Note: Descriptions are shown in the official language in which they were submitted.
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Death Domain Containing Receptor 5
Background of the Invention
Field of the Invention
The present invention relates to a novel member of the tumor necrosis
factor family of receptors. More specifically, isolated nucleic acid molecules
are
provided encoding human Death Domain Containing Receptor 5, or simply
"DRS." DRS polypeptides are also provided, as are vectors, host cells, and
recombinant methods for producing the same. The invention further relates to
screening methods for identifying agonists and antagonists of DRS activity.
Related Art
Numerous biological actions, for instance, response to certain stimuli and
natural biological processes, are controlled by factors, such as cytokines.
Many
cytokines act through receptors by engaging the receptor and producing an
intra-
cellular response.
For example, tumor necrosis factors (TNF) alpha and beta are cytokines,
which act through TNF receptors to regulate numerous biological processes,
including protection against infection and induction of shock and inflammatory
disease. The TNF molecules belong to the "TNF-ligand" superfamily, and act
together with their receptors or counter-ligands, the "TNF-receptor"
superfamily.
So far, nine members of the TNF ligand superfamily have been identified and
ten
members of the TNF-receptor superfamily have been characterized.
Among the ligands, there are included TNF-a, lymphotoxin-a (LT- a, also
known as TNF-(3), LT-(3 (found in complex heterotrimer LT-a2-~3), FasL,
CD40L, CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor (NGF). The
superfamily of TNF receptors includes the p55TNF receptor, p75TNF receptor,
TNF receptor-related protein, FAS antigen or APO-1, CD40, CD27, CD30,
4-1BB, OX40, low affinity p75 and NGF-receptor (Meager, A., Biologicals,
22:291-295 ( 1994)).
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Many members of the TNF-ligand superfamily are expressed by activated
T-cells, implying that they are necessary for T-cell interactions with other
cell
types which underlie cell ontogeny and functions. (Meager, A., supra).
Considerable insight into the essential functions of several members of the
TNF receptor family has been gained from the identification and creation of
mutants that abolish the expression of these proteins. For example, naturally
occurring mutations in the FAS antigen and its ligand cause
lymphoproliferative
disease (Watanabe-Fukunaga, R., et al., Nature 356:314 ( 1992)), perhaps
reflecting a failure of programmed cell death. Mutations of the CD40 ligand
cause
an X-linked immunodeficiency state characterized by high levels of
immunoglobulin M and low levels of immunoglobulin G in plasma, indicating
faulty T-cell-dependent B-cell activation (Allen, R.C. et al., Science 259:990
(1993)). Targeted mutations of the low affinity nerve growth factor receptor
cause a disorder characterized by faulty sensory innovation of peripheral
structures (Lee, K.F. et al., Cell 69:737 (1992)).
TNF and LT-a, are capable ofbinding to two TNF receptors (the 55- and
75-kd TNF receptors). A large number of biological effects elicited by TNF and
LT-a, acting through their receptors, include hemorrhagic necrosis
oftransplanted
tumors, cytotoxicity, a role in endotoxic shock, inflammation,
immunoregulation,
proliferation and anti-viral responses, as well as protection against the
deleterious
effects of ionizing radiation. TNF and LT-a, are involved in the pathogenesis
of
a wide range of diseases, including endotoxic shock, cerebral malaria, tumors,
autoimmune disease, AIDS and graft-host rejection (Beutler, B. and Von Huffel,
C., Science 264:667-668 (1994)). Mutations in the p55 Receptor cause increased
susceptibility to microbial infection.
Moreover, an about 80 amino acid domain near the C-terminus of TNFR-1
(p55) and Fas was reported as the "death domain," which is responsible for
transducing signals for programmed cell death (Tartaglia et al., Cell 74:845
(1993)).
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Apoptosis, or programmed cell death, is a physiologic process essential for
the normal development and homeostasis of multicellular organisms (H. Steller,
Science 267:1445-1449 (1995)). Derangements of apoptosis contribute to the
pathogenesis of several human diseases including cancer, neurodegenerative
disorders, and acquired immune deficiency syndrome (C.B. Thompson, Science
267:1456-1462 (1995)). Recently, much attention has focused on the signal
transduction and biological function of two cell surface death receptors,
Fas/APO-1 and TNFR-1 (J.L. Cleveland etal., Cel181:479-482 (1995); A. Fraser,
et al., Cell 85:781-784 (1996); S. Nagata et al., Science 267:1449-56 (1995)).
Both are members of the TNF receptor family which also include TNFR-2, low
afFmity NGFR, CD40, and CD30, among others (C.A. Smith et al., Science
248:1019-23 ( 1990); M. Tewari et al., in Modular Texts in Molecular and Cell
Biology M. Purton, Heldin, Carl, Ed. (Chapman and Hall, London, 1995). While
family members are defined by the presence of cysteine-rich repeats in their
extracellular domains, Fas/APO-1 and TNFR-1 also share a region of
intracellular
homology, appropriately designated the "death domain", which is distantly
related
to the Drosophila suicide gene, reaper (P. Golstein, et al., Cell 81:185-186
(1995); K. White et al., Science 264:677-83 (1994)). This shared death domain
suggests that both receptors interact with a related set of signal transducing
molecules that, until recently, remained unidentified. Activation of Fas/APO-1
recruits the death domain-containing adapter molecule FADD/MORTI (A.M.
Chinnaiyan et al., Cell 81: 505-12 (1995); M. P. Boldin et al., .l. Biol Chem
270: 7795-8 (1995); F.C. Kischkel et al., EMBO 14:5579-5588 (1995)), which in
turn binds and presumably activates FLICE/MACH1, a member of the
ICE/CED-3 family of pro-apoptotic proteases (M. Muzio et al.., Cell85: 817-827
(1996); M.P. Boldin et al., Cell 85:803-815 (1996)). While the central role of
Fas/APO-1 is to trigger cell death, TNFR-1 can signal an array of diverse
biological activities-many of which stem from its ability to activate NF-kB
(L.A.
Tartagliaetal., Immunol Today 13:151-3 (1992)). Accordingly, TNFR-1 recruits
the multivalent adapter molecule TRADD, which like FADD, also contains a
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death domain (H. Hsu eJ al., Cel181:495-504 ( 1995); H. Hsu, et al.,
Cel184:299
308 (1996)). Through its associations with a number of signaling molecules
including FADD, TRAF2, and RIP, TRADD can signal both apoptosis and NF-kB
activation (H. Hsuetal., Ced184:299-308 (1996); H. Hsu, etal., Immunity4:387
396 (1996)).
Recently, a new apoptosis -inducing TNF ligand has been discovered.
S.R. Wiley et al. (Immunity 3:673-682 (1995)) named the molecule - "TNF-
related apoptosis-inducing ligand" or simply "TRAIL." The molecule was also
called "Apo-2 ligand" or "Apo-2L." R.M. Pitt et al., J. Biol. Chem. 271:12687-
12690 (1996). For convenience, the molecule will be referred to herein as
TRAIL.
Unlike FAS ligand, whose transcripts appear to be largely restricted to
stimulated T-cells, significant levels of TRAIL are detected in many human
tissues
(e.g., spleen, lung, prostate, thymus, ovary, small intestine, colon,
peripheral blood
1 S lymphocytes, placenta, kidney), and is constitutively transcribed by some
cell lines.
It has been shown that TRAIL acts independently from the Fas ligand (Whey et
al., supra). It has also been shown that TRAIL, activates apoptosis rapidly,
within
a time frame that is similar to death signaling by Fas/Apo-1L, but much faster
than
TNF-induced apoptosis. S.A. Marsters etal., CurrentBiology 6:750-752 (1996).
The inability of TRAIL to bind TNFR-1, Fas, or the recently identified DR3,
suggests that TRAIL may interact with a unique receptor(s).
The effects of TNF family ligands and TNF family receptors are varied and
influence numerous functions, both normal and abnormal, in the biological
processes of the mammalian system. There is a clear need, therefore, for
identification and characterization of such receptors and ligands that
influence
biological activity, both normally and in disease states. In particular, there
is a
need to isolate and characterize additional novel receptors that bind TRAIL.
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Summary of the Invention
The present invention provides for isolated nucleic acid molecules
comprising, or alternatively consisting of, nucleic acid sequences encoding
the
amino acid sequence shown in FIG. 1 (SEQ ID N0:2) or the amino acid sequence
encoded by the cDNA deposited as ATCC Deposit No. 97920 on March 7, 1997.
The present invention also provides recombinant vectors, which include
the isolated nucleic acid molecules of the invention, and to host cells
containing
the recombinant vectors, as well as to methods of making such vectors and host
cells and for using them for production of DRS polypeptides or peptides by
recombinant techniques.
The invention further provides an isolated DRS polypeptide having an
amino acid sequence encoded by a polynucleotide described herein.
The present invention also provides diagnostic assays such as quantitative
and diagnostic assays for detecting levels of DRS protein. Thus, for instance,
a
diagnostic assay in accordance with the invention for detecting over-
expression
of DRS, or soluble form thereof, compared to normal control tissue samples may
be used to detect the presence of tumors.
Tumor Necrosis Factor (TNF) family ligands are known to be among the
most pleiotropic cytokines, inducing a large number of cellular responses,
including cytotoxicity, anti-viral activity, immunoregulatory activities, and
the
transcriptional regulation of several genes. Cellular response to TNF-family
ligands include not only normal physiological responses, but also diseases
associated with increased apoptosis or the inhibition of apoptosis. Apoptosis -
programmed cell death - is a physiological mechanism involved in the deletion
of
peripheral T lymphocytes of the immune system, and its dysregulation can lead
to
a number of different pathogenic processes. Diseases associated with increased
cell survival, or the inhibition of apoptosis, include cancers, autoimmune
disorders,
viral infections, inflammation, graft versus host disease, acute graft
rejection, and
chronic graft rejection. Diseases associated with increased apoptosis include
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AIDS, neurodegenerative disorders, myelodysplastic syndromes, ischemic injury,
toxin-induced liver disease, septic shock, cachexia and anorexia.
Thus, the invention further provides a method for enhancing apoptosis
induced by a TNF-family ligand, which involves administering to a cell which
expresses the DRS polypeptide an effective amount of an agonist capable of
increasing DRS mediated signaling. Preferably, DRS mediated signaling is
increased to treat and/or prevent a disease wherein decreased apoptosis is
exhibited.
In a further aspect, the present invention is directed to a method for
inhibiting apoptosis induced by a TNF-family ligand, which involves
administering
to a cell which expresses the DRS polypeptide an effective amount of an
antagonist capable of decreasing DRS mediated signaling. Preferably, DRS
mediated signaling is decreased to treat and/or prevent a disease wherein
increased
apoptosis is exhibited.
Whether any candidate "agonist" or "antagonist" of the present invention
can enhance or inhibit apoptosis can be determined using art-known TNF-family
ligand/receptor cellular response assays, including those described in more
detail
below. Thus, in a further aspect, a screening method is provided for
determining
whether a candidate agonist or antagonist is capable of enhancing or
inhibiting a
cellular response to a TNF-family ligand. The method involves contacting cells
which express the DRS polypeptide with a candidate compound and a TNF-family
ligand, assaying a cellular response, and comparing the cellular response to a
standard cellular response, the standard being assayed when contact is made
with
the ligand in absence of the candidate compound, whereby an increased cellular
response over the standard indicates that the candidate compound is an agonist
of
the ligand/receptor signaling pathway and a decreased cellular response
compared
to the standard indicates that the candidate compound is an antagonist of the
ligand/receptor signaling pathway. By the invention, a cell expressing the DRS
polypeptide can be contacted with either an endogenous or exogenously
administered TNF-family ligand.
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Brief Description of the Figures
FIG. 1 shows the nucleotide (SEQ ID NO:1 ) and deduced amino acid
sequence (SEQ ID N0:2) of DRS. It is predicted that amino acids from about 1
to about 51 (underlined) constitute the signal peptide (amino acid residues
from
about -51 to about -I in SEQ ID N0:2); amino acids from about 52 to about 184
constitute the extracellular domain (amino acid residues from about 1 to about
133 in SEQ ID N0:2); amino acids from about 84 to about 179 constitute the
cysteine rich domain (amino acid residues from about 33 to 128 in SEQ ID
N0:2);
amino acids from about 185 to about 208 (underlined) constitute the
transmembrane domain (amino acid residues from about 134 to about 157 in SEQ
ID N0:2); and amino acids from about 209 to about 411 constitute the
intracellular domain (amino acid residues from about 158 to about 360 in SEQ
ll~
N0:2), of which amino acids from about 324 to about 391 (italicized)
constitute
the death domain (amino acid residues from about 273 to about 340 in SEQ ID
N0:2).
FIG. 2 shows the regions of similarity between the amino acid sequences
of DRS (HL,YBX88), human tumor necrosis factor receptor 1 (h TNFR-1 ) (SEQ
TD N0:3), human Fas protein (SEQ ID N0:4), and the death domain containing
receptor 3 (SEQ 117 NO:S). The comparison was created with the Megalign
program which is contained in the DNA Star suite of programs, using the
Clustal
method. Residues that match the consensus are shaded.
FIG. 3 shows an analysis of the DRS amino acid sequence. Alpha, beta,
turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions;
flexible regions; antigenic index and surface probability are shown, as
predicted
for the amino acid sequence depicted in FIG. 1 using the default parameters of
the
recited computer program. In the "Antigenic Index - Jameson-Wolf' graph, amino
acid residues about 62 to about 110, about 119 to about 164, about 224 to
about
271, and about 275 to about 370 as depicted in FIG. 1 correspond to the shown
highly antigenic regions ofthe DRS protein. These highly antigenic fragments
in
FIG. 1 correspond to the following fragments, respectively, in SEQ ID N0:2:
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amino acid residues from about 11 to about S9, from about 68 to about 113,
from
about 173 to about 220, and from about 224 to about 319.
FIG. 4 shows the nucleotide sequences (HAPBU 13R and HSBBU76R) of
two cDNA molecules which are related to the nucleotide sequence shown in FIG.
S 1 (SEQ ID NO: l ).
FIG. SA is a bar graph showing that overexpression of DRS induced
apoptosis in MCF7 human breast carcinoma cells. FIG. SB is a bar graph showing
that overexpression of DRS induced apoptosis in human epitheloid carcinoma
(HeLa) cells. FIG. SC is a bar graph showing that DRS-induced apoptosis was
blocked by caspase inhibitors, CrmA and z-VAD-fink, but dominant negative
FADD was without effect. FIG. SD is an immunoblot showing that, like DR4,
DRS did not interact with FADD and TRADD in vivo. FIG. SE is a bar graph
showing that a dominant negative version of a newly identified FLICE-like
molecule, FLICE2 (Vincent, C. et al., .~. Biol. Chem. 272:6578 (1997)),
1 S efficiently blocked DRS-induced apoptosis, while dominant negative FLICE
had
only partial effect under conditions it blocked. It also shows that TNFR-1
blocked
apoptosis effectively.
FIG. 6A is an immunoblot showing that DRS-Fc (as well as DR4 and
TRID) specifically bound TRAIL, but not the related cytotoxic ligand TNFa. The
bottom panel of FIG. 6A shows the input Fc-fusions present in the binding
assays.
FIG. 6B is a bar graph showing that DRS-Fc blocked the ability of TRAIL to
induce apoptosis. The data (mean ~ SD) shown in FIG. 6B are the percentage of
apoptotic nuclei among total nuclei counted (n=4). FIG. 6C is a bar graph
showing that DRS-Fc had no effect on apoptosis TNFa-induced cell death under
2S conditions where TNFR-1-Fc completely abolished TNFa killing.
Detailed Description of the Preferred Embodiments
The present invention provides isolated nucleic acid molecules comprising,
3 0 or alternatively consisting of, a polynucleotide encoding a DRS
polypeptide having
the amino acid sequence shown in FIG. 1 (SEQ ID N0:2), or a fragment of this
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polypeptide. The DRS polypeptide of the present invention shares sequence
homology with other known death domain containing receptors of the TNFR
family including human TNFR-1, DR3 and Fas (FIG. 2). The nucleotide sequence
shown in FIG. 1 (SEQ 117 NO:I) was obtained by sequencing cDNA clones such
as HLYBX88, which was deposited on March 7, 1997 at the American Type
Culture, 10801 University Boulevard, Manassas, Virginia, 20110-2209, and given
Accession Number 97920. The deposited cDNA is contained in the pSport 1
plasmid (Life Technologies, Gaithersburg, MD).
Nucleic Acid Molecules
Unless otherwise indicated, all nucleotide sequences determined by
sequencing a DNA molecule herein were determined using an automated DNA
sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino
acid sequences of polypeptides encoded by DNA molecules determined herein
were predicted by translation of a DNA sequence determined as above.
Therefore, as is known in the art for any DNA sequence determined by this
automated approach, any nucleotide sequence determined herein may contain
some errors. Nucleotide sequences determined by automation are typically at
least
about 90% identical, more typically at least about 95% to at least about 99.9%
identical to the actual nucleotide sequence of the sequenced DNA molecule. The
actual sequence can be more precisely determined by other approaches including
manual DNA sequencing methods well known in the art. As is also known in the
art, a single insertion or deletion in a determined nucleotide sequence
compared
to the actual sequence will cause a frame shift in translation of the
nucleotide
sequence such that the predicted amino acid sequence encoded by a determined
nucleotide sequence will be completely different from the amino acid sequence
actually encoded by the sequenced DNA molecule, beginning at the point of such
an insertion or deletion.
Using the information provided herein, such as the nucleic acid sequence
set out in SEQ ID NO: l, a nucleic acid molecule ofthe present invention
encoding
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a DRS polypeptide may be obtained using standard cloning and screening
procedures, such as those for cloning cDNAs using mRNA as starting material.
Illustrative of the invention, the nucleic acid molecule of the invention has
been
identified in cDNA libraries of the following tissues: primary dendritic
cells,
endothelial tissue, spleen, chronic lymphocytic leukemia, and human thymus
stromal cells.
The determined nucleotide sequence of the DRS cDNA of SEQ ID NO:1
contains an open reading frame encoding a protein of about 411 amino acid
residues whose initiation codon is at position 130-132 ofthe nucleotide
sequence
shown in FIG. 1 (SEQ ID NO. l), with a leader sequence of about 51 amino acid
residues. Of known members of the TNF receptor family, the DRS polypeptide
of the invention shares the greatest degree of homology with human TNFR-l, FAS
and DR3 polypeptides shown in FIG. 2, including significant sequence homology
over multiple cysteine-rich domains. The homology DR5 shows to other death
domain containing receptors strongly indicates that DRS is also a death domain
containing receptor with the ability to induce apoptosis. DRS has also now
been
shown to bind TRAIL.
As indicated, the present invention also provides the mature forms) of the
DRS protein ofthe present invention. According to the signal hypothesis,
proteins
secreted by mammalian cells have a signal or secretory leader sequence which
is
cleaved from the mature protein once export of the growing protein chain
across
the rough endoplasmic reticulum has been initiated. Most mammalian cells and
even insect cells cleave secreted proteins with the same specificity. However,
in
some cases, cleavage of a secreted protein is not entirely uniform, which
results
in two or more mature species on the protein. Further, it has long been known
that the cleavage specificity of a secreted protein is ultimately determined
by the
primary structure of the complete protein, that is, it is inherent in the
amino acid
sequence of the polypeptide.
Therefore, the present invention provides a nucleotide sequence encoding
the mature DRS polypeptide having the amino acid sequence encoded by the
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cDNA contained in the plasmid identified as ATCC Deposit No. 97920, and as
shown in FIG. 1 (SEQ ID N0:2). By the mature DRS protein having the amino
acid sequence encoded by the cDNA contained in the plasmid identified as ATCC
Deposit No. 97920, is meant the mature forms) of the DRS protein produced by
S expression in a mammalian cell (e.g., COS cells, as described below) of the
complete open reading frame encoded by the human cDNA contained in the
deposited plasmid. As indicated below, the mature DRS having the amino acid
sequence encoded by the cDNA contained in ATCC Deposit No. 97920, may or
may not differ from the predicted "mature" DRS protein shown in SEQ ID N0:2
(amino acids from about 1 to about 360) depending on the accuracy of the
predicted cleavage site based on computer analysis.
Methods for predicting whether a protein has a secretory leader as well as
the cleavage point for that leader sequence are available. For instance, the
method
of McGeoch (Tlirus Res. 3:271-286 (1985)) and von Heinje (Nucleic Acids Res.
14:4683-4690 ( 1986)) can be used. The accuracy of predicting the cleavage
points of known mammalian secretory proteins for each of these methods is in
the
range of 75-80%. von Heinje, .supra. However, the two methods do not always
produce the same predicted cleavage points) for a given protein.
In the present case, the predicted amino acid sequence of the complete
DRS polypeptide of the present invention was analyzed by a computer program
("PSORT"). See, K. Nakai and M. Kanehisa, Genomics 14:897-911 (1992).
PSORT is an expert system for predicting the cellular location of a protein
based
on the amino acid sequence. As part of this computational prediction of
localization, the methods of McGeoch and von Heinje are incorporated. The
analysis by the PSORT program predicted the cleavage sites between amino acids
51 and 52 in FIG. 1 (-1 and 1 in SEQ ID N0:2). Thereafter, the complete amino
acid sequences were further analyzed by visual inspection, applying a simple
form
of the (-1,-3) rule of von Heinje. von Heinje, supra. Thus, the leader
sequence
for the DRS protein is predicted to consist of amino acid residues from about
1 to
about 51, underlined in FIG. 1 (corresponding to amino acid residues about -S1
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to about 1 in SEQ ID N0:2), while the predicted mature DRS protein consists of
residues from about 52 to about 411 in FIG. 1 (corresponding to amino acid
residues about 1 to about 360 in SEQ ID N0:2).
As one of ordinary skill would appreciate, due to the possibility of
sequencing errors, as well as the variability of cleavage sites for leaders in
different
known proteins, the predicted DRS receptor polypeptide encoded by the
deposited
cDNA comprises about 411 amino acids, but may be anywhere in the range of
401-421 amino acids; and the predicted leader sequence of this protein is
about
51 amino acids, but may be anywhere in the range of about 41 to about 61 amino
acids. It will further be appreciated that, the domains described herein have
been
predicted by computer analysis, and accordingly, that depending on the
analytical
criteriaused for identifying various functional domains, the exact "address"
of, for
example, the extracelluar domain, intracellular domain, death domain, cysteine-
rich motifs, and transmembrane domain of DRS may differ slightly. For example,
the exact location of the DRS extracellular domain in FIG. 1 (SEQ ID N0:2) may
vary slightly (e.g., the address may "shift" by about 1 to about 20 residues,
more
likely about 1 to about S residues) depending on the criteria used to define
the
domain. In any event, as discussed further below, the invention further
provides
polypeptides having various residues deleted from the N-terminus and/or
C-terminus of the complete DRS, including polypeptides lacking one or more
amino acids from the N-termini of the extracellular domain described herein,
which constitute soluble forms of the extracellular domain of the DRS
polypeptides.
As indicated, nucleic acid molecules of the present invention may be in the
form of RNA, such as mRNA, or in the form of DNA, including, for instance,
cDNA and genomic DNA obtained by cloning or produced synthetically. The
DNA may be double-stranded or single-stranded. Single-stranded DNA may be
the coding strand, also known as the sense strand, or it may be the non-coding
strand, also referred to as the anti-sense strand.
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By "isolated" nucleic acid molecules) is intended a nucleic acid molecule,
DNA or RNA, which has been removed from its native environment For example,
recombinant DNA molecules contained in a vector are considered isolated for
the
purposes of the present invention. Further examples of isolated DNA molecules
S include recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
However, a nucleic acid molecule contained in a clone that is a member of
a mixed clone library (e.g., a genomic or cDNA library) and that has not been
isolated from other clones of the library (e.g., in the form of a homogeneous
solution containing the clone without other members of the library) or a
chromosome isolated or removed from a cell or a cell lysate (e.g., a
"chromosome
spread", as in a karyotype), is not "isolated" for the purposes of this
invention.
Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA
molecules of the present invention. Isolated nucleic acid molecules according
to
1 S the present invention further include such molecules produced
synthetically.
Isolated nucleic acid molecules ofthe present invention include DRS DNA
molecules comprising, or alternatively consisting of, an open reading frame
(ORF)
shown in SEQ >D NO:1; DNA molecules comprising, or alternatively consisting
of, the coding sequence for the mature DRS protein; and DNA molecules which
comprise, or alternatively consist of, a sequence substantially different from
those
described above, but which, due to the degeneracy of the genetic code, still
encode the DRS protein. Of course, the genetic code is well known in the art.
Thus, it would be routine for one skilled in the art to generate such
degenerate
variants.
2S In addition, the invention provides nucleic acid molecules having
nucleotide sequences related to extensive portions of SEQ ID NO:1 which have
been determined from the following related cDNAs: HAPBU13R (SEQ ID N0:6)
and HSBBU76R (SEQ ID N0:7). The nucleotide sequences ofHAPBU13R and
HSBBU76R are shown in FIG. 4.
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The nucleotide sequence of an additional related polynucleotide which has
been assigned GenBank Accession number 266083 is shown in SEQ ID N0:14.
In another aspect, the invention provides isolated nucleic acid molecules
encoding the DRS polypeptide having an amino acid sequence encoded by the
cDNA contained in the plasmid deposited as ATCC Deposit No. 97920 on March
7, 1997. In a further embodiment, nucleic acid molecules are provided that
encode the mature DRS polypeptide or the full length DRS polypeptide lacking
the
N-terminal methionine. The invention further provides an isolated nucleic acid
molecule having the nucleotide sequence shown in SEQ ID NO:1 or the
nucleotide sequence of the DRS cDNA contained in the above-described
deposited plasmid, or a nucleic acid molecule having a sequence complementary
to one of the above sequences. Such isolated molecules, particularly DNA
molecules, have uses which include, but are not limited to, as probes for gene
mapping by in .situ hybridization with chromosomes, and for detecting
expression
of the DRS gene in human tissue, for instance, by Northern blot analysis.
The present invention is further directed to fragments of the isolated
nucleic acid molecules described herein. By fragments of an isolated DNA
molecule having the nucleotide sequence shown in SEQ ID NO:1 or having the
nucleotide sequence of the deposited cDNA (the cDNA contained in the plasmid
deposited as ATCC Deposit No. 97920) are intended DNA fragments at least 20
nt, and more preferably at least 30 nt in length, and even more preferably, at
least
about 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,
750, 800, 850, 900, 950, 1000, 1050, 1100, 11 S0, or 1200 nucleotides in
length,
which are useful as DNA probes as discussed above. Of course, DNA fragments
corresponding to most, if not all, of the nucleotide sequence shown in SEQ ID
NO:1 are also useful as DNA probes. By a fragment at least about 20 nt in
length,
for example, is intended fragments which include 20 or more contiguous bases
from the nucleotide sequence of the deposited DNA or the nucleotide sequence
as shown in SEQ ID NO:1. In this context "about" includes the particularly
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recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at
either
terminus or at both termini.
Representative examples ofDRS polynucleotide fragments ofthe invention
include, for example, fragments that comprise, or alternatively consist of, a
sequence from about nucleotide 1-130, 130-180, 181-231, 232-282, 283-333,
334-384, 385-435, 436-486, 487-537, 538-588, 589-639, 640-681, 682-732, 733-
753, 754-804, 805-855, 856-906, 907-957, 958-1008, 1009-1059, 1060-1098,
1099-1149, 1150-1200, 1201-1251, 1252-1302, 1303-1353, 1354-1362, and
1363 to the end of SEQ ID NO:1, or the complementary DNA strand thereto, or
the cDNA contained in the deposited plasmid. In this context "about" includes
the
particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1 )
nucleotides, at either terminus or at both termini. Polypeptides encoded by
these
polynucleotides are also encompassed by the invention.
The present invention is further directed to polynucleotides comprising, or
alternatively consisting of, isolated nucleic acid molecules which encode
domains
of DRS. In one aspect, the invention provides polynucleotides comprising, or
alternatively consisting of, nucleic acid molecules which encode beta-sheet
regions
of DR5 protein set out in Table I. Representative examples of such
polynucleotides include nucleic acid molecules which encode a polypeptide
comprising, or alternatively consisting of, one, two, three, four, five, or
more
amino acid sequences selected from the group consisting o~ amino acid residues
from about -16 to about -2, amino acid residues from about 2 to about 9, amino
acid residues from about 60 to about 67, amino acid residues from about 135 to
about 151, amino acid residues from about 193 to about 199, and amino acid
residues from about 302 to about 310 in SEQ ID N0:2. In this context "about"
includes the particularly recited value and values larger or smaller by
several (5,
4, 3, 2, or 1 ) amino acid residues. Polypeptides encoded by these
polynucleotides
are also encompassed by the invention.
In specific embodiments, the polynucleotide fragments of the invention
encode a polypeptide which demonstrates a DR5 functional activity. By a
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polypeptide demonstrating a DRS "functional activity" is meant, a polypeptide
capable of displaying one or more known functional activities associated with
a
complete (full-length) or mature DRS polypeptide, as well as secreted forms of
DRS. Such functional activities include, but are not limited to, biological
activity
(e.g., ability to induce apoptosis in cells expressing the polypeptide (see
e.g.,
Example 5)), antigenicity (ability to bind (or compete with a DRS polypeptide
for
binding) to an anti-DRS antibody), immunogenicity (ability to generate
antibody
which binds to a DRS polypeptide), ability to form multimers, and ability to
bind
to a receptor or ligand for a DRS polypeptide (e.g., TRAIL,; Wiley et al,
Immunity
3, 673-682 (1995)).
The functional activity of DRS polypeptides, and fragments, variants
derivatives, and analogs thereof, can be assayed by various methods.
For example, in one embodiment where one is assaying for the ability to
bind or compete with full-length (complete) DRS polypeptide for binding to
anti
DRS antibody, various immunoassays known in the art can be used, including but
not limited to, competitive and non-competitive assay systems using techniques
such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich"~ immunoassays, immunoradiometric assays, gel diffusion
precipitation
reactions, immunodifFusion assays, in situ immunoassays (using colloidal gold,
enzyme or radioisotope labels, for example), western blots, precipitation
reactions,
agglutination assays (e.g., gel agglutination assays, hemagglutination
assays),
complement fixation assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. In one embodiment, antibody binding is
detected by detecting a label on the primary antibody. In another embodiment,
the
primary antibody is detected by detecting binding of a secondary antibody or
reagent to the primary antibody. In a further embodiment, the secondary
antibody
is labeled. Many means are known in the art for detecting binding in an
immunoassay and are within the scope of the present invention.
In another embodiment, where a DRS ligand is identified (e.g. TRAIL), or
the ability of a polypeptide fragment, variant or derivative of the invention
to
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multimerize is being evaluated, binding can be assayed, e.g., by means well-
known
in the art, such as, for example, reducing and non-reducing gel
chromatography,
protein affinity chromatography, and affinity blotting. See generally,
Phizicky, et
al., Microbiol. Rev. 59:94-123 (1995). In another embodiment, physiological
S correlates of DRS binding to its substrates (signal transduction) can be
assayed.
In addition, assays described herein (see Examples S and 6), and otherwise
known in the art may routinely be applied to measure the ability of DRS
polypeptides and fragments, variants derivatives and analogs thereof to elicit
DRS
related biological activity (e.g., ability to induce apoptosis in cells
expressing the
polypeptide (see e.g., Example S), and the ability to bind a ligand, e.g.,
TRAIL
(see, e.g., Example 6) in vitro or in vivo). For example, biological activity
can
routinely be measured using the cell death assays performed essentially as
previously described (Chinnaiyan et al., Cell 81:505-S 12 (1995); Boldin et
al., J.
Biol. Chem. 270:7795-8(1995); Kischkel et al., EMBO 14:5579-SS88 (1995);
1S Chinnaiyan et al., J. Biol. Chem. 271:4961-4965 (1996)) and as set forth in
Example S below. In one embodiment involving MCF7 cells, plasmids encoding
full-length DRS or a candidate death domain containing receptor are co
transfected with the pLantern reporter construct encoding green fluorescent
protein. Nuclei of cells transfected with DRS will exhibit apoptotic
morphology
as assessed by DAPI staining.
Other methods will be known to the skilled artisan and are within the
scope of the invention.
Preferred nucleic acid fragments of the present invention include, but are
not limited to, a nucleic acid molecule encoding a polypeptide comprising, or
alternatively consisting of, one, two, three, four, five, or more amino acid
sequences selected from the group consisting of: a polypeptide comprising, or
alternatively consisting of, the DRS extracellular domain (amino acid residues
from about S2 to about 184 in FIG. 1 (amino acid residues from about 1 to
about
133 in SEQ ID N0:2)); a polypeptide comprising, or alternatively consisting
of,
the DRS transmembrane domain (amino acid residues from about 185 to about
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208 in FIG. 1 (amino acid residues from about 134 to about 157 in SEQ >D
N0:2)); a polypeptide comprising, or alternatively consisting of, the cysteine
rich
domain of DRS (amino acid residues from about 84 to about 179 in FIG. 1 (from
about 33 to about 128 in SEQ ID N0:2)); a polypeptide comprising, or
alternatively consisting of, the DRS intracellular domain (amino acid residues
from
about 209 to about 411 in FIG. 1 (amino acid residues from about 158 to about
360 in SEQ ID N0:2)); a polypeptide comprising, or alternatively consisting
of,
a fragment of the predicted mature DRS polypeptide, wherein the fragment has a
DRS functional activity (e.g., antigenic activity or biological activity); a
polypeptide comprising, or alternatively consisting of, the DRS receptor
extracellular and intracellular domains with all or part of the transmembrane
domain deleted; a polypeptide comprising, or alternatively consisting of, the
DRS
death domain (amino acid residues from about 324 to about 391 in FIG. 1 (from
about 273 to about 340 in SEQ >D N0:2)); and a polypeptide comprising, or
alternatively consisting of, one, two, three, four or more, epitope bearing
portions
of the DRS receptor protein. In additional embodiments, the polynucleotide
fragments of the invention encode a polypeptide comprising, or alternatively
consisting of, any combination of 1, 2, 3, 4, S, 6, 7, or all 8 ofthe above
members.
Since the location of these domains have been predicted by computer graphics,
one of ordinary skill would appreciate that the amino acid residues
constituting
these domains may vary slightly (e.g., by about 1 to 15 residues) depending on
the
criteria used to define each domain. Polypeptides encoded by these nucleic
acid
molecules are also encompassed by the invention.
It is believed one or both of the extracellular cysteine rich motifs of DRS
disclosed in FIG. 1 is important for interactions between DRS and its ligands
(e.g.,
TRAIL). Accordingly, specific embodiments of the invention are directed to
polynucleotides encoding a polypeptide comprising, or alternatively consisting
of,
one or both amino acid sequences selected from the group consisting o~ amino
acid residues 84 to 131, and/or 132 to 179 of the DRS sequence shown in FIG.
1 (amino acid residues 33 to 80, and/or 81 to 128 in SEQ ID N0:2). In a
specific
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embodiment the polynucleotides encoding DRS polypeptides of the invention
comprise, or alternatively consist of, both of the extracellular cysteine-rich
motifs
disclosed in FIG. 1.
In certain embodiments, polynucleotides of the invention comprise, or
alternatively consist of, a polynucleotide sequence at least 80%, 85%, 90%,
92%,
95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding
the cysteine-rich domain described above. The present invention also
encompasses the above polynucleotide sequences fused to a heterologous
polynucleotide sequence. Polypeptides encoded by these polynucleotides are
also
encompassed by the invention. Methods to measure the percent identity of a
polynucleotide sequence to a reference polynucleotide sequence are described
infra.
In another embodiment, the invention provides an isolated nucleic acid
molecule comprising, or alternatively consisting of, a polynucleotide which
hybridizes under stringent hybridization conditions to nucleic acids
complementary
to the cysteine-rich domain encoding polynucleotides described above. The
meaning of the phrase "stringent conditions" as used herein is described
infra.
Polypeptides encoded by such polynucleotides are also contemplated by the
invention.
Preferred nucleic acid fragments of the invention encode a full-length DRS
polypeptide lacking the nucleotides encoding the amino-terminal methionine
(nucleotides 130-132 in SEQ ID NO:1) as it is known that the methionine is
cleaved naturally and such sequences maybe useful in genetically engineering
DRS
expression vectors. Polypeptides encoded by such polynucleotides are also
contemplated by the invention.
In additional embodiments, the polynucleotides of the invention encode
functional attributes of DRS. Preferred embodiments of the invention in this
regard include fragments that comprise, or alternatively consist of, one, two,
three, four, or more of the following functional domains: alpha-helix and
alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet
forming
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regions ("beta-regions"), turn and turn-forming regions ("turn-regions"), coil
and
coil-forming regions ("coil-regions"), hydrophilic regions, hydrophobic
regions,
alpha-amphipathic regions, beta-amphipathic regions, flexible regions,
surface-forming regions and high antigenic index regions of DRS.
The data representing the structural or functional attributes of DRS set
forth in FIG. 3 and/or Table I, as described above, were generated using the
various identified modules and algorithms of the DNA*STAR set on default
parameters. In a preferred embodiment, the data presented in columns VIII, IX,
XIII, and XIV of Table I can be used to determine regions of DRS which exhibit
a high degree of potential for antigenicity. Regions of high antigenicity are
determined from the data presented in columns VIII, IX, XIII, and/or XIV by
choosing values which represent regions of the polypeptide which are likely to
be
exposed on the surface of the polypeptide in an environment in which antigen
recognition may occur in the process of initiation of an immune response.
Certain preferred regions in these regards are set out in FIG. 3, but may,
as shown in Table I, be represented or identified by using tabular
representations
of the data presented in FIG. 3. The DNA*STAR computer algorithm used to
generate FIG. 3 (set on the original default parameters) was used to present
the
data in FIG. 3 in a tabular format (See Table I). The tabular format of the
data in
FIG. 3 may be used to easily determine specific boundaries of a preferred
region.
The above-mentioned preferred regions set out in FIG. 3 and in Table I
include, but are not limited to, regions of the aforementioned types
identified by
analysis of the amino acid sequence set out in SEQ ff~ N0:2. As set out in
FIG.
3 and in Table I, such preferred regions include Gamier-Robson alpha-regions,
beta-regions, turn-regions, and coil-regions (columns I, III, V, and VII in
Table
I), Chou-Fasman alpha-regions, beta-regions, and turn-regions (columns II, IV,
and VI in Table I), Kyte-Doolittle hydrophilic regions (column VIII in Table
I),
Hopp-Woods hydrophobic regions (column IX in Table I), Eisenberg alpha- and
beta-amphipathic regions (columns X and XI in Table I), Karplus-Schulz
flexible
regions (column XII in Table I), Jameson-Wolf regions of high antigenic index
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(column XIII in Table I), and Emini surface-forming regions (column XIV in
Table I).
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00 O~ OD H l~ M O N 01 W 00 rl N l0 41 01 l0 Lf1 M M N 10 ~f1 lf1 ~ d' lW f1
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OD O M l0 N Lf1 01 l0 d0 00 aD 01 O L~ 01 M L~ ~ dl O rl Lf1 Lf1 00 l0 l0 OD
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M M M M L~ H a0 V' N d' a1 \O O 10 10 N V' M dl O rl OD tf1 VW -i OD Lf1 I~ [~
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l0 00 O N l0 01 M 1D O O 1f1 O W 01 O N o0 M O O rl N r N Lf1 M l0 10 r r N V'
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H~. ~ ~ ~ O O ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ O O O O O O O
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l0 N M ~ l0 R7 If7 M l0 L~ O 00 N H aD l0 ~O 01 l~ 01 1D M a0 V' 01 M Lf1 N T
d' M N V~
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aD U1 OD O N O W dl rl N ~O M M M ~ 00 I~ dl
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Among highly preferred fragments in this regard are those that comprise,
or alternatively consist of, regions of DRS that combine several structural
features, such as several of the features set out above. Preferred nucleic
acid
fragments of the present invention further include nucleic acid molecules
encoding a polypeptide comprising, or alternatively consisting of, one, two,
three, four, five, or more epitope-bearing portions of the DRS protein. In
particular, such nucleic acid fragments of the present invention include, but
are
not limited to, nucleic acid molecules encoding a polypeptide comprising, or
alternatively consisting of, one, two, three, or more amino acid sequences
selected from the group consisting of: amino acid residues from about 62 to
about 110 in FIG. 1 (amino acid residues from about 11 to about 59 in SEQ m
N0:2); a polypeptide comprising, or alternatively consisting of, amino acid
residues from about 119 to about 164 in FIG. 1 (amino acid residues from about
68 to about 113 in SEQ ID N0:2); a polypeptide comprising, or alternatively
consisting of, amino acid residues from about 224 to about 271 in FIG. 1
(amino
acid residues from about 173 to about 220 in SEQ ID N0:2); and a polypeptide
comprising, or alternatively consisting of, amino acid residues from about 275
to
about 370 in FIG. 1 (amino acid residues from about 224 to about 319 in SEQ
ID N0:2). The inventors have determined that the above polypeptide fragments
are antigenic regions of the DRS protein. Methods for determining other such
epitope-bearing portions ofthe DRS protein are described in detail below. In
this
context "about" includes the particularly recited value and values larger or
smaller by several (S, 4, 3, 2, or 1) amino acid residues. Polypeptides
encoded
by these nucleic acids are also encompassed by the invention.
Further, the invention includes a polynucleotide comprising, or
alternatively consisting of, any portion of at least about 30 nucleotides,
preferably
at least about 50 nucleotides, of SEQ ID NO:1 from residue 283 to 1,362,
preferably from 283 to 681. Polypeptides encoded by these polynucleotides are
also encompassed by the invention.
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In specific embodiments, the polynucleotides of the invention are less
than 100000 kb, 50000 kb, 10000 kb, 1000 kb, 500 kb, 400 kb, 350 kb, 300 kb,
250 kb, 200 kb, 175 kb, 150 kb, 125 kb, 100 kb, 75 kb, 50 kb, 40 kb, 30 kb, 25
kb, 20 kb, 15 kb, 10 kb, 7.5 kb, or 5 kb in length.
In further embodiments, polynucleotides of the invention comprise, or
alternatively consisting of, at least 15, at least 30, at least 50, at least
100, or at
least 250, at least 500, or at least 1000 contiguous nucleotides of DRS coding
sequence, but consist of less than or equal to 1000 kb, 500 kb, 250 kb, 200
kb,
1 SO kb, 100 kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kb of
genomic DNA that flanks the 5' or 3' coding nucleotide set forth in FIG. 1
(SEQ
ID NO: l). In further embodiments, polynucleotides of the invention comprise,
or alternatively consist of, at least 15, at least 30, at least 50, at least
100, or at
least 250, at least 500, or at least 1000 contiguous nucleotides of DRS coding
sequence, but do not comprise, or alternatively consist of, all or a portion
of any
DRS intron. In another embodiment, the nucleic acid comprising, or
alternatively
consisting of, DRS coding sequence does not contain coding sequences of a
genomic flanking gene (i.e., 5' or 3' to the DRS gene in the genome). In other
embodiments, the polynucleotides of the invention do not contain the coding
sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or
1
genomic flanking gene(s).
In another embodiment, the invention provides an isolated nucleic acid
molecule comprising, or alternatively consisting of, a polynucleotide which
hybridizes under stringent hybridization conditions to a portion of the
polynucleotide in a nucleic acid molecule of the invention described above,
for
instance, the sequence complementary to the coding and/or noncoding (i.e.,
transcribed, untranslated) sequence depicted in SEQ ID NO:I, the cDNA
contained in ATCC Deposit No. 97920, and the sequence encoding a DR5
domain, or a polynucleotide fragment as described herein. By "stringent
hybridization conditions" is intended overnight incubation at 42°C in a
solution
comprising, or alternatively consisting of: 50% formamide, Sx SSC (750 mM
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NaCI, 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), Sx
Denhardt's solution, 10% dextran sulfate, and 20 pg/ml denatured, sheared
salmon sperm DNA, followed by washing the filters in 0. lx SSC at about 65
° C.
Polypeptides encoded by these polynucleotides are also encompassed by the
invention.
By a polynucleotide which hybridizes to a "portion" of a polynucleotide
is intended a polynucleotide (either DNA or RNA) hybridizing to at least about
nucleotides (nt), and more preferably at least about 20 nt, still more
preferably
at least about 30 nt, and even more preferably about 30-70 or 80-150 nt, or
the
10 entire length of the reference polynucleotide. By a portion of a
polynucleotide
of "at least about 20 nt in length," for example, is intended 20 or more
contiguous nucleotides from the nucleotide sequence of the reference
polynucleotide (e.g., the deposited cDNA or the nucleotide sequence as shown
in SEQ ID NO:1 ). In this context "about" includes the particularly recited
size,
15 larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either
terminus or at
both termini. These have uses, which include, but are not limited to, as
diagnostic probes and primers as discussed above and in more detail below.
Of course, a polynucleotide which hybridizes only to a poly A sequence
(such as the 3' terminal poly(A) tract of the DRS cDNA shown in FIG. 1 (SEQ
ID NO:1)), or to a complementary stretch of T (or U) resides, would not be
included in a polynucleotide of the invention used to hybridize to a portion
of a
nucleic acid ofthe invention, since such a polynucleotide would hybridize to
any
nucleic acid molecule containing a poly (A) stretch or the complement thereof
(e.g., practically any double-stranded cDNA generated from an oligo-dT primed
cDNA library).
As indicated, nucleic acid molecules of the present invention which
encode a DRS polypeptide may include, but are not limited to, the coding
sequence for the mature polypeptide, by itself; the coding sequence for the
mature polypeptide and additional sequences, such as those encoding a leader
or
secretory sequence, such as a pre-, pro- or prepro- protein sequence; the
coding
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sequence of the mature polypeptide, with or without the aforementioned
additional coding sequences, together with additional, non-coding sequences,
including for example, but not limited to introns and non-coding S' and 3'
sequences, such as the transcribed, non-translated sequences that play a role
in
transcription, mRNA processing - including splicing and polyadenylation
signals,
for example - ribosome binding and stability of mRNA; additional coding
sequence which codes for additional amino acids, such as those which provide
additional functionalities. Thus, for instance, the polypeptide may be fused
to a
marker sequence, such as a peptide, which facilitates purification of the
fused
polypeptide. In certain preferred embodiments ofthis aspect ofthe invention,
the
marker sequence is a hexa-histidine peptide, such as the tag provided in a pQE
vector (Qiagen, Inc.), among others, many ofwhich are commercially available.
As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989),
for
instance, hexa-histidine provides for convenient purification ofthe fusion
protein.
The "HA" tag is another peptide useful for purification which corresponds to
an
epitope derived from the influenza hemagglutinin protein, which has been
described by Wilson et al., Cell 37:767 -778(1984). As discussed below, other
such fusion proteins include the DRS receptor fused to Fc at the N- or C-
terminus.
The present invention further relates to variants of the nucleic acid
molecules of the present invention, which encode portions, analogs, or
derivatives of the DRS receptor. Variants may occur naturally, such as a
natural
allelic variant. By an "allelic variant" is intended one of several alternate
forms
of a gene occupying a given locus on a chromosome of an organism. Genes II,
Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring
variants may be produced using art-known mutagenesis techniques.
Such variants include those produced by nucleotide substitutions,
deletions or additions which may involve one or more nucleotides. The variants
may be altered in coding or non-coding regions or both. Alterations in the
coding regions may produce conservative or non-conservative amino acid
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substitutions, deletions or additions. Especially preferred among these are
silent
substitutions, additions, and deletions, which do not alter the properties and
activities of the DR5 receptor or portions thereof. Also especially preferred
in
this regard are conservative substitutions.
Further embodiments of the invention include isolated nucleic acid
molecules that are at least 80% identical, and more preferably at least 85%,
90%,
92%, 95%, 96%, 97%, 98% or 99% identical, to (a) a nucleotide sequence
encoding the polypeptide comprising, or alternatively consisting of, the amino
acid sequence in SEQ ID N0:2; (b) a nucleotide sequence encoding the
polypeptide comprising, or alternatively consisting of, the amino acid
sequence
in SEQ ID N0:2, but lacking the amino terminal methionine; (c) a nucleotide
sequence encoding the polypeptide comprising, or alternatively consisting of,
the
amino acid sequence at positions from about 1 to about 360 in SEQ ID N0:2;
(d) a nucleotide sequence encoding the polypeptide comprising, or
alternatively
consisting of, the amino acid sequence encoded by the cDNA contained in ATCC
Deposit No. 97920; (e) a nucleotide sequence encoding the mature DR5
polypeptide comprising, or alternatively consisting of, the amino acid
sequence
encoded by the cDNA contained in ATCC Deposit No. 97920; (f) a nucleotide
sequence that encodes the DR5 extracellular domain comprising, or
alternatively
consisting of, the amino acid sequence at positions from about 1 to about 133
in
SEQ ID N0:2, or the DR5 extracellular domain encoded by the cDNA contained
in ATCC Deposit No. 97920; (g) a nucleotide sequence that encodes the DR5
cysteine rich domain comprising, or alternatively consisting of, the amino
acid
sequence at positions from about 33 to about 128 in SEQ 117 N0:2, or the DR5
cysteine rich domain encoded by the cDNA contained in ATCC Deposit No.
97920; (h) a nucleotide sequence that encodes the DR5 transmembrane domain
comprising, or alternatively consisting of, the amino acid sequence at
positions
from about 134 to about 157 of SEQ ID N0:2, or the DR5 transmembrane
domain encoded by the cDNA contained in ATCC Deposit No. 97920; (i) a
nucleotide sequence that encodes the DR5 intracellular domain comprising, or
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alternatively consisting of, the amino acid sequence at positions from about
158
to about 360 of SEQ ID N0:2, or the DRS intracellular domain encoded by the
cDNA contained in ATCC Deposit No. 97920; (j) a nucleotide sequence that
encodes the DRS receptor extracellular and intracellular domains with all or
part
of the transmembrane domain deleted; (k) a nucleotide sequence that encodes
the DRS death domain comprising, or alternatively consisting of, the amino
acid
sequence at positions from about 273 to about 340 of SEQ ID N0:2, or the DRS
death domain encoded by the cDNA contained in ATCC Deposit No. 97920; (1)
a nucleotide sequence that encodes a fragment of the polypeptide of (c) having
DRS functional activity (e.g., antigenic or biological activity); and (m) a
nucleotide sequence complementary to any of the nucleotide sequences in (a),
(b), (c), (d), (e), (f), (g), (h), (i), (j), (k), or (I) above. Polypeptides
encoded by
these polynucleotides are also encompassed by the invention.
By a polynucleotide having a nucleotide sequence at least, for example,
95% "identical" to a reference nucleotide sequence encoding a DRS polypeptide
is intended that the nucleotide sequence of the polynucleotide is identical to
the
reference sequence except that the polynucleotide sequence may include up to
five mismatches per each 100 nucleotides of the reference nucleotide sequence
encoding the DRS polypeptide. In other words, to obtain a polynucleotide
having a nucleotide sequence at least 95% identical to a reference nucleotide
sequence, up to 5% of the nucleotides in the reference sequence may be deleted
or substituted with another nucleotide, or a number of nucleotides up to 5% of
the total nucleotides in the reference sequence may be inserted into the
reference
sequence. The reference (query) sequence may be the entire DRS nucleotide
sequence shown in FIG. 1 (SEQ ID NO:1) or any polynucleotide fragment (e.g.,
a polynucleotide encoding the amino acid sequence of a DRS N and/or C
terminal deletion described herein) as described herein.
