Title: Monoclonal blocking
antibody to human RANKL
United States Patent: 7,411,050
Issued: August 12, 2008
Inventors: Anderson; Dirk
M. (Seattle, WA)
Corporation (Thousand Oaks, CA)
Appl. No.: 10/802,133
Filed: March 16, 2004
Training Courses -- Pharm/Biotech/etc.
Disclosed herein are kits for detecting
RANKL protein or nucleic acids, the kits comprising isolated human RANKL
polypeptides, RANKL nucleic acids and/or antibodies specific for RANKL.
Description of the
SUMMARY OF THE INVENTION
The present invention provides a counterstructure, or ligand, for a novel
receptor referred to as RANK (for receptor activator of NF-.kappa.B), that
is a member of the TNF superfamily. The ligand, which is referred to as
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
RANK is a Type I transmembrane protein having 616 amino acid residues that
is a member of the TNFR superfamily, and interacts with TRAF3. Triggering of
RANK by over-expression, co-expression of RANK and membrane bound RANKL, or
by 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
DETAILED DESCRIPTION OF THE INVENTION
A novel partial cDNA insert with a predicted open reading frame having some
similarity to CD40 was identified in a database containing sequence
information from cDNAs generated from human bone marrow-derived dendritic
cells (DC). The insert was used to hybridize to colony blots generated from
a DC cDNA library containing full-length cDNAs. Several colony
hybridizations were performed, and two clones (SEQ ID NOs: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.
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.
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
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, RANKL, will allow further definition
of this signaling pathway, and development of diagnostic and therapeutic
modalities for use in the area of autoimmune and/or inflammatory disease.
DNAs, Proteins and Analogs
The present invention provides isolated RANKL polypeptides and analogs (or
muteins) thereof having an activity exhibited by the native molecule (i.e,
RANKL muteins that bind specifically to a RANK expressed on cells or
immobilized on a surface or to RANKL-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
RANKL 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 RANKL
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-
Derivatives of RANKL 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 RANKL,
as well as other proteins that bind RANKL or homologs thereof.
Soluble forms of RANKL are also within the scope of the invention. The
nucleotide and predicted amino acid sequence of the RANKL is shown in SEQ ID
Nos:11 and 13 (murine and human, respectively). Computer analysis indicated
that the RANKL is a Type 2 transmembrane protein; murine RANKL contains a
predicted 48 amino acid intracellular domain, 21 amino acid transmembrane
domain and 247 amino acid extracellular domain, and human RANKL contains a
predicted 47 amino acid intracellular domain, 21 amino acid transmembrane
domain and 249 amino acid extracellular domain.
Soluble RANKL comprises a signal peptide and the extracellular domain or a
fragment thereof. An exemplary signal peptide is that shown in SEQ ID NO:9;
other signal (or leader) peptides are well-known in the art, and include
that of murine Interleukin-7 or human growth hormone. RANKL is similar to
other members of the TNF family in having a region of amino acids between
the transmembrane domain and the receptor binding region that does not
appear to be required for biological activity; this is referred to as a
`spacer` region. Amino acid sequence alignment indicates that the receptor
binding region is from about amino acid 162 of human RANKL to about amino
acid 317 (corresponding to amino acid 139 through 294 of murine RANKL, SEQ
ID NO:11), beginning with an Ala residue that is conserved among many
members of the family (amino acid 162 of SEQ ID NO:13).
Moreover, fragments of the extracellular domain will also provide soluble
forms of RANKL. Those skilled in the art will recognize that the actual
receptor binding region may be different than that predicted by computer
analysis. Thus, the N-terminal amino acid of a soluble RANKL is expected to
be within about five amino acids on either side of the conserved Ala
residue. Alternatively, all or a portion of the spacer region may be
included at the N-terminus of a soluble RANKL, as may be all or a portion of
the transmembrane and/or intracellular domains, provided that the resulting
soluble RANKL is not membrane-associated. Accordingly, a soluble RANKL will
have an N-terminal amino acid selected from the group consisting of amino
acids 1 through 162 of SEQ ID NO:13 (1 though 139 of SEQ ID NO:11).
Preferably, the amino terminal amino acid is between amino acids 69 and 162
of SEQ ID NO:13 (human RANKL; amino acids 48 and 139 of SEQ ID NO:11).
Similarly, the carboxy terminal amino acid can be between amino acid 313 and
317 of SEQ ID NO:13 (human RANKL; corresponding to amino acids 290 through
294 of SEQ ID NO:11). Those skilled in the art can prepare these and
additional soluble forms through routine experimentation.
