Note: Descriptions are shown in the official language in which they were submitted.
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LDL RELATED PROTEIN AND USES THEREOF
BACKGROUND OF THE INVENTION
Members of the low density lipoprotein (LDL)
receptor gene family include: the LDL receptor, the very
low density lipoprotein (VLDL) receptor, LDL receptor-
related protein 2 (LRP-2/megalin/gp330), LDL receptor-
related protein 3 (LRP-3/LRp-105), LDL receptor-related
protein 5 (LRP-5), LDL receptor-related protein 6 (LRP-
6), and LRBB
Common to many members of the LDL receptor family
is the endocytic uptake of ligands. The ligands bound by
the members of the LDL receptor family are diverse. For
example, LDL receptor is thought to bind lipoproteins
containing apoB or apoE; VLDL receptor is thought to bind
lipoproteins containing apoE; and LRP-2 is thought to
bind apoE-containing lipoproteins, plasminogen,
lactoferrin, and lipoprotein lipase. Significantly, LRP-
2 is also thought to mediate the uptake of complexes of
clusterin/ApoJ and the amyloid beta protein.
Summary of the Invention
The present invention is based; at least in part,
on the discovery of cDNA molecules encoding TANGO 136, a
type I membrane protein. Described below are cDNA
molecules encoding both human and murine TANGO 136.
These proteins, fragments, derivatives, and variants
thereof are collectively referred to as "polypeptides of
the invention" or "proteins of the invention.".Nucleic
acid molecules encoding polypeptides of the invention are
collectively referred to as "nucleic acids of the
invention."
The nucleic acids and polypeptides of the present
invention are useful as modulating agents in regulating a
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variety of cellular processes. Accordingly, in one
aspect, this invention provides isolated nucleic acid
molecules encoding a polypeptide of the invention or a
biologically active portion thereof. The present
invention also provides nucleic acid molecules which are
suitable as primers or hybridization probes for the
detection of nucleic acids encoding a polypeptide of the
invention.
The invention features nucleic acid molecules
which are at least 45% (or 55%, 65%, 75%, 85%, 95%, or
98%) identical to the nucleotide sequence of SEQ ID NO:1,
3, 4 or 6, or the nucleotide sequence of the cDNA insert
of a clone deposited with ATCC as Accession Number 98880
(the "cDNA of ATCC 98880"), or a complement thereof.
The invention features nucleic acid molecules
which include a fragment of at least 300 (325, 350, 375,
400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000,
or 1200) nucleotides of the nucleotide sequence of SEQ ID
NO:1, 3, 4, or 6, or the nucleotide sequence of the cDNA
of ATCC Accession Number 98880, or a complement thereof.
The invention also features nucleic acid molecules
which include a nucleotide sequence encoding a protein
having an amino acid sequence that is at least 45% (or
55%, 65%, 75%, 85%, 95%, or 98%) identical to the amino
acid sequence of SEQ ID N0:2 or 5, or the amino acid
sequence encoded by the cDNA of ATCC Accession Number
98880, or a complement thereof.
In various embodiments, the nucleic acid molecules
have the nucleotide sequence of SEQ ID NO:1, 3, 4, or 6,
or the nucleotide sequence of the cDNA of ATCC Accession
Number 98880.
Also within the invention are nucleic acid
molecules which encode a fragment of a polypeptide having
the amino acid sequence of SEQ ID N0:2 or 5, the fragment
including at least 15 (25, 30, 50, 100, 150, 300, or 400)
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contiguous amino acids of SEQ ID N0:2 or 5 or the amino
acid sequence encoded by the cDNA of ATCC Accession
Number 98880.
The invention includes nucleic acid molecules
which encode a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID
N0:2 or 5 or the amino acid sequence encoded by the cDNA
of ATCC Accession Number 98880, wherein the nucleic acid
molecule hybridizes to a nucleic acid molecule comprising
l0 the nucleic acid sequence encoding SEQ ID N0:2 or 5, or a
complement thereof under stringent conditions.
Also within the invention are isolated
polypeptides or proteins having an amino acid sequence
that is at least about 65%, preferably 75%, 85%, 95%, or
98~ identical to the amino acid sequence of SEQ ID N0:2
or 5.
Also within the invention are isolated
polypeptides or proteins which are encoded by a nucleic
acid molecule having a nucleotide sequence that is at
least about 65%, preferably 75%, 85%, or 95% identical
the nucleic acid sequence encoding SEQ ID N0:2 or 5 and
isolated polypeptides or proteins which are encoded by a
nucleic acid molecule having a nucleotide sequence which
hybridizes under stringent hybridization conditions to a
nucleic acid molecule having the nucleotide sequence of
SEQ ID NO:1, 3, 4, or 6, or a complement thereof or the
non-coding strand of the cDNA of ATCC Accession Number
98880.
Also within the invention are polypeptides which
are naturally occurring allelic variants of a polypeptide
that includes the amino acid sequence of SEQ ID NO:2 or 5
or the amino acid sequence encoded by the cDNA of ATCC
Accession Number 98880, wherein the polypeptide is
encoded by a nucleic acid molecule which hybridizes to a
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nucleic acid molecule comprising SEQ ID NO:1, 3, 4, or 6,
or a complement thereof under stringent conditions.
The invention also features nucleic acid molecules
that hybridize under stringent conditions to a nucleic
acid molecule comprising the nucleotide sequence of SEQ
ID NO: 1, 3, 4, or 6, or the cDNA of ATCC Accession
Number 98880, or a complement thereof. In other
embodiments, the nucleic acid molecules are at least 300
(325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700,
800, 900, 1000, or 1290) nucleotides in length and
hybridize under stringent conditions to a nucleic acid
molecule comprising the nucleotide sequence of SEQ ID
NO: l, 3, 4, or 6, or the cDNA of ATCC Accession Number
98880, or a complement thereof.
In other embodiments, the isolated nucleic acid
molecules encode one or more domains, e.g., a cytoplasmic
domain (SEQ ID NO:11 or 18), a transmembrane domain (SEQ
ID N0:10 or 17), or an extracellular domain (SEQ ID N0:9
or 16), of a polypeptide of the invention or a complement
thereof. In another embodiment, the invention provides
an isolated nucleic acid molecule which is antisense to
the coding strand of a nucleic acid of the invention.
Another aspect of the invention provides vectors,
e.g., recombinant expression vectors, comprising a
nucleic acid molecule of the invention. In another
embodiment, the invention provides host cells containing
such a vector. The invention also provides methods for
producing a polypeptide of the invention by culturing, in
a suitable medium, a host cell of the invention
containing a recombinant expression vector such that a
polypeptide is produced.
Another aspect of the invention features isolated
or recombinant proteins and polypeptides of the
invention. Preferred proteins and polypeptides possess
at least one biological activity possessed by the
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corresponding naturally-occurring human polypeptide. An
activity, a biological activity, and a functional
activity of a polypeptide of the invention refers to an
activity exerted by a protein, polypeptide or nucleic
acid molecule of the invention on a responsive cell as
determined in vivo, or in vitro, according to standard
techniques. Such activities can be a direct activity,
such as an association with or an enzymatic activity on a
second protein or an indirect activity, such as a
cellular signaling activity mediated by interaction of
the protein with a second protein. Thus, such activities
include, e.g., (1) the ability to form protein-protein
interactions with proteins in the signaling pathway of
the naturally-occurring polypeptide; (2) the ability to
bind a ligand of the naturally-occurring polypeptide; (3)
the ability to bind to an intracellular target of the
naturally-occurring polypeptide. Other activities
include: (1) the ability to modulate cellular
proliferation; (2) the ability to modulate cellular
differentiation; and (3) the ability to modulate cell
death.
In one embodiment, a polypeptide of the invention
has an amino acid sequence sufficiently identical to an
identified domain of a polypeptide of the invention. As
used herein, the term "sufficiently identical" refers to
a first amino acid or nucleotide sequence which contains
a sufficient or minimum number of identical or equivalent
(e.g., with a similar side chain) amino acid residues or
nucleotides to a second amino acid or nucleotide sequence
such that the first and second amino acid or nucleotide
sequences have a common structural domain and/or common
functional activity. For example, amino acid or
nucleotide sequences which contain a common structural
domain having about 65% identity, preferably 75%
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identity, more preferably 85~, 95~, or 98~ identity are
defined herein as sufficiently identical.
In one embodiment, the isolated polypeptides lack
both a transmembrane and a cytoplasmic domain. In
another embodiment the polypeptide lacks both a
transmembrane domain and a cytoplasmic domain and is
soluble under physiological conditions.
The polypeptides of the present invention, or
biologically active portions thereof, can be operably
linked to a heterologous amino acid sequence to form
fusion proteins. The invention further features
antibodies that specifically bind a polypeptide of the
invention such as monoclonal or polyclonal antibodies.
In addition, the polypeptides of the invention or
biologically active portions thereof can be incorporated
into pharmaceutical compositions, which optionally
include pharmaceutically acceptable carriers.
In another aspect, the present invention provides
methods for detecting the presence of the activity or
expression of a polypeptide of the invention in a
biological sample by contacting the biological sample
with an agent capable of detecting an indicator of
activity such that the presence of activity is detected
in the biological sample.
In another aspect, the invention provides methods
for modulating activity of a polypeptide of the invention
comprising contacting a cell with an agent that modulates
(inhibits or stimulates) the activity or expression of a
polypeptide of the invention such that activity or
expression in the cell is modulated. In one embodiment,
the agent is an antibody that specifically binds to a
polypeptide of the invention.
In another embodiment, the agent modulates
expression of a polypeptide of the invention by
modulating transcription, splicing, or translation of an
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mRNA encoding a polypeptide of the invention. In yet
another embodiment, the agent is a nucleic acid molecule
having a nucleotide sequence that is antisense to the
coding strand of an mRNA encoding a polypeptide of the
invention.
The present invention also provides methods to
treat a subject having a disorder characterized by
aberrant activity of a polypeptide of the invention or
aberrant expression of a nucleic acid of the invention
by administering an agent which is a modulator of the
activity of a polypeptide of the invention or a modulator
of the expression of a nucleic acid of the invention to
the subject. In one embodiment, the modulator is a
protein of the invention. In another embodiment, the
modulator is a nucleic acid of the invention. In other
embodiments, the modulator is a peptide, peptidomimetic,
or other small molecule.
The present invention also provides diagnostic
assays for identifying the presence or absence of a
genetic lesion or mutation characterized by at least one
of: (i) aberrant modification or mutation of a gene
encoding a polypeptide of the invention, (ii) mis-
regulation of a gene encoding a polypeptide of the
invention, and (iii) aberrant post-translational
modification of the invention wherein a wild-type form of
the gene encodes a protein having the activity of the
polypeptide of the invention.
In another aspect, the invention provides a method
for identifying a compound that binds to or modulates the
activity of a polypeptide of the invention. In general,
such methods entail measuring a biological activity of
the polypeptide in the presence and absence of a teat
compound and identifying those compounds which alter the
activity of the polypeptide.
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The invention also features methods for
identifying a compound which modulates the expression of
a polypeptide or nucleic acid of the invention by
measuring the expression of the polypeptide or nucleic
acid in the presence and absence of the compound.
Other features and advantages of the invention
will be apparent from the following detailed description
and claims.
Brief Description of the Drawincts
~ Figure 1 depicts a partial cDNA sequence (SEQ ID
NO:1) and predicted partial amino acid sequence (SEQ ID
N0:2) of murine TANGO 136. The open reading frame of SEQ
ID NO:1 extends from nucleotide 89 to nucleotide 1813
(SEQ ID N0:3).
Figure 2 depicts a hydropathy plot of a portion of
murine TANGO 136. Relatively hydrophobic residues are
above the horizontal line, and relatively hydrophilic
residues are below the horizontal line. The cysteine
residues (cys) and potential N-glycosylation sites (Ngly)
are indicated by short vertical lines just below the
hydropathy trace.
Figure 3 depicts the cDNA sequence (SEQ ID N0:4)
and predicted amino acid sequence (SEQ ID N0:5) of human
TANGO 136. The open reading frame of SEQ ID N0:4 extends
from nucleotide 541 to 2679 (SEQ ID N0:6).
Figure 4 depicts a hydropathy plot of human TANGO
136. Relatively hydrophobic residues are above the
horizontal line, and relatively hydrophilic residues are
below the horizontal line. The cysteine residues (cys)
and potential N-glycosylation sites (Ngly) are indicated
by short vertical lines just below the hydropathy trace.
Figure 5 depicts an alignment of the amino acid
sequences of murine TANGO 136 (partial sequence; SEQ ID
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N0:2), human TANGO 136 (SEQ ID N0:5), human LRp105 (SEQ
ID NO:11) and rat LRp105 (SEQ ID N0:13).
Figure 6 depicts an alignment of the nucleic acid
sequences of murine TANGO 136 (partial sequence; SEQ ID
NO: l) and human TANGO 136 (SEQ ID N0:4).
Figure 7 depicts an alignment of the amino acid
sequences of murine TANGO 136 (partial sequence; SEQ ID
N0:2) and human TANGO 136 (SEQ ID N0:5).
Figure 8 depicts alignments of the CUB-like
domains of murine TANGO 136 with a consensus CUB domain.
In these alignments an uppercase letter between the two
sequences indicates an exact match, and a "+" indicates a
similarity.
Figure 9 depicts alignments of the CUB-like
domains of human TANGO 136 with a consensus CUB domain.
In these alignments an uppercase letter between the two
sequences indicates an exact match, and a "+" indicates a
similarity.
Figure 10 depicts alignments of the LDL class A
domains of human TANGO 136 with a consensus LDL class A
domain. In these alignments an uppercase letter between
the two sequences indicates an exact match, and a "x"
indicates a similarity.
Detailed Description of the Invention
The present invention is based on the discovery of
cDNA molecules encoding murine and human TANGO 136, a
transmembrane protein. Various features of murine and
human TANGO 136 are summarized below.
Murine TANGO 136
A cDNA encoding a portion of murine TANGO 136 was
identified using a screening process'which selects for
nucleotide sequences which encode secreted proteins. A
detailed description of this method, called "signal
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trapping" is provided in PCT Publication No. WO 98/22491,
published May 28, 1998. In brief, a randomly primed cDNA
library was prepared using cDNA prepared from mRNA
extracted from lipopolysaccharide-stimulated mouse
macrophages. To prepare this library, the cDNA was
inserted into the mammalian expression vector pMEAP
adjacent to a cDNA encoding placental alkaline
phosphatase which lacks a secretory signal. Next, the
cDNA library was amplified in bacteria. The amplified
cDNA was then isolated and transfected into human 293T
cells. After 28 hours, cell supernatants were collected
and assayed for alkaline phosphatase activity. Clones
giving rise to detectable alkaline phosphatase activity
in the supernatant of transfected cells were isolated and
analyzed further by sequencing and the novel clones
subjected to further sequencing
Once such clone, murine TANGO 136, was identified.
This clone includes a 1813 nucleotide cDNA (Figure 1; SEQ
ID NO:1). The open reading frame of this cDNA,
nucleotides 89 to 1813 (SEQ ID N0:3), encodes a 575 amino
acid putative type I membrane protein (Figure 1; SEQ ID
N0:2). Because no translation stop codon occurs at the
end of the open reading frame, this cDNA is likely to be
a partial cDNA which does not encode the most carboxy
terminal portion of murine TANGO 136.
The signal peptide prediction program SIGNALP
(Nielsen et al. (1997) Protein Engineering 10:1-6)
predicted that murine TANGO 136 includes a 17 amino acid
signal peptide (amino acid 1 to about amino acid 17 of
SEQ ID N0:2; SEQ ID N0:7) preceding the 558 amino acid
(partial) mature protein (about amino acid 18 to amino
acid 575; SEQ ID N0:8). Mature murine TANGO 136 has an
extracellular domain (amino acids 18 to 441 of SEQ ID
N0:2; SEQ ID N0:9); a transmembrane domain (amino acids
442 to 462 of SEQ ID N0:2; SEQ ID NO:10); and a
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cytoplasmic domain (amino acids 463 to 575 of SEQ ID
N0:2; SEQ ID N0:11).
The extracellular region of murine TANGO 136
includes two CUB-like domains (amino acids 32 to 86 and
amino acids 193 to 306 of SEQ ID N0:2). CUB domains are
extracellular domains found in a number of functionally
diverse, developmentally regulated proteins including the
dorsal-ventral patterning protein tolloid, bone
morphogenetic protein 1, a family of spermadhesins,
complement subcomponents Cls/Clr and the neuronal
recognition molecule A5. The majority of CUB domains
contain four conserved cysteines which are thought to
form two disulfide bridges (C1-C2 and C3-C4) (Bork et al.
(1993) J. Mol. Biol. 231:539-545). The first CUB-like
domain of murine TANGO 136 (amino acids 32 to 86)
includes two cysteines, and the second CUB-like domain of
murine TANGO 136 (amino aicds 193 to 306) includes two
cysteines. Alignments of the CUB-like domains of murine
TANGO 136 with a CUB domain consensus sequence are
depicted in Figure 8.
Figure 2 depicts a hydropathy plot of a portion of
murine TANGO 136. Relatively hydrophobic residues are
above the horizontal line, and relatively hydrophilic
residues are below the horizontal line. The cysteine
residues (cys) and potential N-glycosylation sites (Ngly)
are indicated by short vertical lines just below the
hydropathy trace.
Human TANGO 136
The murine TANGO 136 cDNA described above was used
to screen a human placental cDNA library to identify
human clones encoding TANGO 136. One clone identified by
this screening was sequenced fully. This human TANGO 136
cDNA (Figure 4; SEQ ID N0:4) includes an open reading
frame (nucleotides 541 to 2679 of SEQ ID N0:4; SEQ ID
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N0:6) encoding a 713 amino acid putative type I
transmembrane protein (Figure 4; SEQ ID N0:5).
