Note: Descriptions are shown in the official language in which they were submitted.
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
TITLE
RECEPTOR ACTIVATOR OF NF-KAPPA B, RECEPTOR IS MEMBER OF TNF RECEPTOR
SUPERFAMILY
TECHNICAL FIELD OF THE INVENTION
The present invention relates generally to the field of cytokine receptors,
and more
specifically to cytokine receptor/ligand pairs having immunoregulatory
activity.
BACKGROUND OF THE INVENTION
Efficient functioning of the immune system requires a fine balance between
cell
proliferation and differentiation and cell death, to ensure that the immune
system is capable
of reacting to foreign, but not self antigens. Integral to the process of
regulating the
immune and inflammatory response are various members of the Tumor Necrosis
Factor
(TNF) Receptor/Nerve Growth Factor Receptor superfamily (Smith et al., Science
248:1019; 1990). This family of receptors includes two different TNF receptors
(Type I
and Type II; Smith et al., supra; and Schall et al., Cell 61:361, 1990), nerve
growth factor
receptor (Johnson et al., Cell 47:545, 1986), B cell antigen CD40 (Stamenkovic
et al.,
EMBO J. 8:1403, 1989), CD27 (Camerini et al., J. Immunol. 147:3165, 1991),
CD30
(Durkop et al., Cell 68:421, 1992), T cell antigen OX40 (Mallett et al., EMBO
J. 9:1063,
1990), human Fas antigen (Itoh et al., Cell 66:233, 1991), murine 4-1BB
receptor (Kwon
et al., Proc. Natl. Acad. Sci. USA 86:1963, 1989) and a receptor referred to
as Apoptosis-
Inducing Receptor (AIR; USSN 08/720,864, filed October 4, 1996).
CD40 is a receptor present on B lymphocytes, epithelial cells and some
carcinoma
cell lines that interacts with a ligand found on activated T cells, CD40L
(USSN
08/249,189, filed May 24, 1994). The interaction of this ligand/receptor pair
is essential
for both the cellular and humoral immune response. Signal transduction via
CD40 is
mediated through the association of the cytoplasmic domain of this molecule
with members
of the TNF receptor-associated factors (TRAFs; Baker and Reddy, Oncogene 12:1,
1996).
It has recently been found that mice that are defective in TRAF3 expression
due to a
targeted disruption in the gene encoding TRAF3 appear normal at birth but
develop
progressive hypoglycemia and depletion of peripheral white cells, and die by
about ten days
of age (Xu et al., Immunity 5:407, 1996). The immune responses of chimeric
mice
reconstituted with TRAF3-/- fetal liver cells resemble those of CD40-deficient
mice,
although TRAF34- B cells appear to be functionally normal.
The critical role of TRAF3 in signal transduction may be in its interaction
with one
of the other members of the TNF receptor superfamily, for example, CD30 or
CD27,
which are present on T cells. Alternatively, there may be other, as yet
unidentified
1
SUBSTITUTE SHEET (RULE 26)
CA 02274803 2005-08-03
72249-164
members of this family of receptors that interact with TRAF3 and play an
important role in
postnatal development as well as in the development of a competent immune
system.
Identifying additional members of the TNF receptor superfamily would provide
an
additional means of regulating the immune and inflammatory response, as well
as
potentially providing further insight into post-natal development in mammals.
SUMMARY OF THE INVENTION
The present invention provides a novel receptor, referred to as RANK (for
receptor
activator of NF-KB), that is a member of the TNF receptor superfamily. RANK is
a Type I
transmembrane protein having 616 amino acid residues that interacts with
TRAF3.
Triggering of RANK by over-expression, co-expression of RANK and membrane
bound
RANK ligand (RANKL), and with addition of soluble RANKL or agonistic
antibodies to
RANK results in the upregulation of the transcription factor NF-KB, a
ubiquitous
transcription factor that is most extensively utilized in cells of the immune
system.
Soluble forms of the receptor can be prepared and used to interfere with
signal
transduction through membrane-bound RANK, and hence upregulation of NF-KB;
accordingly, pharmaceutical compositions comprising soluble forms of the novel
receptor
are also provided. Inhibition of NF-xB by RANK antagonists may be useful in
ameliorating negative effects of an inflammatory response that result from
triggering of
RANK, for example in treating toxic shock or sepsis, graft-versus-host
reactions, or acute
inflammatory reactions. Soluble forms of the receptor will also be useful in
vitro to screen
for agonists or antagonists of RANK activity.
The cytoplasmic domain of RANK will be useful in developing assays for
inhibitors of signal transduction, for example, for screening for molecules
that inhibit
interaction of RANK with TRAF2 or TRAF3. Deleted forms and fusion proteins
comprising the novel receptor are also disclosed.
The present invention also identifies a counterstructure, or ligand, for RANK,
referred to as RANKL. RANKL is a Type 2 transmembrane protein with an
intracellular
domain of less than about 50 amino acids, a transmembrane domain and an
extracellular
domain of from about 240 to 250 amino acids. Similar to other members of the
TNF
family to which it belongs, RANKL has a `spacer' region between the
transmembrane
domain and the receptor binding domain that is not necessary for receptor
binding.
Accordingly, soluble forms of RANKL can comprise the entire extracellular
domain or
fragments thereof that include the receptor binding region.
2
CA 02274803 2010-11-12
72249-164
One specific aspect of the invention relates to an
isolated receptor activator of NF-KB (RANK) polypeptide
capable of binding receptor activator of NF-KB ligand
(RANKL) or binding a Tumor Necrosis Factor receptor-
associated factor (TRAF) selected from the group consisting
of (a) a polypeptide comprising the amino acid sequence as
set forth in SEQ ID NO:6; and (b) a polypeptide comprising
the amino acid sequence x to y of SEQ ID NO:6, wherein x is
any one of amino acids 1-33 and y is any one of amino acids
196-616; wherein RANKL consists of the amino acid sequence
of SEQ ID NO:13.
Another specific aspect of the invention relates
to an isolated receptor activator of NF-KB (RANK)
polypeptide encoded by a DNA capable of hybridizing to the
complement of nucleotides 33-213 of SEQ ID NO:5 under the
conditions of hybridizing at 6XSSC at 63 C and washing in
3XSSC at 55 C, wherein the RANK polypeptide is capable of
binding receptor activator of NF-KB ligand (RANKL) that
comprises the amino acid sequence of SEQ ID NO:13 or binding
a Tumor Necrosis Factor receptor-associated factor (TRAF).
Another specific aspect of the invention relates
to an isolated receptor activator of NF-KB (RANK)
polypeptide comprising amino acids 339-421, inclusive, of
SEQ ID NO:6, wherein said polypeptide is capable of binding
TRAF6.
Another specific aspect of the invention relates
to an isolated receptor activator of NF-KB (RANK)
polypeptide comprising amino acids 544-616, inclusive, of
SEQ ID NO:6, wherein said polypeptide is capable of binding
a TRAF selected from the group consisting of TRAF1, TRAF2,
TRAF3, TRAF5 and TRAF6.
2a
CA 02274803 2010-11-12
72249-164
Another specific aspect of the invention relates
to an isolated receptor activator of NF-KB (RANK)
polypeptide consisting of the amino acid sequence according
to SEQ ID NO:6 or a fragment thereof that is capable of
binding a receptor activator of NF-KB ligand (RANKL) protein
that consists of the amino acid sequence shown in SEQ ID
NO:13, wherein said polypeptide or fragment is capable of
inducing an antibody that binds specifically with a
polypeptide consisting of the amino acid sequence set forth
in SEQ ID NO:6.
Another specific aspect of the invention relates
to a receptor activator of NF-KB (RANK) fusion polypeptide
capable of binding a receptor activator of NF-KB ligand
(RANKL) polypeptide consisting of the amino acid sequence of
SEQ ID NO:13, said fusion polypeptide comprising the RANK
polypeptide according to any one of claims 1 to 19 fused to
an Fc region of a human immunoglobulin protein or a GST
polypeptide.
Another specific aspect of the invention relates
to an isolated receptor activator of NF-KB (RANK)
polypeptide capable of binding receptor activator of NF-KB
ligand (RANKL) or binding a Tumor Necrosis Factor receptor-
associated factor (TRAF) selected from the group consisting
of: (a) a polypeptide comprising the amino acid sequence as
set forth in SEQ ID NO:15; and (b) a polypeptide comprising
the amino acid sequence x to y of SEQ ID NO: 15, wherein x
is amino acid 1 or any one of amino acids 25-35 and y is any
one of amino acids 197-214, wherein RANKL consists of the
amino acid sequence of SEQ ID NO:11 or 13.
2b
CA 02274803 2010-11-12
72249-164
Another specific aspect of the invention relates
to a receptor activator of NF-KB (RANK) fusion polypeptide
comprising amino acids x to 213 of SEQ ID NO:15, wherein x
is amino acid 1 or 35 of SEQ ID NO:15, and amino acids 3-232
of SEQ ID NO:8, wherein the RANK fusion polypeptide is
capable of binding a receptor activator of NF-KB ligand
(RANKL) polypeptide consisting of the amino acid sequence of
SEQ ID NO:13 or a TRAF protein.
Another specific aspect of the invention relates
to an isolated receptor activator of NF-KB (RANK)
polypeptide encoded by a DNA capable of hybridizing to the
complement of the nucleotide sequence set forth in SEQ ID
N0:5 under stringent conditions of hybridizing at 6XSSC at
63 C and washing in 3XSSC at 55 C, and wherein the
polypeptide is capable of binding a TRAF selected from the
group consisting of TRAF1, TRAF2, TRAF3, TRAF5 and TRAF6.
Another specific aspect of the invention relates
to an isolated receptor activator of NF-KB (RANK)
polypeptide produced by a process comprising culturing a
host cell transformed or transfected with a recombinant
expression vector containing the nucleotide sequence set
forth in SEQ ID NO:5 or containing a nucleotide sequence
capable of hybridizing to the complement of the nucleotide
sequence set forth in SEQ ID NO:5 under the stringent
conditions of hybridizing in 6XSSC at 63 C and washing in
3XSSC at 55 C, and isolating the polypeptide resulting from
the expression of said nucleotide sequence, said polypeptide
being capable of binding a receptor activator of NF-KB
ligand (RANKL) protein that consists of the amino acid
sequence shown in SEQ ID NO:13.
2c
CA 02274803 2010-11-12
72249-164
Another specific aspect of the invention relates
to an isolated receptor activator of NF-KB (RANK)
polypeptide which is at least 80% identical in sequence to
amino acids 544-616 of SEQ ID NO:6, wherein said polypeptide
is capable of binding a TRAF selected from the group
consisting of TRAF1, TRAF2, TRAF3, TRAF5 and TRAF6.
Another specific aspect of the invention relates
to an isolated receptor activator of NF-KB (RANK)
polypeptide that is at least 70% identical in amino acid
sequence to a polypeptide with the amino acid sequence x to
y of SEQ ID N0:6, wherein x is any one of amino acids 1-33
and y is any one of amino acids 213-616, wherein said
isolated polypeptide is capable of binding a receptor
activator of NF-KB ligand (RANKL) polypeptide that consists
of the amino acid sequence shown in SEQ ID NO:13.
Another specific aspect of the invention relates
to an isolated receptor activator of NF-KB (RANK)
polypeptide comprising an amino acid sequence with at least
80% sequence identity to SEQ ID NO:6, wherein said isolated
polypeptide is capable of binding a receptor activator of
NF-KB ligand (RANKL) polypeptide that consists of the amino
acid sequence shown in SEQ ID NO:13.
Another specific aspect of the invention relates
to an isolated receptor activator of NF-KB (RANK)
polypeptide comprising an amino acid sequence with at least
80% sequence identity to SEQ ID NO:15, wherein said isolated
polypeptide is capable of binding to a receptor activator of
NF-KB ligand (RANKL) polypeptide consisting of SEQ ID NO:11.
2d
CA 02274803 2010-11-12
72249-164
Another specific aspect of the invention relates
to a soluble receptor activator of NF-KB (RANK) polypeptide
comprising the amino acid sequence x to y of SEQ ID NO:6,
wherein x is any one of amino acids 1-33 and y is any one of
amino acids 196-213 or fragments thereof, wherein the
soluble RANK is capable of binding to a receptor activator
of NF-KB ligand (RANKL) protein that consists of the amino
acid sequence of SEQ ID NO:13.
Another specific aspect of the invention relates
to a primer comprising at least 17 contiguous nucleotides of
SEQ ID NO:5 and which binds specifically to the complement
of the nucleotide sequence set forth as SEQ ID NO:5.
Another specific aspect of the invention relates
to a primer which comprises at least 17 contiguous
nucleotides of SEQ ID NO:5 and which binds specifically to
the complement of the nucleotide sequence set forth as SEQ
ID NO:5.
Another specific aspect of the invention relates
to a primer which comprises at least 17 contiguous
nucleotides of SEQ ID NO:14 and which binds specifically to
the complement of the nucleotide sequence set forth as SEQ
ID NO:5.
Another specific aspect of the invention relates
to a DNA molecule capable of hybridization to the complement
of the nucleotide sequence set forth in SEQ ID NO:5 under
stringent conditions of hybridizing at 6XSSC at 63 C and
washing in 3XSSC at 55 C, and which encodes receptor
activator of NF-KB (RANK) that is capable of binding to
2e
CA 02274803 2010-11-12
72249-164
receptor activator of NF-KB ligand (RANKL) consisting of the
amino acid sequence of SEQ ID NO:13.
Another specific aspect of the invention relates
to a DNA molecule capable of hybridizing to the complement
of nucleotides 33-213 of SEQ ID NO:5 under stringent
conditions of hybridizing at 6XSSC at 63 C and washing in
3XSSC at 55 C, and which encodes receptor activator of NF-KB
(RANK) that is capable of binding to receptor activator of
NF-KB ligand (RANKL) consisting of the amino acid sequence
of SEQ ID NO:13.
Another specific aspect of the invention relates
to an isolated antibody that specifically binds a RANK
polypeptide of amino acids 1-616 of SEQ ID NO:6; or amino
acids 33-213 of SEQ ID NO:6.
Another specific aspect of the invention relates
to a method for preparing an antibody, comprising immunizing
a non-human mammal with a polypeptide comprising amino acids
544-616 of SEQ ID NO:6 in an amount effective to produce
antibodies which bind specifically to the polypeptide and
harvesting the antibodies.
Another specific aspect of the invention relates
to a method for preparing an antibody, comprising immunizing
a non-human mammal with a polypeptide comprising amino acids
339-421 of SEQ ID NO:6 in an amount effective to produce
antibodies which bind specifically to the polypeptide and
harvesting the antibodies.
Another specific aspect of the invention relates
to a method for preparing an antibody, comprising immunizing
a non-human mammal with a polypeptide comprising amino acids
33-213 of SEQ ID NO:6 in an amount effective to produce
2f
CA 02274803 2010-11-12
72249-164
antibodies which bind specifically to the polypeptide and
harvesting the antibodies.
Another specific aspect of the invention relates
to use of a RANK:Fc polypeptide comprising amino acids x to
213 of SEQ ID NO:6, wherein x is amino acid 1, or any one of
amino acids 24-33, inclusive, of SEQ ID NO:61 and an Fc from
a human IgG1 immunoglobulin for inhibiting RANK activity in a
subject having a tumor or neoplastic disease, wherein RANK
activity is the ability of RANK to bind RANK ligand,
andwherein said RANK:FC polypeptide is in an amount
sufficient to inhibit RANK signal transduction in said
subject, wherein RANK comprises SEQ ID NO:6.
Another specific aspect of the invention relates
to use of a fusion protein comprising a receptor activator
of NF-KB (RANK) polypeptide linked to a human immunoglobulin
Fc region, wherein said RANK polypeptide has at least 90%
amino acid sequence identity with amino acids 33 to 213 of
SEQ ID NO:6 and is capable of binding a RANKL protein
consisting of the amino acid sequence shown in SEQ ID NO:13,
and further wherein said RANK polypeptide is in an amount
sufficient to inhibit RANK signal transduction in a subject
having a tumor or neoplastic disease.
Another specific aspect of the invention relates
to use of a composition that comprises a receptor activator
of NF-KB (RANK) polypeptide and a physiologically acceptable
carrier, excipient or diluent for treating a subject having
a tumor or neoplastic disease, wherein the RANK polypeptide
has an at least 90% amino acid sequence identity with amino
acids 33-213 of SEQ ID NO:6; is capable of binding a
receptor activator of NF-KB ligand (RANKL) polypeptide that
2g
CA 02274803 2010-11-12
72249-164
consists of the amino acid sequence shown in SEQ ID NO:13;
is in an amount sufficient to inhibit RANK signal
transduction in said subject.
Another specific aspect of the invention relates
to a kit comprising a RANK:Fc polypeptide comprising amino
acids x to 213 of SEQ ID NO:6, wherein x is amino acid 1, or
any one of amino acids 24-33, inclusive, of SEQ ID NO:6, and
an Fc from a human immunoglobulin; together with
instructions for inhibiting RANK activity in a subject
having a tumor or neoplastic disease, wherein said RANK:Fc
polypeptide is in an amount sufficient to inhibit RANK
signal transduction in said subject.
Another specific aspect of the invention relates
to a kit comprising a fusion protein comprising a RANK
polypeptide linked to a human immunoglobulin Fc region,
wherein said RANK polypeptide has at least 90% amino acid
sequence identity with amino acids 33 to 213 of SEQ ID NO:6
and is capable of binding a RANKL protein consisting of the
amino acid sequence shown in SEQ ID NO:13, and further
wherein said RANK polypeptide is in an amount sufficient to
inhibit RANK signal transduction in said subject, together
with instructions for inhibiting RANK activity in a subject
having a tumor or neoplastic disease.
Another specific aspect of the invention relates
to a kit comprising a composition that comprises a receptor
activator of NF-KB (RANK) polypeptide capable of binding a
RANKL polypeptide that consists of the amino acid sequence
shown in SEQ ID NO:13, wherein the RANK polypeptide has an
at least 90% amino acid sequence identity with amino acids
33-213 of SEQ ID NO:6 and a physiologically acceptable
2h
CA 02274803 2010-11-12
72249-164
carrier, excipient or diluent together with instructions for
treating a subject having a tumor or neoplastic disease.
Another specific aspect of the invention relates
to a method of screening a molecule for its capacity to
antagonize or agonize receptor activator of NF-KB (RANK),
which comprises SEQ ID NO:6, said method comprising: a)
contacting a RANK polypeptide or RANK fusion polypeptide as
described herein with a receptor activator of NF-KB ligand
(RANKL) polypeptide, which comprises SEQ ID NO: 13, or a
fragment thereof that binds said RANK polypeptide, in the
presence of the molecule under conditions that permit
binding of the RANK and RANKL polypeptides; (b) detecting
the binding of the RANK and RANKL polypeptides; (c)
determining that the molecule is a RANK antagonist if the
amount of RANK-RANKL binding detected is decreased when the
molecule is present; and (d) determining that the molecule
is a RANK agonist if the amount of RANK-RANKL binding
detected is increased when the molecule is present.
