Indian Patents
Title of Invention

ANTIBODIES OR FRAGMENTS THEREOF HAVING SCLEROTIN BINDING AND SCLEROTIN NEUTRALIZING ACTIVITY
Abstract Compositions and methods relating to epitopes of selerostin protein, and selerostin binding agents, such as antibodies capable of binding to selerostin, are provided.
Full Text WO 2006/119107 PCT/US2006/016441
BINDING AGENTS
TECHNICAL FIELD
The present invention relates generally to epitopes of sclerostin protein, including
human sclerostin protein, and binding agents (such as antibodies) capable of binding to
sclerostin or fragments thereof.
BACKGROUND OF THE INVENTION
Two or three distinct phases of changes to bone mass occur over the life of an
individual (see Riggs, West J. Med. 154:63-77 (1991)). The first phase occurs in both men and
women and proceeds to attainment of a peak bone mass. This first phase is achieved through
linear growth of the endochondral growth plates and radial growth due to a rate of periosteal
apposition. The second phase begins around age 30 for trabecular bone (flat bones such as the
vertebrae and pelvis) and about age 40 for cortical bone (e.g., long bones found in the limbs) and
continues to old age. This phase is characterized by slow bone loss and occurs in both men and
women. In women, a third phase of bone loss also occurs, most likely due to postmenopausal
estrogen deficiencies. During this phase alone, women may lose an additional bone mass from
the cortical bone and from the trabecular compartment (see Riggs, supra).
Loss of bone mineral content can be caused by a wide variety of conditions and
may result in significant medical problems. For example, osteoporosis is a debilitating disease
in humans and is characterized by marked decreases in skeletal bone mass and mineral density,
structural deterioration of bone, including degradation of bone microarchitecture and
corresponding increases in bone fragility (i. e., decreases in bone strength), and susceptibility to
fracture in afflicted individuals. Osteoporosis in humans is generally preceded by clinical
osteopenia (bone mineral density that is greater than one standard deviation but less than 2.5
standard deviations below the mean value for young adult bone), a condition found in
approximately 25 million people in the United States. Another 7-8 million patients in the United
States have been diagnosed with clinical osteoporosis (defined as bone mineral content greater
than 2.5 standard deviations below that of mature young adult bone). The frequency of
osteoporosis in the human population increases with age. Among Caucasians, osteoporosis is
predominant in women who, in the United States, comprise 80% of the osteoporosis patient
pool. The increased fragility and susceptibility to fracture of skeletal bone in the aged is
aggravated by the greater risk of accidental falls in this population. Fractured hips, wrists, and
vertebrae are among the most common injuries associated with osteoporosis. Hip fractures in
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particular are extremely uncomfortable and expensive for the patient, and for women, correlate
with high rates of mortality and morbidity.
Although osteoporosis has been regarded as an increase in the risk of fracture due
to decreased bone mass, few of the presently available treatments for skeletal disorders can
increase the bone density of adults, and most of the presently available treatments work
primarily by inhibiting further bone resorption rather than stimulating new bone formation.
Estrogen is now being prescribed to retard bone loss. However, some controversy exists over
whether patients gain any long-term benefit and whether estrogen has any effect on patients
over 75 years old. Moreover, use of estrogen is believed to increase the risk of breast and
endometrial cancer. Calcitonin, osteocalcin with vitamin K, or high doses of dietary calcium,
with or without vitamin D, have also been suggested for postmenopausal women. High doses of
calcium, however, often have undesired gastrointestinal side effects, and serum and urinary
calcium levels must be continuously monitored (e.g., Khosla and Riggs, Mayo Clin. Proc.
70:978982, 1995).
Other current therapeutic approaches to osteoporosis include bisphosphonates
(e.g., Fosamax™, Actonel™, Bonviva™, Zometa™, olpadronate, neridronate, skelid, bonefos), .-■■
parathyroid hormone, calcilytics, calcimimetics (e.g., cinacalcet). statins, anabolic steroids,
lanthanum and strontium salts, and sodium fluoride. Such therapeutics, however, are often
associated with undesirable side effects {see Khosla and Riggs, supra).
Sclerostin, the product of the SOST gene, is absent in sclerosteosis, a skeletal
disease characterized by bone overgrowth and strong dense bones (Brunkow et al., Am. J. Hum.
Genet., 68:577-589,2001; Balemans et al., Hum. Mol. Genet., 10:537-543,2001). The amino
acid sequence of human sclerostin is reported by Brunkow et al. ibid and is disclosed herein as
SEQ ID NO:1.
BRIEF SUMMARY OF THE INVENTION
Disclosed herein are compositions and methods that can be used to increase at
least one of bone formation, bone mineral density, bone mineral content, bone mass, bone
quality and bone strength, and that therefore may be used to treat a wide variety of conditions in
which an increase in at least one of bone formation, bone mineral density, bone mineral content,
bone mass, bone quality and bone strength is desirable. The present invention also offers other
related advantages described herein.
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The invention relates to regions (epitopes) of human sclerostin recognized by the
binding agents disclosed herein, methods of using these epitopes, and methods of making such
epitopes.
The invention also relates to epitopes specific to the region of sclerostin
identified as Loop 2, and binding agents which specifically bind to that region.
The invention also relates to epitopes specific to the cystine-knot region of
sclerostin, and binding agents such as antibodies specifically binding to that region.
The invention relates to binding agents, such as antibodies, that specifically bind
to sclerostin. The binding agents can be characterized by their ability to cross-block the binding
of at least one antibody disclosed herein to sclerostin and/or to be cross-blocked from binding
sclerostin by at least one antibody disclosed herein. The antibodies and other binding agents can
also be characterized by their binding pattern to human sclerostin peptides in a "human
sclerostin peptide epitope competition binding assay" as disclosed herein.
The invention relates to binding agents, such as antibodies, that can increase at
least one of bone formation, bone mineral density, bone mineral content, bone mass, bone
quality and bone strength in a mammal.
The invention relates to binding agents, such as antibodies, that can biock the
inhibitory effect of sclerostin in a cell based mineralization assay.
The invention further relates to polypeptide constructs comprising two, three, or
four polypeptide fragments linked by at least one disulfide bond, representing a core region of
the cystine-knot of sclerostin, and antibodies capable of specifically binding thereto.
The invention relates to methods of obtaining epitopes suitable for use as
immunogens for generating, in mammals, binding agents, such as antibodies capable of binding
specifically to sclerostin; in certain embodiments the binding agents generated are capable of
neutralizing sclerostin activity in vivo.
The invention relates to a composition for eliciting an antibody specific for
sclerostin when the composition is administered to an animal, the composition comprising a
polypeptide having the amino acid sequence of SEQ ID NO:6, SEQ ID NO:63, SEQ ID NO:64,
SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69.
The invention also relates to a composition for eliciting an antibody specific for
sclerostin when the composition is administered to an animal, the composition comprising at
least one polypeptide consisting essentially of the amino acid sequence of SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4 or SEQ ID NO:5; the composition may comprise at least two or at least
three of the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID
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NO:5, and the composition may comprise all four of the amino acid sequences of SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
,The invention further relates to a composition for eliciting an antibody specific
for sclerostin when the composition is administered to an animal, the composition comprising a
polypeptide having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4
and SEQ ID NO:5, wherein SEQ ID NO:2 and 4 are joined by a disulfide bond at amino acid
positions 57 and 111 with reference to SEQ ID NO:1, and SEQ IDNO:3 and 5 are joined by at
least one of (a) a disulfide bond at amino acid positions 82 and 142 with reference to SEQ ID
NO:1, and (b) a disulfide bond at amino acid positions 86 and 144 with reference to SEQ ID
NO: 1; the polypeptide may retain the tertiary structure of the corresponding polypeptide region
of human sclerostin of SEQ ID NO:1.
The invention also relates to polypeptide T20.6 consisting essentially of a
multiply truncated human sclerostin protein of SEQ ID NO: 1, wherein amino acids 1-50, 65-72,
91-100, 118-137, and 150-190 of SEQ ID NO:1 are absent from the polypeptide; this
polypeptide may be obtained by tryptic digestion of human sclerostin, and the protein may be
isolated by HPLC fractionation. i
'•' The invention further relates to immunogenic portion T20.6 of human sclerostin
comprising amino acids 51-64,73-90, 101-117, and 138-149 of SEQ ID NO:1, wherein the
immunogenic portion comprises at least one of:
(a) a disulfide bond between amino acids 57 and 111;
(b) a disulfide bond between amino acids 82 and' 142; and
(c) a disulfide bond between amino acids 86 and 144;
the immunogenic portion may have at least two of these disulfide bonds; and the
immunogenic portion may have all three disulfide bonds.
The invention further relates to an immunogenic portion T20.6 derivative of
human sclerostin comprising amino acids 57-64, 73-86, 111-117, and 138-144 of SEQ ID NO:1,
wherein the immunogenic portion comprises at least one of:
(a) a disulfide bond between amino acids 57 and 111;
(b) a disulfide bond between amino acids 82 and 142; and
(c) a disulfide bond between amino acids 86 and 144;
the immunogenic portion may have at least two of these disulfide bonds; and the
immunogenic portion may have all three disulfide bonds.
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The invention yet further relates to a polypeptide consisting essentially of a
human sclerostin protein of SEQ ID NO:1 truncated at the C-terminal and N-terminal ends,
wherein amino acids 1-85 and 112-190 of SEQ ID NO:1 are absent from the polypeptide.
The invention also relates to an immunogenic portion of human sclerostin,
comprising amino acids 86-111 of SEQ ID NO:1; the immunogenic portion may consist
essentially of contiguous amino acids CGPARLLPNAIGRGKWWRPSGPDFRC (SEQ ID
NO:6).
The invention further relates to an immunogenic portion of rat sclerostin,
comprising amino acids 92-109 of SEQ ID NO:98; the immunogenic portion may consist
essentially of contiguous amino acids PNAIGRVKWWRPNGPDFR (SEQ ID NO:96).
The invention still further relates to an immunogenic portion of rat sclerostin,
comprising amino acids 99-120 of SEQ ID NO:98; the immunogenic portion may consist
essentially of contiguous amino acids KWWRPNGPDFRCEPDRYRAQRV (SEQ ID NO:97).
The invention relates to a method of producing an immunogenic portion of
human sclerostin, comprising the steps of:
(a) treating human sclerostin to achieve complete tryptic digestion;
(b) collecting the tryptic digest sample having average molecular weight of
7,122.0 Daltons (theoretical mass 7121.5 Daltons)or retention time of about
20.6 minutes as determined by elution from a reverse-phase HPLC column
with linear gradient from 0.05% trifluoroacetic acid to 90% acetonitrile in
0.05% TFA at a flow rate of 0.2ml/min; and
(c) purifying the immunogenic portion.
The invention relates to a method of generating an antibody capable of
specifically binding to sclerostin, comprising:
(a) immunizing an animal with a composition comprising a polypeptide of SEQ
ID NO:6, SEQ ID NO.63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,
SEQ ID NO:67, SEQ ID NO.68, SEQ ID NO:69, SEQ ID NO:96, or SEQ ID
NO:97;
(b) collecting sera from the animal; and
(c) isolating from the sera an antibody capable of specifically binding to
sclerostin.
The invention also relates to a method of generating an antibody capable of
specifically binding to sclerostin, the method comprising:
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(a) immunizing an animal with a composition comprising polypeptide T20.6 or a
derivative of T20.6;
(b) collecting sera from the animal; and
(c) isolating from the sera an antibody capable of specifically binding to
sclerostin.
The invention further relates to a method of detecting an anti-sclerostin antibody
in a biological sample, comprising the steps of
" (a) contacting the biological sample with a polypeptide consisting essentially of
SEQ ID NO:6, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NC-.65, SEQ ID
NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:96, or
SEQ ID NO:97 under conditions allowing a complex to form between the
antibody and the polypeptide; and
(b) detecting the presence or absence of the complex,
wherein the presence of the complex indicates that the biological sample contains an anti-
sclerostin antibody.
The invention also relates to a method of detecting an anti-sclerostin antibody in
a biological sample, comprising the steps of
(a) contacting the biological sample with polypeptide T20.6 or a derivative of
T20.6 under conditions allowing a complex to form between the antibody and
the polypeptide; and
(b) detecting the presence or absence of the complex,
wherein the presence of the complex indicates that the biological sample contains an anti-
sclerostin antibody.
The invention further relates to a sclerostin binding agent, such as an antibody,
that cross-blocks the binding of at least one of antibodies Ab-A, Ab-B, Ab-C, or Ab-D to a
sclerostin protein. The sclerostin binding agent may also be cross-blocked from binding to
sclerostin by at least one of antibodies Ab-A, Ab-B, Ab-C, or Ab-D. 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.
The invention further relates to a sclerostin binding agent, such as an antibody,
that is cross-blocked from binding to sclerostin by at least one of antibodies Ab-A, Ab-B, Ab-C,
or Ab-D. 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.
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The invention further relates to a sclerostin binding agent, such as an isolated
antibody, that cross-blocks the binding of at least one of antibodies 1-24 (Ab-1 to Ab-24) to a
sclerostin protein. The sclerostin binding agent may also be cross-blocked from binding to
sclerostin by at least one of antibodies 1-24 (Ab-1 to Ab-24). The isolated antibody, or an
antigen-binding fragment thereof, may be a polyclonal antibody, a monoclonal antibody, a
humanized antibody, a human antibody, or a chimeric antibody.
The invention further relates to a sclerostin binding agent, such as an isolated
antibody, that is cross-blocked from binding to sclerostin by at least one of antibodies 1-24 (Ab-
1 to Ab-24); the isolated antibody, or an antigen-binding fragment thereof, may be a polyclonal
antibody, a monoclonal antibody, a humanized antibody, a human antibody, or a chimeric
antibody.
The invention further relates to a binding agent, such as an isolated antibody that
exhibits a similar binding pattern to human sclerostin peptides in a "human sclerostin peptide
epitope competition binding assay" as that exhibited by at least one of the antibodies Ab-A, Ab-
B, Ab-C or Ab-D; the isolated antibody, or an antigen-binding fragment thereof, may be a
polyclonal antibody, a monoclonal antibody; a humanized antibody, a human antibody, or a
chimeric antibody.
The invention still further relates to a method for treating a bone disorder
associated with at least one of low bone formation, low bone mineral density, low bone mineral
content, low bone mass, low bone quality and low bone strength in a mammalian subject which
comprises providing to a subject in need of such treatment an amount of an anti-sclerostin
binding agent sufficient to increase at least one of bone formation, bone mineral density, bone
mineral content, bone mass, bone quality and bone strength wherein the anti-sclerostin binding
agent comprises an antibody, or sclerostin-binding fragment thereof.
The invention also relates to an isolated sclerostin polypeptide or fragments
thereof, wherein the polypeptide contains 6 conserved cysteine residues and the fragments
thereof comprise from 7 to 14 amino acids of SEQ ID NO:2; 8 to 17 ammo acids of SEQ ID
NO:3; 8 to 18 residues of SEQ ID NO:4; and 6 to 12 residues of SEQ D}NO:5, and the
polypeptide or fragments thereof are stabilized by disulfide bonds between SEQ ID NO:2 and 4,
and between SEQ ID NO:3 and 5; the polypeptide or fragments may comprise 10-14 amino
acids of SEQ ID NO:2; 14 to 17 amino acids of SEQ ID NO:3; 13 to 18 amino acids of SEQ ID
NO:4;, and 8 to 12 residues of SEQ ID NO:5; and the polypeptide or fragments may comprise
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
Provided herein are antibodies that specifically bind to human sclerostin. The
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antibodies are characterized by their ability to cross-block the binding of at least one antibody
disclosed herein to human sclerostin and/or to be cross-blocked from binding human sclerostin
by at least one antibody disclosed herein.
Also provided is an isolated antibody, or an antigen-binding fragment thereof,
that can increase at least one of bone formation, bone mineral density, bone mineral content,
bone mass, bone quality and bone strength in a mammal.
Also provided in an isolated antibody, or an antigen-binding fragment thereof,
that can block'the inhibitory effect of sclerostin in a cell based mineralization assay.
Also provided is a binding agent, such as an antibody, that specifically binds to
human sclerostin and has at least one CDR sequence selected from SEQ ID NOs: 39,40,41,42,
43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 78,79, 80, 81,99,
100,101,102,103, 104, 105, 106, 107,108, 109, 110, 111, 112,113, 114,115,116,237,238,
239, 240,241,242, 243,244, 245, 246,247, 248,249,250, 251,252, 253, 254,255, 256,257,
258, 259,260,261, 262, 263, 264, 265,266, 267,268, 269, 270,271, 272,273,274,275,276,
277, 278,279,280, 281, 282,283, 284,285, 286,287,288, 289>.29O,291,292,293, 294,295,
296, 297,298, 351, 352, 353, 358, 359, and 360, and variants thereof, wherein the antibody or
antigen-binding fragment thereof neutralizes sclerostin.
Also provided is a binding agent, such as an antibody, that specifically binds to
human sclerostin and has at least one CDR sequence selected from SEQ ED NOs:39, 40,41, 42,
x 43,44, 45,46, 47,48, 49, 50, 51, 52, 53,. 54, 55, 56, 57, 58, 59, 60, 61, 62, 78,79, 80, 81, 99,
100,101,102,103, 104,105, 106, 107,108,109,110, 111, 112,113,114,115,116,237,238,
239, 240, 241,242, 243,244,245, 246,247, 248, 249,250, 251,252,253,254,255,256,257,
258,259,260,261, 262, 263,264, 265,266, 267, 268,269, 270,271, 272,273,274,275, 276,
277,278, 279,280, 281,282, 283, 284,285, 286, 287,288,289, 290,291,292,293,294, 295,
296,297, 298, 351, 352, 353, 358, 359, and 360, and variants thereof.
Also provided are regions of human sclerostin which are important for the in vivo
activity of the protein.
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.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the amino acid sequences of the mature form (signal peptides
cleaved off) of the light chain (Figure 1 A) (SEQ ID NO:23) and heavy chain (Figure IB) (SEQ
ID NO:27) for the anti-human sclerostin and anti-mouse sclerostin antibody Ab-A.
Figure 2 depicts the amino acid sequences of the mature form (signal peptides
cleaved off) of the light chain (Figure 2A) (SEQ ID NO:31) and heavy chain (Figure 2B) (SEQ
ID NO:35) for the anti-human sclerostin and anti-mouse sclerostin antibody Ab-B.
Figure 3 depicts the amino acid sequences of the mature form (signal peptides
cleaved off) of the light chain (Figure 3 A) (SEQ ID NO: 15) and heavy chain (Figure 3B) (SEQ
ID NO: 19) for the anti-human sclerostin and anti-mouse sclerostin antibody Ab-C.
Figure 4 depicts the amino acid sequences of the mature form (signal peptides
cleaved off) of the light chain (Figure 4A) (SEQ ED NO:7) and heavy chain (Figure 4B) (SEQ
ID NO:11) for the anti-human sclerostin and anti-mouse sclerostin antibody Ab-D.
Figure 5 depicts bone mineral density in mice measured at two skeletal sites
(lumbar vertebrae and tibial metaphysis) after 3 weeks of treatment with vehicle, PTH (1-34),
Ab-A or Ab-B.
Figure 6 shows bone mineral density in mice measured at two skeletal sites
(lumbar vertebrae and tibial metaphysis) after 2 weeks of treatment with vehicle, PTH (1-34) or
Ab-C.
Figure 7 depicts bone mineral density ia mice measured at two skeletal sites
(lumbar vertebrae and tibial metaphysis) after 3 weeks of treatment with vehicle or Ab-D.
Figure 8 depicts the amino acid sequence of the mature form (signal peptide
cleaved off) of human sclerostin (SEQ ID NO:1). Also depicted is the nucleotide sequence of
the human sclerostin coding region that encodes the mature form of human sclerostin. The eight
cysteines are numbered Cl through C8. The cystine-knot is formed by three disulfide bonds
(C1-C5; C3-C7; C4-C8). C2 and C6 also form a disulfide bond, however this disulfide is not
part of the cystine-knot.
Figure 9 depicts a schematic of the basic structure of human sclerostin. There is
an N-terminal arm (from the first Q to Cl) and a C-terminal arm (from C8 to the terminal Y). In
between these arms there is the cystine-knot structure (formed by three disulfides: C1-C5; C3-
C7; C4-C8) and three loops which are designated Loopl, Loop 2 and Loop 3. The distal regions
of Loop 1 and Loop 3 are linked by the C2-C6 disulfide. Potential trypsin cleavage sites are
indicated (arginine=R and lysine=K). Some of the potential AspN cleavage sites are indicated
(only aspartic acid (D) residues are shown).
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Figure 10 depicts the HPLC peptide maps of human sclerostin after digestion
with either trypsin or AspN. The human sclerostin peptides generated by trypsin digestion are
indicated (T19.2, T20, T20.6 and T21-22) as are the human sclerostin peptides generated by
AspN digestion (AspN14.6, AspN18.6 and AspN22.7-23.5).
Figure 11 depicts sequence and mass information for the isolated human
sclerostin disulfide linked peptides generated by trypsin digestion. Seq. pos. = sequence
position. Obs. = observed. Observed mass was determined by ESI-LC-MS analysis.
" Figure 12 depicts sequence and mass information for the isolated human
sclerostin peptides generated by AspN digestion. The AspN22.7-23.5 peptide contains the 4
disulfide bonds. Seq. pos. = sequence position. Obs. = observed. Observed mass was
determined by ESI-LC-MS analysis.
Figure 13 shows a linear schematic of four human sclerostin peptides (T19.2,
T20, T20.6 and T21-22) generated by trypsin digestion.
Figure 14 shows a linear schematic of five human sclerostin peptides (AspN14.6,
AspN18.6 and AspN22.7-23.5) generated by AspN digestion. The AspN14.6 HPLC peak is
composed of three peptides not linked by any disulfide bonds.
Figure 15 shows the resonance unit (Ru) signal from the Biacore-based "human
sclerostin peptide epitope competition binding assay." Relative Mab binding to various human sclerostin-peptides (in solution) versus Mab binding to intact mature form human sclerostin
(immobilized on Biacore chip) was assessed. Data shown is for Ab-A. Human sclerostin
peptides used were T19.2, T20, T20.6, T21-22, AspN14.6, AspN18.6 and AspN22.7-23.5.
Figure 16 shows the resonance unit (Ru) signal from the Biacore-based "human
sclerostin peptide epitope competition binding assay!" Relative Mab binding to various human
sclerostin-peptides (in solution) versus Mab binding to intact mature form human sclerostin
(immobilized on Biacore chip) was assessed. Data shown is for Ab-B. Human sclerostin
peptides used were T19.2, T20, T20.6,121-22, AspN14.6, AspN18.6 and AspN22.7-23.5.
Figure 17 shows the resonance unit (Ru) signal from the Biacore-based "human
sclerostin peptide epitope competition binding assay." Relative Mab binding to various human
sclerostin-peptides (in solution) versus Mab binding to intact mature form human sclerostin
(immobilized on Biacore chip) was assessed. Data shown is for Ab-C. Human sclerostin
peptides used were T19.2, T20, T20.6, T21-22, AspN14.6, AspN18.6 and AspN22.7-23.5.
Figure 18 shows the resonance unit (Ru) signal from Biacore-based "human
sclerostin peptide epitope competition binding assay." Relative Mab binding to various human
sclerostin-peptides (in solution) versus Mab binding to intact mature form human sclerostin
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(immobilized on Biacore chip) was assessed. Data shown is for Ab-D. Human sclerostin
peptides used were T19.2, T20, T20.6, T21-22, AspN14.6, AspN18.6 and AspN22.7-23.5.
Figure 19 shows two Mab binding epitopes of human sclerostin. Figure 19A
shows sequence of the Loop 2 epitope for binding of Ab-A and Ab-B to human sclerostin (SEQ
ID NO:6). Figure 19B shows sequence, disulfide bonding and schematic of the T20.6 epitope
for binding of Ab-C and Ab-D to human sclerostin (SEQ ID NO:2-5).
Figure 20 depicts the HPLC peptide maps of human sclerostin after digestion
with trypsin. Figure 20A shows digestion of the human sclerostin Ab-D complex. Figure 20B
shows digestion of human sclerostin alone. The T19.2, T20, T20.6 and T21-22 peptide peaks
are indicated.
Figure 21 shows the sequence, disulfide bonding and schematic of the
"T20.6 derivative 1 (cystine-knot + 4 arms)" epitope for binding of Ab-D to human
sclerostin. (SEQ ID NO:70-73).
Figure 22 shows results from the MC3T3-E1-BF osteoblast cell line
mineralization assay used for identifying anti-sclerostin neutralizing Mabs. Mouse
sclerostin (Scl) was used at 1 µg/ml. Monoclonal antibodies were used at 10 and 5 µg/ml.
Extent of mineraiization (various types of insoluble calcium phosphate) was quantitated by
measuring calcium. .. . .
Figure 23 depicts results from the MC3T3-E1-BF osteoblast cell line
mineralization assay used for identifying anti-sclerostin neutralizing Mabs. Human sclerostin
(Scl) was used at 1 µg/ml. Monoclonal antibodies were used at 8 and 4 µg/ml. Extent of
mineralization (various types of insoluble calcium phosphate) was quantitated by measuring
calcium.
Figure 24 shows results from the MC3T3-E1-BF osteoblast cell line
mineralization assay used for identifying anti-sclerostin neutralizing Mabs. Human sclerostin
(Scl) was used at 1 µg/ml. Monoclonal antibodies were used at 10 µg/ml. Extent of
mineralization (various types of insoluble calcium phosphate) was quantitated by measuring
calcium.
Figure 25 depicts results from an inflammation-induced bone loss SCDD mouse
model. Ab-A treatment protected mice from inflammation-related bone loss associated with
colitis when measured as total bone mineral density (Figure 25A), vertebral bone density (Figure
25B), and femur bone density (Figure 25C).
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DETAILED DESCRIPTION
The present invention relates to regions of the human sclerostin protein that
contain epitopes recognized by antibodies that also bind to full-length sclerostin, and methods of
making and using these epitopes. The invention also provides binding agents (such as
antibodies) that specifically bind to sclerostin or portions of sclerostin, and methods for using
such binding agents. The binding agents are useful to block or impair binding of human
sclerostin to One or more ligand.
Recombinant human sclerostin/SOST is commercially available from R&D
Systems (Minneapolis, MN, USA; 2006 cat# 1406-ST-025). Additionally, recombinant mouse
sclerostin/SOST is commercially available from R&D Systems (Minneapolis, MN, USA; 2006
cat# 1589-ST-025). Research grade sclerostin binding monoclonal antibodies arc commercially
available from R&D Systems (Minneapolis, MN, USA; mouse monoclonal: 2006 cat#
MAB1406; rat monoclonal: 2006 cat# MAB1589). U.S. PatentNos. 6,395,511 and 6,803,453,
and U.S. Patent Publications 20040009535 and 20050106683 refer to anti-sclerostin antibodies
generally.
As used herein, the term human scierostin is intended to include the protein of
SEQ ID NO:1 and allelic variants thereof. Sclerostin can be purified from 293T host cells that
have been transfected by a gene encoding sclerostin by elution of filtered supernatant of host cell
culture fluid using a Heparin HP column, using a salt gradient. The preparation and further
purification using cation exchange chromatography are described in Examples 1 and 2.
Binding agents of the invention are preferably antibodies, 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')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, 9,1126-1136). Antibody
polypeptides are also disclosed in U. S. Patent No. 6,703,199, including fibronectin polypeptide
monobodies. Other antibody polypeptides are disclosed in U.S. Patent Publication
2005/0238646, which are single-chain polypeptides.
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
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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')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. Patent
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.
An antibody fragment may also be any synthetic or genetically engineered
protein. For example, antibody fragments inciude 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).
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 region") 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)).
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 CDR's as
described herein. The binding agent further may comprise at least one variable region domain of
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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 sclerostin, for example CDR-H1, CDR-H2, CDR-H3 and/or the light chain CDRs
specifically described herein and which is 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 sclerostin with an affinity at least equal to 1 x 10-7M or less as describedbelow.
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 VH). 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 (scVH).
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 createdj for exampie, 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.
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 CHI
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 CHI and CK domain, respectively. The CHI 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 farther domains, such as antibody CH2 and CH3
domains.
As described herein, binding agents comprise at least one of these CDRs. For
example, one or more CDR may be incorporated into known antibody framework regions (IgGl,
IgG2, etc.), or conjugated to a suitable vehicle to enhance the half-life thereof. Suitable vehicles
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WO 2006/119107 PCT/US2006/016441
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.
In certain preferred 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 oxjde/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 polymer 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.
It will be appreciated 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, that do not destroy the sclerostin 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 araino acid residue with a normative residue such that there is little or no effect on the
polarity or charge of the ammo acid residue at that position.
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 non-homologous regions of the molecule.
Moreover, one skilled in the art may generate test variants containing a single
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WO 2006/119107 PCT/US2006/016441
amino acid substitution at each desired amino acid residue. The variants can then be screened
using activity assays known to those skilled in the art. 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.
' 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.
Additionally, one skilled in the art can review structure-function studies
identifying residues in similar polypeplides thai 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.
One skilled in the art can also analyze the three-dimensional structure and amino
acid sequence in relation to that structure in similar polypeptides. In view of such information,
one skilled in the art may predict the alignment of amino acid residues of an antibody with
respect to its three dimensional structure. In certain embodiments, one skilled in the art may
choose not to make radical changes to amino acid residues predicted to be on the surface of the
protein, since such residues may be involved in important interactions with other molecules.
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 Mol. 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
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WO 2006/119107 PCT/US2006/016441
proteins which have a sequence identity of greater than 30%, or 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 at., Nucl. Acid. Res.,
27(l):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.
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)).
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.
Desired 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 sclerostin, or to increase or decrease the affinity of the antibodies to sclerostin described
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WO 2006/119107 PCT/US2006/016441
herein.
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.
In certain embodiments, binding agents of the invention may be chemically
bonded with polymers, lipids, or other moieties.
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 that bind to an antigen (e.g., CDRs, a variable region,
etc.) 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.
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, LACI-D1,
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WO 2006/119107 PCT/US2006/016441
Z domain and tendramisat domains may be used (See e.g., .Nygren and Uhlen, 1997, Current
Opinion in Structural Biology, 7, 463-469).
In preferred embodiments, it will be appreciated that the binding agents of the
invention include the humanized antibodies described herein.. Humanized antibodies such as
those described herein can be produced using techniques known to those skilled in the art
(Zhang, W., et al, Molecular Immunology. 42(72):1445-1451,2005; Hwang W. et al, Methods.
36(1):35-A2, 2005; Dall'Acqua WF, et al., Methods 36(J):43-6Q, 2005; and Clark, M.,
Immunology Today. 21(8):391-402, 2000).
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-
L1, CDR-L2 and CDR-L3 as specifically disclosed herein. At least one of the regions of CDR-
Hl, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 may have at least one ammo acid
substitution, provided that the binding agent retains the binding specificity of the non-substituted
CDR. 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 sclerostin and/or
. neutralizes sclerostin. The non-CDR portion of the binding agent may be a non-protein
molecule in which the binding agent exhibits a similar binding pattern to human sclerostin
peptides in a "human sclerostin peptide epitope competition binding assay" as that exhibited by
at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-
7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-125 Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-
20, Ab-21, Ab-22, Ab-23, and Ab-24, and/or neutralizes sclerostin. 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 sclerostin and/or neutralizes sclerostin. 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 sclerostin peptides in the human sclerostin peptide epitope competition binding
assay (described hereinbelow) as that exhibited by at least one of the antibodies Ab-A, Ab-B,
Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12,
Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24,
and/or neutralizes sclerostin.
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
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WO 2006/119107 PCT/US2006/016441
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®. Each of the above-mentioned CDRs will be typically located in a variable region
framework at positions 31-35 (CDR-H1), 50-65 (CDR-H2) and 95-102 (CDR-H3) of the heavy
chain and positions 24-34 (CDR-L1), 50-56 (CDR-L2) and 89-97 (CDR-L3) of the light chain
according to the Kabat numbering system (Kabat et al., 1987 in Sequences of Proteins of
Immunological Interest, U.S. Department of Health and Human Services, NIH, USA).
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, Schizosaccharomycespombe, and Pichiapastoris), 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.
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. In order 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 Sangeref 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
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WO 2006/119107 PCT/US2006/016441
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).
Where it is desired to improve the affinity of antibodies according to the
invention containing one or more of the above-mentioned CDRs can be obtained by a number of
affinity maturation protocols including maintaining the CDRs (Yang et al, J. Mol. Biol, 254,
392-403, 1995), chain shuffling (Marks et al, Bio/Technology, 10, 779-783, 1992), use of
mutation strains of E. coll (Low et al, J. Mol. Biol, 250, 350-368, 1996), DNA shuffling
(Patten et al, Curr. Opin. Biotechnol, 8, 724-733, 1997), phage display (Thompson et al, J.
Mol. 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).
Other 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. Patent
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..
Monoclonal antibodies can be obtained by injecting an animal, for example, a rat,
hamster, a rabbit, or preferably a mouse, including for example a transgenic or a knock-out, as
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known in the art, with an immunogen comprising human sclerostin of SEQ ID NO:1, or a
fragment thereof, according to methods known in the art and described herein. 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 sclerostin 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 hybridoraa 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 sclerostin, 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 sclerostin are
selected and cultured. The monoclonal antibodies from the hybridoma cultures may be isolated
from the supematants 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 selected 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, ananticonstant region
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WO 2006/119107 PCT/US2006/016441
(light chain or heavy chain) antibody, an anti-idiotype antibody, and a TGF-beta binding protein,
or fragment or variant thereof.
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, 6:519, 1994; U.S. Patent 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 sclerostin. 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.
Another method for generating human antibodies of the invention includes
immortalizing human peripheral blood cells by EBV transformation. See, e.g., U.S. Patent No.
4,464,456. Such an immortalized B cell line (or lymphoblastoid cell line) producing a
monoclonal antibody that specifically binds to sclerostin 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-sclerostin 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.,
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WO 2006/119107 PCT/US2006/016441
Hybridama 8:377-89 (1989)). Still another method to generate human monoclonal antibodies is
in vifro immunization, which includes priming human splenic B cells with human sclerostin,
followed by fusion of primed B cells with a heterohybrid fusion partner. See, e.g., Boerner et al,
1991 /. Immunol. 147:86-95.
In certain embodiments, a B cell that is producing an anti-human sclerostin
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; US patent
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
sclerostin. 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 sclerostin. 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.
An additional method for obtaining 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. Human or murine immunoglobulin variable region gene combinatorial
libraries may be created in phage vectors that can be screened to select Ig fragments (Fab, Fv,
sFv, or multimers thereof) that bind specifically to TGF-beta binding protein or variant or
fragment thereof. See, e.g., U.S. Patent 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 7. Molec. Biol 227:381-388; Schlebusch et al., 1997 Hybridoma
16:47-52 and references cited therein. 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 M13 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.
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WO 2006/119107 PCT/US2006/016441
According to certain embodiments, immunoglobulin Fab fragments may also be displayed on a
phage particle (see, e.g., U.S. Patent No. 5,698,426).
Heavy and light chain immunoglobulin cDNA expression libraries may also be
prepared in lambda phage, for example, using MmmunoZap™(H) and MmmunoZap™(L)
vectors (Stratagene, La Jolla, California). Briefly, mRNA is isolated from a B cell population,
and used to create heavy and light chain immunoglobulin cDNA expression libraries in the
XImmunoZap(H) and WmmunoZap(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.
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 maybe
synthesized by one of ordinary skill in the art, or may be purchased from commercially available
sources. (See, e.g., Stratagene (La jolla, California), 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 Irnrnur.oZAP™H or ImmunoZAP™L (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).
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.
Preferably the binding agents specifically bind to sclerostin. 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 sclerostin, preferably
human sclerostin, 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 sclerostin
25