The signal peptide prediction program SIGNALP
(Nielsen et al. (1997) Protein Engineering 10:1-6)
predicted that human TANGO 136 includes a 16 amino acid
signal peptide (amino acid 1 to about amino acid 16 of
SEQ ID N0:5; SEQ ID N0:14) preceding the 697 amino acid
mature protein (about amino acid 17 to amino acid 713 of
SEQ ID N0:5; SEQ ID N0:15). Human TANGO 136 has an
extracellular domain (amino acids 17 to 440 of SEQ ID
N0:5; SEQ ID N0:16); a transmembrane domain (amino acids
441 to 461 of SEQ ID N0:5; SEQ ID N0:17); and a
cytoplasmic domain (amino acids 462 to 713 of SEQ ID
N0:5; SEQ ID N0:18).
A clone, pT136, which encodes human TANGO 136 was
deposited with the American Type Culture Collection
(10801 University Boulevard, Manassas, VA 20110-2209) on
September 11, 1998 and assigned Accession Number 98880.
This deposit will be maintained under the terms of the
Budapest Treaty on the International Recognition of the
Deposit of Microorganisms for the Purposes of Patent
Procedure. This deposit was made merely as a convenience
for those of skill in the art and is not an admission
that a deposit is required under 35 U.S.C. X112.
The extracellular region of human TANGO 136
includes two CUB-like domains (amino acids 31 to 136 and
amino acids 192 to 305 of SEQ ID N0:5). Both of the CUB-
like domains of human TANGO 136 include two cysteines.
Alignments of the CUB-like domains of human TANGO 136
with a CUB domain consensus sequence are depicted in
Figure 9.
The extracellular region of human TANGO 136 also
includes four LDL receptor class A domains (amino acids
138 to 176, amino acids 328 to 355; amino acids 380 to
398; and amino acids 399 to 435 of SEQ ID N0:5). The
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LDL receptor class A domain is an approximately 40 amino
acid cysteine-rich domain having a found in LDL receptor
and other members of the LDL receptor family. Repeats of
this domain are thought to involved in ligand binding
(Yamamoto et al. (1984) CeII 39:27-38; and Fass et al.
(1997) Nature 388:691-693). The LDL receptor class A
domain extending from amino acid 380 to 398 of human
TANGO 136 has relatively weak homology to the consensus
LDL receptor type A domain compared to the other three
LDL receptor class A domains. Alignments of the LDL
receptor class A domains of human TANGO 136 with a LDL
receptor class A domain consensus sequence are depicted
in Figure 10.
Figure 4 depicts a hydropathy plot of human TANGO
136. Relatively hydrophobic residues are above the
horizontal line, and relatively hydrophilic residues are
below the horizontal line. The cysteine residues (cys)
and potential N-glycosylation sites (Ngly) are indicated
by short vertical lines just below the hydropathy trace.
Mature human TANGO 136 has a predicted MW of
76.7 kDa (78.4 kDa for immature human TANGO 136), not
including post-translational modifications.
Human TANGO 136 maps to chromosome 14 near
D14S283.
The amino acid sequence of human TANGO 136 (SEQ ID
N0:5) was used to search public databases (using BLASTP;
Altschul et al. (1990) J. Mol. Hiol. 215:403-410) in
order to identify proteins having homology to human TANGO
136. This analysis revealed that both murine and human
TANGO 136 has considerable homology to human LDL receptor
related protein LRp105/LRP-3 (Ishii et al. (1998)
Genomics 51:132-135). Figure 5 depicts an alignment of
the amino acids sequences of murine.TANGO 136, human
TANGO 136, human LRp105/LRP-3, and rat Lrp105/LRP-3.
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When compared using the algorithm of Myers and
Miller ((1988) CABIOS 4:11-17; PAM120 scoring matrix, -12
gap opening penalty, -4 gap extension penalty) murine
TANGO 136 is 34.4% identical to human LRp105/LRP-3 and
34% identical to rat LRp105/LRP-3; human TANGO 136 is 38%
identical to human LRp105/LRP-3 and 37.6% identical to
rat Lrp105/LRP-3; and human TANGO 136 is 72.6 identical
to murine TANGO 136.
The full length human TANGO 136 nucleotide
sequence is 86.1% identical (FASTA version 2.Ou53;
Pearson and Lipman (1988) Proc. Natl Acad. Sci. USA
85:2444-2448) to the partial murine TANGO 136 nucleotide
sequence (Figure 6). The full length human TANGO 136
amino acid sequence is 90.8% identical (FASTA version
2.Ou53; Pearson and Lipman (1988) Proc. Natl Acad. Sci.
85:2444-2448) to the partial murine TANGO 136 amino acid
sequence ( Figure 7). As shown in Figure 7, the protein
domain structure (described above) is highly conserved
between the human and murine proteins.
Human multiple tissue northern (MTN) blots
(Clontech, Palo Alto, CA), containing 2 ~.g of poly A+
RNA per lane were probed with a murine TANGO 136 cDNA
probe. This analysis revealed that TANGO 136 mRNA is
relatively highly expressed in spleen, prostate, uterus,
peripheral blood leukocytes, heart, placenta, kidney and
pancreas. This analysis also revealed that TANGO 136
mRNA is expressed at a somewhat lower level in thymus,
testis, colon, lung, liver and skeletal muscle. TANGO
136 nucleic acids, polypeptides, agonists, and
antagonists can be used to modulate the activities of the
tissues in which it is expressed and thus treat disorders
of these tissues. For example, TANGO 136 is expressed in
prostate and testis and may be involved in
spermatogenesis.
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Use of TANGO 136 Nucleic Acids Polvneptides and TANGO
136 Aaonists or Antaaonists
Due to the homology between TANGO 136 and
LRp105/LRP-3, TANGO 136 is predicted to be a member of
the low density lipoprotein receptor family, which
includes LDLR, LRP-2 (megalin/gp330), LRP-3 (LRp105),
LRP-5, LRP-6, and LRBB. Members of this family are
endocytic receptors that bind and internalize ligands
from the circulation and extracellular space. Since
TANGO 136 is predicted to be a member of the low density
lipoprotein receptor family, it may function similarly to
other members of the low density lipoprotein receptor
family.
LDLR binds plasma lipoproteins that contain
apolipoprotein B-100 (apoB-100) or apoE on their surface.
LDLR is critical for the uptake of these lipoproteins,
and mutations in LDLR are the cause of familial
hypercholesterolemia, a disorder characterized by high
levels of cholesterol-rich LDL in the plasma. The
elevation of plasma cholesterol levels in patients
afflicted with familial hypercholesterolemia leads to
atherosclerosis and increased risk for myocardial
infarction. TANGO 136 potentially plays a role in
disorders of lipoprotein metabolism and transport, e.g.,
cardiovascular diseases such as atherosclerosis.
Accordingly, TANGO 136 nucleic acids, polypeptides and
TANGO 136 antagonists and agonists are useful for
treatment of disorders of lipoprotein metabolism and
transport, e.g., cardiovascular diseases such as
atherosclerosis.
In vitro studies have shown that LRP-2 is capable
of binding and mediating the cellular uptake of a large
number of different ligands including apoE-enriched very
low density lipoproteins (Willnow et al. (1992) aT. Biol.
Chem. 267:26172-26180), complexes of urokinase
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plasminogen activator and plasminogen activator
inhibitor-1 (tPA:PAI-1) (Willnow et al., supra),
lipoprotein lipase (Willnow et al., supra), and
lactoferrin. A receptor associated protein known as RAP
(Orlando et al. (1992) Proc. Natl Acad. Sci. 89:6698-
6702) inhibits the binding of these ligands to LRP-2.
Some or all of these ligands may bind TANGO 136.
Accordingly, TANGO 136 nucleic acids, polypeptides,
antagonists and agonists are useful for treatment of
clotting disorders, e.g., inhibiting clot formation or
dissolving clots.
A few specific and physiologically relevant
ligands for LRP-2 have been identified, including
apolipoprotein J(apoJ)/clusterin (Kounnas et al. (1995)
J. Biol. Chem. 22:13070-13075) and thyroglobulin (Zheng
et al. (1998) Endocrinology 139:1462-1465). ApoJ has
been reported to bind several proteins, including the aA4
peptide of the Alzheimer's precursor protein, a subclass
of high density lipoprotein, and the complement membrane
attack complex C5-C9 (Kounnas et al., supra). The
clearance of apoJ complexed with these and other
molecules is expected to occur via LRP-2. Thus, LRP-2
may play an important functional role in the clearance of
these complexes. For example, LRP-2 may function to
target lipoproteins for clearance or may inhibit the
cytolytic activity of the complement membrane C5b-C9 by
clearing the apoJ/C5b-C9 complex. The fact that LRP-2
can bind the apoJ/amyloid-~i complex suggests that LRP-2
may be involved in regulating the pathogenesis of
Alzheimer's disease. A role for LRP-2 in Alzheimer's
disease is further supported by another study that showed
that LRP-2 may be involved in transporting the
apoJ/amyloid a complex across the blood-brain-barrier
(Zlokovic et al. (1996) Proc. Natl Acad. Sci. 93:4229-
4234). Thus, TANGO 136 nucleic acids, proteins,
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agonists, and antagonists are useful for the treatment
of Alzheimer's disease and other neurodegenerative
disorders, e.g., Huntington's disease and Parkinson's
disease.
LRP-2 is involved in participating in the
endocytosis of thyroglobulin, which results in the
release of thyroid hormones (Zheng et al. (1998)
Endocrinolgy 139:1462-65). TANGO 136 may also be
involved in the regulating the release of thyroid
hormones. Thus, TANGO 136 nucleic acids, proteins,
agonists, and antagonists are useful for the treatment
of thyroid disorders, e.g., thyroid hormone release
disorders..
LRP-2 is also predicted to play a role as a drug
receptor and is thought to be involved in the uptake of
polybasic drugs, e.g., aprotinin, aminoglycosides and
polymyxin B. The uptake of polybasic drugs can be toxic,
e.g., the administration of aminoglycosides is often
associated with nephro- and ototoxicity. TANGO 136 may
also mediate uptake of polybasic drugs, and TANGO 136
nucleic acids, proteins, agonists, and antagonists are
useful for the modulating the uptake of such drugs.
TANGO 136 can also be used to design less toxic versions
of such drugs.
In addition, LRP-2 is involved in the pathogenesis
of Heymann Nephritis nephropathy (HN), an autoimmune
glomerular disease, which is similar to human membranous
nephropathy. It is thought that LRP-2 is the major
pathogenic antigen and forms an antigen-antibody complex
between the glomular basement membrane and the foot
processes of glomerular epithelial cells. The presence
of the antigen-antibody complex leads to extensive damage
of the basement membrane and proteinuria (Farquhar et al.
(1994) Ann. N.Y. Acad. Sci. 97-106). Similar to LRP-2,
TANGO 136 may play a pathogenic role in autoimmune
ii
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glomerular disease. Thus, TANGO 136 nucleic acids,
proteins, agonists, and antagonists are useful for the
treatment of autoimmune glomerular disease.
LRP-5 and LRP-6 are thought to function in
endocytosis. Based on genetic evidence, LRP-5 and
possibly LRP-6 are thought to play a role in the
molecular pathogenesis of type I diabetes (Brown et al.
(1998) Biochem. Biophys. Res. Comm. 248:879-888). TANGO
136 is also likely plays a role in type I diabetes.
Thus, TANGO 136 nucleic acids, proteins, agonists, and
antagonists are useful for the treatment of type I
diabetes.
LRBB is expressed in brain and might be involved
in brain-specific lipid transport. Brain-specific lipid
transport may involve apoE4, which is associated with
Alzheimer's disease. TANGO 136 may also be involved in
brain-specific lipid transport, and TANGO 136 nucleic
acids, proteins, agonists, and antagonists are useful
for the treatment of Alzheimer's disease.
In general, TANGO 136 nucleic acids, proteins,
agonists, and antagonists may be useful for the
treatment of neurological disorders, e.g.,
neurodegenerative disorders and neuropsychiatric
disorders. Examples of neurodegenerative disorders
include Alzheimer's disease, Parkinson's disease, and
Huntington's disease. Examples of neuropsychiatric
disorders include schizophrenia, attention deficit
disorder, unipolar affective (mood) disorder, bipolar
affective (mood) disorders (e. g., severe bipolar
affective disorder (BP-I) and bipolar affective disorder
with hypomania and major depression (BP-II)), and
schizoaffective disorders.
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TABLE 1: Summary of Murine arid Human TANGO 136 Sequence
Information
Gene cDNA ORF Protein Figure Accession
No.
MURINE SEQ ID N0:1 SEQ ID N0:3 SEQ ID N0:2 Fig.
1
TANGO 136
HUMAN SEQ ID N0:4 SEQ ID N0:5 SEQ ID N0:5 Fig. ATCC 98880
3
TANGO 136
TABLE 2: Summary of Domains of Murine and Human TANGO 136
Protein
1 Protein Signal Mature ExtracelluTransmemhranCytoplasmic
0 Sequence Protein lar a Domain
Domain Domain
MURINE as 1-1? as 18-575 as 18-441 as 442-462 as 463-575
TANGO 136
SEQ ID SEQ ID N0:7SEQ ID SEQ ID SEQ ID NO:10SEQ ID
N0:8
N0:2 N0:9 N0:11
1 HUMAN as 1-16 as 17-713 as 17-440 as 441-461 as 462-713
5
TANGO 136
SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID N0:17SEQ ID
N0:5 N0:14 N0:15 N0:16 N0:18
Various aspects of the invention are described in
20 further detail in the following subsections
I. Isolated Nucleic Acid Molecules
One aspect of the invention pertains to isolated
nucleic acid molecules that encode a polypeptide of the
invention or a biologically active portion thereof, as
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well as nucleic acid molecules sufficient for use as
hybridization probes to identify nucleic acid molecules
encoding a polypeptide of the invention and fragments of
such nucleic acid molecules suitable for use as PCR
primers for the amplification or mutation of nucleic acid
molecules. As used herein, the term "nucleic acid
molecule" is intended to include DNA molecules (e. g.,
cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and
analogs of the DNA or RNA generated using nucleotide
analogs. The nucleic acid molecule can be single-
stranded or double-stranded, but preferably is double-
stranded DNA.
An "isolated" nucleic acid molecule is one which
is separated from other nucleic acid molecules which are
present in the natural source of the nucleic acid
molecule. An "isolated" nucleic acid molecule is free of
sequences (preferably protein encoding sequences) which
naturally flank the nucleic acid (i.e., sequences located
at the 5' and 3' ends of the nucleic acid) in the genomic
DNA of the organism from which the nucleic acid is
derived. For example, in various embodiments, the
isolated nucleic acid molecule can contain less than
about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of
nucleotide sequences which naturally flank the nucleic
acid molecule in genomic DNA of the cell from which the
nucleic acid is derived. Moreover, an "isolated" nucleic
acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture
medium when produced by recombinant techniques, or
substantially free of chemical precursors or other
chemicals when chemically synthesized.
A nucleic acid molecule of the present invention,
e.g., a nucleic acid molecule having the nucleotide
sequence of SEQ ID NO:1, 3, 4, or 6 or the cDNA of ATCC
98880, or a complement thereof, can be isolated using
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standard molecular biology techniques and the sequence
information provided herein. Using all or a portion of
the nucleic acid sequences of SEQ ID NO:1, 3, 4, or 6 or
the cDNA of ATCC 98880 as a hybridization probe, nucleic
acid molecules of the invention can be isolated using
standard hybridization and cloning techniques (e.g., as
described in Sambrook et al., eds., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY, 1989).
A nucleic acid molecule of the invention can be
amplified using cDNA, mRNA or genomic DNA as a template
and appropriate oligonucleotide primers according to
standard PCR amplification techniques. The nucleic acid
so amplified can be cloned into an appropriate vector and
characterized by DNA sequence analysis. Furthermore,
oligonucleotides corresponding to all or a portion of a
nucleic acid molecule of the invention can be prepared by
standard synthetic techniques, e.g., using an automated
DNA synthesizer.
In another embodiment, an isolated nucleic acid
molecule of the invention comprises a nucleic acid
molecule which is a complement of the nucleotide sequence
of SEQ iD NO:1, 3, 4, or 6 or the cDNA of ATCC 98880, or
a portion thereof. A nucleic acid molecule which is
complementary to a given nucleotide sequence is one which
is sufficiently complementary to the given nucleotide
sequence that it can hybridize to the given nucleotide
sequence thereby forming a stable duplex.
Moreover, a nucleic acid~molecule of the invention
can comprise only a portion of a nucleic acid sequence
encoding a full length polypeptide of the invention for
example, a fragment which can be used as a probe or
primer or a fragment encoding a biologically active
portion of a polypeptide of the invention. The nucleotide
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sequence determined from the cloning one gene allows for
the generation of probes and primers designed for use in
identifying and/or cloning homologues in other cell
types, e.g., from other tissues, as well as homologues
from other mammals. The probe/primer typically comprises
substantially purified oligonucleotide. The
oligonucleotide typically comprises a region of
nucleotide sequence that hybridizes under stringent
conditions to at least about 12, preferably about 25,
more preferably about 50, 75, 100, 125, 150, 175, 200,
250, 300, 350 or 400 consecutive nucleotides of the sense
or anti-sense sequence of SEQ ID NO:1, 3, 4, or 6, or the
cDNA of ATCC 98880 or of a naturally occurring mutant of
SEQ ID N0:1, 3, 4, or 6 or the cDNA of ATCC 98880.
Probes based on the sequence of a nucleic acid
molecule of the invention can be used to detect
transcripts or genomic sequences encoding the same
protein molecule encoded by a selected nucleic acid
molecule. The probe comprises a label group attached
thereto, e.g., a radioisotope, a fluorescent compound, an
enzyme, or an enzyme co-factor. Such probes can be used
as part of a diagnostic test kit for identifying cells or
tissues which mis-express the protein, such as by
measuring levels of a nucleic acid molecule encoding the
protein in a sample of cells from a subject, e.g.,
detecting mRNA levels or determining whether a gene
encoding the protein has been mutated or deleted.
