U.S. patent application number 14/065133 was filed with the patent office on 2014-07-03 for receptor activator of nf-kappab.
This patent application is currently assigned to IMMUNEX CORPORATION. The applicant listed for this patent is IMMUNEX CORPORATION. Invention is credited to DIRK M. ANDERSON.
Application Number | 20140186887 14/065133 |
Document ID | / |
Family ID | 27369779 |
Filed Date | 2014-07-03 |
United States Patent
Application |
20140186887 |
Kind Code |
A1 |
ANDERSON; DIRK M. |
July 3, 2014 |
RECEPTOR ACTIVATOR OF NF-kappaB
Abstract
Isolated receptors, DNAs encoding such receptors, and
pharmaceutical compositions made therefrom, are disclosed. The
isolated receptors can be used to regulate an immune response. The
receptors are also useful in screening for inhibitors thereof.
Inventors: |
ANDERSON; DIRK M.; (Port
Townsend, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IMMUNEX CORPORATION |
Thousand Oaks |
CA |
US |
|
|
Assignee: |
IMMUNEX CORPORATION
Thousand Oaks
CA
|
Family ID: |
27369779 |
Appl. No.: |
14/065133 |
Filed: |
October 28, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13424179 |
Mar 19, 2012 |
8569456 |
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14065133 |
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13092718 |
Apr 22, 2011 |
8153775 |
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13424179 |
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11881911 |
Jul 30, 2007 |
7932375 |
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13092718 |
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10405878 |
Apr 1, 2003 |
7262274 |
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11881911 |
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09871291 |
May 30, 2001 |
6562948 |
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10405878 |
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09577800 |
May 24, 2000 |
6479635 |
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09871291 |
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09466496 |
Dec 17, 1999 |
6528482 |
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09577800 |
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08996139 |
Dec 22, 1997 |
6017729 |
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09466496 |
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60064671 |
Oct 14, 1997 |
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60077181 |
Mar 7, 1997 |
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60059978 |
Dec 23, 1996 |
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Current U.S.
Class: |
435/69.1 ;
435/252.3; 435/252.31; 435/252.33; 435/252.34; 435/252.35;
435/254.2; 435/254.21; 435/254.23; 435/320.1; 435/325; 435/348;
435/352; 435/361; 435/365; 435/367; 536/23.5 |
Current CPC
Class: |
C07K 2319/73 20130101;
C07K 2317/76 20130101; C07K 14/52 20130101; C12Q 1/6876 20130101;
C07K 14/525 20130101; C07K 2319/30 20130101; A61P 29/00 20180101;
A61K 38/00 20130101; C07K 16/241 20130101; C07K 2319/32 20130101;
A61P 37/00 20180101; C07K 14/7151 20130101; C07K 2319/00 20130101;
C07K 16/2875 20130101; A61P 43/00 20180101; C07K 14/70575 20130101;
C07K 14/70578 20130101; C07K 14/435 20130101; C07K 2319/02
20130101; C07K 2317/73 20130101; A61P 37/02 20180101; A61P 35/00
20180101 |
Class at
Publication: |
435/69.1 ;
435/325; 435/348; 435/352; 435/361; 435/365; 435/367; 435/252.3;
435/252.31; 435/252.33; 435/252.34; 435/252.35; 435/254.2;
435/254.21; 435/254.23; 435/320.1; 536/23.5 |
International
Class: |
C07K 14/715 20060101
C07K014/715 |
Claims
1. An isolated DNA encoding a polypeptide comprising the amino acid
sequence of amino acids 33 through 196 of SEQ ID NO:6 or a fragment
thereof, wherein the polypeptide or fragment can bind a RANKL
polypeptide consisting of the amino acid sequence set forth in SEQ
ID NO:13.
2. An isolated DNA encoding a RANK polypeptide, wherein the amino
acid sequence of the polypeptide is at least 80% identical to the
amino acid sequence as set forth in SEQ ID NO:6, and wherein the
RANK polypeptide can bind a RANKL polypeptide consisting of the
amino acid sequence set forth in SEQ ID NO:13.
3. A vector comprising the DNA of claim 1.
4. A vector comprising the DNA of claim 2.
5. A host cell comprising the vector of claim 3.
6. A host cell comprising the vector of claim 4.
7. A process for preparing a polypeptide comprising culturing the
host cell of claim 5 under conditions promoting expression of the
polypeptide.
8. A process for preparing a polypeptide comprising culturing the
host cell of claim 6 under conditions promoting expression of the
polypeptide.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/424,179 filed Mar. 19, 2012, which is a
continuation of U.S. patent application Ser. No. 13/092,718 filed
Apr. 22, 2011 (now U.S. Pat. No. 8,153,775), which is a
continuation of U.S. patent application Ser. No. 11/881,911 filed
Jul. 30, 2007 (now U.S. Pat. No. 7,932,375), which is a divisional
of U.S. patent application Ser. No. 10/405,878 filed Apr. 1, 2003
(now U.S. Pat. No. 7,262,274), which is a continuation of U.S.
patent application Ser. No. 09/871,291 filed May 30, 2001 (now U.S.
Pat. No. 6,562,948), which is a divisional of U.S. patent
application Ser. No. 09/577,800 filed May 24, 2000 (now U.S. Pat.
No. 6,479,635), which is a continuation of U.S. patent application
Ser. No. 09/466,496 filed Dec. 17, 1999 (now U.S. Pat. No.
6,528,482), which is a continuation of U.S. patent application Ser.
No. 08/996,139 filed Dec. 22, 1997 (now U.S. Pat. No. 6,017,729),
which claims the benefit of U.S. provisional application No.
60/064,671 filed Oct. 14, 1997, U.S. provisional application No.
60/077,181 filed Mar. 7, 1997, and U.S. provisional application No.
60/059,978, filed Dec. 23, 1996.
REFERENCE TO THE SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled Seq_List_ST25.txt, created Oct. 21, 2013, which is
77,824 bytes in size. The information in the electronic format of
the Sequence Listing is incorporated hereby reference in its
entirety.
TECHNICAL FIELD OF THE INVENTION
[0003] 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
[0004] 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; U.S. Ser. No. 08/720,864, filed
Oct. 4, 1996).
[0005] 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 (U.S. Ser. No. 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.sup.-/- fetal liver cells
resemble those of CD40-deficient mice, although TRAF3.sup.-/- B
cells appear to be functionally normal.
[0006] 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
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
[0007] The present invention provides a novel receptor, referred to
as RANK (for receptor activator of NF-.kappa.B), that is a member
of the TNF receptor superfamily. RANK is a Type I transmembrane
protein having 616 amino acid residues 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-.kappa.B, a ubiquitous transcription
factor that is most extensively utilized in cells of the immune
system.
[0008] Soluble forms of the receptor can be prepared and used to
interfere with signal transduction through membrane-bound RANK, and
hence upregulation of NF-.kappa.B; accordingly, pharmaceutical
compositions comprising soluble forms of the novel receptor are
also provided. Inhibition of NF-.kappa.B 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.
[0009] 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.
[0010] 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.
[0011] These and other aspects of the present invention will become
evident upon reference to the following detailed description of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 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.
[0013] FIG. 2 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 [.sup.3H]-thymidine and the cells harvested onto
glass fiber sheets for counting. Values represent the
mean.+-.standard deviation (SD) of triplicate cultures.
DETAILED DESCRIPTION OF THE INVENTION
[0014] 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:1 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:1 and 3.
[0015] 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 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.
[0016] 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.
[0017] 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
[0018] 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.
[0019] 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 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.
[0020] 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:1 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.
[0021] 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.
[0022] 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.
[0023] Other derivatives of the RANK proteins within the scope of
this invention include covalent or aggregative conjugates of the
proteins or their fragments with other proteins or polypeptides,
such as by synthesis in recombinant culture as N-terminal or
C-terminal fusions. For example, the conjugated peptide may be a
signal (or leader) polypeptide sequence at the N-terminal region of
the protein which co-translationally or post-translationally
directs transfer of the protein from its site of synthesis to its
site of function inside or outside of the cell membrane or wall
(e.g., the yeast .alpha.-factor leader).
