U.S. patent application number 15/477204 was filed with the patent office on 2018-02-22 for actriib binding agents and uses thereof.
The applicant listed for this patent is Acceleron Pharma Inc., Dyax Corp.. Invention is credited to David Buckler, Janja Cosic, Monique Davies, Asya Grinberg, Rachel Kent, Ravindra Kumar, Diana Martik.
Application Number | 20180051088 15/477204 |
Document ID | / |
Family ID | 48172683 |
Filed Date | 2018-02-22 |
United States Patent
Application |
20180051088 |
Kind Code |
A1 |
Kumar; Ravindra ; et
al. |
February 22, 2018 |
ACTRIIB BINDING AGENTS AND USES THEREOF
Abstract
The disclosure provides, among other aspects, neutralizing
antibodies and portions thereof that bind to ActRIIB and uses for
same.
Inventors: |
Kumar; Ravindra; (Acton,
MA) ; Grinberg; Asya; (Lexington, MA) ;
Davies; Monique; (Harpswell, ME) ; Martik; Diana;
(Cambridge, MA) ; Cosic; Janja; (Arlington,
MA) ; Kent; Rachel; (Boxborough, MA) ;
Buckler; David; (Chester, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Acceleron Pharma Inc.
Dyax Corp. |
Cambridge
Burlington |
MA
MA |
US
US |
|
|
Family ID: |
48172683 |
Appl. No.: |
15/477204 |
Filed: |
April 3, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15234581 |
Aug 11, 2016 |
9624301 |
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15477204 |
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14508988 |
Oct 7, 2014 |
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15234581 |
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13828419 |
Mar 14, 2013 |
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14508988 |
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13283552 |
Oct 27, 2011 |
8765385 |
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13828419 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/00 20180101;
C07K 2317/52 20130101; A61P 1/16 20180101; C07K 2319/30 20130101;
C07K 2317/92 20130101; A61P 21/00 20180101; C07K 2317/21 20130101;
A61P 35/00 20180101; A61P 25/28 20180101; C07K 2317/76 20130101;
A61P 25/16 20180101; A61P 13/12 20180101; A61P 7/06 20180101; C07K
2317/55 20130101; C07K 16/2863 20130101; C07K 16/40 20130101; C07K
2317/56 20130101; C07K 2317/565 20130101; C07K 2317/71
20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1. An anti-ActRIIB antibody or functional fragment thereof that
binds to ActRIIB between amino acids 19-134 of SEQ ID NO:1.
2. The anti-ActRIIB antibody or functional fragment thereof
according to claim 1, wherein the anti-ActRIIB antibody or
functional fragment thereof binds to ActRIIB with a K.sub.D of 1 nM
or less.
3. The anti-ActRIIB antibody or functional fragment thereof
according to claim 1, wherein the anti-ActRIIB antibody or
functional fragment thereof inhibits myostatin binding to
ActRIIB
4. The anti-ActRIIB antibody or functional fragment thereof
according to claim 1, wherein the anti-ActRIIB antibody or
functional fragment thereof inhibits myostatin induced signaling as
measured by a Smad dependent reporter gene assay.
5. The anti-ActRIIB antibody or functional fragment thereof
according to claim 1, wherein the anti-ActRIIB antibody or
functional fragment thereof binds to ActRIIB with a 10-fold or
greater affinity that it binds to ActRIIA.
6. The anti-ActRIIB antibody or functional fragment thereof
according to claim 1, wherein the anti-ActRIIB antibody or
functional fragment thereof binds to an epitope selected from the
group consisting of: amino acids 77-83 of SEQ ID NO:1, amino acids
60-64 of SEQ ID NO:1, 73-74 of SEQ ID NO:1, amino acids 73-83 of
SEQ ID NO:1, amino acids 98-101 of SEQ ID NO:1; amino acids 35-39
of SEQ ID NO:1 and amino acids 52-55 of SEQ ID NO:1.
7. The anti-ActRIIB antibody or functional fragment thereof
according to claim 1, wherein the anti-ActRIIB antibody or
functional fragment thereof is of the IgG1 isotype.
8. The anti-ActRIIB antibody or functional fragment thereof
according to claim 1, wherein the anti-ActRIIB antibody or
functional fragment thereof is characterized by an altered effector
function through mutation of the Fc region.
9. An isolated polynucleotide sequence encoding the antibody or
functional fragment thereof according to claim 1.
10. A cloning or expression vector comprising the isolated
polynucleotide sequence according to claim 9.
11. An isolated host cell comprising the vector according to claim
10.
12. A process for the production of an antibody or functional
fragment thereof that binds to ActRIIB between amino acids 19-1344
of SEQ ID NO:1, comprising culturing the host cell of claim 11 and
isolating the antibody or functional fragment thereof.
13. A pharmaceutical composition comprising the anti-ActRIIB
antibody or functional fragment thereof according to claim 1
14. The pharmaceutical composition of claim 13, where the
composition further comprises a pharmaceutically acceptable diluent
or carrier.
15. The antibody or fragment thereof of claim 2 that comprises at
least one, at least two, at least three, at least four, at least
five or at least six CDR sequences having at least 80% identity to
a CDR selected from the group consisting of SEQ ID NOs: 37-42.
16. The antibody or fragment thereof according to claim 15 wherein
said percent identity is at least 85%.
17. The antibody or fragment thereof according to claim 15 wherein
said percent identity is at least 90%.
18. The antibody or fragment thereof according to claim 15 wherein
said percent identity is 95%.
19. The antibody or fragment thereof according to claim 15 wherein
said percent identity is 100%.
Description
RELATED APPLICATION
[0001] This application is a continuation of and claims priority to
U.S. application Ser. No. 13/283,552, filed Oct. 27, 2011. 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 in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on October 7, 2014, is named PHPH064103 Seq.txt and is 26,546 bytes
in size.
BACKGROUND OF THE INVENTION
[0003] The transforming growth factor-beta (TGF-beta) superfamily
contains a variety of growth factors that share common sequence
elements and structural motifs. These proteins are known to exert
biological effects on a large variety of cell types in both
vertebrates and invertebrates. Members of the superfamily perform
important functions during embryonic development in pattern
formation and tissue specification and can influence a variety of
differentiation processes, including adipogenesis, myogenesis,
chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and
epithelial cell differentiation. The superfamily includes proteins
that are variously described as Growth and Differentiation Factors
(GDFs), Bone Morphogenetic Proteins (BMPs), activins and
inhibins.
[0004] By manipulating the activity of a member of the TGF-beta
superfamily, it is often possible to cause significant
physiological changes in an organism. For example, GDF8 (myostatin)
is a well-known regulator of skeletal muscle mass and strength. The
Piedmontese and Belgian Blue cattle breeds carry a loss-of-function
mutation in the 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. Other members of
the superfamily, such as activin A, are also reported to regulate
skeletal muscle. Modulators of GDF8 and activin are in clinical
development for the treatment of muscle conditions. Antibodies
directed to myostatin promote muscle formation in animal models,
although results in human clinical trials have not demonstrated
prominent increases in skeletal muscle mass or strength, raising a
question as to whether other members of the TGF-beta superfamily
may regulate muscle growth. Walker et al., Ann Neurol. 2008,
63:561-71. The activin receptor type IIB (ActRIIB, also known as
ACVR2B), is a high affinity receptor for myostatin, activin A and
other members of the TGF-beta superfamily, including GDF11 and
other activins. Lee et al. Proc Natl Acad Sci USA 2005,
102:18117-22; Mathews et al. Science 1992 255:1702-5; WO 00/43781;
WO 2006/012627. An ActRIIB-Fc fusion protein acts as a high
affinity antagonist to each of these ligands and promotes
substantial muscle growth in animal models as well as humans. Lee
et al. Proc Natl Acad Sci USA 2005, 102:18117-22; WO 2004/039948;
WO 2006/012627; WO 2008/097541. ActRIIB-Fc is also known to promote
bone formation and, in some cases, affect other tissues. WO
95/10611; Hamrick et al. Calcif Tissue Int 2002, 71:63-68; WO
2006/012627; WO 2008/097541. Similarly, it has been proposed that
antibodies that bind to ActRIIB and disrupt ligand binding and/or
signaling (e.g., neutralizing antibodies) can be used to promote
muscle or bone formation and treat a variety of disorders. U.S.
Pat. No. 6,656,475; WO 2006/012627; WO 2008/097541.
[0005] Thus, it is an object of this disclosure to provide
antibodies that bind to ActRIIB and uses for same.
SUMMARY OF THE INVENTION
[0006] The disclosure provides, among other aspects, antibodies and
fragments thereof that bind to ActRIIB and inhibit ActRIIB-mediated
signaling. A variety of uses for such proteins are described
herein. For example, the antibodies may be used to treat a variety
of diseases, including disorders of skeletal muscle and bone, and
as part of assays to identify known and novel ActRIIB-binding
agents. In a further embodiment, antibodies that bind to ActRIIB
and inhibit ligand binding may be used in assays to detect and
characterize antibodies that may be generated in a human in
response to administration of a polypeptide comprising a part or
all of the extracellular domain of ActRIIB, such as an ActRIIB-Fc
fusion protein.
[0007] In certain embodiments, the disclosure relates to
antibodies, and fragments of antibodies (e.g., Fab, scFv) that
specifically bind to ActRIIB The binding agents can be
characterized by their ability to inhibit binding to or signaling
through ActRIIB by one or more ligands, such as myostatin, GDF11,
activin A, activin B or others described in the art. Binding agents
may cross-block the binding of at least one antibody disclosed
herein, such as Ab-17G05, to ActRIIB and/or to be cross-blocked
from binding ActRIIB by at least one of said antibodies. In certain
aspects, the anti-ActRIIB antibody is a therapeutic antibody or
functional fragment thereof. An anti-ActRIIB antibody or functional
fragment thereof may bind to a ligand-binding domain of ActRIIB,
the boundaries and attributes of which are described herein. An
anti-ActRIIB antibody or functional fragment thereof may bind to
ActRIIB between amino acids 19-134 of SEQ ID NO: 1. Any of the
antibodies and fragments described herein may bind to ActRIIB with
a KD (dissociation constant) of 1 nM, 100 pM, 50 pM, 20 pM, 10 pM
or less. An anti-ActRIIB antibody may inhibit the binding of one or
more ligands to ActRIIB, including myostatin, activin A, GDF11,
activin B, BMP9 or BMP10. Because both activins and myostatin, and
possibly GDF11, act as negative regulators of skeletal muscle mass,
an anti-ActRIIB antibody may be selected so as to inhibit the
binding of two of more of the aforementioned ligands to ActRIIB An
anti-ActRIIB antibody may inhibit the signaling caused by an
ActRIIB ligand, such as myostatin, and such signaling may be
measured by a Smad dependent reporter gene assay, such as the A204
assay described in the Examples. Smad activation may also be
assessed by measuring the levels of phosphor-Smads, particularly
Smad2 or Smad3. In certain instances, it will be desirable to
selectively bind ActRIIB with little or no binding to the related
receptor ActRIIA, and accordingly, an anti-ActRIIB antibody or
functional fragment thereof may bind to ActRIIB with a 10-fold,
20-fold, 50-fold, 100-fold, 200-fold, 500-fold or 1000-fold or
greater/better affinity than it binds to ActRIIA. An anti-ActRIIB
antibody or functional fragment thereof may be of any of the known
immunoglobulin isotypes, and particularly IgG1, IgG2 or IgG4, and
may have an altered effector function. An altered effector function
may be achieved by modifying or mutating the Fc region, and this
may be performed to create an antibody having reduced ADCC or CDC
reactivity. An anti-ActRIIB antibody or fragment thereof may
promote skeletal muscle growth in vivo, particularly in a mouse,
non-human primate or a human.
[0008] An anti-ActRIM antibody or fragment thereof may comprise at
least one CDR sequence having at least 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100.degree. A identity to a
CDR selected from the group consisting of SEQ ID NOs: 37-42. The
antibody or fragment thereof may comprise at least two, three,
four, five or six of the foregoing CDR sequences and may, for
example, comprise three CDRs, CDR-H1, CDR-H2, and CDR-H3 wherein
CDR-H1 comprises a sequence that is at least 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID
NO: 37, CDR-H2 comprises a sequence that is at least 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to
SEQ ID NO: 38, and CDR-H3 comprises a sequence that is at least
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 39. The antibody or fragment thereof may
comprise three CDRs, CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1
comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 40,
CDR-H2 comprises a sequence that is at least 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID
NO: 41, and CDR-H3 comprises a sequence that is at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical
to SEQ ID NO: 42. Other combinations and permutations of the
foregoing CDR sequences and variants are included within the
disclosure. An anti-ActRIIB antibody may comprise a heavy chain
wherein said heavy chain comprises a polypeptide having at least
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
100.degree. A identity to the sequence given in SEQ ID NO: 15. An
anti-ActRIIB antibody may comprise a light chain wherein said light
chain comprises a polypeptide having at least 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the
sequence given in SEQ ID NO: 16. An anti-ActRIIB antibody may
comprise any combination of the foregoing heavy chain and light
chain sequences.
[0009] The anti-ActRIIB antibodies and functional fragments
described may be formulated as pharmaceutical compositions
comprising such antibody or functional fragment. A pharmaceutical
composition may comprise a pharmaceutically acceptable diluent or
carrier.
[0010] In certain aspects the disclosure provides isolated
polynucleotide sequences encoding the antibody or functional
fragment thereof described herein, including any of the
polynucleotides encoding each of the heavy and light chains
described, as well as the variable domains and the respective CDR
portions. The disclosure further provides cloning or expression
vectors comprising any of the foregoing isolated polynucleotide
sequences, and cells, particularly host cells such as CHO or NSO
cells comprising any of the above nucleic acids or vectors. Such
host cells may be used to produce the anti-ActRIIB antibodies
described. In certain aspects the disclosure provides processes for
the production of an antibody or functional fragment thereof
described herein, comprising culturing a host cell comprising a
cloning or expression vector encoding the antibody or functional
fragment thereof, and isolating the antibody or functional fragment
thereof.
[0011] A variety of uses for binding agents that neutralize ActRIIB
have been described, and thus in certain aspects, the disclosure
provides methods for using anti-ActRIIB antibodies or functional
fragments. For example, such agents, and pharmaceutical
preparations containing same, may be used in a method of treating a
patient suffering from a musculoskeletal disease or disorder; acute
and/or chronic renal disease or failure; cancer; breast cancer;
Parkinson's Disease; conditions associated with neuronal death;
ALS; brain atrophy; dementia; anemia; liver, kidney and pulmonary
fibrosis; one or more age-related condition; rhabdomyosarcoma;
bone-loss inducing cancer. Such methods may comprise the step of:
administering an effective dose of an antibody disclosed herein or
functional fragment thereof to said patient. Examples of
musculoskeletal diseases or disorders include muscle atrophy,
myopathy, myotonia, a congential myopathy, nemalene myopathy,
multi/minicore myopathy and myotubular (centronuclear) myopathy,
mitochondrial myopathy, familial periodic paralysis, inflammatory
myopathy, metabolic myopathy, a glycogen or lipid storage disease,
dermatomyositisis, polymyositis, inclusion body myositis, myositis
ossificans, rhabdomyolysis and myoglobinurias; a dystrophy,
including Duchenne, Becker, myotonic, fascioscapulohumeral,
Emery-Dreifuss, oculopharyngeal, scapulohumeral, limb girdle,
Fukuyama, a congenital muscular dystrophy, or hereditary distal
myopathy; osteoporosis; a bone fracture; short stature; dwarfism;
prolonged bed rest; voluntary inactivity; and/or involuntary
inactivity. As further examples, a patient being treated may be
elderly, may have spent time in a zero gravity environment or may
have undergone a period of inactivity, and treatment may be
initiated prior to the aforementioned event. Such a patient may
have a fracture to a limb or joint or have undergone or be about to
undergo hip or knee replacement surgery.
[0012] In certain aspects, the disclosure provides methods for
using anti-ActRIIB antibodies to detect ActRIIB in cells and
tissues, and also in assays designed to detect or assess other
antibodies that bind to ActRIIB Reagents that include the
ligand-binding portion of ActRIIB (e.g., an ActRIIB-Fc fusion
protein) are in development as therapeutic agents, and as with all
biologic products, it is of interest to determine whether such
agents cause the production in patients of antibodies against the
therapeutic protein and whether such antibodies are neutralizing.
Accordingly, a method described herein for detecting or
characterizing anti-ActRIIB antibodies in blood may comprise a step
of contacting an ActRIIB polypeptide (e.g. a polypeptide comprising
an ActRIIB ligand binding domain) with a neutralizing anti-ActRIIB
antibody. In an embodiment, such a method may comprise (i) forming
a mixture comprising a sample (e.g., a blood or serum sample from a
patient treated with an ActRIIB-Fc fusion protein or a placebo), an
ActRIIB polypeptide and a control antibody that is a known
neutralizing anti-ActRIIB antibody; and (ii) measuring the amount
of control antibody that is bound to the ActRIIB polypeptide,
wherein the ActRIIB polypeptide is a polypeptide comprising a
ligand binding domain of ActRIIB A decrease in the amount of
control antibody bound to the ActRIIB polypeptide relative to a
standard indicates that the sample contains a neutralizing
anti-ActRIIB antibody. The standard may be a mixture comprising the
ActRIIB polypeptide and the control antibody. The standard may
further comprise a sample that is known to contain no substantial
amount of neutralizing anti-ActRIIB antibody, and may further
comprise a sample that contains a known amount of neutralizing
anti-ActRIIB antibody. In another format, the assay may comprise
(i) forming a mixture comprising the sample, an ActRIIB polypeptide
and a ligand that binds to ActRIIB; (ii) measuring the amount of
ligand that is bound to the ActRIIB polypeptide and comparing the
amount of ligand that is bound with a standard, wherein the
standard is measured amount of ligand bound to the ActRIIB
polypeptide in a mixture comprising the ActRIIB polypeptide, the
ligand and a control antibody that is a known neutralizing
anti-ActRIIB antibody, and wherein the ActRIIB polypeptide is a
polypeptide comprising a ligand binding domain of ActRIIB A
comparison of the amount of ligand bound to the ActRIIB polypeptide
in the mixture versus the amount of ligand bound to the ActRIIB
polypeptide in the standard may be used to assess the presence or
absence of a neutralizing anti-ActRIIB antibody in the sample, with
decreased ligand binding indicating higher levels of neutralizing
anti-ActRIIB antibody. The ligand may be any known ligand for
ActRIIB and may be selected from the group consisting of: activin
A, activin B, myostatin and GDF11. In each assay format, the sample
may contain blood or a blood product, optionally from a patient
treated with an ActRIIB-Fc fusion protein or a patient that has not
been treated with an ActRIIB-Fc fusion protein. The neutralizing
anti-ActRIIB antibody may be any of the anti-ActRIII3 antibodies
disclosed herein and may, for example comprise at least one CDR
sequence having at least 80% identity to a CDR selected from the
group consisting of SEQ ID NOs: 37-42 or may be the Ab-17G05.