WO 2006/119107 PCT/US2006/016441
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 sclerostin of less
than or equal to 1 x 10-7M, less than or equal to 1 x 10-8M, less than or equal to 1 x 10-9M, less
than or equal to 1 x 10-10M, less than or equal to 1 x 10-11 M, or less than or equal to 1 x 10-12 M.
Affinity may be determined by an affinity ELISA assay. In certain embodiments,
affinity may be determined by a BIAcore 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.
Sclerostin binding agents of the present invention preferably modulate sclerostin
function in the cell-based assay described herein and/or the in vivo assay described herein and/or
bind to one or more of the epitopes described herein and/or cross-block the binding of one of the
antibodies described in this application and/or are cross-blocked from binding sclerostin by one
of the antibodies described in this application. Accordingly such binding agents can be
identified using the assays described herein.
In certain embodiments, binding agents are generated by first identifying
antibodies that bind to one more of the epitopes provided herein and/or neutralize in the cell-
based and/or in vivo assays described herein and/or cross-block the antibodies described in this
application and/or are cross-blocked from binding sclerostin by one of the antibodies described
in this application. The CDR regions from these antibodies are then used to insert into
appropriate biocompatible frameworks to generate sclerostin binding agents. The non-CDR
portion of the binding agent may be composed of amino acids, or may be a non-protein
molecule. The assays described herein allow the characterization of binding agents. Preferably
the binding agents of the present invention are antibodies as defined herein.
It will be understood by one skilled in the art that some proteins, such as
antibodies, may undergo a variety of posttranslational modifications. 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
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, RJ. Journal of Chromatography
705:129-134,1995).
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WO 2006/119107 PCT/US2006/016441
Antibodies referred to as Ab-A, Ab-B, Ab-C, Ab-D and Ab-1 are described
below. "HC" refers to the heavy chain and "LC" refers to the light chain. For some antibodies
below, the CDRs are box shaded and the constant (C) regions are shown in bold italics.
Ab-D
Antibody D (also referred to herein as Ab-D and Mab-D) is a mouse antibody
which exhibits high affinity binding to sclerostin. The BIAcore binding pattern of Ab-D is
shown in Figure 18.
The amino acid sequence of the mature form (signal peptide removed) of Ab-D

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WO 2006/119107 PCT/US2006/016441
The CDR (complementarity determining region) sequences in the variable region
of the heavy chain of Ab-D are as follows:
CDR-H1: DHYMS (SEQ ID NO:39)
CDR-H2: DINPYSGETTYNQKFKG (SEQ ID NC-.40)
CDR-H3: DDYDASPFAY (SEQ ID NO:41)
The light chain variable region CDR sequences of Ab-D are:
CDR-L1: QASQGTSMLN (SEQ ID NO:42)
CDR-L2: GSSNLED (SEQ ID NO:43)
CDR-L3: LQHSYLPYT (SEQ ED NO:44)
Ab-C
Antibody C (also referred to herein as Ab-C and Mab-C) is a mouse antibody
which exhibits high affinity binding to sclerostin. The BIAcore binding pattern of Ab-C is
shown in Figure 17. The amino acid sequence of the mature form (signal peptide removed) of
Ab-C Light Chain is as follows:

■■ 30

WO 2006/119107 PCT/US2006/016441
151 SGGASWCFL NNFYPKDINV KWKIDGSERQ NGVLNSWTDQ DSKDSTYSMS
201 STLTLTKDEY ERHNSYTCEA THKTSTSPIV KSFNRNEC (SEQ ID NO: 17)
The nucleic acid sequence of Ab-C LC including signal peptide encoding

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WO 2006/119107 PCT/US2006/016441

The CDR (complementarity determining region) sequences in the variable region
of the heavy chain of Ab-C are as follows:
CDR-H1: DCYMN (SEQ ID NO.45)
CDR-H2: DINPFNGGTTYNQKFKG (SEQ ID NO:46)
CDR-H3: SHYYFDGRVPWDAMDY (SEQ ID NO:47)
The light chain variable region CDR sequences of Ab-C are:
CDR-L1: KASQSVDYDGDSYMN (SEQ ID NO:48) ■
CDR-L2: AASNLES (SEQ ID NO:49)
CDR-L3: QQSNEDPWT (SEQ ID NO:50) .
Ab-A
Antibody A (also referred to herein as Ab-A and Mab-A) is a rabbit-mouse
chimeric antibody which exhibits high affinity binding to sclerostin. The BIAcore binding
pattern of Ab-A is shown in Figure 15.
Ab-A Light Chain

The nucleic acid sequence encoding the mature form (signal peptide removed) of
Ab-A LC:
1 GCGCAAGTGC TGACCCAGAC TCCAGCCTCC GTGTCTGCAG CTGTGGGAGG
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WO 2006/119107 PCT/US2006/016441

The CDR (complementarity determining region) sequences in the variable region
of trie heavy chain of Ab-A are as follows:
CDR-H1: SYWMN(SEQ ID NO:51)
CDR-H2: TIDSGGRTDYASWAKG (SEQ ED NO:52)
CDR-H3: NWNL (SEQ ED NO:53)
The light chain variable region CDR sequences of Ab-A are:
CDR-L1: QSSQSVYDNNWLA (SEQ ID NO:54)
CDR-L2: DASDLAS (SEQ ID NO:55)
CDR-L3: QGAYNDVIYA (SEQ ID NO: 5 6)
i
Ab-A was humanized, and is referred to as Antibody 1 (also referred to herein as
Ab-1), having the following sequences:
The nucleic acid sequence of the Ab-1 LC variable region including signal
peptide encoding sequence is
ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGT
GCCACATTTGCTCAAGTTCTGACCCAGAGTCCAAGCAGTCTCTCCGCCAGCGTAGGC
36

WO 2006/119107 PCT/US2006/016441

The CDR (complementarity determining region) sequences in the variable region
of the heavy chain of Ab-1 are as follows:
CDR-H1: SYWMN(SEQ ID NO:51)
CDR-H2: TIDSGGRTDYAS WAKG (SEQ ID NO:52)
CDR-H3: NWNL (SEQ ID NO:53)
The light chain variable region CDR sequences of Ab-1 are:
CDR-L1: QSSQSVYDNNWLA (SEQ ED NO:54)
CDR-L2: DASDLAS (SEQ ID NO:55)
CDR-L3: QGAYNDVIYA (SEQ ID NO:56)
Ab-B
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WO 2006/119107 PCT/US2006/016441
Antibody B (also referred to herein as Ab-B and Mab-B) is a mouse antibody
which exhibits high affinity binding to sclerostin. The BIAcore binding pattern of Ab-B is
shown in Figure 16.
Ab-B Light Chain

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WO 2006/119107 PCT/US2006/016441
The CDR (complementarity determining region) sequences in the variable region
of the heavy chain of Ab-B are as follows:
CDR-H1: TSGMGVG (SEQ ID NO:57)
CDR-H2: HIWWDDVKRYNPVLKS (SEQ ID NO: 5 8)
CDR-H3: EDFDYDEEYYAMDY (SEQ ID NO:59)
The light chain variable region CDR sequences of Ab-B are:
CDR-L1: SASSSVSFVD (SEQ ID NO:60)
CDR-L2: RTSNLGF (SEQ ID NO: 61)
CDR-L3: QQRSTYPPT (SEQ ID NO:62)
Antibodies disclosed herein bind to regions of human sclerostin which are
important for the in vivo activity of the protein. Binding of an antibody to sclerostin can be
correlated with increases in, for example, the bone mineral density achieved by use of the
antibody in vivo such as described in Examples 5 and 9 (mice) and Example 12 (monkey).
Increases in at least one of bone formation, bone mineral content, bone mass, bone quality and
bone strength can also be achieved by use of the antibody in vivo such as described in Examples
5 and 9 (mice) and Example 12 (monkey). Since the binding of an antibody to sclerostin is
primarily determined by its CDR sequences, an antibody for practicing the invention may be
generated with all or some of the disclosed CDR sequences in an appropriate framework,
wherein the antibody retains the ability to bind specifically to.sclerostin, and can be expected to
achieve increases in, for example, bone mineral density. Such antibodies are useful in the
treatment of human or animal conditions that are caused by, associated with, or result in at least
one of low bone formation, low bone mineral density, low bone mineral content, low bone mass,
low bone quality and low bone strength. Methods of constructing and expressing antibodies and
fragments thereof comprising CDR's of the present invention are known to those of skill in the
art.
The present invention therefore relates in one embodiment to an isolated
antibody, including Ab-A, or an antigen binding fragment thereof, which specifically binds to
sclerostin and wherein the variable domain of the heavy chain comprises at least one CDR
having the sequences given in SEQ ID NO:51 for CDR-H1, SEQ ID NO:52 for CDR-H2 and
SEQ ID NO:53 for CDR-H3. The antibody or antigen binding fragment thereof may comprise a
heavy chain variable domain in which the CDRs consist of at least one of the peptides of SEQ
ID NO:51 for CDR-H1, SEQ ID NO:52 for CDR-H2 and SEQ ID NO:53 for CDR-H3.
When in antibodies of the invention a light chain is present the light chain may be
any suitable complementary chain and may in particular be selected from a light chain wherein
41