[0024] Protein fusions can comprise peptides added to facilitate
purification or identification of RANK proteins and homologs (e.g.,
poly-His). The amino acid sequence of the inventive proteins can
also be linked to an identification peptide such as that described
by Hopp et al., Bio/Technology 6:1204 (1988). 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.
[0025] Fusion proteins further comprise the amino acid sequence of
a RANK linked to an immunoglobulin Fc region. An exemplary Fc
region is a human IgG.sub.1 having 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 Fc.gamma.RI. Canfield and Morrison (J. Exp.
Med. 173:1483; 1991) reported that Leu.sub.(234) and Leu.sub.(235)
were critical to high affinity binding of IgG.sub.3 to Fc.gamma.RI
present on U937 cells. Similar results were obtained by Lund et al.
(J. Immunol. 147:2657, 1991; Molecular Immunol. 29:53, 1991). Such
mutations, alone or in combination, can be made in an IgG.sub.1 Fc
region to decrease the affinity of IgG.sub.1 for FcR. Depending on
the portion of the Fc region used, a fusion protein may be
expressed as a dimer, through formation of interchain disulfide
bonds. If the fusion proteins are made with both heavy and light
chains of an antibody, it is possible to form a protein oligomer
with as many as four RANK regions.
[0026] 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 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.
[0027] 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 oligomerization of heat-shock transcription factors
(Rabindran et al., Science 259:230, 1993).
[0028] 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).sub.n
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.
[0029] 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
.alpha.-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.
[0030] 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 (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.
[0031] 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, Calif.). 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.
[0032] 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 signaling, for example using
the immunoprecipitation techniques described herein, or another
technique such as a yeast two-hybrid system (Rothe et al.,
supra).
[0033] 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.sub.1-Z, where
A.sub.1 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 Asn or for residue Z, deleting
Asn or Z, or inserting a non-Z amino acid between A.sub.1 and Z, or
an amino acid other than Asn between Asn and A.sub.1.
[0034] 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).
[0035] 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.
[0036] 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-.kappa.B 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.
[0037] 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-.kappa.B 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
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 Gln and Asn. Other such conservative substitutions, for
example, substitutions of entire regions having similar
hydrophobicity characteristics, are well known.
[0038] 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.
[0039] 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, incorporated herein by
reference), 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.
[0040] 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.
[0041] 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.
Pat. Nos. 4,518,584 and 4,737,462 disclose suitable techniques, and
are incorporated by reference herein.
[0042] 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.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and
hybridization conditions of 50.degree. C., 5.times.SSC, overnight)
to the DNA sequences encoding RANK, or more preferably under
stringent conditions (for example, hybridization in 6.times.SSC at
63.degree. C. overnight; washing in 3.times.SSC at 55.degree. C.),
and other sequences which are degenerate to those which encode the
RANK. In one embodiment, RANK polypeptides are at least about 70%
identical in amino acid sequence to the amino acid sequence of
native RANK protein as set forth in SEQ ID NO: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.
[0043] Percent identity may be determined using a computer program,
for example, the GAP computer program described by Devereux et al.
(Nuel. 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
[0044] 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.
[0045] 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 25 nucleotides,
more preferably at least 30 consecutive nucleotides, of the RANK
DNA 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).
[0046] 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.
[0047] 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
[0048] 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-.kappa.B 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.
[0049] 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.
[0050] 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-.kappa.B 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.
[0051] 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
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.
[0052] 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
[0053] 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.
[0054] 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.
[0055] 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 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.
[0056] 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 pGEM1 (Promega Biotec, Madison, Wis., 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.
[0057] Promoters commonly used in recombinant microbial expression
vectors include the .beta.-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 .lamda. P.sub.L
promoter and cI857ts thermolabile repressor. Plasmid vectors
available from the American Type Culture Collection which
incorporate derivatives of the .lamda. P.sub.L promoter include
plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and
pPLc28, resident in E. coli RR1 (ATCC 53082).
[0058] 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.
[0059] Preferred yeast vectors can be assembled using DNA sequences
from pBR322 for selection and replication in E. coli (Amp.sup.r
gene and origin of replication) and yeast DNA sequences including a
glucose-repressible ADH2 promoter and .alpha.-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 .alpha.-factor leader, which directs secretion 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.
[0060] 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).
[0061] 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.
[0062] 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.
Host Cells
[0063] 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.
[0064] 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), the relevant
disclosure of which is hereby incorporated by reference.
[0065] 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.
[0066] 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 2.mu. 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 trp1 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 trp1 lesion in the yeast host cell genome then provides an
effective environment for detecting transformation by growth in the
absence of tryptophan.
[0067] 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.sup.+ transformants in a selective medium consisting of 0.67%
yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 .mu.g/ml
adenine and 20 .mu.g/ml uracil. Host strains transformed by vectors
comprising the ADH2 promoter may be grown for expression in a rich
medium consisting of 1% yeast extract, 2% peptone, and 1% glucose
supplemented with 80 .mu.g/ml adenine and 80 .mu.g/ml uracil.
Derepression of the ADH2 promoter occurs upon exhaustion of medium
glucose. Crude yeast supernatants are harvested by filtration and
held at 4.degree. C. prior to further purification.
[0068] 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, Bio/Technology 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-1/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
[0069] Purified RANK, and homologs or analogs thereof are prepared
by culturing suitable host/vector systems to express the
recombinant translation products of the DNAs of the present
invention, which are then purified from culture media or cell
extracts. For example, supernatants from systems which secrete
recombinant protein into culture media can be first concentrated
using a commercially available protein concentration filter, for
example, an Amicon or Millipore Pellicon ultrafiltration unit.
[0070] Following the concentration step, the concentrate can be
applied to a suitable purification matrix. For example, a suitable
affinity matrix can comprise a counter structure protein or lectin
or antibody molecule bound to a suitable support. Alternatively, an
anion exchange resin can be employed, for example, a matrix or
substrate having pendant diethylaminoethyl (DEAE) groups. The
matrices can be acrylamide, agarose, dextran, cellulose or other
types commonly employed in protein purification. Alternatively, a
cation exchange step can be employed. Suitable cation exchangers
include various insoluble matrices comprising sulfopropyl or
carboxymethyl groups. Sulfopropyl groups are preferred. Gel
filtration chromatography also provides a means of purifying the
inventive proteins.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
Uses and Administration of RANK Compositions
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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-.kappa.B
activity. NF-.kappa.B 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-.kappa.B in cellular
transformation. Tumor cells are more responsive to radiation when
their NF-.kappa.B 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.
[0080] The following examples are offered by way of illustration,
and not by way of limitation. Those skilled in the art will
recognize that variations of the invention embodied in the examples
can be made, especially in light of the teachings of the various
references cited herein, the disclosures of which are incorporated
by reference.
Example 1
[0081] 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 al., 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 .sup.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 5.times.SSC, 50% formamide at 42.degree. C.
overnight, washing in 0.5.times.SSC at 63.degree. 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, N.Y.; 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:1 and 3.
[0082] The encoded protein was designated RANK, for receptor
activator of NF-.kappa.B. The cDNA encodes a predicted Type 1
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 (pBluescriptSK-) containing human RANK
sequence, designated pBluescript:huRANK (in E. coli DH10B), was
deposited with the American Type Culture Collection, Manassas, Va.
(ATCC) on Dec. 20, 1996, under terms of the Budapest Treaty, and
given accession number 98285.
Example 2
[0083] 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.sub.1 was constructed in the
mammalian expression vector pDC409 (U.S. Ser. No. 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.sub.1 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.sub.1
mutein was also prepared. Both expression vectors were found to
induce high levels of expression of the RANK/Fc fusion protein in
transfected cells.
[0084] 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 sepharose 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
.about.55 kDa in the presence of a reducing agent, and at a
molecular weight of .about.110 kDa in the absence of a reducing
agent.
[0085] N-terminal amino acid sequencing of the purified protein
made using the CMV 827080 leader showed 60% cleavage after Ala20,
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 Gln25 (80% after Gln25 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.
[0086] 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 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
[0087] 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. Pat. No.
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.
[0088] 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,
Mont.), and injected in amounts ranging from 10-100 .mu.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.
[0089] 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., NS1 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.
[0090] 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.
Pat. No. 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 (>1 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.