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 TNF
family (of which RANKL is a member) and selecting forms similar to those
prepared for other family members. Alternatively, unique restriction sites
or PCR techniques that are known in the art can be used to prepare numerous
truncated forms which can be expressed and analyzed for activity.
Other derivatives of the RANKL 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
Protein fusions can comprise peptides added to facilitate purification or
identification of RANKL 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.
Fusion proteins further comprise the amino acid sequence of a RANKL 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 IgG3 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
In another embodiment, RANKL 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.
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).
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.
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.
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.
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.
The present invention also includes RANKL 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 RANKL 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.
RANKL protein derivatives may also be obtained by mutations of the native
RANKL or subunits thereof. A RANKL mutated protein, as referred to herein,
is a polypeptide homologous to a native RANKL protein, 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 RANKL analogs, muteins or
derivatives can be determined by any of the assays described herein (for
example, induction of NF-.kappa.B activation).
Analogs of the inventive proteins may be constructed by, for example, making
various substitutions of residues or sequences or deleting terminal or
internal residues or sequences not needed for biological activity. For
example, cysteine residues can be deleted or replaced with other amino acids
to prevent formation of incorrect intramolecular disulfide bridges upon
renaturation. Other approaches to mutagenesis involve modification of
adjacent dibasic amino acid residues to enhance expression in yeast systems
in which KEX2 protease activity is present.
When a deletion or insertion strategy is adopted, the potential effect of
the deletion or insertion on biological activity should be considered.
Subunits of the inventive proteins may be constructed by deleting terminal
or internal residues or sequences. Soluble forms of RANKL can be readily
prepared and tested for their ability to induce 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 RANKL to proteins that have
similar structures, as well as by performing structural analysis of the
inventive RANKL proteins.
Generally, substitutions should be made conservatively; i.e., the most
preferred substitute amino acids are those which do not affect the
biological activity of RANKL (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 induce a
RANKL signal, or ability to induce NF-.kappa.B activation). 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
Mutations in nucleotide sequences constructed for expression of analog
proteins or fragments thereof must, of course, preserve the reading frame
phase of the coding sequences and preferably will not create complementary
regions that could hybridize to produce secondary mRNA structures such as
loops or hairpins which would adversely affect translation of the mRNA.
Not all mutations in the nucleotide sequence which encodes a RANKL 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.
Although a mutation site may be predetermined, it is not necessary that the
nature of the mutation per se be predetermined. For example, in order to
select for optimum characteristics of mutants, random mutagenesis may be
conducted and the expressed mutated proteins screened for the desired
activity. Mutations can be introduced at particular loci by synthesizing
oligonucleotides containing a mutant sequence, flanked by restriction sites
enabling ligation to fragments of the native sequence. Following ligation,
the resulting reconstructed sequence encodes an analog having the desired
amino acid insertion, substitution, or deletion.
Alternatively, oligonucleotide-directed site-specific mutagenesis procedures
can be employed to provide an altered gene having particular codons altered
according to the substitution, deletion, or insertion required. Exemplary
methods of making the alterations set forth above are disclosed by Walder et
al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques,
January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and
Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462
disclose suitable techniques, and are incorporated by reference herein.
Additional embodiments of the inventive proteins include RANKL polypeptides
encoded by DNAs capable of hybridizing to the DNAS of SEQ ID NO:10 or 12
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 RANKL, 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
RANKL. In one embodiment, RANKL polypeptides are at least about 70%
identical in amino acid sequence to the amino acid sequence of native RANKL
protein as set forth in SEQ ID NOs:10 and 12. In a preferred embodiment,
RANKL polypeptides are at least about 80% identical in amino acid sequence
to the native form of RANKL; most preferred polypeptides are those that are
at least about 90% identical to native RANKL.
Percent identity may be determined using a computer program, for example,
the GAP computer program described by Devereux et al. (Nucl. Acids Res.
12:387, 1984) and available from the University of Wisconsin Genetics
Computer Group (UWGCG). For fragments derived from the RANKL protein, the
identity is calculated based on that portion of the RANKL protein that is
present in the fragment
The biological activity of RANKL analogs or muteins can be determined by
testing the ability of the analogs or muteins to induce a signal through
RANK, for example, activation of transcription as described in the Examples
herein. Alternatively, suitable assays, for example, an enzyme immunoassay
or a dot blot, employing an antibody that binds native RANKL, or a soluble
form of RANK, can be used to assess the activity of RANKL analogs or muteins.