WO 2006/119107 PCT/US2006/016441
the variable domain comprises at least one CDR having the sequences given in SEQ ID NO.54
for CDR-L1, SEQ ID NO:55 for CDR-L2 and SEQ ID NO:56 for CDR-L3. The antibody or
antigen binding fragment thereof may comprise a light chain variable domain in which the
CDRs consist of at least one of the peptides of SEQ ID NO:54 for CDR-L1, SEQ ID NO.55 for
CDR-L2 and SEQ ID NO:56 for CDR-L3.
The present invention further relates to an isolated antibody, including
Ab-B, or an antigen binding fragment hereof, which specifically binds to sclerostin and wherein
the variable domain of the heavy chain comprises at least one CDR having the sequences given
in SEQ ID NO:57 for CDR-H1, SEQ ID NO:58 for CDR-H2 and SEQ ID NO:59 for CDR-H3.
The antibody or antigen binding fragment thereof may comprise a heavy chain variable domain
in which the CDRs consist of at least one of the peptides of SEQ ID NO:57 for CDR-H1, SEQ
ID NO:58 for CDR-H2 and SEQ ID NO:59 for CDR-H3.
When in antibodies of the invention a light chain is present the light chain may be
any suitable complementary chain and may in particular be selected from a light chain wherein
the variable domain comprises at least one CDR having the sequences given in SEQ ID NO:60
for CDR-L1, SEQ ID NO:61 for CDR-L2 and SEQ ID NO:62 for CDR-L3. The antibody or
antigen binding fragment thereof may comprise a light chain variable domain in which the
CDRs consist of at least one of the peptides of SEQ ID NO:60 for CDR-L1, SEQ ID NO:61 for
CDR-L2 and SEQ ID NO:62 for CDR-L3.
The present invention still further relates to an isolated antibody, including Ab-C,
or an antigen binding fragment hereof, which specifically binds to sclerostin and wherein the
variable domain of the heavy chain comprises at least one CDR having the sequences given in
SEQ ID NO:45 for CDR-H1, SEQ ID NO:46 for CDR-H2 and SEQ ID NO:47 for CDR-H3.
The antibody or antigen binding fragment thereof may comprise a heavy chain variable domain
in which the CDRs consist of at least one of the peptides of SEQ ID NO:45 for CDR-H1, SEQ
ID NO:46 for CDR-H2 and SEQ ID NO:47 for CDR-H3.
When in antibodies of the invention a light chain is present the light chain may be
any suitable complementary chain and may in particular be selected from a light chain wherein
the variable domain comprises at least one CDR having the sequences given in SEQ ID NO:48
for CDR-L1, SEQ ID NO:49 for CDR-L2 and SEQ ID NO:50 for CDR-L3. The antibody or
antigen binding fragment thereof may comprise a light chain variable domain in which the
CDRs consist of at least one of the peptides of SEQ ID NO:48 for CDR-L1, SEQ ED NO:49 for
CDR-L2 and SEQ ID NO:50 for CDR-L3.
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WO 2006/119107 PCT/US2006/016441
The present invention also relates to an isolated antibody, including Ab-D, or an
antigen binding fragment hereof, which specifically binds to sclerostin and wherein the variable
domain of the heavy chain comprises at least one CDR having the sequences given in SEQ ED
NO:39 for CDR-H1, SEQ ID NO:40 for CDR-H2 and SEQ ID NO.41 for CDR-H3. The
antibody or antigen binding fragment thereof may comprise a heavy chain variable domain in
which the CDRs consist of at least one of the peptides of SEQ ID NO :3 9 forCDR-Hl, SEQ ID
NO:40 for CDR-H2 and SEQ ID NO:41 for CDR-H3.
When in antibodies of the invention a light chain is present the light chain may be
any suitable complementary chain and may in particular be selected from a light chain wherein
the variable domain comprises at least one CDR having the sequences given in SEQ ID NO:42
for CDR-L1, SEQ ID NO:43 for CDR-L2 and SEQ ED NO:44 for CDR-L3. The antibody or
antigen binding fragment thereof may comprise a light chain variable domain in which the
CDRs consist of at least one of the peptides of SEQ ID NO:42 for CDR-L1, SEQ ID NO:43 for
CDR-L2 and SEQ ID NO:44 for CDR-L3.
Additional anti-sclerostin antibodies are described below. For some of the amino acid
sequences the complementarity-determining regions (CDRs) are boxed-shaded and the constant
regions are in bold-italics.
Ab-2 '
The sequences of the Antibody 2 (also referred to as Ab-2) LC and HC are as follows:

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WO 2006/119107 PCT/US2006/016441
1401 A (SEQ ID NO: 140)
Ab-4 was humanized to generate Ab-5.
Ab-5
The sequences of the Antibody 5 (also referred to herein as Ab-5) LC and HC are as follows:
Ab-5 Light Chain:

52



























































WO 2006/119107 PCT/US2006/016441
(SEQ ID NO:220)"
The CDR sequences in the variable region of the heavy chain of Ab-14 are:
CDR-H1: DYYMN (SEQ ID NO:296)
CDR-H2: DINPYNDDTTYNHKFKG (SEQ ID NO:297)
CDR-H3: ETAVITTNAMD (SEQ ID NO:298)
The light chain variable region CDR sequences of Ab-14 are:
CDR-L1: RASSSVTSSYLN (SEQ ID NO:284)
CDR-L2: STSNLAS (SEQ ID NO.285)
CDR-L3: ■ QQYDFFPST (SEQ ID NO:286)
Ab-14 Variable domains:

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WO 2006/119107 PCT/US2006/01644I
301 GCCGTTATTACTACTAACGC TATGG ATTAC TGGGGTCAAG GAACCACTGT
351 TACCGTCTCTAGT(SEQIDNO:383) - - -
Ab-3 was humanized to generate Ab-15.
Ab-15
The sequences of the Antibody 15 (also referred to herein as Ab-15) LC and HC are as follows:
Ab-15 Light Chain:
Amino acid sequence of the mature form (signal peptide removed) of the Ab-15 LC:

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WO 2006/119107 PCT/US2006/016441

The CDR sequences in the variable region of the heavy chain of Ab-15 are:
CDR-H1: DFYLH (SEQ ID NO:290)'.
CDR-H2: RIDPENGDTLYDPKFQD (SEQIDNO:291)
CDR-H3: EADYFHDGTSYWYFDV (SEQ ID NO:292)
The light chain variable region CDR sequences of Ab-15 are:
CDR-L1: SVSST-ISSNHLH (SEQ ID NO:278)
CDR-L2: GTSNLAS (SEQ ID NO.279)
CDR-L3: QQWSSYPLT (SEQ ID NO:280)
Ab-15 Variable domains:
Ab-15 light chain variable domain amino acid sequence (without signal sequence):

86







































WO 2006/119107 PCT/US2006/016441
CDR-H2: MIHPSASEIRLDQKFKD (SEQ ID NO:359)
CDR-H3: SGEWGSMDY (SEQ ID NO:360)
Table I below provides the SEQ ID NOs and amino acid sequences of the CDR's
5 of Ab-A, Ab-B, Ab-C, Ab-D, Ab-l, Ab-2, Ab-3, Ab-4, Abo, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10,
Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-l6, Ab-17, Ab-l8, Ab-l9, Ab-20, Ab-21, Ab-22, Ab-
23, and Ab-24. LI, L2, and L3 refer to light chain CDR's 1,2, and 3, and HI, H2, and H3 refer
to heavy chain CDR's 1,2, and 3 according to the Kabat numbering system (Kabat et al, 1987
in Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human
10 Services, NTH, USA). "_
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WO 2006/119107 PCT/US2006/016441