[0091] 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
[0092] This example illustrates the induction of NF-.kappa.B
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-.kappa.B 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-.kappa.B
activity by a gel retardation assay using a 25 base pair
oligonucleotide spanning the NF-.kappa.B 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.
[0093] Nuclear extracts were prepared by solubilization of isolated
nuclei with 400 mM NaCl (Yao et al., supra). Oligonucleotides
containing an NF-.kappa.B binding site were annealed and end
labeled with .sup.32P using T4 DNA polynucleotide kinase. Mobility
shift reactions contained 10 .mu.g of nuclear extract, 4 .mu.g 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.times. Tris-borate-EDTA buffer.
[0094] Overexpression of RANK resulted in induction of NF-.kappa.B
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.
Example 5
[0095] 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-.alpha.) is known to be dependent
upon intact NF-.kappa.B 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.
[0096] 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.times.10.sup.4 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.
[0097] In one such experiment, the 293/EBNA 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 DEAE 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.
[0098] For the mIL-4R-specific RIA, a monoclonal antibody reactive
with mIL-4R is labeled with .sup.125I via a Chloramine T
conjugation method; the resulting specific activity is typically
1.5.times.10.sup.16 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.degree. C./5%
CO.sub.2 in a tissue culture incubator for one hour. The blocking
media is decanted and binding buffer containing .sup.125I
anti-mIL-4R (clone M1; rat IgG1) 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 (2.times.) and twice with
phosphate-buffered saline (PBS). Cells are lysed in 1 ml of 0.5M
NaOH, and total radioactivity is measured with a gamma counter.
[0099] Using this assay, 293/EBNA co-transfected with DNAs encoding
RANK demonstrated transcriptional activation, as shown by detection
of muIL-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
[0100] 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.RTM.; 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-40. 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.RTM. epitope. TRAFs 2 and 3 did
not immunoprecipitate with anti-RANK in the absence of RANK
expression.
Example 7
[0101] 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 U.S. Ser. No. 08/249,189, filed
May 24, 1994 (the relevant disclosure of which is incorporated by
reference herein), 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 CV1-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.
[0102] 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 DH10B), was deposited with the American
Type Culture Collection, Manassas, Va. (ATCC) on Dec. 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 U.S. Pat. No. 5,599,905, issued Feb. 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.
[0103] 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 BIAcore assays
essentially as described in U.S. Ser. No. 08/249,189, 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-.kappa.B activation and
thus protecting cells that express RANK from apoptosis.
Example 8
[0104] 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 85% identical to
the corresponding region of murine RANK ligand, confirming that the
fragment was from human RANKL.
[0105] 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, Calif.), substantially as described in U.S.
Pat. No. 5,599,905, issued Feb. 4, 1997. Several positive
hybridizing plaques were identified and purified, their inserts
subcloned into pBluescript SK.sup.- (Stratagene, La Jolla, Calif.),
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.
[0106] 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
Mar. 11, 1997 under terms of the Budapest Treaty, and given
accession number 98354.
[0107] Murine and human RANKL are Type 2 transmembrane proteins.
Murine RANKL contains a predicted 48 amino acid intracellular
domain, 21 amino acid transmembrane domain and 247 amino acid
extracellular domain. Human RANKL contains a predicted 47 amino
acid intracellular domain, 21 amino acid transmembrane domain and
249 amino acid extracellular domain.
Example 9
[0108] 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 PCRable DNA kit from BIOS Laboratories
(New Haven, Conn.), 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 Genebridge 4 Radiation Hybrid Panel
(Research Genetics, Huntsville, Ala.; described in Walter, M A et
al., Nature Genetics 7:22-28, 1994). Data from this analysis was
then submitted electronically to the MIT Radiation Hybrid Mapper
following the instructions contained therein. This analysis yielded
specific genetic marker names which, when submitted electronically
to the NCBI Entrez browser, yielded the specific map locations.
RANK mapped to chromosome 18q22.1, and RANKL mapped to chromosome
13q14.
Example 10
[0109] 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 U.S. Pat.
No. 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 diagnostic or research assays for RANKL or RANKL
activity, or in affinity purification of RANKL.
[0110] 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,
Mont.), and injected in amounts ranging from 10-100 .mu.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.
[0111] 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., NS1 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.
[0112] 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 U.S. Pat. No. 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 (>1 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.
Example 11
[0113] 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-.beta.
(TGF-.beta.) and other commercially available cytokines
(IL1-.alpha., IL-2, IL-3, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15,
IFN-.gamma., TNF-.alpha.). 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-US-00001 TABLE 1 Upregulation of RANK by Cytokines Cytokine
(concentration) Results: IL-4 (50 ng/ml) + TGF-.beta. (5 ng/ml) +
to ++ IL-4 (50 ng/ml) + TGF-.beta. (5 ng/ml) ++++ IL1-.alpha. (10
ng/ml) - IL-2 (20 ng/ml) - IL-3 (25 ng/ml) - IL-7 (20 ng/ml) - IL-8
(10 ng/ml) - IL-10 (50 ng/ml) - IL-12 (10 ng/ml) - IL-15 (10 ng/ml)
- IFN-.gamma. (100 U/ml) -
[0114] Of the cytokines tested, IL-4 and TGF-.beta. 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-.beta.
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.
Example 12
[0115] This example illustrates the influence of RANK.Fc and hRANKL
on activated T cell growth. The addition of TGF.beta. 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 TGF.beta.-treated T cells by adding
RANK.Fc or soluble human RANKL to T cell cultures.
[0116] Human peripheral blood T cells (7.times.10.sup.5 PBT) were
cultured for six days on anti-CD3 (OKT3, 5 .mu.g/ml) and anti-Flag
(M1, 5 .mu.g/ml) coated 24 well plates in the presence of TGF.beta.
(1 ng/ml) and IL-4 (10 ng/ml), with or without recombinant
FLAG-tagged soluble hRANKL (1 .mu.g/ml) or RANK.Fc (10 .mu.g/ml).
Viable T cell recovery was determined by triplicate trypan blue
countings.
[0117] 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 (FIG. 1). Thus,
endogenous or exogenous RANKL enhances the number of viable T cells
generated in the presence of TGF.beta.. TGF.beta., along with IL-4,
has been implicated in immune response regulation when secreted by
the T.sub.H3/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, TGF.beta. 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
[0118] This example illustrates the influence of the interaction of
RANK on CD1a.sup.+ dendritic cells (DC). Functionally mature
dendritic cells (DC) were generated in vitro from CD34.sup.+ bone
marrow (BM) progenitors. Briefly, human BM cells from normal
healthy volunteers were density fractionated using Ficoll medium
and CD34.sup.+ cells immunoaffinity isolated using an anti-CD34
matrix column (Ceprate, CellPro). The CD34.sup.+ BM cells were then
cultured in human GM-CSF (20 ng/ml), human IL-4 (20 ng/ml), human
TNF-.alpha. (20 ng/ml), human CHO-derived Flt3L (FL; 100 ng/ml) in
Super McCoy's medium supplemented with 10% fetal calf serum in a
fully humidified 37.degree. C. incubator (5% CO.sub.2) for 14 days.
CD1a.sup.+, HLA-DR.sup.+ DC were then sorted using a FACStar
Plus.TM., and used for biological evaluation of RANK
[0119] On human CD1a.sup.+ DC derived from CD34.sup.+ bone marrow
cells, only a subset (20-30%) of CD1a.sup.+ DC expressed RANK at
the cell surface as assessed by flow cytometric analysis. However,
addition of CD40L to the DC cultures resulted in RANK surface
expression on the majority of CD1a.sup.+ 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
[0120] 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. Sorted human
CD1a.sup.+ DC were cultured in a cytokine cocktail (GM-CSF, IL-4,
TNF-.alpha. and FL), in cocktail plus CD40L (1 .mu.g/ml), in
cocktail plus RANKL (1 .mu.g/ml), or in cocktail plus heat
inactivated (.DELTA.H) RANKL (1 .mu.g/ml) 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, CD11a, CD54 or CD58.
[0121] The addition of RANKL to CD1a.sup.+ DC enhanced their
allo-stimulatory capacity in a mixed lymphocyte reaction (MLR) by
at least 3- to 10-fold, comparable to CD40L-cultured DC (FIG. 2).