[0013] These and other aspects of the present invention will become
apparent upon reference to the following detailed description and
attached drawings. All references disclosed herein are hereby
incorporated by reference in their entireties as if each was
incorporated individually.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0015] FIG. 1 depicts kinetic characterization of Fab-17G05 binding
to hActRIM-hFc as determined by BIACORE.TM.-based analysis at
25.degree. C. (A) or 37.degree. C. (B). hActRIIB-hFc was captured
on a chip with covalently immobilized antibody against human IgG1
Fc and then exposed to Fab-17G05 at concentrations ranging from
0.0195 .mu.g/m1 to 5 .mu.g/ml. RU, response units. As determined by
nonlinear regression, the K.sub.D was 5.5.times.10.sup.-10 at
25.degree. C. and 1.9.times.10.sup.-9 at 37.degree. C.
[0016] FIG. 2 depicts kinetic characterization of Ab-17G05 binding
to hActRIIB-mFc as determined by BIACORE.TM.-based analysis at
25.degree. C. (A) or 37.degree. C. (B). hActRIIB-mFc was captured
on a chip with covalently immobilized antibody against murine IgG2a
Fc and then exposed to Ab-17G05 at concentrations ranging from
0.0195 pg/m1 to 5 pg/ml. The K.sub.D was 2.8.times.10.sup.-11 at
25.degree. C. and 9.2.times.10.sup.-11 at 37.degree. C.
[0017] FIG. 3 depicts neutralizing activity of Ab-17G05 or
Fab-17G05 in a cell-based reporter gene assay. Included are assay
responses with activin A alone (5 ng/ml) and combined activin A and
ActRIIB-Fc (50 ng/ml). The potency of Ab-17G05
(IC.sub.50.apprxeq.0.04 nM) in neutralizing the interaction between
activin A and ActRIIB-Fc was approximately two orders of magnitude
higher than that of Fab-17G05 (IC.sub.50 2.6 nM).
DETAILED DESCRIPTION OF THE INVENTION
1. Overview
[0018] Activins are dimeric polypeptide growth factors that belong
to the TGF-beta superfamily. There are three principal activin
forms (A, B, and AB) that are homo/heterodimers of two closely
related .beta. subunits (.beta..sub.A.beta..sub.A,
.beta..sub.B.beta..sub.B, and .beta..sub.A.beta..sub.B). The human
genome also encodes an activin C and an activin E, which are
primarily expressed in the liver. In the TGF-beta superfamily,
activins are unique and multifunctional factors that can stimulate
hormone production in ovarian and placental cells, support neuronal
cell survival, influence cell-cycle progress positively or
negatively depending on cell type, and induce mesodermal
differentiation at least in amphibian embryos (DePaolo et al.,
1991, Proc Soc Ep Biol Med. 198:500-512; Dyson et al., 1997, Curr
Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol. 55:953-963). In
several tissues, activin signaling is antagonized by its related
heterodimer, inhibin. For example, during the release of
follicle-stimulating hormone (FSH) from the pituitary, activin
promotes FSH secretion and synthesis, while inhibin prevents FSH
secretion and synthesis. Other proteins that may regulate activin
bioactivity and/or bind to activin include follistatin (FS),
follistatin-related protein (FSRP), a.sub.2-macroglobulin,
Cerberus, and endoglin. Together with myostatin, activin has been
implicated as a negative regulator of skeletal muscle mass. He et
al. 2005 Anat Embryol (Berl) 209:401-407; Link and Nishi, Exp. Cell
Res. 1997 233:350-62.
[0019] TGF-.beta. superfamily 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.
[0020] Two related type II receptors, ActRIIA and ActRIIB, have
been identified as the type II receptors for activins (Mathews and
Vale, 1991, Cell 65:973-982; Attisano et al., 1992, Cell 68:
97-108). Besides activins, ActRIIA and ActRIIB can interact
biochemically with several other TGF-.beta. family proteins,
including BMP7, Nodal, BMP9, BMP10, GDF8, and GDF11 (Yamashita et
al., 1995, J. Cell Biol. 130:217-226; Lee and McPherron, 2001,
Proc. Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol.
Cell 7: 949-957; Oh et al., 2002, Genes Dev. 16:2749-54). ALK4 is
the primary type I receptor for activins, particularly for activin
A, and ALK-7 may serve as a receptor for activins as well,
particularly for activin B.
[0021] Inhibitors of the activin signaling pathway have been
proposed for treatment of a variety of disorders, including bone
loss, various tumors including multiple myeloma and breast cancer,
and anemia. Inhibitors of myostatin and GDF11 signaling have
likewise been proposed for the treatment of a variety of disorders,
including muscle disorders, neurological disorders and bone
disorders. Neutralizing anti-ActRIIB antibodies that interfere with
signaling by any or all of activin A, activin B, GDF8 or GDF11 may
be used in a variety of indications for the treatment of muscle
loss or insufficient muscle growth, including myopathies, muscular
dystrophies, muscular atrophy, cachexia, and age-related conditions
such as sarcopenia as well as for the treatment of bone disorders
and various cancers.
[0022] 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.
[0023] "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.
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.
[0024] 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. Unless indicated otherwise, BLAST shall be the
default algorithm for comparisons.
[0025] "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.
[0026] 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.
[0027] 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.
[0028] As used herein, the term "ActRIIB" refers to a family of
activin receptor type IIB (ActRIIB) proteins from any species.
Reference to ActRIIB herein is understood to be a reference to any
one of the currently identified forms. Members of the ActRIIB
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.
[0029] The human ActRIIB precursor protein sequence is as follows
(NCBI Reference Sequence NM 001106.3), with the underlined sequence
corresponding to the literature-reported mature extracellular
domain, within which are epitopes targeted by neutralizing
anti-ActRIIB antibodies and other ActRIIB binding agents.
TABLE-US-00001 (SEQ ID NO: 1)
MTAPWVALALLWGSLCAGSGRGEAETRECIYYNANWELERTNQSGLERCE
GEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVY
FCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTLLTVLAYSLLPIGGLS
LIVLLAFWMYRHRKPPYGHVDIHEDPGPPPPSPLVGLKPLQLLEIKARGR
FGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFSTPGMKHENLLQFIAA
EKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMSRGLSY
LHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGK
PPGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRC
KAADGPVDEYMLPFEEEIGQHPSLEELQEVVVHKKMRPTIKDHWLKHPGL
AQLCVTIEECWDHDAEARLSAGCVEERVSLIRRSVNGTTSDCLVSLVTSV TNVDLPPKESSI
[0030] A protein comprising an extracellular domain of ActRIIB
(ECD) may comprise amino acids 19-134 of SEQ ID NO:1, or smaller
portions, such as amino acids 20-134, 25-131 or any polypeptide
comprising a portion of SEQ ID NO:1 beginning at any of amino acids
19-29 and ending at any of amino acids 129-134. Each of the
foregoing has been demonstrated to retain ligand binding activity.
A protein comprising an extracellular domain of ActRIIB may
comprise a polypeptide that is at least 80, 85, 90, 95, 96, 97, 98,
99 or 100% identical to any of the foregoing amino acid sequence
portions of SEQ ID NO:1. An ActRIIB-Fc fusion protein is any
protein comprising any of the foregoing extracellular domains of
ActRIIB and an Fc portion of an immunoglobulin (e.g., IgG1, IgG2,
IgG4), optionally including an interposed linker between the
ActRIIB portion and the Fc portion.
[0031] The nucleic acid sequence encoding human ActRIIB precursor
protein is as follows (nucleotides 25-1560 of Genbank NM
001106.3):
TABLE-US-00002 (SEQ ID NO: 2) atgacggcgc cctgggtggc cctcgccctc
ctctggggat cgctgtgcgc cggctctggg cgtggggagg ctgagacacg ggagtgcatc
tactacaacg ccaactggga gctggagcgc accaaccaga gcggcctgga gcgctgcgaa
ggcgagcagg acaagcggct gcactgctac gcctcctggc gcaacagctc tggcaccatc
gagctcgtga agaagggctg ctggctagat gacttcaact gctacgatag gcaggagtgt
gtggccactg aggagaaccc ccaggtgtac ttctgctgct gtgaaggcaa cttctgcaac
gaacgcttca ctcatttgcc agaggctggg ggcccggaag tcacgtacga gccacccccg
acagccccca ccctgctcac ggtgctggcc tactcactgc tgcccatcgg gggcctttcc
ctcatcgtcc tgctggcctt ttggatgtac cggcatcgca agccccccta cggtcatgtg
gacatccatg aggaccctgg gcctccacca ccatcccctc tggtgggcct gaagccactg
cagctgctgg agatcaaggc tcgggggcgc tttggctgtg tctggaaggc ccagctcatg
aatgactttg tagctgtcaa gatcttccca ctccaggaca agcagtcgtg gcagagtgaa
cgggagatct tcagcacacc tggcatgaag cacgagaacc tgctacagtt cattgctgcc
gagaagcgag gctccaacct cgaagtagag ctgtggctca tcacggcctt ccatgacaag
ggctccctca cggattacct caaggggaac atcatcacat ggaacgaact gtgtcatgta
gcagagacga tgtcacgagg cctctcatac ctgcatgagg atgtgccctg gtgccgtggc
gagggccaca agccgtctat tgcccacagg gactttaaaa gtaagaatgt attgctgaag
agcgacctca cagccgtgct ggctgacttt ggcttggctg ttcgatttga gccagggaaa
cctccagggg acacccacgg acaggtaggc acgagacggt acatggctcc tgaggtgctc
gagggagcca tcaacttcca gagagatgcc ttcctgcgca ttgacatgta tgccatgggg
ttggtgctgt gggagcttgt gtctcgctgc aaggctgcag acggacccgt ggatgagtac
atgctgccct ttgaggaaga gattggccag cacccttcgt tggaggagct gcaggaggtg
gtggtgcaca agaagatgag gcccaccatt aaagatcact ggttgaaaca cccgggcctg
gcccagcttt gtgtgaccat cgaggagtgc tgggaccatg atgcagaggc tcgcttgtcc
gcgggctgtg tggaggagcg ggtgtccctg attcggaggt cggtcaacgg cactacctcg
gactgtctcg tttccctggt gacctctgtc accaatgtgg acctgccccc taaagagtca
agcatc
2. ActRIIB Binding Agents
[0032] The disclosure provides binding agents (such as antibodies)
that specifically bind to ActRIIB or portions of ActRIIB, and
methods for using such binding agents. The binding agents are
useful to block or impair the binding of human ActRIIB to one or
more ligand(s) and to interfere with its biological activity.
[0033] It will be understood by one of skill in the art that there
is a high degree of sequence identity between the orthologs of
ActRIIB For example, a murine ortholog of human ActRIIB has been
described (NCBI Ref. Seq.: NP 031423) that differs by only one
amino acid substitution in the mature ActRIIB extracellular domain
(119 amino acids). Accordingly, agents binding to human ActRIIB
will be expected to bind to murine ActRIIB in cases where the
recognition site of the binding agent, e.g., an antibody binding
site such as an epitope, is highly conserved and in particular
nearly or completely identical to the human sequence. Thus, when
the term "specific binding to ActRIIB" is used, it is understood to
include binding to multiple species of ActRIIB where the sequences
between species are conserved.
[0034] Given the known structure of ActRIIB and the highly
characterized ligand binding interface (see, e.g., Weber et al.
2007, BMC Structural Biology 7:6; Thompson et al. 2003 EMBO J.
22:1555-1566; WO 2006/012627), it is understood that neutralizing
anti-ActRIIB antibodies will bind to amino acids within one or more
of the following strings of amino acids of ActRIIB as follows
(numbering is relative to SEQ ID NO:1): amino acids 77-83 of SEQ ID
NO:1, amino acids 60-64 of SEQ ID NO:1, 73-74 of SEQ ID NO:1, amino
acids 73-83 of SEQ ID NO:1, amino acids 98-101 of SEQ ID NO:1;
amino acids 35-39 of SEQ ID NO:1 and/or amino acids 52-55 of SEQ ID
NO:1.
[0035] Examples of binding agents according to the invention
include the antibody 17G05 (Ab-17G05) and the corresponding
Fab-17G05. As used herein, Ab-17G05 comprises the polypeptides
expressed by the nucleotides shown in SEQ ID NOs: 17 and 18.
[0036] Binding agents of the invention are typically antibodies or
fragments thereof, as defined herein. The term "antibody" refers to
an intact antibody, or a binding fragment thereof. An antibody may
comprise a complete antibody molecule (including polyclonal,
monoclonal, chimeric, humanized, or human versions having
full-length heavy and/or light chains), or comprise an
antigen-binding fragment thereof. Antibody fragments include
F(ab').sub.2, Fab, Fab', Fv, Fc, and Fd fragments, and can be
incorporated into single-domain antibodies, single-chain
antibodies, maxibodies, minibodies, intrabodies, diabodies,
triabodies, tetrabodies, v-NAR and bis-scFv (See e.g., Hollinger
and Hudson, 2005, Nature Biotechnology, 23, 9, 1 126-1136).
Antibody-like polypeptides are also disclosed in U.S. Pat. No.
6,703,199 ["Artificial Antibody Polypeptides", assigned to Research
Corp Technologies], including fibronectin polypeptide monobodies.
Other antibody-like polypeptides are disclosed in U.S. patent
publication 2005/0238646, which are single-chain polypeptides. As
used herein, the isolated antibody or an antigen-binding fragment
thereof may be a polyclonal antibody, a monoclonal antibody, a
humanized antibody, a human antibody, a chimeric antibody, or the
like. In each of these types of binding agents, it is generally
expected that one would insert one or more CDRs from the antibodies
disclosed herein to produce an alternative ActRIIB binding
agent.
[0037] An antibody according to the present invention may belong to
any immunoglobin class, for example IgG, IgE, IgM, IgD, or IgA. It
may be obtained from or derived from an animal, for example, birds
(e.g., chicken) and mammals, which include but are not limited to a
mouse, rat, hamster, rabbit, cow, horse, sheep, goat, camel, human,
or other primate. The antibody may be an internalizing antibody.
Within the human IgG class, classes IgG1, IgG2 and IgG4 are
particularly useful. An anti-ActRIIB antibody of the IgG2 or IgG4
class may be particularly useful as a therapeutic as these classes
will diminish the action of the immune system against cells to
which the anti-ActRIIB antibody binds.
[0038] Antigen binding fragments derived from an antibody can be
obtained, for example, by proteolytic hydrolysis of the antibody,
for example, pepsin or papain digestion of whole antibodies
according to conventional methods. By way of example, antibody
fragments can be produced by enzymatic cleavage of antibodies with
pepsin to provide a 5S fragment termed F(ab').sub.2. This fragment
can be further cleaved using a thiol reducing agent to produce 3.5S
Fab monovalent fragments. Optionally, the cleavage reaction can be
performed using a blocking group for the sulfhydryl groups that
result from cleavage of disulfide linkages. As an alternative, an
enzymatic cleavage using papain produces two monovalent Fab
fragments and an Fc fragment directly. These methods are described,
for example, by Goldenberg, U.S. Pat. No. 4,331,647, Nisonoff et
al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J.
73:119, 1959; Edelman et al., in Methods in Enzymology 1 :422
(Academic Press 1967); and by Andrews, S. M. and Titus, J. A. in
Current Protocols in Immunology (Coligan J. E., et al., eds), John
Wiley & Sons, New York (2003), pages 2.8.1-2.8.10 and
2.10A.1-2.10A.5. Other methods for cleaving antibodies, such as
separating heavy chains to form monovalent light-heavy chain
fragments (Fd), further cleaving of fragments, or other enzymatic,
chemical, or genetic techniques may also be used, so long as the
fragments bind to the antigen that is recognized by the intact
antibody.
[0039] An antibody fragment may also be any synthetic or
genetically engineered protein. For example, antibody fragments
include isolated fragments consisting of the light chain variable
region, "Fv" fragments consisting of the variable regions of the
heavy and light chains, recombinant single-chain polypeptide
molecules in which light and heavy variable regions are connected
by a peptide linker (scFv proteins).
[0040] Another form of an antibody fragment is a peptide comprising
one or more complementarity determining regions (CDRs) of an
antibody. CDRs (also termed "minimal recognition units", or
"hypervariable regions") can be obtained by constructing
polynucleotides that encode the CDR of interest. Such
polynucleotides are prepared, for example, by using the polymerase
chain reaction to synthesize the variable region using mRNA of
antibody-producing cells as a template (see, for example, Larrick
et al., Methods: A Companion to Methods in Enzymology 2:106, 1991;
Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in
Monoclonal Antibodies. Production, Engineering and Clinical
Application, Ritter et al. (eds.), page 166 (Cambridge University
Press 1995); and Ward et al., "Genetic Manipulation and Expression
of Antibodies," in Monoclonal Antibodies: Principles and
Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc.
1995)).
[0041] Thus, in one embodiment, the binding agent comprises at
least one CDR as described herein. The binding agent may comprise
at least two, three, four, five or six CDRs, as described herein.
The binding agent further may comprise at least one variable region
domain of an antibody described herein. The variable region domain
may be of any size or amino acid composition and will generally
comprise at least one CDR sequence responsible for binding to human
ActRIIB, for example CDR-H1, CDR-H2, CDR-H3, and/or the light chain
CDRs specifically described herein and which are adjacent to or in
frame with one or more framework sequences. In general terms, the
variable (V) region domain may be any suitable arrangement of
immunoglobulin heavy (VH) and/or light (VL) chain variable domains.
Thus, for example, the V region domain may be monomeric and be a VH
or VL domain, which is capable of independently binding human
ActRIIB with an affinity at least equal to 1.times.10.sup.-7M or
less as described below. Alternatively, the V region domain may be
dimeric and contain VH-VH, VH-VL, or VL-VL dimers. The V region
dimer comprises at least one VH and at least one VL chain that may
be non-covalently associated (hereinafter referred to as FV). If
desired, the chains may be covalently coupled either directly, for
example via a disulfide bond between the two variable domains, or
through a linker, for example a peptide linker, to form a single
chain Fv (scFV).
[0042] The variable region domain may be any naturally occurring
variable domain or an engineered version thereof. By engineered
version is meant a variable region domain that has been created
using recombinant DNA engineering techniques. Such engineered
versions include those created, for example, from a specific
antibody variable region by insertions, deletions, or changes in or
to the amino acid sequences of the specific antibody. Particular
examples include engineered variable region domains containing at
least one CDR and optionally one or more framework amino acids from
a first antibody and the remainder of the variable region domain
from a second antibody.
[0043] The variable region domain may be covalently attached at a
C-terminal amino acid to at least one other antibody domain or a
fragment thereof. Thus, for example, a VH domain that is present in
the variable region domain may be linked to an immunoglobulin CH1
domain, or a fragment thereof. Similarly a VL domain may be linked
to a CK domain or a fragment thereof. In this way, for example, the
antibody may be a Fab fragment wherein the antigen binding domain
contains associated VH and VL domains covalently linked at their
C-termini to a CH1 and CK domain, respectively. The CH1 domain may
be extended with further amino acids, for example to provide a
hinge region or a portion of a hinge region domain as found in a
Fab fragment, or to provide further domains, such as antibody CH2
and CH3 domains.