An oligopeptide or polypeptide is within the scope of the invention if it has 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 least one of the CDR's of Table 1 above; and/or to a CDR of a sclerostin binding
agent that cross-blocks the binding of at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1,
Ab-2, Ab-3, Ab-4, Ab-S, Ab-6, Ab-7, Ab-8,Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15,
Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24 to sclerostin, and/or is
cross-blocked from binding to sclerostin by at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D,
Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14,
Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24; and/or to a
CDR of a sclerostin binding agent wherein the binding agent can block the inhibitory effect of
sclerostin in a cell based mineralization assay (i.e. a sclerostin neutralizing binding agent);
and/or to a CDR of a sclerostin binding agent that binds to a Loop 2 epitope; and/or to a CDR of
a sclerostin binding agent that binds to a T20.6 epitope; and/or to a CDR of a sclerostin binding
agent that binds to a "T20.6 derivative (cystine-knot + 4 arms)" epitope.
Sclerostin binding agent polypeptides and antibodies are within the scope of the
invention if they have amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%,
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WO 2006/119107 PCT/US2006/016441
91%, 92%, 93%794%^ 95%, 96%, 97%, 98% or 99% identical to a variable region of at least
one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8,
Ab-9, Ab-10, Ab-11, Ab-12, Ab4-3, Ab-14, Ab-15, Ab-16, Ab-W7-Ab-WrAb-t9rAb-20, Ab-
21, Ab-22, Ab-23, and Ab-24, and cross-block the binding of at least one of antibodies Ab-A,
Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11,
Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and
Ab-24 to sclerostin, and/or are cross-blocked from binding to sclerostin by at least one of
antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9,
Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-
22, Ab-23, and Ab-24; and/or can block the inhibitory effect of sclerostin in a cell based
mineralization assay (i.e. a sclerostin neutralizing binding agent); and/or bind to a Loop 2
epitope; and/or bind to a T20.6 epitope; and/or bind to a "T20.6 derivative (cystine-knot + 4
arms)" epitope.
Polynucleotides encoding sclerostin binding agents are within the scope of the
invention if they 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 at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2,
Ab-3, Ab-4, Ah-5: Ab-6; Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-
16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24, and wherein the encoded
sclerostin binding agents cross-block the binding of at least one of antibodies Ab-A, Ab-B, Ab-
C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-l 1, Ab-12, Ab-
13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24 to
sclerostin, and/or are cross-blocked from binding to sclerostin by at least one of antibodies Ab-
A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11,
Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and
Ab-24; and/or can block the inhibitory effect of sclerostin in a cell based mineralization
assay(i.e. a sclerostin neutralizing binding agent); and/or bind to a Loop 2 epitope; and/or bind
to a T20.6 epitope; and/or bind to a "T20.6 derivative (cystine-knot + 4 arms)" epitope.
Antibodies according to the invention may have a binding; affinity for human
sclerostin of less than or equal to 1 x 10-7M, less than or equal to 1 x 10-8M, less than or equal to
1 x 10-9M, less than or equal to 1 x 10-10M, less than or equal to 1 x 1O-11 M, or less than or equal
to 1x 10-12M.
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
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one of ordinary skill in the art using conventional techniques, for example those described by
Scatchard et al. (Ann. N.Y. Acad Sci. 51:660-672 (1949)) or by surface plasmon resonance
(SPR; BIAcore, Biosensor, Piscataway, NJ).Eor-sur-face plasmon resonance target moteeules
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)).
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, fowl (e.g., chicken) and mammals, which includes but is not limited to a
mouse, rat, hamster, rabbit, or other rodent, cow, horse, sheep, goat, camel, human, or other
primate. The antibody may be an internalizing antibody. Production of antibodies is disclosed
generally in U.S. Patent Publication No. 2004/0146888 Al.
Characterization Assays
In the methods described above to generate antibodies according to the invention,
including the manipulation of the specific Ab-A, Ab-B, Ab-C, Ab-D, and Antibody l-24(Ab-l
to Ab-24) 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
sclerosttn; cross-blocking assays; Biacore-based "human sclerostin peptide epitope competition
binding assay;" MC3T3-E1 cell based assay; in vivo assays).
Epitope Binding Assays
Mature form human sclerostin is a 190 amino acid glycoprotein with a
cystine-knot structure (Figures 8 and 9). In addition to the cystine-knot structure, the protein is
characterized as having three loops designated as Loop 1, Loop 2 and Loop 3. Human sclerostin
was subjected to proteolytic digestion to produce fragments. Briefly, using different proteases,
including trypsin, aspN, and lysC, fragments with various cleavage sites and sizes were
generated. The sequences and mass for various human sclerostin peptides were determined.
Antibody protection was evaluated to determine the effect on accessibility for proteolysis,
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including clipped site masking and peptide shifting. Finally, a BIAcore-based "human sclerostin
peptide epitope competition assay" was performed.
Exposure of sclerosti to trypsin cleavage resulted in a pattern of peptide
fragments as summarized in Figure 13. The fragments are referred to as T19.2, T20, T20.6, and
T21-22. As shown schematically in Figure 19B, the T20.6 epitope is a complex of four separate
peptide sequences which are joined by the three disulfide bonds of the cystine-knot region. Two
of the peptides are joined by two disulfide bonds. The other two peptides are linked by one
disulfide bond that, schematically, bisects the first two polypeptides.
The T20.6 epitope that was generated by trypsin digestion retains the
cystine-knot structure of the native polypeptide and is recognized by antibodies Ab-C and Ab-D.
A derivative of epitope T20.6 consists of the cystine-knot region and amino acids 58-64, 73-81,
112-117 and 138-141 in sequence position with reference to SEQ ID NO.l. This derivative
epitope is shown in Figure 21. An epitope comprising the cystine-knot region may have one or
more amino acids that is present in the T20.6 epitope (Figure 19B) but not present in the T20.6
derivative epitope (Figure 21).
Another epitope-containing region was identified in the Loop 2 region of human
sclerostin (Figure 19A) and is recognized by antibodies Ab-A and Ab-B. A Loop 2 epitope
comprises amino acids 86-111 of SEQ ID NO:1 (C4GPARLLPNAIGRGKWWRPSGPDFRC5,
SEQ ID NO:6). Sterically, with reference to full-length sclerostin of SEQ ID NO:1, the Loop 2-
containing structure is defined at one end by. a disulfide bond between cysteine at position 86
(C4) and cysteine at position 144 (C8), and at the other end by a disulfide bond between cysteine
at position 111 (C5) and cysteine at position 57 (Cl).
The peptides generated by aspN cleavage of human sclerostin are shown in
Figure 12. In the Figure, these peptides are designated as AspNl4.6, AspN18.6, and AspN22.7-
23.5, and are also referred to herein as N14.6, N18.6, and N22.7-23.5, respectively.
One group of antibodies exhibits a specific pattern of binding to certain epitopes
as evidenced by a Biacore-based "human sclerostin peptide epitope competition binding assay."
Briefly, the antibody is preincubated with the epitope to be tested, at concentrations that will
saturate the epitope-binding sites on the antibody. The antibody is then exposed to sclerostin
bound to a chip surface. After the appropriate incubation and washing procedures, a pattern of
competitive binding is established. As shown in Figure 18, exemplary antibody Ab-D bound to
sclerostin molecules attached to the surface of the chip. Preincubation of antibody Ab-D with
sclerostin decreased the binding of the antibody to the sclerostin on the chip to close to zero.
Preincubation with a peptide consisting of epitope T19.2 showed that T19.2 did not compete
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with sclerostin for antibody binding. However, preincubation with any one of the epitopes
designated T20, T20.6, T21-22, or N22.7-23.5 abolished a large proportion of the binding of
antibody to sclerostin on the chip. In contrast, preincubation of the antibody with any one of the
epitopes designated T19.2, N14.6 or N18.6 did not abolish the ability of the antibody to bind to
sclerostin. A second exemplary antibody with this binding profile (Fig. 17) is Ab-C.
Antibody Ab-D therefore is exemplary and representative of a group of
antibodies that bind to the epitopes T20, T20.6, T21-22, and N22.7-23.5, and have minimal
detectable binding to epitopes T19.2, N14.6 and N18.6, as measured by the ability to block
antibody binding to sclerostin. Antibodies having this characteristic binding pattern may or may
not share amino acid sequence in one or more regions of the antibody molecule. Antibody
similarity is determined functionally such as by the ability to bind to sclerostin following
preincubation with each of the epitopes described above. Antibodies that exhibit a binding
pattern similar or identical to that of antibody Ab-D are included in the invention. By "similar
to" is meant, for example, the antibody will exhibit binding to each of the polypeptides T20,
T20.6, T21-22 andN22.7-23.5 whereby this binding will specifically compete out at least 50%
of the antibody's binding to sclerostin that would otherwise occur in the absence of
preincubation with sclerostin or a sclerostin peptide. The antibody will also exhibit little or no
detectable binding to polypeptides Tl 9.2, N14.6 and N18.6, resulting in a reduction of 30% or
less of the binding that would occur in the absence of preincubation with sclerostin or a
sclerostin peptide.
For example, without being bound by a particular mechanism, the antibody
binding pattern of Figure 18 suggests that the epitope space to which antibody Ab-D and other
antibodies having the epitope binding pattern of Ab-D bind consists of a polypeptide comprising
the cystine-knot region of sclerostin.
Thus, as disclosed herein and with reference to Figure 19B, an exemplary T20.6
epitope comprises four peptide chains attached via three separate disulfide bonds. Peptide chain
SAKPVTELVC3SGQC4GPAR (SEQ ID NO.3) is attached to peptide chain
LVASC7KC8KRLTR (SEQ ID NO:5) by disulfide bonds from C3 to C7, and from C4 to C8.
Peptide chain DVSEYSC1RELHFTR (SEQ ID NO:2) is attached to peptide chain
WWRPSGPDFRC5IPDRYR(SEQ ID NO:4) by a disulfide bond from Cl to C5. The
polypeptides of SEQ ID NOs:3 and 5 remain associated with the polypeptides of SEQ ID NOs:2
and 4 through a steric construct whereby the C1-C5 bond crosses the plane of the C4-C8 and
C3-C7 bonds and is located between them, as illustrated in Figure 19B.
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As disclosed herein and with reference to Figure 21, an exemplary derivative
epitope of T20.6 comprises four peptide chains attached via three separate disulfide bonds.
Peptide chain SAKPVTELVC3SGQC4 (SEQ ID NO.70) is attached to peptide chain
LVASC7KC8 (SEQ ID NO:71) by disulfide bonds from C3 to C7, and from C4 to C8. Peptide
chain C1RELHFTR (SEQ ID NO:72) is attached to peptide chain C5IPDRYR (SEQ ID NO:73)
by a disulfide bond from Cl to C5. The polypeptides of SEQ ID NOs:70 and 71 remain
associated with the polypeptides of SEQ ED NOs:72 and 73 through a steric construct whereby
the C1-C5 bond crosses the plane of the C4-C8 and C3-C7 bonds and is located between them,
as illustrated in Figure 21.
Antibody Ab-A is exemplary and representative of a second group of antibodies
that have a characteristic binding pattern to human sclerostin peptides that is distinct from that
obtained for antibodies Ab-C and Ab-D. Ab-A and the group of antibodies it represents bind to
the N22.7-23.5 epitope and have minimal detectable binding to epitopes T19.2, T20, T20.6,
T21-22, N14.6 or N18.6, as measured by the ability to block antibody binding to sclerostin (Fig
15). A second exemplary antibody with this binding profile (Fig. 16) is Ab-B. Antibodies
having this characteristic binding pattern may or may not share amino acid sequence in one or
more regions of the antibody molecule. Antibody similarity is determined functionally such as
by the ability to bind to sclerostin following preincubation with each of the epitopes described
above. Antibodies that exhibit a binding pattern similar or identical to that of antibody Ab-A are
included in the invention. By "similar to" is meant, for example, the antibody will exhibit
binding to the N22.7-23.5 polypeptide whereby this binding will specifically compete out at
least 50% of the antibody's binding to sclerostin that would otherwise occur in the absence of
preincubation with sclerostin or a sclerostin peptide. The antibody will also exhibit little or no
detectable binding to polypeptides T19.2, T20, T20.6, T21-22, N14.6 and N18.6, resulting in a
reduction of 30% or less of the binding that would occur in the absence of preincubation with
sclerostin or a sclerostin peptide.
For example, without being bound by a particular mechanism, the antibody
binding pattern of Figure 15 suggests that the epitope space to which antibody Ab-A and other
antibodies having the epitope binding pattern of Ab-A bind consists of a polypeptide comprising
the Loop 2 region of sclerostin. Thus, as disclosed herein and with reference to Figure 19 A, the
Loop 2 region can be described as a linear peptide, but it acquires a tertiary structure when it is
present in native sclerostin or a cystine-knot-containing portion of sclerostin in which the native
disulfide bond structure is maintained. The linear or tertiary structure of the Loop 2 epitope can
affect antibody binding thereto, as discussed in the Examples. A Loop 2 region can comprise
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the following amino acid sequence: C4GPARLLPNAIGRGKWWRPSGPDFRC5 (SEQ ID
NO:6). "C4" refers to a cysteine residue located at position 86 with reference to SEQ ID NO: 1.
"C5" refers to a cysteine residue located at position 111 with reference to SEQ ID NO: I. In
native sclerostin protein, C4 is linked to a cysteine at position 144 (C8) by a disulfide bond, and
C5 is linked to a cysteine at position 57 (Cl) by a disulfide bond. Epitopes derived from the
Loop 2 region include CGPARLLPNAIGRGKWWRPS (SEQ ID NO:63);
GPARLLPNAIGRGKWWRPSG (SEQ ID NO:64); PARLLPNAIGRGKWWRPSGP (SEQ ID
NO:65); ARLLPNAIGRGKWWRPSGPD (SEQ ID NO:66); RLLPNAIGRGKWWRPSGPDF
(SEQ ID NO:67); LLPNAIGRGKWWRPSGPDFR (SEQ ID NO:68); and
LPNAIGRGKWWRPSGPDFRC (SEQ ID NO:69)
CROSS-BLOCKING ASSAYS
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 sclerostin.
The extent to which an antibody or other binding agent is able to interfere with the
binding of another to sclerostin, 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 machine which can measure the extent of interactions using
surface plasmon resonance technology. 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 sclerostin.
BIACORE CROSS-BLOCKING ASSAY
The following generally describes a suitable Biacore 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 sclerostin binding agents described herein.
The Biacore machine (for example the Biacore 3000) is operated in line with the manufacturer's
recommendations.
Thus in one cross-blocking assay, sclerostin is coupled to a CM5 Biacore chip
using standard amine coupling chemistry to generate a sclerostin-coated surface. Typically 200-
800 resonance units of sclerostin would be coupled to the chip (an amount that gives easily
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measurable levels of binding but that is readily saturable by the concentrations of test reagent
being used).
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 sclerostin binding sites on that antibody.
The concentration of each antibody in the test mix should be high enough to
readily saturate the binding sites for that antibody on the sclerostin molecules captured on the
Biacore 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).
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.
The test mixture is passed over the sclerostin-coated Biacore 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 sclerostin. Typically this is done by treating the
chip with 30 raM HC1 for 60 seconds.
The solution of antibody A* alone is then passed over the sclerostin-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 sclerostin.
The solution of antibody B* alone is then passed over the sclerostin-coated
surface and the amount of binding recorded.
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
sclerostin 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.
Thus, in general, a cross-blocking antibody or other binding agent according to
the invention is one which will bind to sclerostin in the above Biacore 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
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theoretical binding (as just defined above) of the two antibodies or binding agents in
combination.
The Biacore assay described above is a primary 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 sclerostin coupled via
amine chemistry to a CM5 Biacore chip (this usually occurs when the relevant binding site on
sclerostin is masked or destroyed by the coupling to the chip). In such cases cross-blocking can
be determined using a tagged version of Sclerostin, for example N-terminal His-tagged
Sclerostin (R & D Systems, Minneapolis, MN, USA; 2005 cat# 1406-ST-025). In this particular
format, an anti-His antibody would be coupled to the Biacore chip and then the His-tagged
Sclerostin 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 sclerostin would be loaded back onto the anti-His
antibody coated surface. In addition to the example given using N-terminal His-tagged
Sclerostin, C-terminal His-tagged sclerostin could alternatively be used. Furthermore, 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).
ELISA-BASED CROSS-BLOCKING ASSAY
The following generally describes an ELISA assay for determining whether an
anti-sclerostin antibody or other sclerostin binding agent cross-blocks or is capable of cross-
blocking according to the invention. For convenience, reference is made to two antibodies (Ab-
X and Ab-Y), but it will be appreciated that the assay can be used with any of the sclerostin
binding agents described herein.
The general principal of the assay is to have an anti-sclerostin antibody coated
onto the wells of an ELISA plate. An excess amount of a second, potentially cross-blocking,
anti-sclerostin antibody is added in solution (i.e. not bound to the ELISA plate). A limited
amount of sclerostin is then added to the wells. The coated antibody and the antibody in
solution compete for binding of the limited number of sclerostin molecules. The plate is washed
to remove sclerostin 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 sclerostin. The amount of bound sclerostin is then measured using an
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appropriate sclerostin 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 sclerostin molecules that the
coated antibody can bind relative to the number of sclerostin molecules that the coated antibody
can bind in the absence of the second, solution phase, antibody.
This assay is described in more detail further below for Ab-X and Ab-Y. In the
instance where Ab-X 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-Y is
then added to the ELISA plate such that the moles of Ab-Y sclerostin binding sites per well are
at least 10 fold higher than the moles of Ab-X sclerostin binding sites that were used, per well,
during the coating of the ELISA plate. Sclerostin is then added such that the moles of sclerostin
added per well are at least 25-fold lower than the moles of Ab-X sclerostin binding sites that
were used for coating each well. Following a suitable incubation period the ELISA plate is
washed and a sclerostin detection reagent is added to measure the amount of sclerostin
specifically bound by the coated anti-sclerostin antibody (in this case Ab-X). The background
signal for the assay is defined as the signal obtained in wells with the coated antibody (in this
case Ab-X), second solution phase antibody (in this case Ab-Y), sclerostin buffer only (i.e. no
sclerostin) and sclerostin 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-X), second solution
phase antibody buffer only (i.e. no second solution phase antibody), sclerostin and sclerostin
detection reagents. The ELISA assay needs to be run in such a manner so as to have the positive
control signal be at least 6 times the background signal.
To avoid any artifacts (e.g. significantly different affinities between Ab-X and
Ab-Y for sclerostin) resulting from the choice of which antibody to use as the coating antibody
and which to use as the second (competitor) antibody, the cross-blocking assay needs to be run
in two formats:
1) format 1 is where Ab-X is the antibody that is coated onto the ELISA plate and Ab-Y is
the competitor antibody that is in solution
and
2) format 2 is where Ab-Y is the antibody that is coated onto the ELISA plate and Ab-X is
the competitor antibody that is in solution.
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Ab-X and Ab-Y are defined as cross-blocking if, either in format 1 or in format 2,
the solution phase anti-sclerostin antibody is able to cause a reduction of between 60% and
100%, specifically between 70% and 100%, and more specifically between 80% and 100%, of
the sclerostin detection signal (i.e. the amount of sclerostin bound by the coated antibody) as
compared to the sclerostin detection signal obtained in the absence of the solution phase anti-
sclerostin antibody {i.e. the positive control wells).
An example of such an ELISA-based cross blocking assay can be found in
Example 7 ("ELISA-based cross-blocking assay").
CELL BASED NEUTRALIZATION ASSAY
Mineralization by osteoblast-lineage cells in culture, either primary cells or cell
lines, is used as an in vitro model of bone formation. Mineralization takes from about one to six
weeks to occur beginning with the induction of osteoblast-lineage cell differentiation by one or
more differentiation agents. The overall sequence of events involves cell proliferation,
differentiation, extracellular matrix production, matrix maturation and finally deposition of
mineral, which refers to crystallization and/or deposition of calcium phosphate. This sequence
of events starting with cell proliferation and differentiation, and ending with deposition of
mineral is referred to herein as mineralization. Measurement of calcium (mineral) is the output
of the assay.
MC3T3-E1 cells (Sudo H, Kodama H-A, Amagai Y, Yamamoto S, Kasai S.
1983. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from
newborn mouse calvaria. J. Cell Biol. 96:191-198) and subclones of the original cell line can
form mineral in culture upon growth in the presence of differentiating agents. Such subclones
include MC3T3-E1-BF (Smith E, Redman R, Logg C, Coetzee G, KasaharaN, Frenkel B. 2000.
Glucocorticoids inhibit developmental stage-specific osteoblast cell cycle. J. Biol. Chem.
275:19992-20001). For both the MC3T3-E1-BF subclone as well as the original MC3T3-E1
cells, sclerostin can inhibit one or more of the sequence of events leading up to and including
mineral deposition (i.e. sclerostin inhibits mineralization). Anti-sclerostin antibodies that are
able to neutralize sclerostin's inhibitory activity allow for mineralization of the culture in the
presence of sclerostin such that there is a statistically significant increase in deposition of
calcium phosphate (measured as calcium) as compared to the amount of calcium measured in the
sclerostin-only (i.e. no antibody) treatment group. The antibodies used in the cell based
mineralization assay experiments shown in Figures 22,23 and 24 have molecular weights of
about 145 Kd and have 2 sclerostin binding sites per antibody molecule,
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When funning the assay with the goal of determining whether a particular anti-
sclerostin antibody or anti-sclerostin binding agent can neutralize sclerostin (i.e., is a sclerostin
neutralizing antibody or derivative thereof, or is a sclerostin neutralizing binding agent), the
amount of sclerostin used in the assay needs to be the minimum amount of sclerostin that causes
at least a 70%, statistically significant, reduction in deposition of calcium phosphate (measured
as calcium) in the sclerostin-only group, as compared to the amount of calcium measured in the
no sclerostin group. An anti-sclerostin neutralizing antibody or an anti-sclerostin neutralizing
binding agent is defined as one that causes a statistically significant increase in deposition of
calcium phosphate (measured as calcium) as compared to the amount of calcium measured in the
sclerostin-only (i.e. no antibody, no binding agent) treatment group. To determine whether an
anti-sclerostin antibody or an anti-sclerostin binding agent is neutralizing or not, the amount of
anti-sclerostin antibody or anti-sclerostin binding agent used in the assay needs to be such that
there is an excess of moles of sclerostin binding sites per well as compared to the number of
moles of sclerostin per well. Depending on the potency of the antibody, the fold excess that may
be required can be 24, 18, 12, 6, 3, or 1.5, and one of skill is familiar with the routine practice of
testing more than one concentration of binding agent. For example, a very potent anti-sclerostin
neutralizing antibody or anti-sclerostin neutralizing binding agent will be able to neutralize
sclerostin even when there is less than a 6-fold excess of moles, of sclerostin binding sites per
well as compared to the number of moles of sclerostin per weii. A less potent anti-sclerostir.
neutralizing antibody or anti-sclerostin neutralizing binding agent will be able to neutralize
sclerostin only at a 12,18 or 24 fold excess. Sclerostin binding agents within this full range of
potencies are suitable as neutralizing sclerostin binding agents. Exemplary cell based
mineralization assays are described in detail in Example 8.
Anti-sclerostin antibodies and derivatives thereof that can neutralize human
sclerostin, and sclerostin binding agents that can neutralize human sclerostin may be of use in
the treatment of human conditions/disorders that are caused by, associated with, or result in at
least one of low bone formation, low bone mineral density, low bone mineral content, low bone
mass, low bone quality and low bone strength.
IN VIVO NEUTRALIZATION ASSAY
Increases in various parameters associated with, or that result from, the
stimulation of new bone formation can be measured as an output from in vivo testing of
sclerostin binding agents in order to identify those binding agents that are able to neutralize
sclerostin and thus able to cause stimulation of new bone formation. Such parameters include
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various serurn anabolic markers [e.g. osteocalcin, P1NP (n-terminal propeptide of type 1
procollagen)], histomorphoraetric markers of bone formation (e.g. osteoblast surface/bone
surface; bone formation rate/bone surface; trabecular thickness), bone mineral density, bone
mineral content, bone mass, bone quality and bone strength. A sclerostin neutralizing binding
agent is defined as one capable of causing a statistically significant increase, as compared to
vehicle treated animals, in any parameter associated with, or that results from, the stimulation of
new bone formation. Such in vivo testing can be performed in any suitable mammal (e.g.
mouse, rat, monkey). An example of such in vivo testing can be found in Example 5 (In vivo
testing of anti-sclerostin monoclonal antibodies").
Although the amino acid sequence of scterostin is not 100% identical across
mammalian species (e.g. mouse sclerostin is not 100% identical to human sclerostin), it will be
appreciated by one skilled in the art that a sclerostin binding agent that can neutralize, in vivo,
the sclerostin of a certain species (e.g. mouse) and that also can bind human sclerostin in vitro is
very likely to be able to neutralize human sclerostin in vivo. Thus, such a human sclerostin
binding agent (e.g. anti-human sclerostin antibody) may be of use in the treatment of human
conditions/disorders that are caused by, associated with, or result in at least one of low bone
formation, low bone mineral density, low bone mineral content, low bone mass, low bone
quality and low bone strength. Mice in which homologous recombination had been used to
delete the mouse sclerostin gene and insert the human sclerostin gene in its place (i.e. human
sclerostin gene knock-in mice or human SOST knock-in mice) would be an example of an
additional in vivo system.
Pharmaceutical compositions are provided, comprising one of the
above-described binding agents such as at least one of antibody Ab-A, Ab-B, Ab-C, Ab-D and
Ab-1 toAb-24 to human sclerostin, along with a pharmaceutically or physiologically acceptable
carrier, excipient, or diluent. Pharmaceutical compositions and methods of treatment are
disclosed in copending Application Serial No. 10/868,497, filed June 16,2004, which claims
priority to Serial No. 60/478,977, both of which are incorporated by reference herein.
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.
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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.
In certain circumstances it will be desirable to deliver the pharmaceutical
compositions disclosed herein subcutaneously, parentsrally, 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. Patent No. 5,543,158; U.S. Patent No. 5,641,515 and
U.S. Patent No. 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.
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. Patent 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. The 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.
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
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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.
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.
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.
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
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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.
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. Patent No. 5,567,434; U.S. Patent No. 5,552,157; U.S. Patent No.
5,565,213; U.S. Patent No. 5,738,868 and U.S. Patent No. 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)).
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 0.1µm) 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(l):l-20,1988; zurMuhien et al., Eur. J. Pharm. Biopharm. 45(2):149-55, 1998; Zambauxet
al., 7. Controlled Release 50(1-3):31-40, 1998; and U.S. Patent No. 5,145,684.
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. •
The dose administered may range from 0.01 mg/kg to 100 mg/kg of body weight.
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
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desired response, the "condition of the patient, and so forth. Typically, the compositions may be
administered by a variety of techniques, as noted above.
Increases in bone mineral content and/or bone mineral density may be
determined directly through the use of X-rays (e.g., Dual Energy X-ray Absorptometry or
"DEXA"), or by inference through the measurement of 1) markers of bone formation and/or
