Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL MOLECULES OF THE TNF RECEPTOR SUPERFAMILY
AND USES THEREFOR
Background of the Invention
5 The tumor necrosis factor receptor (TNFR) superfamily of proteins
encompasses
over a dozen members, most of which are type I transmembrane proteins, related
by the
presence of conserved cysteine-rich repeats {CRRs) in their N-terminal
cysteine-rich
domains (CRDs). Members of the TNFR superfamily include TNFRl (p55), TNFR2
(p75), TNFR3 (TNF-RP), Fas (also known as CD95 and Apol), OX-40, 41-BB, CD40,
10 CD30, CD27, OPG, and p75 NGFR. (Smith et al. (1993) Cell 76:959-962;
Armitage,
R.J. (1994) Curr. Opin. Immunol. 6:407-413; Gruss et al. (1995) Blood 85, 3378-
3404;
Baker et al. (1996) Oncogene 12:1-9; and Simonet et al. (1997) Cell 89:309-
319.) A
TNFR superfamily member is typically a membrane-bound, trimeric or multimeric
complex which is stabilized via intracysteine disulfide bonds that are formed
between
15 the cysteine-rich domains of individual subunit members (Banner et al.
(1993) Cell
73:431-445). The proteins themselves do not have intrinsic catalytic activity,
rather they
function via association with other proteins to transduce cellular signals.
A functional TNFR superfamily protein can also exist in a soluble form.
Soluble
versions of the superfamily bind cognate ligands and influence
bioavailability. For
20 instance, the osteoprotegerin protein family exists as a soluble protein.
(Simonet et al.
{1997) Cell 89:309-319.) Many soluble forms of the TNFR have been identified.
Certain soluble TNFRs are elevated in disease states such as lupus and
rheumatoid
arthritis. (Gabay et al. ( 1997) J. Rheumatol. 24(2):303-308.) The soluble
superfamily
members lack the transmembrane domain characteristic of the majority of
superfamily
25 members due to either proteolytic cleavage or, at least in one instance, to
alternative
splicing (truss et al. (1995) Blood 85, 3378-3404.)
Summar~r of the Invention
The present invention is based, at least in part, on the discovery of novel
30 molecules of the TNF receptor superfamily, referred to herein as "STRIFE"
nucleic acid
and protein molecules. Two splice forms of the "STRIFE" nucleic acid molecule
have
been identified and are referred to herein as the "STRIFE1" and "STRIFE2"
nucleic acid
and protein molecules. The STRIFEl and STRIFE2 molecules of the present
invention
are useful as modulating agents in regulating a variety of cellular processes.
35 Accordingly, in one aspect, this invention provides isolated nucleic acid
molecules
encoding STRIFE1 and STRIFE2 proteins or biologically active portions thereof,
as
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well as nucleic acid fragments suitable as primers or hybridization probes for
the
detection of STRIFE1 and STRIFE2-encoding nucleic acids.
In one embodiment, a STRIFE1 nucleic acid molecule is at least about 60%,
65%, 70%, 71 %, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the
nucleotide sequence shown in SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:4, or a
complement thereof. In yet another embodiment, a STRIFE2 nucleic acid molecule
is at
least about 60%, 65%, 70%, 71 %, 75%, 80%, 85%, 90%, 95%, 98% or more
homologous to the nucleotide sequence shown in SEQ ID NO:S, SEQ ID N0:7, SEQ
ID
N0:8, or a complement thereof. In a preferred embodiment, an isolated STRIFEl
10 nucleic acid molecule has the nucleotide sequence shown SEQ ID N0:3, or a
complement thereof. In another embodiment, a STRIFEl nucleic acid molecule
further
comprises nucleotides 1-106 of SEQ ID NO:1. In yet another preferred
embodiment, a
STRIFE1 nucleic acid molecule further comprises nucleotides 751-981 of SEQ ID
NO:1. In another preferred embodiment, an isolated STRIFE1 nucleic acid
molecule
has the nucleotide sequence shown in SEQ ID NO:1.
In another preferred embodiment, an isolated STRIFE2 nucleic acid molecule
has the nucleotide sequence shown SEQ ID N0:7, or a complement thereof. In
another
embodiment, a STRIFE2 nucleic acid molecule further comprises nucleotides 1-
109 of
SEQ ID NO:S. In yet another preferred embodiment, a STRIFE2 nucleic acid
molecule
20 further comprises nucleotides 562-655 of SEQ ID NO:S. In another preferred
embodiment, an isolated STRIFE2 nucleic acid molecule has the nucleotide
sequence
shown in SEQ ID NO:S.
In another embodiment, a STRIFE1 or a STRIFE2 nucleic acid molecule include
a nucleotide sequence encoding a protein having an amino acid sequence
sufficiently
homologous to the amino acid sequence of SEQ ID N0:2, or SEQ ID N0:6,
respectively. In another preferred embodiment, a STRIFE1 or a STRIFE2 nucleic
acid
molecule include a nucleotide sequence encoding a protein having an amino acid
sequence 60%, 65%, 70%, 75%, 80%, 85%, 86%, 90%, 95%, 98% or more homologous
to the amino acid sequence of SEQ ID N0:2, or SEQ ID N0:6, respectively.
30 In another embodiment, an isolated nucleic acid molecule of the present
invention encodes a STRIFE 1 protein which includes a cysteine-rich domain,
optionally
a signal sequence, and is membrane bound. In another embodiment, an isolated
nucleic
acid molecule of the present invention encodes a STRIFE1 protein which
includes a
signal sequence and a cysteine-rich domain, wherein the cysteine-rich domain
comprises
at least one module, and is membrane bound. In yet another embodiment, a
STRIFE1
nucleic acid molecule encodes a STRIFE1 protein and is a naturally occurring
nucleotide sequence.
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In another embodiment, an isolated nucleic acid molecule of the present
invention encodes a STRIFE2 protein which includes a cysteine-rich domain,
optionally
a signal sequence, and is secreted. In another embodiment, an isolated nucleic
acid
molecule of the present invention encodes a STRIFE2 protein which includes a
signal
5 sequence and a cysteine-rich domain, wherein the cysteine-rich domain
comprises at
least one module, and is secreted. In yet another embodiment, a STRIFE2
nucleic acid
molecule encodes a STRIFE2 protein and is a naturally occurring nucleotide
sequence.
Another embodiment of the invention features STRIFE1 or STRIFE2 nucleic
acid molecules which specifically detect STRIFE1 or STRIFE2 nucleic acid
molecules,
respectively, relative to nucleic acid molecules encoding non-STRIFE1 or non-
STRIFE2
proteins. For example, in one embodiment, a STRIFE1 or STRIFE2 nucleic acid
molecule hybridizes under stringent conditions to a nucleic acid molecule
comprising
the nucleotides 107-751, 1-16, 413-602, or 711-981 of the nucleotide sequence
shown in
SEQ ID NO:1, or to nucleotides 110-562; 1-16, 416-489, or 519-655 of
nucleotide
15 sequence shown in SEQ ID NO:S, respectively. In another embodiment, the
STRIFE1
or STRIFE2 nucleic acid molecule is at least 450 nucleotides in length and
hybridizes
under stringent conditions to a nucleic acid molecule comprising the
nucleotide
sequence shown in SEQ ID NO:1, or SEQ ID NO:S, respectively, or a complement
thereof.
Another embodiment of the invention provides an isolated nucleic acid molecule
which is antisense to the coding strand of a STRIFE1 or a STRIFE2 nucleic acid
molecule.
Another aspect of the invention provides a vector comprising a STRiFEI or a
STRIFE2 nucleic acid molecule. In certain embodiments, the vector is a
recombinant
25 expression vector. In another embodiment, the invention provides a host
cell containing
a vector of the invention. The invention also provides a method for producing
a
STRIFE1 or a STRIFE2 protein by culturing in a suitable medium, a host cell of
the
invention containing a recombinant expression vector such that a STRIFE1 or a
STRIFE2 protein, respectively, is produced.
30 Another aspect of this invention features isolated or recombinant STRIFE1
or
STRIFE2 proteins and polypeptides. In one embodiment, an isolated STRIFE1
protein
has a cysteine-rich domain, optionally a signal sequence, and is membrane
bound. In
another embodiment, an isolated STRIFE2 protein has a cysteine-rich domain,
optionally a signal sequence, and is secreted. In yet another embodiment, an
isolated
35 STRIFE1 or STRIFE2 protein has an amino acid sequence sufficiently
homologous to
the amino acid sequence of SEQ ID N0:2, or SEQ ID N0:6, respectively. In a
preferred
embodiment, a STRIFE1 protein has an amino acid sequence at least about 60%,
65%,
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70%, 75%, 80%, 85%, 86%, 90%, 95%, 98% or more homologous to the amino acid
sequence of SEQ ID N0:2. in another preferred embodiment, a STRIFE2 protein
has an
amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 90%,
95%,
98% or more homologous to the amino acid sequence of SEQ ID N0:6. In another
embodiment, a STRIFE1 or a STRIFE2 protein has the amino acid sequence of SEQ
ID
N0:2, or SEQ ID N0:6, respectively.