[0044] As described herein, binding agents may comprise at least
one of these CDRs. For example, one or more CDRs may be
incorporated into known antibody framework regions (IgG1, IgG2,
etc.), or conjugated to a suitable vehicle to enhance the half-life
thereof. Suitable vehicles include, but are not limited to Fc,
polyethylene glycol (PEG), albumin, transferrin, and the like.
These and other suitable vehicles are known in the art. Such
conjugated CDR peptides may be in monomeric, dimeric, tetrameric,
or other form. In one embodiment, one or more water-soluble polymer
is bonded at one or more specific position, for example at the
amino terminus, of a binding agent.
[0045] In certain embodiments, a binding agent comprises one or
more water soluble polymer attachments, including, but not limited
to, polyethylene glycol, polyoxyethylene glycol, or polypropylene
glycol. See, e.g., U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144,
4,670,417, 4,791,192 and 4,179,337. In certain embodiments, a
derivative binding agent comprises one or more of
monomethoxy-polyethylene glycol, dextran, cellulose, or other
carbohydrate-based polymers, poly-(N-vinyl
pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a
polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated
polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures
of such polymers. In certain embodiments, one or more water-soluble
polymers is randomly attached to one or more side chains. In
certain embodiments, PEG can act to improve the therapeutic
capacity for a binding agent, such as an antibody. Certain such
methods are discussed, for example, in U.S. Pat. No. 6,133,426,
which is hereby incorporated by reference for any purpose.
[0046] Antibodies according to the invention may be obtained by
conventional immunization and cell fusion procedures as described
herein and known in the art. Monoclonal antibodies of the invention
may be generated using a variety of known techniques. In general,
monoclonal antibodies that bind to specific antigens may be
obtained by methods known to those skilled in the art (see, for
example, Kohler et al., Nature 256:495, 1975; Coligan et al.
(eds.), Current Protocols in Immunology, 1 :2.5.12.6.7 (John Wiley
& Sons 1991); U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439,
and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension
in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol
(eds.) (1980); and Antibodies: A Laboratory Manual, Harlow and Lane
(eds.), Cold Spring Harbor Laboratory Press (1988); Picksley et
al., "Production of monoclonal antibodies against proteins
expressed in E. coli," in DNA Cloning 2: Expression Systems, 2nd
Edition, Glover et al. (eds.), page 93 (Oxford University Press
1995)). Antibody fragments may be derived therefrom using any
suitable standard technique such as proteolytic digestion, or
optionally, by proteolytic digestion (for example, using papain or
pepsin) followed by mild reduction of disulfide bonds and
alkylation. Alternatively, such fragments may also be generated by
recombinant genetic engineering techniques as described herein.
[0047] Monoclonal antibodies can be obtained by injecting an
animal, for example, a rat, hamster, rabbit, or mouse, with an
immunogen comprising human ActRIIB of SEQ ID NO: 1, or a fragment
thereof, according to methods known in the art and described
herein. A polypeptide comprising amino acids 19-134 or 20-134 of
SEQ ID NO:1 is particularly useful for generating antibodies that
bind to the extracellular domain of ActRIIB, which includes the
ligand binding portion. The presence of specific antibody
production may be monitored after the initial injection and/or
after a booster injection by obtaining a serum sample and detecting
the presence of an antibody that binds to human ActRIIB or peptide
using any one of several immunodetection methods known in the art
and described herein. From animals producing the desired
antibodies, lymphoid cells, most commonly cells from the spleen or
lymph node, are removed to obtain B-lymphocytes. The B lymphocytes
are then fused with a drug-sensitized myeloma cell fusion partner,
preferably one that is syngeneic with the immunized animal and that
optionally has other desirable properties (e.g., inability to
express endogenous Ig gene products, e.g., P3X63-Ag 8.653 (ATCC No.
CRL 1580); NSO, SP20) to produce hybridomas, which are immortal
eukaryotic cell lines. The lymphoid (e.g., spleen) cells and the
myeloma cells may be combined for a few minutes with a membrane
fusion-promoting agent, such as polyethylene glycol or a nonionic
detergent, and then plated at low density on a selective medium
that supports the growth of hybridoma cells but not unfused myeloma
cells. A preferred selection media is HAT (hypoxanthine,
aminopterin, thymidine). After a sufficient time, usually about one
to two weeks, colonies of cells are observed. Single colonies are
isolated, and antibodies produced by the cells may be tested for
binding activity to human ActRIIB, using any one of a variety of
immunoassays known in the art and described herein. The hybridomas
are cloned (e.g., by limited dilution cloning or by soft agar
plaque isolation) and positive clones that produce an antibody
specific to ActRIIB are selected and cultured. The monoclonal
antibodies from the hybridoma cultures may be isolated from the
supernatants of hybridoma cultures. An alternative method for
production of a murine monoclonal antibody is to inject the
hybridoma cells into the peritoneal cavity of a syngeneic mouse,
for example, a mouse that has been treated (e.g., pristane-primed)
to promote formation of ascites fluid containing the monoclonal
antibody. Monoclonal antibodies can be isolated and purified by a
variety of well-established techniques. Such isolation techniques
include affinity chromatography with protein-A Sepharose,
size-exclusion chromatography, and ion-exchange chromatography
(see, for example, Coligan at pages 2.7.1-2.7.12 and pages
2.9.1-2.9.3; Baines et al., "Purification of Immunoglobulin G
(IgG)," in Methods in Molecular Biology, Vol. 10, pages 79- 104
(The Humana Press, Inc. 1992)). Monoclonal antibodies may be
purified by affinity chromatography using an appropriate ligand
whose selection is based on particular properties of the antibody
(e.g., heavy- or light-chain isotype, binding specificity, etc.).
Examples of a suitable ligand, immobilized on a solid support,
include Protein A, Protein G, an anti-constant region (light chain
or heavy chain) antibody, an anti-idiotype antibody, or fragment or
variant thereof.
[0048] It will be appreciated by one of skill in the art that a
binding agent of the present invention may have at least one amino
acid substitution, providing that the binding agent retains binding
specificity. Therefore, modifications to the binding agent
structures are encompassed within the scope of the invention. These
may include amino acid substitutions, which may be conservative or
non-conservative and that do not destroy the ActRIIB binding
capability of a binding agent. Conservative amino acid
substitutions may encompass non-naturally occurring amino acid
residues, which are typically incorporated by chemical peptide
synthesis rather than by synthesis in biological systems. These
include peptidomimetics and other reversed or inverted forms of
amino acid moieties. A conservative amino acid substitution may
also involve a substitution of a native amino acid residue with a
normative residue such that there is little or no effect on the
polarity or charge of the amino acid residue at that position.
[0049] Conservative substitutions are shown in Table 1 under the
heading of "preferred substitutions". If such substitutions result
in a change in biological activity, then more substantial changes,
denominated "exemplary substitutions" in Table 1, or as further
described below in reference to amino acid classes, may be
introduced and the products screened.
TABLE-US-00003 TABLE 1 Conservative Substitutions Original
Preferred Residue Exemplary Substitutions Substitutions Ala (A)
Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp;
Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;
Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Arg; Asn; Gln;
Lys Arg Ile (I) Leu; Val; Met; Ala; Phe; Leu Norleucine Leu (L)
Ile; Norleucine; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg
Met (M) Leu; Phe; Ile Leu Phe (F) Tyr; Trp; Leu; Val; Ile; Ala Tyr
Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser; Val Ser Trp (W) Tyr;
Phe Tyr Tyr (Y) Phe; Trp; Thr; Ser Phe Val (V) Leu; Ile;
Norleucine; Met; Phe; Ala Leu
[0050] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties:
[0051] (1) hydrophobic: Met, Ala, Val, Leu, Ile, Norleucine;
[0052] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0053] (3) acidic: Asp, Glu;
[0054] (4) basic: His, Lys, Arg;
[0055] (5) residues that influence chain orientation: Gly, Pro;
and
[0056] (6) aromatic: Trp, Tyr, Phe.
[0057] Non-conservative substitutions may involve the exchange of a
member of one class of amino acids or amino acid mimetics for a
member from another class with different physical properties (e.g.
size, polarity, hydrophobicity, charge). Such substituted residues
may be introduced into regions of the human antibody that are
homologous with non-human antibodies, or into the nonhomologous
regions of the molecule.
[0058] Moreover, one skilled in the art may generate test variants
containing a single amino acid substitution at each desired amino
acid residue. The variants can then be screened using activity
assays as described herein. Such variants could be used to gather
information about suitable variants. For example, if one discovered
that a change to a particular amino acid residue resulted in
destroyed, undesirably reduced, or unsuitable activity, variants
with such a change may be avoided. In other words, based on
information gathered from such routine experiments, one skilled in
the art can readily determine the amino acids where further
substitutions should be avoided either alone or in combination with
other mutations.
[0059] A skilled artisan will be able to determine suitable
variants of the polypeptide as set forth herein using well-known
techniques. In certain embodiments, one skilled in the art may
identify suitable areas of the molecule that may be changed without
destroying activity by targeting regions not believed to be
important for activity. In certain embodiments, one can identify
residues and portions of the molecules that are conserved among
similar polypeptides. In certain embodiments, even areas that may
be important for biological activity or for structure may be
subject to conservative amino acid substitutions without destroying
the biological activity or without adversely affecting the
polypeptide structure.
[0060] Additionally, one skilled in the art can review
structure-function studies identifying residues in similar
polypeptides that are important for activity or structure. In view
of such a comparison, one can predict the importance of amino acid
residues in a protein that correspond to amino acid residues which
are important for activity or structure in similar proteins. One
skilled in the art may opt for chemically similar amino acid
substitutions for such predicted important amino acid residues.
[0061] A number of scientific publications have been devoted to the
prediction of secondary structure. See Moult J., Curr. Op. in
Biotech., 7(4):422-427 (1996), Chou et al., Biochemistry,
13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222
(1974); Chou et al., Adv. Enzymol. Relat. Areas MoI. Biol., 47:
45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276 and Chou
et al., Biophys. J., 26:367-384 (1979). Moreover, computer programs
are currently available to assist with predicting secondary
structure. One method of predicting secondary structure is based
upon homology modeling. For example, two polypeptides or proteins
which have a sequence identity of greater than 30%, or sequence
similarity greater than 40% often have similar structural
topologies. The recent growth of the protein structural database
(PDB) has provided enhanced predictability of secondary structure,
including the potential number of folds within a polypeptide's or
protein's structure. See Holm et al., Nucl. Acid. Res.,
27(0:244-247 (1999). It has been suggested (Brenner et al., Curr.
Op. Struct. Biol., 7(3):369-376 (1997)) that there are a limited
number of folds in a given polypeptide or protein and that once a
critical number of structures have been resolved, structural
prediction will become dramatically more accurate.
[0062] Additional methods of predicting secondary structure include
"threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87
(1997); Sippl et al., Structure, 4(1):15-19 (1996)), "profile
analysis" (Bowie et al., Science, 253:164- 170 (1991); Gribskov et
al., Meth. Enzym., 183:146-159 (1990); Gribskov et al., Proc. Nat.
Acad. Sci., 84(13):4355-4358 (1987)), and "evolutionary linkage"
(See Holm, supra (1999), and Brenner, supra (1997)).
[0063] It will be understood by one skilled in the art that some
proteins, such as antibodies, may undergo a variety of
posttranslational modifications during expression and secretion
from host cells. The type and extent of these modifications often
depends on the host cell line used to express the protein as well
as the culture conditions. Such modifications may include
variations in glycosylation, methionine or tryptophan oxidation,
diketopiperizine formation, aspartate isomerization and asparagine
deamidation. A frequent modification is the loss of a
carboxy-terminal basic residue (such as lysine or arginine) due to
the action of carboxypeptidases (as described in Harris, R J.
Journal of Chromatography 705:129-134, 1995). Once the proteins
have been expressed and processed they are in a `mature` form. Thus
it is understood that the invention includes mature antibodies that
result from expression of the DNAs of the invention.
[0064] In certain embodiments, variants of binding agents include
glycosylation variants wherein the number and/or type of
glycosylation site has been altered compared to the amino acid
sequences of a parent polypeptide. In certain embodiments, variants
comprise a greater or a lesser number of N-linked glycosylation
sites than the native protein. An N-linked glycosylation site is
characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the
amino acid residue designated as X may be any amino acid residue
except proline. The substitution of amino acid residues to create
this sequence provides a potential new site for the addition of an
N-linked carbohydrate chain. Alternatively, substitutions which
eliminate this sequence will remove an existing N-linked
carbohydrate chain. Also provided is a rearrangement of N-linked
carbohydrate chains wherein one or more N-linked glycosylation
sites (typically those that are naturally occurring) are eliminated
and one or more new N-linked sites are created. Additional
preferred antibody variants include cysteine variants wherein one
or more cysteine residues are deleted from or substituted for
another amino acid (e.g., serine) as compared to the parent amino
acid sequence. Cysteine variants may be useful when antibodies must
be refolded into a biologically active conformation such as after
the isolation of insoluble inclusion bodies. Cysteine variants
generally have fewer cysteine residues than the native protein, and
typically have an even number to minimize interactions resulting
from unpaired cysteines.
[0065] Amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired. In certain embodiments,
amino acid substitutions can be used to identify important residues
of antibodies to ActRIIB, or to increase or decrease the affinity
of the antibodies to ActRIIB described herein.
[0066] According to certain embodiments, preferred amino acid
substitutions are those which: (1) reduce susceptibility to
proteolysis, (2) reduce susceptibility to oxidation, (3) alter
binding affinity for forming protein complexes, (4) alter binding
affinities, and/or (4) confer or modify other physiochemical or
functional properties on such polypeptides. According to certain
embodiments, single or multiple amino acid substitutions (in
certain embodiments, conservative amino acid substitutions) may be
made in the naturally-occurring sequence (in certain embodiments,
in the portion of the polypeptide outside the domain(s) forming
intermolecular contacts). In certain embodiments, a conservative
amino acid substitution typically may not substantially change the
structural characteristics of the parent sequence (e.g., a
replacement amino acid should not tend to break a helix that occurs
in the parent sequence, or disrupt other types of secondary
structure that characterizes the parent sequence). Examples of
art-recognized polypeptide secondary and tertiary structures are
described in Proteins, Structures and Molecular Principles
(Creighton, Ed., W. H. Freeman and Company, New York (1984));
Introduction to Protein Structure (C. Branden and J. Tooze, eds.,
Garland Publishing, New York, N.Y. (1991)); and Thornton et al.
Nature 354:105 (1991), which are each incorporated herein by
reference.
[0067] In certain embodiments, binding agents of the invention may
be chemically bonded with polymers, lipids, or other moieties.
[0068] The binding agents may comprise at least one of the CDRs
described herein incorporated into a biocompatible framework
structure. In one example, the biocompatible framework structure
comprises a polypeptide or portion thereof that is sufficient to
form a conformationally stable structural support, or framework, or
scaffold, which is able to display one or more sequences of amino
acids (e.g., CDRs, a variable region, etc.) that bind to an antigen
in a localized surface region. Such structures can be a naturally
occurring polypeptide or polypeptide "fold" (a structural motif),
or can have one or more modifications, such as additions, deletions
or substitutions of amino acids, relative to a naturally occurring
polypeptide or fold. These scaffolds can be derived from a
polypeptide of any species (or of more than one species), such as a
human, other mammal, other vertebrate, invertebrate, plant,
bacteria, or virus.
[0069] Typically the biocompatible framework structures are based
on protein scaffolds or skeletons other than immunoglobulin
domains. For example, those based on fibronectin, ankyrin,
lipocalin, neocarzinostain, cytochrome b, CP1 zinc finger, PST1,
coiled coil, LAC1-D1, Z domain and tendramisat domains may be used
(See e.g., Nygren and Uhlen, 1997, Current Opinion in Structural
Biology, 7, 463-469).
[0070] In preferred embodiments, it will be appreciated that the
binding agents of the invention include humanized antibodies, which
can be produced using techniques known to those skilled in the art
(Zhang, W., et al., Molecular Immunology. 42(12): 1445-1451, 2005;
Hwang W. et al., Methods. 36(1):35-42, 2005; Dall'Acqua W F, et
al., Methods 36(1):43-60, 2005; and Clark, M., Immunology Today.
21(8):397-402, 2000).
[0071] An antibody of the present invention may also be a human
monoclonal antibody. Human monoclonal antibodies may be generated
by any number of techniques with which those having ordinary skill
in the art will be familiar. Such methods include, but are not
limited to, Epstein Barr Virus (EBV) transformation of human
peripheral blood cells (e.g., containing B lymphocytes), in vitro
immunization of human B cells, fusion of spleen cells from
immunized transgenic mice carrying inserted human immunoglobulin
genes, isolation from human immunoglobulin V region phage
libraries, or other procedures as known in the art and based on the
disclosure herein. For example, human monoclonal antibodies may be
obtained from transgenic mice that have been engineered to produce
specific human antibodies in response to antigenic challenge.
Methods for obtaining human antibodies from transgenic mice are
described, for example, by Green et al., Nature Genet. 7:13, 1994;
Lonberg et al., Nature 368:856, 1994; Taylor et al., Int. Immun.
6:579, 1994; U.S. Pat. No. 5,877,397; Bruggemann et al., 1997 Curr.
Opin. Biotechnol. 8:455-58; Jakobovits et al., 1995 Ann. N. Y Acad.
Sci. 764:525-35. In this technique, elements of the human heavy-
and light-chain locus are introduced into strains of mice derived
from embryonic stem cell lines that contain targeted disruptions of
the endogenous heavy-chain and light-chain loci (see also
Bruggemann et al., Curr. Opin. Biotechnol. 8:455-58 (1997)). For
example, human immunoglobulin transgenes may be mini-gene
constructs, or transloci on yeast artificial chromosomes, which
undergo B cell-specific DNA rearrangement and hypermutation in the
mouse lymphoid tissue. Human monoclonal antibodies may be obtained
by immunizing the transgenic mice, which may then produce human
antibodies specific for ActRIIB Lymphoid cells of the immunized
transgenic mice can be used to produce human antibody-secreting
hybridomas according to the methods described herein. Polyclonal
sera containing human antibodies may also be obtained from the
blood of the immunized animals.
[0072] Another method for generating human antibodies of the
invention includes immortalizing human peripheral blood cells by
EBV transformation. See, e.g., U.S. Pat. No. 4,464,456. Such an
immortalized B cell line (or lymphoblastoid cell line) producing a
monoclonal antibody that specifically binds to ActRIIB can be
identified by immunodetection methods as provided herein, for
example, an ELISA, and then isolated by standard cloning
techniques. The stability of the lymphoblastoid cell line producing
an anti-ActRIIB antibody may be improved by fusing the transformed
cell line with a murine myeloma to produce a mouse-human hybrid
cell line according to methods known in the art (see, e.g., Glasky
et al., Hybridoma 8:377-89 (1989)). Still another method to
generate human monoclonal antibodies is in vitro immunization,
which includes priming human splenic B cells with human ActRIIB,
followed by fusion of primed B cells with a heterohybrid fusion
partner. See, e.g., Boerner et al., 1991, J. Immunol.
147:86-95.