A nucleic acid fragment encoding a "biologically
active portion" of a polypeptide of the invention can be
prepared by isolating a portion of any of SEQ ID NOS:3 or
6 or the nucleotide sequence of thecDNA of ATCC 98880
which encodes a polypeptide having a biological activity,
expressing the encoded portion of the polypeptide protein
(e. g., by recombinant expression in vitro) and assessing
the activity of the encoded portion of the polypeptide.
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The invention further encompasses nucleic acid
molecules that differ from the nucleotide sequence of SEQ
ID N0:1, 3, 4, or 6 or the cDNA of ATCC 98880 due to
degeneracy of the genetic code and thus encode the same
protein as that encoded by the nucleotide sequence of SEQ
ID N0:3 or 6 or the cDNA of ATCC 98880.
In addition to the nucleotide sequences of SEQ ID
NOs:3 and 6 and present in cDNA of ATCC 98880, it will be
appreciated by those skilled in the art that DNA sequence
polymorphisms that lead to changes in the amino acid
sequence may exist within a population (e. g., the human
population). Such genetic polymorphisms may exist among
individuals within a population due to natural allelic
variation. An allele is one of a group of genes which
occur alternatively at a given genetic locus. As used
herein, the phrase "allelic variant" refers to a
nucleotide sequence which occurs at a given locus or to a
polypeptide encoded by the nucleotide sequence. As used
herein, the terms "gene" and "recombinant gene" refer to
nucleic acid molecules comprising an open reading frame
encoding a polypeptide of the invention. Such natural
allelic variations can typically result in 1-5~ variance
in the nucleotide sequence of a given gene. Alternative
alleles can be identified by sequencing the gene of
interest in a number of different individuals. This can
be readily carried out by using hybridization probes to
identify the same genetic locus in a variety of
individuals. Any and all such nucleotide variations and
resulting amino acid polymorphisms or variations that are
the result of natural allelic variation and that do not
alter the functional activity are intended to be within
the scope of the invention.
Moreover, nucleic acid molecules encoding proteins
of the invention from other species (homologues), which
have a nucleotide sequence which differs from that of the
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human protein described herein are intended to be within
the scope of the invention. Nucleic acid molecules
corresponding to natural allelic variants and homologues
of a cDNA of the invention can be isolated based on their
identity to the human nucleic acid molecule disclosed
herein using the human cDNAs, or a portion thereof, as a
hybridization probe according to standard hybridization
techniques under stringent hybridization conditions. For
example, a cDNA encoding a soluble form of a membrane-
bound protein of the invention isolated based on its
hybridization to a nucleic acid molecule encoding all or
part of the membrane-bound form. Likewise, a cDNA
encoding a membrane-bound form can be isolated based on
its hybridization to a nucleic acid molecule encoding all
or part of the soluble form.
Accordingly, in another embodiment, an isolated
nucleic acid molecule of the invention is at least 300
(325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700,
800, 900, 1000, or 1290) nucleotides in length and
hybridizes under stringent conditions to the nucleic acid
molecule comprising the nucleotide sequence, preferably
the coding sequence, of SEQ ID NO:l, 3, 4, or 6, or the
cDNA of ATCC 98880, or a complement thereof.
As used herein, the term "hybridizes under
stringent conditions" is intended to describe conditions
for hybridization and washing under which nucleotide
sequences at least 60% (65%; 70%, preferably 75%)
identical to each other typically remain hybridized to
each other. Such stringent conditions are known to those
skilled in the art and can be found in Current Protocols
in Molecular Bio.Iogy, John Wiley & Sons, N.Y. (1989),
6.3.1-6.3.6. A non-limiting example of stringent
hybridization conditions are hybridization in 6X sodium
chloride/sodium citrate (SSC) at about 45°C, followed by
one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C.
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Preferably, an isolated nucleic acid molecule of the
invention that hybridizes under stringent conditions to
the sequence of SEQ ID Nail, 3, 4, or 6, or the cDNA of
ATCC 98880, or the complement thereof, corresponds to a
naturally-occurring nucleic acid molecule. As used
herein, a "naturally-occurring" nucleic acid molecule
refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e. g., encodes a natural
protein).
In addition to naturally-occurring allelic
variants of a nucleic acid molecule of the invention
sequence that may exist in the population, the skilled
artisan will further appreciate that changes can be
introduced by mutation thereby leading to changes in the
amino acid sequence of the encoded protein, without
altering the biological activity of the protein. For
example, one can make nucleotide substitutions leading to
amino acid substitutions at "non-essential" amino acid
residues. A "non-essential" amino acid residue is a
residue that can be altered from the wild-type sequence
without altering the biological activity; whereas an
"essential" amino acid residue is required for biological
activity. For example, amino acid residues that are not
conserved or only semi-conserved among homologues of
various species may be non-essential for activity and
thus would be likely targets for alteration.
Alternatively, amino acid residues that are conserved
among the homologues of various species (e.g., murine and
human) may be essential for activity and thus would not
be likely targets for alteration.
Accordingly, another aspect of the invention
pertains to nucleic acid molecules encoding a polypeptide
of the invention that contain changes in amino acid
residues that are not essential for activity. Such
polypeptides differ in amino acid sequence from SEQ ID
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N0:2, 5, 8, or 15 yet retain biological activity. In one
embodiment, the isolated nucleic acid molecule includes a
nucleotide sequence encoding a protein that includes an
amino acid sequence that is at least about 45~ identical,
65~, 75~, 85~, 95~, or 98~ identical to the amino acid
sequence of SEQ ID N0:2, 5, 8, or 15.
An isolated nucleic acid molecule encoding a
variant protein can be created by introducing one or more
nucleotide substitutions, additions or deletions into the
nucleotide sequence of SEQ ID NO:1, 3, 4, or 6 or the
cDNA of ATCC 98880 such that one or more amino acid
substitutions, additions or deletions are introduced into
the encoded protein. Mutations can be introduced by
standard techniques, such as site-directed mutagenesis
and PCR-mediated mutagenesis. Preferably, conservative
amino acid substitutions are made at one or more
predicted non-essential amino acid residues. A
"conservative amino acid substitution" is one in which
the amino acid residue is replaced with an amino acid
residue having a similar side chain. Families of amino
acid residues having similar side chains have been
defined in the art. These families include amino acids
with basic side chains (e. g., lysine, arginine,
histidine), acidic side chains (e. g., aspartic acid,
glutamic acid), uncharged polar side chains (e. g.,
glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine), nonpolar side chains (e. g., alanine,
valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), beta-branched side chains (e. g.,
threonine, valine, isoleucine) and aromatic side chains
(e. g., tyrosine, phenylalanine, tryptophan, histidine).
Alternatively, mutations can be introduced randomly along
all or part of the coding sequence, such as by
saturation mutagenesis, and the resultant mutants can be
screened for biological activity to identify mutants that
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retain activity. Following mutagenesis, the encoded
protein can be expressed recombinantly and the activity
of the protein can be determined.
In one embodiment, a mutant polypeptide that is a
variant of a polypeptide of the invention can be assayed
for: (1) the ability to form protein: protein interactions
with proteins in a signaling pathway of the polypeptide
of the invention; (2) the ability to bind a ligand of the
polypeptide of the invention; or (3) the ability to bind
to an intracellular target protein of the polypeptide of
the invention. In yet another embodiment, the mutant
polypeptide can be assayed for the ability to modulate
cellular proliferation or cellular differentiation.
The present invention encompasses antisense
nucleic acid molecules, i.e., molecules which are
complementary to a sense nucleic acid encoding a
polypeptide of the invention, e.g., complementary to the
coding strand of a double-stranded cDNA molecule or
complementary to an mRNA sequence. Accordingly, an
antisense nucleic acid can hydrogen bond to a sense
nucleic acid. The antisense nucleic acid can be
complementary to an entire coding strand, or to only a
portion thereof, e.g., all or part of the protein coding
region (or open reading frame). An antisense nucleic
acid molecule can be antisense to all or part of a non-
coding region of the coding strand of a nucleotide
sequence encoding a polypeptide of the invention. The
non-coding regions ("5' and 3' untranslated regions") are
the 5' and 3' sequences which flank the coding region and
are not translated into amino acids.
An antisense oligonucleotide can be, for example,
about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides
in length. An antisense nucleic acid of the invention
can be constructed using chemical synthesis and enzymatic
ligation reactions using procedures known in the art.
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For example, an antisense nucleic acid (e.g., an
antisense oligonucleotide) can be chemically synthesized
using naturally occurring nucleotides or variously
modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical
stability of the duplex formed between the antisense and
sense nucleic acids, e.g., phosphorothioate derivatives
and acridine substituted nucleotides can be used.
Examples of modified nucleotides which can be used to
generate the antisense nucleic acid include 5-
fluorouracil, 5-bromouracil, 5-chlorouracil, 5-
iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-
(carboxyhydroxylmethyl) uracil, 5-
carboxymethylaminomethyl-2-thiouridine, 5-
carboxymethylaminomethyTuracil, 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, 5-methyl-2-thiouracil, 2-thiouracil, 4-
thiouracil, 5-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. Alternatively, the
antisense nucleic acid can be produced biologically using
an expression vector into which a nucleic acid has been
subcloned in an antisense orientation (i.e., RNA
transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of
interest, described further in the following subsection).
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The antisense nucleic acid molecules of the
invention are typically administered to a subject or
generated in situ such that they hybridize with or bind
to cellular mRNA and/or genomic DNA encoding a selected
polypeptide of the invention to thereby inhibit
expression, e.g., by inhibiting transcription and/or
translation. The hybridization can be by conventional
nucleotide complementarity to form a stable duplex, or,
for example, in the case of an antisense nucleic acid
molecule which binds to DNA duplexes, through specific
interactions in the major groove of the double helix. An
example of a route of administration of antisense nucleic
acid molecules of the invention includes direct injection
at a tissue site. Alternatively, antisense nucleic acid
molecules can be modified to target selected cells and
then administered systemically. For example, for
systemic administration, antisense molecules can be
modified such that they specifically bind to receptors or
antigens expressed on a selected cell surface, e.g., by
linking the antisense nucleic acid molecules to peptides
or antibodies which bind to cell surface receptors or
antigens. The antisense nucleic acid molecules can also
be delivered to cells using the vectors described herein.
To achieve sufficient intracellular concentrations of the
antisense molecules, vector constructs in which the
antisense nucleic acid molecule is placed under the
control of a strong pol II or pol III promoter are
preferred.
An antisense nucleic acid molecule of the
invention can be an a-anomeric nucleic acid molecule. An
a-anomeric nucleic acid molecule forms specific double-
stranded hybrids with complementary RNA in which,
contrary to the usual /3-units, the strands run parallel
to each other (Gaultier et al. (1987) Nucleic Acids Res.
15:6625-6641). The antisense nucleic acid molecule can
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also comprise a 2'-o-methylribonucleotide (Inoue et al.
(1987) Nucleic Acids Res. 15:6131-6148) or a chimeric
RNA-DNA analogue (moue et al. (1987) FEBS Lett. 215:327-
330) .
The invention also encompasses ribozymes.
Ribozymes are catalytic RNA molecules with ribonuclease
activity which are capable of cleaving a single-stranded
nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e. g., hammerhead
ribozymes (described in Haselhoff and Gerlach (1988)
Nature 334:585-591)) can be used to catalytically cleave
mRNA transcripts to thereby inhibit translation of the
protein encoded by the mRNA. A ribozyme having
specificity for a nucleic acid molecule encoding a
polypeptide of the invention can be designed based upon
the nucleotide sequence of a cDNA disclosed herein. For
example, a derivative of a Tetrahymena L-19 IVS RNA can
be constructed in which the nucleotide sequence of the
active site is complementary to the nucleotide sequence
to be cleaved in a Cech et al. U.S. Patent No. 4,987,071;
and Cech et al. U.S. Patent No. 5,116,742.
Alternatively, an mRNA encoding a polypeptide of the
invention can be used to select a catalytic RNA having a
specific ribonuclease activity from a pool of RNA
molecules. See, e.g., Bartel and Szostak (1993) Science
261:1411-1418.
The invention also encompasses nucleic acid
molecules which form triple helical structures. For
example, expression of a polypeptide of the invention can
be inhibited by targeting nucleotide sequences
complementary to the regulatory region of the gene
encoding the polypeptide (e. g., the promoter and/or
enhancer) to form triple helical structures that prevent
transcription of the gene in target cells. See generally
Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene
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(1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992)
Bioassays 14(12):807-15.
In various embodiments, the nucleic acid molecules
of the invention can be modified at the base moiety,
sugar moiety or phosphate backbone to improve, e.g., the
stability, hybridization, or solubility of the molecule.
For example, the deoxyribose phosphate backbone of the
nucleic acids can be modified to generate peptide nucleic
acids (see Hyrup et al. (1996) Bioorganic & Medicinal
Chemistry 4(1): 5-23). As used herein, the terms
"peptide nucleic acids" or "PNAs" refer to nucleic acid
mimics, e.g., DNA mimics, in which the deoxyribose
phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are
retained. The neutral backbone of PNAs has been shown to
allow for specific hybridization to DNA and RNA under
conditions of low ionic strength. The synthesis of PNA
oligomers can be performed using standard solid phase
., peptide synthesis protocols as described in Hyrup et al.
(1996), supra; Perry-0'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93: 14670-675.
PNAs can be used in therapeutic and diagnostic
applications. For example, PNAs can be used as antisense
or antigene agents for sequence-specific modulation of
gene expression by, e.g., inducing transcription or
translation arrest or inhibiting replication. PNAs can
also be used, e.g., in the analysis of single base pair
mutations in a gene by, e.g., PNA directed PCR clamping;
as artificial restriction enzymes when used in
combination with other enzymes, e.g., S1 nucleases (Hyrup
(1996), supra; or as probes or primers for DNA sequence
and hybridization (Hyrup (1996), supra; Perry-0'Keefe et
al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675).
In another embodiment, PNAs can be modified, e.g.,
to enhance their stability or cellular uptake, by
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attaching lipophilic or other helper groups to PNA, by
the formation of PNA-DNA chimeras, or by the use of
liposomes or other techniques of drug delivery known in
the art. For example, PNA-DNA chimeras can be generated
which may combine the advantageous properties of PNA and
DNA. Such chimeras allow DNA recognition enzymes, e.g.,
RNAse H and DNA polymerases, to interact with the DNA
portion while the PNA portion would provide high binding
affinity and specificity. PNA-DNA chimeras can be linked
using linkers of appropriate lengths selected in terms of
base stacking, number of bonds between the nucleobases,
and orientation (Hyrup (1996), supra). The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup
(1996}, supra, and Finn et al. {1996) Nucleic Acids Res.
24:3357-63. For example, a DNA chain can be synthesized
on a solid support using standard phosphoramidite
coupling chemistry and modified nucleoside analogs.
Compounds such as 5'-(4-methoxytrityl)amino-5'-deoxy-
thymidine phosphoramidite can be used as a link between
the PNA and the 5' end of DNA (Mag et al. (1989) Nucleic
Acids Res. 17:5973-88). PNA monomers are then coupled in
a stepwise manner to produce a chimeric molecule with a
5' PNA segment and a 3' DNA segment (Finn et al. (1996)
Nucleic Acids Res. 24(17):3357-63). Alternatively,
chimeric molecules can be synthesized with a 5' DNA
segment and a 3' PNA segment (Peterser et al. (1975}
Bioorganic Med. Chem. Lett. 5:1119-11124).
In other embodiments, 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. (1989} Proc. Natl. Acad. Sci. USA
86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad.
Sci. USA 84:648-652; PCT Publication No. WO 88/09810) or
the blood-brain barrier (see, e.g., PCT Publication No.
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WO 89/10134). In addition, oligonucleotides can be
modified with hybridization-triggered cleavage agents
(see, e.g., Krol et al. (1988) Bio/Techniques 6:958-976}
or intercalating agents (see, e:g., Zon (1988) Pharm.
Res. 5:539-549). 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.
II. Isolated Proteins and Antibodies
One aspect of the invention pertains to isolated
proteins, and biologically active portions thereof, as
well as polypeptide fragments suitable for use as
immunogens to raise antibodies directed against a
polypeptide of the invention. In one embodiment, the
native polypeptide can be isolated from cells or tissue
sources by an appropriate purification scheme using
standard protein purification techniques. In another
embodiment, polypeptides of the invention are produced by
recombinant DNA techniques. Alternative to recombinant
expression, a polypeptide of the invention can be
synthesized chemically using standard peptide synthesis
techniques.
An "isolated" or "purified" protein or
biologically active portion thereof is substantially free
of cellular material or other contaminating proteins from
the cell or tissue source from which the protein is
derived, or substantially free of chemical precursors or
other chemicals when chemically synthesized. The
language "substantially free of cellular material"
includes preparations of protein in which the protein is
separated from cellular components of the cells from
which it is isolated or recombinantly produced. Thus,
protein that is substantially free of cellular material
includes preparations of protein having less than about
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30%, 20%, 10%, or 5% (by dry weight) of heterologous
protein (also referred to herein as a "contaminating
protein"). When the protein or biologically active
portion thereof is recombinantly produced, it is also
preferably substantially free of culture medium, i.e.,
culture medium represents less than about 20%, 10%, or 5%
of the volume of the protein preparation. When the
protein is produced by chemical synthesis, it is
preferably substantially free of chemical precursors or
other chemicals, i.e., it is separated from chemical
precursors or other chemicals which are involved in the
synthesis of the protein. Accordingly such preparations
of the protein have less than about 30%, 20%, 10%, 5% (by
dry weight) of chemical precursors or compounds other
than the polypeptide of interest.
Biologically active portions of a polypeptide of
the invention include polypeptides comprising amino acid
sequences sufficiently identical to or derived from the
amino acid sequence of the protein (e. g., the amino acid
sequence shown in any of SEQ ID Nos:2, 5, 8, and 15),
which include fewer amino acids than the full length
protein, and exhibit at least one activity of the
corresponding full-length protein. Typically,
biologically active portions comprise a domain or motif
with at least one activity of the corresponding protein.