Another embodiment of the invention features an isolated STRIFE1 protein
which is encoded by a nucleic acid molecule having a nucleotide sequence at
least about
60% homologous to a nucleotide sequence of SEQ ID NO:1, or a complement
thereof.
10 Another embodiment of the invention features an isolated STRIFE2 protein
which is
encoded by a nucleic acid molecule having a nucleotide sequence at least about
60%
homologous to a nucleotide sequence of SEQ ID NO:S, or a complement thereof.
This
invention further features an isolated STRIFE1 or STRIFE2 protein which is
encoded by
a nucleic acid molecule having a nucleotide sequence which hybridizes under
stringent
15 hybridization conditions to a nucleic acid molecule comprising the
nucleotide sequence
of SEQ ID NO:1, or SEQ ID NO:S, respectively, or a complement thereof.
The STRIFE1 and STRIFE2 proteins of the present invention, or biologically
active portions thereof, can be operatively linked to a non-STRIFE1 and a non-
STRIFE2
polypeptide to form STRIFE1 and STRIFE2 fusion proteins. The invention further
20 features antibodies that specifically bind STRIFE1 and STRIFE2 proteins,
such as
monoclonal or polyclonal antibodies. In addition, the STRIFE1 and STRIFE2
proteins
or biologically active portions thereof can be incorporated into
pharmaceutical
compositions, which optionally include pharmaceutically acceptable carriers.
In another aspect, the present invention provides a method for detecting
25 STRIFE 1 and STRIFE2 expression in a biological sample by contacting the
biological
sample with an agent capable of detecting a STRIFE1 and a STRIFE2 nucleic acid
molecule, protein or polypeptide such that the presence of a STRIFE 1 and a
STRIFE2
nucleic acid molecule, protein or polypeptide is detected in the biological
sample.
In another aspect, the present invention provides a method for detecting the
30 presence of STRIFEI and STRIFE2 activity in a biological sample by
contacting the
biological sample with an agent capable of detecting an indicator of STRIFE1
and
STRIFE2 activity such that the presence of STRIFE1 and STRIFE2 activity is
detected
in the biological sample.
In another aspect, the invention provides a method for modulating STRIFE 1 and
35 STRIFE2 activity comprising contacting the cell with an agent that
modulates STRIFEI
and/or STRIFE2 activity such that STRIFE1 and/or STRIFE2 activity in the cell
is
modulated. In one embodiment, the agent inhibits STRIFE1 and/or STRIFE2
activity.
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In another embodiment, the agent stimulates STRIFE1 and/or STRIFE2 activity.
In one
embodiment, the agent is an antibody that specifically binds to a STRIFE1
and/or a
STRIFE2 protein. In another embodiment, the agent modulates expression of
STRIFE1
and STRIFE2 by modulating transcription of a STRIFE! and a STRIFE2 gene or
translation of a STRIFE1 and a STRIFE2 mRNA. In yet another embodiment, the
agent
is a nucleic acid molecule having a nucleotide sequence that is antisense to
the coding
strand of a STRIFE1 and a STRIFE2 mRNA or a STRIFE gene.
In one embodiment, the methods of the present invention are used to treat a
subject having a disorder characterized by aberrant STRIFE1 and/or STRIFE2
protein or
10 nucleic acid expression or activity by administering an agent which is a
STRIFE1 and/or
STRIFE2 modulator to the subject. In one embodiment, the STRIFE1 and STRIFE2
modulator is a STRIFE1 and a STRIFE2 protein, respectively. In another
embodiment
the STRIFE1 or STRIFE2 modulator is a STRIFE1 or a STRIFE2 nucleic acid
molecule, respectively. In yet another embodiment, the STRIFE 1 and the
STRIFE2
15 modulator is a peptide, peptidomimetic, or other small molecule. In a
preferred
embodiment, the disorder characterized by aberrant STRIFE1 and/or STRIFE2
protein
or nucleic acid expression is a developmental, differentiative, or
proliferative disorder.
The present invention also provides a diagnostic assay for identifying the
presence or absence of a genetic alteration characterized by at least one of
(i) aberrant
20 modification or mutation of a gene encoding a STRIFE1 and/or a STRIFE2
protein; (ii)
mis-regulation of said gene; and (iii) aberrant post-translational
modification of a
STRIFE1 and/or a STRIFE2 protein, wherein a wild-type form of said gene
encodes an
protein with a STRIFE1 and a STRIFE2 activity, respectively.
In another aspect the invention provides a method for identifying a compound
25 that binds to or modulates the activity of a STRIFE1 or a STRIFE2 protein,
by providing
an indicator composition comprising a STRIFE! and/or STRIFE2 protein having
STRIFE 1 and/or STRIFE2 activity, respectively, contacting the indicator
composition
with a test compound, and determining the effect of the test compound on
STRIFE 1 or
STRIFE2 activity in the indicator composition to identify a compound that
modulates
30 the activity of a STRIFE1 or a STRIFE2 protein, respectively.
Other features and advantages of the invention will be apparent from the
following detailed description and claims.
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Brief Description of the Drawings
Figure 1 depicts the cDNA sequence and predicted amino acid sequence of
marine STRIFE1. The nucleotide sequence corresponds to nucleic acids 1 to 981
of
SEQ ID NO:1. The amino acid sequence corresponds to amino acids 1 to 214 of
SEQ
5 ID N0:2.
Figure 2 depicts the cDNA sequence and predicted amino acid sequence of
marine STRIFE2. The nucleotide sequence corresponds to nucleic acids 1 to 655
of
SEQ ID NO:S. The amino acid sequence corresponds to amino acids 1 to 150 of
SEQ
ID N0:6.
10 Figure 3 depicts an alignment of the amino acid sequences of marine STRIFE1
(also refered to herein as "Tango127a" or "T127a"), STRIFE2 (also refered to
herein as
"Tango127b" or "T127b"), and marine OX40 (Accesssion Number P47741). Amino
acid residues which are conserved between marine STRIFE1 and STRIFE2 family
members are highlighted.
1 S Figure 4 depicts the results from a FASTA search using the amino acid
sequence
of STRIFE 1 as a Query.
Figure S depicts the results froma FASTA search using the nucleotide sequence
of STRIFE 1 as a query.
20 Detailed Description of the Invention
The present invention is based, at least in part, on the discovery of novel
TNF
receptor family members, referred to herein as "STRIFE1" and "STRIFE2" nucleic
acid
and protein molecules. TNF receptors are typically membrane-bound, trimeric or
multimeric complexes which are stabilized via intracysteine disulfide bonds
that are
25 formed between the cysteine-rich domains of individual subunit members
(Banner et al.
(1993) Cell 73:431-445). Functional TNF receptors can also exist in a soluble
form.
Soluble members of the superfamily bind cognate ligands and influence
bioavailability.
The soluble superfamily members lack the transmembrane domain characteristic
of the
majority of superfamily members due to either proteolytic cleavage or, at
least in one
30 instance, to alternative splicing (Grass et al. (1995) Blood 85, 3378-
3404).
TNF receptors are the sole mediators of Tumor Necrosis Factor (TNF) signaling.
TNF is a cytokine that is capable of acting independently or in conjunction
with other
factors to affect various different body functions. In vitro, TNF has diverse
biological
effects, including killing of transformed cells, stimulation of granulocytes
and
35 fibroblasts, damage to endothelial cells, and anti-parasitic effects. In
vivo, TNF plays a
key role as an endogenous mediator of inflammatory, immune, and host defense
functions. In addition, TNF plays a role in various neoplastic disease states.
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The STRIFE1 and STRIFE2 molecules of the present invention having
homology to the TNF receptors may also be TNF receptors involved in TNF
signaling.
Thus, the STRIFE1 and STRIFE2 molecules of the present invention may play a
role in
mediating inflammatory, immune, and host defense functions. In addition, the
STRIFE 1
5 and STRIFE2 molecules of the present invention may play a role in various
neoplastic
disease states. Thus, the STRIFE 1 and STRIFE2 molecules may be useful as
targets for
developing novel diagnostic and therapeutic agents to treat TNF-associated
disorders
and TNF receptor-associated disorders.
As used herein, the terms "TNF-associated disorder" and "TNF receptor-
10 associated disorder" include any disorder, disease, or condition which is
associated with
an abnormal or undesired TNF or TNF receptor function or an abnormal or
undesired
TNF or TNF receptor level, e.g., plasma, tissue, or cellular levels or
concentration.