[0073] In certain embodiments, a B cell that is producing an
anti-human ActRIIB antibody is selected and the light chain and
heavy chain variable regions are cloned from the B cell according
to molecular biology techniques known in the art (WO 92/02551 ;
U.S. Pat. No. 5,627,052; Babcook et al., Proc. Natl. Acad. Sci. USA
93:7843-48 (1996)) and described herein. B cells from an immunized
animal may be isolated from the spleen, lymph node, or peripheral
blood sample by selecting a cell that is producing an antibody that
specifically binds to ActRIIB B cells may also be isolated from
humans, for example, from a peripheral blood sample. Methods for
detecting single B cells that are producing an antibody with the
desired specificity are well known in the art, for example, by
plaque formation, fluorescence-activated cell sorting, in vitro
stimulation followed by detection of specific antibody, and the
like. Methods for selection of specific antibody-producing B cells
include, for example, preparing a single cell suspension of B cells
in soft agar that contains human ActRIIB Binding of the specific
antibody produced by the B cell to the antigen results in the
formation of a complex, which may be visible as an
immunoprecipitate. After the B cells producing the desired antibody
are selected, the specific antibody genes may be cloned by
isolating and amplifying DNA or mRNA according to methods known in
the art and described herein.
[0074] Additionally, one skilled in the art will recognize that
suitable binding agents include portions of these antibodies, such
as one or more of CDR-H1, CDR-H2, CDR-H3, CDR-Ll, CDR-L2 and
CDR-L3, as specifically disclosed herein. At least one of the
regions of CDR-H1, CDR-H2, CDR-H3, CDR-Ll, CDR-L2 and CDR-L3 may
have at least one amino acid substitution, provided that the
binding agent retains the binding specificity of the
non-substituted CDR. CDRs may be altered to increase or decrease
length as well, and thus changes that are characterized as
substitutions, insertions and deletions are all contemplated. The
non-CDR portion of the binding agent may be a non-protein molecule,
wherein the binding agent cross-blocks the binding of an antibody
disclosed herein to ActRIIB and/or neutralizes ActRIIB The non-CDR
portion of the binding agent may be composed of amino acids,
wherein the binding agent is a recombinant binding protein or a
synthetic peptide, and the recombinant binding protein cross-blocks
the binding of an antibody disclosed herein to ActRIIB and/or
neutralizes ActRIIB The non-CDR portion of the binding agent may be
composed of amino acids, wherein the binding agent is a recombinant
binding protein, and the recombinant binding protein exhibits a
similar binding pattern to human ActRIIB peptides in the human
ActRIIB peptide epitope competition binding assay (described
hereinbelow) as that exhibited by antibody Ab-17G05, and/or
neutralizes ActRIIB
[0075] In one embodiment, it is contemplated that one can use the
antibody heavy chain as `bait` in a library screen where the
library is composed of human antibody light chains, to identify
complementing human light chains where the reconstituted antibody
binds to ActRIIB In this embodiment, the heavy chain is from an
antibody specific to ActRIIB and is mouse, chimeric, or
humanized.
[0076] Where an antibody comprises one or more of CDR-H1, CDR-H2,
CDR-H3, CDR-L1, CDR-L2, and CDR-L3, as described above, it may be
obtained by expression from a host cell containing DNA coding for
these sequences. A DNA coding for each CDR sequence may be
determined on the basis of the amino acid sequence of the CDR and
synthesized together with any desired antibody variable region
framework and constant region DNA sequences using oligonucleotide
synthesis techniques, site-directed mutagenesis and polymerase
chain reaction (PCR) techniques as appropriate. DNA coding for
variable region frameworks and constant regions is widely available
to those skilled in the art from genetic sequences databases such
as GenBank.RTM..
[0077] Once synthesized, the DNA encoding an antibody of the
invention or fragment thereof may be propagated and expressed
according to any of a variety of well-known procedures for nucleic
acid excision, ligation, transformation, and transfection using any
number of known expression vectors. Thus, in certain embodiments
expression of an antibody fragment may be preferred in a
prokaryotic host, such as Escherichia coli (see, e.g., Pluckthun et
al., 1989 Methods Enzymol. 178:497-515). In certain other
embodiments, expression of the antibody or a fragment thereof may
be preferred in a eukaryotic host cell, including yeast (e.g.,
Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia
pastoris), animal cells (including mammalian cells) or plant cells.
Examples of suitable animal cells include, but are not limited to,
myeloma (such as a mouse NSO line), COS, CHO, or hybridoma cells.
Examples of plant cells include tobacco, corn, soybean, and rice
cells.
[0078] One or more replicable expression vectors containing DNA
encoding an antibody variable and/or constant region may be
prepared and used to transform an appropriate cell line, for
example, a non-producing myeloma cell line, such as a mouse NSO
line or a bacteria, such as E. coli, in which production of the
antibody will occur. To obtain efficient transcription and
translation, the DNA sequence in each vector should include
appropriate regulatory sequences, particularly a promoter and
leader sequence operatively linked to the variable domain sequence.
Particular methods for producing antibodies in this way are
generally well-known and routinely used. For example, basic
molecular biology procedures are described by Maniatis et al.
(Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring
Harbor Laboratory, New York, 1989; see also Maniatis et al, 3rd
ed., Cold Spring Harbor Laboratory, New York, (2001)). DNA
sequencing can be performed as described in Sanger et al. (PNAS
74:5463, (1977)) and the Amersham International pic sequencing
handbook, and site directed mutagenesis can be carried out
according to methods known in the art (Kramer et al., Nucleic Acids
Res. 12:9441, (1984); Kunkel Proc. Natl. Acad. Sci. USA 82:488-92
(1985); Kunkel et al., Methods in Enzymol. 154:367-82 (1987); the
Anglian Biotechnology Ltd handbook). Additionally, numerous
publications describe techniques suitable for the preparation of
antibodies by manipulation of DNA, creation of expression vectors,
and transformation and culture of appropriate cells (Mountain A and
Adair, J R in Biotechnology and Genetic Engineering Reviews (ed.
Tombs, M P, 10, Chapter 1 , 1992, Intercept, Andover, UK); "Current
Protocols in Molecular Biology", 1999, F. M. Ausubel (ed.), Wiley
Interscience, New York).
[0079] Where it is desired to improve the affinity of antibodies
according to the invention containing one or more of the
above-mentioned CDRs, improved antibodies can be obtained by a
number of affinity maturation protocols including maintaining the
CDRs (Yang et al., J. MoI. Biol., 254, 392-403, 1995), chain
shuffling (Marks et al., Bio/Technology, 10, 779-783, 1992), use of
mutation strains of E. coli. (Low et al., J. MoI. Biol., 250,
350-368, 1996), DNA shuffling (Patten et al., Curr. Opin.
Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J.
MoI. Biol., 256, 7-88, 1996) and sexual PCR (Crameri, et al.,
Nature, 391, 288-291, 1998). All of these methods of affinity
maturation are discussed by Vaughan et al. (Nature Biotechnology,
16, 535-539, 1998).
[0080] An additional method for obtaining or maturing antibodies of
the invention is by phage display. See, e.g., Winter et al., 1994
Annu. Rev. Immunol. 12:433-55; Burton et al., 1994 Adv. Immunol.
57:191-280. See also the methodology described in the Examples.
Combinatorial libraries of human or murine immunoglobulin
variable-region genes may be created in phage vectors that can be
screened to select Ig fragments (Fab, Fv, sFv, or multimers
thereof) that bind specifically to ActRIIB or variant or fragment
thereof. See, e.g., U.S. Pat. No. 5,223,409; Huse et al., 1989
Science 246:1275-81; Sastry et al., Proc. Natl. Acad. Sci. USA
86:5728-32 (1989); Alting- Mees et al., Strategies in Molecular
Biology 3:1-9 (1990); Kang et al., 1991 Proc. Natl. Acad. Sci. USA
88:4363-66; Hoogenboom et al., 1992 J. Molec. Biol. 227:381-388;
Schlebusch et al., 1997 Hybridoma 16:47-52 and references cited
therein; and Hoet et al., 2005, Nat Biotechnol 23:344-348. For
example, a library containing a plurality of polynucleotide
sequences encoding Ig variable region fragments may be inserted
into the genome of a filamentous bacteriophage, such as M 13 or a
variant thereof, in frame with the sequence encoding a phage coat
protein. A fusion protein may be a fusion of the coat protein with
the light chain variable-region domain and/or with the heavy chain
variable-region domain. According to certain embodiments,
immunoglobulin Fab fragments may also be displayed on a phage
particle (see, e.g., U.S. Pat. No. 5,698,426).
[0081] Heavy and light chain immunoglobulin cDNA expression
libraries may also be prepared in lambda phage, for example, using
lambda ImmunoZap TM (H) and lambda ImmunoZap TM (L) vectors
(Stratagene, La Jolla, Calif.). Briefly, mRNA is isolated from a B
cell population, and used to create heavy and light chain
immunoglobulin cDNA expression libraries in the lambda ImmunoZap(H)
and lambda ImmunoZap(L) vectors. These vectors may be screened
individually or co-expressed to form Fab fragments or antibodies
(see Huse et al., supra; see also Sastry et al., supra). Positive
plaques may subsequently be converted to a non-lytic plasmid that
allows high level expression of monoclonal antibody fragments from
E. coli.
[0082] In one embodiment in a hybridoma, the variable regions of a
gene expressing a monoclonal antibody of interest are amplified
using nucleotide primers. These primers may be synthesized by one
of ordinary skill in the art, or may be purchased from commercially
available sources. (See, e.g., Stratagene (La Jolla, Calif), which
sells primers for mouse and human variable regions including, among
others, primers for VHa, VHb, VHc, VHd, CHI, VL and CL regions.)
These primers may be used to amplify heavy or light chain variable
regions, which may then be inserted into vectors such as ImmunoZAP
TM H or ImmunoZAP TM (Stratagene), respectively. These vectors may
then be introduced into E. coli, yeast, or mammalian-based systems
for expression. Large amounts of a single-chain protein containing
a fusion of the VH and VL domains may be produced using these
methods (see Bird et al., Science 242:423-426, 1988).
[0083] Once cells producing antibodies according to the invention
have been obtained using any of the above-described immunization
and other techniques, the specific antibody genes may be cloned by
isolating and amplifying DNA or mRNA therefrom according to
standard procedures as described herein. The antibodies produced
therefrom may be sequenced and the CDRs identified and the DNA
coding for the CDRs may be manipulated as described previously to
generate other antibodies according to the invention.
[0084] Preferably the binding agents bind specifically to ActRIIB
As with all binding agents and binding assays, one of skill in this
art recognizes that the various moieties to which a binding agent
should not detectably bind in order to be therapeutically effective
and suitable would be exhaustive and impractical to list.
Therefore, for a binding agent disclosed herein, the term
"specifically binds" refers to the ability of a binding agent to
bind to ActRIIB, preferably human ActRIIB, with greater affinity
than it binds to an unrelated control protein. Preferably the
control protein is hen egg white lysozyme. Preferably the binding
agents bind to ActRIIB with an affinity that is at least, 50, 100,
250, 500, 1000, or 10,000 times greater than the affinity for a
control protein. A binding agent may have a binding affinity for
human ActRIIB of less than or equal to 1.times.10.sup.-7M, less
than or equal to 1.times.10.sup.-8M, less than or equal to
1.times.10.sup.-9M, less than or equal to 1.times.10.sup.-10M.sup.,
less than or equal to 1.times.10.sup.-11M, or less than or equal to
1.times.10.sup.-12M. Antibodies having improved affinity may be
generated by any of a variety of known maturation techniques, such
as those described above. Affinity may be assessed at different
temperatures, using any of the techniques described herein.
Temperatures of 20 deg. C, 25 deg. C or 37 deg. C may be used.
[0085] Affinity may be determined by an affinity ELISA assay. In
certain embodiments, affinity may be determined by a BIACORE.TM.
assay. In certain embodiments, affinity may be determined by a
kinetic method. In certain embodiments, affinity may be determined
by an equilibrium/solution method. Such methods are described in
further detail herein or known in the art.
[0086] The affinity of a binding agent such as an antibody or
binding partner, as well as the extent to which a binding agent
(such as an antibody) inhibits binding, can be determined by one of
ordinary skill in the art using conventional techniques, for
example by surface plasmon resonance (SPR; BIACORE.TM., Biosensor,
Piscataway, N.J.) or according to methods described by Scatchard et
al. (Ann. N.Y. Acad. Sci. 51 :660-672 (1949)). For surface plasmon
resonance, target molecules are immobilized on a solid phase and
exposed to ligands in a mobile phase running along a flow cell. If
ligand binding to the immobilized target occurs, the local
refractive index changes, leading to a change in SPR angle, which
can be monitored in real time by detecting changes in the intensity
of the reflected light. The rates of change of the SPR signal can
be analyzed to yield apparent rate constants for the association
and dissociation phases of the binding reaction. The ratio of these
values gives the apparent equilibrium constant (affinity) (see,
e.g., Wolff et al., Cancer Res. 53:2560-65 (1993)).
[0087] An oligopeptide or polypeptide is within the scope of the
invention if it comprises an amino acid sequence that is at least
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to at least one of the CDRs depicted in the Examples (SEQ
ID NOs: 19-42); and/or to a CDR of an ActRIIB binding agent that
cross-blocks the binding of Ab-17G05 to ActRIIB, and/or is
cross-blocked from binding to ActRIIB by Ab-17G05; and/or to a CDR
of an ActRIIB binding agent wherein the binding agent can block the
effect of ActRIIB in a cell-based assay (i.e. an ActRIIB
neutralizing binding agent).
[0088] Examples of ActRIIB binding agent polypeptides and
antibodies that are within the scope of the invention are those
that have amino acid sequences that are at least 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to a variable region of Ab-17G05 (SEQ ID NOs: 15 and 16),
and cross-block the binding of Ab-17G05 to ActRIIB, and/or are
cross-blocked from binding to ActRIIB by Ab-17G05; and/or can block
the inhibitory effect of ActRIIB in a cell-based assay (i.e. an
ActRIIB neutralizing binding agent).
[0089] Examples of polynucleotides encoding ActRIIB binding agents
that are within the scope of the invention are those that have
polynucleotide sequences that are at least 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a
polynucleotide encoding a variable region of Ab-17G05 (SEQ ID NOs:
17 and 18), and wherein the encoded ActRIIB binding agents
cross-block the binding of Ab-17G05 to ActRIIB, and/or are
cross-blocked from binding to ActRIIB by Ab-17G05; and/or can block
the inhibitory effect of ActRIIB in a cell-based assay (i.e. an
ActRIIB neutralizing binding agent).
[0090] ActRIIB binding agents of the present invention preferably
modulate ActRIIB function in the cell-based assay described herein
and/or the in vivo assay described herein and/or cross-block the
binding of antibody Ab-17G05 described in this application and/or
are cross-blocked from binding ActRIIB by the antibody Ab-17G05
described in this application. Accordingly, such binding agents can
be identified using the assays described herein.
[0091] In certain embodiments, binding agents are generated by
first identifying antibodies that neutralize ActRIIB in the
cell-based and/or in vivo assays described herein and/or
cross-block the antibody Ab-17G05 described in this application
and/or are cross-blocked from binding ActRIIB by antibody Ab-17G05
described in this application. The CDR regions from these
antibodies are then used to insert into appropriate biocompatible
frameworks to generate ActRIIB binding agents. The non-CDR portion
of the binding agent may be composed of amino acids, or may be a
nonprotein molecule. The assays described herein allow the
characterization of binding agents. Preferably the binding agents
of the present invention are antibodies as defined herein.
[0092] In the methods described herein to generate antibodies
according to the invention, including the manipulation of the
specific Ab-17G05 CDRs into new frameworks and/or constant regions,
appropriate assays are available to select the desired antibodies
or binding agents (i.e. assays for determining binding affinity to
ActRIIB; cross-blocking assays such as the BIACORE.TM.-based human
ActRIIB peptide competition binding assays described in Example 2
below; A204 cell-based assay; in vivo assays).
[0093] The terms "cross-block", "cross-blocked" and
"cross-blocking" are used interchangeably herein to mean the
ability of an antibody or other binding agent to interfere with the
binding of other antibodies or binding agents to ActRIIB
[0094] The extent to which an antibody or other binding agent is
able to interfere with the binding of another to ActRIIB, and
therefore whether it can be said to cross-block according to the
invention, can be determined using competition binding assays. One
particularly suitable quantitative assay uses a BIACORE.TM.
instrument which can measure the extent of interactions using
surface plasmon resonance technology. Example 2 provides methods
for conducting a BIACORE.TM. based cross-blocking assays. Another
suitable quantitative cross-blocking assay uses an ELISA-based
approach to measure competition between antibodies or other binding
agents in terms of their binding to ActRIIB
[0095] The following generally describes a suitable BIACORE.TM.
assay for determining whether an antibody or other binding agent
cross-blocks or is capable of cross-blocking according to the
invention. For convenience, reference is made to two antibodies,
but it will be appreciated that the assay can be used with any of
the ActRIIB binding agents described herein. The BIACORE.TM.
instrument (for example the BIACORE.TM. 3000) is operated according
to the manufacturer's recommendations.
[0096] Thus, in one cross-blocking assay, ActRIIB-mFc fusion
protein is captured on a CM5 BIACORE.TM. chip by previously
attached anti-mFc IgG to generate an ActRIIB-coated surface.
Typically 200-800 resonance units of ActRIIB-mFc (dimeric) would be
coupled to the chip (an amount that gives easily measurable levels
of binding but that is readily saturable by the concentrations of
test reagent being used).
[0097] The two antibodies (termed A* and B*) to be assessed for
their ability to cross-block each other are mixed at a one to one
molar ratio of binding sites in a suitable buffer to create the
test mixture. When calculating the concentrations on a binding site
basis the molecular weight of an antibody is assumed to be the
total molecular weight of the antibody divided by the number of
ActRIIB binding sites on that antibody.
[0098] The concentration of each antibody in the test mix should be
high enough to readily saturate the binding sites for that antibody
on the ActRIIB-mFc molecules captured on the BIACORE.TM. chip. The
antibodies in the mixture are at the same molar concentration (on a
binding basis) and that concentration would typically be between
1.00 and 1.5 micromolar (on a binding site basis).
[0099] Separate solutions containing antibody A* alone and antibody
B* alone are also prepared. Antibody A* and antibody B* in these
solutions should be in the same buffer and at the same
concentration as in the test mix.
[0100] The test mixture is passed over the ActRIIB-mFc-coated
BIACORE.TM. chip and the total amount of binding recorded. The chip
is then treated in such a way as to remove the bound antibodies
without damaging the chip-bound ActRIIB-mFc. Typically, this is
done by treating the chip with 30 mM HCl for 60 seconds.
[0101] The solution of antibody A* alone is then passed over the
ActRIIB-mFc-coated surface and the amount of binding recorded. The
chip is again treated to remove all of the bound antibody without
damaging the chip-bound ActRIIB-mFc.
[0102] The solution of antibody B* alone is then passed over the
ActRIIB-mFc-coated surface and the amount of binding recorded.