A biologically active portion of a protein of the
invention can be a polypeptide which is, for example, 10,
25, 50, 100 or more amino acids in length. Moreover,
other biologically active portions, in which other
regions of the protein are deleted, can be prepared by
recombinant techniques and evaluated for one or more of
the functional activities of the native form of a
polypeptide of the invention.
Polypeptides can have the amino acid sequence of
SEQ ID N0:2, 5, 7-11, or 14-18. Other useful proteins
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are substantially identical (e.g., at least about 45%,
preferably 55%, 65%, 75%, 85%, 95%, or 99%) to any of SEQ
ID N0:2, 5, 7-11, or 14-18 and retain the functional
activity of the protein of the corresponding naturally-
occurring protein yet differ in amino acid sequence due
to natural allelic variation or mutagenesis.
To determine the percent identity of two amino
acid sequences or of two nucleic acids, the sequences are
aligned for optimal comparison purposes (e.g., gaps can
be introduced in the sequence of a first amino acid or
nucleic acid sequence for optimal alignment with a second
amino or nucleic acid sequence). The amino acid residues
or nucleotides at corresponding amino acid positions or
nucleotide positions are then compared. When a position
in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in
the second sequence, then the molecules are identical at
that position. The percent identity between the two
sequences is a function of the number of identical
positions shared by the sequences (i.e., ~ identity = #
of identical positions/total # of positions (e. g.,
overlapping positions) x 100). In one embodiment the two
sequences are the same length.
The determination of percent identity between two
sequences can be accomplished using a mathematical
algorithm. A preferred, non-limiting example of a
mathematical algorithm utilized for the comparison of two
sequences is the algorithm of Karlin and Altschul (1990)
Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA
90:5873-5877. Such an algorithm is incorporated into the
NBLAST and XBLAST programs of Altschul, et al. (1990) J.
Mol. Biol. 215:403-410. BLAST nucleotide searches can be
performed with the NBLAST program, score = 100,
wordlength = 12 to obtain nucleotide sequences homologous
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to a nucleic acid molecules of the invention. BLAST
protein searches can be performed with the XBLAST
program, score = 50, wordlength = 3 to obtain amino acid
sequences homologous to a protein molecules of the
invention. To obtain gapped alignments for comparison
purposes, Gapped BLAST can be utilized as described in
Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
Alternatively, PSI-Blast can be used to perform an
iterated search which detects distant relationships
between molecules. Id. When utilizing BLAST, Gapped
BLAST, and PSI-Blast programs, the default parameters of
the respective programs (e.g., XBLAST and NBLAST) can be
used. See http://www.ncbi.nlm.nih.gov. Another
preferred, non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the
algorithm of Myers and Miller, (1988) CABIOS 4:11-17.
Such an algorithm is incorporated into the ALIGN program
(version 2.0) which is part of the GCG sequence alignment
software package. When utilizing the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue
table, a gap length penalty of 12, and a gap penalty of 4
can be used.
The percent identity between two sequences can be
determined using techniques similar to those described
above, with or without allowing gaps. In calculating
percent identity, only exact matches are counted.
The invention also provides chimeric or fusion
proteins. As used herein, a "chimeric protein" or
"fusion protein" comprises all or part (preferably
biologically active) of a polypeptide of the invention
operably linked to a heterologous polypeptide (i.e., a
polypeptide other than the same polypeptide of~the
invention). Within the fusion protein, the term
"operably linked" is intended to indicate that the
polypeptide of the invention and the heterologous
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polypeptide are fused in-frame to each other. The
heterologous polypeptide can be fused to the N-terminus
or C-terminus of the polypeptide of the invention.
One useful fusion protein is a GST fusion protein in
which the polypeptide of the invention is fused to the C-
terminus of GST sequences. Such fusion proteins can
facilitate the purification of a recombinant polypeptide
of the invention.
In another embodiment, the fusion protein contains
a heterologous signal sequence at its N-terminus. For
example, the native signal sequence of a polypeptide of
the invention can be removed and replaced with a signal
sequence from another protein. For example, the gp67
secretory sequence of the baculovirus envelope protein
can be used as a heterologous signal sequence (Current
Protocols in Molecular Biology, Ausubel et al., eds.,
John Wiley & Sons, 1992). Other examples of eukaryotic
heterologous signal sequences include the secretory
sequences of melittin and human placental alkaline
phosphatase (Stratagene; La Jolla, California). In yet
another example, useful prokaryotic heterologous signal
sequences include the phoA secretory signal (Sambrook et
al., supra) and the protein A secretory signal (Pharmacia
Biotech; Piscataway, New Jersey).
In yet another embodiment, the fusion protein is
an immunoglobulin fusion protein in which all or part of
a polypeptide of the invention is fused to sequences
derived from a member of the immunoglobulin protein
family. The immunoglobulin fusion proteins of the
invention can be incorporated into pharmaceutical
compositions and administered to a subject to inhibit an
interaction between a ligand (soluble or membrane-bound)
and a protein on the surface of a cell (receptor), to
thereby suppress signal transduction in vivo. The
immunoglobulin fusion protein can be used to affect the
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bioavailability of a cognate ligand of a polypeptide of
the. invention. Inhibition of ligand/receptor interaction
may be useful therapeutically, both for treating
proliferative and differentiative disorders and for
modulating (e. g. promoting or inhibiting) cell survival.
Moreover, the immunoglobulin fusion proteins of the
invention can be used as immunogens to produce antibodies
directed against a polypeptide of the invention in a
subject, to purify ligands and in screening assays to
identify molecules which inhibit the interaction of
receptors with ligands.
Chimeric and fusion proteins of the invention can
be produced by standard recombinant DNA techniques. In
another embodiment, the fusion gene can be synthesized by
conventional techniques including automated DNA
synthesizers. Alternatively, PCR amplification of gene
fragments can be carried out using anchor primers which
give rise to complementary overhangs between two
consecutive gene fragments which can subsequently be
annealed and reamplified to generate a chimeric gene
sequence (see, e.g., Ausubel et al., supra). Moreover,
many expression vectors are commercially available that
already encode a fusion moiety (e. g., a GST polypeptide).
A nucleic acid encoding a polypeptide of the invention
can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to the polypeptide
of the invention.
A signal sequence of a polypeptide of the
invention (SEQ ID N0:7 or 14) can be used to facilitate
secretion and isolation of the secreted protein or other
proteins of interest. Signal sequences are typically
characterized by a core of hydrophobic amino acids which
are generally cleaved from the mature protein during
secretion in one or more cleavage events. Such signal
peptides contain processing sites that allow cleavage of
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the signal sequence from the mature proteins as they pass
through the secretory pathway. Thus, the invention
pertains to the described polypeptides having a signal
sequence, as well as to the signal sequence itself and to
the polypeptide in the absence of the signal sequence
(i.e., the cleavage products). In one embodiment, a
nucleic acid sequence encoding a signal sequence of the
invention can be operably linked in an expression vector
to a protein of interest, such as a protein which is
ordinarily not secreted or is otherwise difficult to
isolate. The signal sequence directs secretion of the
protein, such as from a eukaryotic host into which the
expression vector is transformed, and the signal sequence
is subsequently or concurrently cleaved. The protein can
then be readily purified from the extracellular medium by
art recognized methods. Alternatively, the signal
sequence can be linked to the protein of interest using a
sequence which facilitates purification, such as with a
GST domain.
In another embodiment, the signal sequences of the
present invention can be used to identify regulatory
sequences, e.g., promoters, enhancers, repressors. Since
signal sequences are the most amino-terminal sequences of
a peptide, it is expected that the nucleic acids which
flank the signal sequence on its amino-terminal side will
be regulatory sequences which affect transcription.
Thus, a nucleotide sequence which encodes all or a
portion of a signal sequence can be used as a probe to
identify and isolate signal sequences and their flanking
regions, and these flanking regions can be studied to
identify regulatory elements therein.
The present invention also pertains to variants of
the polypeptides of the invention. Such variants have an
altered amino acid sequence which can function as either
agonists (mimetics) or as antagonists. Variants can be
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generated by mutagenesis, e.g., discrete point mutation
or truncation. An agonist can retain substantially the
same, or a subset, of the biological activities of the
naturally occurring form of the protein. An antagonist
of a protein can inhibit one or more of the activities of
the naturally occurring form of the protein by, for
example, competitively binding to a downstream or
upstream member of a cellular signaling cascade which
includes the protein of interest. Thus, specific
biological effects can be elicited by treatment with a
variant of limited function. Treatment of a subject with
a variant having a subset of the biological activities of
the naturally occurring form of the protein can have
fewer side effects in a subject relative to treatment
with the naturally occurring form of the protein.
Variants of a protein of the invention which
function as either agonists (mimetics) or as antagonists
can be identified by screening combinatorial libraries of
mutants, e.g., truncation mutants, of the protein of the
invention for agonist or antagonist activity. In one
embodiment, a variegated library of variants is generated
by combinatorial mutagenesis at the nucleic acid level
and is encoded by a variegated gene library. A
variegated library of variants can be produced by, for
example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a
degenerate set of potential protein sequences is
expressible as individual polypeptides, or alternatively,
as a set of larger fusion proteins (e. g., for phage
display). There are a variety of methods which can be
used to produce libraries of potential variants of the
polypeptides of the invention from a degenerate
oligonucleotide sequence. Methods for synthesizing
degenerate oligonucleotides are known in the art (see,
e.g., Narang (1983) Tetrahedron 39:3; Itakura et al.
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(1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984}
Science 198:1056; Ike et al. (1983) Nucleic Acid Res.
11:477) .
In addition, libraries of fragments of the coding
sequence of a polypeptide of the invention can be used to
generate a variegated population of polypeptides far
screening and subsequent selection of variants. For
example, a library of coding sequence fragments can be
generated by treating a double stranded PCR fragment of
the coding sequence of interest with a nuclease under
conditions wherein nicking occurs only about once per
molecule, denaturing the double stranded DNA, renaturing
the DNA to form double stranded DNA which can include
sense/antisense pairs from different nicked products,
removing single stranded portions from reformed duplexes
by treatment with S1 nuclease, and ligating the resulting
fragment library into an expression vector. By this
method, an expression library can be derived which
encodes N-terminal and internal fragments of various
sizes of the protein of interest.
Several techniques are known in the art for
screening gene products of combinatorial libraries made
by point mutations or truncation, and for screening cDNA
libraries for gene products having a selected property.
The most widely used techniques, which are amenable to
high through-put analysis, for screening large gene
libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate
cells with the resulting library of vectors, and
expressing the combinatorial genes under conditions in
which detection of a desired activity facilitates
isolation of the vector encoding the gene whose product
was detected. Recursive ensemble mutagenesis (REM), a
technique which enhances the frequency of functional
mutants in the libraries, can be used in combination with
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the screening assays to identify variants of a protein of
the. invention (Arkin and Yourvan (1992) Proc. Natl. Acad.
Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein
Engineering 6:327-331).
An isolated polypeptide of the invention, or a
fragment thereof, can be used as an immunogen to generate
antibodies using standard techniques for polyclonal and
monoclonal antibody preparation. The full-length
polypeptide or protein can be used or, alternatively, the
invention provides antigenic peptide fragments for use as
immunogens. The antigenic peptide of a protein of the
invention comprises at least 8 (preferably 10, 15, 20, or
30) amino acid residues of the amino acid sequence of SEQ
ID N0:2 or 5 and encompasses an epitope of the protein
such that an antibody raised against the peptide forms a
specific immune complex with the protein.
Preferred epitopes encompassed by the antigenic
peptide are regions that are located on the surface of
the protein, e.g., hydrophilic regions. Figures 2 and 4
are hydrophobicity plots of the proteins of the
invention. These plots or similar analyses can be used
to identify hydrophilic regions.
An immunogen typically is used to prepare
antibodies by immunizing a suitable subject, (e. g.,
rabbit, goat, mouse or other mammal). An appropriate
immunogenic preparation can contain, for example,
recombinantly expressed or chemically synthesized
polypeptide. The preparation can further include an
adjuvant, such as Freund's complete or incomplete
adjuvant, or similar immunostimulatory agent.
Accordingly, another aspect of the invention
pertains to antibodies directed against a polypeptide of
the invention. The term ~~antibody~~ as used herein refers
to immunoglobulin molecules and immunologically active
portions of immunoglobulin molecules, i.e., molecules
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that contain an antigen binding site which specifically
binds an antigen, such as a polypeptide of the invention.
A molecule which specifically binds to a given
polypeptide of the invention is a molecule which binds
the polypeptide, but does not substantially bind other
molecules in a sample, e.g., a biological sample, which
naturally contains the polypeptide. Examples of
immunologically active portions of immunoglobulin
molecules include Flab) and F(ab')2 fragments which can be
generated by treating the antibody with an enzyme such as
pepsin. The invention provides polyclonal and monoclonal
antibodies. The term "monoclonal antibody" or
"monoclonal antibody composition", as used herein, refers
to a population of antibody molecules that contain only
one species of an antigen binding site capable of
immunoreacting with a particular epitope.
Polyclonal antibodies can be prepared as described
above by immunizing a suitable subject with a polypeptide
of the invention as an immunogen. The antibody titer in
the immunized subject can be monitored over time by
standard techniques, such as with an enzyme linked
immunosorbent assay (ELISA) using immobilized
polypeptide. If desired, the antibody molecules can be
isolated from the mammal (e.g., from the blood) and
further purified by well-known techniques, such as
protein A chromatography to obtain the IgG fraction. At
an appropriate time after immunization, e.g., when the
specific antibody titers are highest, antibody-producing
cells can be obtained from the subject and used to
prepare monoclonal antibodies by standard techniques,
such as the hybridoma technique originally described by
Kohler and Milstein (1975) Nature 256:495-497, the human
B cell hybridoma technique (Kozbor et al. (1983) Immunol.
Today 4:72), the EBV-hybridoma technique (Cole et al.
(1985), Monoclonal Antibodies and Cancer Therapy, Alan R.
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Liss, Inc., pp. 77-96) or trioma techniques. The
technology for producing hybridomas is well known (see
generally Current Protocols in Immunology (1994) Coligan
et al. (eds.) John Wiley & Sons, Inc., New York, NY).
Hybridoma cells producing a monoclonal antibody of the
invention are detected by screening the hybridoma culture
supernatants for antibodies that bind the polypeptide of
interest, e.g., using a standard ELISA assay.
Alternative to preparing monoclonal antibody-
secreting hybridomas, a monoclonal antibody directed
against a polypeptide of the invention can be identified
and isolated by screening a recombinant combinatorial
immunoglobulin library (e. g., an antibody phage display
.. library) with the polypeptide of interest. Kits for
generating and screening phage display libraries are
commercially available (e. g., the Pharmacia Recombinant
Phage Antibody System, Catalog No. 27-9400-01; and the
Stratagene SurfZAPT"' Phage Display Ki t, Catalog No .
240612). Additionally, examples of methods and reagents
particularly amenable for use in generating and screening
antibody display library can be found in, for example,
U.S. Patent No. 5,223,409; PCT Publication No. WO
92/18619; PCT Publication No. WO 91/17271; PCT
Publication No. WO 92/20791; PCT Publication No. WO
92/15679; PCT Publication No. WO 93/01288; PCT
Publication No. WO 92/01047; PCT Publication No. WO
92/09690; PCT Publication No. WO 90/02809; Fuchs et al.
(1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum..
Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science
246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-
734.
Additionally, recombinant antibodies, such as
chimeric and humanized monoclonal antibodies, comprising
both human and non-human portions, which can be made
using standard recombinant DNA techniques, are within the
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scope of the invention. Such chimeric and humanized
monoclonal antibodies can be produced by recombinant DNA
techniques known in the art, for example using methods
described in PCT Publication No. WO 87/02671; European
Patent Application 184,187; European Patent Application
171,496; European Patent Application 173,494; PCT
Publication No. WO 86/01533; U.S. Patent No. 4,816,567;
European Patent Application 125,023; Better et al. (1988)
Science 240:1041-1043; Liu et al. (1987) Proc. Nato.
lO Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.
Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl..
Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Canc.
Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449;
and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-
1559); Morrison (1985) Science 229:1202-1207; Oi et al.
(1986) Bio/Techniques 4:214; U.S. Patent 5,225,539; Jones
et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988)
Science 239:1534; and Beidler et al. (1988) J. Immuno~.
141:4053-4060.
Completely human antibodies are particularly
desirable for therapeutic treatment of human patients.
Such antibodies can be produced using transgenic mice
which are incapable of expressing endogenous
immunoglobulin heavy and light chains genes, but which
can express human heavy and light chain genes. 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 using
conventional hybridoma technology. The human
immunoglobulin transgenes harbored by the 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 and IgE antibodies. For
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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., U.S. Patent 5,625,126; U.S. Patent
5,633,425; U.S. Patent 5,569,825; U.S. Patent 5,661,016;
and U.S. Patent 5,545,806: In addition, companies such
as Abgenix, Inc. (Freemont, 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 murine
antibody, is used to guide the selection of a completely
human antibody recognizing the same epitope (Jespers et
al. (1994) Bio/techno.Iogy 12:899-903).
An antibody directed against a polypeptide of the
invention (e.g., monoclonal antibody) can be used to
isolate the polypeptide by standard techniques, such as
affinity chromatography or immunoprecipitation.
Moreover, such an antibody can be used to detect the
protein (e. g., in a cellular lysate or cell supernatant)
in order to evaluate the abundance and pattern of
expression of the polypeptide. The antibodies can also
be used diagnostically to monitor protein levels in
tissue as part of a clinical testing procedure, e.g., to,
for example, determine the efficacy of a given treatment
regimen. 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, and radioactive materials.
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Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, /3-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic
group complexes include streptavidin/biotin 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 of
bioluminescent materials include luciferase, luciferin,
and aequorin, and examples of suitable radioactive
material include l2sI , 131I , ass or 3H .
III. Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to
vectors; preferably expression vectors, containing a
nucleic acid encoding a polypeptide of the invention (or
a portion thereof). As used herein, the term "vector"
refers to a nucleic acid molecule capable of transporting
another nucleic acid to which it has been linked. One
type of vector is a "plasmid", which refers to a circular
double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a
viral vector, wherein additional DNA segments can be
ligated into the viral genome. Certain vectors are
capable of autonomous replication in a host cell into
which they are introduced (e. g., bacterial vectors having
a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e. g., non-episomal mammalian
vectors) are integrated into the genome of a host cell
upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain
vectors, expression vectors, are capable of directing the
expression of genes to which they are operably linked.
In general, expression vectors of utility in recombinant
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DNA techniques are often in the form of plasmids
(vectors). However, the invention is intended to include
such other forms of expression vectors, such as viral
vectors (e. g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve
equivalent functions.
The recombinant expression vectors of the
invention comprise a nucleic acid of the invention in a
form suitable for expression of the nucleic acid in a
host cell. This means that the recombinant expression
vectors include one or more regulatory sequences,
selected on the basis of the host cells to be used for
expression, which is operably linked to the nucleic acid
sequence to be expressed. Within a recombinant
expression vector, "operably linked" is intended to mean
that the nucleotide sequence of interest is linked to the
regulatory sequences) in a manner which allows for
expression of the nucleotide sequence (e.g., in an in
vitro transcription/translation system or in a host cell
when the vector is introduced into the host cell). The
term "regulatory sequence" is intended to include
promoters, enhancers and other expression control
elements (e. g., polyadenylation signals). Such
regulatory sequences are described, for example, in
Goeddel, Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, CA (1990).
Regulatory sequences include those which direct
constitutive expression of a nucleotide sequence in many
types of host cell and those which direct expression of
the nucleotide sequence only in certain host cells (e. g.,
tissue-specific regulatory sequences). It will.be
appreciated by those skilled in the art that the design
of the expression vector can depend on such factors as
the choice of the host cell to be transformed, the level
of expression of protein desired, etc. The expression
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vectors of the invention can be introduced into host
cells to thereby produce proteins or peptides, including
fusion proteins or peptides, encoded by nucleic acids as
described herein.
The recombinant expression vectors of the
invention can be designed for expression of a polypeptide
of the invention in prokaryotic or eukaryotic cells,
e.g., bacterial cells such as E. coli, insect cells
(using baculovirus expression vectors), yeast cells or
mammalian cells. Suitable host cells are discussed
further in Goeddel, supra. Alternatively, the
recombinant expression vector can be transcribed and
translated is vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most
often carried out in E. coli with vectors containing
constitutive or inducible promoters directing the
expression of either fusion or non-fusion proteins.
Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the
recombinant protein. Such fusion vectors typically serve
three purposes: 1) to increase expression of recombinant
protein; 2) to increase the solubility of the recombinant
protein; and 3) to aid in the purification of the
recombinant protein by acting as a ligand in affinity
purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction
of the fusion moiety and the recombinant protein to
enable separation of the recombinant protein from the
fusion moiety subsequent to purification of the fusion
protein. Such enzymes, and their cognate recognition
sequences, include Factor Xa, thrombin and enterokinase.
Typical fusion expression vectors include pGEX (Pharmacia
Biotech Inc; Smith and Johnson (1988) Gene 67:31-40),
pMAL (New England Biolabs, Beverly, MA) and pRIT5
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(Pharmacia, Piscataway, NJ) which fuse glutathione S-
transferase (GST), maltose E binding protein, or protein
A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli
expression vectors include pTrc (Amann et al., (1988)
Gene 69:301-315) and pET lld (Studier et al., Gene
Expression Technology: Methods in Enzymo~ogy 185,
Academic Press, San Diego, California (1990) 60-89).
Target gene expression from the pTrc vector relies on
host RNA polymerase transcription from a hybrid trp-lac
fusion promoter. Target gene expression from the pET 11d
vector relies on transcription from a T7 gnl0-lac fusion
promoter mediated by a coexpressed viral RNA polymerase
(T7 gnl). This viral polymerase is supplied by host
strains BL21(DE3) or HMS174(DE3) from a resident ~
prophage harboring a T7 gnl gene under the
transcriptional control of the lacW 5 promoter.
One strategy to maximize recombinant protein
expression in E. coli is to express the protein in a host
bacteria with an impaired capacity to proteolytically
cleave the recombinant protein (Gottesman, Gene
Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, California (1990) 119-128).
Another strategy is to alter the nucleic acid sequence of
the nucleic acid to be inserted into an expression vector
so that the individual codons for each amino acid are
those preferentially utilized in E. coli (Wada et al.
(1992) Nucleic Acids Res. 20:2111-2118). Such alteration
of nucleic acid sequences of the invention can be carried
out by standard DNA synthesis techniques.
In another embodiment, the expression vector is a
yeast expression vector. Examples of vectors for
expression in yeast S. cerivisae include pYepSecl
(Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan
and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz
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et al. (1987) Gene 54:113-123), pYES2 (Invitrogen
Corporation, San Diego, CA), and pPicZ (Invitrogen Corp,
San Diego, CA).
Alternatively, the expression vector is a
baculovirus expression vector. Baculovirus vectors
available for expression of proteins in cultured insect
cells (e.g., Sf 9 cells) include the pAc series (Smith et
al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL
series (Lucklow and Summers (1989) Virology 170:31-39).
In yet another embodiment, a nucleic acid of the
invention is expressed in mammalian cells using a
mammalian expression vector. Examples of mammalian
expression vectors include pCDMB (Seed (1987) Nature
329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-
195). When used in mammalian cells, the expression
vector's control functions are often provided by viral
regulatory elements. For example, commonly used
promoters are derived from polyoma, Adenovirus 2,
cytomegalovirus and Simian Virus 40. For other suitable
expression systems for both prokaryotic and eukaryotic
cells see chapters 16 and 17 of Sambrook et al., supra.
In another embodiment, the recombinant mammalian
expression vector is capable of directing expression of
the nucleic acid preferentially in a particular cell type
(e.g., tissue-specific regulatory elements are used to
express the nucleic acid). Tissue-specific regulatory
elements are known in the art. Non-limiting examples of
suitable tissue-specific promoters include the albumin
promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and
Eaton (1988) Adv. Immunol. 43:235-275), in particular
promoters of T cell receptors (Winoto and Baltimore
(1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et
al. (1983) Cel~ 33:729-740; Queen and Baltimore (1983)
Cell 33:741-748), neuron-specific promoters (e.g., the
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neurofilament promoter; Byrne and Ruddle (1989) Proc.
Natl. Acad. Sci. USA 86:5473-5477}, pancreas-specific
promoters (Edlund et al. (1985) Science 230:912-916). and
mammary gland-specific promoters (e. g., milk whey
promoter; U.S. Patent No. 4,873,316 and European
Application Publication No. 264,166). Developmentally-
regulated promoters are also encompassed, for example the
murine hox promoters (Kessel and Gruss (1990} Science
249:374-379) and the a-fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3:537-546).
The invention further provides a recombinant
expression vector comprising a'DNA molecule of the
invention cloned into the expression vector in an
antisense orientation. That is, the DNA molecule is
operably linked to a regulatory sequence in a manner
which allows for expression (by transcription of the DNA
molecule) of an RNA molecule which is antisense to the
mRNA encoding a polypeptide of the invention.
Regulatory sequences operably linked to a nucleic acid
cloned in the antisense orientation can be chosen which
direct the continuous expression of the antisense RNA
molecule in a variety of cell types, for instance viral
promoters and/or enhancers, or regulatory sequences can
be chosen which direct constitutive, tissue specific or
cell type specific expression of antisense RNA. The
antisense expression vector can be in the form of a
recombinant plasmid, phagemid or attenuated virus in
which antisense nucleic acids are produced under the
control of a high efficiency regulatory region, the
activity of which can be determined by the cell type into
which the vector is introduced. For a discussion of the
regulation of gene expression using antisense genes see
Weintraub et al. (Reviews - Trends in Genetics, Vol. 1(1)
1986 ) .
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Another aspect of the invention pertains to host
cells into which a recombinant expression vector of the
invention has been introduced. The terms "host cell" and
"recombinant host cell" are used interchangeably herein.
It is understood that such terms refer not only to the
particular subject cell but to the progeny or potential
progeny of such a cell. Because certain modifications
may occur in succeeding generations due to either
mutation or environmental influences, such progeny may
not, in fact, be identical to the parent cell, but are
still included within the scope of the term as used
herein.
A host cell can be any prokaryotic cell (e.g., E.
coli) or any eukaryotic cell (e.g., an insect cell, a
yeast cell, or a mammalian cell).
Vector DNA can be introduced into prokaryotic or
eukaryotic cells via conventional transformation or
transfection techniques. As used herein, the terms
"transformation" and "transfection" are intended to refer
to a variety of art-recognized techniques for introducing
foreign nucleic acid into a host cell, including calcium
phosphate or calcium chloride co-precipitation, DEAE-
dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or
transfecting host cells can be found in Sambrook, et al.
(supra), and other laboratory manuals.
For stable transfection of mammalian cells, it is
known that, depending upon the expression vector and
transfection technique used, only a small fraction of
cells may integrate the foreign DNA into their genome.
In order to identify and select these integrants, a gene
that encodes a selectable marker (e.g., for resistance to
antibiotics) is generally introduced into the host cells
along with the gene of interest. Preferred selectable
markers include those which confer resistance to drugs,
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such as 6418, hygromycin and methotrexate. Cells stably
transfected with the introduced nucleic acid can be
identified by drug selection (e. g., cells that have
incorporated the selectable marker gene will survive,
while the other cells die).
A host cell of the invention, such as a
prokaryotic or eukaryotic host cell in culture, can be
used to produce a polypeptide of the invention.
Accordingly, the invention further provides methods for
producing a polypeptide of the invention using the host
cells of the invention. In one embodiment, the method
comprises culturing the host cell of invention (into
which a recombinant expression vector encoding a
polypeptide of the invention has been introduced) in a
suitable medium such that the polypeptide is produced.
In another embodiment, the method further comprises
isolating the polypeptide from the medium or the host
cell.
The host cells of the invention can also be used
to produce nonhuman transgenic animals. For example, in
one embodiment, a host cell of the invention is a
fertilized oocyte or an embryonic stem cell into which a
sequences encoding a polypeptide of the invention have
been introduced. Such host cells can then be used to
create non-human transgenic animals in which exogenous
sequences encoding a polypeptide of the invention have
been introduced into their genome or homologous
recombinant animals in which endogenous encoding a
polypeptide of the invention sequences have been altered.
Such animals are useful for studying the function and/or
activity of the polypeptide and for identifying and/or
evaluating modulators of polypeptide activity. As used
herein, a "transgenic animal" is a non-human animal,
preferably a mammal, more preferably a rodent such as a
rat or mouse, in which one or more of the cells of the
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animal includes a transgene. Other examples of
transgenic animals include non-human primates, sheep,
dogs, cows, goats, chickens, amphibians, etc. A
transgene is exogenous DNA which is integrated into the
genome of a cell from which a transgenic animal develops
and which remains in the genome of the mature animal,
thereby directing the expression of an encoded gene
product in one or more cell types or tissues of the
transgenic animal. As used herein, an "homologous
recombinant animal" is a non-human animal, preferably a
mammal, more preferably a mouse, in which an endogenous
gene has been altered by homologous recombination between
the endogenous gene and an exogenous DNA molecule
introduced into a cell of the animal, e.g., an embryonic
cell of the animal, prior to development of the animal.
A transgenic animal of the invention can be
created by introducing nucleic acid encoding a
polypeptide of the invention (or a homologue thereof)
into the male pronuclei of a fertilized oocyte, e.g., by
microinjection, retroviral infection, and allowing the
oocyte to develop in a pseudopregnant female foster
animal. Intronic sequences and polyadenylation signals
can also be included in the transgene to increase the
efficiency of expression of the transgene. A tissue-
specific regulatory sequences) can be operably~linked to
the transgene to direct expression of the polypeptide of
the invention to particular cells. Methods for
generating transgenic animals via embryo manipulation and
microinjection, particularly animals such as mice, have
become conventional in the art and are described, for
example, in U.S. Patent NOS. 4,736,866 and 4,870,009,
U.S. Patent No. 4,873,191 and in Hogan, Manipulating the
Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1986). Similar methods are used for
production of other transgenic animals. A transgenic
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founder animal can be identified based upon the presence
of the transgene in its genome and/or expression of mRNA
encoding the transgene in tissues or cells of the
animals. A transgenic founder animal can then be used to
breed additional animals carrying the transgene.
Moreover, transgenic animals carrying the transgene can
further be bred to other transgenic animals carrying
other transgenes.
To create an homologous recombinant animal, a
vector is prepared which contains at least a portion of a
gene encoding a polypeptide of the invention into which a
deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the gene. In
a preferred embodiment, the vector is designed such that,
upon homologous recombination, the endogenous gene is
functionally disrupted (i.e., no longer encodes a
functional protein; also referred to as a "knock out"
vector). Alternatively, the vector can be designed such
that, upon homologous recombination, the endogenous gene
is mutated or otherwise altered but still encodes
functional protein (e. g., the upstream regulatory region
can be altered to thereby alter the expression of the
endogenous protein). In the homologous recombination
vector, the altered portion of the gene is flanked at its
5' and 3' ends by additional nucleic acid of the gene to
allow for homologous recombination to occur between the
exogenous gene carried by the vector and an endogenous
gene in an embryonic stem cell. The additional flanking
nucleic acid sequences are of sufficient length for
successful homologous recombination with the endogenous
gene. Typically, several kilobases of flanking DNA (both
at the 5' and 3' ends) are included in the vector (see,
e.g., Thomas and Capecchi (1987) Cell 51:503 for a
description of homologous recombination vectors). The
vector is introduced into an embryonic stem cell line
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(e.g., by electroporation) and cells in which the
introduced gene has homologously recombined with the
endogenous gene are selected (see, e.g., Li et al. (1992)
Cell 69:915). The selected cells are then injected into
a blastocyst of an animal (e. g., a mouse) to form
aggregation chimeras (see, e.g., Bradley in
Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, Robertson, ed. (IRL, Oxford, 1987) pp. 113-
152). A chimeric embryo can then be implanted into a
suitable pseudopregnant female foster animal and the
embryo brought to term. Progeny harboring the
homologously recombined DNA in their germ cells can be
used to breed animals in which all cells of the animal
contain the homologously recombined DNA by germline
transmission of the transgene. Methods for constructing
homologous recombination vectors and homologous
recombinant animals are described further in Bradley
(1991) Current Opinion in Bio/Technology 2:823-829 and in
PCT Publication NOS. WO 90/11354, WO 91/01140, WO
92/0968, and WO 93/04169.
In another embodiment, transgenic non-human
animals can be produced which contain selected systems
which allow for regulated expression of the transgene.
One example of such a system is the cre/loxP recombinase
system of bacteriophage P1. For a description of the
cre/loxP recombinase system, see, e.g., Lakso et al.
(1992) Proc. Natl. Acad. Sci. USA 89:6232-6236. Another
example of a recombinase system is the FLP recombinase
syatem of Saccharomyces cerevisiae (O'Gorman et al.
(1991) Science 251:1351-1355. If a cre/loxP recombinase
system is used to regulate expression of the transgene,
animals containing transgenes encoding both the Cre
recombinase and a selected protein are required. Such
animals can be provided through the construction of
"double" transgenic animals, e.g., by mating two
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transgenic animals, one containing a transgene encoding a
selected protein and the other containing a transgene
encoding a recombinase.
Clones of the non-human transgenic animals
described herein can also be produced according to the
methods described in Wilmut et al. (1997) Nature 385:810-
813 and PCT Publication NOS. WO 97/07668 and WO 97/07669.
_IV Pharmaceutical Compositions
The nucleic acid molecules, polypeptides, and
antibodies (also referred to herein as "active
compounds") of the invention can be incorporated into
pharmaceutical compositions suitable for administration.
Such compositions typically comprise the nucleic acid
molecule, protein, or antibody and a pharmaceutically
acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to
include any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and
agents for pharmaceutically active substances is well
known in the art. Except insofar as any conventional
media or agent is incompatible with the active compound,
use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated
into the compositions.
The invention includes methods for preparing
pharmaceutical compositions for modulating the expression
or activity of a polypeptide or nucleic acid of the
invention. Such methods comprise formulating a
pharmaceutically acceptable carrier with an agent which
modulates expression or activity of a polypeptide or
nucleic acid of the invention. Such compositions can
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further include additionl active agents. Thus, the
invention further includes methods for preparing a
pharmaceutical composition by formulating a
pharmaceutically acceptable carrier with an agent which
modulates expression or activity of a polypeptide or
nucleic acid of the invention and one or more additional
active compounds.
A pharmaceutical composition of the invention is
formulated to be compatible with its intended route of
administration. Examples of routes of administration
include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral (e. g., inhalation), transdermal
(topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the
following components: a sterile diluent such as water for
injection, saline solution,,fixed oils, polyethylene
glycols, glycerine, propylene glycol or other synthetic
solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or
sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the
adjustment of tonicity such as sodium chloride or
dextrose. pH can be adjusted with acids or bases, such
as hydrochloric acid or sodium hydroxide. The parenteral
preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for
injectable use include sterile aqueous solutions (where
water soluble) or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable
solutions or dispersions. For intravenous
administration, suitable carriers include physiological
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saline, bacteriostatic water, Cremophor EL'" (BASF;
Parsippany, NJ) or phosphate buffered saline (PBS). In
all cases, the composition must be sterile and should be
fluid to the extent that easy syringability exists. It
must be stable under the conditions of manufacture and
storage and must be preserved against the contaminating
action of microorganisms such as bacteria and fungi. The
carrier can be a solvent or dispersion medium containing,
for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyetheylene
glycol, and the like), and suitable mixtures thereof.