Another specific aspect of the invention relates
to a method of screening a molecule for its capacity to
agonize or antagonize RANK, said method comprising the steps
of: (a) incubating the molecule with a cell that expresses a
RANK polypeptide on its surface, wherein said RANK
polypeptide has an amino acid sequence selected from the
group consisting of: (i) amino acids x to y of SEQ ID NO:6,
wherein x is any one of amino acids 1-33 and y is amino acid
616; and (ii) a polypeptide that is at least 80% identical
to amino acids 33-616 of SEQ ID NO:6 and that is capable of
binding a RANKL polypeptide consisting of amino acids 1-317
of SEQ ID NO:13; (b) measuring a biological activity
2i
CA 02274803 2010-11-12
72249-164
associated with the RANK polypeptide, wherein said
biological activity is selected from the group consisting
of: (i) binding a RANKL polypeptide, which comprises SEQ ID
NO: 13, or a RANK-binding fragment thereof; and (ii) ability
of the RANK polypeptide to bind TRAF1, TRAF2, TRAF3, TRAF5
or TRAF6; and (c) determining that the molecule is a RANK
agonist if the biological activity is increased in the
presence of the molecule or that the molecule is a RANK
antagonist if the biological activity is decreased in the
presence of the molecule.
2j
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 demonstrates the influence of RANK.Fc and hRANKL on activated T cell
growth. Human peripheral blood T cells were cultured as described in Example
12; viable
T cell recovery was determined by triplicate trypan blue countings.
Figure 2 illustrates the ability of RANKL to induce human DC cluster
formation.
Functionally mature dendritic cells (DC) were generated in vitro from CD34+
bone marrow
(BM) progenitors and cultured as described in Example 13. CD 1 a+ DC were
cultured in a
cytokine cocktail alone (Figure 2A), in cocktail plus CD40L (Figure 2B), RANKL
(Figure
2C), or heat inactivated (oH) RANKL (Figure 2D), and then photographed using
an
inversion microscope.
Figure 3 demonstrates that RANKL enhances DC allo-stimulatory capacity.
Allogeneic T cells were incubated with varying numbers of irradiated DC
cultured as
described in Example 13. The cultures were pulsed with [3H]-thymidine and the
cells
harvested onto glass fiber sheets for counting. Values represent the mean
standard
deviation (SD) of triplicate cultures.
Figure 4 presents an alignment of human RANK with other TNFR family members
in the region of structurally conserved extracellular cysteine-rich
pseudorepeats. Predicted
disulfide linkages (DS 1-DS3) are indicated. RANK and CD40 contain identical
amino acid
substitutions (CAH, CAG) eliminating DS2 in the second pseudorepeat.
Figure 5 presents an alignment of human RANKL with other TNF family members.
DETAILED DESCRIPTION OF THE INVENTION
A novel partial cDNA insert with a predicted open reading frame having some
similarity to CD40 was identified in a database containing sequence
information from
cDNAs generated from human bone marrow-derived dendritic cells (DC). The
insert was
used to hybridize to colony blots generated from a DC cDNA library containing
full-length
cDNAs. Several colony hybridizations were performed, and two clones (SEQ ID
NOs: I
and 3) were isolated. SEQ ID NO:5 shows the nucleotide and amino acid sequence
of a
predicted full-length protein based on alignment of the overlapping sequences
of SEQ ID
NOs:I and 3.
RANK is a member of the TNF receptor superfamily; it most closely resembles
CD40 in the extracellular region. Similar to CD40, RANK associates with TRAF2
and
TRAF3 (as determined by co-immunoprecipitation assays substantially as
described by
Rothe et al., Cell 83:1243, 1995). TRAFs are critically important in the
regulation of the
immune and inflammatory response. Through their association with various
members of
the TNF receptor superfamily, a signal is transduced to a cell. That signal
results in the
proliferation, differentiation or apoptosis of the cell, depending on which
receptor(s) is/are
triggered and which TRAF(s) associate with the receptor(s); different signals
can be
3
SUBSTITUTE SHEET (RULE 26)
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
transduced to a cell via coordination of various signaling events. Thus, a
signal transduced
through one member of this family may be proliferative, differentiative or
apoptotic,
depending on other signals being transduced to the cell, and/or the state of
differentiation of
the cell. Such exquisite regulation of this proliferative/apoptotic pathway is
necessary to
develop and maintain protection against pathogens; imbalances can result in
autoimmune
disease.
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.
Moreover, the post-natal lethality of mice having a targeted disruption of the
TRAF3
gene demonstrates the importance of this molecule not only in the immune
response but in
development. The isolation of RANK, as a protein that associates with TRAF3,
and its
ligand will allow further definition of this signaling pathway, and
development of
diagnostic and therapeutic modalities for use in the area of autoimmune and/or
inflammatory
disease.
DNAs. Proteins and Analogs
The present invention provides isolated RANK polypeptides and analogs (or
muteins) thereof having an activity exhibited by the native molecule (i.e,
RANK muteins
that bind specifically to a RANK ligand expressed on cells or immobilized on a
surface or
to RANK-specific antibodies; soluble forms thereof that inhibit RANK ligand-
induced
signaling through RANK). Such proteins are substantially free of contaminating
endogenous materials and, optionally, without associated native-pattern
glycosylation.
Derivatives of RANK within the scope of the invention also include various
structural
forms of the primary proteins which retain biological activity. Due to the
presence of
ionizable amino and carboxyl groups, for example, a RANK protein may be in the
form of
acidic or basic salts, or may be in neutral form. Individual amino acid
residues may also be
modified by oxidation or reduction. The primary amino acid structure may be
modified by
forming covalent or aggregative conjugates with other chemical moieties, such
as glycosyl
groups, lipids, phosphate, acetyl groups and the like, or by creating amino
acid sequence
mutants. Covalent derivatives are prepared by linking particular functional
groups to amino
acid side chains or at the N- or C-termini.
Derivatives of RANK may also be obtained by the action of cross-linking
agents,
such as M-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, at
cysteine and
4
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
lysine residues. The inventive proteins may also be covalently bound through
reactive side
groups to various insoluble substrates, such as cyanogen bromide-activated,
bisoxirane-
activated, carbonyldiimidazole-activated or tosyl-activated agarose
structures, or by
adsorbing to polyolefin surfaces (with or without glutaraldehyde cross-
linking). Once
bound to a substrate, the proteins may be used to selectively bind (for
purposes of assay or
purification) antibodies raised against the proteins or against other proteins
which are
similar to RANK or RANKL, as well as other proteins that bind RANK or RANKL or
homologs thereof.
Soluble forms of RANK are also within the scope of the invention. The
nucleotide
and predicted amino acid sequence of the RANK is shown in SEQ ID NOs:I through
6.
Computer analysis indicated that the protein has an N-terminal signal peptide;
the predicted
cleavage site follows residue 24. Those skilled in the art will recognize that
the actual
cleavage site may be different than that predicted by computer analysis. Thus,
the N-
terminal amino acid of the cleaved peptide is expected to be within about five
amino acids
on either side of the predicted, preferred cleavage site following residue 24.
Moreover a
soluble form beginning with amino acid 33 was prepared; this soluble form
bound
RANKL. The signal peptide is predicted to be followed by a 188 amino acid
extracellular
domain, a 21 amino acid transmembrane domain, and a 383 amino acid cytoplasmic
tail.
Soluble RANK comprises the signal peptide and the extracellular domain
(residues
1 to 213 of SEQ ID NO:6) 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:6. Moreover, fragments of the extracellular domain will also provide
soluble forms
of RANK. 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 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.
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.
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
5
CA 02274803 1999-06-15
WO 98/28424 PCTIUS97/23866
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 (X-factor
leader).
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., BiolTechnology 6:1204 (1988). 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. Fusion proteins capped with such peptides
may also be
resistant to intracellular degradation in E. coli.
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, having a
nucleotide
an amino acid sequence set forth in SEQ ID NO:8. 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 FcyRI. Canfield and Morrison
(J. Exp. Med.
173:1483; 1991) reported that Leu(234) and Leu(235)were critical to high
affinity binding of
IgG3 to FcyRI 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, Fc region to decrease the affinity of
IgG, 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.
In another embodiment, RANK proteins further comprise an oligomerizing peptide
such as a leucine zipper domain. Leucine zippers were originally identified in
several
DNA-binding proteins (Landschulz et al., Science 240:1759, 1988). Leucine
zipper
domain is a term used to refer to a conserved peptide domain present in these
(and other)
proteins, which is responsible for dimerization of the proteins. The leucine
zipper domain
(also referred to herein as an oligomerizing, or oligomer-forming, domain)
comprises a
repetitive heptad repeat, with four or five leucine residues interspersed with
other amino
acids. Examples of leucine 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
leucine 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
6
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
and jun comprise leucine zipper domains preferentially form a heterodimer
(O'Shea et al.,
Science 245:646, 1989; Turner and Tjian, Science 243:1689, 1989). The leucine
zipper
domain is necessary for biological activity (DNA binding) in these proteins.
The fusogenic proteins of several different viruses, including paramyxovirus,
coronavirus, measles virus and many retroviruses, also possess leucine zipper
domains
(Buckland and Wild, Nature 338:547,1989; Britton, Nature 353:394, 1991;
Delwart and
Mosialos, AIDS Research and Human Retroviruses 6:703, 1990). The leucine
zipper
domains in these fusogenic viral proteins are near the transmembrane region of
the proteins;
it has been suggested that the leucine zipper domains could contribute to the
oligomeric
structure of the fusogenic proteins. Oligomerization of fusogenic viral
proteins is involved
in fusion pore formation (Spruce et al, Proc. Natl. Acad. Sci. U.S.A. 88:3523,
1991).
Leucine zipper domains have also been recently reported to play a role in
oigomerization of
heat-shock transcription factors (Rabindran et al., Science 259:230, 1993).
Leucine zipper domains fold as short, parallel coiled coils. (O'Shea et al.,
Science
254:539; 1991) The general architecture of the parallel coiled coil has been
well
characterized, with a "knobs-into-holes" packing as proposed by Crick in 1953
(Acta
Crystallogr. 6:689). The dimer formed by a leucine zipper domain is stabilized
by the
heptad repeat, designated (abcdefg)õ according to the notation of McLachlan
and Stewart
(J. Mol. Biol. 98:293; 1975), in which residues a and d are generally
hydrophobic
residues, with d being a leucine, which line up on the same face of a helix.
Oppositely-
charged residues commonly occur at positions g and e. Thus, in a parallel
coiled coil
formed from two helical leucine zipper domains, the "knobs" formed by the
hydrophobic
side chains of the first helix are packed into the "holes" formed between the
side chains of
the second helix.
The leucine residues at position d contribute large hydrophobic stabilization
energies, and are important for dimer formation (Krystek et al., Int. J.
Peptide Res.
38:229, 1991). Lovejoy et al. recently reported the synthesis of a triple-
stranded a-helical
bundle in which the helices run up-up-down (Science 259:1288, 1993). Their
studies
confirmed that hydrophobic stabilization energy provides the main driving
force for the
formation of coiled coils from helical monomers. These studies also indicate
that
electrostatic interactions contribute to the stoichiometry and geometry of
coiled coils.
Several studies have indicated that conservative amino acids may be
substituted for
individual leucine residues with minimal decrease in the ability to dimerize;
multiple
changes, however, usually result in loss of this ability (Landschulz et al.,
Science
243:1681, 1989; Turner and Tjian, Science 243:1689, 1989; Hu et al., Science
250:1400,
1990). van Heekeren et al. reported that a number of different amino residues
can be
substituted for the leucine residues in the leucine zipper domain of GCN4, and
further
found that some GCN4 proteins containing two leucine substitutions were weakly
active
7
CA 02274803 2005-08-03
72249-164
(Nucl. Acids Res. 20:3721, 1992). Mutation of the first and second heptadic
leucines of
the leucine zipper domain of the measles virus fusion protein (MVF) did not
affect
syncytium formation (a measure of virally-induced cell fusion); however,-
mutation of all
four leucine residues prevented fusion completely (Buckland et al., J. Gen.
Virol. 73:1703,
1992). None of the mutations affected the ability of MVF to form a tetramer.
Amino acid substitutions in the a and d residues of a synthetic peptide
representing
the GCN4 leucine zipper domain have been found to change the oligomerization
properties
of the leucine zipper domain (Alber, Sixth Symposium of the Protein Society,
San Diego,
CA). When all residues at position a are changed to isoleucine, the leucine
zipper still
forms a parallel dimer. When, in addition to this change, all leucine residues
at position d
are also'changed to isoleucine, the resultant peptide spontaneously forms a
trimeric parallel'
coiled coil in solution. Substituting all amino acids at position d with
isoleucine and at
position a with leucine results in a peptide that tetramerizes. Peptides
containing these
substitutions are still referred to as leucine zipper domains.
Also included within the scope of the invention are fragments or derivatives
of the
intracellular domain of RANK. Such fragments are prepared by any of the herein-
mentioned techniques, and include peptides that are identical to the
cytoplasmic domain of
RANK as shown in SEQ ID NO:6, or of murine RANK as shown in SEQ ID NO:15, and
those that comprise a portion of the cytoplasmic region. All techniques used
in preparing
soluble forms may also be used in preparing fragments or analogs of the
cytoplasmic
domain (i.e., RT-PCR techniques or use of selected restriction enzymes to
prepare
truncations). DNAs encoding all or a fragment of the intracytoplasmic domain
will be
useful in identifying other proteins that are associated with RANK signalling,
for example
using the immunoprecipitation techniques described herein, or another
technique such as a
yeast two-hybrid system (Rothe et al., supra).
The present invention also includes RANK with or without associated native-
pattern
glycosylation. Proteins expressed in yeast or mammalian expression systems,
e.g., COS-7
cells, may be similar or slightly different in molecular weight and
glycosylation pattern than
the native molecules, depending upon the expression system. Expression of DNAs
encoding the inventive proteins in bacteria such as E. coli provides non-
glycosylated
molecules. Functional mutant analogs of RANK protein having inactivated N-
glycosylation sites can be produced by oligonucleotide synthesis and ligation
or by site-
specific mutagenesis techniques. These analog proteins can be produced in a
homogeneous, reduced-carbohydrate form in good yield using yeast expression
systems.
N-glycosylation sites in eukaryotic proteins are characterized by the amino
acid triplet Asn-
A,-Z, where A, is any amino acid except Pro, and Z is Ser or Thr. In this
sequence,
asparagine provides a side chain amino group for covalent attachment of
carbohydrate.
Such a site can be eliminated by substituting another amino acid for Asti or
for residue Z,
8
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
deleting Asn or Z, or inserting a non-Z amino acid between Al and Z, or an
amino acid
other than Asn between Asn and A,.
RANK protein derivatives may also be obtained by mutations of the native RANK
or subunits thereof. A RANK mutated protein, as referred to herein, is a
polypeptide
homologous to a native RANK protein, respectively, but which has an amino acid
sequence
different from the native protein because of one or a plurality of deletions,
insertions or
substitutions. The effect of any mutation made in a DNA encoding a mutated
peptide may
be easily determined by analyzing the ability of the mutated peptide to bind
its
counterstructure in a specific manner. Moreover, activity of RANK analogs,
muteins or
derivatives can be determined by any of the assays described herein (for
example, inhibition
of the ability of RANK to activate transcription).
Analogs of the inventive proteins may be constructed by, for example, making
various substitutions of residues or sequences or deleting terminal or
internal residues or
sequences not needed for biological activity. For example, cysteine residues
can be deleted
or replaced with other amino acids to prevent formation of incorrect
intramolecular disulfide
bridges upon renaturation. Other approaches to mutagenesis involve
modification of
adjacent dibasic amino acid residues to enhance expression in yeast systems in
which
KEX2 protease activity is present.
When a deletion or insertion strategy is adopted, the potential effect of the
deletion
or insertion on biological activity should be considered. Subunits of the
inventive proteins
may be constructed by deleting terminal or internal residues or sequences.
Soluble forms
of RANK can be readily prepared and tested for their ability to inhibit RANK-
induced NF-
KB activation. Polypeptides corresponding to the cytoplasmic regions, and
fragments
thereof (for example, a death domain) can be prepared by similar techniques.
Additional
guidance as to the types of mutations that can be made is provided by a
comparison of the
sequence of RANK to proteins that have similar structures, as well as by
performing
structural analysis of the inventive RANK proteins.
Generally, substitutions should be made conservatively; i.e., the most
preferred
substitute amino acids are those which do not affect the biological activity
of RANK (i.e.,
ability of the inventive proteins to bind antibodies to the corresponding
native protein in
substantially equivalent a manner, the ability to bind the counterstructure in
substantially the
same manner as the native protein, the ability to transduce a RANK signal, or
ability to
induce NF-KB activation upon overexpression in transient transfection systems.
for
example). Examples of conservative substitutions include substitution of amino
acids
outside of the binding domain(s) (either ligand/receptor or antibody binding
areas for the
extracellular domain, or regions that interact with other, intracellular
proteins for the
cytoplasmic domain), and substitution of amino acids that do not alter the
secondary and/or
9
CA 02274803 2005-08-03
72249-164
tertiary structure of the native protein. Additional examples include;
substituting one
aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another,
or substitutions
of one polar residue for another, such as between Lys and Arg; Glu and Asp; or
Gin and
Asn. Other such conservative substitutions, for example, substitutions of
entire regions
having similar hydrophobicity characteristics, are well known.
Mutations in nucleotide sequences constructed for expression of analog
proteins or
fragments thereof must, of course, preserve the reading frame phase of the
coding
sequences and preferably will not create complementary regions that could
hybridize to
produce secondary mRNA structures such as loops or hairpins which would
adversely
affect translation of the mRNA.
Not all mutations in the nucleotide sequence which encodes a RANK protein or
fragments thereof will be expressed in the final product, for example,
nucleotide
substitutions may be made to enhance expression, primarily to avoid secondary
structure
loops in the transcribed mRNA (see EPA 75,444A) or to
provide codons that are more readily translated by the selected host, e.g.,
the well-known
E. coli preference codons for E. coli expression.
Although a mutation site may be predetermined, it is not necessary that the
nature of
the mutation per se be predetermined. For example, in order to select for
optimum
characteristics of mutants, random mutagenesis may be conducted and the
expressed
mutated proteins screened for the desired activity. Mutations can be
introduced at particular
loci by synthesizing oligonucleotides containing a mutant sequence, flanked by
restriction
sites enabling ligation to fragments of the native sequence. Following
ligation, the resulting
reconstructed sequence encodes an analog having the desired amino acid
insertion,
substitution, or deletion.
Alternatively, oligonucleotide-directed site-specific mutagenesis procedures
can be
employed to provide an altered gene having particular codons altered according
to the
substitution, deletion, or insertion required. Exemplary methods of making the
alterations
set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et
al. (,Gene
37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.
(Genetic
Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patent NOs.
4,518,584 and 4,737,462 disclose suitable techniques.