As a practical matter, whether any particular nucleic acid molecule is at
least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for
instance, the nucleotide sequence shown in SEQ ID NO:1 or to the nucleotide
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sequence of the deposited cDNA can be determined conventionally using known
computer programs such as the Bestfit program (Wisconsin Sequence Analysis
Package, Version 8 for Unix, Genetics Computer Group, University Research
Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology
algorithm of Smith and Waterman, Advances in AppliedMathematics 2:482-489
( 1981 ), to find the best segment of homology between two sequences. When
using Bestfit or any other sequence alignment program to determine whether a
particular sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of course, such
that
the percentage of identity is calculated over the full length of the reference
nucleotide sequence and that gaps in homology of up to 5% of the total number
of nucleotides in the reference sequence are allowed.
In a specific embodiment, the identity between a reference (query)
sequence (a sequence of the present invention) and a subject sequence, also
referred to as a global sequence alignment, is determined using the FASTDB
computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci.
6:237-245 (1990)). Preferred parameters used in a FASTDB alignment ofDNA
sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4,
Mismatch
Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff
Score=1, Gap Penalty=S, Gap Size Penalty 0.05, Window Size=500 or the length
of the subject nucleotide sequence, whichever is shorter. According to this
embodiment, if the subject sequence is shorter than the query sequence because
of 5' or 3' deletions, not because of internal deletions, a manual correction
is
made to the results to take into consideration the fact that the FASTDB
program
does not account for 5' and 3' truncations of the subject sequence when
calculating percent identity. For subject sequences truncated at the 5' or 3'
ends,
relative to the query sequence, the percent identity is corrected by
calculating the
number of bases of the query sequence that are 5' and 3' of the subject
sequence,
which are not matched/aligned, as a percent of the total bases of the query
sequence. A determination of whether a nucleotide is matched/aligned is
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determined by results of the FASTDB sequence alignment. This percentage is
then subtracted from the percent identity, calculated by the above FASTDB
program using the specified parameters, to arrive at a final percent identity
score.
This corrected score is what is used for the purposes of this embodiment. Only
bases outside the 5' and 3' bases of the subject sequence, as displayed by the
FASTDB alignment, which are not matched/aligned with the query sequence, are
calculated for the purposes of manually adjusting the percent identity score.
For
example, a 90 base subject sequence is aligned to a 100 base query sequence to
determine percent identity. The deletions occur at the 5' end of the subject
sequence and therefore, the FASTDB alignment does not show a
matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases
represent 10% of the sequence (number of bases at the 5' and 3' ends not
matched/total number of bases in the query sequence) so 10% is subtracted from
the percent identity score calculated by the FASTDB program. If the remaining
90 bases were perfectly matched the final percent identity would be 90%. In
another example, a 90 base subject sequence is compared with a 100 base query
sequence. This time the deletions are internal deletions so that there are no
bases
on the 5' or 3' of the subject sequence which are not matched/aligned with the
query. In this case the percent identity calculated by FASTDB is not manually
corrected. Once again, only bases 5' and 3' ofthe subject sequence which are
not
matched/aligned with the query sequence are manually corrected for. No other
manual corrections are made for the purposes of this embodiment.
The present application is directed to nucleic acid molecules at least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid
sequence shown in SEQ ID NO:1, the nucleic acid sequence of the deposited
cDNAs, or fragments thereof, irrespective of whether they encode a polypeptide
having DRS functional activity. This is because even where a particular
nucleic
acid molecule does not encode a polypeptide having DRS functional activity,
one
of skill in the art would still know how to use the nucleic acid molecule, for
instance, as a hybridization probe or a polymerase chain reaction (PCR)
primer.
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Uses of the nucleic acid molecules of the present invention that do not encode
a polypeptide having DR5 functional activity include, inter alias ( 1 )
isolating the
DR5 gene or allelic variants thereof in a cDNA library; (2) in situ
hybridization
(e.g., "FISH") to metaphase chromosomal spreads to provide precise
chromosomal location of the DR5 gene, as described in Verma et al., Human
Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York
(1988); and (3) Northern Blot analysis for detecting DR5 mRNA expression in
specific tissues.
Preferred, however, are nucleic acid molecules having sequences at least
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic
acid sequence shown in SEQ ID NO:l, the nucleic acid sequence of the
deposited cDNAs, or fragments thereof, which do, in fact, encode a polypeptide
having DR5 protein functional activity. By "a polypeptide having DR5
functional
activity" is intended polypeptides exhibiting activity similar, but not
necessarily
identical, to a functional activity of the DR5 protein of the invention
(either the
full-length (i.e., complete) protein or, preferably, the mature protein), as
measured in a particular biological assay. For example, DR5 polypeptide
functional activity can be measured by the ability of a polypeptide sequence
described herein to form multimers (e.g., homodimers and homotrimers) with
complete DRS, and to bind a DR5 ligand (e.g., TRAIL,). DR5 polypeptide
functional activity can be also be measured, for example, by determining the
ability of a polypeptide of the invention to induce apoptosis in cells
expressing
the polypeptide. These functional assays can be routinely performed using
techniques described herein and otherwise known in the art.
For example, DR5 protein functional activity (e.g., biological activity) can
be measured using the cell death assays performed essentially as previously
described (A.M. Chinnaiyan, et al., Cell 81:505-12 (1995); M.P. Boldin, et
al.,
JBiol Chem 270:7795-8 (1995); F.C. Kischkel, et al., EMBO 14:5579-5588
(1995); A.M. Chinnaiyan, et al., JBiol Chem 271:4961-4965 (1996)) and as set
forth in Example 5, below. In MCF7 cells, plasmids encoding full-length DRS
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or a candidate death domain containing receptor are co-transfected with the
pLantern reporter construct encoding green fluorescent protein. Nuclei of
cells
transfected with DRS will exhibit apoptotic morphology as assessed by DAPI
staining. Similar to TNFR-1 and Fas/APO-1 (M. Muzio, et al., Cel185:817-827
(1996); M. P. Boldin, et al., Cell 85:803-815 (1996); M. Tewari, et al., JBiol
Chem 270:3255-60 (1995)), DRS-induced apoptosis is preferably blocked by the
inhibitors of ICE-like proteases, CrmA and z-VAD-fmk.
Of course, due to the degeneracy of the genetic code, one of ordinary
skill in the art will immediately recognize that a large number of the nucleic
acid
molecules having a sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%,
98% or 99% identical to for example, the nucleic acid sequence of the
deposited
cDNA, the nucleic acid sequence shown in SEQ ID NO: l, or fragments thereof,
will encode a polypeptide "having DRS protein functional activity." In fact,
since
degenerate variants of these nucleotide sequences all encode the same
polypeptide, in many instances, this will be clear to the skilled artisan even
without performing the above described comparison assay. It will be further
recognized in the art that, for such nucleic acid molecules that are not
degenerate
variants, a reasonable number will also encode a polypeptide having DRS
protein
functional activity. This is because the skilled artisan is fully aware of
amino acid
substitutions that are either less likely or not likely to significantly
effect protein
function (e.g., replacing one aliphatic amino acid with a second aliphatic
amino
acid), as further described below.
For example, guidance concerning how to make phenotypically silent
amino acid substitutions is provided in Bowie, J.U. et al., "Deciphering the
Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science
247:1306-1310 (1990), wherein the authors indicate that proteins are
surprisingly tolerant of amino acid substitutions.
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Polynucleotide Assays
This invention is also related to the use of the DRS polynucleotides to
detect complementary polynucleotides such as, for example, as a diagnostic
reagent. Detection of a mutated form of DRS associated with a dysfunction will
provide a diagnostic tool that can add or define a diagnosis of a disease or
susceptibility to a disease which results from under-expression, over-
expression,
or altered expression of DRS or a soluble form thereof, such as, for example,
tumors or autoimmune disease.
Individuals carrying mutations in the DRS gene may be detected at the
DNA level by a variety of techniques. Nucleic acids for diagnosis may be
obtained from a patient's cells, such as from blood, urine, saliva, tissue
biopsy
and autopsy material. The genomic DNA may be used directly for detection or
may be amplified enzymatically by using PCR prior to analysis. (Saiki et al.,
Nature 324:163-166 (1986)). RNA or cDNA may also be used in the same
ways. As an example, PCR primers complementary to the nucleic acid encoding
DRS can be used to identify and analyze DRS expression and mutations. For
example, deletions and insertions can be detected by a change in size of the
amplified product in comparison to the normal genotype. Point mutations can
be identified by hybridizing amplified DNA to radiolabeled DRS RNA or
alternatively, radiolabeled DRS antisense DNA sequences. Perfectly matched
sequences can be distinguished from mismatched duplexes by RNase A digestion
or by differences in melting temperatures.
Sequence differences between a reference gene and genes having
mutations also may be revealed by direct DNA sequencing. In addition, cloned
DNA segments may be employed as probes to detect specific DNA segments.
The sensitivity of such methods can be greatly enhanced by appropriate use of
PCR or another amplification method. For example, a sequencing primer is used
with double-stranded PCR product or a single-stranded template molecule
generated by a modified PCR. The sequence determination is performed by
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conventional procedures with radiolabeled nucleotide orby automatic sequencing
procedures with fluorescent-tags.
Genetic testing based on DNA sequence differences may be achieved by
detection of alteration in electrophoretic mobility ofDNA fragments in gels,
with
or without denaturing agents. Small sequence deletions and insertions can be
visualized by high resolution gel electrophoresis. DNA fragments of different
sequences may be distinguished on denaturing formamide gradient gels in which
the mobilities of different DNA fragments are retarded in the gel at different
positions according to their specific melting or partial melting temperatures
(see,
e.g., Myers et al., Science 230:1242 (1985)).
Sequence changes at specific locations also may be revealed by nuclease
protection assays, such as RNase and Sl protection or the chemical cleavage
method (e.g., Cotton et al., Proc. Natl Acad. Sci. USA 85: 4397-4401 (1985)).
Thus, the detection of a specific DNA sequence may be achieved by
methods which include, but are not limited to, hybridization, RNase
protection,
chemical cleavage, direct DNA sequencing or the use of restriction enzymes,
(e.g., restriction fragment length polymorphisms ("RFLP") and Southern
blotting
of genomic DNA).
In addition to more conventional gel-electrophoresis and DNA
sequencing, mutations also can be detected by in situ analysis.
Vectors and Host Cells
The present invention also relates to vectors which include DNA
molecules of the present invention, host cells which are genetically
engineered
with vectors ofthe invention and the production ofpolypeptides ofthe invention
by recombinant techniques.
Host cells can be genetically engineered to incorporate nucleic acid
molecules and express polypeptides of the present invention. The
polynucleotides may be introduced alone or with other polynucleotides. Such
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other polynucleotides may be introduced independently, co-introduced or
introduced joined to the polynucleotides of the invention.
In accordance with this aspect of the invention the vector may be, for
example, a plasmid vector, a single or double-stranded phage vector, a single
or
double-stranded RNA or DNA viral vector. Such vectors may be introduced into
cells as polynucleotides, preferably DNA, by well known techniques for
introducing DNA and RNA into cells. Viral vectors may be replication
competent or replication defective. In the latter case viral propagation
generally
will occur only in complementing host cells.
Preferred among vectors, in certain respects, are those for expression of
polynucleotides and polypeptides of the present invention. Generally, such
vectors comprise ci.s-acting control regions effective for expression in a
host
operatively linked to the polynucleotide to be expressed. Appropriate trans-
acting factors either are supplied by the host, supplied by a complementing
vector
or supplied by the vector itself upon introduction into the host.
A great variety of expression vectors can be used to express a
polypeptide of the invention. Such vectors include chromosomal, episomal and
virus-derived vectors e.g., vectors derived from bacterial plasmids, from
bacteriophage, from yeast episomes, from yeast chromosomal elements, from
viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses,
adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and
vectors derived from combinations thereof, such as those derived from plasmid
and bacteriophage genetic elements, such as cosmids and phagemids, all may be
used for expression in accordance with this aspect of the present invention.
Generally, any vector suitable to maintain, propagate or express
polynucleotides
to express a polypeptide in a host may be used for expression in this regard.
The DNA sequence in the expression vector is operatively linked to
appropriate expression control sequence(s)), including, for instance, a
promoter
to direct mRNA transcription. Representatives of such promoters include the
phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40
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early and late promoters and promoters of retroviral LTRs, to name just a few
ofthe well-known promoters. In general, expression constructs will contain
sites
for transcription, initiation and termination, and, in the transcribed region,
a
ribosome binding site for translation. The coding portion of the mature
transcripts expressed by the constructs will include a translation initiating
AUG
at the beginning and a termination codon (UAA, UGA or UAG) appropriately
positioned at the end of the polypeptide to be translated.
In addition, the constructs may contain control regions that regulate as
well as engender expression. Generally, such regions will operate by
controlling
transcription, such as repressor binding sites and enhancers, among others.
Vectors for propagation and expression generally will include selectable
markers. Such markers also may be suitable for amplification or the vectors
may
contain additional markers for this purpose. In this regard, the expression
vectors preferably contain one or more selectable marker genes to provide a
phenotypic trait for selection of transformed host cells. Such markers
include,
but are not limited to, dihydrofolate reductase or neomycin resistance for
eukaryotic cell culture, and tetracycline or ampicillin resistance genes for
culturing E. coli and other bacteria.
The vector containing the appropriate DNA sequence as described
elsewhere herein, as well as an appropriate promoter, and other appropriate
control sequences, may be introduced into an appropriate host using a variety
of
well known techniques suitable to expression therein of a desired polypeptide.
Representative examples of appropriate hosts include bacterial cells, such as
E
coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as
yeast
cells; insect cells such as Drosophila S2 and Spodoptera S~ cells; animal
cells
such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate
culture mediums and conditions for the above-described host cells are known in
the art.
Among vectors preferred for use in bacteria are pQE70, pQE60 and
pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript
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vectors, pNH8A, pNHl6a, pNHl8A, pNH46A, available from Stratagene; and
ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS available from Pharmacia.
Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI
and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL
available from Pharmacia. These vectors are listed solely by way of
illustration
ofthe many commercially available and well known vectors available to those of
skill in the art.
Selection of appropriate vectors and promoters for expression in a host
cell is a well known procedure and the requisite techniques for expression
vector
construction, introduction of the vector into the host and expression in the
host
are routine skills in the art.
The present invention also relates to host cells containing the above-
described vector constructs described herein, and additionally encompasses
host
cells containing nucleotide sequences of the invention that are operably
associated with one or more heterologous control regions (e.g., promoter
and/or
enhancer) using techniques known of in the art. The host cell can be a higher
eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a
lower
eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic
cell,
such as a bacterial cell. The host strain may be chosen which modulates the
expression of the inserted gene sequences, or modifies and processes the gene
product in the specific fashion desired. Expression from certain promoters can
be elevated in the presence of certain inducers; thus expression of the
genetically
engineered polypeptide may be controlled. Furthermore, difFerent host cells
have
characteristics and specific mechanisms for the translational and post-
translational processing and modification (e.g., phosphorylation, cleavage) of
proteins. Appropriate cell lines can be chosen to ensure the desired
modifications
and processing of the foreign protein expressed.
Introduction ofthe construct into the host cell can be effected by calcium
phosphate transfection, DEAF-dextran mediated transfection, cationic lipid-
3 0 mediated transfection, electroporation, transduction, infection or other
methods.
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Such methods are described in many standard laboratory manuals, such as Davis
et al., Basic Melhods in Molecular Biology (1986).
In addition to encompassing host cells containing the vector constructs
discussed herein, the invention also encompasses primary, secondary, and
immortalized host cells of vertebrate origin, particularly mammalian origin,
that
have been engineered to delete or replace endogenous genetic material (e.g.,
DRS coding sequence), and/or to include genetic material (e.g., heterologous
polynucleotide sequences) that is operably associated with DRS polynucleotides
of the invention, and which activates, alters, and/or amplifies endogenous DRS
polynucleotides. For example, techniques known in the art may be used to
operably associate heterologous control regions (e.g., promoter and/or
enhancer)
and endogenous DRS polynucleotide sequences via homologous recombination
(see, e.g., US Patent Number 5,641,670, issued June 24, 1997; International
Publication Number WO 96/29411, published September 26, 1996; International
Publication Number WO 94/12650, published August 4, 1994; Koller et al.,
Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature
342:435-438 (1989), the disclosures of each of which are incorporated by
reference in their entireties).
The polypeptide may be expressed in a modified form, such as a fusion
protein (comprising the polypeptide joined via a peptide bond to a
heterologous
protein sequence (of a different protein)), and may include not only secretion
signals but also additional heterologous functional regions. Such a fusion
protein
can be made by ligating polynucleotides of the invention and the desired
nucleic
acid sequence encoding the desired amino acid sequence to each other, by
methods known in the art, in the proper reading frame, and expressing the
fusion
protein product by methods known in the art. Alternatively, such a fusion
protein
can be made by protein synthetic techniques, e.g., by use of a peptide
synthesizer.
Thus, for instance, a region of additional amino acids, particularly charged
amino
acids, may be added to the N-terminus ofthe polypeptide to improve stability
and
persistence in the host cell, during purification or during subsequent
handling and
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storage. Also, region also may be added to the polypeptide to facilitate
purification. Such regions may be removed prior to final preparation of the
polypeptide. For example, in one embodiment, polynucleotides encoding DRS
polypeptides of the invention may be fused to the pelB pectate lyase signal
sequence to increase the efficiency to expression and purification of such
polypeptides in Gram-negative bacteria. See, US Patent Nos. 5,576,195 and
5,846,818, the contents of which are herein incorporated by reference in their
entireties.
Alternatively, such a fusion protein can be made by protein synthetic
techniques, e.g., by use of a peptide synthesizer. Thus, for instance, a
region of
additional amino acids, particularly charged amino acids, may be added to the
N-
terminus ofthe polypeptide to improve stability and persistence in the host
cell,
during purification or during subsequent handling and storage. Additionally, a
region also may be added to the polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the polypeptide. The
addition of peptide moieties to polypeptides to engender secretion or
excretion,
to improve stability and to facilitate purification, among others, are
familiar and
routine techniques in the art. A preferred fusion protein comprises a
heterologous region from immunoglobulin that is useful to solubilize proteins.
For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion
proteins comprising various portions of constant region of immunoglobin
molecules together with another human protein or part thereof. In many cases,
the Fc part in a fusion protein is thoroughly advantageous for use in therapy
and
diagnosis and thus results, for example, in improved pharmacokinetic
properties
(EP-A 0232 262). On the other hand, for some uses it would be desirable to be
able to delete the Fc part after the fusion protein has been expressed,
detected
and purified in the advantageous manner described. This is the case when the
Fc
portion proves to be a hindrance to use in therapy and diagnosis, for example
when the fusion protein is to be used as an antigen for immunizations. In drug
discovery, for example, human proteins, such as the hIL-5-receptor, have been
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fused with Fc portions for the purpose of high-throughput screening assays to
identify antagonists of hIL-5. See, D. Bennett et al., Journal of Molecular
Recognition, 8:52-58 (1995) and K. Johanson et al., The Journal of Biological
Chemistry, 270:9459-9471 (1995).
Polypeptides of the present invention include naturally purified products,
products of chemical synthetic procedures, and products produced by
recombinant techniques from a prokaryotic or eukaryotic host, including, for
example, bacterial, yeast, higher plant, insect and mammalian cells. Depending
upon the host employed in a recombinant production procedure, the polypeptides
of the present invention may be glycosylated or may be non-glycosylated. In
addition, polypeptides of the invention may also include an initial modified
methionine residue, in some cases as a result of host-mediated processes.
Transgenics and "Knock-outs"
The DRS polypeptides of the invention can also be expressed in
transgenic animals. Animals of any species, including, but not limited to,
mice,
rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and
non-
human primates, e.g., baboons, monkeys, and chimpanzees may be used to
generate transgenic animals. In a specific embodiment, techniques described
herein or otherwise known in the art, are used to express polypeptides of the
invention in humans, as part of a gene therapy protocol.
Any technique known in the art may be used to introduce the transgene
(i.e., nucleic acids of the invention) into animals to produce the founder
lines of
transgenic animals. Such techniques include, but are not limited to,
pronuclear
microinjection (Patersonetal., Appl. Microbiol. Biotechnol. 40:691-698 (1994);
Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al.,
Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., US Patent Number
4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der
Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts
or
embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-
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321 (1989)); electroporation of cells or embryos (Lo, Mol Cell. Biol. 3:1803-
1814 (1983)); introduction of the polynucleotides of the invention using a
gene
gun (see, e.g., Ulmer et al.., Science 259:1745 (1993); introducing nucleic
acid
constructs into embryonic pleuripotent stem cells and transferring the stem
cells
back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al.,
Cell
57:717-723 (1989); etc. For a review of such techniques, see Cordon,
"Transgenic Animals," Intl. Rev. Cytol. 115:171-229 (1989), which is
incorporated by reference herein in its entirety. See also, U.S. Patent No.
5,464,764 (Capecchi, et al., Positive-Negative Selection Methods and Vectors);
U. S. Patent No. 5,631,153 (Capecchi, et al., Cells and Non-Human Organisms
Containing Predetermined Genomic Modifications and Positive-Negative
Selection Methods and Vectors for Making Same); U.S. Patent No. 4,736,866
(Leder, et al.., Transgenic Non-Human Animals); and U. S. Patent No. 4,
873,191
(Wagner, et al., Genetic Transformation of Zygotes); each of which is hereby
incorporated by reference in its entirety. Further, the contents of each of
the
documents recited in this paragraph is herein incorporated by reference in its
entirety.
Any technique known in the art may be used to produce transgenic clones
containing polynucleotides of the invention, for example, nuclear transfer
into
enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells
induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al.,
Nature 385:810-813 (1997)), each ofwhich is herein incorporated by reference
in its entirety).
The present invention provides for transgenic animals that carry the
transgene in all their cells, as well as animals which carry the transgene in
some,
but not all their cells, i.e., mosaic animals or chimeric animals. The
transgene
may be integrated as a single transgene or as multiple copies such as in
concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene
may also be selectively introduced into and activated in a particular cell
type by
following, for example, the teaching of Lasko et al. (Proc. Natl. Acad. Sci.
USA
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89:6232-6236 (1992)). The regulatory sequences required for such a cell-type
specific activation will depend upon the particular cell type of interest, and
will
be apparent to those of skill in the art. When it is desired that the
polynucleotide
transgene be integrated into the chromosomal site of the endogenous gene, gene
targeting is preferred. Briefly, when such a technique is to be utilized,
vectors
containing some nucleotide sequences homologous to the endogenous gene are
designed for the purpose of integrating, via homologous recombination with
chromosomal sequences, into and disrupting the function of the nucleotide
sequence of the endogenous gene. The transgene may also be selectively
introduced into a particular cell type, thus inactivating the endogenous gene
in
only that cell type, by following, for example, the teaching of Gu et al.
(Science
265:103-106 (1994)). The regulatory sequences required for such a cell-type
specific inactivation will depend upon the particular cell type of interest,
and will
be apparent to those of skill in the art. The contents of each of the
documents
1 S recited in this paragraph is herein incorporated by reference in its
entirety.
Once transgenic animals have been generated, the expression of the
recombinant gene may be assayed utilizing standard techniques. Initial
screening
may be accomplished by Southern blot analysis or PCR techniques to analyze
animal tissues to verify that integration of the transgene has taken place.
The
level of mRNA expression of the transgene in the tissues of the transgenic
animals may also be assessed using techniques which include, but are not
limited
to, Northern blot analysis of tissue samples obtained from the animal, in situ
hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of
transgenic gene-expressing tissue may also be evaluated immunocytochemically
or immunohistochemically using antibodies specific for the transgene product.
Once the founder animals are produced, they may be bred, inbred,
outbred, or crossbred to produce colonies of the particular animal. Examples
of
such breeding strategies include, but are not limited to: outbreeding of
founder
animals with more than one integration site in order to establish separate
lines;
inbreeding of separate lines in order to produce compound transgenics that
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express the transgene at higher levels because of the effects of additive
expression of each transgene; crossing of heterozygous transgenic animals to
produce animals homozygous for a given integration site in order to both
augment expression and eliminate the need for screening of animals by DNA
analysis; crossing of separate homozygous lines to produce compound
heterozygous or homozygous lines; and breeding to place the transgene on a
distinct background that is appropriate for an experimental model of interest.
Transgenic and "knock-out" animals of the invention have uses which
include, but are not limited to, animal model systems useful in elaborating
the
biological function of DRS polypeptides, studying conditions and/or disorders
associated with aberrant DRS expression, and in screening for compounds
effective in ameliorating such conditions and/or disorders.
In further embodiments of the invention, cells that are genetically
engineered to express the proteins of the invention, or alternatively, that
are
genetically engineered not to express the proteins of the invention (e.g.,
knockouts) are administered to a patient in vivo. Such cells may be obtained
from the patient (i.e., animal, including human) or an MHC compatible donor
and can include, but are not limited to fibroblasts, bone marrow cells, blood
cells
(e.g., lymphocytes), adipocytes, muscle cells, endothelial cells, etc. The
cells are
genetically engineered in vitro using recombinant DNA techniques to introduce
the coding sequence of polypeptides of the invention into the cells, or
alternatively, to disrupt the coding sequence and/or endogenous regulatory
sequence associated with the polypeptides of the invention, e.g., by
transduction
(using viral vectors, and preferably vectors that integrate the transgene into
the
cell genome) or transfection procedures, including, but not limited to, the
use of
plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The
coding sequence of the polypeptides of the invention can be placed under the
control of a strong constitutive or inducible promoter or promoter/enhancer to
achieve expression, and preferably secretion, ofthe polypeptides ofthe
invention.
The engineered cells which express and preferably secrete the polypeptides of
the
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invention can be introduced into the patient systemically, e.g., in the
circulation,
or intraperitoneally. Alternatively, the cells can be incorporated into a
matrix and
implanted in the body, e.g., genetically engineered fibroblasts can be
implanted
as part of a skin graft; genetically engineered endothelial cells can be
implanted
as part of a lymphatic or vascular graft. (See, for example, Anderson et al.
US
Patent Number 5,399,349; and Mulligan & Wilson, US Patent Number
5,460,959, each of which is incorporated by reference herein in its entirety).
When the cells to be administered are non-autologous or non-MHC
compatible cells, they can be administered using well known techniques which
prevent the development of a host immune response against the introduced
cells.
For example, the cells may be introduced in an encapsulated form which, while
allowing for an exchange of components with the immediate extracellular
environment, does not allow the introduced cells to be recognized by the host
immune system.
DRS Proteins and Fragments
The invention further provides for the proteins containing polypeptide
sequences encoded by the polynucleotides of the invention.
The DRS proteins of the invention may be in monomers or multimers
(i. e. , dimers, trimers, tetramers, and higher multimers). Accordingly, the
present
invention relates to monomers and multimers of the DRS proteins of the
invention, their preparation, and compositions (preferably, pharmaceutical
compositions) containing them. In specific embodiments, the polypeptides of
the
invention are monomers, dimers, trimers or tetramers. In additional
embodiments, the multimers of the invention are at least dimers, at least
trimers,
or at least tetramers.
Multimers encompassed by the invention may be homomers or
heteromers. As used herein, the term homomer, refers to a multimer containing
only DRS proteins of the invention (including DRS fragments, variants, and
fusion proteins, as described herein). These homomers may contain DRS
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proteins having identical or different polypeptide sequences. In a specific
embodiment, a homomer of the invention is a multimer containing only DRS
proteins having an identical polypeptide sequence. In another specific
embodiment, a homomer of the invention is a multimer containing DRS proteins
having different polypeptide sequences. In specific embodiments, the multimer
of the invention is a homodimer (e.g., containing DRS proteins having
identical
or different polypeptide sequences) or a homotrimer (e.g., containing DRS
proteins having identical or different polypeptide sequences). In additional
embodiments, the homomeric multimer of the invention is at least a homodimer,
at least a homotrimer, or at least a homotetramer.
As used herein, the term heteromer refers to a multimer containing
heterologous proteins (i. e., proteins containing only polypeptide sequences
that
do not correspond to a polypeptide sequences encoded by the DRS gene) in
addition to the DRS proteins of the invention. In a specific embodiment, the
multimer of the invention is a heterodimer, a heterotrimer, or a
heterotetramer.
In additional embodiments, the heteromeric multimer of the invention is at
least
a heterodimer, at least a heterotrimer, or at least a heterotetramer.
Multimers of the invention may be the result of hydrophobic, hydrophilic,
ionic and/or covalent associations and/or may be indirectly linked, by for
example, liposome formation. Thus, in one embodiment, multimers of the
invention, such as, for example, homodimers or homotrimers, are formed when
proteins of the invention contact one another in solution. In another
embodiment, heteromultimers of the invention, such as, for example,
heterotrimers or heterotetramers, are formed when proteins of the invention
contact antibodies to the polypeptides of the invention (including antibodies
to
the heterologous polypeptide sequence in a fusion protein of the invention) in
solution. In other embodiments, multimers of the invention are formed by
covalent associations with and/or between the DRS proteins of the invention.
Such covalent associations may involve one or more amino acid residues
contained in the polypeptide sequence of the protein (e.g., the polypeptide
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sequence recited in SEQ ID N0:2 or the polypeptide encoded by the deposited
cDNA). In one instance, the covalent associations are cross-linking between
cysteine residues located within the polypeptide sequences of the proteins
which
interact in the native (i.e., naturally occurring) polypeptide. In another
instance,
the covalent associations are the consequence of chemical or recombinant
manipulation. Alternatively, such covalent associations may involve one or
more
amino acid residues contained in the heterologous polypeptide sequence in a
DRS fusion protein. In one example, covalent associations are between the
heterologous sequence contained in a fusion protein of the invention (see,
e.g.,
US Patent Number 5,478,925). In a specific example, the covalent associations
are between the heterologous sequence contained in a DRS-Fc fusion protein of
the invention (as described herein). In another specific example, covalent
associations of fusion proteins of the invention are between heterologous
polypeptide sequences from another TNF family ligand/receptor member that is
capable of forming covalently associated multimers, such as for example,
oseteoprotegerin (see, e.g., International Publication No. WO 98/49305, the
contents of which are herein incorporated by reference in its entirety). In
another
embodiment, two or more DRS polypeptides of the invention are joined through
synthetic linkers (e.g., peptide, carbohydrate or soluble polymer linkers).
Examples include, but are not limited to, those peptide linkers described in
U. S.
Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising
multiple DRS polypeptides separated by peptide linkers may be produced using
conventional recombinant DNA technology.
Another method for preparing multimer DRS polypeptides of the
invention involves use of DRS polypeptides fused to a leucine zipper or
isoleucine zipper polypeptide sequence. Leucine zipper domains and isoleucine
zipper domains are polypeptides that promote multimerization of the proteins
in
which they are found. Leucine zippers were originally identified in several
DNA-
binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since
been found in a variety of different proteins. Among the known leucine zippers
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are naturally occurring peptides and derivatives thereof that dimerize or
trimerize. Examples of leucine zipper domains suitable for producing soluble
multimeric DRS proteins are those described in PCT application WO 94/10308,
hereby incorporated by reference. Recombinant fusion proteins comprising a
soluble DRS polypeptide fused to a peptide that dimerizes or trimerizes in
solution are expressed in suitable host cells, and the resulting soluble
multimeric
DRS is recovered from the culture supernatant using techniques known in the
art.
Certain members of the TNF family of proteins are believed to exist in
trimeric form (Beutler and Huffel, Science 264:667, 1994; Banner et al., Cell
73:431 (1993)). Thus, trimeric DRS may offer the advantage of enhanced
biological activity. Preferred leucine zipper moieties are those that
preferentially
form trimers. One example is a leucine zipper derived from lung surfactant
protein D (SPD), as described in Hoppe et al. (TEBS Letters 344:191, (1994))
and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by
reference. Other peptides derived from naturally occurring trimeric proteins
may
be employed in preparing trimeric DRS.
In another example, proteins of the invention are associated by
interactions between Flag~ polypeptide sequence contained in Flag~-DRS or
Flag~-DRS fusion proteins of the invention. In a further embodiment,
associations proteins of the invention are associated by interactions between
heterologous polypeptide sequence contained in Flag~-DRS or Flag~-DRS
fusion proteins of the invention and anti-Flag~ antibody.
The multimers of the invention may be generated using chemical
techniques known in the art. For example, proteins desired to be contained in
the
multimers ofthe invention may be chemically cross-linked using linker
molecules
and linker molecule length optimization techniques known in the art (see,
e.g.,
US Patent Number 5,478,925, which is herein incorporated by reference in its
entirety). Additionally, multimers of the invention may be generated using
techniques known in the art to form one or more inter-molecule cross-links
between the cysteine residues located within the polypeptide sequence of the
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proteins desired to be contained in the multimer (see, e.g., US Patent Number
5,478,925, which is herein incorporated by reference in its entirety).
Further, proteins of the invention may be routinely modified by the
addition of cysteine or biotin to the C- terminus or N-terminus of the
polypeptide
sequence of the protein and techniques known in the art may be applied to
generate multimers containing one or more ofthese modified proteins (see,
e.g.,
US Patent Number 5,478,925, which is herein incorporated by reference in its
entirety). Additionally, techniques known in the art may be applied to
generate
liposomes containing the protein components desired to be contained in the
multimer of the invention (see, e.g., US Patent Number 5,478,925, which is
herein incorporated by reference in its entirety).
Alternatively, multimers of the invention may be generated using genetic
engineering techniques known in the art. In one embodiment, proteins contained
in multimers of the invention are produced recombinantly using fusion protein
technology described herein or otherwise known in the art (see, e.g., US
Patent
Number 5,478,925, which is herein incorporated by reference in its entirety).
In
a specific embodiment, polynucleotides coding for a homodimer of the invention
are generated by ligating a polynucleotide sequence encoding a polypeptide of
the invention to a sequence encoding a linker polypeptide and then further to
a
synthetic polynucleotide encoding the translated product ofthe polypeptide in
the
reverse orientation from the original C-terminus to the N-terminus (lacking
the
leader sequence) (see, e.g., US Patent Number 5,478,925, which is herein
incorporated by reference in its entirety). In another embodiment, recombinant
techniques described herein or otherwise known in the art are applied to
generate
recombinant polypeptides of the invention which contain a transmembrane
domain and which can be incorporated by membrane reconstitution techniques
into liposomes (see, e.g., US Patent Number 5,478,925, which is herein
incorporated by reference in its entirety).
The polypeptides of the present invention are preferably provided in an
isolated form. By "isolated polypeptide" is intended a polypeptide removed
from
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its native environment. Thus, a polypeptide produced and/or contained within
a recombinant host cell is considered isolated for purposes of the present
invention. Also intended as an "isolated polypeptide" are polypeptides that
have
been purified, partially or substantially, from a recombinant host cell. For
example, a recombinantly produced version of the DRS polypeptide can be
substantially purified by the one-step method described in Smith and Johnson,
Gene 67:31-40 (1988).
In one embodiment, the invention provides an isolated DRS polypeptide
having the amino acid sequence encoded by the deposited cDNA, or the amino
acid sequence in SEQ >D N0:2, or a polypeptide or peptide comprising, or
alternatively consisting of, a portion (i.e., fragment) of the above
polypeptides.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
Polypeptide fragments of the present invention include polypeptides
comprising, or alternatively consisting of, an amino acid sequence contained
in
SEQ ID N0:2, encoded by the cDNA contained in the deposited plasmid, or
encoded by nucleic acids which hybridize (e.g., under stringent hybridization
conditions) to the nucleotide sequence contained in the deposited plasmid, or
shown in FIG. 1 (SEQ ID NO:1) or the complementary strand thereto. Protein
fragments may be "free-standing," or comprised within a larger polypeptide of
which the fragment forms a part or region, most preferably as a single
continuous
region. Representative examples of polypeptide fragments of the invention,
include, for example, fragments that comprise, or alternatively consist of, a
member selected from the group consisting of from about amino acid residues
-51 to -1, 1 to 27, 28 to 40, 41 to 60, 61 to 83, 84 to 100, 101 to 127, 128
to
133, 134 to 157, 158 to 167, 168 to 180, 181 to 200, 201 to 220, 221 to 240,
241 to 260, 261 to 272, 273 to 310, 311 to 340, and 341 to 360 of SEQ >D
N0:2, as well as isolated polynucleotides which encode these polypeptides.
Additional representative examples of polypeptide fragments of the invention,
include, for example, fragments that comprise, or alternatively consist of, a
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member selected from the group consisting of from about amino acid residues
1-60, 11-70, 21-80, 31-90, 41-100, 51-110, 61-120, 71-130, 81-140, 91-150,
101-160, 111-170, 121-180, 131-190, 141-200, 151-210, 161-220, 171-230,
181-240, 191-250, 201-260, 211-270, 221-280, 231-290, 241-300, 251-310,
261-320, 271-330, 281-340, 291-350, and 301-360 of SEQ ID N0:2, as well as
isolated polynucleotides which encode these polypeptides.
Moreover, polypeptide fragments can be at least about 10, 20, 30, 40, 50,
60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this
context "about" includes the particularly recited value, larger or smaller by
several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
Preferred polypeptide fragments of the present invention include a
polypeptide comprising, or alternatively consisting of, one, two, three, four,
five
or more amino acid sequences selected from the group consisting o~ a
polypeptide comprising, or alternatively consisting of, the DRS receptor
extracellular domain (predicted to constitute amino acid residues from about 1
to about 133 in SEQ ID N0:2); a polypeptide comprising, or alternatively
consisting of, the DRS cysteine rich domain (predicted to constitute amino
acid
residues from about 33 to about 128 in SEQ ID N0:2); a polypeptide
comprising, or alternatively consisting of, the DRS receptor transmembrane
domain (predicted to constitute amino acid residues from about 134 to about
157
in SEQ ID N0:2); a polypeptide comprising, or alternatively consisting of,
fragment of the predicted mature DRS polypeptide, wherein the fragment has a
DRS functional activity (e.g., antigenic activity or biological activity); a
polypeptide comprising, or alternatively consisting of, the DRS receptor
intracellular domain (predicted to constitute amino acid residues from about
158
to about 360 in SEQ ID N0:2); a polypeptide comprising, or alternatively
consisting of, the DRS receptor extracellular and intracellular domains with
all
or part of the transmembrane domain deleted; a polypeptide comprising, or
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alternatively consisting of, the DRS receptor death domain (predicted to
constitute amino acid residues from about 273 to about 340 in SEQ ID N0:2);
and a polypeptide comprising, or alternatively consisting of, one, two, three,
four
or more epitope bearing portions of the DRS receptor protein. In additional
S embodiments, the polypeptide fragments of the invention comprise, or
alternatively consist of, any combination of 1, 2, 3, 4, S, 6, 7, or all 8 of
the
above members. As above, with the leader sequence, the amino acid residues
constituting the DRS receptor extracellular, transmembrane and intracellular
domains have been predicted by computer analysis. Thus, as one of ordinary
skill
would appreciate, the amino acid residues constituting these domains may vary
slightly (e.g., by about 1 to about 1 S amino acid residues) depending on the
criteriaused to define each domain. Polynucleotides encoding these
polypeptides
are also encompassed by the invention.
As discussed above, it is believed that one or both of the extracellular
1 S cysteine-rich motifs of DRS is important for interactions between DRS and
its
ligands. Accordingly, in preferred embodiments, polypeptide fragments of the
invention comprise, or alternatively consist of, amino acid residues 33 to 80,
and/or 81 to 128 of SEQ ID N0:2. In a specific embodiment the polypeptides
of the invention comprise, or alternatively consist of, both of the
extracellular
cysteine-rich motifs disclosed in SEQ >D N0:2. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
Among the especially preferred fragments of the invention are fragments
comprising, or alternatively consisting of, structural or functional
attributes of
DRS. Such fragments include amino acid residues that comprise, or
alternatively
2S consisting of, one, two, three, four or more of the following functional
domains:
alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and
beta-sheet-forming regions ("beta-regions"), turn and turn-forming regions
("turn-regions"), coil and coil-forming regions ("coil-regions"), hydrophillic
regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic
regions, surface forming regions, and high antigenic index regions (i.e.,
regions
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of polypeptides consisting of amino acid residues having an antigenic index of
or
equal to greater than 1.5, as identified using the default parameters of the
Jameson-Wolf program) of DRS.
Certain preferred regions are those, disclosed in FIG. 3 and Table I and
include, but are not limited to, regions of the aforementioned types
identified by
analysis of the amino acid sequence depicted in FIG. 1, such preferred regions
include; Gamier-Robson predicted alpha-regions, beta-regions, turn-regions,
and
coil-regions; Chou-Fasman predicted alpha-regions, beta-regions, and turn-
regions; Kyte-Doolittle predicted hydrophilic regions and Hopp-Woods predicted
hydrophobic regions; Eisenberg alpha and beta amphipathic regions; Emini
surface-forming regions; and Jameson-Wolf high antigenic index regions, as
predicted using the default parameters of these computer programs.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
In another aspect, the invention provides a peptide or polypeptide
comprising, or alternatively consisting of, one, two, three, four, five or
more
epitope-bearing portions of a polypeptide of the invention. The epitope of
this
polypeptide portion is an immunogenic or antigenic epitope of a polypeptide
described herein. Polynucleotides encoding these polypeptides are also
encompassed by the invention.
As to the selection of peptides or polypeptides bearing an antigenic
epitope (i. e., that contain a region of a protein molecule to which an
antibody can
bind), it is well known in that art that relatively short synthetic peptides
that
mimic part of a protein sequence are routinely capable of eliciting an
antiserum
that reacts with the partially mimicked protein. See, for instance, J.G.
Sutcliffe
et al., "Antibodies That React With Predetermined Sites on Proteins," Science
219:660-666 (1983). Peptides capable of eliciting protein-reactive sera are
frequently represented in the primary sequence of a protein, can be
characterized
by a set of simple chemical rules, and are confined neither to immunodominant
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regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or
carboxyl terminals.
Antigenic epitope-bearing peptides and polypeptides ofthe invention are
therefore usefixl to raise antibodies, including monoclonal antibodies, that
bind
specifically to a polypeptide of the invention. See, for instance, Wilson et
al.,
Cell. 37:767-778 (1984) at 777. Antigenic epitope-bearing peptides and
polypeptides of the invention preferably contain a sequence of at least seven,
more preferably at least nine and most preferably between at least about 15 to
about 30 amino acids contained within the amino acid sequence of a polypeptide
of the invention. In the present invention, antigenic epitopes preferably
contain
a sequence of at least 4, at least 5, at least 6, at least 7, more preferably
at least
8, at least 9, at least 10, at least 15, at least 20, at least 25, and, most
preferably,
between about 15 to about 30 amino acids. Preferred polypeptides comprising,
or alternatively consisting of, immunogenic or antigenic epitopes are at least
10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100
amino
acid residues in length. Polynucleotides encoding these polypeptides are also
encompassed by the invention
Antigenic epitopes are useful, for example, to raise antibodies, including
monoclonal antibodies, that specifically bind the epitope. Further, antigenic
epitopes can be used as the target molecules in immunoassays. (See, for
instance, Wilson etal., Cel137:767-778 (1984); Sutcliffe etal., Science
219:660-
666 (1983)).
Non-limiting examples of antigenic polypeptides or peptides that can be
used to generate DRS receptor-specific antibodies include: a polypeptide
comprising, or alternatively consisting of, amino acid residues from about 11
to
about 59 in SEQ ID N0:2, from about 68 to about 113 in SEQ ID N0:2, from
about 173 to about 220 in SEQ ID N0:2, and from about 224 to about 319 in
SEQ ID N0:2. In this context "about" includes the particularly recited ranges,
larger or smaller by several (5, 4, 3, 2, or 1 ) amino acid residues, at
either
terminus or at both termini. As indicated above, the inventors have determined
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that the above polypeptide fragments are antigenic regions of the DRS receptor
protein. Polynucleotides encoding these polypeptides are also encompassed by
the invention.
The epitope-bearing peptides and polypeptides of the invention may be
produced by any conventional means. R.A. Houghten, "General Method for the
Rapid Solid-Phase Synthesis of Large Numbers of Peptides: Specificity of
Antigen-Antibody Interaction at the Level of Individual Amino Acids," Proc.
Natl. Acad. Sci. USA 82:5131-5135 (1985). This "Simultaneous Multiple
Peptide Synthesis (SMPS)" process is further described in U.S. Patent No.
4,631,211 to Houghten et al. (1986).
Immunogenic epitopes can be used, for example, to induce antibodies
according to methods well known in the art. (See, for instance, Sutcliffe et
al.,
supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-
914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). A preferred
immunogenic epitope includes the secreted protein. The polypeptides comprising
one or more immunogenic epitopes may be presented for eliciting an antibody
response together with a carrier protein, such as an albumin, to an animal
system
(such as, for example, rabbit or mouse), or, if the polypeptide is of
sufficient
length (at least about 25 amino acids), the polypeptide may be presented
without
a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino
acids have been shown to be sufficient to raise antibodies capable of binding
to,
at the very least, linear epitopes in a denatured polypeptide (e.g., in
Western
blotting).
Epitope-bearing polypeptides of the present invention may be used to
induce antibodies according to methods well known in the art including, but
not
limited to, in vivo immunization, in vitro immunization, and phage display
methods. See, e.g., Sutcliffe et al., supra; Wilson et al., .supra, and Bittle
et al.,
J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals
may be immunized with free peptide; however, anti-peptide antibody titer may
be boosted by coupling the peptide to a macromolecular carrier, such as
keyhole
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limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing
cysteine residues may be coupled to a carrier using a linker such as
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may
be coupled to carriers using a more general linking agent such as
glutaraldehyde.
Animals such as, for example, rabbits, rats, and mice are immunized with
either
free or carrier-coupled peptides, for instance, by intraperitoneal and/or
intradermal injection of emulsions containing about 100 micrograms of peptide
or carrier protein and Freund's adjuvant or any other adjuvant known for
stimulating an immune response. Several booster injections may be needed, for
instance, at intervals of about two weeks, to provide a useful titer of anti-
peptide
antibody that can be detected, for example, by ELISA assay using free peptide
adsorbed to a solid surface. The titer of anti-peptide antibodies in serum
from
an immunized animal may be increased by selection of anti-peptide antibodies,
for instance, by adsorption to the peptide on a solid support and elution of
the
selected antibodies according to methods well known in the art.
As one of skill in the art will appreciate, DRS receptor polypeptides of
the present invention and the epitope-bearing fragments thereof described
herein
(e.g., corresponding to a portion of the extracellular domain, such as, for
example, amino acid residues 1 to 133 of SEQ 117 N0:2) can be combined with
heterologous polypeptide sequences. For example, the polypeptides of the
present invention may be fused with the constant domain of immunoglobulins
(IgA, IgE, IgG, IgM) or portions thereof (CH 1, CH2, CH3, and any combination
thereof, including both entire domains and portions thereof), resulting in
chimeric
polypeptides. These fusion proteins facilitate purification and show an
increased
half life. This has been shown, e.g., for chimeric proteins consisting of the
first
two domains ofthe human CD4-polypeptide and various domains ofthe constant
regions of the heavy or light chains of mammalian immunoglobulins (EPA
394,827; Trauneckeretal., Nature 331:84-86 (1988)). Fusion proteins that have
a disulfide-linked dimeric structure due to the IgG part can also be more
effcient
in binding and neutralizing other molecules than the monomeric DRS protein or
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protein fragment alone (Fountoulakis et al., J. Biochem. 270:3 95 8-3 964 (
1995)).
Nucleic acids encoding the above epitopes can also be recombined with a gene
of interest as an epitope tag (e.g., the hemagglutinin ("HA") tag or flag tag)
to
aid in detection and purification of the expressed polypeptide. For example, a
system described by Janknecht et al. allows for the ready purification of non-
denatured fusion proteins expressed in human cell lines (Janknecht et al.,
1991,
Proc. Natl. Acad. Sci. USA 88:8972- 897). In this system, the gene of interest
is subcloned into a vaccinia recombination plasmid such that the open reading
frame of the gene is translationally fused to an amino-terminal tag consisting
of
six histidine residues. The tag serves as a matrix-binding domain for the
fusion
protein. Extracts from cells infected with the recombinant vaccinia virus are
loaded onto Niz+ nitriloacetic acid-agarose column and histidine-tagged
proteins
can be selectively eluted with imidazole-containing buffers. Polynucleotides
encoding these fusion proteins are also encompassed by the invention.
The techniques of gene-shuffling, motif shuffling, exon-shuffling, and/or
codon-shuffling (collectively referred to as "DNA shuffling") may be employed
to modulate the activities of DRS thereby effectively generating agonists and
antagonists of DRS. See generally, U.S. Patent Nos. 5,605,793, 5,811,238,
5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et ad., Curr. Opinion
Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82
(1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo,
M. M. and Blasco, R. BioTechniques 24(2):308-13 (1998) (each ofthese patents
and publications are hereby incorporated by reference). In one embodiment,
alteration of DRS polynucleotides and corresponding polypeptides may be
achieved by DNA shuffling. DNA shuffling involves the assembly of two or
more DNA segments into a desired DRS molecule by homologous, or site
specific, recombination. In another embodiment, DRS polynucleotides and
corresponding polypeptides may be altered by being subjected to random
mutagenesis by error-prone PCR, random nucleotide insertion or other methods
prior to recombination.
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In another embodiment, one ormore components, motifs, sections, parts,
domains, fragments, etc., of DRS may be recombined with one or more
components, motifs, sections, parts, domains, fragments, etc. of one or more
heterologous molecules. In preferred embodiments, the heterologous molecules
are, for example, TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-
beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, Fast,
CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International
Publication No. WO 96/14328), AlM-I (International Publication No. WO
97/33899), AIM-II (International Publication No. WO 97/34911), APRIL (J.
Exp. Med. 188(6):1185-1190), endokine-alpha (International Publication No.
WO 98/07880), Neutrokine-alpha (International Publication No. WO 98/ 18921 ),
OPG, nerve growth factor (NGF), DR3 (International Publication No. WO
97/33904), DR4 (International Publication No. WO 98/32856), TRS
(International Publication No. WO 98/30693), TR6 (International Publication
No. WO 98/30694), TRANK, TR9 (International Publication No. WO
98/56892), TR10 (International Publication No. WO 98/54202),312C2
(International Publication No. WO 98/06842), TR12, TNF-R1,
TRAMP/DR3/APO-3/WSL/LARD, TRAIL,-R1/DR4/APO-2, TRAIL,-R2/DRS,
DcRl/TRAIL,-R3/TR117/LIT, DcR2/TRAIL-R4, CAD, TRAIL, TRAMP, and
v-FLIP. In additional preferred embodiments, the heterologous molecules are,
for example, soluble forms of Fas, CD30, CD27, CD40 and 4-IBB.
In further preferred embodiments, the heterologous molecules are any
member of the TNF family.
To improve or alter the characteristics of DRS polypeptides, protein
engineering may be employed. Recombinant DNA technology known to those
skilled in the art can be used to create novel mutant proteins or "muteins
including single or multiple amino acid substitutions, deletions, additions or
fusion proteins. Such modified polypeptides can show, e.g., enhanced activity
or increased stability. In addition, they may be purified in higher yields and
show
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better solubility than the corresponding natural polypeptide, at least under
certain
purification and storage conditions.
For instance, for many proteins, including the extracellular domain of a
membrane associated protein or the mature forms) of a secreted protein, it is
known in the art that one or more amino acids may be deleted from the N
terminus or C-terminus without substantial loss ofbiological function.