osteoblast activity, such as, but not limited to, osteoblast specific alkaline phosphatase,
osteocalcin, type 1 procollagen C propeptide (PICP), total alkaline phosphatase (see Cornier,
Curr. Opin. in Rheu. 7:243(1995)) and serum procollagen 1 N-terminal propeptide (P1NP)
and/or 2) markers of bone resorption and/or osteoclast activity including, but not limited to,
pyridinoline, deoxypryridinoline, N-telopeptide, urinary hydroxyproline, plasma tartrate-
resistant acid phosphatases, and galactosyl hydroxylysine; (see Cornier, id), serum TRAP 5b
(tartrate-resistant acid phosphatase isoform 5b) and serum cross-linked C-telopeptide (sCTXI).
The amount of bone mass may also be calculated from body weights or by using other methods
(see Guinness-Hey, Metab. Bone Dis. Relat. Res. 5:177-181, 1984).Animals and particular
animal models are used in the art for testing the effect of the compositions and methods of the
invention on, for example, parameters of bone loss, bone resorption, bone formation, bone
strength or bone mineralization that mimic conditions of human disease such as osteoporosis and
osteopenias. Examples of such models include the ovariectomized rat model (Kalu, D.N., The
ovariectomized rat model of postmenopausal hone loss. Bone and Mineral 15:175-192 (1991);
Frost, H.M. and Jee, W.S.S. On the rat model of human osteopenias and osteoporosis. Bone and
Mineral 18:227-236 (1992); and Jee, W.S.S. and Yao, W., Overview: animal models of
osteopenia and osteoporosis. J. Musculoskel. Neuron. Interact. 1:193-207 (2001)).
Particular conditions which may be treated by the compositions of the present
invention include dysplasias, wherein growth or development of bone is abnormal and a wide
variety of causes of osteopenia, osteoporosis and bone loss. Representative examples of such
conditions include achondroplasia, cleidocranial dysostosis, enchondromatosis, fibrous
dysplasia, Gaucher's Disease, hypophosphatemic rickets, Marfan's syndrome, multiple
hereditary exotoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteopoikilosis,
sclerotic lesions, pseudoarthrosis, and pyogenic osteomyelitis, periodontal disease, anti-epileptic
drug induced bone loss, primary and secondary hyperparathyroidism, familial
hyperparathyroidism syndromes, weightlessness induced bone loss, osteoporosis in men,
postmenopausal bone loss, osteoarthritis, renal osteodystrophy, infiltrative disorders of bone,
oral bone loss, osteonecrosis of the jaw, juvenile Paget's disease, melorheostosis, metabolic
bone diseases, mastocytosis, sickle cell anemia/disease, organ transplant related bone loss,
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kidney transplant related bone loss, systemic lupus erythematosus, ankylosing spondylitis,
epilepsy, juvenile arthritides, thalassemia, mucopolysaccharidoses, fabry disease, turner
syndrome, Down Syndrome, Klinefelter Syndrome, leprosy, Perthes' Disease, adolescent
idiopathic scoliosis, infantile onset multi-system inflammatory disease, Winchester Syndrome,
Menkes Disease, Wilson's Disease, ischemic bone disease (such as Legg-Calve-Perthes disease,
regional migratory osteoporosis), anemic states, conditions caused by steroids, glucocorticoid-
induced bone loss, heparin-induced bone loss, bone marrow disorders, scurvy, malnutrition,
calcium deficiency, idiopathic osteopenia or osteoporosis, congenital osteopenia or osteoporosis,
alcoholism, chronic liver disease, postmenopausal state, chronic inflammatory conditions,
rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory colitis,
Crohn's disease, oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus, hyperthyroidism,
thyroid disorders, parathyroid disorders, Cushing's disease, acromegaly, hypogonadism,
immobilization or disuse, reflex sympathetic dystrophy syndrome, regional osteoporosis,
osteomalacia, bone loss associated with joint replacement, HIV associated bone loss, bone loss
associated with loss of growth hormone, bone loss associated with cystic fibrosis, fibrous
dysplasia, chemotherapy associated bone loss, tumor induced bone loss, cancer-related bone
loss, hormone ablative bone loss, multiple myeloma, drug-induced bone loss, anorexia nervosa,
disease associated facial bone loss, disease associated cranial, .bone loss, disease associated bone
loss of the jaw, disease associated bone loss of the skull, and bone loss associated with space
travel. Further conditions relate to bone loss associated with aging, including facial bone loss
associated with aging, cranial bone loss associated with aging, jaw bone loss associated with
aging, and skull bone loss associated with aging.
Compositions of the present invention may also be useful for improving
outcomes in orthopedic procedures, dental procedures, implant surgery, joint replacement, bone
grafting, bone cosmetic surgery and bone repair such as fracture healing, nonunion healing,
delayed union healing and facial reconstruction. One or more compositions may be
administered before, during and/or after the procedure, replacement, graft, surgery or repair.
The invention also provides a diagnostic kit comprising at least one anti-
sclerostin binding agent according to the present invention. The binding agent may be an
antibody. In addition, such a kit may optionally comprise one or more of the following:
(1) instructions for using the one or more binding agent(s) for screening,
diagnosis, prognosis, therapeutic monitoring or any combination of these
applications;
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(2)"a labeled binding partner to the anti-sclerostin binding agent(s);
(3) a solid phase (such as a reagent strip) upon which the anti-sclerostin binding
agent(s) is immobilized; and
(4) a label or insert indicating regulatory approval for screening, diagnostic,
prognostic or therapeutic use or any combination thereof.
If no labeled binding partner to the binding agent(s) is provided, the binding agent(s) itself can
be labeled with one or more of a detectable marlcer(s), e.g. a chemiluminescent, enzymatic,
fluorescent, or radioactive moiety.
The following examples are offered by way of illustration, and not by way of
limitation.
EXAMPLES
EXAMPLE 1
Recombinant Expression of Sclerostin
Recombinant human sclerostin/SOST is commercially available from R&D
Systems (Minneapolis, MN, USA; 2006 cat# 1406-ST-025). Additionally, recombinant mouse
sclerostin/SOST is commercially available from R&D Systems (Minneapolis, MN, USA; 2006
cat# 1589-ST-025). , .
Alternatively, the different species of sclerostin can be expressed transiently in
serum-free suspension adapted 293T or 293EBNA cells. Transfections can be performed as 500
mL or 1L cultures. The following reagents and materials are available from Gibco BRL (now
Invitrogen, Carlsbad, CA). Catalog numbers are listed in parentheses: serum-free DMEM
(21068-028); DMEM/F12 (3:1) (21068/11765); IX Insulin-Transferrin-Selenium Supplement
(51500-056); IX Pen Strep Glut (10378-016); 2mM 1-Glutamine (25030-081); 20 mM HEPES
(15630-080); 0.01% Pluronic F68 (24040-032). Briefly, the cell inoculum (5.0-10.0 X 105
cells/mL X culture volume) is centrifuged at 2,500 RPM for 10 minutes at 4°C to remove the
conditioned medium.
The cells are resuspended in serum-free DMEM and centrifuged again at 2,500
RPM for 10 minutes at 4°C. After aspirating the wash solution, the cells are resuspended in
growth medium [DMEM/F12 (3:1) + IX Insulin-Transferrin-Selenium Supplement + IX Pen
Strep Glut + 2mM L-Glutamine + 20 mM HEPES + 0.01% Pluronic F68] in a 1L or 3L spinner
flask culture. The spinner flask culture is maintained on magnetic stir plate at 125 RPM which
is placed in a humidified incubator maintained at 37°C and 5% CO2. The mammalian
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expression plasmid UNA (e.g. pcDNA3.1, pCEP4, Invitrogen Life Technologies, Carlsbad,
CA), containing the complete coding region (and stop codon) of sclerostin with a Kozak
consensus sequence (e.g., CCACC) directly 5' of the start site ATG, is complexed to the
transfection reagent in a 50 mL conical tube.
The DNA-transfection reagent complex can be prepared in 5-10% of the final
culture volume in serum-free DMEM or OPTI-MEM. The transfection reagents that can be used
for this purpose include X-tremeGene RO-1539 (Roche Applied Science, Indianapolis, IN),
FuGene6 (Roche Applied Science, Indianapolis, IN), Lipofectamine 2000 (Invitrogen,
Carlsbad, CA) and 293fectin (Invitrogen, Carlsbad, CA). 1-5 ug piasmid DNA/mL culture is
first added to serum-free DMEM, followed by 1-5 µl transfection reagent/mL culture. The
complexes can be incubated at room temperature for approximately 10-30 minutes and then
added to the cells in the spinner flask. The transfection/expression can be performed for 4-7
days, after which the conditioned medium (CM) is harvested by centrifugation at 4,000 RPM for
60 minutes at 4°C.
EXAMPLE 2
PURIFICATION OF RECOMBINANT SCLEROSTIN
Recombinant sclerostin was purified from mammalian host cells as follows. All
purification processes were carried out at room temperature. One purification scheme was used
to purify various species of sclerostin, including murine and human sclerostin. The purification
scheme used affinity chromatography followed by cation exchange chromatography.
Heparin Chromatography
The mammalian host cell conditioned medium (CM) was centrifuged in a
Beckman J6-M1 centrifuge at 4000 rpm for lhour at 4°C to remove cell debris. The CM
supernatant was then filtered through a sterile 0.2 urn filter. (At this point the sterile filtered CM
may be optionally stored frozen until purification.) If the CM was frozen, it was thawed at the
following temperatures, or combination thereof: 4°C, room temperature or,warm water.
Following thawing the CM was filtered through a sterile 0.2 urn filter and optionally
concentrated by tangential flow ultrafiltration (TFF) using a 10 kD molecular weight cut-off
membrane. The CM concentrate was filtered through a sterile 0.2 µm filter and then loaded onto
a Heparin High Performance (Heparin HP) column (GE Healthcare, formerly Amersham
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Biosciences) reqquired in PBS. Alternatively, the filtered CM supernatant may be loaded
directly onto the Heparin HP column equilibrated in PBS.
After loading, the Heparin HP column was washed with PBS until the absorbance
at 280 nm of the flow-through returned to baseline (i.e., absorbance measured before loading
CM supernatant). The sclerostin was then eluted from the column using a linear gradient from
150 mM to 2M sodium chloride in PBS. The absorbance at 280 nm of the eluate was monitored
and fractions containing protein were collected. The fractions were then assayed by Coomassie-
stained SDS-PAGE to identify fractions containing a polypeptide that migrates at the size of
glycosylated sclerostin. The appropriate fractions from the column were combined to make the
Heparin HP pool.
Cation Exchange Chromatography
The sclerostin eluted from the Heparin HP column was further purified by cation
exchange chromatography using SP High Performance (SPHP) chromatography media (GE
Healthcare, formerly Amersham Biosciences). The Heparin HP pool was buffer exchanged into
PBS by dialysis using 10,000 MWCO membranes (Pierce Slide-A-Lyzer). The dialyzed
Heparin HP pool was then loaded onto an SPHP column equilibrated in PBS. After loading, the
column was washed with PBS until the absorbance at 280 nm of the flow-through returned to
baseline. The sclerostin was then eluted from the SPHP column using a linear gradient from 150
mM to 1.M sodium chloride in PBS. The absorbance at 280 nm of the eluate was monitored and
the eluted sclerostin was collected in fractions. The fractions were then assayed by Coomassie-
stained SDS-PAGE to identify fractions containing a polypeptide that migrates at the size of
glycosylated sclerostin. The appropriate fractions from the column were combined to make the
SPHP pool.
Formulation
Following purification, the SPHP pool was formulated in PBS by dialysis using
10,000 MWCO membranes (Pierce Slide-A-Lyzer). If concentration of sclerostin was
necessary, a centrifugal device (Amicon Centricon or Centriprep) with a 10;000 MWCO
membrane was used. Following formulation the sclerostin was filtered through a sterile 0.2 µm
filter and stored at 4°C or frozen.
EXAMPLE 3
PEPTIDE BINDING ELISA
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A series of overlapping peptides (each peptide being approximately 20-25 amino acids
long) were synthesized based on the known amino acid sequence of rat sclerostin (SEQ ID
NO:98). The peptides were designed such that they all contained a reduced cysteine residue; an
additional cysteine was included at the C-terminus of each peptide which did not already contain
one in its sequence. This enabled the peptides to be bound to the assay plates by covalent
coupling, using commercially available sulfhydryl binding plates (Costar), at a concentration of
lµg/ml, in phosphate buffered saline (PBS: pH 6.5) containing I mM EDTA. Following
incubation for 1 hour at room temperature, the plates were washed three times with PBS
containing 0.5% Tween 20. The plates were blocked by incubation with a PBS solution
containing 0.5% fish skin gelatin (Sigma) for 30 minutes at room temperature and then washed
three times in PBS containing 0.5% Tween 20.
Antibodies to be tested were diluted to lµg/ml in PBS containing 0.5%
fish skin gelatin and incubated with the peptide-coated plates for 1 hour at room temperature.
Excess antibody was removed by three washes with PBS, 0.5% Tween 20. The plates were then
incubated with an appropriate secondary antibody conjugated to horseradish peroxidase (diluted
appropriately in PBS containing 0.5% Tween 20) and capable of binding to the antibody of
interest. The plates were then washed three times: once with PBS containing 0.5% Tween 20,
and twice with PBS. Finally the plates were incubated with a horseradish peroxidase
chromogenic substrate (TMB-Stable Stop, RDI) for 5 minutes at room temperature, the color
development was stopped with acid, and the plates' optical density measured at 450nm.
Materials
Costar's Sulfhydryl Binding Plates (VWR # 29442-278)
Coating Buffer: 1XPBS PH 6.5 + lmM EDTA
Blocking Buffer: IX PBS + 0.5% Fish Skin Gelatin (PBS from CS; FSG from Sigma# G
7765)
Wash Buffer: IX PBS + 0.5% Tween 20
Rat Sclerostin peptides
Antibody Samples: Transient Ab, Purified recombinant Ab, rabbit Serum, etc.
Appropriate secondary Ab: Goat-anti-Rabbit/Mouse-HRP (Jackson Immuno Research, 115-
036-072)
TMB-Stable Stop (RDI# RDI-TMBSX-1L)
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0.5M HCl
Methods were as follows:
1. Coat plates with 100µl/well of rat sclerostin peptide diluted in 1XPBS PH 6.5 + lmM
EDTA at lµg/ml. Incubate plates 1 hour at room temperature. (Plates should be used
within 30 minutes of opening).
2. Wash plates 3X with wash buffer.
3. Block plates with 200ul/welI blocking buffer. Incubate plates 30 minutes at room temp.
4. Repeat washing as described in (2).
5. Incubate plates with 50ul/well of samples diluted in blocking buffer - Serum titers
starting at 1:100; Transient Recombinant Ab use neat; Purified recombinant Ab use at
lµg/ml (all samples run in duplicates). Incubate plates 1h at room temp.
6. Wash plates as described in (2).
7. Incubate plates with 50ul/well of appropriate Secondary Antibody (HRP labeled) diluted
1:1600 in Blocking Buffer. Incubate plates 1 hour at room temperature.
8. Wash plates 1X wash buffer, 2x PBS
9. Incubate plates with 50µl/well of TMB, 5 minutes at room temp.
10. Stop reaction with 50µl/well 0.5M HC1.
11. Read plates at 450 nm wavelength.
The following peptides sequences were screened as described above:
QGWQAFKNDATEIIPGLREYPEPP(SEQ ID NO:82)
TEIIPGLREYPEPPQELENN (SEQ ID NO:83)
PEPPQELENNQTMNRAENGG (SEQ ID NO:84)
ENGGRPPHHPYDTKDVSEYS (SEQ ID NO:85)
CRELHYTRFVTDGP (SEQ ID NO:86)
CRELHYTRFVTDGPSRSAKPVTELV (SEQ ID NO:87)
CRSAKPVTELVSSGQSGPRARLL (SEQ ED NO:88)
CGPARLLPNAIGRVKWWRPNGPDFR (SEQ ID NO:89)
RAQRVQLLCPGGAAPRSRKV (SEQ ID NO.90)
PGGAAPRSRKVRLVAS (SEQ ID NO:91)
KRLTRFHNQSELKDFGPETARPQ (SEQ ID NO:92)
IPDRYAQRVQLLSPGG (SEQ ID NQ:93)
SELKDFGPETARPQKGRKPRPRAR (SEQ ID NO:94)
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KGRKPRPRARGAKANQAELENAY (SEQ ID NO.95)
PNAIGRVKWWRPNGPDFR (SEQ ID NO:96)
KWWRPNGPDFRCIPDRYRAQRV (SEQ ID NO:97).
A high-affinity neutralizing antibody (Ab-19) bound to two
overlapping peptide sequences: PNAIGRVKWWRPNGPDFR (SEQ ID NO:96) and
KWWRPNGPDFRCIPDRYRAQRV (SEQ ID NO:97).
This procedure allows the recognition of epitopes for antibodies that
react with apparent linear epitopes. Peptides that contain all or part of the antibody binding site
will bind antibody and thus be detected.
EXAMPLE 4
IDENTIFICATION OF HUMAN SCLEROSTIN EPITOPES
Sclerostin structure
Mature form (signal peptide removed) human sclerostin is a 190 amino acid
protein (Figure 8). Figure 9 shows a schematic of the general structure of sclerostin with an N-
terminal arm (from the N-terminal Q to Cysteinel) and a C-terminal arm (from Cysteine8 to the
terminal Y). Sandwiched in between these two arms there is the cystine-knot structure and three
loops which are designated Loopl, Loop2 and Loop 3. The four disulfide bonds in sclerostin
are Cysl at sequence position 57 linked to Cys5 at sequence position 111 (referred to as C1-C5),
Cys2 at sequence position 71 linked to Cys6 at sequence position 125 (referred to as C2-C6),
Cys3 at sequence position 82 linked to Cys7 at sequence position 142 (referred to as C3-C7),
Cys4 at sequence position 86 linked to Cys8 at sequence position 144 (referred to as C4-C8).
The eight-membered ring structure is formed via C3-C7 and C4-C8 disulfide bonding. This ring
structure, together with the C1-C5 disulfide bond penetrating through the ring, forms a typical
cystine-knot. C2-C6, which is not part of the cystine-knot, brings two large loop structures, loop
1 (residues 57 to 82) and loop 3 (residues 111 to 142) close together. Loop 2 goes from C4
(residue 86) to C5 (residue 111).
Experimental
The general approach for characterizing the epitopes bound by anti-sclerostin
monoclonal antibodies involved fragmenting human Sclerostin into peptides with different
proteases, determining the sequence of the various human sclerostin peptides, isolating these
peptides and testing each of them for their ability to bind to a particular monoclonal antibody
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using a Biacore based human sclerostin peptide epitope competition binding assay.". The
resulting data permitted the location of the binding epitope to be determined.
The peptide digests were subjected to HPLC peptide mapping; the individual
peaks were collected, and the peptides identified and mapped by matrix assisted laser desorption
mass spectrometry (MALDI-MS) and electrospray ionization LC-MS (ESI-LC-MS) analyses
and/or by N-terminal sequencing. All HPLC analyses for these studies were performed using a
reverse-phase C8 column (2.1 mm i.d. x 15 cm length). HPLC peptide mapping was performed
with a linear gradient from 0.05% trifloroacetic acid (mobile phase A) to 90% acetonitrile in
0.05% trifuoroacetic acid. Columns were developed over 50 minutes at a flow rate of 0.2
ml/min.
Trvpsin and AspN Endoproteinase Digestions
Mature form human sclerostin was digested with trypsin, which cleaves after
arginine and lysine, or with AspN. About 200 µg of sclerostin at 0.5-1.0 mg/ml was incubated
in PBS (pH 7.2) for 20 hrs at 37°C with 8 µg of either trypsin or AspN.
Trvpsin digestion
HPLC chromatography of the trypsin digests yielded several major peaks
(Fig. 10A). Sequence analysis was conducted on the peptide peaks recovered from HPLC after
trypsin digestion. On-line ESI LC-MS analysis of the peptide digest was also performed to
determine the precise mass of the peptides that were separated by HPLC. The identity of the
peptides present in the peptide peaks was thus determined (Fig. 11). Figure 13 shows the
alignment of various peptide sequences (T19.2, T20, T20.6, T21-22) along the sclerostin
sequence. The number following each T (e.g., T19.2) reflects the retention time. T19.2 contains
two peptides (one from loop 1 and one from loop 3) linked by the C2-C6 disulfide bond. T20
contains two peptides held together by the cystine-knot structure, with intact loops 1 and 3 held
together by the C2-C6 disulfide and with most of loop 2 absent. T20.6 contains four sequences
held together by the cystine-knot structure, but is missing part of loop 1 and 3 (the T19.2 part)
and is missing most of loop 2. T21-22 is almost identical to T20 but has 3 additional amino
acids in the loop 2 region.
AspN Digestion
HPLC chromatography of the AspN digests yielded several major peaks
(Fig. 10B). Sequence analysis was conducted on the peptide peaks recovered from HPLC. On-
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line ESILC-MS analysis of the peptide digest was also performed to determine the precise mass
of the peptides that were separated by HPLC. The identity of the peptides present in the peptide
peaks from the AspN digestion was thus determined (Fig. 12). Figure 14 shows the alignment
of various peptide sequences (AspN14.6, AspN18.6, AspN22.7-23.5) along the sclerostin
sequence. The number following each AspN (e.g. AspN18.6) reflects the retention time.
AspN14.6 contains three short peptides from both the N- and C-terminal arms of sclerostin,
while AspN18.6 is a larger peptids from the N-terminal arm of sclerostin. AspN22.7-23.5
contains a single peptide fragment of 104 amino acids the encompasses all eight cysteines (the
four disulfide bonds), the cystine-knot and all of loops 1,2 and 3.
The strategy for characterizing the epitopes was to use these various trypsin and
AspN generated human sclerostin peptides and determine which peptides could still be bound by
the various Antibodies (Ab-A, Ab-B, Ab-C and Ab-D). Specifically this was tested in a
Biacore-based "human sclerostin peptide epitope competition binding assay" where the binding
of a particular monoclonal antibody to human sclerostin immobilized on the Biacore chip was
determine in the presence or absence of each of the various isolated trypsin and AspN HPLC
peptide fractions. In the absence of any competing peptides, the particular monoclonal antibody
was able to bind the human sclerostin on the chip and produce a resonance unit, RU, response.
Preincubation of the particular monoclonal antibody with intact human sclerostin in solution,
followed by testing of binding to the chip, demonstrated that the binding of the Mab to human
sclerostin in solution prevented the binding of the Mab to the human sclerostin on the chip, thus
validating the general principal of this competition assay.
This general procedure was repeated individually for each peptide. A robust RU
response was taken to indicate that the particular peptide being tested could not bind the Mab in
solution (hence the Mab was free to bind the human sclerostin that had been immobilized on the
chip). Conversely, the absence of a robust RU response indicated that the Mab was able to bind
the sclerostin peptide in solution. These binding patterns, couple with the known identity of the
various sclerostin peptides, were used to determine the epitopes of sclerostin that were bound by
anti-sclerostin antibodies Ab-A, Ab-B, Ab-C and Ab-D.
BIACORE-BASED HUMAN SCLEROSTIN PEPTIDE EPITOPE COMPETITION BINDING ASSAY
Preparation of human sclerostin surface:
Immobilization of mature form human sclerostin to a BIAcore sensor chip (CM5)
surface was performed according to manufacturer's instructions. Briefly, carboxyl groups on the
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sensor chip surfaces were activated by injecting 60 µL of a mixture containing 0.2 M N-ethyl-
N'-(dimethylaminopropyl) carbodiimide (EDC) and 0.05 M N-hydroxysuccinimide (NHS).
Human sclerostin was diluted in 10 mM sodium acetate, pH 4.0 at a concentration of 20 ug/mL
followed by injecting over the activated CMS surface. Excess reactive groups on the surfaces
were deactivated by injecting 60 uL of 1 M ethanolamine. Final immobilized levels were ~
5000 resonance units (RU) for the human sclerostin surface. A blank, mock-coupled reference
surface was also prepared on the sensor chips.
Binding specificity analysis:
IX Phosphate-buffered saline without calcium chloride or magnesium chloride
was from Gibco/Invitrogen, Carlsbad, CA. Bovine serum albumin, fraction V, IgG-free was
from Sigma-Aldrich, St. Louis, MO. Each Mab (2 nM) was separately incubated with 20 nM
human sclerostin or a particular human sclerostin peptide (note: there are 3 unlinked peptides in '
AspN14.6) in sample buffer (IX PBS + 0.005% P-20 + 0.1 mg/mL BSA) before injection over
the immobilized human sclerostin surface. The flow rate for sample injection was 5 uL/min
followed by surface regeneration using 1 M NaCl in 8 mM Glycine, pH 2.0 at 30 uL/min for 30
seconds. The data was analyzed using BIAevaluation 3.2r and is presented in Figure 15 (Ab-A),
Figure 16 (Ab-B), Figure 17 (Ab-C) and Figure 18 (Ab-D).
Loop 2 and T20.6 epitopes:
The sclerostin peptide binding pattern for two representative antibodies (Ab-A
and Ab-B) were virtually identical (Fig. 15 and Fig. 16) and showed that both of these
Antibodies could only bind the AspN22.7-23.5 peptide. The unique difference between
AspN22.7-23.5 and all the other sclerostin peptides is that AspN22.7-23.5 contains an intact
loop 2. This shows that Ab-A and Ab-B bind the loop 2 region of sclerostin thus defining the
loop 2 epitope (Fig. 19A). The sclerostin peptide binding pattern for Ab-C and Ab-D were
virtually identical to each other (Fig. 17 and Fig. 18) but completely distinct from that found for
Ab-A and Ab-B. Of the peptides tested in this Example, the most diminutive peptide that Ab-C
and Ab-D could bind to was the T20.6 peptide. This result defines the T20-6 epitope (Fig. 19B).
Protease protection assay:
The general principle of this assay is that binding of a Mab to sclerostin can result
in protection of certain specific protease cleavage sites and this information can be used to
determine the region of sclerostin to where the Mab binds.
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"T20.6 derivative I (cystine-knot + 4 arms)" epitope:
Figure 20 shows the HPLC peptide maps for a human sclerostin Ab-D complex
(Fig 20A: human sclerostin was preincubated at a 1:1 molar ratio with Ab-D prior to digestion
with trypsin as described above) and human sclerostin alone (Fig 20B: human sclerostin was
digested with trypsin as described above). The peptide peaks of T19.2 and T20.6 in Figure 20A
showed a clear reduction in their respective peak height, as compared to Figure 20B. This
reduction in peak heights was accompanied by an increase in peak height for peptides T20 and
T21-22. These data indicate that basic amino acid residues in loop 1 and loop 3, which in the
absence of Ab-D were cleaved by trypsin to generate peptides T19.2 and T20.6, were resistant to
cleavage by trypsin when Ab-D was prebound to sclerostin. The presence of T20, T20.6 and
T21-22 indicates that loop 2 was still cleaved efficiently when Ab-D was prebound to sclerostin.
These data indicate that Ab-D bound on the loop 1 and loop 3 side of the T20.6 epitope thus
defining the smaller "T20.6 derivative 1 (cystine-knot + 4 arms)" epitope shown in Figure 21.
EXAMPLE 5
IN VIVO TESTING OF ANTI-SCLEROSTIN MONOCLONAL ANTIBODIES IN MICE
Four week-old BDF1 male mice were obtained from Charles River Laboratories
(Raleigh, NC) and housed in clean caging, five animals per cage. Room temperature was
maintained between 68 and 72°F, and relative humidity was maintained between 34 and 73%.
The laboratory housing the cages had a 12-hour light/dark cycle and met all AAALAC
specifications. Clinical observations of all mice on study occurred once daily.
Purified anti-sclerostin monoclonal antibodies (Ab-A Fig. 1; Ab-B Fig.2; Ab-C
Fig.3; Ab-D Fig.4) were diluted in sterile Dulbecco's phosphate buffered saline. Mice were
injected with anti-sclerostin Antibodies or PBS vehicle subcutaneously at 21 u.1 per gram body
weight, two times per week (Monday and Thursday) at 25 mg/kg. Human PTH (1-34) was
diluted in PTH buffer (0.001 N HC1,0.15 M NaCl, 2% BSA), and dosed subcutaneously at 21 ui
per gram body weight five times per week (Monday, Tuesday, Wednesday, Thursday, Friday) at
100 ug/kg as a positive control (Figures 5 and 6). Number of mice per group was N=5 in Fig 5
and 6, and N=6 in Figure 7.
PIXImus in vivo Bone Densitometry
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"Hone mineral density (BMD) was determined weekly at the proximal tibial
metaphysis and lumbar vertebrae by peripheral Dual Energy X-ray Absorptometry (pDEXA)
with the PIXImus2 system from GE/Lunar Medical Systems, Madison, WI. A 25mm2 region of
interest (ROI) was placed to include the proximal articular surface, the epiphysis, and the
proximal end on the metaphysis of the tibia. A region of interest (ROI) was placed to include
the lumbar vertebrae (L1-L5). The proximal tibia and lumbar regions were analyzed to
determine total bone mineral density. Group means were reported ± Standard Deviation and
compared to the vehicle treatment group for statistical analysis.
Statistical analysis
Statistical analysis was performed with a Dunnett's and Tukey-Kramer (using
MS Excel and JMP v. 5.0. for the BMD data). Group means for each data set were considered
significantly different when the P value was less than 0.05 (P Sclerostin neutralizing activity of antibodies
The statistically significant increases in BMD as compared to vehicle seen for
each of Ab-A (Figure 5), Ab-B (Figure 5), Ab-C (Figure 6) and Ab-D (Figure 7) demonstrates
that these four antibodies are sclerostin neutralizing antibodies. Furthermore this data shows
that, for anti-sclerostin antibodies that bind mouse sclerostin, treatment and analysis of mice as
described above can be used to identify sclerostin neutralizing antibodies.
• , EXAMPLE 6
SCREENING ASSAY FOR ANTIBODIES THAT BLOCK BINDING OF AN ANTIBODY
TO HUMAN SCLEROSTIN
Human sclerostin was coupled to a CM5 Biacore chip using standard amine
coupling chemistry to generate a sclerostin coated surface. 300 resonance units of sclerostin
were coupled to the surface.
The antibodies to be-tested were diluted to a concentration of 200ug/ml in HBS-
EP buffer (being 10 mM HEPES pH 7.4,150 mM NaCl, 3 mM EDTA, 0.065% (v/v) Surfactant
P20) and then mixed in a one to one molar ratio (on a binding site basis) to generate the test
mixture. This test mixture thus contained each antibody at a concentration of lOOug/ml (1.3um
on a binding site basis). Separate solutions containing each of the antibodies in the test mix
alone were also prepared. These solutions contained the individual antibodies in HBS-EP buffer
at a concentration of 100µg/ml (1.3um on a binding site basis).
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20µL of the test mixture was passed over the sclerostin-coated chip at a flow rate
of 10 µL/min and the amount of binding recorded. The chip was then treated with two 60
second pulses of 30 mM HC1 to remove all of the bound antibody. A solution containing only
one of the antibodies of the test mixture (at L.3µM in the same buffer as the test mixture on a
binding site basis) was then passed over the chip in the same manner as the test mixture and the
amount of binding recorded. The chip was again treated to remove all of the bound antibody and
finally a solution containing the other antibody from the test mixture alone (at 1.3µM in the
same buffer as the test mixture on a binding site basis) was passed over the chip and the amount
of binding recorded.
The table below show the resu its irom cross-blocking assays on a range of
different antibodies. The values in each square of the table represent the amount of binding (in
RU) seen when the antibodies (at 1 .3µM on a binding site basis) or buffer indicated in the top
row of the table were mixed with the antibodies (at 1.3µM on a binding site basis) or buffer
indicated in the first column of the table.