Other embodiments of the inventive proteins include RANK polypeptides encoded
by DNAs capable of hybridizing to the DNA of SEQ ID NO:5, under moderately
stringent
conditions (prewashing solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and
hybridization conditions of 50 C, 5 X SSC, overnight) to the DNA sequences
encoding
RANK, or more preferably under, stringent conditions (for example,
hybridization in 6 X
SSC at 63 C overnight; washing in 3 X SSC at 55 C), and other sequences which
are
CA 02274803 2005-08-03
72249-164
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.6. 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.
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 -
The biological activity of RANK analogs or muteins can be determined by
testing
the ability of the analogs or muteins to inhibit activation of transcription,
for example as
described in the Examples herein. Alternatively, suitable assays, for example,
an enzyme
immunoassay or a dot blot, employing an antibody that binds native RANK, or a
soluble
form of RANKL, can be used to assess the activity of RANK analogs or muteins,
as can
assays that employ cells expressing RANKL. Suitable assays also include, for
example,
signal transduction assays and methods that evaluate the ability of the
cytoplasmic region of
RANK to associate with other intracellular proteins (i.e., TRAFs 2 and 3)
involved in
signal transduction will also be useful to assess the activity of RANK analogs
or muteins.
Such methods are well known in the art.
Fragments of the RANK nucleotide sequences are also useful. In one embodiment,
such fragments comprise at least about 17 consecutive nucleotides, preferably
at least about
nucleotides, more preferably at least 30 consecutive nucleotides, of the RANK
DNA
25 disclosed herein. DNA and RNA complements of such fragments are provided
herein,
along with both single-stranded and double-stranded forms of the RANK DNA of
SEQ ID
NO:5, and those encoding the aforementioned polypeptides. A fragment of RANK
DNA
generally comprises at least about 17 nucleotides, preferably from about 17 to
about 30
nucleotides. Such nucleic acid fragments (for example, a probe corresponding
to the
extracellular domain of RANK) are used as a probe or as primers in a
polymerase chain
reaction (PCR).
The probes also find use in detecting the presence of RANK nucleic acids in
in vitro assays and in such procedures as Northern and Southern blots. Cell
types
expressing RANK can be identified as well. Such procedures are well known, and
the
skilled artisan can choose a probe of suitable length, depending on the
particular intended
application. For PCR, 5' and 3' primers corresponding to the termini of a
desired RANK
DNA sequence are employed to amplify that sequence, using conventional
techniques.
11
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
Other useful fragments of the RANK nucleic acids are antisense or sense
oligonucleotides comprising a single-stranded nucleic acid sequence (either
RNA or DNA)
capable of binding to target RANK mRNA (sense) or RANK DNA (antisense)
sequences.
The ability to create an antisense or a sense oligonucleotide, based upon a
cDNA sequence
for a given protein is described in, for example, Stein and Cohen, Cancer Res.
48:2659,
1988 and van der Krol et al., BioTechniques 6:958, 1988.
Uses of DNAs, Proteins and Analogs
The RANK DNAs, proteins and analogs described herein will have numerous uses,
including the preparation of pharmaceutical compositions. For example, soluble
forms of
RANK will be useful as antagonists of RANK-mediated NF-KB activation, as well
as to
inhibit transduction of a signal via RANK. RANK compositions (both protein and
DNAs)
will also be useful in development of both agonistic and antagonistic
antibodies to RANK.
The inventive DNAs are useful for the expression of recombinant proteins, and
as probes
for analysis (either quantitative or qualitative) of the presence or
distribution of RANK
transcripts.
The inventive proteins will also be useful in preparing kits that are used to
detect
soluble RANK or RANKL, or monitor RANK-related activity, for example, in
patient
specimens. RANK proteins will also find uses in monitoring RANK-related
activity in
other samples or compositions, as is necessary when screening for antagonists
or mimetics
of this activity (for example, peptides or small molecules that inhibit or
mimic, respectively,
the interaction). A variety of assay formats are useful in such kits,
including (but not
limited to) ELISA, dot blot, solid phase binding assays (such as those using a
biosensor),
rapid format assays and bioassays.
The purified RANK according to the invention will facilitate the discovery of
inhibitors of RANK, and thus, inhibitors of an inflammatory response (via
inhibition of
NF-KB activation). The use of a purified RANK polypeptide in the screening for
potential
inhibitors is important and can virtually eliminate the possibility of
interfering reactions with
contaminants. Such a screening assay can utilize either the extracellular
domain of RANK,
the intracellular domain, or a fragment of either of these polypeptides.
Detecting the
inhibiting activity of a molecule would typically involve use of a soluble
form of RANK
derived from the extracellular domain in a screening assay to detect molecules
capable of
binding RANK and inhibiting binding of, for example, an agonistic antibody or
RANKL,
or using a polypeptide derived from the intracellular domain in an assay to
detect inhibition
of the interaction of RANK and other, intracellular proteins involved in
signal transduction.
Moreover, in vitro systems can be used to ascertain the ability of molecules
to
antagonize or agonize RANK activity. Included in such methods are uses of RANK
chimeras, for example, a chimera of the RANK intracellular domain and an
extracellular
12
*rB
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
domain derived from a protein having a known ligand. The effects on signal
transduction
of various molecule can then be monitored by utilizing the known ligand to
transduce a
signal.
In addition, RANK polypeptides can also be used for structure-based design of
RANK-inhibitors. Such structure-based design is also known as "rational drug
design."
The RANK polypeptides can be three-dimensionally analyzed by, for example, X-
ray
crystallography, nuclear magnetic resonance or homology modeling, all of which
are well-
known methods. The use of RANK structural information in molecular modeling
software
systems to assist in inhibitor design is also encompassed by the invention.
Such computer-
assisted modeling and drug design may utilize information such as chemical
conformational
analysis, electrostatic potential of the molecules, protein folding, etc. A
particular method
of the invention comprises analyzing the three dimensional structure of RANK
for likely
binding sites of substrates, synthesizing a new molecule that incorporates a
predictive
reactive site, and assaying the new molecule as described above.
Expression of Recombinant RANK
The proteins of the present invention are preferably produced by recombinant
DNA
methods by inserting a DNA sequence encoding RANK protein or an analog thereof
into a
recombinant expression vector and expressing the DNA sequence in a recombinant
expression system under conditions promoting expression. DNA sequences
encoding the
proteins provided by this invention can be assembled from cDNA fragments and
short
oligonucleotide linkers, or from a series of oligonucleotides, to provide a
synthetic gene
which is capable of being inserted in a recombinant expression vector and
expressed in a
recombinant transcriptional unit.
Recombinant expression vectors include synthetic or cDNA-derived DNA
fragments encoding RANK, or homologs, muteins or bioequivalent analogs
thereof,
operably linked to suitable transcriptional or translational regulatory
elements derived from
mammalian, microbial, viral or insect genes. Such regulatory elements include
a
transcriptional promoter, an optional operator sequence to control
transcription, a sequence
encoding suitable mRNA ribosomal binding sites, and sequences which control
the
termination of transcription and translation, as described in detail below.
The ability to
replicate in a host, usually conferred by an origin of replication, and a
selection gene to
facilitate recognition of transformants may additionally be incorporated.
DNA regions are operably linked when they are functionally related to each
other.
For example, DNA for a signal peptide (secretory leader) is operably linked to
DNA for a
polypeptide if it is expressed as a precursor which participates in the
secretion of the
polypeptide; a promoter is operably linked to a coding sequence if it controls
the
transcription of the sequence; or a ribosome binding site is operably linked
to a coding
13
CA 02274803 1999-06-15
WO 98/28424 PCTIUS97/23866
sequence if it is positioned so as to permit translation. Generally, operably
linked means
contiguous and, in the case of secretory leaders, contiguous and in reading
frame. DNA
sequences encoding RANK, or homologs or analogs thereof which are to be
expressed in a
microorganism will preferably contain no introns that could prematurely
terminate
transcription of DNA into mRNA.
Useful expression vectors for bacterial use can comprise a selectable marker
and
bacterial origin of replication derived from commercially available plasmids
comprising
genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such
commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala,
Sweden) and pGEMI (Promega Biotec, Madison, WI, USA). These pBR322 "backbone"
sections are combined with an appropriate promoter and the structural sequence
to be
expressed. E. coli is typically transformed using derivatives of pBR322, a
plasmid derived
from an E. coli species (Bolivar et al., Gene 2:95, 1977). pBR322 contains
genes for
ampicillin and tetracycline resistance and thus provides simple means for
identifying
transformed cells.
Promoters commonly used in recombinant microbial expression vectors include
the
P-lactamase (penicillinase) and lactose promoter system (Chang et al., Nature
275:615,
1978; and Goeddel et al., Nature 281:544, 1979), the tryptophan (trp) promoter
system
(Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EPA 36,776) and tac
promoter
(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, p.
412, 1982). A particularly useful bacterial expression system employs the
phage ~PL
promoter and c1857ts thermolabile repressor. Plasmid vectors available from
the American
Type Culture Collection which incorporate derivatives of the k PL promoter
include
plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and pPLc28,
resident in
E. coli RR1 (ATCC 53082).
Suitable promoter sequences in yeast vectors include the promoters for
metallothionein, 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; and
Holland et al., Biochem. 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. Suitable vectors and
promoters
for use in yeast expression are further described in R. Hitzeman et al., EPA
73,657.
Preferred yeast vectors can be assembled using DNA sequences from pBR322 for
selection and replication in E. coli (Ampr gene and origin of replication) and
yeast DNA
sequences including a glucose-repressible ADH2 promoter and a-factor secretion
leader.
The ADH2 promoter has been described by Russell et al. (J. Biol. Chem.
258:2674, 1982)
and Beier et al. (Nature 300:724, 1982). The yeast a-factor leader, which
directs secretion
14
*rB
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
of heterologous proteins, can be inserted between the promoter and the
structural gene to be
expressed. See, e.g., Kurjan et al., Cell 30:933, 1982; and Bitter et al.,
Proc. Natl. Acad.
Sci. USA 81:5330, 1984. The leader sequence may be modified to contain, near
its 3' end,
one or more useful restriction sites to facilitate fusion of the leader
sequence to foreign
genes.
The transcriptional and translational control sequences in expression vectors
to be
used in transforming vertebrate cells may be provided by viral sources. For
example,
commonly used promoters and enhancers are derived from Polyoma, Adenovirus 2,
Simian
Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the
SV40
viral genome, for example, SV40 origin, early and late promoter, enhancer,
splice, and
polyadenylation sites may be used to provide the other genetic elements
required for
expression of a heterologous DNA sequence. The early and late promoters are
particularly
useful because both are obtained easily from the virus as a fragment which
also contains the
SV40 viral origin of replication (Fiers et al., Nature 273:113, 1978). Smaller
or larger
SV40 fragments may also be used, provided the approximately 250 bp sequence
extending
from the Hind III site toward the BglI site located in the viral origin of
replication is
included. Further, viral genomic promoter, control and/or signal sequences may
be
utilized, provided such control sequences are compatible with the host cell
chosen.
Exemplary vectors can be constructed as disclosed by Okayama and Berg (Mol.
Cell. Biol.
3:280, 1983).
A useful system for stable high level expression of mammalian receptor cDNAs
in
C127 murine mammary epithelial cells can be constructed substantially as
described by
Cosman et al. (Mol. Immunol. 23:935, 1986). A preferred eukaryotic vector for
expression of RANK DNA is referred to as pDC406 (McMahan et al., EMBO J.
10:2821,
1991), and includes regulatory sequences derived from SV40, human
immunodeficiency
virus (HIV), and Epstein-Barr virus (EBV). Other preferred vectors include
pDC409 and
pDC410, which are derived from pDC406. pDC410 was derived from pDC406 by
substituting the EBV origin of replication with sequences encoding the SV40
large T
antigen. pDC409 differs from pDC406 in that a Bgl II restriction site outside
of the
multiple cloning site has been deleted, making the Bgl II site within the
multiple cloning site
unique.
A useful cell line that allows for episomal replication of expression vectors,
such as
pDC406 and pDC409, which contain the EBV origin of replication, is CV-1/EBNA
(ATCC
CRL 10478). The CV-1/EBNA cell line was derived by transfection of the CV-1
cell line
with a gene encoding Epstein-Barr virus nuclear antigen-1 (EBNA-1) and
constitutively
express EBNA-1 driven from human CMV immediate-early enhancer/promoter.
CA 02274803 2005-08-03
72249-164
Host Cells
Transformed host cells are cells which have been transformed or transfected
with
expression vectors constructed using recombinant DNA techniques and which
contain
sequences encoding the proteins of the present invention. Transformed host
cells may
express the desired protein (RANK, or homologs or analogs thereof), but host
cells
transformed for purposes of cloning or amplifying the inventive DNA do not
need to
express the protein. Expressed proteins will preferably be secreted into the
culture
supernatant, depending on the DNA selected, but may be deposited in the cell
membrane.
Suitable host cells for expression of proteins include prokaryotes, yeast or
higher
eukaryotic cells under the control of appropriate promoters. Prokaryotes
include gram
negative or gram positive organisms, for example E. coli or Bacillus spp.
Higher
eukaryotic cells include established cell lines of mammalian origin as
described below.
Cell-free translation systems could also be employed to produce proteins using
RNAs
derived from the DNA constructs disclosed herein. Appropriate cloning and
expression
vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts
are described by
Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York,
1985).
Prokaryotic expression hosts may be used for expression of RANK, or homologs
or analogs thereof that do not require extensive proteolytic and disulfide
processing.
Prokaryotic expression vectors generally comprise one or more phenotypic
selectable
markers, for example a gene encoding proteins conferring antibiotic resistance
or supplying
an autotrophic requirement, and an origin of replication recognized by the
host to ensure
amplification within the host. Suitable prokaryotic hosts for transformation
include E. coli,
Bacillus subtilis, Salmonella typhimurium, and various species within the
genera
Pseudomonas, Streptomyces, and Staphylococcus, although others may also be
employed
as a matter of choice.
Recombinant RANK may also be expressed in yeast hosts, preferably from the
Saccharomyces species, such as S. cerevisiae. Yeast of other genera, such as
Pichia or
Kluyveromyces may also be employed. Yeast vectors will generally contain an
origin of
replication from the 2g yeast plasmid or an autonomously replicating sequence
(ARS),
promoter, DNA encoding the protein, sequences for polyadenylation and
transcription
termination and a selection gene. Preferably, yeast vectors will include an
origin of
replication and selectable marker permitting transformation of both yeast and
E. coli, e.g.,
the ampicillin resistance gene of E. coli and S. cerevisiae trpl gene, which
provides a
selection marker for a mutant strain of yeast lacking the ability to grow in
tryptophan, and a
promoter derived from a highly expressed yeast gene to induce transcription of
a structural
sequence downstream. The presence of the trpl lesion in the yeast host cell
genome then
16
CA 02274803 2005-08-03
72249-164
provides an effective environment for detecting transformation by growth in
the absence of
tryptophan.
Suitable yeast transformation protocols are known to those of skill in the
art; an
exemplary technique is described by Hinnen et al., Proc. Natl. Acad. Sci. USA
75:1929,
1978, selecting for Trp+ transformants in a selective medium consisting of
0.67% yeast
nitrogen base, 0.5% casamino acids, 2% glucose, 10 g/ml adenine and 20.tg/ml
uracil.
Host strains transformed by vectors comprising the ADH2 promoter may be grown
for
expression in a rich medium consisting of I % yeast extract, 2% peptone, and
I% glucose
supplemented with 80 jig/nil adenine and 80 .tg/ml uracil. Derepression of the
ADH2
promoter occurs upon exhaustion of medium glucose. Crude yeast supernatants
are
fiarvested by filtration and held at 4 C prior to further purification.
Various mammalian or insect cell culture systems can be employed to express
recombinant protein. Baculovirus systems for production of heterologous
proteins in insect
cells are reviewed by Luckow and Summers, Biolfechnology 6:47 (1988). Examples
of
suitable mammalian host cell lines include the COS-7 lines of monkey kidney
cells,
described by Gluzman (Cell 23:175, 1981), and other cell lines capable of
expressing an
appropriate vector including, for example, CV-I/EBNA (ATCC CRL 10478), L
cells,
C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines. Mammalian
expression vectors may comprise nontranscribed elements such as an origin of
replication,
a suitable promoter and enhancer linked to the gene to be expressed, and other
5' or 3'
flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such
as
necessary ribosome binding sites, a polyadenylation site, splice donor and
acceptor sites,
and transcriptional termination sequences.
Purification of Recombinant RANK
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
AmiconTM or Millipore PelliconTM ultrafiltration unit.
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
17
CA 02274803 1999-06-15
WO 98/28424 PCTIUS97/23866
matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups
are
preferred. Gel filtration chromatography also provides a means of purifying
the inventive
proteins.
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.
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.
Some or all of the foregoing purification steps, in various combinations, can
also be
employed to provide a homogeneous recombinant protein.
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. Secreted recombinant protein resulting from a
large-scale
fermentation can be purified by methods analogous to those disclosed by Urdal
et al. (J.
Chromatog. 296:171, 1984). This reference describes two sequential, reversed-
phase
HPLC steps for purification of recombinant human GM-CSF on a preparative HPLC
column.
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.
18
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
Uses and Administration of RANK Compositions
The present invention provides methods of using therapeutic compositions
comprising an effective amount of a protein and a suitable diluent and
carrier, and methods
for regulating an immune or inflammatory response. The use of RANK in
conjunction
with soluble cytokine receptors or cytokines, or other immunoregulatory
molecules is also
contemplated.
For therapeutic use, purified protein is administered to a patient, preferably
a
human, for treatment in a manner appropriate to the indication. Thus, for
example, RANK
protein compositions administered to regulate immune 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.
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.
Soluble forms of RANK and other RANK antagonists such as antagonistic
monoclonal antibodies can be administered for the purpose of inhibiting RANK-
induced
induction of NF-KB activity. NF-KB is a transcription factor that is utilized
extensively by
cells of the immune system, and plays a role in the inflammatory response.
Thus,
inhibitors of RANK signalling will be useful in treating conditions in which
signalling
through RANK has given rise to negative consequences, for example, toxic or
septic
shock, or graft-versus-host reactions. They may also be useful in interfering
with the role
of NF-KB in cellular transformation. Tumor cells are more responsive to
radiation when
their NF-KB is blocked; thus, soluble RANK (or other antagonists of RANK
signalling)
will be useful as an adjunct therapy for disease characterized by neoplastic
cells that express
RANK.
19
CA 02274803 2005-08-03
72249-164
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.
EXAMPLE]
The example describes the identification and isolation of a DNA encoding a
novel
member of the TNF receptor superfamily. A partial cDNA insert with a predicted
open
reading frame having some similarity to CD40 (a cell-surface antigen present
on the surface
of both normal and neoplastic human B cells that has been shown to play an
important role
in B-cell proliferation and differentiation; Stamenkovic et a]., EMBO J.