However,
even if deletion of one or more amino acids from the N-terminus or C-terminus
of a protein results in modification or loss of one or more biological
functions of
the protein, other DRS functional activities may still be retained. For
example,
in many instances, the ability of the shortened protein to induce and/or bind
to
antibodies which recognize DRS (preferably antibodies that bind specifically
to
DRS) will retained irrespective of the size or location of the deletion. In
fact,
polypeptides composed of as few as six DRS amino acid residues may often
evoke an immune response. Whether a particular polypeptide lacking N-terminal
and/or C-terminal residues of a complete protein retains such immunologic
activities can readily be determined by routine methods described herein and
otherwise known in the art.
As mentioned above, even if deletion of one or more amino acids from
the N-terminus of a protein results in modification or loss of one or more
biological functions of the protein, other functional activities (e.g.,
biological
activities, ability to multimerize, ability to bind DRS ligand) may still be
retained.
For example, the ability of shortened DRS muteins to induce and/or bind to
antibodies which recognize the complete or mature forms of the polypeptides
generally will be retained when less than the majority of the residues of the
complete or mature polypeptide are removed from the N-terminus. Whether a
particular polypeptide lacking N-terminal residues of a complete polypeptide
retains such immunologic activities can readily be determined by routine
methods
described herein and otherwise known in the art. It is not unlikely that a DRS
mutein with a large number of deleted N-terminal amino acid residues may
retain
some biological or immunogenic activities.
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It will be recognized in the art that some amino acid sequence of DRS can
be varied without significant effect on the structure or function of the
protein.
If such differences in sequence are contemplated, it should be remembered that
there will be critical areas on the protein which determine activity. Such
areas
S will usually comprise residues which make up the ligand binding site or the
death
domain, or which form tertiary structures which affect these domains.
Accordingly, the present invention further provides polypeptides having
one or more residues deleted from the amino terminus of the DRS amino acid
sequence shown in FIG. 1, up to the alanine residue at position number 406 and
polynucleotides encoding such polypeptides. In particular, the present
invention
provides polypeptides comprising, or alternatively consisting of, the amino
acid
sequence of residues n'-411 of FIG. 1, where ni is an integer from 2 to 406
corresponding to the position of the amino acid residue in FIG. 1 (which is
identical to the sequence shown as SEQ ID N0:2, with the exception that the
1 S amino acid residues in FIG. 1 are numbered consecutively from 1 through
411
from the N-terminus to the C-terminus, while the amino acid residues in SEQ ID
N0:2 are numbered consecutively from -S 1 through 360 to reflect the position
of the predicted signal peptide).
More in particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, the amino acid
sequence
of a member selected from the group consisting of residues: E-2 to S-411; Q-3
to S-411; R-4 to S-411; G-S to S-411; Q-6 to S-411; N-7 to S-411; A-8 to 5-
411; P-9 to S-411; A-10 to S-411; A-11 to S-411; S-12 to S-411; G-13 to 5-
411; A-14 to S-411; R-1 S to S-411; K-16 to S-411; R-17 to S-41 l; H-18 to S-
2S 411; G-19 to S-411; P-20 to S-411; G-21 to S-411; P-22 to S-411; R-23 to S-
411; E-24 to S-411; A-2S to S-411; R-26 to S-41 l; G-27 to S-411; A-28 to S-
411; R-29 to S-411; P-30 to S-411; G-31 to S-411; P-32 to S-411; R-33 to S-
411; V-34 to S-411; P-3S to S-411; K-36 to S-411; T-37 to S-411; L-38 to 5-
411; V-39 to S-411; L-40 to S-411; V-41 to S-41 l; V-42 to S-411; A-43 to S-
411; A-44 to S-411; V-4S to S-411; L-46 to S-411; L-47 to S-411; L-48 to S-
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411; V-49 to S-411; S-50 to S-411; A-51 to S-411; E-52 to S-411; S-53 to 5-
411; A-54 to S-411; L-55 to S-411; I-56 to S-411; T-57 to S-411; Q-58 to 5-
411; Q-59 to S-411; D-60 to S-411; L-61 to S-411; A-62 to S-411; P-63 to 5-
411; Q-64 to S-411; Q-65 to S-411; R-66 to S-411; A-67 to S-411; A-68 to S-
411; P-69 to S-411; Q-70 to S-411; Q-71 to S-411; K-72 to S-411; R-73 to S-
411; S-74 to S-411; S-75 to S-411; P-76 to S-411; S-77 to S-411; E-78 to 5-
411; G-79 to S-411; L-80 to S-411; C-81 to S-411; P-82 to S-411; P-83 to 5-
411; G-84 to S-411; H-85 to S-411; H-86 to S-411; I-87 to S-411; S-88 to 5-
411; E-89 to S-411; D-90 to S-411; G-91 to S-411; R-92 to S-411; D-93 to S-
411; C-94 to S-411; I-95 to S-411; S-96 to S-411; C-97 to S-411; K-98 to S-
411; Y-99 to S-411; G-100 to S-411; Q-101 to S-411; D-102 to S-411; Y-103
to S-411; S-104 to S-411; T-105 to S-411; H-106 to S-411; W-107 to S-411;
N-108 to S-411; D-109 to S-411; L-110 to S-411; L-111 to S-411; F-112 to 5-
411; C-113 to S-411; L-114 to S-411; R-115 to S-411; C-116 to S-411; T-117
to S-411; R-118 to S-411; C-119 to S-411; D-120 to S-411; S-121 to S-411; G-
122 to S-411; E-123 to S-411; V-124 to S-411; E-125 to S-411; L-126 to 5-
411; S-127 to S-411; P-128 to S-411; C-129 to S-411; T-130 to S-411; T-131
to S-411; T-132 to S-411; R-133 to S-411; N-134 to S-411; T-135 to S-41 l; V-
136 to S-411; C-137 to S-411; Q-138 to S-411; C-139 to S-411; E-140 to S-
411; E-141 to S-41 l; G-142 to S-41 l; T-143 to S-411; F-144 to S-411; R-145
to S-411; E-146 to S-411; E-147 to S-411; D-148 to S-41 l; S-149 to S-411; P-
150 to S-411; E-151 to S-411; M-152 to S-411; C-153 to S-411; R-154 to 5-
411; K-1 SS to S-411; C-156 to S-41 l; R-157 to S-411; T-158 to S-411; G-159
to S-411; C-160 to S-411; P-161 to S-411; R-162 to S-411; G-163 to S-411; M-
164 to S-411; V-165 to S-411; K-166 to S-411; V-167 to S-411; G-168 to S-
411; D-169 to S-41 l; C-170 to S-411; T-171 to S-41 l; P-172 to S-41 l; W-173
to S-411; S-174 to S-411; D-175 to S-411; I-176 to S-411; E-177 to S-41 l; C-
178 to S-411; V-179 to S-411; H-180 to S-411; K-181 to S-41 l; E-182 to S-
41 l; S-183 to S-411; G-184 to S-411; I-185 to S-411; I-186 to S-41 l; I-187
to
S-411; G-188 to S-411; V-189 to S-411; T-190 to S-41 l; V-191 to S-41 l; A-
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192 to S-411; A-193 to S-411; V-194 to S-411; V-195 to S-41 l; L-196 to S-
411; I-197 to S-411; V-198 to S-411; A-199 to S-411; V-200 to S-41 l; F-201
to S-411; V-202 to S-41 l; C-203 to S-41 l; K-204 to S-411; S-205 to S-411; L-
206 to S-41 l; L-207 to S-411; W-208 to S-411; K-209 to S-41 l; K-210 to S-
411; V-211 to S-41 l; L-212 to S-411; P-213 to S-411; Y-214 to S-411; L-215
to S-41 I; K-216 to S-411; G-217 to S-411; I-218 to S-411; C-219 to S-411; 5-
220 to S-41 l; G-221 to S-41 l; G-222 to S-411; G-223 to S-411; G-224 to 5-
411; D-225 to S-411; P-226 to S-411; E-227 to S-411; R-228 to S-41 l; V-229
to S-411; D-230 to S-411; R-231 to S-411; S-232 to S-41 l; S-233 to S-411; Q-
234 to S-411; R-235 to S-411; P-236 to S-411; G-237 to S-411; A-238 to S-
411; E-239 to S-411; D-240 to S-41 I; N-241 to S-41 I; V-242 to S-411; L-243
to S-411; N-244 to S-411; E-245 to S-411; I-246 to S-411; V-247 to S-411; 5-
248 to S-411; I-249 to S-411; L-250 to S-411; Q-251 to S-411; P-252 to S-41 l;
T-253 to S-411; Q-254 to S-411; V-255 to S-411; P-256 to S-411; E-257 to S-
41 l; Q-258 to S-41 l; E-259 to S-411; M-260 to S-411; E-261 to S-411; V-262
to S-411; Q-263 to S-411; E-264 to S-411; P-265 to S-41 l; A-266 to S-411; E-
267 to S-411; P-268 to S-411; T-269 to S-411; G-270 to S-411; V-271 to 5-
411; N-272 to S-411; M-273 to S-411; L-274 to S-411; S-275 to S-411; P-276
to S-41 l; G-277 to S-411; E-278 to S-411; S-279 to S-41 I; E-280 to S-411; H-
281 to S-411; L-282 to S-41 l; L-283 to S-411; E-284 to S-411; P-285 to S-41
l;
A-286 to S-41 I; E-287 to S-41 l; A-288 to S-411; E-289 to S-411; R-290 to 5-
411; S-291 to S-411; Q-292 to S-411; R-293 to S-411; R-294 to S-411; R-295
to S-411; L-296 to S-411; L-297 to S-411; V-298 to S-411; P-299 to S-411; A-
300 to S-411; N-301 to S-411; E-302 to S-411; G-303 to S-411; D-304 to S-
41 l; P-305 to S-411; T-306 to S-411; E-307 to S-411; T-308 to S-411; L-309
to S-411; R-310 to S-411; Q-311 to S-411; C-312 to S-411; F-313 to S-41 I; D-
314 to S-41 l; D-315 to S-411; F-316 to S-411; A-317 to S-411; D-318 to S-
41 l; L-319 to S-41 l; V-320 to S-411; P-321 to S-411; F-322 to S-411; D-323
to S-411; S-324 to S-411; W-325 to S-411; E-326 to S-411; P-327 to S-411; L-
328 to S-411; M-329 to S-411; R-330 to S-411; K-331 to S-411; L-332 to S-
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411; G-333 to S-411; L-334 to S-41 l; M-335 to S-411; D-336 to S-411; N-337
to S-411; E-338 to S-411; I-339 to S-411; K-340 to S-411; V-341 to S-411; A-
342 to S-411; K-343 to S-411; A-344 to S-411; E-345 to S-411; A-346 to S-
411; A-347 to S-411; G-348 to S-411; H-349 to S-411; R-350 to S-411; D-351
to S-411; T-352 to S-411; L-353 to S-411; Y-354 to S-41 I; T-355 to S-411; M-
356 to S-411; L-357 to S-41 I; I-358 to S-411; K-359 to S-411; W-360 to S-
411; V-361 to S-411; N-362 to S-411; K-363 to S-411; T-364 to S-41 l; G-365
to S-411; R-366 to S-41 I; D-367 to S-411; A-368 to S-411; S-369 to S-411; V-
370 to S-411; H-371 to S-411; T-372 to S-41 l; L-373 to S-411; L-374 to S-
411; D-375 to S-411; A-376 to S-411; L-377 to S-411; E-378 to S-411; T-379
to S-411; L-380 to S-411; G-381 to S-411; E-382 to S-411; R-383 to S-411; L-
384 to S-411; A-385 to S-411; K-386 to S-411; Q-387 to S-411; K-388 to 5-
411; I-389 to S-411; E-390 to S-411; D-391 to S-41 I; H-392 to S-41 I; L-393
to S-411; L-394 to S-411; S-395 to S-411; S-396 to S-41 I; G-397 to S-411; K-
398 to S-411; F-399 to S-411; M-400 to S-41 l; Y-401 to S-411; L-402 to S-
411; E-403 to S-411; G-404 to S-411; N-405 to S-411; and A-406 to S-411 of
the DRS sequence shown in FIG. 1 (which is identical to the sequence shown as
SEQ m N0:2, with the exception that the amino acid residues in FIG. 1 are
numbered consecutively from I through 411 from the N-terminus to the
C-terminus, while the amino acid residues in SEQ ID N0:2 are numbered
consecutively from -51 through 360 to reflect the position ofthe predicted
signal
peptide).
The present invention is also directed to nucleic acid molecules
comprising, or alternatively consisting of, a polynucleotide sequence at least
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the
polynucleotide sequences encoding the polypeptides described above. The
invention is further directed to nucleic acid molecules comprising, or
alternatively
consisting of, polynucleotide sequences which encode polypeptides that are at
least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the
polypeptides described above. The present invention also encompasses the
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above polynucleotide sequences fused to a heterologous polynucleotide
sequence. Polypeptides encoded by these polynucleotides are also encompassed
by the invention.
In another embodiment, N-terminal deletions of the DRS polypeptide can
be described by the general formula n2 to 184 where nz is a number from 1 to
179
corresponding to the amino acid sequence identified in FIG. 1 (or where nz is
a
number from -51 to 128 corresponding to the amino acid sequence identified in
SEQ ID N0:2). In specific embodiments, N-terminal deletions ofthe DRS ofthe
invention comprise, or alternatively consist of, a member selected from the
group
consisting of amino acid residues: E-2 to G-184; Q-3 to G-184; R-4 to G-184;
G-5 to G-184; Q-6 to G-184; N-7 to G-184; A-8 to G-184; P-9 to G-184; A-10
to G-184; A-11 to G-184; S-12 to G-184; G-13 to G-184; A-14 to G-184; R-15
to G-184; K-16 to G-184; R-17 to G-184; H-18 to G-184; G-19 to G-184; P-20
to G-184; G-21 to G-184; P-22 to G-184; R-23 to G-184; E-24 to G-184; A-25
to G-184; R-26 to G-184; G-27 to G-184; A-28 to G-184; R-29 to G-184; P-30
to G-184; G-31 to G-184; P-32 to G-184; R-33 to G-184; V-34 to G-184; P-35
to G-184; K-36 to G-184; T-37 to G-184; L-38 to G-184; V-39 to G-184; L-40
to G-184; V-41 to G-184; V-42 to G-184; A-43 to G-184; A-44 to G-184; V-45
to G-184; L-46 to G-184; L-47 to G-184; L-48 to G-184; V-49 to G-184; S-50
to G-184; A-51 to G-184; E-52 to G-184; S-53 to G-184; A-54 to G-184; L-55
to G-184; I-56 to G-184; T-57 to G-184; Q-58 to G-184; Q-59 to G-184; D-60
to G-184; L-61 to G-184; A-62 to G-184; P-63 to G-184; Q-64 to G-184; Q-65
to G-184; R-66 to G-184; A-67 to G-184; A-68 to G-184; P-69 to G-184; Q-70
to G-184; Q-71 to G-184; K-72 to G-184; R-73 to G-184; S-74 to G-184; S-75
to G-184; P-76 to G-184; S-77 to G-184; E-78 to G-184; G-79 to G-184; L-80
to G-184; C-81 to G-184; P-82 to G-184; P-83 to G-184; G-84 to G-184; H-85
to G-184; H-86 to G-184; I-87 to G-184; S-88 to G-184; E-89 to G-184; D-90
to G-184; G-91 to G-184; R-92 to G-184; D-93 to G-184; C-94 to G-184; I-95
to G-184; S-96 to G-184; C-97 to G-184; K-98 to G-184; Y-99 to G-184; G-
100 to G-184; Q-101 to G-184; D-102 to G-184; Y-103 to G-184; S-104 to G-
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184; T-105 to G-184; H-106 to G-184; W-107 to G-184; N-108 to G-184; D-
109 to G-184; L-110 to G-184; L-111 to G-184; F-112 to G-184; C-113 to 6-
184; L-114 to G-184; R-115 to G-184; C-116 to G-184; T-117 to G-184; R-118
to G-184; C-119 to G-184; D-120 to G-184; S-121 to G-184; G-122 to G-184;
E-123 to G-184; V-124 to G-184; E-125 to G-184; L-126 to G-184; S-127 to
G-184; P-128 to G-184; C-129 to G-184; T-130 to G-184; T-131 to G-184; T-
132 to G-184; R-133 to G-184; N-134 to G-184; T-135 to G-184; V-136 to 6-
184; C-137 to G-184; Q-138 to G-184; C-139 to G-184; E-140 to G-184; E-141
to G-184; G-142 to G-184; T-143 to G-184; F-144 to G-184; R-145 to G-184;
E-146 to G-184; E-147 to G-184; D-148 to G-184; S-149 to G-184; P-150 to
G-184; E-151 to G-184; M-152 to G-184; C-153 to G-184; R-154 to G-184; K-
155 to G-184; C-156 to G-184; R-157 to G-184; T-158 to G-184; G-159 to 6-
184; C-160 to G-184; P-161 to G-184; R-162 to G-184; G-163 to G-184; M-
164 to G-184; V-165 to G-184; K-166 to G-184; V-167 to G-184; G-168 to G-
184; D-169 to G-184; C-170 to G-184; T-171 to G-184; P-172 to G-184; W-
173 to G-184; S-174 to G-184; D-175 to G-184; I-176 to G-184; E-177 to 6-
184; C-178 to G-184; and V-179 to G-184 of the DRS extracellular domain
sequence shown in FIG. 1 (which is identical to the sequence shown as SEQ >D
N0:2, with the exception that the amino acid residues in FIG. 1 are numbered
consecutively from 1 through 411 from the N-terminus to the C-terminus, while
the amino acid residues in SEQ ID N0:2 are numbered consecutively from -51
through 360 to reflect the position of the predicted signal peptide).
The present invention is also directed to nucleic acid molecules
comprising, or alternatively consisting of, a polynucleotide sequence at least
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the
polynucleotide sequences encoding the polypeptides described above. The
invention is further directed to nucleic acid molecules comprising, or
alternatively
consisting of, polynucleotide sequences which encode polypeptides that are at
least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the
polypeptides described above. The present invention also encompasses the
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above polynucleotide sequences fused to a heterologous polynucleotide
sequence. Polypeptides encoded by these polynucleotides are also encompassed
by the invention.
Also as mentioned above, even if deletion of one or more amino acids
from the C-terminus of a protein results in modification of loss of one or
more
biological functions of the protein, other functional activities (e.g.,
biological
activities, ability to multimerize, ability to bind DRS ligand (e.g., TRAIL,))
may
still be retained. For example, the ability ofthe shortened DRS mutein to
induce
and/or bind to antibodies which recognize the complete or mature forms of the
polypeptide generally will be retained when less than the majority of the
residues
of the complete or mature polypeptide are removed from the C-terminus.
Whether a particular polypeptide lacking C-terminal residues of a complete
polypeptide retains such immunologic activities can readily be determined by
routine methods described herein and otherwise known in the art. It is not
unlikely that a DRS mutein with a large number of deleted C-terminal amino
acid
residues may retain some biological or immunogenic activities. In fact,
peptides
composed of as few as six DRS amino acid residues may often evoke an immune
response.
Accordingly, the present invention further provides polypeptides having
one or more residues deleted from the carboxy terminus of the amino acid
sequence of the DRS polypeptide shown in FIG. 1 (SEQ ID N0:2), up to the
glutamic acid residue at position number 52, and polynucleotides encoding such
polypeptides. In particular, the present invention provides polypeptides
comprising, or alternatively consisting of, the amino acid sequence of
residues
52-m' of FIG. 1 (i.e., SEQ ID N0:2), where m' is an integer from 57 to 410
corresponding to the position of the amino acid residue in FIG. 1 (or where m'
is an integer from 6 to 360 corresponding to the position of the amino acid
residue in SEQ B7 N0:2). More in particular, the invention provides
polynucleotides encoding polypeptides comprising, or alternatively consisting
of,
a member selected from the group consisting of residues: E-52 to M-410; E-52
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to A-409; E-S2 to S-408; E-S2 to D-407; E-S2 to A-406; E-52 to N-405; E-S2
to G-404; E-S2 to E-403; E-S2 to L-402; E-S2 to Y-401; E-52 to M-400; E-S2
to F-399; E-S2 to K-398; E-S2 to G-397; E-S2 to S-396; E-S2 to S-395; E-S2
to L-394; E-S2 to L-393; E-S2 to H-392; E-S2 to D-391; E-S2 to E-390; E-S2
S to I-389; E-S2 to K-388; E-S2 to Q-387; E-S2 to K-386; E-S2 to A-385; E-S2
to L-384; E-S2 to R-383; E-S2 to E-382; E-S2 to G-381; E-S2 to L-380; E-S2
to T-379; E-52 to E-378; E-S2 to L-377; E-S2 to A-376; E-S2 to D-375; E-S2
to L-374; E-S2 to L-373; E-S2 to T-372; E-S2 to H-371; E-S2 to V-370; E-S2
to S-369; E-S2 to A-368; E-S2 to D-367; E-S2 to R-366; E-S2 to G-365; E-52
to T-364; E-S2 to K-363; E-S2 to N-362; E-S2 to V-361; E-S2 to W-360; E-S2
to K-359; E-S2 to I-358; E-S2 to L-357; E-S2 to M-356; E-S2 to T-3SS; E-S2
to Y-354; E-S2 to L-353; E-S2 to T-352; E-S2 to D-351; E-S2 to R-350; E-S2
to H-349; E-52 to G-348; E-S2 to A-347; E-S2 to A-346; E-S2 to E-345; E-S2
to A-344; E-S2 to K-343; E-S2 to A-342; E-S2 to V-341; E-S2 to K-340; E-S2
to I-339; E-S2 to E-338; E-S2 to N-337; E-S2 to D-336; E-S2 to M-335; E-S2
to L-334; E-S2 to G-333; E-S2 to L-332; E-S2 to K-331; E-S2 to R-330; E-S2
to M-329; E-52 to L-328; E-S2 to P-327; E-S2 to E-326; E-S2 to W-325; E-S2
to S-324; E-S2 to D-323; E-52 to F-322; E-S2 to P-321; E-S2 to V-320; E-S2
to L-319; E-S2 to D-318; E-S2 to A-317; E-S2 to F-316; E-S2 to D-315; E-S2
to D-314; E-S2 to F-313; E-S2 to C-312; E-S2 to Q-311; E-S2 to R-310; E-S2
to L-309; E-S2 to T-308; E-S2 to E-307; E-S2 to T-306; E-S2 to P-305; E-S2
to D-304; E-S2 to G-303; E-52 to E-302; E-S2 to N-301; E-S2 to A-300; E-S2
to P-299; E-S2 to V-298; E-S2 to L-297; E-S2 to L-296; E-S2 to R-295; E-S2
to R-294; E-S2 to R-293; E-S2 to Q-292; E-S2 to S-291; E-S2 to R-290; E-S2
2S to E-289; E-S2 to A-288; E-S2 to E-287; E-S2 to A-286; E-S2 to P-285; E-S2
to E-284; E-S2 to L-283; E-S2 to L-282; E-S2 to H-281; E-S2 to E-280; E-S2
to S-279; E-52 to E-278; E-S2 to G-277; E-S2 to P-276; E-S2 to S-275; E-S2
to L-274; E-52 to M-273; E-S2 to N-272; E-S2 to V-271; E-52 to G-270; E-S2
to T-269; E-S2 to P-268; E-S2 to E-267; E-S2 to A-266; E-S2 to P-265; E-S2
to E-264; E-S2 to Q-263; E-S2 to V-262; E-S2 to E-261; E-S2 to M-260; E-52
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to E-259; E-52 to Q-258; E-52 to E-257; E-52 to P-256; E-52 to V-255; E-52
to Q-254; E-52 to T-253; E-52 to P-252; E-52 to Q-251; E-52 to L-250; E-52
to I-249; E-52 to S-248; E-52 to V-247; E-52 to I-246; E-52 to E-245; E-52 to
N-244; E-52 to L-243; E-52 to V-242; E-52 to N-241; E-52 to D-240; E-52 to
E-239; E-52 to A-238; E-52 to G-237; E-52 to P-236; E-52 to R-235; E-52 to
Q-234; E-52 to S-233; E-52 to S-232; E-52 to R-231; E-52 to D-230; E-52 to
V-229; E-52 to R-228; E-52 to E-227; E-52 to P-226; E-52 to D-225; E-52 to
G-224; E-52 to G-223; E-52 to G-222; E-52 to G-221; E-52 to S-220; E-52 to
C-219; E-52 to I-218; E-52 to G-217; E-52 to K-216; E-52 to L-215; E-52 to
Y-214; E-52 to P-213; E-52 to L-212; E-52 to V-211; E-52 to K-210; E-52 to
K-209; E-52 to W-208; E-52 to L-207; E-52 to L-206; E-52 to S-205; E-52 to
K-204; E-52 to C-203; E-52 to V-202; E-52 to F-201; E-52 to V-200; E-52 to
A-199; E-52 to V-198; E-52 to I-197; E-52 to L-196; E-52 to V-195; E-52 to
V-194; E-52 to A-193; E-52 to A-192; E-52 to V-191; E-52 to T-190; E-52 to
V-189; E-52 to G-188; E-52 to I-187; E-52 to I-186; E-52 to I-185; E-52 to G-
184; E-52 to S-183; E-52 to E-182; E-52 to K-181; E-52 to H-180; E-52 to V-
179; E-52 to C-178; E-52 to E-177; E-52 to I-176; E-52 to D-175; E-52 to 5-
174; E-52 to W-173; E-52 to P-172; E-52 to T-171; E-52 to C-170; E-52 to D-
169; E-52 to G-168; E-52 to V-167; E-52 to K-166; E-52 to V-165; E-52 to M-
164; E-52 to G-163; E-52 to R-162; E-52 to P-161; E-52 to C-160; E-52 to G-
159; E-52 to T-158; E-52 to R-157; E-52 to C-156; E-52 to K-155; E-52 to 8-
154; E-52 to C-153; E-52 to M-152; E-52 to E-1 S 1; E-52 to P-150; E-52 to 5-
149; E-52 to D-148; E-52 to E-147; E-52 to E-146; E-52 to R-145; E-52 to F-
144; E-52 to T-143; E-52 to G-142; E-52 to E-141; E-52 to E-140; E-52 to C-
139; E-52 to Q-138; E-52 to C-137; E-52 to V-136; E-52 to T-135; E-52 to N-
134; E-52 to R-133; E-52 to T-132; E-52 to T-131; E-52 to T-130; E-52 to C-
129; E-52 to P-128; E-52 to S-127; E-52 to L-126; E-52 to E-125; E-52 to V-
124; E-52 to E-123; E-52 to G-122; E-52 to S-121; E-52 to D-120; E-52 to C-
1 I 9; E-52 to R-118; E-52 to T-117; E-52 to C-116; E-52 to R-115; E-52 to L-
114; E-52 to C-113; E-52 to F-112; E-52 to L-111; E-52 to L-110; E-52 to D-
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109; E-52 to N-108; E-52 to W-107; E-52 to H-106; E-52 to T-105; E-52 to 5-
104; E-52 to Y-103; E-52 to D-102; E-52 to Q-101; E-52 to G-100; E-52 to Y-
99; E-52 to K-98; E-52 to C-97; E-52 to S-96; E-52 to I-95; E-52 to C-94; E-52
to D-93; E-52 to R-92; E-52 to G-91; E-52 to D-90; E-52 to E-89; E-52 to S-
88; E-52 to I-87; E-52 to H-86; E-52 to H-85; E-52 to G-84; E-52 to P-83; E-52
to P-82; E-52 to C-81; E-52 to L-80; E-52 to G-79; E-52 to E-78; E-52 to S-77;
E-52 to P-76; E-52 to S-75; E-52 to S-74; E-52 to R-73; E-52 to K-72; E-52 to
Q-71; E-52 to Q-70; E-52 to P-69; E-52 to A-68; E-52 to A-67; E-52 to R-66;
E-52 to Q-65; E-52 to Q-64; E-52 to P-63; E-52 to A-62; E-52 to L-61; E-52
to D-60; E-52 to Q-59; E-52 to Q-58; and E-52 to T-57; of the DRS sequence
shown in FIG. 1 (which is identical to the sequence shown as SEQ ID N0:2,
with the exception that the amino acid residues in FIG. 1 are numbered
consecutively from 1 through 411 from the N-terminus to the C-terminus, while
the amino acid residues in SEQ ID N0:2 are numbered consecutively from -51
1 S through 360 to reflect the position of the predicted signal peptide).
The present invention is also directed to nucleic acid molecules
comprising, or alternatively consisting of, a polynucleotide sequence at least
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the
polynucleotide sequences encoding the polypeptides described above. The
invention is further directed to nucleic acid molecules comprising, or
alternatively
consisting of, polynucleotide sequences which encode polypeptides that are at
least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the
polypeptides described above. The present invention also encompasses the
above polynucleotide sequences fused to a heterologous polynucleotide
sequence. Polypeptides encoded by these polynucleotides are also encompassed
by the invention.
In another embodiment, C-terminal deletions ofthe DRS polypeptide can
be described by the general formula 52-m2 where m2 is a number from 57 to 183
corresponding to the amino acid sequence identified in FIG. 1 (SEQ >D N0:2).
In specific embodiments, C-terminal deletions of the DRS of the invention
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comprise, or alternatively, consist of, a member selected from the group
consisting of residues: E-52 to S-183; E-52 to E-182; E-52 to K-181; E-52 to
H-180; E-52 to V-179; E-52 to C-178; E-52 to E-177; E-52 to I-176; E-52 to
D-175; E-52 to S-174; E-52 to W-173; E-52 to P-172; E-52 to T-171; E-52 to
C-170; E-52 to D-169; E-52 to G-168; E-52 to V-167; E-52 to K-166; E-52 to
V-165; E-52 to M-164; E-52 to G-163; E-52 to R-162; E-52 to P-161; E-52 to
C-160; E-52 to G-159; E-52 to T-158; E-52 to R-157; E-52 to C-156; E-52 to
K-155; E-52 to R-154; E-52 to C-153; E-52 to M-152; E-52 to E-151; E-52 to
P-150; E-52 to S-149; E-52 to D-148; E-52 to E-147; E-52 to E-146; E-52 to
R-145; E-52 to F-144; E-52 to T-143; E-52 to G-142; E-52 to E-141; E-52 to
E-140; E-52 to C-139; E-52 to Q-138; E-52 to C-137; E-52 to V-136; E-52 to
T-135; E-52 to N-134; E-52 to R-133; E-52 to T-132; E-52 to T-131; E-52 to
T-130; E-52 to C-129; E-52 to P-128; E-52 to S-127; E-52 to L-126; E-52 to
E-125; E-52 to V-124; E-52 to E-123; E-52 to G-122; E-52 to S-121; E-52 to
D-120; E-52 to C-119; E-52 to R-118; E-52 to T-117; E-52 to C-116; E-52 to
R-115; E-52 to L-114; E-52 to C-113; E-52 to F-112; E-52 to L-111; E-52 to
L-110; E-52 to D-109; E-52 to N-108; E-52 to W-107; E-52 to H-106; E-52 to
T-105; E-52 to S-104; E-52 to Y-103; E-52 to D-102; E-52 to Q-101; E-52 to
G-100; E-52 to Y-99; E-52 to K-98; E-52 to C-97; E-52 to S-96; E-52 to I-95;
E-52 to C-94; E-52 to D-93; E-52 to R-92; E-52 to G-91; E-52 to D-90; E-52
to E-89; E-52 to S-88; E-52 to I-87; E-52 to H-86; E-52 to H-85; E-52 to G-84;
E-52 to P-83; E-52 to P-82; E-52 to C-81; E-52 to L-80; E-52 to G-79; E-52 to
E-78; E-52 to S-77; E-52 to P-76; E-52 to S-75; E-52 to S-74; E-52 to R-73;
E-52 to K-72; E-52 to Q-71; E-52 to Q-70; E-52 to P-69; E-52 to A-68; E-52
to A-67; E-52 to R-66; E-52 to Q-65; E-52 to Q-64; E-52 to P-63; E-52 to A-
62; E-52 to L-61; E-52 to D-60; E-52 to Q-59; E-52 to Q-58; and E-52 to T-57
of the DRS extracellular domain sequence shown in FIG. 1 (SEQ ID N0:2).
The present invention is also directed to nucleic acid molecules
comprising, or alternatively consisting of, a polynucleotide sequence at least
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% Identical to the
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polynucleotide sequences encoding the polypeptides described above. The
invention is further directed to nucleic acid molecules comprising, or
alternatively
consisting of, polynucleotide sequences which encode polypeptides that are at
least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the
polypeptides described above. The present invention also encompasses the
above polynucleotide sequences fused to a heterologous polynucleotide
sequence. Polypeptides encoded by these polynucleotides are also encompassed
by the invention.
The invention also provides polypeptides having one or more amino acids
IO deleted from both the amino and the carboxyl termini of a DR5 polypeptide,
which may be described generally as having residues n'- m' and/or n2- m2 of
FIG.
I (i. e., SEQ ID N0:2), where n', n2, m', and m2 are integers as described
above.
Also included are a nucleotide sequence encoding a polypeptide
consisting of a portion of the complete DRS amino acid sequence encoded by the
cDNA contained in ATCC Deposit No. 97920, where this portion excludes from
1 to about 78 amino acids from the amino terminus of the complete amino acid
sequence encoded by the cDNA contained in ATCC Deposit No. 97920, or from
1 to about 233 amino acids from the carboxy terminus, or any combination of
the
above amino terminal and carboxy terminal deletions, ofthe complete amino acid
sequence encoded by the cDNA contained in ATCC Deposit No. 97920.
Polynucleotides encoding all ofthe above deletion mutant polypeptide forms
also
are provided.
Preferred amongst the N- and C-terminal deletion mutants are those
comprising, or alternatively consisting of, only a portion of the
extracellular
domain; i.e., within residues 52-184, since any portion therein is expected to
be
soluble.
It will be recognized in the art that some amino acid sequence of DRS can
be varied without significant effect of the structure or function of the
protein. If
such differences in sequence are contemplated, it should be remembered that
there will be critical areas on the protein which determine activity. Such
areas
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will usually comprise residues which make up the ligand binding site or the
death
domain, or which form tertiary structures which affect these domains.
Thus, the invention further includes variations of the DRS protein which
show substantial DRS protein activity or which include regions of DRS, such as
the protein portions discussed below. Such mutants include deletions,
insertions,
inversions, repeats, and type substitutions. As indicated above, guidance
concerning which amino acid changes are likely to be phenotypically silent can
be found in Bowie, J.U. et al., Science 247:1306-1310 (1990).
Thus, the fragment, derivative, or analog of the polypeptide of SEQ 1D
N0:2, or that encoded by the deposited cDNA, may be (i) one in which at least
one or more of the amino acid residues are substituted with a conserved or non-
conserved amino acid residue (preferably a conserved amino acid residue(s),
and
more preferably at least one but less than ten conserved amino acid residues)
and
such substituted amino acid residue may or may not be one encoded by the
genetic code, or (ii) one in which one or more of the amino acid residues
includes
a substituent group, or (iii) one in which the mature polypeptide is fused
with
another compound, such as a compound to increase the half life of the
polypeptide (for example, polyethylene glycol), or (iv) one in which the
additional amino acids are fused to the mature polypeptide, such as an IgG Fc
fusion region peptide or leader or secretory sequence or a sequence which is
employed for purification of the mature polypeptide or a proprotein sequence.
Such fragments, derivatives and analogs are deemed to be within the scope of
those skilled in the art from the teachings herein. Polynucleotides encoding
these
fragments, derivatives or analogs are also encompassed by the invention.
Of particular interest are substitutions of charged amino acids with
another charged amino acids and with neutral or negatively charged amino
acids.
The latter results in proteins with reduced positive charge to improve the
characteristics of the DRS protein. Additionally, one or more of the amino
acid
residues of the polypeptides of the invention (e.g., arginine and lysine
residues)
may be deleted or substituted with another residue to eliminate undesired
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processing by proteases such as, for example, furins or kexins. The prevention
of aggregation is highly desirable. Aggregation of proteins not only results
in a
loss of activity but can also be problematic when preparing pharmaceutical
formulations, because they can be immunogenic. (Pinckard et al., Clin Exp.
Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987);
Cleland etal. Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993)).
The replacement of amino acids can also change the selectivity of binding
to cell surface receptors. Ostade et al., Nature 361:266-268 (1993) describes
certain mutations resulting in selective binding of TNF-alpha to only one of
the
two known types of TNF receptors. Thus, the DRS receptor of the present
invention may include one or more amino acid substitutions, deletions or
additions, either from natural mutations or human manipulation.
As indicated, changes are preferably of a minor nature, such as
conservative amino acid substitutions that do not significantly affect the
folding
1 S or activity of the protein (see Table II).
TABLE II. Conservative Amino Acid Substitution
Aromatic Phenylalanine
Tryptophan
Tyrosine
Hydrophobic Leucine
Isoleucine
Valine
Polar ~ Glutamine
Asparagine
Basic Arginine
3 0 Lysine
Histidine
Acidic Aspartic Acid
Glutamic Acid
Small Alanine
Serine
Threonine
Methionine
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In specific embodiments, the number of substitutions, additions or
deletions in the amino acid sequence of FIG. 1 and/or any of the polypeptide
fragments described herein (e.g., the extracellular domain or intracellular
domain)
is 75, 70, 60, 50, 40, 35, 30, 25, 20, 1 S, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or
30-20, 20-
15, 20-10, 15-10, 10-1, S-10, 1-5, 1-3 or 1-2. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
Amino acids in the DRS protein of the present invention that are essential
for function can be identified by methods known in the art, such as site-
directed
mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science
244:1081-1085 (1989)). The latter procedure introduces single alanine
mutations at every residue in the molecule. The resulting mutant molecules are
then tested for biological activity such as receptor binding or in vitro, or
in vitro
proliferative activity. Sites that are critical for ligand-receptor binding
can also
be determined by structural analysis such as crystallization, nuclear magnetic
resonance or photoa~nity labeling (Smith et al., J. Mol. Biol. 224:899-904
(1992) and de Vos et al. Science 255:306-312 (1992)).
Additionally, protein engineering may be employed to improve or alter
the characteristics of DRS polypeptides. Recombinant DNA technology known
to those skilled in the art can be used to create novel mutant proteins or
muteins
including single or multiple amino acid substitutions, deletions, additions or
fusion proteins. Such modified polypeptides can show, e.g., enhanced activity
or increased stability. In addition, they may be purified in higher yields and
show
better solubility than the corresponding natural polypeptide, at least under
certain
purification and storage conditions.
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques, which include, but are not limited to oligonucleotide
mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed
mutagenesis (see e.g., Carter et al., Nucl. AcidsRes. 13:4331 (1986); and
Zoller
et al., Nucd. Acids Res. 10:6487 (1982)), cassette mutagenesis (see e.g.,
Wells
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et al., Gene 34:315 (1985)), and restriction selection mutagenesis (see e.g.,
Wells et al., Philos. Traps. R. Soc. London SerA 317:415 (1986)).
Thus, the invention also encompasses DRS derivatives and analogs that
have one or more amino acid residues deleted, added, or substituted to
generate
DRS polypeptides that are better suited for expression, scale up, etc., in the
host
cells chosen. For example, cysteine residues can be deleted or substituted
with
another amino acid residue in order to eliminate disulfide bridges; N-linked
glycosylation sites can be altered or eliminated to achieve, for example,
expression of a homogeneous product that is more easily recovered and purified
from yeast hosts which are known to hyperglycosylate N-linked sites. To this
end, a variety of amino acid substitutions at one or both of the first or
third
amino acid positions on any one or more of the glycosylation recognitions
sequences in the DRS polypeptides of the invention, and/or an amino acid
deletion at the second position of any one or more such recognition sequences
will prevent glycosylation of the DRS at the modified tripeptide sequence
(see,
e.g., Miyajimo et al., ENIBO J 5(6):1193-1197).
The polypeptides of the present invention also include a polypeptide
comprising, or alternatively consisting of, one, two, three, four, five or
more
amino acid sequences selected from the group consisting of: the polypeptide
encoded by the deposited cDNA (the deposit having ATCC Accession Number
97920) including the leader; the mature polypeptide encoded by the deposited
the
cDNA minus the leader (i.e., the mature protein); a polypeptide comprising, or
alternatively consisting of, amino acids from about -51 to about 360 in SEQ ID
N0:2; a polypeptide comprising, or alternatively consisting of, amino acids
from
about -50 to about 360 in SEQ TD N0:2; a polypeptide comprising, or
alternatively consisting of, amino acids from about 1 to about 360 in SEQ ID
N0:2; a polypeptide comprising, or alternatively consisting of, the DRS
extracellular domain; a polypeptide comprising, or alternatively consisting
of, the
DRS cysteine rich domain; a polypeptide comprising, or alternatively
consisting
of, the DRS transmembrane domain; a polypeptide comprising, or alternatively
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consisting of, the DRS intracellular domain; a polypeptide comprising, or
alternatively consisting of, the extracellular and intracellular domains with
all or
part of the transmembrane domain deleted; and a polypeptide comprising, or
alternatively consisting of, the DRS death domain; as well as polypeptides
which
S are at least 80% identical, more preferably at least 90% or 95% identical,
still
more preferably at least 96%, 97%, 98%, or 99% identical to the polypeptides
described above, and also include portions of such polypeptides with at least
30
amino acids and more preferably at least 50 amino acids. Polynucleotides
encoding these polypeptides are also encompassed by the invention.
By a polypeptide having an amino acid sequence at least, for example,
95% "identical" to a reference amino acid sequence of a DRS polypeptide is
intended that the amino acid sequence of the polypeptide is identical to the
reference sequence except that the polypeptide sequence may include up to five
amino acid alterations per each 100 amino acids of the reference amino acid of
the DRS polypeptide. In other words, to obtain a polypeptide having an amino
acid sequence at least 95% identical to a reference amino acid sequence, up to
5% of the amino acid residues in the reference sequence may be deleted or
substituted with another amino acid, or a number of amino acids up to 5% of
the
total amino acid residues in the reference sequence may be inserted into the
reference sequence. These alterations ofthe reference sequence may occur at
the
amino or carboxy terminal positions of the reference amino acid sequence or
anywhere between those terminal positions, interspersed either individually
among residues in the reference sequence or in one or more contiguous groups
within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the
amino acid sequence shown in FIG. 1 (SEQ ID N0:2), the amino acid sequence
encoded by the deposited cDNA, or fragments thereof, can be determined
conventionally using known computer programs such the Bestfit program
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer
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Group, University Research Park, 575 Science Drive, Madison, WI 53711).
When using Bestfit or any other sequence alignment program to determine
whether a particular sequence is, for instance, 95% identical to a reference
sequence according to the present invention, the parameters are set, of
course,
such that the percentage of identity is calculated over the full length of the
reference amino acid sequence and that gaps in homology of up to 5% of the
total number of amino acid residues in the reference sequence are allowed.
In a specific embodiment, the identity between a reference (query)
sequence (a sequence of the present invention) and a subject sequence, also
referred to as a global sequence alignment, is determined using the FASTDB
computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci.
6:237-245 (1990)). Preferred parameters used in a FASTDB amino acid
alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining
Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window
Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window
Size=500 or the length of the subject amino acid sequence, whichever is
shorter.
According to this embodiment, if the subject sequence is shorter than the
query
sequence due to N- or C-terminal deletions, not because of internal deletions,
a
manual correction is made to the results to take into consideration the fact
that
the FASTDB program does not account for N- and C-terminal truncations of the
subject sequence when calculating global percent identity. For subject
sequences
truncated at the N- and C-termini, relative to the query sequence, the percent
identity is corrected by calculating the number of residues of the query
sequence
that are N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent of the
total
bases of the query sequence. A determination of whether a residue is
matched/aligned is determined by results of the FASTDB sequence alignment.
This percentage is then subtracted from the percent identity, calculated by
the
above FASTDB program using the specified parameters, to arrive at a final
percent identity score. This final percent identity score is what is used for
the
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purposes of this embodiment. Only residues to the N- and C-termini of the
subject sequence, which are not matched/aligned with the query sequence, are
considered for the purposes of manually adjusting the percent identity score.
That is, only query residue positions outside the farthest N- and C-terminal
residues of the subject sequence. For example, a 90 amino acid residue subject
sequence is aligned with a 100 residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject sequence and
therefore, the FASTDB alignment does not show a matching/alignment of the
first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of
the sequence (number of residues at the N- and C-termini not matched/total
number of residues in the query sequence) so 10% is subtracted from the
percent
identity score calculated by the FASTDB program. If the remaining 90 residues
were perfectly matched the final percent identity would be 90%. In another
example, a 90 residue subject sequence is compared with a 100 residue query
sequence. This time the deletions are internal deletions so there are no
residues
at the N- or C-termini of the subject sequence which are not matched/aligned
with the query. In this case the percent identity calculated by FASTDB is not
manually corrected. Once again, only residue positions outside the N- and C-
terminal ends of the subject sequence, as displayed in the FASTDB alignment,
which are not matched/aligned with the query sequence are manually corrected
for. No other manual corrections are made for the purposes of this embodiment.
The polypeptide of the present invention have uses that include, but are
not limited to, use as a molecular weight marker on SDS-PAGE gels or on
molecular sieve gel filtration columns and as a source for generating
antibodies
that bind the polypeptides of the invention, using methods well known to those
of skill in the art.
The present application is also directed to proteins containing
polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%
identical to the DRS polypeptide sequence set forth herein as nl-m', and/or nz-
mz.
In preferred embodiments, the application is directed to proteins containing
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polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%
identical to polypeptides having the amino acid sequence of the specific DRS N-
and C-terminal deletions recited herein. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
In certain preferred embodiments, DRS proteins of the invention
comprise fusion proteins as described above wherein the DRS polypeptides are
those described as n'-m', and n2-m2, herein. In preferred embodiments, the
application is directed to nucleic acid molecules at least 80%, 85%, 90%, 92%,
95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences encoding
polypeptides having the amino acid sequence of the specific N- and C-terminal
deletions recited herein. Polynucleotides encoding these polypeptides are also
encompassed by the invention.
The present inventors have discovered that the DRS polypeptide is a 411
residue protein exhibiting three main structural domains. First, the ligand
binding
1 S domain (extracellular domain) was identified within residues from about 52
to
about 184 in FIG. 1 (amino acid residues from about 1 to about 133 in SEQ ID
N0:2). Second, the transmembrane domain was identified within residues from
about 185 to about 208 in FIG. 1 (amino acid residues from about 134 to about
157 in SEQ 117 N0:2). Third, the intracellular domain was identified within
residues from about 209 to about 411 in FIG. 1 (amino acid residues from about
158 to about 360 in SEQ ID N0:2). Importantly, the intracellular domain
includes a death domain at residues from about 324 to about 391 (amino acid
residues from about 273 to about 340 in SEQ ID N0:2). Further preferred
fragments of the polypeptide shown in FIG. 1 include the mature protein from
residues about 52 to about 411 (amino acid residues from about 1 to about 360
in SEQ ID N0:2), and soluble polypeptides comprising all or part of the
extracellular and intracellular domains but lacking the transmembrane domain.
The invention further provides DRS polypeptides encoded by the
deposited cDNA including the leader and DRS polypeptide fragments selected
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from the mature protein, the extracellular domain, the transmembrane domain,
the intracellular domain, the death domain, and all combinations thereof.
In addition, proteins of the invention can be chemically synthesized using
techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures
and
S Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller, M., et al.,
Nature 310:105-111 (1984)). For example, a peptide corresponding to a
fragment of the DRS polypeptides of the invention can be synthesized by use of
a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or
chemical amino acid analogs can be introduced as a substitution or addition
into
the DRS polypeptide sequence. Non-classical amino acids include, but are not
limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric
acid,
a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-
Abu,
e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic
acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,
homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as
b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino
acid analogs in general. Furthermore, the amino acid can be D (dextrorotary)
or
L (levorotary).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques, which include, but are not limited to oligonucleotide
mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed
mutagenesis(.oee, e.g., Carter etal.., Nucl. AcidsRes. 13:4331 (1986);
andZoller
et al., Nucl. Acids Res. 10:6487 (1982)), cassette mutagenesis (see, e.g.,
Wells
e1 al., Gene 34:315 (1985)), restriction selection mutagenesis (see, e.g.,
Wells
et al., Phidos. Traps. R. Soc. London SerA 317:415 (1986)).
The invention additionally, encompasses DRS polypeptides which are
dif~'erentially modified during or after translation, e.g., by glycosylation,
acetylation, phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to an antibody
molecule
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or other cellular ligand, etc. Any of numerous chemical modifications may be
carried out by known techniques, including but not limited to, specific
chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease,
NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in
the
presence of tunicamycin; etc.
Additional post-translational modifications encompassed by the invention
include, for example, e.g., N-linked or O-linked carbohydrate chains,
processing
of N-terminal or C-terminal ends), attachment of chemical moieties to the
amino
acid backbone, chemical modifications of N-linked or O-linked carbohydrate
chains, and addition or deletion of an N-terminal methionine residue as a
result
of procaryotic host cell expression. The polypeptides may also be modified
with
a detectable label, such as an enzymatic, fluorescent, isotopic or affinity
label to
allow for detection and isolation of the protein.
Also provided by the invention are chemically modified derivatives of
DRS which may provide additional advantages such as increased solubility,
stability and circulating time of the polypeptide, or decreased immunogenicity
(see U. S. Patent No. 4,179,337). The chemical moieties for derivitization may
be selected from water soluble polymers such as polyethylene glycol, ethylene
glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl
alcohol and the like. The polypeptides may be modified at random positions
within the molecule, or at predetermined positions within the molecule and may
include one, two, three or more attached chemical moieties.
The polymer may be of any molecular weight, and may be branched or
unbranched. For polyethylene glycol, the preferred molecular weight is between
about 1 kDa and about 100 kDa (the term "about" indicating that in
preparations
of polyethylene glycol, some molecules will weigh more, some less, than the
stated molecular weight) for ease in handling and manufacturing. Other sizes
may be used, depending on the desired therapeutic profile (e.g., the duration
of
sustained release desired, the effects, if any on biological activity, the
ease in
handling, the degree or lack of antigenicity and other known effects of the
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polyethylene glycol to a therapeutic protein or analog). For example, the
polyethylene glycol may have an average molecular weight of about 200, 500,
1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500,
7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000,
12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500,
17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000,
35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000,
85,000, 90,000, 95,000, or 100,000 kDa.
As noted above, the polyethylene glycol may have a branched structure.
Branched polyethylene glycols are described, for example, in U.S. Patent No.
5,643,575; Morpurgo et al., Appd. Biochem. Biotechnol. 56:59-72 (1996);
Vorobjev et al., Nucleoside.sNucleotides 18:2745-2750 (1999); and Caliceti et
al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are
incorporated herein by reference.
The polyethylene glycol molecules (or other chemical moieties) should
be attached to the protein with consideration of effects on functional or
antigenic
domains of the protein. There are a number of attachment methods available to
those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference
(coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035
(1992) (reporting pegylation of GM-CSF using tresyl chloride). For example,
polyethylene glycol may be covalently bound through amino acid residues via a
reactive group, such as, a free amino or carboxyl group. Reactive groups are
those to which an activated polyethylene glycol molecule may be bound. The
amino acid residues having a free amino group may include lysine residues and
the N-terminal amino acid residues; those having a free carboxyl group may
include aspartic acid residues glutamic acid residues and the C-terminal amino
acid residue. Sulfhydryl groups may also be used as a reactive group for
attaching the polyethylene glycol molecules. Preferred for therapeutic
purposes
is attachment at an amino group, such as attachment at the N-terminus or
lysine
group.