[0103] The maximum theoretical binding of the mixture of antibody
A* and antibody B* is next calculated, and is the sum of the
binding of each antibody when passed over the ActRIIB surface
alone. If the actual recorded binding of the mixture is less than
this theoretical maximum then the two antibodies are cross-blocking
each other.
[0104] Thus, in general, a cross-blocking antibody or other binding
agent according to the invention is one which will bind to ActRIIB
in the above BIACORE.TM. cross-blocking assay such that during the
assay and in the presence of a second antibody or other binding
agent of the invention the recorded binding is between 80% and 0.1%
(e.g. 80% to 4%) of the maximum theoretical binding, specifically
between 75% and 0.1% (e.g. 75% to 4%) of the maximum theoretical
binding, and more specifically between 70% and 0.1% (e.g. 70% to
4%) of maximum theoretical binding (as just defined above) of the
two antibodies or binding agents in combination.
[0105] The BIACORE.TM. assay described above is an assay used to
determine if antibodies or other binding agents cross-block each
other according to the invention. On rare occasions, particular
antibodies or other binding agents may not bind to ActRIIB-mFc
coupled via anti-mFc IgG to a CMS BIACORE.TM. chip (this might
occur when the relevant binding site on ActRIIB is masked or
destroyed by ActRIIB linkage to mFc). In such cases, cross-blocking
can be determined using a tagged version of ActRIIB, for example
C-terminal His-tagged ActRIIB In this particular format, an
anti-His antibody would be coupled to the BIACORE.TM. chip and then
the His-tagged ActRIIB would be passed over the surface of the chip
and captured by the anti-His antibody. The cross-blocking analysis
would be carried out essentially as described above, except that
after each chip regeneration cycle, new His-tagged ActRIIB would be
loaded back onto the surface coated with anti-His antibody.
Moreover, various other tags and tag binding protein combinations
that are known in the art could be used for such a cross-blocking
analysis (e.g. HA tag with anti-HA antibodies; FLAG tag with
anti-FLAG antibodies; biotin tag with streptavidin).
[0106] The following generally describes an ELISA assay for
determining whether an anti-ActRIIB antibody or other ActRIIB
binding agent cross-blocks or is capable of cross-blocking
according to the invention. For convenience, reference is made to
two antibodies, but it will be appreciated that the assay can be
used with any of the ActRIIB binding agents described herein.
[0107] The general principle of the assay is to have an
anti-ActRIIB antibody coated onto the wells of an ELISA plate. An
excess amount of a second, potentially cross-blocking, anti-ActRIIB
antibody is added in solution (i.e. not bound to the ELISA plate).
A limited amount of ActRIIB (or alternatively ActRIIB-mFc) is then
added to the wells. The coated antibody and the antibody in
solution compete for binding of the limited number of ActRIIB (or
ActRIIB-mFc) molecules. The plate is washed to remove ActRIIB that
has not been bound by the coated antibody and to also remove the
second, solution-phase antibody as well as any complexes formed
between the second, solution-phase antibody and ActRIIB The amount
of bound ActRIIB is then measured using an appropriate ActRIIB
detection reagent. An antibody in solution that is able to
cross-block the coated antibody will be able to cause a decrease in
the number of ActRIIB molecules that the coated antibody can bind
relative to the number of ActRIIB molecules that the coated
antibody can bind in the absence of the second, solution-phase
antibody.
[0108] This assay is described here in more detail for Ab-17G05 and
a theoretical antibody Ab-XX. In the instance where Ab-17G05 is
chosen to be the immobilized antibody, it is coated onto the wells
of the ELISA plate, after which the plates are blocked with a
suitable blocking solution to minimize non-specific binding of
reagents that are subsequently added. An excess amount of Ab-XX is
then added to the ELISA plate such that the moles of Ab-XX ActRIIB
binding sites per well are at least 10-fold higher than the moles
of Ab-17G05 ActRIIB binding sites that were used, per well, during
the coating of the ELISA plate.
[0109] ActRIIB is then added such that the moles of ActRIIB added
per well are at least 25-fold lower than the moles of Ab-17G05
ActRIIB binding sites that were used for coating each well.
Following a suitable incubation period the ELISA plate is washed
and an ActRIIB detection reagent is added to measure the amount of
ActRIIB specifically bound by the coated anti-ActRIIB antibody (in
this case Ab-17G05). The background signal for the assay is defined
as the signal obtained in wells with the coated antibody (in this
case Ab-17G05), solution-phase antibody (in this case Ab-XX),
ActRIIB buffer only (i.e. no ActRIIB) and ActRIIB detection
reagents. The positive control signal for the assay is defined as
the signal obtained in wells with the coated antibody (in this case
Ab-17G05), solution-phase antibody buffer only (i.e. no
solution-phase antibody), ActRIIB and ActRIIB detection reagents.
The ELISA assay needs to be run in such a manner so as to have the
positive control signal at least 3 times the background signal.
[0110] As a control for methodologic artifacts, the cross-blocking
assay may be run in the format just described and also reversed,
with Ab-XX as the coated antibody and Ab-17G05 as the
solution-phase antibody.
[0111] A reporter gene assay in A204 cells may be used to determine
the ability of anti-ActRIIB Fabs and recombinant antibodies to
neutralize ActRIIB This assay is based on a human rhabdomyosarcoma
cell line transfected with a pGL3(CAGA)12 reporter plasmid (Dennler
et al, 1998, EMBO 17: 3091-3100) as well as a Renilla reporter
plasmid (pRLCMV) to control for transfection efficiency. The CAGA12
motif is present in TGF-beta responsive genes (PAI-1 gene), so this
vector is of general use for factors signaling through Smad2 and
Smad3. Since the A204 cell line expresses primarily ActRIIA rather
than ActRIIB, it is not possible to directly test antibodies for
potential ActRIIB neutralizing ability. Instead, this assay is
designed to detect the ability of test articles to neutralize the
inhibitory effect of the soluble fusion protein ActRIIB-Fc on
activation of endogenous ActRIIA by ligands (such as activin A,
myostatin or GDF11) that can bind with high affinity to both
ActRIIA and ActRIIB Thus, in this assay, ligand-mediated activation
of ActRIIA will occur despite the presence of ActRIIB-Fc if the
anti-ActRIIB Fab or antibody is neutralizing.
[0112] On the first day of the assay, A204 cells (ATCC HTB-82) are
distributed in 48-well plates at 10.sup.5 cells per well. On the
second day, a solution containing 10 .mu.g pGL3(CAGA)12, 1 .mu.g
pRLCMV, 30 .mu.l Fugene 6 (Roche Diagnostics), and 970 .mu.l
OptiMEM (Invitrogen) is preincubated for 30 min, then added to
McCoy's growth medium, which is applied to the plated cells (500
.mu.1/well) for incubation overnight at room temperature. On the
third day, medium is removed, and cells are incubated for 6 h at
37.degree. C. with a mixture of ligands and inhibitors prepared as
described below.
[0113] To evaluate the neutralizing potency of Fabs or recombinant
antibodies, a serial dilution of the test article is made in a
48-well plate in a 200 .mu.l volume of assay buffer (McCoy's medium
+0.1% BSA). An equal volume of ActRIIB-Fc (200 .mu.g/ml) in assay
buffer is then added. The test solutions are incubated at
37.degree. C. for 30 minutes, then 400 .mu.l of GDF11 (10 ng/ml) or
activin A (10 ng/ml) is added to all wells, and 350.sub.11.1 of
this mixture is added to each well of the 48-well plate of A204
cells. Each concentration of Fab or antibody is tested in
duplicate. The final concentration of ActRIIB-Fc is 50 ng/ml (which
is the IC50 for this inhibitor of activin A signaling when the
final concentration of activin A is 5 ng/ml). After incubation with
test solutions for 6 h, cells are rinsed with phosphate-buffered
saline containing 0.1% BSA, then lysed with passive lysis buffer
(Promega E1941) and stored overnight at -70.degree. C. On the
fourth and final day, plates are warmed to room temperature with
gentle shaking. Cell lysates are transferred in duplicate to a
chemoluminescence plate (96-well) and analyzed in a luminometer
with reagents from a Dual-Luciferase Reporter Assay system (Promega
E1980) to determine normalized luciferase activity.
[0114] Antibodies disclosed herein bind to regions of human ActRIIB
which are important for the in vivo activity of the protein,
thereby inhibiting the activity of ActRIIB Ab-17G05 binds to an
epitope within the sequence of amino acids 20-134 of SEQ ID NO:l.
Binding of an antibody to ActRIIB can be correlated with changes in
biomarkers associated with ActRIIB-mediated signaling, for example,
serum FSH levels, bone density, muscle dimensions (or mass or
strength), or body weight.
[0115] Pharmacodynamic parameters dependent on ActRIIB signaling
can be measured as endpoints for in vivo testing of ActRIIB binding
agents in order to identify those binding agents that are able to
neutralize ActRIIB and provide a therapeutic benefit. An ActRIIB
neutralizing binding agent is defined as one capable of causing a
statistically significant change, as compared to vehicle-treated
animals, in such a pharmacodynamic parameter. Such in vivo testing
can be performed in any suitable mammal (e.g. mouse, rat,
monkey).
3. Screening Assays and Other Biochemical Uses
[0116] In certain aspects, the present invention relates to the use
of the subject ActRIIB binding agents to identify compounds
(agents) which are agonist or antagonists of ActRIIB Compounds
identified through this screening can be tested to assess their
ability to modulate ActRIIB-mediated signaling in vivo or in vitro.
These compounds can be tested, for example, in animal models.
[0117] 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.
[0118] 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.
[0119] In many drug screening programs which test libraries of
compounds and natural extracts, high throughput assays are
desirable in order to maximize the number of compounds surveyed in
a given period of time. Assays which are performed in cell-free
systems, such as may be derived with purified or semi-purified
proteins, are often preferred as "primary" screens in that they can
be generated to permit rapid development and relatively easy
detection of an alteration in a molecular target which is mediated
by a test compound. Moreover, the effects of cellular toxicity or
bioavailability of the test compound can be generally ignored in
the in vitro system, the assay instead being focused primarily on
the effect of the drug on the molecular target as may be manifest
in an alteration of binding affinity between an ActRIIB binding
agent and an ActRIIB polypeptide.
[0120] Merely to illustrate, in an exemplary screening assay of the
present invention, the compound of interest is contacted with an
isolated and purified ActRIIB binding agent which is ordinarily
capable of binding to an ActRIIB polypeptide, as appropriate for
the intention of the assay. To the mixture of the compound and
ActRIIB binding agent is then added a composition containing an
ActRIIB polypeptide. Detection and quantification of complexes
between ActRIIB polypeptide and ActRIIB binding agent provides a
means for determining the compound's efficacy at inhibiting (or
potentiating) complex formation between the ActRIIB polypeptide and
ActRIIB binding agent. The efficacy of the compound can be assessed
by generating dose response curves from data obtained using various
concentrations of the test compound. Moreover, a control assay can
also be performed to provide a baseline for comparison. For
example, in a control assay, isolated and purified ActRIIB binding
agent is added to a composition containing an ActRIIB polypeptide,
and the formation of complexes between ActRIIB polypeptide and
ActRIIB binding agent 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.
[0121] Complex formation between ActRIIB polypeptide and ActRIIB
binding agent may be detected by a variety of techniques. For
instance, modulation of the formation of complexes can be
quantitated using, for example, detectably labeled proteins such as
radiolabeled (e.g., .sup.32P, .sup.35S, .sup.14C or .sup.3H),
fluorescently labeled (e.g., FITC), or enzymatically labeled
ActRIIB polypeptide or ActRIIB binding agent, by immunoassay, or by
chromatographic detection.
4. Formulation and Delivery of Therapeutics
[0122] Pharmaceutical compositions are provided, comprising one of
the above-described binding agents such as antibody Ab-17G05 or a
humanized version thereof, along with a pharmaceutically or
physiologically acceptable carrier, excipient, or diluent.
[0123] The development of suitable dosing and treatment regimens
for using the particular compositions described herein in a variety
of treatment regimens, including e.g., subcutaneous, oral,
parenteral, intravenous, intranasal, and intramuscular
administration and formulation, is well known in the art, some of
which are briefly discussed below for general purposes of
illustration.
[0124] In certain applications, the pharmaceutical compositions
disclosed herein may be delivered via oral administration to an
animal. As such, these compositions may be formulated with an inert
diluent or with an assimilable edible carrier, or they may be
enclosed in hard- or soft-shell gelatin capsule, or they may be
compressed into tablets, or they may be incorporated directly with
the food of the diet.
[0125] In certain circumstances it will be desirable to deliver the
pharmaceutical compositions disclosed herein subcutaneously,
parenterally, intravenously, intramuscularly, or even
intraperitoneally. Such approaches are well known to the skilled
artisan, some of which are further described, for example, in U.S.
Pat. Nos. 5,543,158; 5,641,515 and 5,399,363. In certain
embodiments, solutions of the active compounds as free base or
pharmacologically acceptable salts may be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions may also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations generally will
contain a preservative to prevent the growth of microorganisms.
[0126] Illustrative pharmaceutical forms suitable for injectable
use include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions (for example, see U.S. Pat. No.
5,466,468). In all cases the form must be sterile and must be fluid
to the extent that easy syringability exists. It must be stable
under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g.,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and/or vegetable oils. Proper
fluidity may be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and/or by the use of surfactants.
Prevention of the action of microorganisms can be facilitated by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0127] In one embodiment, for parenteral administration in an
aqueous solution, the solution should be suitably buffered if
necessary and the liquid diluent first rendered isotonic with
sufficient saline or glucose. These particular aqueous solutions
are especially suitable for intravenous, intramuscular,
subcutaneous and intraperitoneal administration. In this
connection, a sterile aqueous medium that can be employed will be
known to those of skill in the art in light of the present
disclosure. For example, one dosage may be dissolved in 1 ml of
isotonic NaCl solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, Remington's Pharmaceutical Sciences, 15th ed.,
pp. 1035-1038 and 1570-1580). Some variation in dosage will
necessarily occur depending on the condition of the subject being
treated. Moreover, for human administration, preparations will of
course preferably meet sterility, pyrogenicity, and the general
safety and purity standards as required by FDA Office of Biologies
standards.
[0128] In another embodiment of the invention, the compositions
disclosed herein may be formulated in a neutral or salt form.
Illustrative pharmaceutically-acceptable salts include the acid
addition salts (formed with the free amino groups of the protein)
and which are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic, tartaric, mandelic, and the like. Salts formed with the
free carboxyl groups can also be derived from inorganic bases such
as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. Upon formulation,
solutions will be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically
effective.
[0129] The carriers can further comprise any and all solvents,
dispersion media, vehicles, coatings, diluents, antibacterial and
antifungal agents, isotonic and absorption delaying agents,
buffers, carrier solutions, suspensions, colloids, and the like.
The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions. The phrase
"pharmaceutically-acceptable" refers to molecular entities and
compositions that do not produce an allergic or similar untoward
reaction when administered to a human.
[0130] In certain embodiments, liposomes, nanocapsules,
microparticles, lipid particles, vesicles, and the like, are used
for the introduction of the compositions of the present invention
into suitable host cells/organisms. In particular, the compositions
of the present invention may be formulated for delivery either
encapsulated in a lipid particle, a liposome, a vesicle, a
nanosphere, or a nanoparticle or the like. Alternatively,
compositions of the present invention can be bound, either
covalently or non-covalently, to the surface of such carrier
vehicles.
[0131] The formation and use of liposome and liposome-like
preparations as potential drug carriers is generally known to those
of skill in the art (see for example, Lasic, Trends Biotechnol.
16(7):307-21, 1998; Takakura, Nippon Rinsho 56(3):691-95, 1998;
Chandran et al., Indian J. Exp. Biol. 35(8):801-09, 1997; Margalit,
Crit. Rev. Ther. Drug Carrier Syst. 12(2-3):233-61, 1995; U.S. Pat.
Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587, each
specifically incorporated herein by reference in its entirety). The
use of liposomes does not appear to be associated with autoimmune
responses or unacceptable toxicity after systemic delivery. In
certain embodiments, liposomes are formed from phospholipids that
are dispersed in an aqueous medium and spontaneously form
multilamellar concentric bilayer vesicles (also termed
multilamellar vesicles (MLVs)).
[0132] Alternatively, in other embodiments, the invention provides
for pharmaceutically-acceptable nanocapsule formulations of the
compositions of the present invention. Nanocapsules can generally
entrap compounds in a stable and reproducible way (see, for
example, Quintanar-Guerrero et al., Drug Dev. Ind. Pharm. 24(12):
1113-28, 1998). To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
um) may be designed using polymers able to be degraded in vivo.
Such particles can be made as described, for example, by Couvreur
et al., Crit. Rev. Ther. Drug Carrier Syst. 5(1):1-20, 1988; zur
Muhlen et al., Eur. J. Pharm. Biopharm. 45(2):149-55, 1998; Zambaux
et al., J Controlled Release 50(1-3):31-40, 1998; and U.S. Pat. No.
5,145,684.
[0133] In addition, pharmaceutical compositions of the present
invention may be placed within containers, along with packaging
material that provides instructions regarding the use of such
pharmaceutical compositions. Generally, such instructions will
include a tangible expression describing the reagent concentration,
as well as within certain embodiments, relative amounts of
excipient ingredients or diluents (e.g., water, saline or PBS) that
may be necessary to reconstitute the pharmaceutical
composition.
[0134] The dose administered may range from 0.01 mg/kg to 200 mg/kg
of body weight, and optionally between 0.5 mg/kg and 20 mg/kg.
However, as will be evident to one of skill in the art, the amount
and frequency of administration will depend, of course, on such
factors as the nature and severity of the indication being treated,
the desired response, the condition of the patient, and so forth.
Typically, the compositions may be administered by a variety of
techniques, as noted above.
5. Therapeutic Uses of ActRIIB Binding Agents
[0135] In certain embodiments, ActRIIB binding agents of the
present invention can be used for treating or preventing a disease
or condition that is associated with abnormal activity of ActRIIB
and/or an ActRIIB ligand (e.g., activin A, GDF8, or GDF11). These
diseases, disorders or conditions are generally referred to herein
as "ActRIIB-associated conditions." In certain embodiments, the
present invention provides methods of treating or preventing a
disease, disorder, or condition in an individual in need thereof
through administering to the individual a therapeutically effective
amount of an ActRIIB binding agent as described above. These
methods are particularly aimed at therapeutic and prophylactic
treatments of animals, and more particularly, humans.
[0136] 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.
[0137] ActRIIB and ActRIIB-ligand complexes play essential roles in
tissue growth as well as early developmental processes such as the
correct formation of various structures or in one or more
post-developmental capacities including sexual development,
pituitary hormone production, and creation of bone and cartilage.
Thus, ActRIIB-associated conditions include abnormal tissue growth
and developmental defects. In addition, ActRIIB-associated
conditions include, but are not limited to, disorders of cell
growth and differentiation such as inflammation, allergy,
autoimmune diseases, infectious diseases, and tumors.