8:1403, 1989),
was identified in a database containing sequence information from cDNAs
generated from
human bone marrow-derived dendritic cells (DC). The insert was excised from
the vector
by restriction endonuclease digestion, gel purified. labeled with 32P, and
used to hybridize
to colony blots generated from a DC cDNA library containing larger cDNA
inserts using
high stringency hybridization and washing techniques (hybridization in 5xSSC,
50%
formamide at 42 C overnight, washing in 0.5xSSC at 63 C); other suitable high
stringency
conditions are disclosed in Sambrook et al. in Molecular Cloning: A Laboratory
Manual,
2nd ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; 1989), 9.52-
9.55.
Initial experiments yielded a clone referred to as 9D-8A (SEQ ID NO: 1);
subsequent
analysis indicated that this clone contained all but the extreme 5' end of a
novel cDNA, with
predicted intron sequence at the extreme 5' end (nucleotides 1-92 of SEQ ID
NO: 1).
Additional colony hybridizations were performed, and a second clone was
isolated. The
second clone, referred to as 9D-15C (SEQ ID NO:3), contained the 5' end
without intron
interruption but not the full 3'end. SEQ ID NO:5 shows the nucleotide and
amino acid
sequence of a predicted full-length protein based on alignment of the
overlapping sequences
of SEQ ID NOs: I and 3.
The encoded protein was designated RANK, for receptor activator of NF-icB. The
cDNA encodes a predicted Type I transmembrane protein having 616 amino acid
residues,
with a predicted 24 amino acid signal sequence (the computer predicted
cleavage site is after
Leu24), a 188 amino acid extracellular domain, a 21 amino acid transmembrane
domain,
and a 383 amino acid cytoplasmic tail. The extracellular region of RANK
displayed
significant amino acid homology (38.5% identity, 52.3% similarity) to CD40. A
cloning
vector (pB]uescriptSKTM-) containing human RANK sequence, designated
pBluescript:huRANK (in E. coli DHIOB), was deposited with the American Type
Culture
Collection, Manassas, VA (ATCC) on December 20, 1996, under terms of the
Budapest
Treaty, and given accession number 98285.
CA 02274803 2005-08-03
72249-164
EXAMPLE 2
This example describes construction of a RANK DNA construct to express a
RANK/Fc fusion protein. A soluble form of RANK fused to the Fc region of human
IgG,
was constructed in the mammalian expression vector pDC409 (USSN 08/571,579).
This
expression vector encodes the leader sequence of the Cytomegalovirus (CMV)
open reading
frame R27080 (SEQ ID NO:9), followed by amino acids 33-213 of RANK, followed
by a
mutated form of the constant domain of human IgG, that exhibits reduced
affinity for Fc
receptors (SEQ ID NO:8; for the fusion protein, the Fc portion of the
construct consisted of
Arg3 through Lys232). An alternative expression vector encompassing amino
acids 1-213
of RANK (using the native leader sequence) followed by the IgG, mutein was
also
-prepared. - Both expression vectors were found to induce high levels of
expression of the
RANK/Fc fusion protein in transfected cells.
To obtain RANK/Fc protein, a RANK/Fc expression plasmid is transfected into
CV-1/EBNA cells, and supernatants are collected for about one week. The
RANK/Fc
fusion protein is purified by means well-known in the art for purification of
Fc fusion
proteins, for example, by protein A sepharoseTM column chromatography
according to
manufacturer's recommendations (i.e., Pharmacia, Uppsala, Sweden). SDS-
polyacrylamide gel electrophoresis analysis indicted that the purified RANK/Fc
protein
migrated with a molecular weight of -55kDa in the presence of a reducing
agent, and at a
molecular weight of -I I OkDa in the absence of a reducing agent.
N-terminal amino acid sequencing of the purified protein made using the CMV
R27080 leader showed 60% cleavage after Ala2O, 20% cleavage after Pro22 and
20%
cleavage after Arg28 (which is the Furin cleavage site; amino acid residues
are relative to
SEQ ID NO:9); N-terminal amino acid analysis of the fusion protein expressed
with the
native leader showed cleavage predominantly after G1n25 (80% after G1n25 and
20% after
Arg23; amino acid residues are relative to SEQ ID NO:6, full-length RANK).
Both fusion
proteins were able to bind a ligand for RANK is a specific manner (i.e., they
bound to the
surface of various cell lines such as a murine thymoma cell line, EL4),
indicating that the
presence of additional amino acids at the N-terminus of RANK does not
interfere with its
ability to bind RANKL. Moreover, the construct comprising the CMV leader
encoded
RANK beginning at amino acid 33; thus, a RANK peptide having an N-terminus at
an
amino acid between Arg23 and Pro33, inclusive, is expected to be able to bind
a ligand for
RANK in a specific manner.
Other members of the TNF receptor superfamily have a region of amino acids
between the transmembrane domain and the ligand binding domain that is
referred to as a
'spacer' region, which is not necessary for ligand binding. In RANK, the amino
acids
between 196 and 213 are predicted to form such a spacer region. Accordingly, a
soluble
form of RANK that terminates with an amino acid in this region is expected to
retain the
21
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
ability to bind a ligand for RANK in a specific manner. Preferred C-terminal
amino acids
for soluble RANK peptides are selected from the group consisting of amino
acids 213 and
196 of SEQ ID NO:6, although other amino acids in the spacer region may be
utilized as a
C-terminus.
EXAMPLE 3
This example illustrates the preparation of monoclonal antibodies against
RANK.
Preparations of purified recombinant RANK, for example, or transfected cells
expressing
high levels of RANK, are employed to generate monoclonal antibodies against
RANK
using conventional techniques, such as those disclosed in U.S. Patent
4,411,993. DNA
encoding RANK can also be used as an immunogen, for example, as reviewed by
Pardoll
and Beckerleg in Immunity 3:165, 1995. Such antibodies are likely to be useful
in
interfering with RANK-induced signaling (antagonistic or blocking antibodies)
or in
inducing a signal by cross-linking RANK (agonistic antibodies), as components
of
diagnostic or research assays for RANK or RANK activity, or in affinity
purification of
RANK.
To immunize rodents, RANK immunogen is emulsified in an adjuvant (such as
complete or incomplete Freund's adjuvant, alum, or another adjuvant, such as
Ribi
adjuvant R700 (Ribi, Hamilton, MT), and injected in amounts ranging from 10-
100 Ag
subcutaneously into a selected rodent, for example, BALB/c mice or Lewis rats.
DNA may
be given intradermally (Raz et al., Proc. Natl. Acad. Sci. USA 91:9519, 1994)
or
intamuscularly (Wang et al., Proc. Natl. Acad. Sci. USA 90:4156, 1993); saline
has been
found to be a suitable diluent for DNA-based antigens. Ten days to three weeks
days later,
the immunized animals are boosted with additional immunogen and periodically
boosted
thereafter on a weekly, biweekly or every third week immunization schedule.
Serum samples are periodically taken by retro-orbital bleeding or tail-tip
excision for
testing by dot-blot assay (antibody sandwich), ELISA (enzyme-linked
immunosorbent
assay), immunoprecipitation, or other suitable assays, including FACS
analysis.
Following detection of an appropriate antibody titer, positive animals are
given an
intravenous injection of antigen in saline. Three to four days later, the
animals are
sacrificed, splenocytes harvested, and fused to a murine myeloma cell line
(e.g., NS I or
preferably Ag 8.653 [ATCC CRL 1580]). Hybridoma cell lines generated by this
procedure are plated in multiple microtiter plates in a selective medium (for
example, one
containing hypoxanthine, aminopterin, and thymidine, or HAT) to inhibit
proliferation of
non-fused cells, myeloma-myeloma hybrids, and splenocyte-splenocyte hybrids.
Hybridoma clones thus generated can be screened by ELISA for reactivity with
RANK, for example, by adaptations of the techniques disclosed by Engvall et
al.,
Immunochem. 8:871 (1971) and in U.S. Patent 4,703,004. A preferred screening
22
CA 02274803 1999-06-15
WO 98/28424 PCTIUS97/23866
technique is the antibody capture technique described by Beckman et al., J.
Immunol.
144:4212 (1990). Positive clones are then injected into the peritoneal
cavities of syngeneic
rodents to produce ascites containing high concentrations (>I mg/ml) of anti-
RANK
monoclonal antibody. The resulting monoclonal antibody can be purified by
ammonium
sulfate precipitation followed by gel exclusion chromatography. Alternatively,
affinity
chromatography based upon binding of antibody to protein A or protein G can
also be
used, as can affinity chromatography based upon binding to RANK protein.
Monoclonal antibodies were generated using RANK/Fc fusion protein as the
immunogen. These reagents were screened to confirm reactivity against the RANK
protein. Using the methods described herein to monitor the activity of the
mAbs, both
blocking (i.e., antibodies that bind RANK and inhibit binding of a ligand to
RANK) and
non-blocking (i.e., antibodies that bind RANK and do not inhibit ligand
binding) were
isolated.
EXAMPLE 4
This example illustrates the induction of NF-KB activity by RANK in 293/EBNA
cells (cell line was derived by transfection of the 293 cell line with a gene
encoding Epstein-
Barr virus nuclear antigen-1 (EBNA-1) that constitutively express EBNA-1
driven from
human CMV immediate-early enhancer/promoter). Activation of NF-KB activity was
measured in 293/EBNA cells essentially as described by Yao et al. (Immunity
3:811,
1995). Nuclear extracts were prepared and analyzed for NF-KB activity by a gel
retardation
assay using a 25 base pair oligonucleotide spanning the NF-KB binding sites.
Two million
cells were seeded into 10 cm dishes two days prior to DNA transfection and
cultured in
DMEM-F12 media containing 2.5% FBS (fetal bovine serum). DNA transfections
were
performed as described herein for the IL-8 promoter/reporter assays.
Nuclear extracts were prepared by solubilization of isolated nuclei with 400
mM
NaCl (Yao et al., supra). Oligonucleotides containing an NF-xB binding site
were
annealed and endlabeled with 32P using T4 DNA polynucleotide kinase. Mobility
shift
reactions contained 10.tg of nuclear extract, 4 tg of poly(dI-dC) and 15,000
cpm labeled
double-stranded oligonucleotide and incubated at room temperature for 20
minutes.
Resulting protein-DNA complexes were resolved on a 6% native polyacrylamide
gel in
0.25 X Tris-borate-EDTA buffer.
Overexpression of RANK resulted in induction of NF-KB activity as shown by an
appropriate shift in the mobility of the radioactive probe on the gel. Similar
results were
observed when RANK was triggered by a ligand that binds RANK and transduces a
signal
to cells expressing the receptor (i.e., by co-transfecting cells with human
RANK and
murine RANKL DNA; see Example 7 below), and would be expected to occur when
triggering is done with agonistic antibodies.
23
CA 02274803 1999-06-15
WO 98/28424 - PCT/US97/23866
EXAMPLE 5
This example describes a gene promoter/reporter system based on the human
Interleukin-8 (IL-8) promoter used to analyze the activation of gene
transcription in vivo.
The induction of human IL-8 gene transcription by the cytokines Interleukin-1
(IL-1) or
tumor necrosis factor-alpha (TNF-a) is known to be dependent upon intact NF-KB
and
NF-IL-6 transcription factor binding sites. Fusion of the cytokine-responsive
IL-8
promoter with a cDNA encoding the murine IL-4 receptor (mIL-4R) allows
measurement
of promoter activation by detection of the heterologous reporter protein (mIL-
4R) on the
cell surface of transfected cells.
Human kidney epithelial cells (293/EBNA) are transfected (via the
DEAE/DEXTRAN method) with plasmids encoding: 1). the reporter/promoter
construct
(referred to as pIL-8rep), and 2). the cDNA(s) of interest. DNA concentrations
are always
kept constant by the addition of empty vector DNA. The 293/EBNA cells are
plated at a
density of 2.5 x 104 cells/ml (3 ml/ well) in a 6 well plate and incubated for
two days prior
to transfection. Two days after transfection, the mIL-4 receptor is detected
by a
radioimmunoassay (RIA) described below.
In one such experiment, the 293JEBNA cells were co-transfected with DNA
encoding RANK and with DNA encoding RANKL (see Example 7 below). Co-expression
of this receptor and its counterstructure by cells results in activation of
the signaling process
of RANK. For such co-transfection studies, the DNA concentration/well for the
DEAF
transfection were as follows: 40 ng of pIL-8rep [pBluescriptSK- vector
(Stratagene)]; 0.4
ng CD40 (DNA encoding CD40, a control receptor; pCDM8 vector); 0.4 ng RANK
(DNA
encoding RANK; pDC409 vector), and either 1-50 ng CD40L (DNA encoding the
ligand
for CD40, which acts as a positive control when co-transfected with CD40 and
as a
negative control when co-transfected with RANK; in pDC304) or RANKL (DNA
encoding
a ligand for RANK; in pDC406). Similar experiments can be done using soluble
RANKL
or agonistic antibodies to RANK to trigger cells transfected with RANK.
For the mIL-4R-specific RIA, a monoclonal antibody reactive with mIL-4R is
labeled with 1211 via a Chloramine T conjugation method; the resulting
specific activity is
typically 1.5 x 1016 cpm/nmol. After 48 hours, transfected cells are washed
once with
media (DMEM/F12 5% FBS). Non-specific binding sites are blocked by the
addition of
pre-warmed binding media containing 5% non-fat dry milk and incubation at 37
C/5% CO2
in a tissue culture incubator for one hour. The blocking media is decanted and
binding
buffer containing 125I anti-mlL-4R (clone M 1; rat IgG 1) is added to the
cells and incubated
with rocking at room temperature for 1 hour. After incubation of the cells
with the radio-
labeled antibody, cells are washed extensively with binding buffer (2X) and
twice with
24
CA 02274803 2005-08-03
72249-164
phosphate-buffered saline (PBS). Cells are lysed in I ml of 0.5M NaOH, and
total
radioactivity is measured with a gamma counter.
Using this assay, 293/EBNA coo-transfected with DNAs encoding RANK
demonstrated transcriptional activation, as shown by detection of mulL-4R on
the cell
surface. Overexpression of RANK resulted in transcription of muIL-4R, as did
triggering
of the RANK by RANKL. Similar results are observed when RANK is triggered by
agonistic antibodies.
EXAMPLE 6
This example illustrates the association of RANK with TRAF proteins.
Interaction
of RANK with cytoplasmic TRAF proteins was demonstrated by co-
immunoprecipitation
assays essentially as described by Hsu et al. (Cell 84:299; 1996). Briefly,
293/EBNA cells
were co-transfected with plasmids that direct the synthesis of RANK and
epitope-tagged
(FLAG ; SEQ ID NO:7) TRAF2 or TRAF3. Two days after transfection, surface
proteins
were labeled with biotin-ester, and cells were lysed in a buffer containing
0.5% NP-40TH.
RANK and proteins associated with this receptor were immunoprecipitated with
anti-
RANK, washed extensively, resolved by electrophoretic separation on a 6-10%
SDS
polyacrylamide gel and electrophoretically transferred to a nitrocellulose
membrane for
Western blotting. The association of TRAF2 and TRAF3 proteins with RANK was
visualized by probing the membrane with an antibody that specifically
recognizes the
FLAG epitope. TRAFs 2 and 3 did not immunopreciptitate with anti-RANK in the
absence of RANK expression.
EXAMPLE 7
This example describes isolation of a ligand for RANK, referred to as RANKL,
by
direct expression cloning. The ligand was cloned essentially as described in
US Patent
No. 5,961,974, filed May 24, 1994,
for CD40L. Briefly, a library was prepared from a clone of a mouse
thymoma cell line EL-4 (ATCC TIB 39), called EL-40.5, derived by sorting five
times with
biotinylated CD40/Fc fusion protein in a FACS (fluorescence activated cell
sorter). The
cDNA library was made using standard methodology; the plasmid DNA was isolated
and
transfected into sub-confluent CVI-EBNA cells using a DEAE-dextran method.
Transfectants were screened by slide autoradiography for expression of RANKL
using a
two-step binding method with RANK/Fc fusion protein as prepared in Example 2
followed
by radioiodinated goat anti-human IgG antibody.
A clone encoding a protein that specifically bound RANK was isolated and
sequenced; the clone was referred to as 11H. An expression vector containing
murine
RANKL sequence, designated pDC406:muRANK-L (in E. coli DH I OB), was deposited
CA 02274803 2005-08-03
72249-164
with the American Type Culture Collection, Manassas, VA (ATCC) on December 20,
1996, under terms of the Budapest Treaty, and given accession number 98284.
The
nucleotide sequence and predicted amino acid sequence of this clone are
illustrated in SEQ
ID NO: 10. This clone did not contain an initiator methionine; additional,
full-length clones
were obtained from a 7B9 library (prepared substantially as described in US
patent
5,599,905, issued February 4, 1997); the 5' region was found to be identical
to that of
human RANKL as shown in SEQ ID NO: 12, amino acids 1 through 22, except for
substitution of a Gly for a Thr at residue 9.
This ligand is useful for assessing the ability of RANK to bind RANKL by a
number of different assays. For example, transfected cells expressing RANKL
can be used
in a FACS assay (or similar assay) to evaluate the ability of soluble RANK to
bind
RANKL. Moreover, soluble forms of RANKL can be prepared and used in assays
that are
known in the art (i.e., ELISA or BIAcoreTM assays essentially as described in
US Patent
No. 5,961,974, filed May 24, 1994). RANKL is also useful in affinity
purification of RANK,
and as a reagent in methods to measure the levels of RANK in a sample. Soluble
RANKL
is also useful in inducing NF-KB activation and thus protecting cells that
express RANK
from apoptosis.
EXAMPLE 8
This example describes the isolation of a human RANK ligand (RANKL) using a
PCR-based technique. Murine RANK ligand-specific oligonucleotide primers were
used in
PCR reactions using human cell line-derived first strand cDNAs as templates.
Primers
corresponded to nucleotides 478-497 and to the complement of nucleotides 858-
878 of
murine RANK ligand (SEQ ID NO:10). An amplified band approximately 400 bp in
length
from one reaction using the human epidermoid cell line KB (ATCC CCL- 17) was
gel
purified, and its nucleotide sequence determined; the sequence was 859b
identical to the
corresponding region of murine RANK ligand, confirming that the fragment was
from
human RANKL.
To obtain full-length human RANKL cDNAs, two human RANKL-specific
oligonucleotides derived from the KB PCR product nucleotide sequence were
radiolabeled
and used as hybridization probes to screen a human PBL cDNA library prepared
in lambda
gt10 (Stratagene, La Jolla, CA), substantially as described in US patent
5,599,905, issued
February 4, 1997 . Several positive hybridizing plaques were identified and
purified, their
inserts subcloned into pBluescript SK- (Stratagene, La Jolla, CA), and their
nucleotide
sequence determined. One isolate, PBL3, was found to encode most of the
predicted
human RANKL, but appeared to be missing approximately 200 bp of 5' coding
region. A
second isolate, PBL5 was found to encode much of the predicted human RANKL,
including the entire 5' end and an additional 200 bp of 5' untranslated
sequence.
26
CA 02274803 2005-08-03
72249-164
The 5' end of PBL5 and the 3' end of PBL3 were ligated together to form a full
length cDNA encoding human RANKL. The nucleotide and predicted amino acid
sequence
of the full-length human RANK ligand is shown in SEQ ID NO: 12. Human RANK
ligand
shares 83% nucleotide and 84% amino acid identity with murine RANK ligand. A
plasmid
vector containing human RANKL sequence, designated pBluescript:huRANK-L (in E.
coli
DH10B), was deposited with the American Type Culture Collection, Manassas, VA
.