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As suggested above, polyethylene glycol may be attached to proteins via
linkage to any of a number of amino acid residues. For example, polyethylene
glycol can be linked to a proteins via covalent bonds to lysine, histidine,
aspartic
acid, glutamic acid,~or cysteine residues. One or more reaction chemistries
may
be employed to attach polyethylene glycol to specific amino acid residues
(e.g.,
lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein
or to
more than one type of amino acid residue (e.g., lysine, histidine, aspartic
acid,
glutamic acid, cysteine and combinations thereof) of the protein.
One may specifically desire proteins chemically modified at the
N-terminus. Using polyethylene glycol as an illustration of the present
composition, one may select from a variety of polyethylene glycol molecules
(by
molecular weight, branching, etc.), the proportion of polyethylene glycol
molecules to protein (or peptide) molecules in the reaction mix, the type of
pegylation reaction to be performed, and the method of obtaining the selected
N-terminally pegylated protein. The method of obtaining the N-terminally
pegylated preparation (i.e., separating this moiety from other monopegylated
moieties if necessary) may be by purification of the N-terminally pegylated
material from a population of pegylated protein molecules. Selective proteins
chemically modified at the N-terminus modification may be accomplished by
reductive alkylation which exploits differential reactivity of different types
of
primary amino groups (lysine versus the N-terminal) available for
derivatization
in a particular protein. Under the appropriate reaction conditions,
substantially
selective derivatization of the protein at the N-terminus with a carbonyl
group
containing polymer is achieved.
As indicated above, pegylation of the proteins of the invention may be
accomplished by any number of means. For example, polyethylene glycol may
be attached to the protein either directly or by an intervening linker.
Linkerless
systems for attaching polyethylene glycol to proteins are described in Delgado
et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al.,
Intern. J. ofHematol. 68:1-18 (1998); U.S. Patent No. 4,002,531; U.S. Patent
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No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of
which are incorporated herein by reference.
One system for attaching polyethylene glycol directly to amino acid
residues of proteins without an intervening linker employs tresylated MPEG,
which is produced by the modification of monmethoxy polyethylene glycol
(MPEG) using tresylchloride (C1SOZCHZCF3). Upon reaction of protein with
tresylated MPEG, polyethylene glycol is directly attached to amine groups
ofthe
protein. Thus, the invention includes protein-polyethylene glycol conjugates
produced by reacting proteins of the invention with a polyethylene glycol
molecule having a 2,2,2-trifluoreothane sulphonyl group.
Polyethylene glycol can also be attached to proteins using a number of
different intervening linkers. For example, U. S. Patent No. 5,612,460, the
entire
disclosure ofwhich is incorporated herein by reference, discloses urethane
linkers
for connecting polyethylene glycol to proteins. Protein-polyethylene glycol
conjugates wherein the polyethylene glycol is attached to the protein by a
linker
can also be produced by reaction of proteins with compounds such as MPEG-
succinimidylsuccinate, MPEG activated with 1,1'-carbonyldiimidazole, MPEG-
2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various
MPEG-succinate derivatives. A number additional polyethylene glycol
derivatives and reaction chemistries for attaching polyethylene glycol to
proteins
are described in WO 98/32466, the entire disclosure of which is incorporated
herein by reference. Pegylated protein products produced using the reaction
chemistries set out herein are included within the scope of the invention.
The number of polyethylene glycol moieties attached to each protein of
the invention (i. e., the degree of substitution) may also vary. For example,
the
pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4,
5, 6,
7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly,
the
average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7,
6-8,
7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-
20 polyethylene glycol moieties per protein molecule. Methods for determining
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the degree of substitution are discussed, for example, in Delgado et al.,
Crit. Rev.
Thera. Drug Carrier Sys. 9:249-304 (1992).
As mentioned, DRS polypeptides may be modified by either natural
processes, such as posttranslational processing, or by chemical modification
techniques which are well known in the art. It will be appreciated that the
same
type of modification may be present in the same or varying degrees at several
sites in a given DRS polypeptide. Also, a given DRS polypeptide may contain
many types of modifications. DRS polypeptides may be branched, for example,
as a result of ubiquitination, and they may be cyclic, with or without
branching.
Cyclic, branched, and branched cyclic DRS polypeptides may result from
posttranslation natural processes or may be made by synthetic methods.
Modifications include acetylation, acylation, ADP-ribosylation, amidation,
covalent attachment of flavin, covalent attachment of a heme moiety, covalent
attachment of a nucleotide or nucleotide derivative, covalent attachment of a
lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-
linking, cyclization, disulfide bond formation, demethylation, formation of
covalent cross-links, formation of cysteine, formation of pyroglutamate,
formylation, gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation,
proteolytic processing, phosphorylation, prenylation, racemization,
selenoylation,
sulfation, transfer-RNA mediated addition of amino acids to proteins such as
arginylation, and ubiquitination. (See, for instance, PROTEINS - STRUCTURE
AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman
and Company, New York (1993); POSTTRANSLATIONAL COVALENT
MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New
York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990);
Rattan et al., Ann NY Acad Sci 663:48-62 (1992)).
The DRS polypeptides can be recovered and purified from chemical
synthesis and recombinant cell cultures by standard methods which include, but
are not limited to, ammonium sulfate or ethanol precipitation, acid
extraction,
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anion or canon exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most preferably,
high performance liquid chromatography ("HPLC") is employed for purification.
Well known techniques for refolding protein may be employed to regenerate
active conformation when the polypeptide is denatured during isolation and/or
purification.
DRS polynucleotides and polypeptides may be used in accordance with
the present invention for a variety of applications, particularly those that
make
use of the chemical and biological properties of DRS. Among these are
applications in the treatment and/or prevention of tumors, parasitic
infections,
bacterial infections, viral infections, restenosis, and graft vs. host
disease; to
induce resistance to parasites, bacteria and viruses; to induce proliferation
of T-
cells, endothelial cells and certain hematopoietic cells; to regulate anti-
viral
1 S responses; and to treat and/or prevent certain autoimmune diseases after
stimulation of DRS by an agonist. Additional applications relate to diagnosis,
treatment, and/or prevention of disorders of cells, tissues and organisms.
These
aspects of the invention are discussed further below.
The present invention encompasses polypeptides comprising, or
alternatively consisting of, an epitope of the polypeptide having an amino
acid
sequence of SEQ ID N0:2, or an epitope of the polypeptide sequence encoded
by a polynucleotide sequence contained in the cDNA deposited as ATCC
Deposit No. 97920 or encoded by a polynucleotide that hybridizes to the
complement of the sequence of SEQ ID NO:1 or contained in the cDNA
deposited as ATCC Deposit No. 97920 under stringent hybridization conditions
or lower stringency hybridization conditions as defined supra. The present
invention further encompasses polynucleotide sequences encoding an epitope of
a polypeptide sequence of the invention (such as, for example, the sequence
disclosed in SEQ B7 NO:1), polynucleotide sequences of the complementary
strand of a polynucleotide sequence encoding an epitope of the invention, and
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polynucleotide sequences which hybridize to the complementary strand under
stringent hybridization conditions or lower stringency hybridization
conditions
defined supra.
In another aspect, the invention provides a peptide or polypeptide
comprising an epitope-bearing portion of a polypeptide described herein. The
epitope of this polypeptide portion is an immunogenic or antigenic epitope of
a
polypeptide of the invention. The term "epitopes," as used herein, refers to
portions of a polypeptide having antigenic or immunogenic activity in an
animal,
preferably a mammal, and most preferably in a human. In a preferred
embodiment, the present invention encompasses a polypeptide comprising an
epitope, as well as the polynucleotide encoding this polypeptide. An
"immunogenic epitope" is defined as a part of a protein that elicits an
antibody
response when the whole protein is the immunogen. On the other hand, a region
of a protein molecule to which an antibody can bind is defined as an
"antigenic
epitope." The number of immunogenic epitopes of a protein generally is less
than the number of antigenic epitopes. See, for instance, Geysen et al., Proc.
Natl. Acad. Sci. USA 81:3998- 4002 (1983).
Fragments that function as epitopes may be produced by any conventional
means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-S 135 (1985),
further described in U.S. Patent No. 4,631,21 I).
As to the selection of peptides or polypeptides bearing an antigenic
epitope (i.e., that contain a region of a protein molecule to which an
antibody can
bind), it is well known in that art that relatively short synthetic peptides
that
mimic part of a protein sequence are routinely capable of eliciting an
antiserum
that reacts with the partially mimicked protein. See, for instance, Sutcli~e,
J. G.,
Shinnick, T. M., Green, N. and Learner, R.A., "Antibodies That React With
Predetermined Sites on Proteins," Science 219:660-666 (1983). Peptides
capable of eliciting protein-reactive sera are frequently represented in the
primary
sequence of a protein, can be characterized by a set of simple chemical rules,
and
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are confined neither to immunodominant regions of intact proteins (i.e.,
immunogenic epitopes) nor to the amino or carboxyl terminals.
Non-limiting examples of antigenic polypeptides or peptides that can be
used to generate DRS-specific antibodies include: a polypeptide comprising, or
S alternatively consisting of, amino acid residues from about 62 to about 110
in
FIG. 1 (about 11 to about 59 in SEQ ID N0:2); a polypeptide comprising, or
alternatively consisting of, amino acid residues from about 119 to about 164
in
FIG. 1 (about 68 to about 113 in SEQ ID N0:2); a polypeptide comprising, or
alternatively consisting of, amino acid residues from about 224 to about 271
in
FIG. 1 (about 173 to about 220 in SEQ ID N0:2); and a polypeptide comprising,
or alternatively consisting of, amino acid residues from about 275 to about
370
in FIG. 1 (about 224 to about 319 in SEQ ID N0:2). As indicated above, the
inventors have determined that the above polypeptide fragments are antigenic
regions of the DRS protein.
The epitope-bearing peptides and polypeptides of the invention may be
produced by any conventional means. Hougthen, R.A., "General Method for the
Rapid Solid-Phase Synthesis of Large Numbers of Peptides: Specificity of
Antigen-Antibody Interaction at the Level of Individual Amino Acids," Proc.
Natl. Acad. Sci. USA 82:5131-5135 (1985). This "Simultaneous Multiple
Peptide Synthesis (SMPS)" process is further described in U.S. Patent No.
4,631,211 to Hougthen et al. (1986). As one of skill in the art will
appreciate,
DRS polypeptides of the present invention and the epitope-bearing fragments
thereof described above can be combined with parts of the constant domain of
immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion
proteins facilitate purification and show an increased half life in vivo. This
has
been shown, e.g. , for chimeric proteins consisting of the first two domains
of the
human CD4-polypeptide and various domains of the constant regions of the
heavy or light chains ofmammalian immunoglobulins (EPA 394,827; Traunecker
et al., Nature 331:84- 86 (1988)). Fusion proteins that have a disulfide-
linked
dimeric structure due to the IgG part can also be more efficient in binding
and
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neutralizing other molecules than the monomeric DRS protein or protein
fragment alone (Fountoulakis et al., JBiochem. 270:3958-3964 (1995)).
Antibodies
S
The present invention further relates to antibodies and T-cell antigen
receptors (TCR) which immunospecifically bind a polypeptide, preferably an
epitope, of the present invention (as determined by immunoassays well known
in the art for assaying specific antibody-antigen binding). Antibodies of the
invention include, but are not limited to, polyclonal, monoclonal,
multispecific,
human, humanized or chimeric antibodies, single chain antibodies, Fab
fragments,
F(ab') fragments, fragments produced by a Fab expression library, anti-
idiotypic
(anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the
invention), and epitope-binding fragments of any of the above. The term
"antibody," as used herein, refers to immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules, i.e., molecules
that contain an antigen binding site that immunospecifically binds an antigen.
The immunoglobulin molecules of the invention can be of any type (e.g., IgG,
IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAI and
IgA2) or subclass of immunoglobulin molecule.
Most preferably the antibodies are human antigen-binding antibody
fragments of the present invention and include, but are not limited to, Fab,
Fab'
and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-
linked
Fvs (sdFv) and fragments comprising either a Vr, or VH domain. Antigen-binding
antibody fragments, including single-chain antibodies, may comprise the
variable
regions) alone or in combination with the entirety or a portion of the
following:
hinge region, CH1, CH2, and CH3 domains. Also included in the invention are
antigen-binding fragments also comprising any combination ofvariable regions)
with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the
invention may be from any animal origin including birds and mammals.
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Preferably, the antibodies are human, murine, donkey, ship rabbit, goat,
guinea
pig, camel, horse, or chicken. As used herein, "human" antibodies include
antibodies having the amino acid sequence of a human immunoglobulin and
include antibodies isolated from human immunoglobulin libraries or from
animals
transgenic for one or more human immunoglobulin and that do not express
endogenous immunoglobulins, as described infra and, for example in, U.S.
Patent No. 5,939,598 by Kucherlapati et al.
The antibodies of the present invention may be monospecific, bispecific,
trispecific or of greater multispecificity. Multispecific antibodies may be
specific
for different epitopes of a polypeptide of the present invention or may be
specific
for both a polypeptide of the present invention as well as for a heterologous
epitope, such as a heterologous polypeptide or solid support material. See,
e.g.,
PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793;
Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Patent Nos. 4,474,893;
4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.
148:1547-1553 (1992).
Antibodies ofthe present invention may be described or specified in terms
of the epitope(s) or portions) of a polypeptide of the present invention that
they
recognize or specifically bind. The epitope(s) or polypeptide portions) may be
specified as described herein, e.g., by N-terminal and C-terminal positions,
by
size in contiguous amino acid residues, or listed in the Tables and Figures.
Antibodies that specifically bind any epitope or polypeptide of the present
invention may also be excluded. Therefore, the present invention includes
antibodies that specifically bind polypeptides of the present invention, and
allows
for the exclusion of the same.
Antibodies of the present invention may also be described or specified in
terms of their cross-reactivity. Antibodies that do not bind any other analog,
ortholog, or homolog of a polypeptide of the present invention are included.
Antibodies that bind polypeptides with at least 95%, at least 90%, at least
85%,
at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least
55%,
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and at least 50% identity (as calculated using methods known in the art and
described herein) to a polypeptide of the present invention are also included
in
the present invention. Antibodies that do not bind polypeptides with less than
95%, less than 90%, less than 85%, less than 80%, less than 75%, less than
70%,
S less than 65%, less than 60%, less than 55%, and less than 50% identity (as
calculated using methods known in the art and described herein) to a
polypeptide
of the present invention are also included in the present invention. Further
included in the present invention are antibodies that bind polypeptides
encoded
by polynucleotides which hybridize to a polynucleotide of the present
invention
under stringent hybridization conditions (as described herein). Antibodies of
the
present invention may also be described or specified in terms of their binding
affnity to a polypeptide of the invention. Preferred binding affinities
include
those with a dissociation constant or Kd less than SX10-ZM, 10-ZM, SX10-3M,
10-3M, SX10-4M, 10-4M, SX10-SM, 10-SM, SX10-~M, 10-6M, SX10-'M, 10-'M,
5 X 10-gM, 10-gM, 5 X 10-9M, 10~9M, 5 X 10-' °M, 10-' °M, 5 X 10-
"M, 10-"M,
SX10-'ZM, 10-'zM, SX10-"M, 10-'3M, SX10-'4M, 10-'4M, SX10-'SM, and 10-'SM.
The invention also provides antibodies that competitively inhibit binding
of an antibody to an epitope of the invention as determined by any method
known in the art for determining competitive binding, for example, the
immunoassays described herein. In preferred embodiments, the antibody
competitively inhibits binding to the epitope by at least 90%, at least 80%,
at
least 70%, at least 60%, or at least 50%.
Antibodies of the present invention may act as agonists or antagonists of
the polypeptides of the present invention. For example, the present invention
includes antibodies which disrupt the receptor/ligand interactions with the
polypeptides of the invention either partially or fully. The invention
features both
receptor-specific antibodies and ligand-specific antibodies. The invention
also
features receptor-specific antibodies which do not prevent ligand binding but
prevent receptor activation. Receptor activation (i.e., signaling) may be
determined by techniques described herein or otherwise known in the art. For
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example, receptor activation can be determined by detecting the
phosphorylation
(e.g., tyrosine or serine/threonine) of the receptor or its substrate by
immunoprecipitation followed by western blot analysis (for example, as
described
supra). In specific embodiments, antibodies are provided that inhibit ligand
or
receptor activity by at least 90%, at least 80%, at least 70%, at least 60%,
or at
least 50% of the activity in absence of the antibody.
The invention also features receptor-specific antibodies which both
prevent ligand binding and receptor activation as well as antibodies that
recognize the receptor-ligand complex, and, preferably, do not specifically
recognize the unbound receptor or the unbound ligand. Likewise, included in
the
invention are neutralizing antibodies which bind the ligand and prevent
binding
of the ligand to the receptor, as well as antibodies which bind the ligand,
thereby
preventing receptor activation, but do not prevent the ligand from binding the
receptor. Further included in the invention are antibodies which activate the
receptor. These antibodies may act as receptor agonists, i.e., potentiate or
activate either all or a subset of the biological activities of the ligand-
mediated
receptor activation. The antibodies may be specified as agonists, antagonists
or
inverse agonists for biological activities comprising the specific biological
activities of the peptides of the invention disclosed herein. Thus, the
invention
further relates to antibodies which act as agonists or antagonists of the
polypeptides of the present invention. The above antibody agonists can be made
using methods known in the art. See, e.g., PCT publication WO 96/40281; U. S.
Patent No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al.,
Cancer Re.s. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.
161(4):1786-1794 (1998); Zhu et al.., Cancer Res. 58(15):3209-3214 (1998);
Yoon et al., .l. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci.
lll(Pt2):237-247 (1998); Pitard et al., .I. Immunol. Methods 205(2):177-190
(1997); Liautard et al., Cytokirte 9(4):233-241 (1997); Carlson et al., .l.
Biol.
Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762
(1995); Mulleretal., Structure 6(9):1153-1167 (1998); Bartuneketal., Cytokine
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8(1):14-20 (1996) (which are all incorporated by reference herein in their
entireties).
Antibodies of the present invention may be used, for example, but not
limited to, to purify, detect, and target the polypeptides of the present
invention,
including both in vitro and in vivo diagnostic and therapeutic methods. For
example, the antibodies have use in immunoassays for qualitatively and
quantitatively measuring levels of the polypeptides of the present invention
in
biological samples. S'ee, e.g., Harlow et al., Antibodies: A Laboratory
Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference
herein in its entirety).
As discussed in more detail below, the antibodies of the present invention
may be used either alone or in combination with other compositions. The
antibodies may further be recombinantly fused to a heterologous polypeptide at
the N- or C-terminus or chemically conjugated (including covalently and
non-covalently conjugations) to polypeptides or other compositions. For
example, antibodies of the present invention may be recombinantly fused or
conjugated to molecules useful as labels in detection assays and effector
molecules such as heterologous polypeptides, drugs, or toxins. See, e.g., PCT
publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No.
5,314,995; and EP 396,387.
The antibodies of the invention include derivatives that are modified, i. e,
by the covalent attachment of any type of molecule to the antibody such that
covalent attachment does not prevent the antibody from generating an
anti-idiotypic response. For example, but not by way of limitation, the
antibody
derivatives include antibodies that have been modified, e.g., by
glycosylation,
acetylation, pegylation, phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand
or
other protein, etc. Any of numerous chemical modifications may be carried out
by known techniques, including, but not limited to specific chemical cleavage,
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acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Additionally,
the derivative may contain one or more non-classical amino acids.
The antibodies of the present invention may be generated by any suitable
method known in the art. Polyclonal antibodies to an antigen of interest can
be
produced by various procedures well known in the art. For example, a
polypeptide of the invention can be administered to various host animals
including, but not limited to, rabbits, mice, rats, etc. to induce the
production of
sera containing polyclonal antibodies specific for the antigen. Various
adjuvants
may be used to increase the immunological response, depending on the host
species, and include but are not limited to, Freund's (complete and
incomplete),
mineral gels such as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are
also well known in the art.
Monoclonal antibodies can be prepared using a wide variety oftechniques
known in the art including the use of hybridoma, recombinant, and phage
display
technologies, or a combination thereof. For example, monoclonal antibodies can
be produced using hybridoma techniques including those known in the art and
taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold
Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in:
Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)
(said references incorporated by reference in their entireties). The term
"monoclonal antibody" as used herein is not limited to antibodies produced
through hybridoma technology. The term "monoclonal antibody" refers to an
antibody that is derived from a single clone, including any eukaryotic,
prokaryotic, or phage clone, and not the method by which it is produced. Thus,
the term "monoclonal antibody" is not limited to antibodies produced through
hybridoma technology. Monoclonal antibodies can be prepared using a wide
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variety of techniques known in the art including the use of hybridoma and
recombinant and phage display technology.
Methods for producing and screening for specific antibodies using
hybridoma technology are routine and well-known in the art and are discussed
in detail in Example 11. Briefly, mice can be immunized with a polypeptide of
the invention or a cell expressing such peptide. Once an immune response is
detected, e.g., antibodies specific for the antigen are detected in the mouse
serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes
are then fused by well-known techniques to any suitable myeloma cells, for
example cells from cell line SP20 available from the ATCC. Hybridomas are
selected and cloned by limited dilution. The hybridoma clones are then assayed
by methods known in the art for cells that secrete antibodies capable of
binding
a polypeptide of the invention. Ascites fluid, which generally contains high
levels
of antibodies, can be generated by immunizing mice with positive hybridoma
1 S clones.
Accordingly, the present invention provides methods of generating
monoclonal antibodies as well as antibodies produced by the method comprising
culturing a hybridoma cell secreting an antibody of the invention wherein,
preferably, the hybridoma is generated by fusing splenocytes isolated from a
mouse immunized with an antigen of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma clones that
secrete an antibody able to bind a polypeptide of the invention.
Antibody fragments that recognize specific epitopes may be generated by
known techniques. For example, Fab and F(ab')2 fragments of the invention
may be produced by proteolytic cleavage of immunoglobulin molecules, using
enzymes such as papain (to produce Fab fragments) or pepsin (to produce
F(ab')2 fragments). F(ab')2 fragments contain the variable region, the light
chain
constant region and the CH1 domain of the heavy chain.
For example, the antibodies ofthe present invention can also be generated
using various phage display methods known in the art. In phage display
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methods, functional antibody domains are displayed on the surface of phage
particles which carry the polynucleotide sequences encoding them. In a
particular, such phage can be utilized to display antigen-binding domains
expressed from a repertoire or combinatorial antibody library (e.g., human or
murine). Phage expressing an antigen binding domain that binds the antigen of
interest can be selected or identified with antigen, e.g., using labeled
antigen or
antigen bound or captured to a solid surface or bead. Phage used in these
methods are typically filamentous phage including fd and M13 binding domains
expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII protein.
Examples
of phage display methods that can be used to make the antibodies of the
present
invention include those disclosed in Brinkman et al., .~ Immunol. Methods
182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);
Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene
187:9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994);
PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO
91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225;
5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by
reference in its entirety.
As described in the above references, after phage selection, the antibody
coding regions from the phage can be isolated and used to generate whole
antibodies, including human antibodies, or any other desired antigen binding
fragment, and expressed in any desired host, including mammalian cells, insect
cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments
can also be employed using methods known in the art such as those disclosed in
PCT publication WO 92/22324; Mullinax et al., BioTechniyues 12(6):864-869
(1992); and Sawai et al., A.IRI 34:26-34 (1995); and Better et al., Science
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240:1041-1043 (1988) (said references incorporated by reference in their
entireties).
Examples of techniques which can be used to produce single-chain Fvs
and antibodies include those described in U.S. Patents 4,946,778 and
5,258,498;
Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS
90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For
some uses, including in vivo use of antibodies in humans and in vitro
detection
assays, it may be preferable to use chimeric, humanized, or human antibodies.
A chimeric antibody is a molecule in which different portions of the antibody
are
derived from different animal species, such as antibodies having a variable
region
derived from a murine monoclonal antibody and a human immunoglobulin
constant region. Methods for producing chimeric antibodies are known in the
art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques
4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.
Patent Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein
by reference in their entireties. Humanized antibodies are antibody molecules
from non-human species antibody that binds the desired antigen having one or
more complementarity determining regions (CDRs) from the non-human species
and framework regions from a human immunoglobulin molecule. Often,
framework residues in the human framework regions will be substituted with the
corresponding residue from the CDR donor antibody to alter, preferably
improve, antigen binding. These framework substitutions are identified by
methods well known in the art, e.g., by modeling of the interactions of the
CDR
and framework residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework residues at
particular positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089;
Riechmann et al., Nature 332:323 (1988), which are incorporated herein by
reference in their entireties.) Antibodies can be humanized using a variety of
techniques known in the art including, for example, CDR-grafting (EP 239,400;
PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101; and
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5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan,
Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein
Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)),
and chain shuffling (U.S. Patent No. 5,565,332).
Completely human antibodies are particularly desirable for therapeutic
treatment of human patients. Human antibodies can be made by a variety of
methods known in the art including phage display methods described above using
antibody libraries derived from human immunoglobulin sequences. See also,
U. S. Patent Nos. 4,444, 887 and 4,716,111; and PCT publications WO 98/46645,
WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735,
and WO 91/10741; each of which is incorporated herein by reference in its
entirety.
Human antibodies can also be produced using transgenic mice which are
incapable of expressing functional endogenous immunoglobulins, but which can
express human immunoglobulin genes. For example, the human heavy and light
chain immunoglobulin gene complexes may be introduced randomly or by
homologous recombination into mouse embryonic stem cells. Alternatively, the
human variable region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and light chain
genes. The mouse heavy and light chain immunoglobulin genes may be rendered
non-functional separately or simultaneously with the introduction of human
immunoglobulin loci by homologous recombination. In particular, homozygous
deletion of the JH region prevents endogenous antibody production. The
modified embryonic stem cells are expanded and microinjected into blastocysts
to produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring that express human antibodies. The transgenic mice are
immunized in the normal fashion with a selected antigen, e.g., all or a
portion of
a polypeptide of the invention. Monoclonal antibodies directed against the
antigen can be obtained from the immunized, transgenic mice using conventional
hybridoma technology. The human immunoglobulin transgenes harbored by the
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transgenic mice rearrange during B-cell differentiation, and subsequently
undergo
class switching and somatic mutation. Thus, using such a technique, it is
possible
to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an
overview of this technology for producing human antibodies, see Lonberg and
Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this
technology for producing human antibodies and human monoclonal antibodies
and protocols for producing such antibodies, .see, e.g., PCT publications WO
98/24893; WO 96/34096; WO 96/33735; U.S. Patent Nos. 5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and
5,939,598, which are incorporated by reference herein in their entirety. In
addition, companies such as Abgenix, Inc. (Freemont, CA) and GenPharm (San
Jose, CA) can be engaged to provide human antibodies directed against a
selected antigen using technology similar to that described above.
Completely human antibodies which recognize a selected epitope can be
generated using a technique referred to as "guided selection." In this
approach
a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to
guide the selection of a completely human antibody recognizing the same
epitope. (Jespers et al.., Bioltechnology 12:899-903 (1988)).
Further, antibodies to the polypeptides of the invention can, in turn, be
utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the
invention using techniques well known to those skilled in the art. (See, e.g.,
Greenspan & Bona, FASEB J. 7(5):437-444 (1989) and Nissinoff, J. Immunol.
147(8) :2429-243 8 ( 1991 )). For example, antibodies which bind to and
competitively inhibit polypeptide multimerization and/or binding of a
polypeptide
of the invention to a ligand can be used to generate anti-idiotypes that
"mimic"
the polypeptide multimerization and/or binding domain and, as a consequence,
bind to and neutralize polypeptide and/or its ligand. Such neutralizing
anti-idiotypes or Fab fragments of such anti-idiotypes can be used in
therapeutic
regimens to neutralize polypeptide ligand. For example, such anti-idiotypic
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antibodies can be used to bind a polypeptide of the invention and/or to bind
its
ligands/receptors, and thereby block its biological activity.
A. Polynucleotides Encoding Antibodies.
The invention further provides polynucleotides comprising a nucleotide
sequence encoding an antibody of the invention and fragments thereof. The
invention also encompasses polynucleotides that hybridize under stringent or
lower stringency hybridization conditions, e.g., as defined supra, to
polynucleotides that encode an antibody, preferably, that specifically binds
to a
polypeptide of the invention, preferably, an antibody that binds to a
polypeptide
having the amino acid sequence of SEQ >D N0:2.
The polynucleotides may be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. For example, if
the nucleotide sequence of the antibody is known, a polynucTeotide encoding
the
antibody may be assembled from chemically synthesized oligonucleotides (e.g.,
as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly,
involves the synthesis of overlapping oligonucleotides containing portions
ofthe
sequence encoding the antibody, annealing and ligation ofthose
oligonucleotides,
and then amplification of the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an antibody may be generated
from nucleic acid from a suitable source. If a clone containing a nucleic acid
encoding a particular antibody is not available, but the sequence of the
antibody
molecule is known, a nucleic acid encoding the immunoglobulin may be obtained
from a suitable source (e.g., an antibody cDNA library, or a cDNA library
generated from, or nucleic acid, preferably polyA+ RNA, isolated from, any
tissue or cells expressing the antibody, such as hybridoma cells selected to
express an antibody of the invention) by PCR amplification using synthetic
primers hybridizable to the 3' and 5' ends of the sequence or by cloning using
an
oligonucleotide probe specific for the particular gene sequence to identify,
e.g.,
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a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic
acids generated by PCR may then be cloned into replicable cloning vectors
using
any method well known in the art.
Once the nucleotide sequence and corresponding amino acid sequence of
the antibody is determined, the nucleotide sequence of the antibody may be
manipulated using methods well known in the art for the manipulation of
nucleotide sequences, e.g., recombinant DNA techniques, site directed
mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook
et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998,
Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both
incorporated by reference herein in their entireties), to generate antibodies
having
a different amino acid sequence, for example to create amino acid
substitutions,
deletions, and/or insertions.
In a specific embodiment, the amino acid sequence of the heavy and/or
light chain variable domains may be inspected to identify the sequences of the
complementarity determining regions (CDRs) by methods that are well know in
the art, e.g., by comparison to known amino acid sequences of other heavy and
light chain variable regions to determine the regions of sequence
hypervariability.
Using routine recombinant DNA techniques, one or more of the CDRs may be
inserted within framework regions, e.g., into human framework regions to
humanize a non-human antibody, as described supra. The framework regions
may be naturally occurring or consensus framework regions, and preferably
human framework regions (see, e.g., Chothia et al., .7. Mol. Biol. 278:457-479
( 1998) for a listing of human framework regions). Preferably, the
polynucleotide
generated by the combination of the framework regions and CDRs encodes an
antibody that specifically binds a polypeptide of the invention. Preferably,
as
discussed supra, one or more amino acid substitutions may be made within the
framework regions, and, preferably, the amino acid substitutions improve
binding
of the antibody to its antigen. Additionally, such methods may be used to make
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amino acid substitutions or deletions of one or more variable region cysteine
residues participating in an intrachain disulfide bond to generate antibody
molecules lacking one or more intrachain disulfide bonds. Other alterations to
the polynucleotide are encompassed by the present invention and within the
skill
of the art.
In addition, techniques developed for the production of "chimeric
antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855;
Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature
314:452-454) by splicing genes from a mouse antibody molecule of appropriate
antigen specificity together with genes from a human antibody molecule of
appropriate biological activity can be used. As described supra, a chimeric
antibody is a molecule in which different portions are derived from dii~erent
animal species, such as those having a variable region derived from a murine
monoclonal antibody and a human immunoglobulin constant region, e.g.,
humanized antibodies.
Alternatively, techniques described for the production of single chain
antibodies (U. S. Patent No. 4,694,778; Bird, 1988, Science 242:423-42; Huston
et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989,
Nature 334:544-554) can be adapted to produce single chain antibodies. Single
chain antibodies are formed by linking the heavy and light chain fragments of
the
Fv region via an amino acid bridge, resulting in a single chain polypeptide.
Techniques for the assembly of functional Fv fragments in E coli may also be
used (Skerra et al., 1988, Science 242:1038- 1041).
B. Methods of Producing Antibodies
The antibodies of the invention can be produced by any method known
in the art for the synthesis of antibodies, in particular, by chemical
synthesis or
preferably, by recombinant expression techniques.
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Recombinant expression of an antibody of the invention, or fragment,
derivative or analog thereof, e.g., a heavy or light chain of an antibody of
the
invention, requires construction of an expression vector containing a
polynucleotide that encodes the antibody. Once a polynucleotide encoding an
antibody molecule or a heavy or light chain of an antibody, or portion thereof
(preferably containing the heavy or light chain variable domain), of the
invention
has been obtained, the vector for the production of the antibody molecule may
be produced by recombinant DNA technology using techniques well known in
the art. Thus, methods for preparing a protein by expressing a polynucleotide
containing an antibody encoding nucleotide sequence are described herein.
Methods which are well known to those skilled in the art can be used to
construct expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals. These methods
include, for example, in vitro recombinant DNA techniques, synthetic
techniques,
and in vivo genetic recombination. The invention, thus, provides replicable
vectors comprising a nucleotide sequence encoding an antibody molecule of the
invention, or a heavy or light chain thereof, or a heavy or light chain
variable
domain, operably linked to a promoter. Such vectors may include the nucleotide
sequence encoding the constant region of the antibody molecule (see, e.g., PCT
Publication WO 86/05807; PCT Publication WO 89/01036; and U. S. Patent No.
5,122,464) and the variable domain of the antibody may be cloned into such a
vector for expression of the entire heavy or light chain.
The expression vector is transferred to a host cell by conventional
techniques and the transfected cells are then cultured by conventional
techniques
to produce an antibody of the invention. Thus, the invention includes host
cells
containing a polynucleotide encoding an antibody of the invention, or a heavy
or light chain thereof, operably linked to a heterologous promoter. In
preferred
embodiments for the expression of double-chained antibodies, vectors encoding
both the heavy and light chains may be co-expressed in the host cell for
expression of the entire immunoglobulin molecule, as detailed below.
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A variety of host-expression vector systems may be utilized to express the
antibody molecules of the invention. Such host-expression systems represent
vehicles by which the coding sequences of interest may be produced and
subsequently purified, but also represent cells which may, when transformed or
transfected with the appropriate nucleotide coding sequences, express an
antibody molecule of the invention in situ. These include but are not limited
to
microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing antibody coding sequences; yeast (e.g., Saccharomyces,
Pichia) transformed with recombinant yeast expression vectors containing
antibody coding sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing antibody coding sequences;
plant cell systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed
1 S with recombinant plasmid expression vectors (e.g., Ti plasmid) containing
antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK,
293, 3T3 cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g., metallothionein
promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the
vaccinia virus 7.SK promoter). Preferably, bacterial cells such as Escherichia
coli, and more preferably, eukaryotic cells, especially for the expression
ofwhole
recombinant antibody molecule, are used for the expression of a recombinant
antibody molecule. For example, mammalian cells such as Chinese hamster
ovary cells (CHO), in conjunction with a vector such as the major intermediate
early gene promoter element from human cytomegalovirus is an effective
expression system for antibodies (Foecking et al., 1986, Gene 45:101; Cockett
et al., 1990, BiolTechnology 8:2).
In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the antibody
molecule being expressed. For example, when a large quantity of such a protein
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is to be produced, for the generation of pharmaceutical compositions of an
antibody molecule, vectors which direct the expression of high levels of
fusion
protein products that are readily purified may be desirable. Such vectors
include,
but are not limited, to the E. coli expression vector pUR278 (Ruther et al.,
1983,
S EMBO .l. 2:1791), in which the antibody coding sequence may be ligated
individually into the vector in frame with the lacZ coding region so that a
fusion
protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.
13:3101-3109; VanHeeke& Schuster, 1989, J. Biol. Chem. 24:5503-5509); and
the like. pGEX vectors may also be used to express foreign polypeptides as
fusion proteins with glutathione S-transferase (GST). In general, such fusion
proteins are soluble and can easily be purified from lysed cells by adsorption
and
binding to a matrix glutathione-agarose beads followed by elution in the
presence
of free glutathione. The pGEX vectors are designed to include thrombin or
factor Xa protease cleavage sites so that the cloned target gene product can
be
released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus
(AcNPV) is used as a vector to express foreign genes. The virus grows in
Spodoptera frugiperda cells. The antibody coding sequence may be cloned
individually into non-essential regions (for example the polyhedrin gene) of
the
virus and placed under control of an AcNPV promoter (for example the
polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may
be utilized. In cases where an adenovirus is used as an expression vector, the
antibody coding sequence of interest may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter and
tripartite
leader sequence. This chimeric gene may then be inserted in the adenovirus
genome by in vitro or in vivo recombination. Insertion in a non- essential
region
of the viral genome (e.g., region E 1 or E3) will result in a recombinant
virus that
is viable and capable of expressing the antibody molecule in infected hosts.
(e.g.,
see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific
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initiation signals may also be required for efficient translation of inserted
antibody
coding sequences. These signals include the ATG initiation codon and adjacent
sequences. Furthermore, the initiation codon must be in phase with the reading
frame of the desired coding sequence to ensure translation of the entire
insert.
These exogenous translational control signals and initiation codons can be of
a
variety of origins, both natural and synthetic. The efficiency of expression
may
be enhanced by the inclusion of appropriate transcription enhancer elements,
transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymod.
153:51-544).
In addition, a host cell strain may be chosen which modulates the
expression of the inserted sequences, or modifies and processes the gene
product in the specific fashion desired. Such modifications (e.g.,
glycosylation)
and processing (e.g., cleavage) of protein products may be important for the
function of the protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification of proteins
and gene products. Appropriate cell lines or host systems can be chosen to
ensure the correct modification and processing of the foreign protein
expressed.
To this end, eukaryotic host cells which possess the cellular machinery for
proper
processing of the primary transcript, glycosylation, and phosphorylation of
the
gene product may be used. Such mammalian host cells include but are not
limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in
particular, breast cancer cell lines such as, for example, BT483, Hs578T,
HTB2,
BT20 and T47D, and normal mammary gland cell line such as, for example,
CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable
expression is preferred. For example, cell lines which stably express the
antibody
molecule may be engineered. Rather than using expressionvectors which contain
viral origins of replication, host cells can be transformed with DNA
controlled
by appropriate expression control elements (e.g., promoter, enhancer,
sequences,
transcription terminators, polyadenylation sites, etc.), and a selectable
marker.
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Following the introduction of the foreign DNA, engineered cells may be allowed
to grow for 1-2 days in an enriched media, and then are switched to a
selective
media. The selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into their
chromosomes and grow to form foci which in turn can be cloned and expanded
into cell lines. This method may advantageously be used to engineer cell lines
which express the antibody molecule. Such engineered cell lines may be
particularly useful in screening and evaluation of compounds that interact
directly
or indirectly with the antibody molecule.
A number of selection systems may be used, including but not limited to
the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell. 11:223),
hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192,
Proc. Nail. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase
(Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt-
cells, respectively. Also, antimetabolite resistance can be used as the basis
of
selection for the following genes: dhfr, which confers resistance to
methotrexate
(Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc.
Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic
acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which
confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505;
Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and
Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH
11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et
al., 1984, Gene 30:147). Methods commonly known in the art of recombinant
DNA technology which can be used are described in Ausubel et al. (eds.), 1993,
Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler,
1990, Gene Transfer and L'xpression, A Laboratory Manual, Stockton Press,
NY; and in Chapters 12 and 13, Dracopoli et al.. (eds), 1994, Current
Protocols
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in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J.
Mol. Biol. 150:1, which are incorporated by reference herein in their
entireties.
The expression levels of an antibody molecule can be increased by vector
amplification (for a review, see Bebbington and Hentschel, The use of vectors
based on gene amplification for the expression of cloned genes in mammalian
cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a
marker in the vector system expressing antibody is amplifiable, increase in
the
level of inhibitor present in culture of host cell will increase the number of
copies
of the marker gene. Since the amplified region is associated with the antibody
gene, production of the antibody will also increase (Grouse et al., 1983, Mol.
Cell. Biol. 3:257).
The host cell may be co-transfected with two expression vectors of the
invention, the first vector encoding a heavy chain derived polypeptide and the
second vector encoding a light chain derived polypeptide. The two vectors may
contain identical selectable markers which enable equal expression of heavy
and
light chain polypeptides. Alternatively, a single vector may be used which
encodes both heavy and light chain polypeptides. In such situations, the light
chain should be placed before the heavy chain to avoid an excess of toxic free
heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad.
Sci. USA 77:2197). The coding sequences for the heavy and light chains may
comprise cDNA or genomic DNA.
Once an antibody molecule of the invention has been recombinantly
expressed, it may be purified by any method known in the art for purification
of
an immunoglobulin molecule, for example, by chromatography (e.g., ion
exchange, affinity, particularly by affnity for the specific antigen after
Protein
A, and sizing column chromatography), centrifugation, differential solubility,
or
by any other standard technique for the purification of proteins.
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C. Antibody Conjugates
The present invention encompasses antibodies recombinantly fused or
chemically conjugated (including both covalently and non-covalently
conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20
or
50 amino acids of the polypeptide) of the present invention to generate fusion
proteins. The fusion does not necessarily need to be direct, but may occur
through linker sequences. The antibodies may be specific for antigens other
than
polypeptides (or portion thereof, preferably at least 10, 20 or 50 amino acids
of
the polypeptide) of the present invention. For example, antibodies may be used
to target the polypeptides of the present invention to particular cell types,
either
in vitro or in vivo, by fusing or conjugating the polypeptides of the present
invention to antibodies specific for particular cell surface receptors.
Antibodies
fused or conjugated to the polypeptides of the present invention may also be
used
in in vitro immunoassays and purification methods using methods known in the
art. See e.g., Harbor et al.., supra, and PCT publication WO 93/21232; EP
439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Patent
5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.
146:2446-2452 (1991), which are incorporated by reference in their entireties.
The present invention further includes compositions comprising the
polypeptides of the present invention fused or conjugated to antibody domains
other than the variable regions. For example, the polypeptides of the present
invention may be fused or conjugated to an antibody Fc region, or portion
thereof. The antibody portion fused to a polypeptide of the present invention
may comprise the constant region, hinge region, CHl domain, CH2 domain, and
CH3 domain or any combination of whole domains or portions thereof. The
polypeptides may also be fused or conjugated to the above antibody portions to
form multimers. For example, Fc portions fused to the polypeptides of the
present invention can form dimers through disulfide bonding between the Fc
portions. Higher multimeric forms can be made by fusing the polypeptides to
portions of IgA and IgM. Methods for fusing or conjugating the polypeptides
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of the present invention to antibody portions are known in the art. See, e.g.,
U. S.
PatentNos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946;
EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570;
Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et
al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci.
USA 89:11337- 11341(1992) (said references incorporated by reference in their
entireties).
As discussed, supra, the polypeptides of the present invention may be
fused or conjugated to the above antibody portions to increase the in vivo
half
life of the polypeptides or for use in immunoassays using methods known in the
art. Further, the polypeptides of the present invention may be fused or
conjugated to the above antibody portions to facilitate purification. One
reported
example describes chimeric proteins consisting of the first two domains of the
human CD4-polypeptide and various domains of the constant regions of the
heavy or light chains of mammalian immunoglobulins. (EP 394, 827; Traunecker
et al., Nature 331:84-86 ( 1988). The polypeptides of the present invention
fused
or conjugated to an antibody having disulfide- linked dimeric structures (due
to
the IgG) may also be more eWcient in binding and neutralizing other molecules,
than the monomeric secreted protein or protein fragment alone. (Fountoulakis
et ad., J. Biochem. 270:3958-3964 (1995)). In many cases, the Fc part in a
fusion protein is beneficial in therapy and diagnosis, and thus can result in,
for
example, improved pharmacokinetic properties. (EP A 232,262). Alternatively,
deleting the Fc part after the fusion protein has been expressed, detected,
and
purified, would be desired. For example, the Fc portion may hinder therapy and
diagnosis if the fusion protein is used as an antigen for immunizations. In
drug
discovery, for example, human proteins, such as hIL-S receptor, have been
fused
with Fc portions for the purpose of high-throughput screening assays to
identify
antagonists of hIL-5. (See, D. Bennett ef al., J. Molecular Recognition 8:52-
58
(1995); K. Johanson et al., J Biol. Chem. 270:9459-9471 (1995).
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Moreover, the antibodies or fragments thereof of the present invention
can be fused to marker sequences, such as a peptide to facilitates their
purification. In preferred embodiments, the marker amino acid sequence is a
hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN,
Inc.,
9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are
commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci.
USA
86:821-824 (1989), for instance, hexa-histidine provides for convenient
purification of the fusion protein. Other peptide tags useful for purification
include, but are not limited to, the "HA" tag, which corresponds to an epitope
derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767
(1984)) and the "flag" tag.
The present invention further encompasses antibodies or fragments
thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be
used diagnostically to, for example, monitor the development or progression of
a tumor as part of a clinical testing procedure to, e.g., determine the
efficacy of
a given treatment and/or prevention regimens. Detection can be facilitated by
coupling the antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, radioactive materials,
positron
emitting metals using various positron emission tomographies, and
nonradioactive paramagnetic metal ions. See, for example, U. S. Patent No.
4,741,900 for metal ions which can be conjugated to antibodies for use as
diagnostics according to the present invention. Examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase, [3-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group complexes include
streptavidinlbiotin and avidin/biotin; examples of suitable fluorescent
materials
include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an
example
of a luminescent material includes luminol; examples ofbioluminescent
materials
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include luciferase, luciferin, and aequorin; and examples of suitable
radioactive
material include'ZSI, '3'h lIn or 99Tc.
Further, an antibody or fragment thereof may be conjugated to a
therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent,
a
therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent
includes any agent that is detrimental to cells. Examples include paclitaxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,
propranolol, and puromycin and analogs or homologs thereof. Therapeutic
agents include, but are not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine),
alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum
(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin)
and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),
bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
The conjugates of the invention can be used for modifying a given
biological response, the therapeutic agent or drug moiety is not to be
construed
as limited to classical chemical therapeutic agents. For example, the drug
moiety
may be a protein or polypeptide possessing a desired biological activity. Such
proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas
exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor,
a-interferon, 13-interferon, nerve growth factor, platelet derived growth
factor,
tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent,
e.g., angiostatin or endostatin; or, biological response modifiers such as,
for
example, lymphokines, interleukin-1 ("IL,-1 "), interleukin-2 ("IL-2"),
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interleukin-6 ("IL-6"), granulocyte macrophase colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth
factors.
Antibodies may also be attached to solid supports, which are particularly
useful for immunoassays or purification of the target antigen. Such solid
supports include, but are not limited to, glass, cellulose, polyacrylamide,
nylon,
polystyrene, polyvinyl chloride or polypropylene.
Techniques for conjugating such therapeutic moiety to antibodies are well
known, see, e.g., Arnon etal., "Monoclonal Antibodies For Immunotargeting Of
Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy,
Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et
al.,
"Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.),
Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in
1 S Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera
et
al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of
The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in
Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.
(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation
And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev.
62:119-58 (1982).
Alternatively, an antibody can be conjugated to a second antibody to form
an antibody heteroconjugate as described by Segal in U. S. Patent No.
4,676,980,
which is incorporated herein by reference in its entirety.
An antibody, with or without a therapeutic moiety conjugated to it,
administered alone or in combination with cytotoxic factors) and/or
cytokine(s)
can be used as a therapeutic.
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D. Assays For Antibody Binding
The antibodies of the invention may be assayed for immunospecific
binding by any method known in the art. The immunoassays which can be used
include but are not limited to competitive and non-competitive assay systems
using techniques such as western blots, radioimmunoassays, ELISA (enzyme
linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion
assays, agglutination assays, complement-fixation assays, immunoradiometric
assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
Such assays are routine and well known in the art (see, e.g., Ausubel et al.,
eds,
1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,
New York, which is incorporated by reference herein in its entirety).
Exemplary
immunoassays are described briefly below (but are not intended by way of
limitation).
Immunoprecipitation protocols generally comprise lysing a population of
cells in a lysis buffer such as RIPA buffer ( 1 % NP-40 or Triton X-100, 1
sodium deoxycholate, 0.1 % SDS, 0.1 S M NaCI, 0.01 M sodium phosphate at pH
7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease
inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody
of interest to the cell lysate, incubating for a period of time (e.g., I-4
hours) at
4° C, adding protein A and/or protein G sepharose beads to the cell
lysate,
incubating for about an hour or more at 4° C, washing the beads in
lysis buffer
and resuspending the beads in SDS/sample buffer. The ability of the antibody
of
interest to immunoprecipitate a particular antigen can be assessed by, e.g.,
western blot analysis. One of skill in the art would be knowledgeable as to
the
parameters that can be modified to increase the binding of the antibody to an
antigen and decrease the background (e.g., pre-clearing the cell lysate with
sepharose beads). For further discussion regarding immunoprecipitation
protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular
Biology, Vol. l, John Wiley & Sons, Inc., New York at 10.16.1.
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Western blot analysis generally comprises preparing protein samples,
electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20%
SDS-PAGE depending on the molecular weight of the antigen), transferring the
protein sample from the polyacrylamide gel to a membrane such as
nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution
(e.g.,
PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer
(e.g., PBS-Tween 20), blocking the membrane with primary antibody (the
antibody ofinterest) diluted in blocking buffer, washing the membrane in
washing
buffer, blocking the membrane with a secondary antibody (which recognizes the
primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic
substrate (e.g., horseradish peroxidase or alkaline phosphatase) or
radioactive
molecule (e.g., 3zP or lzsl) diluted in blocking buffer, washing the membrane
in
wash buffer, and detecting the presence of the antigen. One of skill in the
art
would be knowledgeable as to the parameters that can be modified to increase
the signal detected and to reduce the background noise. For further discussion
regarding western blot protocols see, e.g., Ausubel et al., eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96 well
microtiter plate with the antigen, adding the antibody of interest conjugated
to
a detectable compound such as an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) to the well and incubating for a period of
time, and detecting the presence of the antigen. In ELISAs the antibody of
interest does not have to be conjugated to a detectable compound; instead, a
second antibody (which recognizes the antibody of interest) conjugated to a
detectable compound may be added to the well. Further, instead of coating the
well with the antigen, the antibody may be coated to the well. In this case, a
second antibody conjugated to a detectable compound may be added following
the addition of the antigen of interest to the coated well. One of skill in
the art
would be knowledgeable as to the parameters that can be modified to increase
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the signal detected as well as other variations of ELISAs known in the art.
For
further discussion regarding ELISAs see, e.g., Ausubel et al., eds, 1994,
Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
11.2.1.
S The binding affnity of an antibody to an antigen and the ofd rate of an
antibody-antigen interaction can be determined by competitive binding assays.
One example of a competitive binding assay is a radioimmunoassay comprising
the incubation of labeled antigen (e.g., 3H or'z5I) with the antibody of
interest in
the presence of increasing amounts of unlabeled antigen, and the detection of
the
antibody bound to the labeled antigen. The ai~mity of the antibody of interest
for
a particular antigen and the binding off-rates can be determined from the data
by
scatchard plot analysis. Competition with a second antibody can also be
determined using radioimmunoassays. In this case, the antigen is incubated
with
antibody of interest is conjugated to a labeled compound (e.g., 3H or'zsI) in
the
presence of increasing amounts of an unlabeled second antibody.