The proper fluidity can be maintained, for example, by
the use of a coating such as lecithin, by the maintenance
of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various
antibacterial and antifungal agents, for example,
parabens, chlorobutanol, phenol, ascorbic acid,
thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example,
sugars, polyalcohols such as mannitol, sorbitol, sodium
chloride in the composition. Prolonged absorption of the
injectable compositions can be brought about by including
in the composition an agent which delays absorption, for
example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a polypeptide or
antibody) in the required amount in an appropriate
solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle
which contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the
case of sterile powders for the preparation of sterile
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PCT/US99I25178
injectable solutions, the preferred methods of
preparation are vacuum drying and freeze-drying which
yields a powder of the active ingredient plus any
additional desired ingredient from a previously sterile-
filtered solution thereof.
Oral compositions generally include an inert
diluent or an edible carrier. They can be enclosed in
gelatin capsules or compressed into tablets. For the
purpose of oral therapeutic administration, the active
compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules. Oral
compositions can also be prepared using a fluid carrier
for use as a mouthwash, wherein the compound in the fluid
carrier is applied orally and swished and expectorated or
swallowed.
Pharmaceutically compatible binding agents, and/or
adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and
the like can contain any of the following ingredients, or
compounds of a similar nature: a binder such as
microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating
agent such as alginic acid, Primogel, or corn starch; a
lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening
agent such as sucrose or saccharin; or a flavoring agent
such as peppermint, methyl salicylate, or orange
flavoring.
For administration by inhalation, the compounds
are delivered in the form of an aerosol spray from a
pressurized container or dispenser which contains a
suitable propellant, e.g., a gas such as carbon dioxide,
or a nebulizer.
Systemic administration can also be by
transmucosal or transdermal means. For transmucosal or
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transdermal administration, penetrants appropriate to the
barrier to be permeated are used in the formulation.
Such penetrants are generally known in the art, and
include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives.
Transmucosal administration can be accomplished through
the use of nasal sprays or suppositories. For
transdermal administration, the active compounds are
formulated into ointments, salves, gels, or creams as
generally known in the art.
The compounds can also be prepared in the form of
suppositories (e. g., with conventional suppository bases
such as cocoa butter and other glycerides) or retention
enemas for rectal delivery.
In one embodiment, the active compounds are
prepared with carriers that will protect the compound
against rapid elimination from the body, such as a
controlled release formulation, including implants and
microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene
vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Method's
for preparation of such formulations will be apparent to
those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including
liposomes targeted to infected cells with monoclonal
antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be
prepared according to methods known to those skilled in
the art, for example, as described in U.S. Patent No.
4,522,811.
It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit
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form as used herein refers to physically discrete units
suited as unitary dosages for the subject to be treated;
each unit containing a predetermined quantity of active
compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical
carrier. The specification for the dosage unit forms of
the invention are dictated by and directly dependent on
the unique characteristics of the active compound and the
particular therapeutic effect to be achieved, and the
limitations inherent in the art of compounding such an
active compound fox the treatment of individuals.
For antibodies, the preferred dosage is 0.1 mg/kg
to 100 mg/kg of body weight (generally 10 mg/kg to 20
mg/kg). If the antibody is to act in the brain, a dosage
of 50 mg/kg to 100 mg/kg is usually appropriate.
Generally, partially human antibodies and fully human
antibodies have a longer half-life within the human body
than other antibodies. Accordingly, lower dosages and
less frequent administration is often possible.
Modifications such as lipidation can be used to stabilize
antibodies and to enhance uptake and tissue penetration
(e.g., into the brain). A method for lipidation of
antibodies is described by Cruikshank et al. ((1997) J.
Acquired Immune Deficiency Syndromes and Human
Retrovirology 14:193).
The nucleic acid molecules of the invention can be
inserted into vectors and used as gene therapy vectors.
Gene therapy vectors can be delivered to a subject by,
for example, intravenous injection, local administration
(U. S. Patent 5,328,470) or by stereotactic injection
(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA
91:3054-3057). The pharmaceutical preparation~of the
gene therapy vector can include the gene therapy vector
in an acceptable diluent, or can comprise a slow release
matrix in which the gene delivery vehicle is imbedded.
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Alternatively, where the complete gene delivery vector
can be produced intact from recombinant cells, e.g.
retroviral vectors, the pharmaceutical preparation can
include one or more cells which produce the gene delivery
system.
The pharmaceutical compositions can be included in
a container, pack, or dispenser together with
instructions for administration.
V. Uses and Methods of the Invention
The nucleic acid molecules, proteins, protein
homologues, and antibodies described herein can be used
in one or more of the following methods: a) screening
assays; b) detection assays (e. g., chromosomal mapping,
tissue typing, forensic biology); c) predictive medicine
(e.9., diagnostic assays, prognostic assays, monitoring
clinical trials, and pharmacogenomics); and d) methods of
treatment (e.g., therapeutic and prophylactic). For
example, polypeptides of the invention can to used to (i)
modulate cellular proliferation; (ii) modulate cellular
differentiation; and (iii) modulate cell survival. The
isolated nucleic acid molecules of the invention can be
used to express proteins (e. g., via a recombinant
expression vector in a host cell in gene therapy
applications), to detect mRNA (e. g., in a biological
sample) or a genetic lesion, and to modulate activity of
a polypeptide of the invention. In addition, the
polypeptides of the invention can be used to screen drugs
or compounds which modulate activity or expression of a
polypeptide of the invention as well as to treat
disorders characterized by insufficient or excessive
production of a protein of the invention or production
of a form of a protein of the invention which has
decreased or aberrant activity compared to the wild type
protein. In addition, the antibodies of the invention
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can be used to detect and isolate a protein of the and
modulate activity of a protein of the invention.
This invention further pertains to novel agents
identified by the above-described screening assays and
uses thereof for treatments as described herein.
A. Scr nu Assavs
The invention provides a method (also referred to
herein as a "screening assay") for identifying
modulators, i.e., candidate or test compounds or agents
l0 (e.g., peptides, peptidomimetics, small molecules or
other drugs) which bind to polypeptide of the invention
or have a stimulatory or inhibitory effect on, for
example, expression or activity of a polypeptide of the
invention.
In one embodiment, the invention provides assays
for screening candidate or test compounds which bind to
or modulate the activity of the membrane-bound form of a
polypeptide of the invention or biologically active
portion thereof. The test compounds of the present
invention can be obtained using any of the numerous
approaches in combinatorial library methods known in the
art, including: biological libraries; spatially
addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring
deconvolution; the "one-bead one-compound" library
method; and synthetic library methods using affinity
chromatography selection. The biological library
approach is limited to peptide libraries, while the other
four approaches are applicable to peptide, non-peptide
oligomer or small molecule libraries of compounds (Lam
(1997) Anticancer Drug Des. 12:145) .
Examples of methods for the synthesis of molecular
libraries can be found in the art, for example in:
DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909;
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Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;
Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et
al. (1993) Science 261:1303; Carrell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J.
Med. Chem. 37:1233.
Libraries of compounds may be presented in
solution (e. g., Houghten (1992) Bio/Techniques 13:412-
421), or on beads (Lam (1991) Nature 354:82-84), chips
(Fodor (1993) Nature 364:555-556), bacteria (U. S. Patent
No. 5,223,409), spores (Patent NOS. 5,571,698; 5,403,484;
and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl.
Acad. Sci. USA 89:1865-1869) or phage (Scott and Smith
(1990) Science 249:386-390; Devlin (1990) Science
249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci.
USA 87:6378-6382; and Felici (1991) J. Mol. Biol.
222:301-310).
In one embodiment, an assay is a cell-based assay
in which a cell which expresses a membrane-bound form of
a polypeptide of the invention, or a biologically active
portion thereof, on the cell surface is contacted with a
test compound and the ability of the test compound to
bind to the polypeptide determined. The cell, for
example, can be a yeast cell or a cell of mammalian
origin. Determining the ability of the test compound to
bind to the polypeptide can be accomplished, for example,
by coupling the test compound with a radioisotope or
enzymatic label such that binding of the test compound to
the polypeptide or biologically active portion thereof
can be determined by detecting the labeled compound in a
complex. For example, test compounds can be labeled with
~asl ~ asS ~ 14C ~ or 3H, either directly or indirectly, and
the radioisotope detected by direct counting of
radioemmission or by scintillation counting.
Alternatively, test compounds can be enzymatically
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labeled with, for example, horseradish peroxidase,
alkaline phosphatase, or luciferase, and the enzymatic
label detected by determination of conversion of an
appropriate substrate to product. In a preferred
embodiment, the assay comprises contacting a cell which
expresses a membrane-bound form of a polypeptide of the
invention, or a biologically active portion thereof, on
the cell surface with a known compound which binds the
polypeptide to form an assay mixture, contacting the
assay mixture with a test compound, and determining the
ability of the test compound to interact with the
polypeptide, wherein determining the ability of the test
compound to interact with the polypeptide comprises
determining the ability of the test compound to
preferentially bind to the polypeptide or a biologically
active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based
assay comprising contacting a cell expressing a membrane-
bound form of a polypeptide of the invention, or a
biologically active portion thereof, on the cell surface
with a test compound and determining the ability of the
test compound to modulate (e. g., stimulate or inhibit)
the activity of the polypeptide or biologically active
portion thereof. Determining the ability of the test
compound to modulate the activity of the polypeptide or a
biologically active portion thereof can be accomplished,
for example, by determining the ability of the
polypeptide protein to bind to or interact with a target
molecule.
Determining the ability of a polypeptide of the
invention to bind to or interact with a target molecule
can be accomplished by one of the methods described above
for determining direct binding. As used herein, a
"target molecule" is a molecule with which a selected
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polypeptide (e. g., a polypeptide of the invention binds
or interacts with in nature, for example, a molecule on
the surface of a cell which expresses the selected
protein, a molecule on the surface of a second cell, a
molecule in the extracellular milieu, a molecule
associated with the internal surface of a cell membrane
or a. cytoplasmic molecule. A target molecule can be a
polypeptide of the invention or some other polypeptide or
protein. For example, a target molecule can be a
component of a signal transduction pathway which
facilitates transduction of an extracellular signal
(e.g., a signal generated by binding of a compound to a
polypeptide of the invention) through the cell membrane
and into the cell or a second intercellular protein which
has catalytic activity or a protein which facilitates the
association of downstream signaling molecules with a
polypeptide of the invention. Determining the ability of
a polypeptide of the invention to bind to or interact
with a target molecule can be accomplished by determining
the activity of the target molecule. For example, the
activity of the target molecule can be determined by
detecting induction of a cellular second messenger of the
target (e. g., intracellular Ca2+, diacylglycerol, IP3,
etc.), detecting catalytic/enzymatic activity of the
target on an appropriate substrate, detecting the
induction of a reporter gene (e. g., a regulatory element
that is responsive to a polypeptide of the invention
operably linked to a nucleic acid encoding a detectable
marker, e.g. luciferase), or detecting a cellular
response, for example, cellular differentiation, or cell
proliferation.
In yet another embodiment, an assay of the present
invention is a cell-free assay comprising contacting a
polypeptide of the invention or biologically active
portion thereof with a test compound and determining the
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ability of the test compound to bind to the polypeptide
or biologically active portion thereof. Binding of the
test compound to the polypeptide can be determined either
directly or indirectly as described above. In a
preferred embodiment, the assay includes contacting the
polypeptide of the invention or biologically active
portion thereof with a known compound which binds the
polypeptide to form an assay mixture, contacting the
assay mixture with a test compound, and determining the
ability of the test compound to interact with the
polypeptide, wherein determining the ability of the test
compound to interact with the polypeptide comprises
determining the ability of the test compound to
preferentially bind to the polypeptide or biologically
active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-free
assay comprising contacting a polypeptide of the
invention or biologically active portion thereof with a
test compound and determining the ability of the test
compound to modulate (e.g., stimulate or inhibit) the
activity of the polypeptide or biologically active
portion thereof. Determining the ability of the test
compound to modulate the activity of the polypeptide can
be accomplished, for example, by determining the ability
of the polypeptide to bind to a target molecule by one of
the methods described above for determining direct
binding. In an alternative embodiment, determining the
ability of the test compound to modulate the activity of
the polypeptide can be accomplished by determining the
ability of the polypeptide of the invention to further
modulate the target molecule. For example, the
catalytic/enzymatic activity of the target molecule on an
appropriate substrate can be determined as previously
described.
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In yet another embodiment, the cell-free assay
comprises contacting a polypeptide of the invention or
biologically active portion thereof with a known compound
which binds the polypeptide to form an assay mixture,
contacting the assay mixture with a test compound, and
determining the ability of the test compound to interact
with the polypeptide, wherein determining the ability of
the test compound to interact with the polypeptide
comprises determining the ability of the polypeptide to
preferentially bind to or modulate the activity of a
target molecule.
The cell-free assays of the present invention are
amenable to use of both a soluble form or the membrane-
bound form of a polypeptide of the invention. In the
case of cell-free assays comprising the membrane-bound
form of the polypeptide, it may be desirable to utilize a
solubilizing agent such that the membrane-bound form of
the polypeptide is maintained in solution. Examples of
such solubilizing agents include non-ionic detergents
such as n-octylglucoside, n-dodecylglucoside, n-
octylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-
methylglucamide, Triton X-100, Triton X-114, Thesit,
Isotridecypoly(ethylene glycol ether)n, 3-[(3-
cholamidopropyl)dimethylamminio]-1-propane sulfonate
(CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-
hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl=N,N-
dimethyl-3-ammonio-1-propane sulfonate.
In more than one embodiment of the above assay
methods of the present invention, it may be desirable to
immobilize either the polypeptide of the invention or its
target molecule to facilitate separation of coinplexed
from uncomplexed forms of one or both of the proteins, as
well as to accommodate automation of the assay. Binding
of a test compound to the polypeptide, or interaction of
the polypeptide with a target molecule in the presence
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and absence of a candidate compound, can be accomplished
in any vessel suitable for containing the reactants.
Examples of such vessels include microtitre plates, test
tubes, and micro-centrifuge tubes. In one embodiment, a
fusion protein can be provided which adds a domain that
allows one or both of the proteins to be bound to a
matrix. For example, glutathione-S-transferase fusion
proteins or glutathione-S-transferase fusion proteins can
be adsorbed onto glutathione sepharose beads (Sigma
Chemical; St. Louis, MO) or glutathione derivatized
microtitre plates, which are then combined with the test
compound or the test compound and either the non-adsorbed
target protein or A polypeptide of the invention, and the
mixture incubated under conditions conducive to complex
formation (e.g., at physiological conditions for salt and
pH). Following incubation, the beads or microtitre plate
wells are washed to remove any unbound components and
complex formation is measured either directly or
indirectly, for example, as described above.
Alternatively, the complexes can be dissociated from the
matrix, and the level of binding or activity of the
polypeptide of the invention can be determined using
standard techniques.
Other techniques for immobilizing proteins on
matrices can also be used in the screening assays of the
invention. For example, either the polypeptide of the
invention or its target molecule can be immobilized
utilizing conjugation of biotin and streptavidin.
Biotinylated polypeptide of the invention or target
molecules can be prepared from biotin-NHS (N-hydroxy-
succinimide) using techniques well known in the art
(e. g., biotinylation kit, Pierce Chemicals; Rockford,
IL), and immobilized in the wells of streptavidin-coated
96 well plates (Pierce Chemical). Alternatively,
antibodies reactive with the polypeptide of the invention
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or target molecules but which do not interfere with
binding of the polypeptide of the invention to its target
molecule can be derivatized to the wells of the plate,
and unbound target or polypeptidede of the invention
trapped in the wells by antibody conjugation. Methods
for detecting such complexes, in addition to those
described above for the GST-immobilized complexes,
include immunodetection of complexes using antibodies
reactive with the polypeptide of the invention or target
20 molecule, as well as enzyme-linked assays which rely on
detecting an enzymatic activity associated with the
polypeptide of the invention or target molecule.
In another embodiment, modulators of expression of
a polypeptide of the invention are identified in a method
in which a cell is contacted with a candidate compound
and the expression of the selected mRNA or protein (i.e.,
the mRNA or protein corresponding to a polypeptide or
nucleic acid of the invention) in the cell is determined.
The level of expression of the selected mRNA or protein
in the presence of the candidate compound is compared to
the level of expression of the selected mRNA or protein
in the absence of the candidate compound. The candidate
compound can then be identified as a modulator of
expression of the polypeptide of the invention based on
this comparison. For example, when expression of the
selected mRNA or protein is greater (statistically
significantly greater) in the presence of the candidate
compound than in its absence, the candidate compound is
identified as a stimulator of the selected mRNA or
protein expression. Alternatively, when expression of
the selected mRNA or protein is less (statistically
significantly less) in the presence of the candidate
compound than in its absence, the candidate compound is
identified as an inhibitor of the selected mRNA or
protein expression. The level of the selected mRNA or
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protein expression in the cells can be determined by
methods described herein.
In yet another aspect of the invention, a
polypeptide of the inventions can be used as "bait
proteins" in a two-hybrid assay or three hybrid assay
(aee, e.g., U.S. Patent No. 5,283,317; Zervos et al.
(1993) Cell 72:223-232; Madura et al. (1993) J. Biol.
Chem. 268:12046-12054; Bartel et al. (1993)
Bio/Techniques 14:920-924; Iwabuchi et al. (1993)
Oncogene 8:1693-1696; and PCT Publication No. WO
94/10300), to identify other proteins, which bind to or
interact with the polypeptide of the invention and
modulate activity of the polypeptide of the invention.
Such binding proteins are also likely to be involved in
the propagation of signals by the polypeptide of the
inventions as, for example, upstream or downstream
elements of a signaling pathway involving the polypeptide
of the invention.
This invention further pertains to novel agents
identified by the above-described screening assays and
uses thereof for treatments as described herein.