Using the mean binding value (in RU) for each combination of antibodies in the
above table (since each combination appears twice) it is possible to calculate the percentage of
the theoretical binding shown by each combination of antibodies. The theoretical binding being
calculated as the sum of the average values for the components of each test mixture when
assayed alone (i.e., antibody and buffer).

From the above data it is clear that Ab-4, Ab-A and Ab-19 cross-block each
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other. Similarly Ab-13 and Ab-3 cross block each other.
EXAMPLE 7
ELISA-BASED CROSS-BLOCKING ASSAY
Liquid volumes used in this example would be those typically used in 96-well
plate ELISAs (e.g. 50-200 µl/well). Ab-X and Ab-Y, in this example are assumed to have
molecular weights of about 145 Kd and to have 2 sclerostin binding sites per antibody molecule.
An anti-sclerostin antibody (Ab-X) is coated (e.g. 50u of 1 µg/ml) onto a 96-well ELISA plate
[e.g. Corning 96 Well EIA/RIA Flat Bottom Microplate (Product # 3590), Corning Inc., Acton,
MA] for at least one hour. After this coating step the antibody solution is removed, the plate is
washed once or twice with wash solution (e.g., PBS and 0.05% Tween 20) and is then blocked
using an appropriate blocking solution (e.g., PBS, 1% BSA, 1% goat serum and 0.5% Tween 20)
and procedures known in the art. Blocking solution is then removed from the ELISA plate and a
second anti-sclerostin antibody (Ab-Y), which is being tested for it's ability to cross-block the
coated antibody, is added in excess (e.g. 50µl of 10µg/ml) in blocking solution to the appropriate
wells of the ELISA plate. Following this, a limited amount (e.g. 50 µl of 10 ng/ml) of sclerostin
in blocking solution is then added to the appropriate wells and the plate is incubated for at least
one hour at room temperature while shaking. The plate is then washed 2-4 times with wash
solution. An appropriate amount of a sclerostin detection reagent [e.g., biotinylated anti-
sclerostin polyclonal antibody that has been pre-complexed with an appropriate amount of a
streptavidin-horseradish peroxidase (HRP) conjugate] in blocking solution is added to the
ELISA plate and incubated for at least one hour at room temperature. The plate is then washed
at least 4 times with wash solution and is developed with an appropriate reagent [e.g. HRP
substrates such as TMB (colorimetric) or various HRP luminescent substrates]. The background
signal for the assay is defined as the signal obtained in wells with the coated antibody (in this
case Ab-X), second solution phase antibody (in this case Ab-Y), sclerostin buffer only (i.e. no
sclerostin) and sclerostin 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-X), second solution
phase antibody buffer only (i.e. no second solution phase antibody), sclerostin and sclerostin
detection reagents. The ELISA assay needs to be run in such a manner so as to have the positive
control signal be at least 6 times the background signal.
To avoid any artifacts (e.g. significantly different affinities between Ab-X and
Ab-Y for sclerostin) resulting from the choice of which antibody touse as the coating antibody
and which to use as the second (competitor) antibody, the cross-blocking assay needs to be run
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in two rormats:
1) format 1 is where Ab-X is the antibody that is coated onto the ELISA plate and
Ab-Y is the competitor antibody that is in solution
and
2) format 2 is where Ab-Y is the antibody that is coated onto the ELISA plate and
Ab-X is the competitor antibody that is in solution.
Ab-X and Ab-Y are defined as cross-blocking if, either in format I or in format 2,
the solution phase anti-sclerostin antibody is able to cause a reduction of between 60% and
100%, specifically between 70% and 100%, and more specifically between 80% and 100%, of
the sclerostin detection signal (i.e. the amount of sclerostin bound by the coated antibody) as
compared to the sclerostin detection signal obtained in the absence of the solution phase anti-
sclerostin antibody (i.e. the positive control wells).
In the event that a tagged version of sclerostin is used in the ELISA, such as a N-
terminal His-tagged Sclerostin (R&D Systems, Minneapolis, MN, USA; 2005 cat# 1406-ST-
025) then an appropriate type of sclerostin detection reagent would include an HRP labeled anti-
His antibody. In addition to using N-terminal His-tagged Sclerostin, one could also use C-
terminal His-tagged Sclerostin. Furthermore, various other tags and tag binding protein
combinations that are known in the art could be used in this ELISA-based cross-blocking assay
(e.g.; HA tag with anti-HA antibodies; FLAG tag with anti-FLAG antibodies; biotin tag with
streptavidin). ■ .
EXAMPLE 8
CELL BASED MINERALIZATION ASSAY FOR IDENTIFYING AGENTS
ABLE TO ANTAGONIZE SCLEROSTIN ACTIVITY
Introduction
Mineralization by osteoblast-lineage cells in culture, either primary cells or cell
lines, is used as an in vitro model of bone formation. Mineralization takes from about one to six
weeks to occur beginning with the induction of osteoblast-lineage cell differentiation by one or
more differentiation agents. The overall sequence of events involves cell proliferation,
differentiation, extracellular matrix production, matrix maturation and finally deposition of
mineral, which refers to crystallization and/or deposition of calcium phosphate. This sequence
of events starting with cell proliferation and differentiation, and ending with deposition of
mineral is referred to herein as mineralization. Measurement of calcium .(mineral) is the output
of the assay.
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Deposition of mineral has a strong biophysical characteristic, in that once mineral
"seeds" begin to form, the total amount of mineral that will be deposited in the entire culture can
sometimes be deposited quite rapidly, such as within a few days thereafter. The timing and
extent of mineral deposition in culture is influenced, in part, by the particular osteoblast-lineage
cells/cell-line being used, the growth conditions, the choice of differentiation agents and the
particular lot number of serum used in the cell culture media. For osteoblast-lineage cell/cell-
line mineralization cultures, at least eight to fifteen serum lots from more than one supplier
should be tested in order to identify a particular serum lot that allows for mineralization to take
place.
MC3T3-E1 cells (Sudo H et al., In vitro differentiation and calcification in a new
clonal osteogenic cell line derived from newborn mouse calvaria. J. Cell Biol. 96:191-198) and
subclones of the original cell line can form mineral in culture upon Growth in the presence of
differentiating agents. Such subclones include MC3T3-E1-BF (Smith E, Redman R, Logg C,
Coetzee G, Kasahara N, Frenkel B. 2000. Glucocorticoids inhibit developmental stage-specific
osteoblast cell cycle. J Biol Chem 275:19992-20001).
Identification of Sclerostin Neutralizing Antibodies
MC3T3-E1-BF cells were used for the mineralization assay. Ascorbic acid and
B-glycerophosphate were used to induce MC3T3-E1-BF cell differentiation leading to mineral
deposition. The specific screening protocol, in 96-well format, involved plating cells on a
Wednesday, followed by seven media changes (as described further below) over a 12-day period
with most of the mineral .deposition taking place in the final approximately eighteen hours (e.g.
Sunday night through Monday). For any given treatment, 3 wells were used (N=3). The
specific timing, and extent, of mineral deposition may vary depending, in part, on the particular
serum lot number being used. Control experiments will allow such variables to be accounted
for, as is well know in the art of cell culture experimentation generally.
In this assay system sclerostin inhibited one or more of the sequence of events
leading up to and including mineral deposition (i.e., sclerostin inhibited mineralization). Anti-
sclerostin antibodies that were able to neutralize sclerostin's inhibitory activity allowed for
mineralization of the culture in the presence of sclerostin such that there was a statistically
significant increase in deposition of calcium phosphate (measured as calcium) as compared to
the amount of calcium measured in the sclerostin-only (i.e., no antibody) treatment group. For
statistical analysis (using MS Excel and JMP) a 1-way-ANOVA followed by Dunnett's
comparison was used to determine differences between groups. Group means for each data set
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were considered significantly different when the P value was less than 0.05 (P representative result from running this assay is shown in Figure 22. In the absence of
recombinant mouse sclerostin, the sequence of events leading up to and including mineral.
deposition proceeded normally. Calcium levels in each treatment group are shown as means ±
Standard Error of the Mean (SEM). In this exemplary experiment calcium levels from the
calcium assay were -31 µg/ml. However, addition of recombinant mouse sclerostin caused
inhibition of mineralization, and calcium was reduced by ~ 85%. Addition of anti-sclerostin
monoclonal antibody Ab-19 or Ab-4 along with the recombinant sclerostin resulted in a
statistically significant increase in mineral deposition, as compared to the sclerostin-only group,
because the inhibitory activity of sclerostin was neutralized by either antibody. The results from
this experiment indicate that Ab-19 and Ab-4 are sclerostin neutralizing monoclonal antibodies
(Mabs).
Figure 23 shows a very similar result using recombinant human sclerostin and
two humanized anti-sclerostin Mabs. Figure 24 also shows a very similar result using
recombinant human sclerostin and mouse and humanized anti-sclerostin Mabs as indicated.
The antibodies used for the experiments shown in Fig 22, 23 and 24 have
molecular weights of about 145 Kd and have 2 sclerostin binding sites per antibody molecule.
A detailed MC3T3-E1-BF cell culture protocol is described below.
Reagents and Medias
Reagents Company Catalog #
Alpha-MEM Gibco-Invitrogen 12571-048
Ascorbic acid Sigma A4544
Beta-glycerophosphate Sigma G6376
100X PenStrepGlutamine Gibco-Invitrogen 10378-016
Dimethylsulphoxide (DMSO) Sigma D5879 or D2650
Fetal bovine serum (FBS) Cansera CS-C08-500 (lot # SF50310)
or Fetal bovine serum (FBS) TerraCell Int. CS-C08-1000A (lot # SF-20308)
Alpha-MEM is usually manufactured with a 1 year expiration date. Alpha-MEM that was not
older than 6-months post-manufacture date was used for the cell culture.
Expansion Medium (Alpha-MEM/10%FBS/PenStrepGlu) was prepared as follows:
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A 500 mL bottLe of FBS was thawed and filter sterilized through a 0.22 micron filter.
100 mis of this FBS was added to 1 liter of Alpha-MEM followed by the addition of 10 mis of
100x PenStrepGlutamine. Unused FBS was aliquoted and refrozen for later use.
Differentiation Medium (Alpha-MEM/10%FBS/PenStrepGlu, + 50 µg/ml ascorbic acid, + 10
mM beta-glycerophosphate) was prepared as follows:
100 mis of Differentiation Medium was prepared by supplementing 100 mis of Expansion
Medium with ascorbic acid and beta-glycerophosphate as follows:
Stock cone Volume Final Cone.
(see below)
Ascorbic acid 10mg/ml 0.5 mis 100 µg/ml (50ug/ml + 50µg/ml)
P-glycerophosphate 1M 1.0 mis 10 mM
Differentiation Medium was made by supplementing Expansion Medium only on
the day that the Differentiation media was going to be used for cell culture. The final
concentration of ascorbic acid in Differentiation medium is 100 µg/ml because Alpha-MEM
already contains 50 µg/ml ascorbic acid. Ascorbic acid stock solution (10 mg/ml) was made and
aliquoted for freezing at -80°C. Each aliquot was only used once (i.e. not refrozen). Beta-
glycerophosphate stock solution (1 M) was made and aliquoted for freezing at -20°C. Each
aliquot was frozen and thawed a maximum of 5 times before being discarded.
Cell Culture for expansion of MC3T3-E1-BF cells.
Cell culture was performed at 37°C and 5% CO2. A cell bank was generated for
the purposes of screening for sclerostin neutralizing antibodies. The cell bank was created as
follows:
One vial of frozen MC3T3-E1-BF cells was thawed by agitation in a 37°C water
bath. The thawed cells were put into 10 mis of Expansion Medium (Alpha-
MEM/10%FBS/PenStrepGlu) in a 50 ml tube and gently spun down for 5 minutes. The cells
were then resuspended in 4 mis of Alpha-MEM/10%FBS/PenStrepGlu. After determining the
number of cells using trypan blue and hemacytometer, 1 x 106 cells were plated in 50 mis
Alpha-MEM/10%FBS/PenStrepGlu media in one TI75 flask.
When this passage was confluent (at approximately 7 days), the cells were
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trypsinized witH trypsin/EDTA (0.05% Trypsin; 0.53 raM EDTA), gently spun down for 5
minutes and then resuspended in 5 mls Alpha-MEM/10%FBS/PenStrepGlu. After determining
the number of cells using trypan blue and hemacytometer, cells were plated at 1 x 106 cells in 50
mis Alpha-MEM/10%FBS/PenStrepGlu media per one T175 flask. The number of T175 flasks
used for plating at this point depended upon the total cell number available and the desired
number of flasks that were to be taken forward to the next passage. Extra cells were frozen
down at l-2x106 live cells/ml in 90%FBS/10%DMSO.
When this passage was confluent (about 3-4 days), the cells were trypsinized with
trypsin/EDTA (0.05% Trypsin; 0-53 mM EDTA), gently spun down for 5 minutes and then
resuspended in 5 mls Alpha-MEM/10%FBS/PenStrepGlu. After determining the number of
cells using trypan blue and hemacytometer, cells were plated at 1 x 106 cells in 50 mls Alpha-
MEM/10%FBS/PenStrepGlu media per one-T175 flask. The number of T175 flasks used for
plating at this point depended upon the total cell number available and the desired number of
flasks that were to be taken forward to the next passage. Extra cells were frozen down at 1-
2xl06 live cells/ml in 90%FBS/10%DMSO.
When this passage was confluent (about 3-4 days), the cells were trypsinized with
trypsin/EDTA (0.05% Trypsin; 0.53 mM EDTA), gently spun down for 5 minutes and then
resuspended in 5 mls Alpha-MEM/10%FBS/PenStrepGlu. After determining the number of
cells using trypan blue and hemacytometer, cells were plated at 1 x 106 cells in 50 mls Alpha-
MEM/10%FBS/PenStrepGlu media per one T175 flask. The number of T175 flasks used for
plating at this point depended upon the total cell number available and the desired number of
flasks that were to be taken forward to the next passage. Extra cells were frozen down at 1-
2x106 live cells/ml in 90%FBS/10%DMSO.
When this passage was confluent (about 3-4 days), the cells were trypsinized with
trypsin/EDTA (0.05% Trypsin; 0.53 mM EDTA), gently spun down for 5 minutes and then
resuspended in 5 mls Alpha-MEM/10%FBS/PenStrepGlu. After determining the number of
cells using trypan blue and hemacytometer, the cells were frozen down at 1-2x10 live cells/ml
in 90%FBS/10%DMSO. This "final passage" of frozen cells was the passage that was used for
the screening assay.
Cell Culture for mineralizing MC3T3-E1-BF cells.
Cell culture was performed at 37°C and 5% CO2. It is desirable to minimize
temperature and % CO2 fluctuations during the mineralization cell culture procedure. This can
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be achieved by minimizing the time that plates spend out of the incubator during feeding and
also by minimizing the number of times the incubator door is opened and closed during the
mineralization cell culture procedure. In this regard having a tissue culture incubator that is .
dedicated exclusively for the mineralization cell culture (and thus not opened and closed more
than is necessary) can be helpful.
An appropriate number of "final passage" vials prepared as described above were
thawed by agitation in a 37°C water bath. The thawed cells were put into 10 mls of Expansion
Medium (Alpha-MEM/10%FBS/PenStrepGlu) in a 50 ml tube and gently spun down for 5
minutes. The cells were then resuspended in 4 mls of Alpha-MEM/10%FBS/PenStrepGlu.
After determining the number of cells by trypan blue and hemacytometer, 2500 cells were plated
in 200 microliters of Expansion media per well on collagen I coated 96-well plates (Becton
Dickinson Labware, cat # 354407).
To avoid a mineralization plate-edge effect, cells were not plated in the outermost
row/column all the way around the plate. Instead 200 microliters of PBS was added to these
wells.
Exemplary cell culture procedure
In the following procedure, the starting day for plating the cells is indicated to be
a Wednesday. If a different day of the week is used as the starting day for plating the cells, that
day will trigger the daily schedule for removing and adding media, during the entire process as
indicated below. For example, if the cells are plated on a Tuesday, media should not be
removed and added on the first Friday and Saturday, nor on the second Friday and Saturday.
With a Tuesday start, the plates would be prepared for the calcium assay on the final Sunday.
Cells were plated on a Wednesday at 2500 cells in 200 µl of Expansion media.
On Thursday all of the Expansion media was removed and 200 ul of differentiation Media was
added.
On Friday 100 µl of media was removed and 100 µl of fresh Differentiation Media was added.
On Monday 100 µl of media was removed and 100 µl of fresh Differentiation Media was added.
On Tuesday 100 µl of media was removed and 100 µl of fresh Differentiation Media was added.
On Wednesday 100 µl of media was removed and 100 µl of fresh Differentiation Media was
added.
On Thursday 100 µl of media was removed and 100 ul of fresh Differentiation Media was
added.
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On Friday 100 µl of media was removed and 100 µl of fresh Differentiation Media was added.
On the following Monday plates were prepared for the calcium assay as follows:
Plates were washed once with 10 mM Tris, HC1 pH 7-8.
Working under a fume hood, 200 u.1 of 0.5 N HCl was added per well. Plates were then frozen
at-80°C.
Just prior to measuring calcium, the plates were freeze-thawed twice, and then
trituration with a multichannel pipette was used to disperse the contents of the plate. The
contents of the plate was then allowed to settle at 4oC for 30 minutes at which point an
appropriate amount of supernatant was removed for measuring calcium using a commercially
available calcium kit. An exemplary and not-limiting kit is Calcium (CPC) Liquicolor, Cat. No.
0150-250, Stanbio Laboratory, Boerne, TX.
In this cell based assay, sclerostin inhibits one or more of the sequence of events
leading up to and including mineral deposition (i.e. sclerostin inhibits mineralization). Thus, in
experiments where sclerostin was included in the particular cell culture experiment, the
recombinant sclerostin was added to the media starting on the first Thursday and every feeding
day thereafter. In cases where an anti-sclerostin monoclonal antibody (Mab) was being tested
for the ability to neutralize sclerostin. i.e. allow for mineralization by neutralizing sclerostin's
ability to inhibit mineralization, the Mab was added to the media starting on the first Thursday
and every feeding day thereafter. According to the protocol, this was accomplished as follows:
the Mab was preincubated with the recombinant sclerostin in Differentiation media for 45-60
minutes at 37°C and then this media was used for feeding the cells.
Described above is a 12-day mineralization protocol for MC3T3-E1-BF cells.
Using the same reagents and feeding protocol, the original MC3T3-E1 cells (Sudo H, Kodama
H-A, Amagai Y, Yamamoto S, Kasai S. 1983. In vitro differentiation and calcification in a new
clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 96:191-198) which
we obtained from the RIKEN Cell Bank (RCB 1126, RIKEN BioResource Center 3-1-1
Koyadai,Tsukuba-shi, Ibaraki 305-0074 Japan) took longer to mineralize (20 days total for
mineralization) than the MC3T3-E1-BF cells. Mineralization of the original MC3T3-E1 cells
was inhibited by recombinant sclerostin and this inhibition was blocked using a sclerostin
neutralizing antibody.
EXAMPLE 9
ANTI-SCLEROSTIN ANTIBODY PROTECTS FROM INFLAMMATION-INDUCED BONE LOSS IN THE CD4
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CD45RB"' TRANSFER MODEL OF COLITIS IN SCID MICE
Summary of model
Injection of the CD45RBhigh subset of CD4+ T cells into C.B-17 scid mice results
in chronic intestinal inflammation with characteristics similar to those of human inflammatory
bowel disease (D3D). Diarrhoea and wasting disease is noted 3-5 weeks after cell transfer with
severe leukocyte infiltration into the colon accompanied by epithelial cell hyperplasia and
granuloma formation. C.B-17 scid mice which receive the reciprocal subset of CD4+ cells, those
which express CD45RBlow, do not exhibit colitis and have a weight gain indistinguishable from
uninjected scid mice. In addition to colitis symptoms, the CD4+ CD45RBhigh T cell transfer
model of colitis is accompanied by a reduction in bone mineral density (BMD), thought to be
primarily through inflammatory mechanisms rather than dietary malabsorption (Byrne, F. R. et
al., Gut 54:78-86,2005).
Induction of colitis and inflammation-induced bone loss
Spleens were taken from female balb/c mice and disrupted through a 70µm cell
strainer. The CD4+ population was then enriched by negative selection with Dynabeads using
antibodies against B220, MAC-1, CD8 and I-Ad. The enriched population was then stained with
FITC conjugated anti-CD4 and PE conjugated anti-CD45RB and fractionated into
CD4+CD45RBhigh and CD4+CD45RBlow populations by two-color sorting on a Moflo
(Dakocytomation). The CD45RBhigh and CD45RBlow populations were defined as the brightest
staining 40% and the dullest staining 20% of CD4+ cells respectively. 5 x 105 cells were then
injected i.p. into C.B-17 scid mice on day 0 and the development of colitis was monitored
through the appearance of soft stools or diarrhoea and weight loss. Bone mineral density
measurements were taken at the termination of the study (day 88).
Effect of anti-Sclerostin treatment on colitis symptoms and BMP
Ab-A IgG was dosed at 10mg/kg s.c. from the day prior to CD4+CD45RBhigh cell
transfer and compared with mice which received the negative control antibody 101.4 also dosed
at lOmg/kg s.c. The antibodies were dosed weekly thereafter. A group of mice which received
non-pathogenic CD4+CD45RBlow cells and were dosed with 10mg/kg 101.4 was studied as a
control. At the termination of the study (day 88) the bone mineral density was measured and
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sections of the colon taken for analysis of cell infiltration and assessment of histological
damage.
a) No effect on colitis symptoms
Typical colitis symptoms such as weight loss and infiltration of inflammatory
cells into the colon were unaffected by treatment with Ab-A. Similarly there was no
improvement of histological damage to the colon after treatment with Ab-A.
b) Inhibition of inflammation-induced loss of bone mineral density.
On day 88 after transfer of cells into C.B-17 scid mice, the bone mineral density
was measured (total BMD, vertebrae BMD and femur BMD). In comparison to control mice
which received CD4+CD45RBlow non-pathogenic cells, mice which received CD4+ CD45RBhigh
T cells and the negative control antibody 101.4 had reduced bone mineral density, as shown in
Figure 25. In contrast, no reduction in BMD was noted after treatment with Ab-A. Total,
vertebrae and femur measurements of BMD were significantly higher in mice receiving CD4+
CD45RBhigh T cells and treated with Ab-A than mice receiving CD4+ CD45RBhigh T cells and
treated with 101.4 (P EXAMPLE 10
KINEXA-BASED DETERMINATION OF AFFINITY (KD) OF ANTI-SCLEROSTIN ANTIBODIES FOR HUMAN
SCLEROSTIN
The affinity of several anti-sclerostin antibodies to human sclerostin was assessed by a solut-
equilibrium binding analysis using KinExA® 3000 (Sapidyne Instruments Inc., Boise, ID). For thes
measurements, Reacti-Gel 6x beads (Pierce, Rockford, IL) were pre-coated with 40 µg/ml human
sclerostin in 50 mM Na2CO3, pH 9.6 at 4°C overnight. The beads were then blocked with 1 mg/m
BSA in 1 M Tris-HCl, pH 7.5 at 4°C for two hours. 10 pM, 30 pM, or 100 pM of the antibody was
mixed with various concentrations of human sclerostin, ranging in concentration from 0.1 pM to 11
and equilibrated at room temperature for over 8 hours in PBS with 0.1 mg/ml BSA and 0.005% P2(
The mixtures were then passed over the human sclerostin coated beads. The amount of bead-bounc
anti-sclerostin antibody was quantified using fluorescent Cy5-labeled goat anti-mouse-IgG or
fluorescent Cy5-labeled goat anti-human-IgG antibodies (Jackson Immuno Research, West Grove,
for the mouse or human antibody samples, respectively. The amount of fluorescent signal measure
was proportional to the concentration of free anti-sclerostin antibody in each reaction mixture at
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equilibrium. The dissociation equilibrium constant (KD) was obtained from nonlinear regression of tt
competition curves using a n-curve one-site homogeneous binding model provided in the KinExA Prc
software. Results of the KinExA assays for the selected antibodies are summarized in the table below
Antibodies Antigen KD (pM) 95% confidence interval
Ab-13 Human Sclerostin 0.6 0.4- 0.8 pM
Ab-4 Human Sclerostin 3 1.8 ~ 4 pM
Ab-19 Human Sclerostin 3 1.7-4 pM
Ab-14 Human Sclerostin 1 0.5-2 pM
Ab-5 Human Sclerostin 6 4.3-8 pM
Ab-23 Human Sclerostin 4 2.1-8 pM
EXAMPLE 11
BLACORE METHOD FOR DETERMINING THE AFFINITY OF HUMANISED ANTI-SCLEROSTIN ANTIBODIES
FOR HUMAN SCLEROSTIN.
The BIAcore technology monitors the binding between biomolecules in real time and
without the requirement for labelling. One of the interactants, termed the ligand, is either :
immobilised directly or captured on the immobilised surface while the other, termed die analyte,
flows in solution over the captured surface. The sensor detects the change in mass on the sensor
surface as the analyte binds to the ligand to form a complex on the surface. This corresponds to
the association process. The dissociation process is monitored when the analyte is replaced by
buffer. In the affinity BIAcore assay, the ligand is the anti-sclerostin antibody and the analyte is
sclerostin.
Instrument
Biacore ® 3000, Biacore AB, Uppsala, Sweden
Sensor chip
CM5 (research grade) Catalogue Number: BR-1001-14, Biacore AB, Uppsala, Sweden. Chips
were stored at 4 °C.
BIAnormalising solution
70% (w/w) Glycerol. Part of BIAmaintenance Kit Catalogue Number: BR-1002-51, Biacore
AB, Uppsala, Sweden. The BIAmaintenance kit was stored at 4 °C.
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Amine Coupling Kit
Catalogue Number: BR-1000-50, Biacore AB, Uppsala, Sweden.
Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Made up to 75 mg/mL in
distilled water and stored in 200 u.L aliquots at-70 °C.
N-Hydroxysuccinimide (NHS). Made up to 11.5 mg/mL in distilled water and stored in 200 uX
aliquots at -70 °C.
1 M Ethanolamine hydrochloride-NaOH pH8.5. Stored in 200 uL aliquots at-70°C.
Buffers
Running buffer for immobilising capture antibody. HBS-EP (being 0.01 M HEPES pH 7.4, 0.15
MNaCl, 3 mMEDTA, 0.005 % Surfactant P20). Catalogue Number: BR-1001-88, Biacore AB,
Uppsala, Sweden. Buffer stored at 4 °C.
Immobilisation buffer: Acetate 5.0 (being 10 mM sodium acetate pH 5.0). Catalogue number:
BR-1003-51, Biacore AB, Uppsala, Sweden. Buffer stored at 4 °C.
Running buffer for binding assay: HBS-EP (being 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 raM
EDTA, 0.005 % Surfactant P20, Catalogue Number: BR-1001-88, Biacore AB, Uppsaia,
Sweden) with CM-Dextran added at 1 mg/mL (Catalogue Number 27560, Fluka BioChemika,
Bucbs, Switzerland). Buffer stored at 4 °C.
Ligand capture
Affinipure F(ab')2 fragment goat anti-human IgG, Fc fragment specific. Jackson
ImmunoResearch Inc (Pennsylvania, USA) Catalogue number: 109-006-098. Reagent stored at
4°C.
Ligand
Humanised anti-human sclerostin antibodies Ab5, Abl4 and Ab20.
Analvte
Recombinant human sclerostin. Aliquots stored at -70°C and thawed once for each assay.
Regeneration Solution
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40 mM HC1 prepared by dilution with distilled water from an 11.6 M stock solution (BDH,
Poole, England. Catalogue number: 101254H).
5 mM NaOH prepared by dilution with distilled water from a 50 mM stock solution. Catalogue
number: BR-1003-58, Biacore AB, Uppsala, Sweden.
Assay Method
The assay format was capture of the anti-sclerostin antibody by immobilised anti-human IgG-Fc
then titration of the sclerostin over the captured surface.
An example of the procedure is given below:
BIA (Biamolecular Interaction Analysis) was performed using a BIAcore 3000 (BIAcore AB).
Affinipure F(ab')2 Fragment goat anti-human IgG, Fc fragment specific (Jackson
ImmunoResearch) was immobilised on a CM5 Sensor Chip via amine coupling chemistry to a
capture level of ~4000 response units (RUs). HBS-EP buffer (10mM HEPES pH 7.4, 0.15 M
NaCl, 3 mM EPTA, 0.005 % Surfactant P20, BIAcore AB) containing 1 mg/mL CM-Dextran
was used as the running buffer with a flow rate of 10 µl/min, A 10 µl injection of the anti-
sclerostin antibody at ~5 µg/mL was used for capture by the immobilised anti-human IgG-Fc.
Antibody capture levels were typically 100-200 RU. Sclerostin was titrated over the captured
anti-sclerostin antibody at various concentrations at a flow rate of 30 µL/min. The surface was
regenerated by two 10 µL, injections of 40 mM HC1, followed by a 5 µL injection of 5 mM
NaOH at a flowrate of 10µL/min.
Background subtraction binding curves were analysed using the BIAevaluation software
(version 3.2) following standard procedures. Kinetic parameters were determined from the
fitting algorithm.
The kinetic data and calculated dissociation constants are given in Table 2.
TABLE 2: Affinity of anti-sclerostin antibodies for sclerostin