(ATCC) on March 11, 1997 under terms of the Budapest Treaty, and given
accession
number 98354.
Morin 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.
EXAMPLE 9
This example describes the chromosomal mapping of human RANK using PCR-
based mapping strategies. Initial human chromosomal assignments were made
using
RANK and RANKL-specific PCR primers and a BIOS Somatic Cell Hybrid PCRableTM
DNA kit from BIOS Laboratories (New Haven, CT), following the manufacturer's
instructions. RANK mapped to human chromosome 18; RANK ligand mapped to human
chromosome 13. More detailed mapping was performed using a radiation hybrid
mapping
panel GenebridgeTM 4 Radiation Hybrid Panel (Research Genetics, Huntsville,
AL;
described in Walter, MA et al., Nature Genetics 7:22-28, 1994). RANK mapped to
chromosome 18q22. 1, and RANKL mapped to chromosome 13814.
EXAMPLE 10
This example illustrates the preparation of monoclonal antibodies against
RANKL.
Preparations of purified recombinant RANKL, for example, or transfixed cells
expressing
high levels of RANKL, are employed to generate monoclonal antibodies against
RANKL
using conventional techniques, such as those disclosed in US Patent 4,411,993.
DNA
encoding RANKL can also be used as an immunogen, for example, as reviewed by
Pardoll
and Beckerleg in Immunity 3:165, 1995. Such antibodies are likely to be useful
in
interfering with RANKL signaling (antagonistic or blocking antibodies), as
components of
27
CA 02274803 1999-06-15
WO 98/28424 - PCT/US97/23866
diagnostic or research assays for RANKL or RANKL activity, or in affinity
purification of
RANKL.
To immunize rodents, RANKL immunogen is emulsified in an adjuvant (such as
complete or incomplete Freund's adjuvant, alum, or another adjuvant, such as
Ribi
adjuvant R700 (Ribi, Hamilton, MT), and injected in amounts ranging from 10-
100 p.g
subcutaneously into a selected rodent, for example, BALB/c mice or Lewis rats.
DNA may
be given intradermally (Raz et al., Proc. Natl. Acad. Sci. USA 91:9519, 1994)
or
intamuscularly (Wang et al., Proc. Natl. Acad. Sci. USA 90:4156, 1993); saline
has been
found to be a suitable diluent for DNA-based antigens. Ten days to three weeks
days later,
the immunized animals are boosted with additional immunogen and periodically
boosted
thereafter on a weekly, biweekly or every third week immunization schedule.
Serum samples are periodically taken by retro-orbital bleeding or tail-tip
excision for
testing by dot-blot assay (antibody sandwich), ELISA (enzyme-linked
immunosorbent
assay), immunoprecipitation, or other suitable assays, including FACS
analysis.
Following detection of an appropriate antibody titer, positive animals are
given an
intravenous injection of antigen in saline. Three to four days later, the
animals are
sacrificed, splenocytes harvested, and fused to a murine myeloma cell line
(e.g., NS 1 or
preferably Ag 8.653 [ATCC CRL 1580]). Hybridoma cell lines generated by this
procedure are plated in multiple microtiter plates in a selective medium (for
example, one
containing hypoxanthine, aminopterin, and thymidine, or HAT) to inhibit
proliferation of
non-fused cells, myeloma-myeloma hybrids, and splenocyte-splenocyte hybrids.
Hybridoma clones thus generated can be screened by ELISA for reactivity
with RANKL, for example, by adaptations of the techniques disclosed by Engvall
et al.,
Immunochem. 8:871 (1971) and in US Patent 4,703,004. A preferred screening
technique
is the antibody capture technique described by Beckman et al., J. Immunol.
144:4212
(1990). Positive clones are then injected into the peritoneal cavities of
syngeneic rodents to
produce ascites containing high concentrations (>I mg/ml) of anti-RANK
monoclonal
antibody. The resulting monoclonal antibody can be purified by ammonium
sulfate
precipitation followed by gel exclusion chromatography. Alternatively,
affinity
chromatography based upon binding of antibody to protein A or protein G can
also be
used, as can affinity chromatography based upon binding to RANKL protein.
Using the
methods described herein to monitor the activity of the mAbs, both blocking
(i.e.,
antibodies that bind RANKL and inhibit binding to RANK) and non-blocking
(i.e.,
antibodies that bind RANKL and do not inhibit binding) are isolated.
28
CA 02274803 1999-06-15
WO 98/28424 - PCTIUS97/23866
EXAMPLE 11
This example demonstrates that RANK expression can be up-regulated. Human
peripheral blood T cells were purified by flow cytometry sorting or by
negative selection
using antibody coated beads, and activated with anti-CD3 (OKT3, Dako) coated
plates or
phytohemagglutinin in the presence or absence of various cytokines, including
Interleukin-
4 (IL-4),Transforming Growth Factor-B (TGF-B) and other commercially available
cytokines ( IL 1-a, IL-2, IL-3, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IFN-y,
TNF- cc).
Expression of RANK was evaluated by FACS in a time course experiment for day 2
to day
8, using a mouse monoclonal antibody mAb144 (prepared as described in Example
3), as
shown in the table below. Results are expressed as `+' to `++++' referring to
the relative
increase in intensity of staining with anti-RANK. Double labeling experiments
using both
anti-RANK and anti-CD8 or anti-CD4 antibodies were also performed.
Table 1: Upregulation of RANK by Cytokines
Cytokine (concentration) Results:
IL-4 (50 ng/ml) +
TGF-B (5 ng/ml) + to ++
IL-4 (50 ng/ml) +TGF-B (5 ng/ml) ++++
IL 1-a (l Ong/ml) -
IL-2 (20ng/ml) -
IL-3 (25ng/ml) -
IL-7 (20ng/ml) -
IL-8 (lOng/ml) -
IL-10 (50ng/ml) -
IL-12 (1 Ong/ml) -
IL-15 (l Ong/ml) -
IFN-y (100U/ml) -
TNF-a (l Ong/ml) -
Of the cytokines tested, IL-4 and TGF-B increased the level of RANK expression
on both CD8+ cytotoxic and CD4+ helper T cells from day 4 to day 8. The
combination of
IL-4 and TGF-B acted synergistically to upregulate expression of this receptor
on activated
T cells. This particular combination of cytokines is secreted by suppresser T
cells, and is
believed to be important in the generation of tolerance (reviewed in Mitchison
and Sieper,
Z. Rheumatol. 54:141, 1995), implicating the interaction of RANK in regulation
of an
immune response towards either tolerance or induction of an active immune
response.
29
CA 02274803 2005-08-03
72249-164
EXAMPLE 12
This example illustrates the influence of RANK.Fc and hRANKL on activated T
cell growth. The addition of TGFB to anti-CD3 activated human peripheral blood
T
lymphocytes induces proliferation arrest and ultimately death of most
lymphocytes within
the first few days of culture. We tested the effect of RANK:RANKL interactions
on TGFB-
treated T cells by adding RANK.Fc or soluble human RANKL to T cell cultures.
Human peripheral blood T cells (7 x IO 5 PBT) were cultured for six days on
anti-
CD3 (OKT3, 5 g/ml) and anti-Flag (M1, 5 g/ml) coated 24 well plates in the
presence of
TGFB (l ng/ml) and IL-4 (l 0ng/ml ), with or without recombinant FLAG-tagged
soluble
hRANKL (1 g/ml) or RANK.Fc (10 g/ml). Viable T cell recovery was determined by
triplicate trypan blue countings.
The addition of RANK.Fc significantly reduced the number of viable T cells
recovered after six days, whereas soluble RANKL greatly increased the recovery
of viable
T cells (Figure 1). Thus, endogenous or exogenous RANKL enhances the number of
viable T cells generated in the presence of TGFB. TGFB, along with IL-4, has
been
implicated in immune response regulation when secreted by the TH3/regulatory T
cell
subset. These T cells are believed to mediate bystander suppression of
effector T cells.
Accordingly, RANK and its ligand may act in an auto/paracrine fashion to
influence T cell
tolerance. Moreover, TGFB is known to play a role in the evasion of the immune
system
effected by certain pathogenic or opportunistic organisms. In addition to
playing a role in
the development of tolerance, RANK may also play a role in immune system
evasion by
pathogens.
EXAMPLE 13
This example illustrates the influence of the interaction of RANK on CDla+
dendritic cells (DC). Functionally mature dendritic cells (DC) were generated
in vitro from
CD34+ bone marrow (BM) progenitors. Briefly, human BM cells from normal
healthy
volunteers were density fractionated using FicollTM medium and CD34+ cells
immunoaffinity isolated using an anti-CD34 matrix column (CeprateTM, Cell
Pro). The
CD34+ BM cells were then cultured in human GM-CSF (20 ng/ml), human IL-4
(20 ng/ml), human TNF-a (20 ng/ml), human CHO-derived FIt3L (FL; 100 ng/ml) in
Super McCoy's medium supplemented with 10% fetal calf serum in a fully
humidified
37~C incubator (5% CO2) for 14 days. CD1a+, HLA-DR+ DC were then sorted using
a
FACStar P1usTM, and used for biological evaluation of RANK.
On human CDIa+ DC derived from CD34+ bone marrow cells, only a subset (20-
30%) of CDIa+ DC expressed RANK at the cell surface as assessed by flow
cytometric
CA 02274803 1999-06-15
WO 98/28424 PCTIUS97/23866
analysis. However, addition of CD40L to the DC cultures resulted in RANK
surface
expression on the majority of CDIa+ DC. CD40L has been shown to activate DC by
enhancing in vitro cluster formation, inducing DC morphological changes and
upregulating
HLA-DR, CD54, CD58, CD80 and CD86 expression
Addition of RANKL to DC cultures significantly increased the degree of DC
aggregation and cluster formation above control cultures, similar to the
effects seen with
CD40L (Figure 2). Sorted human CDla+ DC were cultured in a cytokine cocktail
(GM-
CSF, IL-4, TNF-a and FL) (upper left panel), in cocktail plus CD40L (l g/ml)
(upper
right), in cocktail plus RANKL (1 g/ml) (lower left), or in cocktail plus heat
inactivated
(AH) RANKL (1 gg/ml) (lower right) in 24-well flat bottomed culture plates in
1 ml culture
media for 48-72 hours and then photographed using an inversion microscope. An
increase
in DC aggregation and cluster formation above control cultures was not evident
when heat
inactivated RANKL was used, indicating that this effect was dependent on
biologically
active protein. However, initial phenotypic analysis of adhesion molecule
expression
indicated that RANKL-induced clustering was not due to increased levels of
CD2, CD 11 a,
CD54 or CD58.
The addition of RANKL to CDIa+ DC enhanced their allo-stimulatory capacity in
a
mixed lymphocyte reaction (MLR) by at least 3- to 10-fold, comparable to CD40L-
cultured
DC (Figure 3). Allogeneic T cells (1x105) were incubated with varying numbers
of
irradiated (2000 rad) DC cultured as indicated above for Figure 2 in 96-well
round
bottomed culture plates in 0.2 ml culture medium for four days. The cultures
were pulsed
with 0.5 mCi [3H]-thymidine for eight hours and the cells harvested onto glass
fiber sheets
for counting on a gas phase (i counter. The background counts for either T
cells or DC
cultured alone were <100 cpm. Values represent the mean SD of triplicate
cultures. Heat
inactivated RANKL had no effect. DC allo-stimulatory activity was not further
enhanced
when RANKL and CD40L were used in combination, possibly due to DC functional
capacity having reached a maximal level with either cytokine alone. Neither
RANKL nor
CD40L enhanced the in vitro growth of DC over the three day culture period.
Unlike
CD40L, RANKL did not significantly increase the levels of HLA-DR expression
nor the
expression of CD80 or CD86.
RANKL can enhance DC cluster formation and functional capacity without
modulating known molecules involved in cell adhesion (CD 18, CD54), antigen
presentation (HLA-DR) or costimulation (CD86), all of which are regulated by
CD40/CD40L signaling. The lack of an effect on the expression of these
molecules
suggests that RANKL may regulate DC function via an alternate pathway(s)
distinct from
CD40/CD40L. Given that CD40L regulates RANK surface expression on in vitro-
generated DC and that CD40L is upregulated on activated T cells during DC-T
cell
31
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
interactions, RANK and its ligand may form an important part of the activation
cascade that
is induced during DC-mediated T cell expansion. Furthermore, culture of DC in
RANKL
results in decreased levels of CDlb/c expression, and increased levels of
CD83. Both of
these molecules are similarly modulated during DC maturation by CD40L (Caux et
al. J.
Exp. Med. 180:1263; 1994), indicating that RANKL induces DC maturation.
Dendritic cells are referred to as "professional" antigen presenting cells,
and have a
high capacity for sensitizing MHC-restricted T cells. There is growing
interest in using
dendritic cells ex vivo as tumor or infectious disease vaccine adjuvants (see,
for example,
Romani, et al., J. Exp. Med., 180:83, 1994). Therefore, an agent such as RANKL
that
induces DC maturation and enhances the ability of dendritic cells to stimulate
an immune
response is likely to be useful in immunotherapy of various diseases.
EXAMPLE 14
This example describes the isolation of the murine homolog of RANK, referred
to
as muRANK. MuRANK was isolated by a combination of cross-species PCR and
colony
hybridization. The conservation of Cys residues in the Cys-rich pseudorepeats
of the
extracellular domains of TNFR superfamily member proteins was exploited to
design
human RANK-based PCR primers to be used on murine first strand cDNAs from
various
sources. Both the sense upstream primer and the antisense downstream primer
were
designed to have their 3' ends terminate within Cys residues.
The upstream sense primer encoded nucleotides 272-295 of SEQ ID NO:5 (region
encoding amino acids 79-86); the downstream antisense primer encoded the
complement of
nucleotides 409-427 (region encoding amino acids 124-130). Standard PCR
reactions
were set up and run, using these primers and first strand cDNAs from various
murine cell
line or tissue sources. Thirty reaction cycles of 94 C for 30 seconds, 50 C
for 30 seconds,
and 72 C for 20 seconds were run. PCR products were anlyzed by
electrophoresis, and
specific bands were seen in several samples. The band from one sample was gel
purified
and DNA sequencing revealed that the sequence between the primers was
approximately
85% identical to the corresponding human RANK nucleotide sequence.
A plasmid based cDNA library prepared from the murine fetal liver epithelium
line
FLE18 (one of the cell lines identified as positive in the PCR screen) was
screened for full-
length RANK cDNAs using murine RANK-specific oligonucleotide probes derived
from
the murine RANK sequence determined from sequencing the PCR product. Two
cDNAs,
one encoding the 5' end and one encoding the 3' end of full-length murine RANK
(based
on sequence comparison with the full-length human RANK) were recombined to
generate a
full-length murine RANK cDNA. The nucleotide and amino acid seqeunce of muRANK
are shown in SEQ ID Nos: 14 and 15.
32
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
The cDNA encodes a predicted Type 1 transmembrane protein having 625 amino
acid residues, with a predicted 30 amino acid signal sequence, a 184 amino
acid
extracellular domain, a 21 amino acid transmembrane domain, and a 390 amino
acid
cytoplasmic tail. The extracellular region of muRANK displayed significant
amino acid
homology (69.7% identity, 80.8% similarity) to huRANK. Those of skill in the
art will
recognize that the actual cleavage site can be different from that predicted
by computer;
accordingly, the N-terminal of RANK may be from amino acid 25 to amino acid
35.
Other members of the TNF receptor superfamily have a region of amino acids
between the transmembrane domain and the ligand binding domain that is
referred to as a
`spacer' region, which is not necessary for ligand binding. In muRANK, the
amino acids
between 197 and 214 are predicted to form such a spacer region. Accordingly, a
soluble
form of RANK that terminates with an amino acid in this region is expected to
retain the
ability to bind a ligand for RANK in a specific manner. Preferred C-terminal
amino acids
for soluble RANK peptides are selected from the group consisting of amino
acids 214, and
197 of SEQ ID NO:14, although other amino acids in the spacer region may be
utilized as a
C-terminus.
EXAMPLE 15
This example illustrates the preparation of several different soluble forms of
RANK
and RANKL. Standard techniques of restriction enzyme cutting and ligation, in
combination with PCR-based isolation of fragments for which no convenient
restriction
sites existed, were used. When PCR was utilized, PCR products were sequenced
to
ascertain whether any mutations had been introduced; no such mutations were
found.
In addition to the huRANK/Fc described in Example 2, another RANK/Fc fusion
protein was prepared by ligating DNA encoding amino acids 1-213 of SEQ ID
NO:6, to
DNA encoding amino acids 3-232 of the Fc mutein described previously (SEQ ID
NO:8).
A similar construct was prepared for murine RANK, ligating DNA encoding amino
acids 1-
213 of full-length murine RANK (SEQ ID NO: 15) to DNA encoding amino acids 3-
232 of
the Fc mutein (SEQ ID NO:8).
A soluble, tagged, poly-His version of huRANKL was prepared by ligating DNA
encoding the leader peptide from the immunoglobulin kappa chain (SEQ ID NO:
16) to
DNA encoding a short version of the FLAGTM tag (SEQ ID NO:17), followed by
codons
encoding Gly Ser, then a poly-His tag (SEQ ID NO: 18), followed by codons
encoding Gly
Thr Ser, and DNA encoding amino acids 138-317 of SEQ ID NO: 13. A soluble,
poly-His
tagged version of murine RANKL was prepared by ligating DNA encoding the CMV
leader
(SEQ ID NO:9) to codons encoding Arg Thr Ser, followed by DNA encoding poly-
His
(SEQ ID NO: 18) followed by DNA encoding amino acids 119-294 of SEQ ID NO: 11.
33
CA 02274803 2005-08-03
72249-164
A soluble, oligomeric form of huRANKL was prepared by ligating DNA encoding
the CMV leader (SEQ ID NO:9) to a codon encoding Asp followed by DNA ending a
trimer-former "leucine" zipper (SEQ ID NO:19), then by codons encoding Thr Arg
Ser
followed by amino acids 138-317 of SEQ ID NO:13.
These and other constructs are prepared by routine experimentation. The
various
DNAs are then inserted into a suitable expression vector, and expressed.
Particularly
preferred expression vectors are those which can be used in mammalian cells.
For
example, pDC409 and pDC304, described herein, are useful for transient
expression. For
stable transfection, the use of CHO cells is preferred; several useful vectors
are described in
US Patent No. 6,027,915, for example, one of the 2A5-3 A-derived expression
vectors
discussed therein.
This example demonstrates that RANKL expression can be up-regulated on murine
T cells. Cells were obtained from mesenteric lymph nodes of C57BL/6 mice, and
activated
with anti-CD3 coated plates, Concanavalin A (ConA) or phorbol rnyristate
acetate in
combination with ionomycin (anti-CD3: 500A2; Immunex Corporation, Seattle WA;
ConA,
PMA, ionomycin, Sigma, St. Louis, MO) substantially as described herein, and
cultured
from about 2 to 5 days. Expression of RANKL was evaluated in a three color
analysis by
FACS, using antibodies to the T cell markers CD4, CD8 and CD45RB, and RANK/Fc,
prepared as described herein.