Examples of TNF-associated and TNF receptor-associated disorders include, but
are not
limited to, sepsis syndrome, including cachexia; circulatory collapse and
shock resulting
15 from acute or chronic bacterial infection; acute and chronic parasitic or
infectious
processes, including bacterial, viral and fungal infections; acute and
chronic.immune and
autoimmune pathologies, such as systemic lupus erythematosus and rheumatoid
arthritis; alcohol-induced hepatitis; chronic inflammatory pathologies such as
sarcoidosis and Crohn's pathology; vascular inflammatory pathologies such as
20 disseminated intravascular coagulation; graft-versus-host pathology;
Rawasaki's
pathology; malignant pathologies involving TNF-secreting tumors; cerebral
malaria;
and multiple sclerosis.
The term "family" when referring to the protein and nucleic acid molecules of
the invention is intended to mean two or more proteins or nucleic acid
molecules having
25 a common structural domain and having sufficient amino acid or nucleotide
sequence
homology as defined herein. Such family members can be naturally occurring and
can
be from either the same or different species. For example, a family can
contain a first
protein of human origin, as well as other, distinct proteins of human origin
or
alternatively, can contain homologues of non-human origin. Members of a family
may
30 also have common functional characteristics.
In one embodiment, a STRIFE1 and a STRIFE2 family member is identified
based on the presence of at least one "cysteine-rich domain" in the protein or
corresponding nucleic acid molecule. As used herein, the term "cysteine-rich
domain"
refers to a protein domain of about 110-160 amino acid residues in length,
preferably
35 about 100-1 SO amino acid residues in length, more preferably about 90-140
amino acid
residues in length, and even more preferably at least about 80-130 amino acid
residues in
length, of which at least about 10-30, preferably about 10-20, and more
preferably about
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12, 13, 14, or 15 amino acid residues are cysteine residues. In a preferred
embodiment,
a cysteine-rich domain is located in the N-terminal region of a STRIFE 1 and
STRIFE2
protein and includes about amino acid residues 34 through 114 of SEQ ID N0:2
and
SEQ ID N0:6, respectively. Preferred cysteine rich domains contain at least
about two,
5 three, or four modules or motifs, wherein each module is a region of about
20-60 amino
acid residues in length, preferably 30-50 amino acid residues in length, more
preferably
40 amino acid residues in length and includes about 3-10 cysteines, preferably
5-7
cysteines, and more preferably 6 cysteines. In one embodiment, the module has
the
following motif
C-Xaal(4-14~C-Xaa2(0-2~C-Xaa3(2-4)-C-Xaa4(6-12~C-XaaS(6-10)-C(SEQ ID
N0:17),
wherein "C" is the amino acid cysteine and "Xaal-XaaS" can be any amino acid
residue.
In a preferred embodiment, Xaal is between 4-6, 6-8, 8-10, 10-12, or 12-14
amino acid
residues; Xaa4 is between 6-8, 8-10, or 10-12 amino acid residues; and XaaS is
between
15 6-8 or 8-10 amino acid residues. In another preferred embodiment, Xaal is 4-
6 amino
acid residues, of which at least one is the amino acid phenylalanine, at least
one is the
amino acid tyrosine, and/or at least one is the amino acid histidine. In yet
another
preferred embodiment, XaaS is 6-10 amino acid residues, of which at least one
is the
amino acid aspartic acid, at least one is the amino acid asparagine, at least
one is the
20 amino acid glutamic acid, at least one is the amino acid glutamine, at
least one is the
amino acid serine, at least one is the amino acid lysine, and/or at least one
is the amino
acid proline. In another embodiment, the module has the following motif:
C-Xaal(4,6~FYH-Xaa2(5,10)-C-Xaa3(0,2~C-Xaa4(2,3~C-XaaS(7,11~C-Xaa6(4,6)
DNEQSKP-Xaa7(2rC(SEQ ID N0:16).
25 For example, in one embodiment, a STRIFE1 protein contains a cysteine-rich
domain
including a first module containing about amino acids 34-72 of SEQ ID N0:2
(shown
separately as SEQ ID NO:11 ) having 6 cysteine residues at positions indicated
by the
aforementioned motifs, and a second module containing about amino acids 75-114
of
SEQ ID N0:2 (shown separately as SEQ ID N0:12) having 6 cysteine residues at
30 positions indicated by the aforementioned motifs. In another embodiment, a
STRIFE2,
protein contains a cysteine rich domain including a first module containing
about amino
acids 34-72 of SEQ ID N0:6 (shown separately as SEQ ID N0:14) having 6
cysteine
residues at positions indicated by the aforementioned motifs, and a second
module
containing about amino acids 75-114 of SEQ ID N0:6 (shown separately as SEQ ID
35 NO:15) having 6 cysteine residues at positions indicated by the
aforementioned motifs.
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In another embodiment of the invention, a STRIFE1 and STRIFE2 protein has at
least one cysteine-rich domain and a signal sequence. As used herein. a
"signal
sequence" refers to a peptide containing about 20 amino acids which occurs at
the N-
terminus of secretory and integral membrane proteins and which contains a
large
5 number of hydrophobic amino acid residues. For example, a signal sequence
contains at
least about 14-28 amino acid residues, preferably about 16-26 amino acid
residues, more
preferably about 18-24 amino acid residues, and more preferably about 20-22
amino
acid residues, and has at least about 40-70%, preferably about 50-65%, and
more
preferably about 55-60% hydrophobic amino acid residues (e.g., Alanine,
Valine,
Leucine, Isoleucine, Phenylalanine, Tyrosine, Tryptophan, or Proline). Such a
"signal
sequence", also referred to in the art as a "signal peptide", series to direct
a protein
containing such a sequence to a lipid bilayer. For example, in one embodiment,
a
STRIFE1 protein contains a signal sequence of about amino acids 1-29 of SEQ ID
N0:2
(shown separately as SEQ ID N0:9). In another embodiment, a STRIFE2 protein
contains a signal sequence of about amino acids I-29 of SEQ ID N0:6 (shown
separately as SEQ ID N0:13).
Accordingly, one embodiment of the invention features a STRIFE1 or a
STRIFE2 protein having at least one cysteine-rich domain. Another embodiment
features a STRIFE1 or a STRIFE2 protein having at least one cysteine-rich
domain,
20 wherein the cysteine-rich domain includes at least one module having the
predicted
motif of SEQ ID N0:16. Another embodiment features a STRIFE1 or a STRIFE2
protein having at least one cysteine-rich domain: wherein the cysteine-rich
domain
includes at least two modules. Another embodiment features a protein having
20, 30,
40, 50, 60, 70, 80, 90, 95, or 99% homology to a cysteine-rich domain of a
STRIFE1 or
a STRIFE2 protein of the invention.
Yet another embodiment of the invention features a STRIFE 1 or a STRIFE2
protein
having at least one cysteine-rich domain and a signal peptide. Another
embodiment
features a STRIFE 1 or a STRIFE2 protein having at least one cysteine-rich
domain and a
signal peptide, wherein the cysteine-rich domain includes at least one module
having the
predicted motif of SEQ ID N0:16.
In yet another embodiment of the invention, a STRIFE 1 protein has a
transmembrane domain. As used herein, the term "transmembrane domain" refers
to a
structural amino acid motif which includes a hydrophobic helix that spans the
plasma
membrane. A transmembrane domain preferably includes a series of hydrophobic
residues,
35 such as leucine, valine, and tyrosine residues. For example, a STRIFE 1
protein contains a
transmembrane domain containing amino acids 169-193 of SEQ ID N0:2 (shown
seperately as SEQ ID NO:10).
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Preferred STRIFE 1 or STRIFE2 molecules of the present invention have an amino
acid sequence sufficiently homologous to the amino acid sequence of SEQ ID
N0:2, or
SEQ ID N0:6, respectively. As used herein, the term "sufficiently homologous"
refers to a
first amino acid or nucleotide sequence which contains a sufficient or minimum
number of
5 identical or equivalent (e.g., an amino acid residue which has 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 share common structural
