr 100 105 110 Gly Gly Gly
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 115 120 125 Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 130 135
140 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
145 150 155 160 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu 165 170 175 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr 180 185 190 Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn 195 200 205 Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 210 215 220 Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 225 230 235 240 Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 245 250 255
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 260
265 270 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro 275 280 285 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr 290 295 300 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val 305 310 315 320 Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 325 330 335 Ser Pro Gly Lys 340
424PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 42Thr Gly Gly Gly 1 434PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 43Ser
Gly Gly Gly 1 444PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 44Gly Gly Gly Gly 1 455PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 45Thr
Gly Gly Gly Gly 1 5 465PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 46Ser Gly Gly Gly Gly 1 5
476PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6xHis tag 47His His His His His His 1 5
48225PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMOD_RES(43)..(43)Asp or
AlaMOD_RES(100)..(100)Lys or AlaMOD_RES(212)..(212)Asn or Ala 48Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 1 5 10
15 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
20 25 30 Arg Thr Pro Glu Val Thr Cys Val Val Val Xaa Val Ser His
Glu Asp 35 40 45 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 50 55 60 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 65 70 75 80 Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu 85 90 95 Tyr Lys Cys Xaa Val Ser
Asn Lys Ala Leu Pro Val Pro Ile Glu Lys 100 105 110 Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 115 120 125 Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 130 135 140
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 145
150 155 160 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu 165 170 175 Asp Ser Asp Gly Pro Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 180 185 190 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 195 200 205 Ala Leu His Xaa His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 210 215 220 Lys 225
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