[0138] Exemplary ActRIIB-associated conditions include
neuromuscular disorders (e.g., muscular dystrophy and muscle
atrophy), congestive obstructive pulmonary disease or pulmonary
emphysema (and associated muscle wasting), muscle wasting syndrome,
sarcopenia, cachexia, adipose tissue disorders (e.g., obesity),
type 2 diabetes, and bone degenerative disease (e.g.,
osteoporosis). Other exemplary ActRIIB-associated conditions
include musculodegenerative and neuromuscular disorders, tissue
repair (e.g., wound healing), neurodegenerative diseases (e.g.,
amyotrophic lateral sclerosis), immunologic disorders (e.g.,
disorders related to abnormal proliferation or function of
lymphocytes), and obesity or disorders related to abnormal
proliferation of adipocytes.
[0139] In certain embodiments, ActRIIB binding agents of the
invention are used as part of a treatment for a muscular dystrophy.
The term "muscular dystrophy" refers to a group of degenerative
muscle diseases characterized by gradual weakening and
deterioration of skeletal muscles and sometimes the heart and
respiratory muscles. Muscular dystrophies are genetic disorders
characterized by progressive muscle wasting and weakness that begin
with microscopic changes in the muscle. As muscles degenerate over
time, the person's muscle strength declines. Exemplary muscular
dystrophies that can be treated with a regimen including the
subject ActRIIB binding agents include: Duchenne muscular dystrophy
(DMD), Becker muscular dystrophy (BMD), Emery-Dreifuss muscular
dystrophy (EDMD), limb-girdle muscular dystrophy (LGMD),
fascioscapulohumeral muscular dystrophy (FSH or FSHD) (also known
as Landouzy-Dejerine), myotonic muscular dystrophy (MMD) (also
known as Steinert's Disease), oculopharyngeal muscular dystrophy
(OPMD), distal muscular dystrophy (DD), congenital muscular
dystrophy (CMD), and scapulohumeral muscular dystrophy (SMD).
[0140] Duchenne muscular dystrophy (DMD) was first described by the
French neurologist Guillaume Benjamin Amand Duchenne in the 1860s.
Becker muscular dystrophy (BMD) is named after the German doctor
Peter Emil Becker, who first described this variant of DMD in the
1950s. DMD is one of the most frequent inherited diseases in males,
affecting one in 3,500 boys. DMD occurs when the dystrophin gene,
located on the short arm of the X chromosome, is broken. Since
males only carry one copy of the X chromosome, they only have one
copy of the dystrophin gene. Without the dystrophin protein, muscle
is easily damaged during cycles of contraction and relaxation.
While early in the disease muscle compensates by regeneration,
later on muscle progenitor cells cannot keep up with the ongoing
damage and healthy muscle is replaced by non-functional fibro-fatty
tissue.
[0141] BMD results from different mutations in the dystrophin gene.
BMD patients have some dystrophin, but it is either insufficient in
quantity or poor in quality. Having some dystrophin protects the
muscles of those with BMD from degenerating as badly or as quickly
as those of people with DMD.
[0142] For example, studies demonstrate that blocking or
eliminating function of GDF8 (an ActRIIB ligand) in vivo can
effectively treat at least certain symptoms in DMD and BMD
patients. Thus, the subject ActRIIB binding agents may act as GDF8
inhibitors (antagonists), and constitute an alternative means of
blocking the functions of GDF8 and/or ActRIIB in vivo in DMD and
BMD patients.
[0143] In other embodiments, ActRIIB binding agents may also be
used to treat or prevent muscular atrophy due to myopathies,
examples of which include inflammatory myopathy, metabolic
myopathy, and myotonia. Subject ActRIIB binding agents have
application in treating congenital myopathies such as myotubular
myopathy, nemalene myopathy, and mitochondrial myopathy. The
subject ActRIIB binding agents may be used to treat inclusion body
myositis, myoglobinurias, rhabdomyolysis, myositis ossificans,
polymyositis, or dermatomyositis. In addition, ActRIIB binding
agents may treat or prevent muscle atrophy arising from
glucocorticoid treatment, sarcopenia, prolonged bed rest, skeletal
immobilization, sepsis, or congestive heart failure.
[0144] The subject ActRIIB binding agents provide an effective
means to increase muscle mass in other neuromuscular diseases or
conditions that are in need of muscle growth. For example,
amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's
disease or motor neuron disease) is a chronic, incurable, and
unstoppable CNS disorder that attacks the motor neurons, components
of the CNS that connect the brain to the skeletal muscles. In ALS,
the motor neurons deteriorate and eventually die, and though a
person's brain normally remains fully functioning and alert, the
command to move cannot reach the muscles. Most people who develop
ALS are between 40 and 70 years old. The first motor neurons that
weaken are those leading to the arms or legs. Those with ALS may
have trouble walking, they may drop things, fall, slur their
speech, and laugh or cry uncontrollably. Eventually the muscles in
the limbs begin to atrophy from disuse. This muscle weakness will
become debilitating and a person will need a wheel chair or become
unable to function out of bed. Most ALS patients die from
respiratory failure or from complications of ventilator assistance
like pneumonia, 3-5 years from disease onset. Other neuromuscular
diseases in which ActRIIB binding agents may be useful include
paralysis due to spinal cord injury or stroke; denervation due to
trauma or degenerative, metabolic, or inflammatory neuropathy;
adult motor neuron disease; autoimmune motor neuropathy with
multifocal conductor block; and infantile or juvenile spinal
muscular atrophy.
[0145] Increased muscle mass induced by ActRIIB binding agents
might also benefit those suffering from muscle wasting diseases.
Gonzalez-Cadavid et al. (1998, Proc. Natl. Acad. Sci. USA
95:14938-43) reported that that GDF8 expression correlates
inversely with fat-free mass in humans and that increased
expression of the GDF8 gene is associated with weight loss in men
with AIDS wasting syndrome. By inhibiting the function of GDF8 in
AIDS patients, at least certain symptoms of AIDS may be alleviated,
if not completely eliminated, thus significantly improving quality
of life in AIDS patients.
[0146] The cancer anorexia-cachexia syndrome is among the most
debilitating and life-threatening aspects of cancer. Progressive
weight loss in cancer anorexia-cachexia syndrome is a common
feature of many types of cancer and is responsible not only for a
poor quality of life and poor response to chemotherapy, but also a
shorter survival time than is found in patients with comparable
tumors without weight loss. Associated with anorexia, fat and
muscle tissue wasting, psychological distress, and a lower quality
of life, cachexia arises from a complex interaction between the
cancer and the host. It is one of the most common causes of death
among cancer patients and is present in 80% at death. It is a
complex example of metabolic chaos effecting protein, carbohydrate,
and fat metabolism. Tumors produce both direct and indirect
abnormalities, resulting in anorexia and weight loss. Currently,
there is no treatment to control or reverse the process. Cancer
anorexia-cachexia syndrome affects cytokine production, release of
lipid-mobilizing and proteolysis-inducing factors, and alterations
in intermediary metabolism. Although anorexia is common, a
decreased food intake alone is unable to account for the changes in
body composition seen in cancer patients, and increasing nutrient
intake is unable to reverse the wasting syndrome. Cachexia is
generally suspected in patients with cancer if an involuntary
weight loss of greater than five percent of premorbid weight occurs
within a six-month period.
[0147] Since systemic overexpression of GDF8 in adult mice was
found to induce profound muscle and fat loss analogous to that seen
in human cachexia syndromes (Zimmers et al., 2002, Science
296:1486-1488), the subject ActRIIB binding agents as
pharmaceutical compositions can be beneficially used to prevent,
treat, or alleviate the symptoms of the cachexia syndrome, where
muscle growth is desired. This would include cachexia associated
with cancer as well as cachexia associated with rheumatoid
arthritis.
[0148] In other embodiments, the present invention provides methods
of inducing bone and/or cartilage formation, preventing bone loss,
increasing bone mineralization or preventing the demineralization
of bone. For example, the subject ActRIIB binding agents identified
in the present invention have application in treating osteoporosis
and the healing of bone fractures and cartilage defects in humans
and other animals. ActRIIB binding agents may be useful in patients
that are diagnosed with subclinical low bone density, as a
protective measure against the development of osteoporosis.
[0149] In one specific embodiment, methods and compositions of the
present invention may find medical utility in the healing of bone
fractures and cartilage defects in humans and other animals. The
subject methods and compositions may also have prophylactic use in
closed as well as open fracture reduction and also in the improved
fixation of artificial joints. De novo bone formation induced by an
osteogenic agent contributes to the repair of congenital,
trauma-induced, or oncologic resection induced craniofacial
defects, and also is useful in cosmetic plastic surgery. Further,
methods and compositions of the invention may be used in the
treatment of periodontal disease, and in other tooth repair
processes. In certain cases, the subject ActRIIB binding agents may
provide an environment to attract bone-forming cells, stimulate
growth of bone-forming cells or induce differentiation of
progenitors of bone-forming cells. ActRIIB binding agents of the
invention may also be useful in the treatment of osteoporosis.
Further, ActRIIB binding agents may be used in cartilage defect
repair and prevention/reversal of osteoarthritis.
[0150] In another specific embodiment, the invention provides a
therapeutic method and composition for repairing fractures and
other conditions related to cartilage and/or bone defects or
periodontal diseases. The invention further provides therapeutic
methods and compositions for wound healing and tissue repair. The
types of wounds include, but are not limited to, burns, incisions
and ulcers. See e.g., PCT Publication No. WO84/01106. Such
compositions comprise a therapeutically effective amount of at
least one of the ActRIIB binding agents of the invention in
admixture with a pharmaceutically acceptable vehicle, carrier or
matrix.
[0151] In another specific embodiment, methods and compositions of
the invention can be applied to conditions causing bone loss such
as osteoporosis, hyperparathyroidism, Cushing's disease,
thyrotoxicosis, chronic diarrheal state or malabsorption, renal
tubular acidosis, or anorexia nervosa. Many people know that being
female, having a low body weight, and leading a sedentary lifestyle
are risk factors for osteoporosis (loss of bone mineral density,
leading to fracture risk). However, osteoporosis can also result
from the long-term use of certain medications. Osteoporosis
resulting from drugs or another medical condition is known as
secondary osteoporosis. In a condition known as Cushing's disease,
the excess amount of cortisol produced by the body results in
osteoporosis and fractures. The most common medications associated
with secondary osteoporosis are the corticosteroids, a class of
drugs that act like cortisol, a hormone produced naturally by the
adrenal glands. Although adequate levels of thyroid hormones (which
are produced by the thyroid gland) are needed for the development
of the skeleton, excess thyroid hormone can decrease bone mass over
time. Antacids that contain aluminum can lead to bone loss when
taken in high doses by people with kidney problems, particularly
those undergoing dialysis. Other medications that can cause
secondary osteoporosis include phenytoin (Dilantin) and
barbiturates that are used to prevent seizures; methotrexate
(Rheumatrex, Immunex, Folex PFS), a drug for some forms of
arthritis, cancer, and immune disorders; cyclosporine (Sandimmune,
Neoral), a drug used to treat some autoimmune diseases and to
suppress the immune system in organ transplant patients;
luteinizing hormone-releasing hormone agonists (Lupron, Zoladex),
used to treat prostate cancer and endometriosis; heparin
(Calciparine, Liquaemin), an anticlotting medication; and
cholestyramine (Questran) and colestipol (Colestid), used to treat
high cholesterol. Gum disease causes bone loss because these
harmful bacteria in our mouths force our bodies to defend against
them. The bacteria produce toxins and enzymes under the gum-line,
causing a chronic infection.
[0152] In a further embodiment, the present invention provides
methods and therapeutic agents for treating diseases or disorders
associated with abnormal or unwanted bone growth. For example,
patients having the disease known as Fibrodysplasia Ossificans
Progressiva (FOP) grow an abnormal "second skeleton" that prevents
any movement. Additionally, abnormal bone growth can occur after
hip replacement surgery and thus ruin the surgical outcome. This is
a more common example of pathological bone growth and a situation
in which the subject methods and compositions may be
therapeutically useful. The same methods and compositions may also
be useful for treating other forms of abnormal bone growth (e.g.,
pathological growth of bone following trauma, burns or spinal cord
injury), and for treating or preventing the undesirable conditions
associated with the abnormal bone growth seen in connection with
metastatic prostate cancer or osteosarcoma. Examples of these
therapeutic agents include, but are not limited to, ActRIIB binding
agents that specifically bind to an ActRIIB receptor such that an
ActRIIB ligand cannot bind to the ActRIIB receptor.
[0153] In other embodiments, the present invention provides
compositions and methods for regulating body fat content in an
animal and for treating or preventing conditions related thereto,
and particularly, health-compromising conditions related thereto.
According to the present invention, to regulate (control) body
weight can refer to reducing or increasing body weight, reducing or
increasing the rate of weight gain, or increasing or reducing the
rate of weight loss, and also includes actively maintaining, or not
significantly changing body weight (e.g., against external or
internal influences which may otherwise increase or decrease body
weight). One embodiment of the present invention relates to
regulating body weight by administering to an animal (e.g., a
human) in need thereof an ActRIIB binding agent.
[0154] In one specific embodiment, the present invention relates to
methods and ActRIIB binding agents for reducing body weight and/or
reducing weight gain in an animal, and more particularly, for
treating or ameliorating obesity in patients at risk for or
suffering from obesity. Loss of GDF8 (an ActRIIB ligand) function
is associated with fat loss without diminution of nutrient intake
(McPherron et al., 1997, Proc. Natl. Acad. Sci. USA,
94:12457-12461). In another specific embodiment, the present
invention is directed to methods and compounds for treating an
animal that is unable to gain or retain weight (e.g., an animal
with a wasting syndrome). Such methods are effective to increase
body weight and/or mass, or to reduce weight and/or mass loss, or
to improve conditions associated with or caused by undesirably low
(e.g., unhealthy) body weight and/or mass. The subject ActRIIB
binding agents may further be used as a therapeutic agent for
slowing or preventing the development of type II diabetes and
metabolic syndrome.
EXEMPLIFICATION
[0155] 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
Selection, Prescreening, and Sequencing of ActRIIB-Binding Fabs
[0156] A multi-round selection procedure was used to screen for
Fabs that displace ligand binding from human ActRIIB with high
affinity. Dyax's Fab310 phage-display library (Hoet et al., 2005,
Nat Biotechnol 23:344-348) was screened with both a biotinylated
human ActRIIB ECD target and a fusion protein target consisting of
human ActRIIB ECD linked to biotinylated human IgG1 Fc
(ActRIIB-Fc), each target previously immobilized on magnetic
streptavidin beads and validated by SDS-polyacrylamide gel
electrophoresis. To increase the probability of obtaining
ligand-blocking Fabs, an additional selection strategy involved
multiple rounds in which the Fab library was `depleted` by exposure
to a complex consisting of biotinylated activin A bound to
histidine-tagged ActRIIB ECD. In this way, Fabs which bound to
regions outside the ActRIIB ligand-binding site were preferentially
removed from the library. Three rounds of such library depletion
alternated with three rounds of positive selection using either of
the aforementioned targets.
[0157] Individual selected clones were tested in a prescreening
phage ELISA. In this assay, amplified phage supernatant from each
clone was added to ELISA plates onto which biotinylated ActRIIB had
been immobilized, and bound M13 phage was detected with a
horseradish-peroxidase-conjugated antibody against the P8 major
coat protein. Selection outputs yielding positive data from this
prescreen ELISA analysis, defined as signal greater than three
times background, were carried forward for reformatting to generate
soluble sFab-producing clones. Reformatting involves excision of
gIII-encoding DNA from isolated phagemid vector to convert Fab
cassette DNA to a vector format suitable for sFab expression in E.
coli. Specifically, polyclonal phagemid DNA was isolated from each
selection output; gIII DNA was removed from circular,
double-stranded phagemid DNA by restriction digestion with MluI;
the linearized DNA was purified and religated; and host E. coli was
transformed with ligation product to obtain clonal transformants
containing vector expressing sFab.
[0158] To identify clones expressing sFabs that bind ActRIIB, the
reformatted clones were subjected to high-throughput plating,
picking, and additional screening by ELISA. This ELISA screening
format differed from that described above for the Fab-phage ELISA
in that supernatant from sFab cultures was added to the ELISA wells
and sFab bound to immobilized ActRIIB was detected by anti-Fab
antibody. Those reformatted clones displaying a sFab ELISA signal
greater than two times background were rearrayed and subjected to
confirmatory ELISA and high-throughput DNA sequencing of the VH and
VL regions. Ninety-five sFab clones were initially identified as
most promising leads based on the sFab ELISA analysis and
sequencing results described above.
Example 2
Characterization and Production of Lead ActRIIB-Binding Fabs
[0159] The 95 sFab clones were cultured on a small scale,
affinity-purified with protein A, and subjected to additional
characterization. At Dyax, purified sFabs were immobilized on a
surface plasmon resonance (SPR) microarray chip and exposed to
ActRIIB ECD or ActRIIB-Fc fusion protein in a high-throughput assay
to determine approximate on- and off-rates. sFabs were ranked by
off-rate (k.sub.d) rather than equilibrium dissociation constant
(K.sub.D) due to bivalency of the target protein (confounding
avidity effects), and thirteen sFabs were obtained with off-rates
less than 10.sup.-4s.sup.-1, as well as an additional 50 sFabs with
off-rates less than 10.sup.-3s.sup.-1. A competition assay was also
performed to identify sFabs that compete for the ligand-binding
site on ActRIIB Specifically, immobilized sFabs were exposed to a
complex of ActRIIB ECD or ActRIIB-Fc fusion protein with activin A
(in a ratio of 1 .mu.M:100 nM) and the signal associated with sFab
binding to this complex was compared with that of sFab binding to
ActRIIB alone. sFabs whose signal was reduced at least 50% by the
presence of activin A were classified as competitors, and 72 of 95
sFabs met this criterion. Potential cross-reactivity with
ActRIIA-Fc was also evaluated by SPR and was found to occur for 11
of 95 sFabs.
[0160] Affinity-purified sFabs were also screened in two SPR-based
competition assays at Acceleron. The first assay evaluated the
effect of sFab pretreatment on binding of GDF11 to immobilized
ActRIIB-Fc. Specifically, biotinylated ActRIIB-Fc was immobilized
on a BIACORE.TM. streptavidin chip and exposed to GDF11 (500 ng/ml)
to determine the SPR signal associated with maximum activin binding
(Emax). This signal was compared with a second signal (residual
GDF11 binding) resulting from GDF11 binding to immobilized
ActRIIB-Fc that had first been exposed to sFab (40 .mu.g/ml). In
this assay, a Fab was considered neutralizing if residual GDF11
binding was less than 50% of Emax, and five sFabs (17A07, 17A11,
17C09, 17G01, and 17G05) of the 95 screened met this criterion. A
second assay evaluated the effect of sFab pretreatment on ActRIIB
binding to immobilized activin A. In this case, biotinylated
activin A was immobilized on a BIACORE.TM. streptavidin chip and
exposed to ActRIIB-Fc (1 .mu.g/ml) to determine the SPR signal
associated with maximum activin A binding (Emax). This signal was
compared with residual binding of activin A to a complex of sFab
with ActRIIB-Fc, which was formed by premixing these proteins in a
20:1 ratio (20 .mu.g/ml Fab and 1 .mu.g/ml ActRIIB-Fc). sFabs were
ranked according to residual binding, and 19 of 95 sFabs screened
exhibited residual binding less than 50% of Emax in this assay.