domains
and/or a common functional activity. For example, amino acid or nucleotide
sequences
which share common structural domains have at least about 40% homology,
preferably
10 50% homology, more preferably 60%-70% homology across the amino acid
sequences of
the domains and contain at least one, preferably two, more preferably three,
and even more
preferably four, five or six structural domains, are defined herein as
sufficiently
homologous. Furthermore, amino acid or nucleotide sequences which share at
least 40%,
preferably 50%, more preferably 60, 70, or 80% homology and share a common
fiulctional
1 S activity are defined herein as sufficiently homologous.
As used interchangeably herein, a "STRIFE1 and a STRIFE2 activity",
"biological activity of STRIFE 1 and STRIFE2" or "fimctional activity of
STRIFE 1 and
STRIFE2", refers to an activity exerted by a STRIFE1 and a STRIFE2 protein,
polypeptide or nucleic acid molecule on a STRIFE1 or a STRIFE2 responsive cell
as
20 determined in vivo, or in vitro, according to standard techniques. In one
embodiment, a
STRIFE1 and a STRIFE2 activity is a direct activity, such as an association
with a
STRIFE1 or a STRIFE2-target molecule. As used herein, a "target molecule" is a
molecule with which a STRIFE 1 and a STRIFE2 protein binds or interacts in
nature,
such that STRIFE 1 or STRIFE2-mediated function is achieved. A STRIFE 1 or a
25 STRIFE2 target molecule can be a non-STRIFE1 and a non-STRIFE2 molecule or
a
STRIFE1 or STRIFE2 protein or polypeptide of the present invention. In an
exemplary
embodiment, a STRIFE2 target molecule is a membrane-bound protein (e.g., a
"STRIFE2 receptor") or a modified form of such a protein which has been
altered such
that the protein is soluble (e.g., recombinantly produced such that the
protein does not
30 express a membrane-binding domain). In another embodiment, a STRIFE 1 or a
STRIFE2 target is a second soluble protein molecule (e.g., a "STRIFE1 or a
STRIFE2
binding partner" or a "STRIFE1 and STRIFE2 substrate"). In such an exemplary
embodiment, a STRIFE 1 and a STRIFE2 binding partner, can be a soluble non-
STRIFE 1
and non-STRIFE2 protein or a second STRIFE 1 and a STRIFE2 protein molecule of
the
35 present invention. Alternatively, a STRIFE1 and a STRIFE2 activity is an
indirect
activity, such as a cellular signaling activity mediated by interaction of the
STRIFE1 and
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the STRIFE2 protein with a second protein (e.g., a STRIFE1 ligand or a STRIFE2
receptor).
In a preferred embodiment, a STRIFE 1 activity is at least one or more of the
following activities: (i) interaction of a STRIFE1 protein on the cell surface
with a
5 second non-STRIFE1 protein molecule on the surface of the same cell; (ii)
interaction of
a STRIFE1 protein on the cell surface with a second non-STRIFE1 protein
molecule on
the surface of a different cell; (iii) complex formation between a membrane-
bound
STRIFE1 protein and a cytokine, e.g., TNF; (iv) interaction of a STRIFE1
protein with
an intracellular protein including SH2 domain-containing proteins or
cytoskeletal
10 proteins; (v) formation of a homogeneous multimeric signaling complex with
STRIFE1-
like proteins; and (vi) formation of a heterogeneous multimeric signaling
complex with
other TNFR superfamily proteins.
In another preferred embodiment, a STRIFE2 activity is at least one or more of
the following activities: (i) interaction of a STRIFE2 protein with a membrane-
bound
15 STRIFE2 receptor; (ii) interaction of a STRIFE2 protein with a soluble form
of a
STRIFE2 receptor; (iii) interaction of a STRIFE2 protein with an intracellular
protein
via a membrane-bound STRIFE2 receptor; (iv) complex formation between a
soluble
STRIFE2 protein and a second soluble STRIFE2 binding partner; (v) complex
formation
between a soluble STRIFE2 protein and a second soluble STRIFE2 binding
partner,
20 wherein the STRIFE2 binding partner is a non-STRIFE2 protein molecule; and
(vi)
complex formation between a soluble STRIFE2 protein and a second soluble
STRIFE2
binding partner, wherein the STRIFE2 binding partner is a second STRIFE2
protein
molecule.
In yet another preferred embodiment, a STRIFE1 or a STRiFE2 activity is at
25 least one or more of the following activities: (i) modulation of cellular
signal
transduction, either in vitro or in vivo; (ii) regulation of gene
transcription in a cell
involved in development or differentiation, either in vitro or in vivo; (iii)
modulation of
cellular signal transduction; (iv) regulation of cellular proliferation; (v)
regulation of
cellular differentiation; and (vi) regulation of cell survival.
30 Accordingly, another embodiment of the invention features isolated STRIFE1
and STRIFE2 proteins and polypeptides having a STRIFE 1 and/or STRIFE2
activity,
respectively. Preferred STRIFE1 and STRIFE2 proteins have at least one
cysteine-rich
domain and a STRIFE 1 and/or a STRIFE2 activity. In another preferred
embodiment,
the STRIFE1 and STRIFE2 protein has at least one cysteine-rich domain, wherein
the
35 cysteine-rich domain comprises at least one module, and a STRIFE1 and
STRIFE2
activity, respectively. In another preferred embodiment, the STRIFEI and
STRIFE2
protein has at least one cysteine-rich domain, wherein the cysteine-rich
domain
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comprises at least two modules, and a STRIFE l and STRIFE2 activity,
respectively. In
yet another preferred embodiment, a STRIFE1 and a STRIFE2 protein further
comprises
a signal sequence. In still another preferred embodiment, a STRIFE1 and a
STRIFE2
protein has a cysteine-rich.domain, a STRIFE1 and a STRIFE2 activity, and an
amino
5 acid sequence sufficiently homologous to an amino acid sequence of SEQ ID
N0:2, or
SEQ ID N0:6, respectively.
The marine STRIFE1 cDNA, which is approximately 981 nucleotides in length,
encodes a protein which is approximately 214 amino acid residues in length.
The
marine STRIFE 1 protein contains an N-terminal signal sequence and a cysteine-
rich
10 domain comprising two modules. A STRIFE1 cysteine-rich domain can be found
at
least, for example, from about amino acids 34-114 of SEQ ID N0:2. The STRIFE1
cysteine-rich domain comprises a first module from about amino acids 34-72 of
SEQ ID
N0:2 (shown separately as SEQ ID NO:11 ) and a second module from about amino
acids 75-114 of SEQ ID N0:2 (shown separately as SEQ ID N0:12). The marine
15 STRIFE1 protein is a membrane bound protein which contains a transmembrane
domain
at about amino acids 169-193 of SEQ ID N0:2 (shown seperately as SEQ ID NO:10)
and a signal sequence at about amino acids 1-29 of SEQ ID N0:2 (shown
separately as
SEQ ID N0:9). The prediction of such a signal peptide can be made, for
example,
utilizing the computer algorithm SIGNALP (Henrik, et al. (1997) Protein
Engineering
20 10:1-6).
The marine STRIFE2 cDNA, which is approximately 655 nucleotides in length,
encodes a protein which is approximately 1 SO amino acid residues in length.
The
marine STRIFE2 protein contains an N-terminal signal sequence and a cysteine-
rich
domain comprising two modules. A STRIFE2 cysteine-rich domain can be found at
25 least, for example, from about amino acids 34-114 of SEQ ID N0:6. The
STRIFE2
cysteine-rich domain comprises a first module from about amino acids 34-72 of
SEQ ID
N0:6 (shown separately as SEQ ID N0:14) and a second module from about amino
acids 75-114 of SEQ ID N0:6 (shown separately as SEQ ID NO:1 S). The marine
STRIFE2 protein is a secreted protein which further contains a signal sequence
at about
30 amino acids 1-29 of SEQ ID N0:6 (shown separately as SEQ ID N0:13).
Various aspects of the invention are described in further detail in the
following
subsections:
I. Isolated Nucleic Acid Molecules
35 One aspect of the invention pertains to isolated nucleic acid molecules
which
encode STRIFE1 and STRIFE2 proteins or biologically active portions thereof,
as well
as nucleic acid fragments suiTlcient for use as hybridization probes to
identify STRIFE1
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and STRIFE2-encoding nucleic acids (e.g., STRIFE1 and STRIFE2 mRNA) and
fragments for use as PCR primers for the amplification or mutation of STRIFE1
and
STRIFE2 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
5 (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.
Preferably, an
10 "isolated" nucleic acid is free of 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 STRIFE1 and STRIFE2 nucleic acid molecule can contain less than
about 5
kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which
naturally flank
15 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.
20 A nucleic acid molecule of the present invention, e.g., a nucleic acid
molecule
having the nucleotide sequence of SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:4, SEQ
ID
N0:5, SEQ ID N0:7, or SEQ ID N0:8 or a portion thereof, can be isolated using
standard molecular biology techniques and the sequence information provided
herein.
Using all or a portion of the nucleic acid sequence of SEQ ID NO:1, SEQ ID
N0:3, SEQ
25 ID N0:4, SEQ ID N0:5, SEQ ID N0:7, or SEQ ID N0:8 as a hybridization probe,
STRIFEI and STRIFE2 nucleic acid molecules can be isolated using standard
hybridization and cloning techniques {e.g., as described in Sambrook, J.,
Fritsh, E. F.,
and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
30 1989).
Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID
NO:1, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:7, or SEQ ID N0:8
can be isolated by the polymerase chain reaction (PCR) using synthetic
oligonucleotide
primers designed based upon the sequence of SEQ ID NO:1, SEQ ID N0:3, SEQ ID
35 N0:4, SEQ ID NO:S, SEQ ID N0:7, or SEQ ID N0:8.
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A nucleic acid of the invention can be amplified using cDNA, mRNA or
alternatively, genomic DNA, as a template and appropriate oligonucleotide
primers
according to standard PCR amplification techniques. The nucleic acid so amplif
ed can
be cloned into an appropriate vector and characterized by DNA sequence
analysis.