EXAMPLE 12
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IN VIVO TESTING OF ANTI-SCLEROSTIN MONOCLONAL ANTIBODIES IN CYNOMOLGOUS MONKEYS
Thirty-three, approximately 3-5 year old, female cynomolgus monkeys (Macaca
fascicularis) were used in this 2- month study. The study contained 11 groups:
Group 1: vehicle (N=4)
Group 2: Ab-23 (N=2, dose 3 mg/kg)
Group 3: Ab-23 (N=3, dose 10 mg/kg)
Group 4: Ab-23 (N=3, dose 30 mg/kg)
Group 5: Ab-5 (N=3, dose 3 mg/kg)
Group 6: Ab-5 (N=3, dose 10 mg/kg)
Group 7: Ab-5 (N=3, dose 30 mg/kg)
Group 8: Ab-14 (N=3, dose 3 mg/kg)
Group 9: Ab-14 (N=3, dose 10 mg/kg)
Group 10: Ab-14 (N=3, dose 30 mg/kg)
Group 11: Parathyroid Hormone (1-34) [PTH (1-34)] (N=3, dose 10 ug/kg)
All dosing was subcutaneous. PTH (1-34) was dosed everyday, monoclonal antibodies (Mabs)
were dosed twice (first dose at the beginning of the study and second dose at the one month time
point). For assessment of bone parameters (e.g. bone mineral density) pQCT (peripheral
quantitative computed tomography) and DXA (dual energy X-ray absorptiometry) scans were
performed prior to the beginning of the study (to obtain baseline values) and after a month (prior
to the second dose of Mab) and finally at the end of the study (2-month time point) at which
point the monkeys were necropsied for further analysis (e.g. histomorphometric analysis).
Animals were fiuorochrome labeled (days 14,24, 47, and 57) for dynamic histomorphometry.
Serum was collected at various time points during the study [day 1 pre-dose (the day of the first
Mab dose), day 1 twelve hours post-dose, day 2, day 3, day 5, day 7, day 14, day 21, day 28, day
29 twelve hours post-dose (day 29 was the day of the second and final Mab dose), day 30, day
31, day 33, day 35, day 42, day 49 and day 56].
Three bone-related serum biomarkers were measured using commercially available kits:
Osteocalcin (OCD (DSL Osteocalcin Radioimmunoassay Kit; Diagnostic Systems Laboratories,
Inc.,Webster,TX,USA)
N-terminal Propeptide of Type 1 Procollagen (P1NP) (PINP Radioimmunoassay Kit; Orion
Diagnostica, Espoo, Finland)
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C-telopeptide fragments of collagen type I al chains (sCTXD (Serum CrossLaps® ELISA;
Nordic Bioscience Diagnostics A/S, Herlev, Denmark).
pQCT and DXA scans yielded data on various bone parameters (including bone
mineral density (BMD) and bone mineral content) across numerous skeletal sites (including
tibial metaphysis and diaphysis, radial metaphysis and diaphysis, femur neck, lumbar vertebrae).
Analysis of this bone data (percent change from baseline for each animal) and the anabolic (OC,
P1NP) serum biomarker data (percent change from baseline for each animal) revealed
statistically significant increases, versus the vehicle group, in some parameters at some of the
time points and doses for each Mab. This bone parameter data, serum biomarker data, as well as
the histomorphometric data, indicated that each of the 3 Mabs (Ab-23, Ab-5 and Ab-14) was
able to neutralize sclerostin in cynomolgous monkeys. This activity was most robust for Ab-23
and Ab-5, particularly at the highest dose (30 mg/kg), with a clear increase in bone formation
(anabolic effect) as well as net gains in bone (e.g. BMD). Statistically significant increases in
bone parameters and anabolic histomorphometric parameters were also found for the positive
control group (PTH (1-34)).
Serum bone formation markers (P1NP, osteocalcin) were increased (p vehicle (VEH)) at various time points and doses, but particularly in the 30 mg/kg groups for Ab-
23 and Ab-5. Histomorphometric analysis revealed dramatic increases (p formation rates in cancellous bone at lumbar vertebra and proximal tibia (up to 5-fold increase),
as well as at the endocortical surface of the femur midshaft (up to 10-fold increase) at the higher
doses of Ab-23 and Ab-5. Trabecular thickness was increased with high dose Ab-23 and Ab-5
in lumbar vertebrae (>60%, p change from baseline, was increased (p 23, 30 mg/kg), and lumbar vertebrae (Ab-5, 30 mg/kg). The increases in areal BMD at the
lumbar vertebrae were accompanied by increases in vertebral strength (97% increase in vertebral
maximal load for Ab-23, 30 mg/kg; p prior to Mab dosing were statistically similar across all groups. In summary, short-term
administration of sclerostin-neutralizing Mabs in cynomolgous monkeys resulted, in part, in
increases in bone formation, BMD and vertebral bone strength.
From the foregoing, although specific embodiments of the invention have been
described herein for purposes of illustration, various modifications may be made without
deviating from the spirit and scope of the invention. Accordingly, the invention is not limited
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except as by the appended claims. All publications, published patent applications, and patent
documents disclosed herein are hereby incorporated by reference.
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CLAIMS
What is claimed is:
1. A sclerostin binding agent that cross-blocks the binding of at least one of
antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9,
Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-
22, Ab-23, and Ab-24 to sclerostin.
2. The sclerostin binding agent of claim t wherein said sclerostin binding
agent is cross-blocked from binding to sclerostin by at least one of antibodies Ab-A, Ab-B, Ab-
C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-
13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24.
3. A sclerostin binding agent that is cross-blocked from binding to sclerostin
by at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6,
Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19,
Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24.
4. The sclerostin binding agent of claim 1 or 3 wherein the ability of said
sclerostin binding agent to cross-block or to be cross-blocked is detected in a Biacore assay.
5. The sclerostin binding agent of claim 1 or 3 wherein the ability of said
sclerostin binding agent to cross-block or to be cross-blocked is detected in an ELISA. assay.
6. The sclerostin binding agent of claim 1 or 3 wherein said sclerostin
binding agent is an antibody.
7. The sclerostin binding agent of claim 1 or 3 wherein said sclerostin
binding agent can increase at least one of bone formation, bone mineral density, bone mineral
content, bone mass, bone quality and bone strength in a mammal.
8. The sclerostin binding agent of claim 1 or 3 wherein said sclerostin
binding agent can block the inhibitory effect of sclerostin in a cell based mineralization assay.
9. A sclerostin binding agent wherein said sclerostin binding agent can block
the inhibitory effect of sclerostin in a cell based mineralization assay.

10. A sclerostin binding agent that binds to a Loop 2 epitope.
11. A sclerostin binding agent that binds to a T20.6 epitope.
12. A sclerostin binding agent that binds to a "T20.6 derivative 1 (cystine-
knot + 4 arms)" epitope.
13. The sclerostin binding agent of any one of claims 7-12 wherein said
sclerostin binding agent is an antibody.
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14. A sclerostin binding agent that comprises at least one CDR sequence
having at least 75% identity to a CDR selected from SEQ ID NOs:39,40,41,42, 43,44,45,46,
47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 78,79, 80, 81, 99,100, 101,102,
103,104,105,106,107,108, 109,110, 111, 112, 113,114, 115,116,237,238,239,240,241,
242, 243,244,245,246,247, 248, 249,250, 251,252,253, 254,255,256,257, 258,259, 260,
261,262,263,264, 265,266, 267, 268, 269,270, 271,272, 273,274,275,276, 277, 278, 279,
280,281,282,283, 284,285, 286,287, 288, 289,290,291, 292, 293,294,295,296,297, 298,
351, 352, 353,358, 359, and 360.
15. The sclerostin binding agent of claim 14 comprising at least two of said
CDR's.
16. The sclerostin binding agent of claim 14 comprising six of said CDR's.
17. The sclerostin binding agent according to claim 14 wherein said percent
identity is 85%.
18. The sclerostin binding agent according to claim 14 wherein said percent
identity is 95%.
19. The sclerostin binding agent according to claim 14 comprising:

a) CDR sequences of SEQ ID NOs:39, 40, and 41;
b) CDR sequences of SEQ ID NOs:42, 43, and 44;
c) CDR sequences of SEQ ID NOs:45, 46, and 47;
d) CDR sequences of SEQ ID NOs:48, 49, and 50;
e) CDR sequences of SEQ ID NOs:51, 52, and 53;
f) CDR sequences of SEQ ID NOs:54, 55, and 56;
g) CDR sequences of SEQ ID NOs:57, 58, and 59;
h) . CDR sequences of SEQ ED NOs:60, 61, and 62;
i) CDR sequences of SEQ ID NOs:275,276, and 277; .
j) CDR sequences of SEQ ID NOs:287,288, and 289;
k) CDR sequences of SEQ ID NOs:278,279, and 280;
1) CDR sequences of SEQ ID NOs:290,291, and 292;
m) CDR sequences of SEQ ID NOs:78,79, and 80;
n) CDR sequences of SEQ ID NOs:245,246, and 247;
o) CDR sequences of SEQ ID NOs:81, 99, and 100;
p) CDR sequences of SEQ ID NOs:248,249, and 250
q) CDR sequences of SEQ ID NOs: 101,102, and 103;
r) CDR sequences of SEQ ID NOs:251,252, and 253;
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s) CDR sequences of SEQ ID NOs:104,105, and 106
t) CDR sequences of SEQ ID NOs:254,255, and 256;
u) CDR sequences of SEQ ID NQs:107, 108, and 109
v) CDR sequences of SEQ ID NOs:257,258, and 259
w) CDRsequences ofSEQ IDNOs:110, 111, and 112;
x) CDR sequences of SEQ ID NOs:260, 261, and 262;
y) CDR sequences of SEQ ID NOs:28l, 282, and 283;
z) CDR sequences of SEQ ID NOs:293,294, and 295;
aa) CDRsequences of SEQ IDNOs:113, 114, and 115;
bb) CDR sequences of SEQ ID NOs:263, 264, and 265;
cc) CDR sequences of SEQ ID NQs:284,285, and 286;
dd) CDR sequences of SEQ ID NOs:296, 297, and 298;
ee) CDR sequences of SEQ ID NOs:l 16,237, and 238;
ff) CDR sequences of SEQ ID NOs:266, 267, and 268;
gg) CDR sequences of SEQ ID NOs:239, 240, and 241;
hh) CDR sequences of SEQ ED NOs:269, 270, and 271;
ii) CDRsequences of SEQ ID NOs:272, 273, and 274;
jj) CDR sequences of SEQ ID NOs:242, .243, and 244;
kk) CDR sequences of SEQ ID NOs:351, 352, and 353; or
11) CDR sequences of SEQ ID NOs:358, 359, and 360.
20. The sclerostin binding agent according to claim 14 comprising:
a) ' CDR sequences of SEQ ID NOs:54, 55, and 56 and CDR
sequences of SEQ ID NOs:51, 52, and 53;
b) CDR sequences of SEQ ID NOs:60, 61, and 62 and CDR
sequences of SEQ ID NOs:57, 58, and 59;
c) CDR sequences of SEQ ID NOs:48,49, and 50and CDR
sequences of SEQ ID NOs:45,46, and 47;
d) CDR sequences of SEQ ID NOs:42, 43, and 44 and CDR
sequences of SEQ ID NOs:39,40, and 41;
e) CDR sequences of SEQ ID NOs:275, 276, and 277 and CDR
sequences of SEQ ID NOs:287,288, and 289;
f) CDR sequences of SEQ ID NOs:278,279, and 280 and CDR
sequences of SEQ ID NOs:290,291, and 292;
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g) CDR sequences of SEQ ID NOs:78,79, and 80 and CDR
sequences of SEQ ID NOs: 245,246, and 247;
h) CDR sequences of SEQ ID NOs:81,99, and 100 and CDR '
sequences of SEQ ID NOs:248, 249, and 250;
i) CDR sequences of SEQ ID NOs:101, 102, and 103 and CDR
sequences of SEQ ID NOs:251, 252, and 253;
j) CDR sequences of SEQ ID NOs: 104,105, and 106 and CDR
sequences of SEQ ID NOs:254,255, and 256;
k) CDR sequences of SEQ ID NOs: 107,108, and 109 and CDR
sequences of SEQ ID NOs:257, 258, and 259;
1) CDR sequences of SEQ ID NQs: 110,111, and 112 and CDR
sequences of SEQ IDNOs:260, 261, and 262;
m) CDR sequences of SEQ ID NOs:281,282, and 283 and CDR
sequences of SEQ ID NOs:293,294, and 295;
n) CDR sequences of SEQ ID NOs: 113, 114, and 115 and CDR
sequences of SEQ ID NOs:263,264, and 265;
o) CDR sequences of SEQ ID NOs:284, 285, and 286 and CDR
sequences of SEQ ID NOs:296,297, and 298;
p) CDR sequences of SEQ ID NOs:l 16,237, and 238 and CDR
sequences of SEQ ID NOs:266,267, and 268;
q) CDR sequences of SEQ ID NOs:239,240, and 241 and CDR
sequences of SEQ ID NOs:269,270, and 271;
r) CDR sequences of SEQ ID NOs:242,243, and 244 and CDR
sequences of SEQ ID NOs:272,273, and 274; or
s) CDR sequences of SEQ ID NOs:351, 352, and 353 and CDR
sequences of SEQ ID NOs:358,359, and 360.
21. The sclerostin binding agent of any one of claims 14-20 wherein said
sclerostin binding agent is an antibody.
22. A pharmaceutical composition comprising a sclerostin binding agent
according to any one of claims 1-12 and 14-20.
23. The composition of claim 22 wherein said sclerostin binding agent is an
antibody.
24. A sclerostin binding agent comprising at least one CDR sequence having
at least 75% identity to a CDR selected from SEQ ID NQs:245,246,247,78,79, 80,269,270,
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271, 239,240 and 241.
25. A sclerostin binding agent comprising at least one CDR sequence haying
at least 75% identity to a CDR selected from CDR-H1, CDR-H2, CDR-H3, CDR-Ll, CDR-L2
and CDR-L3 wherein CDR-H1 has the sequence given in SEQ ED NO:245 or SEQ ID NQ:269,
CDR-H2 has the sequence given in SEQ ID NQ:246 or SEQ ID NO:270, CDR-H3 has the
sequence given in SEQ ID NO:247 or SEQ ID NQ:271, CDR-Ll has the sequence given in SEQ
ID NO:78 or SEQ ID NO:239, CDR-L2 has the sequence given in SEQ ID NO:79 or SEQ ID
NO-.240 and CDR-L3 has the sequence given ia SEQ ID NO.80 or SEQ ID NO 241.
26. A sclerostin binding agent according to claim 25 comprising three CDRs,
CDR-H1, CDR-H2 and CDR-H3 wherein

(a) CDR-H1 is SEQ ID NO:245, CDR-H2 is SEQ ID NO.246 and
CDR-H3 is SEQ ID NO:247 or
(b) CDR-H1 is SEQ ID NO:269, CDR-H2 is SEQ ID NQ-.270 and
CDR-H3 is SEQ ID NO:271.
27. A sclerostin binding agent according to claim 25 comprising three CDRs,
CDR-Ll, CDR-L2 and CDR-L3 wherein
(a) CDRL1 is SEQ ID NO:78, CDR-L2 is SEQ ID NQ:79 and CDR-L3 is
SEQ ID NO: 80; or
(b)CDRL1 is SEQ ID NO:239,CDR-L2 is SEQ ID NO:240 and CDR-L3
is SEQ ID NO:24l.
28. A sclerostin binding agent according to claim 25 comprising six CDRs,
CDRH-1, CDR-H2, CDR-H3, CDR-Ll CDRTL2 and CDR-L3 wherein
(a) CDR-H1 is SEQ ID NO:245, CDR-H2 is SEQ ID NO-.246, CDR-H3 is
SEQ ID NO:247, CDR-Ll is SEQ ED NQ:78, CDR-L2 is SEQ ID NO:79 and CDR-L3 is SEQ
ID NO:80;or
(b) CDR-H1 is SEQ ID NO-.269, CDR-H2 is SEQ ID NO:270, CDR-H3
is SEQ ID NO:271, CDR-L1 is SEQ ID NO:239, CDR-L2 is SEQ ID NO:240 and CDR-L3 is
SEQIDNO:241.
29. The sclerostin binding agent of any one of claims 24-28 which is an
antibody.
30. The sclerostin binding agent of claim 29 comprising a heavy chain
wherein said heavy chain comprises a polypeptide having at least 85% identity to the sequence
given in SEQ ID NO:333; SEQ ID NO:378; SEQ ID NO:327; SEQ ID NO:329; or SEQ ID
NO:366.
159.