[0161] Based on the foregoing analyses, 24 sFab clones with
preferred characteristics (such as slow off-rate, inhibition of
ligand binding, and low cross-reactivity with ActRIIA) were chosen
for scaled-up production. Protein levels of approximately 50 .mu.g
were obtained for the majority of clones after one-step
purification with protein A. Purified sFab proteins were subjected
to confirmatory characterization by ELISA and SDS-polyacrylamide
gel electrophoresis before transfer to Acceleron in these larger
quantities. DNA sequences of the 24 preferred clones were also
confirmed at this stage by standard methods.
Example 3
Sequences of Lead ActRIIB-Binding Fabs
[0162] Of the 24 sFabs with preferred characteristics, four were
selected for more detailed characterization. Shown below are the
amino acid sequences of VH and VL, respectively, for Fab-17A11 (CDR
sequences are underlined).
TABLE-US-00004 (SEQ ID NO: 3) 1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS
TYAMMWVRQA PGKGLEWVSR 51 IYPSGGTTTY ADSVKGRFTI SRDNSKNTLY
LQMNSLRAED TAVYYCARGS 101 AASSYWGQGT LVTVSS (SEQ ID NO: 4) 1
QDIQMTQSPS FLSASVGDRV TITCRASQGI SNYLAWYQQK PGKAPKLLIY 51
AASTLQSGVP SRFSGSGSGT EFTLTISSLQ PEDIGTYYCQ QLISYPFTFG 101
PGTKVDIK
Shown below are nucleotide sequences encoding VH and VL,
respectively, for Fab-17A11.
TABLE-US-00005 (SEQ ID NO: 5) 1 GAAGTTCAAT TGTTAGAGTC TGGTGGCGGT
CTTGTTCAGC CTGGTGGTTC 51 TTTACGTCTT TCTTGCGCTG CTTCCGGATT
CACTTTCTCT ACTTACGCTA 101 TGATGTGGGT TCGCCAAGCT CCTGGTAAAG
GTTTGGAGTG GGTTTCTCGT 151 ATCTATCCTT CTGGTGGCAC TACTACTTAT
GCTGACTCCG TTAAAGGTCG 201 CTTCACTATC TCTAGAGACA ACTCTAAGAA
TACTCTCTAC TTGCAGATGA 251 ACAGCTTAAG GGCTGAGGAC ACGGCCGTGT
ATTACTGTGC GAGGGGATCA 301 GCTGCCAGCT CCTACTGGGG CCAGGGAACC
CTGGTCACCG TCTCAAGC (SEQ ID NO: 6) 1 CAAGACATCC AGATGACCCA
GTCTCCATCC TTCCTGTCTG CATCTGTTGG 51 AGACAGGGTC ACCATCACTT
GCCGGGCCAG TCAGGGCATT AGCAATTATT 101 TAGCCTGGTA TCAGCAAAAA
CCAGGGAAAG CCCCTAAGCT CCTGATCTAT 151 GCTGCATCCA CTTTGCAAAG
TGGGGTCCCA TCAAGGTTCA GCGGCAGTGG 201 ATCTGGGACA GAATTCACTC
TCACAATCAG CAGCCTGCAG CCTGAAGATA 251 TTGGAACTTA TTACTGTCAA
CAGCTTATTA GTTACCCATT CACTTTCGGC 301 CCTGGGACCA AAGTGGATAT CAA
Shown below are the amino acid sequences of VH and VL,
respectively, for Fab-17C09 (CDR sequences are underlined).
TABLE-US-00006 (SEQ ID NO: 7) 1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS
QYNMTWVRQA PGKGLEWVSS 51 IYSSGGVTPY ADSVKGRFTI SRDNSKNTLY
LQMNSLRAED TAVYYCARGR 101 LLFDYWGQGT LVTVSS (SEQ ID NO: 8) 1
QDIQMTQSPS SLSASVGDRV TITCRASQSI SNYLNWYQQR PGKPPKLLIY 51
AASSLQSGVP SRFSGSGSGT DFSLSISILQ PEDFATYYCQ QGYTAPRSFG 101
QGTKVEIK
Shown below are nucleotide sequences encoding VH and VL,
respectively, for Fab-17C09.
TABLE-US-00007 (SEQ ID NO: 9) 1 GAAGTTCAAT TGTTAGAGTC TGGTGGCGGT
CTTGTTCAGC CTGGTGGTTC 51 TTTACGTCTT TCTTGCGCTG CTTCCGGATT
CACTTTCTCT CAGTACAATA 101 TGACTTGGGT TCGCCAAGCT CCTGGTAAAG
GTTTGGAGTG GGTTTCTTCT 151 ATCTATTCTT CTGGTGGCGT TACTCCTTAT
GCTGACTCCG TTAAAGGTCG 201 CTTCACTATC TCTAGAGACA ACTCTAAGAA
TACTCTCTAC TTGCAGATGA 251 ACAGCTTAAG GGCTGAGGAC ACGGCCGTGT
ATTACTGTGC GAGAGGTCGC 301 CTCCTCTTTG ACTACTGGGG CCAGGGAACC
CTGGTCACCG TCTCAAGC (SEQ ID NO: 10) 1 CAAGACATCC AGATGACCCA
GTCTCCATCC TCCCTGTCTG CATCTGTCGG 51 AGACAGAGTC ACCATCACTT
GCCGGGCAAG TCAGAGCATT AGCAACTATT 101 TAAATTGGTA TCAGCAGAGA
CCAGGGAAAC CCCCTAAGCT CCTGATCTAT 151 GCTGCATCCA GTTTGCAAAG
TGGGGTCCCA TCAAGGTTTA GCGGCAGTGG 201 ATCTGGGACA GATTTCAGTC
TCTCCATCAG CATTCTGCAA CCTGAAGATT 251 TTGCAACTTA CTACTGTCAA
CAGGGTTACA CTGCCCCTCG CAGTTTTGGC 301 CAGGGGACCA AGGTGGAGAT CAA
Shown below are the amino acid sequences of VH and VL,
respectively, for Fab-17G01 (CDR sequences are underlined).
TABLE-US-00008 (SEQ ID NO: 11) 1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS
NYQMDWVRQA PGKGLEWVSY 51 IGPSGGRTKY ADSVKGRFTI SRDNSKNTLY
LQMNSLRAED TATYYCARGL 101 YSFDYWGQGT LVTVSS (SEQ ID NO: 12) 1
QDIQMTQSPS SLSASVGDRV TITCRAGQSI SNFLNWYQHT PGTGPKVLIY 51
AASSLQSGVP SRFSGSGSGT EFTLTITNLQ PEDFATYYCQ QSYSTPFTFG 101
PGTKVDIK
Shown below are nucleotide sequences encoding VH and VL,
respectively, for Fab-17G01.
TABLE-US-00009 (SEQ ID NO: 13) 1 GAAGTTCAAT TGTTAGAGTC TGGTGGCGGT
CTTGTTCAGC CTGGTGGTTC 51 TTTACGTCTT TCTTGCGCTG CTTCCGGATT
CACTTTCTCT AATTACCAGA 101 TGGATTGGGT TCGCCAAGCT CCTGGTAAAG
GTTTGGAGTG GGTTTCTTAT 151 ATCGGTCCTT CTGGTGGCCG TACTAAGTAT
GCTGACTCCG TTAAAGGTCG 201 CTTCACTATC TCTAGAGACA ACTCTAAGAA
TACTCTCTAC TTGCAGATGA 251 ACAGCTTAAG GGCTGAGGAC ACAGCCACAT
ATTACTGTGC GAGAGGATTG 301 TACTCGTTTG ACTACTGGGG CCAGGGAACC
CTGGTCACCG TCTCAAGC (SEQ ID NO: 14) 1 CAAGACATCC AGATGACCCA
GTCTCCATCC TCCCTGTCTG CATCTGTAGG 51 AGACAGAGTC ACCATCACTT
GCCGGGCAGG TCAGAGCATT AGCAACTTTT 101 TAAATTGGTA TCAGCATACA
CCAGGGACAG GCCCTAAAGT CCTGATCTAT 151 GCTGCATCCA GTTTGCAAAG
TGGGGTCCCA TCACGGTTCA GTGGCAGTGG 201 ATCTGGGACA GAATTCACTC
TCACCATCAC CAATCTGCAA CCTGAAGATT 251 TTGCAACTTA CTACTGTCAA
CAGAGTTACA GTACCCCATT CACTTTCGGC 301 CCTGGGACCA AAGTGGATAT CAG
Shown below are the amino acid sequences of VH and VL,
respectively, for Fab-17G05 (CDR sequences are underlined).
TABLE-US-00010 (SEQ ID NO: 15) 1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS
NYWMGWVRQA PGKGLEWVSY 51 IRSSGGLTHY ADSVKGRFTI SRDNSKNTLY
LQMNSLRAED TATYYCAKGL 101 YSFDYWGQGT LVTVSS (SEQ ID NO: 16) 1
QDIQMTQSPS SLSASVGDRV TITCRASQGV NNFLAWYQQK PGKAPRLLIY 51
AASTLQSGVP SRFSGSGSGT DFTLSISNLQ PEDFATYYCQ QSYSTPRGFG 101
QGTKVEIK
Shown below are nucleotide sequences encoding VH and VL,
respectively, for Fab-17G05.
TABLE-US-00011 (SEQ ID NO: 17) 1 GAAGTTCAAT TGTTAGAGTC TGGTGGCGGT
CTTGTTCAGC CTGGTGGTTC 51 TTTACGTCTT TCTTGCGCTG CTTCCGGATT
CACTTTCTCT AATTACTGGA 101 TGGGTTGGGT TCGCCAAGCT CCTGGTAAAG
GTTTGGAGTG GGTTTCTTAT 151 ATCCGTTCTT CTGGTGGCCT TACTCATTAT
GCTGACTCCG TTAAAGGTCG 201 CTTCACTATC TCTAGAGACA ACTCTAAGAA
TACTCTCTAC TTGCAGATGA 251 ACAGCTTAAG GGCTGAGGAC ACAGCCACAT
ATTACTGTGC GAAAGGACTA 301 TATTCCTTTG ACTACTGGGG CCAGGGAACC
CTGGTCACCG TCTCAAGC (SEQ ID NO: 18) 1 CAAGACATCC AGATGACCCA
GTCTCCATCT TCCCTGTCTG CTTCTGTAGG 51 AGACAGAGTC ACCATCACTT
GCCGGGCCAG TCAGGGCGTT AACAATTTTT 101 TAGCCTGGTA TCAGCAAAAA
CCAGGGAAGG CCCCTAGGCT CCTGATCTAT 151 GCTGCATCCA CTTTGCAGAG
TGGGGTCCCA TCAAGGTTCA GCGGCAGTGG 201 ATCTGGGACA GATTTCACTC
TCTCCATCAG CAACCTGCAG CCTGAAGACT 251 TTGCAACTTA TTACTGTCAA
CAGAGTTACA GTACCCCTCG GGGGTTCGGC 301 CAAGGGACCA AGGTGGAAAT CAA
Listed below are CDR sequences for Fab-17A11.
TABLE-US-00012 CDR-H1 (SEQ ID NO: 19) TYAMM CDR-H2 (SEQ ID NO: 20)
RIYPSGGTTTYADSVKG CDR-H3 (SEQ ID NO: 21) GSAASSY CDR-L1 (SEQ ID NO:
22) RASQGISNYLA CDR-L2 (SEQ ID NO: 23) AASTLQS CDR-L3 (SEQ ID NO:
24) QQLISYPFT
Listed below are CDR sequences for Fab-17C09.
TABLE-US-00013 CDR-H1 (SEQ ID NO: 25) QYNMT CDR-H2 (SEQ ID NO: 26)
SIYSSGGVTPYADSVKG CDR-H3 (SEQ ID NO: 27) GRLLFDY CDR-L1 (SEQ ID NO:
28) RASQSISNYLN CDR-L2 (SEQ ID NO: 29) AASSLQS CDR-L3 (SEQ ID NO:
30) QQGYTAPRS
Listed below are CDR sequences for Fab-17G01.
TABLE-US-00014 CDR-H1 (SEQ ID NO: 31) NYQMD CDR-H2 (SEQ ID NO: 32)
YIGPSGGRTKYADSVKG CDR-H3 (SEQ ID NO: 33) GLYSFDY CDR-L1 (SEQ ID NO:
34) RAGQSISNFLN CDR-L2 (SEQ ID NO: 35) AASSLQS CDR-L3 (SEQ ID NO:
36) QQSYSTPFT
Listed below are CDR sequences for Fab-17G05.
TABLE-US-00015 CDR-H1 (SEQ ID NO: 37) NYWMG CDR-H2 (SEQ ID NO: 38)
YIRSSGGLTHYADSVKG CDR-H3 (SEQ ID NO: 39) GLYSFDY CDR-L1 (SEQ ID NO:
40) RASQGVNNFLA CDR-L2 (SEQ ID NO: 41) AASTLQS CDR-L3 (SEQ ID NO:
42) QQSYSTPRG
Example 4
Characterization of Lead Fab Binding to ActRIIB
[0163] Applicants used SPR (BIACORE.TM.-based analysis) to more
fully characterize binding of lead sFabs to ActRIIB FIG. 1 shows
kinetic characterization of Fab-17G05 binding to human ActRIIB-hFc
(dimeric protein), and the binding parameters for several lead
sFabs are indicated below.
TABLE-US-00016 25.degree. C. 37.degree. C. Fab k.sub.a
(M.sup.-1s.sup.-1) k.sub.d (s.sup.-1) K.sub.D (M) k.sub.a
(M.sup.-1s.sup.-1) k.sub.d (s.sup.-1) K.sub.D (M) 17G05 16 .times.
10.sup.5 8.7 .times. 10.sup.-4 5.5 .times. 10.sup.-10 26 .times.
10.sup.5 3.1 .times. 10.sup.-3 1.9 .times. 10.sup.-9 575A-M31-E07
8.7 .times. 10.sup.5 1.6 .times. 10.sup.-4 18 .times. 10.sup.-10 11
.times. 10.sup.5 5.3 .times. 10.sup.-3 5 .times. 10.sup.-9 17G01
4.6 .times. 10.sup.5 18 .times. 10.sup.-4 38 .times. 10.sup.-10 5.8
.times. 10.sup.5 12 .times. 10.sup.-3 21 .times. 10.sup.-9 17A11 12
.times. 10.sup.5 1.9 .times. 10.sup.-4 16 .times. 10.sup.-10 14
.times. 10.sup.5 14 .times. 10.sup.-3 9.9 .times. 10.sup.-9
Among the lead sFabs analyzed, Fab-17G05 displayed the best kinetic
parameters for off-rate and K.sub.D at 37.degree. C., and for
K.sub.D at 25.degree. C.
Example 5
Reporter Gene Assay in A204 Cells
[0164] A reporter gene assay in A204 cells was used to determine
the ability of anti-ActRIIB Fabs and recombinant antibodies to
neutralize ActRIIB This assay is based on a human rhabdomyosarcoma
cell line transfected with a pGL3(CAGA)12 reporter plasmid (Dennler
et al, 1998, EMBO 17: 3091-3100) as well as a Renilla reporter
plasmid (pRLCMV) to control for transfection efficiency. The CAGA12
motif is present in TGF-beta responsive genes (PAI-1 gene), so this
vector is of general use for factors signaling through Smad2 and
Smad3. Since the A204 cell line expresses primarily ActRIIA rather
than ActRIIB, it is not possible to directly test antibodies for
potential ActRIIB neutralizing ability. Instead, this assay was
designed to detect the ability of test articles to neutralize the
inhibitory effect of the soluble fusion protein ActRIIB-Fc on
activation of endogenous ActRIIA by ligands (such as activin A,
GDF11 or myostatin) that can bind with high affinity to both
ActRIIA and ActRIIB Thus, in this assay, ligand-mediated activation
of ActRIIA will occur despite the presence of ActRIIB-Fc if the
anti-ActRIIB Fab or antibody is neutralizing.
[0165] On the first day of the assay, A204 cells (ATCC HTB-82) were
distributed in 48-well plates at 10.sup.5 cells per well. On the
second day, a solution containing 10 .mu.g pGL3(CAGA)12, 1 .mu.g
pRLCMV, 30 .mu.l Fugene 6 (Roche Diagnostics), and 970 .mu.l
OptiMEM (Invitrogen) was preincubated for 30 min, then added to
McCoy's growth medium, which was applied to the plated cells (500
.mu.1/well) for incubation overnight at room temperature. On the
third day, medium was removed, and cells were incubated for 6 h at
37.degree. C. with a mixture of ligands and inhibitors prepared as
described below.
[0166] To evaluate the neutralizing potency of Fabs or recombinant
antibodies, a serial dilution of the test article was made in a
48-well plate in a 200 .mu.l volume of assay buffer (McCoy's medium
+0.1% BSA). An equal volume of ActRIIB-Fc (200 .mu.g/ml) in assay
buffer was then added. The test solutions were incubated at
37.degree. C. for 30 minutes, then 400 .mu.l of GDF11 (10 ng/ml) or
activin A (10 ng/ml) was added to all wells, and 350 .mu.l of this
mixture was added to each well of the 48-well plate of A204 cells.
Each concentration of Fab or antibody was tested in duplicate. The
final concentration of ActRIIB-Fc was 50 ng/ml (which is the IC50
for this inhibitor of activin A signaling when the final
concentration of activin A is 5 ng/ml). After incubation with test
solutions for 6 h, cells were rinsed with phosphate-buffered saline
containing 0.1% BSA, then lysed with passive lysis buffer (Promega
E1941) and stored overnight at -70.degree. C. On the fourth and
final day, plates were warmed to room temperature with gentle
shaking. Cell lysates were transferred in duplicate to a
chemoluminescence plate (96-well) and analyzed in a luminometer
with reagents from a Dual-Luciferase Reporter Assay system (Promega
E1980) to determine normalized luciferase activity.
[0167] This reporter gene assay was used to screen several of the
lead sFabs. In two different assays with GDF11 as ligand, Fab-17G05
was a more potent neutralizer of cellular signaling than were other
sFabs tested, including Fab-17A11 and Fab-17G01.