5 Furthermore, oligonucleotides corresponding to STRIFE1 and STRIFE2
nucleotide
sequences can be prepared by standard synthetic techniques, e.g., using an
automated
DNA synthesizer.
In a preferred embodiment, an isolated nucleic acid molecule of the invention
comprises the nucleotide sequence shown in SEQ ID NO:1. The sequence of SEQ ID
10 NO:1 corresponds to the marine STRIFE1 cDNA. This cDNA comprises sequences
encoding the marine STRIFE1 protein (i.e., "the coding region", from
nucleotides 107-
751 ), as well as 5' untranslated sequences (nucleotides 1 to 106) and 3'
untranslated
sequences (nucleotides 752-981 ). Alternatively, the nucleic acid molecule can
comprise
only the coding region of SEQ ID NO:1 (e.g., nucleotides 107-751,
corresponding to
15 SEQ ID N0:3).
In another preferred embodiment, an isolated nucleic acid molecule of the
invention comprises the nucleotide sequence shown in SEQ ID NO:S. The sequence
of
SEQ ID NO:S corresponds to the marine STRIFE2 cDNA. This cDNA comprises
sequences encoding the marine STRIFE2 protein (i.e., "the coding region", from
20 nucleotides 110-562), as well as S' untranslated sequences (nucleotides 1-
109) and 3'
untranslated sequences (nucleotides 563-655). Alternatively, the nucleic acid
molecule
can comprise only the coding region of SEQ ID NO:S (e.g., nucleotides 110-562,
corresponding to SEQ ID N0:7).
In another preferred embodiment, an isolated nucleic acid molecule of the
25 invention comprises a nucleic acid molecule which is a complement of the
nucleotide
sequence shown in SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ
ID N0:7, or SEQ ID , or a portion of either of these nucleotide sequences. A
nucleic
acid molecule which is complementary to the nucleotide sequence shown in SEQ
ID
NO:1, SEQ ID N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID N0:7, or SEQ ID N0:8 is
30 one which is sufficiently complementary to the nucleotide sequence shown in
SEQ ID
NO:1, SEQ ID N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID N0:7, or SEQ ID N0:8
such that it can hybridize to the nucleotide sequence shown in SEQ ID NO:1,
SEQ ID
N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID N0:7, or SEQ ID N0:8, thereby forming
a stable duplex.
35 In still another preferred embodiment, an isolated nucleic acid molecule of
the
present invention comprises a nucleotide sequence which is at least about 60%,
65%,
70%, 71 %, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the nucleotide
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sequences show in SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID
N0:7, or SEQ ID N0:8, or a portion of either of these nucleotide sequences
larger than
450 bp.
Moreover, the nucleic acid molecule of the invention can comprise only a
portion
of the nucleic acid sequence of SEQ ID NO:1, or SEQ ID NO:S, for example a
fragment
which can be used as a probe or primer or a fragment encoding a biologically
active
portion of a STRIFE1 or a STRIFE2 protein. The nucleotide sequence determined
from
the cloning of the marine STRIFE1 and STRIFE2 genes allows for the generation
of
probes and primers designed for use in identifying and/or cloning STRIFE
homologues
10 in other cell types, e.g., from other tissues, as well as STRIFE homologues
from other
mammals including humans. The probe/primer typically comprises a 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 40, 50 or 75 consecutive nucleotides of a sense
sequence of
15 SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID N0:7, or SEQ ID
N0:8, of an antisense sequence of SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:4, SEQ
ID
NO:S, SEQ ID N0:7, or SEQ ID N0:8, or of a naturally occurring mutant of SEQ
ID
NO:1, SEQ ID N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID N0:7, or SEQ ID N0:8.
In an exemplary embodiment, a nucleic acid molecule of the present invention
20 comprises a nucleotide sequence which hybridizes under stringent
hybridization
conditions to a nucleic acid molecule comprising nucleotides 1-16, 413-602, or
711-981
of SEQ ID NO:1 or to a nucleic acid molecule comprising nucleotides 1-16, 416-
489, or
519-655 of SEQ ID NO:S.
Probes based on the marine STRIFE 1 and STRIFE2 nucleotide sequence can be
25 used to detect transcripts or genomic sequences encoding the same or
homologous
proteins. In preferred embodiments, the probe further comprises a label group
attached
thereto, e.g., the label group can be a radioisotope, a fluorescent compound,
an enzyme,
or an enzyme co-factor. Such probes can be used as a part of a diagnostic test
kit for
identifying cells or tissue which misexpress a STRIFE1 or a STRIFE2 protein,
such as
30 by measuring a level of a STRIFE1 or a STRIFE2-encoding nucleic acid in a
sample of
cells from a subject e.g., detecting STRIFE1 or STRIFE2 mRNA levels or
determining
whether a genomic STRIFE 1 or STRIFE2 gene has been mutated or deleted.
A nucleic acid fragment encoding a "biologically active portion of a STRIFE 1
or
a STRIFE2 protein" can be prepared by isolating a portion of SEQ ID NO:1, SEQ
ID
35 N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID N0:7, or SEQ ID N0:8 which encodes a
polypeptide having a STRIFE1 or a STRIFE2 biological activity (the biological
activities of the STRIFE 1 and STRIFE2 proteins include biological activities
attributed
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to the TNFR super-family of proteins), expressing the encoded portion of the
STRIFE1
or the STRIFE2 protein (e.g., by recombinant expression in vitro) and
assessing the
activity of the encoded portion of the STRIFE1 or STRIFE2 protein.
The invention further encompasses nucleic acid molecules that differ from the
S nucleotide sequence shown in SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:4, SEQ ID
NO:S, SEQ ID N0:7, or SEQ ID NO:8 due to degeneracy of the genetic code and
thus
encode the same STRIFE1 or STRIFE2 proteins as those encoded by the nucleotide
sequence shown in SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ
ID N0:7, or SEQ ID N0:8, respectively. In another embodiment, an isolated
nucleic
10 acid molecule of the invention has a nucleotide sequence encoding a protein
having an
amino acid sequence shown in SEQ ID N0:2 or SEQ ID N0:6.
In addition to the marine. STRIFE1 and STRIFE2 nucleotide sequences shown
in SEQ ID NO:1 and SEQ ID NO:S, respectively, it will be appreciated by those
skilled
in the art that DNA sequence polymorphisms that lead to changes in the amino
acid
15 sequences of the STRIFE1 and STRIFE2 proteins may exist within a population
(e.g.,
the human population). Such genetic polymorphism in the STRIFE1 or STRIFE2
genes
may exist among individuals within a population due to natural allelic
variation. As
used herein, the terms "gene" and "recombinant gene" refer to nucleic acid
molecules
comprising an open reading frame encoding a STRIFE1 or STRIFE2 protein,
preferably
20 a mammalian STRIFE1 or STRIFE2 protein. Such natural allelic variations can
typically result in 1-5% variance in the nucleotide sequence of a STRIFE1 or a
STRIFE2
gene. Any and all such nucleotide variations and resulting amino acid
polymorphisms in
STRIFE1 or STRIFE2 genes that are the result of natural allelic variation and
that do
not alter the functional activity of a STRIFE1 or STRIFE2 protein are intended
to be
25 within the scope of the invention.
Moreover, nucleic acid molecules encoding STRIFE1 and STRIFE2 proteins
from other species, and thus which have a nucleotide sequence which differs
from the
marine sequence of SEQ ID NO:1 and SEQ ID NO:S are intended to be within the
scope
of the invention.
30 Nucleic acid molecules corresponding to natural allelic variants and
homologues
of the STRIFEI or STRIFE2 cDNAs of the invention can be isolated based on
their
homology to the marine STRIFE1 or STRIFE2 nucleic acids disclosed herein using
the
marine cDNA, or a portion thereof, as a hybridization probe according to
standard
hybridization techniques under stringent hybridization conditions.
35 Accordingly, in another embodiment, an isolated nucleic acid molecule of
the
invention is at least 15 nucleotides in length and hybridizes under stringent
conditions to
the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,
SEQ ID
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N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID N0:7, or SEQ ID N0:8. In another
embodiment, the nucleic acid is at least 30, 50, 100, 250 or 500 nucleotides
in length.
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%
homologous to each other typically remain hybridized to each other.
Preferably, the
conditions are such that sequences at least about 70%, more preferably at
least about
80%, even more preferably at least about 85% or 90% homologous 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 Biology,
John
Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, 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.