Suitable assays also include, for example, assays that measure the ability
of a RANKL peptide or mutein to bind cells expressing RANK, and/or the
biological effects thereon. Such methods are well known in the art.
Fragments of the RANKL 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 RANKL DNA disclosed herein. DNA and
RNA complements of such fragments are provided herein, along with both
single-stranded and double-stranded forms of the RANKL DNAs of SEQ ID NOs:10
and 12, and those encoding the aforementioned polypeptides. A fragment of
RANKL 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 RANKL) are used as a
probe or as primers in a polymerase chain reaction (PCR).
The probes also find use in detecting the presence of RANKL nucleic acids in
in vitro assays and in such procedures as Northern and Southern blots. Cell
types expressing RANKL 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 RANKL DNA sequence are employed to
amplify that sequence, using conventional techniques.
Other useful fragments of the RANKL nucleic acids are antisense or sense
oligonucleotides comprising a single-stranded nucleic acid sequence (either
RNA or DNA) capable of binding to target RANKL mRNA (sense) or RANKL DNA (antisense)
sequences. The ability to create an antisense or a sense oligonucleotide,
based upon a cDNA sequence for a given protein is described in, for example,
Stein and Cohen, Cancer Res. 48:2659, 1988 and van der Krol et al.,
BioTechniques 6:958, 1988.
Uses of DNAs, Proteins and Analogs
The RANKL DNAs, proteins and analogs described herein will have numerous
uses, including the preparation of pharmaceutical compositions. For example,
soluble forms of RANKL will be useful to transduce signal via RANK. RANKL
compositions (both protein and DNAs) will also be useful in development of
antibodies to RANKL, both those that inhibit binding to RANK and those that
do not. 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 RANKL transcripts.
The inventive proteins will also be useful in preparing kits that are used
to detect soluble RANK or RANKL, or monitor RANK-related activity, for
example, in patient specimens. RANKL proteins will also find uses in
monitoring RANK-related activity in other samples or compositions, as is
necessary when screening for antagonists or mimetics of this activity (for
example, peptides or small molecules that inhibit or mimic, respectively,
the interaction). A variety of assay formats are useful in such kits,
including (but not limited to) ELISA, dot blot, solid phase binding assays
(such as those using a biosensor), rapid format assays and bioassays.
The purified RANKL 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 RANKL
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 RANKL, or a fragment thereof. Detecting the inhibiting activity of
a molecule would typically involve use of a soluble form of RANKL derived
from the extracellular domain in a screening assay to detect molecules
capable of binding RANK and inhibiting binding of the RANKL.
In addition, RANKL polypeptides can also be used for structure-based design
of RANKL-inhibitors. Such structure-based design is also known as "rational
drug design." The RANKL 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 RANKL 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 RANKL for likely binding sites of substrates, synthesizing a
new molecule that incorporates a predictive reactive site, and assaying the
new molecule as described above.
Moreover, as shown in the Examples herein, soluble forms of RANKL will be
useful to induce maturation of dendritic cells (DC), and to enhance their
allo-stimulatory capacity. Accordingly, RANKL proteins will be useful in
augmenting an immune response, and can be used for these purposes either ex
vivo (i.e., in obtaining cells such as DC from an individual, exposing them
to antigen and cytokines ex vivo, and re-administering them to the
individual) or in vivo (i.e., as a vaccine adjuvant that will augment
humoral and/or cellular immunity). RANKL will also be useful promoting
viability of T cells in the presence of TGF.beta., which will also be
helpful in regulating an immune response.
Expression of Recombinant RANKL
The proteins of the present invention are preferably produced by recombinant
DNA methods by inserting a DNA sequence encoding RANKL protein or an analog
thereof into a recombinant expression vector and expressing the DNA sequence
in a recombinant expression system under conditions promoting expression.
DNA sequences encoding the proteins provided by this invention can be
assembled from cDNA fragments and short oligonucleotide linkers, or from a
series of oligonucleotides, to provide a synthetic gene which is capable of
being inserted in a recombinant expression vector and expressed in a
recombinant transcriptional unit.