B. Detection Assavs
Portions or fragments of the cDNA sequences
identified herein (and the corresponding complete gene
sequences) can be used in numerous ways as polynucleotide
reagents. For example, these sequences can be used to:
(i) map their respective genes on a chromosome and, thus,
locate gene regions associated with genetic disease; (ii)
identify an individual from a minute biological sample
(tissue typing); and (iii) aid in forensic identification
of a biological sample. These applications are described
in the subsections below.
1. Chromosome Mapping
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Once the sequence (or a portion of the sequence)
of a gene has been isolated, this sequence can be used to
map the location of the gene on a chromosome.
Accordingly, nucleic acid molecules described herein or
fragments thereof, can be used to map the location of the
corresponding genes on a chromosome. The mapping of the
sequences to chromosomes is an important first step in
correlating these sequences with genes associated with
disease.
Briefly, genes can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 by in length)
from the sequence of a gene of the invention. Computer
analysis of the sequence of a gene of the invention can
be used to rapidly select primers that do not span more
than one exon in the genomic DNA, thus complicating the
amplification process. These primers can then be used
for PCR screening of somatic cell hybrids containing
individual human chromosomes. Only those hybrids
containing the human gene corresponding to the gene
sequences will yield an amplified fragment. For a review
of this technique, see D'Eustachio et al. ((1983) Science
220:919-924).
PCR mapping of somatic cell hybrids is a rapid
procedure for assigning a particular sequence to a
particular chromosome. Three or -more sequences can be
assigned per day using a single thermal cycler. Using
the nucleic acid sequences of the invention to design
oligonucleotide primers, sublocalization can be achieved
with panels of fragments from specific chromosomes.
Other mapping strategies which can similarly be used to
map a gene to its chromosome include in situ
hybridization (described in Fan et al. (1990) Proc. Natl.
Acad. Sci. USA 87:6223-27), pre-screening with labeled
flow-sorted chromosomes (CITE), and pre-selection by
hybridization to chromosome specific cDNA libraries.
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Fluorescence in situ hybridization (FISH) of a DNA
sequence to a metaphase chromosomal spread can further be
used to provide a precise chromosomal location in one
step. For a review of this technique, see Verma et al.,
(Human Chromosomes: A Manual of Basic Techniques
(Pergamon Press, New York, 1988)).
Reagents for chromosome mapping can be used
individually to mark a single chromosome or a single site
on that chromosome, or panels of reagents can be used for
marking multiple sites and/or multiple chromosomes.
Reagents corresponding to noncoding regions of the genes
actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene
families, thus increasing the chance of cross
hybridizations during chromosomal mapping.
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 disease, mapped to the
same chromosomal region, can then be identified through
linkage analysis (co-inheritance of physically adjacent
genes), described in, e.g., Egeland et al. (1987) Nature
325:783-787.
Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease
associated with a gene of the invention can be
determined. If a mutation is observed in some or all of
the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the
causative agent of the particular disease. Comparison of
affected and unaffected individuals generally involves
first looking for structural alterations in the
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chromosomes such as deletions or translocations that are
visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete
sequencing of genes from several individuals can be
performed to confirm the presence of a mutation and to
distinguish mutations from polymorphisms.
2. Tissue Typing
The nucleic acid sequences of the present
invention can also be used to identify individuals from
minute biological samples. The United States military,
for example, is considering the use of restriction
fragment length polymorphism (RFLP) for identification of
its personnel. In this technique, an individual's
genomic DNA is digested with one or more restriction
enzymes, and probed on a Southern blot to yield unique
bands for identification. This method does not suffer
from the current limitations of "Dog Tags" which can be
lost, switched, or stolen, making positive identification
difficult. The sequences of the present invention are
useful as additional DNA markers for RFLP (described in
U.S. Patent 5,272,057).
Furthermore, the sequences of the present
invention can be used to provide an alternative technique
which determines the actual base-by-base DNA sequence of
selected portions of an individual's genome. Thus, the
nucleic acid sequences described herein can be used to
prepare two PCR primers from the 5' and 3' ends of the
sequences. These primers can then be used to amplify an
individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from
individuals, prepared in this manner, can provide unique
individual identifications, as each individual will have
a unique set of such DNA sequences due to allelic
differences. The sequences of the present invention can
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be used to obtain such identification sequences from
individuals and from tissue. The nucleic acid sequences
of the invention uniquely represent portions of the human
genome. Allelic variation occurs to some degree in the
coding regions of these sequences, and to a greater
degree in the noncoding regions. It is estimated that
allelic variation between individual humans occurs with a
frequency of about once per each 500 bases. Each of the
sequences described herein can, to some degree, be used
as a standard against which DNA from an individual can be
compared for identification purposes. Because greater
numbers of polymorphisms occur in the noncoding regions,
fewer sequences are necessary to differentiate
individuals. The noncoding sequences of SEQ ID NO:1 or 4
can comfortably provide positive individual
identification with a panel of perhaps 20 to 1,000
primers which each yield a noncoding amplified sequence
of 100 bases. If predicted coding sequences, such as
those in SEQ ID N0:3 or 6 are used, a more appropriate
number of primers for positive individual identification
would be 500-2,000.
If a panel of reagents from the nucleic acid
sequences described herein is used to generate a unique
identification database for an individual, those same
reagents can later be used to identify tissue from that
individual. Using the unique identification database,
positive identification of the individual, living or
dead, can be made from extremely small tissue samples.
3. Use of Partial Gene Seauences in Forensic Biolo
DNA-based identification techniques can also be
used in forensic biology. Forensic biology is a
scientific field employing genetic typing of biological
evidence found at a crime scene as a means for positively
identifying, for example, a perpetrator of a crime. To
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make such an identification, PCR technology can be used
to amplify DNA sequences taken from very small biological
samples such as tissues, e.g., hair or skin, or body
fluids, e.g., blood, saliva, or semen found at a crime
scene. The amplified sequence can then be compared to a
standard, thereby allowing identification of the origin
of the biological sample.
The sequences of the present invention can be used
to provide polynucleotide reagents, e.g., PCR primers,
targeted to specific loci in the human genome, which can
enhance the reliability of DNA-based forensic
identifications by, for example, providing another
"identification marker" (i.e. another DNA sequence that
is unique to a particular individual). As mentioned
above, actual base sequence information can be used for
identification as an accurate alternative to patterns
formed by restriction enzyme generated fragments.
Sequences targeted to noncoding regions are particularly
appropriate for this use as greater numbers of
polymorphisms occur in the noncoding regions, making it
easier to differentiate individuals using this technique.
Examples of polynucleotide reagents include the nucleic
acid sequences of the invention or portions thereof,
e.g., fragments derived from noncoding regions having a
length of at least 20 or 30 bases.
The nucleic acid sequences described herein can
further be used to provide polynucleotide reagents, e.g.,
labeled or labelable probes which can be used in, for
example, an in situ hybridization technique, to identify
a specific tissue, e.g., brain tissue. This can be very
useful in cases where a forensic pathologist is presented
with a tissue of unknown origin. Panels of such probes
can be used to identify tissue by species and/or by organ
type.
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C. Predictive Medicine
The present invention also pertains to the field
of predictive medicine in which diagnostic assays,
prognostic assays, pharmacogenomics, and monitoring
clinical trails are used for prognostic (predictive)
purposes to thereby treat an individual prophylactically.
Accordingly, one aspect of the present invention relates
to diagnostic assays for determining expression of a
polypeptide or nucleic acid of the invention and/or
activity of a polypeptide of the invention, in the
context of a biological sample (e. g., blood; serum,
cells, tissue) to thereby determine whether an individual
is afflicted with a disease or disorder, or is at risk of
developing a disorder, associated with aberrant
expression or activity of a polypeptide of the invention.
The invention also provides for prognostic (or
predictive) assays for determining whether an individual
is at risk of developing a disorder associated with
aberrant expression or activity of a polypeptide of the
invention. For example, mutations in a gene of the
invention can be assayed in a biological sample. Such
assays can be used for prognostic or predictive purpose
to thereby prophylactically treat an individual prior to
the onset of a disorder characterized by or associated
with aberrant expression or activity of a polypeptide of
the invention.
Another aspect of the invention provides methods
for expression of a nucleic acid or polypeptide of the
invention or activity of a polypeptide of the invention
in an individual to thereby select appropriate
therapeutic or prophylactic agents for that individual
(referred to herein as ~~pharmacogenomics ~~ ) .
Pharmacogenomics allows for the selection of agents
(e. g., drugs) for therapeutic or prophylactic treatment
of an individual based on the genotype of the individual
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(e.g., the genotype of the individual examined to
determine the ability of the individual to respond to a
particular agent).
Yet another aspect of the invention pertains to
monitoring the influence of agents (e. g., drugs or other
compounds) ow the expression or activity of a polypeptide
of the invention in clinical trials. These and other
agents are described in further detail in the following
sections.
1. Diagnostic Assays
An exemplary method for detecting the presence or
absence of a polypeptide or nucleic acid of the invention
in a biological sample involves obtaining a biological
sample from a test subject and contacting the biological
sample with a compound or an agent capable of detecting a
polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of
the invention such that the presence of a polypeptide or
nucleic acid of the invention is detected in the
biological sample. A preferred agent for detecting mRNA
or genomic DNA encoding a polypeptide of the invention is
a labeled nucleic acid probe capable of hybridizing to
mRNA or genomic DNA encoding a polypeptide of the
invention. The nucleic acid probe can be, for example, a
full-length cDNA, such as the nucleic acid of SEQ ID NO:
1, 3, 4, or 6, or a portion thereof, such as an
oligonucleotide of at least 15, 30, 50, 100, 250 or 500
nucleotides in length and sufficient to specifically
hybridize under stringent conditions to a mRNA or genomic
DNA encoding a polypeptide of the invention. Other
suitable probes for use in the diagnostic assays of the
invention are described herein.
A preferred agent for detecting a polypeptide of
the invention is an antibody capable of binding to a
polypeptide of the invention, preferably an antibody with
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a detectable label. Antibodies can be polyclonal, or
more preferably, monoclonal. An intact antibody, or a
fragment thereof (e.g., Fab or F(ab')2) can be used. The
term "labeled", with regard to the probe or antibody, is
intended to encompass direct labeling of the probe or
antibody by coupling (i.e., physically linking) a
detectable substance to the probe or antibody, as well as
indirect labeling of the probe or antibody by reactivity
with another reagent that is directly labeled. Examples
of indirect labeling include detection of a primary
antibody using a fluorescently labeled secondary antibody
and end-labeling of a DNA probe with biotin such that it
can be detected with fluorescently labeled streptavidin.
The term "biological sample" is intended to include
tissues, cells and biological fluids isolated from a
subject, as well as tissues, cells and fluids present
within a subject. That is, the detection method of the
invention can be used to detect mRNA, protein, or genomic
DNA in a biological sample in vitro as well as in vivo.
For example, in vitro techniques for detection of mRNA
include Northern hybridizations and in situ
hybridizations. In vitro techniques for detection of a
polypeptide of the invention include enzyme linked
immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. In vitro
techniques for detection of genomic DNA include Southern
hybridizations. Furthermore, in vivo techniques for
detection of a polypeptide of the invention include
introducing into a subject a labeled antibody directed
against the polypeptide. For example, the antibody can
be labeled with a radioactive marker whose presence and
location in a subject can be detected by standard imaging
techniques.
In one embodiment, the biological sample contains
protein molecules from the test subject. Alternatively,
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the biological sample can contain mRNA molecules from the
test subject or genomic DNA molecules from the test
subject. A preferred biological sample is a peripheral
blood leukocyte sample isolated by conventional means
from a subject.
In another embodiment, the methods further involve
obtaining a control biological sample from a control
subject, contacting the control sample with a compound or
agent capable of detecting a polypeptide of the invention
or mRNA or genomic DNA encoding a polypeptide of the
invention, such that the presence of the polypeptide or
mRNA or genomic DNA encoding the polypeptide is detected
in the biological sample, and comparing the presence of
the polypeptide or mRNA or genomic DNA encoding the
polypeptide in the control sample with the presence of
the polypeptide or mRNA or genomic DNA encoding the
polypeptide in the test sample.
The invention also encompasses kits for detecting
the presence of a polypeptide or nucleic acid of the
invention in a biological sample (a test sample). Such
kits can be used to determine if a subject is suffering
from or is at increased risk of developing a disorder
associated with aberrant expression of a polypeptide of
the invention (e.g., a disorder of lipid metabolism or
transport). For example, the kit can comprise a labeled
compound or agent capable of detecting the polypeptide or
mRNA encoding the polypeptide in a biological sample and
means for determining the amount of the polypeptide or
mRNA in the sample (e.g., an antibody which binds the
polypeptide or an oligonucleotide probe which binds to
DNA or mRNA encoding the polypeptide). Kits may also
include instructions for observing that the tested
subject is suffering from or is at risk of developing a
disorder associated with aberrant expression of the
polypeptide if the amount of the polypeptide or mRNA
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encoding the polypeptide is above or below a normal
level.
For antibody-based kits, the kit may comprise, for
example: (1) a first antibody (e. g., attached to a solid
support) which binds to a polypeptide of the invention;
and, optionally, (2) a second, different antibody which
binds to either the polypeptide or the first antibody and
is conjugated to a detectable agent.
For oligonucleotide-based kits, the kit may
comprise, for example: (1) an oligonucleotide, e.g., a
detestably labeled oligonucleotide, which hybridizes to a
nucleic acid sequence encoding a polypeptide of the
invention or (2) a pair of primers useful for amplifying
a nucleic acid molecule encoding a polypeptide of the
invention.
The kit may also comprise, e.g., a buffering agent, a
preservative, or a protein stabilizing agent. The kit
may also comprise components necessary for detecting the
detectable agent (e.g., an enzyme or a substrate). The
kit may also contain a control sample or a series of
control samples which can be assayed and compared to the
test sample contained. Each component of the kit is
usually enclosed within an individual container and all
of the various containers are within a single package
along with instructions for observing whether the tested
subject is suffering from or is at risk of developing a
disorder associated with aberrant expression of the
polypeptide.
2. Proctnostic Assays
The methods described herein can furthermore be
utilized as diagnostic or prognostic assays to identify
subjects having or at risk of developing a disease or
disorder associated with aberrant expression or activity
of a polypeptide of the invention. For example, the
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assays described herein, such as the preceding diagnostic
assays or the following assays, can be utilized to
identify a subject having or at risk of developing a
disorder associated with aberrant expression or activity
of a polypeptide of the invention. Alternatively, the
prognostic assays can be utilized to identify a subject
having or at risk for developing such a disease or
disorder. Thus, the present invention provides a method
in which a test sample is obtained from a subject and a
polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of
the invention is detected, wherein the presence of the
polypeptide or nucleic acid is diagnostic for a subject
having or at risk of developing a disease or disorder
associated with aberrant expression or activity of the
polypeptide. As used herein, a "test sample" refers to a
biological sample obtained from a subject of interest.
For example, a test sample can be a biological fluid
(e. g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described
herein can be used to determine whether a subject can be
administered an agent (e. g., an agonist, antagonist,
peptidomimetic, protein, peptide, nucleic acid, small
molecule, or other drug candidate) to treat a disease or
disorder associated with aberrant expression or activity
of a polypeptide of the invention. For example, such
methods can be used to determine whether a subject can be
effectively treated with a specific agent or class of
agents (e.g., agents of a type which decrease activity of
the polypeptide). Thus, the present invention provides
methods for determining whether a subject can be
effectively treated with an agent for a disorder
associated with aberrant expression or activity of a
polypeptide of the invention in which a test sample is
obtained and the polypeptide or nucleic acid encoding the
polypeptide is detected (e.g., wherein the presence of
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the polypeptide or nucleic acid is diagnostic for a
subject that can be administered the agent to treat a
disorder associated with aberrant expression or activity
of the polypeptide).
The methods of the invention can also be used to
detect genetic lesions or mutations in a gene of the
invention, thereby determining if a subject with the
lesioned gene is at risk for a disorder characterized
aberrant expression or activity of a polypeptide of the
invention. In preferred embodiments, the methods include
detecting, in a sample of cells from the subject, the
presence or absence of a genetic lesion or mutation
characterized by at least one of an alteration affecting
the integrity of a gene encoding the polypeptide of the
invention, or the mis-expression of the gene encoding the
polypeptide of the invention. For example, such genetic
lesions or mutations can be detected by ascertaining the
existence of at least one of: 1) a deletion of one or
more nucleotides from the gene; 2) an addition of one or
more nucleotides to the gene; 3) a substitution of one or
more nucleotides of the gene; 4) a chromosomal
rearrangement of the gene; 5) an alteration in the level
of a messenger RNA transcript of the gene; 6) an aberrant
modification of the gene, such as of the methylation
pattern of the genomic DNA; 7) the presence of a non-wild
type splicing pattern of a messenger RNA transcript of
the gene; 8) a non-wild type level of a the protein
encoded by the gene; 9) an allelic loss of the gene; and
10) an inappropriate post-translational modification of
the protein encoded by the gene. As described herein,
there are a large number of assay techniques known in the
art which can be used for detecting lesions in a gene.
In certain embodiments, detection of the lesion
involves the use of a probe/primer in a polymerase chain
reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and
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4,683,202), such as anchor PCR or RACE PCR, or,
alternatively, in a ligation chain reaction (LCR) (see,
e.g., Landegran et al. (1988) Science 241:1077-1080; and
Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-
364), the latter of which can be particularly useful for
detecting point mutations in a gene (see, e.g., Abravaya
et al. (1995} Nucleic Acids Res. 23:675-682). This
method can include the steps of collecting a sample of
cells from a patient, isolating nucleic acid (e. g.,
genomic, mRNA or both) from the cells of the sample,
contacting the nucleic acid sample with one or more
primers which specifically hybridize to the selected gene
under conditions such that hybridization and
amplification of the gene (if present) occurs, and
detecting the presence or absence of an amplification
product, or detecting the size of the amplification
product and comparing the length to a control sample. It
is anticipated that PCR and/or LCR may be desirable to
use as a preliminary amplification step in conjunction
with any of the techniques used for detecting mutations
described herein.