Allogeneic T cells (1.times.10.sup.5) were incubated with varying
numbers of irradiated (2000 rad) DC cultured as indicated above in
96-well round bottomed culture plates in 0.2 ml culture medium for
four days. The cultures were pulsed with 0.5 mCi
[.sup.3H]-thymidine for eight hours and the cells harvested onto
glass fiber sheets for counting on a gas phase .beta. 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.
[0122] RANKL can enhance DC cluster formation and functional
capacity without modulating known molecules involved in cell
adhesion (CD18, 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
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 CD1b/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.
[0123] 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
[0124] 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.
[0125] 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.degree. C. for 30 seconds, 50.degree. C. for 30 seconds, and
72.degree. C. for 20 seconds were run. PCR products were analyzed
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.
[0126] 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 sequence of muRANK
are shown in SEQ ID Nos:14 and 15.
[0127] 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.
[0128] 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
[0129] 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.
[0130] 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).
[0131] 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
FLAG.TM. 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.
[0132] 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.
[0133] 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 U.S. Ser. No.
08/785,150, now allowed, for example, one of the 2A5-3
.lamda.-derived expression vectors discussed therein.
Example 16
[0134] 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 myristate acetate in
combination with ionomycin (anti-CD3: 500A2; Immunex Corporation,
Seattle Wash.; 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.
[0135] 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.sup.+/CD45RB.sup.Lo
and CD4.sup.+/CD45RB.sup.Hi 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
[0136] 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.
[0137] RAW cells constitutively produce small amounts of
TNF-.alpha.. Incubation with either human or murine RANKL enhanced
production of TNF-.alpha. by these cells in a dose dependent
manner. The results were not due to contamination of RANKL
preparations with endotoxin, since boiling RANKL for 10 minutes
abrogated TNF-.alpha. production, whereas a similar treatment of
purified endotoxin (LPS) did not affect the ability of the LPS to
stimulate TNF-.alpha. production. Despite the fact that RANKL
activated the macrophage cell line RAW T64.7 for TNF-.alpha.
production, neither human RANKL nor murine RANKL stimulated nitric
oxide production by these cells.
Example 18
[0138] 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 1
.mu.g, and doubling the dose about every four days, for a final
dosage of 4 .mu.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.
[0139] 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
[0140] 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.
[0141] 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 2
below. Sequencing confirmed that no undesired mutations had been
introduced in the constructs.
[0142] Radio-labeled (.sup.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)
[0143] 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+.beta.-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-US-00002 TABLE 2 Binding of Various TRAF Proteins to the
Cytoplasmic Domain of RANK C terminal E206- E206- E206- E206- Full
Truncations: S339 Y421 M476 G544 length TRAF1 - - - - ++ TRAF2 - -
- - ++ TRAF3 - - - - ++ TRAF4 - - - - - TRAF5 - - - - + TRAF6 - + +
+ ++
[0144] 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.
TABLE-US-00003 SEQUENE LISTING <160> NUMBER OF SEQ ID NOS: 19
<210> SEQ ID NO 1 <211> LENGTH: 3115 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (93)..(1868)
<400> SEQUENCE: 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 10 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 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 Gln
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 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 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
<210> SEQ ID NO 2 <211> LENGTH: 591 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 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 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
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 <210> SEQ ID NO 3
<211> LENGTH: 1391 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (39)..(1391) <400> SEQUENCE: 3
ccgctgaggc cgcggcgccc gccagcctgt cccgcgcc atg gcc ccg cgc gcc cgg
56 Met Ala Pro Arg Ala Arg 1 5 cgg cgc cgc ccg ctg ttc gcg ctg ctg
ctg ctc tgc gcg ctg ctc gcc 104 Arg Arg Arg Pro Leu Phe Ala Leu Leu
Leu Leu Cys Ala Leu Leu Ala 10 15 20 cgg ctg cag gtg gct ttg cag
atc gct cct cca tgt acc agt gag aag 152 Arg Leu Gln Val Ala Leu Gln
Ile Ala Pro Pro Cys Thr Ser Glu Lys 25 30 35 cat tat gag cat ctg
gga cgg tgc tgt aac aaa tgt gaa cca gga aag 200 His Tyr Glu His Leu
Gly Arg Cys Cys Asn Lys Cys Glu Pro Gly Lys 40 45 50 tac atg tct
tct aaa tgc act act acc tct gac agt gta tgt ctg ccc 248 Tyr Met Ser
Ser Lys Cys Thr Thr Thr Ser Asp Ser Val Cys Leu Pro 55 60 65 70 tgt
ggc ccg gat gaa tac ttg gat agc tgg aat gaa gaa gat aaa tgc 296 Cys
Gly Pro Asp Glu Tyr Leu Asp Ser Trp Asn Glu Glu Asp Lys Cys 75 80
85 ttg ctg cat aaa gtt tgt gat aca ggc aag gcc ctg gtg gcc gtg gtc
344 Leu Leu His Lys Val Cys Asp Thr Gly Lys Ala Leu Val Ala Val Val
90 95 100 gcc ggc aac agc acg acc ccc cgg cgc tgc gcg tgc acg gct
ggg tac 392 Ala Gly Asn Ser Thr Thr Pro Arg Arg Cys Ala Cys Thr Ala
Gly Tyr 105 110 115 cac tgg agc cag gac tgc gag tgc tgc cgc cgc aac
acc gag tgc gcg 440 His Trp Ser Gln Asp Cys Glu Cys Cys Arg Arg Asn
Thr Glu Cys Ala 120 125 130 ccg ggc ctg ggc gcc cag cac ccg ttg cag
ctc aac aag gac aca gtg 488 Pro Gly Leu Gly Ala Gln His Pro Leu Gln
Leu Asn Lys Asp Thr Val 135 140 145 150 tgc aaa cct tgc ctt gca ggc
tac ttc tct gat gcc ttt tcc tcc acg 536 Cys Lys Pro Cys Leu Ala Gly
Tyr Phe Ser Asp Ala Phe Ser Ser Thr 155 160 165 gac aaa tgc aga ccc
tgg acc aac tgt acc ttc ctt gga aag aga gta 584 Asp Lys Cys Arg Pro
Trp Thr Asn Cys Thr Phe Leu Gly Lys Arg Val 170 175 180 gaa cat cat
ggg aca gag aaa tcc gat gcg gtt tgc agt tct tct ctg 632 Glu His His
Gly Thr Glu Lys Ser Asp Ala Val Cys Ser Ser Ser Leu 185 190 195 cca
gct aga aaa cca cca aat gaa ccc cat gtt tac ttg ccc ggt tta 680 Pro
Ala Arg Lys Pro Pro Asn Glu Pro His Val Tyr Leu Pro Gly Leu 200 205
210 ata att ctg ctt ctc ttc gcg tct gtg gcc ctg gtg gct gcc atc atc
728 Ile Ile Leu Leu Leu Phe Ala Ser Val Ala Leu Val Ala Ala Ile Ile
215 220 225 230 ttt ggc gtt tgc tat agg aaa aaa ggg aaa gca ctc aca
gct aat ttg 776 Phe Gly Val Cys Tyr Arg Lys Lys Gly Lys Ala Leu Thr
Ala Asn Leu 235 240 245 tgg cac tgg atc aat gag gct tgt ggc cgc cta
agt gga gat aag gag 824 Trp His Trp Ile Asn Glu Ala Cys Gly Arg Leu
Ser Gly Asp Lys Glu 250 255 260 tcc tca ggt gac agt tgt gtc agt aca
cac acg gca aac ttt ggt cag 872 Ser Ser Gly Asp Ser Cys Val Ser Thr
His Thr Ala Asn Phe Gly Gln 265 270 275 cag gga gca tgt gaa ggt gtc
tta ctg ctg act ctg gag gag aag aca 920 Gln Gly Ala Cys Glu Gly Val
Leu Leu Leu Thr Leu Glu Glu Lys Thr 280 285 290 ttt cca gaa gat atg
tgc tac cca gat caa ggt ggt gtc tgt cag ggc 968 Phe Pro Glu Asp Met
Cys Tyr Pro Asp Gln Gly Gly Val Cys Gln Gly 295 300 305 310 acg tgt
gta gga ggt ggt ccc tac gca caa ggc gaa gat gcc agg atg 1016 Thr
Cys Val Gly Gly Gly Pro Tyr Ala Gln Gly Glu Asp Ala Arg Met 315 320
325 ctc tca ttg gtc agc aag acc gag ata gag gaa gac agc ttc aga cag
1064 Leu Ser Leu Val Ser Lys Thr Glu Ile Glu Glu Asp Ser Phe Arg
Gln 330 335 340 atg ccc aca gaa gat gaa tac atg gac agg ccc tcc cag
ccc aca gac 1112 Met Pro Thr Glu Asp Glu Tyr Met Asp Arg Pro Ser
Gln Pro Thr Asp 345 350 355 cag tta ctg ttc ctc act gag cct gga agc
aaa tcc aca cct cct ttc 1160 Gln Leu Leu Phe Leu Thr Glu Pro Gly
Ser Lys Ser Thr Pro Pro Phe 360 365 370 tct gaa ccc ctg gag gtg ggg
gag aat gac agt tta agc cag tgc ttc 1208 Ser Glu Pro Leu Glu Val
Gly Glu Asn Asp Ser Leu Ser Gln Cys Phe 375 380 385 390 acg ggg aca
cag agc aca gtg ggt tca gaa agc tgc aac tgc act gag 1256 Thr Gly
Thr Gln Ser Thr Val Gly Ser Glu Ser Cys Asn Cys Thr Glu 395 400 405
ccc ctg tgc agg act gat tgg act ccc atg tcc tct gaa aac tac ttg
1304 Pro Leu Cys Arg Thr Asp Trp Thr Pro Met Ser Ser Glu Asn Tyr
Leu 410 415 420
caa aaa gag gtg gac agt ggc cat tgc ccg cac tgg gca gcc agc ccc
1352 Gln Lys Glu Val Asp Ser Gly His Cys Pro His Trp Ala Ala Ser
Pro 425 430 435 agc ccc aac tgg gca gat gtc tgc aca ggc tgc cgg aac
1391 Ser Pro Asn Trp Ala Asp Val Cys Thr Gly Cys Arg Asn 440 445
450 <210> SEQ ID NO 4 <211> LENGTH: 451 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
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 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 <210> SEQ
ID NO 5 <211> LENGTH: 3136 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (39)..(1886) <400> SEQUENCE: 5
ccgctgaggc cgcggcgccc gccagcctgt cccgcgcc atg gcc ccg cgc gcc cgg
56 Met Ala Pro Arg Ala Arg 1 5 cgg cgc cgc ccg ctg ttc gcg ctg ctg
ctg ctc tgc gcg ctg ctc gcc 104 Arg Arg Arg Pro Leu Phe Ala Leu Leu
Leu Leu Cys Ala Leu Leu Ala 10 15 20 cgg ctg cag gtg gct ttg cag
atc gct cct cca tgt acc agt gag aag 152 Arg Leu Gln Val Ala Leu Gln
Ile Ala Pro Pro Cys Thr Ser Glu Lys 25 30 35 cat tat gag cat ctg
gga cgg tgc tgt aac aaa tgt gaa cca gga aag 200 His Tyr Glu His Leu
Gly Arg Cys Cys Asn Lys Cys Glu Pro Gly Lys 40 45 50 tac atg tct
tct aaa tgc act act acc tct gac agt gta tgt ctg ccc 248 Tyr Met Ser
Ser Lys Cys Thr Thr Thr Ser Asp Ser Val Cys Leu Pro 55 60 65 70 tgt
ggc ccg gat gaa tac ttg gat agc tgg aat gaa gaa gat aaa tgc 296 Cys
Gly Pro Asp Glu Tyr Leu Asp Ser Trp Asn Glu Glu Asp Lys Cys 75 80
85 ttg ctg cat aaa gtt tgt gat aca ggc aag gcc ctg gtg gcc gtg gtc
344 Leu Leu His Lys Val Cys Asp Thr Gly Lys Ala Leu Val Ala Val Val
90 95 100 gcc ggc aac agc acg acc ccc cgg cgc tgc gcg tgc acg gct
ggg tac 392 Ala Gly Asn Ser Thr Thr Pro Arg Arg Cys Ala Cys Thr Ala
Gly Tyr 105 110 115 cac tgg agc cag gac tgc gag tgc tgc cgc cgc aac
acc gag tgc gcg 440 His Trp Ser Gln Asp Cys Glu Cys Cys Arg Arg Asn
Thr Glu Cys Ala 120 125 130 ccg ggc ctg ggc gcc cag cac ccg ttg cag
ctc aac aag gac aca gtg 488 Pro Gly Leu Gly Ala Gln His Pro Leu Gln
Leu Asn Lys Asp Thr Val 135 140 145 150 tgc aaa cct tgc ctt gca ggc
tac ttc tct gat gcc ttt tcc tcc acg 536 Cys Lys Pro Cys Leu Ala Gly
Tyr Phe Ser Asp Ala Phe Ser Ser Thr 155 160 165 gac aaa tgc aga ccc
tgg acc aac tgt acc ttc ctt gga aag aga gta 584 Asp Lys Cys Arg Pro
Trp Thr Asn Cys Thr Phe Leu Gly Lys Arg Val 170 175 180 gaa cat cat
ggg aca gag aaa tcc gat gcg gtt tgc agt tct tct ctg 632 Glu His His
Gly Thr Glu Lys Ser Asp Ala Val Cys Ser Ser Ser Leu 185 190 195 cca
gct aga aaa cca cca aat gaa ccc cat gtt tac ttg ccc ggt tta 680 Pro
Ala Arg Lys Pro Pro Asn Glu Pro His Val Tyr Leu Pro Gly Leu 200 205
210 ata att ctg ctt ctc ttc gcg tct gtg gcc ctg gtg gct gcc atc atc
728 Ile Ile Leu Leu Leu Phe Ala Ser Val Ala Leu Val Ala Ala Ile Ile
215 220 225 230 ttt ggc gtt tgc tat agg aaa aaa ggg aaa gca ctc aca
gct aat ttg 776 Phe Gly Val Cys Tyr Arg Lys Lys Gly Lys Ala Leu Thr
Ala Asn Leu 235 240 245 tgg cac tgg atc aat gag gct tgt ggc cgc cta
agt gga gat aag gag 824 Trp His Trp Ile Asn Glu Ala Cys Gly Arg Leu
Ser Gly Asp Lys Glu 250 255 260 tcc tca ggt gac agt tgt gtc agt aca
cac acg gca aac ttt ggt cag 872 Ser Ser Gly Asp Ser Cys Val Ser Thr
His Thr Ala Asn Phe Gly Gln 265 270 275 cag gga gca tgt gaa ggt gtc
tta ctg ctg act ctg gag gag aag aca 920 Gln Gly Ala Cys Glu Gly Val
Leu Leu Leu Thr Leu Glu Glu Lys Thr 280 285 290 ttt cca gaa gat atg
tgc tac cca gat caa ggt ggt gtc tgt cag ggc 968 Phe Pro Glu Asp Met
Cys Tyr Pro Asp Gln Gly Gly Val Cys Gln Gly 295 300 305 310 acg tgt
gta gga ggt ggt ccc tac gca caa ggc gaa gat gcc agg atg 1016 Thr
Cys Val Gly Gly Gly Pro Tyr Ala Gln Gly Glu Asp Ala Arg Met 315 320
325 ctc tca ttg gtc agc aag acc gag ata gag gaa gac agc ttc aga cag
1064 Leu Ser Leu Val Ser Lys Thr Glu Ile Glu Glu Asp Ser Phe Arg
Gln 330 335 340 atg ccc aca gaa gat gaa tac atg gac agg ccc tcc cag
ccc aca gac 1112 Met Pro Thr Glu Asp Glu Tyr Met Asp Arg Pro Ser
Gln Pro Thr Asp 345 350 355 cag tta ctg ttc ctc act gag cct gga agc
aaa tcc aca cct cct ttc 1160 Gln Leu Leu Phe Leu Thr Glu Pro Gly
Ser Lys Ser Thr Pro Pro Phe 360 365 370 tct gaa ccc ctg gag gtg ggg
gag aat gac agt tta agc cag tgc ttc 1208 Ser Glu Pro Leu Glu Val
Gly Glu Asn Asp Ser Leu Ser Gln Cys Phe 375 380 385 390 acg ggg aca
cag agc aca gtg ggt tca gaa agc tgc aac tgc act gag 1256 Thr Gly
Thr Gln Ser Thr Val Gly Ser Glu Ser Cys Asn Cys Thr Glu 395 400 405
ccc ctg tgc agg act gat tgg act ccc atg tcc tct gaa aac tac ttg
1304 Pro Leu Cys Arg Thr Asp Trp Thr Pro Met Ser Ser Glu Asn Tyr
Leu 410 415 420 caa aaa gag gtg gac agt ggc cat tgc ccg cac tgg gca
gcc agc ccc 1352 Gln Lys Glu Val Asp Ser Gly His Cys Pro His Trp
Ala Ala Ser Pro 425 430 435 agc ccc aac tgg gca gat gtc tgc aca ggc
tgc cgg aac cct cct ggg 1400
Ser Pro Asn Trp Ala Asp Val Cys Thr Gly Cys Arg Asn Pro Pro Gly 440
445 450 gag gac tgt gaa ccc ctc gtg ggt tcc cca aaa cgt gga ccc ttg
ccc 1448 Glu Asp Cys Glu Pro Leu Val Gly Ser Pro Lys Arg Gly Pro
Leu Pro 455 460 465 470 cag tgc gcc tat ggc atg ggc ctt ccc cct gaa
gaa gaa gcc agc agg 1496 Gln Cys Ala Tyr Gly Met Gly Leu Pro Pro
Glu Glu Glu Ala Ser Arg 475 480 485 acg gag gcc aga gac cag ccc gag
gat ggg gct gat ggg agg ctc cca 1544 Thr Glu Ala Arg Asp Gln Pro
Glu Asp Gly Ala Asp Gly Arg Leu Pro 490 495 500 agc tca gcg agg gca
ggt gcc ggg tct gga agc tcc cct ggt ggc cag 1592 Ser Ser Ala Arg
Ala Gly Ala Gly Ser Gly Ser Ser Pro Gly Gly Gln 505 510 515 tcc cct
gca tct gga aat gtg act gga aac agt aac tcc acg ttc atc 1640 Ser
Pro Ala Ser Gly Asn Val Thr Gly Asn Ser Asn Ser Thr Phe Ile 520 525
530 tcc agc ggg cag gtg atg aac ttc aag ggc gac atc atc gtg gtc tac
1688 Ser Ser Gly Gln Val Met Asn Phe Lys Gly Asp Ile Ile Val Val
Tyr 535 540 545 550 gtc agc cag acc tcg cag gag ggc gcg gcg gcg gct
gcg gag ccc atg 1736 Val Ser Gln Thr Ser Gln Glu Gly Ala Ala Ala
Ala Ala Glu Pro Met 555 560 565 ggc cgc ccg gtg cag gag gag acc ctg
gcg cgc cga gac tcc ttc gcg 1784 Gly Arg Pro Val Gln Glu Glu Thr
Leu Ala Arg Arg Asp Ser Phe Ala 570 575 580 ggg aac ggc ccg cgc ttc
ccg gac ccg tgc ggc ggc ccc gag ggg ctg 1832 Gly Asn Gly Pro Arg
Phe Pro Asp Pro Cys Gly Gly Pro Glu Gly Leu 585 590 595 cgg gag ccg
gag aag gcc tcg agg ccg gtg cag gag caa ggc ggg gcc 1880 Arg Glu
Pro Glu Lys Ala Ser Arg Pro Val Gln Glu Gln Gly Gly Ala 600 605 610
aag gct tgagcgcccc ccatggctgg gagcccgaag ctcggagcca gggctcgcga 1936
Lys Ala 615 gggcagcacc gcagcctctg ccccagcccc ggccacccag ggatcgatcg
gtacagtcga 1996 ggaagaccac ccggcattct ctgcccactt tgccttccag
gaaatgggct tttcaggaag 2056 tgaattgatg aggactgtcc ccatgcccac
ggatgctcag cagcccgccg cactggggca 2116 gatgtctccc ctgccactcc
tcaaactcgc agcagtaatt tgtggcacta tgacagctat 2176 ttttatgact
atcctgttct gtgggggggg ggtctatgtt ttccccccat atttgtattc 2236
cttttcataa cttttcttga tatctttcct ccctcttttt taatgtaaag gttttctcaa
2296 aaattctcct aaaggtgagg gtctctttct tttctctttt cctttttttt
ttcttttttt 2356 ggcaacctgg ctctggccca ggctagagtg cagtggtgcg
attatagccc ggtgcagcct 2416 ctaactcctg ggctcaagca atccaagtga
tcctcccacc tcaaccttcg gagtagctgg 2476 gatcacagct gcaggccacg
cccagcttcc tccccccgac tccccccccc cagagacacg 2536 gtcccaccat
gttacccagc ctggtctcaa actccccagc taaagcagtc ctccagcctc 2596
ggcctcccaa agtactggga ttacaggcgt gagcccccac gctggcctgc tttacgtatt
2656 ttcttttgtg cccctgctca cagtgtttta gagatggctt tcccagtgtg
tgttcattgt 2716 aaacactttt gggaaagggc taaacatgtg aggcctggag
atagttgcta agttgctagg 2776 aacatgtggt gggactttca tattctgaaa
aatgttctat attctcattt ttctaaaaga 2836 aagaaaaaag gaaacccgat
ttatttctcc tgaatctttt taagtttgtg tcgttcctta 2896 agcagaacta
agctcagtat gtgaccttac ccgctaggtg gttaatttat ccatgctggc 2956
agaggcactc aggtacttgg taagcaaatt tctaaaactc caagttgctg cagcttggca
3016 ttcttcttat tctagaggtc tctctggaaa agatggagaa aatgaacagg
acatggggct 3076 cctggaaaga aagggcccgg gaagttcaag gaagaataaa
gttgaaattt taaaaaaaaa 3136 <210> SEQ ID NO 6 <211>
LENGTH: 616 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 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 Asn 35 40 45 Lys Cys Glu
Pro Gly Lys Tyr Met Ser Ser Lys Cys Thr Thr Thr Ser 50 55 60 Asp
Ser Val Cys Leu Pro Cys Gly Pro Asp Glu Tyr Leu Asp Ser Trp 65 70
75 80 Asn Glu Glu Asp Lys Cys Leu Leu His Lys Val Cys Asp Thr Gly
Lys 85 90 95 Ala Leu Val Ala Val Val Ala Gly Asn Ser Thr Thr Pro
Arg Arg Cys 100 105 110 Ala Cys Thr Ala Gly Tyr His Trp Ser Gln Asp
Cys Glu Cys Cys Arg 115 120 125 Arg Asn Thr Glu Cys Ala Pro Gly Leu
Gly Ala Gln His Pro Leu Gln 130 135 140 Leu Asn Lys Asp Thr Val Cys
Lys Pro Cys Leu Ala Gly Tyr Phe Ser 145 150 155 160 Asp Ala Phe Ser
Ser Thr Asp Lys Cys Arg Pro Trp Thr Asn Cys Thr 165 170 175 Phe Leu
Gly Lys Arg Val Glu His His Gly Thr Glu Lys Ser Asp Ala 180 185 190
Val Cys Ser Ser Ser Leu Pro Ala Arg Lys Pro Pro Asn Glu Pro His 195
200 205 Val Tyr Leu Pro Gly Leu Ile Ile Leu Leu Leu Phe Ala Ser Val
Ala 210 215 220 Leu Val Ala Ala Ile Ile Phe Gly Val Cys Tyr Arg Lys
Lys Gly Lys 225 230 235 240 Ala Leu Thr Ala Asn Leu Trp His Trp Ile
Asn Glu Ala Cys Gly Arg 245 250 255 Leu Ser Gly Asp Lys Glu Ser Ser
Gly Asp Ser Cys Val Ser Thr His 260 265 270 Thr Ala Asn Phe Gly Gln
Gln Gly Ala Cys Glu Gly Val Leu Leu Leu 275 280 285 Thr Leu Glu Glu
Lys Thr Phe Pro Glu Asp Met Cys Tyr Pro Asp Gln 290 295 300 Gly Gly
Val Cys Gln Gly Thr Cys Val Gly Gly Gly Pro Tyr Ala Gln 305 310 315
320 Gly Glu Asp Ala Arg Met Leu Ser Leu Val Ser Lys Thr Glu Ile Glu
325 330 335 Glu Asp Ser Phe Arg Gln Met Pro Thr Glu Asp Glu Tyr Met
Asp Arg 340 345 350 Pro Ser Gln Pro Thr Asp Gln Leu Leu Phe Leu Thr
Glu Pro Gly Ser 355 360 365 Lys Ser Thr Pro Pro Phe Ser Glu Pro Leu
Glu Val Gly Glu Asn Asp 370 375 380 Ser Leu Ser Gln Cys Phe Thr Gly
Thr Gln Ser Thr Val Gly Ser Glu 385 390 395 400 Ser Cys Asn Cys Thr
Glu Pro Leu Cys Arg Thr Asp Trp Thr Pro Met 405 410 415 Ser Ser Glu
Asn Tyr Leu Gln Lys Glu Val Asp Ser Gly His Cys Pro 420 425 430 His
Trp Ala Ala Ser Pro Ser Pro Asn Trp Ala Asp Val Cys Thr Gly 435 440
445 Cys Arg Asn Pro Pro Gly Glu Asp Cys Glu Pro Leu Val Gly Ser Pro
450 455 460 Lys Arg Gly Pro Leu Pro Gln Cys Ala Tyr Gly Met Gly Leu
Pro Pro 465 470 475 480 Glu Glu Glu Ala Ser Arg Thr Glu Ala Arg Asp
Gln Pro Glu Asp Gly 485 490 495 Ala Asp Gly Arg Leu Pro Ser Ser Ala
Arg Ala Gly Ala Gly Ser Gly 500 505 510 Ser Ser Pro Gly Gly Gln Ser
Pro Ala Ser Gly Asn Val Thr Gly Asn 515 520 525 Ser Asn Ser Thr Phe
Ile Ser Ser Gly Gln Val Met Asn Phe Lys Gly 530 535 540 Asp Ile Ile
Val Val Tyr Val Ser Gln Thr Ser Gln Glu Gly Ala Ala 545 550 555 560
Ala Ala Ala Glu Pro Met Gly Arg Pro Val Gln Glu Glu Thr Leu Ala 565
570 575 Arg Arg Asp Ser Phe Ala Gly Asn Gly Pro Arg Phe Pro Asp Pro
Cys 580 585 590 Gly Gly Pro Glu Gly Leu Arg Glu Pro Glu Lys Ala Ser
Arg Pro Val 595 600 605 Gln Glu Gln Gly Gly Ala Lys Ala 610 615
<210> SEQ ID NO 7 <211> LENGTH: 8 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: FLAG peptide <400> SEQUENCE: 7
Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> SEQ ID NO 8
<211> LENGTH: 232 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 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
<210> SEQ ID NO 9 <211> LENGTH: 31 <212> TYPE:
PRT <213> ORGANISM: Cytomegalovirus <400> SEQUENCE: 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 <210> SEQ ID NO 10 <211> LENGTH: 1630
<212> TYPE: DNA <213> ORGANISM: Mus musculus
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (3)..(884) <400> SEQUENCE: 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 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 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 <210> SEQ ID NO 11 <211> LENGTH: 294 <212>
TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:
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 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 <210> SEQ ID
NO 12 <211> LENGTH: 954 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (1)..(951) <400> SEQUENCE: 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 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 <210> SEQ ID NO 13 <211> LENGTH: 317 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
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 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 <210> SEQ ID NO 14 <211>
LENGTH: 1878 <212> TYPE: DNA <213> ORGANISM: Murine
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (1)..(1875) <400> SEQUENCE: 14 atg gcc ccg cgc gcc
cgg cgg cgc cgc cag ctg ccc gcg ccg ctg ctg 48 Met Ala Pro Arg Ala
Arg Arg Arg Arg Gln Leu Pro Ala Pro Leu Leu 1 5 10 15 gcg ctc tgc
gtg ctg ctc gtt cca ctg cag gtg act ctc cag gtc act 96 Ala Leu Cys
Val Leu Leu Val Pro Leu Gln Val Thr Leu Gln Val Thr 20 25 30 cct
cca tgc acc cag gag agg cat tat gag cat ctc gga cgg tgt tgc 144 Pro
Pro Cys Thr Gln Glu Arg His Tyr Glu His Leu Gly Arg Cys Cys 35 40
45 agc aga tgc gaa cca gga aag tac ctg tcc tct aag tgc act cct acc
192 Ser Arg Cys Glu Pro Gly Lys Tyr Leu Ser Ser Lys Cys Thr Pro Thr
50 55 60 tcc gac agt gtg tgt ctg ccc tgt ggc ccc gat gag tac ttg
gac acc 240 Ser Asp Ser Val Cys Leu Pro Cys Gly Pro Asp Glu Tyr Leu
Asp Thr 65 70 75 80 tgg aat gaa gaa gat aaa tgc ttg ctg cat aaa gtc
tgt gat gca ggc 288 Trp Asn Glu Glu Asp Lys Cys Leu Leu His Lys Val
Cys Asp Ala Gly 85 90 95 aag gcc ctg gtg gcg gtg gat cct ggc aac
cac acg gcc ccg cgt cgc 336 Lys Ala Leu Val Ala Val Asp Pro Gly Asn
His Thr Ala Pro Arg Arg 100 105 110 tgt gct tgc acg gct ggc tac cac
tgg aac tca gac tgc gag tgc tgc 384 Cys Ala Cys Thr Ala Gly Tyr His
Trp Asn Ser Asp Cys Glu Cys Cys 115 120 125 cgc agg aac acg gag tgt
gca cct ggc ttc gga gct cag cat ccc ttg 432 Arg Arg Asn Thr Glu Cys
Ala Pro Gly Phe Gly Ala Gln His Pro Leu 130 135 140 cag ctc aac aag
gat acg gtg tgc aca ccc tgc ctc ctg ggc ttc ttc 480 Gln Leu Asn Lys
Asp Thr Val Cys Thr Pro Cys Leu Leu Gly Phe Phe 145 150 155 160 tca
gat gtc ttt tcg tcc aca gac aaa tgc aaa cct tgg acc aac tgc 528 Ser
Asp Val Phe Ser Ser Thr Asp Lys Cys Lys Pro Trp Thr Asn Cys 165 170
175 acc ctc ctt gga aag cta gaa gca cac cag ggg aca acg gaa tca gat
576 Thr Leu Leu Gly Lys Leu Glu Ala His Gln Gly Thr Thr Glu Ser Asp
180 185 190 gtg gtc tgc agc tct tcc atg aca ctg agg aga cca ccc aag
gag gcc 624 Val Val Cys Ser Ser Ser Met Thr Leu Arg Arg Pro Pro Lys
Glu Ala 195 200 205 cag gct tac ctg ccc agt ctc atc gtt ctg ctc ctc
ttc atc tct gtg 672 Gln Ala Tyr Leu Pro Ser Leu Ile Val Leu Leu Leu
Phe Ile Ser Val 210 215 220 gta gta gtg gct gcc atc atc ttc ggc gtt
tac tac agg aag gga ggg 720 Val Val Val Ala Ala Ile Ile Phe Gly Val
Tyr Tyr Arg Lys Gly Gly 225 230 235 240 aaa gcg ctg aca gct aat ttg
tgg aat tgg gtc aat gat gct tgc agt 768 Lys Ala Leu Thr Ala Asn Leu
Trp Asn Trp Val Asn Asp Ala Cys Ser 245 250 255 agt cta agt gga aat
aag gag tcc tca ggg gac cgt tgt gct ggt tcc 816 Ser Leu Ser Gly Asn
Lys Glu Ser Ser Gly Asp Arg Cys Ala Gly Ser 260 265 270 cac tcg gca
acc tcc agt cag caa gaa gtg tgt gaa ggt atc tta cta 864 His Ser Ala
Thr Ser Ser Gln Gln Glu Val Cys Glu Gly Ile Leu Leu
275 280 285 atg act cgg gag gag aag atg gtt cca gaa gac ggt gct gga
gtc tgt 912 Met Thr Arg Glu Glu Lys Met Val Pro Glu Asp Gly Ala Gly
Val Cys 290 295 300 ggg cct gtg tgt gcg gca ggt ggg ccc tgg gca gaa
gtc aga gat tct 960 Gly Pro Val Cys Ala Ala Gly Gly Pro Trp Ala Glu
Val Arg Asp Ser 305 310 315 320 agg acg ttc aca ctg gtc agc gag gtt
gag acg caa gga gac ctc tcg 1008 Arg Thr Phe Thr Leu Val Ser Glu
Val Glu Thr Gln Gly Asp Leu Ser 325 330 335 agg aag att ccc aca gag
gat gag tac acg gac cgg ccc tcg cag cct 1056 Arg Lys Ile Pro Thr
Glu Asp Glu Tyr Thr Asp Arg Pro Ser Gln Pro 340 345 350 tcg act ggt
tca ctg ctc cta atc cag cag gga agc aaa tct ata ccc 1104 Ser Thr
Gly Ser Leu Leu Leu Ile Gln Gln Gly Ser Lys Ser Ile Pro 355 360 365
cca ttc cag gag ccc ctg gaa gtg ggg gag aac gac agt tta agc cag
1152 Pro Phe Gln Glu Pro Leu Glu Val Gly Glu Asn Asp Ser Leu Ser
Gln 370 375 380 tgt ttc acc ggg act gaa agc acg gtg gat tct gag ggc
tgt gac ttc 1200 Cys Phe Thr Gly Thr Glu Ser Thr Val Asp Ser Glu
Gly Cys Asp Phe 385 390 395 400 act gag cct ccg agc aga act gac tct
atg ccc gtg tcc cct gaa aag 1248 Thr Glu Pro Pro Ser Arg Thr Asp
Ser Met Pro Val Ser Pro Glu Lys 405 410 415 cac ctg aca aaa gaa ata
gaa ggt gac agt tgc ctc ccc tgg gtg gtc 1296 His Leu Thr Lys Glu
Ile Glu Gly Asp Ser Cys Leu Pro Trp Val Val 420 425 430 agc tcc aac
tca aca gat ggc tac aca ggc agt ggg aac act cct ggg 1344 Ser Ser
Asn Ser Thr Asp Gly Tyr Thr Gly Ser Gly Asn Thr Pro Gly 435 440 445
gag gac cat gaa ccc ttt cca ggg tcc ctg aaa tgt gga cca ttg ccc
1392 Glu Asp His Glu Pro Phe Pro Gly Ser Leu Lys Cys Gly Pro Leu
Pro 450 455 460 cag tgt gcc tac agc atg ggc ttt ccc agt gaa gca gca
gcc agc atg 1440 Gln Cys Ala Tyr Ser Met Gly Phe Pro Ser Glu Ala
Ala Ala Ser Met 465 470 475 480 gca gag gcg gga gta cgg ccc cag gac
agg gct gat gag agg gga gcc 1488 Ala Glu Ala Gly Val Arg Pro Gln
Asp Arg Ala Asp Glu Arg Gly Ala 485 490 495 tca ggg tcc ggg agc tcc
ccc agt gac cag cca cct gcc tct ggg aac 1536 Ser Gly Ser Gly Ser
Ser Pro Ser Asp Gln Pro Pro Ala Ser Gly Asn 500 505 510 gtg act gga
aac agt aac tcc acg ttc atc tct agc ggg cag gtg atg 1584 Val Thr
Gly Asn Ser Asn Ser Thr Phe Ile Ser Ser Gly Gln Val Met 515 520 525
aac ttc aag ggt gac atc atc gtg gtg tat gtc agc cag acc tcg cag
1632 Asn Phe Lys Gly Asp Ile Ile Val Val Tyr Val Ser Gln Thr Ser
Gln 530 535 540 gag ggc ccg ggt tcc gca gag ccc gag tcg gag ccc gtg
ggc cgc cct 1680 Glu Gly Pro Gly Ser Ala Glu Pro Glu Ser Glu Pro
Val Gly Arg Pro 545 550 555 560 gtg cag gag gag acg ctg gca cac aga
gac tcc ttt gcg ggc acc gcg 1728 Val Gln Glu Glu Thr Leu Ala His
Arg Asp Ser Phe Ala Gly Thr Ala 565 570 575 ccg cgc ttc ccc gac gtc
tgt gcc acc ggg gct ggg ctg cag gag cag 1776 Pro Arg Phe Pro Asp
Val Cys Ala Thr Gly Ala Gly Leu Gln Glu Gln 580 585 590 ggg gca ccc
cgg cag aag gac ggg aca tcg cgg ccg gtg cag gag cag 1824 Gly Ala
Pro Arg Gln Lys Asp Gly Thr Ser Arg Pro Val Gln Glu Gln 595 600 605
ggt ggg gcg cag act tca ctc cat acc cag ggg tcc gga caa tgt gca
1872 Gly Gly Ala Gln Thr Ser Leu His Thr Gln Gly Ser Gly Gln Cys
Ala 610 615 620 gaa tga 1878 Glu 625 <210> SEQ ID NO 15
<211> LENGTH: 625 <212> TYPE: PRT <213> ORGANISM:
Murine <400> SEQUENCE: 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 Gly Pro
Val Cys Ala Ala Gly Gly Pro Trp Ala Glu Val Arg Asp Ser 305 310 315
320 Arg Thr Phe Thr Leu Val Ser Glu Val Glu Thr Gln Gly Asp Leu Ser
325 330 335 Arg Lys Ile Pro Thr Glu Asp Glu Tyr Thr Asp Arg Pro Ser
Gln Pro 340 345 350 Ser Thr Gly Ser Leu Leu Leu Ile Gln Gln Gly Ser
Lys Ser Ile Pro 355 360 365 Pro Phe Gln Glu Pro Leu Glu Val Gly Glu
Asn Asp Ser Leu Ser Gln 370 375 380 Cys Phe Thr Gly Thr Glu Ser Thr
Val Asp Ser Glu Gly Cys Asp Phe 385 390 395 400 Thr Glu Pro Pro Ser
Arg Thr Asp Ser Met Pro Val Ser Pro Glu Lys 405 410 415 His Leu Thr
Lys Glu Ile Glu Gly Asp Ser Cys Leu Pro Trp Val Val 420 425 430 Ser
Ser Asn Ser Thr Asp Gly Tyr Thr Gly Ser Gly Asn Thr Pro Gly 435 440
445 Glu Asp His Glu Pro Phe Pro Gly Ser Leu Lys Cys Gly Pro Leu Pro
450 455 460 Gln Cys Ala Tyr Ser Met Gly Phe Pro Ser Glu Ala Ala Ala
Ser Met 465 470 475 480 Ala Glu Ala Gly Val Arg Pro Gln Asp Arg Ala
Asp Glu Arg Gly Ala 485 490 495 Ser Gly Ser Gly Ser Ser Pro Ser Asp
Gln Pro Pro Ala Ser Gly Asn 500 505 510 Val Thr Gly Asn Ser Asn Ser
Thr Phe Ile Ser Ser Gly Gln Val Met 515 520 525 Asn Phe Lys Gly Asp
Ile Ile Val Val Tyr Val Ser Gln Thr Ser Gln 530 535 540 Glu Gly Pro
Gly Ser Ala Glu Pro Glu Ser Glu Pro Val Gly Arg Pro 545 550 555 560
Val Gln Glu Glu Thr Leu Ala His Arg Asp Ser Phe Ala Gly Thr Ala 565
570 575 Pro Arg Phe Pro Asp Val Cys Ala Thr Gly Ala Gly Leu Gln Glu
Gln 580 585 590 Gly Ala Pro Arg Gln Lys Asp Gly Thr Ser Arg Pro Val
Gln Glu Gln 595 600 605 Gly Gly Ala Gln Thr Ser Leu His Thr Gln Gly
Ser Gly Gln Cys Ala 610 615 620 Glu 625 <210> SEQ ID NO 16
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 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
20
<210> SEQ ID NO 17 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: shortened FLAG peptide <400>
SEQUENCE: 17 Asp Tyr Lys Asp Glu 1 5 <210> SEQ ID NO 18
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: poly-His peptide <400> SEQUENCE: 18 His His His
His His His 1 5 <210> SEQ ID NO 19 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: leucine zipper
peptide <400> SEQUENCE: 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
* * * * *