RANKL was not expressed on unstimulated murine T cells. T cells stimulated
with
either anti-CD3, ConA, or PMA/ionomycin, showed differential expression of
RANKL:
CD4+/CD45RBL0 and CD4'/CD45RBHi cells were positive for RANKL, but CD8+ cells
were not. RANKL was not observed on B cells, similar to results observed with
human
cells.
EXAMPLE 17
This example illustrates the effects of murine RANKL on cell proliferation and
activation. Various cells or cell lines representative of cells that play a
role in an immune
response (murine spleen, thymus and lymphnode) were evaluated by culturing
them under
conditions promoting their viability, in the presence or absence of RANKL.
RANKL did
not stimulate any of the tested cells to proliferate. One cell line, a
macrophage cell line
referred to as RAW 264.7 (ATCC accession number TIB 71) exhibited some signs
of
activation.
RAW cells constitutively produce small amounts of TNF-a. Incubation with
either
human or murine RANKL enhanced production of TNF-a by these cells in a dose
34
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
dependent manner. The results were not due to contamination of RANKL
preparations
with endotoxin, since boiling RANKL for 10 minutes abrogated TNF-a production,
whereas a similar treatment of purified endotoxin (LPS) did not affect the
ability of the LPS
to stimulate TNF-a production. Despite the fact that RANKL activated the
macrophage cell
line RAW T64.7 for TNF-a production, neither human RANKL nor murine RANKL
stimulated nitric oxide production by these cells.
EXAMPLE 18
This example illustrates the effects of murine RANKL on growth and development
of the thymus in fetal mice. Pregnant mice were injected with 1 mg of RANK/Fc
or vehicle
control protein (murine serum albumin; MSA) on days 13, 16 and 19 of
gestation. After
birth, the neonates continued to be injected with RANK/Fc intraperitoneally
(IP) on a daily
basis, beginning at a dose of I g, and doubling the dose about every four
days, for a final
dosage of 4 g. Neonates were taken at days 1, 8 and 15 post birth, their
thymuses and
spleens harvested and examined for size, cellularity and phenotypic
composition.
A slight reduction in thymic size at day 1 was observed in the neonates born
to the
female injected with RANK/Fc; a similar decrease in size was not observed in
the control
neonates. At day 8, thymic size and cellularity were reduced by about 50% in
the
RANK/Fc-treated animals as compared to MSA treated mice. Phenotypic analysis
demonstrated that the relative proportions of different T cell populations in
the thymus were
the same in the RANK/Fc mice as the control mice, indicating that the
decreased cellularity
was due to a global depression in the number of thymic T cells as opposed to a
decrease in
a specific population(s). The RANK/Fc-treated neonates were not significantly
different
from the control neonates at day 15 with respect to either size, cellularity
or phenotype of
thymic cells. No significant differences were observed in spleen size,
cellularity or
composition at any of the time points evaluated. The difference in cellularity
on day 8 and
not on day 15 may suggest that RANK/Fc may assert its effect early in thymic
development.
EXAMPLE 19
This example demonstrates that the C-terminal region of the cytoplasmic domain
of
RANK is important for binding of several different TRAF proteins. RANK
contains at
least two recognizable PXQX(X)T motifs that are likely TRAF docking sites.
Accordingly,
the importance of various regions of the cytoplasmic domain of RANK for TRAF
binding
was evaluated. A RANK/GST fusion protein was prepared substantially as
described in
Smith and Johnson, Gene 67:31 (1988), and used in the preparation of various
truncations
as described below.
CA 02274803 1999-06-15
WO 98/28424 PCTIUS97/23866
Comparison of the nucleotide sequence of murine and human RANK indicated that
there were several conserved regions that could be important for TRAF binding.
Accordingly, a PCR-based technique was developed to facilitate preparation of
various C-
terminal truncations that would retain the conserved regions. PCR primers were
designed
to introduce a stop codon and restriction enzyme site at selected points,
yielding the
truncations described in Table 1 below. Sequencing confirmed that no undesired
mutations
had been introduced in the constructs.
Radio-labeled (35S-Met, Cys) TRAF proteins were prepared by in vitro
translation
using a commercially available reticulocyte lysate kit according to
manufacturer's
instructions (Promega). Truncated GST fusion proteins were purified
substantially as
described in Smith and Johnson (supra). Briefly, E. coli were transfected with
an
expression vector encoding a fusion protein, and induced to express the
protein. The
bacteria were lysed, insoluble material removed, and the fusion protein
isolated by
precipitation with glutathione-coated beads (Sepahrose 4B, Pharmacia, Uppsala
Sweden)
The beads were washed, and incubated with various radiolabeled TRAF proteins.
After incubation and wash steps, the fusion protein/TRAF complexes were
removed from
the beads by boiling in 0.1% SDS + 13-mercaptoethanol, and loaded onto 12% SDS
gels
(Novex). The gels were subjected to autoradiography, and the presence or
absence of
radiolabeled material recorded. The results are shown in Table 2 below.
Table 2: Binding of Various TRAF Proteins to the Cytoplasmic Domain of RANK
C terminal
Truncations: E206-S339 E206-Y421 E206-M476 E206-G544 Full length
TRAF1 - - - - ++
TRAF2 - - - - ++
TRAF3 - - - - ++
TRAF4 - - - - -
TRAF5 - - - - +
TRAF6 - + + + ++
These results indicate that TRAF1, TRAF2, TRAF3, TRAF 5 and TRAF6 bind to
the most distal portion of the RANK cytoplasmic domain (between amino-acid
G544 and
A616). TRAF6 also has a binding site between S339 and Y421. In this
experiment,
TRAF5 also bound the cytoplasmic domain of RANK.
36
CA 02274803 1999-11-23
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: IMMUNEY CORPORATION
(ii) TITLE OF INVENTION: RECEPTOR ACTIVATOR OF NF-KAPPA B, RECEPTOR IS
MEMBER OF TNF RECEPTOR SUPERFAMILY
(iii) NUMBER OF SEQUENCES: 19
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SMART & BIGGAR
(B) STREET: P.O. BOX 2999, STATION D
(C) CITY: OTTAWA
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE;. ASCII (text)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,274,803
(B) FILING DATE: 22-DEC-1997
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/059,978
(B) FILING DATE: 23--DEC-1996
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/813,509
(B) FILING DATE: 07-MAR-1997
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/064,671
(B) FILING DATE: 14-OCT-1997
(viii) ATTORNEY/AGENT INFORMATION:
37
CA 02274803 1999-11-23
(A) NAME: SMART & BIGGAR
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 72249-88
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (6131-232-2486
(B) TELEFAX: (613)-232-8440
(2) INFORMATION FOR SEQ ID NO::i:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3115 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
37a
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: BONE-MARROW DERIVED DENDRITIC CELLS
(B) CLONE: 9D-8A
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 93..1868
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GCTGCTGCTG CTCTGCGCGC TGCTCGCCCG GCTGCAGTTT TATCCAGAAA GAGCTGTGTG 60
GACTCTCTGC CTGACCTCAG TGTTCTTTTC AG GTG GCT TTG CAG ATC GCT CCT 113
Val Ala Leu Gln Ile Ala Pro
1 5
CCA TGT ACC AGT GAG AAG CAT TAT GAG CAT CTG GGA CGG TGC TGT AAC 161
Pro Cys Thr Ser Glu Lys His Tyr Glu His Leu Gly Arg Cys Cys Asn
15 20
AAA TGT GAA CCA GGA AAG TAC ATG TCT TCT AAA TGC ACT ACT ACC TCT 209
Lys Cys Glu Pro Gly Lys Tyr Met Ser Ser Lys Cys Thr Thr Thr Ser
25 30 35
GAC AGT GTA TGT CTG CCC TGT GGC CCG GAT GAA TAC TTG GAT AGC TGG 257
Asp Ser Val Cys Leu Pro Cys Gly Pro Asp Glu Tyr Leu Asp Ser Trp
40 45 50 55
AAT GAA GAA GAT AAA TGC TTG CTG CAT AAA GTT TGT GAT ACA GGC AAG 305
Asn Glu Glu Asp Lys Cys Leu Leu His Lys Val Cys Asp Thr Gly Lys
60 65 70
GCC CTG GTG GCC GTG GTC GCC GGC AAC AGC ACG ACC CCC CGG CGC TGC 353
Ala Leu Val Ala Val Val Ala Gly Asn Ser Thr Thr Pro Arg Arg Cys
75 80 85
GCG TGC ACG GCT GGG TAC CAC TGG AGC CAG GAC TGC GAG TGC TGC CGC 401
Ala Cys Thr Ala Gly Tyr His Trp Ser Gln Asp Cys Glu Cys Cys Arg
90 95 100
CGC AAC ACC GAG TGC GCG CCG GGC CTG GGC GCC CAG CAC CCG TTG CAG 449
Arg Asn Thr Glu Cys Ala Pro Gly Leu Gly Ala Gln His Pro Leu Gln
105 110 115
CTC AAC AAG GAC ACA GTG TGC AAA CCT TGC CTT GCA GGC TAC TTC TCT 497
Leu Asn Lys Asp Thr Val Cys Lys Pro Cys Leu Ala Gly Tyr Phe Ser
120 125 130 135
38
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
GAT GCC TTT TCC TCC ACG GAC AAA TGC AGA CCC TGG ACC AAC TGT ACC 545
Asp Ala Phe Ser Ser Thr Asp Lys Cys Arg Pro Trp Thr Asn Cys Thr
140 145 150
TTC CTT GGA AAG AGA GTA GAA CAT CAT GGG ACA GAG AAA TCC GAT GCG 593
Phe Leu Gly Lys Arg Val Glu His His Gly Thr Glu Lys Ser Asp Ala
155 160 165
GTT TGC AGT TCT TCT CTG CCA GCT AGA AAA CCA CCA AAT GAA CCC CAT 641
Val Cys Ser Ser Ser Leu Pro Ala Arg Lys Pro Pro Asn Glu Pro His
170 175 180
GTT TAC TTG CCC GGT TTA ATA ATT CTG CTT CTC TTC GCG TCT GTG GCC 689
Val Tyr Leu Pro Gly Leu Ile Ile Leu Leu Leu Phe Ala Ser Val Ala
185 190 195
CTG GTG GCT GCC ATC ATC TTT GGC GTT TGC TAT AGG AAA AAA GGG AAA 737
Leu Val Ala Ala Ile Ile Phe Gly Val Cys Tyr Arg Lys Lys Gly Lys
200 205 210 215
GCA CTC ACA GCT AAT TTG TGG CAC TGG ATC AAT GAG GCT TGT GGC CGC 785
Ala Leu Thr Ala Asn Leu Trp His Trp Ile Asn Glu Ala Cys Gly Arg
220 225 230
CTA AGT GGA GAT AAG GAG TCC TCA GGT GAC AGT TGT GTC AGT ACA CAC 833
Leu Ser Gly Asp Lys Glu Ser Ser Gly Asp Ser Cys Val Ser Thr His
235 240 245
ACG GCA AAC TTT GGT CAG CAG GGA GCA TGT GAA GGT GTC TTA CTG CTG 881
Thr Ala Asn Phe Gly Gln Gln Gly Ala Cys Glu Gly Val Leu Leu Leu
250 255 260
ACT CTG GAG GAG AAG ACA TTT CCA GAA GAT ATG TGC TAC CCA GAT CAA 929
Thr Leu Glu Glu Lys Thr Phe Pro Glu Asp Met Cys Tyr Pro Asp Gln
265 270 275
GGT GGT GTC TGT CAG GGC ACG TGT GTA GGA GGT GGT CCC TAC GCA CAA 977
Gly Gly Val Cys Gln Gly Thr Cys Val Gly Gly Gly Pro Tyr Ala Gln
280 285 290 295
GGC GAA GAT GCC AGG ATG CTC TCA TTG GTC AGC AAG ACC GAG ATA GAG 1025
Gly Glu Asp Ala Arg Met Leu Ser Leu Val Ser Lys Thr Glu Ile Glu
300 305 310
GAA GAC AGC TTC AGA CAG ATG CCC ACA GAA GAT GAA TAC ATG GAC AGG 1073
Glu Asp Ser Phe Arg Gln Met Pro Thr Glu Asp Glu Tyr Met Asp Arg
315 320 325
CCC TCC CAG CCC ACA GAC CAG TTA CTG TTC CTC ACT GAG CCT GGA AGC 1121
Pro Ser Gln Pro Thr Asp Gin Leu Leu Phe Leu Thr Glu Pro Gly Ser
330 335 340
AAA TCC ACA CCT CCT TTC TCT GAA CCC CTG GAG GTG GGG GAG AAT GAC 1169
Lys Ser Thr Pro Pro Phe Ser Glu Pro Leu Glu Val Gly Glu Asn Asp
345 350 355
AGT TTA AGC CAG TGC TTC ACG GGG ACA CAG AGC ACA GTG GGT TCA GAA 1217
Ser Leu Ser Gln Cys Phe Thr Gly Thr Gln Ser Thr Val Gly Ser Glu
360 365 370 375
39
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
AGC TGC AAC TGC ACT GAG CCC CTG TGC AGG ACT GAT TGG ACT CCC ATG 1265
Ser Cys Asn Cys Thr Glu Pro Leu Cys Arg Thr Asp Trp Thr Pro Met
380 385 390
TCC TCT GAA AAC TAC TTG CAA AAA GAG GTG GAC AGT GGC CAT TGC CCG 1313
Ser Ser Glu Asn Tyr Leu Gln Lys Glu Val Asp Ser Gly His Cys Pro
395 400 405
CAC TGG GCA GCC AGC CCC AGC CCC AAC TGG GCA GAT GTC TGC ACA GGC 1361
His Trp Ala Ala Ser Pro Ser Pro Asn Trp Ala Asp Val Cys Thr Gly
410 415 420
TGC CGG AAC CCT CCT GGG GAG GAC TGT GAA CCC CTC GTG GGT TCC CCA 1409
Cys Arg Asn Pro Pro Gly Glu Asp Cys Glu Pro Leu Val Gly Ser Pro
425 430 435
AAA CGT GGA CCC TTG CCC CAG TGC GCC TAT GGC ATG GGC CTT CCC CCT 1457
Lys Arg Gly Pro Leu Pro Gln Cys Ala Tyr Gly Met Gly Leu Pro Pro
440 445 450 455
GAA GAA GAA GCC AGC AGG ACG GAG GCC AGA GAC CAG CCC GAG GAT GGG 1505
Glu Glu Glu Ala Ser Arg Thr Glu Ala Arg Asp Gln Pro Glu Asp Gly
460 465 470
GCT GAT GGG AGG CTC CCA AGC TCA GCG AGG GCA GGT GCC GGG TCT GGA 1553
Ala Asp Gly Arg Leu Pro Ser Ser Ala Arg Ala Gly Ala Gly Ser Gly
475 480 485
AGC TCC CCT GGT GGC CAG TCC CCT GCA TCT GGA AAT GTG ACT GGA AAC 1601
Ser Ser Pro Gly Gly Gln Ser Pro Ala Ser Gly Asn Val Thr Gly Asn
490 495 500
AGT AAC TCC ACG TTC ATC TCC AGC GGG CAG GTG ATG AAC TTC AAG GGC 1649
Ser Asn Ser Thr Phe Ile Ser Ser Gly Gln Val Met Asn Phe Lys Gly
505 510 515
GAC ATC ATC GTG GTC TAC GTC AGC CAG ACC TCG CAG GAG GGC GCG GCG 1697
Asp Ile Ile Val Val Tyr Val Ser Gln Thr Ser Gln Glu Gly Ala Ala
520 525 530 535
GCG GCT GCG GAG CCC ATG GGC CGC CCG GTG CAG GAG GAG ACC CTG GCG 1745
Ala Ala Ala Glu Pro Met Gly Arg Pro Val Gln Glu Glu Thr Leu Ala
540 545 550
CGC CGA GAC TCC TTC GCG GGG AAC GGC CCG CGC TTC CCG GAC CCG TGC 1793
Arg Arg Asp Ser Phe Ala Gly Asn Gly Pro Arg Phe Pro Asp Pro Cys
555 560 565
GGC GGC CCC GAG GGG CTG CGG GAG CCG GAG AAG GCC TCG AGG CCG GTG 1841
Gly Gly Pro Glu Gly Leu Arg Glu Pro Glu Lys Ala Ser Arg Pro Val
570 575 580
CAG GAG CAA GGC GGG GCC AAG GCT TGA GCGCCCCCCA TGGCTGGGAG 1888
Gln Glu Gln Gly Gly Ala Lys Ala
585 590
CCCGAAGCTC GGAGCCAGGG CTCGCGAGGG CAGCACCGCA GCCTCTGCCC CAGCCCCGGC 1948
CACCCAGGGA TCGATCGGTA CAGTCGAGGA AGACCACCCG GCATTCTCTG CCCACTTTGC 2008
CTTCCAGGAA ATGGGCTTTT CAGGAAGTGA ATTGATGAGG ACTGTCCCCA TGCCCACGGA 2068
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