Preferably, an isolated nucleic acid molecule of the invention that hybridizes
under
stringent conditions to the sequence of SEQ ID NO:1 corresponds to a naturally-
1 S 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 the STRIFE 1 or STRIFE2
sequences that may exist in the population, the skilled artisan will further
appreciate that
changes can be introduced by mutation into the nucleotide sequences of SEQ ID
NO:1,
or SEQ ID NO:S, thereby leading to changes in the amino acid sequence of the
encoded
STRIFE1 or STRIFE2 proteins, without altering the functional ability of the
STRIFE1
or STRIFE2 proteins. For example, nucleotide substitutions leading to amino
acid
substitutions at "non-essential" amino acid residues can be made in the
sequence of SEQ
ID NO:1, or SEQ ID NO:S. A "non-essential" amino acid residue is a residue
that can
be altered from the wild-type sequence of STRIFE1 or STRIFE2 (e.g., the
sequence of
SEQ ID N0:2 or SEQ ID N0:6) without altering the biological activity, whereas
an
"essential" amino acid residue is required for biological activity. For
example, .amino
acid residues that are conserved among the STRIFE1 or STRIFE2 proteins of the
present
invention, are predicted to be particularly unamenable to alteration.
Furthermore, amino
acid residues that are conserved between STRIFE1 or STRIFE2 protein and other
proteins having cysteine-rich domains are not likely to be amenable to
alteration.
Accordingly, another aspect of the invention pertains to nucleic acid
molecules
encoding STRIFE1 or STRIFE2 proteins that contain changes in amino acid
residues
that are not essential for activity. Such STRIFE1 or STRIFE2 proteins differ
in amino
acid sequence from SEQ ID N0:2 or SEQ ID N0:6 yet retain biological activity.
In one
embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence
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encoding a protein, wherein the protein comprises an amino acid sequence at
least about
60% homologous to the amino acid sequence of SEQ ID N0:2, SEQ ID N0:6, SEQ ID
N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID
N0:14, or SEQ ID NO:1~5. Preferably, the protein encoded by the nucleic acid
molecule
is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 90%, 95%, 98%
or more homologous to SEQ ID N0:2, SEQ ID N0:6, SEQ ID N0:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, or SEQ ID NO:15.
An isolated nucleic acid molecule encoding a STRIFE1 or STRIFE2 protein
homologous to the protein of SEQ ID N0:2 or SEQ ID NO:6, respectively, can be
10 created by introducing one or more nucleotide substitutions, additions or
deletions into
the nucleotide sequence of SEQ ID NO:1, SEQ ID N0:3, SEQ ID NO:S, or SEQ ID
N0:7, such that one or more amino acid substitutions, additions or deletions
are
introduced into the encoded protein. Mutations can be introduced into SEQ ID
NO:1,
SEQ ID N0:3, SEQ ID NO:S, or SEQ ID N0:7 by standard techniques, such as site-
15 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
20 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
25 (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted
nonessential
amino acid residue in a STRIFE1 or STRIFE2 protein is preferably replaced with
another amino acid residue from the same side chain family. Alternatively, in
another
embodiment, mutations can be introduced randomly along all or part of a
STRIFE1 or
STRIFE2 coding sequence, such as by saturation mutagenesis, and the resultant
mutants
30 can be screened for STRIFE1 or STRIFE2 biological activity to identify
mutants that
retain activity. Following mutagenesis of SEQ ID NO:1, SEQ ID N0:3, SEQ ID
N0:4,
SEQ ID NO:S, SEQ ID N0:7, or SEQ ID N0:8, the encoded protein can be expressed
recombinantly and the activity of the protein can be determined.
In a preferred embodiment, a mutant STRIFE 1 or STRIFE2 protein can be
35 assayed for the ability to (i) modulate cellular signal transduction,
either in vitro or in
vivo; (ii) regulate gene transcription in a cell involved in development or
differentiation,
either in vitro or in vivo; (iii) modulate cellular signal transduction; (iv)
regulate cellular
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proliferation; (v) regulate cellular differentiation; (vi) regulate cell
survival; and (vii)
modulate a cell involved in the immune response.
In addition to the nucleic acid molecules encoding STRIFEI or STRIFE2
proteins described above, another aspect of the invention pertains to isolated
nucleic acid
molecules which are antisense thereto. An "antisense" nucleic acid comprises a
nucleotide sequence which is complementary to a "sense" nucleic acid encoding
a
protein, 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 STRIFEI or STRIFE2 coding strand, or to only a
portion
thereof. In one embodiment, an antisense nucleic acid molecule is antisense to
a "coding
region" of the coding strand of a nucleotide sequence encoding STRIFE 1 or
STRIFE2.
The term "coding region" refers to the region of the nucleotide sequence
comprising
codons which are translated into amino acid residues (e.g., the coding region
of marine
I 5 STRIFE I corresponds to SEQ ID N0:3 and the coding region of marine
STRIFE2
corresponds to SEQ ID N0:7). In another embodiment, the antisense nucleic acid
molecule is antisense to a "noncoding region" of the coding strand of a
nucleotide
sequence encoding STRIFEI or STRIFE2. The term "noncoding region" refers to 5'
and
3' sequences which flank the coding region that are not translated into amino
acids (i.e.,
also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding STRIFEI or STRIFE2 disclosed
herein (e.g., SEQ ID N0:3 or SEQ ID N0:7), antisense nucleic acids of the
invention
can be designed according to the rules of Watson and Crick base pairing. The
antisense
nucleic acid molecule can be complementary to the entire coding region of
STRIFE1 or
STRIFE2 mRNA, but more preferably is an oligonucleotide which is antisense to
only a
portion of the coding or noncoding region of STRIFE1 or STRIFE2 mRNA. For
example, the antisense oligonucleotide can be complementary to the region
surrounding
the translation start site of STRIFE1 or STRIFE2 mRNA. 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. 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 S-
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fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xantine, 4-
acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-
thiouridine, S-carboxymethylaminomethyluracil, dihydrouracil, beta-D-
galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-
methylinosine,
5 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-
10 methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-S-
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
15 the inserted nucleic acid will be of an antisense orientation to a target
nucleic acid of
interest, described further in the following subsection).
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 STRIFE1 or STRIFE2 protein to thereby inhibit
20 expression of the protein, 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
25 invention include 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
30 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.
35 In yet another embodiment, the antisense nucleic acid molecule of the
invention
is 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
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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 also comprise a 2'-
0-
methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or
a
chimeric RNA-DNA analogue (moue et a1. ( 1987) FEBS Lett. 215:327-330).
In still another embodiment, an antisensewucleic acid of the invention is a
ribozyme. 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
i 0 catalytically cleave STRIFE 1 or STRIFE2 mRNA transcripts to thereby
inhibit
translation of STRIFE 1 or STRIFE2 mRNA. A ribozyme having specificity for a
STRIFE1 or STRIFE2-encoding nucleic acid can be designed based upon the
nucleotide
sequence of a STRIFE1 or STRIFE2 cDNA disclosed herein (i.e., SEQ ID NO:1, SEQ
ID N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID N0:7, or SEQ ID N0:8). For
15 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 STRIFE1 or STRIFE2-encoding mRNA. See, e.g., Cech et al. U.S.
Patent
No. 4,987,071; and Cech et a!. U.S. Patent No. 5,116,742. Alternatively,
STRIFE1 or
STRIFE2 mRNA can be used to select a catalytic RNA having a specific
ribonuclease
20 activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak,
J.W. (1993)
Science 261:1411-1418.
Alternatively, STRIFE1 or STRIFE2 gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory region of the
STRIFE 1
or STRIFE2 (e.g., the STRIFE1 or STRIFE2 promoter and/or enhancers) to form
triple
25 helical structures that prevent transcription of the STRIFE 1 or STRIFE2
gene in target
cells. See generally, Helene, C. (1991 ) Anticancer Drug Des. 6(6):569-84;
Helene, C. et
al. (1992) Ann. N. Y. Acad. Sci. 660:27-36; and Maher, L.J. (1992) Bioassays
14(12):807-1 S.
In yet another embodiment, the STRIFEI or STRIFE2 nucleic acid molecules of
30 the present 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 acid molecules can
be
modified to generate peptide nucleic acids (see Hyrup B. et al. (1996)
Bioorganic &
Medicinal Chemistry 4 ( 1 ): 5-23). As used herein, the terms "peptide nucleic
acids" or
35 "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
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specific hybridisation 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 B. et al. (1996) supra; Pent'-
O'Keefe et al.
PNAS 93: 14670-675.
5 PNAs of STRIFE1 or STRIFE2 nucleic acid molecules can be used for
therapeutic and diagnostic applications. For example, PNAs can be used as
antisense or
antigene agents for sequence-specific modulation of gene expression by, for
example,
inducing transcription or translation arrest or inhibiting replication. PNAs
of STRIFE1
or STRIFE2 nucleic acid molecules can also be used in the analysis of single
base pair
10 mutations in a gene, (e.g., by PNA-directed PCR clamping); as 'artificial
restriction
enzymes' when used in combination with other enzymes, (e.g., S 1 nucleases
(Hyrup B.
(1996) supra)); or as probes or primers for DNA sequencing or hybridization
(Hyrup B.
et al. ( 1996) supra; Perry-O'Keefe supra).