Example 6
Generation of Ab-17G05 by Fab Conversion
[0168] On the basis of the foregoing results, Fab-17G05 was
selected for conversion to an antibody. Construction of vectors for
expression of human IgG heavy and light chains was based on Persic
et al. (1997, Gene 187:9-18). Both vectors use an IgG secretory
leader containing a unique restriction site (BssHII) to clone VH
and VL at their 5' end. Heavy-chain vector incorporates an adjacent
VH linker containing a BstEII site, which is conserved across all
VH subgroups, for cloning VH at the 3' end. Thus, VH from Fab-17G05
was generated by PCR and inserted into digested pAID4 human IgG1
heavy-chain vector (BssHII 5' and BstEII 3'). To accommodate the
full range of VL subgroups at their 3' boundary, light-chain vector
incorporates a VL linker containing a XhoI site, which is available
in some VL subgroups, followed by a short intron containing a PacI
site, which can be used for cloning all other VL subgroups. Thus,
VL from Fab-17G05 was generated by PCR and inserted into digested
pAID4 human kappa light-chain vector (BssHII 5' and PacI 3'). The
completed constructs underwent confirmatory sequencing and were
transiently cotransfected into COS cells. COS conditioned medium
was analyzed by Western blot to confirm antibody size and by ELISA
for human Fc domain to determine antibody concentration. Antibody
was also produced in stably transfected CHO cells. Purification of
antibody protein from COS or CHO cell conditioned medium was
achieved by protein A chromatography (e.g. MabSelect SuRe.TM.,
General Electric, Piscataway, N.J.), dialysis, viral filtration,
and buffer exchange. The N-terminus of purified VH protein was
confirmed by N-terminal sequencing to be EVQLLESGGG (SEQ ID NO:
43).
Example 7
Characterization of Ab-17G05 Binding to ActRIIB
[0169] BIACORE.TM.-based analysis was used to characterize binding
of Ab-17G05 to ActRIIB For this analysis, Ab-17G05 was purified
from COS-cell-conditioned media by one-step protein G
chromatography. FIG. 2 shows kinetic characterization of Ab-17G05
binding to hActRIIB-mFc (dimeric protein), and binding parameters
are listed below compared with those of Fab-17G05.
TABLE-US-00017 25.degree. C. 37.degree. C. k.sub.a
(M.sup.-1s.sup.-1) k.sub.d (s.sup.-1) K.sub.D (M) k.sub.a
(M.sup.-1s.sup.-1) k.sub.d (s.sup.-1) K.sub.D (M) Ab-17G05 9.7
.times. 10.sup.5 2.7 .times. 10.sup.-5 2.8 .times. 10.sup.-11 1.5
.times. 10.sup.6 1.4 .times. 10.sup.-4 9.2 .times. 10.sup.-11
Fab-17G05 16 .times. 10.sup.5 87 .times. 10.sup.-5 55 .times.
10.sup.-11 2.6 .times. 10.sup.6 31 .times. 10.sup.-4 190 .times.
10.sup.-11
Conversion of Fab-17G05 to an antibody resulted in improvements of
20-fold or greater in off-rate and K.sub.D at both 25.degree. C. or
37.degree. C.
Example 8
Neutralization by Ab-17G05 in a Cell-Based Assay
[0170] Ab-17G05 was evaluated for its ability to neutralize binding
of activin A and ActRIIB in the cell-based reporter gene assay
described in Example 4. In this assay, ligand-mediated activation
of endogenous ActRIIA will occur despite the presence of exogenous
ActRIIB-Fc if the anti-ActRIIB antibody or Fab is neutralizing.
Results obtained for Ab-R17G05 are based on conditioned media from
COS cells (quantitated by ELISA) and for Fab-R17G05 on material
purified with protein A. As shown in FIG. 3, Ab-17G05 was a potent
stimulator of activin A signaling through ActRIIA, thus indicating
that Ab-17G05 can neutralize ActRIIB binding to activin A in a
cell-based system. Unconverted Fab-17G05 also displayed
neutralizing capability; however, the potency of Ab-17G05 (IC50
0.04 nM) was two orders of magnitude higher than that of Fab-17G05
(IC50 2.6 nM).
[0171] Taken together, the foregoing findings demonstrate the
generation of an antibody(17G05) capable of binding to ActRIIB with
high affinity and potently neutralizing ActRIIB-mediated signaling.
Consistent with these findings, Ab-17G05 increases muscle mass in
vivo.
Example 9
Detection of Human Anti-ActRIIB Antibodies in Serum
[0172] In the course of clinical development of an ActRIIB-Fc
fusion protein (known as ACE-031), an ELISA method has been
developed to detect neutralizing antibodies to the ActRIIB portion
of ActRIIB-Fc in human serum. Briefly, ACE-031 is coated on the
microplate followed by control antibody (murine or human
anti-ActRIIB; 17G05) and sample incubation. After washing, samples
are incubated with biotinylated Activin A and bound ligand is
detected with streptavidin horseradish peroxidase (HRP) and
tetramethylbenzidine (TMB) substrate. Biotinylated-Activin A
binding to ACE-031 in the absence of neutralizing antibody is
recorded as max signal, the difference between the max signal and
signal obtained in the presence of bound neutralizing antibody
(inhibition) is proportional to the amount of neutralization
activity against ACE-031 in the sample. Serum samples from human
patients treated with ActRIIB-Fc and suspected of having an immune
reaction to the ActRIIB-Fc may be evaluated by the same protocol,
replacing the control antibody with the serum sample and comparing
the measured signal against the standard values generated with the
control antibody.
INCORPORATION BY REFERENCE
[0173] 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.
[0174] 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
431512PRTHomo sapiens 1Met Thr Ala Pro Trp Val Ala Leu Ala Leu Leu
Trp Gly Ser Leu Cys 1 5 10 15 Ala Gly Ser Gly Arg Gly Glu Ala Glu
Thr Arg Glu Cys Ile Tyr Tyr 20 25 30 Asn Ala Asn Trp Glu Leu Glu
Arg Thr Asn Gln Ser Gly Leu Glu Arg 35 40 45 Cys Glu Gly Glu Gln
Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg 50 55 60 Asn Ser Ser
Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp 65 70 75 80 Asp
Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn 85 90
95 Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg
100 105 110 Phe Thr His Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr
Glu Pro 115 120 125 Pro Pro Thr Ala Pro Thr Leu Leu Thr Val Leu Ala
Tyr Ser Leu Leu 130 135 140 Pro Ile Gly Gly Leu Ser Leu Ile Val Leu
Leu Ala Phe Trp Met Tyr 145 150 155 160 Arg His Arg Lys Pro Pro Tyr
Gly His Val Asp Ile His Glu Asp Pro 165 170 175 Gly Pro Pro Pro Pro
Ser Pro Leu Val Gly Leu Lys Pro Leu Gln Leu 180 185 190 Leu Glu Ile
Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Gln 195 200 205 Leu
Met Asn Asp Phe Val Ala Val Lys Ile Phe Pro Leu Gln Asp Lys 210 215
220 Gln Ser Trp Gln Ser Glu Arg Glu Ile Phe Ser Thr Pro Gly Met Lys
225 230 235 240 His Glu Asn Leu Leu Gln Phe Ile Ala Ala Glu Lys Arg
Gly Ser Asn 245 250 255 Leu Glu Val Glu Leu Trp Leu Ile Thr Ala Phe
His Asp Lys Gly Ser 260 265 270 Leu Thr Asp Tyr Leu Lys Gly Asn Ile
Ile Thr Trp Asn Glu Leu Cys 275 280 285 His Val Ala Glu Thr Met Ser
Arg Gly Leu Ser Tyr Leu His Glu Asp 290 295 300 Val Pro Trp Cys Arg
Gly Glu Gly His Lys Pro Ser Ile Ala His Arg 305 310 315 320 Asp Phe
Lys Ser Lys Asn Val Leu Leu Lys Ser Asp Leu Thr Ala Val 325 330 335
Leu Ala Asp Phe Gly Leu Ala Val Arg Phe Glu Pro Gly Lys Pro Pro 340
345 350 Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro
Glu 355 360 365 Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe
Leu Arg Ile 370 375 380 Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu
Leu Val Ser Arg Cys 385 390 395 400 Lys Ala Ala Asp Gly Pro Val Asp
Glu Tyr Met Leu Pro Phe Glu Glu 405 410 415 Glu Ile Gly Gln His Pro
Ser Leu Glu Glu Leu Gln Glu Val Val Val 420 425 430 His Lys Lys Met
Arg Pro Thr Ile Lys Asp His Trp Leu Lys His Pro 435 440 445 Gly Leu
Ala Gln Leu Cys Val Thr Ile Glu Glu Cys Trp Asp His Asp 450 455 460
Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Glu Glu Arg Val Ser Leu 465
470 475 480 Ile Arg Arg Ser Val Asn Gly Thr Thr Ser Asp Cys Leu Val
Ser Leu 485 490 495 Val Thr Ser Val Thr Asn Val Asp Leu Pro Pro Lys
Glu Ser Ser Ile 500 505 510 21536DNAHomo sapiens 2atgacggcgc
cctgggtggc cctcgccctc ctctggggat cgctgtgcgc cggctctggg 60cgtggggagg
ctgagacacg ggagtgcatc tactacaacg ccaactggga gctggagcgc
120accaaccaga gcggcctgga gcgctgcgaa ggcgagcagg acaagcggct
gcactgctac 180gcctcctggc gcaacagctc tggcaccatc gagctcgtga
agaagggctg ctggctagat 240gacttcaact gctacgatag gcaggagtgt
gtggccactg aggagaaccc ccaggtgtac 300ttctgctgct gtgaaggcaa
cttctgcaac gaacgcttca ctcatttgcc agaggctggg 360ggcccggaag
tcacgtacga gccacccccg acagccccca ccctgctcac ggtgctggcc
420tactcactgc tgcccatcgg gggcctttcc ctcatcgtcc tgctggcctt
ttggatgtac 480cggcatcgca agccccccta cggtcatgtg gacatccatg
aggaccctgg gcctccacca 540ccatcccctc tggtgggcct gaagccactg
cagctgctgg agatcaaggc tcgggggcgc 600tttggctgtg tctggaaggc
ccagctcatg aatgactttg tagctgtcaa gatcttccca 660ctccaggaca
agcagtcgtg gcagagtgaa cgggagatct tcagcacacc tggcatgaag
720cacgagaacc tgctacagtt cattgctgcc gagaagcgag gctccaacct
cgaagtagag 780ctgtggctca tcacggcctt ccatgacaag ggctccctca
cggattacct caaggggaac 840atcatcacat ggaacgaact gtgtcatgta
gcagagacga tgtcacgagg cctctcatac 900ctgcatgagg atgtgccctg
gtgccgtggc gagggccaca agccgtctat tgcccacagg 960gactttaaaa
gtaagaatgt attgctgaag agcgacctca cagccgtgct ggctgacttt
1020ggcttggctg ttcgatttga gccagggaaa cctccagggg acacccacgg
acaggtaggc 1080acgagacggt acatggctcc tgaggtgctc gagggagcca
tcaacttcca gagagatgcc 1140ttcctgcgca ttgacatgta tgccatgggg
ttggtgctgt gggagcttgt gtctcgctgc 1200aaggctgcag acggacccgt
ggatgagtac atgctgccct ttgaggaaga gattggccag 1260cacccttcgt
tggaggagct gcaggaggtg gtggtgcaca agaagatgag gcccaccatt
1320aaagatcact ggttgaaaca cccgggcctg gcccagcttt gtgtgaccat
cgaggagtgc 1380tgggaccatg atgcagaggc tcgcttgtcc gcgggctgtg
tggaggagcg ggtgtccctg 1440attcggaggt cggtcaacgg cactacctcg
gactgtctcg tttccctggt gacctctgtc 1500accaatgtgg acctgccccc
taaagagtca agcatc 15363116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 3Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met
Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Arg Ile Tyr Pro Ser Gly Gly Thr Thr Thr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Ala Ala Ser Ser Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115
4108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 4Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Phe
Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Ser Asn 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Ala Ser
Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro
Glu Asp Ile Gly Thr Tyr Tyr Cys Gln Gln Leu Ile Ser Tyr Pro 85 90
95 Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105
5348DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 5gaagttcaat tgttagagtc tggtggcggt
cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct
acttacgcta tgatgtgggt tcgccaagct 120cctggtaaag gtttggagtg
ggtttctcgt atctatcctt ctggtggcac tactacttat 180gctgactccg
ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240ttgcagatga acagcttaag ggctgaggac acggccgtgt attactgtgc
gaggggatca 300gctgccagct cctactgggg ccagggaacc ctggtcaccg tctcaagc
3486324DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 6caagacatcc agatgaccca gtctccatcc
ttcctgtctg catctgttgg agacagggtc 60accatcactt gccgggccag tcagggcatt
agcaattatt tagcctggta tcagcaaaaa 120ccagggaaag cccctaagct
cctgatctat gctgcatcca ctttgcaaag tggggtccca 180tcaaggttca
gcggcagtgg atctgggaca gaattcactc tcacaatcag cagcctgcag
240cctgaagata ttggaactta ttactgtcaa cagcttatta gttacccatt
cactttcggc 300cctgggacca aagtggatat caaa 3247116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
7Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gln
Tyr 20 25 30 Asn Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Tyr Ser Ser Gly Gly Val Thr Pro
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Leu
Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser
Ser 115 8108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 8Gln Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Ser Ile Ser Asn 20 25 30 Tyr Leu Asn Trp Tyr
Gln Gln Arg Pro Gly Lys Pro Pro Lys Leu Leu 35 40 45 Ile Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly
Ser Gly Ser Gly Thr Asp Phe Ser Leu Ser Ile Ser Ile Leu Gln 65 70
75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Ala
Pro 85 90 95 Arg Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 9348DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 9gaagttcaat tgttagagtc tggtggcggt
cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct
cagtacaata tgacttgggt tcgccaagct 120cctggtaaag gtttggagtg
ggtttcttct atctattctt ctggtggcgt tactccttat 180gctgactccg
ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240ttgcagatga acagcttaag ggctgaggac acggccgtgt attactgtgc
gagaggtcgc 300ctcctctttg actactgggg ccagggaacc ctggtcaccg tctcaagc
34810324DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 10caagacatcc agatgaccca gtctccatcc
tccctgtctg catctgtcgg agacagagtc 60accatcactt gccgggcaag tcagagcatt
agcaactatt taaattggta tcagcagaga 120ccagggaaac cccctaagct
cctgatctat gctgcatcca gtttgcaaag tggggtccca 180tcaaggttta
gcggcagtgg atctgggaca gatttcagtc tctccatcag cattctgcaa
240cctgaagatt ttgcaactta ctactgtcaa cagggttaca ctgcccctcg
cagttttggc 300caggggacca aggtggagat caaa 32411116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
11Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Tyr 20 25 30 Gln Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Tyr Ile Gly Pro Ser Gly Gly Arg Thr Lys
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Tyr
Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser
Ser 115 12108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 12Gln Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile
Thr Cys Arg Ala Gly Gln Ser Ile Ser Asn 20 25 30 Phe Leu Asn Trp
Tyr Gln His Thr Pro Gly Thr Gly Pro Lys Val Leu 35 40 45 Ile Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60
Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Thr Asn Leu Gln 65
70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser
Thr Pro 85 90 95 Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 105 13348DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 13gaagttcaat tgttagagtc
tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt
cactttctct aattaccaga tggattgggt tcgccaagct 120cctggtaaag
gtttggagtg ggtttcttat atcggtcctt ctggtggccg tactaagtat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa
tactctctac 240ttgcagatga acagcttaag ggctgaggac acagccacat
attactgtgc gagaggattg 300tactcgtttg actactgggg ccagggaacc
ctggtcaccg tctcaagc 34814324DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 14caagacatcc
agatgaccca gtctccatcc tccctgtctg catctgtagg agacagagtc 60accatcactt
gccgggcagg tcagagcatt agcaactttt taaattggta tcagcataca
120ccagggacag gccctaaagt cctgatctat gctgcatcca gtttgcaaag
tggggtccca 180tcacggttca gtggcagtgg atctgggaca gaattcactc
tcaccatcac caatctgcaa 240cctgaagatt ttgcaactta ctactgtcaa
cagagttaca gtaccccatt cactttcggc 300cctgggacca aagtggatat caag
32415116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 15Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Gly Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Arg Ser
Ser Gly Gly Leu Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90
95 Ala Lys Gly Leu Tyr Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110 Thr Val Ser Ser 115 16108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
16Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1
5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Val Asn
Asn 20 25 30 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Arg Leu Leu 35 40 45 Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Ser Ile Ser Asn Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Tyr Ser Thr Pro 85 90 95 Arg Gly Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 17348DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
17gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60tcttgcgctg cttccggatt cactttctct aattactgga tgggttgggt tcgccaagct
120cctggtaaag gtttggagtg ggtttcttat atccgttctt ctggtggcct
tactcattat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca
actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac
acagccacat attactgtgc gaaaggacta 300tattcctttg actactgggg
ccagggaacc ctggtcaccg tctcaagc 34818324DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
18caagacatcc agatgaccca gtctccatct tccctgtctg cttctgtagg agacagagtc
60accatcactt gccgggccag tcagggcgtt aacaattttt tagcctggta tcagcaaaaa
120ccagggaagg cccctaggct cctgatctat gctgcatcca ctttgcagag
tggggtccca 180tcaaggttca gcggcagtgg atctgggaca gatttcactc
tctccatcag caacctgcag 240cctgaagact ttgcaactta ttactgtcaa
cagagttaca gtacccctcg ggggttcggc 300caagggacca aggtggaaat caaa
324195PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptide 19Thr Tyr Ala Met Met 1 5 2017PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Arg
Ile Tyr Pro Ser Gly Gly Thr Thr Thr Tyr Ala Asp Ser Val Lys 1 5 10
15 Gly 217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Gly Ser Ala Ala Ser Ser Tyr 1 5
2211PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 22Arg Ala Ser Gln Gly Ile Ser Asn Tyr Leu Ala 1 5
10 237PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 23Ala Ala Ser Thr Leu Gln Ser 1 5
249PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 24Gln Gln Leu Ile Ser Tyr Pro Phe Thr 1 5
255PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 25Gln Tyr Asn Met Thr 1 5 2617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 26Ser
Ile Tyr Ser Ser Gly Gly Val Thr Pro Tyr Ala Asp Ser Val Lys 1 5 10
15 Gly 277PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Gly Arg Leu Leu Phe Asp Tyr 1 5
2811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Arg Ala Ser Gln Ser Ile Ser Asn Tyr Leu Asn 1 5
10 297PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 29Ala Ala Ser Ser Leu Gln Ser 1 5
309PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Gln Gln Gly Tyr Thr Ala Pro Arg Ser 1 5
315PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Asn Tyr Gln Met Asp 1 5 3217PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 32Tyr
Ile Gly Pro Ser Gly Gly Arg Thr Lys Tyr Ala Asp Ser Val Lys 1 5 10
15 Gly 337PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33Gly Leu Tyr Ser Phe Asp Tyr 1 5
3411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 34Arg Ala Gly Gln Ser Ile Ser Asn Phe Leu Asn 1 5
10 357PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 35Ala Ala Ser Ser Leu Gln Ser 1 5
369PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 36Gln Gln Ser Tyr Ser Thr Pro Phe Thr 1 5
375PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 37Asn Tyr Trp Met Gly 1 5 3817PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 38Tyr
Ile Arg Ser Ser Gly Gly Leu Thr His Tyr Ala Asp Ser Val Lys 1 5 10
15 Gly 397PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 39Gly Leu Tyr Ser Phe Asp Tyr 1 5
4011PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 40Arg Ala Ser Gln Gly Val Asn Asn Phe Leu Ala 1 5
10 417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 41Ala Ala Ser Thr Leu Gln Ser 1 5
429PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 42Gln Gln Ser Tyr Ser Thr Pro Arg Gly 1 5
4310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 43Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 1 5
10
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