U.S. patent application number 13/367760 was filed with the patent office on 2012-06-07 for wnt antagonists and their use in the diagnosis and treatment of wnt-mediated disorders.
Invention is credited to Venita I. DeAlmeida, James A. Ernst, Paul Polakis, Bonnee Rubinfeld.
Application Number | 20120141481 13/367760 |
Document ID | / |
Family ID | 39092004 |
Filed Date | 2012-06-07 |
United States Patent
Application |
20120141481 |
Kind Code |
A1 |
Ernst; James A. ; et
al. |
June 7, 2012 |
WNT ANTAGONISTS AND THEIR USE IN THE DIAGNOSIS AND TREATMENT OF
WNT-MEDIATED DISORDERS
Abstract
The present invention provides for chimeric Wnt antagonists
comprising a Frz domain component derived from a Frizzled protein,
a secreted Frizzled related protein or Ror protein and an Fc
immunoglobulin component, and their use in the treatment and
diagnostic detection of cellular Wnt signaling and Wnt-mediated
disorders, including cancer.
Inventors: |
Ernst; James A.; (San
Francisco, CA) ; Polakis; Paul; (Mill Valley, CA)
; Rubinfeld; Bonnee; (Danville, CA) ; DeAlmeida;
Venita I.; (San Carlos, CA) |
Family ID: |
39092004 |
Appl. No.: |
13/367760 |
Filed: |
February 7, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12826194 |
Jun 29, 2010 |
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13367760 |
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11851596 |
Sep 7, 2007 |
7947277 |
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12826194 |
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60951175 |
Jul 20, 2007 |
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60825063 |
Sep 8, 2006 |
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Current U.S.
Class: |
424/134.1 ;
530/350; 530/387.3; 604/20 |
Current CPC
Class: |
A61P 35/00 20180101;
Y02A 50/30 20180101; A61P 35/04 20180101; C07K 14/71 20130101; C07K
14/4702 20130101; A61K 38/00 20130101; G01N 2333/71 20130101; A61P
43/00 20180101; Y02A 50/473 20180101 |
Class at
Publication: |
424/134.1 ;
530/387.3; 530/350; 604/20 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61M 37/00 20060101 A61M037/00; A61P 35/00 20060101
A61P035/00; C07K 16/18 20060101 C07K016/18; C07K 14/705 20060101
C07K014/705 |
Claims
1.-32. (canceled)
33. A soluble receptor comprising (a) a fragment of an
extracellular domain of a human Frizzled (Frz) receptor and (b) a
human Fc domain, wherein the fragment of the extracellular domain
of the human Frz receptor consists essentially of an amino acid
sequence selected from the group consisting of amino acid residues
1 to 156 of SEQ ID NO:42, amino acid residues 1 to 129 of SEQ ID
NO:25, amino acid residues 1 to 155 of SEQ ID NO:39; and amino acid
residues 1 to 129 of SEQ ID NO:22, wherein the soluble receptor has
a longer half-life in vivo than a soluble receptor comprising the
extracellular domain of the Frz receptor and the human Fc
domain.
34. The soluble receptor of claim 33 wherein the human Fc is human
IgG1 Fc comprising the amino acid sequence of SEQ ID NO:67.
35. A pharmaceutical composition comprising the soluble receptor of
claim 33.
36. A kit comprising the soluble receptor of claim 33.
37. The soluble receptor of claim 33, wherein the soluble receptor
inhibits the Wnt-dependant growth of solid tumor cells.
38. The soluble receptor of claim 33, wherein the soluble receptor
inhibits the Wnt-dependant growth of breast tumor cells.
39. An isolated polypeptide comprising an amino acid sequence
having at least 95% sequence identity to an amino acid sequence
selected from the group consisting of amino acid residues 1 to 129
of SEQ ID NO:25 and amino acid residues 1 to 129 of SEQ ID NO:22,
wherein said polypeptide is a soluble receptor that inhibits the
Wnt-dependent growth of solid tumor cells.
40. A soluble receptor comprising (a) a fragment of an
extracellular domain of a human Frizzled (Frz) receptor and (b) a
human Fc domain, wherein the fragment of the extracellular domain
of the human Frz receptor consists essentially of an amino acid
sequence selected from the group consisting of amino acid residues
1 to 156 of SEQ ID NO:42 and amino acid residues 1 to 155 of SEQ ID
NO:39, and wherein the soluble receptor has a half-life in vivo of
at least 24 hours in mice following i.p. injection.
41. A soluble receptor comprising (a) a fragment of an
extracellular domain of a human Frizzled (Frz) receptor and (b) a
human Fc domain, wherein the fragment of the extracellular domain
of the human Frz receptor consists essentially of an amino acid
sequence selected from the group consisting of amino acid residues
1 to 156 of SEQ ID NO:42 and amino acid residues 1 to 155 of SEQ ID
NO:39, and wherein the soluble receptor is detectable in serum at
least 24 hours following i.p. injection in mice.
42. A method of inhibiting the growth of solid tumor cells in a
subject in need thereof, the method comprising administering to the
subject the soluble receptor of claim 33 in an amount effective to
inhibit the Wnt-dependent growth of solid tumor cells.
43. A method of inhibiting the growth of solid tumor cells in a
subject in need thereof, the method comprising administering to the
subject the soluble receptor of claim 40 in an effective amount to
inhibit the Wnt-dependent growth of solid tumor cells.
44. A method of inhibiting the growth of solid tumor cells in a
subject in need thereof, the method comprising administering to the
subject the soluble receptor of claim 41 in an effective amount to
inhibit the Wnt-dependent growth of solid tumor cells.
45. The method of claim 42, wherein the soluble receptor is
administered with radiation therapy.
46. The method of claim 42, wherein the soluble receptor is
administered with chemotherapy.
47. The method of claim 42, wherein the solid tumor cells are from
a breast tumor, colorectal tumor, lung tumor, pancreatic tumor,
prostate tumor, or a head and neck tumor.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/851,596 filed Sep. 7, 2007 which in turn claims priority to
U.S. Provisional Application Ser. No. 60/825,063, filed Sep. 8,
2006, and U.S. Provisional Application Ser. No. 60/951,175, filed
Jul. 20, 2007, both of which are incorporated by reference in their
entirety herein.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the regulation of
cell growth. More specifically, the present invention relates to
inhibitors of the Wnt pathway as well as to their use in the
diagnosis and treatment of disorders characterized by the
activation of Wnt pathway signaling, as well as to the modulation
of cellular events mediated by Wnt pathway signaling.
BACKGROUND OF THE INVENTION
[0003] The Wnt signaling pathway's association with carcinogenesis
began as a result of early observations and experiments in certain
murine mammary tumors. Wnt-1 proto-oncogene (Int-1) was originally
identified from mammary tumors induced by mouse mammary tumor virus
(MMTV) due to an insertion of a viral DNA sequence. Nusse et al.,
Cell 1982; 31: 99-109. The result of such viral integration was
unregulated expression of Int-1 resulting in the formation of
tumors. Vanooyen, A. et al., Cell 1984; 39: 233-240; Nusse, R. et
al., Nature 1984; 307: 131-136; Tsukamoto et al., Cell 1988; 55:
619-625. Subsequent sequence analysis demonstrated that the Int-1
was a mammalian homolog of the Drosophila gene Wingless (Wg), which
was implicated in development, and the terms were then combined to
create "Wnt" to identify this family of proteins.
[0004] The human Wnt gene family of secreted ligands has now grown
to at least 19 members (e.g., Wnt-1 (RefSeq.: NM.sub.--005430),
Wnt-2 (RefSeq.: NM.sub.--003391), Wnt-2B (Wnt-13) (RefSeq.:
NM.sub.--004185), Wnt-3 (RefSeq.: NM.sub.--030753), Wnt3a (RefSeq.:
NM.sub.--033131), Wnt-4 (RefSeq.: NM.sub.--030761), Wnt-5A
(RefSeq.: NM.sub.--003392), Wnt-5B (RefSeq.: NM.sub.--032642),
Wnt-6 (RefSeq.: NM.sub.--006522), Wnt-7A (RefSeq.:
NM.sub.--004625), Wnt-7B (RefSeq.: NM.sub.--058238), Wnt-8A
(RefSeq.: NM.sub.--058244), Wnt-8B (RefSeq.: NM.sub.--003393),
Wnt-9A (Wnt-14) (RefSeq.: NM.sub.--003395), Wnt-9B (Wnt-15)
(RefSeq.: NM.sub.--003396), Wnt-10A (RefSeq.: NM.sub.--025216),
Wnt-10B (RefSeq.: NM.sub.--003394), Wnt-11 (RefSeq.:
NM.sub.--004626), Wnt-16 (RefSeq.: NM.sub.--016087)). Each member
has varying degrees of sequence identity but all contain 23-24
conserved cysteine residues which show highly conserved spacing.
McMahon, A P et al., Trends Genet. 1992; 8: 236-242; Miller, J R.
Genome Biol. 2002; 3(1): 3001.1-3001.15. The Wnt proteins are small
(i.e., 39-46 kD) acylated, secreted glycoproteins which play key
roles in both embryogenesis and mature tissues. During
embryological development, the expression of Wnt proteins is
important in patterning through control of cell proliferation and
determination of stem cell fate. The Wnt molecules are also
palmitoylated, and thus are more hydrophobic than would be
otherwise predicted by analysis of the amino acid sequence alone.
Willert, K. et al., Nature 2003; 423: 448-52. The site or sites of
palmitoylation are also believed to be essential for function.
[0005] The Wnt proteins act as ligands to activate the Frizzled
(Frz) family of seven-pass transmembrane receptors. Ingham, P. W.
Trends Genet. 1996; 12: 382-384; YangSnyder, J. et al., Curr. Biol.
1996; 6: 1302-1306; Bhanot, P. et al., Nature 1996; 382: 225-230.
There are ten known members of the Frz family (e.g., Frz1, Frz2,
Frz3 . . . Frz10), each characterized by the presence of a cysteine
rich domain (CRD). Huang et al., Genome Biol. 2004; 5: 234.1-234.8.
There is a great degree of promiscuity between the various
Wnt-Frizzled interactions, but Wnt-Frz binding must also
incorporate the LDL receptor related proteins (LRP5 or LRP6) and
the membrane and the cytoplasmic protein Dishevelled (Dsh) to form
an active signaling complex.
[0006] The binding of Wnt to Frizzled can activate signaling via
either the canonical Wnt signaling pathway, thereby resulting in
stabilization and increased transcriptional activity of
f.beta.-catenin [Peifer, M. et al., Development 1994; 120: 369-380;
Papkoff, J. et al., Mol. Cell. Biol. 1996; 16: 2128-2134] or
non-canonical signaling, such as through the Wnt/planar cell
polarity (Wnt/PCP) or Wnt-calcium (Wnt/Ca.sup.2+) pathway. Veeman,
M. T. et al., Dev. Cell 2003; 5: 367-377.
[0007] The canonical Wnt signaling pathway is the most relevant of
the Wnt signaling pathways to the development of cancer. Ilyas, M.
J. Pathol. 2005; 205: 130-144. Normal activation of this pathway
begins a series of downstream events culminating in the
stabilization and increased levels of the protein .beta.-catenin.
This protein is normally an inactive cytoplasmic protein, and is
found at the cell membrane bound to proteins including e-cadherin.
In the absence of Wnt ligand, phosphorylated cytoplasmic
.beta.-catenin is normally rapidly degraded. Upon activation of the
canonical pathway, unphosphorylated .beta.-catenin is transported
to the nucleus where it further results in transcriptional
activation of various target genes. The subsequent upregulation in
transcription of these target genes leads to changes in the cell,
and continuous, unregulated expression of such target genes results
in tumor development. Since aberrant Wnt signaling appears to be a
necessary precursor in carcinogenesis, effective inhibitors of Wnt
signaling are of great interest as cancer therapeutics.
[0008] The use of soluble receptors as antagonists to
ligand-receptor interactions is known in the art. Such molecules
can be effective therapeutic antagonists if they bind the free
ligand in a manner so as to prevent the initial receptor activation
step of the signaling pathway. Soluble minimal extracellular domain
(ECD) fragments of the cysteine-rich domain (CRD) of a Frizzled
receptor which exhibit binding to Wnt have been identified, based
on crystallography data. Dann et al., Nature 412: 86-90 (2001).
However, while such Frizzled fragments did exhibit binding to Wnt
ligand, such fragments are unsuitable for therapeutics because of
their rapid degradation in vivo.
[0009] The use of a soluble Frizzled domain coupled to an
immunoglobulin Fc as a potential Wnt antagonist has been proposed.
Therapeutic Opportunities of the Wnt Signaling Pathway in Cancer,
New York Academy of Sciences, Oct. 25, 2005; Hsieh, J-C. et al.,
PNAS, 96: 3546-3551 (1999). However, prior to the present
invention, attempts at creating a soluble Frizzled receptor-Fc
fusion therapeutic were not successful. For example, one such
chimera based on residues 1-173 of the Frz8 CRD (Frz (173)-Fc, SEQ
ID NO: 113) had suboptimal efficacy (FIG. 12), and was unstable in
vivo (FIG. 11). Moreover, the Frz (173)-Fc chimera only reduced the
rate of increase in tumor volume (as opposed to shrinking starting
tumor volume). Additionally, while the creation of Fc fusions is
generally known as one technique to improve the in vivo stability
of the resulting construct, the creation of effective therapeutic
Fc constructs can be difficult owing to a number of problems,
including improper protein folding of the new protein construct and
steric hindrance of the fusion construct to the target.
[0010] Thus, a need exits for a Wnt antagonist therapeutic with
enhanced in vivo stability that acts to inhibit Wnt ligand induced
cellular signaling.
SUMMARY OF THE INVENTION
[0011] The invention provides for compositions and their use in
methods of diagnosing and treating Wnt-mediated disorder, such as
cancer, and in inhibiting cellular Wnt signaling. Specifically, the
invention provides for Wnt antagonists that are chimeric molecules
comprising a Frizzled domain component, such as a polypeptide
derived from a Frizzled (Frz) protein, a Frizzled related protein
(sFRP) or another protein (e.g., Ror-1, -2, etc.), and an
immunoglobulin Fc domain, and their use in methods of diagnosing
and treating Wnt-mediated disorders and in inhibiting cellular Wnt
signaling.
[0012] One aspect of the invention provides for a Wnt antagonist
comprising a Frizzled domain component and a Fc domain. The
Frizzled domain component of the Wnt antagonist comprises a
polypeptide derived from a Frz protein, a FRP protein, or a Ror
protein. In one embodiment, the Wnt antagonist is active in vivo
for at least 1 hour. In another embodiment, the Wnt antagonist is
active in vivo for at least 5 hours. In another embodiment, the Wnt
antagonist has an in vivo half-life of at least 1 day. In yet
another embodiment, the Wnt antagonist has an in vivo half-life of
at least 2 days.
[0013] In a further embodiment, the Frizzled domain component
comprises a minimal CRD (ECD) domain from a Frz polypeptide
selected from the group consisting of hFrz1 (SEQ ID NO: 18), hFrz2
(SEQ ID NO: 19), hFrz3 (SEQ ID NO: 20), hFrz4 (SEQ ID NO: 21),
hFrz5 (SEQ ID NO: 22), hFrz6 (SEQ ID NO: 23), hFrz7 (SEQ ID NO:
24), hFrz8 (SEQ ID NO: 25), hFrz9 (SEQ ID NO: 26), and hFrz10 (SEQ
ID NO: 27), and active variants thereof. In yet a further
embodiment, the Frizzled domain component comprises a minimal CRD
(ECD) domain from a sFRP polypeptide selected from the group
consisting of sFRP1 (SEQ ID NO: 28), sFRP2 (SEQ ID NO: 29), sFRP3
(SEQ ID NO: 30), sFRP4 (SEQ ID NO: 31), and sFRP5 (SEQ ID NO: 32),
and active variants thereof. In yet a further embodiment, the
Frizzled domain component comprises a minimal CRD (ECD) domain from
a Ror polypeptide selected from the group consisting of hRorl (SEQ
ID NO: 33), and hRor2 (SEQ ID NO: 34), and active variants
thereof.
[0014] In yet a further embodiment, the Frizzled domain component
comprises a mature Frz polypeptide selected from the group
consisting of hFrz1 (SEQ ID NO: 50), hFrz2 (SEQ ID NO: 51), hFrz3
(SEQ ID NO: 52), hFrz4 (SEQ ID NO: 53), hFrz5 (SEQ ID NO: 54),
hFrz6 (SEQ ID NO: 55), hFrz7 (SEQ ID NO: 56), hFrz8 (SEQ ID NO:
57), hFrz9 (SEQ ID NO: 58), and hFrz10 (SEQ ID NO: 59), and active
variants thereof, or a mature sFrp polypeptide selected from the
group consisting of sFRP1 (SEQ ID NO: 60), sFRP2 (SEQ ID NO: 61),
sFRP3 (SEQ ID NO: 62), sFRP4 (SEQ ID NO: 63), and sFRP5 (SEQ ID NO:
64), and active variants thereof, or a mature Ror polypeptide
selected from the group consisting of hRorl (SEQ ID NO: 65), and
hRor2 (SEQ ID NO: 66), and active variants thereof.
[0015] In a still further embodiment, the Frizzled domain component
comprises a pro-Frz polypeptide selected from the group consisting
of hFrz1 (SEQ ID NO: 35), hFrz2 (SEQ ID NO: 36), hFrz3 (SEQ ID NO:
37), hFrz4 (SEQ ID NO: 38), hFrz5 (SEQ ID NO: 39), hFrz6 (SEQ ID
NO: 40), hFrz7 (SEQ ID NO: 41), hFrz8 (SEQ ID NO: 42), hFrz9 (SEQ
ID NO: 43), and hFrz10 (SEQ ID NO: 44), and active variants
thereof, or a pro-sFrp polypeptide selected from the group
consisting of sFRP1 (SEQ ID NO: 45), sFRP2 (SEQ ID NO: 46), sFRP3
(SEQ ID NO: 47), sFRP4 (SEQ ID NO: 48), and sFRP5 (SEQ ID NO: 49),
and active variants thereof.
[0016] In one embodiment, the Wnt antagonist comprises a Fc
component derived from an immunoglobulin selected from the group
consisting of IgGl, IgG2, IgG3 and IgG4. In another embodiment, the
Fc is derived from an IgGl immunoglobulin. In yet another
embodiment the Fc sequence comprises the Fc shown in SEQ ID NO: 67
or SEQ ID NO: 68.
[0017] In one embodiment, the Wnt antagonist further comprises a
linker connecting the Frizzled domain component to the Fc domain.
In one such embodiment, the linker is a peptide linker such as
ESGGGGVT (SEQ ID NO: 69), LESGGGGVT (SEQ ID NO: 70), GRAQVT (SEQ ID
NO: 71), WRAQVT (SEQ ID NO: 72), and ARGRAQVT (SEQ ID NO: 73).
[0018] In particular embodiments, the Wnt antagonist comprises a
polypeptide selected from the group consisting of in Frz8-Fc (SEQ
ID NO: 74), FrzS-Fc (SEQ ID NO: 75), Frz1-Fc (SEQ ID NO: 76),
Frz2-Fc (SEQ ID NO: 77), Frz3-Fc (SEQ ID NO: 78), Frz4-Fc (SEQ ID
NO: 79), Frz6-Fc (SEQ ID NO: 80), Frz7-Fc (SEQ ID NO: 81), Frz9-Fc
(SEQ ID NO: 82), Frz10-Fc (SEQ ID NO: 83), sFRP1-Fc (SEQ ID NO:
84), sFRP2 (SEQ ID NO: 85), sFRP3-Fc (SEQ ID NO: 86), sFRP4-Fc (SEQ
ID NO: 87), and sFRP5-Fc (SEQ ID NO: 88).
[0019] Another aspect of the invention provides for a composition
comprising at least one pharmaceutically acceptable carrier or
excipient and a Wnt antagonist as described above.
[0020] Yet another aspect of the invention provides for a nucleic
acid sequence encoding any of the Wnt antagonists described above.
In one embodiment, the nucleic acid encoding a Wnt antagonist
further comprises a vector containing control sequences to which
the nucleic acid is operably linked. In another embodiment, the
vector is contained in host cells, such as a mammalian, insect, E.
coli or yeast cell.
[0021] Another aspect of the invention provides for an article of
manufacture comprising a composition comprising at least one
pharmaceutically acceptable carrier or excipient and a Wnt
antagonist as described above and a container, wherein the Wnt
antagonist is contained within the container and the container
further comprises (a) a label affixed to the container, or (b) a
package insert inside the container referring to the use of the Wnt
antagonist indicating use of the composition for the therapeutic
treatment or diagnostic detection of a Wnt-mediated disorder.
[0022] Yet another aspect of the invention provides for a method of
inhibiting Wnt signaling in a cell comprising contacting the cell
with an effective amount of a Wnt antagonist comprising a Frizzled
domain component and a Fc domain. The Frizzled domain component of
the Wnt antagonist comprises a polypeptide derived from a Frz
protein, a FRP protein, or a Ror protein. In one embodiment, the
Wnt antagonist is active in vivo for at least 1 hour. In another
embodiment, the Wnt antagonist is active in vivo for at least 5
hours. In another embodiment, the Wnt antagonist has an in vivo
half-life of at least 1 day. In yet another embodiment, the Wnt
antagonist has an in vivo half-life of at least 2 days.
[0023] In a further embodiment of this aspect, the Frizzled domain
component comprises a minimal CRD (ECD) domain from a Frz
polypeptide selected from the group consisting of hFrz1 (SEQ ID NO:
18), hFrz2 (SEQ ID NO: 19), hFrz3 (SEQ ID NO: 20), hFrz4 (SEQ ID
NO: 21), hFrz5 (SEQ ID NO: 22), hFrz6 (SEQ ID NO: 23), hFrz7 (SEQ
ID NO: 24), hFrz8 (SEQ ID NO: 25), hFrz9 (SEQ ID NO: 26), and
hFrz10 (SEQ ID NO: 27), and active variants thereof. In yet a
further embodiment, the Frizzled domain component comprises a
minimal CRD (ECD) domain from a sFRP polypeptide selected from the
group consisting of sFRP1 (SEQ ID NO: 28), sFRP2 (SEQ ID NO: 29),
sFRP3 (SEQ ID NO: 30), sFRP4 (SEQ ID NO: 31), and sFRP5 (SEQ ID NO:
32), and active variants thereof. In yet a further embodiment, the
Frizzled domain component comprises a minimal CRD (ECD) domain from
a Ror polypeptide selected from the group consisting of hRorl (SEQ
ID NO: 33), and hRor2 (SEQ ID NO: 34), and active variants
thereof.
[0024] In yet a further embodiment, the Frizzled domain component
comprises a mature Frz polypeptide selected from the group
consisting of: (SEQ ID NO: 50), hFrz2 (SEQ ID NO: 51), hFrz3 (SEQ
ID NO: 52), hFrz4 (SEQ ID NO: 53), hFrz5 (SEQ ID NO: 54), hFrz6
(SEQ ID NO: 55), hFrz7 (SEQ ID NO: 56), hFrz8 (SEQ ID NO: 57),
hFrz9 (SEQ ID NO: 58), and hFrz10 (SEQ ID NO: 59), and active
variants thereof, or a mature sFrp polypeptide selected from the
group consisting of sFRP1 (SEQ ID NO: 60), sFRP2 (SEQ ID NO: 61),
sFRP3 (SEQ ID NO: 62), sFRP4 (SEQ ID NO: 63), and sFRP5 (SEQ ID NO:
64), and active variants thereof, or a mature Ror polypeptide
selected from the group consisting of hRorl (SEQ ID NO: 65), and
hRor2 (SEQ ID NO: 66), and active variants thereof.
[0025] In a still further embodiment, the Frizzled domain component
comprises a pro-Frz polypeptide selected from the group consisting
of hFrz1 (SEQ ID NO: 35), hFrz2 (SEQ ID NO: 36), hFrz3 (SEQ ID NO:
37), hFrz4 (SEQ ID NO: 38), hFrz5 (SEQ ID NO: 39), hFrz6 (SEQ ID
NO: 40), hFrz7 (SEQ ID NO: 41), hFrz8 (SEQ ID NO: 42), hFrz9 (SEQ
ID NO: 43), and hFrz10 (SEQ ID NO: 44), and active variants
thereof, or a pro-sFrp polypeptide selected from the group
consisting of sFRP1 (SEQ ID NO: 45), sFRP2 (SEQ ID NO: 46), sFRP3
(SEQ ID NO: 47), sFRP4 (SEQ ID NO: 48), and sFRP5 (SEQ ID NO: 49),
and active variants thereof.
[0026] In one embodiment, the Wnt antagonist comprises a Fc
component derived from an immunoglobulin selected from the group
consisting of IgG1, IgG2, IgG3 and IgG4. In another embodiment, the
Fc is derived from an IgG1 immunoglobulin. In yet another
embodiment the Fc sequence shown in SEQ ID NO: 67 or SEQ ID NO:
68.
[0027] In one embodiment, Wnt antagonist further comprises a linker
connecting the Frizzled domain component to the Fc domain. In one
embodiment, the linker is a peptide linker such as ESGGGGVT (SEQ ID
NO: 69), LESGGGGVT (SEQ ID NO: 70), GRAQVT (SEQ ID NO: 71), WRAQVT
(SEQ ID NO: 72), and ARGRAQVT (SEQ ID NO: 73).
[0028] In particular embodiments, the Wnt antagonist comprises a
polypeptide selected from the group consisting of Frz8-Fc (SEQ ID
NO: 74), FrzS-Fc (SEQ ID NO: 75), Frz1-Fc (SEQ ID NO: 76), Frz2-Fc
(SEQ ID NO: 77), Frz3-Fc (SEQ ID NO: 78), Frz4-Fc (SEQ ID NO: 79),
Frz6-Fc (SEQ ID NO: 80), Frz7-Fc (SEQ ID NO: 81), Frz9-Fc (SEQ ID
NO: 82), Frz10-Fc (SEQ ID NO: 83), sFRP1-Fc (SEQ ID NO: 84), sFRP2
(SEQ ID NO: 85), sFRP3-Fc (SEQ ID NO: 86), sFRP4-Fc (SEQ ID NO:
87), and sFRP5-Fc (SEQ ID NO: 88).
[0029] In one embodiment of this method, the cell is contained
within a mammal and the amount administered is a therapeutically
effective amount. In another embodiment, the Wnt signaling results
from activation of a Wnt signaling component through somatic
mutation. In another embodiment, the inhibition of Wnt signaling
results in the inhibition of growth of the cell. In yet another
embodiment, the cell is a cancer cell.
[0030] Another aspect of the invention provides for a method of
treating a Wnt-mediated disorder in a mammal suffering therefrom,
comprising administering to the mammal a therapeutically effective
amount of a Wnt antagonist comprising a Frizzled domain component
and a Fc domain. The Frizzled domain component of the Wnt
antagonist comprises a polypeptide derived from a Frz protein, a
FRP protein, or a Ror protein. In one embodiment, the Wnt
antagonist is active in vivo for at least 1 hour. In another
embodiment, the Wnt antagonist is active in vivo for at least 5
hours. In another embodiment, the Wnt antagonist has an in vivo
half-life of at least 1 day. In yet another embodiment, the Wnt
antagonist has an in vivo half-life of at least 2 days.
[0031] In a further embodiment of this aspect, the Frizzled domain
component comprises a minimal CRD (ECD) domain from a Frz
polypeptide selected from the group consisting of hFrz1 (SEQ ID NO:
18), hFrz2 (SEQ ID NO: 19), hFrz3 (SEQ ID NO: 20), hFrz4 (SEQ ID
NO: 21), hFrz5 (SEQ ID NO: 22), hFrz6 (SEQ ID NO: 23), hFrz7 (SEQ
ID NO: 24), hFrz8 (SEQ ID NO: 25), hFrz9 (SEQ ID NO: 26), and
hFrz10 (SEQ ID NO: 27), and active variants thereof. In yet a
further embodiment, the Frizzled domain component comprises a
minimal CRD (ECD) domain from a sFRP polypeptide selected from the
group consisting of sFRP1 (SEQ ID NO: 28), sFRP2 (SEQ ID NO: 29),
sFRP3 (SEQ ID NO: 30), sFRP4 (SEQ ID NO: 31), and sFRP5 (SEQ ID NO:
32), and active variants thereof. In yet a further embodiment, the
Frizzled domain component comprises a minimal CRD (ECD) domain from
a Ror polypeptide selected from the group consisting of hRorl (SEQ
ID NO: 33), and hRor2 (SEQ ID NO: 34), and active variants
thereof.
[0032] In yet a further embodiment, the Frizzled domain component
comprises a mature Frz polypeptide selected from the group
consisting of: (SEQ ID NO: 50), hFrz2 (SEQ ID NO: 51), hFrz3 (SEQ
ID NO: 52), hFrz4 (SEQ ID NO: 53), hFrz5 (SEQ ID NO: 54), hFrz6
(SEQ ID NO: 55), hFrz7 (SEQ ID NO: 56), hFrz8 (SEQ ID NO: 57),
hFrz9 (SEQ ID NO: 58), and hFrz10 (SEQ ID NO: 59), and active
variants thereof, or a mature sFrp polypeptide selected from the
group consisting of sFRP1 (SEQ ID NO: 60), sFRP2 (SEQ ID NO: 61),
sFRP3 (SEQ ID NO: 62), sFRP4 (SEQ ID NO: 63), and sFRP5 (SEQ ID NO:
64), and active variants thereof, or a mature Ror polypeptide
selected from the group consisting of hRorl (SEQ ID NO: 65), and
hRor2 (SEQ ID NO: 66), and active variants thereof.
[0033] In still further embodiments, the Frizzled domain component
comprises a pro-Frz polypeptide selected from the group consisting
of hFrz1 (SEQ ID NO: 35), hFrz2 (SEQ ID NO: 36), hFrz3 (SEQ ID NO:
37), hFrz4 (SEQ ID NO: 38), hFrz5 (SEQ ID NO: 39), hFrz6 (SEQ ID
NO: 40), hFrz7 (SEQ ID NO: 41), hFrz8 (SEQ ID NO: 42), hFrz9 (SEQ
ID NO: 43), and hFrz10 (SEQ ID NO: 44), and active variants
thereof, or a pro-sFrp polypeptide selected from the group
consisting of sFRP1 (SEQ ID NO: 45), sFRP2 (SEQ ID NO: 46), sFRP3
(SEQ ID NO: 47), sFRP4 (SEQ ID NO: 48), and sFRP5 (SEQ ID NO: 49),
and active variants thereof.
[0034] In one embodiment, the Wnt antagonist comprises a Fc
component derived from an immunoglobulin selected from the group
consisting of IgG1, IgG2, IgG3 and IgG4. In another embodiment, the
Fc is derived from an IgG1 immunoglobulin. In yet another
embodiment the Fc sequence shown in SEQ ID NO: 67 or SEQ ID NO:
68.
[0035] In one embodiment, Wnt antagonist further comprises a linker
connecting the Frizzled domain component to the Fc domain. In one
embodiment, the linker is a peptide linker such as ESGGGGVT (SEQ ID
NO: 69), LESGGGGVT (SEQ ID NO: 70), GRAQVT (SEQ ID NO: 71), WRAQVT
(SEQ ID NO: 72), and ARGRAQ VT (SEQ ID NO: 73).
[0036] In particular embodiments, the Wnt antagonist comprises a
polypeptide selected from the group consisting of Frz8-Fc (SEQ ID
NO: 74), FrzS-Fc (SEQ ID NO: 75), Frz1-Fc (SEQ ID NO: 76), Frz2-Fc
(SEQ ID NO: 77), Frz3-Fc (SEQ ID NO: 78), Frz4-Fc (SEQ ID NO: 79),
Frz6-Fc (SEQ ID NO: 80), Frz7-Fc (SEQ ID NO: 81), Frz9-Fc (SEQ ID
NO: 82), Frz10-Fc (SEQ ID NO: 83), sFRP1-Fc (SEQ ID NO: 84), sFRP2
(SEQ ID NO: 85), sFRP3-Fc (SEQ ID NO: 86), sFRP4-Fc (SEQ ID NO:
87), and sFRP5-Fc (SEQ ID NO: 88).
[0037] In one embodiment of this method, the disorder is a cell
proliferative disorder associated with aberrant Wnt signaling
activity. In another embodiment, the aberrant Wnt signaling
activity results from increased expression of a Wnt protein. In yet
another embodiment, the cell proliferative disorder is cancer, such
as of colon cancer, colorectal cancer, breast cancer, leukemia,
gliomas, or medulloblastomas.
[0038] Yet another aspect of the invention provides for a method
for detecting the presence of a Wnt protein, comprising contacting
the sample with a Wnt antagonist as described above, where the
presence of a complex, or the binding level between the Wnt
antagonist and Wnt protein is indicative of the presence of a Wnt
protein and/or signaling. In one embodiment, the method further
comprises determining if the level of Wnt signaling is aberrant,
the method further comprising comparing the level of binding in the
sample to the level in a second sample known to have
physiologically normal Wnt signaling. A level of binding in the
sample that is higher or lower than that of the second sample is
indicative of aberrant Wnt signaling. In yet another embodiment,
the aberrant Wnt signaling is further indicative of the presence of
a Wnt-mediated disorder, such as cancer.
[0039] Another aspect of the invention provides for a method of
modulating the expression of a Wnt target gene in a cell
characterized by activated or excessive Wnt signaling, comprising
contact the cell with an effective amount of a Wnt antagonist
described above.
[0040] Yet another aspect of the invention provides for a method of
therapeutically treating a Wnt-mediated cancer, comprising
administering a therapeutically effective amount of a Wnt
antagonist comprising a Frizzled domain component and a Fc domain.
The Frizzled domain component of the Wnt antagonist comprises a
polypeptide derived from a Frz protein, a FRP protein, or a Ror
protein. In one embodiment, the Wnt antagonist is active in vivo
for at least 1 hour. In another embodiment, the Wnt antagonist is
active in vivo for at least 5 hours. In another embodiment, the Wnt
antagonist has an in vivo half-life of at least 1 day. In yet
another embodiment, the Wnt antagonist has an in vivo half-life of
at least 2 days.
[0041] In a further embodiment of this aspect, the Frizzled domain
component comprises a minimal CRD (ECD) domain from a Frz
polypeptide selected from the group consisting of hFrz1 (SEQ ID NO:
18), hFrz2 (SEQ ID NO: 19), hFrz3 (SEQ ID NO: 20), hFrz4 (SEQ ID
NO: 21), hFrz5 (SEQ ID NO: 22), hFrz6 (SEQ ID NO: 23), hFrz7 (SEQ
ID NO: 24), hFrz8 (SEQ ID NO: 25), hFrz9 (SEQ ID NO: 26), and
hFrz10 (SEQ ID NO: 27), and active variants thereof. In yet a
further embodiment, the Frizzled domain component comprises a
minimal CRD (ECD) domain from a sFRP polypeptide selected from the
group consisting of sFRP1 (SEQ ID NO: 28), sFRP2 (SEQ ID NO: 29),
sFRP3 (SEQ ID NO: 30), sFRP4 (SEQ ID NO: 31), and sFRP5 (SEQ ID NO:
32), and active variants thereof. In yet a further embodiment, the
Frizzled domain component comprises a minimal CRD (ECD) domain from
a Ror polypeptide selected from the group consisting of hRorl (SEQ
ID NO: 33), and hRor2 (SEQ ID NO: 34), and active variants
thereof.
[0042] In yet a further embodiment, the Frizzled domain component
comprises a mature Frz polypeptide selected from the group
consisting of: (SEQ ID NO: 50), hFrz2 (SEQ ID NO: 51), hFrz3 (SEQ
ID NO: 52), hFrz4 (SEQ ID NO: 53), hFrz5 (SEQ ID NO: 54), hFrz6
(SEQ ID NO: 55), hFrz7 (SEQ ID NO: 56), hFrz8 (SEQ ID NO: 57),
hFrz9 (SEQ ID NO: 58), and hFrz10 (SEQ ID NO: 59), and active
variants thereof, or a mature sFrp polypeptide selected from the
group consisting of sFRP1 (SEQ ID NO: 60), sFRP2 (SEQ ID NO: 61),
sFRP3 (SEQ ID NO: 62), sFRP4 (SEQ ID NO: 63), and sFRP5 (SEQ ID NO:
64), and active variants thereof, or a mature Ror polypeptide
selected from the group consisting of hRorl (SEQ ID NO: 65), and
hRor2 (SEQ ID NO: 66), and active variants thereof.
[0043] In a still further embodiment, the Frizzled domain component
comprises a pro-Frz polypeptide selected from the group consisting
of hFrz1 (SEQ ID NO: 35), hFrz2 (SEQ ID NO: 36), hFrz3 (SEQ ID NO:
37), hFrz4 (SEQ ID NO: 38), hFrz5 (SEQ ID NO: 39), hFrz6 (SEQ ID
NO: 40), hFrz7 (SEQ ID NO: 41), hFrz8 (SEQ ID NO: 42), hFrz9 (SEQ
ID NO: 43), and hFrz10 (SEQ ID NO: 44), and active variants
thereof, or a pro-sFrp polypeptide selected from the group
consisting of sFRP1 (SEQ ID NO: 45), sFRP2 (SEQ ID NO: 46), sFRP3
(SEQ ID NO: 47), sFRP4 (SEQ ID NO: 48), and sFRP5 (SEQ ID NO: 49),
and active variants thereof.
[0044] In one embodiment, the Wnt antagonist comprises a Fc
component derived from an immunoglobulin selected from the group
consisting of IgG1, IgG2, IgG3 and IgG4. In another embodiment, the
Fc is derived from an IgG1 immunoglobulin. In yet another
embodiment the Fc sequence shown in SEQ ID NO: 67 or SEQ ID NO:
68.
[0045] In one embodiment, Wnt antagonist further comprises a linker
connecting the Frizzled domain component to the Fc domain. In one
embodiment, the linker is a peptide linker such as ESGGGGVT (SEQ ID
NO: 69), LESGGGGVT (SEQ ID NO: 70), GRAQVT (SEQ ID NO: 71), WRAQVT
(SEQ ID NO: 72), and ARGRAQVT (SEQ ID NO: 73).
[0046] In particular embodiments, the Wnt antagonist comprises a
polypeptide selected from the group consisting of Frz8-Fc (SEQ ID
NO: 74), FrzS-Fc (SEQ ID NO: 75), Frz1-Fc (SEQ ID NO: 76), Frz2-Fc
(SEQ ID NO: 77), Frz3-Fc (SEQ ID NO: 78), Frz4-Fc (SEQ ID NO: 79),
Frz6-Fc (SEQ ID NO: 80), Frz7-Fc (SEQ ID NO: 81), Frz9-Fc (SEQ ID
NO: 82), Frz10-Fc (SEQ ID NO: 83), sFRP1-Fc (SEQ ID NO: 84), sFRP2
(SEQ ID NO: 85), sFRP3-Fc (SEQ ID NO: 86), sFRP4-Fc (SEQ ID NO:
87), and sFRP5-Fc (SEQ ID NO: 88).
[0047] The administration of the antagonist arrests any subsequent
increase in size or advancement in severity of the cancer. In one
embodiment, the administration of the Wnt antagonist results in the
reduction in size or severity of the cancer. In another embodiment,
the administration of the Wnt antagonist reduces the tumor burden
of the cancer. In yet another embodiment, the administration of the
Wnt antagonist kills the cancer.
[0048] Another aspect of the invention provides for the use of a
Wnt antagonist in the manufacture of a medicament for the treatment
of a cell proliferative disorder. Wnt antagonist comprises a
Frizzled domain component and a Fc domain. The Frizzled domain
component of the Wnt antagonist comprises a polypeptide derived
from a Frz protein, a FRP protein, or a Ror protein. In one
embodiment, the Wnt antagonist is active in vivo for at least 1
hour. In another embodiment, the Wnt antagonist is active in vivo
for at least 5 hours. In another embodiment, the Wnt antagonist has
an in vivo half-life of at least 1 day. In yet another embodiment,
the Wnt antagonist has an in vivo half-life of at least 2 days.
[0049] In a further embodiment of this aspect, the Frizzled domain
component comprises a minimal CRD (ECD) domain from a Frz
polypeptide selected from the group consisting of hFrz1 (SEQ ID NO:
18), hFrz2 (SEQ ID NO: 19), hFrz3 (SEQ ID NO: 20), hFrz4 (SEQ ID
NO: 21), hFrz5 (SEQ ID NO: 22), hFrz6 (SEQ ID NO: 23), hFrz7 (SEQ
ID NO: 24), hFrz8 (SEQ ID NO: 25), hFrz9 (SEQ ID NO: 26), and
hFrz10 (SEQ ID NO: 27), and active variants thereof. In yet a
further embodiment, the Frizzled domain component comprises a
minimal CRD (ECD) domain from a sFRP polypeptide selected from the
group consisting of sFRP1 (SEQ ID NO: 28), sFRP2 (SEQ ID NO: 29),
sFRP3 (SEQ ID NO: 30), sFRP4 (SEQ ID NO: 31), and sFRP5 (SEQ ID NO:
32), and active variants thereof. In yet a further embodiment, the
Frizzled domain component comprises a minimal CRD (ECD) domain from
a Ror polypeptide selected from the group consisting of hRorl (SEQ
ID NO: 33), and hRor2 (SEQ ID NO: 34), and active variants
thereof.
[0050] In yet a further embodiment, the Frizzled domain component
comprises a mature Frz polypeptide selected from the group
consisting of: (SEQ ID NO: 50), hFrz2 (SEQ ID NO: 51), hFrz3 (SEQ
ID NO: 52), hFrz4 (SEQ ID NO: 53), hFrz5 (SEQ ID NO: 54), hFrz6
(SEQ ID NO: 55), hFrz7 (SEQ ID NO: 56), hFrz8 (SEQ ID NO: 57),
hFrz9 (SEQ ID NO: 58), and hFrz10 (SEQ ID NO: 59), and active
variants thereof, or a mature sFrp polypeptide selected from the
group consisting of sFRP1 (SEQ ID NO: 60), sFRP2 (SEQ ID NO: 61),
sFRP3 (SEQ ID NO: 62), sFRP4 (SEQ ID NO: 63), and sFRP5 (SEQ ID NO:
64), and active variants thereof, or a mature Ror polypeptide
selected from the group consisting of hRorl (SEQ ID NO: 65), and
hRor2 (SEQ ID NO: 66), and active variants thereof.
[0051] In a still further embodiment, the Frizzled domain component
comprises a pro-Frz polypeptide selected from the group consisting
of hFrz1 (SEQ ID NO: 35), hFrz2 (SEQ ID NO: 36), hFrz3 (SEQ ID NO:
37), hFrz4 (SEQ ID NO: 38), hFrz5 (SEQ ID NO: 39), hFrz6 (SEQ ID
NO: 40), hFrz7 (SEQ ID NO: 41), hFrz8 (SEQ ID NO: 42), hFrz9 (SEQ
ID NO: 43), and hFrz10 (SEQ ID NO: 44), and active variants
thereof, or a pro-sFrp polypeptide selected from the group
consisting of sFRP1 (SEQ ID NO: 45), sFRP2 (SEQ ID NO: 46), sFRP3
(SEQ ID NO: 47), sFRP4 (SEQ ID NO: 48), and sFRP5 (SEQ ID NO: 49),
and active variants thereof.
[0052] In one embodiment, the Wnt antagonist comprises a Fc
component derived from an immunoglobulin selected from the group
consisting of IgG1, IgG2, IgG3 and IgG4. In another embodiment, the
Fc is derived from an IgG1 immunoglobulin. In yet another
embodiment the Fc sequence shown in SEQ ID NO: 67 or SEQ ID NO:
68.
[0053] In one embodiment, Wnt antagonist further comprises a linker
connecting the Frizzled domain component to the Fc domain. In one
embodiment, the linker is a peptide linker such as ESGGGGVT (SEQ ID
NO: 69), LESGGGGVT (SEQ ID NO: 70), GRAQVT (SEQ ID NO: 71), WRAQVT
(SEQ ID NO: 72), and ARGRAQVT (SEQ ID NO: 73).
[0054] In particular embodiments, the Wnt antagonist comprises a
polypeptide selected from the group consisting of Frz8-Fc (SEQ ID
NO: 74), Frz5-Fc (SEQ ID NO: 75), Frz1-Fc (SEQ ID NO: 76), Frz2-Fc
(SEQ ID NO: 77), Frz3-Fc (SEQ ID NO: 78), Frz4-Fc (SEQ ID NO: 79),
Frz6-Fc (SEQ ID NO: 80), Frz7-Fc (SEQ ID NO: 81), Frz9-Fc (SEQ ID
NO: 82), Frz10-Fc (SEQ ID NO: 83), sFRP1-Fc (SEQ ID NO: 84), sFRP2
(SEQ ID NO: 85), sFRP3-Fc (SEQ ID NO: 86), sFRP4-Fc (SEQ ID NO:
87), and sFRP5-Fc (SEQ ID NO: 88).
[0055] In one embodiment, the cell proliferative disorder is cancer
such as colon cancer, colorectal cancer, breast cancer, leukemia,
gliomas, or medulloblastomas.
DESCRIPTION OF THE FIGURES
[0056] FIG. 1 is an abbreviated summary of the canonical Wnt
signaling pathway both in the "off" or inactive state as well as
the "on" or active state.
[0057] FIG. 2 is a schematic diagram representing a Frizzled
extracellular domain linked to the Fc region of a human
immunoglobulin domain.
[0058] FIG. 3 is an alignment of the 17 known Frizzled protein
extracellular domains. FIG. 3A shows an alignment of the
extracellular domains of the 10 pro-Frizzled proteins (SEQ ID NOs:
35-44) and the 5 pro-sFRP proteins (SEQ ID NOs: 45-49), while FIG.
3B shows an alignment of the extracellular domains of 10 mature
Frizzled proteins (SEQ ID NOs: 50-59), and 5 mature sFRP proteins
(SEQ ID NOs: 60-64), as well as the extracellular domains of the
mature Ror proteins (SEQ ID NOs: 65-66). Similar residues are boxed
in gray, identical residues are indicated by asterisks. Similar
residues are grouped as acidic, basic, polar and non-polar. In FIG.
3B, the minimal CRD (ECD) domains are indicated between the two
boxed arrowed lines (SEQ ID NOs: 18-34).
[0059] FIG. 4 shows the sequences of the Frz (156)-Fc and Frz
(173)-Fc chimeric constructs. FIG. 4A shows the longer Frz (173)-Fc
sequence (SEQ ID NO: 113). Shown in bold (i.e., first 24 N-terminal
amino acid residues) is the leader signal sequence. Residues 25-27
are alanine residues that may be present or absent in the mature
protein. Shown in boxed text (i.e., residues 157-173) are the
additional sequences of the Frz8 receptors that distinguish the
longer (Frz173) from the shorter (Frz156) chimeric constructs. The
linker sequence (i.e., residues 174-182) is underlined, while the
Fc domain sequence is shown in italics (i.e., residues 183-409).
FIG. 4B shows the shorter Frz (156)-Fc (SEQ ID NO: 74). In bold
(i.e., first 24 N-terminal amino acid residues) is the leader
signal sequence. Residues 25-27 are alanine residues that may be
present or absent in the mature protein. The linker sequence (i.e.,
residues 157-164) is underlined, while the Fc domain sequence is
shown in italics (i.e., residues 165-391).
[0060] FIG. 5A-5H shows the nucleic acid sequence encoding several
Wnt antagonist chimeric constructs (Frz1-Fc (SEQ ID NO: 115),
Frz2-Fc (SEQ ID NO: 116), Frz3-Fc (SEQ ID NO: 117), Frz4-Fc (SEQ ID
NO: 118), Frz5-Fc (SEQ ID NO: 119), Frz6-Fc (SEQ ID NO: 120),
Frz7-Fc (SEQ ID NO: 121), Frz8-Fc (SEQ ID NO: 122), Frz9-Fc (SEQ ID
NO: 123), Frz10-Fc (SEQ ID NO: 124), sFRP1-Fc (SEQ ID NO: 125),
sFRP2-Fc (SEQ ID NO: 126), sFRP3-Fc (SEQ ID NO: 127), sFRP4-Fc (SEQ
ID NO: 128), and sFRP5-Fc (SEQ ID NO:129)).
[0061] FIG. 6A-6E shows the full length amino acid sequences of the
human Frz, sFRP, and Ror proteins.
[0062] FIG. 7 (A, B, and C) shows the amino acid sequences of
several Wnt antagonist chimeric constructs (Frz1-Fc (SEQ ID NO:
76), Frz2-Fc (SEQ ID NO: 77), Frz3-Fc (SEQ ID NO: 78), Frz4-Fc (SEQ
ID NO: 79), Frz5-Fc (SEQ ID NO: 75), Frz6-Fc (SEQ ID NO: 80),
Frz7-Fc (SEQ ID NO: 81), Frz8-Fc (SEQ ID NO: 74), Frz9-Fc (SEQ ID
NO: 82), Frz10-Fc (SEQ ID NO: 83), sFRP1-Fc (SEQ ID NO: 84),
sFRP2-Fc (SEQ ID NO: 85), sFRP3-Fc (SEQ ID NO: 86), sFRP4-Fc (SEQ
ID NO: 87), and sFRP5-Fc (SEQ ID NO: 88)). The bold text for
Frz1-Fc (first 28 N-terminal amino acid residues), Frz2-Fc (first
31 N-terminal amino acid residues), Frz3-Fc (first 31 N-terminal
amino acid residues), Frz4-Fc (first 31 N-terminal amino acid
residues) Frz5-Fc (first 31 N-terminal amino acid residues), and
sFRP3-Fc (first 31 N-terminal amino acid residues) indicates a
non-native leader sequence. The linker is underlined and the Fc
domain, following the linker, is shown in italics.
[0063] FIG. 8 shows an alignment of Frizzled extracellular domains
where black shows conserved residues across all receptors and gray
represents residues conserved across homologous groups.
[0064] FIG. 9 shows Frizzleds grouped into families based on both
full-length and extracellular domain sequence identities.
[0065] FIG. 10 depicts samples of purified Frizzled-Fc fusion
proteins expressed and purified from CHO cells. Samples were
separated on non-reducing SDS-PAGE gels and imaged by Coomassie
staining.
[0066] FIG. 11 show a comparison of serum stability of the two
different Frz8-Fc chimeras Frz8(173)-Fc and Frz(156)-Fc. FIG. 11A
is an immunoblot for human FC used to detect the chimeric proteins
present at increasing time points in serum of athymic nude mice
injected with the chimeras. FIG. 11B shows the Wnt inhibitory
activity of the chimeric proteins assayed by measuring TOPglow
activity shown on the Y axis as relative luciferase activity.
[0067] FIG. 12 is a graph of tumor volume over time resulting from
treatment with Frz8(173)-Fc chimera.
[0068] FIG. 13 shows pharmacokinetic (PK) data for Frz8-Fc
following administration of a single dose of the protein. FIG. 13A
is an immunoblot of a neat serum from mice treated with Frz8-Fc
showing detection in serum at 7 days and beyond from both 20 or 5
mg/kg I.V. or 20 mg/kg I.P. FIGS. 13B and 13C are a graphical
summary of Frz8-Fc serum levels as determined from the
pharmacokinetic study. FIG. 13D is a summary of the parameters for
a biphasic model of Frz8-Fc pharmacokinetics.
[0069] FIG. 14 demonstrate the enhanced ability of Frz8-ECD to
block Wnt3a signaling when linked to a dimeric Fc domain. FIG. 14A
is an IC.sub.50 graph of a Wnt3a inhibition assay of two different
preparations of Frz8(156)-FC. FIG. 14B is a gel confirming the
purity of the isolated Frz8(156) CRD (ECD). Shown are: (a)
non-reduced Frz8 ECD (Lane 1); (b) molecular weight markers (Lane
2); and reduced Frz8 ECD (Lane 3).
[0070] FIG. 15 demonstrates direct binding of Wnt3a to the Frz8-Fc
chimera. FIG. 15A is BIAcore sensogram demonstrating binding of
purified soluble Wnt3a to immobilized Frz8-Fc. FIG. 15B is an
immunoprecipitation of a purified soluble Wnt3a by immobilized
Frz8-Fc.
[0071] FIG. 16 demonstrates direct binding of several Frz-Fc
chimeras to Wnt ligands as measured using the OCTET.TM. system.
FIG. 16A shows data from the binding of Wnt3a to the Frz1-Frz10-Fc
chimeras, FIG. 16B shows data from the binding of Wnt3a to sFRP-Fc
chimeras. FIG. 16C shows data from the binding of Wnt5a to the
Frz1-Frz10-Fc chimeras and sFRP-Fc chimeras.
[0072] FIG. 17 shows the effect of the Wnt antagonists on
Wnt-stimulated cells transiently transfected with TOPglow
luciferase TCF reporter plasmid. FIG. 17A shows cells stimulated
with Wnt3a and FIG. 17B shows cells stimulated with Wnt-5a. Cells
to be treated with Wnt5a were transfected with Frz4 and Lrp5 in
addition to the reporter. 293 (human kidney) cells were activated
with 100 ng/ml Wnt3a or 1 ug/ml Wnt5a. Cells then left untreated,
treated with control Fc, or treated with purified Frz-Fc protein in
PBS and assayed for luciferase response.
[0073] FIG. 18 shows inhibition of Wnt signaling by the Wnt
antagonists in U2OS (human osteosarcoma) cells stably transfected
with a luciferase TCF reporter plasmid. Initial Wnt signaling in
cells was obtained with Wnt3a activation.
[0074] FIG. 19 shows the effect of Frz8-Fc on expression of
Wnt-target genes in cultured teratoma cells and tumor xenografts.
FIG. 19A shows expression of Wnt-target genes in PA-1 cell lines
treated with Wnt3a and Frz8-Fc. RNA isolated from PA-1 cells that
were treated with Wnt3a, Frz8-Fc, or control Fc protein was subject
to microarray analysis and the change in expression levels of the
indicated genes in response to exogenously added Wnt3a, Frz8-Fc,
and control Fc protein was plotted. Columns, mean expression level
from three wells; bars, standard error (S). FIG. 19B shows the
relative expression of Wnt target genes APCDD1, Gad-1, and Fzd5 in
NTera-2 tumors from mice given PBS, CD4-Fc, or Frz8-Fc relative to
PBS control. The data represents the mean expression level from the
indicated number of tumors and is representative of at least two
independent qRT-PCR experiments done in duplicate. Regulation of
expression of each gene by the addition of purified Wnt3a to the
corresponding cultured cells is also presented.
[0075] FIG. 20 shows the accession number and sequence of primers
and probes used for real-time quantitative PCR analysis of gene
expression shown in FIG. 19 (Example 9).
[0076] FIG. 21 is a linear schematic describing the vector
construct used in the transfection to create the Wnt animal
model.
[0077] FIG. 22 illustrates the efficacy of Frz8-Fc against MMTV-Wnt
tumor transplants in athymic nude mice by intraperitoneal (IP)
dosing. FIG. 22A is a graph showing data from nude mice hosting
MMTV-Wnt-1 tumor transplants were administered PBS, CD4-Fc (10
mg/kg/day) or Frz8-Fc (10 mg/kg/day) by intraperitoneal injection
twice weekly. Mean tumor volume is plotted over time and the
treatment days are indicated by arrows on the X-axis. FIG. 22B is
tabular summary of mean tumor volume and mean % change in tumor
volume over time in the four treatment groups.
[0078] FIG. 23 illustrates the efficacy of Frz8-Fc against MMTV-Wnt
tumor transplant in athymic nude mice by intravenous (IV) dosing.
FIG. 23A is a graph showing data from nude mice hosting MMTV-Wnt-1
tumor transplants were administered PBS, CD4-Fc (10 mg/kg/day) or
Frz8-Fc (10 mg/kg/day) by intravenous injection three times weekly.
Mean tumor volume is plotted over time and the treatment days are
indicated by arrows on the X-axis. FIG. 23B is a tabular summary of
mean tumor volume and mean % change in tumor volume over time in
the four treatment groups.
[0079] FIG. 24 is a bar graph showing the Wnt signaling antagonist
activity in the TOPglow assay of various Wnt antagonists in serum
isolated from the MMTV Wnt tumor study. The X-axis samples appear
in groups A-E (FIG. 24A) or A-F (FIG. 24B) according to treatment,
mouse study number and dilution. The relative luciferase activity
in the TOPGLOW gene reporter assay is shown on the Y-axis. All
samples are treated with .about.40 ng/ml purified Wnt3a except for
NA (control). All other protein controls are present in the medium
at 5 .mu.g/ml. FIG. 24A shows the testing results of serum isolated
from IP treated mice, while the IV treated ones appear in FIG.
24B.
[0080] FIG. 25 shows Wnt signaling antagonist activity in the
TOPglow assay of various Wnt antagonists in the indicated
teratacarcinoma cell lines in the absence (FIG. 25A) or presence
(FIG. 25B) of exogenously added Wnt3a. For each cell line, activity
was expressed relative to that observed in the absence of any
treatment (NA); representative of at least two independent
experiments. Relative luciferase activity (Y-axis) were measured
from TOPglow assays from various cancer cell lines in the presence
or absence or Wnt inhibitors.
[0081] FIG. 26 demonstrates the anti-tumor efficacy of Frz8
(156)-Fc treatment on the growth of NTera2 tumor xenografts in
athymic nude mice. FIG. 26A is procedural flow chart, while FIG.
26B is a graph plotting mean tumor volume over time, wherein the
treatment days are indicated by arrows on the X-axis. FIG. 26C is a
bar graph plotting the mean tumor weights at sacrifice of all
animals in the group at day 20 of the study. FIGS. 26D and 26E are
tabular summaries of mean tumor volume and mean % change in tumor
volume, respectively.
[0082] FIG. 27 is a bar graph showing Wnt signaling antagonist
activity of serum isolated from various animals in the NTera2 tumor
study as determined by the TOPglow assay. The Y-axis shows relative
luciferase activity (Y-axis) from the TOPglow assay for the
controls and Frz8-Fc Wnt antagonist. No additional purified Wnt or
Wnt conditioned media was added to the cells.
[0083] FIG. 28 shows the anti-tumor efficacy of Frz8 (156)-Fc
treatment on the growth of PA-1 tumor xenografts in athymic nude
mice. FIG. 28A is a procedural flow chart, while FIG. 28B is a
graph plotting mean tumor volume over time. FIG. 28C is a graph of
mean tumor weight at sacrifice. The mean tumor weight.+-.SEM is
plotted as a function of the group. FIGS. 28D and 28E are tabular
summaries of mean tumor volume and mean % change in tumor volume,
respectively.
[0084] FIG. 29 shows Wnt signaling inhibition in mice treated with
Frz8-Fc or Frz5-Fz as determined by the TOPglow assay. The Y-axis
shows relative luciferase activity (Y-axis) from the TOPglow assay
for the controls and Frz8-Fc and Frz5-Fc Wnt antagonists.