E. Antibody Based Therapies
The present invention is further directed to antibody-based therapies
which involve administering antibodies of the invention to an animal,
preferably
a mammal, and most preferably a human, patient for treating and/or preventing
one or more of the disorders or conditions described herein. Therapeutic
compounds of the invention include, but are not limited to, antibodies of the
invention (including fragments, analogs and derivatives thereof as described
herein) and nucleic acids encoding antibodies of the invention (including
fragments, analogs and derivatives thereof as described herein).
While not intending to be bound to theory, DRS receptors are believed
to induce programmed cell death by a process which involves the
association/cross-linking of death domains between different receptor
molecules.
Further, DRS ligands (e.g., TRAIL) which induce DRS mediated programmed
cell death are believed to function by causing the association/cross-linking
of
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DRS death domains. Thus, agents (e.g., antibodies) which prevent
association/cross-linking of DRS death domains will prevent DRS mediated
programmed cell death, and agents (e.g., antibodies) which facilitate the
association/cross-linking of DRS death domains will induce DRS mediated
S programmed cell death.
As noted above, DRS receptors have been shown to bind TRAIL. DRS
receptors are also known to be present in a number of tissues and on the
surfaces
of a number of cell types. These tissues and cell types include primary
dendritic
cells, endothelial tissue, spleen, lymphocytes ofpatients with chronic
lymphocytic
leukemia, and human thymus stromal cells. Further, as explained in more detail
below, TRAIL has been shown to induce apoptosis and to inhibit the growth of
tumor cells in vivo. Additionally, TRAIL activities are believed to be
modulated,
at least in part, through interaction with DR4 and DRS receptors.
TRAIL, is a member of the TNF family of cytokines which has been
1 S shown to induce apoptotic cell death in a number of tumor cell lines and
appears
to mediate its apoptosis inducing effects through interaction with DR4 and DRS
receptors. These death domain containing receptors are believed to form
membrane-bound self activating signaling complexes which initiate apoptosis
through cleavage of caspases.
In addition to DR4 and DRS receptors, TRAIL also binds to several
receptors proposed to be "decoy" receptors, DcR2 (a receptor with a truncated
death domain), DcRI (a GPI-anchored receptor), and OPG (a secreted protein
which binds to another member of the TNF family, RANKL,).
Further, recent studies have shown that the rank-order of amities of
2S TRAIL for the recombinant soluble forms ofits receptors is strongly
temperature
dependent. In particular, at 37° C, DRS has the highest afFmity for
TRAIL and
OPG having the lowest affinity.
The DR4 and DRS receptor genes, as well as genes encoding two decoy
receptors, have been shown to be located on human chromosome 8p21-22.
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Further, this region of the human genome is frequently disrupted in head and
neck cancers.
It has recently been found that the FaDu nasopharyngeal cancer cell line
contains an abnormal chromosome 8p21-22 region. (Ozoren et al., Int. J.
S Oncol. 16:917-925 (2000).) In particular, a homozygous deletion involving
DR4,
but not DRS, has been found in these cells. (Ozoren et al., Int. J.
Oncol.16:917-925 (2000).) The homozygous loss within the DR4 receptor gene
in these FaDu cells encompasses the DR4 receptor death domain. This
disruption of the DR4 receptor death domain is associated with resistance to
TRAIL-mediated cytotoxicity. Further, re-introduction of a wild-type DR4
receptor gene has been shown to both lead to apoptosis and restoration of
TRAIL sensitivity of FaDu cells. (Ozoren et al., Int. J. Oncol.l6:917-925
(2000).) These data indicate that the DR4 receptor gene may be inactivated in
human cancers and DR4 receptor gene disruption may contribute to resistance
to TRAIL, therapy. It is expected that similar results would be found in cells
having analogous deletions in the DRS gene.
It has also been shown that overexpression of the cytoplasmic domain of
the DR4 receptor in human breast, lung, and colon cancer cell lines leads to
p53-independent apoptotic cell death which involves the cleavage of caspases.
(Xu et al., Biochem. Biophys. Res. Commun. 269:179-190 (2000).) Further,
DR4 cytoplasmic domain overexpression has also been shown to result in
cleavage of both poly(ADP-ribose) polymerase (PARP) and a DNA
fragmentation factor (i.e., ICAD-DFF45). (Xu et al., Biochem. Biophys. Res.
Commun. 269:179-190 (2000).) In addition, despite similar levels of DR4
cytoplasmic domain protein as compared to cancer cells tested, normal lung
fibroblasts have been shown to be resistant to DR4 cytoplasmic domain
overexpression and show no evidence of caspase-cleavage. (Xu et al., Biochem.
Biophys. Res. Commun. 269:179-190 (2000).) Again, similar results are
expected with cells that overexpress the cytoplasmic domain of DRS. Thus, the
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cytoplasmic domains of the DR4 and DRS receptors are useful as agents for
inducing apoptosis, for example, in cancer cells.
Further, overexpression of the cyclin-dependent kinase inhibitor
p21 (WAF 1 /CIP 1 ), as well as the N-terminal 91 amino acids of this protein,
has
S cell cycle-inhibitory activity and inhibits DR4 cytoplasmic domain-dependent
caspase cleavage. Thus, DR4 receptors are also involved in the regulation of
cell
cycle progression. As above, similar results are expected with the DRS
receptor.
Thus, the DR4 and DRS receptors, as well as agonists and antagonists of these
receptors, are useful for regulating cell cycle progression.
Antibodies which bind to DRS receptors are useful for treating and/or
preventing diseases and conditions associated with increased or decreased
DRS-induced apoptotic cells death. Further, these antibodies vary in the
effect
they have on DRS receptors. These effects direr based on the specific portions
of the DRS receptor to which the antibodies bind, the three-dimensional
1 S conformation of the antibody molecules themselves, and/or the manner in
which
they interact with the DRS receptor. Thus, antibodies which bind to the
extracellular domain of a DRS receptor can either stimulate or inhibit DRS
activities (e.g., the induction of apoptosis). Antibodies which stimulate DRS
receptor activities (e.g., by facilitating the association between DRS
receptor
death domains) are DRS agonists, and antibodies which inhibit DRS receptor
activities (e.g., by blocking the binding of TRAIL and/or preventing the
association between DRS receptor death domains) are DRS antagonists.
Antibodies ofthe invention which function as agonists and antagonists of
DRS receptors include antigen-binding antibody fragments such as Fab and
2S F(ab')Z fragments, Fd, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv)
and
fragments comprising either a VL or VH domain, as well as polyclonal,
monoclonal and humanized antibodies. Divalent antibodies are preferred as
agonists. Each of these antigen-binding antibody fragments and antibodies are
described in more detail elsewhere herein.
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In view of the above, antibodies of the invention, as well as other
agonists, are useful for stimulating DRS death domain activity to promote
apoptosis in cells which express DRS receptors (e.g., cancer cells).
Antibodies
of this type are useful for prevention and/or treating diseases and conditions
S associated with increased cell survival and/or insensitivity to apoptosis-
inducing
agents (e.g., TRAIL), such as solid tissue cancers (e.g., skin cancer, head
and
neck tumors, breast tumors, endothelioma, lung cancer, osteoblastoma,
osteoclastoma, and Kaposi's sarcoma) and leukemias.
Antagonists of the invention (e.g., anti-DRS antibodies) function by
preventing DRS mediated apoptosis and are useful for preventing and/or
treating
diseases associated with increased apoptotic cell death. Examples of such
diseases include diabetes mellitus, AIDS, neurodegenerative disorders,
myelodysplastic syndromes, ischemic injury, toxin-induced liver disease,
septic
shock, cachexia and anorexia.
As noted above, DRS receptors are present on the surfaces of T-cells.
Thus, agonists of the invention (e.g., anti-DRS receptor antibodies) are also
useful for inhibiting T-cell mediated immune responses, as well as preventing
and/or treating diseases and conditions associated with increased T-cell
proliferation. Diseases and conditions associated with T-cell mediated immune
responses and increased T-cell proliferation include graft-v-host responses
and
diseases, osteoarthritis, psoriasis, septicemia, inflammatory bowel disease,
inflammation in general, autoimmune diseases, and T-cell leukemias.
When an agonist of the invention is administered to an individual for the
treatment and/or prevention of a disease or condition associated with
increased
T-cell populations or increased cell proliferation (e.g., cancer), the
antagonist
may be co-administered with another agent which induces apoptosis (e.g.,
TRAIL,) or otherwise inhibits cell proliferation (e.g., an anti-cancer drug).
Combination therapies of this nature, as well as other combination therapies,
are
discussed below in more detail.
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Further, antagonists ofthe invention (e.g., anti-DRS receptor antibodies)
are also useful for enhancing T-cell mediated immune responses, as well as
preventing and/or treating diseases and conditions associated with decreased
T-cell proliferation. Antibodies of the invention which block the binding of
DRS
receptor ligands to DRS receptors or interfere with DRS receptor
conformational
changes associated with membrane signal transduction can inhibit DRS mediated
T-cell apoptosis. The inhibition of DRS mediated apoptosis can, for examples,
either result in an increase in the expansion rate of in vivo T-cell
populations or
prevent a decrease in the size of such populations. Thus, antagonists of the
invention can be used to prevent and/or treat diseases or conditions
associated
with decreased or decreases in T-cell populations. Examples of such diseases
and conditions included acquired immune deficiency syndrome (AIDS) and
related afflictions (e.g., AIDS related complexes), T-cell immunodeficiencies,
radiation sickness, and T-cell depletion due to radiation and/or chemotherapy.
When an antagonist of the invention is administered to an individual for
the treatment and/or prevention of a disease or condition associated with
decreased T-cell populations, the antagonist may be co-administered with an
agent which activates and/or induces lymphocyte proliferation (e.g., a
cytokine).
Combination therapies of this nature, as well as other combination therapies,
are
discussed below in more detail.
Similarly, agonists and antagonists of the invention (e.g., anti-DRS
receptor antibodies) are also useful when administered alone or in combination
with another therapeutic agent for either inhibiting or enhancing B-cell
mediated
immune responses, as well as preventing and/or treating diseases and
conditions
associated with increased or decreased B-cell proliferation.
Anti-DRS antibodies are thus useful for treating and/or preventing
malignancies, abnormalities, diseases and/or conditions involving tissues and
cell
types which express DRS receptors (e.g., endothelial cells). Further,
malignancies, abnormalities, diseases and/or conditions which can be treated
and/or prevented by the induction of programmed cell death in cells which
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express DRS receptors can be treated and/or prevented using DRS receptor
agonists ofthe invention. Similarly, malignancies, abnormalities, diseases
and/or
conditions which can be treated and/or prevented by inhibiting programmed cell
death in cells which express DRS receptors can be treated and/or prevented
using
S DRS receptor antagonists of the invention.
Further, antibodies of the invention, as well as other agonists, are useful
for stimulating DRS death domain activity in endothelial cells, resulting in
anti-angiogenic activity. Antibodies of this type are useful for prevention
and/or
treating diseases and conditions associated with hypervascularization and
neovascularization, such as rheumatoid arthritis and solid tissue cancers
(e.g.,
skin cancer, head and neck tumors, breast tumors, endothelioma, osteoblastoma,
osteoclastoma, and Kaposi's sarcoma), as well as diseases and conditions
associated with chronic inflammation.
Diseases and conditions associated with chronic inflammation, such as
1 S ulcerative colitis and Crohn's disease, often show histological changes
associated
with the ingrowth of new blood vessels into the inflamed tissues. Agonists of
the
invention which stimulate the activity of DRS death domains will induce
apoptosis in endothelial cells which express these receptors. As a result,
agonists
of the invention can inhibit the formation of blood and lymph vessels and,
thus,
can be used to prevent and/or treat diseases and conditions associated with
hypervascularization and neovascularization.
Other diseases and conditions associated with angiogenesis which can be
prevented and/or treated using agonists ofthe invention include hypertrophic
and
keloid scarring, proliferative diabetic retinopathy, arteriovenous
malformations,
2S atherosclerotic plaques, hemophilic joints, nonunion fractures, Osler-Weber
syndrome, psoriasis, pyogenic granuloma, scleroderma, tracoma, menorrhagia,
and vascular adhesions.
Further, agents which inhibit DRS death domain activity (e.g., DRS
antagonists) are also useful for preventing and/or treating a number of
diseases
and conditions associated with decreased vascularization. As indicated above,
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examples of antagonists of DRS receptor activity include anti-DRS receptor
antibodies. These antibodies can function, for examples, by either binding to
DRS receptors and blocking the binding of ligands which stimulate DRS death
domain activity (e.g., TRAIL,) or inhibiting DRS receptor conformational
changes associated with membrane signal transduction.
An example of a condition associated with decreased vascularization that
can be treated using antagonists of the invention is delayed wound healing.
The
elderly, in particular, often heal at a slower rate than younger individuals.
Antagonists of the invention can thus prevent and/or inhibit apoptosis from
occurring in endothelial cells at wound sites and thereby promote wound
healing
in healing impaired individuals, as well as in individuals who heal at
"normal"
rates. Thus, antagonists of the invention can be used to promote and/or
accelerate wound healing. Antagonists of the invention are also useful for
treating and/or preventing other diseases and conditions including restenosis,
myocardial infarction, peripheral arterial disease, critical limb ischemia,
angina,
atherosclerosis, ischemia, edema, liver cirrhosis, osteoarthritis, and
pulmonary
fibrosis.
A number of additional malignancies, abnormalities, diseases and/or
conditions which can be treated using the agonists and antagonists of the
invention are set out elsewhere herein, for example, in the section below
entitled
"Therapeutics".
The antibodies of the present invention may be used therapeutically in a
number of ways. For example, antibodies which bind polynucleotides or
polypeptides of the present invention can be administered to an individual
(e.g.,
a human) either locally or systemically. Further, these antibodies can be
administered alone, in combination with another therapeutic agent, or
associated
with or bound to a toxin.
Anti-DRS antibodies may be utilized in combination with other
monoclonal or chimeric antibodies, or with lymphokines, tumor necrosis factors
or TNF-related molecules (e.g., TNF-a, TNF-(3, TNF-y, TNF-y-a, TNF-y-Vii,
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and TRAIL), or hematopoietic growth factors (e.g., IL-2, IL-3 and IL-7). For
example, agonistic anti-DRS antibodies may be administered in conjunction with
TRAIL when one seeks to induce DRS mediated cell death in cells which express
DR5 receptors of the invention. Combination therapies of this nature, as well
as
other combination therapies, are discussed below in more detail.
The antibodies of the invention may be administered alone or in
combination with other types of treatments (e.g., radiation therapy,
chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).
Generally, administration of products of a species origin or species
reactivity (in
the case of antibodies) that is the same species as that of the patient is
preferred.
Thus, in a preferred embodiment, human antibodies, fragments derivatives,
analogs, or nucleic acids, are administered to a human patient for therapy or
prophylaxis.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or
neutralizing antibodies against polypeptides or polynucleotides of the present
invention, fragments or regions thereof, for both immunoassays directed to and
therapy of disorders related to polynucleotides or polypeptides, including
fragments thereof, of the present invention. Such antibodies, fragments, or
regions, will preferably have an affinity for polynucleotides or polypeptides,
including fragments thereof. Preferred binding affinities include those with a
dissociation constant or Kd less than SX10-6M, 10-6M, SX10-'M, 10-'M,
5 X 10-gM, 10-gM, 5 X 10-9M, 10-9M, 5 X 10-' °M, 10-' °M, 5 X 10-
"M, 10-"M,
SX10-'ZM, 10-'ZM, SX10-'3M, 10~'3M, SX10-'4M, 10-'4M, SX10-'SM, and 10-'SM.
Polypeptirle Assays
The present invention also relates to diagnostic assays such as
quantitative and diagnostic assays for detecting levels of DRS protein, or the
soluble form thereof, in cells and tissues, including determination of normal
and
abnormal levels. Thus, for instance, a diagnostic assay in accordance with the
invention for detecting over-expression of DRS, or soluble form thereof,
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compared to normal control tissue samples may be used to detect the presence
of tumors, for example. Assay techniques that can be used to determine levels
of a protein, such as a DRS protein of the present invention, or a soluble
form
thereof, in a sample derived from a host are well-known to those of skill in
the
art. Such assay methods include radioimmunoassays, competitive-binding assays,
Western Blot analysis, and ELISA assays.
Assaying DRS protein levels in a biological sample can occur using any
art-known method. By "biological sample" is intended any biological sample
obtained from an individual, cell line, tissue culture, or other source
containing
DRS receptor protein or mRNA. Preferred for assaying DRS protein levels in a
biological sample are antibody-based techniques. For example, DRS protein
expression in tissues can be studied with classical immunohistological
methods.
(Jalkanen, M. et al., .l. Cell. Biol. 101:976-985 (1985); Jalkanen, M. et al.,
.I.
Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for
detecting DRS protein gene expression include immunoassays, such as the
enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Suitable labels are known in the art and include enzyme labels, such as
glucose oxidase, radioisotopes, such as iodine ('zsl, '2'I), carbon ('4C),
sulphur
(35s) tritium (3H), indium ("ZIn), and technetium (~9"'Tc), and fluorescent
labels,
such as fluorescein and rhodamine, and biotin.
Therapeutics
The Tumor Necrosis Factor (TNF) family ligands are known to be among
the most pleiotropic cytokines, inducing a large number of cellular responses,
including cytotoxicity, anti-viral activity, immunoregulatory activities, and
the
transcriptional regulation of several genes (Goeddel, D.V. et al., "Tumor
Necrosis Factors: Gene Structure and Biological Activities," Symp. Quant.
Biol.
51:597-609 ( 1986), Cold Spring Harbor; Beutler, B., and Cerami, A., Annu.
Rev.
Biochem. 57:505-518 (1988); Old, L.J., .fci. Am. 258:59-75 (1988); Fiers, W.,
FEBSLett. 285:199-224 (1991)). The TNF-family ligands induce such various
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cellular responses by binding to TNF-family receptors, including the DRS of
the
present invention.
DRS polynucleotides, polypeptides, agonists and/or antagonists of the
invention may be administered to a patient (e.g., mammal, preferably human)
afflicted with any disease or disorder mediated (directly or indirectly) by
defective, or deficient levels of, DRS. Alternatively, a gene therapy approach
may be applied to treat and/or prevent such diseases or disorders. In one
embodiment of the invention, DRS polynucleotide sequences are used to detect
mutein DRS genes, including defective genes. Mutein genes may be identified
in in vitro diagnostic assays, and by comparison ofthe DRS nucleotide sequence
disclosed herein with that of a DRS gene obtained from a patient suspected of
harboring a defect in this gene. Defective genes may be replaced with normal
DRS-encoding genes using techniques known to one skilled in the art.
In another embodiment, the DRS polypeptides, polynucleotides, agonists
and/or antagonists of the present invention are used as research tools for
studying
the phenotypic effects that result from inhibiting TRAIL/DRS interactions on
various cell types. DRS polypeptides and antagonists (e.g. monoclonal
antibodies
to DRS) also may be used in in vitro assays for detecting TRAIL or DRS or the
interactions thereof.
It has been reported that certain ligands of the TNF family (of which
TRAIL is a member) bind to more than one distinct cell surface receptor
protein.
For example, a receptor protein designated DR4 reportedly binds TRAIL,, but is
distinct from the DRS ofthe present invention (Pan et al., Science 276:111-
113,
(1997); hereby incorporated by reference). In another embodiment, a purified
DRS polypeptide, agonist and/or antagonist is used to inhibit binding of TRAIL
to endogenous cell surface TRAIL. By competing for TRAIL binding, soluble
DRS polypeptides of the present invention may be employed to inhibit the
interaction of TRAIL, not only with cell surface DRS, but also with TRAIL,
receptor proteins distinct from DRS. Thus, in a further embodiment, DRS
polynucleotides, polypeptides, agonists and/or antagonists of the invention
are
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used to inhibit a functional activity of TRAIL, in in vitro or in vivo
procedures.
By inhibiting binding of TRAIL to cell surface receptors, DRS also inhibits
biological effects that result from the binding of TRAIL to endogenous
receptors.
Various forms of DRS may be employed, including, for example, the
S above-described DRS fragments, derivatives, and variants that are capable of
binding TRAIL,. In a preferred embodiment, a soluble DRS, is employed to
inhibit a functional activity of TRAIL, e.g., to inhibit TRAIL-mediated
apoptosis
of cells susceptible to such apoptosis. Thus, in an additional embodiment, DRS
is administered to a mammal (e.g., a human) to treat and/or prevent a
TRAIL-mediated disorder. Such TRAIL-mediated disorders include conditions
caused (directly or indirectly) or exacerbated by TRAIL,.
Cells which express the DRS polypeptide and are believed to have a
potent cellular response to DRS ligands include primary dendritic cells,
endothelial tissue, spleen, chronic lymphocytic leukemia, and human thymus
stromal cells. By "a cellular response to a TNF-family ligand" is intended any
genotypic, phenotypic, and/or morphologic change to a cell, cell line, tissue,
tissue culture or patient that is induced by a TNF-family ligand. As
indicated,
such cellular responses include not only normal physiological responses to TNF-
family ligands, but also diseases associated with increased apoptosis or the
inhibition of apoptosis. Apoptosis (programmed cell death) is a physiological
mechanism involved in the deletion of peripheral T lymphocytes of the immune
system, and its dysregulation can lead to a number of different pathogenic
processes (Ameisen, J.C., AIDS 8:1197-1213 (1994); Krammer, P.H. et al.,
Curr. Opirt. Immunol. 6:279-289 (1994)).
Diseases associated with increased cell survival, or the inhibition of
apoptosis, that may be treated, prevented, diagnosed and/or prognosed with the
DRS polynucleotides, polypeptides and/or agonists or antagonists of the
invention include, but are not limited to, include cancers (such as follicular
lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors
including, but not limited to colon cancer, cardiac tumors, pancreatic cancer,
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melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer,
testicular
cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma,
endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma,
adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer);
S autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome,
Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,
polymyositis, systemic lupus erythematosus and immune-related
glomerulonephritis and rheumatoid arthritis) and viral infections (such as
herpes
viruses, pox viruses and adenoviruses), inflammation, graft v. host disease,
acute
graft rejection, and chronic graft rejection. In preferred embodiments, DRS
polynucleotides, polypeptides, and/or antagonists of the invention are used to
inhibit growth, progression, and/or metastasis of cancers, in particular those
listed above.
Additional diseases or conditions associated with increased cell survival
1 S that may be treated, prevented, diagnosed and/or prognosed with the DRS
polynucleotides, polypeptides and/or agonists or antagonists of the invention
include, but are not limited to, progression, and/or metastases of
malignancies
and related disorders such as leukemia (including acute leukemias (e.g., acute
lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic,
promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic
leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic
lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease
and non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, heavy chain disease, and solid tumors including, but not
limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate
cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat
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gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic
carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical
cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder
carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic
neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma.
Diseases associated with increased apoptosis that may be treated,
prevented, diagnosed and/or prognosed with the DRS polynucleotides,
polypeptides and/or agonists or antagonists of the invention include, but are
not
limited to, AIDS; neurodegenerative disorders (such as Alzheimer's disease,
Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa,
Cerebellar degeneration and brain tumor or prior associated disease);
autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome,
Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,
polymyositis, systemic lupus erythematosus and immune-related
glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such
as aplastic anemia), graft v. host disease, ischemic injury (such as that
caused by
myocardial infarction, stroke and reperfusion injury), liver injury (e.g.,
hepatitis
related liver injury, ischemia/reperfusion injury, cholestosis (bile duct
injury) and
liver cancer); toxin-induced liver disease (such as that caused by alcohol),
septic
shock, cachexia and anorexia. In preferred embodiments, DRS polynucleotides,
polypeptides and/or agonists are used to treat and/or prevent the diseases and
disorders listed above.
The state of Immunodeficiency that defines A>DS is secondary to a
decrease in the number and function of CD4+ T-lymphocytes. Recent reports
estimate the daily loss of CD4+ T-cells to be between 3.5 X 10' and 2 X 10'
cells
(Wei X. etal., Nature 373:117-122 (1995)). One cause of CD4+ T-cell depletion
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in the setting of HIV infection is believed to be HIV-induced apoptosis (see,
for
example, Meyaard et al.., Science 257:217-219, 1992; Groux et al., J Exp.
Med.,
175:331, 1992; and Oyaizu et al., in Cell Activation and Apoptosis in HITI
Infection, Andrieu and Lu, Eds., Plenum Press, New York, 1995, pp. 101-114).
Indeed, HIV-induced apoptotic cell death has been demonstrated not only in
vitro but also, more importantly, in infected individuals (Ameisen, J.C., AIDS
8:1197-1213 (1994); Finkel, T.H., and Banda, N.K., Curr. Opin. Immunol.
6:605-615(1995); Muro-Cacho, C.A. etal., J. Immunol. 154:5555-5566 (1995)).
Furthermore, apoptosis and CD4+ T-lymphocyte depletion is tightly correlated
in dii~erent animal models of AIDS (Brunner, T., et al., Nature 373:441-444
(1995); Gougeon, M.L., etal., AIDSRes. Hum. Retroviruses9:553-563 (1993))
and, apoptosis is not observed in those animal models in which viral
replication
does not result in AIDS (Gougeon, M.L. et al., AIDS Res. Hum. Retroviruses
9:553-563 ( 1993)). Further data indicates that uninfected but primed or
activated
T lymphocytes from HIV-infected individuals undergo apoptosis after
encountering the TNF-family ligand Fast. Using monocytic cell lines that
result
in death following HIV infection, it has been demonstrated that infection of
U93 7
cells with HIV results in the de novo expression of Fast and that Fast
mediates
HIV-induced apoptosis (Badley, A.D. et al., J. Tirol. 70:199-206 (1996)).
Further the TNF-family ligand was detectable in uninfected macrophages and its
expression was upregulated following HIV infection resulting in selective
killing
of uninfected CD4 T-lymphocytes (Badley, A.D et al., J. Tlirol. 70:199-206
(1996)). Further, additional studies have implicated Fas-mediated apoptosis in
the loss of T-cells in HIV individuals (Katsikis et al., J. Exp. Med.
181:2029-2036, 1995).
Thus, by the invention, a method for treating and/or preventing HIV+
individuals is provided which involves administering DRS, DRS antagonists,
and/or DRS agonists of the present invention to reduce selective killing of
CD4+
T-lymphocytes. Modes of administration and dosages are discussed in detail
below.
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In rejection of an allograft, the immune system ofthe recipient animal has
not previously been primed to respond because the immune system for the most
part is only primed by environmental antigens. Tissues from other members of
the same species have not been presented in the same way that, for example,
viruses and bacteria have been presented. In the case of allograft rejection,
immunosuppressive regimens are designed to prevent the immune system from
reaching the efFector stage. However, the immune profile ofxenograft rejection
may resemble disease recurrence more than allograft rejection. In the case of
disease recurrence, the immune system has already been activated, as evidenced
by destruction of the native islet cells. Therefore, in disease recurrence the
immune system is already at the effector stage. Agonists of the present
invention
are able to suppress the immune response to both allografts and xenografts
because lymphocytes activated and differentiated into effector cells will
express
the DRS polypeptide, and thereby are susceptible to compounds which enhance
apoptosis. Thus, the present invention further provides a method for creating
immune privileged tissues.
DRS antagonists or agonists of the invention may be useful for treating
and/or preventing inflammatory diseases, such as rheumatoid arthritis,
osteoarthritis, psoriasis, septicemia, and inflammatory bowel disease.
In addition, due to lymphoblast expression of DRS, soluble DRS agonist
or antagonist mABs may be used to treat and/or prevent this form of cancer.
Further, soluble DRS or neutralizing mABs may be used to treat and/or prevent
various chronic and acute forms of inflammation such as rheumatoid arthritis,
osteoarthritis, psoriasis, septicemia, and inflammatory bowel disease.
Polynucleotides and/or polypeptides of the invention and/or agonists
and/or antagonists thereof are useful in the diagnosis, prognosis, treatment
and/or
prevention of a wide range of diseases and/or conditions. Such diseases and
conditions include, but are not limited to, cancer (e.g., immune cell related
cancers, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma,
glioblastoma, cancer associated with mutation or alteration of p53, brain
tumor,
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bladder cancer, uterocervical cancer, colon cancer, colorectal cancer, non-
small
cell carcinoma of the lung, small cell carcinoma of the lung, stomach cancer,
etc.), lymphoproliferative disorders (e.g., lymphadenopathy and lymphomas
(e.g.,
Hodgkin's disease)), microbial (e.g., viral, bacterial, etc.) infection (e.g.,
HIV-1
infection, HIV-2 infection, herpesvirus infection (including, but not limited
to,
HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus infection,
poxvirus infection, human papilloma virus infection, hepatitis infection
(e.g.,
HAV, HBV, HCV, etc.),Helicobacterpylori infection, invasiveSfaphylococcia,
etc.), parasitic infection, nephritis, bone disease (e.g., osteoporosis),
atherosclerosis, pain, cardiovascular disorders (e.g., neovascularization,
hypovascularization or reduced circulation (e.g., ischemic disease (e.g.,
myocardial infarction, stroke, etc.)), AIDS, allergy, inflammation,
neurodegenerative disease (e.g., Alzheimer's disease, Parkinson's disease,
amyotrophic lateral sclerosis, pigmentary retinitis, cerebellar degeneration,
etc.),
graft rejection (acute and chronic), graft vs. host disease, diseases due to
osteomyelodysplasia (e.g., aplastic anemia, etc.), joint tissue destruction in
rheumatism, liver disease (e.g., acute and chronic hepatitis, liver injury,
and
cirrhosis), autoimmune disease (e.g.,. multiple sclerosis, myasthenia gravis,
rheumatoid arthritis, systemic lupus erythematosus, immune complex
glomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenic
purpura, Grave's disease, Hashimoto's thyroiditis, inflammatory autoimmune
diseases, etc.), cardiomyopathy (e.g., dilated cardiomyopathy), diabetes,
diabetic
complications (e.g., diabetic nephropathy, diabetic neuropathy, diabetic
retinopathy), influenza, asthma, psoriasis, osteomyelitis, glomerulonephritis,
septic shock, and ulcerative colitis.
Polynucleotides and/or polypeptides of the invention and/or agonists
and/or antagonists thereof are useful in promoting regulating hematopoiesis,
regulating (e.g., promoting) angiogenesis, wound healing (e.g., wounds, burns,
and bone fractures), and regulating bone formation.
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DRS polynucleotides or polypeptides, or agonists of DRS, can be used
in the treatment and/or prevention of infectious agents. For example, by
increasing the immune response, particularly increasing the proliferation and
differentiation of B-cells in response to an infectious agent, infectious
diseases
S may be treated and/or prevented. The immune response may be increased by
either enhancing an existing immune response, or by initiating a new immune
response. Alternatively, DRS polynucleotides or polypeptides, or agonists or
' antagonists of DRS, may also directly inhibit the infectious agent, without
necessarily eliciting an immune response.
Viruses are one example of an infectious agent that can cause disease or
symptoms that can be treated and/or prevented by DRS polynucleotides or
polypeptides, or agonists of DRS. Examples of viruses, include, but are not
limited to the following DNA and RNA viruses and viral families: arbovirus,
adenoviridae, arenaviridae, arterivirus, birnaviridae, bunyaviridae,
caliciviridae,
circoviridae, coronaviridae, Dengue virus, HIV-1, HIV-2, flaviviridae,
hepadnaviridae (e.g., hepatitis B virus), herpesviridae (e.g.,
cytomegalovirus,
herpes simplex viruses 1 and 2, varicella-zoster virus, Epstein-Barr virus
(EBV),
herpes B virus, and human herpesviruses 6, 7, and 8), morbillivirus,
rhabdoviridae (e.g., rabies virus), orthomyxoviridae (e.g., influenza A virus,
and
influenza B), paramyxoviridae (e.g., parainfluenza virus), papilloma virus,
papovaviridae, parvoviridae, picornaviridae (e.g., EMCV and poliovirus),
poxviridae (e.g., variola or vaccinia virus), reoviridae (e.g., rotavirus),
retroviridae (HTLV-I, HTLV-II, lentivirus), and togaviridae (e.g., rubivirus).
These viruses and virus families can cause a variety of diseases or symptoms,
including, but not limited to: arthritis, bronchiollitis, respiratory
diseases,
encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic
fatigue
syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B
encephalitis,
Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, smallpox,
opportunistic infections (e.g., AIDS, Kaposi's sarcoma), pneumonia, Burkitt's
lymphoma, chickenpox, zoster, hemorrhagic fever, measles, mumps,
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parainfluenza, rabies, the common cold, polio, leukemia, rubella, sexually
transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. DRS
polynucleotides or polypeptides, or agonists or antagonists of DRS, can be
used
to treat, prevent, and/or detect any of these symptoms or diseases. In
specific
embodiments, DRS polynucleotides, polypeptides, or agonists are used to treat
and/or prevent: meningitis, Dengue, EBV, and/or hepatitis. In an additional
specific embodiment DRS polynucleotides, polypeptides, or agonists are used to
treat patients non-responsive to one or more other commercially available
hepatitis vaccines. In a further specific embodiment, DRS polynucleotides,
polypeptides, or agonists are used to treat AIDS.
Similarly, bacteria and fungi that can cause disease or symptoms and that
can be treated and/or prevented by DRS polynucleotides or polypeptides, or
agonists or antagonists of DRS, include, but are not limited to the following
organisms. Bacteria include, but are not limited to Actinomyces, Bacillus
(e.g.,
B. anthracis), Bacteroides, Bordetella, Bartonella, Borrelia (e.g., B.
burgdorferi), Brucella, Campylobacter, Capnocytophaga, Chlamydia,
Clostridium, Corynebacterium, Coxiella, Dermatophilus, Enterococcus,
Ehrlichia, Escherichia (e.g., enterotoxigenic E. coli and enterohemorrhagic E.
coli), Francisella, Fusobacterium, Haemobartonella, Haemophilus (e.g., H.
infZuenzae type b), Helicobacter, Klebsiella, L-form bacteria, Legionella,
L epto.spira, Listeria, Mycobacteria (e.g., M. leprae and M. tuberculosis),
Mycoplasma, Neisseria (e.g., N. gonorrheae and N. meningitidis),
Neorickettsia, Nocardia, Pasteurella, Peptococcus, Peptostreptococcus,
Pneumococcus, Proteus, Pseudomonas, Rickettsia, Rochalimaea, Salmonella
(e.g., S. typhimurium and S. typhi), Seratia, Shigella, Staphylococcus (e.g.,
S.
aureus), Streptococcus (e.g., S. pyogene.s, S. pneumoniae, and Group B
streptococcus), Streptomyces, Treponema, T~ibrio (e.g., hibrio cholerae) and
Yersinia (e.g., Y. pestis). Fungi include, but are not limited to: Absidia,
Acremonium, Alternaria, Aspergillus, Basidiobolus, Bipolaris, Blastomyces,
Candida (e.g., C. albicans), Coccidioides, Conidiobolus, Cryptococcus (e.g.,
C.
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neoformans), Curvalaria, Erysipelothrix, Epidermophyton, Exophiala,
Geotrichum, Histoplasma, Madurella, Madassezia, Microsporum, Moniliella,
Mortierella, Mucor, Paecilomyces, Penicillium, Phialemonium, Phialophora,
Prototheca, Pseudallescheria, Pseudomicrodochium, Pythium, Rhinosporidium,
S Rhizopus, Scolecobasidium, Sporothrix, Stemphylium, Trichophyton,
Trichosporon, and Xylohypha. These and other bacteria or fungi can cause
diseases or symptoms including, but not limited to: bacteremia, endocarditis,
eye
infections (conjunctivitis, uveitis), gingivitis, opportunistic infections
(e.g., AIDS
related infections), paronychia, prosthesis-related infections, Reiter's
Disease,
respiratory tract infections, such as whooping cough or emphysema, sepsis,
Lyme Disease, cat-scratch disease, dysentery, paratyphoid fever, food
poisoning,
typhoid, pneumonia, gonorrhea, meningitis, chlamydia, syphilis, diphtheria,
leprosy, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus,
impetigo, rheumatic fever, scarlet fever, sexually transmitted diseases, skin
diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract
infections, and
wound infections. DRS polynucleotides or polypeptides, or agonists or
antagonists of DRS, can be used to treat, prevent and/or detect any of these
symptoms or diseases. In specific embodiments, DRS polynucleotides,
polypeptides, or agonists thereof are used to treat and/or prevent: tetanus,
diphtheria, botulism, and/or meningitis type B.
Moreover, parasites causing parasitic diseases or symptoms that can be
treated and/or prevented by DRS polynucleotides or polypeptides, or agonists
of
DRS, include, but are not limited to: protozoan parasites including, but not
limited to, Babesia, Balantidium, Besnoitia, Cryptosporidium, Eimeria,
Encephalitozoon, Entamoeba, Giardia, Hammondia, Hepatozoon, Isospora,
Leishmania, Microsporidia, Neospora, Nosema, Pentatrichomonas, Plasmodium
(e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and
Plasmodium ovale), Pneumocystis, Sarcocystis, Schistosoma, Theileria,
Toxoplasma, and Trypanosoma; and helminth parasites including, but not limited
to, Acanthocheilonema, Aelurostrongylus, Ancylostoma, Angiostrongylu.s,
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A.scari,s, Brugia, Bunostomum, Capillaria, Chabertia, C'ooperia, Crenosoma,
Dictyocaulus, Dioctophyme, Dipetalonema, Diphyllobothrium, Diplydium,
Dirofilaria, Dracunculu,s, Enterobius, Filaroides, Haemonchus,
Lagochilascaris, Loa, Mansonella, Muellerius, Nanophyetus, Necator,
Nematodirus, Oe.sophagoslomum, Onchocerca, Opi.sthorchis, Ostertagia,
Parafilaria, Paragonimu,s, Parascaris, Physaloptera, Protostrongylus, Setaria,
Spirocerca, Spirometra, Stephanoftlaria, Strongyloides, Strongylus, Thelazia,
Toxascaris, Toxocara, Trichinella, Trichostrongylus, Trichuris, Uncinaria, and
Wuchereria. These parasites can cause a variety of diseases or symptoms,
including, but not limited to: scabies, trombiculiasis, eye infections (e.g.,
river
blindness), elephantiasis, intestinal disease (e.g., dysentery, giardiasis),
liver
disease, lung disease, opportunistic infections (e.g., AIDS related), malaria,
pregnancy complications, and toxoplasmosis. DRS polynucleotides or
polypeptides, or agonists or antagonists of DRS, can be used to treat, prevent
and/or detect any of these symptoms or diseases. In specific embodiments, DRS
polynucleotides, polypeptides, or agonists thereof are used to treat and/or
prevent malaria.
Polynucleotides and/or polypeptides of the invention and/or agonists
and/or antagonists thereof are also useful as a vaccine adjuvant to enhance
immune responsiveness to specific antigen, tumor-specific, and/or anti-viral
immune responses.
An adjuvant to enhance anti-viral immune responses. Anti-viral immune
responses that may be enhanced using the compositions of the invention as an
adjuvant, include virus and virus associated diseases or symptoms described
herein or otherwise known in the art. In specific embodiments, the
compositions
of the invention are used as an adjuvant to enhance an immune response to a
virus, disease, or symptom selected from the group consisting of: AIDS,
meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B). In another
specific
embodiment, the compositions of the invention are used as an adjuvant to
enhance an immune response to a virus, disease, or symptom selected from the
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group consisting o~ HIV/AIDS, Respiratory syncytial virus, Dengue, Rotavirus,
Japanese B encephalitis, Influenza A and B, Parainfluenza, Measles,
Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever, Herpes simplex
virus, and yellow fever.
Anti-bacterial or anti-fungal immune responses that may be enhanced
using the compositions ofthe invention as an adjuvant, include bacteria or
fungus
and bacteria or fungus associated diseases or symptoms described herein or
otherwise known in the art. In specific embodiments, the compositions of the
invention are used as an adjuvant to enhance an immune response to a bacteria
or fungus, disease, or symptom selected from the group consisting of: tetanus,
diphtheria, botulism, and meningitis type B. In another specific embodiment,
the
compositions of the invention are used as an adjuvant to enhance an immune
response to a bacteria selected from the group consisting of: hibrio cholerae,
Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Nei,sseria
meningitides, Streptococcuspneumoniae, Group B streptococcus, Shigella spp.,
enterotoxigenic E. coli, enterohemorrhagic E. coli, and Borrelia burgdorferi.
Anti-parasitic immune responses that may be enhanced using the
compositions of the invention as an adjuvant, include parasite and parasite
associated diseases or symptoms described herein or otherwise known in the
art.
In specific embodiments, the compositions of the invention are used as an
adjuvant to enhance an immune response to a parasite. In another specific
embodiment, the compositions of the invention are used as an adjuvant to
enhance an immune response to Plasmodium spp. (malaria).
More generally, DRS polynucleotides and/or polypeptides of the
invention and/or agonists and/or antagonists thereof are useful in regulating
(i. e.,
elevating or reducing) immune response. For example, polynucleotides and/or
polypeptides of the invention may be useful in preparation or recovery from
surgery, trauma, radiation therapy, chemotherapy, and transplantation.
Further,
polynucleotides and/or polypeptides of the invention may be may be used to
boost immune response and/or accelerate recovery in the elderly and
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immunocompromised individuals, or as an agent that elevates the immune status
of an individual prior to their receipt of immunosuppressive therapies. Also,
polynucleotides and/or polypeptides of the invention may be useful as an agent
to induce higher affinity antibodies, or to increase serum immunoglobulin
concentrations.
In one embodiment, DRS polynucleotides and/or polypeptides of the
invention and/or agonists thereof may be used as an immune system enhancer
prior to, during, or after bone marrow transplant and/or other transplants
(e.g.,
allogenic or xenogenic organ transplantation). With respect to
transplantation,
compositions of the invention may be administered prior to, concomitant with,
and/or after transplantation. In a specific embodiment, compositions of the
invention are administered after transplantation, prior to the beginning of
recovery of T-cell populations. In another specific embodiment, compositions
of the invention are first administered after transplantation after the
beginning of
recovery of T-cell populations, but prior to full recovery of B-cell
populations.
In another embodiment, DRS polynucleotides and/or polypeptides of the
invention and/or agonists thereof may be used as an agent to boost
immunoresponsiveness among B-cell immunodeficient individuals. B-cell
immunodeficiencies that may be ameliorated or treated and/or prevented by
administering the DRS polypeptides or polynucleotides of the invention, or
agonists thereof, include, but are not limited to, severe combined immune
deficiency (SCID), congenital agammaglobulinemia, common variable
immunodeficiency, Wiskott-Aldrich Syndrome, and X-linked immunodeficiency
with hyper IgM.
Additionally, DRS polynucleotides and/or polypeptides of the invention
and/or agonists thereof may be used as an agent to boost immunoresponsiveness
among individuals having an acquired loss of B-cell function. Conditions
resulting in an acquired loss of B-cell function that may be ameliorated,
treated,
and/or prevented by administering the DRS polypeptides or polynucleotides of
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the invention, or agonists thereof, include, but are not limited to, HIV
Infection,
AIDS, bone marrow transplant, and B-cell chronic lymphocytic leukemia (CLL).
Furthermore, DRS polynucleotides and/or polypeptides of the invention
and/or agonists thereof may be used as an agent to boost immunoresponsiveness
S among individuals having a temporary immune deficiency. Conditions resulting
in a temporary immune deficiency that may be ameliorated, treated, and/or
prevented by administering the DRS polypeptides or polynucleotides of the
invention, or agonists thereof, include, but are not limited to, recovery from
viral
infections (e.g., influenza), conditions associated with malnutrition,
recovery
from infectious mononucleosis, or conditions associated with stress, recovery
from measles, recovery from blood transfusion, recovery from surgery.
DRS polynucleotides and/or polypeptides ofthe invention and/or agonists
thereof may also be used as a regulator of antigen presentation by monocytes,
dendritic cells, and/or B-cells. In one embodiment, DRS (in soluble, membrane-
bound or transmembrane forms) enhances antigen presentation or antagonizes
antigen presentation in vitro or in vivo.
In related embodiments, said enhancement or antagonization of antigen
presentation may be useful as an anti-tumor treatment or to modulate the
immune
system. For example, DRS polynucleotides and/or polypeptides of the invention
and/or agonists thereof may be used as an agent to direct an individuals
immune
system towards development of a humoral response (i.e. TH2) as opposed to a
TH1 cellular response. Also, DRS polynucleotides and/or polypeptides of the
invention and/or agonists thereof may be used as a stimulator of B-cell
production in pathologies such as AIDS, chronic lymphocyte disorder and/or
Common Variable Immunodeficiency.
In another embodiment, DRS polynucleotides and/or polypeptides of the
invention and/or agonists thereof may be used as a means to induce tumor
proliferation and thus make the tumor more susceptible to anti-neoplastic
agents.
For examples multiple myeloma is a slowly dividing disease and is thus
refractory
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to virtually all anti-neoplastic regimens. If these cells were forced to
proliferate
more rapidly their susceptibility profile would likely change.
Other embodiments where DRS polynucleotides and/or polypeptides of
the invention and/or agonists thereof may be used include, but are not limited
to:
as a stimulator of B-cell production in pathologies such as AIDS, chronic
lymphocyte disorder and/or Common Variable Immunodeficiency; as a therapy
for generation and/or regeneration oflymphoid tissues following surgery,
trauma
or genetic defect; as a gene-based therapy for genetically inherited disorders
resulting in immuno-incompetence such as observed among SCI17 patients; as an
antigen for the generation of antibodies to inhibit or enhance DRS mediated
responses; as a means of activating T-cells; as pretreatment of bone marrow
samples prior to transplant (such treatment would increase B-cell
representation
and thus accelerate recovery); as a means of regulating secreted cytokines
that
are elicited by DRS; to modulate IgE concentrations in vitro or in vivo; and
to
treat and/or prevent IgE-mediated allergic reactions including, but are not
limited
to, asthma, rhinitis, and eczema.
Alternatively, DRS polynucleotides and/or polypeptides of the invention
and/or agonists and/or antagonists thereof are useful as immunosuppressive
agents, for example in the treatment and/or prevention of autoimmune
disorders.
In specific embodiments, polynucleotides and/or polypeptides ofthe invention
are
used to treat and/or prevent chronic inflammatory, allergic or autoimmune
conditions, such as those described herein or are otherwise known in the art.
Preferably, treatment using DRS polynucleotides or polypeptides, or
agonists of DRS, could either be by administering an effective amount of DRS
polypeptide to the patient, or by removing cells from the patient, supplying
the
cells with DRS polynucleotide, and returning the engineered cells to the
patient
(ex vivo therapy). Moreover, as further discussed herein, the DRS polypeptide
or polynucleotide can be used as an adjuvant in a vaccine to raise an immune
response against infectious disease.
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Additional preferred embodiments of the invention include, but are not
limited to, the use of DRS polypeptides and functional agonists in the
following
applications: administration to an animal (e.g., mouse, rat, rabbit, hamster,
guinea
pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-
human primate, and human, most preferably human) to boost the immune system
to produce increased quantities of one or more antibodies (e.g., IgG, IgA,
IgM,
and IgE), to induce higher affinity antibody production (e.g., IgG, IgA, IgM,
and
IgE), and/or to increase an immune response; or administration to an animal
(including, but not limited to, those listed above, and also including
transgenic
animals) incapable of producing functional endogenous antibody molecules or
having an otherwise compromised endogenous immune system, but which is
capable of producing human immunoglobulin molecules by means of a
reconstituted or partially reconstituted immune system from another animal
(see,
e.g., published PCT Application Nos. W098/24893, W096/34096,
W096/33735, and W091/10741.
Antagonists of DRS include binding and/or inhibitory antibodies,
antisense nucleic acids, ribozymes or soluble forms of the DRS receptor(s).
These would be expected to reverse many of the activities of herein, as well
as
find clinical or practical application including, but not limited to the
following
applications. DRS antagonists may be used as a means of blocking various
aspects of immune responses to foreign agents or self, for example, autoimmune
disorders such as lupus, and arthritis, as well as immunoresponsiveness to
skin
allergies, inflammation, bowel disease, injury and pathogens. Although our
current data speaks directly to the potential role of DR5 in B-cell and T-cell
related pathologies, it remains possible that other cell types may gain
expression
or responsiveness to DRS. Thus, DRS may, like CD40 and its ligand, may be
regulated by the status of the immune system and the microenvironment in which
the cell is located. DRS antagonists may be used as a therapy for preventing
the
B-cell proliferation and Ig secretion associated with autoimmune diseases such
as idiopathic thrombocytopenic purpura, systemic lupus erythramatosus and; as
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an inhibitor of graft versus host disease or transplant rejection; as a
therapy for
B-cell malignancies such as ALL, Hodgkins disease, non-Hodgkins lymphoma,
Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's
lymphoma, and EBV-transformed diseases; as a therapy for chronic
hypergammaglobulinemeia evident in such diseases as monoclonalgammopathy
ofundetermined significance (MGUS), Waldenstrom's disease, related idiopathic
monoclonalgammopathies, and plasmacytomas; as a therapy for decreasing
cellular proliferation of Large B-cell Lymphomas; as a means of decreasing the
involvement of B-cells and Ig associated with Chronic Myelogenous Leukemia;
or as an immunosuppressive agent.
Furthermore, DRS polypeptides or polynucleotides of the invention, or
antagonists thereof may be used to modulate IgE concentrations in vitro or in
vivo, or to treat and/or prevent IgE-mediated allergic reactions including,
but not
limited to, asthma, rhinitis, and eczema.
All of the therapeutic applications of DRS polynucleotides and/or
polypeptides of the invention and/or agonists and/or antagonists thereof
described herein may, in addition to their uses in human medicine, be used in
veterinary medicine. The present invention includes treatment of companion
animals, including, but not limited to dogs, cats, ferrets, birds, and horses;
food
animals, including, but not limited to cows, pigs, chickens, and sheep; and
exotic
animals, e.g., zoo animals.
The above-recited applications have uses in a wide variety ofhosts. Such
hosts include, but are not limited to, human, murine, rabbit, goat, guinea
pig,
camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep,
dog, cat, non-human primate, and human. In specific embodiments, the host is
a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or
cat.
In preferred embodiments, the host is a mammal. In most preferred
embodiments, the host is a human.
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DRS polynucleotides and/or polypeptides ofthe invention and/or agonists
and/or antagonists thereof described herein may be employed in a composition
with a pharmaceutically acceptable carrier, e.g., as described herein.
In one aspect, the present invention is directed to a method for enhancing
apoptosis induced by a TNF-family ligand, which involves administering to a
cell
which expresses the DRS polypeptide an effective amount of DRS ligand, analog
or an agonist capable of increasing DR5 mediated signaling. Preferably, DRS
mediated signaling is increased to treat and/or prevent a disease wherein
decreased apoptosis or decreased cytokine and adhesion molecule expression is
exhibited. An agonist can include soluble forms of DRS and monoclonal
antibodies directed against the DRS polypeptide.
In a further aspect, the present invention is directed to a method for
inhibiting apoptosis induced by a TNF-family ligand, which involves
administering to a cell which expresses the, DRS polypeptide an effective
amount
of an antagonist capable of decreasing DRS mediated signaling. Preferably, DRS
mediated signaling is decreased to treat and/or prevent a disease wherein
increased apoptosis or NF-kB expression is exhibited. An antagonist can
include
soluble forms of DRS (e.g., polypeptides containing all or a portion of the
DRS
extracellular domain) and monoclonal antibodies directed against the DRS
polypeptide.
By "agonist" is intended naturally occurring and synthetic compounds
capable of enhancing or potentiating apoptosis. By "antagonist" is intended
naturally occurring and synthetic compounds capable of inhibiting apoptosis.