In another embodiment, PNAs of STRIFE1 or STRIFE2 can be modified, (e.g.,
15 to enhance their stability or cellular uptake), by attaching lipophilic or
other helper
groups to PNA, by the formation of PNA-DNA chimeras, or by the use of
Iiposomes or
other techniques of drug delivery known in the art. For example, PNA-DNA
chimeras
of STRIFE 1 or STRIFE2 nucleic acid molecules can be generated which may
combine
the advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition
20 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 B:
(1996)
supra). The synthesis of PNA-DNA chimeras can be performed as described in
Hyrup
25 B. (1996) supra and Finn P.J. et al. (1996) Nucleic Acids Res. 24 (17):
3357-63. For
example, a DNA chain can be synthesized on a solid support using standard
phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-
(4-
methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used as a
between the
PNA and the 5' end of DNA (Mag, M. et al. ( 1989) Nucleic Acid Res. 17: 5973-
88).
30 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 P.J. et al. ( 1996) supra).
Alternatively, chimeric moleclues can be synthesized with a 5' DNA segment and
a 3'
PNA segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-
11124).
In other embodiments, the oligonucleotide may include other appended groups
35 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. US 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-
652;
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PCT Publication No. W088/09810, published December 1 S, 1988) or the blood-
brain
barrier (see, e.g., PCT Publication No. W089/10134, published April 25, 1988).
In
addition, oligonucleotides can be modified with hybridization-triggered
cleavage agents
(See, e.g., Krol et al. (1988) BioTechniques 6:958-976) or intercalating
agents. (See,
5 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, or hybridization-triggered cleavage agent).
II. Isolated STRIFE1 and STRIFE2 Proteins and Anti-STRIFE1 and -STRIFE2
Antibodies
One aspect of the invention pertains to isolated STRIFE1 and STRIFE2 proteins,
and biologically active portions thereof, as well as polypeptide fragments
suitable for
use as immunogens to raise anti-STRIFE1 and STRIFE2 antibodies. In one
embodiment, native STRIFE 1 or STRIFE2 proteins can be isolated from cells or
tissue
15 sources by an appropriate purification scheme using standard protein
purification
techniques. In another embodiment, STRIFEI or STRIFE2 proteins are produced by
recombinant DNA techniques. Alternative to recombinant expression, a STRIFE1
or
STRIFE2 protein or polypeptide can be synthesized chemically using standard
peptide
synthesis techniques.
20 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 STRIFE 1 or STRIFE2 protein is derived, or
substantially
free from chemical precursors or other chemicals when chemically synthesized.
The
language "substantially free of cellular material" includes preparations of
STRIFE1 or
25 STRIFE2 protein in which the protein is separated from cellular components
of the cells
from which it is isolated or recombinantly produced. In one embodiment, the
language
"substantially free of cellular material" includes preparations of STRIFE1 or
STRIFE2
protein having less than about 30% (by dry weight) of non-STRIFE1 or STRIFE2
protein (also referred to herein as a "contaminating protein"), more
preferably less than
30 about 20% of non-STRIFE 1 or non-STRIFE2 protein, still more preferably
less than
about 10% of non-STRIFE 1 or non-STRIFE2 protein, and most preferably less
than
about 5% non-STRIFE1 or non-STRIFE2 protein. When the STRIFE1 or STRIFE2
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
35 about 20%, more preferably less than about 10%, and most preferably less
than about
5% of the volume of the protein preparation.
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The language "substantially free of chemical precursors or other chemicals"
includes preparations of STRIFE1 or STRIFE2 protein in which the protein is
separated
from chemical precursors or other chemicals which are involved in the
synthesis of the
protein. In one embodiment, the language "substantially free of chemical
precursors or
5 other chemicals" includes preparations of STRIFE1 or STRIFE2 protein having
less than
about 30% (by dry weight) of chemical precursors or non-STRIFE1 or non-STRIFE2
chemicals, more preferably less than about 20% chemical precursors or non-
STRIFE 1 or
non-STRIFE2 chemicals, still more preferably less than about 10% chemical
precursors
or non-STRIFE1 or non-STRIFE2 chemicals, and most preferably less than about
5%
10 chemical precursors or non-STRIFE 1 or non-STRIFE2 chemicals.
Biologically active portions of a STRIFE 1 or STRIFE2 protein include peptides
comprising amino acid sequences sufficiently homologous to or derived from the
amino
acid sequence of the STRIFE1 or STRIFE2 protein, e.g., the amino acid sequence
shown
in SEQ ID N0:2 or SEQ ID N0:6, which include less amino acids than the full
length
15 STRIFE 1 or STRIFE2 proteins, and exhibit at least one activity of a STRIFE
1 or
STRIFE2 protein. Typically, biologically active portions comprise a domain or
motif
with at least one activity of the STRIFE1 or STRIFE2 protein. A biologically
active
portion of a STRIFE1 or STRIFE2 protein can be a polypeptide which is, for
example,
10, 25, 50, 100 or more amino acids in length.
20 In one embodiment, a biologically active portion of a STRIFE 1 or STRIFE2
protein comprises at least a cysteine-rich domain. In another embodiment, a
biologically active portion of a STRIFE1 or STRIFE2 protein comprises at least
a
cysteine-rich domain, wherein the cysteine-domain includes at least one
module. In yet
another embodiment, a biologically active portion of a STRIFE 1 or STRIFE2
protein
25 comprises at least a signal sequence. In yet a further embodiment, a
biologically active
portion of a STRIFE 1 or STRIFE2 protein comprises at least a cysteine-rich
domain and
a signal sequence.
In an alternative embodiment, a biologically active portion of a STRIFE 1 or
STRIFE2 protein comprises a STRIFE 1 or STRIFE2 amino acid sequence lacking a
30 signal sequence. In another alternative embodiment, a biologically active
portion of a
STRIFE1 or STRIFE2 protein comprises a STRIFE1 or STRIFE2 amino acid sequence
lacking a cysteine-rich domain.
It is to be understood that a preferred biologically active portion of a
STRIFE 1
or STRIFE2 protein of the present invention may contain at least one of the
above-
35 identified structural domains. Another preferred biologically active
portion of a
STRIFE1 or STRIFE2 protein may contain at least two of the above-identified
structural
domains. Another more prefetTed biologically active portion of a STRIFE1 or
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STRIFE2 protein may contain at least three or more of the above-identified
structural
domains.
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 a native STRIFE1 or STRIFE2 protein.
In a preferred embodiment, the STRIFE 1 or STRIFE2 protein has an amino acid
sequence shown in SEQ ID N0:2 or SEQ ID N0:6, respectively. In other
embodiments,
the STRIFE1 or STRIFE2 protein is substantially homologous to SEQ ID N0:2 or
SEQ
ID N0:6, and retains the functional activity of the protein of SEQ ID N0:2 or
SEQ ID
10 N0:6, respectively, yet differs in amino acid sequence due to natural
allelic variation or
mutagenesis, as described in detail in subsection I above. Accordingly, in
another
embodiment, the STRIFE 1 or STRIFE2 protein is a protein which comprises an
amino
acid sequence at least about 60% homologous to the amino acid sequence of SEQ
ID
N0:2 or SEQ ID N0:6 and preferably retains a functional activity of the STRIFE
1 or
15 STRIFE2 protein of SEQ ID N0:2 or SEQ ID N0:6, respectively. Preferably,
the
protein is at least about 70% homologous to SEQ ID N0:2 or SEQ ID N0:6, more
preferably at least about 80% homologous to SEQ ID N0:2 or SEQ ID N0:6, even
more
preferably at least about 90% homologous to SEQ ID N0:2 or SEQ ID N0:6, and
most
preferably at least about 95% or more homologous to SEQ ID N0:2 or SEQ ID
N0:6.
20 To determine the percent identity of two amino acid sequences or of two
nucleic
acid sequences, the sequences are aligned for optimal comparison purposes
(e.g., gaps
can be introduced in one or both of a first and a second amino acid or nucleic
acid
sequence for optimal alignment and non-homologous sequences can be disregarded
for
comparison purposes). In a preferred embodiment, the length of a reference
sequence
25 aligned for comparison purposes is at least 30%, preferably at least 40%,
more
preferably at least 50%, even more preferably at least 60%, and even more
preferably at
least 70%, 80%, or 90% of the length of the reference sequence (e.g., when
aligning a
second sequence to the STRIFE 1 and STRIFE2 amino acid sequence of SEQ ID N0:2
or SEQ ID N0:6 having 177 amino acid residues, at least 80, preferably at
least 100,
30 more preferably at least 120, even more preferably at least 140, and even
more
preferably at least 150, 160 or 170 amino acid residues are aligned). 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
35 molecules are identical at that position (as used herein amino acid or
nucleic acid
"identity" is equivalent to amino acid or nucleic acid "homology"). The
percent identity
between the two sequences is a function of the number of identical positions
shared by
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the sequences, taking into account the number of gaps, and the length of each
gap, which
need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity between two
sequences can be accomplished using a mathematical algorithm. In a preferred
5 embodiment, the percent identity between two amino acid sequences is
determined using
the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which
has
been incorporated into the GAP program in the GCG software package (available
at
http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and
a gap
weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or
6. In yet
10 another preferred embodiment, the percent identity between two nucleotide
sequences is
determined using the GAP program in the GCG software package (available at
http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, S0,
60,
70, or 80 and a length weight of 1, 2, 3, 4, S, or 6. In another embodiment,
the percent
identity between two amino acid or nucleotide sequences is determined using
the
15 algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has
been
incorporated into the ALIGN program (version 2.0), using a PAM 120 weight
residue
table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences of the present invention can further be
used as a "query sequence" to perform a search against public databases to,
for example,
20 identify other family members or related sequences. Such searches can be
performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990)
J.