[0085] FIG. 30 shows the reduced Axin2 expression in Frz8-Fc and
Frz5-Fz treated tumor with FIG. 30A showing expression normalized
to expression of GAPDH and FIG. 30B showing expression normalized
to expression of rpl19.
[0086] FIG. 31 shows immunohistochemistry (IHC) photomicrographs
for IHC staining of .beta.-catenin and demonstrate that Frz8-Fc
treatment on regenerative tissues such as intestine and skin appear
normal. FIG. 31A shows IHC for .beta.-catenin in small intestine of
PBS (A-1) control protein (A-2) and Frz8-Fc (A-3) treated mice.
FIG. 31B shows IHC for .beta.-catenin in skin of PBS (B-1) control
protein (B-2) and Frz8-Fc (B-3) treated mice.
[0087] FIG. 32 is an illustration of active Wnt signaling in human
breast cancer. FIG. 32A shows Wnt-1 expression (as shown by in
vitro hybridization) in normal (A-1), low grade (A-2) and high
grade (A-3) human breast tumor initially reported in Wong et al.,
J. Pathol. 196: 145 (2002). FIG. 32B shows nuclear (B-1) and
cytoplasmic (B-2) localization (as shown by IHC) of .beta.-catenin
in breast cancer patients. Also shown is a Kaplan-Meier survival
plot (B-3) showing patient survival probability that correlates
with the indicated .beta.-catenin expression pattern. This data was
initially reported in Lin et al., P.N.A.S. (USA) 97(8): 4262-66
(2000). FIG. 32C is a microarray analysis of Wnt-1 expression in a
normal breast from a patient without cancer in comparison with
tissue isolated from a patient with infiltrating ductal carcinoma,
her-2 negative.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0088] A "Wnt protein" is a ligand of the Wnt signaling pathway
component which binds to a Frizzled receptor so as to activate Wnt
signaling. Specific examples of Wnt proteins include at least 19
members, including: Wnt-1 (RefSeq.: NM.sub.--005430), Wnt-2
(RefSeq.: NM.sub.--003391), Wnt-2B (Wnt-13) (RefSeq.:
NM.sub.--004185), Wnt-3 (ReSeq.: NM.sub.--030753), Wnt3a (RefSeq.:
NM.sub.--033131), Wnt-4 (RefSeq.: NM.sub.--030761), Wnt-5A
(RefSeq.: NM.sub.--003392), Wnt-5B (RefSeq.: NM.sub.--032642),
Wnt-6 (RefSeq.: NM.sub.--006522), Wnt-7A (RefSeq.:
NM.sub.--004625), Wnt-7B (RefSeq.: NM.sub.--058238), Wnt-8A
(RefSeq.: NM.sub.--058244), Wnt-8B (RefSeq.: NM.sub.--003393),
Wnt-9A (Wnt-14) (RefSeq.: NM.sub.--003395), Wnt-9B (Wnt-15)
(RefSeq.: NM.sub.--003396), Wnt-10A (RefSeq.: NM.sub.--025216),
Wnt-10B (RefSeq.: NM.sub.--003394), Wnt-11 (RefSeq.:
NM.sub.--004626), Wnt-16 (RefSeq.: NM.sub.--016087)). While each
member has varying degrees of sequence identity, each contain 23-24
conserved cysteine residues which show highly conserved spacing.
McMahon, A P et al., Trends Genet. 8: 236-242 (1992); Miller J R.,
Genome Biol. 3(1): 3001.1-3001.15 (2002). For purposes of this
invention, a Wnt protein and active variants thereof is a protein
that binds to a Frizzled ECD or the CRD component of such an Frz
ECD.
[0089] A "Frizzled" (Frz) protein is a Wnt signaling pathway
component that is a seven-pass transmembrane receptors that binds
to a Wnt protein, and further complexes with other
membrane-associated Wnt signaling components, so as to transmit Wnt
signaling to downstream intracellular components. Frz proteins
include Frz1, Frz2, Frz3, Frz4, Frz5, Frz6, Frz7, Frz8, Frz9, and
Frz10. Examples of human full length Frz proteins are hFrz1
(NP.sub.--003496) (SEQ ID NO: 1), hFrz2 (NP.sub.--001457) (SEQ ID
NO: 2), hFrz3 (NP.sub.--059108) (SEQ ID NO: 3), hFrz4
(NP.sub.--036325) (SEQ ID NO: 4), hFrz5 (NP.sub.--003459) (SEQ ID
NO: 5), hFrz6 (NP.sub.--003497) (SEQ ID NO: 6), hFrz7
(NP.sub.--003498) (SEQ ID NO: 7), hFrz8 (NP.sub.--114072) (SEQ ID
NO: 8), hFrz9 (NP.sub.--003499) (SEQ ID NO: 9), and hFrz10
(NP.sub.--009128) (SEQ ID NO: 10) (FIGS. 6A-6C).
[0090] A "secreted Frizzled related protein" (sFRP) is a Wnt
signaling pathway component that is a secreted extracellular
polypeptide that binds to a Wnt protein. sFRP proteins include
sFRP1, sFRP2, sFRP3, sFRP4, and sFRP5. Examples of human full
length sFRP proteins are sFRP1 (NP.sub.--003003) (SEQ ID NO: 11),
sFRP2 (NP.sub.--003004) (SEQ ID NO: 12), sFRP3 (NP.sub.--001454)
(SEQ ID NO: 13), sFRP4 (NP.sub.--003005) (SEQ ID NO: 14), and sFRP5
(NP.sub.--003006) (SEQ ID NO: 15) (FIGS. 6C-6D).
[0091] The "Ror" protein, includes the mammalian homologs, Rorl and
Ror2, which are characterized by extracellular Frizzled-like
cysteine-rich domains (CRDs) as well as membrane proximal kringle
domains. Ror proteins play crucial roles in developmental
morphogenesis and are associated with different components of the
cytoskeleton. Rorl co-localizes with F-actin along stress fibers,
while Ror2 partially colocalizes with microtubules. Rorl and Ror2
share about 58% overall sequence identity. Ror2 associates with the
melanoma-associated antigen (MAGE) family protein Dlxin-1 and
regulates its intracellular distribution. Rorl proteins include
Rorl and Ror2. Examples of human full length Ror proteins are hRorl
(NP.sub.--005003) (SEQ ID NO: 16), and hRor2 (NP.sub.--004551) (SEQ
ID NO: 17) (FIGS. 6D-6E).
[0092] A "Frz domain component" is a polypeptide derived from a Frz
protein, a sFRP protein, a Ror protein, or other protein, that is
capable of binding with a Wnt protein. A polypeptide "derived from"
a protein means a polypeptide that has an amino acid sequence that
can be found within the reference protein sequence or within the
sequence of active variants of the protein. Examples of a Frz
domain component include a minimal cysteine rich domain (CRD) of an
extracellular domain "CRD (ECD)" of a Frz protein, a sFRP protein,
or a Ror protein, such as the CRD (ECD) of Frz1, Frz2, Frz3, Frz4,
Frz5, Frz6, Frz7, Frz8, Frz9, Frz10, sFRP1, sFRP2, sFRP3, sFRP4,
sFRP5, Rorl, or Ror2, and active variants thereof. The CRD (ECD) is
a conserved structural motif of 100 to 250 amino acids and is
defined by highly conserved cysteines. Particular examples of human
CRD (ECD)s are shown in boxed text in FIG. 3B and presented as SEQ
ID NOs: hFrz1 (SEQ ID NO: 18), hFrz2 (SEQ ID NO: 19), hFrz3 (SEQ ID
NO: 20), hFrz4 (SEQ ID NO: 21), hFrz5 (SEQ ID NO: 22), hFrz6 (SEQ
ID NO: 23), hFrz7 (SEQ ID NO: 24), hFrz8 (SEQ ID NO: 25), hFrz9
(SEQ ID NO: 26), hFrz10 (SEQ ID NO: 27), sFRP1 (SEQ ID NO: 28),
sFRP2 (SEQ ID NO: 29), sFRP3 (SEQ ID NO: 30), sFRP4 (SEQ ID NO:
31), sFRP5 (SEQ ID NO: 32), hRorl (SEQ ID NO: 33), and hRor2 (SEQ
ID NO: 34).
[0093] Additional examples of a Frz domain component include a
pro-Frz domain derived from a pro-Frz or pro-sFRP protein such as
Frz1, Frz2, Frz3, Frz4, Frz5, Frz6, Frz7, Frz8, Frz9, Frz10, sFRP1,
sFRP2, sFRP3, sFRP4, or sFRP5, and active variants thereof.
Particular examples of human pro-Frz domains are shown in FIG. 3A
and presented as SEQ ID NOs: hFrz1 (SEQ ID NO: 35), hFrz2 (SEQ ID
NO: 36), hFrz3 (SEQ ID NO: 37), hFrz4 (SEQ ID NO: 38), hFrz5 (SEQ
ID NO: 39), hFrz6 (SEQ ID NO: 40), hFrz7 (SEQ ID NO: 41), hFrz8
(SEQ ID NO: 42), hFrz9 (SEQ ID NO: 43), hFrz10 (SEQ ID NO: 44),
sFRP1 (SEQ ID NO: 45), sFRP2 (SEQ ID NO: 46), sFRP3 (SEQ ID NO:
47), sFRP4 (SEQ ID NO: 48), and sFRP5 (SEQ ID NO: 49).
[0094] Additional examples of a Frz domain component include a
mature Frz domain derived from a mature Frz, sFRP, or Ror protein,
such as Frz1, Frz2, Frz3, Frz4, Frz5, Frz6, Frz7, Frz8, Frz9,
Frz10, sFRP1, sFRP2, sFRP3, sFRP4, sFRP5, Rorl, or Ror2 and active
variants thereof. Particular examples of human mature Frz domains
are shown in FIG. 3B and presented as SEQ ID NOs: hFrz1 (SEQ ID NO:
50), hFrz2 (SEQ ID NO: 51), hFrz3 (SEQ ID NO: 52), hFrz4 (SEQ ID
NO: 53), hFrz5 (SEQ ID NO: 54), hFrz6 (SEQ ID NO: 55), hFrz7 (SEQ
ID NO: 56), hFrz8 (SEQ ID NO: 57), hFrz9 (SEQ ID NO: 58), hFrz10
(SEQ ID NO: 59), sFRP1 (SEQ ID NO: 60), sFRP2 (SEQ ID NO: 61),
sFRP3 (SEQ ID NO: 62), sFRP4 (SEQ ID NO: 63), sFRP5 (SEQ ID NO:
64), hRorl (SEQ ID NO: 65), and hRor2 (SEQ ID NO: 66).
[0095] A "Wnt antagonist" is a chimeric polypeptide comprising a
Frz domain component and an immunoglobulin Fc domain that binds to
a Wnt protein and is active by attenuating cellular Wnt signaling,
or a physiological symptom resulting therefrom.
[0096] In certain embodiments, the Fc domain is a human IgG1, IgG2,
IgG3 or IgG4 Fc domain. In one embodiment, the Fc domain is a human
IgG1 Fc domain. Specific examples of Fc domains are shown in FIGS.
4, 5, and FIG. 7 and in SEQ ID NO: 67 and SEQ ID NO: 68.
[0097] In some embodiments, the Frz domain component and the Fc
domain are fused by a linker. The term "linker" refers to a
component that tethers together the Frz domain component to the Fc
domain. Linkers that are suitable for use in the invention exhibit
minimal or no interference with expression, secretion and folding
of the protein domains of the Wnt antagonist molecules and provide
minimal or no interference with either the effector function of the
Fc domain or Wnt protein interaction function of the Frz domain
(e.g., binding to a Wnt protein) through steric or other means. In
particular embodiments, the linker is short peptide sequence. A
linker sequence may also include additional amino acid residues
from either the Frz domain component or Fc domain outside the
minimal residues needed for activity. Preferred linkers will also
provide for good serum stability and are resistant to protease
cleavage. Specific examples of useful linkers appear in FIG. 4,
FIG. 5, and FIG. 7, including the sequences ESGGGGVT (SEQ ID NO:
69), LESGGGGVT (SEQ ID NO: 70), GRAQVT (SEQ ID NO: 71), WRAQVT (SEQ
ID NO: 72), and ARGRAQVT (SEQ ID NO: 73). As noted above, these
linkers may include additional amino acid residues from either the
Frz domain component or the Fc domain outside the minimal residues
needed for activity. These linkers may also comprise additional
amino acid residues other than those from the Frz domain component
or Fc domain component.
[0098] A "Wnt signaling pathway component" is a component that
transduces a signal originating from an interaction between a Wnt
protein and an Frz receptor. As the Wnt signaling pathway is
complex, and involves extensive feedback regulation, there are
numerous and likely not yet discovered members of the Wnt signaling
pathway. Example Wnt signaling pathway components include the
membrane associated proteins LRP5 and LRP6, Axin, and Dishevelled,
the extracellular Wnt interactive proteins sFRP, WIF-1, the LRP
inactivating proteins Dkk and Krn, the cytoplasmic protein
.beta.-catenin, members of the .beta.-catenin "degradation complex"
APC, GSK3.beta., CKI.alpha. and PP2A, the nuclear transport
proteins APC, pygopus and bcl9/legless, and the transcription
factors TCF/LEF, Groucho and various histone acetylases such as
CBP/p300 and Brg-1.
[0099] A "Wnt-mediated disorder" is a disorder, condition, or
disease state characterized by aberrant Wnt signaling. In a
specific aspect, the aberrant Wnt signaling is a level of Wnt
signaling in a cell or tissue suspected of being diseased that
exceeds the level of Wnt signaling in a similar non-diseased cell
or tissue. In a specific aspect, a Wnt-mediated disorder includes
cancer.
[0100] The term "cancer" refers to the physiological condition in
mammals that is typically characterized by unregulated cell
growth/proliferation. Examples of cancer include, but are not
limited to: carcinoma, lymphoma, blastoma, and leukemia. More
particular examples of cancers include, but are not limited to:
chronic lymphocytic leukemia (CLL), lung, including non small cell
(NSCLC), breast, ovarian, cervical, endometrial, prostate,
colorectal, intestinal carcinoid, bladder, gastric, pancreatic,
hepatic (hepatocellular), hepatoblastoma, esophageal, pulmonary
adenocarcinoma, mesothelioma, synovial sarcoma, osteosarcoma, head
and neck squamous cell carcinoma, juvenile nasopharyngeal
angiofibromas, liposarcoma, thyroid, melanoma, basal cell carcinoma
(BCC), medulloblastoma and desmoid.
[0101] The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0102] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0103] An "active" polypeptide, variant polypeptide, or fragments
thereof, retain a biological activity of native or
naturally-occurring component of the active polypeptide. Biological
activity refers to a function mediated by the native or
naturally-occurring counterpart of the active polypeptide. For
example, binding or a protein-protein interaction constitutes a
biological activity. In a specific sense, an active Wnt signaling
pathway component is one which can effectively transduce a signal
through interaction with other Wnt signaling pathway components. In
another specific sense, an active Wnt antagonist is one which
detectably attenuates Wnt signaling or a physiological condition
resulting therefrom, relative to the level prior to administration
of the Wnt antagonist.
[0104] "Stringency" of hybridization reactions is readily
determinable by one of ordinary skill in the art, and generally is
an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes
require higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured DNA to reanneal when complementary strands
are present in an environment below their melting temperature. The
higher the degree of desired homology between the probe and
hybridizable sequence, the higher the relative temperature which
can be used. As a result, it follows that higher relative
temperatures would tend to make the reaction conditions more
stringent, while lower temperatures less so. For additional details
and explanation of stringency of hybridization reactions, see
Ausubel et al., Current Protocols in Molecular Biology, Wiley
Interscience Publishers, (1995).
[0105] "High stringency conditions", as defined herein, may be
identified by those that: (1) employ low ionic strength and high
temperature for washing, for example 0.015 M sodium chloride/0.0015
M sodium citrate/0.1% sodium dodecyl sulfate at 50.degree. C.; (2)
employ during hybridization a denaturing agent, such as formamide,
for example, 50% (v/v) formamide with 0.1% bovine serum
albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM
sodium citrate at 42.degree. C.; or (3) overnight hybridization in
a solution that employs 50% formamide, 5.times.SSC (0.75 M NaCl,
0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%
sodium pyrophosphate, 5.times.Denhardt's solution, sonicated salmon
sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at
42.degree. C., with a 10 minute wash at 42.degree. C. in
0.2.times.SSC (sodium chloride/sodium citrate) followed by a 10
minute high-stringency wash consisting of 0.1.times.SSC containing
EDTA at 55.degree. C.
[0106] "Moderately stringent conditions" may be identified as
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Press, 1989, and include the
use of washing solution and hybridization conditions (e.g.,
temperature, ionic strength and % SDS) less stringent that those
described above. An example of moderately stringent conditions is
overnight incubation at 37.degree. C. in a solution comprising: 20%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,
followed by washing the filters in 1.times.SSC at about
37-50.degree. C. The skilled artisan will recognize how to adjust
the temperature, ionic strength, etc. as necessary to accommodate
factors such as probe length and the like.
[0107] The term "epitope tagged" refers to a polypeptide that is
fused to a "tag polypeptide." The tag polypeptide has enough
residues to provide an epitope against which an antibody can be
made, yet is short enough such that it does not interfere with
activity of the polypeptide to which it is fused. The tag
polypeptide preferably also is fairly unique so that the antibody
does not substantially cross-react with other epitopes. Suitable
tag polypeptides generally have at least six amino acid residues
and usually between about 8 and 50 amino acid residues (preferably,
between about 10 and 20 amino acid residues). Example epitope tag
sequences include HA, GD, c-myc, poly-His and FLAG.
[0108] "Treating" or "treatment" or "alleviation" refers to both
therapeutic treatment and prophylactic or preventative measures,
wherein the object is to prevent or slow down (lessen) the targeted
pathologic disease or condition or disorder. Those in need of
treatment include those already with the disorder as well as those
prone to having the disorder or those in whom the disorder is to be
prevented (prophylaxis). When the Wnt-mediated disorder is cancer,
a subject or mammal is successfully "treated" or shows a reduced
tumor burden if, after receiving a therapeutic amount of a Wnt
antagonist according to the methods of the present invention, the
patient shows observable and/or measurable reduction in or absence
of one or more of the following: reduction in the number of cancer
cells or absence of the cancer cells; reduction in the tumor size;
inhibition (i.e., slow to some extent and preferably stop) of
cancer cell infiltration into peripheral organs including the
spread of cancer into soft tissue and bone; inhibition (i.e., slow
to some extent and preferably stop) of tumor metastasis;
inhibition, to some extent, of tumor growth; and/or relief to some
extent, one or more of the symptoms associated with the specific
cancer; reduced morbidity and mortality, and improvement in quality
of life issues. To the extent the Wnt antagonist may prevent growth
and/or kill existing cancer cells, it may be cytostatic and/or
cytotoxic. Reduction of these signs or symptoms may also be felt by
the patient.
[0109] The above parameters for assessing successful treatment and
improvement in the disorder are readily measurable by routine
procedures familiar to a physician. For cancer therapy, efficacy
can be measured, for example, by assessing the time to disease
progression (TDP) and/or determining the response rate (RR).
Metastasis can be determined by staging tests and by bone scan and
tests for calcium level and other enzymes to determine spread to
the bone. CT scans can also be done to look for spread to the
pelvis and lymph nodes in the area. Chest X-rays and measurement of
liver enzyme levels by known methods are used to look for
metastasis to the lungs and liver, respectively. Other routine
methods for monitoring the disease include transrectal
ultrasonography (TRUS) and transrectal needle biopsy (TRNB).
[0110] "Chronic" administration refers to administration of the
agent(s) in a continuous mode as opposed to an acute mode, so as to
maintain the initial therapeutic effect (activity) for an extended
period of time. "Intermittent" administration is treatment that is
cyclic, or subject to periodic interruptions, as opposed to
continuous or consecutive.
[0111] "Mammal" refers to any animal classified as a mammal,
including humans, domestic and farm animals, and zoo, sports, or
pet animals, such as dogs, cats, cattle, horses, sheep, pigs,
goats, rabbits, etc. Preferably, the mammal is human.
[0112] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0113] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers which are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.RTM., polyethylene glycol (PEG), and PLURONIC S.RTM..
[0114] An "effective amount" of a Wnt antagonist is an amount
sufficient to carry out a specifically stated purpose. An
"effective amount" may be determined empirically and in a routine
manner, in relation to the stated purpose.
[0115] The term "therapeutically effective amount" refers to an
amount of a Wnt antagonist effective to "treat" a Wnt-mediated
disorder in a subject or mammal. In the case of cancer, the
therapeutically effective amount of the drug may reduce the number
of cancer cells; reduce the tumor size; inhibit (i.e., slow to some
extent and preferably stop) cancer cell infiltration into
peripheral organs; inhibit (i.e., slow to some extent and
preferably stop) tumor metastasis; inhibit, to some extent, tumor
growth; and/or relieve to some extent one or more of the symptoms
associated with the cancer. See the definition herein of
"treating". To the extent the drug may prevent growth and/or kill
existing cancer cells, it may be cytostatic and/or cytotoxic.
[0116] A "growth inhibitory amount" of a Wnt antagonist is an
amount capable of inhibiting the growth of a cell, especially
tumor, e.g., cancer cell, either in vitro or in vivo. A "growth
inhibitory amount" of a Wnt antagonist for purposes of inhibiting
neoplastic cell growth may be determined empirically and in a
routine manner.
[0117] A "cytotoxic amount" of a Wnt antagonist is an amount
capable of causing the destruction of a cell, especially tumor,
e.g., cancer cell, either in vitro or in vivo. A "cytotoxic amount"
of a Wnt antagonist for purposes of inhibiting neoplastic cell
growth may be determined empirically and in a routine manner.
[0118] The terms "antibody" and "immunoglobulin" are used
interchangeably, and in the broadest sense, including monoclonal
antibodies (e.g., full length or intact monoclonal antibodies),
polyclonal antibodies, multivalent antibodies, multispecific
antibodies (e.g., bispecific antibodies exhibiting the desired
biological activity) and may also include certain antibody
fragments, as described herein in greater detail. An antibody can
be chimeric, human, humanized or affinity matured.
[0119] The light chain from any vertebrate species can be assigned
to one of two clearly distinct types, called kappa (.kappa.) and
lambda (.lamda.), based on the amino acid sequences of their
constant domains. Depending on the amino acid sequence of the
constant domain of their heavy chains (C.sub.H), immunoglobulins
can be assigned to different classes or isotypes. There are five
classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having
heavy chains designated .alpha., .delta., .epsilon., .gamma., and
.mu., respectively. The .gamma. and .alpha. classes are further
divided into subclasses on the basis of relatively minor
differences in C.sub.H sequence and function, e.g., humans express
the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
The subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known and described
generally in, for example, Abbas et al., Cellular and Molecular
Biology, 4.sup.th Ed. (2000). An antibody may be part of a larger
fusion molecule, formed by covalent or non-covalent associated of
the antibody with one or more other proteins or peptides.
[0120] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2 and Fv fragments, diabodies, linear antibodies (U.S.
Pat. No. 5,641,870); Zapata et al., Protein Eng. 8(10): 1057-1062
(1995), single chain antibody molecules and multispecific
antibodies formed from antibody fragments.
[0121] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, and a residual
"Fc" fragment, a designation reflecting the ability to crystallize
readily. The Fab fragment consists of an entire light chain along
with the variable region domain of the heavy chain (V.sub.H), and
the first constant domain of one heavy chain (C.sub.H1). Each Fab
fragment is monovalent with respect to antigen binding, i.e., it
has a single antigen-binding site. Pepsin treatment of an antibody
yields a single large F(ab').sub.2 fragment which roughly
corresponds to two disulfide linked Fab fragments having divalent
antigen-binding activity and is still capable of cross-linking
antigen. Fab' fragments differ from Fab fragments by having
additional few residues at the carboxy terminus of the C.sub.H1
domain including one or more cysteines from the antibody hinge
region. Fab'-SH is the designation herein for Fab' in which the
cysteine residue(s) of the constant domains bear a free thiol
group. F(ab').sub.2 antibody fragments originally were produced as
pairs of Fab' fragments which have hinge cysteines between them.
Other chemical couplings of antibody fragments are also known.
[0122] The Fc fragment comprises the carboxy-terminal portions of
both heavy chains held together by disulfides. The effector
functions of antibodies are determined by sequences in the Fc
region, which region is also the part recognized by Fc receptors
(FcR) found on certain types of cells.
[0123] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This fragment
consists of a dimer of one heavy- and one light-chain variable
region domain in tight, non-covalent association.
[0124] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogenous
antibodies, i.e. the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigen. In contract to polyclonal antibody preparations that
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen.
[0125] The term "chimeric" antibody, specifically included within
the definition of monoclonal antibody, means antibodies in which a
portion of the heavy and/or light chain is identical with or
homologous to corresponding sequences in antibodies derived from a
particular species or belonging to a particular antibody class or
subclass, while the remainder of the chain(s) is identical with or
homologous to corresponding sequences derived from another species
or belonging to another antibody class or subclass, as well as
fragment of such antibodies, so long as they exhibit the desired
biological activity U.S. Pat. No. 4,816,567; Morrison et al.,
P.N.A.S. USA 81: 6851-6855 (1984).
[0126] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from the
non-human antibody. For the most part, humanized antibodies are
human immunoglobulins (recipient antibody) in which residues from a
hypervariable region of the recipient are replaced by residues from
a hypervariable region of a non-human species (donor antibody) such
as mouse, rat, rabbit or non-human primate having the desired
antibody specificity, affinity, and capability. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also,
Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115
(1998); Harris, Biochem. Soc. Trans. 23: 1035-1038 (1995); Hurle
and Gross, Curr. Op. Biotech. 5: 428-433 (1994).
[0127] "Polynucleotide" or "nucleic acid" are used interchangeably
herein, and refer to polymers of nucleotides of any length,
including, but are not limited to DNA and RNA. The nucleotides can
be deoxyribonucleotides, ribonucleotides, modified nucleotides or
bases, and/or their analogs, or any substrate that can be
incorporated into a polymer by DNA or RNA polymerase, or by a
synthetic reaction. A polynucleotide may comprise modified
nucleotides, such as methylated nucleotides and their analogs. If
present, modification to the nucleotide structure may be imported
before or after assembly of the polymer. The sequence of
nucleotides may be interrupted by non-nucleotide components. A
polynucleotide may be further modified after synthesis, such as by
conjugation with a label. Other types of modifications include, for
example, "caps", substitution of one or more of the naturally
occurring nucleotides with an analog, internucleotide modifications
such as, for example: uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.);
charged linkages (e.g., phosphorothioates, phosphorodithioates,
etc.); pendant moieties, such as, for example, proteins (e.g.,
nucleases, toxins, antibodies, signal peptides, poly-L-lysine,
etc.); intercalators (e.g., acridine, psoralen, etc.); chelators
(e.g., metals, radioactive metals, boron, oxidative metal, etc.),
alkylators, modified linkages (e.g., alpha anomeric nucleic acids,
etc.). Further, any of the hydroxyl groups ordinarily present in
the sugars may be replaced, for example, by phosphonate groups,
phosphate groups, protected by standard protecting groups, or
activated to prepare additional linkages to additional nucleotides,
or may be conjugated to solid or semi-solid supports. The 5' and 3'
terminal OH can be phosphorylated or substituted with amines or
organic capping group moieties of from 1 to 20 carbon atoms. Other
hydroxyls may also be derivatized to standard protecting groups.
Polynucleotides can also contain analogous forms of ribose or
deoxyribose sugars that are generally known in the art, including,
for example, 2'-O-methyl, 2'-O-allyl, 2'-fluoro- or
2'-azido-ribose, carbocyclic sugar analogs, .alpha.-anomeric
sugars, epimeric sugars such as arabinose, xylose or lyxose,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and abasic nucleoside analogs such as methyl riboside. One or more
phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include, but are not
limited to, embodiments wherein phosphate is replace by
P(O)--S-(thioate), P(S)--S-(dithioate)-, (O)NR.sub.2-amidate,
P(O)R, P(O)OR', CO or CH.sub.2-(formacetal), in which each R or R'
is independently H or substituted or unsubstituted C.sub.1-20
alkyl, optionally containing an ether, aryl, alkenyl, cycloalkenyl
or aralkyl linkage. Not all linkages in a polynucleotide need be
identical. The preceding description applies to all polynucleotides
referred to herein, including RNA and DNA.
[0128] The term "peptide" generally refers to a contiguous and
relatively short sequence of amino acids linked by peptidyl bonds.
Typically, but not necessarily, a peptide has length of about 2 to
50 amino acids, 4-40 amino acids or 10-30 amino acids. Although the
term "protein" generally refers to longer forms of a "polypeptide,"
the two terms can be and are used interchangeably in some contexts
herein, and refer to amino acid sequences that are generally longer
and perhaps more complex (e.g., multiple sequence, secondary and
higher structure).
[0129] A "region" of a polypeptide is a contiguous sequence of 2 or
more amino acid residues. In alternative embodiments, a region is
at least about 3, 5, 10, 15 or more contiguous amino acid
residues.
[0130] "C-terminal region", "C-terminal sequence" and variations
thereof, as used herein, refer to an amino acid sequence that is
located at or in close proximity to the C-terminal (generally 3')
end. Generally, the sequence includes an amino acid that has a free
carboxyl group. In one embodiment, a C-terminal regions or sequence
refers to a region of a polypeptide that includes about 1-15
residues located closest to the C-terminus.
[0131] "N-terminal region", "N-terminal sequence", and variations
thereof, as used herein, refer to an amino acid sequence that is
located at or in close proximity to the N-terminal (generally 5')
end. Generally, the sequence includes an amino acid that has free
amino group. In one embodiment, an N-terminal region or sequence
refers to a region of a polypeptide that includes about 1-15
residues located closest to the N terminus of the polypeptide.
[0132] "Internal region" or "internal sequence", and variations
thereof, refer to an amino acid sequence that is located within a
polypeptide and is flanked on both its N- and C-termini by one or
more amino acids that are not part of the sequence. Generally, the
sequence does not include an amino acid with either a free carboxyl
or amino group.
[0133] A "ligand" refers to a naturally-occurring or synthetic
molecule or moiety that is capable of a binding interaction with a
specific site on a protein or other molecule, such as a receptor. A
Wnt ligand is a molecule that specifically interacts with a
Frizzled receptor. A "receptor" is often, but need not be located
on the cell surface or membrane.
[0134] A "fusion protein" refers to a polypeptide having two
portions covalently linked together, where each of the portions is
derived from different proteins. The two portions may be linked
directly by a single peptide bond or through a peptide linker
containing one or more amino acid residues. Generally, the two
portions and the linker will be in reading frame with each other
and are produced using recombinant techniques.
[0135] A Wnt antagonist that "inhibits the growth of tumor cells"
or a "growth inhibitory" Wnt antagonist is one which results in
measurable growth inhibition of tumor cells having aberrant Wnt
signaling activity. Preferred growth inhibitory Wnt antagonists
inhibit growth of tumor cells having aberrant Wnt signaling
activity by greater than 20%, preferably from about 20% to about
50%, and even more preferably, by greater than 50% (e.g., from
about 50% to about 100%) as compared to the appropriate control,
the control typically being cancer cells not treated with the Wnt
antagonist molecule being tested. In one embodiment, growth
inhibition can be measured at a Wnt antagonist concentration of
about 0.1 to 30 .mu.g/ml or about 0.5 nM to 200 nM in cell culture,
where the growth inhibition is determined 1-10 days after exposure
of the tumor cells to the Wnt antagonist. Growth inhibition of
tumor cells in vivo can be determined in various ways such as is
described in the Experimental Examples section below. The Wnt
antagonist is growth inhibitory in vivo if administration of the
Wnt antagonist at about 1 .mu.g/kg to about 100 mg/kg body weight
results in reduction in tumor size or cell proliferation within
about 5 days to 3 months from the first administration of the
antibody, preferably within about 5 to 30 days. In a specific
aspect, the tumor size is reduced relative to its size at the start
of therapy.
[0136] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one embodiment, the cell
proliferative disorder is cancer.
[0137] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues.
[0138] A Wnt antagonist molecule which "induces cell death" is one
which causes a viable cell to become nonviable. The cell is one
having aberrant Wnt signaling activity as compared to a normal cell
of the same tissue type. Preferably, the cell is a cancer cell, as
defined herein. Cell death in vitro may be determined in the
absence of complement and immune effector cells to distinguish cell
death induced by antibody-dependent cell-mediated cytotoxicity
(ADCC) or complement dependent cytotoxicity (CDC). Thus, the assay
for cell death may be performed using heat inactivated serum (i.e.,
in the absence of complement) and in the absence of immune effector
cells. To determine whether the Wnt antagonist is able to induce
cell death, loss of membrane integrity as evaluated by uptake of
propidium iodide (PI), trypan blue (see Moore et al. Cvtotechnology
17:1-11 (1995)) or 7AAD can be assessed relative to untreated
cells. Preferred cell death-inducing antibodies, oligopeptides or
other organic molecules are those which induce PI uptake in the PI
uptake assay in BT474 cells.
[0139] The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the antibody, oligopeptide or other organic molecule so as to
generate a "labeled" antibody, oligopeptide or other organic
molecule. The label may be detectable by itself (e.g. radioisotope
labels or fluorescent labels) or, in the case of an enzymatic
label, may catalyze chemical alteration of a substrate compound or
composition which is detectable.
[0140] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, and
radioactive isotopes of Lu), chemotherapeutic agents, enzymes and
fragments thereof such as nucleolytic enzymes, antibiotics, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof, and the various antitumor or anticancer
agents disclosed below. Other cytotoxic agents are described below.
A tumoricidal agent causes destruction of tumor cells.
[0141] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,
MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic
acid; a camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gammal I and calicheamicin omegall (see, e.g., Agnew, Chem. Intl.
Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A;
an esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antiobiotic chromophores), aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defo
famine; demecolcine; diaziquone; elformithine; elliptinium acetate;
an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;
lonidainine; maytansinoids such as maytansine and ansamitocins;
mitoguazone; mitoxantrone; mopidanmol; nitraerine; p entostatin;
phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;
procarbazine; PSK.RTM. polysaccharide complex (JHS Natural
Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine
(ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
("Ara-C"); thiotepa; taxoids, e.g., TAXOL.RTM. paclitaxel
(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE.TM.
Cremophor-free, albumin-engineered nanoparticle formulation of
paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),
and TAXOTERE.RTM. doxetaxel (Rhone-Poulenc Rorer, Antony, France);
chloranbucil; gemcitabine (GEMZAR.RTM.); 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine (VELBAN.RTM.); platinum; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN.RTM.);
oxaliplatin; leucovovin; vinorelbine (NAVELBINE.RTM.); novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid; capecitabine (XELODA.RTM.); pharmaceutically
acceptable salts, acids or derivatives of any of the above; as well
as combinations of two or more of the above such as CHOP, an
abbreviation for a combined therapy of cyclophosphamide,
doxorubicin, vincristine, and prednisolone, and FOLFOX, an
abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovovin.
[0142] Also included in this definition are anti-hormonal agents
that act to regulate, reduce, block, or inhibit the effects of
hormones that can promote the growth of cancer, and are often in
the form of systemic, or whole-body treatment. They may be hormones
themselves. Examples include anti-estrogens and selective estrogen
receptor modulators (SERMs), including, for example, tamoxifen
(including NOLVADEX.RTM. tamoxifen), EVISTA.RTM. raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and FARESTON.RTM. toremifene; anti-progesterones;
estrogen receptor down-regulators (ERDs); agents that function to
suppress or shut down the ovaries, for example, leutinizing
hormone-releasing hormone (LHRH) agonists such as LUPRON.RTM. and
ELIGARD.RTM. leuprolide acetate, goserelin acetate, buserelin
acetate and tripterelin; other anti-androgens such as flutamide,
nilutamide and bicalutamide; and aromatase inhibitors that inhibit
the enzyme aromatase, which regulates estrogen production in the
adrenal glands, such as, for example, 4(5)-imidazoles,
aminoglutethimide, MEGASE.RTM. megestrol acetate, AROMASIN.RTM.
exemestane, formestanie, fadrozole, RIVISOR.RTM. vorozole,
FEMARA.RTM. letrozole, and ARIMIDEX.RTM. anastrozole. In addition,
such definition of chemotherapeutic agents includes bisphosphonates
such as clodronate (for example, BONEFOS.RTM. or OSTAC.RTM.),
DIDROCAL.RTM. etidronate, NE-58095, ZOMETA.RTM. zoledronic
acid/zoledronate, FOSAMAX.RTM. alendronate, AREDIA.RTM.
pamidronate, SKELID.RTM. tiludronate, or ACTONEL.RTM. risedronate;
as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine
analog); antisense oligonucleotides, particularly those that
inhibit expression of genes in signaling pathways implicated in
abherant cell proliferation, such as, for example, PKC-alpha, Raf,
H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such
as THERATOPE.RTM. vaccine and gene therapy vaccines, for example,
ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM.
vaccine; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM.
rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase
small-molecule inhibitor also known as GW572016); and
pharmaceutically acceptable salts, acids or derivatives of any of
the above.
[0143] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
a cancer cell having Wnt signaling activity, either in vitro or in
vivo. Thus, the growth inhibitory agent may be one which
significantly reduces the percentage of such cells in S phase.
Examples of growth inhibitory agents include agents that block cell
cycle progression (at a place other than S phase), such as agents
that induce G1 arrest and M-phase arrest. Classical M-phase
blockers include the vincas (vincristine and vinblastine), taxanes,
and topoisomerase II inhibitors such as doxorubicin, epirubicin,
daunorubicin, etoposide, and bleomycin. Those agents that arrest G1
also spill over into S-phase arrest, for example, DNA alkylating
agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine,
cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further
information can be found in The Molecular Basis of Cancer,
Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle
regulation, oncogenes, and antineoplastic drugs" by Murakami et al.
(WB Saunders: Philadelphia, 1995), especially p. 13. The taxanes
(paclitaxel and docetaxel) are anticancer drugs both derived from
the yew tree. Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer),
derived from the European yew, is a semisynthetic analogue of
paclitaxel (TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and
docetaxel promote the assembly of microtubules from tubulin dimers
and stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
[0144] "Doxorubicin" is an anthracycline antibiotic. The full
chemical name of doxorubicin is
(8S-cis)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexapyranosyl)oxy]-7,-
8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-napht-
hacenedione.
[0145] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications and/or warnings
concerning the use of such therapeutic products.
II. Description of Specific Embodiments
[0146] The Wnt antagonists described herein are capable of binding
to Wnt ligands in vitro and are capable of inhibiting or
suppressing Wnt stimulated cell signaling. Additionally, the Wnt
antagonists have a long in vivo half life and exhibit anti-tumor
activity in vivo, inhibiting the growth of Wnt-1 driven tumors in a
mouse MMTV breast tumor model. The Wnt antagonists are also capable
of inhibiting the growth in mice of tumor xenografts derived from
human teratoma cell lines. Regenerative tissues taken from mice
that were treated with a Wnt antagonist appear to be within
physiological norms. The Wnt antagonists are also capable of
inhibiting autocrine Wnt signaling in human tumor cell lines in
vitro.
[0147] The Frizzled receptor proteins can be grouped into families
based on both full-length and extracellular domain sequence
identities. This grouping is illustrated in the alignments shown in
FIGS. 8 and 9. The underlined residues in this figure are conserved
across all the Frz receptors and the shadowed residues are
conserved across homologous groupings. The Frz proteins can be
grouped into the following families 1) Frz1, Frz2, and Frz7 having
a shared homology of 68-77% for the full length sequence and 90%
for the ECD; 2) Frz5 and Frz8 having a shared homology of 57% for
the full length sequence and 80% for the ECD; 3) Frz9 and Frz10
having a shared homology of 61% for the full length sequence and
74% for the ECD; 4) Frz3 and Frz6 having a shared homology of 49%
for the full length sequence and 50% for the ECD; and 5) Frz4
(which exhibits a shared homology of 46% for the full length
sequence and 48% for the ECD with Frz10). The family of Frz1, Frz2,
and Frz7 also has significant homology to Drosophila Frz1 and the
family of Frz5 and Frz8 has significant homology to Drosophila
Frz2, shown to be responsible for planar cell polarity and Wnt
signaling, respectively.
[0148] Wnt ligand-Frizzled binding behavior appears to cluster
within Frizzled families. Both Wnt3a and Wnt5a bind Frz5, Frz8, and
Frz4 fastest relative to the other Frz proteins while Wnt3a binds
Frz1, Frz 2, and Frz7 at a slower rate. The amplitude and linear
nature of Wnt5a binding behavior is indicative of lower binding
affinity, relative to Wnt3a binding, as determined by the OCTET.TM.
binding assay. The presence of both high affinity and low affinity
receptors may confer ability for acute and long term signaling.
[0149] The ability of the Wnt antagonists to inhibit Wnt ligand
induced signaling also appears to cluster within Frizzled families.
Both Frz5 and Frz8 show complete inhibition of the Wnt3a signal and
significant inhibition of the Wnt5a signal in a cell-based assay
(Example 7). Frz4, Frz2, and Frz7 show significant inhibition of
the Wnt3a signal. This finding mirrors the observation in
Drosophila that dFrz2 (with homology to Frz 5 and Frz 8) strongly
activates and dFrz1 (with homology to Frz 1, Frz2, and Frz7) can
weakly activate the Wnt pathway.
[0150] While not being bound to a particular theory of action, the
data presented herein indicate that cell-based Wnt signaling
inhibition data generated using the Wnt antagonists correlates with
data obtained by measuring the direct binding of Wnt ligands to the
Wnt antagonists, indicating that the Wnt antagonists bind directly
to Wnt ligands thus blocking them from binding the full-length
Frizzled receptors on the cell. The data presented herein further
provides validation that in vitro activity can be used to predict
in vivo Wnt signaling blocking activity of the Wnt antagonists.
[0151] As indicated in the studies with Fz8-Fc set forth in the
Examples, the Wnt antagonists comprising both a Frizzled domain and
an immunoglobulin FC domain surprisingly exhibit increased binding
affinity to Wnt ligand over the Frizzled domain alone. For example,
FIG. 14 shows that binding affinity increased over two orders of
magnitude when the Fz ECD domain was converted to the Fz (156)-Fc
construct. The finding of the Fz (156)-Fc construct as a stable and
highly efficacious Wnt signaling inhibitor, in which conjugation to
Fc resulted in a two order of magnitude increase in binding
affinity, was greatly unexpected and non-obvious.
A. Compositions and Methods of the Invention
1. Polypeptides
[0152] The present invention is directed toward compositions and
methods for the treatment of Wnt-mediated disorders, including
cancer, and for inhibiting cellular Wnt signaling. One aspect of
the invention provides Wnt antagonists that are chimeric molecules
comprising a Frizzled (Frz) domain component and an immunoglobulin
Fc domain. In particular embodiments of this aspect, the Frz domain
component and Fc domain are fused through a linker. Another aspect
provides for use of these Wnt antagonists for inhibiting cellular
Wnt signaling and for treatment of Wnt-mediated disorders, such as
cancer.
[0153] In one aspect, the invention provides for Wnt antagonists
that are chimeric molecules with a Frz domain component comprising
a minimal cysteine rich domain (CRD) of an extracellular domain
"CRD (ECD)". The CRD (ECD) is a conserved structural motif of 100
to 250 amino acids and is defined by 10 highly conserved cysteines.
This protein domain appears in two classes of the Wnt signaling
family--the integral membrane Wnt receptor proteins known as
Frizzled, and the secreted extracellular proteins known as the
Frizzled related protein (sFrp).
[0154] In one aspect, the invention provides for Wnt antagonists
that are chimeric molecules having a Frz domain component
comprising a CRD (ECD) of a Frizzled protein such as Frz1, Frz2,
Frz3, Frz4, Frz5, Frz6, Frz7, Frz8, Frz9, or Frz10. Examples of
such CRD (ECD)s are provided in FIG. 3B. In specific embodiments,
the Frz domain component is selected from the group consisting of
CRD (ECD)s of hFrz1 (SEQ ID NO: 18), hFrz2 (SEQ ID NO: 19), hFrz3
(SEQ ID NO: 20), hFrz4 (SEQ ID NO: 21), hFrz5 (SEQ ID NO: 22),
hFrz6 (SEQ ID NO: 23), hFrz7 (SEQ ID NO: 24), hFrz8 (SEQ ID NO:
25), hFrz9 (SEQ ID NO: 26), and hFrz10 (SEQ ID NO: 27), and active
variants thereof.
[0155] Alternatively, the Frz domain component comprises, for
example, a CRD (ECD) from a secreted Frizzled related protein
(sFRP) such as sFRP1, sFRP2, sFRP3, sFRP4, or sFRP5. Examples of
such CRD (ECD)s are provided in FIG. 3B. In specific embodiments,
the Frz domain component is selected from the group consisting CRD
(ECD)s of sFRP1 (SEQ ID NO: 28), sFRP2 (SEQ ID NO: 29), sFRP3 (SEQ
ID NO: 30), sFRP4 (SEQ ID NO: 31), sFRP5 (SEQ ID NO: 32), and
active variants thereof.
[0156] Alternatively, the Frz domain component comprises, for
example, a CRD(ECD) of the receptor tyrosine kinases Rorl and Ror2.
Examples of such CRD (ECD)s are provided in FIG. 3B. In specific
embodiments, the Frz domain component is selected from the group
consisting of CRD (ECD)s of hRorl (SEQ ID NO: 33), and hRor2 (SEQ
ID NO: 34), and active variants thereof.
[0157] In another aspect, the Frz domain component is a pro-Frz or
pro-sFrp sequence, examples of which are shown in FIG. 3A. In
specific embodiments, the Frz domain component is selected from the
group consisting of hFrz1 (SEQ ID NO: 35), hFrz2 (SEQ ID NO: 36),
hFrz3 (SEQ ID NO: 37), hFrz4 (SEQ ID NO: 38), hFrz5 (SEQ ID NO:
39), hFrz6 (SEQ ID NO: 40), hFrz7 (SEQ ID NO: 41), hFrz8 (SEQ ID
NO: 42), hFrz9 (SEQ ID NO: 43), hFrz10 (SEQ ID NO: 44), sFRP1 (SEQ
ID NO: 45), sFRP2 (SEQ ID NO: 46), sFRP3 (SEQ ID NO: 47), sFRP4
(SEQ ID NO: 48), and sFRP5 (SEQ ID NO: 49), and active variants
thereof.
[0158] In yet another aspect, the Frz domain component is derived
from a mature Frz, sFRP or hRor sequence, examples of which are
shown in FIG. 3B. In specific embodiments, the Frz domain component
is selected from the group consisting of hFrz1 (SEQ ID NO: 50),
hFrz2 (SEQ ID NO: 51), hFrz3 (SEQ ID NO: 52), hFrz4 (SEQ ID NO:
53), hFrz5 (SEQ ID NO: 54), hFrz6 (SEQ ID NO: 55), hFrz7 (SEQ ID
NO: 56), hFrz8 (SEQ ID NO: 57), hFrz9 (SEQ ID NO: 58), hFrz10 (SEQ
ID NO: 59), sFRP1 (SEQ ID NO: 60), sFRP2 (SEQ ID NO: 61), sFRP3
(SEQ ID NO: 62), sFRP4 (SEQ ID NO: 63), sFRP5 (SEQ ID NO: 64),
hRorl (SEQ ID NO: 65), and hRor2 (SEQ ID NO: 66), and active
variants thereof.
[0159] In particular embodiments, the Frz domain component and the
immunoglobulin Fc domain of the chimeric Wnt antagonist molecules
are fused through a linker. In one embodiment, the linker is a
peptide linker. In another embodiment, the linker is selected from
the group consisting of ESGGGGVT (SEQ ID NO: 69), LESGGGGVT (SEQ ID
NO: 70), GRAQVT (SEQ ID NO: 71), WRAQVT (SEQ ID NO: 72), and
ARGRAQVT (SEQ ID NO: 73). Optionally, the linkers may include
additional amino acid residues from either the Frz domain component
or the Fc domain outside the minimal residues needed for activity.
These linkers may also comprise additional amino acid residues
other than those from the Frz domain component or Fc domain
component.
[0160] In one embodiment, the Wnt antagonist is Frz8-Fc chimera
comprising a Frz8 CRD (ECD) and a Fc domain. In some embodiments,
the Frz8-Fc chimera further comprises a linker, such as a peptide
linker In a further embodiment, the Frz8-Fc further comprises a
leader sequence. In a particular embodiment, the Frz domain
component comprises amino acids 1-156 of the Frz8 protein (SEQ ID
NO: 8). In another embodiment, the Fc component is a human Fc. In a
further embodiment, the Fc component is a human IgG Fc. In yet a
further embodiment, the Frz8-Fc has a Frz domain component
comprising amino acids 1-156 of the Frz8 protein fused with a
linker to a human IgG Fc. In a further embodiment, the Frz8-Fc is a
chimera with the amino acid sequence as shown in FIG. 4B (SEQ ID
NO: 74). As used in the Examples and accompanying Figures, unless
otherwise noted, "Frz8-Fc" refers to the chimera shown in FIG. 4B
(SEQ ID NO: 74).
[0161] In a further embodiment, the Wnt antagonist is FrzS-Fc
chimera comprising a FrzS CRD (ECD) and a Fc domain. In some
embodiments, the FrzS-Fc chimera further comprises a linker, such
as a peptide linker. In a further embodiment, the FrzS-Fc further
comprises a leader sequence. In a particular embodiment, the Frz
domain component comprises amino acids 27-155 of the FrzS protein
(SEQ ID NO: 5). In another embodiment, the Fc component is a human
Fc. In a further embodiment, the Fc component is a human IgG Fc. In
yet a further embodiment, the FrzS-Fc has a leader sequence and a
Frz domain component comprising amino acids 27-155 of a mature FrzS
protein fused with a linker to a human IgG Fc. In a further
embodiment, the FrzS-Fc is a chimera with the amino acid sequence
as shown in FIG. 7A (SEQ ID NO: 75). As used in the Examples and
accompanying Figures, unless otherwise noted, "Frz5-Fc" refers to
the chimera shown in FIG. 7A (SEQ ID NO: 75).
[0162] Similarly, further embodiments include Frz1-Fc, Frz2-Fc,
Frz3-Fc, Frz4-Fc, Frz6-Fc, Frz7-Fc, Frz9-Fc, Frz-10-Fc, sFRP1-Fc,
sFRP2-Fc, sFRP3-Fc, sFRP4-Fc and sFRP5-Fc chimeras comprising a Frz
domain component comprising a Frz CRD (ECD) from each respective
Frz or sFRP protein and a Fc component. In some embodiments, the
Frz-Fc chimera further comprises a linker, such as a peptide
linker. In further embodiments, these chimeras comprise a leader
sequence. In some embodiments, the Fc component is a human Fc. In
further embodiments, the Fc component is a human IgG Fc. In yet
further embodiments, these chimeras have a leader sequence and a
Frz CRD (ECD) fused with a linker to a human IgG Fc. In further
embodiment, these chimeras have the amino acid sequences as shown
in FIGS. 7A, 7B and 7C (SEQ ID NOs: 76-88). As used in the Examples
and accompanying Figures, unless otherwise noted, "Frz1-Fc,
Frz2-Fc, Frz3-Fc, Frz4-Fc, Frz6-Fc, Frz7-Fc, Frz9-Fc, Frz-10-Fc,
sFRP1-Fc, sFRP2-Fc, and sFRP4-Fc" refer to the respective chimeras
shown in FIGS. 7A, 7B and 7C (SEQ ID NOs: 76-85, and 87).
[0163] The Wnt antagonists are stable in vivo. Prior constructs
utilizing a Frizzled domain attached to a Fc component were rapidly
degraded in vivo making them unsuitable for use as therapeutic
compounds (Hsieh, J-C. et al., PNAS, 96: 3546-3551 (1999)). The Wnt
antagonists described herein remain stable in vivo for
substantially longer than the prior constructs. As shown in Example
4 (FIG. 13), the Frz8-Fc Wnt antagonist displayed an in vivo
half-life of about 4 days. Accordingly, the invention provides for
Wnt antagonists that have an in vivo half-life of at least 1 day, 2
days, 3 days, or 4 days after being administered to a mammal.
[0164] Furthermore, as shown in Example 3 (FIG. 11), the Wnt
antagonists retain activity in vivo for substantially longer than
the prior constructs. In one embodiment, the Wnt antagonist is
active for at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours,
5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours,
18 hours, 20 hours, 22 hours, 24 hours, 30 hours, 36 hours, 40
hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours,
68 hours, 72 hours, 80 hours, 90 hours, or 100 hours after being
administered to a mammal. Activity is measured, for example, by
testing the serum of the mammal administered the Wnt antagonist for
the ability to inhibit Wnt signaling as set forth in Examples 3 and
11, or by using other methods known in the art.
2. Nucleic Acids
[0165] One aspect of the invention provides for a nucleic acid
encoding the Wnt antagonists described herein. In specific
embodiments, the nucleic acid encodes a Wnt antagonist comprising a
CRD (ECD)s of hFrz1 (SEQ ID NO: 18), hFrz2 (SEQ ID NO: 19), hFrz3
(SEQ ID NO: 20), hFrz4 (SEQ ID NO: 21), hFrz5 (SEQ ID NO: 22),
hFrz6 (SEQ ID NO: 23), hFrz7 (SEQ ID NO: 24), hFrz8 (SEQ ID NO:
25), hFrz9 (SEQ ID NO: 26), hFrz10 (SEQ ID NO: 27), sFRP1 (SEQ ID
NO: 28), sFRP2 (SEQ ID NO: 29), sFRP3 (SEQ ID NO: 30), sFRP4 (SEQ
ID NO: 31), sFRP5 (SEQ ID NO: 32), hRorl (SEQ ID NO: 33), or hRor2
(SEQ ID NO: 34).
[0166] In other embodiments, the nucleic acid encodes a Wnt
antagonist comprising a pro-Frz or pro-sFrp proteins selected from
among hFrz1 (SEQ ID NO: 35), hFrz2 (SEQ ID NO: 36), hFrz3 (SEQ ID
NO: 37), hFrz4 (SEQ ID NO: 38), hFrz5 (SEQ ID NO: 39), hFrz6 (SEQ
ID NO: 40), hFrz7 (SEQ ID NO: 41), hFrz8 (SEQ ID NO: 42), hFrz9
(SEQ ID NO: 43), hFrz10 (SEQ ID NO: 44), sFRP1 (SEQ ID NO: 45),
sFRP2 (SEQ ID NO: 46), sFRP3 (SEQ ID NO: 47), sFRP4 (SEQ ID NO:
48), and sFRP5 (SEQ ID NO: 49).
[0167] In still other embodiments, the nucleic acid encodes a Wnt
antagonist comprising a mature Frz, sFRP or hRor proteins selected
from among hFrz1 (SEQ ID NO: 50), hFrz2 (SEQ ID NO: 51), hFrz3 (SEQ
ID NO: 52), hFrz4 (SEQ ID NO: 53), hFrz5 (SEQ ID NO: 54), hFrz6
(SEQ ID NO: 55), hFrz7 (SEQ ID NO: 56), hFrz8 (SEQ ID NO: 57),
hFrz9 (SEQ ID NO: 58), hFrz10 (SEQ ID NO: 59), sFRP1 (SEQ ID NO:
60), sFRP2 (SEQ ID NO: 61), sFRP3 (SEQ ID NO: 62), sFRP4 (SEQ ID
NO: 63), sFRP5 (SEQ ID NO: 64), hRorl (SEQ ID NO: 65), and hRor2
(SEQ ID NO: 66).
[0168] In still other embodiments, the nucleic acid encodes a Wnt
antagonist comprising a Frz8-Fc (SEQ ID NO: 74), FrzS-Fc (SEQ ID
NO: 75), Frz1-Fc (SEQ ID NO: 76), Frz2-Fc (SEQ ID NO: 77), Frz3-Fc
(SEQ ID NO: 78), Frz4-Fc (SEQ ID NO: 79), Frz6-Fc (SEQ ID NO: 80),
Frz7-Fc (SEQ ID NO: 81), Frz9-Fc (SEQ ID NO: 82), Frz10-Fc (SEQ ID
NO: 83), sFRP1-Fc (SEQ ID NO: 84), sFRP2 (SEQ ID NO: 85), sFRP3-Fc
(SEQ ID NO: 86), sFRP4-Fc (SEQ ID NO: 87), or sFRP5-Fc (SEQ ID NO:
88).
[0169] In one particular embodiment, the nucleic acid encodes a
Frz8-Fc and comprises the nucleic acid sequence shown in SEQ ID NO:
122 (FIG. 5D). In another embodiment, the nucleic acid encodes a
FrzS-Fc and comprises the nucleic acid sequence shown in SEQ ID NO:
119 (FIG. 5C). In yet further embodiments, the nucleic acid encodes
a Frz1-Fc, Frz2-Fc, Frz3-Fc, Frz4-Fc, Frz6-Fc, Frz7-Fc, Frz9-Fc,
Frz10-Fc, sFRP1-Fc, sFRP2, sFRP3-Fc, sFRP4-Fc, or sFRP5-Fc and
comprises a nucleic acid sequence shown in FIG. 5 (A-H). For
example, the nucleic acid comprises a Frz1-Fc (SEQ ID NO: 115),
Frz2-Fc (SEQ ID NO: 116), Frz3-Fc (SEQ ID NO: 117), Frz4-Fc (SEQ ID
NO: 118), Frz5-Fc (SEQ ID NO: 119), Frz6-Fc (SEQ ID NO: 120),
Frz7-Fc (SEQ ID NO: 121), Frz8-Fc (SEQ ID NO: 122), Frz9-Fc (SEQ ID
NO: 123), Frz10-Fc (SEQ ID NO: 124), sFRP1-Fc (SEQ ID NO: 125),
sFRP2-Fc (SEQ ID NO: 126), sFRP3-Fc (SEQ ID NO: 127), sFRP4-Fc (SEQ
ID NO: 128), or sFRP5-Fc (SEQ ID NO:129).
[0170] Another aspect of the invention provides for nucleic acids
that hybridize under high stringency conditions to the nucleic
acids described above.
3. Wnt Antagonist Variants
[0171] In addition to the Wnt antagonist polypeptides described
herein, it is contemplated that Wnt antagonist variants can be
prepared. Such variants can be prepared by introducing appropriate
nucleotide changes into the encoding DNA, and/or by synthesis of
the desired variant. Those skilled in the art will appreciate that
amino acid changes may alter post-translational processes of the
Wnt antagonist, such as changing the number or position of
glycosylation sites or altering the membrane anchoring
characteristics.