Whether any candidate "agonist" or "antagonist" of the present invention can
enhance or inhibit apoptosis can be determined using art-known TNF-family
ligand/receptor cellular response assays, including those described in more
detail
below.
One such screening procedure involves the use of melanophores which
are transfected to express the receptor of the present invention. Such a
screening
technique is described in PCT WO 92/01810, published February 6, 1992. Such
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an assay may be employed, for example, for screening for a compound which
inhibits (or enhances) activation of the receptor polypeptide of the present
invention by contacting the melanophore cells which encode the receptor with
both a TNF-family ligand and the candidate antagonist (or agonist). Inhibition
S or enhancement of the signal generated by the ligand indicates that the
compound
is an antagonist or agonist of the ligand/receptor signaling pathway.
Other screening techniques include the use of cells which express the
receptor (for example, transfected CHO cells) in a system which measures
extracellular pH changes caused by receptor activation. For example,
compounds may be contacted with a cell which expresses the receptor
polypeptide of the present invention and a second messenger response, e.g.,
signal transduction or pH changes, may be measured to determine whether the
potential compound activates or inhibits the receptor.
Another such screening technique involves introducing RNA encoding
the receptor into Xenopu.s oocytes to transiently express the receptor. The
receptor oocytes may then be contacted with the receptor ligand and a compound
to be screened, followed by detection of inhibition or activation of a calcium
signal in the case of screening for compounds which are thought to inhibit
activation of the receptor.
Another screening technique involves expressing in cells a construct
wherein the receptor is linked to a phospholipase C or D. Such cells include
endothelial cells, smooth muscle cells, embryonic kidney cells, etc. The
screening
may be accomplished as herein above described by detecting activation of the
receptor or inhibition of activation ofthe receptor from the phospholipase
signal.
Another method involves screening for compounds (antagonists) which
inhibit activation of the receptor polypeptide of the present invention by
determining inhibition of binding of labeled ligand to cells which have the
receptor on the surface thereof. Such a method involves transfecting a
eukaryotic cell with DNA encoding the receptor such that the cell expresses
the
receptor on its surface and contacting the cell with a compound in the
presence
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of a labeled form of a known ligand. The ligand can be labeled, e.g., by
radioactivity. The amount of labeled ligand bound to the receptors is
measured,
e.g., by measuring radioactivity of the receptors. If the compound binds to
the
receptor as determined by a reduction of labeled ligand which binds to the
S receptors, the binding of labeled ligand to the receptor is inhibited.
Further screening assays for agonist and antagonist of the present
invention are described in Tartaglia, L.A., and Goeddel, D.V., J. Biol. Chem.
267:43 04-43 07( 1992).
Thus, in a further aspect, a screening method is provided for determining
whether a candidate agonist or antagonist is capable of enhancing or
inhibiting
a cellular response to a TNF-family ligand. The method involves contacting
cells
which express the DRS polypeptide with a candidate compound and a TNF-
family ligand, assaying a cellular response, and comparing the cellular
response
to a standard cellular response, the standard being assayed when contact is
made
with the ligand in absence of the candidate compound, whereby an increased
cellular response over the standard indicates that the candidate compound is
an
agonist of the ligand/receptor signaling pathway and a decreased cellular
response compared to the standard indicates that the candidate compound is an
antagonist of the ligand/receptor signaling pathway. By "assaying a cellular
response" is intended qualitatively or quantitatively measuring a cellular
response
to a candidate compound and/or a TNF-family ligand (e.g., determining or
estimating an increase or decrease in T-cell or B-cell proliferation, or
tritiated
thymidine labeling). By the invention, a cell expressing the DRS polypeptide
can
be contacted with either an endogenous or exogenously administered TNF-family
ligand.
Agonist according to the present invention include naturally occurring
and synthetic compounds such as, for example, TNF family ligand peptide
fragments, transforming growth factor, neurotransmitters (such as glutamate,
dopamine, N methyl-D-aspartate), tumor suppressors (p53), cytolytic T-cells
and
antimetabolites. Preferred agonists include chemotherapeutic drugs such as,
for
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example, cisplatin, doxorubicin, bleomycin, cytosine arabinoside, nitrogen
mustard, methotrexate and vincristine. Others include ethanol and ~3-amyloid
peptide. (Science 267:1457-1458 (1995)). Further preferred agonist include
polyclonal and monoclonal antibodies raised against the DRS polypeptide, or a
fragment thereof. Such agonist antibodies raised against a TNF-family receptor
are disclosed in Tartaglia, L.A., etal., Proc. Natl. Acad Sci. USA 88:9292-
9296
(1991); and Tartaglia, L.A., and Goeddel, D.V., J. Biol. Chem. 267 (7):4304-
4307 (1992) See, also, PCT Application WO 94/09137.
Antagonist according to the present invention include naturally occurring
and synthetic compounds such as, for example, the CD40 ligand, neutral amino
acids, zinc, estrogen, androgens, viral genes (such as Adenovirus EIB,
Baculovirus p35 and IAP, Cowpox virus crmA, Epstein-Barr virus BHRFI,
LMP-1, African swine fever virus LMWS-HL, and Herpesvirus yl 34.5), calpain
inhibitors, cysteine protease inhibitors, and tumor promoters (such as PMA,
Phenobarbital, and alpha-Hexachlorocyclohexane).
Other potential antagonists include antisense molecules. Antisense
technology can be used to control gene expression through antisense DNA or
RNA or through triple-helix formation. Antisense techniques are discussed, for
example, in Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as
Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).
Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids
Research 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et
al., Science 251:1360 (1991). The methods are based on binding of a
polynucleotide to a complementary DNA or RNA.
For example, the 5' coding portion of a polynucleotide that encodes the
mature polypeptide of the present invention may be used to design an antisense
RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA
oligonucleotide is designed to be complementary to a region of the gene
involved
in transcription thereby preventing transcription and the production of the
receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo
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and blocks translation of the mRNA molecule into receptor polypeptide. The
oligonucleotides described above can also be delivered to cells such that the
antisense RNA or DNA may be expressed in vivo to inhibit production of the
DRS receptor.
In one embodiment, the DRS antisense nucleic acid of the invention is
produced intracellularly by transcription from an exogenous sequence. For
example, a vector or a portion thereof, is transcribed, producing an antisense
nucleic acid (RNA) of the invention. Such a vector would contain a sequence
encoding the DRS antisense nucleic acid. Such a vector can remain episomal or
become chromosomally integrated, as long as it can be transcribed to produce
the
desired antisense RNA. Such vectors can be constructed by recombinant DNA
technology methods standard in the art. Vectors can be plasmid, viral, or
others
know in the art, used for replication and expression in vertebrate cells.
Expression of the sequence encoding DRS, or fragments thereof, can be by any
promoter known in the art to act in vertebrate, preferably human cells. Such
promoters can be inducible or a constitutive. Such promoters include, but are
not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature
29:304-310 (1981), the promoter contained in the 3' long terminal repeat
ofRous
sarcoma virus (Yamamoto et al., Cell 22:787-797 ( 1980), the herpes thymidine
promoter (Wagner et al., Proc. Natl. Acad Sci. U.S.A. 78:1441-1445 (1981),
the regulatory sequences of the metallothionein gene (Brinster, et al., Nalure
296:39-42 (1982)), etc.
The antisense nucleic acids of the invention comprise a sequence
complementary to at least a portion of an RNA transcript of a DRS gene.
However, absolute complementarity, although preferred, is not required. A
sequence "complementary to at least a portion of an RNA," referred to herein,
means a sequence having sufficient complementarity to be able to hybridize
with
the RNA, forming a stable duplex; in the case of double stranded DRS antisense
nucleic acids, a single strand of the duplex DNA may thus be tested, or
triplex
formation may be assayed. The ability to hybridize will depend on both the
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degree of complementarity and the length of the antisense nucleic acid.
Generally, the larger the hybridizing nucleic acid, the more base mismatches
with
a DRS RNA it may contain and still form a stable duplex (or triplex as the
case
may be). One skilled in the art can ascertain a tolerable degree of mismatch
by
use of standard procedures to determine the melting point of the hybridized
complex.
Oligonucleotides that are complementary to the 5' end of the message,
e.g., the 5' untranslated sequence up to and including the AUG initiation
codon,
should work most efficiently at inhibiting translation. However, sequences
complementary to the 3' untranslated sequences of mRNAs have been shown to
be effective at inhibiting translation of mRNAs as well. See generally,
Wagner,
R.,Nature 372:333-335 (1994). Thus, oligonucleotides complementary to either
the 5'- or 3'- non- translated, non-coding regions of the DRS shown in FIG. 1
could be used in an antisense approach to inhibit translation of endogenous
DRS
mRNA. Oligonucleotides complementary to the 5' untranslated region of the
mRNA should include the complement of the AUG start codon. Antisense
oligonucleotides complementary to mRNA coding regions are less efficient
inhibitors of translation but could be used in accordance with the invention.
Whether designed to hybridize to the 5'-, 3'-, or coding region of DRS mRNA,
antisense nucleic acids should be at least six nucleotides in length, and are
preferably oligonucleotides ranging from 6 to about 50 nucleotides in length.
In
specific aspects the oligonucleotide is at least 10 nucleotides, at least 17
nucleotides, at least 25 nucleotides or at least 50 nucleotides.
The polynucleotides of the invention can be DNA or RNA or chimeric
mixtures or derivatives or modified versions thereof, single-stranded or
double-
stranded. The oligonucleotide can be modified at the base moiety, sugar
moiety,
or phosphate backbone, for example, to improve stability of the molecule,
hybridization, etc. The oligonucleotide may include other appended groups such
as peptides (e.g., for targeting host cell receptors in vivo), or agents
facilitating
transport across the cell membrane (see, e.g., Letsinger et al., Proc. Natl.
Acad.
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Sci. U.S.A. 86:6553-6556 (1989); Lemaitre etad., Proc. Natl. Acad. Sci. 84:648-
652 (1987); PCT Publication No. W088/09810, published December 15, 1988)
or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134,
published April 25, 1988), hybridization-triggered cleavage agents. (See,
e.g.,
Krol etal., BioTechniques 6:958-976 (1988)) or intercalating agents. (See,
e.g.,
Zon, Pharm. Res. 5:539-549 (1988)). To this end, the oligonucleotide may be
conjugated to another molecule, e.g., a peptide, hybridization triggered cross-
linking agent, transport agent, hybridization-triggered cleavage agent, etc.
The antisense oligonucleotide may comprise at least one modified base
moiety which is selected from the group including, but not limited to,
S-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil,
dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,
2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,
7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-
2-thiouracil, beta-D-mannosylqueosine, 5 -methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid
(v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, S-methyl-2-
thiouracil,
2-thiouracil, 4-thiouracil, S-methyluracil, uracil-5-oxyacetic acid
methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-
carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
The antisense oligonucleotide may also comprise at least one modified
sugar moiety selected from the group including, but not limited to, arabinose,
2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the antisense oligonucleotide comprises at
least one modified phosphate backbone selected from the group including, but
not limited to, a phosphorothioate, a phosphorodithioate, a
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phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or analog
thereof.
In yet another embodiment, the antisense oligonucleotide is an -anomeric
oligonucleotide. An -anomeric oligonucleotide forms specific double-stranded
hybrids with complementary RNA in which, contrary to the usual -units, the
strands run parallel to each other (Gautier et al., Nucl. Acids Res. 15:6625-
6641
(1987)). The oligonucleotide is a 2-0-methylribonucleotide (moue et al., Nucl.
Acids Re s. 15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (moue et
al., FEBSLett. 215:327-330 (1987)).
Potential antagonists according to the invention also include catalytic
RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364,
published October 4, 1990; Sarver et al, Science 247:1222-1225 (1990). While
ribozymes that cleave mRNA at site specific recognition sequences can be used
to destroy DR5 mRNAs, the use of hammerhead ribozymes is preferred.
Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions
that form complementary base pairs with the target mRNA. The sole
requirement is that the target mRNA have the following sequence of two bases:
5'-UG-3'. The construction and production of hammerhead ribozymes is well
known in the art and is described more fully in Haseloff and Gerlach, Nature
334:585-591 (1988). There are numerous potential hammerhead ribozyme
cleavage sites within the nucleotide sequence of DR5 (FIG. 1). Preferably, the
ribozyme is engineered so that the cleavage recognition site is located near
the
5' end of the DRS mRNA; i.e., to increase efficiency and minimize the
intracellular accumulation ofnon-functional mRNA transcripts. DNA constructs
encoding the ribozyme may be introduced into the cell in the same manner as
described above for the introduction of antisense encoding DNA. Since
ribozymes, unlike antisense molecules are catalytic, a lower intracellular
concentration is required for efficiency.
Further antagonist according to the present invention include soluble
forms of DRS, i.e., DR5 fragments that include the ligand binding domain from
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the extracellular region of the full length receptor. Such soluble forms of
the
receptor, which may be naturally occurring or synthetic, antagonize DRS
mediated signaling by competing with the cell surface DRS for binding to TNF-
family ligands. Thus, soluble forms of the receptor that include the ligand
S binding domain are novel cytokines capable of inhibiting apoptosis induced
by
TNF-family ligands. These may be expressed as monomers, but, are preferably
expressed as dimers or trimers, since these have been shown to be superior to
monomeric forms of soluble receptor as antagonists, e.g., IgGFc-TNF receptor
family fusions. Other such cytokines are known in the art and include Fas B (a
soluble form of the mouse Fas receptor) that acts physiologically to limit
apoptosis induced by Fas ligand (Hughes, D.P. and Crispe, LN., J. Exp. Med.
182:1395-1401 (1995)).
As discussed above, the term "antibody" (Ab) or "monoclonal antibody"
(mAb) as used herein is meant to include intact molecules as well as fragments
thereof (such as, for example, Fab, and F(ab')Z fragments) which are capable
of
binding an antigen. Fab, Fab', and F (ab')2 fragments lack the Fc fragment of
intact antibody, clear more rapidly from the circulation, and may have less
non-
specific tissue binding of an intact antibody (Wahl et al., J. Nucl.. Med.
24:316-
325 (1983)).
Antibodies according to the present invention may be prepared by any of
a variety of standard methods using DRS immunogens of the present invention.
As indicated, such DRS immunogens include the full length DRS polypeptide
(which may or may not include the leader sequence) and DRS polypeptide
fragments such as the ligand binding domain, the transmembrane domain, the
intracellular domain and the death domain.
Antibodies of the invention can be used in methods known in the art
relating to the localization and activity of the polypeptide sequences of the
invention, e.g., for imaging these polypeptides, measuring levels thereof in
appropriate physiological samples, etc. The antibodies also have use in
immunoassays and in therapeutics as agonists and antagonists of DRS.
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Proteins and other compounds which bind the DRS domains are also
candidate agonist and antagonist according to the present invention. Such
binding compounds can be "captured" using the yeast two-hybrid system (Fields
and Song, Nature 340:245-246 (1989)). A modified version of the yeast two-
s hybrid system has been described by Roger Brent and his colleagues (Gyuris,
J.
ei al., Cell 75:791-803 (1993); Zervos, A.S. et al., Cell 72:223-232 (1993)).
Preferably, the yeast two-hybrid system is used according to the present
invention to capture compounds which bind to either the DRS ligand binding
domain or to the DRS intracellular domain. Such compounds are good candidate
agonist and antagonist of the present invention.
By a "TNF-family ligand" is intended naturally occurring, recombinant,
and synthetic ligands that are capable of binding to a member of the TNF
receptor family and inducing the ligand/receptor signaling pathway. Members
of the TNF ligand family include, but are not limited to, DRS ligands, TRAIL,
TNF-a, lymphotoxin-a. (LT-a., also known as TNF-(3), LT-(3 (found in complex
heterotrimer LT-a2-(3), Fast, CD40, CD27, CD30, 4-1BB, OX40 and nerve
growth factor (NGF). An example of an assay that can be performed to
determine the ability of DRS and derivatives (including fragments) and analogs
thereof to bind TRAIL is described below in Example 6.
Gene Therapy
In a specific embodiment, nucleic acids comprising sequences encoding
antibodies or functional derivatives thereof, are administered to treat,
inhibit
and/or prevent a disease or disorder associated with aberrant expression
and/or
activity of a polypeptide of the invention, by way of gene therapy. Gene
therapy
refers to therapy performed by the administration to a subject of an expressed
or
expressible nucleic acid. In this embodiment of the invention, the nucleic
acids
produce their encoded protein that mediates a therapeutic effect.
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Any of the methods for gene therapy available in the art can be used
according to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al.,
1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;
Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993,
Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.
62:191-217; May, 1993, T1BTECH 11 (5):155-215). Methods commonly known
in the art of recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John
Wiley
& Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory
Manual, Stockton Press, NY.
In a preferred aspect, the compound comprises nucleic acid sequences
encoding an antibody, said nucleic acid sequences being part of expression
vectors that express the antibody or fragments or chimeric proteins or heavy
or
light chains thereof in a suitable host. In particular, such nucleic acid
sequences
have promoters operably linked to the antibody coding region, said promoter
being inducible or constitutive, and, optionally, tissue- specific. In another
particular embodiment, nucleic acid molecules are used in which the antibody
coding sequences and any other desired sequences are flanked by regions that
promote homologous recombination at a desired site in the genome, thus
providing for intrachromosomal expression of the antibody nucleic acids
(Koller
and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al.,
1989, Nature 342:435-438). In specific embodiments, the expressed antibody
molecule is a single chain antibody; alternatively, the nucleic acid sequences
include sequences encoding both the heavy and light chains, or fragments
thereof,
of the antibody.
Delivery of the nucleic acids into a patient may be either direct, in which
case the patient is directly exposed to the nucleic acid or nucleic acid-
carrying
vectors, or indirect, in which case, cells are first transformed with the
nucleic
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acids in vitro, then transplanted into the patient. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.
In a specific embodiment, the nucleic acid sequences are directly
administered in vivo, where it is expressed to produce the encoded product.
This
can be accomplished by any of numerous methods known in the art, e.g., by
constructing them as part of an appropriate nucleic acid expression vector and
administering it so that they become intracellular, e.g., by infection using
defective or attenuated retrovirals or other viral vectors (see U. S. Patent
No.
4,980,286), or by direct injection of naked DNA, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or
cell-
surface receptors or transfecting agents, encapsulation in liposomes,
microparticles, or microcapsules, or by administering them in linkage to a
peptide
which is known to enter the nucleus, by administering it in linkage to a
ligand
subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, .I.
Biol.
Chem. 262:4429-4432) (which can be used to target cell types specifically
expressing the receptors), etc. In another embodiment, nucleic acid-ligand
complexes can be formed in which the ligand comprises a fusogenic viral
peptide
to disrupt endosomes, allowing the nucleic acid to avoid lysosomal
degradation.
In yet another embodiment, the nucleic acid can be targeted in vivo for cell
specific uptake and expression, by targeting a specific receptor (.see, e.g.,
PCT
Publications WO 92/06180 dated April 16, 1992 (Wu et al.); WO 92/22635
dated December 23, 1992 (Wilson et al.); W092/20316 dated November 26,
1992 (Findeis et ad.); W093/14188 dated July 22, 1993 (Clarke et al.), WO
93/20221 dated October 14, 1993 (Young)). Alternatively, the nucleic acid can
be introduced intracellularly and incorporated within host cell DNA for
expression, by homologous recombination (Koller and Smithies, 1989, Proc.
Natl. Acad. Sci. USA 86:8932-8935; Zijlstraetal., 1989, Nature 342:435-438).
In a specific embodiment, viral vectors that contains nucleic acid
sequences encoding an antibody of the invention are used. For example, a
retroviral vector can be used (see Miller et ad., 1993, Meth. Enzymol. 217:581-
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599). These retroviral vectors have been to delete retroviral sequences that
are
not necessary for packaging of the viral genome and integration into host cell
DNA. The nucleic acid sequences encoding the antibody to be used in gene
therapy are cloned into one or more vectors, which facilitates delivery of the
gene into a patient. More detail about retroviral vectors can be found in
Boesen
et al., 1994, Biotherapy 6:291-302, which describes the use of a retroviral
vector
to deliver the mdrl gene to hematopoietic stem cells in order to make the stem
cells more resistant to chemotherapy. Other references illustrating the use of
retroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin. Invest.
93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg,
1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr.
Opin. in Genetics and Devel. 3:110-114.
Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses are especially attractive vehicles for delivering genes to
respiratory
epithelia. Adenoviruses naturally infect respiratory epithelia where they
cause a
mild disease. Other targets for adenovirus-based delivery systems are liver,
the
central nervous system, endothelial cells, and muscle. Adenoviruses have the
advantage of being capable of infecting non-dividing cells. Kozarsky and
Wilson, 1993, Current Opinion in Genetics and Development 3:499-503 present
a review of adenovirus-based gene therapy. Bout et al., 1994, Human Gene
Therapy 5:3-10 demonstrated the use of adenovirus vectors to transfer genes to
the respiratory epithelia of rhesus monkeys. Other instances of the use of
adenoviruses in gene therapy can be found in Rosenfeld et al., 1991, Science
252:431-434; Rosenfeld etal., 1992, Cell 68:143- 155; Mastrangeli etal., 1993,
J. Clin. Invest. 91:225-234; PCT Publication W094/12649; and Wang et al.,
1995, Gene Therapy 2:775-783. In a preferred embodiment, adenovirus vectors
are used.
Adeno-associated virus (AAA has also been proposed for use in gene
therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; U.S.
Patent No. 5,436,146).
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Another approach to gene therapy involves transferring a gene to cells in
tissue culture by such methods as electroporation, lipofection, calcium
phosphate
mediated transfection, or viral infection. Usually, the method of transfer
includes
the transfer of a selectable marker to the cells. The cells are then placed
under
selection to isolate those cells that have taken up and are expressing the
transferred gene. Those cells are then delivered to a patient.
In this embodiment, the nucleic acid is introduced into a cell prior to
administration in vivo of the resulting recombinant cell. Such introduction
can
be carried out by any method known in the art, including but not limited to
transfection, electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell fusion,
chromosome-mediated gene transfer, microcell-mediated gene transfer,
spheroplast fusion, etc. Numerous techniques are known in the art for the
introduction of foreign genes into cells (see, e.g., Loei~ler and Behr, 1993,
Meth.
Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Cline,
1985, Pharmac. Ther. 29:69-92) and may be used in accordance with the present
invention, provided that the necessary developmental and physiological
functions
of the recipient cells are not disrupted. The technique should provide for the
stable transfer ofthe nucleic acid to the cell, so that the nucleic acid is
expressible
by the cell and preferably heritable and expressible by its cell progeny.
The resulting recombinant cells can be delivered to a patient by various
methods known in the art. Recombinant blood cells (e.g., hematopoietic stem
or progenitor cells) are preferably administered intravenously. The amount of
cells envisioned for use depends on the desired effect, patient state, etc.,
and can
be determined by one skilled in the art.
Cells into which a nucleic acid can be introduced for purposes of gene
therapy encompass any desired, available cell type, and include but are not
limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts,
muscle
cells, hepatocytes; blood cells such as T-lymphocytes, B-lymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
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granulocytes; various stem or progenitor cells, in particular hematopoietic
stem
or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood,
peripheral blood, fetal liver, etc.
In a preferred embodiment, the cell used for gene therapy is autologous
to the patient.
In an embodiment in which recombinant cells are used in gene therapy,
nucleic acid sequences encoding an antibody are introduced into the cells such
that they are expressible by the cells or their progeny, and the recombinant
cells
are then administered in vivo for therapeutic effect. In a specific
embodiment,
stem or progenitor cells are used. Any stem and/or progenitor cells which can
be isolated and maintained in vitro can potentially be used in accordance with
this
embodiment ofthe present invention (see, e.g., PCT Publication WO 94/08598,
dated April 28, 1994; Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald,
1980, Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo Clinic
Proc. 61:771 ).
In a specific embodiment, the nucleic acid to be introduced for purposes
of gene therapy comprises an inducible promoter operably linked to the coding
region, such that expression of the nucleic acid is controllable by
controlling the
presence or absence of the appropriate inducer of transcription.
Modes of Administration
The invention provides methods of treatment, inhibition and prophylaxis
by administration to a subject of an effective amount of a compound or
pharmaceutical composition of the invention, preferably an antibody of the
invention. In a preferred aspect, the compound is substantially purified
(e.g.,
substantially free from substances that limit its effect or produce undesired
side-
effects). The subject is preferably an animal, including but not limited to
animals
such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a
mammal, and most preferably human.
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Formulations and methods of administration that can be employed when
the compound comprises a nucleic acid or an immunoglobulin are described
above; additional appropriate formulations and routes of administration can be
selected from among those described herein below.
The agonist or antagonists described herein can be administered in vitro,
ex viva, or in viva to cells which express the receptor of the present
invention.
By administration of an "effective amount" of an agonist or antagonist is
intended
an amount of the compound that is sufficient to enhance or inhibit a cellular
response to a TNF-family ligand and include polypeptides. In particular, by
administration of an "effective amount" of an agonist or antagonists is
intended
an amount effective to enhance or inhibit DRS mediated apoptosis. Of course,
where it is desired for apoptosis is to be enhanced, an agonist according to
the
present invention can be co-administered with a TNF-family ligand. One of
ordinary skill will appreciate that effective amounts of an agonist or
antagonist
can be determined empirically and may be employed in pure form or in
pharmaceutically acceptable salt, ester or prodrug form. The agonist or
antagonist may be administered in compositions in combination with one or more
pharmaceutically acceptable excipients (i.e., carriers).
It will be understood that, when administered to a human patient, the
total daily usage of the compounds and compositions of the present invention
will be decided by the attending physician within the scope of sound medical
judgement. The specific therapeutically effective dose level for any
particular
patient will depend upon factors well known in the medical arts.
As a general proposition, the total pharmaceutically effective amount of
DRS polypeptide administered parenterally per dose will be in the range of
about
1 ~cg/kg/day to 10 mg/kg/day of patient body weight, although, as noted above,
this will be subject to therapeutic discretion. More preferably, this dose is
at
least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and
1 mg/kg/day for the hormone. If given continuously, the DRS agonists or
antagonists is typically administered at a dose rate of about 1 ~g/kg/hour to
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about 50 ,ug/kg/hour, either by 1-4 injections per day or by continuous
subcutaneous infusions, for example, using a mini-pump. An intravenous bag
solution may also be employed.
Dosaging may also be arranged in a patient specific manner to provide a
predetermined concentration of an agonist or antagonist in the blood, as
determined by the RIA technique. Thus patient dosaging may be adjusted to
achieve regular on-going trough blood levels, as measured by RIA, on the order
of from SO to 1000 ng/ml, preferably 150 to 500 ng/ml.
Pharmaceutical compositions are provided comprising an agonist or
antagonist (including DRS polynucleotides and polypeptides ofthe invention)
and
a pharmaceutically acceptable carrier or excipient, which may be administered
orally, rectally, parenterally, intracistemally, intravaginally,
intraperitoneally,
topically (as by powders, ointments, drops or transdermal patch), bucally, or
as
an oral or nasal spray. Importantly, by co-administering an agonist and a TNF-
1 S family ligand, clinical side effects can be reduced by using lower doses
of both
the ligand and the agonist. It will be understood that the agonist can be "co-
administered" either before, after, or simultaneously with the TNF-family
ligand,
depending on the exigencies of a particular therapeutic application. By
"pharmaceutically acceptable carrier" is meant a non-toxic solid, semisolid or
liquid filler, diluent, encapsulating material or formulation auxiliary of any
type.
In a specific embodiment, "pharmaceutically acceptable" means approved by a
regulatory agency of the federal or a state government or listed in the U.S.
Pharmacopeia or other generally recognized pharmacopeia for use in animals,
and more particularly humans. The term "carrier" refers to a diluent,
adjuvant,
excipient, or vehicle with which the therapeutic is administered. Such
pharmaceutical carriers include sterile liquids, such as water and oils,
including
those of petroleum, animal, vegetable or synthetic origin, such as peanut oil,
soybean oil, mineral oil, sesame oil and the like. Water is a preferred
carrier
when the pharmaceutical composition is administered intravenously. Saline
solutions and aqueous dextrose and glycerol solutions can also be employed as
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liquid carriers, particularly for injectable solutions. Suitable
pharmaceutical
excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice,
flour,
chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The
composition, if desired, can also contain minor amounts ofwetting or
emulsifying
agents, or pH bui~ering agents. These compositions can take the form of
solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-
release formulations and the like. The composition can be formulated as a
suppository, with traditional binders and carriers such as triglycerides. Oral
formulation can include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose,
magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such
compositions will contain a therapeutically effective amount of the compound,
preferably in purified form, together with a suitable amount of carrier so as
to
provide the form for proper administration to the patient. The formulation
should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance
with routine procedures as a pharmaceutical composition adapted for
intravenous
administration to human beings. Typically, compositions for intravenous
administration are solutions in sterile isotonic aqueous bui~er. Where
necessary,
the composition may also include a solubilizing agent and a local anesthetic
such
as lignocaine to ease pain at the site of the injection. Generally, the
ingredients
are supplied either separately or mixed together in unit dosage form, for
example,
as a dry lyophilized powder or water free concentrate in a hermetically sealed
container such as an ampule or sachette indicating the quantity of active
agent.
Where the composition is to be administered by infusion, it can be dispensed
with an infusion bottle containing sterile pharmaceutical grade water or
saline.
Where the composition is administered by injection, an ampule of sterile water
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for injection or saline can be provided so that the ingredients may be mixed
prior
to administration.
The compounds of the invention can be formulated as neutral or salt
forms. Pharmaceutically acceptable salts include those formed with anions such
as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc.,
and those formed with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-
ethylamino ethanol, histidine, procaine, etc.
The term "parenteral" as used herein refers to modes of administration
which include intravenous, intramuscular, intraperitoneal, intrasternal,
subcutaneous and intraarticular injection and infusion.
Various delivery systems are known and can be used to administer a
compound of the invention, e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant cells capable of expressing the compound, receptor-
mediated endocytosis (.see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-
4432), construction of a nucleic acid as part of a retroviral or other vector,
etc.
Methods ofintroduction include but are not limited to intradermal,
intramuscular,
intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral
routes.
The compounds or compositions may be administered by any convenient route,
for example by infusion or bolus injection, by absorption through epithelial
or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.)
and
may be administered together with other biologically active agents.
Administration can be systemic or local. In addition, it may be desirable to
introduce the pharmaceutical compounds or compositions of the invention into
the central nervous system by any suitable route, including intraventricular
and
intrathecal injection; intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir, such as an
Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use
of an inhaler or nebulizer, and formulation with an aerosolizing agent.
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In a specific embodiment, it may be desirable to administer the
pharmaceutical compounds or compositions of the invention locally to the area
in need of treatment; this may be achieved by, for example, and not by way of
limitation, local infusion during surgery, topical application, e.g., in
conjunction
with a wound dressing after surgery, by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant being of a
porous, non-porous, or gelatinous material, including membranes, such as
sialastic membranes, or fibers. Preferably, when administering a protein,
including an antibody, of the invention, care must be taken to use materials
to
which the protein does not absorb.
In another embodiment, the compound or composition can be delivered
in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-
1533;
Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer,
Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-
Berestein, ibid., pp. 317-327; see generally ibid.)
In yet another embodiment, the compound or composition can be
delivered in a controlled release system. In one embodiment, a pump may be
used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201;
Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med.
321:574). In another embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca
Raton, Florida ( 1974); Controlled Drug Bioavailability, Drug Product Design
and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and
Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy
et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351;
Howard
et al., 1989, J. Neurosurg. 71:105). In yet another embodiment, a controlled
release system can be placed in proximity of the therapeutic target, i.e., the
brain, thus requiring only a fraction of the systemic dose (see, e.g.,
Goodson, in
Medical Applications of Controlled Release, supra, vol. 2, pp. 11 S-138
(1984)).
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Other controlled release systems are discussed in the review by Langer
(1990, Science 249:1527-1533).
DRS compositions of the invention are also suitably administered by
sustained-release systems. Suitable examples of sustained-release compositions
include suitable polymeric materials (such as, for example, semi-permeable
polymer matrices in the form of shaped articles, e.g., films, or
microcapsules),
suitable hydrophobic materials (for example as an emulsion in an acceptable
oil)
or ion exchange resins, and sparingly soluble derivatives (such as, for
example,
a sparingly soluble salt).
Sustained-release matrices include polylactides (U. S. Pat. No. 3,773,919,
EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate
(Sidman, U. et al., Biopolymers 22:547-556 (1983)), poly (2- hydroxyethyl
methacrylate) (R. Langer et al., J. Biomed Mater. Res. 15:167-277 (1981), and
R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et
al., Id.) or poly-D- (-)-3-hydroxybutyric acid (EP 133,988).
Sustained-release compositions also include liposomally entrapped
compositions of the invention (see generally, Langer, Science 249:1527-1533
(1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and
Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317 -327 and
353-365 (1989)). Liposomes containing DRS polypeptide my be prepared by
methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci.
(USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA)
77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP
142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and
4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about
200-800 Angstroms) unilamellar type in which the lipid content is greater than
about 30 mol. percent cholesterol, the selected proportion being adjusted for
the
optimal DRS polypeptide therapy.
In a specific embodiment where the compound of the invention is a
nucleic acid encoding a protein, the nucleic acid can be administered in vivo
to
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promote expression of its encoded protein, by constructing it as part of an
appropriate nucleic acid expression vector and administering it so that it
becomes
intracellular, e.g., by use of a retroviral vector (see U. S. Patent No.
4,980,286),
or by direct injection, or by use of microparticle bombardment (e.g., a gene
gun;
Biolistic, Dupont), or coating with lipids or cell-surface receptors or
transfecting
agents, or by administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (.see, e.g., Joliot et al., 1991, Proc. Natl. Acad.
Sci.
USA 88:1864-1868), etc. Alternatively, a nucleic acid can be introduced
intracellularly and incorporated within host cell DNA for expression, by
homologous recombination.
In yet an additional embodiment, the compositions of the invention are
delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed.
Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N.
Engl. J. Med. 321:574 (1989)).
Other controlled release systems are discussed in the review by Langer
(Science 249:1527-1533 (1990)).
In a specific embodiment where the compound of the invention is a
nucleic acid encoding a protein, the nucleic acid can be administered in vivo
to
promote expression of its encoded protein, by constructing it as part of an
appropriate nucleic acid expression vector and administering it so that it
becomes
intracellular, e.g., by use of a retroviral vector (see U. S. Patent No.
4,980,286),
or by direct injection, or by use of microparticle bombardment (e.g., a gene
gun;
Biolistic, Dupont), or coating with lipids or cell-surface receptors or
transfecting
agents, or by administering it in linkage to a homeobox- like peptide which is
known to enter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad.
Sci.
USA 88:1864-1868), etc. Alternatively, a nucleic acid can be introduced
intracellularly and incorporated within host cell DNA for expression, by
homologous recombination.
Pharmaceutical compositions of the present invention for parenteral
injection can comprise pharmaceutically acceptable sterile aqueous or
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nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile
powders for reconstitution into sterile injectable solutions or dispersions
just
prior to use.
In addition to soluble DRS polypeptides, DRS polypeptide containing the
transmembrane region can also be used when appropriately solubilized by
including detergents, such as CHAPS or NP-40, with buffer.
The compounds or pharmaceutical compositions of the invention are
preferably tested in vitro, and then in vivo for the desired therapeutic or
prophylactic activity, prior to use in humans. For example, in vitro assays to
demonstrate the therapeutic or prophylactic utility of a compound or
pharmaceutical composition include, the effect of a compound on a cell line or
a patient tissue sample. The effect of the compound or composition on the cell
line and/or tissue sample can be determined utilizing techniques known to
those
of skill in the art including, but not limited to, rosette formation assays
and cell
lysis assays. In accordance with the invention, in vitro assays which can be
used
to determine whether administration of a specific compound is indicated,
include
in vitro cell culture assays in which a patient tissue sample is grown in
culture,
and exposed to or otherwise administered a compound, and the effect of such
compound upon the tissue sample is observed.
The compositions of the invention may be administered alone or in
combination with other adjuvants. Adjuvants that may be administered with the
compositions of the invention include, but are not limited to, alum, alum plus
deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.),
BCG, and MPL. In a specific embodiment, compositions of the invention are
administered in combination with alum. In another specific embodiment,
compositions of the invention are administered in combination with QS-21.
Further adjuvants that may be administered with the compositions of the
invention include, but are not limited to, Monophosphoryl lipid
immunomodulator, AdjuVax 100a, QS-18, CRL1005, Aluminum salts, MF-59,
and Virosomal adjuvant technology. Vaccines that may be administered with the
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compositions of the invention include, but are not limited to, vaccines
directed
toward protection against N>MR (measles, mumps, rubella), polio, varicella,
tetanus/diphtheria, Hepatitis A, Hepatitis B, Haemophilus infZuenzae type B,
whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera,
yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and
pertussis. Combinations may be administered either concomitantly, e.g., as an
admixture, separately but simultaneously or concurrently; or sequentially.
This
includes presentations in which the combined agents are administered together
as a therapeutic mixture, and also procedures in which the combined agents are
administered separately but simultaneously, e.g., as through separate
intravenous
lines into the same individual. Administration "in combination" further
includes
the separate administration of one of the compounds or agents given first,
followed by the second.
The compositions of the invention may be administered alone or in
combination with other therapeutic agents. Therapeutic agents that may be
administered in combination with the compositions of the invention, include
but
are not limited to, other members of the TNF family, chemotherapeutic agents,
antibiotics, antivirals, steroidal and non-steroidal anti-inflammatories,
conventional immunotherapeutic agents, cytokines, chemokines and/or growth
factors. Combinations may be administered either concomitantly, e.g., as an
admixture, separately but simultaneously or concurrently; or sequentially.
This
includes presentations in which the combined agents are administered together
as a therapeutic mixture, and also procedures in which the combined agents are
administered separately but simultaneously, e.g., as through separate
intravenous
lines into the same individual. Administration "in combination" further
includes
the separate administration of one of the compounds or agents given first,
followed by the second.
In one embodiment, the compositions of the invention are administered
in combination with other members of the TNF family. TNF, TNF-related or
TNF-like molecules that may be administered with the compositions of the
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invention include, but are not limited to, soluble forms of TNF-alpha,
lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in
complex heterotrimer LT-alpha2-beta), OPGL, Fast, CD27L, CD30L, CD40L,
4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO
96/14328), TRAIL,, AIM-II (International Publication No. WO 97/34911),
APRIL (J. Exp. Med. 188(6):1185-1190), endokine-alpha (International
Publication No. WO 98/07880), TR6 (International Publication No. WO
98/30694), OPG and nerve growth factor (NGF), and soluble forms of Fas,
CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO
96/34095), DR3 (International Publication No. WO 97/33904), DR4
(International Publication No. WO 98/32856), TRS (International Publication
No. WO 98/30693), TRANK, TR9 (International Publication No. WO
98/56892), TR10 (International Publication No. WO 98/54202), 312C2
(International Publication No. WO 98/06842), and TR12, and soluble forms of
CD 154, CD70, and CD 153.
In another embodiment, the compositions of the invention are
administered in combination with CD40 ligand (CD40L), a soluble form of
CD40L (e.g., AVRENDT""), biologically active fragments, variants, or
derivatives
of CD40L, anti-CD40L antibodies (e.g., agonistic or antagonistic antibodies),
and/or anti-CD40 antibodies (e.g., agonistic or antagonistic antibodies).
In yet another embodiment, the compositions of the invention are
administered in combination with one, two, three, four, five, or more of the
following compositions: tacrolimus (Fujisawa), thalidomide (e.g., Celgene),
anti-
Tac(Fv)-PE40 (e.g., Protein Design Labs), inolimomab (Biotest), MAK-195F
(Knoll), ASM-981 (Novartis), interleukin-1 receptor (e.g., Immunex),
interleukin-4 receptor (e.g., Immunex), ICM3 (ICOS), BMS-188667 (Bristol-
Myers Squibb), anti-TNF Ab (e.g., Therapeutic antibodies), CG-1088 (Celgene),
anti-B7 monoclonal antibody (e.g., Innogetics), MEDI-507 (BioTransplant),
ABX-CBL (Abgenix).
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According to the invention, a patient susceptible to both Fas ligand
(Fas-L) mediated and TRAIL mediated cell death may be treated with both an
agent that inhibits TRAIL,/TRAIL,-R interactions and an agent that inhibits
Fas-L/Fas interactions. Suitable agents for blocking binding of Fas-L to Fas
include, but are not limited to, soluble Fas polypeptides; oligomeric forms of
soluble Fas polypeptides (e.g., dimers of sFaslFc); anti-Fas antibodies that
bind
Fas without transducing the biological signal that results in apoptosis; anti-
Fas-L
antibodies that block binding of Fas-L to Fas; and muteins of Fas-L that bind
Fas
but do not transduce the biological signal that results in apoptosis.
Preferably,
the antibodies employed according to this method are monoclonal antibodies.
Examples of suitable agents for blocking Fas-L/Fas interactions, including
blocking anti-Fas monoclonal antibodies, are described in WO 95/10540, hereby
incorporated by reference.
In certain embodiments, compositions of the invention are administered
in combination with antiretroviral agents, nucleoside reverse transcriptase
inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease
inhibitors. Nucleoside reverse transcriptase inhibitors that may be
administered
in combination with the compositions of the invention, include, but are not
limited to, RETROVIRT"" (zidovudine/AZT), VIDEXT"' (didanosine/ddI),
HIVIDT"' (zalcitabine/ddC), ZERITT"' (stavudine/d4T), EPIVIRT"'
(lamivudine/3TC), and CONIBIVIRT"" (zidovudine/lamivudine). Non-nucleoside
reverse transcriptase inhibitors that may be administered in combination with
the
compositions of the invention, include, but are not limited to, VnE2AMIJNET"~
(nevirapine), RESCRIPTORT"~ (delavirdine), and SUSTIVAT"" (efavirenz).
Protease inhibitors that may be administered in combination with the
compositions of the invention, include, but are not limited to, CRIXIVANT""
(indinavir), NORVIRT"' (ritonavir), IN VIRASET"" (saquinavir), and VIRACEPTT"~
(nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside
reverse
transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors,
and/or
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protease inhibitors may be used in any combination with compositions of the
invention to treat AIDS and/or to prevent or treat HIV infection.
In other embodiments, compositions ofthe invention may be administered
in combination with anti-opportunistic infection agents. Anti-opportunistic
agents that may be administered in combination with the compositions of the
invention, include, but are not limited to, TRIMETHOPRIM-
SULFAMETHOXAZOLETM, DAPSONETM, PENTAMIDINETM,
ATOVAQUONETM, ISONIAZIDTM, RIFAMPINTM, PYRAZINAMIDETM~
ETHAMBUTOLTM, RIFABUTINTM, CLARITHROMYCINTM,
lO AZITHROMYCINTM, GANCICLOVIRTM, FOSCARNETTM, CIDOFOVIRTM
FLUCONAZOLETM, ITRACONAZOLETM, KETOCONAZOLETM,
ACYCLOVIRTM, FAMCICOLVIRTM, PYRIMETHAMINETM, LEUCOVORINTM,
NEUPOGENTM (filgrastim/G-CSF), and LELTKINETM (sargramostim/GM-CSF).
In a specific embodiment, compositions of the invention are used in any
combination with TRIMETHOPRIM-SULFAMETHOXAZOLETM,
DAPSONETM, PENTAMmINETM, and/or ATOVAQUONETM to prophylactically
treat and/or prevent an opportunistic Pneumocystis carinii pneumonia
infection.
In another specific embodiment, compositions of the invention are used in any
combination with ISONIAZIDTM, RIFAMPINTM, PYRAZINAMIDETM, and/or
ETHAMBUTOLTM to prophylactically treat and/or prevent an opportunistic
Mycobacterium avium complex infection. In another specific embodiment,
compositions of the invention are used in any combination with RIFABUTINTM,
CLARITHROMYCINTM, and/or AZITHROMYCINTM to prophylactically treat
and/or prevent an opportunistic Mycobacterium tuberculosis infection. In
another specific embodiment, compositions of the invention are used in any
combination with GANCICLOVIRTM, FOSCARNETTM, and/or CIDOFOVIRTM
to prophylactically treat and/or prevent an opportunistic cytomegalovirus
infection. In another specific embodiment, compositions of the invention are
used in any combination with FLUCONAZOLETM, ITRACONAZOLETM, and/or
KETOCONAZOLETM to prophylactically treat and/or prevent an opportunistic
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fungal infection. In another specific embodiment, compositions of the
invention
are used in any combination with ACYCLOVIRT"" and/or FAMCICOLVIRT"'to
prophylactically treat and/or prevent an opportunistic herpes simplex virus
type
I and/or type II infection. In another specific embodiment, compositions of
the
invention are used in any combination with PYRIMETHAlV>ZNET"" and/or
LEUCOVORINT"' to prophylactically treat and/or prevent an opportunistic
Toxoplasma gondii infection. In another specific embodiment, compositions of
the invention are used in any combination with LEUCOVORiNT"" and/or
NEUPOGENT"" to prophylactically treat and/or prevent an opportunistic
bacterial
infection.
In a further embodiment, the compositions of the invention are
administered in combination with an antiviral agent. Antiviral agents that may
be
administered with the compositions of the invention include, but are not
limited
to, acyclovir, ribavirin, amantadine, and remantidine.
In a further embodiment, the compositions of the invention are
administered in combination with an antibiotic agent. Antibiotic agents that
may
be administered with the compositions of the invention include, but are not
limited to, amoxicillin, aminoglycosides, beta-lactam (glycopeptide), beta-
lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin,
ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole,
penicillins, quinolones, rifampin, streptomycin, sulfonamide, tetracyclines,
trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.
Conventional nonspecific immunosuppressive agents, that may be
administered in combination with the compositions of the invention include,
but
are not limited to, steroids, cyclosporine, cyclosporine analogs,
cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-
deoxyspergualin, and other immunosuppressive agents that act by suppressing
the function of responding T-cells.
In specific embodiments, compositions of the invention are administered
in combination with immunosuppressants. Immunosuppressants preparations
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that may be administered with the compositions of the invention include, but
are
not limited to, ORTHOCLONETM (OKT3), SANDIMMUNETM/NEORALTM~
SANGDYATM (cyclosporin), PROGRAFTM (tacrolimus), CELLCEPTTM
(mycophenolate), Azathioprine, glucorticosteroids, and RAPAIVIUNETM
(sirolimus). In a specific embodiment, immunosuppressants may be used to
prevent rejection of organ or bone marrow transplantation.
In an additional embodiment, compositions of the invention are
administered alone or in combination with one or more intravenous immune
globulin preparations. Intravenous immune globulin preparations that may be
administered with the compositions of the invention include, but not limited
to,
GAMMARTM, IVEEGAMTM, SANDOGLOBULINTM, GAMMAGARD S~DTM,
and GAMIMUNETM. In a specific embodiment, compositions of the invention
are administered in combination with intravenous immune globulin preparations
in transplantation therapy (e.g., bone marrow transplant).
In an additional embodiment, the compositions of the invention are
administered alone or in combination with an anti-inflammatory agent. Anti-
inflammatory agents that may be administered with the compositions of the
invention include, but are not limited to, glucocorticoids and the
nonsteroidal
anti-inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid
derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic
acid
derivatives, pyrazoles, pyrazolones, salicylic acid derivatives,
thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-
4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome,
difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide,
orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole,
and
tenidap.
In one embodiment, the compositions of the invention are administered
in combination with steroid therapy. Steroids that may be administered in
combination with the compositions of the invention, include, but are not
limited
to, oral corticosteroids, prednisone, and methylprednisolone (e.g., IV
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methylprednisolone). In a specific embodiment, compositions of the invention
are administered in combination with prednisone. In a further specific
embodiment, the compositions of the invention are administered in combination
with prednisone and an immunosuppressive agent. Immunosuppressive agents
that may be administered with the compositions of the invention and prednisone
are those described herein, and include, but are not limited to, azathioprine,
cylophosphamide, and cyclophosphamide IV. In a another specific embodiment,
compositions of the invention are administered in combination with
methylprednisolone. In a further specific embodiment, the compositions of the
invention are administered in combination with methylprednisolone and an
immunosuppressive agent. Immunosuppressive agents that may be administered
with the compositions of the invention and methylprednisolone are those
described herein, and include, but are not limited to, azathioprine,
cylophosphamide, and cyclophosphamide IV.
In another embodiment, the compositions of the invention are
administered in combination with an antimalarial. Antimalarials that may be
administered with the compositions of the invention include, but are not
limited
to, hydroxychloroquine, chloroquine, and/or quinacrine.
In yet another embodiment, the compositions of the invention are
administered in combination with an NSAID.
In a nonexclusive embodiment, the compositions of the invention are
administered in combination with one, two, three, four, five, ten, or more of
the
following drugs: NRD-101 (Hoechst Marion Roussel), diclofenac (Dimethaid),
oxaprozin potassium (Monsanto), mecasermin (Chiron), T-614 (Toyama),
pemetrexed disodium (Eli Lilly), atreleuton (Abbott), valdecoxib (Monsanto),
eltenac (Byk Gulden), campath, AGM-1470 (Takeda), CDP-571 (Celltech
Chiroscience), CM-101 (CarboMed), ML-3000 (Merckle), CB-2431 (KS
Biomedix), CBF-BS2 (KS Biomedix), IL-1Ra gene therapy (Valentis), JTE-522
(Japan Tobacco), paclitaxel (Angiotech), DW-166HC (Dong Wha), darbufelone
mesylate (Warner-Lambert), soluble TNF receptor 1 (synergen; Amgen), IPR-
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6001 (Institute for Pharmaceutical Research), trocade (Hoffman-La Roche), EF-
(Scotia Pharmaceuticals), BIIL,-284 (Boehringer Ingelheim), BIIF-1149
(Boehringer Ingelheim), LeukoVax (Inflammatics), MK-663 (Merck), ST-1482
(Sigma-Tau), and butixocort propionate (WarnerLambert).
5 In yet another embodiment, the compositions of the invention are
administered in combination with one, two, three, four, five or more of the
following drugs: methotrexate, sulfasalazine, sodium aurothiomalate,
auranofin,
cyclosporine, penicillamine, azathioprine, an antimalarial drug (e.g., as
described
herein), cyclophosphamide, chlorambucil, gold, ENBRELTM (Etanercept), anti-
TNF antibody, and prednisolone. In a more preferred embodiment, the
compositions of the invention are administered in combination with an
antimalarial, methotrexate, anti-TNF antibody, ENBRELT"" and/or suflasalazine.
In one embodiment, the compositions of the invention are administered in
combination with methotrexate. In another embodiment, the compositions ofthe
invention are administered in combination with anti-TNF antibody. In another
embodiment, the compositions of the invention are administered in combination
with methotrexate and anti-TNF antibody. In another embodiment, the
compositions of the invention are administered in combination with
suflasalazine.
In another specific embodiment, the compositions of the invention are
administered in combination with methotrexate, anti-TNF antibody, and
suflasalazine. In another embodiment, the compositions of the invention are
administered in combination ENBRELT"~. In another embodiment, the
compositions of the invention are administered in combination with ENBRELT""
and methotrexate. In another embodiment, the compositions of the invention are
administered in combination with ENBRELT"~, methotrexate and suflasalazine.
In another embodiment, the compositions of the invention are administered in
combination with ENBRELT"', methotrexate and suflasalazine. In other
embodiments, one or more antimalarials is combined with one of the above-
recited combinations. In a specific embodiment, the compositions of the
invention are administered in combination with an antimalarial (e.g.,
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hydroxychloroquine), ENBRELT"', methotrexate and suflasalazine. In another
specific embodiment, the compositions of the invention are administered in
combination with an antimalarial (e.g., hydroxychloroquine), sulfasalazine,
anti-
TNF antibody, and methotrexate.
In another embodiment, compostions of the invention are administered
in combination with a chemotherapeutic agent. Chemotherapeutic agents that
may be administered with the compositions of the invention include, but are
not
limited to, antibiotic derivatives (e.g. , doxorubicin, bleomycin,
daunorubicin, and
dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g.,
fluorouracil,
5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid,
plicamycin,
mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNCT,
lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine,
hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine
sulfate); hormones (e.g., medroxyprogesterone, estramustine phosphate sodium,
ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone,
diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen
mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogen
mustard) and thiotepa); steroids and combinations (e.g., bethamethasone sodium
phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine
sulfate, vinblastine sulfate, and etoposide).
In a specific embodiment, compositions of the invention are administered
in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and
prednisone) or any combination of the components of CHOP. In another
embodiment, compositions of the invention are administered in combination with
Rituximab. In a further embodiment, compositions of the invention are
administered with Rituxmab and CHOP, or Rituxmab and any combination ofthe
components of CHOP.
In an additional embodiment, the compositions of the invention are
administered in combination with cytokines. Cytokines that may be administered
with the compositions ofthe invention include, but are not limited to, GM-CSF,
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G-CSF, II,-lalpha, IL,-lbeta, II,-2, IL,-3, IL,-4, IL,-5, IL,-6, IL-7, IL,-8,
IL-9, IL,-
10, lL,-11, IL-12, lL,-13 , IL,-14, IL,-15, IL,-16, IL,-17, IL,-18, IL,-19, IL-
20, IL,-21,
anti-CD40, CD40L, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, and TNF-
beta.
In an additional embodiment, the compositions of the invention are
administered in combination with hematopoietic growth factors. Hematopoietic
growth factors that may be administered with the compositions of the invention
included, but are not limited to, LELTKINET"" (SARGRAMOSTIMT"") and
NEUPOGENr"' (FILGRASTIMT"~).
In an additional embodiment, the compositions of the invention are
administered in combination with angiogenic proteins. Angiogenic proteins that
may be administered with the compositions of the invention include, but are
not
limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European
Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as
disclosed in European Patent Number EP-682110; Platelet Derived Growth
Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317;
Placental Growth Factor (P1GF), as disclosed in International Publication
Number WO 92/06194; Placental Growth Factor-2 (P1GF-2), as disclosed in
Hauser et al., Growth Factors, 4:259-268 (1993); Vascular Endothelial Growth
Factor (VEGF), as disclosed in International Publication Number WO 90/13649;
Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European
Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2),
as disclosed in International Publication Number WO 96/39515; Vascular
Endothelial Growth Factor B-186 (VEGF-B186), as disclosed in International
Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D
(VEGF-D), as disclosed in International Publication Number WO 98/02543;
Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International
Publication Number WO 98/07832; and Vascular Endothelial Growth Factor-E
(VEGF-E), as disclosed in German Patent Number DE19639601. The above
mentioned references are incorporated herein by reference herein.
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In an additional embodiment, the compositions of the invention are
administered in combination with Fibroblast Growth Factors. Fibroblast Growth
Factors that may be administered with the compositions of the invention
include,
but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7,
FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
In one embodiment, the compositions of the invention are administered
in combination with one or more chemokines. In specific embodiments, the
compositions of the invention are administered in combination with an a.(CxC)
chemokine selected from the group consisting of gamma-interferon inducible
protein-10 (yIP-10), interleukin-8 (IL-8), platelet factor-4 (PF4), neutrophil
activating protein (NAP-2), GRO-a, GRO-~3, GRO-y, neutrophil-activating
peptide (ENA-78), granulocyte chemoattractant protein-2 (GCP-2), and stromal
cell-derived factor-1 (SDF-1, or pre-B-cell stimulatory factor (PBSF)); and/or
a (3 (CC) selected from the group consisting o~ RANTES (regulated on
activation, normal T expressed and secreted), macrophage inflammatory protein-
1 alpha (MIP-1 oc), macrophage inflammatory protein-1 beta (MIP-1 ~3),
monocyte
chemotactic protein-1 (MCP-1), monocyte chemotactic protein-2 (MCP-2),
monocyte chemotactic protein-3 (MCP-3), monocyte chemotactic protein-4
(MCP-4) macrophage inflammatory protein-1 gamma (MIP-ly), macrophage
inflammatory protein-3 alpha (MIP-3a,), macrophage inflammatory protein-3
beta (MIP-3 (3), macrophage inflammatory protein-4 (MIP-4/DC-CK-1 /PARC),
eotaxin, Exodus, and I-309; and/or the y(C) chemokine, lymphotactin.
In additional embodiments, the compositions of the invention are
administered in combination with other therapeutic or prophylactic regimens,
such as, for example, radiation therapy.
The present invention also provides pharmaceutical compositions. Such
compositions comprise a therapeutically effective amount of a compound, and
a pharmaceutically acceptable carrier. In a specific embodiment, the term
"pharmaceutically acceptable" means approved by a regulatory agency of the
Federal or a state government or listed in the U.S. Pharmacopeia or other
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generally recognized pharmacopeia for use in animals, and more particularly in
humans.
The amount of the compound of the invention which will be effective in
the treatment, inhibition and prevention of a disease or disorder associated
with
aberrant expression and/or activity of a polypeptide of the invention can be
determined by standard clinical techniques. In addition, in vitro assays may
optionally be employed to help identify optimal dosage ranges. The precise
dose
to be employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and should be
decided according to the judgment of the practitioner and each patient's
circumstances. Effective doses may be extrapolated from dose-response curves
derived from in vitro or animal model test systems.
For antibodies, the dosage administered to a patient is typically 0.1 mg/kg
to 100 mg/kg of the patient's body weight. Preferably, the dosage administered
to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight,
more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally,
human antibodies have a longer half life within the human body than antibodies
from other species due to the immune response to the foreign polypeptides.
Thus, lower dosages of human antibodies and less frequent administration is
often possible. Further, the dosage and frequency of administration of
antibodies
of the invention may be reduced by enhancing uptake and tissue penetration
(e.g. ,
into the brain) of the antibodies by modifications such as, for example,
lipidation.
The invention also provides a pharmaceutical pack or kit comprising one
or more containers filled with one or more of the ingredients of the
pharmaceutical compositions of the invention. Optionally associated with such
containers) can be a notice in the form prescribed by a governmental agency
regulating the manufacture, use or sale ofpharmaceuticals or biological
products,
which notice reflects approval by the agency of manufacture, use or sale for
human administration.
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Diagnosis anrl Imaging
Labeled antibodies, and derivatives and analogs thereof, which
specifically bind to a polypeptide of interest can be used for diagnostic
purposes
to detect, diagnose, or monitor diseases and/or disorders associated with the
aberrant expression and/or activity of a polypeptide of the invention. The
invention provides for the detection of aberrant expression of a polypeptide
of
interest, comprising (a) assaying the expression of the polypeptide of
interest in
cells or body fluid of an individual using one or more antibodies specific to
the
polypeptide interest and (b) comparing the level of gene expression with a
standard gene expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard expression level is
indicative of aberrant expression.
The invention provides a diagnostic assay for diagnosing a disorder,
comprising (a) assaying the expression of the polypeptide of interest in cells
or
body fluid of an individual using one or more antibodies specific to the
polypeptide interest and (b) comparing the level of gene expression with a
standard gene expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard expression level is
indicative of a particular disorder. With respect to cancer, the presence of a
relatively high amount of transcript in biopsied tissue from an individual may
indicate a predisposition for the development of the disease, or may provide a
means for detecting the disease prior to the appearance of actual clinical
symptoms. A more definitive diagnosis of this type may allow health
professionals to employ preventative measures or aggressive treatment earlier
thereby preventing the development or further progression of the cancer.
Antibodies of the invention can be used to assay protein levels in a
biological sample using classical immunohistological methods known to those of
skill in the art (e.g., see Jalkanen, M. et al., J. Cell. Biol. 101:976-985
(1985);
Jalkanen, M. etal., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based
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methods useful for detecting protein gene expression include immunoassays,
such
as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay
(RIA). Suitable antibody assay labels are known in the art and include enzyme
labels, such as, glucose oxidase; radioisotopes, such as iodine ('zsl,'z'I),
carbon
('4C), sulfur (35S), tritium (3H), indium ("zIn), and technetium (99Tc);
luminescent
labels, such as luminol; and fluorescent labels, such as fluorescein and
rhodamine, and biotin.
One aspect of the invention is the detection and diagnosis of a disease or
disorder associated with aberrant expression of a polypeptide of the interest
in
an animal, preferably a mammal and most preferably a human. In one
embodiment, diagnosis comprises: a) administering (for example, parenterally,
subcutaneously, or intraperitoneally) to a subject an effective amount of a
labeled
molecule which specifically binds to the polypeptide of interest; b) waiting
for a
time interval following the administering for permitting the labeled molecule
to
preferentially concentrate at sites in the subject where the polypeptide is
expressed (and for unbound labeled molecule to be cleared to background
level);
c) determining background level; and d) detecting the labeled molecule in the
subject, such that detection of labeled molecule above the background level
indicates that the subject has a particular disease or disorder associated
with
aberrant expression of the polypeptide of interest. Background level can be
determined by various methods including, comparing the amount of labeled
molecule detected to a standard value previously determined for a particular
system.
It will be understood in the art that the size of the subject and the imaging
system used will determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a human subject,
the
quantity of radioactivity injected will normally range from about 5 to 20
millicuries of 99mTc. The labeled antibody or antibody fragment will then
preferentially accumulate at the location of cells which contain the specific
protein. In vivo tumor imaging is described in S.W. Burchiel et al.,
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"Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments."
(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W.
Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
Depending on several variables, including the type of label used and the
mode of administration, the time interval following the administration for
permitting the labeled molecule to preferentially concentrate at sites in the
subject
and for unbound labeled molecule to be cleared to background level is 6 to 48
hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time
interval
following administration is 5 to 20 days or 5 to 10 days.
In an embodiment, monitoring ofthe disease or disorder is carried out by
repeating the method for diagnosing the disease or disease, for example, one
month after initial diagnosis, six months after initial diagnosis, one year
after
initial diagnosis, etc.
Presence of the labeled molecule can be detected in the patient using
methods known in the art for in vivo scanning. These methods depend upon the
type of label used. Skilled artisans will be able to determine the appropriate
method for detecting a particular label. Methods and devices that may be used
in the diagnostic methods of the invention include, but are not limited to,
computed tomography (CT), whole body scan such as position emission
tomography (PET), magnetic resonance imaging (MRI), and sonography.
In a specific embodiment, the molecule is labeled with a radioisotope and
is detected in the patient using a radiation responsive surgical instrument
(Thurston et al., U.S. Patent No. 5,441,050). In another embodiment, the
molecule is labeled with a fluorescent compound and is detected in the patient
using a fluorescence responsive scanning instrument. In another embodiment,
the molecule is labeled with a positron emitting metal and is detected in the
patent using positron emission-tomography. In yet another embodiment, the
molecule is labeled with a paramagnetic label and is detected in a patient
using
magnetic resonance imaging (MRI).
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Kits
The present invention provides kits that can be used in the above
methods. In one embodiment, a kit comprises an antibody of the invention,
preferably a purified antibody, in one or more containers. In a specific
embodiment, the kits of the present invention contain a substantially isolated
polypeptide comprising an epitope which is specifically immunoreactive with an
antibody included in the kit. Preferably, the kits of the present invention
further
comprise a control antibody which does not react with the polypeptide of
interest. In another specific embodiment, the kits ofthe present invention
contain
a means for detecting the binding of an antibody to a polypeptide of interest
(e.g.,
the antibody may be conjugated to a detectable substrate such as a fluorescent
compound, an enzymatic substrate, a radioactive compound or a luminescent
compound, or a second antibody which recognizes the first antibody may be
1 S conjugated to a detectable substrate).
In another specific embodiment of the present invention, the kit is a
diagnostic kit for use in screening serum containing antibodies specific
against
proliferative and/or cancerous polynucleotides and polypeptides. Such a kit
may
include a control antibody that does not react with the polypeptide of
interest.
Such a kit may include a substantially isolated polypeptide antigen comprising
an
epitope which is specifically immunoreactive with at least one anti-
polypeptide
antigen antibody. Further, such a kit includes means for detecting the binding
of
said antibody to the antigen (e.g., the antibody may be conjugated to a
fluorescent compound such as fluorescein or rhodamine which can be detected
by flow cytometry). In specific embodiments, the kit may include a
recombinantly produced or chemically synthesized polypeptide antigen. The
polypeptide antigen of the kit may also be attached to a solid support.
In a more specific embodiment the detecting means of the above-
described kit includes a solid support to which said polypeptide antigen is
attached. Such a kit may also include a non-attached reporter-labeled anti-
human
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antibody. In this embodiment, binding of the antibody to the polypeptide
antigen
can be detected by binding of the said reporter-labeled antibody.
In an additional embodiment, the invention includes a diagnostic kit for
use in screening serum containing antigens of the polypeptide of the
invention.
The diagnostic kit includes a substantially isolated antibody specifically
immunoreactive with polypeptide or polynucleotide antigens, and means for
detecting the binding of the polynucleotide or polypeptide antigen to the
antibody. In one embodiment, the antibody is attached to a solid support. In a
specific embodiment, the antibody may be a monoclonal antibody. The detecting
means of the kit may include a second, labeled monoclonal antibody.
Alternatively, or in addition, the detecting means may include a labeled,
competing antigen.
In one diagnostic configuration, test serum is reacted with a solid phase
reagent having a surface-bound antigen obtained by the methods of the present
invention. After binding with specific antigen antibody to the reagent and
removing unbound serum components by washing, the reagent is reacted with
reporter-labeled anti-human antibody to bind reporter to the reagent in
proportion to the amount of bound anti-antigen antibody on the solid support.
The reagent is again washed to remove unbound labeled antibody, and the
amount of reporter associated with the reagent is determined. Typically, the
reporter is an enzyme which is detected by incubating the solid phase in the
presence of a suitable fluorometric, luminescent or colorimetric substrate
(Sigma,
St. Louis, MO).
The solid surface reagent in the above assay is prepared by known
techniques for attaching protein material to solid support material, such as
polymeric beads, dip sticks, 96-well plate or filter material. These
attachment
methods generally include non-specific adsorption of the protein to the
support
or covalent attachment of the protein, typically through a free amine group,
to
a chemically reactive group on the solid support, such as an activated
carboxyl,
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hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be
used in conjunction with biotinylated antigen(s).
Thus, the invention provides an assay system or kit for carrying out this
diagnostic method. The kit generally includes a support with surface-bound
recombinant antigens, and a reporter-labeled anti-human antibody for detecting
surface-bound anti-antigen antibody.
Chromosome Assays
The nucleic acid molecules of the present invention are also valuable for
chromosome identification. The sequence is specifically targeted to and can
hybridize with a particular location on an individual human chromosome. The
mapping of DNAs to chromosomes according to the present invention is an
important first step in correlating those sequences with genes associated with
disease.
In certain preferred embodiments in this regard, the cDNA and/or
polynucleotides herein disclosed is used to clone genomic DNA of a DRS gene.
This can be accomplished using a variety ofwell known techniques and
libraries,
which generally are available commercially. The genomic DNA is then used for
in situ chromosome mapping using well known techniques for this purpose.
In addition, sequences can be mapped to chromosomes by preparing PCR
primers (preferably 1 S-25 bp) from the cDNA. Computer analysis of the 3'
untranslated region of the gene is used to rapidly select primers that do not
span
more than one exon in the genomic DNA, thus complicating the amplification
process. These primers are then used for PCR screening of somatic cell hybrids
containing individual human chromosomes.
Fluorescence in situ hybridization ("FISH") of a cDNA to a metaphase
chromosomal spread can be used to provide a precise chromosomal location in
one step. This technique can be used with cDNA as short as 50 or 60 bp. For
a review of this technique, see Verma et al., Human Chromosomes: a Manual
of Baric Techniyue.s, Pergamon Press, New York (1988).
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Once a sequence has been mapped to a precise chromosomal location, the
physical position of the sequence on the chromosome can be correlated with
genetic map data. Such data are found, for example, in V. McKusick, Mendelian
Inheritance in Man, available on line through Johns Hopkins University, Welch
Medical Library. The relationship between genes and diseases that have been
mapped to the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic
sequence between affected and unaffected individuals. If a mutation is
observed
in some or all of the affected individuals but not in any normal individuals,
then
the mutation is likely to be the causative agent of the disease.
Having generally described the invention, the same will be more readily
understood by reference to the following examples, which are provided by way
of illustration and are not intended as limiting.
Example 1
Expression and Purification in E. coli
The DNA sequence encoding the mature DRS protein in the deposited
cDNA (ATCC No. 97920) is amplified using PCR oligonucleotide primers
specific to the amino terminal sequences of the DRS protein and to vector
sequences 3' to the gene. Additional nucleotides containing restriction sites
to
facilitate cloning are added to the 5' and 3' sequences respectively.
The following primers are used for expression of DRS extracellular
domain in E. coli: The 5' primer has the sequence:
5'-CGCCCATGGAGTCTGCTCTGATCAC-3' (SEQ ID N0:8) and contains the
underlined NcoI site; and the 3' primer has the sequence:
S'-CGCAAGCTTTTAGCCTGATTCTTTGTGGAC-3' (SEQ ID N0:9) and
contains the underlined HindIII site.
The restriction sites are convenient to restriction enzyme sites in the
bacterial expression vector pQE60, which are used for bacterial expression in
this
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example. (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311). pQE60
encodes ampicillin antibiotic resistance ("Amp"') and contains a bacterial
origin
of replication ("ori"), an IPTG inducible promoter, and a ribosome binding
site
("RB S").
The amplified DR5 DNA and the vector pQE60 both are digested with
NcoI and HindIII and the digested DNAs are then ligated together. Insertion of
the DRS protein DNA into the restricted pQE60 vector places the DR5 protein
coding region downstream of and operably linked to the vector's IPTG-inducible
promoter and in-frame with an initiating AUG appropriately positioned for
translation of DR5 protein.
The ligation mixture is transformed into competent E. coli cells using
standard procedures. Such procedures are described in Sambrook et al.,
Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strain M15/rep4,
containing multiple copies of the plasmid pREP4, which expresses lac repressor
and confers kanamycin resistance ("Kan"'), is used in carrying out the
illustrative
example described herein. This strain, which is only one of many that are
suitable
for expressing DRS protein, is available commercially from Qiagen, supra.
Transformants are identified by their ability to grow on LB plates in the
presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant
colonies and the identity of the cloned DNA confirmed by restriction analysis,
PCR, and DNA sequencing.
Clones containing the desired constructs are grown overnight ("O/N") in
liquid culture in LB media supplemented with both ampicillin (100 ~cg/ml) and
kanamycin (25 ~g/ml). The O/N culture is used to inoculate a large culture, at
a dilution of approximately 1:100 to 1:250. The cells are grown to an optical
density at 600nm ("OD600") of between 0.4 and 0.6. Isopropyl-B-D-
thiogalactopyranoside ("IPTG") is then added to a final concentration of 1 mM
to induce transcription from lac repressor sensitive promoters, by
inactivating the
lacI repressor. Cells subsequently are incubated further for 3 to 4 hours.
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Cells then are harvested by centrifugation and disrupted, by standard
methods. Inclusion bodies are purified from the disrupted cells using routine
collection techniques, and protein is solubilized from the inclusion bodies
into
8M urea. The 8M urea solution containing the solubilized protein is passed
over
a PD-10 column in 2X phosphate-buffered saline ("PB S "), thereby removing the
urea, exchanging the buffer and refolding the protein. The protein is purified
by
a further step of chromatography to remove endotoxin. Then, it is sterile
filtered.
The sterile filtered protein preparation is stored in 2X PBS at a
concentration of
95 ~c/ml.
Example 2
Expression in Mammalian Cells
A typical mammalian expression vector contains the promoter element,
1 S which mediates the initiation of transcription of mRNA, the protein coding
sequence, and signals required for the termination of transcription and
polyadenylation ofthe transcript. Additional elements include enhancers, Kozak
sequences and intervening sequences flanked by donor and acceptor sites for
RNA splicing. Highly efficient transcription can be achieved with the early
and
late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses,
e.g. RSV, HTLVI, HIVI and the early promoter ofthe cytomegalovirus (CMV).
However, cellular signals can also be used (e.g. the human actin promoter).
Suitable expressionvectors for use in practicing the present invention
include, for
example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden),
pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBCI2MI
(ATCC67109). Mammalian host cells that could be used include, human HeLa
293, H9 and Jurkat cells, mouse NIH3T3 and C 127 cells, Cos 1, Cos 7 and CV1,
quail QCl-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
Alternatively, the gene of interest can be expressed in stable cell lines that
contain the gene integrated into a chromosome. Co-transfection with a
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selectable marker such as dhfr, gpt, neomycin, hygromycin allows the
identification and isolation of the transfected cells.
The transfected gene can also be amplified to express large amounts of
the encoded protein. The dihydrofolate reductase (DHFR) marker is useful to
develop cell lines that carry several hundred or even several thousand copies
of
the gene of interest. Another useful selection marker is the enzyme glutamine
synthase (GS) (Murphy et al., Biochem. J. 227:277-279 (1991); Bebbington et
al., BiolTechnology 10:169-175 (1992)). Using these markers, the mammalian
cells are grown in selective medium and the cells with the highest resistance
selected. These cell lines contain the amplified genes) integrated into a
chromosome. Chinese hamster ovary (CHO) cells are often used for the
production of proteins.
The expression vectors pCl and pC4 contain the strong promoter (LTR)
of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology
5:438-447 (March 1985)), plus a fragment ofthe CMV-enhancer (Boshart et al.,
Cel141:521-530 (1985)). Multiple cloning sites, e.g. with the restriction
enzyme
cleavage sites BamHI, XbaI and Asp718, facilitate the cloning of the gene of
interest. The vectors contain in addition the 3' intron, the polyadenylation
and
termination signal of the rat preproinsulin gene.
Cloning and Expression in CHO Cells
The vector pC4 is used for the expression of the DRS polypeptide.
Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No.
3 7146). The plasmid contains the mouse DHFR gene under control of the SV40
early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate
activity that are transfected with these plasmids, can be selected by growing
the
cells in a selective medium (alpha minus MEM, Life Technologies) supplemented
with the chemotherapeutic agent methotrexate (MTX). The amplification of the
DHFR genes in cells resistant to methotrexate (MTX) has been well documented
(see, e.g., Alt, F. W., Kellems, R. M., Bertino, J. R., and Schimke, R. T., J.
Biol.
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Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys.
Acta 1097:107-143 (1990); Page, M. J. and Sydenham, M. A. 1991,
Biotechnology 9:64-68(1991)). Cells grown in increasing concentrations of
MTX develop resistance to the drug by overproducing the target enzyme,
DHFR, as a result of amplification of the DHFR gene. If a second gene is
linked
to the DHFR gene, it is usually co-amplified and over-expressed. It is known
in
the art that this approach may be used to develop cell lines carrying more
than
1,000 copies of the amplified gene(s). Subsequently, when the methotrexate is
withdrawn, cell lines are obtained which contain the amplified gene integrated
into one or more chromosomes) of the host cell.
Plasmid pC4 contains, for expressing the gene of interest, the strong
promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen
et al., Molecular and CellularBiology 5:43 8-447 (March 1985), plus a fragment
isolated from the enhancer of the immediate early gene of human
cytomegalovirus (CMV) (Boshart etal., Cel141:521-530 (1985)). Downstream
of the promoter are the following single restriction enzyme cleavage sites
that
allow the integration of the genes: BamHI, XbaI, and Asp718. Behind these
cloning sites the plasmid contains the 3' intron and polyadenylation site of
the rat
preproinsulin gene. Other high efficiency promoters can also be used for
expression, e.g., the human ~3-actin promoter, the SV40 early or late
promoters
or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
Clontech's Tet-Offand Tet-On gene expression systems and similar systems can
be used to express the DRS polypeptide in a regulated way in mammalian cells
(Gossen, M., & Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992).
For the polyadenylation of the mRNA, other signals, e.g., from the human
growth hormone or globin genes, can be used as well.
Stable cell lines carrying a gene of interest integrated into the
chromosomes can also be selected upon co-transfection with a selectable marker
such as gpt, 6418, or hygromycin. It is advantageous to use more than one
selectable marker in the beginning, e.g., G418 plus methotrexate.
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The plasmid pC4 is digested with the restriction enzyme BamHI and then
dephosphorylated using calf intestinal phosphates by procedures known in the
art. The vector is then isolated from a 1% agarose gel.
The DNA sequence encoding the complete polypeptide is amplified using
PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the
desired portion of the gene. The S' primer containing the underlined BamHI
site,
a Kozak sequence, and an AUG start codon, has the following sequence:
5'-CGCGGATCCGCCATCATGGAACAACGGGGACAGAAC-3' (SEQ ID
NO:10). The 3' primer, containing the underlined Asp718 site, has the
following
sequence:
5'-CGCGGTACCTTAGGACATGGCAGAGTC-3' (SEQ 117 NO:11 ).
The amplified fragment is digested with the endonuclease BamHI and
Asp718 and then purified again on a 1% agarose gel. The isolated fragment and
the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli
HB 101 or XL,-1 Blue cells are then transformed and bacteria are identified
that
contain the fragment inserted into plasmid pC4 using, for instance,
restriction
enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR gene are used for
transfection. Five ~g of the expression plasmid pC4 is cotransfected with 0. 5
~g
of the plasmid pSVneo using the lipofectin method (Felgner et al., .supra).
The
plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5
encoding an enzyme that confers resistance to a group of antibiotics including
6418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml
6418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning
plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50
ng/ml ofmethotrexate plus 1 mg/ml 6418. After about 10-14 days, single clones
are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using
different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800
nM). Clones growing at the highest concentrations of methotrexate are then
transferred to new 6-well plates containing even higher concentrations of
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methotrexate (1 ~M, 2 ,uM, 5 ~M, 10 mM, 20 mM). The same procedure is
repeated until clones are obtained which grow at a concentration of 100 - 200
~cM. Expression of the desired gene product is analyzed, for instance, by SDS-
PAGE and Western blot or by reversed phase HPLC analysis.
Cloning and Expression in COS Cells
The expression plasmid, pDRS-HA, is made by cloning a cDNA encoding
the soluble extracellular domain of the DRS protein into the expression vector
pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen, Inc.). The
expression vector pcDNAI/amp contains: (1) an E. coli origin of replication
effective for propagation in E. coli and other prokaryotic cells; (2) an
ampicillin
resistance gene for selection of plasmid-containing prokaryotic cells; (3) an
SV40
origin of replication for propagation in eukaryotic cells; (4) a CMV promoter,
a
polylinker, an SV40 and a polyadenylation signal arranged so that a cDNA can
be conveniently placed under expression control of the CMV promoter and
operably linked to the SV40 intron and the polyadenylation signal by means of
restriction sites in the polylinker. A DNA fragment encoding the extracelluar
domain of the DRS polypeptide and a HA tag fused in frame to its 3' end is
cloned into the polylinker region of the vector so that recombinant protein
expression is directed by the CMV promoter. The HA tag corresponds to an
epitope derived from the influenza hemagglutinin protein described by Wilson
et
al., Cell 37:767 (1984). The fusion of the HA tag to the target protein allows
easy detection and recovery of the recombinant protein with an antibody that
recognizes the HA epitope.
The plasmid construction strategy is as follows. The DRS cDNA of the
deposited plasmid is amplified using primers that contain convenient
restriction
sites, much as described above for construction ofvectors for expression ofDRS
in E coli.
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To facilitate detection, purification and characterization of the expressed
DRS, one of the primers contains a hemagglutinin tag ("HA tag") as described
above.
Suitable primers include the following, which are used in this example.
The 5' primer, containing the underlined BamHI site has the following
sequence:
5'-CGCGGATCCGCCATCATGGAACAACGGGGACAGAAC-3' (SEQ B7
NO:10). The 3' primer, containing the underlined Asp718 restriction sequence
has the following sequence:
5'-CGCGGTACCTTAGCCTGATTCTTTTGGAC-3' (SEQ ID NO:12).
The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are
digested with BamHI and Asp718 and then ligated. The ligation mixture is
transformed into E. coli strain SURE (available from Stratagene Cloning
Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037), and the
transformed culture is plated on ampicillin media plates which then are
incubated
to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from
resistant colonies and examined by restriction analysis or other means for the
presence of the fragment encoding the extracellular domain of the DRS
polypeptide
For expression of recombinant DRS, COS cells are transfected with an
expression vector, as described above, using DEAF-DEXTRAN, as described,
for instance, in Sambrook et al., Molecular Cloning: a Laboratory Manual,
Cold Spring Laboratory Press, Cold Spring Harbor, New York ( 1989). Cells are
incubated under conditions for expression of DRS by the vector.
Expression ofthe DRS-HA fusion protein is detected by radiolabeling and
immunoprecipitation, using methods described in, for example Harlow et al.,
Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York (1988). To this end, two days after
transfection, the cells are labeled by incubation in media containing 35S-
cysteine
for 8 hours. The cells and the media are collected, and the cells are washed
and
the lysed with detergent-containing RIPA buffer: 150 mM NaCI, 1% NP-40,
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0.1% SDS, 1% NP-40, 0.5% DOC, SO mM TRIS, pH 7.5, as described by
Wilson et al., cited above. Proteins are precipitated from the cell lysate and
from
the culture media using an HA-specific monoclonal antibody. The precipitated
proteins then are analyzed by SDS-PAGE and autoradiography. An expression
product of the expected size is seen in the cell lysate, which is not seen in
negative controls.
The primer sets used for expression in this example are compatible with
pC4 used for CHO expression in this example, pcDNAI/Amp for COS
expression in this example, and pA2 used for baculovirus expression in the
following example. Thus, for example, the complete DRS encoding fragment
amplified for CHO expression could also be ligated into pcDNAI/Amp for COS
expression or pA2 for baculovirus expression.
Example 3
Protein Fusions of DRS
DRS polypeptides ofthe invention are optionally fused to other proteins.
These fusion proteins can be used for a variety of applications. For example,
fusion of DRS polypeptides to His-tag, HA-tag, protein A, IgG domains, and
maltose binding protein facilitates purification. (See EP A 394,827;
Traunecker,
et al., Nature 331:84-86 (1988).) Similarly, fusion to IgG-l, IgG-3, and
albumin
increases the half life time in vivo. Nuclear localization signals fused to
DRS
polypeptides can target the protein to a specific subcellular localization,
while
covalent heterodimer or homodimers can increase or decrease the activity of a
fusion protein. Fusion proteins can also create chimeric molecules having more
than one function. Finally, fusion proteins can increase solubility and/or
stability
of the fused protein compared to the non-fused protein. All of the types of
fusion proteins described above can be made using techniques known in the art
or by using or routinely modifying the following protocol, which outlines the
fusion of a polypeptide to an IgG molecule.
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Briefly, the human Fc portion ofthe IgG molecule can be PCR amplified,
using primers that span the 5' and 3' ends of the sequence described in SEQ ID
N0:13. These primers also preferably contain convenient restriction enzyme
sites that will facilitate cloning into an expression vector, preferably a
mammalian
expression vector.
For example, if the pC4 (Accession No. 209646) expression vector is
used, the human Fc portion can be ligated into the BamHI cloning site. Note
that
the 3' BamHI site should be destroyed. Next, the vector containing the human
Fc portion is re-restricted with BamHI, linearizing the vector, and DRS
polynucleotide, isolated by the PCR protocol described in Example 1, is
ligated
into this BamHI site. Note that the polynucleotide is cloned without a stop
codon, otherwise a fusion protein will not be produced.
If the naturally occurring signal sequence is used to produce the secreted
protein, pC4 does not need a second signal peptide. Alternatively, if the
naturally
1 S occurring signal sequence is not used, the vector can be modified to
include a
heterologous signal sequence. (See, e.g., WO 96/34891.)
Example 4
Cloning anrl Expression of the Soluble Extracellular Domain of DRS in a
Baculovirus Expression System
In this illustrative example, the plasmid shuttle vector pA2 is used to
insert the cDNA encoding the complete DRS protein, including its naturally
associated signal sequence, into a baculovirus to express the DRS protein,
using
standard methods, such as those described in Summers et al., A Manual of
Methods for Baculovirus hectors and Insect Cell Culture Procedures, Texas
Agricultural Experimental Station Bulletin No. 1555 (1987). This expression
vector contains the strong polyhedron promoter of the Autograph californica
nuclear polyhedrosis virus (ACMNP~ followed by convenient restriction sites.
For easy selection of recombinant virus, the plasmid contains the beta-
galactosidase gene from E. coli under control of a weak Drosophila promoter
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in the same orientation, followed by the polyadenylation signal of the
polyhedrin
gene. The inserted genes are flanked on both sides by viral sequences for cell-
mediated homologous recombination with wild-type viral DNA to generate
viable virus that express the cloned polynucleotide.
Many other baculovirus vectors could be used in place of pA2, such as
pAc373, pVL941 and pAcIMl provided, as one skilled in the art would readily
appreciate, that construction provides appropriately located signals for
transcription, translation, secretion, and the like, such as an in-frame AUG
and
a signal peptide, as required. Such vectors are described, for example, in
Luckow et al., Virology 170:31-39 (1989).
The cDNA sequence encoding the soluble extracellular domain of DRS
protein in the deposited plasmid (ATCC Deposit No. 97920) is amplified using
PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the
gene:
The 5' primer for DRS has the sequence:
5'-CGCGGATCCGCCATCATGGAACAACGGGGACAGAAC-3' (SEQ ID
NO:10) containing the underlined BamHI restriction enzyme site. Inserted into
an expression vector, as described below, the 5' end of the amplified fragment
encoding DRS provides an efficient cleavage signal peptide. An efficient
signal
for initiation of translation in eukaryotic cells, as described by Kozak, M.,
J. Mol.
Biol. 196:947-950 (1987) is appropriately located in the vector portion of the
construct.
The 3' primer for DRS has the sequence:
5'-CGCGGTACCTTAGCCTGATTCTTTGTGGAC-3' (SEQ ID N0:12)
containing the underlined Asp718 restriction followed by nucleotides
complementary to the DRS nucleotide sequence in FIG. 1, followed by the stop
codon.
The amplified fragment is isolated from a 1% agarose gel using a
commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.) The
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fragment then is digested with BamHI and Asp718 and again is purified on a 1%
agarose gel. This fragment is designated "Fl."
The plasmid is digested with the restriction enzymes BamHI and Asp718
and optionally can be dephosphorylated using calf intestinal phosphatase,
using
routine procedures known in the art. The DNA is then isolated from a 1%
agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La
Jolla, Ca.). The vector DNA is designated herein "V1."
Fragment F 1 and the dephosphorylated plasmid V 1 are ligated together
with T4 DNA ligase. E. coli HB 1 O 1 cells, or other suitable E. coli hosts
such as
XL-1 Blue (Stratagene Cloning Systems, La Jolla, CA) cells, are transformed
with the ligation mixture and spread on culture plates. Bacteria containing
the
plasmid with the human DRS are identified using the PCR method, in which one
of the primers used to amplify the gene is directed to the DRS sequence and
the
second primer is from well within the vector so that only those bacterial
colonies
containing the DRS gene fragment will show amplification of the DNA. The
sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid
is designated herein pBac DRS.
S ~cg of the plasmid pBac DRS is co-transfected with 1.0 ,ug of a
commercially available linearized baculovirus DNA ("BaculoGoldT~" baculovirus
DNA", Pharmingen, San Diego, CA.), using the lipofectin method described by
Felgner el al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). 1 ~cg of
BaculoGoldT~~ virus DNA and 5 ,ug of the plasmid pBac DRS are mixed in a
sterile well of a microtiter plate containing 50 ,ul of serum free Grace's
medium
(Life Technologies Inc., Gaithersburg, MD). Afterwards 10 /.cl Lipofectin plus
90 ~l Grace's medium are added, mixed and incubated for 15 minutes at room
temperature. Then the transfection mixture is added drop-wise to S~ insect
cells
(ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's
medium without serum. The plate is rocked back and forth to mix the newly
added solution. The plate is then incubated for 5 hours at 27°C. After
5 hours,
the transfection solution is removed from the plate and 1 ml of Grace's insect
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medium supplemented with 10% fetal calf serum is added. The plate is put back
into an incubator and cultivation is continued at 27°C for four days.
After four days, the supernatant is collected and a plaque assay is
performed, as described by Summers and Smith, cited above. An agarose gel
with "Blue Gal" (Life Technologies Inc., Gaithersburg, MD) is used to allow
easy identification and isolation of gal-expressing clones, which produce blue-
stained plaques. (A detailed description of a "plaque assay" of this type can
also
be found in the user's guide for insect cell culture and baculovirology
distributed
by Life Technologies Inc., Gaithersburg, MD, pages 9-10). After appropriate
incubation, blue stained plaques are picked with the tip of a micropipettor
(e.g,
Eppendorf). The agar containing the recombinant viruses is then resuspended in
a microcentrifuge tube containing 200 ~1 of Grace's medium and the suspension
containing the recombinant baculovirus is used to infect Sf~ cells seeded in
35
mm dishes. Four days later, the supernatants of these culture dishes are
harvested and then they are stored at 4°C. The recombinant virus is
called
V-DRS .
To verify expression of the DRS gene, Sfr3 cells are grown in Grace's
medium supplemented with 10% heat-inactivated FBS. The cells are infected
with the recombinant baculovirus V-DRS at a multiplicity of infection ("MOI")
of about 2 (about 1 to about 3). Six hours later, the medium is removed and is
replaced with SF900 II medium minus methionine and cysteine (available from
Life Technologies Inc., Gaithersburg, MD). Ifradiolabeled proteins are
desired,
42 hours later, 5 ,uCi of ;SS-methionine and 5 ~Ci 35S-cysteine (available
from
Amersham) are added. The cells are further incubated for 16 hours and then
they
are harvested by centrifugation. The proteins in the supernatant as well as
the
intracellular proteins are analyzed by SDS-PAGE followed by autoradiography
(if radiolabeled). Microsequencing of the amino acid sequence of the amino
terminus of purified protein may be used to determine the amino terminal
sequence of the mature protein and thus the cleavage point and length of the
secretory signal peptide.
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Example S
DRS Induced Apoptosis in Mammalian Cells
Overexpression of Fas/APO-1 and TNFR-1 in mammalian cells mimics
S receptor activation (M. Muzio et al., Cell85: 817-827 (1996); M. P. Boldin
et
al., Cell 85:803-815 (1996)). Thus, this system was utilized to study the
functional role of DRS in inducing apoptosis. This example demonstrates that
overexpression ofDRS induced apoptosis in both MCF7 human breast carcinoma
cells and in human epitheloid carcinoma (HeLa) cells.
Experimental Design
Cell death assays were performed essentially as previously described
(A.M. Chinnaiyan, et al., Cell 81:505-12 (1995); M.P. Boldin, et al., J Biol
Chem 270: 7795-8 (1995); F.C. Kischkel, et al., EMBO 14:5579-5588 (1995);
A.M. Chinnaiyan, et al.., .IBiol Chem 271: 4961-4965 (1996)). Briefly, MCF-7
human breast carcinoma clonal cell lines and HeLa cells were co-transfected
with
vector, DRS, DR50 (52-411), or TNFR-1, together with a beta-galactosidase
reporter construct.
MCF7 and HeLa cells were transfected using the lipofectamine procedure
(GIBCO-BRL), according to the manufacturer's instructions. 293 cells were
transfected using CaP04 precipitation. Twenty-four hours following
transfection, cells were fixed and stained with X-Gal as previously described
(A.M. Chinnaiyan, et al., Cell 81:505-12 (1995); M.P. Boldin, et al., .I Biol
Chem 270:7795-8 (1995); F.C. Kischkel, et al., EMBO 14:5579-5588 (1995)),
and examined microscopically. The data (mean~SD) presented in FIG. 5
represents the percentage of round, apoptotic cells as a function of total
beta-
galactosidase_positive cells (n=3). Overexpression of DRS induced apoptosis in
both MCF7 (FIG. SA) and HeLa cells (FIG. SB).
MCF7 cells were also transfected with a DRS expression construct in the
presence of z-VAD-fmk (20 pl) (Enzyme Systems Products, Dublin, CA) or co-
transfected with a three-fold excess of CrmA (M. Tewari et al., J Biol Chem
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270: 3255-60 (1995)), or FADD-DN expression construct, or vector alone. The
data presented in FIG. SC shows that apoptosis induced by DRS was attenuated
by caspase inhibitors, but not by dominant negative FADD.
As depicted in FIG. SD, DRS did not associate with FADD or TRADD
in vivo. 293 cells were co-transfected with the indicated expression
constructs
using calcium phosphate precipitation. After transfection (at 40 hours), cell
lysates were prepared and immunoprecipitated with Flag M2 antibody affinity
gel
(IBI, Kodak), and the presence of FADD or myc-tagged TRADD (myc-
TRADD) was detected by immunoblotting with polyclonal antibody to FADD
or horseradishperoxidase(HRP)conjugatedantibodytomyc(BMB)(Baker,S.J.
et al., Oncogene 12:1 (1996); Chinnaiyan, A.M. et al., Science 274:990
(1996)).
As depicted in FIG. SE, FLICE 2-DN blocks DRS-induced apoptosis.
293 cells were co-transfected with DRS or TNFR-1 expression construct and a
fourfold excess of CrmA, FLICE-DN, FLICE 2-DN, or vector alone in the
presence of a beta-galactosidase reported construct as indicated. Cells were
stained and examined 25-30 hours later.
Results
Overexpression of DRS, induced apoptosis in both MCF7 human breast
carcinoma cells (FIG. SA) and in human epitheloid carcinoma (HeLa) cells (FIG.
SB). Most of the transfected cells displayed morphological changes
characteristic ofcellsundergoing apoptosis (Earnshaw, W.C., Curr. Biol. 7:337
(1995)), becoming rounded, condensed and detaching from the dish. Deletion of
the death domain abolished killing ability. Like DR4, DRS-induced apoptosis
was blocked by caspase inhibitors, CrmA and z-VAD-fmk, but dominant
negative FADD was without effect (FIG. SC). Consistent with this, DRS did not
interact with FADD and TRADD in vivo (FIG. SD). A dominant negative
version of a newly identified FLICE-like molecule, FLICE2 (Vincenz, C. et al.,
J. Biol. Chem. 272:6578 (1997)), ef~'iciently blocked DRS-induced apoptosis,
while dominant negative FLICE had only partial e~'ect under conditions it
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blocked. TNFR-1 induced apoptosis effectively (FIG. SE). Taken together, the
evidence suggests that DRS engages an apoptotic program that involves
activation of FLICE2 and downstream caspases, but is independent of FADD.
s Example 6
The Extracellular Domain of DRS Binds the Cytotoxic Ligand, TRAIL,
and Blocks TRAIL-Induced Apoptosis
As discussed above, TRAIL,/Apo2L is a cytotoxic ligand that belongs to
the tumor necrosis factor (TNF) ligand family and induces rapid cell death of
many transformed cell lines, but not normal tissues, despite its death domain
containing receptor, DR4, being expressed on both cell types. This example
shows that the present receptor, DRS, also binds TRAIL.
Given the similarity of the extracellular ligand binding cysteine-rich
1 S domains of DRS and DR4, the present inventors theorized that DRS would
also
bind TRAIL. To confirm this, the soluble extracellular ligand binding domains
of DRS were expressed as fusions to the Fc portion of human immunoglobulin
(IgG). cDNA encoding the amino acids 1 to 129 in SEQ ID N0:2 was obtained
by polymerase chain reaction and cloned into a modified pCMVIFLAG vector
that allowed for in-frame fusion with the Fc portion of human IgG.
As shown in FIG. 6A, DRS-Fc specifically bound TRAIL,, but not the
related cytotoxic ligand TNFa,. In this experiment, the Fc-extracellular
domains
of DRS, DR4, TRID, or TNFR-1 and the corresponding ligands were prepared
and binding assays performed as described in Pan et al., Science 276:111 (
1997).
2S The respective Fc-fusions were precipitated with protein G-Sepharose and co-
precipitated soluble ligands were detected by immunoblotting with anti-Flag
(Babco) or anti-myc-HRP (BMB). The bottom panel of FIG. 6A shows the
input Fc-fusions present in the binding assays.
Additionally, DRS-Fc blocked the ability of TRAIL, to induce apoptosis
(FIG. 6B). MCF7 cells were treated with soluble TRAIL (200 ng/ml) in the
presence of equal amounts of Fc-fusions or Fc alone. Six hours later, cells
were
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fixed and examined as described in Pan et al., Id. The data (mean ~ SD) shown
in FIG. 6B are the percentage of apoptotic nuclei among total nuclei counted
(n=4).
Finally, DRS-Fc had no effect on apoptosis TNFa,-induced cell death
under conditions where TNFR-1-Fc completely abolished TNFa, killing (FIG.
6C). MCF7 cells were treated with TNFa, (40 ng/ml; Genentech, Inc.) in the
presence of equal amounts of Fc-fusions or Fc alone. Nuclei were stained and
examined 11-15 hours later.
The new identification of DRS as a receptor for TRAIL, adds further
complexity to the biology of TRAIL-initiated signal transduction.
F~a~nple 7
Assays to Detect Stimulation or Inhibition of B Cell Proliferation and
Differentiation
Generation offunctional humoral immune responses requires both soluble
and cognate signaling between B-lineage cells and their microenvironment.
Signals may impart a positive stimulus that allows a B-lineage cell to
continue its
programmed development, or a negative stimulus that instructs the cell to
arrest
its current developmental pathway. To date, numerous stimulatory and
inhibitory
signals have been found to influence B-cell responsiveness including IL-2, IL,-
4,
IL-5, IL-6, IL,-7, IL-10, IL-13, IL-14 and IL,-15. Interestingly, these
signals are
by themselves weak effectors but can, in combination with various co-
stimulatory
proteins, induce activation, proliferation, differentiation, homing, tolerance
and
death among B-cell populations. One of the best studied classes of B-cell co-
stimulatory proteins is the TNF-superfamily. Within this family CD40, CD27,
and CD30 along with their respective ligands CD 154, CD70, and CD 153 have
been found to regulate a variety of immune responses. Assays that allow for
the
detection and/or observation of the proliferation and differentiation of these
B-
cell populations and their precursors are valuable tools in determining the
effects
various proteins may have on these B-cell populations in terms of
proliferation
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and differentiation. Listed below are two assays designed to allow for the
detection of the differentiation, proliferation, or inhibition of B-cell
populations
and their precursors.
Experimental Procedure:
In Tjitro assay- Purified DRS protein, or truncated forms thereof, is
assessed for its ability to induce activation, proliferation, differentiation
or
inhibition and/or death in B-cell populations and their precursors. The
activity of
DRS protein on purified human tonsillar B-cells, measured qualitatively over
the
dose range from 0.1 to 10,000 ng/mL, is assessed in a standard B-lymphocyte
co-stimulation assay in which purified tonsillar B-cells are cultured in the
presence of either formalin-fixed Staphylococcus aureus Cowan I (SAC) or
immobilized anti-human IgM antibody as the priming agent. Second signals such
as IL-2 and lL,-I S synergize with SAC and IgM cross-linking to elicit B-cell
proliferation as measured by tritiated-thymidine incorporation. Novel
synergizing
agents can be readily identified using this assay. The assay involves
isolating
human tonsillar B-cells by magnetic bead (MACS) depletion of CD3-positive
cells. The resulting cell population is greater than 95% B-cells as assessed
by
expression of CD45R (B220). Various dilutions of each sample are placed into
individual wells of a 96-well plate to which are added 105 B-cells suspended
in
culture medium (RPMI 1640 containing 10% FBS, 5 X 10-SM ~3-ME, 100U/ml
penicillin, 10 ~g/ml streptomycin, and 10-5 dilution of SAC) in a total volume
of
150 ~1. Proliferation or inhibition is quantitated by a 20h pulse (1 ~Ci/well)
with
3H-thymidine (6.7 Ci/mM) beginning 72 hours post factor addition. The positive
and negative controls are 1L-2 and medium respectively.
In Vivo assay- BALB/c mice are injected (i.p.) twice per day with buffer
only, or with 2 mg/Kg of DRS protein, or truncated forms thereof. Mice receive
this treatment for 4 consecutive days, at which time they are sacrificed and
various tissues and serum collected for analyses. Comparison of H&E sections
from normal and DRS protein-treated spleens identify the results of the
activity
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of DRS protein on spleen cells, such as the diffusion of peri-arterial
lymphatic
sheaths, and/or significant increases in the nucleated cellularity of the red
pulp
regions, which may indicate the activation of the differentiation and
proliferation
of B-cell populations. Immunohistochemical studies using a B-cell marker, anti-
s CD45R (B220), are used to determine whether any physiological changes to
splenic cells, such as splenic disorganization, are due to increased B-cell
representation within loosely defined B-cell zones that infiltrate established
T-cell
regions.
Flow cytometric analyses of the spleens from DRS protein-treated mice
is used to indicate whether DRS protein specifically increases the proportion
of
ThB+, CD45R (B220) dull B-cells over that which is observed in control mice.
Likewise, a predicted consequence of increased mature B-cell
representation in vivo is a relative increase in serum Ig titers. Accordingly,
serum IgM and 1gA levels are compared between buffer and DRS protein-treated
1 S mice.
The studies described in this example test the activity in DRS protein.
However, one skilled in the art could easily modify the exemplified studies to
test
the activity of DRS polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of DRS.
Example 8
T Cell Proliferation Assay
A CD3-induced proliferation assay is performed on PBMCs and is
measured by the uptake of 3H-thymidine. The assay is performed as follows.
Ninety-six well plates are coated with 100 p,l/well of mAb to CD3 (HIT3a,
Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4°C (1
p,g/ml
in O.OSM bicarbonate buffer, pH 9.5), then washed three times with PB S. PBMC
are isolated by F/H gradient centrifugation from human peripheral blood and
added to quadruplicate wells (S x 104/well) of mAb coated plates in RPMI
containing 10% FCS and P/S in the presence of varying concentrations of DRS
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protein (total volume 200 ~1). Relevant protein buffer and medium alone are
controls. After 48 hours at 37°C, plates are spun for 2 minutes at 1000
rpm and
100 ~l of supernatant is removed and stored at -20°C for measurement of
IL-2
(or other cytokines) if an effect on proliferation is observed. Wells are
supplemented with 100 p,l of medium containing 0.5 pCi of 3H-thymidine and
cultured at 37°C for 18-24 hr. Wells are harvested and incorporation of
3H-
thymidine used as a measure of proliferation. Anti-CD3 alone is the positive
control for proliferation. IL-2 (100 U/ml) is also used as a control which
enhances proliferation. Control antibody which does not induce proliferation
of
T-cells is used as the negative controls for the effects of DRS proteins.
The studies described in this example test the activity in DR5 protein.
However, one skilled in the art could easily modify the exemplified studies to
test
the activity of DRS polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of DRS.