Recombinant expression vectors include synthetic or cDNA-derived DNA
fragments encoding RANKL, or homologs, muteins or bioequivalent analogs
thereof, operably linked to suitable transcriptional or translational
regulatory elements derived from mammalian, microbial, viral or insect
genes. Such regulatory elements include a transcriptional promoter, an
optional operator sequence to control transcription, a sequence encoding
suitable mRNA ribosomal binding sites, and sequences which control the
termination of transcription and translation, as described in detail below.
The ability to replicate in a host, usually conferred by an origin of
replication, and a selection gene to facilitate recognition of transformants
may additionally be incorporated.
DNA regions are operably linked when they are functionally related to each
other. For example, DNA for a signal peptide (secretory leader) is operably
linked to DNA for a polypeptide if it is expressed as a precursor which
participates in the secretion of the polypeptide; a promoter is operably
linked to a coding sequence if it controls the transcription of the
sequence; or a ribosome binding site is operably linked to a coding 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 RANKL, or homologs or analogs thereof
which are to be expressed in a microorganism will preferably contain no
introns that could prematurely terminate transcription of DNA into mRNA.
Useful expression vectors for bacterial use can comprise a selectable marker
and bacterial origin of replication derived from commercially available
plasmids comprising genetic elements of the well known cloning vector pBR322
(ATCC 37017). Such commercial vectors include, for example, pKK223-3
(Pharmacia Fine Chemicals, Uppsala, Sweden) and 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.
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
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
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
The transcriptional and translational control sequences in expression
vectors to be used in transforming vertebrate cells may be provided by viral
sources. For example, commonly used promoters and enhancers are derived from
Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.
DNA sequences derived from the SV40 viral genome, for example, SV40 origin,
early and late promoter, enhancer, splice, and polyadenylation sites may be
used to provide the other genetic elements required for expression of a
heterologous DNA sequence. The early and late promoters are particularly
useful because both are obtained easily from the virus as a fragment which
also contains the SV40 viral origin of replication (Fiers et al., Nature
273:113, 1978). Smaller or larger SV40 fragments may also be used, provided
the approximately 250 bp sequence extending from the Hind III site toward
the BglI site located in the viral origin of replication is included.
Further, viral genomic promoter, control and/or signal sequences may be
utilized, provided such control sequences are compatible with the host cell
chosen. Exemplary vectors can be constructed as disclosed by Okayama and
Berg (Mol. Cell. Biol. 3:280, 1983).
A useful system for stable high level expression of mammalian receptor cDNAs
in C127 murine mammary epithelial cells can be constructed substantially as
described by Cosman et al. (Mol. Immunol. 23:935, 1986). A preferred
eukaryotic vector for expression of RANKL DNA is referred to as pDC406
(McMahan et al., EMBO J. 10:2821, 1991), and includes regulatory sequences
derived from SV40, human immunodeficiency virus (HIV), and Epstein-Barr
virus (EBV). Other preferred vectors include pDC409 and pDC410, which are
derived from pDC406. pDC410 was derived from pDC406 by substituting the EBV
origin of replication with sequences encoding the SV40 large T antigen.
pDC409 differs from pDC406 in that a Bgl II restriction site outside of the
multiple cloning site has been deleted, making the Bgl II site within the
multiple cloning site unique.
A useful cell line that allows for episomal replication of expression
vectors, such as pDC406 and pDC409, which contain the EBV origin of
replication, is CV-1/EBNA (ATCC CRL 10478). The CV-1/EBNA cell line was
derived by transfection of the CV-1 cell line with a gene encoding
Epstein-Barr virus nuclear antigen-1 (EBNA-1) and constitutively express
EBNA-1 driven from human CMV immediate-early enhancer/promoter.
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 (RANKL, or homologs
or analogs thereof), but host cells transformed for purposes of cloning or
amplifying the inventive DNA do not need to express the protein. Expressed
proteins will preferably be secreted into the culture supernatant, depending
on the DNA selected, but may be deposited in the cell membrane.
Suitable host cells for expression of proteins include prokaryotes, yeast or
higher eukaryotic cells under the control of appropriate promoters.