[0172] A Wnt antagonist variant includes, for example, a mutation
or amino acid variant in an amino acid residue in one or more
domains, while still retaining biological activity. A Wnt
antagonist variant also includes Wnt antagonists having at least
one amino acid deletion or addition, while still retaining
biological activity. The addition or deletion of the amino acid
residues can particularly occur in the region surrounding the amino
acid sequence where the Frz domain component and Fc domain are
connected, whether or not such region contains a linker. Wnt
antagonist variants have at least 70%, 75%, 80%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino
acid sequence identity with a reference Wnt antagonist polypeptide
sequence. In general such variants exhibit substantially the same
or greater binding affinity to a Wnt protein than the reference
sequence, e.g., at least 0.75.times., 0.8.times., 0.9.times.,
1.0.times., 1.25.times. or 1.5.times., based on an art-accepted
binding assay quantitation unit/metric.
[0173] In specific embodiments, the Wnt antagonist variant is a
chimeric molecule comprising a Frz domain component having at least
70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% amino acid sequence identity with the CRD
(ECD)s of hFrz1 (SEQ ID NO: 18), hFrz2 (SEQ ID NO: 19), hFrz3 (SEQ
ID NO: 20), hFrz4 (SEQ ID NO: 21), hFrz5 (SEQ ID NO: 22), hFrz6
(SEQ ID NO: 23), hFrz7 (SEQ ID NO: 24), hFrz8 (SEQ ID NO: 25),
hFrz9 (SEQ ID NO: 26), hFrz10 (SEQ ID NO: 27), sFRP1 (SEQ ID NO:
28), sFRP2 (SEQ ID NO: 29), sFRP3 (SEQ ID NO: 30), sFRP4 (SEQ ID
NO: 31), sFRP5 (SEQ ID NO: 32), hRorl (SEQ ID NO: 33), or hRor2
(SEQ ID NO: 34).
[0174] In other embodiments, the Wnt antagonist variant is a
chimeric molecule comprising a Frz domain component having at least
70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% amino acid sequence identity with a
pro-Frz or pro-sFrp proteins selected from among hFrz1 (SEQ ID NO:
35), hFrz2 (SEQ ID NO: 36), hFrz3 (SEQ ID NO: 37), hFrz4 (SEQ ID
NO: 38), hFrz5 (SEQ ID NO: 39), hFrz6 (SEQ ID NO: 40), hFrz7 (SEQ
ID NO: 41), hFrz8 (SEQ ID NO: 42), hFrz9 (SEQ ID NO: 43), hFrz10
(SEQ ID NO: 44), sFRP1 (SEQ ID NO: 45), sFRP2 (SEQ ID NO: 46),
sFRP3 (SEQ ID NO: 47), sFRP4 (SEQ ID NO: 48), and sFRP5 (SEQ ID NO:
49).
[0175] In still other embodiments, the Wnt antagonist variant is a
chimeric molecule comprising a Frz domain component having at least
70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% amino acid sequence identity with mature
Frz, sFRP or hRor proteins selected from among hFrz1 (SEQ ID NO:
50), hFrz2 (SEQ ID NO: 51), hFrz3 (SEQ ID NO: 52), hFrz4 (SEQ ID
NO: 53), hFrz5 (SEQ ID NO: 54), hFrz6 (SEQ ID NO: 55), hFrz7 (SEQ
ID NO: 56), hFrz8 (SEQ ID NO: 57), hFrz9 (SEQ ID NO: 58), hFrz10
(SEQ ID NO: 59), sFRP1 (SEQ ID NO: 60), sFRP2 (SEQ ID NO: 61),
sFRP3 (SEQ ID NO: 62), sFRP4 (SEQ ID NO: 63), sFRP5 (SEQ ID NO:
64), hRorl (SEQ ID NO: 65), and hRor2 (SEQ ID NO: 66).
[0176] In still other embodiments, the Wnt antagonist variant is a
chimeric molecule comprising a Frz domain component having at least
70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% amino acid sequence identity with Frz8-Fc
(SEQ ID NO: 74), Frz5-Fc (SEQ ID NO: 75), Frz1-Fc (SEQ ID NO: 76),
Frz2-Fc (SEQ ID NO: 77), Frz3-Fc (SEQ ID NO: 78), Frz4-Fc (SEQ ID
NO: 79), Frz6-Fc (SEQ ID NO: 80), Frz7-Fc (SEQ ID NO: 81), Frz9-Fc
(SEQ ID NO: 82), Frz10-Fc (SEQ ID NO: 83), sFRP1-Fc (SEQ ID NO:
84), sFRP2 (SEQ ID NO: 85), sFRP3-Fc (SEQ ID NO: 86), sFRP4-Fc (SEQ
ID NO: 87), and sFRP5-Fc (SEQ ID NO: 88).
[0177] "Percent (%) amino acid sequence identity" is defined as the
percentage of amino acid residues that are identical with amino
acid residues in a reference (parent) polypeptide sequence when the
two sequences are aligned. To determine % amino acid identity,
sequences are aligned and if necessary, gaps are introduced to
achieve the maximum % sequence identity; conservative substitutions
are not considered as part of the sequence identity. Amino acid
sequence alignment procedures to determine percent identity are
well known to those of skill in the art. Often publicly available
computer software such as BLAST, BLAST2, ALIGN2 or Megalign
(DNASTAR) software is used to align peptide sequences. Those
skilled in the art can determine appropriate parameters for
measuring alignment, including any algorithms needed to achieve
maximal alignment over the full length of the sequences being
compared.
[0178] When amino acid sequences are aligned, the % amino acid
sequence identity of a given amino acid sequence A to, with, or
against a given amino acid sequence B (which can alternatively be
phrased as a given amino acid sequence A that has or comprises a
certain % amino acid sequence identity to, with, or against a given
amino acid sequence B) can be calculated as:
% amino acid sequence identity=X/Y.times.100
[0179] where
[0180] X is the number of amino acid residues scored as identical
matches by the sequence alignment program's or algorithm's
alignment of A and B
[0181] and
[0182] Y is the total number of amino acid residues in B.
[0183] If the length of amino acid sequence A is not equal to the
length of amino acid sequence B, the % amino acid sequence identity
of A to B will not equal the % amino acid sequence identity of B to
A.
[0184] An "isolated" or "purified" peptide, polypeptide, protein or
biologically active fragment is separated and/or recovered from a
component of its natural environment. Contaminant components
include materials that would typically interfere with diagnostic or
therapeutic uses for the polypeptide, and may include enzymes,
hormones, and other proteinaceous or non-proteinaceous materials.
Preparations having preferably less than 30% by dry weight of
non-desired contaminating material (contaminants), preferably less
than 20%, 10%, and preferably less than 5% contaminants are
considered to be substantially isolated. An isolated,
recombinantly-produced peptide/polypeptide or biologically active
portion thereof is preferably substantially free of culture medium,
i.e., culture medium represents preferably less than 20%,
preferably less than about 10%, and preferably less than about 5%
of the volume of a peptide/polypeptide preparation. Examples of
contaminants include cell debris, culture media, and substances
used and produced during in vitro synthesis of the
peptide/polypeptide.
[0185] Variations in the Wnt antagonist described herein, can be
made, for example, using any of the techniques and guidelines for
conservative and non-conservative mutations set forth, for
instance, in U.S. Pat. No. 5,364,934. Variations may be a
substitution, deletion or insertion of one or more codons encoding
the antibody or polypeptide that results in a change in the amino
acid sequence as compared with the native sequence antibody or
polypeptide. Optionally the variation is by substitution of at
least one amino acid with any other amino acid in one or more of
the domains Wnt antagonist. Guidance in determining which amino
acid residue may be inserted, substituted or deleted without
adversely affecting the desired activity may be found by comparing
the sequence of the Wnt antagonist with that of homologous known
protein molecules and minimizing the number of amino acid sequence
changes made in regions of high homology. Amino acid substitutions
can be the result of replacing one amino acid with another amino
acid having similar structural and/or chemical properties, such as
the replacement of a leucine with a serine, i.e., conservative
amino acid replacements. Insertions, deletions or substitutions may
optionally be in the range of about 1 to 5 amino acids. The
variation allowed may be determined by systematically making
insertions, deletions or substitutions of amino acids in the
sequence and testing the resulting variants for activity exhibited
by the full-length or mature native sequence.
[0186] Wnt antagonists may be prepared by any of a number of
conventional techniques. Desired peptide fragments may be
chemically synthesized. An alternative approach involves generating
antibody or polypeptide fragments by enzymatic digestion, e.g., by
treating the protein with an enzyme known to cleave proteins at
sites defined by particular amino acid residues, or by digesting
the DNA with suitable restriction enzymes and isolating the desired
fragment. Yet another suitable technique involves isolating and
amplifying a DNA fragment encoding a desired antibody or
polypeptide fragment, by polymerase chain reaction (PCR).
Oligonucleotides that define the desired termini of the DNA
fragment are employed at the 5' and 3' primers in the PCR.
[0187] In particular embodiments, conservative substitutions of
interest are shown in Table A under the heading of preferred
substitutions. If such substitutions result in a change in
biological activity, then more substantial changes, denominated
exemplary substitutions in Table A, or as further described below
in reference to amino acid classes, are introduced and the products
screened.
TABLE-US-00001 TABLE A Original Preferred Residue Exemplary
Substitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys;
gln; asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn
glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp
Gly (G) ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val;
met; ala; phe; norleucine leu Leu (L) norleucine; ile; val; met;
ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu
Phe (F) leu; val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) thr;
cys cys Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr;
ser phe Val (V) ile; leu; met; phe; ala; norleucine leu
[0188] Substantial modifications in function or immunological
identity of the Wnt antagonist are accomplished by selecting
substitutions that differ significantly in their effect on
maintaining (a) the structure of the polypeptide backbone in the
area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain. Naturally
occurring residues are divided into groups based on common
side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral
hydrophilic: Cys, Ser, Thr; Asn; Gln (3) acidic: Asp, Glu; (4)
basic: H is, Lys, Arg; (5) residues that influence chain
orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
[0189] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Such substituted
residues also may be introduced into the conservative substitution
sites or, more preferably, into the remaining (non-conserved)
sites.
[0190] The variations can be made using methods known in the art
such as oligonucleotide-mediated (site-directed) mutagenesis,
alanine scanning, and PCR mutagenesis. Site-directed mutagenesis
[Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al.,
Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et
al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells
et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or
other known techniques can be performed on the cloned DNA to
produce the Wnt antagonists of the invention.
[0191] Scanning amino acid analysis can also be employed to
identify one or more amino acids along a contiguous sequence. Among
the preferred scanning amino acids are relatively small, neutral
amino acids. Such amino acids include alanine, glycine, serine, and
cysteine. Alanine is typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the
beta-carbon and is less likely to alter the main-chain conformation
of the variant [Cunningham and Wells, Science, 244:1081-1085
(1989)]. Alanine is also typically preferred because it is the most
common amino acid.
[0192] Further, it is frequently found in both buried and exposed
positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.);
Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does
not yield adequate amounts of variant, an isoteric amino acid can
be used.
[0193] Any cysteine residue not involved in maintaining the proper
conformation of the Wnt antagonist may also be substituted,
generally with serine, to improve the oxidative stability of the
molecule and prevent aberrant crosslinking. Conversely, cysteine
bond(s) may be added to the Wnt antagonist to improve its stability
(particularly where the antibody is an antibody fragment such as an
Fv fragment).
[0194] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody (e.g., a humanized or human antibody). Generally,
the resulting variant(s) selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitutional variants involves affinity maturation using phage
display. Briefly, several hypervariable region sites (e.g., 6-7
sites) are mutated to generate all possible amino substitutions at
each site. The antibody variants thus generated are displayed in a
monovalent fashion from filamentous phage particles as fusions to
the gene III product of M13 packaged within each particle. The
phage-displayed variants are then screened for their biological
activity (e.g., binding affinity) as herein disclosed. In order to
identify candidate hypervariable region sites for modification,
alanine scanning mutagenesis can be performed to identify
hypervariable region residues contributing significantly to antigen
binding. Alternatively, or additionally, it may be beneficial to
analyze a crystal structure of the antigen-antibody complex to
identify contact points between the Wnt antagonist and Wnt protein.
Such contact residues and neighboring residues are candidates for
substitution according to the techniques elaborated herein. Once
such variants are generated, the panel of variants is subjected to
screening as described herein and antibodies with superior
properties in one or more relevant assays may be selected for
further development.
[0195] Covalent modifications of Wnt antagonists are included
within the scope of this invention. One type of covalent
modification includes reacting targeted amino acid residues of a
Wnt antagonist with an organic derivatizing agent that is capable
of reacting with selected side chains or the N- or C-terminal
residues of the Wnt antagonist. Derivatization with bifunctional
agents is useful, for instance, for crosslinking the Wnt antagonist
to a water-insoluble support matrix or surface for use in the
method for purifying Wnt antagonists. Commonly used crosslinking
agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,
glutaraldehyde, N-hydroxysuccinimide esters, for example, esters
with 4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0196] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the .alpha.-amino groups of lysine, arginine, and
histidine side chains [T. E. Creighton, Proteins: Structure and
Molecular Properties, W.H. Freeman & Co., San Francisco, pp.
79-86 (1983)], acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
[0197] Another type of covalent modification of the Wnt antagonist
included within the scope of this invention comprises altering the
native glycosylation pattern of the Frz, Wnt or sFRP polypeptide
domains of the Wnt antagonist. "Altering the native glycosylation
pattern" is defined as deleting one or more carbohydrate moieties
found in native sequence of the component domains (either by
removing the underlying glycosylation site or by deleting the
glycosylation by chemical and/or enzymatic means), and/or adding
one or more glycosylation sites that are not present in the native
sequence component domain. In addition, the phrase includes
qualitative changes in the glycosylation of the native proteins,
involving a change in the nature and proportions of the various
carbohydrate moieties present.
[0198] Glycosylation of antibodies and other polypeptides is
typically either N-linked or O-linked N-linked refers to the
attachment of the carbohydrate moiety to the side chain of an
asparagine residue. The tripeptide sequences asparagine-X-serine
and asparagine-X-threonine, where X is any amino acid except
proline, are the recognition sequences for enzymatic attachment of
the carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars N-aceylgalactosamine,
galactose, or xylose to a hydroxyamino acid, most commonly serine
or threonine, although 5-hydroxyproline or 5-hydroxylysine may also
be used.
[0199] Addition of glycosylation sites to the Wnt antagonist is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the sequence of the
original (i.e., pre-variant) Wnt antagonist. This sequence may
optionally be altered through changes at the DNA level,
particularly by mutating the DNA encoding the sequence at
preselected bases such that codons are generated that will
translate into the desired amino acids.
[0200] Another means of increasing the number of carbohydrate
moieties on the Wnt antagonist is by chemical or enzymatic coupling
of glycosides to the polypeptide. Such methods are described in the
art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin and
Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
[0201] Removal of carbohydrate moieties present on the Wnt
antagonist may be accomplished chemically or enzymatically or by
mutational substitution of codons encoding for amino acid residues
that serve as targets for glycosylation. Chemical deglycosylation
techniques are known in the art and described, for instance, by
Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by
Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of
carbohydrate moieties on polypeptides can be achieved by the use of
a variety of endo- and exo-glycosidases as described by Thotakura
et al., Meth. Enzymol., 138:350 (1987).
[0202] Another type of covalent modification of Wnt antagonist
comprises linking the sequence to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol (PEG),
polypropylene glycol, or polyoxyalkylenes, in the manner set forth
in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417;
4,791,192 or 4,179,337. The antibody or polypeptide also may be
entrapped in microcapsules prepared, for example, by coacervation
techniques or by interfacial polymerization (for example,
hydroxymethylcellulose or gelatin-microcapsules and
poly-(methylmethacylate) microcapsules, respectively), in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, micro emulsions, nano-particles and nanocapsules), or
in macroemulsions. Such techniques are disclosed in Remington: The
Science and Practice of Pharmacy, 20th edition, Gennaro, A., Ed.,
(2000).
[0203] The Wnt antagonists of the present invention may also be
modified in a way to form molecules having additional chimeric
nature, comprising a Wnt antagonist (i.e., Frz-, sFRP- or Ror-Fc
chimera) fused to another, heterologous polypeptide or amino acid
sequence.
[0204] In one embodiment, such a chimeric molecule comprises a
fusion of the Wnt antagonist with a tag polypeptide which provides
an epitope to which an anti-tag antibody can selectively bind. The
epitope tag is generally placed at the amino- or carboxyl-terminus
of the Wnt antagonist. The presence of such epitope-tagged forms of
the Wnt antagonist can be detected using an antibody against the
tag polypeptide. Also, provision of the epitope tag enables the Wnt
antagonist to be readily purified by affinity purification using an
anti-tag antibody or another type of affinity matrix that binds to
the epitope tag. Various tag polypeptides and their respective
antibodies are well known in the art. Examples include
poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly)
tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et
al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the
8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al.,
Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes
Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et
al., Protein Engineering, 3(6):547-553 (1990)]. Other tag
polypeptides include the Flag-peptide [Hopp et al., BioTechnology,
6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al.,
Science, 255:192-194 (1992)]; an .alpha.-tubulin epitope peptide
[Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the
T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl.
Acad. Sci. USA, 87:6393-6397 (1990)].
[0205] In alternative embodiments, the Wnt antagonists comprise a
variant Fc component. For example, the Fc region may comprise a
human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc
region) comprising an amino acid modification (e.g, a substitution)
at one or more amino acid positions including that of a hinge
cysteine. In one embodiment, such variants have at least 70%, 75%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% amino acid sequence identity with a reference Fc
polypeptide sequence.
[0206] In one embodiment, the Fc region variant may display altered
neonatal Fc receptor (FcRn) binding affinity. Such variant Fc
regions may comprise an amino acid modification oat any one or more
of amino acid positions 238, 252, 253, 254, 255, 256, 265, 272,
286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362,
376, 378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435,
436, 439 or 447 of the Fc region, wherein the numbering of the
residues in the Fc region is that of the EU index as in Kabat. Fc
region variants with reduced binding to an FcRn may comprise an
amino acid modification at any one or more of amino acid positions
252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435, 436,
439 or 447 of Fc region (EU index/Kabat numbering). Alternatively,
variants displaying increased binding to FcRn may comprise an amino
acid modification at any one or more of amino acid positions 238,
256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360,
362, 376, 378, 380, 382, 413, 424 or 434 of the Fc region (EU
index/Kabat numbering).
[0207] In another embodiment, the Fc region variant may display
reduced binding to an Fc.gamma.R, and comprises amino acid
modifications at positions 238, 239, 248, 249, 252, 254, 265, 268,
269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 298, 301, 303,
322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389,
414, 416, 419, 434, 435, 437, 438 or 439 of the Fc region (EU
index/Kabat numbering).
[0208] In yet another embodiment, the Fc region variant may display
reduced binding to Fc.gamma.RII and comprises amino acid
modifications at any one or more of amino acid positions 238, 265,
269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 335, 338,
373, 376, 414, 416, 419, 435, 438 or 439 of the Fc region (EU
index/Kabat numbering).
[0209] In a further embodiment, the Fc region variant may display
enhanced binding to Fc.gamma.RII, and comprises an amino acid
modification at any one or more of amino acid positions 238, 265,
269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 338, 373,
376, 414, 416, 419, 435, 438 or 439 of the Fc region (EU
index/Kabat numbering).
[0210] In a still further embodiment, the Fc region variant of
interest may display reduced binding to an FcgRIII, and comprises
an amino acid modification at one or more amino acid positions 238,
239, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293, 294,
295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388,
389, 416, 434, 435 or 437 of the Fc region (EU index/Kabat
numbering).
[0211] In a still further embodiment, Fc region variants with
altered (i.e, improved or diminished) Clq binding and/or complement
dependent cytotoxicity (CDC) are described in WO99/51642. Such
variants may comprise an amino acid substitution at one or more of
amino acid positions 270, 322, 326, 327, 329, 331, 333 or 334 of
the Fc region. See also, Duncan and Winter, Nature 322: 738-40
(1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821 and
WO94/29351 concerning Fc region variants.
B. Preparation of Wnt Antagonists
[0212] The description below relates primarily to production of Wnt
antagonist polypeptides by culturing cells transformed or
transfected with a vector containing Wnt antagonist
polypeptide-encoding nucleic acid. It is, of course, contemplated
that alternative methods, which are well known in the art, may be
employed to prepare such Wnt antagonists. For instance, the
appropriate amino acid sequence, or portions thereof, may be
produced by direct peptide synthesis using solid-phase techniques
[see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H.
Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem.
Soc., 85:2149-2154 (1963)]. In vitro protein synthesis may be
performed using manual techniques or by automation. Automated
synthesis may be accomplished, for instance, using an Applied
Biosystems Peptide Synthesizer (Foster City, Calif.) using
manufacturer's instructions. Various portions of the Wnt antagonist
polypeptide may be chemically synthesized separately and combined
using chemical or enzymatic methods to produce the desired
sequence.
1. Isolation of DNA Encoding Wnt Antagonist Polypeptide
[0213] DNA encoding the sequence of the antagonists or any desired
component domains of the Wnt antagonist, such as an Frz, or sFRP
may be obtained from a cDNA library prepared from tissue believed
to possess such sequence and to express it at a detectable level.
Accordingly, a human Frz or sFRP sequence DNA can be conveniently
obtained from a cDNA library prepared from human tissue. The
desired DNA sequence gene may also be obtained from a genomic
library or by known synthetic procedures (e.g., automated nucleic
acid synthesis).
[0214] Libraries can be screened with probes (such as
oligonucleotides of at least about 20-80 bases) designed to
identify the gene of interest or the protein encoded by it.
Screening the cDNA or genomic library with the selected probe may
be conducted using standard procedures, such as described in
Sambrook et al., Molecular Cloning: A Laboratory Manual (New York:
Cold Spring Harbor Laboratory Press, 1989). An alternative means to
isolate the gene encoding Wnt antagonist polypeptide and components
thereof is to use PCR methodology [Sambrook et al., supra;
Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring
Harbor Laboratory Press, 1995)].
[0215] Techniques for screening a cDNA library are well known in
the art. The oligonucleotide sequences selected as probes should be
of sufficient length and sufficiently unambiguous that false
positives are minimized. The oligonucleotide is preferably labeled
such that it can be detected upon hybridization to DNA in the
library being screened. Methods of labeling are well known in the
art, and include the use of radiolabels like .sup.32P-labeled ATP,
biotinylation or enzyme labeling. Hybridization conditions,
including moderate stringency and high stringency, are provided in
Sambrook et al., supra.
[0216] Sequences identified in such library screening methods can
be compared and aligned to other known sequences deposited and
available in public databases such as GenBank or other private
sequence databases. Sequence identity (at either the amino acid or
nucleotide level) within defined regions of the molecule or across
the full-length sequence can be determined using methods known in
the art and as described herein.
[0217] DNA sequence encoding Fc immunoglobulin domains may be
derived from hybridoma cells secreting mAbs of the desired Fc
subtype.
[0218] Nucleic acid having protein coding sequence may be obtained
by screening selected cDNA or genomic libraries using the deduced
amino acid sequence disclosed herein for the first time, and, if
necessary, using conventional primer extension procedures as
described in Sambrook et al., supra, to detect precursors and
processing intermediates of mRNA that may not have been
reverse-transcribed into cDNA.
2. Selection and Transformation of Host Cells
[0219] Host cells are transfected or transformed with expression or
cloning vectors described herein for Wnt antagonist polypeptide
production and cultured in conventional nutrient media modified as
appropriate for inducing promoters, selecting transformants, or
amplifying the genes encoding the desired sequences. The culture
conditions, such as media, temperature, pH and the like, can be
selected by the skilled artisan without undue experimentation. In
general, principles, protocols, and practical techniques for
maximizing the productivity of cell cultures can be found in
Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed.
(IRL Press, 1991) and Sambrook et al., supra.
[0220] Methods of eukaryotic cell transfection and prokaryotic cell
transformation are known to the ordinarily skilled artisan, for
example, CaCl.sub.2, CaPO.sub.4, liposome-mediated and
electroporation. Depending on the host cell used, transformation is
performed using standard techniques appropriate to such cells. The
calcium treatment employing calcium chloride, as described in
Sambrook et al., supra, or electroporation is generally used for
prokaryotes. Infection with Agrobacterium tumefaciens is used for
transformation of certain plant cells, as described by Shaw et al.,
Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. For
mammalian cells without such cell walls, the calcium phosphate
precipitation method of Graham and van der Eb, Virology, 52:456-457
(1978) can be employed. General aspects of mammalian cell host
system transfections have been described in U.S. Pat. No.
4,399,216. Transformations into yeast are typically carried out
according to the method of Van Solingen et al., J. Bact., 130:946
(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829
(1979). However, other methods for introducing DNA into cells, such
as by nuclear microinjection, electroporation, bacterial protoplast
fusion with intact cells, or polycations, e.g., polybrene,
polyornithine, may also be used. For various techniques for
transforming mammalian cells, see Keown et al., Methods in
Enzymology, 185:527-537 (1990) and Mansour et al., Nature,
336:348-352 (1988).
[0221] Suitable host cells for cloning or expressing the DNA in the
vectors herein include prokaryote, yeast, or higher eukaryote
cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as E. coli. Various E. coli
strains are publicly available, such as E. coli K.sub.12 strain
MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain
W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable
prokaryotic host cells include Enterobacteriaceae such as
Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella,
Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g.,
Serratia marcescans, and Shigella, as well as Bacilli such as B.
subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed
in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.
aeruginosa, and Streptomyces. These examples are illustrative
rather than limiting. Strain W3110 is one particularly preferred
host or parent host because it is a common host strain for
recombinant DNA product fermentations. Preferably, the host cell
secretes minimal amounts of proteolytic enzymes. For example,
strain W3110 may be modified to effect a genetic mutation in the
genes encoding proteins endogenous to the host, with examples of
such hosts including E. coli W3110 strain 1A2, which has the
complete genotype tonA; E. coli W3110 strain 9E4, which has the
complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC
55,244), which has the complete genotype tonA ptr3 phoA E15
(argF-lac)169 degP ompT kan.sup.r; E. coli W3110 strain 37D6, which
has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP
ompT rbs7 ilvG kan.sup.r; E. coli W3110 strain 40B4, which is
strain 37D6 with a non-kanamycin resistant degP deletion mutation;
and an E. coli strain having mutant periplasmic protease disclosed
in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990. Alternatively, in
vitro methods of cloning, e.g., PCR or other nucleic acid
polymerase reactions, are suitable.
[0222] Full length antibody, antibody fragments, and antibody
fusion proteins can be produced in bacteria, in particular when
glycosylation and Fc effector function are not needed, such as when
the therapeutic antibody is conjugated to a cytotoxic agent (e.g.,
a toxin) and the immunoconjugate by itself shows effectiveness in
tumor cell destruction. Full length antibodies have greater half
life in circulation. Production in E. coli is faster and more cost
efficient. For expression of antibody fragments and polypeptides in
bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et. al.), U.S.
Pat. No. 5,789,199 (Joly et al.), and U.S. Pat. No. 5,840,523
(Simmons et al.) which describes translation initiation regio (TIR)
and signal sequences for optimizing expression and secretion, these
patents are incorporated herein by reference. After expression, the
antibody is isolated from the E. coli cell paste in a soluble
fraction and can be purified through, e.g., a protein A or G column
depending on the isotype. Final purification can be carried out
similar to the process for purifying antibody expressed e.g, in CHO
cells.
[0223] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for Wnt antagonist polypeptide-encoding vectors. Saccharomyces
cerevisiae is a commonly used lower eukaryotic host microorganism.
Others include Schizosaccharomyces pombe (Beach and Nurse, Nature,
290: 140 [1981]; EP 139,383 published 2 May 1985); Kluyveromyces
hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio/Technology,
9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683,
CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 [1983]),
K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum
(ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)),
K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia
pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol.,
28:265-278 [1988]); Candida; Trichoderma reesia (EP 244,234);
Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA,
76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces
occidentalis (EP 394,538 published 31 Oct. 1990); and filamentous
fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO
91/00357 published 10 Jan. 1991), and Aspergillus hosts such as A.
nidulans (Ballance et al., Biochem. Biophys. Res. Commun.,
112:284-289 [1983]; Tilburn et al., Gene, 26:205-221 [1983]; Yelton
et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 [1984]) and A.
niger (Kelly and Hynes, EMBO J., 4:475-479 [1985]). Methylotropic
yeasts are suitable herein and include, but are not limited to,
yeast capable of growth on methanol selected from the genera
consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces,
Torulopsis, and Rhodotorula. A list of specific species that are
exemplary of this class of yeasts may be found in C. Anthony, The
Biochemistry of Methylotrophs, 269 (1982).
[0224] Suitable host cells for the expression of glycosylated Wnt
antagonist polypeptide are derived from multicellular organisms.
Examples of invertebrate cells include insect cells such as
Drosophila S2 and Spodoptera Sf9, as well as plant cells, such as
cell cultures of cotton, corn, potato, soybean, petunia, tomato,
and tobacco. Numerous baculoviral strains and variants and
corresponding permissive insect host cells from hosts such as
Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito),
Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly),
and Bombyx mori have been identified. A variety of viral strains
for transfection are publicly available, e.g., the L-1 variant of
Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,
and such viruses may be used as the virus herein according to the
present invention, particularly for transfection of Spodoptera
frugiperda cells.
[0225] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol.
36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl.
Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather,
Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL
70); African green monkey kidney cells (VERO-76, ATCC CRL-1587);
human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney
cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC
CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells
(Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);
TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982));
MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
[0226] Host cells are transformed with the above-described
expression or cloning vectors for Wnt antagonist polypeptide
production and cultured in conventional nutrient media modified as
appropriate for inducing promoters, selecting transformants, or
amplifying the genes encoding the desired sequences.
3. Selection and Use of a Replicable Vector
[0227] One aspect of the invention provides for the nucleic acid
(e.g., cDNA or genomic DNA) encoding a Wnt antagonist polypeptide
inserted into a replicable vector for cloning (amplification of the
DNA) or for expression. Various vectors are publicly available. The
vector may, for example, be in the form of a plasmid, cosmid, viral
particle, or phage. The appropriate nucleic acid sequence may be
inserted into the vector by a variety of procedures. In general,
DNA is inserted into an appropriate restriction endonuclease
site(s) using techniques known in the art. Vector components
generally include, but are not limited to, one or more of a signal
sequence, an origin of replication, one or more marker genes, an
enhancer element, a promoter, and a transcription termination
sequence. Construction of suitable vectors containing one or more
of these components employs standard ligation techniques which are
known to the skilled artisan.
[0228] The Wnt antagonist may be produced recombinantly not only
directly, but also as a fusion polypeptide with a heterologous
polypeptide, which may be a signal sequence or other polypeptide
having a specific cleavage site at the N-terminus of the mature
protein or polypeptide. In general, the signal sequence may be a
component of the vector, or it may be a part of the Wnt antagonist
polypeptide-encoding DNA that is inserted into the vector. The
signal sequence may be a prokaryotic signal sequence selected, for
example, from the group of the alkaline phosphatase, penicillinase,
lpp, or heat-stable enterotoxin II leaders. For yeast secretion the
signal sequence may be, e.g., the yeast invertase leader, alpha
factor leader (including Saccharomyces and Kluyveromyces
.alpha.-factor leaders, the latter described in U.S. Pat. No.
5,010,182), or acid phosphatase leader, the C. albicans
glucoamylase leader (EP 362,179 published 4 Apr. 1990), or the
signal described in WO 90/13646 published 15 Nov. 1990. In
mammalian cell expression, mammalian signal sequences may be used
to direct secretion of the protein, such as signal sequences from
secreted polypeptides of the same or related species, as well as
viral secretory leaders.
[0229] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Such sequences are well known for a variety of
bacteria, yeast, and viruses. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative bacteria, the
2.mu. plasmid origin is suitable for yeast, and various viral
origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for
cloning vectors in mammalian cells. Expression and cloning vectors
will typically contain a selection gene, also termed a selectable
marker. Typical selection genes encode proteins that (a) confer
resistance to antibiotics or other toxins, e.g., ampicillin,
neomycin, methotrexate, or tetracycline, (b) complement auxotrophic
deficiencies, or (c) supply critical nutrients not available from
complex media, e.g., the gene encoding D-alanine racemase for
Bacilli.
[0230] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the Wnt antagonist-encoding nucleic acid, such as DHFR
or thymidine kinase. An appropriate host cell when wild-type DHFR
is employed is the CHO cell line deficient in DHFR activity,
prepared and propagated as described by Urlaub et al., Proc. Natl.
Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use
in yeast is the trp1 gene present in the yeast plasmid YRp7
[Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene,
7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1 gene
provides a selection marker for a mutant strain of yeast lacking
the ability to grow in tryptophan, for example, ATCC No. 44076 or
PEP4-1 [Jones, Genetics, 85:12 (1977)].
[0231] Expression and cloning vectors usually contain a promoter
operably linked to the Wnt antagonist-encoding nucleic acid
sequence to direct mRNA synthesis. Promoters recognized by a
variety of potential host cells are well known. Promoters suitable
for use with prokaryotic hosts include the .beta.-lactamase and
lactose promoter systems [Chang et al., Nature, 275:615 (1978);
Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a
tryptophan (tip) promoter system [Goeddel, Nucleic Acids Res.,
8:4057 (1980); EP 36,776], and hybrid promoters such as the tac
promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25
(1983)]. Promoters for use in bacterial systems also will contain a
Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding
the Wnt antagonist polypeptide.
[0232] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman
et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic
enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland,
Biochemistry, 17:4900 (1978)], such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0233] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657.
[0234] Wnt antagonist polypeptide transcription from vectors in
mammalian host cells is controlled, for example, by promoters
obtained from the genomes of viruses such as polyoma virus, fowlpox
virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as
Adenovirus 2), bovine papilloma virus, avian sarcoma virus,
cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus
40 (SV40), from heterologous mammalian promoters, e.g., the actin
promoter or an immunoglobulin promoter, and from heat-shock
promoters, provided such promoters are compatible with the host
cell systems.
[0235] Transcription of a DNA encoding the Wnt antagonist
polypeptide by higher eukaryotes may be increased by inserting an
enhancer sequence into the vector. Enhancers are cis-acting
elements of DNA, usually about from 10 to 300 bp, that act on a
promoter to increase its transcription. Many enhancer sequences are
now known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. The enhancer may be spliced into the vector at a
position 5' or 3' to the Wnt antagonist polypeptide coding
sequence, but is preferably located at a site 5' from the
promoter.
[0236] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding Wnt
antagonist.
[0237] Still other methods, vectors, and host cells suitable for
adaptation to the synthesis of Wnt antagonist polypeptide in
recombinant vertebrate cell culture are described in Gething et
al., Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46
(1979); EP 117,060; and EP 117,058.
4. Culturing the Host Cells
[0238] One aspect of the invention provides for a host cell
comprising the nucleic acid encoding the Wnt antagonists. The host
cells used to produce the Wnt antagonist polypeptide of this
invention may be cultured in a variety of media. Commercially
available media such as Ham's F10 (Sigma), Minimal Essential Medium
((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's
Medium ((DMEM), Sigma) are suitable for culturing the host cells.
In addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. Re. No. 30,985 may be used
as culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
5. Detecting Gene Amplification/Expression
[0239] Gene amplification and/or expression may be measured in a
sample directly, for example, by conventional Southern blotting,
Northern blotting to quantitate the transcription of mRNA [Thomas,
Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Alternatively,
antibodies may be employed that can recognize specific duplexes,
including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes
or DNA-protein duplexes. The antibodies in turn may be labeled and
the assay may be carried out where the duplex is bound to a
surface, so that upon the formation of duplex on the surface, the
presence of antibody bound to the duplex can be detected.
[0240] Gene expression, alternatively, may be measured by
immunological methods, such as immunohistochemical staining of
cells or tissue sections and assay of cell culture or body fluids,
to quantitate directly the expression of gene product. Antibodies
useful for immunohistochemical staining and/or assay of sample
fluids may be either monoclonal or polyclonal, and may be prepared
in any mammal. Conveniently, the antibodies may be prepared against
a Frz, sFRP or Ror sequence identified herein or against a
synthetic peptide based on the DNA sequences provided herein or
against exogenous sequence fused to the Wnt antagonist and encoding
a specific antibody epitope.
6. Purification of Wnt Antagonist
[0241] Forms of Wnt antagonist polypeptide may be recovered from
culture medium or from host cell lysates. If membrane-bound, it can
be released from the membrane using a suitable detergent solution
(e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in
expression of Wnt antagonist polypeptide can be disrupted by
various physical or chemical means, such as freeze-thaw cycling,
sonication, mechanical disruption, or cell lysing agents.
[0242] It may be desired to purify Wnt antagonist polypeptide from
recombinant cell proteins or polypeptides. The following procedures
are exemplary of suitable purification procedures: by fractionation
on an ion-exchange column; ethanol precipitation; reverse phase
HPLC; chromatography on silica or on a cation-exchange resin such
as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate
precipitation; gel filtration using, for example, Sephadex G-75;
protein A Sepharose columns to remove contaminants such as IgG; and
metal chelating columns to bind epitope-tagged forms of the Wnt
antagonist. Various methods of protein purification may be employed
and such methods are known in the art and described for example in
Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein
Purification Principles and Practice, Springer-Verlag, New York
(1982). The purification step(s) selected will depend, for example,
on the nature of the production process used and the particular Wnt
antagonist polypeptide produced.
[0243] When using recombinant techniques, the Wnt antagonist
polypeptide can be produced intracellularly, in the periplasmic
space, or directly secreted into the medium. If the Wnt antagonist
polypeptide is produced intracellularly, as a first step, the
particulate debris, either host cells or lysed fragments, are
removed, for example, by centrifugation or ultrafiltration. Carter
et al., Bio/Technology 10:163-167 (1992) describe a procedure for
isolating antibodies which are secreted to the periplasmic space of
E. coli. Briefly, cell paste is thawed in the presence of sodium
acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF)
over about 30 min. Cell debris can be removed by centrifugation.
Where the Wnt antagonist polypeptide is secreted into the medium,
supernatants from such expression systems are generally first
concentrated using a commercially available protein concentration
filter, for example, an Amicon or Millipore Pellicon
ultrafiltration unit. A protease inhibitor such as PMSF may be
included in any of the foregoing steps to inhibit proteolysis and
antibiotics may be included to prevent the growth of adventitious
contaminants.
[0244] The Wnt antagonist polypeptide composition prepared from the
cells can be purified using, for example, hydroxylapatite
chromatography, gel electrophoresis, dialysis, and affinity
chromatography, with affinity chromatography being the preferred
purification technique. The suitability of protein A as an affinity
ligand depends on the species and isotype of any immunoglobulin Fc
domain that is present in the antibody. Protein A can be used to
purify antibodies that are based on human .gamma.1, .gamma.2 or
.gamma.4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13
(1983)). Protein G is recommended for all mouse isotypes and for
human .gamma.3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix
to which the affinity ligand is attached is most often agarose, but
other matrices are available. Mechanically stable matrices such as
controlled pore glass or poly(styrenedivinyl)benzene allow for
faster flow rates and shorter processing times than can be achieved
with agarose. Where the Wnt antagonist polypeptide comprises a
C.sub.H3 domain, the Bakerbond ABX.TM. resin (J. T. Baker,
Phillipsburg, N.J.) is useful for purification. Other techniques
for protein purification such as fractionation on an ion-exchange
column, ethanol precipitation, Reverse Phase HPLC, chromatography
on silica, chromatography on heparin SEPHAROSE.TM. chromatography
on an anion or cation exchange resin (such as a polyaspartic acid
column), chromatofocusing, SDS-PAGE, and ammonium sulfate
precipitation are also available depending on the antibody to be
recovered.
[0245] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25M salt).
C. Pharmaceutical Formulations
[0246] One aspect of the invention provides for a composition
comprising a Wnt antagonist and at least one pharmaceutically
acceptable carrier or excipient. Therapeutic formulations of the
Wnt antagonists used in accordance with the present invention are
prepared for storage by mixing the Wnt antagonists having the
desired degree of purity with optional pharmaceutically acceptable
carriers, excipients or stabilizers (Remington: The Science and
Practice of Pharmacy, 20th edition, A. Gennaro, Ed. (2000)), in the
form of lyophilized formulations or aqueous solutions. A
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like,
compatible with pharmaceutical administration. Additional examples
of suitable carriers or diluents include, but are not limited to,
water, saline, Finger's solutions, dextrose solution, and 5% human
serum albumin Liposomes and non-aqueous vehicles such as fixed oils
may also be used. Except when a conventional media or agent is
incompatible with an active compound, use of these compositions is
contemplated. Supplementary active compounds can also be
incorporated into the compositions.
[0247] Acceptable carriers, excipients, or stabilizers are nontoxic
to recipients at the dosages and concentrations employed, and
include buffers such as acetate, Tris, phosphate, citrate, and
other organic acids; antioxidants including ascorbic acid and
methionine; preservatives (such as octadecyldimethylbenzyl ammonium
chloride; hexamethonium chloride; benzalkonium chloride,
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; tonicifiers such as
trehalose and sodium chloride; sugars such as sucrose, mannitol,
trehalose or sorbitol; surfactant such as polysorbate; salt-forming
counter-ions such as sodium; metal complexes (e.g., Zn-protein
complexes); and/or non-ionic surfactants such as TWEEN.RTM.,
PLURONICS.RTM. or polyethylene glycol (PEG). The antibody
preferably comprises the antibody at a concentration of between
5-200 mg/ml, preferably between 10-100 mg/ml.
[0248] The formulations herein may also contain more than one
active compound as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. For example, in addition to a
particular Wnt antagonist, it may be desirable to include in the
one formulation, an additional antibody, e.g., which binds a
different epitope on the Wnt protein, to a different Wnt protein
entirely, or an antibody to some other target such as a growth
factor that affects the growth of the Wnt mediated disorder.
Alternatively, or additionally, the composition may further
comprise a chemotherapeutic agent, cytotoxic agent, cytokine,
growth inhibitory agent, anti-hormonal agent, and/or
cardioprotectant. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0249] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington: The Science and Practice of
Pharmacy, 20th edition, A. Gennaro, Ed. (2000).
[0250] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0251] Therapeutic compositions herein generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0252] The route of administration is in accord with known methods,
e.g. injection or infusion by intravenous, intraperitoneal,
intracerebral, intramuscular, intraocular, intraarterial or
intralesional routes, topical administration, or by sustained
release systems.
[0253] Dosages and desired drug concentrations of pharmaceutical
compositions of the present invention may vary depending on the
particular use envisioned. The determination of the appropriate
dosage or route of administration is well within the skill of an
ordinary physician. Animal experiments provide reliable guidance
for the determination of effective doses for human therapy.
Interspecies scaling of effective doses can be performed following
the principles laid down by Mordenti, J. and Chappell, W. "The use
of interspecies scaling in toxicokinetics", In Toxicokinetics and
New Drug Development, Yacobi et al., Eds., Pergamon Press, New York
1989, pp. 42-96.
[0254] When in vivo administration of a substance or molecule of
the invention is employed, normal dosage amounts may vary from
about 10 ng/kg to up to 100 mg/kg of mammal body weight or more per
day, preferably about 1 .mu.g/kg/day to 10 mg/kg/day, depending
upon the route of administration. Guidance as to particular dosages
and methods of delivery is provided in the literature; see, for
example, U.S. Pat. No. 4,657,760; 5,206,344; or 5,225,212. It is
anticipated that different formulations will be effective for
different treatment compounds and different disorders, that
administration targeting one organ or tissue, for example, may
necessitate delivery in a manner different from that to another
organ or tissue.
[0255] Where sustained-release administration of a substance or
molecule is desired in a formulation with release characteristics
suitable for the treatment of any disease or disorder requiring
administration of the substance or molecule, microencapsulation of
the substance or molecule is contemplated. Suitable examples of
sustained-release preparations include semi-permeable matrices of
solid hydrophobic polymers containing the antibody, which matrices
are in the form of shaped articles, e.g., films, or microcapsules.
Microencapsulation of recombinant proteins for sustained release
has been successfully performed with human growth hormone (rhGH),
interferon-.alpha.,.gamma. (rhIFN-.alpha.,-.gamma.), interleukin-2,
and MN rgp120. Johnson et al., Nat. Med., 2:795-799 (1996); Yasuda,
Biomed. Ther., 27:1221-1223 (1993); Hora et al., Bio/Technology,
8:755-758 (1990); Cleland, "Design and Production of Single
Immunization Vaccines Using Polylactide Polyglycolide Microsphere
Systems," in Vaccine Design: The Subunit and Adjuvant Approach,
Powell and Newman, eds, (Plenum Press: New York, 1995), pp.
439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat. No.
5,654,010.
[0256] The sustained-release formulations of these proteins may be
developed using poly-lactic-coglycolic acid (PLGA) polymer due to
its biocompatibility and wide range of biodegradable properties.
The degradation products of PLGA, lactic and glycolic acids, can be
cleared quickly within the human body. Moreover, the degradability
of this polymer can be adjusted from months to years depending on
its molecular weight and composition. Lewis, "Controlled release of
bioactive agents from lactide/glycolide polymer," in: M. Chasin and
R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems
(Marcel Dekker: New York, 1990), pp. 1-41.
[0257] Additional examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.RTM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
D. Methods of Treating Wnt Mediated Disorder
[0258] The invention provides for methods of treating a
Wnt-mediated disorder in a mammal suffering therefrom, comprising
administering to the mammal a therapeutically effective amount of a
Wnt antagonist. In one embodiment, the disorder is a cell
proliferative disorder associated with aberrant, e.g., increased,
expression of activity of Wnt signaling. In another embodiment, the
disorder results from increased expression of a Wnt protein. In yet
another embodiment, the cell proliferative disorder is cancer, such
as for example, colon cancer, colorectal cancer, breast cancer,
cancer associated with various disorders relating to HSC's, such as
leukemias and various other blood related cancers, and cancer
related to neuronal proliferative disorders, including brain
tumors, such as gliomas, astrocytomas, meningiomas, Schwannomas,
pituitary tumors, primitive neuroectodermal tumors (PNET),
medulloblastomas, craniopharyngioma, and pineal region tumors.
[0259] Treatment of the cell proliferative disorder by
administration of a Wnt antagonist results in an observable and/or
measurable reduction in or absence of one or more of the following:
reduction in the number of cancer cells or absence of the cancer
cells; reduction in the tumor size; inhibition (i.e., slow to some
extent and preferably stop) of cancer cell infiltration into
peripheral organs including the spread of cancer into soft tissue
and bone; inhibition (i.e., slow to some extent and preferably
stop) of tumor metastasis; inhibition, to some extent, of tumor
growth; and/or relief to some extent, one or more of the symptoms
associated with the specific cancer; reduced morbidity and
mortality, and improvement in quality of life issues. To the extent
the Wnt antagonist may prevent growth and/or kill existing cancer
cells, it may be cytostatic and/or cytotoxic. Reduction of these
signs or symptoms may also be felt by the patient.
[0260] The above parameters for assessing successful treatment and
improvement in the disease are readily measurable by routine
procedures familiar to a physician. For cancer therapy, efficacy
can be measured, for example, by assessing the time to disease
progression (TDP) and/or determining the response rate (RR).
Metastasis can be determined by staging tests and by bone scan and
tests for calcium level and other enzymes to determine spread to
the bone. CT scans can also be done to look for spread to the
pelvis and lymph nodes in the area. Chest X-rays and measurement of
liver enzyme levels by known methods are used to look for
metastasis to the lungs and liver, respectively. Other routine
methods for monitoring the disease include transrectal
ultrasonography (TRUS) and transrectal needle biopsy (TRNB).
[0261] In a specific embodiment, the administration of Wnt
antagonist decreases tumor burden (e.g., reduces size or severity
of the cancer). In yet another specific embodiment, the
administration of Wnt antagonist kills the cancer.
E. Methods of Inhibiting Wnt-Signaling in a Cell
[0262] The invention provides for a method of inhibiting
Wnt-signaling in a cell comprising contacting the cell with an
effective amount of a Wnt antagonist. In one embodiment, the cell
is contained within a mammal, preferably a human, and the
administered amount is a therapeutically effective amount. In yet
another embodiment, the inhibition of Wnt signaling further results
in the inhibition of the growth of the cell. In a further
embodiment, the cell is a cancer cell.
[0263] Inhibition of cell proliferation is measured using methods
known to those skilled in the art. For example, a convenient assay
for measuring cell proliferation is the CellTiter-Glo.TM.
Luminescent Cell Viability Assay, which is commercially available
from Promega (Madison, Wis.). That assay determines the number of
viable cells in culture based on quantitation of ATP present, which
is an indication of metabolically active cells. See Crouch et al
(1993) J. Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677. The
assay may be conducted in 96- or 384-well format, making it
amenable to automated high-throughput screening (HTS). See Cree et
al (1995) AntiCancer Drugs 6:398-404. The assay procedure involves
adding a single reagent (CellTiter-Glo.RTM. Reagent) directly to
cultured cells. This results in cell lysis and generation of a
luminescent signal produced by a luciferase reaction. The
luminescent signal is proportional to the amount of ATP present,
which is directly proportional to the number of viable cells
present in culture. Data can be recorded by luminometer or CCD
camera imaging device. The luminescence output is expressed as
relative light units (RLU).
F. Methods of Modulating the Expression of a Wnt Target Gene
[0264] The invention provides for a method of modulating the
expression of a Wnt target gene in a cell characterized by
activated or excessive Wnt signaling, comprising contacting the
cell with an effective amount of a Wnt antagonist. In one
embodiment, the Wnt target gene is overexpressed as a result of the
Wnt signaling, and the result of the contact with the Wnt
antagonist reduces expression of the Wnt target gene. In another
embodiment, the Wnt target gene is selected from the group
consisting of: Axin2, APCDD1, Gad1, Sax1, c-myc, cyclin D1,
PPARdelta, gastrin, clusterin, survivin, cyclooxygenase, fra-1,
osteopontin, uPAR, claudin-1, CD44, MMP-7/9/11/14/26, IGFBP-4, Met,
BMP4, sox-9, histone deacetylase 2, VEGF. In yet another
embodiment, the Wnt target gene is underexpressed as a result of
the Wnt signaling, and the result of contact with the Wnt
antagonist restores expression of the Wnt target gene. In a further
embodiment, the Wnt target gene is selected from the group
consisting of: Lefty1, Lefty2, sFRP1, Fzd5, fas antigen, caspase 3,
integrin .beta.7, alpha e integrin, hath 1, fatty acid binding
protein 2, muc-2, kruppel like factor-4, carbonic anhydrase-11,
EphrinB1, EphB2R, EphB3R, muc-3, histocompatibility 2, Q region
locus 1, .beta.2-microglobulin.
[0265] Expression of the target genes is determined using methods
known to those of skill in the art, including those described
herein and set forth in the Examples below.
G. Methods of Detecting the Presence of a Wnt Protein
[0266] The invention provides for a method of detecting the
presence of a Wnt protein in a sample, comprising contacting the
sample with a Wnt antagonist, wherein the presence of a complex or
the level of binding between the Wnt antagonist and the Wnt protein
is indicative of the presence of Wnt protein and/or Wnt signaling.
In one embodiment, the method further comprises determining if the
level of Wnt signaling is aberrant. In this embodiment, the level
of Wnt protein binding in the sample is compared with the level in
a second sample in which Wnt protein expression and/or Wnt
signaling is known to be physiologically normal. The level of
binding in the suspect sample compared to the second sample that is
higher or lower than the physiologically normal sample is
indicative of aberrant Wnt signaling. In another embodiment, the
presence of Wnt signaling or aberrant Wnt signaling is indicative
of the presence of a Wnt-mediated disorder, such as cancer.
H. The Wnt Pathway and Disorders Associated Therewith
1. The Wnt Signaling Pathway:
[0267] The Wnt signaling pathway is an unusually complex signaling
process involving multiple proteins which exert varying levels of
control in the pathway. This multi-level, tight regulation of the
pathway is indicative of its importance in cellular biology.
Despite the complicated regulatory mechanisms, the initial signal
of the pathway is generated by the binding of a Wnt to the Frizzled
(Frz) receptors. Effective signal further requires the presence of
an additional single pass transmembrane molecule of the LRP (LDL
receptor related protein) class, specifically LRP 5 and LRP 6. Wnt
may further bind with LRP to form a trimeric complex with Frizzled.
The cytoplasmic tail of LRP in turn interacts with Axin, another
downstream component. Dishevelled, a cytoplasmic component that
interacts directly with Frizzled, may also directly interact with
Axin, thus forming a tetra-plex complex of Frizzled, LRP, Dsh and
Axin. This interaction with Axin releases .beta.-catenin from the
"degradation complex" (discussed infra) for subsequent downstream
activity in the Wnt signaling pathway.
[0268] Outside the cell, Wnt signaling is inhibited by various
proteins that can bind to Wnt thereby sequestering it from its
receptor. Included in this group are the secreted Frizzled related
proteins (sFRPs, Jones et al., Bioessays 2002; 24: 811-820) and Wnt
inhibitory factor-1 (WIF-1, Hsieh, J. C. et al., Nature 1999; 398:
431-436). In humans, the sFRP family consists of five members
(e.g., sFRP-1, sFRP-2 . . . sFRP-5), each containing a
cysteine-rich domain (CRD) which shares 30-50% sequence homology
with the CRD of Frz receptors. (Melkonyan, H. S. et al., Proc.
Natl. Acad. Sci. USA 1997; 94: 13636-13641). sFRPs are believed to
form function-inhibiting complexes with Frz receptors, and
therefore are natural antagonists, but the biology is complex, and
in some cases, may even act to agonize Wnt activity. (Uren, A. et
al., J. Biol. Chem. 2000; 275: 4374-4382).
[0269] Another class of extracellular Wnt inhibitor is Dickkopf
(Dkk). [Brott, B. K. et al., Mol. Cell. Biol. 2002; 22: 6100-6110;
Fedi, P. et al., J. Biol. Chem. 1999; 274: 19465-19472] The three
members of the Dkk family (e.g., Dkk-1, Dkk-2 and Dkk-4) can
antagonize Wnt signaling through inactivation of the cell surface
receptor LRP-5 and LRP-6, essential components of the canonical
pathway. [Mao, J. H. et al., Mol. Cell. 2001; 7: 801-809; Pinson,
K. I. et al., Nature 2000; 407: 535-538]. Dkk forms a ternary
complex with LRP5/6 and the single pass transmembrane receptors
Kremen 1 (Krm-1) or Kremen 2 (Krm-2) [Mao et al., Gene 2003; 302:
179-183; Mao et al., Nature 2002; 417: 664-667; Mao et al., Nature
2001; 411: 321-325]. This complex in turn undergoes endocytosis,
thereby removing LRP5/6 receptors from the cell surface. As a
result, Dkks can selectively antagonize canonical Wnt signaling,
while not affecting non-canonical signaling.
[0270] The hallmark of canonical Wnt signaling activation is
elevated levels of the protein .beta.-catenin. .beta.-catenin is
constitutively produced and is present in the cytoplasm as pools of
monomeric protein. [Papkoff, J. et al., Mol. Cell. Biol. 1996; 16:
2128-2134]. The primary mechanism for controlling cytoplasmic
levels of .beta.-catenin is through direct physical degradation
upon recruitment into a large multi-protein complex ("degradation
complex"). The central scaffolding of this complex is provided by
Axin, as well as binding sites for .beta.-catenin, adenomatous
polyposis coli (APC), glycogen synthase kinase 3.beta.
(GSK3.beta.), casein kinase I.alpha. (CKI.alpha.) and protein
phophatase 2A (PP2A) [Hinoi, T. et al., J. Biol. Chem. 2000; 275:
34399-34406; Ikeda et al., Oncogene 2000; 19: 537-545; Yamamoto et
al., J. Biol. Chem. 1999; 274: 10681-10684; Kishida et al., J.
Biol. Chem. 1998; 273: 10823-10826; Ikeda et al., EMBO J. 1998; 17:
1371-1384. After formation, the complex is stabilized by the
GSK3.beta.-mediated phosphorylation of Axin and APC, as well as
PP2A. GSK3.beta.- then phosphorylates .beta.-catenin thereby
allowing it be recognized by .beta.-transducin repeat containing
protein (.beta.-TrCP), thereby targeting it for ubiquitination and
proteosomic degradation. [Aberle et al., EMBO J. 1997; 16:
3797-804; Latres et al., Oncogene 1999; 18: 849-54; Liu et al.,
Proc. Natl. Acad. Sci. USA 1999; 96: 6273-8].
[0271] Although complexation with Axin/APC/GSK3.beta. is the
primary mechanism for degradation of .beta.-catenin, an alternative
degradation pathway has been shown involving ubiquitination induced
by complexation with Siah-1 and the C-terminus of APC. [Matsuzawa
et al., Mol. Cell. 2001; 7: 915-926; Liu et al., Mol. Cell. 2001;
7: 927-936]. In addition to its role as a transcription factor,
.beta.-catenin further is involved in cellular adhesion. [Nelson et
al., Science 2004; 303: 1483-1487; Ilyas et al., J. Pathol. 1997;
182: 128-137. .beta.-catenin can be found at the cell surface sites
of intercellular contact known as adherens junctions, where it is
complexed with E-cadherin and .alpha.-catenin. Thus, any increase
in E-cadherin expression will direct .beta.-catenin to the cell
membrane, thereby depleting cytoplasmic levels, and in turn inhibit
Wnt signaling. Moreover, the breakdown of the E-cadherin-catenin
complex can increase cytoplasmic levels of free .beta.-catenin,
thereby stimulating transcriptional activity. [Nelson et al.,
supra.]. Thus, activation of the cell surface receptors cRON,
epidermal growth factor receptor (EGFR) and c-ErbB2, by liberating
.beta.-catenin, can also stimulate canonical Wnt signaling. Other
signaling pathways that can either activate or facilitate the
effects of Wnt signaling. For example, integrin signaling can
result in nuclear transportation of .beta.-catenin [Eger et al.,
Oncogene 2004; 23: 2672-2680], while signaling through insulin-like
growth factor (IGF) can activate Wnt signaling by "soaking up"
available GSK3.beta.--thereby preventing formation of the
"degradation complex."
[0272] In canonical signaling, an initial step involves the binding
of Wnt to Frz in the presence of LRP5/6. [Mao et al., Mol. Cell.
2001; 7: 801-809; Pinson et al., Nature 2000; 407: 535-538]. The
formation of this trimeric complex has two downstream consequences.
First is the recruitment of Dishevelled (Dsh) to the cell surface
and its phosphorylation by casein kinase I.epsilon. (CI.epsilon.)
[Kishida et al., J. Biol. Chem. 2001; 276: 33147-33155]. The
phosphorylated Dsh can form a complex with Frat 1 and GSK3.beta.,
which in turn can inhibit the activity of GSK3.beta.. Second, the
Wnt/Frz/LRP5/6 tri-plex facilitates the LRP5/6 mediated degradation
of Axin. The net effect of this is the destabilization of the
degradation complex responsible for phosphorylating .beta.-catenin.
In the absence of phosphorylation, .beta.-catenin is not
ubiquinated, thereby escaping degradation, thus increasing
intracellular levels and availability for translocation to the
nucleus.
[0273] The manner in which .beta.-catenin is transported to the
nucleus is not entirely clear, but interaction with the nuclear
transport proteins APC [Rosin-Arbesfeld et al., Nature 2000; 406:
1009-1012; Neufeld et al., Proc. Natl. Acad. Sci. USA 2000; 97:
12085-12090], as well as pygopus and Bcl9/legless have been
implicated. [Townsley et al., Nature Cell Biol. 2004; 6:
626-633].
[0274] Once in the nucleus, .beta.-catenin displaces the
transcriptional repressor Groucho for binding with T-cell-specific
transcription factor/lymphoid enhancer-binding factor-1 (TCF/LEF)
DNA binding proteins. In the absence of displacement by
.beta.-catenin, TCF/LEF complexes with Groucho to repress
expression of the Wnt "target genes". The inhibitory effect of
Groucho is further mediated by interactions with various histone
deacetylases (HDAC), which are believed to make DNA refractive to
transcriptional activation. [Cavallo et al., Nature 1998; 395:
604-8; Chen et al., Genes Dev. 1999; 13: 2218-30]. The conversion
of the TCF transcriptional repressor complex into a transcriptional
activation complex further involves recruitment of histone
acetylases such as Creb binding protein (CBP)/p300 as well as other
activating factors such as Brg-1. [Takemaru et al., J. Cell Biol.
2000; 149: 249-54; Barker et al., Cell 2002; 109: 47-60; Brantjes
et al., Biol. Chem. 2002; 383: 255-261; Roose et al., Biochim.
Biophys Acta--Rev. Cancer 1999; 1424: M23-M37]. The interactions
between the .beta.-catenin-TCF complex and chromatin also may be
mediated by Legless (Bcl9) and Pygopus. Kramps et al., Cell 2002;
109: 47-60; Thompson et al., Nat. Cell Biol. 2002; 4: 367-73;
Parker et al., Development 2002; 129: 2565-76.
[0275] An abbreviated summary of the canonical Wnt signaling
pathway both in the "off" or inactive state as well as the "on" or
active state is depicted in FIG. 1.
2. Disorders Associated with Wnt Signaling Activity:
[0276] Deregulation of the Wnt signaling pathway may be caused by
somatic mutations in genes encoding various Wnt signaling pathway
components. For example, aberrant Wnt signaling activity has been
associated with Wnt ligand overexpression in non small cell lung
cancer (NSCLC) [You et al., Oncogene 2004; 23: 6170-6174], chronic
lymphocytic leukemia (CLL)[Lu et al., Proc. Natl. Acad. Sci. USA
2004; 101: 3118-3123], gastric cancer [Kim et al., Exp. Oncol.
2003; 25: 211-215; Saitoh et al., Int. J. Mol. Med. 2002; 9:
515-519], head and neck squamous cell carcinoma (HNSCC) [Rhee et
al., Oncogene 2002; 21: 6598-6605], colorectal cancer [Holcombe et
al., J. Clin. Pathol--Mol. Pathol. 2002; 55: 220-226], ovarian
cancer [Ricken et al., Endocrinology 2002; 143: 2741-2749], basal
cell carcinoma (BCC) [Lo Muzio et al., Anticancer Res. 2002; 22:
565-576] and breast cancer. Moreover, the reduction of various Wnt
ligand regulatory molecules such as sFRP and WIF-1 have been
associated with breast cancer [Klopocki et al., Int. J. Oncol.
2004; 25: 641-649; Ugolini et al., Oncogene 2001; 20: 5810-5817;
Wissmann et al., J. Pathol. 2003; 201: 204-212], bladder cancer
[Stoehr et al., Lab Invest. 2004; 84: 465-478; Wissmann et al.,
supra], mesothelioma [Lee et al., Oncogene 2004; 23: 6672-6676],
colorectal cancer [Suzuki et al., Nature Genet. 2004; 36: 417-422;
Kim et al., Mol. Cancer. Ther. 2002; 1: 1355-1359; Caldwell et al.,
Cancer Res. 2004; 64: 883-888], prostate cancer [Wissman et al.,
supra], NSCLC [Mazieres et al., Cancer Res. 2004; 64: 4717-4720],
and lung cancer [Wissman et al., supra]. Antagonizing Wnt signaling
with the Wnt antagonist molecules of the invention is expected to
therapeutically treat these cancers.
[0277] Continuing, aberrant Wnt signaling resulting from
overexpression of various components of the Frz-LRP receptor
complex have also been associated with certain cancers. For
example, LRP5 overexpression has been associated with osteosarcoma
[Hoang et al., Int. J. Cancer 2004; 109: 106-111], while Frz
overexpression has been associated with cancers such as prostate
[Wissmann et al., supra], HNSCC [Rhee et al., Oncogene 2002; 21:
6598-6605], colorectal [Holcombe et al., supra], ovarian cancer
[Wissman et al, supra], esophageal [Tanaka et al., Proc. Natl.
Acad. Sci. USA 1998; 95: 10164-10169] and gastric [Kirikoshi et
al., Int. J. Oncol. 2001; 19: 111-115]. Additionally,
overexpression of Wnt signaling pathway components such as
Dishevelled have been associated with cancers such as prostate
[Wissman et al., supra], breast [Nagahata et al., Cancer Sci. 2003;
94: 515-518], mesothelioma [Uematsu et al., Cancer Res. 2003; 63:
4547-4551] and cervical [Okino et al., Oncol. Rep. 2003; 10:
1219-1223]. Frat-1 overexpression has been associated with cancers
such as pancreatic, esophageal, cervical, breast and gastric.
[Saitoh et al., Int. J. Oncol. 2002; 20: 785-789; Saitoh et al.,
Int. J. Oncol. 2001; 19: 311-315]. Axin loss of function (LOF)
mutations have been associated with hepatocellular cancer [Satoh et
al., Nature Genet. 2000; 24: 245-250; Taniguchi et al., Oncogene
2002; 21: 4863-4871] and medulloblastoma [Dahmen et al., Cancer
Res. 2001; 61: 7039-7043; Yokota et al., Int. J. Cancer 2002; 101:
198-201]. The blocking of Wnt-Frz interactions with the Wnt
antagonists of the present invention is expected to alleviate
cancers associated with overexpression of Frz or LRPs.
[0278] Finally, a multitude of cancers has been associated with
activating .beta.-catenin through disruption of the "degradation
complex" such as gain-of-function mutations in .beta.-catenin or
loss-of-function mutations in APC. A reduction in the degradation
of .beta.-catenin results in greater amounts of functional
.beta.-catenin in the cell, which then causes increased
transcription of the target genes, resulting in aberrant cell
proliferation. For example, mutations in the gene encoding
.beta.-catenin (i.e., CTNNB1) have been associated with cancers
such as gastric [Clements et al., Cancer Res. 2002; 62: 3503-3506;
Park et al., Cancer Res. 1999; 59: 4257-4260], colorectal [Morin et
al., Science 1997; 275: 1787-1790; Ilyas et al., Proc. Natl. Acad.
Sci. USA 1997; 94: 10330-10334], intestinal carcinoid [Fujimori et
al., Cancer Res. 2001; 61: 6656-6659], ovarian [Sunaga et al.,
Genes Chrom. Cancer 2001; 30: 316-321], pulmonary adenocarcinoma
[Sunaga et al., supra], endometrial [Fukuchi et al., Cancer Res.
1998; 58: 3526-3528; Kobayashi et al., Japan. J. Cancer Res. 1999;
90: 55-59; Mirabelli-Primdahl et al., Cancer Res. 1999; 59:
3346-3351], hepatocellular [Satoh et al., supra.; Wong et al.,
Cancer 2001; 92: 136-145], hepatoblastoma [Koch et al., Cancer Res.
1999; 59: 269-273], medulloblastoma [Koch et al., Int. J. Cancer
2001; 93: 445-449], pancreatic [Abraham et al., Am. J. Pathol.
2002; 160: 1361-1369], thyroid [Garcia-Rostan et al., Cancer Res.
1999; 59: 1811-1815; Garcia-Rostan et al., Am. J. Pathol. 2001;
158: 987-996], prostate [Chesire et al., Prostate 2000; 45:
323-334; Voeller et al., Cancer Res. 1998; 58: 2520-2523], melanoma
[Reifenberger et al., Int. J. Cancer 2002; 100: 549-556],
pilomatricoma [Chan et al., Nature Genet. 1999; 21: 410-413],
Wilms' tumor [Koesters et al., J. Pathol. 2003; 199: 68-76],
pancreatoblastomas [Abraham et al., Am. J. Pathol. 2001; 159:
1619-1627], liposarcomas [Sakamoto et al., Arch. Pathol. Lab Med.
2002; 126: 1071-1078], juvenile nasopharyngeal angiofibromas
[Abraham et al., Am. J. Pathol. 2001; 158: 1073-1078], desmoid
[Tejpar et al., Oncogene 1999; 18: 6615-6620; Miyoshi et al.,
Oncol. Res. 1998; 10: 591-594], synovial sarcoma [Saito et al., J.
Pathol. 2000; 192: 342-350]. While loss-of-function mutations have
been associated with cancers such as colorectal [Fearon et al.,
Cell 1990; 61: 759-767; Rowan et al., Proc. Natl. Acad. Sci. USA
2000; 97: 3352-3357], melanoma [Reifenberger et al., Int. J. Cancer
2002; 100: 549-556; Rubinfeld et al., Science 1997; 275:
1790-1792], medulloblastoma [Koch et al., Int. J. Cancer 2001; 93:
445-449; Huang et al., Am. J. Pathol. 2000; 156: 433-437] and
desmoids [Tejpar et al., Oncogene 1999; 18: 6615-6620; Alman et
al., Am J. Pathol. 1997; 151: 329-334]. Cancers that result from
aberrant activity of .beta.-catenin thereby activating the Wnt
pathway are suitable for treatment with the Wnt antagonists of the
present invention.
3. Wnt Signaling and Carcinogenesis
[0279] The Wnt pathway has many transcriptional endpoints or target
genes. The majority of these are specific to certain types--which
is not unusual in developmental signaling pathways. This is
consistent with a fundamental mechanism of gene control by
extracellular signals in which the cell rather than the signal
determines the nature of the response. However, in addition to cell
type specific genes, Wnt signaling also controls genes that are
more widely induced, including components of the Wnt signaling
pathway and genes that are most likely activated by the
Wnt-.beta.-catenin-TCF cascade.
[0280] The transition of normal cellular physiology into one
characterized by neoplastic change has been the object of intense
study in an effort to better understand the events underlying the
development of cancer. The inappropriate activation of the target
genes by .beta.-catenin thus can result in a disease state in the
organism even though there may not be any somatic mutation in the
target genes themselves. Ilyas has generated a modification of the
Hanahan and Weinberg list of phenotypes that are acquired by most
malignancies; including "Inappropriate stem cell
phenotype/limitless replicative potential", "evasion of apoptosis,"
"tissue invasion and metastasis," "self sufficiency of growth
signals," "insensitivity to growth inhibitors," "failure of
terminal differentiation," "evasion of immune response," and
"sustained angiogenesis." Ilyas, J. Pathol. 2005; 205: 130-144;
Hanahan and Weinberg, Cell 2000; 100: 57-70. Analysis of the genes
modulated by Wnt signaling, including target genes of
.beta.-catenin or altered expression as shown by microarray
analysis shows that the perturbations from aberrant Wnt signaling
either directly or through the effect on target genes can impart
nearly all of these "neoplastic phenotypes." Ilyas, M., J. Pathol.
2005; 205: 130-144. A list of example targets of Wnt signaling is
given in Table 1. Gene targets that are upregulated appear in
boldface, while those which are downregulated are italicized.
Aberrant expression of such target genes due to the result of
activated and/or excessive Wnt signaling may be remedies upon
application of the Wnt antagonists of the invention.
TABLE-US-00002 TABLE 1 Wnt target genes and effects
phenotypiceffects ##STR00001##
[0281] Increasingly, cancer is being viewed as a "stem cell"
disease (Taipale et al., Nature 2001; 411: 349-54--that is, an
inappropriate activation and/or maintenance of stem cells. Wnt
signaling has been shown to be essential for the maintenance of
stem cells [He et al., Nature Genet. 2004; 36: 1117-1121; Reya et
al., Nature 2003; 423: 409-414; Willert et al., Nature 2003; 423:
448-452]. In the intestine, TCF4 is the main nuclear binding factor
for .beta.-catenin and the failure of TCF4 knock out mice to
develop stem cells in the small intestine further supports the role
of canonical Wnt signaling in stem cell maintenance [Korinek et
al., Nat. Genet. 1998; 19: 379-83; Pinto et al., Genes Dev. 2003;
17: 1709-13; Kuhnert et al., Proc. Natl. Acad. Sc. USA 2004; 101:
66-71].
[0282] The effect of Wnt signaling on multiple biological processes
is illustrated by the matrix metalloproteinase genes (MMPs). MMP7,
MMP14 and MMP26 have been shown to direct targets of .beta.-catenin
[Marchenko et al., Int. J. Biochem. Cell Biol. 2004; 36: 942-956;
Takahashi et al., Oncogene 2002; 21: 5861-5867; Brabletz et al.,
Am. J. Pathol. 1999; 155: 1033-1038], while other MMPs were found
expressed directly by intestinal adenomas [Paoni et al., Physiol.
Genomics 2003; 15: 228-235]. The MMPs are proteolytic enzymes that
breakdown stromal collagen thereby allowing tumor cells to acquire
the phenotype "tissue invasion and metastasis." The enzymatic
activity also allows the release of latent growth factors in the
stroma, which together with other growth factors secreted by the
tumor cells themselves will contribute to "self sufficiency of
growth signals." [Coussens et al., Science 2002; 295: 2387-2392;
Egeblad et al., Nature Rev. Cancer 2002; 2: 161-174]. MMPs can also
act on osteopontin (a secondary Wnt-induced target [Paoni et al.,
supra], to release fragments which together with vascular
endothelial growth factor (VEGF), a direct target of
.beta.-catenin, contributes to the feature of "sustained
angiogenesis." [Zhang et al., Cancer Res. 2001; 61: 6050-6054;
Agnihotri et al., J. Bio. Chem. 2001; 276: 28261-28267].
[0283] While the Wnt signaling pathway can be activated at levels
downstream of the ligand receptor interaction, there is strong
evidence to suggest inhibition of the extracellular ligand-receptor
interaction component is effective in reducing the tumorigenicity,
even though the event initiating the Wnt signaling may have
occurred downstream. For example, Ilyas reports in a recent review
that the inhibition of Wnt signals in several colorectal cancer
cell lines results in reduced tumorigenicity. [Ilyas, supra.].
Moreover, the transfection of inoperative frizzled receptor (Frz7
ectodomain) into carcinoma cell line (SK-CO-1) restored a normal
.beta.-catenin phenotype. This cell line has active Wnt signaling
due to a homozygous APC.sup.-/- mutation. Moreover, such cells also
did not demonstrate tumor formation when transferred in vivo.
Vincan et al., Differentiation 2005; 73: 142-153. This demonstrates
that the inhibition of Wnt signaling at the extracellular level can
downregulate Wnt signaling resulting from activation of a
downstream intracellular Wnt signaling pathway component. This
further suggests that inhibitors such as the Wnt antagonists of the
present invention, which inhibit Wnt-Frz interactions, have
therapeutic benefit for any Wnt-mediated disorder, regardless of
the particular manner in which Wnt signaling has been
activated.
4. Aberrant Wnt Signaling in Colon Cancer:
[0284] Defects in the Wnt signaling component APC was originally
discovered to be the key in the hereditary cancer syndrome familial
adenomatous polyposis (FAP). FAP patients who inherit one defective
APC allele develop large number of colon polyps, or adenomas, in
the early years of their life. Such polyps develop as clonal
outgrowths of epithelial cells in which the second APC allele is
inactivated. The cumulative effect of these FAP adenomas inevitably
results in the appearance of adenocarcinomas, evident as a more or
less ordered accumulation of mutations in additional oncogenes or
tumor suppressor genes, such as K-Ras, p53 and Smad4. Moreover, the
loss of APC also occurs in most sporadic colorectal cancers.
Kinzler et al., Cell 87: 159-170 (1996). The mutational
inactivation of APC, by resulting in the stabilization of, and
eventual nuclear transport of .beta.-catenin, and Wnt signaling,
thereby transforms epithelial cells. Interestingly, reporter
plasmids containing concatemerized TCF binding sites such as the
pTOPFLASH, normally transcribed only upon Wnt signaling, are
inappropriately transcribed in APC mutant cancer cells through
constitutive activation of .beta.-catenin/TCF-4 transcription
complexes. In other examples of colorectal cancer in which APC in
not mutated, the scaffolding protein Axin-2 is mutated [Liu et al.,
Nature Genet. 26: 146-147 (2000) or .beta.-catenin is mutated so as
to remove the N-terminal Ser/Thr destruction motif [Morin et al.,
Science 275: 1787-1790 (1997). Thus, colorectal cancer is linked
not only to defects in APC, but to the inappropriate persistence of
.beta.-catenin/TCF-4 transcriptional activation. It has further
been reported that TCF-4 mutations result in activation of the same
target genes (as shown by microarray analysis) in colorectal
cancers, as is observed through defective APC expression in crypt
stem and progenitor cells. Van de Wetering et al., Cell 111:
241-250 (2002). Once the Wnt cascade is activated, the APC.sup.-/-
adenoma cells maintain their progenitor status indefinitely. As a
result, it is likely that the activation of Wnt signaling is a
necessary precursor in the carcinogenesis of colorectal cancer, and
the inhibition of Wnt signaling could be an effective means to
treat and/or prevent the onset of this disorder.
5. Wnt Signaling in Hematopoietic Stem Cells
[0285] Hematopoietic stem cells give rise to the adult blood cells
of the circulatory system in a process of lineage-committed
progenitor cells from multipotential hematopoietic stem cells
(HSC). It is also apparent that Wnt signaling contributes to the
self-renewal and maintenance of HSC's, and that dysfunctional Wnt
signaling is responsible for various disorders resulting from
HSC's, such as leukemias and various other blood related cancers.
Reya et al., Nature 434: 843-850 (2005); Baba et al., Immunity 23:
599-609 (2005); Jamieson et al., N. Engl. J. Med. 351(7): 657-667
(2004). Wnt signaling is normally reduced as stem cells convert to
committed myeloid progenitor cells. Reya et al., Nature 423:
409-414 (2003).
[0286] Not only are Wnt ligands themselves produced by HSC's, but
Wnt signaling is also active, thereby suggesting autocrine or
paracrine regulation. Rattis et al., Curr. Opin. Hematol. 11: 88-94
(2004); Reya et al., Nature 423: 409-414 (2003). Additionally, both
.beta.-catenin and Wnt3a promote self renewal of murine HSCs and
progenitor cells, while application of Wnt-5A to human
hematopoietic progenitors promotes the expansion of
undifferentiated progenitors in vitro. Reya et al., supra.; Willert
et al., Nature 423: 448-452 (2003); Van Den Berg et al., Blood 92:
3189-3202 (1998).
[0287] In addition to HSC's, it is apparent that embryonic stem
cells, epidermal stem cells and epithelial stem cells are
responsive or dependent on Wnt signaling for maintenance in an
undifferentiated, proliferating state. Willert et al., supra;
Korinek et al., Nat. Genet. 19: 379-383 (1998); Sato et al., Nat.
Med. 10: 55-63 (2004); Gat et al., Cell 95: 605-614 (1998); Zhu et
al., Development 126: 2285-2298 (1999). Therefore the inhibition of
Wnt signaling with the Wnt antagonists of the present invention may
be a therapeutic in the treatment of disorders resulting from
dysfunctional hematopoieses, such as leukemias and various blood
related cancers, such as acute, chronic, lymphoid and myelogenous
leukemias, myelodysplastic syndrome and myeloproliferative
disorders. These include myeloma, lymphoma (e.g., Hodgkin's and
non-Hodgkin's) chronic and nonprogressive anemia, progressive and
symptomatic blood cell deficiencies, polycythemia vera, essential
or primary thrombocythemia, idiopathic myelofibrosis, chronic
myelomonocytic leukemia (CMML), mantle cell lymphoma, cutaneous
T-cell lymphoma, Waldenstrom macroglobinemia,
6. Wnt Signaling in Leukemia
[0288] Unregulated activation of the Wnt signaling pathway is a
precursor to the development of leukemia. Reya et al., supra.
Experimental evidence exists supporting the oncogenic growth of
both myeloid and lymphoid lineages as dependent on Wnt signaling.
Wnt signaling has been implicated in regulating both the chronic
and acute forms of myeloid leukemia. Granulocyte-macrophage
progenitors (GMPs) from chronic myelogenous leukemia patients and
blast crisis cells from patients resistant to therapy display
activated Wnt signaling. Jamieson, et al., supra. Moreover,
inhibition of .beta.-catenin through ectopic expression of Axin
decreases the replating capacity of leukemic cells in vitro,
suggesting that chronic myelogenous leukemia precursors are
dependent on Wnt signaling for growth and renewal. Also, Wnt
overexpression caused GMPs to acquire stem-cell-like properties of
long-term self renewal. Jamieson et al., supra. This finding
further support the hypothesis that Wnt signaling is necessary for
the normal development of blood lineages, but that aberrant Wnt
signaling results in the transformation of progenitor cells. The
Wnt antagonists of the present invention would be useful to treat
these types of leukemias.
[0289] Recent studies also suggest that lymphoid neoplasias may
also be influenced by Wnt signaling. Wnt-16 is overexpressed in
pre-B-cell leukemia cell lines carrying the E2A-PbX translocation,
suggesting that autocrine Wnt activity may contribute to
oncogenesis. McWhirter, et al., Proc. Natl. Acad. Sci. USA 96:
11464-11469 (1999). The role of Wnt signaling in the growth and
survival of normal B-cell progenitors further supports this notion.
Reya et al., Immunity 13: 15-24 (2000); Ranheim et al., Blood 105:
2487-2494 (2005). Autocrine dependence on Wnt has also been
proposed for regulating the growth of multiple myeloma, a cancer of
terminally differentiated B-cells. Derksen et al., Proc. Natl.
Acad. Sci. USA 101: 6122-6127 (2004). Primary myelomas and myeloma
cell lines were also found to express stabilized (i.e., independent
of degradation complex). Although no mutations in Wnt signaling
components was present, the overexpression of several components,
including Wnt-5A and Wnt-10B suggest that tumor dependency and
cancer self-renewal is not necessarily dependent on mutations
appearing in Wnt signaling pathway components, but rather only upon
constitutive activation of the pathway itself. Reya et al., supra.
Through binding overexpressed Wnt, the Wnt antagonists of the
present invention would be an effective therapeutic in treating
B-cell leukemias.
[0290] The transition of self-renewing, pluripotent stem cells to
myeloid progenitors is accompanied by the downregulation of Wnt
signaling. Reya et al, Nature 423: 409-414 (2003). Similarly, the
stable expression of .beta.-catenin in lymphoid progenitors
restored multiple differentiation options, albeit such cells lacked
markers typically associated with either cell type. Baba et al.,
Immunity 23: 599-609 (2005). Thus, it is strongly suggested that
the inhibition of Wnt signaling by the Wnt antagonists of the
invention could be an effective therapeutic in treating leukemia,
such as myelolid and lymphoid leukemia, including acute and chronic
myelogenous leukemia as well as acute and chronic lymphoid
leukemias.
7. Aberrant Wnt Signaling in Neural Disorders
[0291] It has also been observed that the activation of Wnt
signaling through .beta.-catenin can increase cycling and expansion
of neural progenitors, and that loss of such signaling can result
in a loss of progenitor compartment. Chenn et al., Science 297:
365-369 (2002); Zechner et al., Dev. Biol. 258: 406-418 (2003).
Just as normal activation of Wnt signaling may promote self-renewal
of neuronal stem cells, aberrant Wnt pathway activation may be
tumorigenic in the nervous system. Experimental evidence supporting
this conclusion is the discovery that medulloblastoma, a pediatric
brain tumor of the cerebellum, contains mutations in both
.beta.-catenin and Axin--thereby suggesting that medulloblastomas
arise from primitive progenitors that become transformed in
response to uncontrolled Wnt signaling. Zurawel et al., Cancer Res.
58: 896-899 (1998); Dahmen et al., Cancer Res. 61: 7039-7043
(2001); Baeza et al., Oncogene 22: 632-636 (2003). Thus, it is
strongly suggested that the inhibition of Wnt signaling by the Wnt
antagonists of the invention may be an effective therapeutic in the
treatment of various neuronal proliferative disorders, including
brain tumors, such as gliomas, astrocytomas, meningiomas,
Schwannomas, pituitary tumors, primitive neuroectodermal tumors
(PNET), medulloblastomas, craniopharyngioma, pineal region tumors,
and non cancerous neurofibromatoses.
8. Aberrant Wnt Signaling in Breast Cancer.
[0292] In mammary tissues where stem cells have yet to be
definitively isolated, a controlling role for Wnt in progenitor
cell fate or maintenance is suggested by studies of Wnt transgenic
mice develop mammary tumors. These tumors have an increased
frequency of individual cells with stem and progenitor properties,
in stark contrast to tumors from mice overexpressing other
oncogenes. [Liu et al., Proc. Natl. Acad. Sci. USA 101: 4158-4163
(2004); Li et al., Proc. Natl. Acad. Sci. USA 100: 15853-15858
(2003)]. This suggests that the Wnt pathway may be unique in its
ability to target stem and progenitor cells for transformation, and
suggests a key role in the self-renewal of normal breast
epithelium. Thus the inhibition of Wnt signaling by the Wnt
antagonists of the invention is likely an effective therapeutic in
the treatment of breast cancer.
[0293] FIG. 32 is an illustration of active Wnt signaling in human
breast cancer. FIG. 32A shows
[0294] Wnt-1 expression (as shown by in vitro hybridization) in
normal (A-1), low grade (A-2) and high grade (A-3) human breast
tumor initially reported in Wong et al., J. Pathol. 196: 145
(2002). FIG. 32B shows nuclear (B-1) and cytoplasmic (B-2)
localization (as shown by IHC) of .beta.-catenin in breast cancer
patients. Also shown is a Kaplan-Meier survival plot (B-3) showing
patient survival probability that correlates with the indicated
.beta.-catenin expression pattern. This data was initially reported
in Lin et al., P.N.A.S. (USA) 97(8): 4262-66 (2000). FIG. 32C is a
microarray analysis of Wnt-1 expression in a normal breast from a
patient without cancer in comparison with tissue isolated from a
patient with infiltrating ductal carcinoma, her-2 negative.
9. Wnt Signaling in Aging
[0295] The Wnt signaling pathway may also play a critical role in
aging and age-related disorders.
[0296] As reported in Brack A S, et al., Science, 317(5839):807-10
(2007), muscle stem cells from aged mice were observed to convert
from a myogenic to a fibrogenic lineage as they begin to
proliferate. This conversion is associated with an increase in
canonical Wnt signaling pathway activity in aged myogenic
progenitors and can be suppressed by Wnt inhibitors. Additionally,
components of serum from aged mice bind to the Frizzled proteins
and may account for the elevated Wnt signaling in aged cells.
Injection of Wnt3A into young regenerating muscle reduced
proliferation and increased deposition of connective tissue.
[0297] The Wnt signaling pathway has been further implicated in
aging process in studies using the Klotho mouse model of
accelerated aging in which it was determined that the Klotho
protein physically interacted with and inhibited Wnt proteins. Liu
H, et al., Science, 317(5839):803-6 (2007). In a cell culture
model, the Wnt-Klotho interaction resulted in the suppression of
Wnt biological activity while tissues and organs from
Klotho-deficient animals showed evidence of increased Wnt
signaling.
[0298] Accordingly, Wnt antagonists could find use as therapeutics
to reduce the effects of aging and to treat age-related
diseases.
I. Modes of Administration Specific Formulations
1. General Considerations
[0299] A pharmaceutical composition is formulated to be compatible
with its intended route of administration, including intravenous,
intradermal, subcutaneous, oral (e.g., inhalation), transdermal
(i.e., topical), transmucosal, and rectal administration. Solutions
or suspensions used for parenteral, intradermal, or subcutaneous
application can include: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid (EDTA); buffers such
as acetates, citrates or phosphates, and agents for the adjustment
of tonicity such as sodium chloride or dextrose. The pH can be
adjusted with acids or bases, such as hydrochloric acid or sodium
hydroxide. The parenteral preparation can be enclosed in ampules,
disposable syringes or multiple dose vials made of glass or
plastic.
2. Injectable Formulations
[0300] Pharmaceutical compositions suitable for injection include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. For intravenous administration,
suitable carriers include physiological saline, bacteriostatic
water, CREMOPHOR EL (BASF, Parsippany, N.J.) or phosphate buffered
saline (PBS). In all cases, the composition must be sterile and
should be fluid so as to be administered using a syringe. Such
compositions should be stable during manufacture and storage and
must be preserved against contamination from microorganisms such as
bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (such as
glycerol, propylene glycol, and liquid polyethylene glycol), and
suitable mixtures. Proper fluidity can be maintained, for example,
by using a coating such as lecithin, by maintaining the required
particle size in the case of dispersion and by using surfactants.
Various antibacterial and antifungal agents; for example, parabens,
chlorobutanol, phenol, ascorbic acid, and thimerosal, can contain
microorganism contamination. Isotonic agents; for example, sugars,
polyalcohols such as manitol, sorbitol, and sodium chloride can be
included in the composition. Compositions that can delay absorption
include agents such as aluminum monostearate and gelatin.
[0301] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., any modulator
substance/molecule of the invention) in the required amount in an
appropriate solvent with one or a combination of ingredients as
required, followed by sterilization. Generally, dispersions are
prepared by incorporating the active compound into a sterile
vehicle that contains a basic dispersion medium, and the other
required ingredients. Sterile powders for the preparation of
sterile injectable solutions, methods of preparation include vacuum
drying and freeze-drying that yield a powder containing the active
ingredient and any desired ingredient from a sterile solutions.
3. Systemic Administration
[0302] Systemic administration can also be transmucosal or
transdermal. For transmucosal or transdermal administration,
penetrants that can permeate the target barrier(s) are selected.
Transmucosal penetrants include, detergents, bile salts, and
fusidic acid derivatives. Nasal sprays or suppositories can be used
for transmucosal administration. For transdermal administration,
the active compounds are formulated into ointments, salves, gels,
or creams.
[0303] The compounds can also be prepared in the form of
suppositories (e.g., with bases such as cocoa butter and other
glycerides) or retention enemas for rectal delivery.
4. Carriers
[0304] In one embodiment, the active compounds are prepared with
carriers that protect the compound against rapid elimination from
the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable or
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Such materials can be obtained commercially from
ALZA Corporation (Mountain View, Calif.) and NOVA Pharmaceuticals,
Inc. (Lake Elsinore, Calif.), or prepared by one of skill in the
art. Liposomal suspensions can also be used as pharmaceutically
acceptable carriers. These can be prepared according to methods
known to those skilled in the art, such as in (Eppstein et al.,
U.S. Pat. No. 4,522,811, 1985).
5. Unit Dosage
[0305] Oral formulations or parenteral compositions in unit dosage
form can be created to facilitate administration and dosage
uniformity. Unit dosage form refers to physically discrete units
suited as single dosages for the subject to be treated, containing
a therapeutically effective quantity of active compound in
association with the required pharmaceutical carrier. The
specification for the unit dosage forms are dictated by, and
directly dependent on, the unique characteristics of the active
compound and the particular desired therapeutic effect, and the
inherent limitations of compounding the active compound.
6. Gene Therapy Compositions
[0306] The nucleic acid molecules can be inserted into vectors and
used as gene therapy vectors. Gene therapy vectors can be delivered
to a subject by, for example, intravenous injection, local
administration (Nabel and Nabel, U.S. Pat. No. 5,328,470, 1994), or
by stereotactic injection (Chen et al., Proc Natl Acad Sci USA.
91:3054-7 (1994)). The pharmaceutical preparation of a gene therapy
vector can include an acceptable diluent, or can comprise a slow
release matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells that
produce the gene delivery system.
7. Dosage
[0307] The pharmaceutical composition and method may further
comprise other therapeutically active compounds that are usually
applied in the administration of the Wnt antagonists.
[0308] In the treatment or prevention of conditions which require
administration of Wnt antagonists, an appropriate dosage level will
generally be about 0.01 to 500 mg per kg patient body weight per
day which can be administered in single or multiple doses.
Preferably, the dosage level will be about 0.1 to about 250 mg/kg
per day; more preferably about 0.5 to about 100 mg/kg per day. A
suitable dosage level may be about 0.01 to 250 mg/kg per day, about
0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within
this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg
per day. For oral administration, the compositions are preferably
provided in the form of tablets containing 1.0 to 1000 milligrams
of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0,
25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0,
600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active
ingredient for the symptomatic adjustment of the dosage to the
patient to be treated. The compounds may be administered on a
regimen of 1 to 4 times per day, preferably once or twice per
day.
[0309] However, the specific dose level and frequency of dosage for
any particular patient may be varied and will depend upon a variety
of factors including the activity of the specific compound
employed, the metabolic stability and length of action of that
compound, the age, body weight, general health, sex, diet, mode and
time of administration, rate of excretion, drug combination, the
severity of the particular condition, and the host undergoing
therapy.
8. Kits for Compositions
[0310] The compositions (e.g., pharmaceutical compositions) can be
included in a kit, container, pack, or dispenser together with
instructions for administration. When supplied as a kit, the
different components of the composition may be packaged in separate
containers and admixed immediately before use. Such packaging of
the components separately may permit long-term storage without
losing the active components' functions.
[0311] Kits may also include reagents in separate containers that
facilitate the execution of a specific test, such as diagnostic
tests or tissue typing.
[0312] (a) Containers or Vessels
[0313] The reagents included in kits can be supplied in containers
of any sort such that the life of the different components are
preserved and are not adsorbed or altered by the materials of the
container. For example, sealed glass ampules may contain
lyophilized modulator substance/molecule and/or buffer that have
been packaged under a neutral, non-reacting gas, such as nitrogen.
Ampules may consist of any suitable material, such as glass,
organic polymers, such as polycarbonate, polystyrene, etc.,
ceramic, metal or any other material typically employed to hold
reagents. Other examples of suitable containers include simple
bottles that may be fabricated from similar substances as ampules,
and envelopes, that may consist of foil-lined interiors, such as
aluminum or an alloy. Other containers include test tubes, vials,
flasks, bottles, syringes, or the like. Containers may have a
sterile access port, such as a bottle having a stopper that can be
pierced by a hypodermic injection needle. Other containers may have
two compartments that are separated by a readily removable membrane
that upon removal permits the components to mix. Removable
membranes may be glass, plastic, rubber, etc.
[0314] (b) Instructional Materials
[0315] Kits may also be supplied with instructional materials.
Instructions may be printed on paper or other substrate, and/or may
be supplied as an electronic-readable medium, such as a floppy
disc, CD-ROM, DVD-ROM, Zip disc, videotape, laserdisc, audio tape,
etc. Detailed instructions may not be physically associated with
the kit; instead, a user may be directed to an internet web site
specified by the manufacturer or distributor of the kit, or
supplied as electronic mail.
9. Combination Therapy
[0316] In certain embodiments, a pharmaceutical formulation
comprising a Wnt antagonist is administered in combination with at
least one additional therapeutic agent and/or adjuvant. In certain
embodiments, the additional therapeutic agent is a chemotherapeutic
agent, growth inhibitory agent, or cytotoxic agent like a toxin,
such as a maytansinoid, calicheamicin, antibiotic, radioactive
isotope, nucleolytic enzyme or the like.
[0317] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate formulations), and separate administration,
in which case, administration of a Wnt antagonist can occur prior
to, simultaneously, and/or following, administration of the
additional therapeutic agent and/or adjuvant. A Wnt antagonist can
also be used in combination with radiation therapy.
10. Medicaments
[0318] The invention provides a Wnt antagonist for a use in the
preparation of a medicament useful for treating a Wnt-mediated
disorder. In a specific aspect, the Wnt-mediated disorder is
cancer.
[0319] The following examples are included to demonstrate preferred
embodiments of the present invention. It should be appreciated by
those of skill in the art that the techniques disclosed in the
examples that follow represent techniques discovered by the
inventors to function well in the practice of the invention, and
thus can be considered to constitute preferred modes for its
practice. However, those of skill in the art should, in light of
the present disclosure, appreciate that many changes can be made in
the specific embodiments that are disclosed and still obtain a like
or similar result without departing from the spirit and scope of
the invention. All references cited throughout the specification
are expressly incorporated by reference in their entirety
herein.
Example 1
General Protocols
Mammalian Cell Culture.
[0320] Human kidney epithelial (HEK) 293 cells (ATCC # CRL-1573),
human ovarian PA1 cells (ATCC # CRL-1572) were grown in 50/50
Dulbecco modified Eagle high glucose medium, Ham's F12 which has
been supplemented with 10% fetal bovine serum. Human teratoma
derived NTer2 (ATCC #CRL-1973) and Tera2 (ATCC#HTB-106) cells were
maintained in McCoy's medium supplemented with 15% fetal bovine
serum and NCCIT cells (ATCC # CRL-2073) were maintained in RPMI
supplemented with 10% fetal bovine serum. All cell lines were
further supplemented with 2 mM glutamine, and 1%
penicillin-streptomycin at 37.degree. C. in 5% CO.sub.2.
Transfection and Luciferase Assays
[0321] In preparation for transfection, (1) 500,000 HEK293 and (2)
100,000 PA1 cells (ATCC # CRL-1572), NCCIT, NTera2 or Tera2 cells
were plated into each well of a 12-well dish (Nuc) 24 hours before
transfections. Cells were transfected with 0.375 .mu.g TOPglow
(Upstate, Cat #21-204), 0.05 mg LEF1, 0.01 mg SV40 RL with Fugene
(Roche) and at 24 hours post transfection. Media was changed and
cells were untreated or treated with Wnt3a alone, Wnt-5a alone, or
with serum samples for an additional 20-24 hours before harvesting.
All dilutions were made in complete media for the indicated cell
lines. Cells were harvested in 50-100 .mu.l of 1.times.SJC lysis
buffer (20 mM Tris pH 8.0, 137 mM NaCl, 1 mM EGTA, 1% Triton X-100,
10% Glycerol, 1.5 mM MgCl.sub.2, 1 mM DTT, 50 mM NaF, 1 mM
NaVO.sub.4 and protease inhibitors) and duplicate 10 .mu.l were
assayed using Dual-Glo.TM. luciferase assay kit (Promega, Part #
TM058) and detected in an Envision Luminometer (Perkin Elmer).
Luciferase activity was normalized against Renilla activity.
Example 2
Construction of Frz-Fc Chimeric Molecules
Cloning and Expression
FrzS(173)-Fc and Frz8(156)-Fc
[0322] FIGS. 4A and B show the sequences of the Frz8 (156)-Fc and
Frz8 (173)-Fc chimeric constructs. FIG. 4A shows the longer
Frz8(173) sequence. Shown in gray (i.e., first 24 N-terminal amino
acid residues) is the leader signal sequence. Shown in underline
(i.e., residues 25-27) are alanine residues that may be present or
absent in the mature protein. Shown in boxed text (i.e., residues
157-173) are the additional sequences of the Frz8 receptors that
distinguish the longer Frz8 (173) from the shorter Frz8(156)
chimeric constructs. Shown in bold (i.e., residues 174-182) is the
linker sequence, while the sequence in italics (i.e., residues
183-409) is the Fc region. FIG. 4B shows the shorter Frz (156)
minimal CRD (ECD) domain sequence. In gray (i.e., first 24
N-terminal amino acid residues) is the leader signal sequence.
Shown in underline (i.e., residues 25-27) are alanine residues that
may be present or absent in the mature protein. Shown in bold
(i.e., residues 157-164) is the linker sequence, while the sequence
in italics (i.e., residues 165-391) is the Fc region.
[0323] The Frz8(156)-Fc construct was constructed as follows. cDNA
encoding Frizzled 8 residues 1-156 were sub-cloned into the EcoR1
and XhoI sites of a pRK-derived plasmid. While native human cDNA is
preferred, alternative sequence encoding identical protein sequence
(e.g., murine) may also be used. In this cloning procedure, the
carboxyl terminus of the Frz8 was fused to the amino terminus of a
human IgG effector domain (Fc) via a short linker region (e.g.,
residues LESGGGGVT) (SEQ ID NO: 70), to create an Frz8-Fc fusion. A
final construct encodes 156 residues of Frz8. The cloning was
performed using standard molecular biology techniques (Ausubel et
al. (eds.), 2003, Current Protocols in Molecular Biology, 4 Vols.,
John Wiley & Sons). Protein was expressed in Chinese Hamster
Ovary (CHO) cells.
[0324] Alternatively, the cDNA encoding a length of Frz8 of a
length different than described previously (e.g., 1-173) may be
used. In addition, an alternative linker sequence (e.g., ESGGGGVT)
(SEQ ID NO: 69) may also be used.
Frz-Fc and sFrp-Fc Constructs
[0325] The constructs for the Wnt antagonists with a Frizzled
domain component comprising Frz1, Frz2, Frz3, Frz4, FrzS, Frz6,
Frz7, Frz9, Frz10, sFRP1, sFRP2, sFRP3, sFRP4, or sFRP5 were
constructed in a manner similar to the procedure described for
Frz8. Frz2, Frz3, Frz4, FrzS, and sFRP3 were subcloned into a
pRK-derived plasmid using XhoI and AscI. Frz1, Frz6, Frz7, Frz9,
Frz10, and sFRP4 were subcloned into a pRK-derived plasmid using
ClaI and XhoI and sFrp1, sFrp2, and sFRP5 were subcloned into a
pRK-derived plasmid using ClaI and AscI. As with the Frz8
constructs, the carboxyl terminus of the Frz domains were fused to
the amino terminus of a human IgG effector domain (Fc) via a short
linker region to create the chimeric Wnt antagonists. FIG. 7 (A, B,
and C) shows exemplary amino acid sequences for these constructs.
The leader signal sequence is shown in bold with italics indicating
a non-native leader sequence. The linker is underlined and the Fc
component is shown in italics.
[0326] FIG. 5 (A-H) (SEQ ID NOs: 115-129) provides exemplary
nucleic acid sequences for these Wnt antagonist constructs.
[0327] Alternative constructs can be made to optimize in vivo
activity or stability or to provide other beneficial
characteristics, such as, for example, increased solubility,
improved binding characteristics. These constructs may include
linkers that are different than the linkers of the above-described
Wnt antagonists. For example, an alternative construct of the
Frz3-Fc chimeric protein (SEQ ID NO: 114) has been made by
subcloning a Frz3 domain into a pRK-derived plasmid using BstXI and
XhoI and using the LESGGGGVT (SEQ ID NO: 70) peptide linker to fuse
the Frz3 domain to the Fc domain.
Protein Isolation
[0328] The Wnt antagonist chimeric proteins were isolated to
>90% purity by affinity capture using a PROSEP.RTM. (Millipore)
protein-A conjugated resin. Higher order aggregates were separated
from dimers by passage over a Superdex 200.RTM. (GE-Healthcare)
gel-filtration column. Protein identity and processing of the amino
terminus to remove the signal sequence were confirmed by Edmund
degradation. Purity of the final protein is estimated to be greater
than 98% (FIG. 10). Endotoxin levels of the material after
purification is complete and less than 1.0 EU/mg.
Example 3
Serum Stability of Frz8-Fc Chimeric Molecules
[0329] Initial studies of the serum stability of the Frz8(173)-Fc
chimeric constructs indicated that the construct had a limited in
vivo half-life. The in vivo instability was likely due to the
presence of protease cleavage sites in the EC domain (ECD) of the
Frizzled receptor component. The Frz8(156)-Fc construct described
in Example 2 exhibited increased serum stability over the
Frz8(173)-Fc. Athymic nude mice were injected i.v. with 10 mg/kg of
either Frz8(173)-Fc or Frz8(156)-Fc. Serum was collected at
specified time points and analyzed for total and active protein.
FIG. 11A shows an immunoblot for human Fc used to detect the
protein present in 1 .mu.L, of serum and compared with 25 .mu.g of
the respective purified protein (P). Frz8(156)-Fc was detectable in
serum 72 h after administration, whereas Frz8(173)-Fc was not
detectable beyond 30 minutes.
[0330] The activity of Frz8(156)-Fc and Frz8(173)-Fc in the
collected serum was assayed by measuring the inhibition of
Wnt3a-dependent TOPglow reporter activity in HEK293 cells. Although
comparable in vitro potency was observed on treatment with purified
Frz8(156)-Fc and Frz8(173)-Fc at 2.5 .mu.g/mL, only partial
inhibitory activity was recovered from the serum of
Frz8(173)-Fc-treated mice collected 30 minutes after protein
administration. In contrast, more potent inhibitory activity could
be recovered from the serum of Frz8(156)-Fc treated mice for up to
24 hours after administration, with detectable levels of inhibition
for at least 72 hours (FIG. 11B). These studies demonstrate that
the Frz8(156) molecule is more stable in vivo than the molecule
based on Frz8(173).
[0331] Additionally, the Frz8(173)-FC had suboptimal efficacy and
acted only to reduce the rate of increase in tumor volume, as
opposed to shrinking starting tumor volume. This suboptimal
efficacy is illustrated in FIG. 12, showing a graph of tumor volume
over time resulting from treatment with various Wnt signaling
component-Fc chimeric antagonists, including the Frz8(173)-FC
molecule. In this assay, the MMTV-WNT-1 tumors were transplanted
into the mammary fat pad of athymic nude mice, and drug was
administered IV at the time points indicated by the arrows on the
X-axis.
Example 4
In Vivo Pharmacokinetics of Frz8(156)-FC
[0332] The in vivo pharmacokinetics of Frz8(156)-FC were tested by
administration of a single dose of this protein at 1, 5, or 20
mg/kg i.v. or at 20 mg/kg i.p. into nude mice. As reported in FIG.
13 and discussed further in this Example below, the Frz8-Fc reagent
displayed biphasic elimination in nude mice at all doses. After a
single IV or IP dose, Frz8-Fc displays: (1) dose proportional
increase in exposure; (2) rapid absorption after IP dosing; (3)
clearance of about 25-30 ml/day; and (4) a half life of about 4
days. Bioavailability coefficient, AUC.sub.IP/AUC.sub.IV=92%.
Animal Protocol
[0333] Female athymic nude mice are separated into 4 groups of 12,
on the basis of quantity of drug administered and manner of
administration. Group 1: Frz8-Fc 1 mg/kg, intravenous (IV); Group
2: Frz8-Fc, 5 mg/kg, IV; Group 3: Frz8-Fc 20 mg/kg, IV; and Group
4: Frz8-Fc, 20 mg/kg, interperitoneal (IP). Each animal received an
IV or IP bolus dose of Frz8-Fc according to the group designation.
The dose volume administered (5-10 mL/kg) varies depending upon the
concentration of the dosing solution and the weight of each animal.
IV dosing is administered via the tail vein.
[0334] About 125 .mu.l of blood is collected from each animal
according to the following procedure. Serum is stored at
-70.degree. C. until assayed by ELISA. Sample are drawn such that
n=3 animals/timepoint. Extra animals are used for predose sample
collection and/or collection of blank mouse serum. Blood is
collected with a retroorbital bleed for the first two timepoints
for each animal, using alternative eyes. For the final timepoint,
blood is collected via a cardiac stick and about 1 ml is aliquoted
into 2 tubes. One sample will be used to determine Frz8-Fc
concentration and the other will be reserved for research use. Each
animal receives an IP bolus of 10 ml saline as fluid replacement
after each blood collection timepoint. Retroorbital bleeds are
performed under isoflorane anesthesia and terminal bleeds occur
under a ketamine/xylazine cocktail. Animals are euthanized via
cervical dislocation under anesthesia after the final blood
draw.
Results
[0335] FIG. 13A is an immunoblot of a neat serum from mice treated
with Frz8-Fc showing detection in serum at 7 days and beyond from
both 20 or 5 mg/kg I.V. or 20 mg/kg I.P. Samples were taken from
individual mice at 4, 7, 10 or 14 days. For controls, serum samples
were taken from untreated mice, Frz8-Fc protein was added to 20
.mu.g/ml and the samples incubated for 2 hours at 37.degree. C. and
the sample was then treated with SDS loading buffer (labeled as 2
h); neat serum from untreated mice was also run as a negative
control (labeled as S).
[0336] FIGS. 13B and 13C are a graphical summary of Frz8-Fc serum
levels as determined from the pharmacokinetic study. Specific
periods of time include evaluation over 16 days (FIG. 13B) and 2
days (FIG. 13C). Frz8-Fc displayed biphasic elimination
administration in nude mice at all doses. Curves represent the
predicted concentrations, while individual data points represent
the average serum levels of Frz8-Fc protein from individual mice as
determined by ELISA. FIG. 13D is a summary of the parameters for a
biphasic model of Frz8-Fc pharmacokinetics. When dosed at 20 mg/kg
by either the i.p. or i.v. route, comparable serum levels of
protein were achieved within a day of injection and the protein was
detectable in serum up to 7 days. After i.p. dosing at 20 mg/kg,
protein was rapidly absorbed with a T.sub.max of .about.8 h and
bioavailability (AUC.sub.IP/AUC.sub.IV) of 92%. The clearance of
the protein was .about.25 to 30 mL/d/kg with a half-life of about 4
days
Example 5
Binding Affinity of Frz-Fc Molecules
[0337] The addition of the FC domain to the Frz8(156) domains
results in an increase in binding affinity for Wnt3a of over two
magnitudes. FIG. 14 demonstrates the enhanced ability of Frz8-ECD
to block Wnt3a signaling when linked to a dimeric Fc domain. FIG.
14A is an IC.sub.50 graph of a Wnt3a inhibition assay of two
different preparations of Frz8(156)-FC. FIG. 14B is a gel
confirming the purity of the isolated Frz8(156) CRD (ECD). Shown
are: (a) non-reduced Frz8(156) ECD (Lane 1); (b) molecular weight
markers (Lane 2); and reduced Frz8 ECD (156) (Lane 3). This gel
indicates that the Frz8 ECD used in the binding assay is intact and
runs at approximately the expected molecular weight.
Example 6
Binding Activity of Frz-FC Chimeras
ELISA
[0338] For PK evaluation of the Wnt antagonist, the wells of a
384-well ELISA micro titer plate (Nunc Maxisorp, Rochester, N.Y.)
were coated with the rabbit anti-human Fc (Jackson Immuno Research,
Westgrove, Pa.) at a concentration of 1 .mu.g/ml in PBS (25
.mu.g/well). After an overnight incubation at 4.degree. C., the
rabbit anti-human Fc solution was decanted, and the plates were
blocked with 40 .mu.l/well of block buffer (PBS containing 0.5% BSA
and 10 ppm proclin). After a 60 minute incubation at room
temperature with gentle agitation, the rabbit anti-human Fc coated
plates were washed three times with wash buffer (PBS 0.05% Tween
20.RTM. and 10 ppm proclin). The Frizzled-Fc standards (a dilution
series with a concentration range of 0.78-100 ng/ml), and the
samples diluted into assay range in assay buffer (PBS containing
0.5% BSA, 0.05% Tween 20.RTM. and 10 ppm proclin) were added to the
assay plate (25 Owen). After a 120 minute incubation at room
temperature with gentle agitation, the assay plates were washed six
times with wash buffer. The remaining bound Frz-Fc was detected
using a horse radish peroxidase (HRP) conjugated goat anti-human
IgG-Fc (Jackson Immuno Research) diluted into assay diluent (25
.mu.l/well). After appropriate color development (10-25 minutes)
the enzymatic reaction was stopped with 1M phosphoric acid (25
Owen). The assay plates were read at a wavelength of 450 nm with a
reference wavelength of 630 nm. Sample concentrations were
determined by comparing the sample OD against the standard curve
fit using a 4-parameter algorithm.
BIAcore
[0339] FIG. 15 demonstrates direct binding by Wnt3a to the
Frz8(1-156)-Fc chimera. This chimera protein was amine coupled to a
Biocore.TM. (BIAcore, Inc. Piscataway, N.J.) CM5 sensor chip at
approximately 1700 response units as described generally in Chen,
Y. et al., J. Mol. Biol. 293: 865-881 (1999). Briefly,
carboxymethylated dextran biosensor chips (CM5, BIAcore.TM. Inc.)
were activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the
supplier's instructions. An injection of 1M ethanolamine was done
to block unreacted groups. Wnt3a was then injected at an estimated
concentration of 0.5 .mu.g/ml and binding was assessed by the
change in response units as a function of time. Wnt3a was found to
bind Frz8-Fc. As shown in FIG. 15, the association of Wnt3a and
Frz-Fc results in a highly significant increase of 1000 response
units over control protein (E. coli expressed non-native
Wnt3a.)
OCTET
[0340] The ability of the Wnt antagonists to interact with the Wnt
ligands Wnt3a and Wnt5a was measured using the OCTET.TM.-QK system.
(ForteBio, Inc., Menlo Park, Calif.). This system allows for the
measurement of protein binding at a biosensor surface. The assays
were conducted by first incubating one of the Wnt antagonist
molecules (20 ug/mL) with anti-human IgG Fc-specific biosensors for
10 minutes in phosphate buffered saline (PBS) with 0.5% CHAPS. The
unbound Wnt antagonist was removed by washing for 1.5 minutes in
PBS 0.5% CHAPS. Either Wnt3a or Wnt5a (5.0 ug/mL) was then added to
the assay and incubated with the Wnt antagonist molecules bound to
the biosensor surface for 5 minutes in PBS with 0.5% CHAPS. The
interaction between the Wnt antagonist molecules and Wnt ligand was
monitored in the same buffer. All assay steps were performed at
room temperature in a volume of 150 uL. FIG. 16 shows the result of
this binding assay with FIG. 16A showing data from the binding of
Wnt3a to the Frz1-Frz10-Fc chimeras, FIG. 16B showing data from the
binding of Wnt3a to sFRP-Fc chimeras, and FIG. 16C showing data
from the binding of Wnt5a to the Frz1-Frz10-Fc chimeras and sFRP-Fc
chimeras.
[0341] The OCTET.TM. assay indicates that both Wnt3a and Wnt-5a
bind Fz8-Fc, Fz5-Fc, and Fz4-Fc the fastest, relative to the other
Frz proteins, with Wnt3a binding Fz1-Fc, Fz2-Fc, and Fz7-Fc at a
slower rate. The amplitude and linear nature of Wnt-5a binding
curves suggest a lower binding affinity relative to Wnt3a binding,
as determined by this binding assay. The amplitude of the OCTET.TM.
binding data suggest that the sFRP-Fc proteins have an affinity for
Wnt3a similar that observed for Frz1, Frz2, and Frz7, and somewhat
lower that observed for Frz5 and Frz8.
Example 7
Inhibition of Wnt Signaling by the Wnt Antagonists--Cellular
Assays
[0342] Cellular assays were performed using 293 (human kidney)
cells transfected with the TOPglow reporter plasmid. In preparation
for transfection approximately 500,000 HEK293 were plated into a
well of a 12-well dish (Nuc) 24 hours before transfections. Cells
were transfected with 0.375 .mu.g TOPglow (Upstate, Cat #21-204),
0.05 mg LEF1, 0.01 mg SV40 RL with Fugene (Roche) and at 24 hours
post transfection. Media was changed and cells were untreated or
treated with Wnt3a alone, Wnt-5a alone, or with a Wnt antagonist
for an additional 20-24 hours before harvesting. All dilutions were
made in complete media for the indicated cell lines. Cells were
harvested in 50-100 .mu.l of 1.times.SJC lysis buffer (20 mM Tris
pH 8.0, 137 mM NaCl, 1 mM EGTA, 1% Triton X-100, 10% Glycerol, 1.5
mM MgCl.sub.2, 1 mM DTT, 50 mM NaF, 1 mM NaVO.sub.4 and protease
inhibitors) and duplicate 10 .mu.l were assayed using Dual-Glo.TM.
luciferase assay kit (Promega, Part # TM058) and detected in an
Envision Luminometer (Perkin Elmer). Luciferase activity was
normalized against Renilla activity.
[0343] Cells to be treated with Wnt5a were transfected with Frz4
and Lrp5 in addition to the reporter. The presence of these
additional components allows Wnt pathway activation by Wnt5a to
proceed as per the canonical pathway. Mikels A J, and Nusse R.,
PLOS Biol. 4:e115 (2006). Wnt3a activated cells were treated with
100 ng/ml Wnt3a and Wnt5a activated cells were treated with 1 ug/ml
Wnt5a.
[0344] As shown in FIGS. 17A and B, the Frz-Fc antagonist inhibited
Wnt signaling to varying degrees. Both Frz5-Fc and Frz8-Fc showed
complete inhibition of the Wnt3a signal and significantly inhibited
the Wnt5a signal. Frz4-Fc, Frz2-Fc, and Frz7-Fc showed significant
inhibition of the Wnt3a signal.
Example 8
Relative IC50s of the Wnt Antagonists
[0345] The relative IC50s of the Wnt antagonists were determined by
measuring inhibition of Wnt signaling by the Wnt antagonists in
U2OS (human osteosarcoma) cells stably transfected with TOPglow
luciferase TCF reporter plasmid as described in Example 7. Initial
Wnt signaling in cells was obtained with Wnt3a activation. A 3-fold
dilution series of Frz-Fcs was applied to cells overnight. (FIG.
18). As determined by this assay, Wnt3a binds to Frz8-Fc, Frz5-Fc,
and Frz4-Fc with sub-nanomolar IC50 (with Frz8-Fc having an IC50 of
0.04 nM, Frz-5Fc having an IC50 of 0.20 nM, and Frz-4 having an
IC50 of 0.48 nM) and to Frz2-Fc and Frz7-Fc with nanomolar IC50
(with Frz2-Fc having an IC50 of 1.2 nM and Frz2-Fc having an IC50
of 1.4 nM.
Example 9
Wnt Target Genes as Pharmacodynamic Markers of Drug Response
[0346] As an alternative to immunohistochemical analysis of
.beta.-catenin, transcriptional targets of Wnt were used to monitor
inhibition of Wnt signaling activity. The cell lines that had
autocrine Wnt signaling showed increased expression of known Wnt
target genes and this expression was regulated by in vitro
treatment with Wnt3a as well as by Frz8-Fc. RNA analysis of NTera-2
cells indicated that Frz8-Fc treatment affects expression of the
Wnt target genes tested. Thus, the expression of these genes can be
followed as an indicator of treatment efficacy. As an extension of
these observations, expression of these Wnt target genes can be
used as a diagnostic tool to identify cancers that are driven by
Wnt signaling and are likely candidates for anti-Wnt therapeutic
agents.
In Vitro
[0347] In vitro comparative gene expression analysis on PA-1 cells
treated with purified Wnt3a, Fz8 CRD-Fc, or a control protein was
performed to determine the suitability of Wnt target genes to
indicate in vivo inhibition of Wnt signaling in teratoma cells. RNA
isolated from PA-1 cells that were treated with Wnt3a, Frz8-Fc, or
control Fc protein was subject to microarray analysis and the
change in expression levels of the indicated genes in response to
exogenously added Wnt3a, Frz8-Fc, and control Fc protein was
determined. For microarray analysis, cells were treated with the
indicated proteins in triplicate and total RNA was isolated using
the RNAeasy kit (Qiagen). Array analysis was done on the Affymetrix
Human Genome U133 Gene Chip set (Rubinfeld B, et al., Nat
Biotechnol 2006; 24:205-9). The specific probes and primer sets are
shown in FIG. 20.
[0348] The expression levels of previously identified targets of
Wnt signaling such as Axin2, APCDD1, and Gad1 were up-regulated by
Wnt3a treatment or down-regulated by Frz8-Fc treatment (FIG. 19A).
Moreover, some genes such as Lefty2 (A), Lefty1 (B), sFRP1, and
Fzd5 were down-regulated by Wnt3a and up-regulated by inhibition of
Wnt signaling with Frz8-Fc (FIG. 19A). Subsequent gene expression
analysis by qRT-PCR showed that these transcripts were similarly
regulated by Wnt3a and Fz8 CRD-Fc in NTera-2, Tera-2, and NCCIT
cells as well.
In Vivo
[0349] APCDD1, Gad1, and Fzd5 were among the most consistently
modulated genes in above described in vitro analyses and were
therefore selected as potential markers of Wnt responsiveness for
in vivo tumor xenograft studies.
[0350] Tumor tissue RNA was purified from xenograft specimens
collected at the end of the efficacy study and quantitative reverse
transcription-PCR (qRT-PCR) analysis of Wnt-responsive transcripts
carried out as previously described Rubinfeld B, et al., Nat
Biotechnol 2006; 24:205-9). Fold induction for each gene was
determined using the .DELTA..DELTA.Ct method and the result
presented relative to glyceraldehyde-3-phosphate dehydrogenase. The
specific probes and primer sets are shown in FIG. 20. All reactions
were done in duplicate and the average of at least two
assays.+-.SEM was plotted.
[0351] Similar to the effects seen in vitro, treatment with
therapeutic doses of Frz8-Fc reduced the expression of genes for
APCDD1 and Gad1 and increased the expression of Fzd5 in tumors from
the NTera-2 xenografts (FIG. 19B). Although there is a general
nonspecific down-regulation of all genes following CD4hFc
treatment, these changes were not statistically significant
compared with those seen in Frz8-Fc-treated tumors. These
observations show that the antitumorigenic effects of Frz8-Fc in
vivo are on target genes and that the expression levels of these
genes can be used to monitor the efficacy of potential anti-Wnt
therapeutic agents.
Example 10
Inhibitory Effect of Wnt Antagonists on Growth of Tumors in Mice
with Allografts and Human Xenografts
[0352] The studies set forth in this Example indicate that the Wnt
antagonists are useful in treating Wnt expressing tumors. The
largely complete tumor regression in the case of Wnt-1-MMTV model
illustrate the effect of the Wnt antagonists on tumors that are
strongly Wnt driven. However, the significant effect of the Wnt
antagonist on the PA-1 and NTer2 tumors also reflects the strong
therapeutic potential to treat tumors that may not be entirely Wnt
driven.
Animals
[0353] Female C57B16 mice (The Jackson Laboratory) were used for
the passaging of MMTV-Wnt1 tumors. Maintenance of mice and in vivo
procedures were carried out using Institutional Animal Care and Use
Committee-approved protocols.
MMTV-Wnt Model--Allografts
[0354] FIG. 21 is a linear schematic describing the vector
construct used in the transfection to create the Wnt animal model.
This construct mimics the constitutive Wnt signaling activation
observed with MMTA viral insertion, as described in Tsujomoto et
al., Cell 55: 619-625 (1988) and Li et al., Oncogene 19: 1002-1009
(2000).
Passaging of MMTV-Wnt1 transgenic tumors in mice.
[0355] The tumors from MMTV-Wnt1 transgenic mice were serially
passaged in C57B16 mice for 6 to 10 passages by surgical
implantation in the mammary fat pad. Tumor tissue was aseptically
collected from the transgenic mouse, rinsed in HBSS and cut into
small pieces. The recipient mice were anaesthetized with a mixture
of ketamine (75-80 mg/kg) and xylazine (7.5-15 mg/kg), the tumor
fragment inserted under the skin rostral to the third mammary fat
pad, and the skin closed using wound clips. Tumors were passaged
for a maximum of 10 passages, and after the first two passages,
tumor tissue was examined histologically to confirm that it was of
mammary origin and continues to express Wnt. Mammary
adenocarcinomas develop in 6-12 months in the mice. Tumors isolated
from these mice were used to create the transplant models described
below.
In Vivo Studies.
[0356] For in vivo studies testing the efficacy of Wnt antagonists
in the MMTV-Wnt model, the tumor cells were introduced by
subcutaneous injection of cells obtained from macerated tumors
tissue. Tumor tissue was aseptically collected from mice
transplanted with tumors from Wnt transgenic mice (described
above), rinsed in PBS or HBSS, cut into smaller pieces and
macerated into HBSS using a cell dissociation kit (Sigma). The
cells were washed twice in sterile HBSS and suspended in a 50%
matrigel solution in HBSS. The cell suspension was inoculated
subcutaneously into the mammary fat pad of athymic nude mice, with
a volume not exceeding 150 .mu.l/mouse.
[0357] For in vivo studies using the NTera2 or PA-1 animals models,
cells were grown as described and harvested when growth is in the
logarithmic phase. The cells were suspended in a 50% matrigel
solution in HBSS and inoculated subsutaneously into athymic nude
mice at a concentration of either 8 million cells/mouse (NTera2) or
10 million cells/mouse (PA-1).
[0358] Tumors were monitored daily and measured after 7-12 days of
inoculation. Animals were separated into groups with identical mean
tumor volumes in the range of 150-250 mm.sup.3. Treatment with the
Wnt antagonist started 1-2 days after grouping and the mice were
dosed intraperitoneally (IP) or intravenously (IV) with 100-200
.mu.l of Wnt antagonist, negative control protein CD4-Fc, or PBS
negative control once daily. Subsequent drug treatments were
repeated 2-3 times weekly and continued for 3-4 weeks. Tumor volume
was measured twice weekly the animals were sacrificed when the
tumor volume reached 2500 mm.sup.3. Blood was collected during the
study by an orbital vein bleed and the serum assayed for levels of
therapeutic agent by SDS-PAGE followed by immunoblot and detection
using HRP or fluorescent conjugated anti-human Fc, and for activity
of the therapeutic agent by its ability to inhibit Wnt3a activation
of TOPglow activity as described in Example 7.
Allograft Tumors
Inhibitory Effect of Wnt Antagonists on Growth of Tumor
Allografts
[0359] Treatment with Frz8-Fc by either the i.p. or i.v. routed
resulted in rapid tumor regression with sustained inhibition during
the course of treatment, whereas the negative control protein
CD4-Fc had no effect relative to the PBS treatment. The treated
mice were monitored for three weeks after termination of treatment
and regrowth of tumors was eventually observed.
[0360] FIG. 22 illustrates the efficacy of Frz8-Fc against MMTV-Wnt
tumor transplants in athymic nude mice by intraperitoneal (IP)
dosing. FIG. 22A is a graph showing that nude mice hosting
MMTV-Wnt-1 tumor transplants were administered PBS, CD4-Fc (10
mg/kg/day) or Frz8-Fc (10 mg/kg/day) by intraperitoneal injection
twice weekly. Each group had 11 mice and the average tumor volume
for the group was 226 mm.sup.3 before the start of treatments. Mean
tumor volume is plotted over time and the treatment days are
indicated by arrows on the X-axis. On day 25, the control groups
were sacrificed and the drug administration to the treatment group
stopped. FIG. 22B is tabular summary of mean tumor volume and mean
% change in tumor volume over time in the four treatment groups.
Note that in FIG. 22B, the mean tumor volume after treatment with
Frz8-Fc antagonist results in a reduction in tumor volume from 226
mm to about 219 mm.sup.3 on the fifth day after start of treatment,
and about 67 mm.sup.3 on the 18.sup.th day. This represents a 4%
and 70%, respectively, reduction in tumor size. In this study,
tumors administered the Frz-Fc antagonist showed regression in
tumor size compared with control animals. This demonstrates that
Frz-Fc antagonists of the invention are tumoricidal as a single
agent and are useful as anti-cancer therapeutics.
[0361] FIG. 23 illustrates the efficacy of Frz8-Fc against MMTV-Wnt
tumor transplant in athymic nude mice by intravenous (IV) dosing.
FIG. 23A is a graph showing that nude mice hosting MMTV-Wnt-1 tumor
transplants were administered PBS, CD4-Fc (10 mg/kg/day) or Frz8-Fc
(10 mg/kg/day) by intravenous injection three times weekly. Each
group had 11 mice and the average tumor volume for the group was
226 mm.sup.3 before the start of treatments. The fourth group (high
bar) in this study included 10 mice with a mean tumor volume of 375
mm.sup.3 at the start of the study that were treated with Frz8-Fc
(10 mg/kg/day) by intravenous injection three times weekly. Mean
tumor volume is plotted over time and the treatment days are
indicated by arrows on the X-axis. On day 25, the control group
animals were sacrificed and drug administration to the treatment
group stopped. FIG. 23B is a tabular summary of mean tumor volume
and mean % change in tumor volume over time in the four treatment
groups. Note that in all mice treated with Frz-Fc that the tumor
burden was reduced from an average of 226 mm.sup.3 to an average
volume of 179 mm.sup.3 on the 4.sup.th day after start of
treatment, and to 73 mm.sup.3 after the 18.sup.th day. This
represents a 21% amd 67% reduction, respectively, in tumor volume.
For the high bar group, tumor volume was reduced from an average of
376 mm.sup.3 to 225 mm.sup.3 on the 4.sup.th day of treatment, and
to 53 mm.sup.3 on the 18.sup.th day. This represents a 39% and 86%
reduction, respectively, in tumor volume.
Inhibitory Effect of Serum Obtained from Treated Mice on Wnt
Signaling
[0362] Inhibition of Wnt signaling from serum isolated from the
treated mice is reported in FIG. 24, with FIG. 24A showing the
results of serum isolated from IP treated mice, while the W treated
ones appear in FIG. 24B. The data is presented as a bar graph
showing the Wnt signaling antagonist activity in the TOPglow assay
(as described in Example 7). The samples appear in groups according
to treatment, mouse study number and dilution. The relative
luciferase activity in the TOPglow gene reporter assay is shown on
the Y-axis. All samples are treated with .about.40 ng/ml purified
Wnt3a except for NA (control). All other protein controls are
present in the medium at 5 .mu.g/ml.
Human Xenograft Tumors
[0363] Inhibition of naturally derived human tumor models by the
Wnt antagonists would serve as a further indicator of their
usefulness in treating human cancer. Human tumor-derived cell lines
were tested for evidence of autocrine wnt signaling, similar to
that seen in the PA-1 teratoma cell line, as an indication of
usefulness in testing Wnt antagonist activity. The teratoma-derived
NTera-2, Tera-2, and NCCIT cell lines exhibited basal Wnt signaling
that could be inhibited by Frz8-Fc, in contrast with 293 cells that
exhibited low basal signaling that was not inhibited by Frz8-Fc
(FIG. 25A). Nevertheless, all four teratoma cell lines seemed to
express Wnt receptors, as signaling was further stimulated by Wnt3a
treatment, which could be blocked by Frz8-Fc (FIG. 25B). These
results indicate that the teratoma cell lines express Wnt(s), which
might contribute to their tumorigenicity. These lines were
therefore evaluated for tumor formation in athymic nude mice and
based on consistency of tumor formation, NTera-2 and PA-1 were
selected for in vivo efficacy studies.
Inhibitory Effect of Wnt Antagonists on Growth of NTera2 Tumor
Xenografts
[0364] Treatment of mice exhibiting NTera2 tumor xenografts with
the Wnt antagonist Frz8-Fc resulted in a reduction of tumor volume
by approximately 50% and reduction tumor mass by approximately 70%,
relative to the control mice.
[0365] FIG. 26 shows the anti-tumor efficacy of Frz8-Fc treatment
on the growth of NTera2 tumor xenografts in athymic nude mice.
Athymic nude mice bearing NTera2 tumor xenografts were administered
an initial dose of PBS, CD4-Fc and Frz8-Fc at 15 mg/kg/day,
followed by subsequent doses of 10 mg/kg/day by intraperitoneal
injection three times weekly. Each group had 20 mice and the
average tumor volume for the group was 200 mm.sup.3 before the
start of treatments. The fourth group of the study included 10 mice
with a mean tumor volume of 336 mm.sup.3 at the start of the study
that were treated with Frz8-Fc (10 mg/kg/day) by intraperitoneal
injection three times weekly. FIG. 26A is an exemplary procedural
flow chart, while FIG. 26B is a graph plotting mean tumor volume
over time, wherein the treatment days are indicated by arrows on
the X-axis. FIG. 26C is a bar graph plotting the mean tumor weights
at sacrifice of all animals in the group at day 20 of the study.
FIGS. 26D and 26E are tabular summaries of mean tumor volume and
mean % change in tumor volume, respectively.
Inhibitory Effect of Serum Obtained from Mice with Ntera2 Tumor
Xenografts on Wnt Signaling
[0366] FIG. 27 is a bar graph showing Wnt signaling antagonist
activity in the TOPglow assay of the Frz8-Fc Wnt antagonist of
serum isolated from various animals in the NTera2 tumor study.
Relative luciferase activity (Y-axis) as measured from TOPglow
assay from the controls and Frz8-Fc Wnt antagonist. No additional
purified Wnt or Wnt conditioned media was added to the cells. These
results demonstrate that reduced Wnt signaling is associated with
reduction in tumor size in these mice treated with Frz8-Fc Wnt
antagonist.
Inhibitory Effect of Wnt Antagonists on Growth of PA-1 Tumor
Xenografts
[0367] Treatment of mice exhibiting PA-1 tumor xenografts with the
Wnt antagonist Frz8-Fc resulted in a significant reduction in tumor
growth within 12 days of treatment. In this model, the tumors were
approximately 50% smaller, with significantly smaller mass than
tumors in the control mice at the end of the treatment period.
[0368] FIG. 28 demonstrates the anti-tumor efficacy of Frz8-Fc
treatment on the growth of PA-1 tumor xenografts in athymic nude
mice. Athymic nude mice bearing PA-1 tumors xenografts were
administered PBS, CD4-Fc or Frz-Fc at 15 mg/kg/day, followed by
subsequent doses of 10 mg/kg/day by intraperitoneal injection three
times weekly. Each group had 13 mice and the average tumor volume
for the group was 168 mm.sup.3 before the start of treatments. FIG.
28A is an exemplary procedural flow chart, while FIG. 28B is a
graph plotting mean tumor volume over time, wherein the treatment
days are indicated by arrows on the X-axis. FIG. 28C is a graph of
mean tumor weight at sacrifice. The mice were sacrificed on day 58
after cell inoculation (day 32 after start of treatments) and
tumors were excised and weighed. The mean tumor weight.+-.SEM is
plotted as a function of the group. FIGS. 28D and 28E are tabular
summaries of mean tumor volume and mean % change in tumor volume,
respectively.
Example 11
Wnt Signaling in Mice Transplanted with MMTV Tumors and Treated
with Frz8-Fc and Frz5-Fc Wnt Antagonists
Effect of Frz8-Fc and Frz5-Fc Wnt Antagonists on Wnt Signaling
[0369] Frz5-Fz inhibits Wnt3a induced signaling as effectively as
Frz8-Fc.
[0370] Athymic nude mice with MMTV tumors (approximately 400-800
cubic millimeters in size) were treated with Frz8-Fc, Frz5-Fz, or
CD4-Fc, as a negative control, at 10 mg/kg. Five hours after
treatment, serum was collected by cardiac puncture from the mice
and analyzed for Wnt inhibiting effect on 293 cells activated with
Wnt3a and transfected with TOPglow as described in Example 7. All
samples are treated with .about.40 ng/ml purified Wnt3a except for
NA (control). All other protein controls are present in the medium
at 5 .mu.g/ml. FIG. 29 shows the level of inhibition in mice
treated with Frz8-Fc or Frz5-Fz. Treatment with Frz8-Fc or Frz5-Fz
resulted in similar levels of inhibition of Wnt 3a induced
signaling.
Effect of Frz8-Fc and Frz5-Fc Wnt Antagonists on Axin2
Expression
[0371] Frz8-Fc and Frz5-Fz compounds inhibit in vivo Wnt signaling
as determined by modulation of the Wnt target gene Axin2.
[0372] Athymic nude mice with MMTV tumors (approximately 400-800
cubic millimeters in size) were treated with Frz8-Fc, Frz5-Fz, or
CD4-Fc, as a negative control, at 10 mg/kg. Five hours after
treatment, serum was collected by cardiac puncture from the mice.
RNA was extracted from the tumor cells using the QIAGEN RNAEASY kit
(Qiagen, Valencia, Calif.) and analyzed for expression of Axin2 as
described in Example 9. Reduced levels of Axin2 was observed in
samples obtained from mice treated with Frz8-Fc or Frz5-Fz
indicating that these compounds are able to inhibit in vivo Wnt
signaling. FIG. 30 shows the reduced Axin2 expression in Frz8-Fc
and Frz5-Fz treated tumor with FIG. 30A showing expression
normalized to expression of GAPDH and FIG. 30B showing expression
normalized to expression of rpl19.
Example 12
Regenerative Tissue Treated with Wnt Antagonist
[0373] Wnt signaling plays a critical role in self-renewal of
regenerating tissue such as skin, intestine, and hematopoietic
cells, and inhibition of Wnt signaling by Dkkl can adversely affect
the architecture of these tissues in adult mice. The following
Example examines whether exposure to Frz8-Fc under the same
conditions used to obtain antitumor efficacy had any effect on
intestine and skin in the mice. Tissues were collected from mice
that were treated in the MMTV-Wnt1 tumor model (described in
Example 10) after 14 treatments, thrice a week, and sections were
stained for .beta.-catenin protein by immunohistochemistry.
Analysis of skin and various intestinal compartments revealed that
the architecture of these tissues appeared morphologically normal
in treated mice of all groups, with typical patterns of cytoplasmic
and nuclear .beta.-catenin staining in intestinal Paneth cells
(FIG. 31A) and skin hair follicles (FIG. 31B). Furthermore,
histologic and immunohistochemical analysis of skin and intestine
collected from animals using the NTera-2 model, after nine
treatments, thrice a week also revealed no differences between
control and treated groups. This suggests that treatment with
Frz8-Fc with the therapeutic regimen that can inhibit tumor growth
does not have adverse effects on tissue renewal of skin and
intestine.
Example 13
[0374] This Example describes various methods of producing the Wnt
antagonists.
Expression of Wnt Antagonist in E. coli
[0375] This example illustrates preparation of an unglycosylated
form of Wnt antagonist by recombinant expression in E. coli.
[0376] The DNA sequence encoding Wnt antagonist is initially
amplified using selected PCR primers. The primers should contain
restriction enzyme sites which correspond to the restriction enzyme
sites on the selected expression vector. A variety of expression
vectors may be employed. An example of a suitable vector is pBR322
(derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)) which
contains genes for ampicillin and tetracycline resistance. The
vector is digested with restriction enzyme and dephosphorylated.
The PCR amplified sequences are then ligated into the vector. The
vector will preferably include sequences which encode for an
antibiotic resistance gene, a trp promoter, a polyhis leader
(including the first six STII codons, polyhis sequence, and
enterokinase cleavage site), the Wnt antagonist coding region,
lambda transcriptional terminator, and an argU gene.
[0377] The ligation mixture is then used to transform a selected E.
coli strain using the methods described in Sambrook et al., supra.
Transformants are identified by their ability to grow on LB plates
and antibiotic resistant colonies are then selected. Plasmid DNA
can be isolated and confirmed by restriction analysis and DNA
sequencing.
[0378] Selected clones can be grown overnight in liquid culture
medium such as LB broth supplemented with antibiotics. The
overnight culture may subsequently be used to inoculate a larger
scale culture. The cells are then grown to a desired optical
density, during which the expression promoter is turned on.
[0379] After culturing the cells for several more hours, the cells
can be harvested by centrifugation. The cell pellet obtained by the
centrifugation can be solubilized using various agents known in the
art, and the solubilized Wnt antagonist protein can then be
purified using a metal chelating column under conditions that allow
tight binding of the protein.
[0380] Wnt antagonist may be expressed in E. coli in a poly-His
tagged form, using the following procedure. The DNA encoding Wnt
antagonist is initially amplified using selected PCR primers. The
primers will contain restriction enzyme sites which correspond to
the restriction enzyme sites on the selected expression vector, and
other useful sequences providing for efficient and reliable
translation initiation, rapid purification on a metal chelation
column, and proteolytic removal with enterokinase.
[0381] The PCR-amplified, poly-His tagged sequences are then
ligated into an expression vector, which is used to transform an E.
coli host based on strain 52 (W3110 fuhA(tonA) lon galE
rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LB
containing 50 mg/ml carbenicillin at 30.degree. C. with shaking
until an O.D.600 of 3-5 is reached. Cultures are then diluted
50-100 fold into CRAP media (prepared by mixing 3.57 g
(NH.sub.4).sub.2SO.sub.4, 0.71 g sodium citrate.2H2O, 1.07 g KCl,
5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL
water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM
MgSO.sub.4) and grown for approximately 20-30 hours at 30.degree.
C. with shaking. Samples are removed to verify expression by
SDS-PAGE analysis, and the bulk culture is centrifuged to pellet
the cells. Cell pellets are frozen until purification and
refolding.
[0382] E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets)
is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH
8 buffer. Solid sodium sulfite and sodium tetrathionate is added to
make final concentrations of 0.1M and 0.02 M, respectively, and the
solution is stirred overnight at 4.degree. C. This step results in
a denatured protein with all cysteine residues blocked by
sulfitolization. The solution is centrifuged at 40,000 rpm in a
Beckman Ultracentifuge for 30 min. The supernatant is diluted with
3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM
Tris, pH 7.4) and filtered through 0.22 micron filters to clarify.
The clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal
chelate column equilibrated in the metal chelate column buffer. The
column is washed with additional buffer containing 50 mM imidazole
(Calbiochem, Utrol grade), pH 7.4. The protein is eluted with
buffer containing 250 mM imidazole. Fractions containing the
desired protein are pooled and stored at 4.degree. C. Protein
concentration is estimated by its absorbance at 280 nm using the
calculated extinction coefficient based on its amino acid
sequence.
[0383] The proteins are refolded by diluting the sample slowly into
freshly prepared refolding buffer consisting of: 20 mM Tris, pH
8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM
EDTA. Refolding volumes are chosen so that the final protein
concentration is between 50 to 100 micrograms/ml. The refolding
solution is stirred gently at 4.degree. C. for 12-36 hours. The
refolding reaction is quenched by the addition of TFA to a final
concentration of 0.4% (pH of approximately 3). Before further
purification of the protein, the solution is filtered through a
0.22 micron filter and acetonitrile is added to 2-10% final
concentration. The refolded protein is chromatographed on a Poros
R1/H reversed phase column using a mobile buffer of 0.1% TFA with
elution with a gradient of acetonitrile from 10 to 80%. Aliquots of
fractions with A280 absorbance are analyzed on SDS polyacrylamide
gels and fractions containing homogeneous refolded protein are
pooled. Generally, the properly refolded species of most proteins
are eluted at the lowest concentrations of acetonitrile since those
species are the most compact with their hydrophobic interiors
shielded from interaction with the reversed phase resin. Aggregated
species are usually eluted at higher acetonitrile concentrations.
In addition to resolving misfolded forms of proteins from the
desired form, the reversed phase step also removes endotoxin from
the samples.
[0384] Fractions containing the desired folded Wnt antagonist
polypeptide are pooled and the acetonitrile removed using a gentle
stream of nitrogen directed at the solution. Proteins are
formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and
4% mannitol by dialysis or by gel filtration using G25 Superfine
(Pharmacia) resins equilibrated in the formulation buffer and
sterile filtered.
Expression of Wnt Antagonist in Mammalian Cells
[0385] This example illustrates preparation of a potentially
glycosylated form of Wnt antagonist by recombinant expression in
mammalian cells.
[0386] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989),
is employed as the expression vector. Optionally, the Wnt
antagonist DNA is ligated into pRK5 with selected restriction
enzymes to allow insertion of the Wnt antagonist DNA using ligation
methods such as described in Sambrook et al., supra. For purposes
of this example, the resulting vector is referred to as
pRK5-WA.
[0387] In one embodiment, the selected host cells may be 293 cells.
Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue
culture plates in medium such as DMEM supplemented with fetal calf
serum and optionally, nutrient components and/or antibiotics. About
10 .mu.g pRK5-WA DNA is mixed with about 1 .mu.g DNA encoding the
VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved
in 500 .mu.l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl.sub.2. To
this mixture is added, dropwise, 500 .mu.l of 50 mM HEPES (pH
7.35), 280 mM NaCl, 1.5 mM NaPO.sub.4, and a precipitate is allowed
to form for 10 minutes at 25.degree. C. The precipitate is
suspended and added to the 293 cells and allowed to settle for
about four hours at 37.degree. C. The culture medium is aspirated
off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The
293 cells are then washed with serum free medium, fresh medium is
added and the cells are incubated for about 5 days.
[0388] Approximately 24 hours after the transfections, the culture
medium is removed and replaced with culture medium (alone) or
culture medium containing 200 .mu.Ci/ml .sup.35S-cysteine and 200
.mu.Ci/ml .sup.35S-methionine. After a 12 hour incubation, the
conditioned medium is collected, concentrated on a spin filter, and
loaded onto a 15% SDS gel. The processed gel may be dried and
exposed to film for a selected period of time to reveal the
presence of Wnt antagonist polypeptide. The cultures containing
transfected cells may undergo further incubation (in serum free
medium) and the medium is tested in selected bioassays.
[0389] In an alternative technique, Wnt antagonist may be
introduced into 293 cells transiently using the dextran sulfate
method described by Somparyrac et al., Proc. Natl. Acad. Sci.,
12:7575 (1981). 293 cells are grown to maximal density in a spinner
flask and 700 .mu.g pRK5-WA DNA is added. The cells are first
concentrated from the spinner flask by centrifugation and washed
with PBS. The DNA-dextran precipitate is incubated on the cell
pellet for four hours. The cells are treated with 20% glycerol for
90 seconds, washed with tissue culture medium, and re-introduced
into the spinner flask containing tissue culture medium, 5 .mu.g/ml
bovine insulin and 0.1 .mu.g/ml bovine transferrin. After about
four days, the conditioned media is centrifuged and filtered to
remove cells and debris. The sample containing expressed Wnt
antagonist can then be concentrated and purified by any selected
method, such as dialysis and/or column chromatography.
[0390] In another embodiment, Wnt antagonist can be expressed in
CHO cells. The pRK5-WA can be transfected into CHO cells using
known reagents such as CaPO.sub.4 or DEAE-dextran. As described
above, the cell cultures can be incubated, and the medium replaced
with culture medium (alone) or medium containing a radiolabel such
as .sup.35S-methionine. After determining the presence of Wnt
antagonist polypeptide, the culture medium may be replaced with
serum free medium. Preferably, the cultures are incubated for about
6 days, and then the conditioned medium is harvested. The medium
containing the expressed Wnt antagonist can then be concentrated
and purified by any selected method.
[0391] Epitope-tagged Wnt antagonist may also be expressed in host
CHO cells. The Wnt antagonist may be subcloned out of the pRK5
vector. The subclone insert can undergo PCR to fuse in frame with a
selected epitope tag such as a poly-his tag into a Baculovirus
expression vector. The poly-his tagged Wnt antagonist insert can
then be subcloned into a SV40 driven vector containing a selection
marker such as DHFR for selection of stable clones. Finally, the
CHO cells can be transfected (as described above) with the SV40
driven vector. Labeling may be performed, as described above, to
verify expression. The culture medium containing the expressed
poly-His tagged Wnt antagonist can then be concentrated and
purified by any selected method, such as by Ni.sup.2+-chelate
affinity chromatography.
[0392] Wnt antagonist may also be expressed in CHO and/or COS cells
by a transient expression procedure or in CHO cells by another
stable expression procedure.
[0393] Stable expression in CHO cells is performed using the
following procedure. The proteins are expressed as an IgG construct
(immunoadhesin), in which the coding sequences for the soluble
forms (e.g. extracellular domains) of the respective proteins are
fused to an IgG1 constant region sequence containing the hinge, CH2
and CH2 domains and/or is a poly-His tagged form.
[0394] Following PCR amplification, the respective DNAs are
subcloned in a CHO expression vector using standard techniques as
described in Ausubel et al., Current Protocols of Molecular
Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression
vectors are constructed to have compatible restriction sites 5' and
3' of the DNA of interest to allow the convenient shuttling of
cDNA's. The vector used expression in CHO cells is as described in
Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses the
SV40 early promoter/enhancer to drive expression of the cDNA of
interest and dihydrofolate reductase (DHFR). DHFR expression
permits selection for stable maintenance of the plasmid following
transfection.
[0395] Twelve micrograms of the desired plasmid DNA is introduced
into approximately 10 million CHO cells using commercially
available transfection reagents SUPERFECTt.RTM. (Quiagen),
DOSPER.RTM. or FUGENE.RTM. (Boehringer Mannheim). The cells are
grown as described in Lucas et al., supra. Approximately
3.times.10.sup.7 cells are frozen in an ampule for further growth
and production as described below.
[0396] The ampules containing the plasmid DNA are thawed by
placement into water bath and mixed by vortexing. The contents are
pipetted into a centrifuge tube containing 10 mLs of media and
centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated
and the cells are resuspended in 10 mL of selective media (0.2
.mu.m filtered PS20 with 5% 0.2 .mu.m diafiltered fetal bovine
serum). The cells are then aliquoted into a 100 mL spinner
containing 90 mL of selective media. After 1-2 days, the cells are
transferred into a 250 mL spinner filled with 150 mL selective
growth medium and incubated at 37.degree. C. After another 2-3
days, 250 mL, 500 mL and 2000 mL spinners are seeded with
3.times.10.sup.5 cells/mL. The cell media is exchanged with fresh
media by centrifugation and resuspension in production medium.
Although any suitable CHO media may be employed, a production
medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992
may actually be used. A 3 L production spinner is seeded at
1.2.times.10.sup.6 cells/mL. On day 0, the cell number pH is
determined. On day 1, the spinner is sampled and sparging with
filtered air is commenced. On day 2, the spinner is sampled, the
temperature shifted to 33.degree. C., and 30 mL of 500 g/L glucose
and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane
emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout
the production, the pH is adjusted as necessary to keep it at
around 7.2. After 10 days, or until the viability dropped below
70%, the cell culture is harvested by centrifugation and filtering
through a 0.22 .mu.m filter. The filtrate was either stored at
4.degree. C. or immediately loaded onto columns for
purification.
[0397] For the poly-His tagged constructs, the proteins are
purified using a Ni-NTA column (Qiagen). Before purification,
imidazole is added to the conditioned media to a concentration of 5
mM. The conditioned media is pumped onto a 6 ml Ni-NTA column
equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl
and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4.degree. C.
After loading, the column is washed with additional equilibration
buffer and the protein eluted with equilibration buffer containing
0.25 M imidazole. The highly purified protein is subsequently
desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl
and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia)
column and stored at -80.degree. C.
[0398] Immunoadhesin (Fc-containing) constructs are purified from
the conditioned media as follows. The conditioned medium is pumped
onto a 5 ml Protein A column (Pharmacia) which had been
equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading,
the column is washed extensively with equilibration buffer before
elution with 100 mM citric acid, pH 3.5. The eluted protein is
immediately neutralized by collecting 1 ml fractions into tubes
containing 275 .mu.L of 1 M Tris buffer, pH 9. The highly purified
protein is subsequently desalted into storage buffer as described
above for the poly-His tagged proteins. The homogeneity is assessed
by SDS polyacrylamide gels and by N-terminal amino acid sequencing
by Edman degradation.
Expression of Wnt Antagonist in Yeast The following method
describes recombinant expression of Wnt antagonist in yeast.
[0399] First, yeast expression vectors are constructed for
intracellular production or secretion of Wnt antagonist from the
ADH2/GAPDH promoter. DNA encoding Wnt antagonist and the promoter
is inserted into suitable restriction enzyme sites in the selected
plasmid to direct intracellular expression of Wnt antagonist. For
secretion, DNA encoding Wnt antagonist can be cloned into the
selected plasmid, together with DNA encoding the ADH2/GAPDH
promoter, a native Wnt antagonist signal peptide or other mammalian
signal peptide, or, for example, a yeast alpha-factor or invertase
secretory signal/leader sequence, and linker sequences (if needed)
for expression of Wnt antagonist.
[0400] Yeast cells, such as yeast strain AB110, can then be
transformed with the expression plasmids described above and
cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by precipitation with 10%
trichloroacetic acid and separation by SDS-PAGE, followed by
staining of the gels with Coomassie Blue stain.
[0401] Recombinant Wnt antagonist can subsequently be isolated and
purified by removing the yeast cells from the fermentation medium
by centrifugation and then concentrating the medium using selected
cartridge filters. The concentrate containing Wnt antagonist may
further be purified using selected column chromatography
resins.
Expression of Wnt Antagonist in Baculovirus-Infected Insect
Cells
[0402] The following method describes recombinant expression of Wnt
antagonist in Baculovirus-infected insect cells.
[0403] The sequence coding for Wnt antagonist is fused upstream of
an epitope tag contained within a baculovirus expression vector.
Such epitope tags include poly-his tags and immunoglobulin tags
(like Fc regions of IgG). A variety of plasmids may be employed,
including plasmids derived from commercially available plasmids
such as pVL1393 (Novagen). Briefly, the sequence encoding Wnt
antagonist or the desired portion of the coding sequence of Wnt
antagonist such as the sequence encoding an extracellular domain of
a transmembrane protein or the sequence encoding the mature protein
if the protein is extracellular is amplified by PCR with primers
complementary to the 5' and 3' regions. The 5' primer may
incorporate flanking (selected) restriction enzyme sites. The
product is then digested with those selected restriction enzymes
and subcloned into the expression vector.
[0404] Recombinant baculovirus is generated by co-transfecting the
above plasmid and BACULOGOLD.TM. virus DNA (Pharmingen) into
Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using
lipofectin (commercially available from GIBCO-BRL). After 4-5 days
of incubation at 28.degree. C., the released viruses are harvested
and used for further amplifications. Viral infection and protein
expression are performed as described by O'Reilley et al.,
Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford
University Press (1994). Expressed poly-his tagged Wnt antagonist
can then be purified, for example, by Ni.sup.2+-chelate affinity
chromatography as follows. Extracts are prepared from recombinant
virus-infected Sf9 cells as described by Rupert et al., Nature,
362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in
sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl.sub.2; 0.1 mM
EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for
20 seconds on ice. The sonicates are cleared by centrifugation, and
the supernatant is diluted 50-fold in loading buffer (50 mM
phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through
a 0.45 .mu.m filter. A Ni.sup.2+-NTA agarose column (commercially
available from Qiagen) is prepared with a bed volume of 5 mL,
washed with 25 mL of water and equilibrated with 25 mL of loading
buffer. The filtered cell extract is loaded onto the column at 0.5
mL per minute. The column is washed to baseline A.sub.280 with
loading buffer, at which point fraction collection is started.
Next, the column is washed with a secondary wash buffer (50 mM
phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes
nonspecifically bound protein. After reaching A.sub.280 baseline
again, the column is developed with a 0 to 500 mM Imidazole
gradient in the secondary wash buffer. One mL fractions are
collected and analyzed by SDS-PAGE and silver staining or Western
blot with Ni.sup.2+-NTA-conjugated to alkaline phosphatase
(Qiagen). Fractions containing the eluted His.sub.10-tagged Wnt
antagonist are pooled and dialyzed against loading buffer.
[0405] Alternatively, purification of the IgG tagged (or Fc tagged)
Wnt antagonist can be performed using known chromatography
techniques, including for instance, Protein A or protein G column
chromatography.
Purification of Wnt Antagonist Polypeptides Using Affinity
Chromatography
[0406] Native or recombinant Wnt Antagonist polypeptides may be
purified by a variety of standard techniques in the art of protein
purification. For example, pro-, mature, or pre-Wnt antagonist
polypeptide is purified by immunoaffinity chromatography using
antibodies specific for the Wnt antagonist polypeptide of interest.
In general, an immunoaffinity column is constructed by covalently
coupling the Wnt antagonist polypeptide to an activated
chromatographic resin. Alternatively, Wnt antagonist which contain
an Fc domain may be purified directly from media using a
immobilized protein A resin such as ProSepA (Millipore).
[0407] Polyclonal immunoglobulins are prepared from immune sera
either by precipitation with ammonium sulfate or by purification on
immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway,
N.J.). Likewise, monoclonal antibodies are prepared from mouse
ascites fluid by ammonium sulfate precipitation or chromatography
on immobilized Protein A. Partially purified immunoglobulin is
covalently attached to a chromatographic resin such as
CnBr-activated SEPHAROSE.TM. (Pharmacia LKB Biotechnology). The
antibody is coupled to the resin, the resin is blocked, and the
derivative resin is washed according to the manufacturer's
instructions.
[0408] Such an immunoaffinity column may be utilized in the
purification of Wnt antagonist polypeptide by preparing a fraction
from cells containing Wnt antagonist in a soluble form. This
preparation is derived by solubilization of the whole cell or of a
subcellular fraction obtained via differential centrifugation by
the addition of detergent or by other methods well known in the
art. Alternatively, soluble Wnt antagonist polypeptide containing a
signal sequence may be secreted in useful quantity into the medium
in which the cells are grown.
[0409] A soluble Wnt antagonist polypeptide-containing preparation
is passed over the immunoaffinity column, and the column is washed
under conditions that allow the preferential absorbance of Wnt
antagonist polypeptide (e.g., high ionic strength buffers in the
presence of detergent). Then, the column is eluted under conditions
that disrupt antibody/Wnt antagonist binding (e.g., a low pH buffer
such as approximately pH 2-3, or a high concentration of a
chaotrope such as urea or thiocyanate ion), and Wnt antagonist
polypeptide is collected.
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Sequence CWU 1
1
1291646PRTHomo sapiens 1Met Ala Glu Glu Glu Ala Pro Lys Lys Ser Arg
Ala Ala Gly Gly Gly1 5 10 15Ala Ser Trp Glu Leu Cys Ala Gly Ala Leu
Ser Ala Arg Leu Ala Glu 20 25 30Glu Gly Ser Gly Asp Ala Gly Gly Arg
Arg Arg Pro Pro Val Asp Pro 35 40 45Arg Arg Leu Ala Arg Gln Leu Leu
Leu Leu Leu Trp Leu Leu Glu Ala 50 55 60Pro Leu Leu Leu Gly Val Arg
Ala Gln Ala Ala Gly Gln Gly Pro Gly65 70 75 80Gln Gly Pro Gly Pro
Gly Gln Gln Pro Pro Pro Pro Pro Gln Gln Gln 85 90 95Gln Ser Gly Gln
Gln Tyr Asn Gly Glu Arg Gly Ile Ser Val Pro Asp 100 105 110His Gly
Tyr Cys Gln Pro Ile Ser Ile Pro Leu Cys Thr Asp Ile Ala 115 120
125Tyr Asn Gln Thr Ile Met Pro Asn Leu Leu Gly His Thr Asn Gln Glu
130 135 140Asp Ala Gly Leu Glu Val His Gln Phe Tyr Pro Leu Val Lys
Val Gln145 150 155 160Cys Ser Ala Glu Leu Lys Phe Phe Leu Cys Ser
Met Tyr Ala Pro Val 165 170 175Cys Thr Val Leu Glu Gln Ala Leu Pro
Pro Cys Arg Ser Leu Cys Glu 180 185 190Arg Ala Arg Gln Gly Cys Glu
Ala Leu Met Asn Lys Phe Gly Phe Gln 195 200 205Trp Pro Asp Thr Leu
Lys Cys Glu Lys Phe Pro Val His Gly Ala Gly 210 215 220Glu Leu Cys
Val Gly Gln Asn Thr Ser Asp Lys Gly Thr Pro Thr Pro225 230 235
240Ser Leu Leu Pro Glu Phe Trp Thr Ser Asn Pro Gln His Gly Gly Gly
245 250 255Gly His Arg Gly Gly Phe Pro Gly Gly Ala Gly Ala Ser Glu
Arg Gly 260 265 270Lys Phe Ser Cys Pro Arg Ala Leu Lys Val Pro Ser
Tyr Leu Asn Tyr 275 280 285His Phe Leu Gly Glu Lys Asp Cys Gly Ala
Pro Cys Glu Pro Thr Lys 290 295 300Val Tyr Gly Leu Met Tyr Phe Gly
Pro Glu Glu Leu Arg Phe Ser Arg305 310 315 320Thr Trp Ile Gly Ile
Trp Ser Val Leu Cys Cys Ala Ser Thr Leu Phe 325 330 335Thr Val Leu
Thr Tyr Leu Val Asp Met Arg Arg Phe Ser Tyr Pro Glu 340 345 350Arg
Pro Ile Ile Phe Leu Ser Gly Cys Tyr Thr Ala Val Ala Val Ala 355 360
365Tyr Ile Ala Gly Phe Leu Leu Glu Asp Arg Val Val Cys Asn Asp Lys
370 375 380Phe Ala Glu Asp Gly Ala Arg Thr Val Ala Gln Gly Thr Lys
Lys Glu385 390 395 400Gly Cys Thr Ile Leu Phe Met Met Leu Tyr Phe
Phe Ser Met Ala Ser 405 410 415Ser Ile Trp Trp Val Ile Leu Ser Leu
Thr Trp Phe Leu Ala Ala Gly 420 425 430Met Lys Trp Gly His Glu Ala
Ile Glu Ala Asn Ser Gln Tyr Phe His 435 440 445Leu Ala Ala Trp Ala
Val Pro Ala Ile Lys Thr Ile Thr Ile Leu Ala 450 455 460Leu Gly Gln
Val Asp Gly Asp Val Leu Ser Gly Val Cys Phe Val Gly465 470 475
480Leu Asn Asn Val Asp Ala Leu Arg Gly Phe Val Leu Ala Pro Leu Phe
485 490 495Val Tyr Leu Phe Ile Gly Thr Ser Phe Leu Leu Ala Gly Phe
Val Ser 500 505 510Leu Phe Arg Ile Arg Thr Ile Met Lys His Asp Gly
Thr Lys Thr Glu 515 520 525Lys Leu Glu Lys Leu Met Val Arg Ile Gly
Val Phe Ser Val Leu Tyr 530 535 540Thr Val Pro Ala Thr Ile Val Ile
Ala Cys Tyr Phe Tyr Glu Gln Ala545 550 555 560Phe Arg Asp Gln Trp
Glu Arg Ser Trp Val Ala Gln Ser Cys Lys Ser 565 570 575Tyr Ala Ile
Pro Cys Pro His Leu Gln Ala Gly Gly Gly Ala Pro Pro 580 585 590His
Pro Pro Met Ser Pro Asp Phe Thr Val Phe Met Ile Lys Tyr Leu 595 600
605Met Thr Leu Ile Val Gly Ile Thr Ser Gly Phe Trp Ile Trp Ser Gly
610 615 620Lys Thr Leu Asn Ser Trp Arg Lys Phe Tyr Thr Arg Leu Thr
Asn Ser625 630 635 640Lys Gln Gly Glu Thr Thr 6452565PRTHomo
sapiens 2Met Arg Pro Arg Ser Ala Leu Pro Arg Leu Leu Leu Pro Leu
Leu Leu1 5 10 15Leu Pro Ala Ala Gly Pro Ala Gln Phe His Gly Glu Lys
Gly Ile Ser 20 25 30Ile Pro Asp His Gly Phe Cys Gln Pro Ile Ser Ile
Pro Leu Cys Thr 35 40 45Asp Ile Ala Tyr Asn Gln Thr Ile Met Pro Asn
Leu Leu Gly His Thr 50 55 60Asn Gln Glu Asp Ala Gly Leu Glu Val His
Gln Phe Tyr Pro Leu Val65 70 75 80Lys Val Gln Cys Ser Pro Glu Leu
Arg Phe Phe Leu Cys Ser Met Tyr 85 90 95Ala Pro Val Cys Thr Val Leu
Glu Gln Ala Ile Pro Pro Cys Arg Ser 100 105 110Ile Cys Glu Ser Ala
Arg Gln Gly Cys Glu Ala Leu Met Asn Lys Phe 115 120 125Gly Phe Gln
Trp Pro Glu Arg Leu Arg Cys Glu His Phe Pro Arg His 130 135 140Gly
Ala Glu Gln Ile Cys Val Gly Gln Asn His Ser Glu Asp Gly Ala145 150
155 160Pro Ala Leu Leu Thr Thr Ala Pro Pro Pro Gly Leu Gln Pro Gly
Ala 165 170 175Gly Gly Thr Pro Gly Gly Pro Gly Gly Gly Gly Ala Pro
Pro Arg Tyr 180 185 190Ala Thr Leu Glu His Pro Phe His Cys Pro Arg
Val Leu Lys Val Pro 195 200 205Ser Tyr Leu Ser Tyr Lys Phe Leu Gly
Glu Arg Asp Cys Ala Ala Pro 210 215 220Cys Glu Pro Ala Arg Pro Asp
Gly Ser Met Phe Phe Ser Gln Glu Glu225 230 235 240Thr Arg Phe Ala
Arg Leu Trp Ile Leu Thr Trp Ser Val Leu Cys Cys 245 250 255Ala Ser
Thr Phe Phe Thr Val Thr Thr Tyr Leu Val Asp Met Gln Arg 260 265
270Phe Arg Tyr Pro Glu Arg Pro Ile Ile Phe Leu Ser Gly Cys Tyr Thr
275 280 285Met Val Ser Val Ala Tyr Ile Ala Gly Phe Val Leu Gln Glu
Arg Val 290 295 300Val Cys Asn Glu Arg Phe Ser Glu Asp Gly Tyr Arg
Thr Val Val Gln305 310 315 320Gly Thr Lys Lys Glu Gly Cys Thr Ile
Leu Phe Met Met Leu Tyr Phe 325 330 335Phe Ser Met Ala Ser Ser Ile
Trp Trp Val Ile Leu Ser Leu Thr Trp 340 345 350Phe Leu Ala Ala Gly
Met Lys Trp Gly His Glu Ala Ile Glu Ala Asn 355 360 365Ser Gln Tyr
Phe His Leu Ala Ala Trp Ala Val Pro Ala Val Lys Thr 370 375 380Ile
Thr Ile Leu Ala Met Gly Gln Ile Asp Gly Asp Leu Leu Ser Gly385 390
395 400Val Cys Phe Val Gly Leu Asn Ser Leu Asp Pro Leu Arg Gly Phe
Val 405 410 415Leu Ala Pro Leu Phe Val Tyr Leu Phe Ile Gly Thr Ser
Phe Leu Leu 420 425 430Ala Gly Phe Val Ser Leu Phe Arg Ile Arg Thr
Ile Met Lys His Asp 435 440 445Gly Thr Lys Thr Glu Lys Leu Glu Arg
Leu Met Val Arg Ile Gly Val 450 455 460Phe Ser Val Leu Tyr Thr Val
Pro Ala Thr Ile Val Ile Ala Cys Tyr465 470 475 480Phe Tyr Glu Gln
Ala Phe Arg Glu His Trp Glu Arg Ser Trp Val Ser 485 490 495Gln His
Cys Lys Ser Leu Ala Ile Pro Cys Pro Ala His Tyr Thr Pro 500 505
510Arg Met Ser Pro Asp Phe Thr Val Tyr Met Ile Lys Tyr Leu Met Thr
515 520 525Leu Ile Val Gly Ile Thr Ser Gly Phe Trp Ile Trp Ser Gly
Lys Thr 530 535 540Leu His Ser Trp Arg Lys Phe Tyr Thr Arg Leu Thr
Asn Ser Arg His545 550 555 560Gly Glu Thr Thr Val 5653666PRTHomo
sapiens 3Met Ala Met Thr Trp Ile Val Phe Ser Leu Trp Pro Leu Thr
Val Phe1 5 10 15Met Gly His Ile Gly Gly His Ser Leu Phe Ser Cys Glu
Pro Ile Thr 20 25 30Leu Arg Met Cys Gln Asp Leu Pro Tyr Asn Thr Thr
Phe Met Pro Asn 35 40 45Leu Leu Asn His Tyr Asp Gln Gln Thr Ala Ala
Leu Ala Met Glu Pro 50 55 60Phe His Pro Met Val Asn Leu Asp Cys Ser
Arg Asp Phe Arg Pro Phe65 70 75 80Leu Cys Ala Leu Tyr Ala Pro Ile
Cys Met Glu Tyr Gly Arg Val Thr 85 90 95Leu Pro Cys Arg Arg Leu Cys
Gln Arg Ala Tyr Ser Glu Cys Ser Lys 100 105 110Leu Met Glu Met Phe
Gly Val Pro Trp Pro Glu Asp Met Glu Cys Ser 115 120 125Arg Phe Pro
Asp Cys Asp Glu Pro Tyr Pro Arg Leu Val Asp Leu Asn 130 135 140Leu
Ala Gly Glu Pro Thr Glu Gly Ala Pro Val Ala Val Gln Arg Asp145 150
155 160Tyr Gly Phe Trp Cys Pro Arg Glu Leu Lys Ile Asp Pro Asp Leu
Gly 165 170 175Tyr Ser Phe Leu His Val Arg Asp Cys Ser Pro Pro Cys
Pro Asn Met 180 185 190Tyr Phe Arg Arg Glu Glu Leu Ser Phe Ala Arg
Tyr Phe Ile Gly Leu 195 200 205Ile Ser Ile Ile Cys Leu Ser Ala Thr
Leu Phe Thr Phe Leu Thr Phe 210 215 220Leu Ile Asp Val Thr Arg Phe
Arg Tyr Pro Glu Arg Pro Ile Ile Phe225 230 235 240Tyr Ala Val Cys
Tyr Met Met Val Ser Leu Ile Phe Phe Ile Gly Phe 245 250 255Leu Leu
Glu Asp Arg Val Ala Cys Asn Ala Ser Ile Pro Ala Gln Tyr 260 265
270Lys Ala Ser Thr Val Thr Gln Gly Ser His Asn Lys Ala Cys Thr Met
275 280 285Leu Phe Met Ile Leu Tyr Phe Phe Thr Met Ala Gly Ser Val
Trp Trp 290 295 300Val Ile Leu Thr Ile Thr Trp Phe Leu Ala Ala Val
Pro Lys Trp Gly305 310 315 320Ser Glu Ala Ile Glu Lys Lys Ala Leu
Leu Phe His Ala Ser Ala Trp 325 330 335Gly Ile Pro Gly Thr Leu Thr
Ile Ile Leu Leu Ala Met Asn Lys Ile 340 345 350Glu Gly Asp Asn Ile
Ser Gly Val Cys Phe Val Gly Leu Tyr Asp Val 355 360 365Asp Ala Leu
Arg Tyr Phe Val Leu Ala Pro Leu Cys Leu Tyr Val Val 370 375 380Val
Gly Val Ser Leu Leu Leu Ala Gly Ile Ile Ser Leu Asn Arg Val385 390
395 400Arg Ile Glu Ile Pro Leu Glu Lys Glu Asn Gln Asp Lys Leu Val
Lys 405 410 415Phe Met Ile Arg Ile Gly Val Phe Ser Ile Leu Tyr Leu
Val Pro Leu 420 425 430Leu Val Val Ile Gly Cys Tyr Phe Tyr Glu Gln
Ala Tyr Arg Gly Ile 435 440 445Trp Glu Thr Thr Trp Ile Gln Glu Arg
Cys Arg Glu Tyr His Ile Pro 450 455 460Cys Pro Tyr Gln Val Thr Gln
Met Ser Arg Pro Asp Leu Ile Leu Phe465 470 475 480Leu Met Lys Tyr
Leu Met Ala Leu Ile Val Gly Ile Pro Ser Val Phe 485 490 495Trp Val
Gly Ser Lys Lys Thr Cys Phe Glu Trp Ala Ser Phe Phe His 500 505
510Gly Arg Arg Lys Lys Glu Ile Val Asn Glu Ser Arg Gln Val Leu Gln
515 520 525Glu Pro Asp Phe Ala Gln Ser Leu Leu Arg Asp Pro Asn Thr
Pro Ile 530 535 540Ile Arg Lys Ser Arg Gly Thr Ser Thr Gln Gly Thr
Ser Thr His Ala545 550 555 560Ser Ser Thr Gln Leu Ala Met Val Asp
Asp Gln Arg Ser Lys Ala Gly 565 570 575Ser Ile His Ser Lys Val Ser
Ser Tyr His Gly Ser Leu His Arg Ser 580 585 590Arg Asp Gly Arg Tyr
Thr Pro Cys Ser Tyr Arg Gly Met Glu Glu Arg 595 600 605Leu Pro His
Gly Ser Met Ser Arg Leu Thr Asp His Ser Arg His Ser 610 615 620Ser
Ser His Arg Leu Asn Glu Gln Ser Arg His Ser Ser Ile Arg Asp625 630
635 640Leu Ser Asn Asn Pro Met Thr His Ile Thr His Gly Thr Ser Met
Asn 645 650 655Arg Val Ile Glu Glu Asp Gly Thr Ser Ala 660
6654537PRTHomo sapiens 4Met Ala Trp Arg Gly Ala Gly Pro Ser Val Pro
Gly Ala Pro Gly Gly1 5 10 15Val Gly Leu Ser Leu Gly Leu Leu Leu Gln
Leu Leu Leu Leu Leu Gly 20 25 30Pro Ala Arg Gly Phe Gly Asp Glu Glu
Glu Arg Arg Cys Asp Pro Ile 35 40 45Arg Ile Ser Met Cys Gln Asn Leu
Gly Tyr Asn Val Thr Lys Met Pro 50 55 60Asn Leu Val Gly His Glu Leu
Gln Thr Asp Ala Glu Leu Gln Leu Thr65 70 75 80Thr Phe Thr Pro Leu
Ile Gln Tyr Gly Cys Ser Ser Gln Leu Gln Phe 85 90 95Phe Leu Cys Ser
Val Tyr Val Pro Met Cys Thr Glu Lys Ile Asn Ile 100 105 110Pro Ile
Gly Pro Cys Gly Gly Met Cys Leu Ser Val Lys Arg Arg Cys 115 120
125Glu Pro Val Leu Lys Glu Phe Gly Phe Ala Trp Pro Glu Ser Leu Asn
130 135 140Cys Ser Lys Phe Pro Pro Gln Asn Asp His Asn His Met Cys
Met Glu145 150 155 160Gly Pro Gly Asp Glu Glu Val Pro Leu Pro His
Lys Thr Pro Ile Gln 165 170 175Pro Gly Glu Glu Cys His Ser Val Gly
Thr Asn Ser Asp Gln Tyr Ile 180 185 190Trp Val Lys Arg Ser Leu Asn
Cys Val Leu Lys Cys Gly Tyr Asp Ala 195 200 205Gly Leu Tyr Ser Arg
Ser Ala Lys Glu Phe Thr Asp Ile Trp Met Ala 210 215 220Val Trp Ala
Ser Leu Cys Phe Ile Ser Thr Ala Phe Thr Val Leu Thr225 230 235
240Phe Leu Ile Asp Ser Ser Arg Phe Ser Tyr Pro Glu Arg Pro Ile Ile
245 250 255Phe Leu Ser Met Cys Tyr Asn Ile Tyr Ser Ile Ala Tyr Ile
Val Arg 260 265 270Leu Thr Val Gly Arg Glu Arg Ile Ser Cys Asp Phe
Glu Glu Ala Ala 275 280 285Glu Pro Val Leu Ile Gln Glu Gly Leu Lys
Asn Thr Gly Cys Ala Ile 290 295 300Ile Phe Leu Leu Met Tyr Phe Phe
Gly Met Ala Ser Ser Ile Trp Trp305 310 315 320Val Ile Leu Thr Leu
Thr Trp Phe Leu Ala Ala Gly Leu Lys Trp Gly 325 330 335His Glu Ala
Ile Glu Met His Ser Ser Tyr Phe His Ile Ala Ala Trp 340 345 350Ala
Ile Pro Ala Val Lys Thr Ile Val Ile Leu Ile Met Arg Leu Val 355 360
365Asp Ala Asp Glu Leu Thr Gly Leu Cys Tyr Val Gly Asn Gln Asn Leu
370 375 380Asp Ala Leu Thr Gly Phe Val Val Ala Pro Leu Phe Thr Tyr
Leu Val385 390 395 400Ile Gly Thr Leu Phe Ile Ala Ala Gly Leu Val
Ala Leu Phe Lys Ile 405 410 415Arg Ser Asn Leu Gln Lys Asp Gly Thr
Lys Thr Asp Lys Leu Glu Arg 420 425 430Leu Met Val Lys Ile Gly Val
Phe Ser Val Leu Tyr Thr Val Pro Ala 435 440 445Thr Cys Val Ile Ala
Cys Tyr Phe Tyr Glu Ile Ser Asn Trp Ala Leu 450 455 460Phe Arg Tyr
Ser Ala Asp Asp Ser Asn Met Ala Val Glu Met Leu Lys465 470 475
480Ile Phe Met Ser Leu Leu Val Gly Ile Thr Ser Gly Met Trp Ile Trp
485 490 495Ser Ala Lys Thr Leu His Thr Trp Gln Lys Cys Ser Asn Arg
Leu Val 500 505 510Asn Ser Gly Lys Val Lys Arg Glu Lys Arg Gly Asn
Gly Trp Val Lys 515 520 525Pro Gly Lys Gly Ser Glu Thr Val Val 530
5355585PRTHomo sapiens 5Met Ala Arg Pro Asp Pro Ser Ala Pro Pro Ser
Leu Leu Leu Leu Leu1 5 10 15Leu Ala Gln Leu Val Gly Arg Ala Ala Ala
Ala Ser Lys Ala Pro Val 20 25 30Cys Gln Glu Ile Thr Val Pro Met Cys
Arg Gly Ile Gly Tyr Asn Leu
35 40 45Thr His Met Pro Asn Gln Phe Asn His Asp Thr Gln Asp Glu Ala
Gly 50 55 60Leu Glu Val His Gln Phe Trp Pro Leu Val Glu Ile Gln Cys
Ser Pro65 70 75 80Asp Leu Arg Phe Phe Leu Cys Ser Met Tyr Thr Pro
Ile Cys Leu Pro 85 90 95Asp Tyr His Lys Pro Leu Pro Pro Cys Arg Ser
Val Cys Glu Arg Ala 100 105 110Lys Ala Gly Cys Ser Pro Leu Met Arg
Gln Tyr Gly Phe Ala Trp Pro 115 120 125Glu Arg Met Ser Cys Asp Arg
Leu Pro Val Leu Gly Arg Asp Ala Glu 130 135 140Val Leu Cys Met Asp
Tyr Asn Arg Ser Glu Ala Thr Thr Ala Pro Pro145 150 155 160Arg Pro
Phe Pro Ala Lys Pro Thr Leu Pro Gly Pro Pro Gly Ala Pro 165 170
175Ala Ser Gly Gly Glu Cys Pro Ala Gly Gly Pro Phe Val Cys Lys Cys
180 185 190Arg Glu Pro Phe Val Pro Ile Leu Lys Glu Ser His Pro Leu
Tyr Asn 195 200 205Lys Val Arg Thr Gly Gln Val Pro Asn Cys Ala Val
Pro Cys Tyr Gln 210 215 220Pro Ser Phe Ser Ala Asp Glu Arg Thr Phe
Ala Thr Phe Trp Ile Gly225 230 235 240Leu Trp Ser Val Leu Cys Phe
Ile Ser Thr Ser Thr Thr Val Ala Thr 245 250 255Phe Leu Ile Asp Met
Glu Arg Phe Arg Tyr Pro Glu Arg Pro Ile Ile 260 265 270Phe Leu Ser
Ala Cys Tyr Leu Cys Val Ser Leu Gly Phe Leu Val Arg 275 280 285Leu
Val Val Gly His Ala Ser Val Ala Cys Ser Arg Glu His Asn His 290 295
300Ile His Tyr Glu Thr Thr Gly Pro Ala Leu Cys Thr Ile Val Phe
Leu305 310 315 320Leu Val Tyr Phe Phe Gly Met Ala Ser Ser Ile Trp
Trp Val Ile Leu 325 330 335Ser Leu Thr Trp Phe Leu Ala Ala Gly Met
Lys Trp Gly Asn Glu Ala 340 345 350Ile Ala Gly Tyr Ala Gln Tyr Phe
His Leu Ala Ala Trp Leu Ile Pro 355 360 365Ser Val Lys Ser Ile Thr
Ala Leu Ala Leu Ser Ser Val Asp Gly Asp 370 375 380Pro Val Ala Gly
Ile Cys Tyr Val Gly Asn Gln Asn Leu Asn Ser Leu385 390 395 400Arg
Gly Phe Val Leu Gly Pro Leu Val Leu Tyr Leu Leu Val Gly Thr 405 410
415Leu Phe Leu Leu Ala Gly Phe Val Ser Leu Phe Arg Ile Arg Ser Val
420 425 430Ile Lys Gln Gly Gly Thr Lys Thr Asp Lys Leu Glu Lys Leu
Met Ile 435 440 445Arg Ile Gly Ile Phe Thr Leu Leu Tyr Thr Val Pro
Ala Ser Ile Val 450 455 460Val Ala Cys Tyr Leu Tyr Glu Gln His Tyr
Arg Glu Ser Trp Glu Ala465 470 475 480Ala Leu Thr Cys Ala Cys Pro
Gly His Asp Thr Gly Gln Pro Arg Ala 485 490 495Lys Pro Glu Tyr Trp
Val Leu Met Leu Lys Tyr Phe Met Cys Leu Val 500 505 510Val Gly Ile
Thr Ser Gly Val Trp Ile Trp Ser Gly Lys Thr Val Glu 515 520 525Ser
Trp Arg Arg Phe Thr Ser Arg Cys Cys Cys Arg Pro Arg Arg Gly 530 535
540His Lys Ser Gly Gly Ala Met Ala Ala Gly Asp Tyr Pro Glu Ala
Ser545 550 555 560Ala Ala Leu Thr Gly Arg Thr Gly Pro Pro Gly Pro
Ala Ala Thr Tyr 565 570 575His Lys Gln Val Ser Leu Ser His Val 580
5856706PRTHomo sapiens 6Met Glu Met Phe Thr Phe Leu Leu Thr Cys Ile
Phe Leu Pro Leu Leu1 5 10 15Arg Gly His Ser Leu Phe Thr Cys Glu Pro
Ile Thr Val Pro Arg Cys 20 25 30Met Lys Met Ala Tyr Asn Met Thr Phe
Phe Pro Asn Leu Met Gly His 35 40 45Tyr Asp Gln Ser Ile Ala Ala Val
Glu Met Glu His Phe Leu Pro Leu 50 55 60Ala Asn Leu Glu Cys Ser Pro
Asn Ile Glu Thr Phe Leu Cys Lys Ala65 70 75 80Phe Val Pro Thr Cys
Ile Glu Gln Ile His Val Val Pro Pro Cys Arg 85 90 95Lys Leu Cys Glu
Lys Val Tyr Ser Asp Cys Lys Lys Leu Ile Asp Thr 100 105 110Phe Gly
Ile Arg Trp Pro Glu Glu Leu Glu Cys Asp Arg Leu Gln Tyr 115 120
125Cys Asp Glu Thr Val Pro Val Thr Phe Asp Pro His Thr Glu Phe Leu
130 135 140Gly Pro Gln Lys Lys Thr Glu Gln Val Gln Arg Asp Ile Gly
Phe Trp145 150 155 160Cys Pro Arg His Leu Lys Thr Ser Gly Gly Gln
Gly Tyr Lys Phe Leu 165 170 175Gly Ile Asp Gln Cys Ala Pro Pro Cys
Pro Asn Met Tyr Phe Lys Ser 180 185 190Asp Glu Leu Glu Phe Ala Lys
Ser Phe Ile Gly Thr Val Ser Ile Phe 195 200 205Cys Leu Cys Ala Thr
Leu Phe Thr Phe Leu Thr Phe Leu Ile Asp Val 210 215 220Arg Arg Phe
Arg Tyr Pro Glu Arg Pro Ile Ile Tyr Tyr Ser Val Cys225 230 235
240Tyr Ser Ile Val Ser Leu Met Tyr Phe Ile Gly Phe Leu Leu Gly Asp
245 250 255Ser Thr Ala Cys Asn Lys Ala Asp Glu Lys Leu Glu Leu Gly
Asp Thr 260 265 270Val Val Leu Gly Ser Gln Asn Lys Ala Cys Thr Val
Leu Phe Met Leu 275 280 285Leu Tyr Phe Phe Thr Met Ala Gly Thr Val
Trp Trp Val Ile Leu Thr 290 295 300Ile Thr Trp Phe Leu Ala Ala Gly
Arg Lys Trp Ser Cys Glu Ala Ile305 310 315 320Glu Gln Lys Ala Val
Trp Phe His Ala Val Ala Trp Gly Thr Pro Gly 325 330 335Phe Leu Thr
Val Met Leu Leu Ala Met Asn Lys Val Glu Gly Asp Asn 340 345 350Ile
Ser Gly Val Cys Phe Val Gly Leu Tyr Asp Leu Asp Ala Ser Arg 355 360
365Tyr Phe Val Leu Leu Pro Leu Cys Leu Cys Val Phe Val Gly Leu Ser
370 375 380Leu Leu Leu Ala Gly Ile Ile Ser Leu Asn His Val Arg Gln
Val Ile385 390 395 400Gln His Asp Gly Arg Asn Gln Glu Lys Leu Lys
Lys Phe Met Ile Arg 405 410 415Ile Gly Val Phe Ser Gly Leu Tyr Leu
Val Pro Leu Val Thr Leu Leu 420 425 430Gly Cys Tyr Val Tyr Glu Gln
Val Asn Arg Ile Thr Trp Glu Ile Thr 435 440 445Trp Val Ser Asp His
Cys Arg Gln Tyr His Ile Pro Cys Pro Tyr Gln 450 455 460Ala Lys Ala
Lys Ala Arg Pro Glu Leu Ala Leu Phe Met Ile Lys Tyr465 470 475
480Leu Met Thr Leu Ile Val Gly Ile Ser Ala Val Phe Trp Val Gly Ser
485 490 495Lys Lys Thr Cys Thr Glu Trp Ala Gly Phe Phe Lys Arg Asn
Arg Lys 500 505 510Arg Asp Pro Ile Ser Glu Ser Arg Arg Val Leu Gln
Glu Ser Cys Glu 515 520 525Phe Phe Leu Lys His Asn Ser Lys Val Lys
His Lys Lys Lys His Tyr 530 535 540Lys Pro Ser Ser His Lys Leu Lys
Val Ile Ser Lys Ser Met Gly Thr545 550 555 560Ser Thr Gly Ala Thr
Ala Asn His Gly Thr Ser Ala Val Ala Ile Thr 565 570 575Ser His Asp
Tyr Leu Gly Gln Glu Thr Leu Thr Glu Ile Gln Thr Ser 580 585 590Pro
Glu Thr Ser Met Arg Glu Val Lys Ala Asp Gly Ala Ser Thr Pro 595 600
605Arg Leu Arg Glu Gln Asp Cys Gly Glu Pro Ala Ser Pro Ala Ala Ser
610 615 620Ile Ser Arg Leu Ser Gly Glu Gln Val Asp Gly Lys Gly Gln
Ala Gly625 630 635 640Ser Val Ser Glu Ser Ala Arg Ser Glu Gly Arg
Ile Ser Pro Lys Ser 645 650 655Asp Ile Thr Asp Thr Gly Leu Ala Gln
Ser Asn Asn Leu Gln Val Pro 660 665 670Ser Ser Ser Glu Pro Ser Ser
Leu Lys Gly Ser Thr Ser Leu Leu Val 675 680 685His Pro Val Ser Gly
Val Arg Lys Glu Gln Gly Gly Gly Cys His Ser 690 695 700Asp
Thr7057574PRTHomo sapiens 7Met Arg Asp Pro Gly Ala Ala Ala Pro Leu
Ser Ser Leu Gly Leu Cys1 5 10 15Ala Leu Val Leu Ala Leu Leu Gly Ala
Leu Ser Ala Gly Ala Gly Ala 20 25 30Gln Pro Tyr His Gly Glu Lys Gly
Ile Ser Val Pro Asp His Gly Phe 35 40 45Cys Gln Pro Ile Ser Ile Pro
Leu Cys Thr Asp Ile Ala Tyr Asn Gln 50 55 60Thr Ile Leu Pro Asn Leu
Leu Gly His Thr Asn Gln Glu Asp Ala Gly65 70 75 80Leu Glu Val His
Gln Phe Tyr Pro Leu Val Lys Val Gln Cys Ser Pro 85 90 95Glu Leu Arg
Phe Phe Leu Cys Ser Met Tyr Ala Pro Val Cys Thr Val 100 105 110Leu
Asp Gln Ala Ile Pro Pro Cys Arg Ser Leu Cys Glu Arg Ala Arg 115 120
125Gln Gly Cys Glu Ala Leu Met Asn Lys Phe Gly Phe Gln Trp Pro Glu
130 135 140Arg Leu Arg Cys Glu Asn Phe Pro Val His Gly Ala Gly Glu
Ile Cys145 150 155 160Val Gly Gln Asn Thr Ser Asp Gly Ser Gly Gly
Pro Gly Gly Gly Pro 165 170 175Thr Ala Tyr Pro Thr Ala Pro Tyr Leu
Pro Asp Leu Pro Phe Thr Ala 180 185 190Leu Pro Pro Gly Ala Ser Asp
Gly Arg Gly Arg Pro Ala Phe Pro Phe 195 200 205Ser Cys Pro Arg Gln
Leu Lys Val Pro Pro Tyr Leu Gly Tyr Arg Phe 210 215 220Leu Gly Glu
Arg Asp Cys Gly Ala Pro Cys Glu Pro Gly Arg Ala Asn225 230 235
240Gly Leu Met Tyr Phe Lys Glu Glu Glu Arg Arg Phe Ala Arg Leu Trp
245 250 255Val Gly Val Trp Ser Val Leu Cys Cys Ala Ser Thr Leu Phe
Thr Val 260 265 270Leu Thr Tyr Leu Val Asp Met Arg Arg Phe Ser Tyr
Pro Glu Arg Pro 275 280 285Ile Ile Phe Leu Ser Gly Cys Tyr Phe Met
Val Ala Val Ala His Val 290 295 300Ala Gly Phe Leu Leu Glu Asp Arg
Ala Val Cys Val Glu Arg Phe Ser305 310 315 320Asp Asp Gly Tyr Arg
Thr Val Ala Gln Gly Thr Lys Lys Glu Gly Cys 325 330 335Thr Ile Leu
Phe Met Val Leu Tyr Phe Phe Gly Met Ala Ser Ser Ile 340 345 350Trp
Trp Val Ile Leu Ser Leu Thr Trp Phe Leu Ala Ala Gly Met Lys 355 360
365Trp Gly His Glu Ala Ile Glu Ala Asn Ser Gln Tyr Phe His Leu Ala
370 375 380Ala Trp Ala Val Pro Ala Val Lys Thr Ile Thr Ile Leu Ala
Met Gly385 390 395 400Gln Val Asp Gly Asp Leu Leu Ser Gly Val Cys
Tyr Val Gly Leu Ser 405 410 415Ser Val Asp Ala Leu Arg Gly Phe Val
Leu Ala Pro Leu Phe Val Tyr 420 425 430Leu Phe Ile Gly Thr Ser Phe
Leu Leu Ala Gly Phe Val Ser Leu Phe 435 440 445Arg Ile Arg Thr Ile
Met Lys His Asp Gly Thr Lys Thr Glu Lys Leu 450 455 460Glu Lys Leu
Met Val Arg Ile Gly Val Phe Ser Val Leu Tyr Thr Val465 470 475
480Pro Ala Thr Ile Val Leu Ala Cys Tyr Phe Tyr Glu Gln Ala Phe Arg
485 490 495Glu His Trp Glu Arg Thr Trp Leu Leu Gln Thr Cys Lys Ser
Tyr Ala 500 505 510Val Pro Cys Pro Pro Gly His Phe Pro Pro Met Ser
Pro Asp Phe Thr 515 520 525Val Phe Met Ile Lys Tyr Leu Met Thr Met
Ile Val Gly Ile Thr Thr 530 535 540Gly Phe Trp Ile Trp Ser Gly Lys
Thr Leu Gln Ser Trp Arg Arg Phe545 550 555 560Tyr His Arg Leu Ser
His Ser Ser Lys Gly Glu Thr Ala Val 565 5708694PRTHomo sapiens 8Met
Glu Trp Gly Tyr Leu Leu Glu Val Thr Ser Leu Leu Ala Ala Leu1 5 10
15Ala Leu Leu Gln Arg Ser Ser Gly Ala Ala Ala Ala Ser Ala Lys Glu
20 25 30Leu Ala Cys Gln Glu Ile Thr Val Pro Leu Cys Lys Gly Ile Gly
Tyr 35 40 45Asn Tyr Thr Tyr Met Pro Asn Gln Phe Asn His Asp Thr Gln
Asp Glu 50 55 60Ala Gly Leu Glu Val His Gln Phe Trp Pro Leu Val Glu
Ile Gln Cys65 70 75 80Ser Pro Asp Leu Lys Phe Phe Leu Cys Ser Met
Tyr Thr Pro Ile Cys 85 90 95Leu Glu Asp Tyr Lys Lys Pro Leu Pro Pro
Cys Arg Ser Val Cys Glu 100 105 110Arg Ala Lys Ala Gly Cys Ala Pro
Leu Met Arg Gln Tyr Gly Phe Ala 115 120 125Trp Pro Asp Arg Met Arg
Cys Asp Arg Leu Pro Glu Gln Gly Asn Pro 130 135 140Asp Thr Leu Cys
Met Asp Tyr Asn Arg Thr Asp Leu Thr Thr Ala Ala145 150 155 160Pro
Ser Pro Pro Arg Arg Leu Pro Pro Pro Pro Pro Gly Glu Gln Pro 165 170
175Pro Ser Gly Ser Gly His Gly Arg Pro Pro Gly Ala Arg Pro Pro His
180 185 190Arg Gly Gly Gly Arg Gly Gly Gly Gly Gly Asp Ala Ala Ala
Pro Pro 195 200 205Ala Arg Gly Gly Gly Gly Gly Gly Lys Ala Arg Pro
Pro Gly Gly Gly 210 215 220Ala Ala Pro Cys Glu Pro Gly Cys Gln Cys
Arg Ala Pro Met Val Ser225 230 235 240Val Ser Ser Glu Arg His Pro
Leu Tyr Asn Arg Val Lys Thr Gly Gln 245 250 255Ile Ala Asn Cys Ala
Leu Pro Cys His Asn Pro Phe Phe Ser Gln Asp 260 265 270Glu Arg Ala
Phe Thr Val Phe Trp Ile Gly Leu Trp Ser Val Leu Cys 275 280 285Phe
Val Ser Thr Phe Ala Thr Val Ser Thr Phe Leu Ile Asp Met Glu 290 295
300Arg Phe Lys Tyr Pro Glu Arg Pro Ile Ile Phe Leu Ser Ala Cys
Tyr305 310 315 320Leu Phe Val Ser Val Gly Tyr Leu Val Arg Leu Val
Ala Gly His Glu 325 330 335Lys Val Ala Cys Ser Gly Gly Ala Pro Gly
Ala Gly Gly Ala Gly Gly 340 345 350Ala Gly Gly Ala Ala Ala Gly Ala
Gly Ala Ala Gly Ala Gly Ala Gly 355 360 365Gly Pro Gly Gly Arg Gly
Glu Tyr Glu Glu Leu Gly Ala Val Glu Gln 370 375 380His Val Arg Tyr
Glu Thr Thr Gly Pro Ala Leu Cys Thr Val Val Phe385 390 395 400Leu
Leu Val Tyr Phe Phe Gly Met Ala Ser Ser Ile Trp Trp Val Ile 405 410
415Leu Ser Leu Thr Trp Phe Leu Ala Ala Gly Met Lys Trp Gly Asn Glu
420 425 430Ala Ile Ala Gly Tyr Ser Gln Tyr Phe His Leu Ala Ala Trp
Leu Val 435 440 445Pro Ser Val Lys Ser Ile Ala Val Leu Ala Leu Ser
Ser Val Asp Gly 450 455 460Asp Pro Val Ala Gly Ile Cys Tyr Val Gly
Asn Gln Ser Leu Asp Asn465 470 475 480Leu Arg Gly Phe Val Leu Ala
Pro Leu Val Ile Tyr Leu Phe Ile Gly 485 490 495Thr Met Phe Leu Leu
Ala Gly Phe Val Ser Leu Phe Arg Ile Arg Ser 500 505 510Val Ile Lys
Gln Gln Asp Gly Pro Thr Lys Thr His Lys Leu Glu Lys 515 520 525Leu
Met Ile Arg Leu Gly Leu Phe Thr Val Leu Tyr Thr Val Pro Ala 530 535
540Ala Val Val Val Ala Cys Leu Phe Tyr Glu Gln His Asn Arg Pro
Arg545 550 555 560Trp Glu Ala Thr His Asn Cys Pro Cys Leu Arg Asp
Leu Gln Pro Asp 565 570 575Gln Ala Arg Arg Pro Asp Tyr Ala Val Phe
Met Leu Lys Tyr Phe Met 580 585 590Cys Leu Val Val Gly Ile Thr Ser
Gly Val Trp Val Trp Ser Gly Lys 595 600 605Thr Leu Glu Ser Trp Arg
Ser Leu Cys Thr Arg Cys Cys Trp Ala Ser 610 615 620Lys Gly Ala Ala
Val Gly Gly Gly Ala Gly Ala Thr Ala Ala Gly Gly625 630 635 640Gly
Gly Gly Pro
Gly Gly Gly Gly Gly Gly Gly Pro Gly Gly Gly Gly 645 650 655Gly Pro
Gly Gly Gly Gly Gly Ser Leu Tyr Ser Asp Val Ser Thr Gly 660 665
670Leu Thr Trp Arg Ser Gly Thr Ala Ser Ser Val Ser Tyr Pro Lys Gln
675 680 685Met Pro Leu Ser Gln Val 6909591PRTHomo sapiens 9Met Ala
Val Ala Pro Leu Arg Gly Ala Leu Leu Leu Trp Gln Leu Leu1 5 10 15Ala
Ala Gly Gly Ala Ala Leu Glu Ile Gly Arg Phe Asp Pro Glu Arg 20 25
30Gly Arg Gly Ala Ala Pro Cys Gln Ala Val Glu Ile Pro Met Cys Arg
35 40 45Gly Ile Gly Tyr Asn Leu Thr Arg Met Pro Asn Leu Leu Gly His
Thr 50 55 60Ser Gln Gly Glu Ala Ala Ala Glu Leu Ala Glu Phe Ala Pro
Leu Val65 70 75 80Gln Tyr Gly Cys His Ser His Leu Arg Phe Phe Leu
Cys Ser Leu Tyr 85 90 95Ala Pro Met Cys Thr Asp Gln Val Ser Thr Pro
Ile Pro Ala Cys Arg 100 105 110Pro Met Cys Glu Gln Ala Arg Leu Arg
Cys Ala Pro Ile Met Glu Gln 115 120 125Phe Asn Phe Gly Trp Pro Asp
Ser Leu Asp Cys Ala Arg Leu Pro Thr 130 135 140Arg Asn Asp Pro His
Ala Leu Cys Met Glu Ala Pro Glu Asn Ala Thr145 150 155 160Ala Gly
Pro Ala Glu Pro His Lys Gly Leu Gly Met Leu Pro Val Ala 165 170
175Pro Arg Pro Ala Arg Pro Pro Gly Asp Leu Gly Pro Gly Ala Gly Gly
180 185 190Ser Gly Thr Cys Glu Asn Pro Glu Lys Phe Gln Tyr Val Glu
Lys Ser 195 200 205Arg Ser Cys Ala Pro Arg Cys Gly Pro Gly Val Glu
Val Phe Trp Ser 210 215 220Arg Arg Asp Lys Asp Phe Ala Leu Val Trp
Met Ala Val Trp Ser Ala225 230 235 240Leu Cys Phe Phe Ser Thr Ala
Phe Thr Val Leu Thr Phe Leu Leu Glu 245 250 255Pro His Arg Phe Gln
Tyr Pro Glu Arg Pro Ile Ile Phe Leu Ser Met 260 265 270Cys Tyr Asn
Val Tyr Ser Leu Ala Phe Leu Ile Arg Ala Val Ala Gly 275 280 285Ala
Gln Ser Val Ala Cys Asp Gln Glu Ala Gly Ala Leu Tyr Val Ile 290 295
300Gln Glu Gly Leu Glu Asn Thr Gly Cys Thr Leu Val Phe Leu Leu
Leu305 310 315 320Tyr Tyr Phe Gly Met Ala Ser Ser Leu Trp Trp Val
Val Leu Thr Leu 325 330 335Thr Trp Phe Leu Ala Ala Gly Lys Lys Trp
Gly His Glu Ala Ile Glu 340 345 350Ala His Gly Ser Tyr Phe His Met
Ala Ala Trp Gly Leu Pro Ala Leu 355 360 365Lys Thr Ile Val Ile Leu
Thr Leu Arg Lys Val Ala Gly Asp Glu Leu 370 375 380Thr Gly Leu Cys
Tyr Val Ala Ser Thr Asp Ala Ala Ala Leu Thr Gly385 390 395 400Phe
Val Leu Val Pro Leu Ser Gly Tyr Leu Val Leu Gly Ser Ser Phe 405 410
415Leu Leu Thr Gly Phe Val Ala Leu Phe His Ile Arg Lys Ile Met Lys
420 425 430Thr Gly Gly Thr Asn Thr Glu Lys Leu Glu Lys Leu Met Val
Lys Ile 435 440 445Gly Val Phe Ser Ile Leu Tyr Thr Val Pro Ala Thr
Cys Val Ile Val 450 455 460Cys Tyr Val Tyr Glu Arg Leu Asn Met Asp
Phe Trp Arg Leu Arg Ala465 470 475 480Thr Glu Gln Pro Cys Ala Ala
Ala Ala Gly Pro Gly Gly Arg Arg Asp 485 490 495Cys Ser Leu Pro Gly
Gly Ser Val Pro Thr Val Ala Val Phe Met Leu 500 505 510Lys Ile Phe
Met Ser Leu Val Val Gly Ile Thr Ser Gly Val Trp Val 515 520 525Trp
Ser Ser Lys Thr Phe Gln Thr Trp Gln Ser Leu Cys Tyr Arg Lys 530 535
540Ile Ala Ala Gly Arg Ala Arg Ala Lys Ala Cys Arg Ala Pro Gly
Ser545 550 555 560Tyr Gly Arg Gly Thr His Cys His Tyr Lys Ala Pro
Thr Val Val Leu 565 570 575His Met Thr Lys Thr Asp Pro Ser Leu Glu
Asn Pro Thr His Leu 580 585 59010581PRTHomo sapiens 10Met Gln Arg
Pro Gly Pro Arg Leu Trp Leu Val Leu Gln Val Met Gly1 5 10 15Ser Cys
Ala Ala Ile Ser Ser Met Asp Met Glu Arg Pro Gly Asp Gly 20 25 30Lys
Cys Gln Pro Ile Glu Ile Pro Met Cys Lys Asp Ile Gly Tyr Asn 35 40
45Met Thr Arg Met Pro Asn Leu Met Gly His Glu Asn Gln Arg Glu Ala
50 55 60Ala Ile Gln Leu His Glu Phe Ala Pro Leu Val Glu Tyr Gly Cys
His65 70 75 80Gly His Leu Arg Phe Phe Leu Cys Ser Leu Tyr Ala Pro
Met Cys Thr 85 90 95Glu Gln Val Ser Thr Pro Ile Pro Ala Cys Arg Val
Met Cys Glu Gln 100 105 110Ala Arg Leu Lys Cys Ser Pro Ile Met Glu
Gln Phe Asn Phe Lys Trp 115 120 125Pro Asp Ser Leu Asp Cys Arg Lys
Leu Pro Asn Lys Asn Asp Pro Asn 130 135 140Tyr Leu Cys Met Glu Ala
Pro Asn Asn Gly Ser Asp Glu Pro Thr Arg145 150 155 160Gly Ser Gly
Leu Phe Pro Pro Leu Phe Arg Pro Gln Arg Pro His Ser 165 170 175Ala
Gln Glu His Pro Leu Lys Asp Gly Gly Pro Gly Arg Gly Gly Cys 180 185
190Asp Asn Pro Gly Lys Phe His His Val Glu Lys Ser Ala Ser Cys Ala
195 200 205Pro Leu Cys Thr Pro Gly Val Asp Val Tyr Trp Ser Arg Glu
Asp Lys 210 215 220Arg Phe Ala Val Val Trp Leu Ala Ile Trp Ala Val
Leu Cys Phe Phe225 230 235 240Ser Ser Ala Phe Thr Val Leu Thr Phe
Leu Ile Asp Pro Ala Arg Phe 245 250 255Arg Tyr Pro Glu Arg Pro Ile
Ile Phe Leu Ser Met Cys Tyr Cys Val 260 265 270Tyr Ser Val Gly Tyr
Leu Ile Arg Leu Phe Ala Gly Ala Glu Ser Ile 275 280 285Ala Cys Asp
Arg Asp Ser Gly Gln Leu Tyr Val Ile Gln Glu Gly Leu 290 295 300Glu
Ser Thr Gly Cys Thr Leu Val Phe Leu Val Leu Tyr Tyr Phe Gly305 310
315 320Met Ala Ser Ser Leu Trp Trp Val Val Leu Thr Leu Thr Trp Phe
Leu 325 330 335Ala Ala Gly Lys Lys Trp Gly His Glu Ala Ile Glu Ala
Asn Ser Ser 340 345 350Tyr Phe His Leu Ala Ala Trp Ala Ile Pro Ala
Val Lys Thr Ile Leu 355 360 365Ile Leu Val Met Arg Arg Val Ala Gly
Asp Glu Leu Thr Gly Val Cys 370 375 380Tyr Val Gly Ser Met Asp Val
Asn Ala Leu Thr Gly Phe Val Leu Ile385 390 395 400Pro Leu Ala Cys
Tyr Leu Val Ile Gly Thr Ser Phe Ile Leu Ser Gly 405 410 415Phe Val
Ala Leu Phe His Ile Arg Arg Val Met Lys Thr Gly Gly Glu 420 425
430Asn Thr Asp Lys Leu Glu Lys Leu Met Val Arg Ile Gly Leu Phe Ser
435 440 445Val Leu Tyr Thr Val Pro Ala Thr Cys Val Ile Ala Cys Tyr
Phe Tyr 450 455 460Glu Arg Leu Asn Met Asp Tyr Trp Lys Ile Leu Ala
Ala Gln His Lys465 470 475 480Cys Lys Met Asn Asn Gln Thr Lys Thr
Leu Asp Cys Leu Met Ala Ala 485 490 495Ser Ile Pro Ala Val Glu Ile
Phe Met Val Lys Ile Phe Met Leu Leu 500 505 510Val Val Gly Ile Thr
Ser Gly Met Trp Ile Trp Thr Ser Lys Thr Leu 515 520 525Gln Ser Trp
Gln Gln Val Cys Ser Arg Arg Leu Lys Lys Lys Ser Arg 530 535 540Arg
Lys Pro Ala Ser Val Ile Thr Ser Gly Gly Ile Tyr Lys Lys Ala545 550
555 560Gln His Pro Gln Lys Thr His His Gly Lys Tyr Glu Ile Pro Ala
Gln 565 570 575Ser Pro Thr Cys Val 58011314PRTHomo sapiens 11Met
Gly Ile Gly Arg Ser Glu Gly Gly Arg Arg Gly Ala Ala Leu Gly1 5 10
15Val Leu Leu Ala Leu Gly Ala Ala Leu Leu Ala Val Gly Ser Ala Ser
20 25 30Glu Tyr Asp Tyr Val Ser Phe Gln Ser Asp Ile Gly Pro Tyr Gln
Ser 35 40 45Gly Arg Phe Tyr Thr Lys Pro Pro Gln Cys Val Asp Ile Pro
Ala Asp 50 55 60Leu Arg Leu Cys His Asn Val Gly Tyr Lys Lys Met Val
Leu Pro Asn65 70 75 80Leu Leu Glu His Glu Thr Met Ala Glu Val Lys
Gln Gln Ala Ser Ser 85 90 95Trp Val Pro Leu Leu Asn Lys Asn Cys His
Ala Gly Thr Gln Val Phe 100 105 110Leu Cys Ser Leu Phe Ala Pro Val
Cys Leu Asp Arg Pro Ile Tyr Pro 115 120 125Cys Arg Trp Leu Cys Glu
Ala Val Arg Asp Ser Cys Glu Pro Val Met 130 135 140Gln Phe Phe Gly
Phe Tyr Trp Pro Glu Met Leu Lys Cys Asp Lys Phe145 150 155 160Pro
Glu Gly Asp Val Cys Ile Ala Met Thr Pro Pro Asn Ala Thr Glu 165 170
175Ala Ser Lys Pro Gln Gly Thr Thr Val Cys Pro Pro Cys Asp Asn Glu
180 185 190Leu Lys Ser Glu Ala Ile Ile Glu His Leu Cys Ala Ser Glu
Phe Ala 195 200 205Leu Arg Met Lys Ile Lys Glu Val Lys Lys Glu Asn
Gly Asp Lys Lys 210 215 220Ile Val Pro Lys Lys Lys Lys Pro Leu Lys
Leu Gly Pro Ile Lys Lys225 230 235 240Lys Asp Leu Lys Lys Leu Val
Leu Tyr Leu Lys Asn Gly Ala Asp Cys 245 250 255Pro Cys His Gln Leu
Asp Asn Leu Ser His His Phe Leu Ile Met Gly 260 265 270Arg Lys Val
Lys Ser Gln Tyr Leu Leu Thr Ala Ile His Lys Trp Asp 275 280 285Lys
Lys Asn Lys Glu Phe Lys Asn Phe Met Lys Lys Met Lys Asn His 290 295
300Glu Cys Pro Thr Phe Gln Ser Val Phe Lys305 31012295PRTHomo
sapiens 12Met Leu Gln Gly Pro Gly Ser Leu Leu Leu Leu Phe Leu Ala
Ser His1 5 10 15Cys Cys Leu Gly Ser Ala Arg Gly Leu Phe Leu Phe Gly
Gln Pro Asp 20 25 30Phe Ser Tyr Lys Arg Ser Asn Cys Lys Pro Ile Pro
Ala Asn Leu Gln 35 40 45Leu Cys His Gly Ile Glu Tyr Gln Asn Met Arg
Leu Pro Asn Leu Leu 50 55 60Gly His Glu Thr Met Lys Glu Val Leu Glu
Gln Ala Gly Ala Trp Ile65 70 75 80Pro Leu Val Met Lys Gln Cys His
Pro Asp Thr Lys Lys Phe Leu Cys 85 90 95Ser Leu Phe Ala Pro Val Cys
Leu Asp Asp Leu Asp Glu Thr Ile Gln 100 105 110Pro Cys His Ser Leu
Cys Val Gln Val Lys Asp Arg Cys Ala Pro Val 115 120 125Met Ser Ala
Phe Gly Phe Pro Trp Pro Asp Met Leu Glu Cys Asp Arg 130 135 140Phe
Pro Gln Asp Asn Asp Leu Cys Ile Pro Leu Ala Ser Ser Asp His145 150
155 160Leu Leu Pro Ala Thr Glu Glu Ala Pro Lys Val Cys Glu Ala Cys
Lys 165 170 175Asn Lys Asn Asp Asp Asp Asn Asp Ile Met Glu Thr Leu
Cys Lys Asn 180 185 190Asp Phe Ala Leu Lys Ile Lys Val Lys Glu Ile
Thr Tyr Ile Asn Arg 195 200 205Asp Thr Lys Ile Ile Leu Glu Thr Lys
Ser Lys Thr Ile Tyr Lys Leu 210 215 220Asn Gly Val Ser Glu Arg Asp
Leu Lys Lys Ser Val Leu Trp Leu Lys225 230 235 240Asp Ser Leu Gln
Cys Thr Cys Glu Glu Met Asn Asp Ile Asn Ala Pro 245 250 255Tyr Leu
Val Met Gly Gln Lys Gln Gly Gly Glu Leu Val Ile Thr Ser 260 265
270Val Lys Arg Trp Gln Lys Gly Gln Arg Glu Phe Lys Arg Ile Ser Arg
275 280 285Ser Ile Arg Lys Leu Gln Cys 290 29513325PRTHomo sapiens
13Met Val Cys Gly Ser Pro Gly Gly Met Leu Leu Leu Arg Ala Gly Leu1
5 10 15Leu Ala Leu Ala Ala Leu Cys Leu Leu Arg Val Pro Gly Ala Arg
Ala 20 25 30Ala Ala Cys Glu Pro Val Arg Ile Pro Leu Cys Lys Ser Leu
Pro Trp 35 40 45Asn Met Thr Lys Met Pro Asn His Leu His His Ser Thr
Gln Ala Asn 50 55 60Ala Ile Leu Ala Ile Glu Gln Phe Glu Gly Leu Leu
Gly Thr His Cys65 70 75 80Ser Pro Asp Leu Leu Phe Phe Leu Cys Ala
Met Tyr Ala Pro Ile Cys 85 90 95Thr Ile Asp Phe Gln His Glu Pro Ile
Lys Pro Cys Lys Ser Val Cys 100 105 110Glu Arg Ala Arg Gln Gly Cys
Glu Pro Ile Leu Ile Lys Tyr Arg His 115 120 125Ser Trp Pro Glu Asn
Leu Ala Cys Glu Glu Leu Pro Val Tyr Asp Arg 130 135 140Gly Val Cys
Ile Ser Pro Glu Ala Ile Val Thr Ala Asp Gly Ala Asp145 150 155
160Phe Pro Met Asp Ser Ser Asn Gly Asn Cys Arg Gly Ala Ser Ser Glu
165 170 175Arg Cys Lys Cys Lys Pro Ile Arg Ala Thr Gln Lys Thr Tyr
Phe Arg 180 185 190Asn Asn Tyr Asn Tyr Val Ile Arg Ala Lys Val Lys
Glu Ile Lys Thr 195 200 205Lys Cys His Asp Val Thr Ala Val Val Glu
Val Lys Glu Ile Leu Lys 210 215 220Ser Ser Leu Val Asn Ile Pro Arg
Asp Thr Val Asn Leu Tyr Thr Ser225 230 235 240Ser Gly Cys Leu Cys
Pro Pro Leu Asn Val Asn Glu Glu Tyr Ile Ile 245 250 255Met Gly Tyr
Glu Asp Glu Glu Arg Ser Arg Leu Leu Leu Val Glu Gly 260 265 270Ser
Ile Ala Glu Lys Trp Lys Asp Arg Leu Gly Lys Lys Val Lys Arg 275 280
285Trp Asp Met Lys Leu Arg His Leu Gly Leu Ser Lys Ser Asp Ser Ser
290 295 300Asn Ser Asp Ser Thr Gln Ser Gln Lys Ser Gly Arg Asn Ser
Asn Pro305 310 315 320Arg Gln Ala Arg Asn 32514346PRTHomo sapiens
14Met Phe Leu Ser Ile Leu Val Ala Leu Cys Leu Trp Leu His Leu Ala1
5 10 15Leu Gly Val Arg Gly Ala Pro Cys Glu Ala Val Arg Ile Pro Met
Cys 20 25 30Arg His Met Pro Trp Asn Ile Thr Arg Met Pro Asn His Leu
His His 35 40 45Ser Thr Gln Glu Asn Ala Ile Leu Ala Ile Glu Gln Tyr
Glu Glu Leu 50 55 60Val Asp Val Asn Cys Ser Ala Val Leu Arg Phe Phe
Phe Cys Ala Met65 70 75 80Tyr Ala Pro Ile Cys Thr Leu Glu Phe Leu
His Asp Pro Ile Lys Pro 85 90 95Cys Lys Ser Val Cys Gln Arg Ala Arg
Asp Asp Cys Glu Pro Leu Met 100 105 110Lys Met Tyr Asn His Ser Trp
Pro Glu Ser Leu Ala Cys Asp Glu Leu 115 120 125Pro Val Tyr Asp Arg
Gly Val Cys Ile Ser Pro Glu Ala Ile Val Thr 130 135 140Asp Leu Pro
Glu Asp Val Lys Trp Ile Asp Ile Thr Pro Asp Met Met145 150 155
160Val Gln Glu Arg Pro Leu Asp Val Asp Cys Lys Arg Leu Ser Pro Asp
165 170 175Arg Cys Lys Cys Lys Lys Val Lys Pro Thr Leu Ala Thr Tyr
Leu Ser 180 185 190Lys Asn Tyr Ser Tyr Val Ile His Ala Lys Ile Lys
Ala Val Gln Arg 195 200 205Ser Gly Cys Asn Glu Val Thr Thr Val Val
Asp Val Lys Glu Ile Phe 210 215 220Lys Ser Ser Ser Pro Ile Pro Arg
Thr Gln Val Pro Leu Ile Thr Asn225 230 235 240Ser Ser Cys Gln Cys
Pro His Ile Leu Pro His Gln Asp Val Leu Ile 245 250 255Met Cys Tyr
Glu Trp Arg Ser Arg Met Met Leu Leu Glu Asn Cys Leu 260 265 270Val
Glu Lys Trp Arg Asp Gln Leu Ser Lys Arg Ser Ile Gln Trp Glu 275 280
285Glu Arg Leu Gln Glu Gln Arg Arg Thr Val Gln Asp Lys Lys Lys Thr
290
295 300Ala Gly Arg Thr Ser Arg Ser Asn Pro Pro Lys Pro Lys Gly Lys
Pro305 310 315 320Pro Ala Pro Lys Pro Ala Ser Pro Lys Lys Asn Ile
Lys Thr Arg Ser 325 330 335Ala Gln Lys Arg Thr Asn Pro Lys Arg Val
340 34515317PRTHomo sapiens 15Met Arg Ala Ala Ala Ala Ala Gly Gly
Val Arg Thr Ala Ala Leu Ala1 5 10 15Leu Leu Leu Gly Ala Leu His Trp
Ala Pro Ala Arg Cys Glu Glu Tyr 20 25 30Asp Tyr Tyr Gly Trp Gln Ala
Glu Pro Leu His Gly Arg Ser Tyr Ser 35 40 45Lys Pro Pro Gln Cys Leu
Asp Ile Pro Ala Asp Leu Pro Leu Cys His 50 55 60Thr Val Gly Tyr Lys
Arg Met Arg Leu Pro Asn Leu Leu Glu His Glu65 70 75 80Ser Leu Ala
Glu Val Lys Gln Gln Ala Ser Ser Trp Leu Pro Leu Leu 85 90 95Ala Lys
Arg Cys His Ser Asp Thr Gln Val Phe Leu Cys Ser Leu Phe 100 105
110Ala Pro Val Cys Leu Asp Arg Pro Ile Tyr Pro Cys Arg Ser Leu Cys
115 120 125Glu Ala Val Arg Ala Gly Cys Ala Pro Leu Met Glu Ala Tyr
Gly Phe 130 135 140Pro Trp Pro Glu Met Leu His Cys His Lys Phe Pro
Leu Asp Asn Asp145 150 155 160Leu Cys Ile Ala Val Gln Phe Gly His
Leu Pro Ala Thr Ala Pro Pro 165 170 175Val Thr Lys Ile Cys Ala Gln
Cys Glu Met Glu His Ser Ala Asp Gly 180 185 190Leu Met Glu Gln Met
Cys Ser Ser Asp Phe Val Val Lys Met Arg Ile 195 200 205Lys Glu Ile
Lys Ile Glu Asn Gly Asp Arg Lys Leu Ile Gly Ala Gln 210 215 220Lys
Lys Lys Lys Leu Leu Lys Pro Gly Pro Leu Lys Arg Lys Asp Thr225 230
235 240Lys Arg Leu Val Leu His Met Lys Asn Gly Ala Gly Cys Pro Cys
Pro 245 250 255Gln Leu Asp Ser Leu Ala Gly Ser Phe Leu Val Met Gly
Arg Lys Val 260 265 270Asp Gly Gln Leu Leu Leu Met Ala Val Tyr Arg
Trp Asp Lys Lys Asn 275 280 285Lys Glu Met Lys Phe Ala Val Lys Phe
Met Phe Ser Tyr Pro Cys Ser 290 295 300Leu Tyr Tyr Pro Phe Phe Tyr
Gly Ala Ala Glu Pro His305 310 31516937PRTHomo sapiens 16Met His
Arg Pro Arg Arg Arg Gly Thr Arg Pro Pro Leu Leu Ala Leu1 5 10 15Leu
Ala Ala Leu Leu Leu Ala Ala Arg Gly Ala Ala Ala Gln Glu Thr 20 25
30Glu Leu Ser Val Ser Ala Glu Leu Val Pro Thr Ser Ser Trp Asn Ile
35 40 45Ser Ser Glu Leu Asn Lys Asp Ser Tyr Leu Thr Leu Asp Glu Pro
Met 50 55 60Asn Asn Ile Thr Thr Ser Leu Gly Gln Thr Ala Glu Leu His
Cys Lys65 70 75 80Val Ser Gly Asn Pro Pro Pro Thr Ile Arg Trp Phe
Lys Asn Asp Ala 85 90 95Pro Val Val Gln Glu Pro Arg Arg Leu Ser Phe
Arg Ser Thr Ile Tyr 100 105 110Gly Ser Arg Leu Arg Ile Arg Asn Leu
Asp Thr Thr Asp Thr Gly Tyr 115 120 125Phe Gln Cys Val Ala Thr Asn
Gly Lys Glu Val Val Ser Ser Thr Gly 130 135 140Val Leu Phe Val Lys
Phe Gly Pro Pro Pro Thr Ala Ser Pro Gly Tyr145 150 155 160Ser Asp
Glu Tyr Glu Glu Asp Gly Phe Cys Gln Pro Tyr Arg Gly Ile 165 170
175Ala Cys Ala Arg Phe Ile Gly Asn Arg Thr Val Tyr Met Glu Ser Leu
180 185 190His Met Gln Gly Glu Ile Glu Asn Gln Ile Thr Ala Ala Phe
Thr Met 195 200 205Ile Gly Thr Ser Ser His Leu Ser Asp Lys Cys Ser
Gln Phe Ala Ile 210 215 220Pro Ser Leu Cys His Tyr Ala Phe Pro Tyr
Cys Asp Glu Thr Ser Ser225 230 235 240Val Pro Lys Pro Arg Asp Leu
Cys Arg Asp Glu Cys Glu Ile Leu Glu 245 250 255Asn Val Leu Cys Gln
Thr Glu Tyr Ile Phe Ala Arg Ser Asn Pro Met 260 265 270Ile Leu Met
Arg Leu Lys Leu Pro Asn Cys Glu Asp Leu Pro Gln Pro 275 280 285Glu
Ser Pro Glu Ala Ala Asn Cys Ile Arg Ile Gly Ile Pro Met Ala 290 295
300Asp Pro Ile Asn Lys Asn His Lys Cys Tyr Asn Ser Thr Gly Val
Asp305 310 315 320Tyr Arg Gly Thr Val Ser Val Thr Lys Ser Gly Arg
Gln Cys Gln Pro 325 330 335Trp Asn Ser Gln Tyr Pro His Thr His Thr
Phe Thr Ala Leu Arg Phe 340 345 350Pro Glu Leu Asn Gly Gly His Ser
Tyr Cys Arg Asn Pro Gly Asn Gln 355 360 365Lys Glu Ala Pro Trp Cys
Phe Thr Leu Asp Glu Asn Phe Lys Ser Asp 370 375 380Leu Cys Asp Ile
Pro Ala Cys Asp Ser Lys Asp Ser Lys Glu Lys Asn385 390 395 400Lys
Met Glu Ile Leu Tyr Ile Leu Val Pro Ser Val Ala Ile Pro Leu 405 410
415Ala Ile Ala Leu Leu Phe Phe Phe Ile Cys Val Cys Arg Asn Asn Gln
420 425 430Lys Ser Ser Ser Ala Pro Val Gln Arg Gln Pro Lys His Val
Arg Gly 435 440 445Gln Asn Val Glu Met Ser Met Leu Asn Ala Tyr Lys
Pro Lys Ser Lys 450 455 460Ala Lys Glu Leu Pro Leu Ser Ala Val Arg
Phe Met Glu Glu Leu Gly465 470 475 480Glu Cys Ala Phe Gly Lys Ile
Tyr Lys Gly His Leu Tyr Leu Pro Gly 485 490 495Met Asp His Ala Gln
Leu Val Ala Ile Lys Thr Leu Lys Asp Tyr Asn 500 505 510Asn Pro Gln
Gln Trp Met Glu Phe Gln Gln Glu Ala Ser Leu Met Ala 515 520 525Glu
Leu His His Pro Asn Ile Val Cys Leu Leu Gly Ala Val Thr Gln 530 535
540Glu Gln Pro Val Cys Met Leu Phe Glu Tyr Ile Asn Gln Gly Asp
Leu545 550 555 560His Glu Phe Leu Ile Met Arg Ser Pro His Ser Asp
Val Gly Cys Ser 565 570 575Ser Asp Glu Asp Gly Thr Val Lys Ser Ser
Leu Asp His Gly Asp Phe 580 585 590Leu His Ile Ala Ile Gln Ile Ala
Ala Gly Met Glu Tyr Leu Ser Ser 595 600 605His Phe Phe Val His Lys
Asp Leu Ala Ala Arg Asn Ile Leu Ile Gly 610 615 620Glu Gln Leu His
Val Lys Ile Ser Asp Leu Gly Leu Ser Arg Glu Ile625 630 635 640Tyr
Ser Ala Asp Tyr Tyr Arg Val Gln Ser Lys Ser Leu Leu Pro Ile 645 650
655Arg Trp Met Pro Pro Glu Ala Ile Met Tyr Gly Lys Phe Ser Ser Asp
660 665 670Ser Asp Ile Trp Ser Phe Gly Val Val Leu Trp Glu Ile Phe
Ser Phe 675 680 685Gly Leu Gln Pro Tyr Tyr Gly Phe Ser Asn Gln Glu
Val Ile Glu Met 690 695 700Val Arg Lys Arg Gln Leu Leu Pro Cys Ser
Glu Asp Cys Pro Pro Arg705 710 715 720Met Tyr Ser Leu Met Thr Glu
Cys Trp Asn Glu Ile Pro Ser Arg Arg 725 730 735Pro Arg Phe Lys Asp
Ile His Val Arg Leu Arg Ser Trp Glu Gly Leu 740 745 750Ser Ser His
Thr Ser Ser Thr Thr Pro Ser Gly Gly Asn Ala Thr Thr 755 760 765Gln
Thr Thr Ser Leu Ser Ala Ser Pro Val Ser Asn Leu Ser Asn Pro 770 775
780Arg Tyr Pro Asn Tyr Met Phe Pro Ser Gln Gly Ile Thr Pro Gln
Gly785 790 795 800Gln Ile Ala Gly Phe Ile Gly Pro Pro Ile Pro Gln
Asn Gln Arg Phe 805 810 815Ile Pro Ile Asn Gly Tyr Pro Ile Pro Pro
Gly Tyr Ala Ala Phe Pro 820 825 830Ala Ala His Tyr Gln Pro Thr Gly
Pro Pro Arg Val Ile Gln His Cys 835 840 845Pro Pro Pro Lys Ser Arg
Ser Pro Ser Ser Ala Ser Gly Ser Thr Ser 850 855 860Thr Gly His Val
Thr Ser Leu Pro Ser Ser Gly Ser Asn Gln Glu Ala865 870 875 880Asn
Ile Pro Leu Leu Pro His Met Ser Ile Pro Asn His Pro Gly Gly 885 890
895Met Gly Ile Thr Val Phe Gly Asn Lys Ser Gln Lys Pro Tyr Lys Ile
900 905 910Asp Ser Lys Gln Ala Ser Leu Leu Gly Asp Ala Asn Ile His
Gly His 915 920 925Thr Glu Ser Met Ile Ser Ala Glu Leu 930
93517943PRTHomo sapiens 17Met Ala Arg Gly Ser Ala Leu Pro Arg Arg
Pro Leu Leu Cys Ile Pro1 5 10 15Ala Val Trp Ala Ala Ala Ala Leu Leu
Leu Ser Val Ser Arg Thr Ser 20 25 30Gly Glu Val Glu Val Leu Asp Pro
Asn Asp Pro Leu Gly Pro Leu Asp 35 40 45Gly Gln Asp Gly Pro Ile Pro
Thr Leu Lys Gly Tyr Phe Leu Asn Phe 50 55 60Leu Glu Pro Val Asn Asn
Ile Thr Ile Val Gln Gly Gln Thr Ala Ile65 70 75 80Leu His Cys Lys
Val Ala Gly Asn Pro Pro Pro Asn Val Arg Trp Leu 85 90 95Lys Asn Asp
Ala Pro Val Val Gln Glu Pro Arg Arg Ile Ile Ile Arg 100 105 110Lys
Thr Glu Tyr Gly Ser Arg Leu Arg Ile Gln Asp Leu Asp Thr Thr 115 120
125Asp Thr Gly Tyr Tyr Gln Cys Val Ala Thr Asn Gly Met Lys Thr Ile
130 135 140Thr Ala Thr Gly Val Leu Phe Val Arg Leu Gly Pro Thr His
Ser Pro145 150 155 160Asn His Asn Phe Gln Asp Asp Tyr His Glu Asp
Gly Phe Cys Gln Pro 165 170 175Tyr Arg Gly Ile Ala Cys Ala Arg Phe
Ile Gly Asn Arg Thr Ile Tyr 180 185 190Val Asp Ser Leu Gln Met Gln
Gly Glu Ile Glu Asn Arg Ile Thr Ala 195 200 205Ala Phe Thr Met Ile
Gly Thr Ser Thr His Leu Ser Asp Gln Cys Ser 210 215 220Gln Phe Ala
Ile Pro Ser Phe Cys His Phe Val Phe Pro Leu Cys Asp225 230 235
240Ala Arg Ser Arg Thr Pro Lys Pro Arg Glu Leu Cys Arg Asp Glu Cys
245 250 255Glu Val Leu Glu Ser Asp Leu Cys Arg Gln Glu Tyr Thr Ile
Ala Arg 260 265 270Ser Asn Pro Leu Ile Leu Met Arg Leu Gln Leu Pro
Lys Cys Glu Ala 275 280 285Leu Pro Met Pro Glu Ser Pro Asp Ala Ala
Asn Cys Met Arg Ile Gly 290 295 300Ile Pro Ala Glu Arg Leu Gly Arg
Tyr His Gln Cys Tyr Asn Gly Ser305 310 315 320Gly Met Asp Tyr Arg
Gly Thr Ala Ser Thr Thr Lys Ser Gly His Gln 325 330 335Cys Gln Pro
Trp Ala Leu Gln His Pro His Ser His His Leu Ser Ser 340 345 350Thr
Asp Phe Pro Glu Leu Gly Gly Gly His Ala Tyr Cys Arg Asn Pro 355 360
365Gly Gly Gln Met Glu Gly Pro Trp Cys Phe Thr Gln Asn Lys Asn Val
370 375 380Arg Met Glu Leu Cys Asp Val Pro Ser Cys Ser Pro Arg Asp
Ser Ser385 390 395 400Lys Met Gly Ile Leu Tyr Ile Leu Val Pro Ser
Ile Ala Ile Pro Leu 405 410 415Val Ile Ala Cys Leu Phe Phe Leu Val
Cys Met Cys Arg Asn Lys Gln 420 425 430Lys Ala Ser Ala Ser Thr Pro
Gln Arg Arg Gln Leu Met Ala Ser Pro 435 440 445Ser Gln Asp Met Glu
Met Pro Leu Ile Asn Gln His Lys Gln Ala Lys 450 455 460Leu Lys Glu
Ile Ser Leu Ser Ala Val Arg Phe Met Glu Glu Leu Gly465 470 475
480Glu Asp Arg Phe Gly Lys Val Tyr Lys Gly His Leu Phe Gly Pro Ala
485 490 495Pro Gly Glu Gln Thr Gln Ala Val Ala Ile Lys Thr Leu Lys
Asp Lys 500 505 510Ala Glu Gly Pro Leu Arg Glu Glu Phe Arg His Glu
Ala Met Leu Arg 515 520 525Ala Arg Leu Gln His Pro Asn Val Val Cys
Leu Leu Gly Val Val Thr 530 535 540Lys Asp Gln Pro Leu Ser Met Ile
Phe Ser Tyr Cys Ser His Gly Asp545 550 555 560Leu His Glu Phe Leu
Val Met Arg Ser Pro His Ser Asp Val Gly Ser 565 570 575Thr Asp Asp
Asp Arg Thr Val Lys Ser Ala Leu Glu Pro Pro Asp Phe 580 585 590Val
His Leu Val Ala Gln Ile Ala Ala Gly Met Glu Tyr Leu Ser Ser 595 600
605His His Val Val His Lys Asp Leu Ala Thr Arg Asn Val Leu Val Tyr
610 615 620Asp Lys Leu Asn Val Lys Ile Ser Asp Leu Gly Leu Phe Arg
Glu Val625 630 635 640Tyr Ala Ala Asp Tyr Tyr Lys Leu Leu Gly Asn
Ser Leu Leu Pro Ile 645 650 655Arg Trp Met Ala Pro Glu Ala Ile Met
Tyr Gly Lys Phe Ser Ile Asp 660 665 670Ser Asp Ile Trp Ser Tyr Gly
Val Val Leu Trp Glu Val Phe Ser Tyr 675 680 685Gly Leu Gln Pro Tyr
Cys Gly Tyr Ser Asn Gln Asp Val Val Glu Met 690 695 700Ile Arg Asn
Arg Gln Val Leu Pro Cys Pro Asp Asp Cys Pro Ala Trp705 710 715
720Val Tyr Ala Leu Met Ile Glu Cys Trp Asn Glu Phe Pro Ser Arg Arg
725 730 735Pro Arg Phe Lys Asp Ile His Ser Arg Leu Arg Ala Trp Gly
Asn Leu 740 745 750Ser Asn Tyr Asn Ser Ser Ala Gln Thr Ser Gly Ala
Ser Asn Thr Thr 755 760 765Gln Thr Ser Ser Leu Ser Thr Ser Pro Val
Ser Asn Val Ser Asn Ala 770 775 780Arg Tyr Val Gly Pro Lys Gln Lys
Ala Pro Pro Phe Pro Gln Pro Gln785 790 795 800Phe Ile Pro Met Lys
Gly Gln Ile Arg Pro Met Val Pro Pro Pro Gln 805 810 815Leu Tyr Val
Pro Val Asn Gly Tyr Gln Pro Val Pro Ala Tyr Gly Ala 820 825 830Tyr
Leu Pro Asn Phe Tyr Pro Val Gln Ile Pro Met Gln Met Ala Pro 835 840
845Gln Gln Val Pro Pro Gln Met Val Pro Lys Pro Ser Ser His His Ser
850 855 860Gly Ser Gly Ser Thr Ser Thr Gly Tyr Val Thr Thr Ala Pro
Ser Asn865 870 875 880Thr Ser Met Ala Asp Arg Ala Ala Leu Leu Ser
Glu Gly Ala Asp Asp 885 890 895Thr Gln Asn Ala Pro Glu Asp Gly Ala
Gln Ser Thr Val Gln Glu Ala 900 905 910Glu Glu Glu Glu Glu Gly Ser
Val Pro Glu Thr Glu Leu Leu Gly Asp 915 920 925Cys Asp Thr Leu Gln
Val Asp Glu Ala Gln Val Gln Leu Glu Ala 930 935 94018127PRTHomo
sapiens 18Ser Val Pro Asp His Gly Tyr Cys Gln Pro Ile Ser Ile Pro
Leu Cys1 5 10 15Thr Asp Ile Ala Tyr Asn Gln Thr Ile Met Pro Asn Leu
Leu Gly His 20 25 30Thr Asn Gln Glu Asp Ala Gly Leu Glu Val His Gln