TGCTCAGCAG CCCGCCGCAC TGGGGCAGAT GTCTCCCCTG CCACTCCTCA AACTCGCAGC 2128
AGTAATTTGT GGCACTATGA CAGCTATTTT TATGACTATC CTGTTCTGTG GGGGGGGGGT 2188
CTATGTTTTC CCCCCATATT TGTATTCCTT TTCATAACTT TTCTTGATAT CTTTCCTCCC 2248
TCTTTTTTAA TGTAAAGGTT TTCTCAAAAA TTCTCCTAAA GGTGAGGGTC TCTTTCTTTT 2308
CTCTTTTCCT TTTTTTTTTC TTTTTTTGGC AACCTGGCTC TGGCCCAGGC TAGAGTGCAG 2368
TGGTGCGATT ATAGCCCGGT GCAGCCTCTA ACTCCTGGGC TCAAGCAATC CAAGTGATCC 2428
TCCCACCTCA ACCTTCGGAG TAGCTGGGAT CACAGCTGCA GGCCACGCCC AGCTTCCTCC 2488
CCCCGACTCC CCCCCCCCAG AGACACGGTC CCACCATGTT ACCCAGCCTG GTCTCAAACT 2548
CCCCAGCTAA AGCAGTCCTC CAGCCTCGGC CTCCCAAAGT ACTGGGATTA CAGGCGTGAG 2608
CCCCCACGCT GGCCTGCTTT ACGTATTTTC TTTTGTGCCC CTGCTCACAG TGTTTTAGAG 2668
ATGGCTTTCC CAGTGTGTGT TCATTGTAAA CACTTTTGGG AAAGGGCTAA ACATGTGAGG 2728
CCTGGAGATA GTTGCTAAGT TGCTAGGAAC ATGTGGTGGG ACTTTCATAT TCTGAAAAAT 2788
GTTCTATATT CTCATTTTTC TAAAAGAAAG AAAAAAGGAA ACCCGATTTA TTTCTCCTGA 2848
ATCTTTTTAA GTTTGTGTCG TTCCTTAAGC AGAACTAAGC TCAGTATGTG ACCTTACCCG 2908
CTAGGTGGTT AATTTATCCA TGCTGGCAGA GGCACTCAGG TACTTGGTAA GCAAATTTCT 2968
AAAACTCCAA GTTGCTGCAG CTTGGCATTC TTCTTATTCT AGAGGTCTCT CTGGAAAAGA 3028
TGGAGAAAAT GAACAGGACA TGGGGCTCCT GGAAAGAAAG GGCCCGGGAA GTTCAAGGAA 3088
GAATAAAGTT GAAATTTTAA AAAAAAA 3115
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 591 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Val Ala Leu Gln Ile Ala Pro Pro Cys Thr Ser Glu Lys His Tyr Glu
1 5 10 15
His Leu Gly Arg Cys Cys Asn Lys Cys Glu Pro Gly Lys Tyr Met Ser
20 25 30
Ser Lys Cys Thr Thr Thr Ser Asp Ser Val Cys Leu Pro Cys Gly Pro
35 40 45
Asp Glu Tyr Leu Asp Ser Trp Asn Glu Glu Asp Lys Cys Leu Leu His
50 55 60
41
CA 02274803 1999-06-15
WO 98/28424 PCTIUS97/23866
Lys Val Cys Asp Thr Gly Lys Ala Leu Val Ala Val Val Ala Gly Asn
65 70 75 80
Ser Thr Thr Pro Arg Arg Cys Ala Cys Thr Ala Gly Tyr His Trp Ser
85 90 95
Gln Asp Cys Glu Cys Cys Arg Arg Asn Thr Glu Cys Ala Pro Gly Leu
100 105 110
Gly Ala Gln His Pro Leu Gln Leu Asn Lys Asp Thr Val Cys Lys Pro
115 120 125
Cys Leu Ala Gly Tyr Phe Ser Asp Ala Phe Ser Ser Thr Asp Lys Cys
130 135 140
Arg Pro Trp Thr Asn Cys Thr Phe Leu Gly Lys Arg Val Glu His His
145 150 155 160
Gly Thr Glu Lys Ser Asp Ala Val Cys Ser Ser Ser Leu Pro Ala Arg
165 170 175
Lys Pro Pro Asn Glu Pro His Val Tyr Leu Pro Gly Leu Ile Ile Leu
180 185 190
Leu Leu Phe Ala Ser Val Ala Leu Val Ala Ala Ile Ile Phe Gly Val
195 200 205
Cys Tyr Arg Lys Lys Gly Lys Ala Leu Thr Ala Asn Leu Trp His Trp
210 215 220
Ile Asn Glu Ala Cys Gly Arg Leu Ser Gly Asp Lys Glu Ser Ser Gly
225 230 235 240
Asp Ser Cys Val Ser Thr His Thr Ala Asn Phe Gly Gln Gln Gly Ala
245 250 255
Cys Glu Gly Val Leu Leu Leu Thr Leu Glu Glu Lys Thr Phe Pro Glu
260 265 270
Asp Met Cys Tyr Pro Asp Gln Gly Gly Val Cys Gln Gly Thr Cys Val
275 280 285
Gly Gly Gly Pro Tyr Ala Gln Gly Glu Asp Ala Arg Met Leu Ser Leu
290 295 300
Val Ser Lys Thr Glu Ile Glu Glu Asp Ser Phe Arg Gln Met Pro Thr
305 310 315 320
Glu Asp Glu Tyr Met Asp Arg Pro Ser Gln Pro Thr Asp Gln Leu Leu
325 330 335
Phe Leu Thr Glu Pro Gly Ser Lys Ser Thr Pro Pro Phe Ser Glu Pro
340 345 350
Leu Glu Val Gly Glu Asn Asp Ser Leu Ser Gln Cys Phe Thr Gly Thr
355 360 365
Gln Ser Thr Val Gly Ser Glu Ser Cys Asn Cys Thr Glu Pro Leu Cys
370 375 380
42
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
Arg Thr Asp Trp Thr Pro Met Ser Ser Glu Asn Tyr Leu Gln Lys Glu
385 390 395 400
Val Asp Ser Gly His Cys Pro His Trp Ala Ala Ser Pro Ser Pro Asn
405 410 415
Trp Ala Asp Val Cys Thr Gly Cys Arg Asn Pro Pro Gly Glu Asp Cys
420 425 430
Glu Pro Leu Val Gly Ser Pro Lys Arg Gly Pro Leu Pro Gln Cys Ala
435 440 445
Tyr Gly Met Gly Leu Pro Pro Glu Glu Glu Ala Ser Arg Thr Glu Ala
450 455 460
Arg Asp Gln Pro Glu Asp Gly Ala Asp Gly Arg Leu Pro Ser Ser Ala
465 470 475 480
Arg Ala Gly Ala Gly Ser Gly Ser Ser Pro Gly Gly Gln Ser Pro Ala
485 490 495
Ser Gly Asn Val Thr Gly Asn Ser Asn Ser Thr Phe Ile Ser Ser Gly
500 505 510
Gln Val Met Asn Phe Lys Gly Asp Ile Ile Val Val Tyr Val Ser Gln
515 520 525
Thr Ser Gln Glu Gly Ala Ala Ala Ala Ala Glu Pro Met Gly Arg Pro
530 535 540
Val Gln Glu Glu Thr Leu Ala Arg Arg Asp Ser Phe Ala Gly Asn Gly
545 550 555 560
Pro Arg Phe Pro Asp Pro Cys Gly Gly Pro Glu Gly Leu Arg Glu Pro
565 570 575
Glu Lys Ala Ser Arg Pro Val Gln Glu Gln Gly Gly Ala Lys Ala
580 585 590
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1391 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: BONE-MARROW DERIVED DENDRITIC CELLS
(B) CLONE: 9D-15C
43
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 39..1391
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CCGCTGAGGC CGCGGCGCCC GCCAGCCTGT CCCGCGCC ATG GCC CCG CGC GCC 53
Met Ala Pro Arg Ala
1 5
CGG CGG CGC CGC CCG CTG TTC GCG CTG CTG CTG CTC TGC GCG CTG CTC 101
Arg Arg Arg Arg Pro Leu Phe Ala Leu Leu Leu Leu Cys Ala Leu Leu
15 20
GCC CGG CTG CAG GTG GCT TTG CAG ATC GCT CCT CCA TGT ACC AGT GAG 149
Ala Arg Leu Gln Val Ala Leu Gln Ile Ala Pro Pro Cys Thr Ser Glu
25 30 35
AAG CAT TAT GAG CAT CTG GGA CGG TGC TGT AAC AAA TGT GAA CCA GGA 197
Lys His Tyr Glu His Leu Gly Arg Cys Cys Asn Lys Cys Glu Pro Gly
40 45 50
AAG TAC ATG TCT TCT AAA TGC ACT ACT ACC TCT GAC AGT GTA TGT CTG 245
Lys Tyr Met Ser Ser Lys Cys Thr Thr Thr Ser Asp Ser Val Cys Leu
55 60 65
CCC TGT GGC CCG GAT GAA TAC TTG GAT AGC TGG AAT GAA GAA GAT AAA 293
Pro Cys Gly Pro Asp Glu Tyr Leu Asp Ser Trp Asn Glu Glu Asp Lys
70 75 80 85
TGC TTG CTG CAT AAA GTT TGT GAT ACA GGC AAG GCC CTG GTG GCC GTG 341
Cys Leu Leu His Lys Val Cys Asp Thr Gly Lys Ala Leu Val Ala Val
90 95 100
GTC GCC GGC AAC AGC ACG ACC CCC CGG CGC TGC GCG TGC ACG GCT GGG 389
Val Ala Gly Asn Ser Thr Thr Pro Arg Arg Cys Ala Cys Thr Ala Gly
105 110 115
TAC CAC TGG AGC CAG GAC TGC GAG TGC TGC CGC CGC AAC ACC GAG TGC 437
Tyr His Trp Ser Gln Asp Cys Glu Cys Cys Arg Arg Asn Thr Glu Cys
120 125 130
GCG CCG GGC CTG GGC GCC CAG CAC CCG TTG CAG CTC AAC AAG GAC ACA 485
Ala Pro Gly Leu Gly Ala Gln His Pro Leu Gln Leu Asn Lys Asp Thr
135 140 145
GTG TGC AAA CCT TGC CTT GCA GGC TAC TTC TCT GAT GCC TTT TCC TCC 533
Val Cys Lys Pro Cys Leu Ala Gly Tyr Phe Ser Asp Ala Phe Ser Ser
150 155 160 165
ACG GAC AAA TGC AGA CCC TGG ACC AAC TGT ACC TTC CTT GGA AAG AGA 581
Thr Asp Lys Cys Arg Pro Trp Thr Asn Cys Thr Phe Leu Gly Lys Arg
170 175 180
GTA GAA CAT CAT GGG ACA GAG AAA TCC GAT GCG GTT TGC AGT TCT TCT 629
Val Glu His His Gly Thr Glu Lys Ser Asp Ala Val Cys Ser Ser Ser
185 190 195
44
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
CTG CCA GCT AGA AAA CCA CCA AAT GAA CCC CAT GTT TAC TTG CCC GGT 677
Leu Pro Ala Arg Lys Pro Pro Asn Glu Pro His Val Tyr Leu Pro Gly
200 205 210
TTA ATA ATT CTG CTT CTC TTC GCG TCT GTG GCC CTG GTG GCT GCC ATC 725
Leu Ile Ile Leu Leu Leu Phe Ala Ser Val Ala Leu Val Ala Ala Ile
215 220 225
ATC TTT GGC GTT TGC TAT AGG AAA AAA GGG AAA GCA CTC ACA GCT AAT 773
Ile Phe Gly Val Cys Tyr Arg Lys Lys Gly Lys Ala Leu Thr Ala Asn
230 235 240 245
TTG TGG CAC TGG ATC AAT GAG GCT TGT GGC CGC CTA AGT GGA GAT AAG 821
Leu Trp His Trp Ile Asn Glu Ala Cys Gly Arg Leu Ser Gly Asp Lys
250 255 260
GAG TCC TCA GGT GAC AGT TGT GTC AGT ACA CAC ACG GCA AAC TTT GGT 869
Glu Ser Ser Gly Asp Ser Cys Val Ser Thr His Thr Ala Asn Phe Gly
265 270 275
CAG CAG GGA GCA TGT GAA GGT GTC TTA CTG CTG ACT CTG GAG GAG AAG 917
Gln Gln Gly Ala Cys Glu Gly Val Leu Leu Leu Thr Leu Glu Glu Lys
280 285 290
ACA TTT CCA GAA GAT ATG TGC TAC CCA GAT CAA GGT GGT GTC TGT CAG 965
Thr Phe Pro Glu Asp Met Cys Tyr Pro Asp Gln Gly Gly Val Cys Gln
295 300 305
GGC ACG TGT GTA GGA GGT GGT CCC TAC GCA CAA GGC GAA GAT GCC AGG 1013
Gly Thr Cys Val Gly Gly Gly Pro Tyr Ala Gln Gly Glu Asp Ala Arg
310 315 320 325
ATG CTC TCA TTG GTC AGC AAG ACC GAG ATA GAG GAA GAC AGC TTC AGA 1061
Met Leu Ser Leu Val Ser Lys Thr Glu Ile Glu Glu Asp Ser Phe Arg
330 335 340
CAG ATG CCC ACA GAA GAT GAA TAC ATG GAC AGG CCC TCC CAG CCC ACA 1109
Gln Met Pro Thr Glu Asp Glu Tyr Met Asp Arg Pro Ser Gln Pro Thr
345 350 355
GAC CAG TTA CTG TTC CTC ACT GAG CCT GGA AGC AAA TCC ACA CCT CCT 1157
Asp Gin Leu Leu Phe Leu Thr Glu Pro Gly Ser Lys Ser Thr Pro Pro
360 365 370
TTC TCT GAA CCC CTG GAG GTG GGG GAG AAT GAC AGT TTA AGC CAG TGC 1205
Phe Ser Glu Pro Leu Glu Val Gly Glu Asn Asp Ser Leu Ser Gln Cys
375 380 385
TTC ACG GGG ACA CAG AGC ACA GTG GGT TCA GAA AGC TGC AAC TGC ACT 1253
Phe Thr Gly Thr Gln Ser Thr Val Gly Ser Glu Ser Cys Asn Cys Thr
390 395 400 405
GAG CCC CTG TGC AGG ACT GAT TGG ACT CCC ATG TCC TCT GAA AAC TAC 1301
Glu Pro Leu Cys Arg Thr Asp Trp Thr Pro Met Ser Ser Glu Asn Tyr
410 415 420
TTG CAA AAA GAG GTG GAC AGT GGC CAT TGC CCG CAC TGG GCA GCC AGC 1349
Leu Gin Lys Glu Val Asp Ser Gly His Cys Pro His Trp Ala Ala Ser
425 430 435
*rB
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
CCC AGC CCC AAC TGG GCA GAT GTC TGC ACA GGC TGC CGG AAC 1391
Pro Ser Pro Asn Trp Ala Asp Val Cys Thr Gly Cys Arg Asn
440 445 450
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 451 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
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
46
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
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
450
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3136 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
47
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
(A) LIBRARY: BONE-MARROW DERIVED DENDRITIC CELLS
(B) CLONE: FULL LENGTH RANK
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 39..1886
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CCGCTGAGGC CGCGGCGCCC GCCAGCCTGT CCCGCGCC ATG GCC CCG CGC GCC 53
Met Ala Pro Arg Ala
1 5
CGG CGG CGC CGC CCG CTG TTC GCG CTG CTG CTG CTC TGC GCG CTG CTC 101
Arg Arg Arg Arg Pro Leu Phe Ala Leu Leu Leu Leu Cys Ala Leu Leu
15 20
GCC CGG CTG CAG GTG GCT TTG CAG ATC GCT CCT CCA TGT ACC AGT GAG 149
Ala Arg Leu Gin Val Ala Leu Gln Ile Ala Pro Pro Cys Thr Ser Glu
25 30 35
AAG CAT TAT GAG CAT CTG GGA CGG TGC TGT AAC AAA TGT GAA CCA GGA 197
Lys His Tyr Glu His Leu Gly Arg Cys Cys Asn Lys Cys Glu Pro Gly
40 45 50
AAG TAC ATG TCT TCT AAA TGC ACT ACT ACC TCT GAC AGT GTA TGT CTG 245
Lys Tyr Met Ser Ser Lys Cys Thr Thr Thr Ser Asp Ser Val Cys Leu
55 60 65
CCC TGT GGC CCG GAT GAA TAC TTG GAT AGC TGG AAT GAA GAA GAT AAA 293
Pro Cys Gly Pro Asp Glu Tyr Leu Asp Ser Trp Asn Glu Glu Asp Lys
70 75 80 85
TGC TTG CTG CAT AAA GTT TGT GAT ACA GGC AAG GCC CTG GTG GCC GTG 341
Cys Leu Leu His Lys Val Cys Asp Thr Gly Lys Ala Leu Val Ala Val
90 95 100
GTC GCC GGC AAC AGC ACG ACC CCC CGG CGC TGC GCG TGC ACG GCT GGG 389
Val Ala Gly Asn Ser Thr Thr Pro Arg Arg Cys Ala Cys Thr Ala Gly
105 110 115
TAC CAC TGG AGC CAG GAC TGC GAG TGC TGC CGC CGC AAC ACC GAG TGC 437
Tyr His Trp Ser Gln Asp Cys Glu Cys Cys Arg Arg Asn Thr Glu Cys
120 125 130
GCG CCG GGC CTG GGC GCC CAG CAC CCG TTG CAG CTC AAC AAG GAC ACA 485
Ala Pro Gly Leu Gly Ala Gln His Pro Leu Gln Leu Asn Lys Asp Thr
135 140 145
GTG TGC AAA CCT TGC CTT GCA GGC TAC TTC TCT GAT GCC TTT TCC TCC 533
Val Cys Lys Pro Cys Leu Ala Gly Tyr Phe Ser Asp Ala Phe Ser Ser
150 155 160 165
ACG GAC AAA TGC AGA CCC TGG ACC AAC TGT ACC TTC CTT GGA AAG AGA 581
Thr Asp Lys Cys Arg Pro Trp Thr Asn Cys Thr Phe Leu Gly Lys Arg
170 175 180
GTA GAA CAT CAT GGG ACA GAG AAA TCC GAT GCG GTT TGC AGT TCT TCT 629
Val Glu His His Gly Thr Glu Lys Ser Asp Ala Val Cys Ser Ser Ser
185 190 195
48
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
CTG CCA GCT AGA AAA CCA CCA AAT GAA CCC CAT GTT TAC TTG CCC GGT 677
Leu Pro Ala Arg Lys Pro Pro Asn Glu Pro His Val Tyr Leu Pro Gly
200 205 ' 210
TTA ATA ATT CTG CTT CTC TTC GCG TCT GTG GCC CTG GTG GCT GCC ATC 725
Leu Ile Ile Leu Leu Leu Phe Ala Ser Val Ala Leu Val Ala Ala Ile
215 220 225
ATC TTT GGC GTT TGC TAT AGG AAA AAA GGG AAA GCA CTC ACA GCT AAT 773
Ile Phe Gly Val Cys Tyr Arg Lys Lys Gly Lys Ala Leu Thr Ala Asn
230 235 240 245
TTG TGG CAC TGG ATC AAT GAG GCT TGT GGC CGC CTA AGT GGA GAT AAG 821
Leu Trp His Trp Ile Asn Glu Ala Cys Gly Arg Leu Ser Gly Asp Lys
250 255 260
GAG TCC TCA GGT GAC AGT TGT GTC AGT ACA CAC ACG GCA AAC TTT GGT 869
Glu Ser Ser Gly Asp Ser Cys Val Ser Thr His Thr Ala Asn Phe Gly
265 270 275
CAG CAG GGA GCA TGT GAA GGT GTC TTA CTG CTG ACT CTG GAG GAG AAG 917
Gln Gln Gly Ala Cys Glu Gly Val Leu Leu Leu Thr Leu Glu Glu Lys
280 285 290
ACA TTT CCA GAA GAT ATG TGC TAC CCA GAT CAA GGT GGT GTC TGT CAG 965
Thr Phe Pro Glu Asp Met Cys Tyr Pro Asp Gln Gly Gly Val Cys Gln
295 300 305
GGC ACG TGT GTA GGA GGT GGT CCC TAC GCA CAA GGC GAA GAT GCC AGG 1013
Gly Thr Cys Val Gly Gly Gly Pro Tyr Ala Gln Gly Glu Asp Ala Arg
310 315 320 325
ATG CTC TCA TTG GTC AGC AAG ACC GAG ATA GAG GAA GAC AGC TTC AGA 1061
Met Leu Ser Leu Val Ser Lys Thr Glu Ile Glu Glu Asp Ser Phe Arg
330 335 340
CAG ATG CCC ACA GAA GAT GAA TAC ATG GAC AGG CCC TCC CAG CCC ACA 1109
Gln Met Pro Thr Glu Asp Glu Tyr Met Asp Arg Pro Ser Gln Pro Thr
345 350 355
GAC CAG TTA CTG TTC CTC ACT GAG CCT GGA AGC AAA TCC ACA CCT CCT 1157
Asp Gln Leu Leu Phe Leu Thr Glu Pro Gly Ser Lys Ser Thr Pro Pro
360 365 370
TTC TCT GAA CCC CTG GAG GTG GGG GAG AAT GAC AGT TTA AGC CAG TGC 1205
Phe Ser Glu Pro Leu Glu Val Gly Glu Asn Asp Ser Leu Ser Gln Cys
375 380 385
TTC ACG GGG ACA CAG AGC ACA GTG GGT TCA GAA AGC TGC AAC TGC ACT 1253
Phe Thr Gly Thr Gln Ser Thr Val Gly Ser Glu Ser Cys Asn Cys Thr