Example 9
Effect of DRS on the Expression of MHC Class 11, Costimulatory and
Adhesion Molecules and Cell Differentiation of monocytes and Monocyte
Derived Human Dendritic Cells
Dendritic cells are generated by the expansion of proliferating precursors
found in the peripheral blood: adherent PBMC or elutriated monocytic fractions
are cultured for 7-10 days with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These
dendritic cells have the characteristic phenotype of immature cells
(expression of
CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with
activating factors, such as TNF-a,, causes a rapid change in surface phenotype
(increased expression of MHC class I and II, costimulatory and adhesion
molecules, downregulation of FCyRII, upregulation of CD83). These changes
correlate with increased antigen-presenting capacity and with functional
maturation of the dendritic cells.
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FACS analysis of surface antigens is performed as follows. Cells are
treated 1-3 days with increasing concentrations of DRS or LPS (positive
control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and
then incubated with 1:20 dilution of appropriate FITC- or PE-labeled
monoclonal
S antibodies for 30 minutes at 4°C. After an additional wash, the
labeled cells are
analyzed by flow cytometry on a FACScan (Becton Dickinson).
Effect on the production of cytokines
Cytokines generated by dendritic cells, in particular IL,-12, are important
in the initiation ofT-cell dependent immune responses. IL-12 strongly
influences
the development of Thl helper T-cell immune response, and induces cytotoxic T
and NK cell function. An ELISA is used to measure the IL,-12 release as
follows. Dendritic cells (106/ml) are treated with increasing concentrations
of
1S DRS for 24 hours. LPS (100 ng/ml) is added to the cell culture as positive
control. Supernatants from the cell cultures are then collected and analyzed
for
IL,-12 content using commercial ELISA kit (e.g., R & D Systems (Minneapolis,
MN)). The standard protocols provided with the kits are used.
Effect on the expression of MHC Class II, costimulatory and adhesion
molecules
Three major families of cell surface antigens can be identified on
monocytes: adhesion molecules, molecules involved in antigen presentation, and
Fc receptor. Modulation of the expression of MHC class II antigens and other
costimulatory molecules, such as B7 and ICAM-1, may result in changes in the
antigen presenting capacity of monocytes and ability to induce T-cell
activation.
Increase expression of Fc receptors may correlate with improved monocyte
cytotoxic activity, cytokine release and phagocytosis.
FACS analysis is used to examine the surface antigens as follows.
Monocytes are treated 1-S days with increasing concentrations of DRS or LPS
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(positive control), washed with PBS containing 1% BSA and 0.02 mM sodium
azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-
labeled
monoclonal antibodies for 30 minutes at 4°C. After an additional wash,
the
labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).
Monocyte activation and/or increased survival.
Assays for molecules that activate (or alternatively, inactivate) monocytes
and/or increase monocyte survival (or alternatively, decrease monocyte
survival)
are known in the art and may routinely be applied to determine whether a
molecule of the invention functions as an inhibitor or activator of monocytes.
DRS, agonists, or antagonists of DRS can be screened using the three assays
described below. For each of these assays, Peripheral blood mononuclear cells
(PBMC) are purified from single donor leukopacks (American Red Cross,
Baltimore, MD) by centrifugation through a Histopaque gradient (Sigma).
Monocytes are isolated from PBMC by counterflow centrifugal elutriation.
1. Monocyte Survival Assay. Human peripheral blood monocytes
progressively lose viability when cultured in absence of serum or other
stimuli.
Their death results from internally regulated process (apoptosis). Addition to
the
culture of activating factors, such as TNF-alpha dramatically improves cell
survival and prevents DNA fragmentation. Propidium iodide (PI) staining is
used
to measure apoptosis as follows. Monocytes are cultured for 48 hours in
polypropylene tubes in serum-free medium (positive control), in the presence
of
100 ng/ml TNF-alpha (negative control), and in the presence of varying
concentrations of the compound to be tested. Cells are suspended at a
concentration of 2 x 106/ml in PBS containing PI at a final concentration of 5
~g/ml, and then incubated at room temperature for 5 minutes before FACScan
analysis. PI uptake has been demonstrated to correlate with DNA fragmentation
in this experimental paradigm.
2. Effect on cytokine release. An important function of
monocytes/macrophages is their regulatory activity on other cellular
populations
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of the immune system through the release of cytokines after stimulation. An
ELISA to measure cytokine release is performed as follows. Human monocytes
are incubated at a density of 5x105 cells/ml with increasing concentrations of
DRS and under the same conditions, but in the absence of DRS. For IL,-12
production, the cells are primed overnight with IFN-y ( 100 U/ml) in presence
of
DRS. LPS (10 ng/ml) is then added. Conditioned media are collected after 24h
and kept frozen until use. Measurement of TNF-a, IL-10, MCP-1 and IL-8 is
then performed using a commercially available ELISA kit (e.g., R & D Systems
(Minneapolis, MN)) and applying the standard protocols provided with the kit.
3. Oxidative burst. Purified monocytes are plated in 96-well plates at 2-
1x105 cell/well. Increasing concentrations of DRS are added to the wells in a
total volume of 0.2 ml culture medium (RPMI 1640 + 10% FCS, glutamine and
antibiotics). After 3 days incubation, the plates are centrifuged and the
medium
is removed from the wells. To the macrophage monolayers, 0.2 ml per well of
phenol red solution (140 mM NaCI, 10 mM potassium phosphate buffer pH 7.0,
5.5 mM dextrose, 0.56 mM phenol red and 19 U/ml ofHRPO) is added, together
with the stimulant (200 nM PMA). The plates are incubated at 37°C for 2
hours
and the reaction is stopped by adding 20 ~1 1N NaOH per well. The absorbance
is read at 610 nm. To calculate the amount of H202 produced by the
macrophages, a standard curve of a H202 solution of known molarity is
performed for each experiment.
The studies described in this example test the activity in DRS protein.
However, one skilled in the art could easily modify the exemplified studies to
test
the activity of DRS polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of DRS.
Example 10
The Effect of DRS on the Growth of Vascular Endothelial Cells
On day 1, human umbilical vein endothelial cells (HUVEC) are seeded
at 2-5x104 cells/3 S mm dish density in M199 medium containing 4% fetal bovine
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serum (FBS), 16 units/ml heparin, and 50 units/ml endothelial cell growth
supplements (ECGS, Biotechnique, Inc. ). On day 2, the medium is replaced with
M199 containing 10% FBS, 8 units/ml heparin. DR5 protein of SEQ ID NO. 2,
and positive controls, such as VEGF and basic FGF (bFGF) are added, at varying
concentrations. On days 4 and 6, the medium is replaced. On day 8, cell number
is determined with a Coulter Counter.
An increase in the number of HUVEC cells indicates that DR5 may
proliferate vascular endothelial cells.
The studies described in this example test the activity in DR5 protein.
However, one skilled in the art could easily modify the exemplified studies to
test
the activity of DR5 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of DRS.
Example 11
Production of an Antibody
A. Hybridoma Technology
The antibodies of the present invention can be prepared by a variety of
methods. (See, Current Protocols, Chapter 2.) As one example of such
methods, cells expressing DR5 are administered to an animal to induce the
production of sera containing polyclonal antibodies. In a preferred method, a
preparation of DR5 protein is prepared and purified to render it substantially
free
of natural contaminants. Such a preparation is then introduced into an animal
in order to produce polyclonal antisera of greater specific activity.
Monoclonal antibodies specific for protein DR5 are prepared using
hybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler et al.,
Eur. J. Immunol. 6:511 (1976); Kohler et al., L'ur. J. Immunol. 6:292 (1976);
Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier,
N.Y., pp. 563-681 (1981)). In general, an animal (preferably a mouse) is
immunized with DR5 polypeptide or, more preferably, with a secreted DRS
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polypeptide-expressing cell. Such polypeptide-expressing cells are cultured in
any suitable tissue culture medium, preferably in Earle's modified Eagle's
medium supplemented with 10% fetal bovine serum (inactivated at about
56°C),
and supplemented with about 10 g/1 of nonessential amino acids, about 1,000
U/ml of penicillin, and about 100 pg/ml of streptomycin.
The splenocytes of such mice are extracted and fused with a suitable
myeloma cell line. Any suitable myeloma cell line may be employed in
accordance with the present invention; however, it is preferable to employ the
parent myeloma cell line (SP20), available from the ATCC. After fusion, the
resulting hybridoma cells are selectively maintained in HAT medium, and then
cloned by limiting dilution as described by Wands et al. (Gastroenterology
80:225-232 (1981). The hybridoma cells obtained through such a selection are
then assayed to identify clones which secrete antibodies capable of binding
the
DRS polypeptide.
Alternatively, additional antibodies capable of binding to DRS
polypeptide can be produced in a two-step procedure using anti-idiotypic
antibodies. Such a method makes use of the fact that antibodies are themselves
antigens, and therefore, it is possible to obtain an antibody which binds to a
second antibody. In accordance with this method, protein specific antibodies
are
used to immunize an animal, preferably a mouse. The splenocytes of such an
animal are then used to produce hybridoma cells, and the hybridoma cells are
screened to identify clones which produce an antibody whose ability to bind to
the DRS protein-specific antibody can be blocked by DRS. Such antibodies
comprise anti-idiotypic antibodies to the DRS protein-specific antibody and
are
used to immunize an animal to induce formation of further DRS protein-specific
antibodies.
For in vivo use of antibodies in humans, an antibody is "humanized".
Such antibodies can be produced using genetic constructs derived from
hybridoma cells producing the monoclonal antibodies described above. Methods
for producing chimeric and humanized antibodies are known in the art and are
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discussed infra. (See, for review, Morrison, Science 229:1202 (1985); Oi et
al.,
BioTechniques 4:214 (1986); Cabilly et al., U.S. Patent No. 4,816,567;
Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO
8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643
(1984); Neuberger et al., Nature 314:268 (1985).)
B. Isolation Of Antibody Fragments Directed Against DRS From
A Library Of scFvs
Naturally occurring V-genes isolated from human PBLs are constructed
into a large library of antibody fragments which contain reactivities against
polypeptides of the present invention to which the donor may or may not have
been exposed (see, e.g., U.S. Patent 5,885,793 incorporated herein in its
entirety
by reference).
Rescue of the library
A library of scFvs is constructed from the RNA of human PBLs as
described in W092/01047. To rescue phage displaying antibody fragments,
approximately 10~ E. coli harboring the phagemid are used to inoculate 50 ml
of
2xTY containing 1% glucose and 100 pg/ml of ampicillin (2xTY-AMP-GLU)
and grown to an O.D. of 0.8 with shaking. Five ml of this culture is used to
innoculate SO ml of 2xTY-AMP-GLU, 2x108 TU of delta gene 3 helper phage
(M13 gene III, see W092/01047) are added and the culture incubated at
37° C
for 45 minutes without shaking and then at 37° C for 45 minutes with
shaking.
The culture is centrifuged at 4000 r.p.m. for 10 minutes and the pellet
resuspended in 2 liters of 2xTY containing 100 ~g/ml ampicillin and 50 pg/ml
kanamycin and grown overnight. Phage are prepared as described in
W092/01047.
M13 gene III is prepared as follows: M13 gene III helper phage does not
encode gene III protein, hence the phage(mid) displaying antibody fragments
have a greater avidity of binding to antigen. Infectious M13 gene III
particles are
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made by growing the helper phage in cells harboring a pUCl9 derivative
supplying the wild type gene III protein during phage morphogenesis. The
culture is incubated for 1 hour at 37° C without shaking and then for a
further
hour at 37° C with shaking. Cells are pelleted (IEC-Centra 8, 4000
revs/min for
10 min), resuspended in 300 ml 2xTY broth containing 100 ~g ampicillin/ml and
25 ~g kanamycin/ml (2xTY-AMP-KAN) and grown overnight, shaking at 37°
C. Phage particles are purified and concentrated from the culture medium by
two PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS and
passed through a 0.45 ~m filter (Minisart NML; Sartorius) to give a final
concentration of approximately 10'3 transducing units/ml (ampicillin-resistant
clones).
Panning of the library
Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100
mg/ml or 10 mg/ml of a polypeptide of the present invention. Tubes are blocked
with 2% Marvel-PBS for 2 hours at 37° C and then washed 3 times in PBS.
Approximately 10'3 TU of phage are applied to the tube and incubated for 30
minutes at room temperature tumbling on an over and under turntable and then
left to stand for another 1.5 hours. Tubes are washed 10 times with PBS 0.1%
Tween-20 and 10 times with PBS. Phage are eluted by adding 1 ml of 100 mM
triethylamine and rotating 15 minutes on an under and over turntable after
which
the solution is immediately neutralized with 0.5 ml of 1.OM Tris-HCI, pH 7.4.
Phage are then used to infect 10 ml of mid-log E. coli TG1 by incubating
eluted
phage with bacteria for 30 minutes at 37° C. The E coli are then plated
on TYE
plates containing 1% glucose and 100 ~g/ml ampicillin. The resulting bacterial
library is then rescued with M13 gene III helper phage as described above to
prepare phage for a subsequent round of selection. This process is then
repeated
for a total of 4 rounds of affnity purification with tube-washing increased to
20
times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.
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Characterization of binders
Eluted phage from the 3rd and 4th rounds of selection are used to infect
E. coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single
colonies for assay. ELISAs are performed with microtiter plates coated with
either 10 pg/ml of the polypeptide of the present invention in 50 mM
bicarbonate
pH 9.6. Clones positive in ELISA are further characterized by PCR
fingerprinting (see e.g., W092/01047) and then by sequencing.
1 o Example 12
Tissue Distribution of DRS Gene Expression
Northern blot analysis was carried out to examine DRS gene expression
in human tissues, using methods described by, among others, Sambrook et al.,
1 S cited above. A cDNA probe containing the entire nucleotide sequence of the
DRS protein (SEQ ID NO:1) was labeled with 3zP using the rediprimeTM DNA
labeling system (Amersham Life Science), according to manufacturer's
instructions. After labeling, the probe was purified using a CHROMA
SPIN-1 OOTM column (Clontech Laboratories, Inc.), according to manufacturer's
20 protocol number PT1200-1. The purified labeled probe was then used to
examine various human tissues for DRS mRNA.
Multiple Tissue Northern (MTN) blots containing various human tissues
(H) or human immune system tissues (IM) were obtained from Clontech (Palo
Alto, CA) and examined with labeled probe using ExpressHybT~~ hybridization
25 solution (Clontech) according to manufacturer's protocol number PT1190-1.
Following hybridization and washing, the blots were mounted and exposed to
film at -70 ° C overnight. The films were developed according to
standard
procedures. Expression of DRS was detected in heart, brain, placenta, lung,
liver,
skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, uterus,
small
30 intestine, colon, peripheral blood leukocytes (PBLs), lymph node, bone
marrow,
and fetal liver.
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Expression of DRS was also assessed by Northern blot in the following
cancer cell lines, HL60 (promyelocytic leukemia), HeLa cell S3, K562 (chronic
myelogeneous leukemia), MOLT4 (lymphoblast leukemia), Raji (Burkitt's
lymphoma), SW480 (colorectal adenocarcinoma), A549 (lung carcinoma), and
6361 (melanoma), and was detected in all of the cell lines tested.
Example 13
Method of Determining Alterations in the DRS Gene
RNA is isolated from entire families or individual patients presenting with
a phenotype of interest (such as a disease). cDNA is then generated from these
RNA samples using protocols known in the art. (See, Sambrook.) The cDNA
is then used as a template for PCR, employing primers surrounding regions of
interest in SEQ ID NO:1. Suggested PCR conditions consist of 3 5 cycles at 95
°
C for 30 seconds; 60-120 seconds at 52-58° C; and 60-120 seconds at
70° C,
using buffer solutions described in Sidransky, D., et al., Science 252:706
(1991).
PCR products are then sequenced using primers labeled at their 5' end
with T4 polynucleotide kinase, employing SequiTherm Polymerase. (Epicentre
Technologies). The intron-exon borders of selected exons of DRS are also
determined and genomic PCR products analyzed to confirm the results. PCR
products harboring suspected mutations in DRS are then cloned and sequenced
to validate the results of the direct sequencing.
PCR products of DRS are cloned into T-tailed vectors as described in
Holton, T. A. and Graham, M. W., Nucleic Acids Research, 19:1156 ( 1991 ) and
sequenced with T7 polymerase (United States Biochemical). Affected
individuals are identified by mutations in DRS not present in unaffected
individuals.
Genomic rearrangements are also observed as a method of determining
alterations in the DRS gene. Genomic clones isolated using techniques known
in the art are nick-translated with digoxigenindeoxy-uridine S'-triphosphate
(Boehringer Mannheim), and FISH performed as described in Johnson, Cg. et al.,
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Methods Cell Biol. 35:73-99 (1991). Hybridization with the labeled probe is
carried out using a vast excess of human cot-1 DNA for specific hybridization
to
the DRS genomic locus.
Chromosomes are counter-stained with 4,6-diamino-2-phenylidole and
propidium iodide, producing a combination of C- and R-bands. Aligned images
for precise mapping are obtained using a triple-band filter set (Chroma
Technology, Brattleboro, VT) in combination with a cooled charge-coupled
device camera (Photometrics, Tucson, AZ) and variable excitation wavelength
filters. (Johnson, Cv. et al., Genet. Anal. Tech. Appl., 8:75 (1991).) Image
collection, analysis and chromosomal fractional length measurements are
performed using the ISee Graphical Program System. (Inovision Corporation,
Durham, NC.) Chromosome alterations of the genomic region of DRS
(hybridized by the probe) are identified as insertions, deletions, and
translocations. These DRS alterations are used as a diagnostic marker for an
associated disease.
Example 14
Method of Detecting Abnormal Levels of DRS in a Biological Sample
DRS polypeptides can be detected in a biological sample, and if an
increased or decreased level of DRS is detected, this polypeptide is a marker
for
a particular phenotype. Methods of detection are numerous, and thus, it is
understood that one skilled in the art can modify the following assay to fit
their
particular needs.
For example, antibody-sandwich ELISAs are used to detect DRS in a
sample, preferably a biological sample. Wells of a microtiter plate are coated
with specific antibodies to DRS, at a final concentration of 0.2 to 10 pg/ml.
The
antibodies are either monoclonal or polyclonal and are produced using
technique
known in the art. The wells are blocked so that non-specific binding of DRS to
the well is reduced.
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The coated wells are then incubated for > 2 hours at RT with a sample
containing DRS. Preferably, serial dilutions of the sample should be used to
validate results. The plates are then washed three times with deionized or
distilled water to remove unbounded DRS.
Next, 50 pl of specific antibody-alkaline phosphatase conjugate, at a
concentration of 25-400 ng, is added and incubated for 2 hours at room
temperature. The plates are again washed three times with deionized or
distilled
water to remove unbounded conjugate.
75 pl of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl
phosphate (NPP) substrate solution is then added to each well and incubated 1
hour at room temperature to allow cleavage of the substrate and fluorescence.
The fluorescence is measured by a microtiter plate reader. A standard curve is
prepared using the experimental results from serial dilutions of a control
sample
with the sample concentration plotted on the X-axis (log scale) and
fluorescence
or absorbance on the Y-axis (linear scale). The DRS polypeptide concentration
in a sample is then interpolated using the standard curve based on the
measured
fluorescence of that sample.
Example 1 S
Method of Treating Decreased Levels of DRS
The present invention relates to a method for treating an individual in
need of a decreased level of DRS biological activity in the body comprising,
administering to such an individual a composition comprising a therapeutically
effective amount of DRS antagonist. Preferred antagonists for use in the
present
invention are DRS-specific antibodies.
Moreover, it will be appreciated that conditions caused by a decrease in
the standard or normal expression level of DRS in an individual can be treated
by
administering DRS, preferably in a soluble and/or secreted form. Thus, the
invention also provides a method of treatment of an individual in need of an
increased level of DR5 polypeptide comprising administering to such an
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individual a pharmaceutical composition comprising an amount of DRS to
increase the biological activity level of DRS in such an individual.
For example, a patient with decreased levels of DRS polypeptide receives
a daily dose 0.1-100 g/kg ofthe polypeptide for six consecutive days.
Preferably,
S the polypeptide is in a soluble and/or secreted form.
Example 16
Method of Treating Increased Levels of DRS
The present invention also relates to a method for treating an individual
in need of an increased level of DRS biological activity in the body
comprising
administering to such an individual a composition comprising a therapeutically
effective amount of DRS or an agonist thereof.
Antisense technology is used to inhibit production of DRS. This
1 S technology is one example of a method of decreasing levels of DRS
polypeptide,
preferably a soluble and/or secreted form, due to a variety of etiologies,
such as
cancer.
For example, a patient diagnosed with abnormally increased levels of
DRS is administered intravenously antisense polynucleotides at O.S, 1.0, 1.5,
2.0
and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest
period if the is determined to be well tolerated.
Example 17
Method of Treatment Using Gene Therapy - Ex Vivo
2S
One method of gene therapy transplants fibroblasts, which are capable of
expressing soluble and/or mature DRS polypeptides, onto a patient. Generally,
fibroblasts are obtained from a subject by skin biopsy. The resulting tissue
is
placed in tissue-culture medium and separated into small pieces. Small chunks
of the tissue are placed on a wet surface of a tissue culture flask,
approximately
ten pieces are placed in each flask. The flask is turned upside down, closed
tight
and left at room temperature over night. After 24 hours at room temperature,
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the flask is inverted and the chunks of tissue remain fixed to the bottom of
the
flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and
streptomycin) is added. The flasks are then incubated at 37 ° C for
approximately
one week.
At this time, fresh media is added and subsequently changed every several
days. After an additional two weeks in culture, a monolayer of fibroblasts
emerge. The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P.T. et ad., DNA, 7:219-25 (1988)), flanked by the
long terminal repeats of the Moloney murine sarcoma virus, is digested with
EcoRI and HindIII and subsequently treated with calf intestinal phosphatase.
The linear vector is fractionated on agarose gel and purified, using glass
beads.
The cDNA encoding DRS can be amplified using PCR primers which
correspond to the 5' and 3' end encoding sequences respectively. Preferably,
the
5' primer contains an EcoRI site and the 3' primer includes a HindIII site.
Equal
1 S quantities of the Moloney murine sarcoma virus linear backbone and the
amplified EcoRI and HindIII fragment are added together, in the presence of T4
DNA ligase. The resulting mixture is maintained under conditions appropriate
for ligation of the two fragments. The ligation mixture is then used to
transform
E. coli HB 101, which are then plated onto agar containing kanamycin for the
purpose of confirming that the vector contains properly inserted DRS.
The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue
culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)
with 10% calf serum (CS), penicillin and streptomycin. The MSV vector
containing the DRS gene is then added to the media and the packaging cells
transduced with the vector. The packaging cells now produce infectious viral
particles containing the DRS gene (the packaging cells are now referred to as
producer cells).
Fresh media is added to the transduced producer cells, and subsequently,
the media is harvested from a 10 cm plate of confluent producer cells. The
spent
media, containing the infectious viral particles, is filtered through a
Millipore
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filter to remove detached producer cells and this media is then used to infect
fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts
and
quickly replaced with the media from the producer cells. This media is removed
and replaced with fresh media. If the titer of virus is high, then virtually
all
fibroblasts will be infected and no selection is required. If the titer is
very low,
then it is necessary to use a retroviral vector that has a selectable marker,
such
as neo or his. Once the fibroblasts have been efficiently infected, the
fibroblasts
are analyzed to determine whether DRS protein is produced.
The engineered fibroblasts are then transplanted onto the host, either
alone or after having been grown to confluence on cytodex 3 microcarrier
beads.
Example 18
Method of Treatment Using Gene Therapy - In Vivo
Another aspect of the present invention is using in vivo gene therapy
methods to treat disorders, diseases and conditions. The gene therapy method
relates to the introduction of naked nucleic acid (DNA, RNA, and antisense
DNA or RNA) DRS sequences into an animal to increase or decrease the
expression ofthe DRS polypeptide. The DRS polynucleotide may be operatively
linked to a promoter or any other genetic elements necessary for the
expression
of the DRS polypeptide by the target tissue. Such gene therapy and delivery
techniques and methods are known in the art, see, for example, W090/11092,
W098/11779; U. S. Patent NO. 5693622, 5705151, 5580859; Tabata H. et al.,
Cardiovasc. Res. 35:470-479 (1997); Chao J. et al., Pharmacod. Res. 35:517-
522 (1997); WoIffJ.A. Neuromuscul. Di.sord. 7:314-318 (1997); Schwartz B.
et al,. Gene Ther. 3:405-411 (1996); Tsurumi Y. et al., Circulation 94:3281-
3290 (1996) (incorporated herein by reference).
The DRS polynucleotide constructs may be delivered by any method that
delivers injectable materials to the cells of an animal, such as, injection
into the
interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and
the like).
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The DRS polynucleotide constructs can be delivered in a pharmaceutically
acceptable liquid or aqueous carrier.
The term "naked" polynucleotide, DNA or RNA, refers to sequences that
are free from any delivery vehicle that acts to assist, promote, or facilitate
entry
into the cell, including viral sequences, viral particles, liposome
formulations,
lipofectin or precipitating agents and the like. However, the DRS
polynucleotides may also be delivered in liposome formulations (such as those
taught in Felgner P.L. et al. Ann. NY Acad. Sci. 772:126-139 (1995), and
Abdallah B. et al. Biol.. Cell 85:1-7 (1995)) which can be prepared by methods
well known to those skilled in the art.
The DRS polynucleotide vector constructs used in the gene therapy
method are preferably constructs that will not integrate into the host genome
nor
will they contain sequences that allow for replication. Any strong promoter
known to those skilled in the art can be used for driving the expression of
DNA.
Unlike other gene therapy techniques, one major advantage of introducing naked
nucleic acid sequences into target cells is the transitory nature of the
polynucleotide synthesis in the cells. Studies have shown that non-replicating
DNA sequences can be introduced into cells to provide production ofthe desired
polypeptide for periods of up to six months.
The DRS polynucleotide construct can be delivered to the interstitial
space of tissues within an animal, including of muscle, skin, brain, lung,
liver,
spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas,
kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum,
nervous
system, eye, gland, and connective tissue. Interstitial space of the tissues
comprises the intercellular fluid, mucopolysaccharide matrix among the
reticular
fibers of organ tissues, elastic fibers in the walls of vessels or chambers,
collagen
fibers of fibrous tissues, or that same matrix within connective tissue
ensheathing
muscle cells or in the lacunae of bone. It is similarly the space occupied by
the
plasma of the circulation and the lymph fluid of the lymphatic channels.
Delivery
to the interstitial space of muscle tissue is preferred for the reasons
discussed
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below. They may be conveniently delivered by injection into the tissues
comprising these cells. They are preferably delivered to and expressed in
persistent, non-dividing cells which are differentiated, although delivery and
expression may be achieved in non-differentiated or less completely
differentiated
cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo
muscle
cells are particularly competent in their ability to take up and express
polynucleotides.
For the naked DRS polynucleotide injection, an effective dosage amount
of DNA or RNA will be in the range of from about 0.05 g/kg body weight to
about 50 mg/kg body weight. Preferably the dosage will be from about 0.005
mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about
5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this
dosage
will vary according to the tissue site of injection. The appropriate and
effective
dosage of nucleic acid sequence can readily be determined by those of ordinary
skill in the art and may depend on the condition being treated and the route
of
administration. The preferred route of administration is by the parenteral
route
of injection into the interstitial space oftissues. However, otherparenteral
routes
may also be used, such as, inhalation of an aerosol formulation particularly
for
delivery to lungs or bronchial tissues, throat or mucous membranes of the
nose.
In addition, naked DRS polynucleotide constructs can be delivered to arteries
during angioplasty by the catheter used in the procedure.
The dose response effects of injected DRS polynucleotide in muscle in
vivo are determined as follows. Suitable DRS template DNA for production of
mRNA coding for DRS polypeptide is prepared in accordance with a standard
recombinant DNA methodology. The template DNA, which may be either
circular or linear, is either used as naked DNA or complexed with liposomes.
The quadriceps muscles of mice are then injected with various amounts of the
template DNA.
Five to six week old female and male Balb/C mice are anesthetized by
intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is
made
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on the anterior thigh, and the quadriceps muscle is directly visualized. The
DRS
template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27
gauge
needle over one minute, approximately 0.5 cm from the distal insertion site of
the
muscle into the knee and about 0.2 cm deep. A suture is placed over the
injection site for future localization, and the skin is closed with stainless
steel
clips.
After an appropriate incubation time (e.g., 7 days) muscle extracts are
prepared by excising the entire quadriceps. Every fifth 15 pm cross-section of
the individual quadriceps muscles is histochemically stained for DRS protein
expression. A time course for DRS protein expression may be done in a similar
fashion except that quadriceps from different mice are harvested at different
times. Persistence of DRS DNA in muscle following inj ection may be determined
by Southern blot analysis after preparing total cellular DNA and HIRT
supernatants from injected and control mice. The results of the above
experimentation in mice can be use to extrapolate proper dosages and other
treatment parameters in humans and other animals using DR5 naked DNA.
Example 19
A DXS-Fc Fusion Protein Inhibits B Cell Proliferation in Yitro in a
Co-stimulatory Assay
A DRS-Fc polypeptide was prepared that consists of a soluble form of
DRS (corresponding to amino acids -51 to 133 of SEQ ID N0:2) linked to the
Fc portion of a human IgGI immunoglobulin molecule. The ability of this
protein to alter the proliferative response of human B-cells was assessed in a
standard co-stimulatory assay. Briefly, human tonsillar B-cells were purified
by
magnetic bead (MACS) depletion of CD3-positive cells. The resulting cell
population was routinely greater than 95% B-cells as assessed by expression of
CD 19 and CD20 staining. Various dilutions of rHuNeutrokine-alpha
(International Application Publication No. WO 98/18921 ) or the control
protein
rHuIL2 were placed into individual wells of a 96-well plate to which was added
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105 B-cells suspended in culture medium (RPMI 1640 containing 10% FBS, 5 X
10-SM 2ME, 100U/ml penicillin, 10 pg/ml streptomycin, and 10-5 dilution of
formalin-fixed Staphylococcus aureus Cowan I (SAC) also known as Pansorbin
(Pan)) in a total volume of 150 pl. DRS-Fc was then added at various
concentrations. Plates were then placed in the incubator (37°C 5% COz,
95%
humidity) for three days. Proliferation was quantitated by a 20 hour pulse
(1 p,Ci/well) of 3H-thymidine (6.7 Ci/mM) beginning 72 hours post factor
addition. The positive and negative controls are IL-2 and medium,
respectively.
The results of this experiment confirmed that DRS-Fc inhibited B-cell
proliferation in the co-stimulatory assay using Staphylococcus Aureus Cowan 1
(SAC) as priming agent and Neutrokine-alpha as a second signal (data not
shown). It is important to note that other Tumor Necrosis Factor Receptors
(TNFR) fusion proteins (e.g., DR4-Fc (International Application PublicationNo.
WO 98/32856), TR6-Fc (International Application Publication No. WO
98/31799), and TR9-Fc (International Application Publication No. WO
98/56892)) did not inhibit proliferation.
It will be clear that the invention may be practiced otherwise than as
particularly described in the foregoing description and examples. Numerous
modifications and variations of the present invention are possible in light of
the
above teachings and, therefore, are within the scope of the appended claims.
The entire disclosure of each document cited (including patents, patent
applications, journal articles, abstracts, laboratory manuals, books, or other
disclosures) in the Background of the Invention, Detailed Description, and
Examples is hereby incorporated herein by reference.
Further, the Sequence Listing submitted herewith, in paper form, is
hereby incorporated by reference in its entirety.
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232.1
Applicant's or agent's file International application No. T
reference number 1488.131PC07
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule l3bis)
A. The indications made below
relate to the microorganism
referred to in the description
on page 5, line 6
B. IDENTIFICATION OF DEPOSIT
Further deposits are identified
on an additional sheet ~
Name of depository institution
American Type Culture Collection
(ATCC)
Address of depository institution
(including postal code and country)
10801 University Boulevard
Manassas, Virginia 20110-2209
United States of America
Date of deposit March 7, 1997 Accession Number 97920
C. ADDITIONAL INDICATIONS (leave
blank if not applicable) This
information is continued on
an additional sheet 0
DNA Plasmid 1989360
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (ijthe
indication., are not f'or alt
de.stguated States)
E. SEPARATE FURNISHING OF INDICATIONS
heave blank if not applicable)
The indications listed below
will be submitted to the international
Bureau later (specify the general
nature of the indications, e.g.,
"Accession Number of Deposit")
For receiving Office use only For International Bureau use only
l~ This sheet was received with0 This sheet was received by the International
the international application Bureau on:
Authoriz~~~ ~~ Authorized officer
't
~
V
L
t'
i
T
E
R
NATIONAL DIVISION
I
N
70 - 0 - 681
Form PCT/RO/134 (luly 1992)
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SEQUENCE LISTING
<110> Human Genome Sciences, Inc.
Ni, Jian
Gentz, Refiner L.
Yu, Guo-liang
Su, Jeffrey
Rosen, Craig A.
<120> Death Domain Containing Receptor 5
<130> 1488.131PC07
<140> To Be Assigned
<141> To Be Assigned
<150> 60/148,939
<151> 1999-08-13
<150> 60/133,238
<151> 1999-05-07
<150> 60/132,498
<151> 1999-05-04
<160> 14
<170> PatentIn Ver. 2.1
<210> 1
<211> 1600
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (130)..(1362)
<220>
<221> mat_peptide
<222> (283)..(1362)
<220>
<221> sig peptide
<222> (130)..(282)
<400> 1
cacgcgtccg cgggcgcggc cggagaaccc cgcaatcttt gcgcccacaa aatacaccga 60
cgatgcccga tctactttaa gggctgaaac ccacgggcct gagagactat aagagcgttc 120
cctaccgcc atg gaa caa cgg gga cag aac gcc ccg gcc get tcg ggg gcc 171
Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala
-50 -45 -40
cgg aaa agg cac ggc cca gga ccc agg gag gcg cgg gga gcc agg cct 219
Arg Lys Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro
-35 -30 -25
ggg ccc cgg gtc ccc aag acc ctt gtg ctc gtt gtc gcc gcg gtc ctg 267
Gly Pro Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu
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-20 -15 -10
ctgttggtctca getgagtct getctgatc acccaacaa gacctaget 315
LeuLeuValSer AlaGluSer AlaLeuIle ThrGlnGln AspLeuAla
-5 -1 1 5 10
ccccagcagaga gcggcccca caacaaaag aggtccagc ccctcagag 363
ProGlnGlnArg AlaAlaPro GlnGlnLys ArgSerSer ProSerGlu
15 20 25
ggattgtgtcca cctggacac catatctca gaagacggt agagattgc 411
GlyLeuCysPro ProGlyHis HisIleSer GluAspGly ArgAspCys
30 35 40
atctcctgcaaa tatggacag gactatagc actcactgg aatgacctc 459
IleSerCysLys TyrGlyGln AspTyrSer ThrHisTrp AsnAspLeu
45 50 55
cttttctgcttg cgctgcacc aggtgtgat tcaggtgaa gtggagcta 507
LeuPheCysLeu ArgCysThr ArgCysAsp SerGlyGlu ValGluLeu
60 65 70 75
agtccctgcacc acgaccaga aacacagtg tgtcagtgc gaagaaggc 555
SerProCysThr ThrThrArg AsnThrVal CysGlnCys GluGluGly
80 85 90
accttccgggaa gaagattct cctgagatg tgccggaag tgccgcaca 603
ThrPheArgGlu GluAspSer ProGluMet CysArgLys CysArgThr
95 100 105
gggtgtcccaga gggatggtc aaggtcggt gattgtaca ccctggagt 651
GlyCysProArg GlyMetVal LysValGly AspCysThr ProTrpSer
110 115 120
gacatcgaatgt gtccacaaa gaatcaggc atcatcata ggagtcaca 699
AspIleGluCys ValHisLys GluSerGly IleIleIle GlyValThr
125 130 135
gttgcagccgta gtcttgatt gtggetgtg tttgtttgc aagtcttta 747
ValAlaAlaVal ValLeuIle ValAlaVal PheValCys LysSerLeu
140 145 150 155
ctgtggaagaaa gtccttcct tacctgaaa ggcatctgc tcaggtggt 795
LeuTrpLysLys ValLeuPro TyrLeuLys GlyIleCys SerGlyGly
160 165 170
ggtggggaccct gagcgtgtg gacagaagc tcacaacga cctgggget 843
GlyGlyAspPro GluArgVal AspArgSer SerGlnArg ProGlyAla
175 180 185
gaggacaatgtc ctcaatgag atcgtgagt atcttgcag cccacccag 891
GluAspAsnVal LeuAsnGlu IleValSer IleLeuGln ProThrGln
190 195 200
gtccctgagcag gaaatggaa gtccaggag ccagcagag ccaacaggt 939
ValProGluGln GluMetGlu ValGlnGlu ProAlaGlu ProThrGly
205 210 215
gtcaacatgttg tcccccggg gagtcagag catctgctg gaaccggca 987
ValAsnMetLeu SerProGly GluSerGlu HisLeuLeu GluProAla
220 225 230 235
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gaagetgaa aggtctcagagg aggaggctg ctggttcca gcaaatgaa 1035
GluAlaGlu ArgSerGlnArg ArgArgLeu LeuValPro AlaAsnGlu
240 245 250
ggtgatccc actgagactctg agacagtgc ttcgatgac tttgcagac 1083
GlyAspPro ThrGluThrLeu ArgGlnCys PheAspAsp PheAlaAsp
255 260 265
ttggtgccc tttgactcctgg gagccgctc atgaggaag ttgggcctc 1131
LeuValPro PheAspSerTrp GluProLeu MetArgLys LeuGlyLeu
270 275 280
atggacaat gagataaaggtg getaaaget gaggcagcg ggccacagg 1179
MetAspAsn GluIleLysVal AlaLysAla GluAlaAla GlyHisArg
285 290 295
gacaccttg tacacgatgctg ataaagtgg gtcaacaaa accgggcga 1227
AspThrLeu TyrThrMetLeu IleLysTrp ValAsnLys ThrGlyArg
300 305 310 315
gatgcctct gtccacaccctg ctggatgcc ttggagacg ctgggagag 1275
AspAlaSer ValHisThrLeu LeuAspAla LeuGluThr LeuGlyGlu
320 325 330
agacttgcc aagcagaagatt gaggaccac ttgttgagc tctggaaag 1323
ArgLeuAla LysGlnLysIle GluAspHis LeuLeuSer SerGlyLys
335 340 345
ttcatgtat ctagaaggtaat gcagactct gccatgtcc taagtgtgat 1372
PheMetTyr LeuGluGlyAsn AlaAspSer AlaMetSer
350 355 360
tctcttcagg aagtgagacc ttccctggtt tacctttttt ctggaaaaag cccaactgga 1432
ctccagtcag taggaaagtg ccacaattgt cacatgaccg gtactggaag aaactctccc 1492
atccaacatc acccagtgga tggaacatcc tgtaactttt cactgcactt ggcattattt 1552
ttataagctg aatgtgataa taaggacact atggaaaaaa aaaaaaaa 1600
<210> 2
<211> 411
<212> PRT
<213> Homo sapiens
<400> 2
Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys
-50 -45 -40
Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly Pro
-35 -30 -25 -20
Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu
-15 -10 -5
Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro Gln
-1 1 5 10
Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu
15 20 25
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Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser
30 35 40 45
Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe
50 55 60
Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro
65 70 75
Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe
80 85 90
Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys
95 100 105
Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile
110 115 120 125
Glu Cys Val His Lys Glu Ser Gly Ile Ile Ile Gly Val Thr Val Ala
130 135 140
Ala Val Val Leu Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp
145 150 155
Lys Lys Val Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly
160 165 170
Asp Pro Glu Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp
175 180 185
Asn Val Leu Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gln Val Pro
190 195 200 205
Glu Gln Glu Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly Val Asn
210 215 220
Met Leu Ser Pro Gly Glu Ser Glu His Leu Leu Glu Pro Ala Glu Ala
225 230 235
Glu Arg Ser Gln Arg Arg Arg Leu Leu Val Pro Ala Asn Glu Gly Asp
240 245 250
Pro Thr Glu Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala Asp Leu Val
255 260 265
Pro Phe Asp Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp
270 275 280 285
Asn Glu Ile Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr
290 295 300
Leu Tyr Thr Met Leu Ile Lys Trp Val Asn Lys Thr Gly Arg Asp Ala
305 310 315
Ser Val His Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly Glu Arg Leu
320 325 330
Ala Lys Gln Lys Ile Glu Asp His Leu Leu Ser Ser Gly Lys Phe Met
335 340 345
Tyr Leu Glu Gly Asn Ala Asp Ser Ala Met Ser
350 355 360
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<210> 3
<211> 455
<212> PRT
<213> Homo sapiens
<400> 3
Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu
1 5 10 15
Glu Leu Leu Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro
20 25 30
His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys
35 40 45
Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys
50 55 60
Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp
65 70 75 80
Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu
85 90 95
Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val
100 105 110
Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg
115 120 125
Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe
130 135 140
Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu
145 150 155 160
Lys Gln Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu
165 170 175
Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr
180 185 190
Lys Leu Cys Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser
195 200 205
Gly Thr Thr Val Leu Leu Pro Leu Val Ile Phe Phe Gly Leu Cys Leu
210 215 220
Leu Ser Leu Leu Phe Ile Gly Leu Met Tyr Arg Tyr Gln Arg Trp Lys
225 230 235 240
Ser Lys Leu Tyr Ser Ile Val Cys Gly Lys Ser Thr Pro Glu Lys Glu
245 250 255
Gly Glu Leu Glu Gly Thr Thr Thr Lys Pro Leu Ala Pro Asn Pro Ser
260 265 270
Phe Ser Pro Thr Pro Gly Phe Thr Pro Thr Leu Gly Phe Ser Pro Val
275 280 285
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Pro Ser Ser Thr Phe Thr Ser Ser Ser Thr Tyr Thr Pro Gly Asp Cys
290 295 300
Pro Asn Phe Ala Ala Pro Arg Arg Glu Val Ala Pro Pro Tyr Gln Gly
305 310 315 320
Ala Asp Pro Ile Leu Ala Thr Ala Leu Ala Ser Asp Pro Ile Pro Asn
325 330 335
Pro Leu Gln Lys Trp Glu Asp Ser Ala His Lys Pro Gln Ser Leu Asp
340 345 350
Thr Asp Asp Pro Ala Thr Leu Tyr Ala Val Val Glu Asn Val Pro Pro
355 360 365
Leu Arg Trp Lys Glu Phe Val Arg Arg Leu Gly Leu Ser Asp His Glu
370 375 380
Ile Asp Arg Leu Glu Leu Gln Asn Gly Arg Cys Leu Arg Glu Ala Gln
385 390 395 400
Tyr Ser Met Leu Ala Thr Trp Arg Arg Arg Thr Pro Arg Arg Glu Ala
405 410 415
Thr Leu Glu Leu Leu Gly Arg Val Leu Arg Asp Met Asp Leu Leu Gly
420 425 430
Cys Leu Glu Asp Ile Glu Glu Ala Leu Cys Gly Pro Ala Ala Leu Pro
435 440 445
Pro Ala Pro Ser Leu Leu Arg
450 455
<210> 4
<211> 335
<212> PRT
<213> Homo Sapiens
<400> 4
Met Leu Gly Ile Trp Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala
1 5 10 15
Arg Leu Ser Ser Lys Ser Val Asn Ala Gln Val Thr Asp Ile Asn Ser
20 25 30
Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn
35 40 45
Leu Glu Gly Leu His His Asp Gly Gln Phe Cys His Lys Pro Cys Pro
50 55 60
Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro
65 70 75 80
Asp Cys Val Pro Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His
85 90 95
Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly
100 105 110
Leu Glu Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg
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_7_
115 120 125
Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp
130 135 140
Pro Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr
145 150 155 160
Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn Leu Gly Trp
165 170 175
Leu Cys Leu Leu Leu Leu Pro Ile Pro Leu Ile Val Trp Val Lys Arg
180 185 190
Lys Glu Val Gln Lys Thr Cys Arg Lys His Arg Lys Glu Asn Gln Gly
195 200 205
Ser His Glu Ser Pro Thr Leu Asn Pro Glu Thr Val Ala Ile Asn Leu
210 215 220
Ser Asp Val Asp Leu Ser Lys Tyr Ile Thr Thr Ile Ala Gly Val Met
225 230 235 240
Thr Leu Ser Gln Val Lys Gly Phe Val Arg Lys Asn Gly Val Asn Glu
245 250 255
Ala Lys Ile Asp Glu Ile Lys Asn Asp Asn Val Gln Asp Thr Ala Glu
260 265 270
Gln Lys Val Gln Leu Leu Arg Asn Trp His Gln Leu His Gly Lys Lys
275 280 285
Glu Ala Tyr Asp Thr Leu Ile Lys Asp Leu Lys Lys Ala Asn Leu Cys
290 295 300
Thr Leu Ala Glu Lys Ile Gln Thr Ile Ile Leu Lys Asp Ile Thr Ser
305 310 315 320
Asp Ser Glu Asn Ser Asn Phe Arg Asn Glu Ile Gln Ser Leu Val
325 330 335
<210> 5
<211> 417
<212> PRT
<213> Homo sapiens
<400> 5
Met Glu Gln Arg Pro Arg Gly Cys Ala Ala Val Ala Ala Ala Leu Leu
1 5 10 15
Leu Val Leu Leu Gly Ala Arg Ala Gln Gly Gly Thr Arg Ser Pro Arg
20 25 30
Cys Asp Cys Ala Gly Asp Phe His Lys Lys Ile Gly Leu Phe Cys Cys
35 40 45
Arg Gly Cys Pro Ala Gly His Tyr Leu Lys Ala Pro Cys Thr Glu Pro
50 55 60
Cys Gly Asn Ser Thr Cys Leu Val Cys Pro Gln Asp Thr Phe Leu Ala
65 70 75 80
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Trp Glu Asn His His Asn Ser Glu Cys Ala Arg Cys Gln Ala Cys Asp
85 90 95
Glu Gln Ala Ser Gln Val Ala Leu Glu Asn Cys Ser Ala Val Ala Asp
100 105 110
Thr Arg Cys Gly Cys Lys Pro Gly Trp Phe Val Glu Cys Gln Val Ser
115 120 125
Gln Cys Val Ser Ser Ser Pro Phe Tyr Cys Gln Pro Cys Leu Asp Cys
130 135 140
Gly Ala Leu His Arg His Thr Arg Leu Leu Cys Ser Arg Arg Asp Thr
145 150 155 160
Asp Cys Gly Thr Cys Leu Pro Gly Phe Tyr Glu His Gly Asp Gly Cys
165 170 175
Val Ser Cys Pro Thr Ser Thr Leu Gly Ser Cys Pro Glu Arg Cys Ala
180 185 190
Ala Val Cys Gly Trp Arg Gln Met Phe Trp Val Gln Val Leu Leu Ala
195 200 205
Gly Leu Val Val Pro Leu Leu Leu Gly Ala Thr Leu Thr Tyr Thr Tyr
210 215 220
Arg His Cys Trp Pro His Lys Pro Leu Val Thr Ala Asp Glu Ala Gly
225 230 235 240
Met Glu Ala Leu Thr Pro Pro Pro Ala Thr His Leu Ser Pro Leu Asp
245 250 255
Ser Ala His Thr Leu Leu Ala Pro Pro Asp Ser Ser Glu Lys Ile Cys
260 265 270
Thr Val Gln Leu Val Gly Asn Ser Trp Thr Pro Gly Tyr Pro Glu Thr
275 280 285
Gln Glu Ala Leu Cys Pro Gln Val Thr Trp Ser Trp Asp Gln Leu Pro
290 295 300
Ser Arg Ala Leu Gly Pro Ala Ala Ala Pro Thr Leu Ser Pro Glu Ser
305 310 315 320
Pro Ala Gly Ser Pro Ala Met Met Leu Gln Pro Gly Pro Gln Leu Tyr
325 330 335
Asp Val Met Asp Ala Val Pro Ala Arg Arg Trp Lys Glu Phe Val Arg
340 345 350
Thr Leu Gly Leu Arg Glu Ala Glu Ile Glu Ala Val Glu Val Glu Ile
355 360 365
Gly Arg Phe Arg Asp Gln Gln Tyr Glu Met Leu Lys Arg Trp Arg Gln
370 375 380
Gln Gln Pro Ala Gly Leu Gly Ala Val Tyr Ala Ala Leu Glu Arg Met
385 390 395 400
Gly Leu Asp Gly Cys Val Glu Asp Leu Arg Ser Arg Leu Gln Arg Gly
405 410 415
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Pro
<210> 6
<211> 507
<212> DNA
<213> Homo sapiens
<400> 6
aattcggcac agctcttcag gaagtcagac cttccctggt ttaccttttt tctggaaaaa 60
gcccaactgg gactccagtc agtaggaaag tgccacaatt gtcacatgac cggtactgga 120
agaaactctc ccatccaaca tcacccagtg gnatgggaac actgatgaac ttttcactgc 180
acttggcatt atttttgtna agctgaatgt gataataagg gcactgatgg aaatgtctgg 240
atcattccgg ttgtgcgtac tttgagattt gngtttgggg atgtncattg tgtttgacag 300
cacttttttn atccctaatg tnaaatgcnt natttgattg tganttgggg gtnaacattg 360
gtnaaggntn cccntntgac acagtagntg gtncccgact tanaatngnn gaanangatg 420
natnangaac ctttttttgg gtgggggggt nncggggcag tnnaangnng nctccccagg 480
tttggngtng caatngngga annntgg 507
<210> 7
<211> 226
<212> DNA
<213> Homo sapiens
<400> 7
ttttttttgt agatggatct tacaatgtag cccaaataaa taaataaagc atttacatta 60
ggataaaaaa gtgctgtgaa aacaatgaca tcccaaacca aatctcaaag tacgcacaaa 120
cggaatgatc cagacatttc cataggtcct tattatcaca ttcagcttat aaaataatgc 180
caagtgcagt gaaaagttac aggatgttcc atccactggg tggatt 226
<210> 8
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 8
cgcccatgga gtctgctctg atcac 25
<210> 9
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 9
cgcaagcttt tagcctgatt ctttgtggac 30
<210> 10
<211> 36
<212> DNA
<213> Artificial Sequence
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WO 00/66156 PCT/US00/12041
-10-
<220>
<223> Description of Artificial Sequence: Primer
<400> 10
cgcggatccg ccatcatgga acaacgggga cagaac 36
<210> 11
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 11
cgcggtacct taggacatgg cagagtc 27
<210> 12
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 12
cgcggtacct tagcctgatt ctttgtggac 30
<210> 13
<211> 733
<212> DNA
<213> Homo Sapiens
<400> 13
gggatccgga gcccaaatct tctgacaaaa ctcacacatg cccaccgtgc ccagcacctg 60
aattcgaggg tgcaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga 120
tctcccggac tcctgaggtc acatgcgtgg tggtggacgt aagccacgaa gaccctgagg 180
tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg 240
aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact 300
ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca acccccatcg 360
agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc 420
catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct 480
atccaagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga 540
ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg 600
acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc 660
acaaccacta cacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccgc 720
gactctagag gat 733
<210> 14
<211> 257
<212> DNA
<213> Homo Sapiens
<400> 14
agggctgaaa cccacgggcc tgagagacta taagagngtt ccctaccgcc atggaacaac 60
ggggacagaa cgccccggnc ncttcggggg cccggaaaag gcacggccca ggacccaggg 120
aggngcgggg agccaggcct gggccccggg tccccaagac ccttgtgctc gttgtcgccg 180
cggtcctgct gttggtgagt ccccgccgcg gtccctggct ggggaagagc gtncctggcg 240
CA 02369371 2001-11-02
WO 00/66156 PCT/US00/12041
-11-
cctggagagg gcaggga 257