Alternative amplification methods include: self
sustained sequence replication (Guatelli et al. (1990)
Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional
amplification system (Kwoh, et al. (1989) Proc. Natl.
Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi
et al. (1988) Bio/Technology 6:1197), or any other
nucleic acid amplification method, followed by the
detection of the amplified molecules using techniques
well known to those of skill in the art. These detection
schemes are especially useful for the detection of
nucleic acid molecules if such molecules are present in
very low numbers.
In an alternative embodiment, mutations in a
selected gene from a sample cell can be identified by
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alterations in restriction enzyme cleavage patterns. For
example, sample and control DNA is isolated, amplified
(optionally), digested with one or more restriction
er_donucleases, and fragment length sizes are determined
by gel electrophoresis and compared. Differences in
fragment length sizes between sample and control DNA
indicates mutations in the sample DNA. Moreover, the use
of sequence specific ribozymes (see, e.g., U.S. Patent
No. 5,498,531) can be used to score for the presence of
specific mutations by development or loss of a ribozyme
cleavage site.
In other embodiments, genetic mutations can be
identified by hybridizing a sample and control nucleic
acids, e.g., DNA or RNA, to high density arrays
containing hundreds or thousands of oligonucleotides
probes (Cronin et al. (1996) Human Mutation 7:244-255;
Kozal et al. (1996) Nature Medicine 2:753-759). For
example, genetic mutations can be identified in two-
dimensional arrays containing light-generated DNA probes
as described in Cronin et al., supra. Briefly, a first
hybridization array of probes can be used to scan through
long stretches of DNA in a sample and control to identify
base changes between the sequences by making linear
arrays of sequential overlapping probes. This step
allows the identification of point mutations. This step
is followed by a second hybridization array that allows
the characterization of specific mutations by using
smaller, specialized probe arrays complementary to all
variants or mutations detected. Each mutation array is
composed of parallel probe sets, one complementary to the
wild-type gene and the other complementary to the mutant
gene.
In yet another embodiment, any of a variety of
sequencing reactions known in the art can be used to
directly sequence the selected gene and detect mutations
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by comparing the sequence of the sample nucleic acids
with the corresponding wild-type (control) sequence.
Examples of sequencing reactions include those based on
techniques developed by Maxim and Gilbert ((1977) Proc.
Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc.
Natl. Acad. Sci. USA 74:5463). It is also contemplated
that any of a variety of automated sequencing procedures
can be utilized when performing the diagnostic assays
((1995) Bio/Techniques 19:448), including sequencing'by
mass spectrometry (see, e.g., PCT Publication No. WO
94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-
162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.
38:147-159).
Other methods for detecting mutations in a
selected gene include methods in which protection from
cleavage agents is used to detect mismatched bases in
RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985)
Science 230:1242). In general, the technique of
"mismatch cleavage" entails providing heteroduplexes
formed by hybridizing (labeled) RNA or DNA containing the
wild-type sequence with potentially mutant RNA or DNA
obtained from a tissue sample. The double-stranded
duplexes are treated with an agent which cleaves single-
stranded regions of the duplex such as which will exist
due to basepair mismatches between the control and sample
strands. RNA/DNA duplexes can be treated with RNase to
digest mismatched regions, and DNA/DNA hybrids can be
treated with S1 nuclease to digest mismatched regions.
In other embodiments, either DNA/DNA or RNA/DNA duplexes
can be treated with hydroxylamine or osmium tetroxide and
with piperidine in order to digest mismatched regions.
After digestion of the mismatched regions, the resulting
material is then separated by size on denaturing
polyacrylamide gels to determine the site of mutation.
See, e.g., Cotton et al. (1988) Proc. Natl. Acad. Sci.
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USA 85:4397; Saleeba et al. (1992) Methods Enzymol.
217:286-295. In a preferred embodiment, the control DNA
or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage
reaction employs one or more proteins that recognize
mismatched base pairs in double-stranded DNA (so called
"DNA mismatch repair" enzymes) in defined systems for
detecting and mapping point mutations in cDNAs obtained
from samples of cells. For example, the mutt enzyme of
E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches
(Hsu et al. (1994) Carcinogenesis 15:1657-1662).
According to an exemplary embodiment, a probe based on a
selected sequence, e.g., a wild-type sequence, is
hybridized to a cDNA or other DNA product from a test
cell(s). The duplex is treated with a DNA mismatch
repair enzyme, and the cleavage products, if any, can be
detected from electrophoresis protocols or the like.
See, e.g., U.S. Patent No. 5,459,039.
In other embodiments, alterations in
electrophoretic mobility will be used to identify
mutations in genes. For example, single strand
conformation polymorphism (SSCP) may be used to detect
differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989) Proc.
Natl. Acad. Sci. USA 86:2766; see also Cotton (1993)
Mutat. Res. 285:125-144; Hayashi (1992) Genet. Anal.
Tech. Appl. 9:73-79). Single-stranded DNA fragments of
sample and control nucleic acids will be denatured and
allowed to renature. The secondary structure of single-
stranded nucleic acids varies according to sequence, and
the resulting alteration in electrophoretic mobility
enables the detection of even a single base change. The
DNA fragments may be labeled or detected with labeled
probes. The sensitivity of the assay may be enhanced by
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using RNA (rather than DNA), in which the secondary
structure is more sensitive to a change in sequence. In
a preferred embodiment, the subject method utilizes
heteroduplex analysis to separate double stranded
heteroduplex molecules on the basis of changes in
electrophoretic mobility (Keen et al. (1991) Trends
Genet. 7:5) .
In yet another embodiment, the movement of mutant
or wild-type fragments in polyacrylamide gels containing
a gradient of denaturant is assayed using denaturing
gradient gel electrophoresis (DGGE) (Myers et al. (1985)
Nature 313:495). When DGGE is used as the method of
analysis, DNA will be modified to insure that it does not
completely denature, for example by adding a 'GC clamp of
approximately 40 by of high-melting GC-rich DNA by PCR.
In a further embodiment, a temperature gradient is used
in place of a denaturing gradient to identify differences
in the mobility of control and sample DNA (Rosenbaum and
Reissner (1987) Biophys. Chem. 265:12753).
Examples of other techniques for detecting point
mutations include, but are not limited to, selective
oligonucleotide hybridization, selective amplification,
or selective primer extension. For example,
oligonucleotide primers may be prepared in which the
known mutation is placed centrally and then hybridized to
target DNA under conditions which permit hybridization
only if a perfect match is found (Saiki et al. (1986)
Nature 324:163); Saiki et al. (1989} Proc. Natl. Acad.
Sci. USA 86:6230). Such allele specific oligonucleotides
are hybridized to PCR amplified target DNA or a number of
different mutations when the oligonucleotides are
attached to the hybridizing membrane and hybridized with
labeled target DNA.
Alternatively, allele specific amplification
technology which depends on selective PCR amplification
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may be used in conjunction with the instant invention.
Oligonucleotides used as primers for specific
amplification may carry the mutation of interest in the
center of the molecule (so that amplification depends on
differential hybridization) (Gibbs et al. (1989) Nucleic
Acids Res. 17:2437-2448) or at the extreme 3' end of one
primer where, under appropriate conditions, mismatch can
prevent or reduce polymerase extension (Prossner (1993)
Tibtech 11:238). In addition, it may be desirable to
introduce a novel restriction site in the region of the
mutation to create cleavage-based detection (Gasparini et
al. (1992) Mol. Cell Probes 6:1). It is anticipated that
in certain embodiments amplification may also be
performed using Taq ligase for amplification (Barany
I5 (1991) Proc. Natl. Acad. Sci. USA 88:189). In such
cases, ligation will occur only if there is a perfect
match at the 3' end of the 5' sequence making it possible
to detect the presence of a known mutation at a specific
site by looking for the presence or absence of
amplification.
The methods described herein may be performed, for
example, by utilizing pre-packaged diagnostic kits
comprising at least one probe nucleic acid or antibody
reagent described herein, which may be conveniently used,
e.g., in clinical settings to diagnose patients
exhibiting symptoms or family history of a disease or
illness involving a gene encoding a polypeptide of the
invention. Furthermore, any cell type or tissue,
preferably peripheral blood leukocytes, in which the
polypeptide of the invention is expressed may be utilized
in the prognostic assays described herein.
3. Pharmacoc~enomics
Agents, or modulators which have a stimulatory or
inhibitory effect on activity or expression of a
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polypeptide of the invention as identified by a screening
assay described herein can be administered to individuals
to treat (prophylactically or therapeutically) disorders
associated with aberrant activity of the polypeptide. In
conjunction with such treatment, the pharmacogenomics
(i.e., the study of the relationship between an
individual's genotype and that individual's response to a
foreign compound or drug) of the individual may be
considered. Differences in metabolism of therapeutics
can lead to severe toxicity or therapeutic failure by
altering the relation between dose and blood
concentration of the pharmacologically active drug. Thus,
the pharmacogenomics of the individual permits the
selection of effective agents (e.g., drugs) for
prophylactic or therapeutic treatments based on a
consideration of the individual's genotype. Such
pharmacogenomics can further be used to determine
appropriate dosages and therapeutic regimens.
Accordingly, the activity of a polypeptide of the
invention, expression of a nucleic acid of the
invention, or mutation content of a gene of the invention
in an individual can be determined to thereby select
appropriate agents) for therapeutic or prophylactic
treatment of the individual.
Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to
altered drug disposition and abnormal action in affected
persons. See, e.g., Linder {1997) Clin. Chem. 43(2):254-
266. In general, two types of pharmacogenetic conditions
can be differentiated. Genetic conditions transmitted as
a single factor altering the way drugs act on the body
are referred to as "altered drug action." Genetic
conditions transmitted as single factors altering the way
the body acts on drugs are referred to as "altered drug
metabolism". These pharmacogenetic conditions can occur
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either as rare defects or as polymorphisms. For example,
glucose-6-phosphate dehydrogenase deficiency (G6PD) is a
common inherited enzymopathy in which the main clinical
complication is haemolysis after ingestion of oxidant
drugs (anti-malarials, sulfonamides, analgesics,
nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of
drug metabolizing enzymes is a major determinant of both
the intensity and duration of drug action. The discovery
of genetic polymorphisms of drug metabolizing enzymes
(e. g., N-acetyltransferase 2 (NAT 2) and cytochrome P450
enzymes CYP2D6 and CYP2C19) has provided an explanation
as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious
toxicity after taking the standard and safe dose of a
drug. These polymorphisms are expressed in two
phenotypes in the population, the extensive metabolizer
(EM) and poor metabolizer (PM). The prevalence of PM is
different among different populations. For example, the
gene coding for CYP2D6 is highly polymorphic and several
mutations have been identified in PM, which all lead to
the absence of functional CYP2D6. Poor metabolizers of
CYP2D6 and CYP2C19 quite frequently experience
exaggerated drug response and side effects when they
receive standard doses. If a metabolite is the active
therapeutic moiety, a PM will show no therapeutic
response, as demonstrated for the analgesic effect of
codeine mediated by its CYP2D6-formed metabolite
morphine. The other extreme are the so called ultra-
rapid metabolizers who do not respond to standard doses.
Recently, the molecular basis of ultra-rapid metabolism
has been identified to be due to CYP2D6 gene
amplification.
Thus, the activity of a polypeptide of the
invention, expression of a nucleic acid encoding the
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polypeptide, or mutation content of a gene encoding the
polypeptide in an individual can be determined to thereby
select appropriate agents) for therapeutic or
prophylactic treatment of the individual. In addition,
pharmacogenetic studies can be used to apply genotyping
of polymorphic alleles encoding drug-metabolizing enzymes
to the identification of an individual's drug
responsiveness phenotype. This knowledge, when applied
to dosing or drug selection, can avoid adverse reactions
or therapeutic failure and thus enhance therapeutic or
prophylactic efficiency when treating a subject with a
modulator of activity or expression of the polypeptide,
such as a modulator identified by one of the exemplary
screening assays described herein.
4. Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e. g., drugs,
compounds) on the expression or activity of a polypeptide
of the invention (e. g., the ability to modulate aberrant
cell proliferation and/or differentiation) can be applied
not only in basic drug screening, but also in clinical
trials. For example, the effectiveness of an agent, as
determined by a screening assay as described herein, to
increase gene expression, protein levels or protein
activity, can be monitored in clinical trials of subjects
exhibiting decreased gene expression, protein levels, or
protein activity. Alternatively, the effectiveness of an
agent, as determined by a screening assay, to decrease
gene expression, protein levels or protein activity, can
be monitored in clinical trials of subjects exhibiting
increased gene expression, protein levels, or protein
activity.
For example, and not by way of limitation, genes,
including those of the invention, that are modulated in
cells by treatment with an agent (e.g., compound, drug or
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small molecule) which modulates activity or expression of
a polypeptide of the invention (e.g., as identified in a
screening assay described herein) can be identified.
Thus, to study the effect of agents on cellular
proliferation disorders, for example, in a clinical
trial, cells can be isolated and RNA prepared and
analyzed for the levels of expression of a gene of the
invention and other genes implicated in the disorder.
The levels of gene expression (i.e., a gene expression
pattern) can be quantified by Northern blot analysis or
RT-PCR, as described herein, or alternatively by
measuring the amount of protein produced, by one of the
methods as described herein, or by measuring the levels
of activity of a gene of the invention or other genes.
I5 In this way, the gene expression pattern can serve as a
marker, indicative of the physiological response of the
cells to the agent. Accordingly, this response state may
be determined before, and at various points during,
treatment of the individual with the agent.
In a preferred embodiment, the present invention
provides a method for monitoring the effectiveness of
treatment of a subject with an agent (e. g., an agonist,
antagonist, peptidomimetic, protein, peptide, nucleic
acid, small molecule, or other drug candidate identified
by the screening assays described herein) comprising the
steps of (i) obtaining a pre-administration sample from a
subject prior to administration of the agent; (ii)
detecting the level of the polypeptide or nucleic acid of
the invention in the preadministration sample; (iii)
obtaining one or more post-administration samples from
the subject; (iv) detecting the level the of the
polypeptide or nucleic acid of the invention in the post-
administration samples; (v) comparing the level of the
polypeptide or nucleic acid of the invention in the pre-
administration sample with the level of the polypeptide
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or nucleic acid of the invention in the post-
administration sample or samples; and (vi) altering the
administration of the agent to the subject accordingly.
For example, increased administration of the agent may be
desirable to increase the expression or activity of the
polypeptide to higher levels than detected, i.e., to
increase the effectiveness of the agent. Alternatively,
decreased administration of the agent may be desirable to
decrease expression or activity of the polypeptide to
lower levels than detected, i.e., to decrease the
effectiveness of the agent.
C. Methods of Treatment
The present invention provides for both
prophylactic and therapeutic methods of.treating a
subject at risk of (or susceptible to) a disorder or
having a disorder associated with aberrant expression or
activity of a polypeptide of the invention. Such
disorders can include, e.g., disorders of lipoprotein
metabolism, disorders of lipoprotein transport,
neurodegenerative disorders, neuropsychiatric disorders,
and clotting disorders.
1. Prophylactic Methods
In one aspect, the invention provides a method for
preventing in a subject, a disease or condition
associated with an aberrant expression or activity of a
polypeptide of the invention, by administering to the
subject an agent which modulates expression or at least
one activity of the polypeptide. Subjects at risk for a
disease which is caused or contributed to by aberrant
expression or activity of a polypeptide of the invention
can be identified by, for example, any or a combination
of diagnostic or prognostic assays as described herein.
Administration of a prophylactic agent can occur prior to
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the manifestation of symptoms characteristic of the
aberrancy, such that a disease or disorder is prevented
or, alternatively, delayed in its progression. Depending
on the type of aberrancy, for example, an agonist or
antagonist agent can be used for treating the subject.
The appropriate agent can be determined based on
screening assays described herein.
2. Therapeutic Methods
Another aspect of the invention pertains to
methods of modulating expression or activity of a
polypeptide of the invention for therapeutic purposes.
The modulatory method of the invention involves
contacting a cell with an agent that modulates one or
more of the activities of the polypeptide. An agent that
modulates activity can be an agent as described herein,
such as a nucleic acid or a protein, a naturally-
occurring cognate ligand of the polypeptide, a peptide, a
peptidomimetic, or other small molecule. In one
embodiment, the agent stimulates one or more of the
biological activities of the polypeptide. Examples of
such stimulatory agents include the active polypeptide of
the invention and a nucleic acid molecule encoding the
polypeptide of the invention that has been introduced
into the cell. In another embodiment, the agent inhibits
one or more of the biological activities of the
polypeptide of the invention. Examples of such
inhibitory agents include antisense nucleic acid
molecules and antibodies. These modulatory methods can
be performed in vitro (e. g., by culturing the cell with
the agent) or, alternatively, in vivo (e.g, by
administering the agent to a subject). As such, the
present invention provides methods of treating an
individual afflicted with a disease or disorder
characterized by aberrant expression or activity of a
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polypeptide of the invention. In one embodiment, the
method involves administering an agent (e. g., an agent
identified by a screening assay described herein), or
combination of agents that modulates (e. g., upregulates
or downregulates) expression or activity. In another
embodiment, the method involves administering a
polypeptide of the invention or a nucleic acid molecule
of the invention as therapy to compensate for reduced or
aberrant expression or activity of the polypeptide.
Stimulation of activity is desirable in situations
in which activity or expression is abnormally low or
downregulated and/or in which increased activity is
likely to have a beneficial effect. Conversely,
inhibition of activity is desirable in situations in
which activity or expression is abnormally high or
upregulated and/or in which decreased activity is likely
to have a beneficial effect.
This invention is further illustrated by the
following examples which should not be construed as
limiting. The contents of all references, patents and
published patent applications cited throughout this
application are hereby incorporated by reference.
Equivalents
Those skilled in the art will recognize, or be
able to ascertain using no more than routine
experimentation, many equivalents to the specific
embodiments of the invention described herein. Such
equivalents are intended to be encompassed by the
following claims.
What is claimed is:
