U.S. patent application number 10/999523 was filed with the patent office on 2005-04-28 for methods of inhibiting osteoclastogenesis.
This patent application is currently assigned to Immunex Corporation. Invention is credited to Anderson, Dirk M., Galibert, Laurent J..
Application Number | 20050089522 10/999523 |
Document ID | / |
Family ID | 26772773 |
Filed Date | 2005-04-28 |
United States Patent
Application |
20050089522 |
Kind Code |
A1 |
Anderson, Dirk M. ; et
al. |
April 28, 2005 |
Methods of inhibiting osteoclastogenesis
Abstract
Provided here are methods of inhibiting RANKL-induced
osteoclastogenesis in patients suffering from specific types of
cancer.
Inventors: |
Anderson, Dirk M.; (Seattle,
WA) ; Galibert, Laurent J.; (Seattle, WA) |
Correspondence
Address: |
IMMUNEX CORPORATION
PATENT OPERATIONS/MS 27-4-A
ONE AMGEN CENTER DRIVE
THOUSAND OAKS
CA
91320-1799
US
|
Assignee: |
Immunex Corporation
Thousand Oaks
CA
|
Family ID: |
26772773 |
Appl. No.: |
10/999523 |
Filed: |
November 30, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10999523 |
Nov 30, 2004 |
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09705985 |
Nov 3, 2000 |
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09705985 |
Nov 3, 2000 |
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PCT/US99/10588 |
May 13, 1999 |
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60110836 |
Dec 3, 1998 |
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60085487 |
May 14, 1998 |
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Current U.S.
Class: |
424/146.1 |
Current CPC
Class: |
C07K 14/70575 20130101;
A61K 2039/505 20130101; C07K 2319/73 20130101; A61K 38/00 20130101;
A61P 19/08 20180101; A61P 43/00 20180101; A61P 35/04 20180101; C07K
2319/00 20130101; A61P 19/10 20180101; C07K 14/70578 20130101; Y10S
530/866 20130101; C07K 2319/30 20130101 |
Class at
Publication: |
424/146.1 |
International
Class: |
A61K 039/395 |
Claims
We claim:
1. A method of inhibiting RANK-induced osteoclastogenesis in a
cancer patient in need thereof, said method comprising
administering to said patient a monoclonal antibody that
specifically binds a RANKL polypeptide comprising amino acids
70-317 of SEQ ID NO:8, wherein said patient suffers from bone
cancer.
2. A method of inhibiting RANK-induced osteoclastogenesis in a
cancer patient in need thereof, said method comprising
administering to said patient a monoclonal antibody that
specifically binds a RANKL polypeptide comprising amino acids
70-317 of SEQ ID NO:8, wherein said patient suffers from multiple
myeloma.
3. A method of inhibiting RANK-induced osteoclastogenesis in a
cancer patient in need thereof, said method comprising
administering to said patient a monoclonal antibody that
specifically binds a RANKL polypeptide comprising amino acids
70-317 of SEQ ID NO:8, wherein said patient suffers from
melanoma.
4. A method of inhibiting RANK-induced osteoclastogenesis in a
cancer patient in need thereof, said method comprising
administering to said patient a monoclonal antibody that
specifically binds a RANKL polypeptide comprising amino acids
70-317 of SEQ ID NO:8, wherein said patient suffers from breast
cancer.
5. A method of inhibiting RANK-induced osteoclastogenesis in a
cancer patient in need thereof, said method comprising
administering to said patient an antibody that specifically binds a
RANKL polypeptide comprising amino acids 70-317 of SEQ ID NO:8,
wherein said patient suffers from squamous cell carcinoma.
6. A method of inhibiting RANK-induced osteoclastogenesis in a
cancer patient in need thereof, said method comprising
administering to said patient an antibody that specifically binds a
RANKL polypeptide comprising amino acids 70-317 of SEQ ID NO:8,
wherein said patient suffers from lung cancer.
7. A method of inhibiting RANK-induced osteoclastogenesis in a
cancer patient in need thereof, said method comprising
administering to said patient an antibody that specifically binds a
RANKL polypeptide comprising amino acids 70-317 of SEQ ID NO:8,
wherein said patient suffers from prostate cancer. that
specifically binds a RANKL polypeptide comprising amino acids
70-317 of SEQ ID NO:8, wherein said patient suffers from
hematologic cancer.
9. A method of inhibiting RANK-induced osteoclastogenesis in a
cancer patient in need thereof, said method comprising
administering to said patient an antibody that specifically binds a
RANKL polypeptide comprising amino acids 70-317 of SEQ ID NO:8,
wherein said patient suffers from head and neck cancer.
10. A method of inhibiting RANK-induced osteoclastogenesis in a
cancer patient in need thereof, said method comprising
administering to said patient an antibody that specifically binds a
RANKL polypeptide comprising amino acids 70-317 of SEQ ID NO:8,
wherein said patient suffers from renal cancer.
Description
[0001] This application is a continuation of U.S. patent
application 09/705,985, filed Nov. 3, 2000, which is a continuation
of international patent application PCT/US99/10588, filed May 13,
1999, which claims benefit of U.S. provisional patent applications
60/110,836, filed Dec. 3, 1998 and 60/085,487 filed May 14, 1998.
The entire disclosures of these applications are relied upon and
incorporated by reference herein.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of
cytokine receptors, and more specifically to cytokine receptor
protein/ligand pairs having osteoclast regulatory activity.
BACKGROUND OF THE INVENTION
[0003] RANK (Receptor Activator of NF-.kappa.B) and its ligand
(RANKL) are a recently-described receptor/ligand pair that play an
important role in an immune response. The cloning of RANK and RANKL
is described in U.S. Ser. No. 08/996,139 and U.S. Ser. No.
08/995,659, respectively. It has recently been found that RANKL
binds to a protein referred to as osteoprotegerin (OPG), a member
of the Tumor Necrosis Factor Receptor (TNFR) family. Yasuda et al.
(Proc. Natl. Acad. Sci. 95:3597; 1998) expression cloned a ligand
for OPG, which they referred to as osteoclastogenesis inhibitory
factor. Their work was repeated by Lacey et al. (Cell 93:165;
1998). In both cases, the ligand they cloned turned out to be
identical to RANKL.
[0004] In osteoclastogenesis, the interaction of an osteoblast or
stromal cell with an osteoclast precursor leads to the
differentiation of the precursor into an osteoclast. OPG was known
to inhibit this differentiation. A model has been proposed in which
RANKL on the osteoblast or stromal cell surface interacts with a
specific receptor on an osteoclast progenitor surface, signaling a
differentiation event. OPG effectively blocks the interaction of
RANKL with a receptor on osteoclast progenitors in vitro, and has
been shown to ameliorate the effects of ovariectomy on bone-loss in
mice. However, OPG is also known to bind other ligands in the TNF
family, which may have a deleterious effect on the activities of
such ligands in vivo. Moreover, the presence of other ligands that
bind OPG in vivo may require high dosages of OPG to be administered
in order to have sufficient soluble OPG available to inhibit
osteoclastogenesis.
SUMMARY OF THE INVENTION
[0005] The present invention provides processes associated with the
use of a novel receptor, referred to as RANK (for receptor
activator of NF-.kappa.B), that is a member of the TNF receptor
superfamily. RANK is a Type I transmembrane protein having 616
amino acid residues, comprising an extracellular domain,
transmembrane region and cytoplasmic domain. RANK interacts with
various TNF Receptor Associated Factors (TRAFs); triggering of RANK
results in the upregulation of the transcription factor
NF-.kappa.B, a ubiquitous transcription factor that is most
extensively utilized in cells of the immune system.
[0006] Soluble forms of the receptor can be prepared and used to
interfere with signal transduction through membrane-bound RANK.
Inhibition of RANKL-mediated signal transduction will be useful in
ameliorating the effects of osteoclastogenesis and osteoclast
activity in disease conditions in which there is excess bone break
down. Examples of such conditions include osteoporosis, Paget's
disease, cancers that may metastasize to bone and induce bone
breakdown (i.e., multiple myeloma, breast cancer, some melanomas;
see also Mundy, C. Cancer Suppl. 80:1546; 1997), and cancers that
do not necessarily metastasize to bone, but result in hypercalcemia
and bone loss (e.g. squamous cell carcinomas).
[0007] Soluble forms of RANK comprise the extracellular domain of
RANK or a fragment thereof that binds RANKL. Fusion proteins of
RANK may be made to allow preparation of soluble RANK. Examples of
such fusion proteins include a RANK/Fc fusion protein, a fusion
protein of a zipper moiety (i.e., a leucine zipper), and various
tags that are known in the art. Other antagonists of the
interaction of RANK and RANKL (i.e., antibodies to RANKL, small
molecules) will also be useful in the inventive methods. These and
other aspects of the present invention will become evident upon
reference to the following detailed description of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0008] A novel partial cDNA insert with a predicted open reading
frame having some similarity to CD40 was identified and was used to
hybridize to colony blots generated from a dendritic cell (DC) cDNA
library containing full-length cDNAs. SEQ ID NO: 1 shows the
nucleotide and amino acid sequence of a predicted full-length
protein.
[0009] RANK is a member of the TNF receptor superfamily; it most
closely resembles CD40 in the extracellular region. RANK is
expressed on epithelial cells, some B cell lines, and on activated
T cells. However, its expression on activated T cells is late,
about four days after activation. This time course of expression
coincides with the expression of Fas, a known agent of apoptosis.
RANK may act as an anti-apoptotic signal, rescuing cells that
express RANK from apoptosis as CD40 is known to do. Alternatively,
RANK may confirm an apoptotic signal under the appropriate
circumstances, again similar to CD40. RANK and its ligand are
likely to play an integral role in regulation of the immune and
inflammatory response. The isolation of a DNA encoding RANK is
described in U.S. Ser. No. 08/996,139, filed Dec. 22 1997, the
disclosure of which is incorporated by reference herein. U.S. Ser.
No. 08/996,139 describes several forms of RANK that are useful in
the present invention.
[0010] Soluble RANK comprises the signal peptide and the
extracellular domain (residues 1 to 213 of SEQ ID NO:2) or a
fragment thereof. Alternatively, a different signal peptide can be
substituted for the native leader, beginning with residue 1 and
continuing through a residue selected from the group consisting of
amino acids 24 through 33 (inclusive) of SEQ ID NO:2. Moreover,
fragments of the extracellular domain will also provide soluble
forms of RANK.
[0011] Fragments can be prepared using known techniques to isolate
a desired portion of the extracellular region, and can be prepared,
for example, by comparing the extracellular region with those of
other members of the TNFR family (of which RANK is a member) and
selecting forms similar to those prepared for other family members.
Alternatively, unique restriction sites or PCR techniques that are
known in the art can be used to prepare numerous truncated forms
which can be expressed and analyzed for activity.
[0012] Other derivatives of the RANK proteins within the scope of
this invention include covalent or aggregative conjugates of the
proteins or their fragments with other proteins or polypeptides,
such as by synthesis in recombinant culture as N-terminal or
C-terminal fusions. For example, the conjugated peptide may be a
signal (or leader) polypeptide sequence at the N-terminal region of
the protein which co-translationally or post-translationally
directs transfer of the protein from its site of synthesis to its
site of function inside or outside of the cell membrane or wall
(e.g., the yeast .alpha.-factor leader).
[0013] Protein fusions can comprise peptides added to facilitate
purification or identification of RANK proteins and homologs (e.g.,
poly-His). The amino acid sequence of the inventive proteins can
also be linked to an identification peptide such as that described
by Hopp et al., Bio/Technology 6:1204 (1988; FLAG.TM.). Such a
highly antigenic peptide provides an epitope reversibly bound by a
specific monoclonal antibody, enabling rapid assay and facile
purification of expressed recombinant protein. The sequence of Hopp
et al. is also specifically cleaved by bovine mucosal enterokinase,
allowing removal of the peptide from the purified protein.
[0014] Fusion proteins further comprise the amino acid sequence of
a RANK linked to an immunoglobulin Fc region. An exemplary Fc
region is a human IgG.sub.1 having an amino acid sequence as set
forth in SEQ ID NO:3. Fragments of an Fc region may also be used,
as can Fc muteins. For example, certain residues within the hinge
region of an Fc region are critical for high affinity binding to
Fc.gamma.RI. Canfield and Morrison (J. Exp. Med. 173:1483; 1991)
reported that Leu.sub.(234) and Leu.sub.(235) were critical to high
affinity binding of IgG.sub.3 to Fc.gamma.RI present on U937 cells.
Similar results were obtained by Lund et al. (J. Immunol. 147:2657,
1991; Molecular Immunol. 29:53, 1991). Such mutations, alone or in
combination, can be made in an IgG.sub.1 Fc region to decrease the
affinity of IgG.sub.1 for FcR. Depending on the portion of the Fc
region used, a fusion protein may be expressed as a dimer, through
formation of interchain disulfide bonds. If the fusion proteins are
made with both heavy and light chains of an antibody, it is
possible to form a protein oligomer with as many as four RANK
regions.
[0015] In another embodiment, RANK proteins further comprise an
oligomerizing peptide such as a zipper domain. Leucine zippers were
originally identified in several DNA-binding proteins (Landschulz
et. al., Science 240:1759, 1988). Zipper domain is a term used to
refer to a conserved peptide domain present in these (and other)
proteins, which is responsible for multimerization of the proteins.
The zipper domain comprises a repetitive heptad repeat, with four
or five leucine, isoleucine or valine residues interspersed with
other amino acids. Examples of zipper domains are those found in
the yeast transcription factor GCN4 and a heat-stable DNA-binding
protein found in rat liver (C/EBP; Landschulz et al., Science
243:1681, 1989). Two nuclear transforming proteins, fos and jun,
also exhibit zipper domains, as does the gene product of the murine
proto-oncogene, c-myc (Landschulz et al., Science 240:1759, 1988).
The products of the nuclear oncogenes fos and jun comprise zipper
domains that preferentially form a heterodimer (O'Shea et al.,
Science 245:646, 1989; Turner and Tjian, Science 243:1689, 1989). A
preferred zipper moiety is that of SEQ ID NO:6 or a fragment
thereof. This and other zippers are disclosed in U.S. Pat. No.
5,716,805.
[0016] Other embodiments of useful proteins include RANK
polypeptides encoded by DNAs capable of hybridizing to the DNA of
SEQ ID NO:1 under moderately stringent conditions (prewashing
solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and
hybridization conditions of 50.degree. C., 5.times.SSC, overnight)
to the DNA sequences encoding RANK, or more preferably under
stringent conditions (for example, hybridization in 6.times.SSC at
63.degree. C. overnight; washing in 3.times.SSC at 55.degree. C.),
and other sequences which are degenerate to those which encode the
RANK. In one embodiment, RANK polypeptides are at least about 70%
identical in amino acid sequence to the amino acid sequence of
native RANK protein as set forth in SEQ ID NO:2 for human RANK and
NO:5 for murine RANK. In a preferred embodiment, RANK polypeptides
are at least about 80% identical in amino acid sequence to the
native form of RANK; most preferred polypeptides are those that are
at least about 90% identical to native RANK.
[0017] Percent identity may be determined using a computer program,
for example, the GAP computer program described by Devereux et al.
(Nucl. Acids Res. 12:387, 1984) and available from the University
of Wisconsin Genetics Computer Group (UWGCG). For fragments derived
from the RANK protein, the identity is calculated based on that
portion of the RANK protein that is present in the fragment
[0018] The biological activity of RANK analogs or muteins can be
determined by testing the ability of the analogs or muteins to bind
human RANKL (SEQ ID NOS:7 and 8), for example as described in the
Examples herein. Suitable assays include, for example, an enzyme
immunoassay or a dot blot, and assays that employ cells expressing
RANKL. Suitable assays also include, for example, inhibition
assays, wherein soluble RANK is used to inhibit the interaction of
RANKL with membrane-bound or solid-phase associated RANK (i.e.,
signal transduction assays). Such methods are well known in the
art.
[0019] Murine and human RANKL are Type 2 transmembrane proteins.
Murine RANKL contains a predicted 48 amino acid intracellular
domain, 21 amino acid transmembrane domain and 247 amino acid
extracellular domain. Human RANKL contains a predicted 47 amino
acid intracellular domain, 21 amino acid transmembrane domain and
249 amino acid extracellular domain.
[0020] RANKL and RANK are important factors in osteoclastogenesis.
RANK is expressed on osteoclasts and interacts with RANK ligand
(RANKL) to mediate the formation of osteoclast-like (OCL)
multinucleated cells. This was shown by treating mouse bone marrow
preparations with M-CSF (CSF-1) and soluble RANKL for 7 days in
culture. No additional osteoclastogenic hormones or factors were
necessary for the generation of the multinucleated cells. Neither
M-CSF nor RANKL alone led to the formation of OCL. The
multinucleated cells expressed tartrate resistant acid phosphatase
and were positive for [.sup.125]- calcitonin binding. The tyrosine
kinase c-src was highly expressed in multinucleated OCL and a
subset of mononuclear cells as demonstrated by immunofluorescence
microscopy. (See Example 2).
[0021] Purification of Recombinant RANK
[0022] Purified RANK, and homologs or analogs thereof are prepared
by culturing suitable host/vector systems to express the
recombinant translation products of the DNAs of the present
invention, which are then purified from culture media or cell
extracts. For example, supernatants from systems which secrete
recombinant protein into culture media can be first concentrated
using a commercially available protein concentration filter, for
example, an Amicon or Millipore Pellicon ultrafiltration unit.
[0023] Following the concentration step, the concentrate can be
applied to a suitable purification matrix. For example, a suitable
affinity matrix can comprise a counter structure protein or lectin
or antibody molecule bound to a suitable support. Alternatively, an
anion exchange resin can be employed, for example, a matrix or
substrate having pendant diethylaminoethyl (DEAE) groups. The
matrices can be acrylamide, agarose, dextran, cellulose or other
types commonly employed in protein purification. Alternatively, a
cation exchange step can be employed. Suitable cation exchangers
include various insoluble matrices comprising sulfopropyl or
carboxymethyl groups. Sulfopropyl groups are preferred. Gel
filtration chromatography also provides a means of purifying the
inventive proteins.
[0024] Affinity chromatography is a particularly preferred method
of purifying RANK and homologs thereof. For example, a RANK
expressed as a fusion protein comprising an immunoglobulin Fc
region can be purified using Protein A or Protein G affinity
chromatography. Moreover, a RANK protein comprising an
oligomerizing zipper domain may be purified on a resin comprising
an antibody specific to the oligomerizing zipper domain. Monoclonal
antibodies against the RANK protein may also be useful in affinity
chromatography purification, by utilizing methods that are
well-known in the art. A ligand may also be used to prepare an
affinity matrix for affinity purification of RANK.
[0025] Finally, one or more reversed-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify a RANK composition. Suitable
methods include those analogous to the method disclosed by Urdal et
al. (J. Chromatog. 296:171, 1984). Some or all of the foregoing
purification steps, in various combinations, can also be employed
to provide a homogeneous recombinant protein.
[0026] Recombinant protein produced in bacterial culture is usually
isolated by initial extraction from cell pellets, followed by one
or more concentration, salting-out, aqueous ion exchange or size
exclusion chromatography steps. Finally, high performance liquid
chromatography (HPLC) can be employed for final purification steps.
Microbial cells employed in expression of recombinant protein can
be disrupted by any convenient method, including freeze-thaw
cycling, sonication, mechanical disruption, or use of cell lysing
agents. Fermentation of yeast which express the inventive protein
as a secreted protein greatly simplifies purification.
[0027] Protein synthesized in recombinant culture is characterized
by the presence of cell components, including proteins, in amounts
and of a character which depend upon the purification steps taken
to recover the inventive protein from the culture. These components
ordinarily will be of yeast, prokaryotic or non-human higher
eukaryotic origin and preferably are present in innocuous
contaminant quantities, on the order of less than about 1 percent
by weight. Further, recombinant cell culture enables the production
of the inventive proteins free of other proteins which may be
normally associated with the proteins as they are found in nature
in the species of origin.
[0028] Uses and Administration of RANK Compositions
[0029] The present invention provides methods of using therapeutic
compositions comprising a protein and a suitable diluent and
carrier. These methods involve the use of therapeutic compositions
of RANK or soluble fragments of RANK for regulating an immune or
inflammatory response. Further included within the present
invention are methods for regulating osteoclast activity by
administering therapeutic compositions of RANK or soluble RANK
fragments to an individual in amounts sufficient to decrease excess
bone resorption. Typically, the individual is inflicted with excess
bone resorption and suffers from the effects of hypercalcemia, has
symptoms of hypercalcemia, or is suffering a disease that involves
excessive bone resorption. In addition to regulating osteoclast
activity, the methods described herein are applicable to inhibiting
osteoclast activity, regulating osteoclast generation and
inhibiting osteoclast generation in individuals inflicted with
excess bone resorption. In connection with the methods described
herein, the present invention contemplates the use of RANK in
conjunction with soluble cytokine receptors or cytokines, or other
osteoclast/osteoblast regulatory molecules.
[0030] In connection with the methods described herein, RANK ligand
(RANKL) on osteoblasts or stromal cells is known to interact with
RANK on osteoclast progenitor surfaces signaling an event that
leads to the differentiation of osteoclast precursors into
osteoclasts. (See Example 2 below.) Thus, RANK, and in particular
soluble forms of RANK, is useful for the inhibition of the
RANKL-mediated signal transduction that leads to the
differentiation of osteoclast precursors into osteoclasts. Soluble
forms of RANK are also useful for the regulation and inhibition of
osteoclast activity, e.g. bone resorption. By interfering with
osteoclast differentiation, soluble forms of RANK are useful in the
amelioration of the effects of osteoclastogenesis in disease
conditions in which there is excess bone break down. Such disease
conditions include Paget's disease, osteoporosis, and cancer. Many
cancers metastasize to bone and induce bone breakdown by locally
disrupting normal bone remodeling. Such cancers can be associated
with enhanced numbers of osteoclasts and enhanced amount of
osteoclastic bone resorption resulting in hypercalcemia. These
cancers include, but are not limited to, breast cancer, multiple
myeloma, melanomas, lung cancer, prostrate, hematologic, head and
neck, and renal. (See Guise et al. Endocrine Reviews, 19(1):18-54,
1998.) Soluble forms of RANK can be administered to such cancer
patients to disrupt the osteoclast differentiation pathway and
result in fewer numbers of osteoclast, less bone resorption, and
relief from the negative effects of hypercalcemia.
[0031] Other cancers do not metastasize to bone, but are known to
act systemically on bone to disrupt bone remodeling and result in
hypercalcemia. (See Guise et al. Endocrine Reviews, 19(1):18-54,
1998.) In accordance with this invention, RANKL has been found on
the surface of certain squamous cells that do not metastasize to
bone but are associated with hypercalcemia. (See Example 3 below)
Squamous cells that are associated with hypercalcemia also express
M-CSF (CSF-1), a cytokine that, together with RANKL, stimulates the
proliferation and differentiation of osteoclast precursors to
osteoclasts. In accordance with the present invention, it has been
discovered that M-CSF directly upregulates RANK on surfaces of
osteoclast precursors. When squamous cells release excessive
amounts of CSF-1, increased expression of RANK occurs on the
surfaces of osteoclast precursors. Thus, there is a higher
probability that RANK will interact with RANKL on osteoblasts or
stromal cells to produce increased numbers of osteoclasts,
resulting in an enhanced amount of bone break down and
hypercalcemia.
[0032] In addition to the ameliorating the effects of cancers that
metastasize to bone, the present invention provides methods for
ameliorating the systemic effects, e.g. hypercalcemia, of cancers
that are associated with excess osteoclast activity (e.g. squamous
cell carcinomas). Such methods include administering soluble forms
of RANK in amounts sufficient to interfere with the RANK/RANKL
signal transduction that leads to the differentiation of osteoclast
precursors into osteoclasts. Fewer osteoclasts lead to reduced bone
resorption and relief from the negative effects of
hypercalcemia.
[0033] For therapeutic use, purified protein is administered to an
individual, preferably a human, for treatment in a manner
appropriate to the indication. Thus, for example, RANK protein
compositions administered to regulate osteoclast function can be
given by bolus injection, continuous infusion, sustained release
from implants, or other suitable technique. Typically, a
therapeutic agent will be administered in the form of a composition
comprising purified RANK, in conjunction with physiologically
acceptable carriers, excipients or diluents. Such carriers will be
nontoxic to recipients at the dosages and concentrations
employed.
[0034] Ordinarily, the preparation of such protein compositions
entails combining the inventive protein with buffers, antioxidants
such as ascorbic acid, low molecular weight (less than about 10
residues) polypeptides, proteins, amino acids, carbohydrates
including glucose, sucrose or dextrins, chelating agents such as
EDTA, glutathione and other stabilizers and excipients. Neutral
buffered saline or saline mixed with conspecific serum albumin are
exemplary appropriate diluents. Preferably, product is formulated
as a lyophilizate using appropriate excipient solutions (e.g.,
sucrose) as diluents. Appropriate dosages can be determined in
trials. The amount and frequency of administration will depend, of
course, on such factors as the nature and severity of the
indication being treated, the desired response, the condition of
the patient, and so forth.
[0035] Soluble forms of RANK and other RANK antagonists such as
antagonistic monoclonal antibodies can be administered for the
purpose of inhibiting RANK-induced osteoclastogenesis. It is
desirable to inhibit osteoclastogenesis in various disease states
in which excess bone loss occurs. Examples include osteoporosis,
Pagett's disease, and various cancers. Various animal models of
these diseases are known in the art; accordingly, it is a matter of
routine experimentation to determine optimal dosages and routes of
administration of soluble RANK, first in an animal model and then
in human clinical trials.
[0036] The following examples are offered by way of illustration,
and not by way of limitation. Those skilled in the art will
recognize that variations of the invention embodied in the examples
can be made, especially in light of the teachings of the various
references cited herein, the disclosures of which are incorporated
by reference.
EXAMPLE 1
[0037] This example describes a plate binding assay useful in
comparing the ability of various ligands to bind receptors. The
assay is performed essentially as described in Smith et al.,
Virology 236:316 (1997). Briefly, 96-well microtiter plates are
coated with an antibody to human Fc (i.e., polyclonal goat anti
human Fc). Receptor/Fc fusion proteins are then added, and after
incubation, the plates are washed. Serial dilutions of the ligands
are then added. The ligands may be directly labeled (i.e., with
.sup.125I), or a detecting reagent that is radioactively labeled
may be used. After incubation, the plates are washed, specifically
bound ligands are released, and the amount of ligand bound
quantified.
[0038] Using this method, RANK/Fc and OPG/Fc were bound to 96-well
plates. In an indirect method, a RANKL/zipper fusion is detected
using a labeled antibody to the zipper moiety. It was found that
human OPG/Fc binds mRANKL at 0.05 nM, and human RANK/Fc binds
mRANKL at 0.1 nM. These values indicate similar binding affinities
of OPG and RANK for RANKL, confirming the utility of RANK as an
inhibitor of osteoclast activity in a manner similar to OPG.
EXAMPLE 2
[0039] The following describes the formation of osteoclast like
cells from bone marrow cell cultures using a soluble RANKL in the
form of soluble RANKL/leucine zipper fusion protein (RANKL LZ).
[0040] Using RANKL LZ at 1 .mu.g/ml, osteoclasts were generated
from murine bone marrow (BM) in the presence of CSF-1. These
osteoclasts are formed by the fusion of macrophage-like cells and
are characterized by their TRAP (tartrate-resistant acid
phosphatase) positivity. No TRAP.sup.+ cells were seen in cultures
containing CSF-1 alone or in cultures containing CSF-1 and TRAIL LZ
(a control for the soluble RANKL LZ). Even though human and monkey
bone marrow contains more contaminating fibroblasts than murine
bone marrow, osteoclasts were generated from murine and monkey bone
marrow with the combination of CSF-1 and soluble RANKL LZ. In a
dose-response study using murine bone marrow and suboptimal amounts
of CSF-1 (40ng/ml), the effects of soluble RANKL LZ plateaued at
about 100 ng/ml.
[0041] The effect of soluble RANKL LZ on proliferation of cells was
studied in the same cultures using Alamar Blue. After 5 days, the
proliferative response was lower in cultures containing CSF-1 and
RANKL LZ than in those containing CSF-1 alone. The supports the
observation that soluble RANKL LZ is inducing osteoclast
differentiation. When CSF-1 and RANKL LZ are washed out of murine
BM cultures at day 7 or 8, cells do not survive if they are
recultured in medium or in RANKL LZ alone. In contrast, cells do
survive if recultured in CSF-1. When RANKL LZ was added to these
cultures there was no added benefit. Thus, the combination of CSF-1
and RANKL are required for the generation of osteoclast.
Additionally, once formed, CSF-1 is sufficient to maintain their
survival in culture.
[0042] Finally, using human bone marrow, soluble anti-human RANK
mAb and immobilized anti-human RANK mAb were compared to RANKL LZ
for the generation of osteoclasts in the presence of CSF-1.
Immobilized M331 and RANKL LZ were found to be equally effective
for osteoclast generation while soluble M331 was superior to both
immobilized antibody and RANKL LZ. This confirms that the
osteoclast differentiating activity of RANKL is mediated through
RANK rather than via an alternative receptor.
[0043] Since osteoclasts cannot readily be harvested and analyzed
by flow cytometry, .sup.125I-labeled calcitonin binding assays were
used to identify osteoclasts (the calcitonin receptor is considered
to be an osteoclast-specific marker). Osteoclasts generated from
murine BM cultured with CSF-1 and RANKL LZ for 9 days showed
binding of radiolabeled calcitonin confirming their osteoclast
identity.
EXAMPLE 3
[0044] In order to determine RANKL expression by either of two
different squamous cell carcinomas, standard Western blot and
RT-PCR studies were performed on MH-85 and OKK cells. One of these
carcinoma cells, the MH-85 cells, is associated with
hypercalcemia.
[0045] The results confirmed that MH-85 and OKK squamous cells
express RANKL. MH-85 cells, in addition to being linked with
hypercalcemia in patients inflicted with this carcinoma, also
express M-CSF (CSF-1). It was also determined that CSF-1
upregulates RANK expression on osteoclast precursors. The enhanced
amount of CSF-1 in MH-85 type squamous cell cancer patients can
lead to an upregulation of RANK and increased RANK interaction with
RANKL. Signals transduced by RANK and RANKL interaction result in
increased numbers of mature osteoclasts and bone breakdown. Since
soluble forms of RANK can inhibit the RAKNK L interaction,
administering a soluble form of RANK (e.g. the extracellular region
of RANK fused to an Fc) to a squamous cell cancer patient provides
relief from adverse effects of this cancer, including
hypercalcemia.
Sequence CWU 1
1
8 1 3136 DNA Homo sapiens CDS (39)..(1886) 1 ccgctgaggc cgcggcgccc
gccagcctgt cccgcgcc atg gcc ccg cgc gcc cgg 56 Met Ala Pro Arg Ala
Arg 1 5 cgg cgc cgc ccg ctg ttc gcg ctg ctg ctg ctc tgc gcg ctg ctc
gcc 104 Arg Arg Arg Pro Leu Phe Ala Leu Leu Leu Leu Cys Ala Leu Leu
Ala 10 15 20 cgg ctg cag gtg gct ttg cag atc gct cct cca tgt acc
agt gag aag 152 Arg Leu Gln Val Ala Leu Gln Ile Ala Pro Pro Cys Thr
Ser Glu Lys 25 30 35 cat tat gag cat ctg gga cgg tgc tgt aac aaa
tgt gaa cca gga aag 200 His Tyr Glu His Leu Gly Arg Cys Cys Asn Lys
Cys Glu Pro Gly Lys 40 45 50 tac atg tct tct aaa tgc act act acc
tct gac agt gta tgt ctg ccc 248 Tyr Met Ser Ser Lys Cys Thr Thr Thr
Ser Asp Ser Val Cys Leu Pro 55 60 65 70 tgt ggc ccg gat gaa tac ttg
gat agc tgg aat gaa gaa gat aaa tgc 296 Cys Gly Pro Asp Glu Tyr Leu
Asp Ser Trp Asn Glu Glu Asp Lys Cys 75 80 85 ttg ctg cat aaa gtt
tgt gat aca ggc aag gcc ctg gtg gcc gtg gtc 344 Leu Leu His Lys Val
Cys Asp Thr Gly Lys Ala Leu Val Ala Val Val 90 95 100 gcc ggc aac
agc acg acc ccc cgg cgc tgc gcg tgc acg gct ggg tac 392 Ala Gly Asn
Ser Thr Thr Pro Arg Arg Cys Ala Cys Thr Ala Gly Tyr 105 110 115 cac
tgg agc cag gac tgc gag tgc tgc cgc cgc aac acc gag tgc gcg 440 His
Trp Ser Gln Asp Cys Glu Cys Cys Arg Arg Asn Thr Glu Cys Ala 120 125
130 ccg ggc ctg ggc gcc cag cac ccg ttg cag ctc aac aag gac aca gtg
488 Pro Gly Leu Gly Ala Gln His Pro Leu Gln Leu Asn Lys Asp Thr Val
135 140 145 150 tgc aaa cct tgc ctt gca ggc tac ttc tct gat gcc ttt
tcc tcc acg 536 Cys Lys Pro Cys Leu Ala Gly Tyr Phe Ser Asp Ala Phe
Ser Ser Thr 155 160 165 gac aaa tgc aga ccc tgg acc aac tgt acc ttc
ctt gga aag aga gta 584 Asp Lys Cys Arg Pro Trp Thr Asn Cys Thr Phe
Leu Gly Lys Arg Val 170 175 180 gaa cat cat ggg aca gag aaa tcc gat
gcg gtt tgc agt tct tct ctg 632 Glu His His Gly Thr Glu Lys Ser Asp
Ala Val Cys Ser Ser Ser Leu 185 190 195 cca gct aga aaa cca cca aat
gaa ccc cat gtt tac ttg ccc ggt tta 680 Pro Ala Arg Lys Pro Pro Asn
Glu Pro His Val Tyr Leu Pro Gly Leu 200 205 210 ata att ctg ctt ctc
ttc gcg tct gtg gcc ctg gtg gct gcc atc atc 728 Ile Ile Leu Leu Leu
Phe Ala Ser Val Ala Leu Val Ala Ala Ile Ile 215 220 225 230 ttt ggc
gtt tgc tat agg aaa aaa ggg aaa gca ctc aca gct aat ttg 776 Phe Gly
Val Cys Tyr Arg Lys Lys Gly Lys Ala Leu Thr Ala Asn Leu 235 240 245
tgg cac tgg atc aat gag gct tgt ggc cgc cta agt gga gat aag gag 824
Trp His Trp Ile Asn Glu Ala Cys Gly Arg Leu Ser Gly Asp Lys Glu 250
255 260 tcc tca ggt gac agt tgt gtc agt aca cac acg gca aac ttt ggt
cag 872 Ser Ser Gly Asp Ser Cys Val Ser Thr His Thr Ala Asn Phe Gly
Gln 265 270 275 cag gga gca tgt gaa ggt gtc tta ctg ctg act ctg gag
gag aag aca 920 Gln Gly Ala Cys Glu Gly Val Leu Leu Leu Thr Leu Glu
Glu Lys Thr 280 285 290 ttt cca gaa gat atg tgc tac cca gat caa ggt
ggt gtc tgt cag ggc 968 Phe Pro Glu Asp Met Cys Tyr Pro Asp Gln Gly
Gly Val Cys Gln Gly 295 300 305 310 acg tgt gta gga ggt ggt ccc tac
gca caa ggc gaa gat gcc agg atg 1016 Thr Cys Val Gly Gly Gly Pro
Tyr Ala Gln Gly Glu Asp Ala Arg Met 315 320 325 ctc tca ttg gtc agc
aag acc gag ata gag gaa gac agc ttc aga cag 1064 Leu Ser Leu Val
Ser Lys Thr Glu Ile Glu Glu Asp Ser Phe Arg Gln 330 335 340 atg ccc
aca gaa gat gaa tac atg gac agg ccc tcc cag ccc aca gac 1112 Met
Pro Thr Glu Asp Glu Tyr Met Asp Arg Pro Ser Gln Pro Thr Asp 345 350
355 cag tta ctg ttc ctc act gag cct gga agc aaa tcc aca cct cct ttc
1160 Gln Leu Leu Phe Leu Thr Glu Pro Gly Ser Lys Ser Thr Pro Pro
Phe 360 365 370 tct gaa ccc ctg gag gtg ggg gag aat gac agt tta agc
cag tgc ttc 1208 Ser Glu Pro Leu Glu Val Gly Glu Asn Asp Ser Leu
Ser Gln Cys Phe 375 380 385 390 acg ggg aca cag agc aca gtg ggt tca
gaa agc tgc aac tgc act gag 1256 Thr Gly Thr Gln Ser Thr Val Gly
Ser Glu Ser Cys Asn Cys Thr Glu 395 400 405 ccc ctg tgc agg act gat
tgg act ccc atg tcc tct gaa aac tac ttg 1304 Pro Leu Cys Arg Thr
Asp Trp Thr Pro Met Ser Ser Glu Asn Tyr Leu 410 415 420 caa aaa gag
gtg gac agt ggc cat tgc ccg cac tgg gca gcc agc ccc 1352 Gln Lys
Glu Val Asp Ser Gly His Cys Pro His Trp Ala Ala Ser Pro 425 430 435
agc ccc aac tgg gca gat gtc tgc aca ggc tgc cgg aac cct cct ggg
1400 Ser Pro Asn Trp Ala Asp Val Cys Thr Gly Cys Arg Asn Pro Pro
Gly 440 445 450 gag gac tgt gaa ccc ctc gtg ggt tcc cca aaa cgt gga
ccc ttg ccc 1448 Glu Asp Cys Glu Pro Leu Val Gly Ser Pro Lys Arg
Gly Pro Leu Pro 455 460 465 470 cag tgc gcc tat ggc atg ggc ctt ccc
cct gaa gaa gaa gcc agc agg 1496 Gln Cys Ala Tyr Gly Met Gly Leu
Pro Pro Glu Glu Glu Ala Ser Arg 475 480 485 acg gag gcc aga gac cag
ccc gag gat ggg gct gat ggg agg ctc cca 1544 Thr Glu Ala Arg Asp
Gln Pro Glu Asp Gly Ala Asp Gly Arg Leu Pro 490 495 500 agc tca gcg
agg gca ggt gcc ggg tct gga agc tcc cct ggt ggc cag 1592 Ser Ser
Ala Arg Ala Gly Ala Gly Ser Gly Ser Ser Pro Gly Gly Gln 505 510 515
tcc cct gca tct gga aat gtg act gga aac agt aac tcc acg ttc atc
1640 Ser Pro Ala Ser Gly Asn Val Thr Gly Asn Ser Asn Ser Thr Phe
Ile 520 525 530 tcc agc ggg cag gtg atg aac ttc aag ggc gac atc atc
gtg gtc tac 1688 Ser Ser Gly Gln Val Met Asn Phe Lys Gly Asp Ile
Ile Val Val Tyr 535 540 545 550 gtc agc cag acc tcg cag gag ggc gcg
gcg gcg gct gcg gag ccc atg 1736 Val Ser Gln Thr Ser Gln Glu Gly
Ala Ala Ala Ala Ala Glu Pro Met 555 560 565 ggc cgc ccg gtg cag gag
gag acc ctg gcg cgc cga gac tcc ttc gcg 1784 Gly Arg Pro Val Gln
Glu Glu Thr Leu Ala Arg Arg Asp Ser Phe Ala 570 575 580 ggg aac ggc
ccg cgc ttc ccg gac ccg tgc ggc ggc ccc gag ggg ctg 1832 Gly Asn
Gly Pro Arg Phe Pro Asp Pro Cys Gly Gly Pro Glu Gly Leu 585 590 595
cgg gag ccg gag aag gcc tcg agg ccg gtg cag gag caa ggc ggg gcc
1880 Arg Glu Pro Glu Lys Ala Ser Arg Pro Val Gln Glu Gln Gly Gly
Ala 600 605 610 aag gct tgagcgcccc ccatggctgg gagcccgaag ctcggagcca
gggctcgcga 1936 Lys Ala 615 gggcagcacc gcagcctctg ccccagcccc
ggccacccag ggatcgatcg gtacagtcga 1996 ggaagaccac ccggcattct
ctgcccactt tgccttccag gaaatgggct tttcaggaag 2056 tgaattgatg
aggactgtcc ccatgcccac ggatgctcag cagcccgccg cactggggca 2116
gatgtctccc ctgccactcc tcaaactcgc agcagtaatt tgtggcacta tgacagctat
2176 ttttatgact atcctgttct gtgggggggg ggtctatgtt ttccccccat
atttgtattc 2236 cttttcataa cttttcttga tatctttcct ccctcttttt
taatgtaaag gttttctcaa 2296 aaattctcct aaaggtgagg gtctctttct
tttctctttt cctttttttt ttcttttttt 2356 ggcaacctgg ctctggccca
ggctagagtg cagtggtgcg attatagccc ggtgcagcct 2416 ctaactcctg
ggctcaagca atccaagtga tcctcccacc tcaaccttcg gagtagctgg 2476
gatcacagct gcaggccacg cccagcttcc tccccccgac tccccccccc cagagacacg
2536 gtcccaccat gttacccagc ctggtctcaa actccccagc taaagcagtc
ctccagcctc 2596 ggcctcccaa agtactggga ttacaggcgt gagcccccac
gctggcctgc tttacgtatt 2656 ttcttttgtg cccctgctca cagtgtttta
gagatggctt tcccagtgtg tgttcattgt 2716 aaacactttt gggaaagggc
taaacatgtg aggcctggag atagttgcta agttgctagg 2776 aacatgtggt
gggactttca tattctgaaa aatgttctat attctcattt ttctaaaaga 2836
aagaaaaaag gaaacccgat ttatttctcc tgaatctttt taagtttgtg tcgttcctta
2896 agcagaacta agctcagtat gtgaccttac ccgctaggtg gttaatttat
ccatgctggc 2956 agaggcactc aggtacttgg taagcaaatt tctaaaactc
caagttgctg cagcttggca 3016 ttcttcttat tctagaggtc tctctggaaa
agatggagaa aatgaacagg acatggggct 3076 cctggaaaga aagggcccgg
gaagttcaag gaagaataaa gttgaaattt taaaaaaaaa 3136 2 616 PRT Homo
sapiens 2 Met Ala Pro Arg Ala Arg Arg Arg Arg Pro Leu Phe Ala Leu
Leu Leu 1 5 10 15 Leu Cys Ala Leu Leu Ala Arg Leu Gln Val Ala Leu
Gln Ile Ala Pro 20 25 30 Pro Cys Thr Ser Glu Lys His Tyr Glu His
Leu Gly Arg Cys Cys Asn 35 40 45 Lys Cys Glu Pro Gly Lys Tyr Met
Ser Ser Lys Cys Thr Thr Thr Ser 50 55 60 Asp Ser Val Cys Leu Pro
Cys Gly Pro Asp Glu Tyr Leu Asp Ser Trp 65 70 75 80 Asn Glu Glu Asp
Lys Cys Leu Leu His Lys Val Cys Asp Thr Gly Lys 85 90 95 Ala Leu
Val Ala Val Val Ala Gly Asn Ser Thr Thr Pro Arg Arg Cys 100 105 110
Ala Cys Thr Ala Gly Tyr His Trp Ser Gln Asp Cys Glu Cys Cys Arg 115
120 125 Arg Asn Thr Glu Cys Ala Pro Gly Leu Gly Ala Gln His Pro Leu
Gln 130 135 140 Leu Asn Lys Asp Thr Val Cys Lys Pro Cys Leu Ala Gly
Tyr Phe Ser 145 150 155 160 Asp Ala Phe Ser Ser Thr Asp Lys Cys Arg
Pro Trp Thr Asn Cys Thr 165 170 175 Phe Leu Gly Lys Arg Val Glu His
His Gly Thr Glu Lys Ser Asp Ala 180 185 190 Val Cys Ser Ser Ser Leu
Pro Ala Arg Lys Pro Pro Asn Glu Pro His 195 200 205 Val Tyr Leu Pro
Gly Leu Ile Ile Leu Leu Leu Phe Ala Ser Val Ala 210 215 220 Leu Val
Ala Ala Ile Ile Phe Gly Val Cys Tyr Arg Lys Lys Gly Lys 225 230 235
240 Ala Leu Thr Ala Asn Leu Trp His Trp Ile Asn Glu Ala Cys Gly Arg
245 250 255 Leu Ser Gly Asp Lys Glu Ser Ser Gly Asp Ser Cys Val Ser
Thr His 260 265 270 Thr Ala Asn Phe Gly Gln Gln Gly Ala Cys Glu Gly
Val Leu Leu Leu 275 280 285 Thr Leu Glu Glu Lys Thr Phe Pro Glu Asp
Met Cys Tyr Pro Asp Gln 290 295 300 Gly Gly Val Cys Gln Gly Thr Cys
Val Gly Gly Gly Pro Tyr Ala Gln 305 310 315 320 Gly Glu Asp Ala Arg
Met Leu Ser Leu Val Ser Lys Thr Glu Ile Glu 325 330 335 Glu Asp Ser
Phe Arg Gln Met Pro Thr Glu Asp Glu Tyr Met Asp Arg 340 345 350 Pro
Ser Gln Pro Thr Asp Gln Leu Leu Phe Leu Thr Glu Pro Gly Ser 355 360
365 Lys Ser Thr Pro Pro Phe Ser Glu Pro Leu Glu Val Gly Glu Asn Asp
370 375 380 Ser Leu Ser Gln Cys Phe Thr Gly Thr Gln Ser Thr Val Gly
Ser Glu 385 390 395 400 Ser Cys Asn Cys Thr Glu Pro Leu Cys Arg Thr
Asp Trp Thr Pro Met 405 410 415 Ser Ser Glu Asn Tyr Leu Gln Lys Glu
Val Asp Ser Gly His Cys Pro 420 425 430 His Trp Ala Ala Ser Pro Ser
Pro Asn Trp Ala Asp Val Cys Thr Gly 435 440 445 Cys Arg Asn Pro Pro
Gly Glu Asp Cys Glu Pro Leu Val Gly Ser Pro 450 455 460 Lys Arg Gly
Pro Leu Pro Gln Cys Ala Tyr Gly Met Gly Leu Pro Pro 465 470 475 480
Glu Glu Glu Ala Ser Arg Thr Glu Ala Arg Asp Gln Pro Glu Asp Gly 485
490 495 Ala Asp Gly Arg Leu Pro Ser Ser Ala Arg Ala Gly Ala Gly Ser
Gly 500 505 510 Ser Ser Pro Gly Gly Gln Ser Pro Ala Ser Gly Asn Val
Thr Gly Asn 515 520 525 Ser Asn Ser Thr Phe Ile Ser Ser Gly Gln Val
Met Asn Phe Lys Gly 530 535 540 Asp Ile Ile Val Val Tyr Val Ser Gln
Thr Ser Gln Glu Gly Ala Ala 545 550 555 560 Ala Ala Ala Glu Pro Met
Gly Arg Pro Val Gln Glu Glu Thr Leu Ala 565 570 575 Arg Arg Asp Ser
Phe Ala Gly Asn Gly Pro Arg Phe Pro Asp Pro Cys 580 585 590 Gly Gly
Pro Glu Gly Leu Arg Glu Pro Glu Lys Ala Ser Arg Pro Val 595 600 605
Gln Glu Gln Gly Gly Ala Lys Ala 610 615 3 232 PRT Homo sapiens
PEPTIDE (1)..(232) 3 Glu Pro Arg Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Ala Glu Gly Ala Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90
95 Asp Trp Leu Asn Gly Lys Asp Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110 Leu Pro Ala Pro Met Gln Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Arg 145 150 155 160 His Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215
220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 4 1878 DNA Murine CDS
(1)..(1875) 4 atg gcc ccg cgc gcc cgg cgg cgc cgc cag ctg ccc gcg
ccg ctg ctg 48 Met Ala Pro Arg Ala Arg Arg Arg Arg Gln Leu Pro Ala
Pro Leu Leu 1 5 10 15 gcg ctc tgc gtg ctg ctc gtt cca ctg cag gtg
act ctc cag gtc act 96 Ala Leu Cys Val Leu Leu Val Pro Leu Gln Val
Thr Leu Gln Val Thr 20 25 30 cct cca tgc acc cag gag agg cat tat
gag cat ctc gga cgg tgt tgc 144 Pro Pro Cys Thr Gln Glu Arg His Tyr
Glu His Leu Gly Arg Cys Cys 35 40 45 agc aga tgc gaa cca gga aag
tac ctg tcc tct aag tgc act cct acc 192 Ser Arg Cys Glu Pro Gly Lys
Tyr Leu Ser Ser Lys Cys Thr Pro Thr 50 55 60 tcc gac agt gtg tgt
ctg ccc tgt ggc ccc gat gag tac ttg gac acc 240 Ser Asp Ser Val Cys
Leu Pro Cys Gly Pro Asp Glu Tyr Leu Asp Thr 65 70 75 80 tgg aat gaa
gaa gat aaa tgc ttg ctg cat aaa gtc tgt gat gca ggc 288 Trp Asn Glu
Glu Asp Lys Cys Leu Leu His Lys Val Cys Asp Ala Gly 85 90 95 aag
gcc ctg gtg gcg gtg gat cct ggc aac cac acg gcc ccg cgt cgc 336 Lys
Ala Leu Val Ala Val Asp Pro Gly Asn His Thr Ala Pro Arg Arg 100 105
110 tgt gct tgc acg gct ggc tac cac tgg aac tca gac tgc gag tgc tgc
384 Cys Ala Cys Thr Ala Gly Tyr His Trp Asn Ser Asp Cys Glu Cys Cys
115 120 125 cgc agg aac acg gag tgt gca cct ggc ttc gga gct cag cat
ccc ttg 432 Arg Arg Asn Thr Glu Cys Ala Pro Gly Phe Gly Ala Gln His
Pro Leu 130 135 140 cag ctc aac aag gat acg gtg tgc aca ccc tgc ctc
ctg ggc ttc ttc 480 Gln Leu Asn Lys Asp Thr Val Cys Thr Pro Cys Leu
Leu Gly Phe Phe 145 150 155 160 tca gat gtc ttt tcg tcc aca gac aaa
tgc aaa cct tgg acc aac tgc 528 Ser Asp Val Phe Ser Ser Thr Asp Lys
Cys Lys Pro Trp Thr Asn Cys 165 170 175 acc ctc ctt gga aag cta gaa
gca cac cag ggg aca acg gaa tca gat 576 Thr Leu Leu Gly Lys Leu Glu
Ala His Gln Gly Thr Thr Glu Ser Asp 180 185 190 gtg gtc tgc agc tct
tcc atg aca ctg agg aga cca ccc aag gag gcc 624 Val Val Cys Ser Ser
Ser Met Thr Leu Arg Arg Pro Pro Lys Glu Ala 195 200 205 cag gct tac
ctg ccc agt ctc atc gtt ctg ctc ctc ttc atc tct gtg 672 Gln Ala Tyr
Leu Pro Ser Leu Ile Val Leu Leu Leu Phe Ile Ser Val 210 215 220 gta
gta gtg gct gcc atc atc ttc ggc gtt tac tac agg aag gga ggg 720 Val
Val Val Ala
Ala Ile Ile Phe Gly Val Tyr Tyr Arg Lys Gly Gly 225 230 235 240 aaa
gcg ctg aca gct aat ttg tgg aat tgg gtc aat gat gct tgc agt 768 Lys
Ala Leu Thr Ala Asn Leu Trp Asn Trp Val Asn Asp Ala Cys Ser 245 250
255 agt cta agt gga aat aag gag tcc tca ggg gac cgt tgt gct ggt tcc
816 Ser Leu Ser Gly Asn Lys Glu Ser Ser Gly Asp Arg Cys Ala Gly Ser
260 265 270 cac tcg gca acc tcc agt cag caa gaa gtg tgt gaa ggt atc
tta cta 864 His Ser Ala Thr Ser Ser Gln Gln Glu Val Cys Glu Gly Ile
Leu Leu 275 280 285 atg act cgg gag gag aag atg gtt cca gaa gac ggt
gct gga gtc tgt 912 Met Thr Arg Glu Glu Lys Met Val Pro Glu Asp Gly
Ala Gly Val Cys 290 295 300 ggg cct gtg tgt gcg gca ggt ggg ccc tgg
gca gaa gtc aga gat tct 960 Gly Pro Val Cys Ala Ala Gly Gly Pro Trp
Ala Glu Val Arg Asp Ser 305 310 315 320 agg acg ttc aca ctg gtc agc
gag gtt gag acg caa gga gac ctc tcg 1008 Arg Thr Phe Thr Leu Val
Ser Glu Val Glu Thr Gln Gly Asp Leu Ser 325 330 335 agg aag att ccc
aca gag gat gag tac acg gac cgg ccc tcg cag cct 1056 Arg Lys Ile
Pro Thr Glu Asp Glu Tyr Thr Asp Arg Pro Ser Gln Pro 340 345 350 tcg
act ggt tca ctg ctc cta atc cag cag gga agc aaa tct ata ccc 1104
Ser Thr Gly Ser Leu Leu Leu Ile Gln Gln Gly Ser Lys Ser Ile Pro 355
360 365 cca ttc cag gag ccc ctg gaa gtg ggg gag aac gac agt tta agc
cag 1152 Pro Phe Gln Glu Pro Leu Glu Val Gly Glu Asn Asp Ser Leu
Ser Gln 370 375 380 tgt ttc acc ggg act gaa agc acg gtg gat tct gag
ggc tgt gac ttc 1200 Cys Phe Thr Gly Thr Glu Ser Thr Val Asp Ser
Glu Gly Cys Asp Phe 385 390 395 400 act gag cct ccg agc aga act gac
tct atg ccc gtg tcc cct gaa aag 1248 Thr Glu Pro Pro Ser Arg Thr
Asp Ser Met Pro Val Ser Pro Glu Lys 405 410 415 cac ctg aca aaa gaa
ata gaa ggt gac agt tgc ctc ccc tgg gtg gtc 1296 His Leu Thr Lys
Glu Ile Glu Gly Asp Ser Cys Leu Pro Trp Val Val 420 425 430 agc tcc
aac tca aca gat ggc tac aca ggc agt ggg aac act cct ggg 1344 Ser
Ser Asn Ser Thr Asp Gly Tyr Thr Gly Ser Gly Asn Thr Pro Gly 435 440
445 gag gac cat gaa ccc ttt cca ggg tcc ctg aaa tgt gga cca ttg ccc
1392 Glu Asp His Glu Pro Phe Pro Gly Ser Leu Lys Cys Gly Pro Leu
Pro 450 455 460 cag tgt gcc tac agc atg ggc ttt ccc agt gaa gca gca
gcc agc atg 1440 Gln Cys Ala Tyr Ser Met Gly Phe Pro Ser Glu Ala
Ala Ala Ser Met 465 470 475 480 gca gag gcg gga gta cgg ccc cag gac
agg gct gat gag agg gga gcc 1488 Ala Glu Ala Gly Val Arg Pro Gln
Asp Arg Ala Asp Glu Arg Gly Ala 485 490 495 tca ggg tcc ggg agc tcc
ccc agt gac cag cca cct gcc tct ggg aac 1536 Ser Gly Ser Gly Ser
Ser Pro Ser Asp Gln Pro Pro Ala Ser Gly Asn 500 505 510 gtg act gga
aac agt aac tcc acg ttc atc tct agc ggg cag gtg atg 1584 Val Thr
Gly Asn Ser Asn Ser Thr Phe Ile Ser Ser Gly Gln Val Met 515 520 525
aac ttc aag ggt gac atc atc gtg gtg tat gtc agc cag acc tcg cag
1632 Asn Phe Lys Gly Asp Ile Ile Val Val Tyr Val Ser Gln Thr Ser
Gln 530 535 540 gag ggc ccg ggt tcc gca gag ccc gag tcg gag ccc gtg
ggc cgc cct 1680 Glu Gly Pro Gly Ser Ala Glu Pro Glu Ser Glu Pro
Val Gly Arg Pro 545 550 555 560 gtg cag gag gag acg ctg gca cac aga
gac tcc ttt gcg ggc acc gcg 1728 Val Gln Glu Glu Thr Leu Ala His
Arg Asp Ser Phe Ala Gly Thr Ala 565 570 575 ccg cgc ttc ccc gac gtc
tgt gcc acc ggg gct ggg ctg cag gag cag 1776 Pro Arg Phe Pro Asp
Val Cys Ala Thr Gly Ala Gly Leu Gln Glu Gln 580 585 590 ggg gca ccc
cgg cag aag gac ggg aca tcg cgg ccg gtg cag gag cag 1824 Gly Ala
Pro Arg Gln Lys Asp Gly Thr Ser Arg Pro Val Gln Glu Gln 595 600 605
ggt ggg gcg cag act tca ctc cat acc cag ggg tcc gga caa tgt gca
1872 Gly Gly Ala Gln Thr Ser Leu His Thr Gln Gly Ser Gly Gln Cys
Ala 610 615 620 gaa tga 1878 Glu 625 5 625 PRT Murine 5 Met Ala Pro
Arg Ala Arg Arg Arg Arg Gln Leu Pro Ala Pro Leu Leu 1 5 10 15 Ala
Leu Cys Val Leu Leu Val Pro Leu Gln Val Thr Leu Gln Val Thr 20 25
30 Pro Pro Cys Thr Gln Glu Arg His Tyr Glu His Leu Gly Arg Cys Cys
35 40 45 Ser Arg Cys Glu Pro Gly Lys Tyr Leu Ser Ser Lys Cys Thr
Pro Thr 50 55 60 Ser Asp Ser Val Cys Leu Pro Cys Gly Pro Asp Glu
Tyr Leu Asp Thr 65 70 75 80 Trp Asn Glu Glu Asp Lys Cys Leu Leu His
Lys Val Cys Asp Ala Gly 85 90 95 Lys Ala Leu Val Ala Val Asp Pro
Gly Asn His Thr Ala Pro Arg Arg 100 105 110 Cys Ala Cys Thr Ala Gly
Tyr His Trp Asn Ser Asp Cys Glu Cys Cys 115 120 125 Arg Arg Asn Thr
Glu Cys Ala Pro Gly Phe Gly Ala Gln His Pro Leu 130 135 140 Gln Leu
Asn Lys Asp Thr Val Cys Thr Pro Cys Leu Leu Gly Phe Phe 145 150 155
160 Ser Asp Val Phe Ser Ser Thr Asp Lys Cys Lys Pro Trp Thr Asn Cys
165 170 175 Thr Leu Leu Gly Lys Leu Glu Ala His Gln Gly Thr Thr Glu
Ser Asp 180 185 190 Val Val Cys Ser Ser Ser Met Thr Leu Arg Arg Pro
Pro Lys Glu Ala 195 200 205 Gln Ala Tyr Leu Pro Ser Leu Ile Val Leu
Leu Leu Phe Ile Ser Val 210 215 220 Val Val Val Ala Ala Ile Ile Phe
Gly Val Tyr Tyr Arg Lys Gly Gly 225 230 235 240 Lys Ala Leu Thr Ala
Asn Leu Trp Asn Trp Val Asn Asp Ala Cys Ser 245 250 255 Ser Leu Ser
Gly Asn Lys Glu Ser Ser Gly Asp Arg Cys Ala Gly Ser 260 265 270 His
Ser Ala Thr Ser Ser Gln Gln Glu Val Cys Glu Gly Ile Leu Leu 275 280
285 Met Thr Arg Glu Glu Lys Met Val Pro Glu Asp Gly Ala Gly Val Cys
290 295 300 Gly Pro Val Cys Ala Ala Gly Gly Pro Trp Ala Glu Val Arg
Asp Ser 305 310 315 320 Arg Thr Phe Thr Leu Val Ser Glu Val Glu Thr
Gln Gly Asp Leu Ser 325 330 335 Arg Lys Ile Pro Thr Glu Asp Glu Tyr
Thr Asp Arg Pro Ser Gln Pro 340 345 350 Ser Thr Gly Ser Leu Leu Leu
Ile Gln Gln Gly Ser Lys Ser Ile Pro 355 360 365 Pro Phe Gln Glu Pro
Leu Glu Val Gly Glu Asn Asp Ser Leu Ser Gln 370 375 380 Cys Phe Thr
Gly Thr Glu Ser Thr Val Asp Ser Glu Gly Cys Asp Phe 385 390 395 400
Thr Glu Pro Pro Ser Arg Thr Asp Ser Met Pro Val Ser Pro Glu Lys 405
410 415 His Leu Thr Lys Glu Ile Glu Gly Asp Ser Cys Leu Pro Trp Val
Val 420 425 430 Ser Ser Asn Ser Thr Asp Gly Tyr Thr Gly Ser Gly Asn
Thr Pro Gly 435 440 445 Glu Asp His Glu Pro Phe Pro Gly Ser Leu Lys
Cys Gly Pro Leu Pro 450 455 460 Gln Cys Ala Tyr Ser Met Gly Phe Pro
Ser Glu Ala Ala Ala Ser Met 465 470 475 480 Ala Glu Ala Gly Val Arg
Pro Gln Asp Arg Ala Asp Glu Arg Gly Ala 485 490 495 Ser Gly Ser Gly
Ser Ser Pro Ser Asp Gln Pro Pro Ala Ser Gly Asn 500 505 510 Val Thr
Gly Asn Ser Asn Ser Thr Phe Ile Ser Ser Gly Gln Val Met 515 520 525
Asn Phe Lys Gly Asp Ile Ile Val Val Tyr Val Ser Gln Thr Ser Gln 530
535 540 Glu Gly Pro Gly Ser Ala Glu Pro Glu Ser Glu Pro Val Gly Arg
Pro 545 550 555 560 Val Gln Glu Glu Thr Leu Ala His Arg Asp Ser Phe
Ala Gly Thr Ala 565 570 575 Pro Arg Phe Pro Asp Val Cys Ala Thr Gly
Ala Gly Leu Gln Glu Gln 580 585 590 Gly Ala Pro Arg Gln Lys Asp Gly
Thr Ser Arg Pro Val Gln Glu Gln 595 600 605 Gly Gly Ala Gln Thr Ser
Leu His Thr Gln Gly Ser Gly Gln Cys Ala 610 615 620 Glu 625 6 33
PRT Artificial Sequence Murine 6 Arg Met Lys Gln Ile Glu Asp Lys
Ile Glu Glu Ile Leu Ser Lys Ile 1 5 10 15 Tyr His Ile Glu Asn Glu
Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu 20 25 30 Arg 7 954 DNA Homo
sapiens; CDS (1)..(951) 7 atg cgc cgc gcc agc aga gac tac acc aag
tac ctg cgt ggc tcg gag 48 Met Arg Arg Ala Ser Arg Asp Tyr Thr Lys
Tyr Leu Arg Gly Ser Glu 1 5 10 15 gag atg ggc ggc ggc ccc gga gcc
ccg cac gag ggc ccc ctg cac gcc 96 Glu Met Gly Gly Gly Pro Gly Ala
Pro His Glu Gly Pro Leu His Ala 20 25 30 ccg ccg ccg cct gcg ccg
cac cag ccc ccc gcc gcc tcc cgc tcc atg 144 Pro Pro Pro Pro Ala Pro
His Gln Pro Pro Ala Ala Ser Arg Ser Met 35 40 45 ttc gtg gcc ctc
ctg ggg ctg ggg ctg ggc cag gtt gtc tgc agc gtc 192 Phe Val Ala Leu
Leu Gly Leu Gly Leu Gly Gln Val Val Cys Ser Val 50 55 60 gcc ctg
ttc ttc tat ttc aga gcg cag atg gat cct aat aga ata tca 240 Ala Leu
Phe Phe Tyr Phe Arg Ala Gln Met Asp Pro Asn Arg Ile Ser 65 70 75 80
gaa gat ggc act cac tgc att tat aga att ttg aga ctc cat gaa aat 288
Glu Asp Gly Thr His Cys Ile Tyr Arg Ile Leu Arg Leu His Glu Asn 85
90 95 gca gat ttt caa gac aca act ctg gag agt caa gat aca aaa tta
ata 336 Ala Asp Phe Gln Asp Thr Thr Leu Glu Ser Gln Asp Thr Lys Leu
Ile 100 105 110 cct gat tca tgt agg aga att aaa cag gcc ttt caa gga
gct gtg caa 384 Pro Asp Ser Cys Arg Arg Ile Lys Gln Ala Phe Gln Gly
Ala Val Gln 115 120 125 aag gaa tta caa cat atc gtt gga tca cag cac
atc aga gca gag aaa 432 Lys Glu Leu Gln His Ile Val Gly Ser Gln His
Ile Arg Ala Glu Lys 130 135 140 gcg atg gtg gat ggc tca tgg tta gat
ctg gcc aag agg agc aag ctt 480 Ala Met Val Asp Gly Ser Trp Leu Asp
Leu Ala Lys Arg Ser Lys Leu 145 150 155 160 gaa gct cag cct ttt gct
cat ctc act att aat gcc acc gac atc cca 528 Glu Ala Gln Pro Phe Ala
His Leu Thr Ile Asn Ala Thr Asp Ile Pro 165 170 175 tct ggt tcc cat
aaa gtg agt ctg tcc tct tgg tac cat gat cgg ggt 576 Ser Gly Ser His
Lys Val Ser Leu Ser Ser Trp Tyr His Asp Arg Gly 180 185 190 tgg gcc
aag atc tcc aac atg act ttt agc aat gga aaa cta ata gtt 624 Trp Ala
Lys Ile Ser Asn Met Thr Phe Ser Asn Gly Lys Leu Ile Val 195 200 205
aat cag gat ggc ttt tat tac ctg tat gcc aac att tgc ttt cga cat 672
Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His 210
215 220 cat gaa act tca gga gac cta gct aca gag tat ctt caa cta atg
gtg 720 His Glu Thr Ser Gly Asp Leu Ala Thr Glu Tyr Leu Gln Leu Met
Val 225 230 235 240 tac gtc act aaa acc agc atc aaa atc cca agt tct
cat acc ctg atg 768 Tyr Val Thr Lys Thr Ser Ile Lys Ile Pro Ser Ser
His Thr Leu Met 245 250 255 aaa gga gga agc acc aag tat tgg tca ggg
aat tct gaa ttc cat ttt 816 Lys Gly Gly Ser Thr Lys Tyr Trp Ser Gly
Asn Ser Glu Phe His Phe 260 265 270 tat tcc ata aac gtt ggt gga ttt
ttt aag tta cgg tct gga gag gaa 864 Tyr Ser Ile Asn Val Gly Gly Phe
Phe Lys Leu Arg Ser Gly Glu Glu 275 280 285 atc agc atc gag gtc tcc
aac ccc tcc tta ctg gat ccg gat cag gat 912 Ile Ser Ile Glu Val Ser
Asn Pro Ser Leu Leu Asp Pro Asp Gln Asp 290 295 300 gca aca tac ttt
ggg gct ttt aaa gtt cga gat ata gat tga 954 Ala Thr Tyr Phe Gly Ala
Phe Lys Val Arg Asp Ile Asp 305 310 315 8 317 PRT Homo sapiens; 8
Met Arg Arg Ala Ser Arg Asp Tyr Thr Lys Tyr Leu Arg Gly Ser Glu 1 5
10 15 Glu Met Gly Gly Gly Pro Gly Ala Pro His Glu Gly Pro Leu His
Ala 20 25 30 Pro Pro Pro Pro Ala Pro His Gln Pro Pro Ala Ala Ser
Arg Ser Met 35 40 45 Phe Val Ala Leu Leu Gly Leu Gly Leu Gly Gln
Val Val Cys Ser Val 50 55 60 Ala Leu Phe Phe Tyr Phe Arg Ala Gln
Met Asp Pro Asn Arg Ile Ser 65 70 75 80 Glu Asp Gly Thr His Cys Ile
Tyr Arg Ile Leu Arg Leu His Glu Asn 85 90 95 Ala Asp Phe Gln Asp
Thr Thr Leu Glu Ser Gln Asp Thr Lys Leu Ile 100 105 110 Pro Asp Ser
Cys Arg Arg Ile Lys Gln Ala Phe Gln Gly Ala Val Gln 115 120 125 Lys
Glu Leu Gln His Ile Val Gly Ser Gln His Ile Arg Ala Glu Lys 130 135
140 Ala Met Val Asp Gly Ser Trp Leu Asp Leu Ala Lys Arg Ser Lys Leu
145 150 155 160 Glu Ala Gln Pro Phe Ala His Leu Thr Ile Asn Ala Thr
Asp Ile Pro 165 170 175 Ser Gly Ser His Lys Val Ser Leu Ser Ser Trp
Tyr His Asp Arg Gly 180 185 190 Trp Ala Lys Ile Ser Asn Met Thr Phe
Ser Asn Gly Lys Leu Ile Val 195 200 205 Asn Gln Asp Gly Phe Tyr Tyr
Leu Tyr Ala Asn Ile Cys Phe Arg His 210 215 220 His Glu Thr Ser Gly
Asp Leu Ala Thr Glu Tyr Leu Gln Leu Met Val 225 230 235 240 Tyr Val
Thr Lys Thr Ser Ile Lys Ile Pro Ser Ser His Thr Leu Met 245 250 255
Lys Gly Gly Ser Thr Lys Tyr Trp Ser Gly Asn Ser Glu Phe His Phe 260
265 270 Tyr Ser Ile Asn Val Gly Gly Phe Phe Lys Leu Arg Ser Gly Glu
Glu 275 280 285 Ile Ser Ile Glu Val Ser Asn Pro Ser Leu Leu Asp Pro
Asp Gln Asp 290 295 300 Ala Thr Tyr Phe Gly Ala Phe Lys Val Arg Asp
Ile Asp 305 310 315
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