Phe Tyr Pro Leu 35 40 45Val Lys Val Gln Cys Ser Ala Glu Leu Lys Phe
Phe Leu Cys Ser Met 50 55 60Tyr Ala Pro Val Cys Thr Val Leu Glu Gln
Ala Leu Pro Pro Cys Arg65 70 75 80Ser Leu Cys Glu Arg Ala Arg Gln
Gly Cys Glu Ala Leu Met Asn Lys 85 90 95Phe Gly Phe Gln Trp Pro Asp
Thr Leu Lys Cys Glu Lys Phe Pro Val 100 105 110His Gly Ala Gly Glu
Leu Cys Val Gly Gln Asn Thr Ser Asp Lys 115 120 12519127PRTHomo
sapiens 19Ser Ile Pro Asp His Gly Phe Cys Gln Pro Ile Ser Ile Pro
Leu Cys1 5 10 15Thr Asp Ile Ala Tyr Asn Gln Thr Ile Met Pro Asn Leu
Leu Gly His 20 25 30Thr Asn Gln Glu Asp Ala Gly Leu Glu Val His Gln
Phe Tyr Pro Leu 35 40 45Val Lys Val Gln Cys Ser Pro Glu Leu Arg Phe
Phe Leu Cys Ser Met 50 55 60Tyr Ala Pro Val Cys Thr Val Leu Glu Gln
Ala Ile Pro Pro Cys Arg65 70 75 80Ser Ile Cys Glu Ser Ala Arg Gln
Gly Cys Glu
Ala Leu Met Asn Lys 85 90 95Phe Gly Phe Gln Trp Pro Glu Arg Leu Arg
Cys Glu His Phe Pro Arg 100 105 110His Gly Ala Glu Gln Ile Cys Val
Gly Gln Asn His Ser Glu Asp 115 120 12520119PRTHomo sapiens 20His
Ser Leu Phe Ser Cys Glu Pro Ile Thr Leu Arg Met Cys Gln Asp1 5 10
15Leu Pro Tyr Asn Thr Thr Phe Met Pro Asn Leu Leu Asn His Tyr Asp
20 25 30Gln Gln Thr Ala Ala Leu Ala Met Glu Pro Phe His Pro Met Val
Asn 35 40 45Leu Asp Cys Ser Arg Asp Phe Arg Pro Phe Leu Cys Ala Leu
Tyr Ala 50 55 60Pro Ile Cys Met Glu Tyr Gly Arg Val Thr Leu Pro Cys
Arg Arg Leu65 70 75 80Cys Gln Arg Ala Tyr Ser Glu Cys Ser Lys Leu
Met Glu Met Phe Gly 85 90 95Val Pro Trp Pro Glu Asp Met Glu Cys Ser
Arg Phe Pro Asp Cys Asp 100 105 110Glu Pro Tyr Pro Arg Leu Val
11521125PRTHomo sapiens 21Glu Arg Arg Cys Asp Pro Ile Arg Ile Ser
Met Cys Gln Asn Leu Gly1 5 10 15Tyr Asn Val Thr Lys Met Pro Asn Leu
Val Gly His Glu Leu Gln Thr 20 25 30Asp Ala Glu Leu Gln Leu Thr Thr
Phe Thr Pro Leu Ile Gln Tyr Gly 35 40 45Cys Ser Ser Gln Leu Gln Phe
Phe Leu Cys Ser Val Tyr Val Pro Met 50 55 60Cys Thr Glu Lys Ile Asn
Ile Pro Ile Gly Pro Cys Gly Gly Met Cys65 70 75 80Leu Ser Val Lys
Arg Arg Cys Glu Pro Val Leu Lys Glu Phe Gly Phe 85 90 95Ala Trp Pro
Glu Ser Leu Asn Cys Ser Lys Phe Pro Pro Gln Asn Asp 100 105 110His
Asn His Met Cys Met Glu Gly Pro Gly Asp Glu Glu 115 120
12522129PRTHomo sapiens 22Ala Ser Lys Ala Pro Val Cys Gln Glu Ile
Thr Val Pro Met Cys Arg1 5 10 15Gly Ile Gly Tyr Asn Leu Thr His Met
Pro Asn Gln Phe Asn His Asp 20 25 30Thr Gln Asp Glu Ala Gly Leu Glu
Val His Gln Phe Trp Pro Leu Val 35 40 45Glu Ile Gln Cys Ser Pro Asp
Leu Arg Phe Phe Leu Cys Ser Met Tyr 50 55 60Thr Pro Ile Cys Leu Pro
Asp Tyr His Lys Pro Leu Pro Pro Cys Arg65 70 75 80Ser Val Cys Glu
Arg Ala Lys Ala Gly Cys Ser Pro Leu Met Arg Gln 85 90 95Tyr Gly Phe
Ala Trp Pro Glu Arg Met Ser Cys Asp Arg Leu Pro Val 100 105 110Leu
Gly Arg Asp Ala Glu Val Leu Cys Met Asp Tyr Asn Arg Ser Glu 115 120
125Ala23119PRTHomo sapiens 23His Ser Leu Phe Thr Cys Glu Pro Ile
Thr Val Pro Arg Cys Met Lys1 5 10 15Met Ala Tyr Asn Met Thr Phe Phe
Pro Asn Leu Met Gly His Tyr Asp 20 25 30Gln Ser Ile Ala Ala Val Glu
Met Glu His Phe Leu Pro Leu Ala Asn 35 40 45Leu Glu Cys Ser Pro Asn
Ile Glu Thr Phe Leu Cys Lys Ala Phe Val 50 55 60Pro Thr Cys Ile Glu
Gln Ile His Val Val Pro Pro Cys Arg Lys Leu65 70 75 80Cys Glu Lys
Val Tyr Ser Asp Cys Lys Lys Leu Ile Asp Thr Phe Gly 85 90 95Ile Arg
Trp Pro Glu Glu Leu Glu Cys Asp Arg Leu Gln Tyr Cys Asp 100 105
110Glu Thr Val Pro Val Thr Phe 11524127PRTHomo sapiens 24Ser Val
Pro Asp His Gly Phe Cys Gln Pro Ile Ser Ile Pro Leu Cys1 5 10 15Thr
Asp Ile Ala Tyr Asn Gln Thr Ile Leu Pro Asn Leu Leu Gly His 20 25
30Thr Asn Gln Glu Asp Ala Gly Leu Glu Val His Gln Phe Tyr Pro Leu
35 40 45Val Lys Val Gln Cys Ser Pro Glu Leu Arg Phe Phe Leu Cys Ser
Met 50 55 60Tyr Ala Pro Val Cys Thr Val Leu Asp Gln Ala Ile Pro Pro
Cys Arg65 70 75 80Ser Leu Cys Glu Arg Ala Arg Gln Gly Cys Glu Ala
Leu Met Asn Lys 85 90 95Phe Gly Phe Gln Trp Pro Glu Arg Leu Arg Cys
Glu Asn Phe Pro Val 100 105 110His Gly Ala Gly Glu Ile Cys Val Gly
Gln Asn Thr Ser Asp Gly 115 120 12525129PRTHomo sapiens 25Ala Ser
Ala Lys Glu Leu Ala Cys Gln Glu Ile Thr Val Pro Leu Cys1 5 10 15Lys
Gly Ile Gly Tyr Asn Tyr Thr Tyr Met Pro Asn Gln Phe Asn His 20 25
30Asp Thr Gln Asp Glu Ala Gly Leu Glu Val His Gln Phe Trp Pro Leu
35 40 45Val Glu Ile Gln Cys Ser Pro Asp Leu Lys Phe Phe Leu Cys Ser
Met 50 55 60Tyr Thr Pro Ile Cys Leu Glu Asp Tyr Lys Lys Pro Leu Pro
Pro Cys65 70 75 80Arg Ser Val Cys Glu Arg Ala Lys Ala Gly Cys Ala
Pro Leu Met Arg 85 90 95Gln Tyr Gly Phe Ala Trp Pro Asp Arg Met Arg
Cys Asp Arg Leu Pro 100 105 110Glu Gln Gly Asn Pro Asp Thr Leu Cys
Met Asp Tyr Asn Arg Thr Asp 115 120 125Leu26125PRTHomo sapiens
26Ala Ala Pro Cys Gln Ala Val Glu Ile Pro Met Cys Arg Gly Ile Gly1
5 10 15Tyr Asn Leu Thr Arg Met Pro Asn Leu Leu Gly His Thr Ser Gln
Gly 20 25 30Glu Ala Ala Ala Glu Leu Ala Glu Phe Ala Pro Leu Val Gln
Tyr Gly 35 40 45Cys His Ser His Leu Arg Phe Phe Leu Cys Ser Leu Tyr
Ala Pro Met 50 55 60Cys Thr Asp Gln Val Ser Thr Pro Ile Pro Ala Cys
Arg Pro Met Cys65 70 75 80Glu Gln Ala Arg Leu Arg Cys Ala Pro Ile
Met Glu Gln Phe Asn Phe 85 90 95Gly Trp Pro Asp Ser Leu Asp Cys Ala
Arg Leu Pro Thr Arg Asn Asp 100 105 110Pro His Ala Leu Cys Met Glu
Ala Pro Glu Asn Ala Thr 115 120 12527125PRTHomo sapiens 27Asp Gly
Lys Cys Gln Pro Ile Glu Ile Pro Met Cys Lys Asp Ile Gly1 5 10 15Tyr
Asn Met Thr Arg Met Pro Asn Leu Met Gly His Glu Asn Gln Arg 20 25
30Glu Ala Ala Ile Gln Leu His Glu Phe Ala Pro Leu Val Glu Tyr Gly
35 40 45Cys His Gly His Leu Arg Phe Phe Leu Cys Ser Leu Tyr Ala Pro
Met 50 55 60Cys Thr Glu Gln Val Ser Thr Pro Ile Pro Ala Cys Arg Val
Met Cys65 70 75 80Glu Gln Ala Arg Leu Lys Cys Ser Pro Ile Met Glu
Gln Phe Asn Phe 85 90 95Lys Trp Pro Asp Ser Leu Asp Cys Arg Lys Leu
Pro Asn Lys Asn Asp 100 105 110Pro Asn Tyr Leu Cys Met Glu Ala Pro
Asn Asn Gly Ser 115 120 12528124PRTHomo sapiens 28Phe Tyr Thr Lys
Pro Pro Gln Cys Val Asp Ile Pro Ala Asp Leu Arg1 5 10 15Leu Cys His
Asn Val Gly Tyr Lys Lys Met Val Leu Pro Asn Leu Leu 20 25 30Glu His
Glu Thr Met Ala Glu Val Lys Gln Gln Ala Ser Ser Trp Val 35 40 45Pro
Leu Leu Asn Lys Asn Cys His Ala Gly Thr Gln Val Phe Leu Cys 50 55
60Ser Leu Phe Ala Pro Val Cys Leu Asp Arg Pro Ile Tyr Pro Cys Arg65
70 75 80Trp Leu Cys Glu Ala Val Arg Asp Ser Cys Glu Pro Val Met Gln
Phe 85 90 95Phe Gly Phe Tyr Trp Pro Glu Met Leu Lys Cys Asp Lys Phe
Pro Glu 100 105 110Gly Asp Val Cys Ile Ala Met Thr Pro Pro Asn Ala
115 12029128PRTHomo sapiens 29Phe Ser Tyr Lys Arg Ser Asn Cys Lys
Pro Ile Pro Ala Asn Leu Gln1 5 10 15Leu Cys His Gly Ile Glu Tyr Gln
Asn Met Arg Leu Pro Asn Leu Leu 20 25 30Gly His Glu Thr Met Lys Glu
Val Leu Glu Gln Ala Gly Ala Trp Ile 35 40 45Pro Leu Val Met Lys Gln
Cys His Pro Asp Thr Lys Lys Phe Leu Cys 50 55 60Ser Leu Phe Ala Pro
Val Cys Leu Asp Asp Leu Asp Glu Thr Ile Gln65 70 75 80Pro Cys His
Ser Leu Cys Val Gln Val Lys Asp Arg Cys Ala Pro Val 85 90 95Met Ser
Ala Phe Gly Phe Pro Trp Pro Asp Met Leu Glu Cys Asp Arg 100 105
110Phe Pro Gln Asp Asn Asp Leu Cys Ile Pro Leu Ala Ser Ser Asp His
115 120 12530123PRTHomo sapiens 30Ala Ala Cys Glu Pro Val Arg Ile
Pro Leu Cys Lys Ser Leu Pro Trp1 5 10 15Asn Met Thr Lys Met Pro Asn
His Leu His His Ser Thr Gln Ala Asn 20 25 30Ala Ile Leu Ala Ile Glu
Gln Phe Glu Gly Leu Leu Gly Thr His Cys 35 40 45Ser Pro Asp Leu Leu
Phe Phe Leu Cys Ala Met Tyr Ala Pro Ile Cys 50 55 60Thr Ile Asp Phe
Gln His Glu Pro Ile Lys Pro Cys Lys Ser Val Cys65 70 75 80Glu Arg
Ala Arg Gln Gly Cys Glu Pro Ile Leu Ile Lys Tyr Arg His 85 90 95Ser
Trp Pro Glu Asn Leu Ala Cys Glu Glu Leu Pro Val Tyr Asp Arg 100 105
110Gly Val Cys Ile Ser Pro Glu Ala Ile Val Thr 115 12031126PRTHomo
sapiens 31Val Arg Gly Ala Pro Cys Glu Ala Val Arg Ile Pro Met Cys
Arg His1 5 10 15Met Pro Trp Asn Ile Thr Arg Met Pro Asn His Leu His
His Ser Thr 20 25 30Gln Glu Asn Ala Ile Leu Ala Ile Glu Gln Tyr Glu
Glu Leu Val Asp 35 40 45Val Asn Cys Ser Ala Val Leu Arg Phe Phe Phe
Cys Ala Met Tyr Ala 50 55 60Pro Ile Cys Thr Leu Glu Phe Leu His Asp
Pro Ile Lys Pro Cys Lys65 70 75 80Ser Val Cys Gln Arg Ala Arg Asp
Asp Cys Glu Pro Leu Met Lys Met 85 90 95Tyr Asn His Ser Trp Pro Glu
Ser Leu Ala Cys Asp Glu Leu Pro Val 100 105 110Tyr Asp Arg Gly Val
Cys Ile Ser Pro Glu Ala Ile Val Thr 115 120 12532125PRTHomo sapiens
32Ser Tyr Ser Lys Pro Pro Gln Cys Leu Asp Ile Pro Ala Asp Leu Pro1
5 10 15Leu Cys His Thr Val Gly Tyr Lys Arg Met Arg Leu Pro Asn Leu
Leu 20 25 30Glu His Glu Ser Leu Ala Glu Val Lys Gln Gln Ala Ser Ser
Trp Leu 35 40 45Pro Leu Leu Ala Lys Arg Cys His Ser Asp Thr Gln Val
Phe Leu Cys 50 55 60Ser Leu Phe Ala Pro Val Cys Leu Asp Arg Pro Ile
Tyr Pro Cys Arg65 70 75 80Ser Leu Cys Glu Ala Val Arg Ala Gly Cys
Ala Pro Leu Met Glu Ala 85 90 95Tyr Gly Phe Pro Trp Pro Glu Met Leu
His Cys His Lys Phe Pro Leu 100 105 110Asp Asn Asp Leu Cys Ile Ala
Val Gln Phe Gly His Leu 115 120 12533142PRTHomo sapiens 33Glu Tyr
Glu Glu Asp Gly Phe Cys Gln Pro Tyr Arg Gly Ile Ala Cys1 5 10 15Ala
Arg Phe Ile Gly Asn Arg Thr Val Tyr Met Glu Ser Leu His Met 20 25
30Gln Gly Glu Ile Glu Asn Gln Ile Thr Ala Ala Phe Thr Met Ile Gly
35 40 45Thr Ser Ser His Leu Ser Asp Lys Cys Ser Gln Phe Ala Ile Pro
Ser 50 55 60Leu Cys His Tyr Ala Phe Pro Tyr Cys Asp Glu Thr Ser Ser
Val Pro65 70 75 80Lys Pro Arg Asp Leu Cys Arg Asp Glu Cys Glu Ile
Leu Glu Asn Val 85 90 95Leu Cys Gln Thr Glu Tyr Ile Phe Ala Arg Ser
Asn Pro Met Ile Leu 100 105 110Met Arg Leu Lys Leu Pro Asn Cys Glu
Asp Leu Pro Gln Pro Glu Ser 115 120 125Pro Glu Ala Ala Asn Cys Ile
Arg Ile Gly Ile Pro Met Ala 130 135 14034142PRTHomo sapiens 34Asp
Tyr His Glu Asp Gly Phe Cys Gln Pro Tyr Arg Gly Ile Ala Cys1 5 10
15Ala Arg Phe Ile Gly Asn Arg Thr Ile Tyr Val Asp Ser Leu Gln Met
20 25 30Gln Gly Glu Ile Glu Asn Arg Ile Thr Ala Ala Phe Thr Met Ile
Gly 35 40 45Thr Ser Thr His Leu Ser Asp Gln Cys Ser Gln Phe Ala Ile
Pro Ser 50 55 60Phe Cys His Phe Val Phe Pro Leu Cys Asp Ala Arg Ser
Arg Thr Pro65 70 75 80Lys Pro Arg Glu Leu Cys Arg Asp Glu Cys Glu
Val Leu Glu Ser Asp 85 90 95Leu Cys Arg Gln Glu Tyr Thr Ile Ala Arg
Ser Asn Pro Leu Ile Leu 100 105 110Met Arg Leu Gln Leu Pro Lys Cys
Glu Ala Leu Pro Met Pro Glu Ser 115 120 125Pro Asp Ala Ala Asn Cys
Met Arg Ile Gly Ile Pro Ala Glu 130 135 14035235PRTHomo sapiens
35Met Ala Glu Glu Glu Ala Pro Lys Lys Ser Arg Ala Ala Gly Gly Gly1
5 10 15Ala Ser Trp Glu Leu Cys Ala Gly Ala Leu Ser Ala Arg Leu Ala
Glu 20 25 30Glu Gly Ser Gly Asp Ala Gly Gly Arg Arg Arg Pro Pro Val
Asp Pro 35 40 45Arg Arg Leu Ala Arg Gln Leu Leu Leu Leu Leu Trp Leu
Leu Glu Ala 50 55 60Pro Leu Leu Leu Gly Val Arg Ala Gln Ala Ala Gly
Gln Gly Pro Gly65 70 75 80Gln Gly Pro Gly Pro Gly Gln Gln Pro Pro
Pro Pro Pro Gln Gln Gln 85 90 95Gln Ser Gly Gln Gln Tyr Asn Gly Glu
Arg Gly Ile Ser Val Pro Asp 100 105 110His Gly Tyr Cys Gln Pro Ile
Ser Ile Pro Leu Cys Thr Asp Ile Ala 115 120 125Tyr Asn Gln Thr Ile
Met Pro Asn Leu Leu Gly His Thr Asn Gln Glu 130 135 140Asp Ala Gly
Leu Glu Val His Gln Phe Tyr Pro Leu Val Lys Val Gln145 150 155
160Cys Ser Ala Glu Leu Lys Phe Phe Leu Cys Ser Met Tyr Ala Pro Val
165 170 175Cys Thr Val Leu Glu Gln Ala Leu Pro Pro Cys Arg Ser Leu
Cys Glu 180 185 190Arg Ala Arg Gln Gly Cys Glu Ala Leu Met Asn Lys
Phe Gly Phe Gln 195 200 205Trp Pro Asp Thr Leu Lys Cys Glu Lys Phe
Pro Val His Gly Ala Gly 210 215 220Glu Leu Cys Val Gly Gln Asn Thr
Ser Asp Lys225 230 23536158PRTHomo sapiens 36Met Arg Pro Arg Ser
Ala Leu Pro Arg Leu Leu Leu Pro Leu Leu Leu1 5 10 15Leu Pro Ala Ala
Gly Pro Ala Gln Phe His Gly Glu Lys Gly Ile Ser 20 25 30Ile Pro Asp
His Gly Phe Cys Gln Pro Ile Ser Ile Pro Leu Cys Thr 35 40 45Asp Ile
Ala Tyr Asn Gln Thr Ile Met Pro Asn Leu Leu Gly His Thr 50 55 60Asn
Gln Glu Asp Ala Gly Leu Glu Val His Gln Phe Tyr Pro Leu Val65 70 75
80Lys Val Gln Cys Ser Pro Glu Leu Arg Phe Phe Leu Cys Ser Met Tyr
85 90 95Ala Pro Val Cys Thr Val Leu Glu Gln Ala Ile Pro Pro Cys Arg
Ser 100 105 110Ile Cys Glu Ser Ala Arg Gln Gly Cys Glu Ala Leu Met
Asn Lys Phe 115 120 125Gly Phe Gln Trp Pro Glu Arg Leu Arg Cys Glu
His Phe Pro Arg His 130 135 140Gly Ala Glu Gln Ile Cys Val Gly Gln
Asn His Ser Glu Asp145 150 15537141PRTHomo sapiens 37Met Ala Met
Thr Trp Ile Val Phe Ser Leu Trp Pro Leu Thr Val Phe1 5 10 15Met Gly
His Ile Gly Gly His Ser Leu Phe Ser Cys Glu Pro Ile Thr 20 25 30Leu
Arg Met Cys Gln Asp Leu Pro Tyr Asn Thr Thr Phe Met Pro Asn 35 40
45Leu Leu Asn His Tyr Asp Gln Gln Thr Ala Ala Leu Ala Met Glu Pro
50 55 60Phe His Pro Met Val Asn Leu Asp Cys Ser Arg Asp Phe Arg Pro
Phe65 70 75 80Leu Cys Ala Leu Tyr Ala Pro Ile Cys Met Glu Tyr Gly
Arg Val Thr 85
90 95Leu Pro Cys Arg Arg Leu Cys Gln Arg Ala Tyr Ser Glu Cys Ser
Lys 100 105 110Leu Met Glu Met Phe Gly Val Pro Trp Pro Glu Asp Met
Glu Cys Ser 115 120 125Arg Phe Pro Asp Cys Asp Glu Pro Tyr Pro Arg
Leu Val 130 135 14038166PRTHomo sapiens 38Met Ala Trp Arg Gly Ala
Gly Pro Ser Val Pro Gly Ala Pro Gly Gly1 5 10 15Val Gly Leu Ser Leu
Gly Leu Leu Leu Gln Leu Leu Leu Leu Leu Gly 20 25 30Pro Ala Arg Gly
Phe Gly Asp Glu Glu Glu Arg Arg Cys Asp Pro Ile 35 40 45Arg Ile Ser
Met Cys Gln Asn Leu Gly Tyr Asn Val Thr Lys Met Pro 50 55 60Asn Leu
Val Gly His Glu Leu Gln Thr Asp Ala Glu Leu Gln Leu Thr65 70 75
80Thr Phe Thr Pro Leu Ile Gln Tyr Gly Cys Ser Ser Gln Leu Gln Phe
85 90 95Phe Leu Cys Ser Val Tyr Val Pro Met Cys Thr Glu Lys Ile Asn
Ile 100 105 110Pro Ile Gly Pro Cys Gly Gly Met Cys Leu Ser Val Lys
Arg Arg Cys 115 120 125Glu Pro Val Leu Lys Glu Phe Gly Phe Ala Trp
Pro Glu Ser Leu Asn 130 135 140Cys Ser Lys Phe Pro Pro Gln Asn Asp
His Asn His Met Cys Met Glu145 150 155 160Gly Pro Gly Asp Glu Glu
16539155PRTHomo sapiens 39Met Ala Arg Pro Asp Pro Ser Ala Pro Pro
Ser Leu Leu Leu Leu Leu1 5 10 15Leu Ala Gln Leu Val Gly Arg Ala Ala
Ala Ala Ser Lys Ala Pro Val 20 25 30Cys Gln Glu Ile Thr Val Pro Met
Cys Arg Gly Ile Gly Tyr Asn Leu 35 40 45Thr His Met Pro Asn Gln Phe
Asn His Asp Thr Gln Asp Glu Ala Gly 50 55 60Leu Glu Val His Gln Phe
Trp Pro Leu Val Glu Ile Gln Cys Ser Pro65 70 75 80Asp Leu Arg Phe
Phe Leu Cys Ser Met Tyr Thr Pro Ile Cys Leu Pro 85 90 95Asp Tyr His
Lys Pro Leu Pro Pro Cys Arg Ser Val Cys Glu Arg Ala 100 105 110Lys
Ala Gly Cys Ser Pro Leu Met Arg Gln Tyr Gly Phe Ala Trp Pro 115 120
125Glu Arg Met Ser Cys Asp Arg Leu Pro Val Leu Gly Arg Asp Ala Glu
130 135 140Val Leu Cys Met Asp Tyr Asn Arg Ser Glu Ala145 150
15540137PRTHomo sapiens 40Met Glu Met Phe Thr Phe Leu Leu Thr Cys
Ile Phe Leu Pro Leu Leu1 5 10 15Arg Gly His Ser Leu Phe Thr Cys Glu
Pro Ile Thr Val Pro Arg Cys 20 25 30Met Lys Met Ala Tyr Asn Met Thr
Phe Phe Pro Asn Leu Met Gly His 35 40 45Tyr Asp Gln Ser Ile Ala Ala
Val Glu Met Glu His Phe Leu Pro Leu 50 55 60Ala Asn Leu Glu Cys Ser
Pro Asn Ile Glu Thr Phe Leu Cys Lys Ala65 70 75 80Phe Val Pro Thr
Cys Ile Glu Gln Ile His Val Val Pro Pro Cys Arg 85 90 95Lys Leu Cys
Glu Lys Val Tyr Ser Asp Cys Lys Lys Leu Ile Asp Thr 100 105 110Phe
Gly Ile Arg Trp Pro Glu Glu Leu Glu Cys Asp Arg Leu Gln Tyr 115 120
125Cys Asp Glu Thr Val Pro Val Thr Phe 130 13541168PRTHomo sapiens
41Met Arg Asp Pro Gly Ala Ala Ala Pro Leu Ser Ser Leu Gly Leu Cys1
5 10 15Ala Leu Val Leu Ala Leu Leu Gly Ala Leu Ser Ala Gly Ala Gly
Ala 20 25 30Gln Pro Tyr His Gly Glu Lys Gly Ile Ser Val Pro Asp His
Gly Phe 35 40 45Cys Gln Pro Ile Ser Ile Pro Leu Cys Thr Asp Ile Ala
Tyr Asn Gln 50 55 60Thr Ile Leu Pro Asn Leu Leu Gly His Thr Asn Gln
Glu Asp Ala Gly65 70 75 80Leu Glu Val His Gln Phe Tyr Pro Leu Val
Lys Val Gln Cys Ser Pro 85 90 95Glu Leu Arg Phe Phe Leu Cys Ser Met
Tyr Ala Pro Val Cys Thr Val 100 105 110Leu Asp Gln Ala Ile Pro Pro
Cys Arg Ser Leu Cys Glu Arg Ala Arg 115 120 125Gln Gly Cys Glu Ala
Leu Met Asn Lys Phe Gly Phe Gln Trp Pro Glu 130 135 140Arg Leu Arg
Cys Glu Asn Phe Pro Val His Gly Ala Gly Glu Ile Cys145 150 155
160Val Gly Gln Asn Thr Ser Asp Gly 16542156PRTHomo sapiens 42Met
Glu Trp Gly Tyr Leu Leu Glu Val Thr Ser Leu Leu Ala Ala Leu1 5 10
15Ala Leu Leu Gln Arg Ser Ser Gly Ala Ala Ala Ala Ser Ala Lys Glu
20 25 30Leu Ala Cys Gln Glu Ile Thr Val Pro Leu Cys Lys Gly Ile Gly
Tyr 35 40 45Asn Tyr Thr Tyr Met Pro Asn Gln Phe Asn His Asp Thr Gln
Asp Glu 50 55 60Ala Gly Leu Glu Val His Gln Phe Trp Pro Leu Val Glu
Ile Gln Cys65 70 75 80Ser Pro Asp Leu Lys Phe Phe Leu Cys Ser Met
Tyr Thr Pro Ile Cys 85 90 95Leu Glu Asp Tyr Lys Lys Pro Leu Pro Pro
Cys Arg Ser Val Cys Glu 100 105 110Arg Ala Lys Ala Gly Cys Ala Pro
Leu Met Arg Gln Tyr Gly Phe Ala 115 120 125Trp Pro Asp Arg Met Arg
Cys Asp Arg Leu Pro Glu Gln Gly Asn Pro 130 135 140Asp Thr Leu Cys
Met Asp Tyr Asn Arg Thr Asp Leu145 150 15543160PRTHomo sapiens
43Met Ala Val Ala Pro Leu Arg Gly Ala Leu Leu Leu Trp Gln Leu Leu1
5 10 15Ala Ala Gly Gly Ala Ala Leu Glu Ile Gly Arg Phe Asp Pro Glu
Arg 20 25 30Gly Arg Gly Ala Ala Pro Cys Gln Ala Val Glu Ile Pro Met
Cys Arg 35 40 45Gly Ile Gly Tyr Asn Leu Thr Arg Met Pro Asn Leu Leu
Gly His Thr 50 55 60Ser Gln Gly Glu Ala Ala Ala Glu Leu Ala Glu Phe
Ala Pro Leu Val65 70 75 80Gln Tyr Gly Cys His Ser His Leu Arg Phe
Phe Leu Cys Ser Leu Tyr 85 90 95Ala Pro Met Cys Thr Asp Gln Val Ser
Thr Pro Ile Pro Ala Cys Arg 100 105 110Pro Met Cys Glu Gln Ala Arg
Leu Arg Cys Ala Pro Ile Met Glu Gln 115 120 125Phe Asn Phe Gly Trp
Pro Asp Ser Leu Asp Cys Ala Arg Leu Pro Thr 130 135 140Arg Asn Asp
Pro His Ala Leu Cys Met Glu Ala Pro Glu Asn Ala Thr145 150 155
16044155PRTHomo sapiens 44Met Gln Arg Pro Gly Pro Arg Leu Trp Leu
Val Leu Gln Val Met Gly1 5 10 15Ser Cys Ala Ala Ile Ser Ser Met Asp
Met Glu Arg Pro Gly Asp Gly 20 25 30Lys Cys Gln Pro Ile Glu Ile Pro
Met Cys Lys Asp Ile Gly Tyr Asn 35 40 45Met Thr Arg Met Pro Asn Leu
Met Gly His Glu Asn Gln Arg Glu Ala 50 55 60Ala Ile Gln Leu His Glu
Phe Ala Pro Leu Val Glu Tyr Gly Cys His65 70 75 80Gly His Leu Arg
Phe Phe Leu Cys Ser Leu Tyr Ala Pro Met Cys Thr 85 90 95Glu Gln Val
Ser Thr Pro Ile Pro Ala Cys Arg Val Met Cys Glu Gln 100 105 110Ala
Arg Leu Lys Cys Ser Pro Ile Met Glu Gln Phe Asn Phe Lys Trp 115 120
125Pro Asp Ser Leu Asp Cys Arg Lys Leu Pro Asn Lys Asn Asp Pro Asn
130 135 140Tyr Leu Cys Met Glu Ala Pro Asn Asn Gly Ser145 150
15545174PRTHomo sapiens 45Met Gly Ile Gly Arg Ser Glu Gly Gly Arg
Arg Gly Ala Ala Leu Gly1 5 10 15Val Leu Leu Ala Leu Gly Ala Ala Leu
Leu Ala Val Gly Ser Ala Ser 20 25 30Glu Tyr Asp Tyr Val Ser Phe Gln
Ser Asp Ile Gly Pro Tyr Gln Ser 35 40 45Gly Arg Phe Tyr Thr Lys Pro
Pro Gln Cys Val Asp Ile Pro Ala Asp 50 55 60Leu Arg Leu Cys His Asn
Val Gly Tyr Lys Lys Met Val Leu Pro Asn65 70 75 80Leu Leu Glu His
Glu Thr Met Ala Glu Val Lys Gln Gln Ala Ser Ser 85 90 95Trp Val Pro
Leu Leu Asn Lys Asn Cys His Ala Gly Thr Gln Val Phe 100 105 110Leu
Cys Ser Leu Phe Ala Pro Val Cys Leu Asp Arg Pro Ile Tyr Pro 115 120
125Cys Arg Trp Leu Cys Glu Ala Val Arg Asp Ser Cys Glu Pro Val Met
130 135 140Gln Phe Phe Gly Phe Tyr Trp Pro Glu Met Leu Lys Cys Asp
Lys Phe145 150 155 160Pro Glu Gly Asp Val Cys Ile Ala Met Thr Pro
Pro Asn Ala 165 17046160PRTHomo sapiens 46Met Leu Gln Gly Pro Gly
Ser Leu Leu Leu Leu Phe Leu Ala Ser His1 5 10 15Cys Cys Leu Gly Ser
Ala Arg Gly Leu Phe Leu Phe Gly Gln Pro Asp 20 25 30Phe Ser Tyr Lys
Arg Ser Asn Cys Lys Pro Ile Pro Ala Asn Leu Gln 35 40 45Leu Cys His
Gly Ile Glu Tyr Gln Asn Met Arg Leu Pro Asn Leu Leu 50 55 60Gly His
Glu Thr Met Lys Glu Val Leu Glu Gln Ala Gly Ala Trp Ile65 70 75
80Pro Leu Val Met Lys Gln Cys His Pro Asp Thr Lys Lys Phe Leu Cys
85 90 95Ser Leu Phe Ala Pro Val Cys Leu Asp Asp Leu Asp Glu Thr Ile
Gln 100 105 110Pro Cys His Ser Leu Cys Val Gln Val Lys Asp Arg Cys
Ala Pro Val 115 120 125Met Ser Ala Phe Gly Phe Pro Trp Pro Asp Met
Leu Glu Cys Asp Arg 130 135 140Phe Pro Gln Asp Asn Asp Leu Cys Ile
Pro Leu Ala Ser Ser Asp His145 150 155 16047155PRTHomo sapiens
47Met Val Cys Gly Ser Pro Gly Gly Met Leu Leu Leu Arg Ala Gly Leu1
5 10 15Leu Ala Leu Ala Ala Leu Cys Leu Leu Arg Val Pro Gly Ala Arg
Ala 20 25 30Ala Ala Cys Glu Pro Val Arg Ile Pro Leu Cys Lys Ser Leu
Pro Trp 35 40 45Asn Met Thr Lys Met Pro Asn His Leu His His Ser Thr
Gln Ala Asn 50 55 60Ala Ile Leu Ala Ile Glu Gln Phe Glu Gly Leu Leu
Gly Thr His Cys65 70 75 80Ser Pro Asp Leu Leu Phe Phe Leu Cys Ala
Met Tyr Ala Pro Ile Cys 85 90 95Thr Ile Asp Phe Gln His Glu Pro Ile
Lys Pro Cys Lys Ser Val Cys 100 105 110Glu Arg Ala Arg Gln Gly Cys
Glu Pro Ile Leu Ile Lys Tyr Arg His 115 120 125Ser Trp Pro Glu Asn
Leu Ala Cys Glu Glu Leu Pro Val Tyr Asp Arg 130 135 140Gly Val Cys
Ile Ser Pro Glu Ala Ile Val Thr145 150 15548144PRTHomo sapiens
48Met Phe Leu Ser Ile Leu Val Ala Leu Cys Leu Trp Leu His Leu Ala1
5 10 15Leu Gly Val Arg Gly Ala Pro Cys Glu Ala Val Arg Ile Pro Met
Cys 20 25 30Arg His Met Pro Trp Asn Ile Thr Arg Met Pro Asn His Leu
His His 35 40 45Ser Thr Gln Glu Asn Ala Ile Leu Ala Ile Glu Gln Tyr
Glu Glu Leu 50 55 60Val Asp Val Asn Cys Ser Ala Val Leu Arg Phe Phe
Phe Cys Ala Met65 70 75 80Tyr Ala Pro Ile Cys Thr Leu Glu Phe Leu
His Asp Pro Ile Lys Pro 85 90 95Cys Lys Ser Val Cys Gln Arg Ala Arg
Asp Asp Cys Glu Pro Leu Met 100 105 110Lys Met Tyr Asn His Ser Trp
Pro Glu Ser Leu Ala Cys Asp Glu Leu 115 120 125Pro Val Tyr Asp Arg
Gly Val Cys Ile Ser Pro Glu Ala Ile Val Thr 130 135 14049170PRTHomo
sapiens 49Met Arg Ala Ala Ala Ala Ala Gly Gly Val Arg Thr Ala Ala
Leu Ala1 5 10 15Leu Leu Leu Gly Ala Leu His Trp Ala Pro Ala Arg Cys
Glu Glu Tyr 20 25 30Asp Tyr Tyr Gly Trp Gln Ala Glu Pro Leu His Gly
Arg Ser Tyr Ser 35 40 45Lys Pro Pro Gln Cys Leu Asp Ile Pro Ala Asp
Leu Pro Leu Cys His 50 55 60Thr Val Gly Tyr Lys Arg Met Arg Leu Pro
Asn Leu Leu Glu His Glu65 70 75 80Ser Leu Ala Glu Val Lys Gln Gln
Ala Ser Ser Trp Leu Pro Leu Leu 85 90 95Ala Lys Arg Cys His Ser Asp
Thr Gln Val Phe Leu Cys Ser Leu Phe 100 105 110Ala Pro Val Cys Leu
Asp Arg Pro Ile Tyr Pro Cys Arg Ser Leu Cys 115 120 125Glu Ala Val
Arg Ala Gly Cys Ala Pro Leu Met Glu Ala Tyr Gly Phe 130 135 140Pro
Trp Pro Glu Met Leu His Cys His Lys Phe Pro Leu Asp Asn Asp145 150
155 160Leu Cys Ile Ala Val Gln Phe Gly His Leu 165 17050163PRTHomo
sapiens 50Gln Ala Ala Gly Gln Gly Pro Gly Gln Gly Pro Gly Pro Gly
Gln Gln1 5 10 15Pro Pro Pro Pro Pro Gln Gln Gln Gln Ser Gly Gln Gln
Tyr Asn Gly 20 25 30Glu Arg Gly Ile Ser Val Pro Asp His Gly Tyr Cys
Gln Pro Ile Ser 35 40 45Ile Pro Leu Cys Thr Asp Ile Ala Tyr Asn Gln
Thr Ile Met Pro Asn 50 55 60Leu Leu Gly His Thr Asn Gln Glu Asp Ala
Gly Leu Glu Val His Gln65 70 75 80Phe Tyr Pro Leu Val Lys Val Gln
Cys Ser Ala Glu Leu Lys Phe Phe 85 90 95Leu Cys Ser Met Tyr Ala Pro
Val Cys Thr Val Leu Glu Gln Ala Leu 100 105 110Pro Pro Cys Arg Ser
Leu Cys Glu Arg Ala Arg Gln Gly Cys Glu Ala 115 120 125Leu Met Asn
Lys Phe Gly Phe Gln Trp Pro Asp Thr Leu Lys Cys Glu 130 135 140Lys
Phe Pro Val His Gly Ala Gly Glu Leu Cys Val Gly Gln Asn Thr145 150
155 160Ser Asp Lys51136PRTHomo sapiens 51Ala Gln Phe His Gly Glu
Lys Gly Ile Ser Ile Pro Asp His Gly Phe1 5 10 15Cys Gln Pro Ile Ser
Ile Pro Leu Cys Thr Asp Ile Ala Tyr Asn Gln 20 25 30Thr Ile Met Pro
Asn Leu Leu Gly His Thr Asn Gln Glu Asp Ala Gly 35 40 45Leu Glu Val
His Gln Phe Tyr Pro Leu Val Lys Val Gln Cys Ser Pro 50 55 60Glu Leu
Arg Phe Phe Leu Cys Ser Met Tyr Ala Pro Val Cys Thr Val65 70 75
80Leu Glu Gln Ala Ile Pro Pro Cys Arg Ser Ile Cys Glu Ser Ala Arg
85 90 95Gln Gly Cys Glu Ala Leu Met Asn Lys Phe Gly Phe Gln Trp Pro
Glu 100 105 110Arg Leu Arg Cys Glu His Phe Pro Arg His Gly Ala Glu
Gln Ile Cys 115 120 125Val Gly Gln Asn His Ser Glu Asp 130
13552119PRTHomo sapiens 52His Ser Leu Phe Ser Cys Glu Pro Ile Thr
Leu Arg Met Cys Gln Asp1 5 10 15Leu Pro Tyr Asn Thr Thr Phe Met Pro
Asn Leu Leu Asn His Tyr Asp 20 25 30Gln Gln Thr Ala Ala Leu Ala Met
Glu Pro Phe His Pro Met Val Asn 35 40 45Leu Asp Cys Ser Arg Asp Phe
Arg Pro Phe Leu Cys Ala Leu Tyr Ala 50 55 60Pro Ile Cys Met Glu Tyr
Gly Arg Val Thr Leu Pro Cys Arg Arg Leu65 70 75 80Cys Gln Arg Ala
Tyr Ser Glu Cys Ser Lys Leu Met Glu Met Phe Gly 85 90 95Val Pro Trp
Pro Glu Asp Met Glu Cys Ser Arg Phe Pro Asp Cys Asp 100 105 110Glu
Pro Tyr Pro Arg Leu Val 11553130PRTHomo sapiens 53Phe Gly Asp Glu
Glu Glu Arg Arg Cys Asp Pro Ile Arg Ile Ser Met1 5 10 15Cys Gln Asn
Leu Gly Tyr Asn Val Thr Lys Met Pro Asn Leu Val Gly 20 25 30His Glu
Leu Gln Thr Asp Ala Glu Leu Gln Leu Thr Thr Phe Thr Pro 35 40 45Leu
Ile Gln Tyr Gly Cys Ser Ser Gln Leu Gln Phe Phe Leu Cys Ser 50 55
60Val Tyr Val Pro Met Cys Thr Glu Lys Ile Asn Ile Pro Ile Gly Pro65
70 75 80Cys Gly Gly Met Cys Leu Ser Val
Lys Arg Arg Cys Glu Pro Val Leu 85 90 95Lys Glu Phe Gly Phe Ala Trp
Pro Glu Ser Leu Asn Cys Ser Lys Phe 100 105 110Pro Pro Gln Asn Asp
His Asn His Met Cys Met Glu Gly Pro Gly Asp 115 120 125Glu Glu
13054129PRTHomo sapiens 54Ala Ser Lys Ala Pro Val Cys Gln Glu Ile
Thr Val Pro Met Cys Arg1 5 10 15Gly Ile Gly Tyr Asn Leu Thr His Met
Pro Asn Gln Phe Asn His Asp 20 25 30Thr Gln Asp Glu Ala Gly Leu Glu
Val His Gln Phe Trp Pro Leu Val 35 40 45Glu Ile Gln Cys Ser Pro Asp
Leu Arg Phe Phe Leu Cys Ser Met Tyr 50 55 60Thr Pro Ile Cys Leu Pro
Asp Tyr His Lys Pro Leu Pro Pro Cys Arg65 70 75 80Ser Val Cys Glu
Arg Ala Lys Ala Gly Cys Ser Pro Leu Met Arg Gln 85 90 95Tyr Gly Phe
Ala Trp Pro Glu Arg Met Ser Cys Asp Arg Leu Pro Val 100 105 110Leu
Gly Arg Asp Ala Glu Val Leu Cys Met Asp Tyr Asn Arg Ser Glu 115 120
125Ala55119PRTHomo sapiens 55His Ser Leu Phe Thr Cys Glu Pro Ile
Thr Val Pro Arg Cys Met Lys1 5 10 15Met Ala Tyr Asn Met Thr Phe Phe
Pro Asn Leu Met Gly His Tyr Asp 20 25 30Gln Ser Ile Ala Ala Val Glu
Met Glu His Phe Leu Pro Leu Ala Asn 35 40 45Leu Glu Cys Ser Pro Asn
Ile Glu Thr Phe Leu Cys Lys Ala Phe Val 50 55 60Pro Thr Cys Ile Glu
Gln Ile His Val Val Pro Pro Cys Arg Lys Leu65 70 75 80Cys Glu Lys
Val Tyr Ser Asp Cys Lys Lys Leu Ile Asp Thr Phe Gly 85 90 95Ile Arg
Trp Pro Glu Glu Leu Glu Cys Asp Arg Leu Gln Tyr Cys Asp 100 105
110Glu Thr Val Pro Val Thr Phe 11556136PRTHomo sapiens 56Gln Pro
Tyr His Gly Glu Lys Gly Ile Ser Val Pro Asp His Gly Phe1 5 10 15Cys
Gln Pro Ile Ser Ile Pro Leu Cys Thr Asp Ile Ala Tyr Asn Gln 20 25
30Thr Ile Leu Pro Asn Leu Leu Gly His Thr Asn Gln Glu Asp Ala Gly
35 40 45Leu Glu Val His Gln Phe Tyr Pro Leu Val Lys Val Gln Cys Ser
Pro 50 55 60Glu Leu Arg Phe Phe Leu Cys Ser Met Tyr Ala Pro Val Cys
Thr Val65 70 75 80Leu Asp Gln Ala Ile Pro Pro Cys Arg Ser Leu Cys
Glu Arg Ala Arg 85 90 95Gln Gly Cys Glu Ala Leu Met Asn Lys Phe Gly
Phe Gln Trp Pro Glu 100 105 110Arg Leu Arg Cys Glu Asn Phe Pro Val
His Gly Ala Gly Glu Ile Cys 115 120 125Val Gly Gln Asn Thr Ser Asp
Gly 130 13557129PRTHomo sapiens 57Ala Ser Ala Lys Glu Leu Ala Cys
Gln Glu Ile Thr Val Pro Leu Cys1 5 10 15Lys Gly Ile Gly Tyr Asn Tyr
Thr Tyr Met Pro Asn Gln Phe Asn His 20 25 30Asp Thr Gln Asp Glu Ala
Gly Leu Glu Val His Gln Phe Trp Pro Leu 35 40 45Val Glu Ile Gln Cys
Ser Pro Asp Leu Lys Phe Phe Leu Cys Ser Met 50 55 60Tyr Thr Pro Ile
Cys Leu Glu Asp Tyr Lys Lys Pro Leu Pro Pro Cys65 70 75 80Arg Ser
Val Cys Glu Arg Ala Lys Ala Gly Cys Ala Pro Leu Met Arg 85 90 95Gln
Tyr Gly Phe Ala Trp Pro Asp Arg Met Arg Cys Asp Arg Leu Pro 100 105
110Glu Gln Gly Asn Pro Asp Thr Leu Cys Met Asp Tyr Asn Arg Thr Asp
115 120 125Leu58139PRTHomo sapiens 58Ala Leu Glu Ile Gly Arg Phe
Asp Pro Glu Arg Gly Arg Gly Ala Ala1 5 10 15Pro Cys Gln Ala Val Glu
Ile Pro Met Cys Arg Gly Ile Gly Tyr Asn 20 25 30Leu Thr Arg Met Pro
Asn Leu Leu Gly His Thr Ser Gln Gly Glu Ala 35 40 45Ala Ala Glu Leu
Ala Glu Phe Ala Pro Leu Val Gln Tyr Gly Cys His 50 55 60Ser His Leu
Arg Phe Phe Leu Cys Ser Leu Tyr Ala Pro Met Cys Thr65 70 75 80Asp
Gln Val Ser Thr Pro Ile Pro Ala Cys Arg Pro Met Cys Glu Gln 85 90
95Ala Arg Leu Arg Cys Ala Pro Ile Met Glu Gln Phe Asn Phe Gly Trp
100 105 110Pro Asp Ser Leu Asp Cys Ala Arg Leu Pro Thr Arg Asn Asp
Pro His 115 120 125Ala Leu Cys Met Glu Ala Pro Glu Asn Ala Thr 130
13559135PRTHomo sapiens 59Ile Ser Ser Met Asp Met Glu Arg Pro Gly
Asp Gly Lys Cys Gln Pro1 5 10 15Ile Glu Ile Pro Met Cys Lys Asp Ile
Gly Tyr Asn Met Thr Arg Met 20 25 30Pro Asn Leu Met Gly His Glu Asn
Gln Arg Glu Ala Ala Ile Gln Leu 35 40 45His Glu Phe Ala Pro Leu Val
Glu Tyr Gly Cys His Gly His Leu Arg 50 55 60Phe Phe Leu Cys Ser Leu
Tyr Ala Pro Met Cys Thr Glu Gln Val Ser65 70 75 80Thr Pro Ile Pro
Ala Cys Arg Val Met Cys Glu Gln Ala Arg Leu Lys 85 90 95Cys Ser Pro
Ile Met Glu Gln Phe Asn Phe Lys Trp Pro Asp Ser Leu 100 105 110Asp
Cys Arg Lys Leu Pro Asn Lys Asn Asp Pro Asn Tyr Leu Cys Met 115 120
125Glu Ala Pro Asn Asn Gly Ser 130 13560144PRTHomo sapiens 60Ala
Ser Glu Tyr Asp Tyr Val Ser Phe Gln Ser Asp Ile Gly Pro Tyr1 5 10
15Gln Ser Gly Arg Phe Tyr Thr Lys Pro Pro Gln Cys Val Asp Ile Pro
20 25 30Ala Asp Leu Arg Leu Cys His Asn Val Gly Tyr Lys Lys Met Val
Leu 35 40 45Pro Asn Leu Leu Glu His Glu Thr Met Ala Glu Val Lys Gln
Gln Ala 50 55 60Ser Ser Trp Val Pro Leu Leu Asn Lys Asn Cys His Ala
Gly Thr Gln65 70 75 80Val Phe Leu Cys Ser Leu Phe Ala Pro Val Cys
Leu Asp Arg Pro Ile 85 90 95Tyr Pro Cys Arg Trp Leu Cys Glu Ala Val
Arg Asp Ser Cys Glu Pro 100 105 110Val Met Gln Phe Phe Gly Phe Tyr
Trp Pro Glu Met Leu Lys Cys Asp 115 120 125Lys Phe Pro Glu Gly Asp
Val Cys Ile Ala Met Thr Pro Pro Asn Ala 130 135 14061142PRTHomo
sapiens 61Leu Gly Ser Ala Arg Gly Leu Phe Leu Phe Gly Gln Pro Asp
Phe Ser1 5 10 15Tyr Lys Arg Ser Asn Cys Lys Pro Ile Pro Ala Asn Leu
Gln Leu Cys 20 25 30His Gly Ile Glu Tyr Gln Asn Met Arg Leu Pro Asn
Leu Leu Gly His 35 40 45Glu Thr Met Lys Glu Val Leu Glu Gln Ala Gly
Ala Trp Ile Pro Leu 50 55 60Val Met Lys Gln Cys His Pro Asp Thr Lys
Lys Phe Leu Cys Ser Leu65 70 75 80Phe Ala Pro Val Cys Leu Asp Asp
Leu Asp Glu Thr Ile Gln Pro Cys 85 90 95His Ser Leu Cys Val Gln Val
Lys Asp Arg Cys Ala Pro Val Met Ser 100 105 110Ala Phe Gly Phe Pro
Trp Pro Asp Met Leu Glu Cys Asp Arg Phe Pro 115 120 125Gln Asp Asn
Asp Leu Cys Ile Pro Leu Ala Ser Ser Asp His 130 135 14062123PRTHomo
sapiens 62Ala Ala Cys Glu Pro Val Arg Ile Pro Leu Cys Lys Ser Leu
Pro Trp1 5 10 15Asn Met Thr Lys Met Pro Asn His Leu His His Ser Thr
Gln Ala Asn 20 25 30Ala Ile Leu Ala Ile Glu Gln Phe Glu Gly Leu Leu
Gly Thr His Cys 35 40 45Ser Pro Asp Leu Leu Phe Phe Leu Cys Ala Met
Tyr Ala Pro Ile Cys 50 55 60Thr Ile Asp Phe Gln His Glu Pro Ile Lys
Pro Cys Lys Ser Val Cys65 70 75 80Glu Arg Ala Arg Gln Gly Cys Glu
Pro Ile Leu Ile Lys Tyr Arg His 85 90 95Ser Trp Pro Glu Asn Leu Ala
Cys Glu Glu Leu Pro Val Tyr Asp Arg 100 105 110Gly Val Cys Ile Ser
Pro Glu Ala Ile Val Thr 115 12063126PRTHomo sapiens 63Val Arg Gly
Ala Pro Cys Glu Ala Val Arg Ile Pro Met Cys Arg His1 5 10 15Met Pro
Trp Asn Ile Thr Arg Met Pro Asn His Leu His His Ser Thr 20 25 30Gln
Glu Asn Ala Ile Leu Ala Ile Glu Gln Tyr Glu Glu Leu Val Asp 35 40
45Val Asn Cys Ser Ala Val Leu Arg Phe Phe Phe Cys Ala Met Tyr Ala
50 55 60Pro Ile Cys Thr Leu Glu Phe Leu His Asp Pro Ile Lys Pro Cys
Lys65 70 75 80Ser Val Cys Gln Arg Ala Arg Asp Asp Cys Glu Pro Leu
Met Lys Met 85 90 95Tyr Asn His Ser Trp Pro Glu Ser Leu Ala Cys Asp
Glu Leu Pro Val 100 105 110Tyr Asp Arg Gly Val Cys Ile Ser Pro Glu
Ala Ile Val Thr 115 120 12564143PRTHomo sapiens 64Arg Cys Glu Glu
Tyr Asp Tyr Tyr Gly Trp Gln Ala Glu Pro Leu His1 5 10 15Gly Arg Ser
Tyr Ser Lys Pro Pro Gln Cys Leu Asp Ile Pro Ala Asp 20 25 30Leu Pro
Leu Cys His Thr Val Gly Tyr Lys Arg Met Arg Leu Pro Asn 35 40 45Leu
Leu Glu His Glu Ser Leu Ala Glu Val Lys Gln Gln Ala Ser Ser 50 55
60Trp Leu Pro Leu Leu Ala Lys Arg Cys His Ser Asp Thr Gln Val Phe65
70 75 80Leu Cys Ser Leu Phe Ala Pro Val Cys Leu Asp Arg Pro Ile Tyr
Pro 85 90 95Cys Arg Ser Leu Cys Glu Ala Val Arg Ala Gly Cys Ala Pro
Leu Met 100 105 110Glu Ala Tyr Gly Phe Pro Trp Pro Glu Met Leu His
Cys His Lys Phe 115 120 125Pro Leu Asp Asn Asp Leu Cys Ile Ala Val
Gln Phe Gly His Leu 130 135 14065162PRTHomo sapiens 65Thr Gly Val
Leu Phe Val Lys Phe Gly Pro Pro Pro Thr Ala Ser Pro1 5 10 15Gly Tyr
Ser Asp Glu Tyr Glu Glu Asp Gly Phe Cys Gln Pro Tyr Arg 20 25 30Gly
Ile Ala Cys Ala Arg Phe Ile Gly Asn Arg Thr Val Tyr Met Glu 35 40
45Ser Leu His Met Gln Gly Glu Ile Glu Asn Gln Ile Thr Ala Ala Phe
50 55 60Thr Met Ile Gly Thr Ser Ser His Leu Ser Asp Lys Cys Ser Gln
Phe65 70 75 80Ala Ile Pro Ser Leu Cys His Tyr Ala Phe Pro Tyr Cys
Asp Glu Thr 85 90 95Ser Ser Val Pro Lys Pro Arg Asp Leu Cys Arg Asp
Glu Cys Glu Ile 100 105 110Leu Glu Asn Val Leu Cys Gln Thr Glu Tyr
Ile Phe Ala Arg Ser Asn 115 120 125Pro Met Ile Leu Met Arg Leu Lys
Leu Pro Asn Cys Glu Asp Leu Pro 130 135 140Gln Pro Glu Ser Pro Glu
Ala Ala Asn Cys Ile Arg Ile Gly Ile Pro145 150 155 160Met
Ala66162PRTHomo sapiens 66Thr Gly Val Leu Phe Val Arg Leu Gly Pro
Thr His Ser Pro Asn His1 5 10 15Asn Phe Gln Asp Asp Tyr His Glu Asp
Gly Phe Cys Gln Pro Tyr Arg 20 25 30Gly Ile Ala Cys Ala Arg Phe Ile
Gly Asn Arg Thr Ile Tyr Val Asp 35 40 45Ser Leu Gln Met Gln Gly Glu
Ile Glu Asn Arg Ile Thr Ala Ala Phe 50 55 60Thr Met Ile Gly Thr Ser
Thr His Leu Ser Asp Gln Cys Ser Gln Phe65 70 75 80Ala Ile Pro Ser
Phe Cys His Phe Val Phe Pro Leu Cys Asp Ala Arg 85 90 95Ser Arg Thr
Pro Lys Pro Arg Glu Leu Cys Arg Asp Glu Cys Glu Val 100 105 110Leu
Glu Ser Asp Leu Cys Arg Gln Glu Tyr Thr Ile Ala Arg Ser Asn 115 120
125Pro Leu Ile Leu Met Arg Leu Gln Leu Pro Lys Cys Glu Ala Leu Pro
130 135 140Met Pro Glu Ser Pro Asp Ala Ala Asn Cys Met Arg Ile Gly
Ile Pro145 150 155 160Ala Glu67227PRTHomo sapiens 67Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65
70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200
205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220Pro Gly Lys22568230PRTHomo sapiens 68Asp Lys Ala Ala Arg
Ser Thr Leu Cys Pro Pro Cys Pro Ala Pro Glu1 5 10 15Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 20 25 30Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 35 40 45Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 50 55 60Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn65 70 75
80Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
85 90 95Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro 100 105 110Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu 115 120 125Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn 130 135 140Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile145 150 155 160Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 165 170 175Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 180 185 190Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 195 200
205Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
210 215 220Ser Leu Ser Pro Gly Lys225 230698PRTArtificial
sequencePeptide linker 69Glu Ser Gly Gly Gly Gly Val Thr1
5709PRTArtificial sequencePeptide linker 70Leu Glu Ser Gly Gly Gly
Gly Val Thr1 5716PRTArtificial sequencePeptide linker 71Gly Arg Ala
Gln Val Thr1 5726PRTArtificial SequencePeptide linker 72Trp Arg Ala
Gln Val Thr1 5738PRTArtificial sequencePeptide linker 73Ala Arg Gly
Arg Ala Gln Val Thr1 574391PRTArtificial sequenceFrz8-Fc chimeric
protein 74Met Glu Trp Gly Tyr Leu Leu Glu Val Thr Ser Leu Leu Ala
Ala Leu1 5 10 15Ala Val Leu Gln Arg Ser Ser Gly Ala Ala Ala Ala Ser
Ala Lys Glu 20 25 30Leu Ala Cys Gln Glu Ile Thr Val Pro Leu Cys Lys
Gly Ile Gly Tyr 35 40 45Asn Tyr Thr Tyr Met Pro Asn Gln Phe Asn His
Asp Thr Gln Asp Glu 50 55 60Ala Gly Leu Glu Val His Gln Phe Trp Pro
Leu Val Glu Ile Gln Cys65 70 75 80Ser Pro Asp Leu Lys Phe Phe Leu
Cys Ser Met Tyr Thr Pro Ile Cys 85 90 95Leu
Glu Asp Tyr Lys Lys Pro Leu Pro Pro Cys Arg Ser Val Cys Glu 100 105
110Arg Ala Lys Ala Gly Cys Ala Pro Leu Met Arg Gln Tyr Gly Phe Ala
115 120 125Trp Pro Asp Arg Met Arg Cys Asp Arg Leu Pro Glu Gln Gly
Asn Pro 130 135 140Asp Thr Leu Cys Met Asp Tyr Asn Arg Thr Asp Leu
Glu Ser Gly Gly145 150 155 160Gly Gly Val Thr Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro 165 170 175Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 180 185 190Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 195 200 205Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 210 215 220Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr225 230
235 240Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 245 250 255Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu 260 265 270Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 275 280 285Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys 290 295 300Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp305 310 315 320Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 325 330 335Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 340 345
350Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
355 360 365Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser 370 375 380Leu Ser Leu Ser Pro Gly Lys385
39075396PRTArtificial sequenceFrz5-Fc chimeric protein 75Met Gly
Gly Thr Ala Ala Arg Leu Gly Ala Val Ile Leu Phe Val Val1 5 10 15Ile
Val Gly Leu His Gly Val Arg Gly Lys Ile Asp Ala Arg Gly Ala 20 25
30Ser Lys Ala Pro Val Cys Gln Glu Ile Thr Val Pro Met Cys Arg Gly
35 40 45Ile Gly Tyr Asn Leu Thr His Met Pro Asn Gln Phe Asn His Asp
Thr 50 55 60Gln Asp Glu Ala Gly Leu Glu Val His Gln Phe Trp Pro Leu
Val Glu65 70 75 80Ile Gln Cys Ser Pro Asp Leu Arg Phe Phe Leu Cys
Ser Met Tyr Thr 85 90 95Pro Ile Cys Leu Pro Asp Tyr His Lys Pro Leu
Pro Pro Cys Arg Ser 100 105 110Val Cys Glu Arg Ala Lys Ala Gly Cys
Ser Pro Leu Met Arg Gln Tyr 115 120 125Gly Phe Ala Trp Pro Glu Arg
Met Ser Cys Asp Arg Leu Pro Val Leu 130 135 140Gly Arg Asp Ala Glu
Val Leu Cys Met Asp Tyr Asn Arg Ser Glu Ala145 150 155 160Gly Arg
Ala Gln Val Thr Asp Lys Ala Ala Arg Ser Thr Leu Cys Pro 165 170
175Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
180 185 190Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val 195 200 205Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe 210 215 220Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro225 230 235 240Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr 245 250 255Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 260 265 270Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 275 280 285Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 290 295
300Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly305 310 315 320Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro 325 330 335Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser 340 345 350Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln 355 360 365Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His 370 375 380Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys385 390 39576470PRTArtificial
sequenceFrz1-Fc chimeric protein 76Met Gly Gly Thr Ala Ala Arg Leu
Gly Ala Val Ile Leu Phe Val Val1 5 10 15Ile Val Gly Leu His Gly Val
Arg Gly Lys Ile Asp Glu Glu Gly Ser 20 25 30Gly Asp Ala Gly Gly Arg
Arg Arg Pro Pro Val Asp Pro Arg Arg Leu 35 40 45Ala Arg Gln Leu Leu
Leu Leu Leu Trp Leu Leu Glu Ala Pro Leu Leu 50 55 60Leu Gly Val Arg
Ala Gln Ala Ala Gly Gln Gly Pro Gly Gln Gly Pro65 70 75 80Gly Pro
Gly Gln Gln Pro Pro Pro Pro Pro Gln Gln Gln Gln Ser Gly 85 90 95Gln
Gln Tyr Asn Gly Glu Arg Gly Ile Ser Val Pro Asp His Gly Tyr 100 105
110Cys Gln Pro Ile Ser Ile Pro Leu Cys Thr Asp Ile Ala Tyr Asn Gln
115 120 125Thr Ile Met Pro Asn Leu Leu Gly His Thr Asn Gln Glu Asp
Ala Gly 130 135 140Leu Glu Val His Gln Phe Tyr Pro Leu Val Lys Val
Gln Cys Ser Ala145 150 155 160Glu Leu Lys Phe Phe Leu Cys Ser Met
Tyr Ala Pro Val Cys Thr Val 165 170 175Leu Glu Gln Ala Leu Pro Pro
Cys Arg Ser Leu Cys Glu Arg Ala Arg 180 185 190Gln Gly Cys Glu Ala
Leu Met Asn Lys Phe Gly Phe Gln Trp Pro Asp 195 200 205Thr Leu Lys
Cys Glu Lys Phe Pro Val His Gly Ala Gly Glu Leu Cys 210 215 220Val
Gly Gln Asn Thr Ser Asp Lys Ala Arg Gly Arg Ala Gln Val Thr225 230
235 240Asp Lys Ala Ala Arg Ser Thr Leu Cys Pro Pro Cys Pro Ala Pro
Glu 245 250 255Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp 260 265 270Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp 275 280 285Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly 290 295 300Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn305 310 315 320Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345
350Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
355 360 365Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn 370 375 380Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile385 390 395 400Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr 405 410 415Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460Ser
Leu Ser Pro Gly Lys465 47077403PRTArtificial sequenceFrz2-Fc
chimeric protein 77Met Gly Gly Thr Ala Ala Arg Leu Gly Ala Val Ile
Leu Phe Val Val1 5 10 15Ile Val Gly Leu His Gly Val Arg Gly Lys Ile
Asp Ala Arg Gly Ala 20 25 30Gln Phe His Gly Glu Lys Gly Ile Ser Ile
Pro Asp His Gly Phe Cys 35 40 45Gln Pro Ile Ser Ile Pro Leu Cys Thr
Asp Ile Ala Tyr Asn Gln Thr 50 55 60Ile Met Pro Asn Leu Leu Gly His
Thr Asn Gln Glu Asp Ala Gly Leu65 70 75 80Glu Val His Gln Phe Tyr
Pro Leu Val Lys Val Gln Cys Ser Pro Glu 85 90 95Leu Arg Phe Phe Leu
Cys Ser Met Tyr Ala Pro Val Cys Thr Val Leu 100 105 110Glu Gln Ala
Ile Pro Pro Cys Arg Ser Ile Cys Glu Arg Ala Arg Gln 115 120 125Gly
Cys Glu Ala Leu Met Asn Lys Phe Gly Phe Gln Trp Pro Glu Arg 130 135
140Leu Arg Cys Glu His Phe Pro Arg His Gly Ala Glu Gln Ile Cys
Val145 150 155 160Gly Gln Asn His Ser Glu Asp Gly Arg Ala Gln Val
Thr Asp Lys Ala 165 170 175Ala Arg Ser Thr Leu Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 180 185 190Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 195 200 205Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 210 215 220Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val225 230 235 240His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 245 250
255Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
260 265 270Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile 275 280 285Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 290 295 300Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser305 310 315 320Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 325 330 335Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 340 345 350Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 355 360 365Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 370 375
380His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser385 390 395 400Pro Gly Lys78388PRTArtificial sequenceFrz3-Fc
chimeric protein 78Met Gly Gly Thr Ala Ala Arg Leu Gly Ala Val Ile
Leu Phe Val Val1 5 10 15Ile Val Gly Leu His Gly Val Arg Gly Lys Ile
Asp Ala Arg Gly His 20 25 30Ser Leu Phe Ser Cys Glu Pro Ile Thr Leu
Arg Met Cys Gln Asp Leu 35 40 45Pro Tyr Asn Thr Thr Phe Met Pro Asn
Leu Leu Asn His Tyr Asp Gln 50 55 60Gln Thr Ala Ala Leu Ala Met Glu
Pro Phe His Pro Met Val Asn Leu65 70 75 80Asp Cys Ser Arg Asp Phe
Arg Pro Phe Leu Cys Ala Leu Tyr Ala Pro 85 90 95Ile Cys Met Glu Tyr
Gly Arg Val Thr Leu Pro Cys Arg Arg Leu Cys 100 105 110Gln Arg Ala
Tyr Ser Glu Cys Ser Lys Leu Met Glu Met Phe Gly Val 115 120 125Pro
Trp Pro Glu Asp Met Glu Cys Ser Arg Phe Pro Asp Cys Asp Glu 130 135
140Pro Tyr Pro Arg Leu Val Asp Leu Gly Arg Ala Gln Val Thr Asp
Lys145 150 155 160Ala Ala Arg Ser Thr Leu Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu 165 170 175Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 180 185 190Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser 195 200 205His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 210 215 220Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr225 230 235 240Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 245 250
255Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
260 265 270Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 275 280 285Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 290 295 300Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val305 310 315 320Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro 325 330 335Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 340 345 350Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 355 360 365Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 370 375
380Ser Pro Gly Lys38579397PRTArtificial sequenceFrz4-Fc chimeric
protein 79Met Gly Gly Thr Ala Ala Arg Leu Gly Ala Val Ile Leu Phe
Val Val1 5 10 15Ile Val Gly Leu His Gly Val Arg Gly Lys Ile Asp Ala
Arg Gly Phe 20 25 30Gly Asp Glu Glu Glu Arg Arg Cys Asp Pro Ile Arg
Ile Ser Met Cys 35 40 45Gln Asn Leu Gly Tyr Asn Val Thr Lys Met Pro
Asn Leu Val Gly His 50 55 60Glu Leu Gln Thr Asp Ala Glu Leu Gln Leu
Thr Thr Phe Thr Pro Leu65 70 75 80Ile Gln Tyr Gly Cys Ser Ser Gln
Leu Gln Phe Phe Leu Cys Ser Val 85 90 95Tyr Val Pro Met Cys Thr Glu
Lys Ile Asn Ile Pro Ile Gly Pro Cys 100 105 110Gly Gly Met Cys Leu
Ser Val Lys Arg Arg Cys Glu Pro Val Leu Lys 115 120 125Glu Phe Gly
Phe Ala Trp Pro Glu Ser Leu Asn Cys Ser Lys Phe Pro 130 135 140Pro
Gln Asn Asp His Asn His Met Cys Met Glu Gly Pro Gly Asp Glu145 150
155 160Glu Gly Arg Ala Gln Val Thr Asp Lys Ala Ala Arg Ser Thr Leu
Cys 165 170 175Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu 180 185 190Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu 195 200 205Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys 210 215 220Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys225 230 235 240Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 245 250 255Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 260 265
270Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
275 280 285Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser 290 295 300Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys305 310 315 320Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln 325 330 335Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly 340 345 350Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 355 360 365Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 370 375 380His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys385 390
39580373PRTArtificial sequenceFrz6-Fc chimeric protein 80Met Glu
Met Phe Thr Phe Leu Leu Thr Cys Ile Phe Leu Pro Leu Leu1 5 10 15Arg
Gly His Ser Leu Phe Thr Cys Glu Pro Ile Thr Val Pro Arg Cys 20 25
30Met Lys Met Ala Tyr Asn Met Thr Phe Phe Pro Asn Leu Met Gly His
35 40 45Tyr Asp Gln Ser Ile Ala Ala Val Glu Met Glu His Phe Leu Pro
Leu 50 55 60Ala Asn Leu Glu Cys Ser Pro Asn Ile Glu Thr Phe Leu Cys
Lys Ala65 70 75 80Phe Val Pro Thr Cys Ile Glu Gln Ile His Val Val
Pro Pro Cys Arg 85 90
95Lys Leu Cys Glu Lys Val Tyr Ser Asp Cys Lys Lys Leu Ile Asp Thr
100 105 110Phe Gly Ile Arg Trp Pro Glu Glu Leu Glu Cys Asp Arg Leu
Gln Tyr 115 120 125Cys Asp Glu Thr Val Pro Val Thr Phe Leu Glu Ser
Gly Gly Gly Gly 130 135 140Val Thr Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu145 150 155 160Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr 165 170 175Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val 180 185 190Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 195 200 205Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 210 215
220Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu225 230 235 240Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala 245 250 255Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro 260 265 270Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln 275 280 285Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 290 295 300Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr305 310 315 320Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 325 330
335Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
340 345 350Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 355 360 365Leu Ser Pro Gly Lys 37081404PRTArtificial
sequenceFrz7-Fc chimeric protein 81Met Arg Asp Pro Gly Ala Ala Ala
Pro Leu Ser Ser Leu Gly Leu Cys1 5 10 15Ala Leu Val Leu Ala Leu Leu
Gly Ala Leu Ser Ala Gly Ala Gly Ala 20 25 30Gln Pro Tyr His Gly Glu
Lys Gly Ile Ser Val Pro Asp His Gly Phe 35 40 45Cys Gln Pro Ile Ser
Ile Pro Leu Cys Thr Asp Ile Ala Tyr Asn Gln 50 55 60Thr Ile Leu Pro
Asn Leu Leu Gly His Thr Asn Gln Glu Asp Ala Gly65 70 75 80Leu Glu
Val His Gln Phe Tyr Pro Leu Val Lys Val Gln Cys Ser Pro 85 90 95Glu
Leu Arg Phe Phe Leu Cys Ser Met Tyr Ala Pro Val Cys Thr Val 100 105
110Leu Asp Gln Ala Ile Pro Pro Cys Arg Ser Leu Cys Glu Arg Ala Arg
115 120 125Gln Gly Cys Glu Ala Leu Met Asn Lys Phe Gly Phe Gln Trp
Pro Glu 130 135 140Arg Leu Arg Cys Glu Asn Phe Pro Val His Gly Ala
Gly Glu Ile Cys145 150 155 160Val Gly Gln Asn Thr Ser Asp Gly Leu
Glu Ser Gly Gly Gly Gly Val 165 170 175Thr Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu 180 185 190Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 195 200 205Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 210 215 220His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu225 230
235 240Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr 245 250 255Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn 260 265 270Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro 275 280 285Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln 290 295 300Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val305 310 315 320Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 325 330 335Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 340 345
350Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
355 360 365Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val 370 375 380Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu385 390 395 400Ser Pro Gly Lys82396PRTArtificial
SequenceFrz9-Fc chimeric protein 82Met Ala Val Ala Pro Leu Arg Gly
Ala Leu Leu Leu Trp Gln Leu Leu1 5 10 15Ala Ala Gly Gly Ala Ala Leu
Glu Ile Gly Arg Phe Asp Pro Glu Arg 20 25 30Gly Arg Gly Ala Ala Pro
Cys Gln Ala Val Glu Ile Pro Met Cys Arg 35 40 45Gly Ile Gly Tyr Asn
Leu Thr Arg Met Pro Asn Leu Leu Gly His Thr 50 55 60Ser Gln Gly Glu
Ala Ala Ala Glu Leu Ala Glu Phe Ala Pro Leu Val65 70 75 80Gln Tyr
Gly Cys His Ser His Leu Arg Phe Phe Leu Cys Ser Leu Tyr 85 90 95Ala
Pro Met Cys Thr Asp Gln Val Ser Thr Pro Ile Pro Ala Cys Arg 100 105
110Pro Met Cys Glu Gln Ala Arg Leu Arg Cys Ala Pro Ile Met Glu Gln
115 120 125Phe Asn Phe Gly Trp Pro Asp Ser Leu Asp Cys Ala Arg Leu
Pro Thr 130 135 140Arg Asn Asp Pro His Ala Leu Cys Met Glu Ala Pro
Glu Asn Ala Thr145 150 155 160Leu Glu Ser Gly Gly Gly Gly Val Thr
Asp Lys Thr His Thr Cys Pro 165 170 175Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe 180 185 190Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 195 200 205Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 210 215 220Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro225 230
235 240Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr 245 250 255Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val 260 265 270Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala 275 280 285Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg 290 295 300Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly305 310 315 320Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 325 330 335Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 340 345
350Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
355 360 365Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His 370 375 380Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys385 390 39583391PRTArtificial sequenceFrz10-Fc chimeric protein
83Met Gln Arg Pro Gly Pro Arg Leu Trp Leu Val Leu Gln Val Met Gly1
5 10 15Ser Cys Ala Ala Ile Ser Ser Met Asp Met Glu Arg Pro Gly Asp
Gly 20 25 30Lys Cys Gln Pro Ile Glu Ile Pro Met Cys Lys Asp Ile Gly
Tyr Asn 35 40 45Met Thr Arg Met Pro Asn Leu Met Gly His Glu Asn Gln
Arg Glu Ala 50 55 60Ala Ile Gln Leu His Glu Phe Ala Pro Leu Val Glu
Tyr Gly Cys His65 70 75 80Gly His Leu Arg Phe Phe Leu Cys Ser Leu
Tyr Ala Pro Met Cys Thr 85 90 95Glu Gln Val Ser Thr Pro Ile Pro Ala
Cys Arg Val Met Cys Glu Gln 100 105 110Ala Arg Leu Lys Cys Ser Pro
Ile Met Glu Gln Phe Asn Phe Lys Trp 115 120 125Pro Asp Ser Leu Asp
Cys Arg Lys Leu Pro Asn Lys Asn Asp Pro Asn 130 135 140Tyr Leu Cys
Met Glu Ala Pro Asn Asn Gly Ser Leu Glu Ser Gly Gly145 150 155
160Gly Gly Val Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
165 170 175Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 180 185 190Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val 195 200 205Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp 210 215 220Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr225 230 235 240Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp 245 250 255Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 260 265 270Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 275 280
285Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
290 295 300Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp305 310 315 320Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys 325 330 335Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 340 345 350Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser 355 360 365Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 370 375 380Leu Ser Leu
Ser Pro Gly Lys385 39084410PRTArtificial sequencesFRP1-Fc chimeric
protein 84Met Gly Ile Gly Arg Ser Glu Gly Gly Arg Arg Gly Ala Ala
Leu Gly1 5 10 15Val Leu Leu Ala Leu Gly Ala Ala Leu Leu Ala Val Gly
Ser Ala Ser 20 25 30Glu Tyr Asp Tyr Val Ser Phe Gln Ser Asp Ile Gly
Pro Tyr Gln Ser 35 40 45Gly Arg Phe Tyr Thr Lys Pro Pro Gln Cys Val
Asp Ile Pro Ala Asp 50 55 60Leu Arg Leu Cys His Asn Val Gly Tyr Lys
Lys Met Val Leu Pro Asn65 70 75 80Leu Leu Glu His Glu Thr Met Ala
Glu Val Lys Gln Gln Ala Ser Ser 85 90 95Trp Val Pro Leu Leu Asn Lys
Asn Cys His Ala Gly Thr Gln Val Phe 100 105 110Leu Cys Ser Leu Phe
Ala Pro Val Cys Leu Asp Arg Pro Ile Tyr Pro 115 120 125Cys Arg Trp
Leu Cys Glu Ala Val Arg Asp Ser Cys Glu Pro Val Met 130 135 140Gln
Phe Phe Gly Phe Tyr Trp Pro Glu Met Leu Lys Cys Asp Lys Phe145 150
155 160Pro Glu Gly Asp Val Cys Ile Ala Met Thr Pro Pro Asn Ala Trp
Arg 165 170 175Ala Gln Val Thr Asp Lys Ala Ala Arg Ser Thr Leu Cys
Pro Pro Cys 180 185 190Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro 195 200 205Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys 210 215 220Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp225 230 235 240Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 245 250 255Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 260 265
270His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
275 280 285Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 290 295 300Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu305 310 315 320Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 325 330 335Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 340 345 350Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 355 360 365Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 370 375 380Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr385 390
395 400Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405
41085396PRTArtificial sequencesFRP2-Fc chimeric protein 85Met Leu
Gln Gly Pro Gly Ser Leu Leu Leu Leu Phe Leu Ala Ser His1 5 10 15Cys
Cys Leu Gly Ser Ala Arg Gly Leu Phe Leu Phe Gly Gln Pro Asp 20 25
30Phe Ser Tyr Lys Arg Ser Asn Cys Lys Pro Ile Pro Ala Asn Leu Gln
35 40 45Leu Cys His Gly Ile Glu Tyr Gln Asn Met Arg Leu Pro Asn Leu
Leu 50 55 60Gly His Glu Thr Met Lys Glu Val Leu Glu Gln Ala Gly Ala
Trp Ile65 70 75 80Pro Leu Val Met Lys Gln Cys His Pro Asp Thr Lys
Lys Phe Leu Cys 85 90 95Ser Leu Phe Ala Pro Val Cys Leu Asp Asp Leu
Asp Glu Thr Ile Gln 100 105 110Pro Cys His Ser Leu Cys Val Gln Val
Lys Asp Arg Cys Ala Pro Val 115 120 125Met Ser Ala Phe Gly Phe Pro
Trp Pro Asp Met Leu Glu Cys Asp Arg 130 135 140Phe Pro Gln Asp Asn
Asp Leu Cys Ile Pro Leu Ala Ser Ser Asp His145 150 155 160Trp Arg
Ala Gln Val Thr Asp Lys Ala Ala Arg Ser Thr Leu Cys Pro 165 170
175Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
180 185 190Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val 195 200 205Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe 210 215 220Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro225 230 235 240Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr 245 250 255Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 260 265 270Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 275 280 285Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 290 295
300Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly305 310 315 320Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro 325 330 335Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser 340 345 350Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln 355 360 365Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His 370 375 380Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys385 390 39586390PRTArtificial
sequencesFRP3-Fc chimeric construct 86Met Gly Gly Thr Ala Ala Arg
Leu Gly Ala Val Ile Leu Phe Val Val1 5 10 15Ile Val Gly Leu His Gly
Val Arg Gly Lys Ile Asp Ala Arg Gly Ala 20 25 30Ala Cys Glu Pro Val
Arg Ile Pro Leu Cys Lys Ser Leu Pro Trp Asn 35 40 45Met Thr Lys Met
Pro Asn His Leu His His Ser Thr Gln Ala Asn Ala 50 55 60Ile Leu Ala
Ile Glu Gln Phe Glu Gly Leu Leu Gly Thr His Cys Ser65 70 75 80Pro
Asp Leu Leu Phe Phe Leu Cys Ala Met Tyr Ala Pro Ile Cys Thr 85 90
95Ile Asp Phe Gln His Glu Pro Ile Lys Pro Cys Lys Ser Val Cys Glu
100 105 110Arg Ala Arg Gln Gly Cys Glu Pro Ile Leu Ile Lys Tyr Arg
His Ser 115 120 125Trp Pro Glu Asn Leu Ala Cys Glu Glu Leu Pro Val
Tyr Asp Arg Gly 130 135 140Val Cys Ile Ser Pro Glu Ala Ile Val Thr
Gly Arg Ala Gln Val Thr145 150 155 160Asp Lys Ala Ala Arg Ser
Thr
Leu Cys Pro Pro Cys Pro Ala Pro Glu 165 170 175Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 180 185 190Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 195 200 205Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 210 215
220Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn225 230 235 240Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp 245 250 255Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro 260 265 270Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu 275 280 285Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 290 295 300Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile305 310 315 320Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 325 330
335Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
340 345 350Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys 355 360 365Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu 370 375 380Ser Leu Ser Pro Gly Lys385
39087380PRTArtificial sequencesFRP4-Fc chimeric protein 87Met Phe
Leu Ser Ile Leu Val Ala Leu Cys Leu Trp Leu His Leu Ala1 5 10 15Leu
Gly Val Arg Gly Ala Pro Cys Glu Ala Val Arg Ile Pro Met Cys 20 25
30Arg His Met Pro Trp Asn Ile Thr Arg Met Pro Asn His Leu His His
35 40 45Ser Thr Gln Glu Asn Ala Ile Leu Ala Ile Glu Gln Tyr Glu Glu
Leu 50 55 60Val Asp Val Asn Cys Ser Ala Val Leu Arg Phe Phe Phe Cys
Ala Met65 70 75 80Tyr Ala Pro Ile Cys Thr Leu Glu Phe Leu His Asp
Pro Ile Lys Pro 85 90 95Cys Lys Ser Val Cys Gln Arg Ala Arg Asp Asp
Cys Glu Pro Leu Met 100 105 110Lys Met Tyr Asn His Ser Trp Pro Glu
Ser Leu Ala Cys Asp Glu Leu 115 120 125Pro Val Tyr Asp Arg Gly Val
Cys Ile Ser Pro Glu Ala Ile Val Thr 130 135 140Leu Glu Ser Gly Gly
Gly Gly Val Thr Asp Lys Thr His Thr Cys Pro145 150 155 160Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170
175Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
180 185 190Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe 195 200 205Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro 210 215 220Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr225 230 235 240Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295
300Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro305 310 315 320Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser 325 330 335Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln 340 345 350Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His 355 360 365Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 370 375 38088406PRTArtificial
sequencesFRP5-Fc chimeric protein 88Met Arg Ala Ala Ala Ala Ala Gly
Gly Val Arg Thr Ala Ala Leu Ala1 5 10 15Leu Leu Leu Gly Ala Leu His
Trp Ala Pro Ala Arg Cys Glu Glu Tyr 20 25 30Asp Tyr Tyr Gly Trp Gln
Ala Glu Pro Leu His Gly Arg Ser Tyr Ser 35 40 45Lys Pro Pro Gln Cys
Leu Asp Ile Pro Ala Asp Leu Pro Leu Cys His 50 55 60Thr Val Gly Tyr
Lys Arg Met Arg Leu Pro Asn Leu Leu Glu His Glu65 70 75 80Ser Leu
Ala Glu Val Lys Gln Gln Ala Ser Ser Trp Leu Pro Leu Leu 85 90 95Ala
Lys Arg Cys His Ser Asp Thr Gln Val Phe Leu Cys Ser Leu Phe 100 105
110Ala Pro Val Cys Leu Asp Arg Pro Ile Tyr Pro Cys Arg Ser Leu Cys
115 120 125Glu Ala Val Arg Ala Gly Cys Ala Pro Leu Met Glu Ala Tyr
Gly Phe 130 135 140Pro Trp Pro Glu Met Leu His Cys His Lys Phe Pro
Leu Asp Asn Asp145 150 155 160Leu Cys Ile Ala Val Gln Phe Gly His
Leu Trp Arg Ala Gln Val Thr 165 170 175Asp Lys Ala Ala Arg Ser Thr
Leu Cys Pro Pro Cys Pro Ala Pro Glu 180 185 190Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 195 200 205Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 210 215 220Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly225 230
235 240Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn 245 250 255Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp 260 265 270Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro 275 280 285Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu 290 295 300Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn305 310 315 320Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 325 330 335Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 340 345
350Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
355 360 365Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys 370 375 380Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu385 390 395 400Ser Leu Ser Pro Gly Lys
4058926DNAArtificial SequencePCR primer 89ctaagcactt acatgtggag
atactg 269024DNAArtificial SequencePCR primer 90acaggacaag
taaacaatga caca 249122DNAArtificial SequenceProbe sequence
91aacctgaggg cagaaagccc aa 229224DNAArtificial SequencePCR primer
92ttcccacact gtcttagaga actt 249327DNAArtificial SequencePCR primer
93ttctgagtat ctacattcaa ttgcttt 279431DNAArtificial SequenceProbe
sequence 94aaacatgcaa atacatgtgg tttctggtga c 319521DNAArtificial
SequencePCR primer 95tgactcgctt agctgaaacc t 219622DNAArtificial
SequencePCR primer 96tgagcctggt cactttatct ga 229726DNAArtificial
SequenceProbe sequence 97tcagaaggtc ttcggaaatg ttgcct
269818DNAArtificial SequencePCR primer 98tgtggttgca gcctgtct
189921DNAArtificial SequencePCR primer 99aaccaactct gcattggatt c
2110038DNAArtificial SequenceProbe sequence 100cctttgaaat
tgttttactc tctgagtttt atatgctg 3810117DNAArtificial SequencePCR
primer 101ctccgtgtgt cgcctat 1710226DNAArtificial SequencePCR
primer 102catgacaaaa gtcattgagt acaaga 2610324DNAArtificial
SequenceProbe sequence 103ttgagggctc aagctttccc ttgt
2410418DNAArtificial SequencePCR primer 104gaacctcgcg ctgtctct
1810519DNAArtificial SequencePCR primer 105acttggtcct gcgattctg
1910621DNAArtificial SequenceProbe sequence 106agcctcaccg
agacgcaggt c 2110721DNAArtificial SequencePCR primer 107ggcaacaatt
tacctttgct t 2110823DNAArtificial SequencePCR primer 108gaaccaagtg
gaacttcatt aca 2310925DNAArtificial SequenceProbe sequence
109cgccaacctt aggattgtaa agccc 2511022DNAArtificial sequencePCR
primer 110cagatacaca ggacatggat ga 2211126DNAArtificial sequencePCR
primer 111caaagctttt gtaagagact taggat 2611235DNAArtificial
SequenceProbe sequence 112ccgtttcctc tagtttcttc ctgtagtact cctct
35113409PRTArtificial sequenceFrz8(1-173)-Fc chimeric protein
113Met Glu Trp Gly Tyr Leu Leu Glu Val Thr Ser Leu Leu Ala Ala Leu1
5 10 15Ala Val Leu Gln Arg Ser Ser Gly Ala Ala Ala Ala Ser Ala Lys
Glu 20 25 30Leu Ala Cys Gln Glu Ile Thr Val Pro Leu Cys Lys Gly Ile
Gly Tyr 35 40 45Asn Tyr Thr Tyr Met Pro Asn Gln Phe Asn His Asp Thr
Gln Asp Glu 50 55 60Ala Gly Leu Glu Val His Gln Phe Trp Pro Leu Val
Glu Ile Gln Cys65 70 75 80Ser Pro Asp Leu Lys Phe Phe Leu Cys Ser
Met Tyr Thr Pro Ile Cys 85 90 95Leu Glu Asp Tyr Lys Lys Pro Leu Pro
Pro Cys Arg Ser Val Cys Glu 100 105 110Arg Ala Lys Ala Gly Cys Ala
Pro Leu Met Arg Gln Tyr Gly Phe Ala 115 120 125Trp Pro Asp Arg Met
Arg Cys Asp Arg Leu Pro Glu Gln Gly Asn Pro 130 135 140Asp Thr Leu
Cys Met Asp Tyr Asn Arg Thr Asp Leu Thr Thr Ala Ala145 150 155
160Pro Ser Pro Pro Arg Arg Leu Pro Pro Pro Pro Pro Pro Leu Glu Ser
165 170 175Gly Gly Gly Gly Val Thr Asp Lys Thr His Thr Cys Pro Pro
Cys Pro 180 185 190Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys 195 200 205Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val 210 215 220Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr225 230 235 240Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 245 250 255Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 260 265 270Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 275 280
285Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
290 295 300Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met305 310 315 320Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro 325 330 335Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn 340 345 350Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu 355 360 365Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 370 375 380Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln385 390 395
400Lys Ser Leu Ser Leu Ser Pro Gly Lys 405114388PRTArtificial
sequenceFrz3-Fc chimeric protein 114Met Gly Gly Thr Ala Ala Arg Leu
Gly Ala Val Ile Leu Phe Val Val1 5 10 15Ile Val Gly Leu His Gly Val
Arg Gly Lys Ile Asp Ala Arg Gly His 20 25 30Ser Leu Phe Ser Cys Glu
Pro Ile Thr Leu Arg Met Cys Gln Asp Leu 35 40 45Pro Tyr Asn Thr Thr
Phe Met Pro Asn Leu Leu Asn His Tyr Asp Gln 50 55 60Gln Thr Ala Ala
Leu Ala Met Glu Pro Phe His Pro Met Val Asn Leu65 70 75 80Asp Cys
Ser Arg Asp Phe Arg Pro Phe Leu Cys Ala Leu Tyr Ala Pro 85 90 95Ile
Cys Met Glu Tyr Gly Arg Val Thr Leu Pro Cys Arg Arg Leu Cys 100 105
110Gln Arg Ala Tyr Ser Glu Cys Ser Lys Leu Met Glu Met Phe Gly Val
115 120 125Pro Trp Pro Glu Asp Met Glu Cys Ser Arg Phe Pro Asp Cys
Asp Glu 130 135 140Pro Tyr Pro Arg Leu Val Asp Leu Leu Glu Ser Gly
Gly Gly Gly Val145 150 155 160Thr Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu 165 170 175Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu 180 185 190Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 195 200 205His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 210 215 220Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr225 230
235 240Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn 245 250 255Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro 260 265 270Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln 275 280 285Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val 290 295 300Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val305 310 315 320Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 325 330 335Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 340 345
350Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
355 360 365Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 370 375 380Ser Pro Gly Lys3851151410DNAArtificial
sequenceFrz1-Fc construct 115atggggggga ctgccgccag gttgggggcc
gtgattttgt ttgtcgtcat agtgggcctc 60catggggtcc gcggcaaaat cgatgaggag
ggcagcgggg acgccggtgg ccgccgccgc 120ccgccagttg acccccggcg
attggcgcgc cagctgctgc tgctgctttg gctgctggag 180gctccgctgc
tgctgggggt ccgggcccag gcggcgggcc aggggccagg ccaggggccc
240gggccggggc agcaaccgcc gccgccgcct cagcagcaac agagcgggca
gcagtacaac 300ggcgagcggg gcatctccgt cccggaccac ggctattgcc
agcccatctc catcccgctg 360tgcacggaca tcgcgtacaa ccagaccatc
atgcccaacc tgctgggcca cacgaaccag 420gaggacgcgg gcctggaggt
gcaccagttc taccctctag tgaaagtgca gtgttccgct 480gagctcaagt
tcttcctgtg ctccatgtac gcgcccgtgt gcaccgtgct agagcaggcg
540ctgccgccct gccgctccct gtgcgagcgc gcgcgccagg gctgcgaggc
gctcatgaac 600aagttcggct tccagtggcc agacacgctc aagtgtgaga
agttcccggt gcacggcgcc 660ggcgagctgt gcgtgggcca gaacacgtcc
gacaaggctc gagggcgcgc ccaggtcacc 720gacaaagctg cgcgctctac
tctgtgccca ccgtgcccag cacctgaact cctgggggga 780ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
840gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 900tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
cgcgggagga gcagtacaac 960agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc aggactggct gaatggcaag 1020gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1080aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaagag
1140atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 1200gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 1260ctggactccg acggctcctt cttcctctac
agcaagctca ccgtggacaa gagcaggtgg 1320cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380cagaagagcc
tctccctgtc tccgggtaaa 14101161209DNAArtificial sequenceFrz2-Fc
construct 116atggggggga ctgccgccag gttgggggcc gtgattttgt ttgtcgtcat
agtgggcctc
60catggggtcc gcggcaaaat cgatgctcga ggggcccagt tccacgggga gaagggcatc
120tccatcccgg accacggctt ctgccagccc atctccatcc cgctgtgcac
ggacatcgcc 180tacaaccaga ccatcatgcc caaccttctg ggccacacga
accaggagga cgcaggccta 240gaggtgcacc agttctatcc gctggtgaag
gtgcagtgct cgcccgaact gcgcttcttc 300ctgtgctcca tgtacgcacc
cgtgtgcacc gtgctggaac aggccatccc gccgtgccgc 360tctatctgtg
agcgcgcgcg ccagggctgc gaagccctca tgaacaagtt cggttttcag
420tggcccgagc gcctgcgctg cgagcacttc ccgcgccacg gcgccgagca
gatctgcgtc 480ggccagaacc actccgagga cgggcgcgcc caggtcaccg
acaaagctgc gcgctctact 540ctgtgcccac cgtgcccagc acctgaactc
ctggggggac cgtcagtctt cctcttcccc 600ccaaaaccca aggacaccct
catgatctcc cggacccctg aggtcacatg cgtggtggtg 660gacgtgagcc
acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg
720cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg
tgtggtcagc 780gtcctcaccg tcctgcacca ggactggctg aatggcaagg
agtacaagtg caaggtctcc 840aacaaagccc tcccagcccc catcgagaaa
accatctcca aagccaaagg gcagccccga 900gaaccacagg tgtacaccct
gcccccatcc cgggaagaga tgaccaagaa ccaggtcagc 960ctgacctgcc
tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat
1020gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga
cggctccttc 1080ttcctctaca gcaagctcac cgtggacaag agcaggtggc
agcaggggaa cgtcttctca 1140tgctccgtga tgcatgaggc tctgcacaac
cactacacgc agaagagcct ctccctgtct 1200ccgggtaaa
12091171164DNAArtificial sequenceFrz3-Fc construct 117atggggggga
ctgccgccag gttgggggcc gtgattttgt ttgtcgtcat agtgggcctc 60catggggtcc
gcggcaaaat cgatgctcga ggccactccc tgttcagctg tgagccaatc
120acccttcgaa tgtgtcagga tctgccttac aataccacct tcatgcctaa
tctgctcaat 180cactacgacc agcaaactgc tgccttggca atggagccct
tccaccctat ggtcaacctg 240gactgtagca gggacttccg tccatttttg
tgtgccttgt atgcacctat ctgtatggag 300tacggccgcg tgacattgcc
ttgtaggagg ctgtgtcagc gagcttacag tgagtgcagc 360aaacttatgg
aaatgtttgg cgtcccctgg ccagaagata tggagtgcag tcggttccca
420gactgtgacg agccataccc tagactggtt gatctcctcg agtcaggagg
aggaggagtc 480accgacaaaa ctcacacatg cccaccgtgc ccagcacctg
aactcctggg gggaccgtca 540gtcttcctct tccccccaaa acccaaggac
accctcatga tctcccggac ccctgaggtc 600acatgcgtgg tggtggacgt
gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 660gacggcgtgg
aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg
720taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg
caaggagtac 780aagtgcaagg tctccaacaa agccctccca gcccccatcg
agaaaaccat ctccaaagcc 840aaagggcagc cccgagaacc acaggtgtac
accctgcccc catcccggga agagatgacc 900aagaaccagg tcagcctgac
ctgcctggtc aaaggcttct atcccagcga catcgccgtg 960gagtgggaga
gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac
1020tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag
gtggcagcag 1080gggaacgtct tctcatgctc cgtgatgcat gaggctctgc
acaaccacta cacgcagaag 1140agcctctccc tgtctccggg taaa
11641181191DNAArtificial sequenceFrz4-Fc construct 118atggggggga
ctgccgccag gttgggggcc gtgattttgt ttgtcgtcat agtgggcctc 60catggggtcc
gcggcaaaat cgatgctcga gggttcgggg acgaggaaga gcggcgctgc
120gaccccatcc gcatctccat gtgccagaac ctcggctaca acgtgaccaa
gatgcccaac 180ctggttgggc acgagctgca gacggacgcc gagctgcagc
tgacaacttt cacaccgctc 240atccagtacg gctgctccag ccagctgcag
ttcttccttt gttctgttta tgtgccaatg 300tgcacagaga agatcaacat
ccccattggc ccatgcggcg gcatgtgtct ttcagtcaag 360agacgctgtg
aacccgtcct gaaggaattt ggatttgcct ggccagagag tctgaactgc
420agcaaattcc caccacagaa cgaccacaac cacatgtgca tggaagggcc
aggtgatgaa 480gaggggcgcg cccaggtcac cgacaaagct gcgcgctcta
ctctgtgccc accgtgccca 540gcacctgaac tcctgggggg accgtcagtc
ttcctcttcc ccccaaaacc caaggacacc 600ctcatgatct cccggacccc
tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 660cctgaggtca
agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag
720ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac
cgtcctgcac 780caggactggc tgaatggcaa ggagtacaag tgcaaggtct
ccaacaaagc cctcccagcc 840cccatcgaga aaaccatctc caaagccaaa
gggcagcccc gagaaccaca ggtgtacacc 900ctgcccccat cccgggaaga
gatgaccaag aaccaggtca gcctgacctg cctggtcaaa 960ggcttctatc
ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac
1020tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta
cagcaagctc 1080accgtggaca agagcaggtg gcagcagggg aacgtcttct
catgctccgt gatgcatgag 1140gctctgcaca accactacac gcagaagagc
ctctccctgt ctccgggtaa a 11911191188DNAArtificial sequenceFrz5-Fc
construct 119atggggggga ctgccgccag gttgggggcc gtgattttgt ttgtcgtcat
agtgggcctc 60catggggtcc gcggcaaaat cgatgctcga ggggcgtcca aggccccggt
gtgccaggaa 120atcacggtgc ccatgtgccg cggcatcggc tacaacctga
cgcacatgcc caaccagttc 180aaccacgaca cgcaggacga ggcgggcctg
gaggtgcacc agttctggcc gctggtggag 240atccaatgct cgccggacct
gcgcttcttc ctatgctcta tgtacacgcc catctgtctg 300cccgactacc
acaagccgct gccgccctgc cgctcggtgt gcgagcgcgc caaggccggc
360tgctcgccgc tgatgcgcca gtacggcttc gcctggcccg agcgcatgag
ctgcgaccgc 420ctcccggtgc tgggccgcga cgccgaggtc ctctgcatgg
attacaaccg cagcgaggcc 480gggcgcgccc aggtcaccga caaagctgcg
cgctctactc tgtgcccacc gtgcccagca 540cctgaactcc tggggggacc
gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 600atgatctccc
ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct
660gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa
gacaaagccg 720cgggaggagc agtacaacag cacgtaccgt gtggtcagcg
tcctcaccgt cctgcaccag 780gactggctga atggcaagga gtacaagtgc
aaggtctcca acaaagccct cccagccccc 840atcgagaaaa ccatctccaa
agccaaaggg cagccccgag aaccacaggt gtacaccctg 900cccccatccc
gggaagagat gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc
960ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga
gaacaactac 1020aagaccacgc ctcccgtgct ggactccgac ggctccttct
tcctctacag caagctcacc 1080gtggacaaga gcaggtggca gcaggggaac
gtcttctcat gctccgtgat gcatgaggct 1140ctgcacaacc actacacgca
gaagagcctc tccctgtctc cgggtaaa 11881201119DNAArtificial
sequenceFrz6-Fc construct 120atggaaatgt ttacattttt gttgacgtgt
atttttctac ccctcctaag agggcacagt 60ctcttcacct gtgaaccaat tactgttccc
agatgtatga aaatggccta caacatgacg 120tttttcccta atctgatggg
tcattatgac cagagtattg ccgcggtgga aatggagcat 180tttcttcctc
tcgcaaatct ggaatgttca ccaaacattg aaactttcct ctgcaaagca
240tttgtaccaa cctgcataga acaaattcat gtggttccac cttgtcgtaa
actttgtgag 300aaagtatatt ctgattgcaa aaaattaatt gacacttttg
ggatccgatg gcctgaggag 360cttgaatgtg acagattaca atactgtgat
gagactgttc ctgtaacttt tctcgagtca 420ggaggaggag gagtcaccga
caaaactcac acatgcccac cgtgcccagc acctgaactc 480ctggggggac
cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc
540cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc
tgaggtcaag 600ttcaactggt acgtggacgg cgtggaggtg cataatgcca
agacaaagcc gcgggaggag 660cagtacaaca gcacgtaccg tgtggtcagc
gtcctcaccg tcctgcacca ggactggctg 720aatggcaagg agtacaagtg
caaggtctcc aacaaagccc tcccagcccc catcgagaaa 780accatctcca
aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc
840cgggaagaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg
cttctatccc 900agcgacatcg ccgtggagtg ggagagcaat gggcagccgg
agaacaacta caagaccacg 960cctcccgtgc tggactccga cggctccttc
ttcctctaca gcaagctcac cgtggacaag 1020agcaggtggc agcaggggaa
cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1080cactacacgc
agaagagcct ctccctgtct ccgggtaaa 11191211212DNAArtificial
sequenceFrz7-Fc construct 121atgcgggacc ccggcgcggc cgctccgctt
tcgtccctgg gcctctgtgc cctggtgctg 60gcgctgctgg gcgcactgtc cgcgggcgcc
ggggcgcagc cgtaccacgg agagaagggc 120atctccgtgc cggaccacgg
cttctgccag cccatctcca tcccgctgtg cacggacatc 180gcctacaacc
agaccatcct gcccaacctg ctgggccaca cgaaccaaga ggacgcgggc
240ctcgaggtgc accagttcta cccgctggtg aaggtgcagt gttctcccga
actccgcttt 300ttcttatgct ccatgtatgc gcccgtgtgc accgtgctcg
atcaggccat cccgccgtgt 360cgttctctgt gcgagcgcgc ccgccagggc
tgcgaggcgc tcatgaacaa gttcggcttc 420cagtggcccg agcggctgcg
ctgcgagaac ttcccggtgc acggtgcggg cgagatctgc 480gtgggccaga
acacgtcgga cggcctcgag tcaggaggag gaggagtcac cgacaaaact
540cacacatgcc caccgtgccc agcacctgaa ctcctggggg gaccgtcagt
cttcctcttc 600cccccaaaac ccaaggacac cctcatgatc tcccggaccc
ctgaggtcac atgcgtggtg 660gtggacgtga gccacgaaga ccctgaggtc
aagttcaact ggtacgtgga cggcgtggag 720gtgcataatg ccaagacaaa
gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 780agcgtcctca
ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc
840tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa
agggcagccc 900cgagaaccac aggtgtacac cctgccccca tcccgggaag
agatgaccaa gaaccaggtc 960agcctgacct gcctggtcaa aggcttctat
cccagcgaca tcgccgtgga gtgggagagc 1020aatgggcagc cggagaacaa
ctacaagacc acgcctcccg tgctggactc cgacggctcc 1080ttcttcctct
acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc
1140tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag
cctctccctg 1200tctccgggta aa 12121221176DNAArtificial
sequenceFrz8-Fc chimeric construct 122atggagtggg gttacctgtt
ggaagtgacc tcgctcctag ccgccttggc ggtgctacag 60cgctctagcg gcgctgccgc
ggcttcggcc aaggagctgg cgtgccaaga gatcacggtg 120ccgttgtgca
aaggcatcgg ttacaactac acttacatgc ccaaccagtt caaccacgac
180acgcaagatg aggcgggcct agaggtgcac cagttttggc cgctggtgga
gatacagtgc 240tccccggacc tcaagttctt tctgtgtagc atgtacacgc
ccatctgcct ggaggactac 300aagaagcctc tgccgccttg tcgctctgtg
tgtgaacgcg ccaaggccgg ctgcgcgccg 360ctcatgcgcc agtacggctt
tgcttggcct gaccgcatgc gctgcgatcg gttgccggag 420cagggcaacc
cggacactct gtgcatggac tacaaccgca ccgacctcga gtcaggagga
480ggaggagtca ccgacaaaac tcacacatgc ccaccgtgcc cagcacctga
actcctgggg 540ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca
ccctcatgat ctcccggacc 600cctgaggtca catgcgtggt ggtggacgtg
agccacgaag accctgaggt caagttcaac 660tggtacgtgg acggcgtgga
ggtgcataat gccaagacaa agccgcggga ggagcagtac 720aacagcacgt
accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc
780aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga
gaaaaccatc 840tccaaagcca aagggcagcc ccgagaacca caggtgtaca
ccctgccccc atcccgggaa 900gagatgacca agaaccaggt cagcctgacc
tgcctggtca aaggcttcta tcccagcgac 960atcgccgtgg agtgggagag
caatgggcag ccggagaaca actacaagac cacgcctccc 1020gtgctggact
ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg
1080tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca
caaccactac 1140acgcagaaga gcctctccct gtctccgggt aaatga
11761231188DNAArtificial sequenceFrz9-Fc construct 123atggccgtgg
cgcctctgcg gggggcgctg ctgctgtggc agctgctggc ggcgggcggc 60gcggcactgg
agatcggccg cttcgacccg gagcgcgggc gcggggctgc gccgtgccag
120gcggtggaga tccccatgtg ccgcggcatc ggctacaacc tgacccgcat
gcccaacctg 180ctgggccaca cgtcgcaggg cgaggcggct gccgagctag
cggagttcgc gccgctggtg 240cagtacggct gccacagcca cctgcgcttc
ttcctgtgct cgctctacgc gcccatgtgc 300accgaccagg tctcgacgcc
cattcccgcc tgccggccca tgtgcgagca ggcgcgcctg 360cgctgcgcgc
ccatcatgga gcagttcaac ttcggctggc cggactcgct cgactgcgcc
420cggctgccca cgcgcaacga cccgcacgcg ctgtgcatgg aggcgcccga
gaacgccacg 480ctcgagtcag gaggaggagg agtcaccgac aaaactcaca
catgcccacc gtgcccagca 540cctgaactcc tggggggacc gtcagtcttc
ctcttccccc caaaacccaa ggacaccctc 600atgatctccc ggacccctga
ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 660gaggtcaagt
tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg
720cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt
cctgcaccag 780gactggctga atggcaagga gtacaagtgc aaggtctcca
acaaagccct cccagccccc 840atcgagaaaa ccatctccaa agccaaaggg
cagccccgag aaccacaggt gtacaccctg 900cccccatccc gggaagagat
gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 960ttctatccca
gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac
1020aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag
caagctcacc 1080gtggacaaga gcaggtggca gcaggggaac gtcttctcat
gctccgtgat gcatgaggct 1140ctgcacaacc actacacgca gaagagcctc
tccctgtctc cgggtaaa 11881241173DNAArtificial sequenceFrz10-Fc
construct 124atgcagcgcc cgggcccccg cctgtggctg gtcctgcagg tgatgggctc
gtgcgccgcc 60atcagctcca tggacatgga gcgcccgggc gacggcaaat gccagcccat
cgagatcccg 120atgtgcaagg acatcggcta caacatgact cgtatgccca
acctgatggg ccacgagaac 180cagcgcgagg cagccatcca gttgcacgag
ttcgcgccgc tggtggagta cggctgccac 240ggccacctcc gcttcttcct
gtgctcgctg tacgcgccga tgtgcaccga gcaggtctct 300acccccatcc
ccgcctgccg ggtcatgtgc gagcaggccc ggctcaagtg ctccccgatt
360atggagcagt tcaacttcaa gtggcccgac tccctggact gccggaaact
ccccaacaag 420aacgacccca actacctgtg catggaggcg cccaacaacg
gctcgctcga gtcaggagga 480ggaggagtca ccgacaaaac tcacacatgc
ccaccgtgcc cagcacctga actcctgggg 540ggaccgtcag tcttcctctt
ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 600cctgaggtca
catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac
660tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga
ggagcagtac 720aacagcacgt accgtgtggt cagcgtcctc accgtcctgc
accaggactg gctgaatggc 780aaggagtaca agtgcaaggt ctccaacaaa
gccctcccag cccccatcga gaaaaccatc 840tccaaagcca aagggcagcc
ccgagaacca caggtgtaca ccctgccccc atcccgggaa 900gagatgacca
agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac
960atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac
cacgcctccc 1020gtgctggact ccgacggctc cttcttcctc tacagcaagc
tcaccgtgga caagagcagg 1080tggcagcagg ggaacgtctt ctcatgctcc
gtgatgcatg aggctctgca caaccactac 1140acgcagaaga gcctctccct
gtctccgggt aaa 11731251230DNAArtificial sequencesFRP1-Fc construct
125atgggcatcg ggcgcagcga ggggggccgc cgcggggcag ccctgggcgt
gctgctggcg 60ctgggcgcgg cgcttctggc cgtgggctcg gccagcgagt acgactacgt
gagcttccag 120tcggacatcg gcccgtacca gagcgggcgc ttctacacca
agccacctca gtgcgtggac 180atccccgcgg acctgcggct gtgccacaac
gtgggctaca agaagatggt gctgcccaac 240ctgctggagc acgagaccat
ggcggaggtg aagcagcagg ccagcagctg ggtgcccctg 300ctcaacaaga
actgccacgc cggcacccag gtcttcctct gctcgctctt cgcgcccgtc
360tgcctggacc ggcccatcta cccgtgtcgc tggctctgcg aggccgtgcg
cgactcgtgc 420gagccggtca tgcagttctt cggcttctac tggcccgaga
tgcttaagtg tgacaagttc 480cccgaggggg acgtctgcat cgccatgacg
ccgcccaatg cctggcgcgc ccaggtcacc 540gacaaagctg cgcgctctac
tctgtgccca ccgtgcccag cacctgaact cctgggggga 600ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
660gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 720tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
cgcgggagga gcagtacaac 780agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc aggactggct gaatggcaag 840gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 900aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaagag
960atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 1020gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 1080ctggactccg acggctcctt cttcctctac
agcaagctca ccgtggacaa gagcaggtgg 1140cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1200cagaagagcc
tctccctgtc tccgggtaaa 12301261188DNAArtificial sequencesFRP2-Fc
construct 126atgctgcagg gccctggctc gctgctgctg ctcttcctcg cctcgcactg
ctgcctgggc 60tcggcgcgcg ggctcttcct ctttggccag cccgacttct cctacaagcg
cagcaattgc 120aagcccatcc ctgccaacct gcagctgtgc cacggcatcg
aataccagaa catgcggctg 180cccaacctgc tgggccacga gaccatgaag
gaggtgctgg agcaggccgg cgcttggatc 240ccgctggtca tgaagcagtg
ccacccggac accaagaagt tcctgtgctc gctcttcgcc 300cccgtctgcc
tcgatgacct agacgagacc atccagccat gccactcgct ctgcgtgcag
360gtgaaggacc gctgcgcccc ggtcatgtcc gccttcggct tcccctggcc
cgacatgctt 420gagtgcgacc gtttccccca ggacaacgac ctttgcatcc
ccctcgctag cagcgaccac 480tggcgcgccc aggtcaccga caaagctgcg
cgctctactc tgtgcccacc gtgcccagca 540cctgaactcc tggggggacc
gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 600atgatctccc
ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct
660gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa
gacaaagccg 720cgggaggagc agtacaacag cacgtaccgt gtggtcagcg
tcctcaccgt cctgcaccag 780gactggctga atggcaagga gtacaagtgc
aaggtctcca acaaagccct cccagccccc 840atcgagaaaa ccatctccaa
agccaaaggg cagccccgag aaccacaggt gtacaccctg 900cccccatccc
gggaagagat gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc
960ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga
gaacaactac 1020aagaccacgc ctcccgtgct ggactccgac ggctccttct
tcctctacag caagctcacc 1080gtggacaaga gcaggtggca gcaggggaac
gtcttctcat gctccgtgat gcatgaggct 1140ctgcacaacc actacacgca
gaagagcctc tccctgtctc cgggtaaa 11881271170DNAArtificial
sequencesFRP3-Fc construct 127atggggggga ctgccgccag gttgggggcc
gtgattttgt ttgtcgtcat agtgggcctc 60catggggtcc gcggcaaaat cgatgctcga
ggggcagcct gtgagcccgt ccgcatcccc 120ctgtgcaagt ccctgccctg
gaacatgact aagatgccca accacctgca ccacagcact 180caggccaacg
ccatcctggc catcgagcag ttcgaaggtc tgctgggcac ccactgcagc
240cccgatctgc tcttcttcct ctgtgccatg tacgcgccca tctgcaccat
tgacttccag 300cacgagccca tcaagccctg taagtctgtg tgcgagcggg
cccggcaggg ctgtgagccc 360atactcatca agtaccgcca ctcgtggccg
gagaacctgg cctgcgagga gctgccagtg 420tacgacaggg gcgtgtgcat
ctctcccgag gccatcgtta ctgggcgcgc ccaggtcacc 480gacaaagctg
cgcgctctac tctgtgccca ccgtgcccag cacctgaact cctgggggga
540ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc
ccggacccct 600gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc
ctgaggtcaa gttcaactgg 660tacgtggacg gcgtggaggt gcataatgcc
aagacaaagc cgcgggagga gcagtacaac 720agcacgtacc gtgtggtcag
cgtcctcacc gtcctgcacc aggactggct gaatggcaag 780gagtacaagt
gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc
840aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc
ccgggaagag 900atgaccaaga accaggtcag cctgacctgc ctggtcaaag
gcttctatcc cagcgacatc 960gccgtggagt gggagagcaa tgggcagccg
gagaacaact acaagaccac gcctcccgtg 1020ctggactccg acggctcctt
cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1080cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
1140cagaagagcc
tctccctgtc tccgggtaaa 11701281140DNAArtificial sequencesFRP4-Fc
construct 128atgttcctct ccatcctagt ggcgctgtgc ctgtggctgc acctggcgct
gggcgtgcgc 60ggcgcgccct gcgaggcggt gcgcatccct atgtgccggc acatgccctg
gaacatcacg 120cggatgccca accacctgca ccacagcacg caggagaacg
ccatcctggc catcgagcag 180tacgaggagc tggtggacgt gaactgcagc
gccgtgctgc gcttcttctt ctgtgccatg 240tacgcgccca tttgcaccct
ggagttcctg cacgacccta tcaagccgtg caagtcggtg 300tgccaacgcg
cgcgcgacga ctgcgagccc ctcatgaaga tgtacaacca cagctggccc
360gaaagcctgg cctgcgacga gctgcctgtc tatgaccgtg gcgtgtgcat
ttcgcctgaa 420gccatcgtca cgctcgagtc aggaggagga ggagtcaccg
acaaaactca cacatgccca 480ccgtgcccag cacctgaact cctgggggga
ccgtcagtct tcctcttccc cccaaaaccc 540aaggacaccc tcatgatctc
ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 600cacgaagacc
ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc
660aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag
cgtcctcacc 720gtcctgcacc aggactggct gaatggcaag gagtacaagt
gcaaggtctc caacaaagcc 780ctcccagccc ccatcgagaa aaccatctcc
aaagccaaag ggcagccccg agaaccacag 840gtgtacaccc tgcccccatc
ccgggaagag atgaccaaga accaggtcag cctgacctgc 900ctggtcaaag
gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg
960gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt
cttcctctac 1020agcaagctca ccgtggacaa gagcaggtgg cagcagggga
acgtcttctc atgctccgtg 1080atgcatgagg ctctgcacaa ccactacacg
cagaagagcc tctccctgtc tccgggtaaa 11401291218DNAArtificial
sequencesFRP5-Fc construct 129atgcgggcgg cggcggcggc ggggggcgtg
cggacggccg cgctggcgct gctgctgggg 60gcgctgcact gggcgccggc gcgctgcgag
gagtacgact actatggctg gcaggccgag 120ccgctgcacg gccgctccta
ctccaagccg ccgcagtgcc ttgacatccc tgccgacctg 180ccgctctgcc
acacggtggg ctacaagcgc atgcggctgc ccaacctgct ggagcacgag
240agcctggccg aagtgaagca gcaggcgagc agctggctgc cgctgctggc
caagcgctgc 300cactcggata cgcaggtctt cctgtgctcg ctctttgcgc
ccgtctgtct cgaccggccc 360atctacccgt gccgctcgct gtgcgaggcc
gtgcgcgccg gctgcgcgcc gctcatggag 420gcctacggct tcccctggcc
tgagatgctg cactgccaca agttccccct ggacaacgac 480ctctgcatcg
ccgtgcagtt cgggcacctg tggcgcgccc aggtcaccga caaagctgcg
540cgctctactc tgtgcccacc gtgcccagca cctgaactcc tggggggacc
gtcagtcttc 600ctcttccccc caaaacccaa ggacaccctc atgatctccc
ggacccctga ggtcacatgc 660gtggtggtgg acgtgagcca cgaagaccct
gaggtcaagt tcaactggta cgtggacggc 720gtggaggtgc ataatgccaa
gacaaagccg cgggaggagc agtacaacag cacgtaccgt 780gtggtcagcg
tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc
840aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa
agccaaaggg 900cagccccgag aaccacaggt gtacaccctg cccccatccc
gggaagagat gaccaagaac 960caggtcagcc tgacctgcct ggtcaaaggc
ttctatccca gcgacatcgc cgtggagtgg 1020gagagcaatg ggcagccgga
gaacaactac aagaccacgc ctcccgtgct ggactccgac 1080ggctccttct
tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac
1140gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca
gaagagcctc 1200tccctgtctc cgggtaaa 1218
* * * * *