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31. The sclerostin binding agent of claim 29 comprising a light chain wherein
said light chain comprises a polypeptide having at least 85% identity to the sequence given in
SEQ ID NO-.332; SEQ ID NO:376; SEQ ID NO:314; SEQ ID NO:328; or SEQ ID NO:364.
32. The sclerostin binding agent of claim 29 comprising both a heavy chain
and a light chain wherein
(a) the heavy chain comprises a polypeptide having at least 85% identity
to the sequence given in SEQ ID NO:333 and the light chain comprises a polypeptide having at
least 85% identity to the sequence given in SEQ ID NO.332; or
(b) the heavy chain comprises a polypeptide having at least 85% identity
to the sequence given in SEQ ID NO:378 and the light chain comprises a polypeptide having at
least 85% identity to the sequence given in SEQ ID NO.376; or
(c) the heavy chain comprises a polypeptide having at least 85% identity
to the sequence given in SEQ ID NO:327 and the light chain comprises a polypeptide having at
least 85% identity to the sequence given in SEQ ID NO:314; or
(d) the heavy chain comprises a polypeptide having at least 85% identity
to the sequence given in SEQ ID NO.329 and the light chain comprises a polypeptide having at
least 85% identity to the sequence given in SEQ ID NO:328; or
(c) the heavy chain comprises a polypeptide having at least 85% identity
to the sequence given in SEQ ID NO:366 and the light chain comprises a polypeptide having at
least 85% identity to the sequence given in SEQ ID NO:364.
33. The sclerostin binding agent of any one of claims 24-32 which comprises
a light chain and/or heavy chain constant region.
34. The sclerostin binding agent of claim 33 which comprises the IgG4 or the
IgG2 constant region.
35. A sclerostin binding agent having a heavy chain comprising CDR's HI,
H2 and H3 and comprising a polypeptide having the sequence provided in SEQ CD NQ:137 or a
variant thereof in which said CDR's are at least 75% identical to SEQ ID NO.245,246 and 247,
respectively, and a light chain comprising CDR's LI, L2 and L3 and comprising a polypeptide
having the sequence provided in SEQ ID NO:133 or a variant thereof in which said CDR's are at
least 75% identical to SEQ ID NO:78, 79 and 80, respectively.
36. A sclerostin binding agent having a heavy chain comprising CDR's HI,
H2 and H3 and comprising a polypeptide having the sequence provided in SEQ ID NO:145 or
392 or a variant thereof in which said CDR's are at least 75% identical to SEQ ID NO:245,246
and 247, respectively, and a light chain comprising CDR's LI, L2 and L3 and comprising a
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polypeptide having the sequence provided in SEQ ID NO: 141 or a variant thereof in which said
CDR's are at least 75% identical to SEQ ID NO:78, 79 and 80, respectively.
37. A sclerostin binding agent having a heavy chain comprising CDR's HI,
H2 and H3 and comprising a polypeptide having the sequence provided in SEQ ID NO:335 or a
variant thereof in which said CDR's are at least 75% identical to SEQ ID NQ:269,270 and 271,
respectively, and a light chain comprising CDR's LI, L2 and L3 and comprising a polypeptide
having the sequence provided in SEQ ID NO:334 or a variant thereof in which said CDR's are at
least 75% identical to SEQ ID NO:239, 240 and 241, respectively.
38. A sclerostin binding agent having a heavy chain comprising CDR's H1,
H2 and H3 and comprising a polypeptide having the sequence provided in SEQ ED NO:331 or a
variant thereof in which said CDR's are at least 75% identical to SEQ ID NO:269,270 and 271,
respectively, and a light chain comprising CDR's LI, L2 and L3 and comprising a polypeptide
having the sequence provided in SEQ ED NO:330 or a variant thereof in which said CDR's are at
least 75% identical to SEQ ID NO:239, 240 and 241, respectively.
39. A sclerostin binding agent having a heavy chain comprising CDR's H1,
H2 and H3 and comprising a polypeptide having the sequence provided in SEQ ID NO:345 or
396 or a variant thereof in which said CDR's are at least 75% identical to SEQ ID NO:269, 270
and 271, respectively, and a light chain comprising CDR's L1, L2 and L3 and comprising a
polypeptide having the sequence provided in SEQ ID NO:341 or a variant thereof in which said
CDR's are at least 75% identical to SEQ ID NO:239, 240 and 241, respectively.
40. A sclerostin binding agent having a heavy chain comprising a polypeptide
having the sequence provided in SEQ ID NO: 137, and a light chain comprising a polypeptide
having the sequence provided in SEQ ID NO: 133.
41. A sclerostin binding agent having a heavy chain comprising a polypeptide
having the sequence provided in SEQ ID NO: 145 or 392, and a light chain comprising a
polypeptide having the sequence provided in SEQ ID NO: 141.
42. A sclerostin binding agent having a heavy chain comprising a polypeptide
having the sequence provided in SEQ ID NO:335, and a light chain comprising a polypeptide
having the sequence provided in SEQ ID NO:334.
43. A sclerostin binding agent having a heavy chain comprising a
polypeptide having the sequence provided in SEQ ID NO:331, and a light chain comprising a
polypeptide having the sequence provided in SEQ ID NO:330.
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44. A sclerostin binding agent having a heavy chain comprising a polypeptide
having the sequence provided in SEQ ID NO:345 or 396, and a light chain comprising a
polypeptide having the sequence provided in SEQ ID NQ:341.
45. A sclerostin binding agent according to any one of claims 24-44 to which
one or more effector or reporter molecule(s) is attached.
46. An isolated polynucleotide sequence encoding the sclerostin binding
agent according to any one of claims 24-44.
47. A cloning or expression vector comprising one or more polynucleotide
sequences according to claim 46.
48. A vector according to claim 47, wherein the vector comprises at least one
sequence given in SEQ ID NO:134,136,138,140,142, 144, 146, 148, 308, 310, 312, 342, 344,
346,348, 349, 365, 367, 373,375, and 379.
49. A host cell comprising one or more cloning or expression vectors
according to claim 47 or claim 48.
50. A process for the production of the sclerostin binding agent of any one of
claims 24-44, comprising culturing the host cell of claim 49 and isolating the sclerostin binding
agent.
51. A pharmaceutical composition comprising a sclerostin binding agent
according to any one of claims 24-45 in combination with one or more of a pharmaceutically
acceptable excipient, diluent or carrier.
52. A pharmaceutical composition according to claim 51, additionally
comprising other active ingredients.
53. A sclerostin binding agent according to any one of claims 24-45 or a
pharmaceutical composition according to claim 51 or claim 52, for use in the treatment or
prophylaxis of a pathological disorder that is mediated by sclerostin or that is associated with an
increased level of sclerostin.
54. A method for treating a bone-related disorder in a mammalian subject
which comprises providing to a subject in need of such treatment a pharmaceutical composition
of claim 22.
55. A method for treating a bone-related disorder in a mammalian subject
which comprises providing to a subject in need of such treatment a pharmaceutical composition
of claim 23.
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56. A method for treating a bone-related disorder in a mammalian subject
which comprises providing to a subject in need of such treatment a pharmaceutical composition
of claim 51.
57. The method according to claim 54, wherein the bone-related disorder is
at least one of achondroplasia, cleidocranial dysostosis, enchondromatosis, fibrous dysplasia,
Gaucher's Disease, hypophosphatemic rickets, Marfan's syndrome, multiple hereditary
exotoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteopoikilosis, sclerotic
lesions, pseudoarthrosis, pyogenic osteomyelitis, periodontal disease, anti-epileptic drug induced
bone loss, primary and secondary hyperparathyroidism, familial hyperparathyroidism
syndromes, weightlessness induced bone loss, osteoporosis in men, postmenopausal bone loss,
osteoarthritis, renal osteodystrophy, infiltrative disorders of bone, oral bone loss, osteonecrosis
of the jaw, juvenile Paget's disease, melorheostosis, metabolic bone diseases, mastocytosis,
sickle cell anemia/disease, organ transplant related bone loss, kidney transplant related bone
loss, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritides,
thalassemia, mucopolysaccharidoses, Fabry Disease, Turner Syndrome, Down Syndrome,
KHnefelter Syndrome, leprosy, Perthes' Disease, adolescent idiopathic scoliosis, infantile onset
multi-system inflammatory disease, Winchester Syndrome, Menkes Disease, Wilson's Disease,
ischemic bone disease (such as Legg-Calve-Perthes disease, regional migratory osteoporosis),
anemic states, conditions caused by steroids,- glucocorticoid-induced bone loss, heparin-induced
bone loss, bone marrow disorders, scurvy, malnutrition, calcium deficiency, osteoporosis,
osteopenia, alcoholism, chronic liver disease, postmenopausal state, chronic inflammatory
conditions, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory
colitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus,
hyperthyroidisra, thyroid disorders, parathyroid disorders, Cushing's disease, acromegaly,
hypogonadism, immobilization or disuse, reflex sympathetic dystrophy syndrome, regional
osteoporosis, osteomalacia, bone loss associated with joint replacement, HTV associated bone
loss, bone loss associated with loss of growth hormone, bone loss associated with cystic
fibrosis, chemotherapy associated bone loss, tumor induced bone loss, cancer-related bone loss,
hormone ablative bone loss, multiple myeloma, drug-induced bone loss, ariorexia nervosa,
disease associated facial bone loss, disease associated cranial bone loss, disease associated bone
loss of the jaw, disease associated bone loss of the skull, bone loss associated with aging, facial
bone loss associated with aging, cranial bone loss associated with aging, jaw bone loss
associated with aging, and skull bone loss associated with aging and bone, loss associated with
space travel.
163

WO 2006/119107 PCT/US2006/016441
58. The method according to claim 55, wherein the bone-related disorder is
at least one of achondroplasia, cleidocranial dysostosis, enchondromatosis, fibrous dysplasia,
Gaucher's Disease, hypophosphatemic rickets, Marfan's syndrome, multiple hereditary
exotoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteopoikilosis, sclerotic
lesions, pseudoarthrosis, pyogenic osteomyelitis, periodontal disease, anti-epileptic drug induced
bone loss, primary and secondary hyperparathyroidism, familial hyperparathyroidism
syndromes, weightlessness induced bone loss, osteoporosis in men, postmenopausal bone loss,
osteoarthritis, renal osteodystrophy, infiltrative disorders of bone, oral bone loss, osteonecrosis
of the jaw, juvenile Paget's disease, melorheostosis, metabolic bone diseases, mastocytosis,
sickle cell anemia/disease, organ transplant related bone loss, kidney transplant related bone
loss, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritides,
thalassemia, mucopolysaccharidoses, Fabry Disease, Turner Syndrome, Down Syndrome,
Klinefelter Syndrome, leprosy, Perthes' Disease, adolescent idiopathic scoliosis, infantile onset
multi-system inflammatory disease, Winchester Syndrome, Menkes Disease, Wilson's Disease,
ischemic bone disease (such as Legg-Calve-Perthes disease, regional migratory osteoporosis),
anemic states, conditions caused by steroids, glucocorticoid-induced bone loss, heparin-induced
bone loss, bone marrow disorders, scurvy, malnutrition, calcium deficiency, osteoporosis,
osteopenia, alcoholism, chronic liver disease, postmenopausal state; chronic inflammatory
conditions, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory
colitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus,
hyperthyroidism, thyroid disorders, parathyroid disorders, Cushing's disease, acromegaly,
hypogonadism, immobilization or disuse, reflex sympathetic dystrophy syndrome, regional
osteoporosis, osteomalacia, bone loss associated with joint replacement, HTV associated bone
loss, bone loss associated with loss of growth hormone, bone loss associated with cystic
fibrosis, chemotherapy associated bone loss, tumor induced bone loss, cancer-related bone loss,
hormone ablative bone loss, multiple myeloma, drug-induced bone loss, anorexia nervosa,
disease associated facial bone loss, disease associated cranial bone loss, disease associated bone
loss of the jaw, disease associated bone loss of the skull, bone loss associated with aging, facial
bone loss associated with aging, cranial bone loss associated with aging, jaw bone loss
associated with aging, skull bone loss associated with aging, and bone loss associated with space
travel.
59. The method according to claim 56, wherein the bone-related disorder is at
least one of achondroplasia, cleidocranial dysostosis, enchondromatosis, fibrous dysplasia,
Gaucher's Disease, hypophosphatemic rickets, Marfan's syndrome, multiple hereditary
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WO 2006/119107 PCT/US2006/016441
exotoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteopoikilosis, sclerotic
lesions, pseudoarthrosis, pyogenic osteomyelitis, periodontal disease, anti-epileptic drug induced
bone loss, primary and secondary hyperparathyroidism, familial hyperparathyroidism
syndromes, weightlessness induced bone loss, osteoporosis in men, postmenopausal bone loss,
osteoarthritis, renal osteodystrophy, infiltrative disorders of bone, oral bone loss, osteonecrosis
of the jaw, juvenile Paget's disease, melorheostosis, metabolic bone diseases, mastocytosis,
sickle cell anemia/disease, organ transplant related bone loss, kidney transplant related bone
loss, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritides,
thalassemia, mucopolysaccharidoses, Fabry Disease, Turner Syndrome, Down Syndrome,
Klinefelter Syndrome, leprosy, Perthes' Disease, adolescent idiopathic scoliosis, infantile onset
multi-system inflammatory disease, Winchester Syndrome, Menlces Disease, Wilson's Disease,
ischemic bone disease (such as Legg-Calve-Perthes disease, regional migratory osteoporosis),
anemic states, conditions caused by steroids, glucocorticoid-induced bone loss, heparin-induced
bone loss, bone marrow disorders, scurvy, malnutrition, calcium deficiency, osteoporosis,
osteopenia, alcoholism, chronic liver disease, postmenopausal state, chronic inflammatory
conditions, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory
colitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus,
hyperthyroidism, thyroid disorders, parathyroid disorders, Cushing's disease, acrornegaly,
hypogonadism, immobilization or disuse, reflex sympathetic dystrophy syndrome, regional
osteoporosis, osteomalacia, bone loss associated with joint replacement, HIV associated bone
loss, bone loss associated with loss of growth hormone, bone loss associated with cystic
fibrosis, chemotherapy associated bone loss, tumor induced bone loss, cancer-related bone loss,
hormone ablative bone loss, multiple myeloma, drug-induced bone loss, anorexia nervosa,
disease associated facial bone loss, disease associated cranial bone loss, disease associated bone
loss of the jaw, disease associated bone loss of the skull, bone loss associated with aging, facial
bone loss associated with aging, cranial bone loss associated with aging, jaw bone loss
associated with aging, skull bone loss associated with aging, and bone loss associated with space
travel.
60. A method of increasing at least one of bone formation, bone mineral
content, bone mass, bone mineral density, bone quality, and bone strength in a mammal
comprising administering to the mammal a pharmaceutical composition of claim 22.
61. A method of increasing at least one of bone formation, bone mineral
content, bone mass, bone mineral density, bone quality, and bone strength in a mammal
comprising administering to the mammal a pharmaceutical composition of claim 23.
165,

WO 2006/119107 PCT/US2006/016441
62. A method of increasing at least one of bone formation, bone mineral
content, bone mass, bone mineral density, bone quality, and bone strength in a mammal
comprising administering to the mammal a pharmaceutical composition of claim 51.
63. A method of improving the outcome in a mammal undergoing one or
more of an orthopedic procedure, dental procedure, implant surgery, joint replacement, bone
grafting, bone cosmetic surgery and bone repair such as fracture healing, nonunion healing,
delayed union healing and facial reconstruction, comprising administering to said mammal a
pharmaceutical composition of claim 22 before, during and/or after said procedure, replacement,
graft, surgery or repair.
64. A method of improving the outcome in a mammal undergoing one or
more of an orthopedic procedure, dental procedure, implant surgery, joint replacement, bone
grafting, bone cosmetic surgery and bone repair such as fracture healing, nonunion healing,
delayed union healing and facial reconstruction, comprising administering to said mammal a
pharmaceutical composition of claim 23 before, during and/or after said procedure, replacement,
graft, surgery or repair.
65. A method of improving the outcome in a mammal undergoing one or
. more of an orthopedic procedure, dental procedure, implant surgery, joint replacement, bone
grafting, bone cosmetic surgery and bone repair such as fracture healing, nonunion healing,
delayed union healing and facial reconstruction, comprising administering to said mammal a
pharmaceutical composition of claim 51 before, during and/or after said procedure, replacement,
graft, surgery or repair.
66. An antibody, wherein said antibody is Ab-A, Ab-B, Ab-C, Ab-D, Ab-1,
Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15,
Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, or Ab-24.
67. A diagnostic kit comprising a sclerostin binding agent according to any
one of claims 1,3, 9-12 and 14-20.
68. A diagnostic kit comprising an antibody according to claim 6.
69. A diagnostic kit comprising an antibody according to claim 21.
70. A diagnostic kit comprising an antibody according to claim 29.
71. A polypeptide comprising at least one of SEQ ID NOs:39,40,41,42, 43,
44,45, 46, 47,48,49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 78, 79, 80, 81,99,100,
101,102,103,104, 105,106,107,108, 109,110, 111, 112, 113,114, 115, 116,237,238,239,
240,241,242,243,244,245,246, 247,248,249, 250,251,252,253, 254,255,256,257,258,
259,260,261,262,263,264,265, 266,267,268, 269,270,271,272,273,274,275,276,277,
166

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278,279,280,281,282, 283,284,285,286,287,288,289,290,291,292,293,294,295,296,
297,298, 351, 352,353, 358,359, and 360.
72. A polypeptide according to claim 71 conjugated to at least one of
Fc, PEG, albumin, and transferrin.
167

Compositions and methods relating to epitopes of selerostin protein, and selerostin binding agents, such as antibodies
capable of binding to selerostin, are provided.

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Patent Number 268876
Indian Patent Application Number 4223/KOLNP/2007
PG Journal Number 39/2015
Publication Date 25-Sep-2015
Grant Date 21-Sep-2015
Date of Filing 02-Nov-2007
Name of Patentee UCB PHARMA S.A.
Applicant Address ALLEE DE LA RECHERCHE 60 B-1070 BRUSSELS
Inventors:
# Inventor's Name Inventor's Address
1 ROBINSON MARTYN KIM NURSERY COTTAGE, HARVEY HILL, WOOBURN GREEN BUCKINGHAMSHIRE HP10 0JH
2 PASZTY CHRISTOPHER 1027 WESTRIDGE DRIVE, VENTURA, CALIFORNIA 93003
3 GRAHAM KEVIN 1627 CHARTERWOOD COURT, THOUSAND OAKS, CALIFORNIA 91362
4 HENRY ALISTAIR JAMES 92 COWLEY ROAD, UXBRIDGE, MIDDLESEX UB8 2LX
5 HOFFMANN KELLY SUE 795 GREEN VALLEY DRIVE, NEWBURY PARK, CALIFORNIA 91320
6 LATHAM JOHN 2409 10TH AVENUE WEST, SEATTLE, WASHINGTON 98119
7 LAWSON ALASTAIR 208 BATH ROAD, SLOUGH, BERKSHIRE SL1 3WE
8 LU HSIENG SEN 2758 RAINFIELD AVENUE, WESTLAKE VILLAGE, CALIFORNIA 91362
9 POPPLEWELL ANDY 208 BATH ROAD, SLOUGH, BERKSHIRE SL1 3WE
10 SHEN WENYAN 2757 AUTUMN RIDGE DRIVE, THOUSAND OAKS, CALIFORNIA 91362
11 WINKLER DAVID 127 GEORGE STREET, ARLINGTON, MASSACHUSETTS 02476-7326
12 WINTERS AARON GEORGE 2501 PIERPONT BOULEVARD, VENTURA, CALIFORNIA 93001
PCT International Classification Number C07K 14/51
PCT International Application Number PCT/US2006/016441
PCT International Filing date 2006-04-28
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/782244 2006-03-13 U.S.A.
2 60/677583 2005-05-03 U.S.A.
3 60/776847 2006-02-24 U.S.A.
4 60/792645 2006-04-17 U.S.A.
5 11/411003 2006-04-25 U.S.A.