Prokaryotes include gram negative or gram positive organisms, for example E.
coli or Bacillus spp. Higher eukaryotic cells include established cell lines
of mammalian origin as described below. Cell-free translation systems could
also be employed to produce proteins using RNAs derived from the DNA
constructs disclosed herein. Appropriate cloning and expression vectors for
use with bacterial, fungal, yeast, and mammalian cellular hosts are
described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier,
N.Y., 1985), the relevant disclosure of which is hereby incorporated by
Prokaryotic expression hosts may be used for expression of RANKL, or
homologs or analogs thereof that do not require extensive proteolytic and
disulfide processing. Prokaryotic expression vectors generally comprise one
or more phenotypic selectable markers, for example a gene encoding proteins
conferring antibiotic resistance or supplying an autotrophic requirement,
and an origin of replication recognized by the host to ensure amplification
within the host. Suitable prokaryotic hosts for transformation include E.
coli, Bacillus subtilis, Salmonella typhimurium, and various species within
the genera Pseudomonas, Streptomyces, and Staphylococcus, although others
may also be employed as a matter of choice.
Recombinant RANKL 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
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.
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
Purification of Recombinant RANKL
Purified RANKL, 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.
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.
Affinity chromatography is a particularly preferred method of purifying
RANKL and homologs thereof. For example, a RANKL expressed as a fusion
protein comprising an immunoglobulin Fc region can be purified using Protein
A or Protein G affinity chromatography. Moreover, a RANKL 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 RANKL 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
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 RANKL composition. Some or all of the foregoing purification steps, in
various combinations, can also be employed to provide a homogeneous
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
Fermentation of yeast which express the inventive protein as a secreted
protein greatly simplifies purification. Secreted recombinant protein
resulting from a large-scale fermentation can be purified by methods
analogous to those disclosed by Urdal et al. (J. Chromatog. 296:171, 1984).
This reference describes two sequential, reversed-phase HPLC steps for
purification of recombinant human GM-CSF on a preparative HPLC column.
Protein synthesized in recombinant culture is characterized by the presence
of cell components, including proteins, in amounts and of a character which
depend upon the purification steps taken to recover the inventive protein
from the culture. These components ordinarily will be of yeast, prokaryotic
or non-human higher eukaryotic origin and preferably are present in
innocuous contaminant quantities, on the order of less than about 1 percent
by weight. Further, recombinant cell culture enables the production of the
inventive proteins free of other proteins which may be normally associated
with the proteins as they are found in nature in the species of origin.
Uses and Administration of RANKL Compositions
The present invention provides methods of using therapeutic compositions
comprising an effective amount of a protein and a suitable diluent and
carrier, and methods for regulating an immune or inflammatory response. The
use of RANKL in conjunction with soluble cytokine receptors or cytokines, or
other immunoregulatory molecules is also contemplated.
For therapeutic use, purified protein is administered to a patient,
preferably a human, for treatment in a manner appropriate to the indication.
Thus, for example, RANKL 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 RANKL, in conjunction with physiologically acceptable
carriers, excipients or diluents. Such carriers will be nontoxic to
recipients at the dosages and concentrations employed.
Ordinarily, the preparation of such protein compositions entails combining
the inventive protein with buffers, antioxidants such as ascorbic acid, low
molecular weight (less than about 10 residues) polypeptides, proteins, amino
acids, carbohydrates including glucose, sucrose or dextrins, chelating
agents such as EDTA, glutathione and other stabilizers and excipients.
Neutral buffered saline or saline mixed with conspecific serum albumin are
exemplary appropriate diluents. Preferably, product is formulated as a
lyophilizate using appropriate excipient solutions (e.g., sucrose) as
diluents. Appropriate dosages can be determined in trials. The amount and
frequency of administration will depend, of course, on such factors as the
nature and severity of the indication being treated, the desired response,
the condition of the patient, and so forth.
As shown hrein, RANKL has beneficial effects on various cells important in
the immune system. Accordingly, RANKL may be adminstered to an individual as
a vaccine adjuvant, or as a therapeutic agent to upregulate an immune
resposne, for example, ininfectious disease. Moreover, NF-.kappa.B has been
found to play a protective role in preventing apoptotic death of cells
induced by TNF-.alpha. or chemotherapy. Accordingly, agonists of RANK (i.e.,
RANKL and agonistic antibodies) will be useful in protecting RANK-expressing
cells from the negative effects of chemotherapy or the presence of high
levels of TNF-.alpha. such as occur in sepsis (see, i.e., Barinaga, Science
274''724, 1996, and the articles by Beg and Baltimore and Wang etal., pages
782 and 784 of that same issue of Science).
Claim 1 of 4 Claims
1. An isolated blocking antibody that
binds to a human RANKL polypeptide as set forth in SEQ ID NO:13 and
inhibits the binding of the human RANKL polypeptide to a human RANK
polypeptide as shown in SEQ ID NO:6.
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