390 395 400 405
GAG CCC CTG TGC AGG ACT GAT TGG ACT CCC ATG TCC TCT GAA AAC TAC 1301
Glu Pro Leu Cys Arg Thr Asp Trp Thr Pro Met Ser Ser Glu Asn Tyr
410 415 420
TTG CAA AAA GAG GTG GAC AGT GGC CAT TGC CCG CAC TGG GCA GCC AGC 1349
Leu Gln Lys Glu Val Asp Ser Gly His Cys Pro His Trp Ala Ala Ser
425 430 435
49
CA 02274803 1999-06-15
WO 98/28424 PCTIUS97/23866
CCC AGC CCC AAC TGG GCA GAT GTC TGC ACA GGC TGC CGG AAC CCT CCT 1397
Pro Ser Pro Asn Trp Ala Asp Val Cys Thr Gly Cys Arg Asn Pro Pro
440 445 450
GGG GAG GAC TGT GAA CCC CTC GTG GGT TCC CCA AAA CGT GGA CCC TTG 1445
Gly Glu Asp Cys Glu Pro Leu Val Gly Ser Pro Lys Arg Gly Pro Leu
455 460 465
CCC CAG TGC GCC TAT GGC ATG GGC CTT CCC CCT GAA GAA GAA GCC AGC 1493
Pro Gln Cys Ala Tyr Gly Met Gly Leu Pro Pro Glu Glu Glu Ala Ser
470 475 480 485
AGG ACG GAG GCC AGA GAC CAG CCC GAG GAT GGG GCT GAT GGG AGG CTC 1541
Arg Thr Glu Ala Arg Asp Gln Pro Glu Asp Gly Ala Asp Gly Arg Leu
490 495 500
CCA AGC TCA GCG AGG GCA GGT GCC GGG TCT GGA AGC TCC CCT GGT GGC 1589
Pro Ser Ser Ala Arg Ala Gly Ala Gly Ser Gly Ser Ser Pro Gly Gly
505 510 515
CAG TCC CCT GCA TCT GGA AAT GTG ACT GGA AAC AGT AAC TCC ACG TTC 1637
Gln Ser Pro Ala Ser Gly Asn Val Thr Gly Asn Ser Asn Ser Thr Phe
520 525 530
ATC TCC AGC GGG CAG GTG ATG AAC TTC AAG GGC GAC ATC ATC GTG GTC 1685
Ile Ser Ser Gly Gln Val Met Asn Phe Lys Gly Asp Ile Ile Val Val
535 540 545
TAC GTC AGC CAG ACC TCG CAG GAG GGC GCG GCG GCG GCT GCG GAG CCC 1733
Tyr Val Ser Gln Thr Ser Gln Glu Gly Ala Ala Ala Ala Ala Glu Pro
550 555 560 565
ATG GGC CGC CCG GTG CAG GAG GAG ACC CTG GCG CGC CGA GAC TCC TTC 1781
Met Gly Arg Pro Val Gln Glu Glu Thr Leu Ala Arg Arg Asp Ser Phe
570 575 580
GCG GGG AAC GGC CCG CGC TTC CCG GAC CCG TGC GGC GGC CCC GAG GGG 1829
Ala Gly Asn Gly Pro Arg Phe Pro Asp Pro Cys Gly Gly Pro Glu Gly
585 590 595
CTG CGG GAG CCG GAG AAG GCC TCG AGG CCG GTG CAG GAG CAA GGC GGG 1877
Leu Arg Glu Pro Glu Lys Ala Ser Arg Pro Val Gln Glu Gln Gly Gly
600 605 610
GCC AAG GCT TGAGCGCCCC CCATGGCTGG GAGCCCGAAG CTCGGAGCCA 1926
Ala Lys Ala
615
GGGCTCGCGA GGGCAGCACC GCAGCCTCTG CCCCAGCCCC GGCCACCCAG GGATCGATCG 1986
GTACAGTCGA GGAAGACCAC CCGGCATTCT CTGCCCACTT TGCCTTCCAG GAAATGGGCT 2046
TTTCAGGAAG TGAATTGATG AGGACTGTCC CCATGCCCAC GGATGCTCAG CAGCCCGCCG 2106
CACTGGGGCA GATGTCTCCC CTGCCACTCC TCAAACTCGC AGCAGTAATT TGTGGCACTA 2166
TGACAGCTAT TTTTATGACT ATCCTGTTCT GTGGGGGGGG GGTCTATGTT TTCCCCCCAT 2226
ATTTGTATTC CTTTTCATAA CTTTTCTTGA TATCTTTCCT CCCTCTTTTT TAATGTAAAG 2286
GTTTTCTCAA AAATTCTCCT AAAGGTGAGG GTCTCTTTCT TTTCTCTTTT CCTTTTTTTT 2346
CA 02274803 1999-06-15
WO 98/28424 PCTIUS97/23866
TTCTTTTTTT GGCAACCTGG CTCTGGCCCA GGCTAGAGTG CAGTGGTGCG ATTATAGCCC 2406
GGTGCAGCCT CTAACTCCTG GGCTCAAGCA ATCCAAGTGA TCCTCCCACC TCAACCTTCG 2466
GAGTAGCTGG GATCACAGCT GCAGGCCACG CCCAGCTTCC TCCCCCCGAC TCCCCCCCCC 2526
CAGAGACACG GTCCCACCAT GTTACCCAGC CTGGTCTCAA ACTCCCCAGC TAAAGCAGTC 2586
CTCCAGCCTC GGCCTCCCAA AGTACTGGGA TTACAGGCGT GAGCCCCCAC GCTGGCCTGC 2646
TTTACGTATT TTCTTTTGTG CCCCTGCTCA CAGTGTTTTA GAGATGGCTT TCCCAGTGTG 2706
TGTTCATTGT AAACACTTTT GGGAAAGGGC TAAACATGTG AGGCCTGGAG ATAGTTGCTA 2766
AGTTGCTAGG AACATGTGGT GGGACTTTCA TATTCTGAAA AATGTTCTAT ATTCTCATTT 2826
TTCTAAAAGA AAGAAAAAAG GAAACCCGAT TTATTTCTCC TGAATCTTTT TAAGTTTGTG 2886
TCGTTCCTTA AGCAGAACTA AGCTCAGTAT GTGACCTTAC CCGCTAGGTG GTTAATTTAT 2946
CCATGCTGGC AGAGGCACTC AGGTACTTGG TAAGCAAATT TCTAAAACTC CAAGTTGCTG 3006
CAGCTTGGCA TTCTTCTTAT TCTAGAGGTC TCTCTGGAAA AGATGGAGAA AATGAACAGG 3066
ACATGGGGCT CCTGGAAAGA AAGGGCCCGG GAAGTTCAAG GAAGAATAAA GTTGAAATTT 3126
TAAAAAAAAA 3136
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 616 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
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 An
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
51
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
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
52
CA 02274803 1999-06-15
WO 98/28424 PCTIUS97/23866
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
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vii) IMMEDIATE SOURCE:
(B) CLONE: FLAG peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 232 amino acids
(B) TYPE: amino acid
53
*rB
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Human
(vii) IMMEDIATE SOURCE:
(B) CLONE: IgOl Fc mutein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
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
54
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
(2) INFORMATION FOR SEQ ID NO:9:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: CMV (R2780 Leader)
(ix) FEATURE:
(D) OTHER INFORMATION: Metl-Arg28 is the actual leader
peptide; Arg29 strengthens the furin cleavage site;
nucleotides encoding Thr30 and Ser3l add a Spel site.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Ala Arg Arg Leu Trp Ile Leu Ser Leu Leu Ala Val Thr Leu Thr
1 5 10 15
Val Ala Leu Ala Ala Pro Ser Gln Lys Ser Lys Arg Arg Thr Ser
20 25 30
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1630 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE: RANKL
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..884
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CC GGC GTC CCA CAC GAG GGT CCG CTG CAC CCC GCG CCT TCT GCA CCG 47
Gly Val Pro His Glu Gly Pro Leu His Pro Ala Pro Ser Ala Pro
1 5 10 15
CA 02274803 1999-06-15
WO 98/28424 PCTIUS97/23866
GCT CCG GCG CCG CCA CCC GCC GCC TCC CGC TCC ATG TTC CTG GCC CTC 95
Ala Pro Ala Pro Pro Pro Ala Ala Ser Arg Ser Met Phe Leu Ala Leu
20 25 30
CTG GGG CTG GGA CTG GGC CAG GTG GTC TGC AGC ATC GCT CTG TTC CTG 143
Leu Gly Leu Gly Leu Gly Gln Val Val Cys Ser Ile Ala Leu Phe Leu
35 40 45
TAC TTT CGA GCG CAG ATG GAT CCT AAC AGA ATA TCA GAA GAC AGC ACT 191
Tyr Phe Arg Ala Gln Met Asp Pro Asn Arg Ile Ser Glu Asp Ser Thr
50 55 60
CAC TGC TTT TAT AGA ATC CTG AGA CTC CAT GAA AAC GCA GAT TTG CAG 239
His Cys Phe Tyr Arg Ile Leu Arg Leu His Glu Asn Ala Asp Leu Gln
65 70 75
GAC TCG ACT CTG GAG AGT GAA GAC ACA CTA CCT GAC TCC TGC AGG AGG 287
Asp Ser Thr Leu Glu Ser Glu Asp Thr Leu Pro Asp Ser Cys Arg Arg
80 85 90 95
ATG AAA CAA GCC TTT CAG GGG GCC GTG CAG AAG GAA CTG CAA CAC ATT 335
Met Lys Gln Ala Phe Gln Gly Ala Val Gln Lys Glu Leu Gln His Ile
100 105 110
GTG GGG CCA CAG CGC TTC TCA GGA GCT CCA GCT ATG ATG GAA GGC TCA 383
Val Gly Pro Gln Arg Phe Ser Gly Ala Pro Ala Met Met Glu Gly Ser
115 120 125
TGG TTG GAT GTG GCC CAG CGA GGC AAG CCT GAG GCC CAG CCA TTT GCA 431
Trp Leu Asp Val Ala Gln Arg Gly Lys Pro Glu Ala Gln Pro Phe Ala
130 135 140
CAC CTC ACC ATC AAT GCT GCC AGC ATC CCA TCG GGT TCC CAT AAA GTC 479
His Leu Thr Ile Asn Ala Ala Ser Ile Pro Ser Gly Ser His Lys Val
145 150 155
ACT CTG TCC TCT TGG TAC CAC GAT CGA GGC TGG GCC AAG ATC TCT AAC 527
Thr Leu Ser Ser Trp Tyr His Asp Arg Gly Trp Ala Lys Ile Ser Asn
160 165 170 175
ATG ACG TTA AGC AAC GGA AAA CTA AGG GTT AAC CAA GAT GGC TTC TAT 575
Met Thr Leu Ser Asn Gly Lys Leu Arg Val Asn Gln Asp Gly Phe Tyr
180 185 190
TAC CTG TAC GCC AAC ATT TGC TTT CGG CAT CAT GAA ACA TCG GGA AGC 623
Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His His Glu Thr Ser Gly Ser
195 200 205
GTA CCT ACA GAC TAT CTT CAG CTG ATG GTG TAT GTC GTT AAA ACC AGC 671
Val Pro Thr Asp Tyr Leu Gln Leu Met Val Tyr Val Val Lys Thr Ser
210 215 220
ATC AAA ATC CCA AGT TCT CAT AAC CTG ATG AAA GGA GGG AGC ACG AAA 719
Ile Lys Ile Pro Ser Ser His Asn Leu Met Lys Gly Gly Ser Thr Lys
225 230 235
AAC TGG TCG GGC AAT TCT GAA TTC CAC TTT TAT TCC ATA AAT GTT GGG 767
Asn Trp Ser Gly Asn Ser Glu Phe His Phe Tyr Ser Ile Asn Val Gly
240 245 250 255
56
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
GGA TTT TTC AAG CTC CGA GCT GGT GAA GAA ATT AGC ATT CAG GTG TCC 815
Gly Phe Phe Lys Leu Arg Ala Gly Glu Glu Ile Ser Ile Gln Val Ser
260 265 270
AAC CCT TCC CTG CTG GAT CCG GAT CAA GAT GCG ACG TAC TTT GGG GCT 863
Asn Pro Ser Leu Leu Asp Pro Asp Gln Asp Ala Thr Tyr Phe Gly Ala
275 280 285
TTC AAA GTT CAG GAC ATA GAC TGAGACTCAT TTCGTGGAAC ATTAGCATGG 914
Phe Lys Val Gln Asp Ile Asp
290
ATGTCCTAGA TGTTTGGAAA CTTCTTAAAA AATGGATGAT GTCTATACAT GTGTAAGACT 974
ACTAAGAGAC ATGGCCCACG GTGTATGAAA CTCACAGCCC TCTCTCTTGA GCCTGTACAG 1034
GTTGTGTATA TGTAAAGTCC ATAGGTGATG TTAGATTCAT GGTGATTACA CAACGGTTTT 1094
ACAATTTTGT AATGATTTCC TAGAATTGAA CCAGATTGGG AGAGGTATTC CGATGCTTAT 1154
GAAAAACTTA CACGTGAGCT ATGGAAGGGG GTCACAGTCT CTGGGTCTAA CCCCTGGACA 1214
TGTGCCACTG AGAACCTTGA AATTAAGAGG ATGCCATGTC ATTGCAAAGA AATGATAGTG 1274
TGAAGGGTTA AGTTCTTTTG AATTGTTACA TTGCGCTGGG ACCTGCAAAT AAGTTCTTTT 1334
TTTCTAATGA GGAGAGAAAA ATATATGTAT TTTTATATAA TGTCTAAAGT TATATTTCAG 1394
GTGTAATGTT TTCTGTGCAA AGTTTTGTAA ATTATATTTG TGCTATAGTA TTTGATTCAA 1454
AATATTTAAA AATGTCTCAC TGTTGACATA TTTAATGTTT TAAATGTACA GATGTATTTA 1514
ACTGGTGCAC TTTGTAATTC CCCTGAAGGT ACTCGTAGCT AAGGGGGCAG AATACTGTTT 1574
CTGGTGACCA CATGTAGTTT ATTTCTTTAT TCTTTTTAAC TTAATAGAGT CTTCAG 1630
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 294 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Gly Val Pro His Glu Gly Pro Leu His Pro Ala Pro Ser Ala Pro Ala
1 5 10 15
Pro Ala Pro Pro Pro Ala Ala Ser Arg Ser Met Phe Leu Ala Leu Leu
20 25 30
Gly Leu Gly Leu Gly Gln Val Val Cys Ser Ile Ala Leu Phe Leu Tyr
35 40 45
Phe Arg Ala Gln Met Asp Pro Asn Arg Ile Ser Glu Asp Ser Thr His
50 55 60
57
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
Cys Phe Tyr Arg Ile Leu Arg Leu His Glu Asn Ala Asp Leu Gln Asp
65 70 75 80
Ser Thr Leu Glu Ser Glu Asp Thr Leu Pro Asp Ser Cys Arg Arg Met
85 90 95
Lys Gln Ala Phe Gln Gly Ala Val Gln Lys Glu Leu Gln His Ile Val
100 105 110
Gly Pro Gln Arg Phe Ser Gly Ala Pro Ala Met Met Glu Gly Ser Trp
115 120 125
Leu Asp Val Ala Gln Arg Gly Lys Pro Glu Ala Gln Pro Phe Ala His
130 135 140
Leu Thr Ile Asn Ala Ala Ser Ile Pro Ser Gly Ser His Lys Val Thr
145 150 155 160
Leu Ser Ser Trp Tyr His Asp Arg Gly Trp Ala Lys Ile Ser Asn Met
165 170 175
Thr Leu Ser Asn Gly Lys Leu Arg Val Asn Gln Asp Gly Phe Tyr Tyr
180 185 190
Leu Tyr Ala Asn Ile Cys Phe Arg His His Glu Thr Ser Gly Ser Val
195 200 205
Pro Thr Asp Tyr Leu Gln Leu Met Val Tyr Val Val Lys Thr Ser Ile
210 215 220
Lys Ile Pro Ser Ser His Asn Leu Met Lys Gly Gly Ser Thr Lys Asn
225 230 235 240
Trp Ser Gly Asn Ser Glu Phe His Phe Tyr Ser Ile Asn Val Gly Gly
245 250 255
Phe Phe Lys Leu Arg Ala Gly Glu Glu Ile Ser Ile Gln Val Ser Asn
260 265 270
Pro Ser Leu Leu Asp Pro Asp Gln Asp Ala Thr Tyr Phe Gly Ala Phe
275 280 285
Lys Val Gln Asp Ile Asp
290
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 954 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
58
CA 02274803 1999-06-15
WO 98/28424 PCTIUS97/23866
(A) ORGANISM: Homo sapiens
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE: huRANKL (full length)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..951
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
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
59
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
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
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 317 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
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
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
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
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1878 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Murine
61
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: Murine Fetal Liver Epithelium
(B) CLONE: muRANK
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1875
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
ATG GCC CCG CGC GCC CGG CGG CGC CGC CAG CTG CCC GCG CCG CTG CTG 48
Net Ala Pro Arg Ala Arg Arg Arg Arg Gin 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 Gin Val Thr Leu Gin 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 Gin 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 Gin His Pro Leu
130 135 140
CAG CTC AAC AAG GAT ACG GTG TGC ACA CCC TGC CTC CTG GGC TTC TTC 480
Gin 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 Gin Gly Thr Thr Glu Ser Asp
180 185 190
62
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
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
63
CA 02274803 1999-06-15
WO 98/28424 PCTIUS97/23866
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
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 625 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
64
CA 02274803 1999-06-15
WO 98/28424 PCTIUS97/23866
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
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
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
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 Gin 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 Gin 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
66
CA 02274803 1999-06-15
WO 98/28424 PCT/US97/23866
Glu
625
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Asp Tyr Lys Asp Glu
5
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
His His His His His His
5
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
67
CA 02274803 1999-06-15
WO 98/28424 PCTIUS97/23866
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
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
68