Mo1 Biol. 215:403-10. BLAST nucleotide searches can be performed with the
NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences
homologous to STRIFE1 or STRIFE2 nucleic acid molecules of the invention.
BLAST
25 protein searches can be performed with the XBLAST program, score = 50,
wordlength =
3 to obtain amino acid sequences homologous to STRIFE1 or STRIFE2 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(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default
30 parameters of the respective programs (e.g., XBLAST and NBLAST) can be
used. See
http://www.ncbi.nlm.nih.gov.
The invention also provides STRIFE1 or STRIFE2 chimeric or fusion proteins.
As used herein, a STRIFE1 or STRIFE2 "chimeric protein" or "fusion protein"
comprises a STRIFE1 or STRIFE2 polypeptide operatively linked to a non-STRIFE1
or
35 non-STRIFE2 polypeptide. A "STRIFE1 or STRIFE2 polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to STRIFE1 or STRIFE2,
whereas a "non-STRIFE1 or non-STRIFE2 polypeptide" refers to a polypeptide
having
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an amino acid sequence corresponding to a protein which is not substantially
homologous to the STRIFE1 or STRIFE2 protein, e.g., a protein which is
different from
the STRIFE1 or STRIFE2 protein and which is derived from the same or a
different
organism. Within a STRIFE1 or STRIFE2 fusion protein the STRIFE1 or STRIFE2
5 polypeptide can correspond to all or a portion of a STRIFE1 or STRIFE2
protein. In a
preferred embodiment, a STRIFE1 or STRIFE2 fusion protein comprises at least
one
biologically active portion of a STRIFE 1 or STRIFE2 protein. In another
preferred
embodiment, a STRIFE1 or STRIFE2 fusion protein comprises at least two
biologically
active portions of a STRIFE1 or STRIFE2 protein. In another preferred
embodiment, a
10 STRIFE1 or STRIFE2 fusion protein comprises at least three biologically
active
portions of a STRIFE1 or STRIFE2 protein. Within the fusion protein, the term
"operatively linked" is intended to indicate that the STRIFE1 or STRIFE2
polypeptide
and the non-STRIFE1 or non-STRIFE2 polypeptide are fused in-frame to each
other.
The non-STRIFE1 or non-STRIFE2 polypeptide can be fused to the N-terminus or C-
15 terminus of the STRIFE1 or STRIFE2 polypeptide.
For example, in one embodiment, the fusion protein is a GST-STRIFE1 or
STRIFE2 fusion protein in which the STRIFE 1 or STRIFE2 sequences are fused to
the
C-terminus of the GST sequences. Such fusion proteins can facilitate the
purification of
recombinant STRIFEI or STRIFE2. In another embodiment, the fusion protein is a
20 STRIFE1 or STRIFE2 protein containing a heterologous signal sequence at its
N-
terminus. For example, the native STRIFE1 or STRIFE2 signal sequence (i.e,
about
amino acids 1-29 of SEQ ID N0:2 or SEQ ID N0:6) can be removed and replaced
with
a signal sequence from another protein. In certain host cells (e.g., mammalian
host
cells), expression and/or secretion of STRIFE 1 or STRIFE2 can be increased
through
25 use of a heterologous signal sequence.
In yet another embodiment, the fusion protein is a STRIFE 1 or STRIFE2-
immunoglobulin fusion protein in which the STRIFE 1 or STRIFE2 sequences
comprising primarily the STRIFE 1 or STRIFE2 cysteine-rich domains are fused
to
sequences derived from a member of the immunoglobulin protein family. Soluble
30 derivatives have also been made of cell surface glycoproteins in the
immunoglobulin
gene superfamily consisting of an extracellular domain of the cell surface
glycoprotein
fused to an immunoglobulin constant (Fc) region (see e.g., Capon, D.J. et al.
(1989)
Nature 337:525-531 and Capon U.S. Patents 5,116,964 and 5,428,130 [CD4-IgGI
constructs]; Linsley, P.S. et al. (1991) J. Exp. Med. 173:721-730 [a CD28-IgGl
35 construct and a B7-1-IgGl construct]; and Linsley, P.S. et al. (1991) J.
Exp. Med
174:561-569 and U.S. Patent 5,434,131[a CTLA4-IgGI]). Such fusion proteins
have
proven useful for modulating receptor-ligand interactions. Soluble derivatives
of cell
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-28-
surface proteins of the tumor necrosis factor receptor (TNFR) superfamily
proteins have
been made consisting of an extracellular domain of the cell surface receptor
fused to an
immunoglobulin constant (Fc) region (see for example Moreland et al. ( 1997)
N. Engl.
J. Med. 337(3):141-147; van der Poll et al. (1997) Blood 89(10):3727-3734; and
Ammann et al. (1997) J. Clin. Invest. 99(7):1699-1703).
The STRIFEI or STRIFE2-immunoglobulin fusion proteins of the invention can
be incorporated into pharmaceutical compositions and administered to a subject
to
inhibit an interaction between a STRIFE1 ligand and a STRIFEI receptor on the
surface
of a cell, or between a STRIFE2 receptor and the STRIFE2 ligand, to thereby
suppress
10 STRIFE 1 or STRIFE2-mediated signal transduction in vivo. The STRIFE 1 or
STRIFE2-immunoglobulin fusion proteins can be used to affect the
bioavailability of a
STRIFE1 or STRIFE2 cognate receptor. Inhibition of the STRIFE1 or STRIFE2
ligand/STRIFE 1 or STRIFE2 interaction may be useful therapeutically for the
treatment
of TNF-associated disorders, e.g., inflammatory, immune, or neoplastic
disorders.
15 Moreover, the STRIFE 1 or STRIFE2-immunoglobulin fusion proteins of the
invention
can be used as immunogens to produce anti-STRIFE1 or STRIFE2 antibodies in a
subject, to purify STRIFE1 or STRIFE2 ligands and in screening assays to
identify
molecules which inhibit the interaction of STRIFE1 or STRIFE2 with a STRIFE1
or
STRIFE2 ligand.
20 Preferably, a STRIFE1 or STRIFE2 chimeric or fusion protein of the
invention is
produced by standard recombinant DNA techniques. For example, DNA fragments
coding for the different polypeptide sequences are ligated together in-frame
in
accordance with conventional techniques, for example by employing blunt-ended
or
stagger-ended termini for ligation, restriction enzyme digestion to provide
for
25 appropriate termini, $Iling-in of cohesive ends as appropriate, alkaline
phosphatase
treatment to avoid undesirable joining, and enzymatic ligation. 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
30 consecutive gene fragments which can subsequently be annealed and
reamplified to
generate a chimeric gene sequence (see, for example, Current Protocols in
Molecular
Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many
expression
vectors are commercially available that already encode a fusion moiety (e.g.,
a GST
polypeptide). A STRIFE1 or STRIFE2-encoding nucleic acid can be cloned into
such
35 an expression vector such that the fusion moiety is linked in-frame to the
STRIFE1 or
STRIFE2 protein.
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The present invention also pertains to variants of the STRIFEI or STRIFE2
proteins which function as either STRIFEI or STRIFE2 agonists (mimetics) or as
STRIFE 1 or STRIFE2 antagonists. Variants of the STRIFE 1 or STRIFE2 proteins
can
be generated by mutagenesis, e.g., discrete point mutation or truncation of a
STRIFE1 or
5 STRIFE2 protein. An agonist of the STRIFE1 or STRIFE2 proteins can retain
substantially the same, or a subset, of the biological activities of the
naturally occurring
form of a STRIFEI or STRIFE2 protein. An antagonist of a STRIFE1 or STRIFE2
protein can inhibit one or more of the activities of the naturally occurring
form of the
STRIFE 1 or STRIFE2 protein by, for example, competitively binding to a
downstream
10 or upstream member of a cellular signaling cascade which includes the
STRIFE1 or
STRIFE2 protein. Thus, specific biological effects can be elicited by
treatment with a
variant of limited function. In one embodiment, treatment of a subject with a
variant
having a subset of the biological activities of the naturally occurring form
of the protein
has fewer side effects in a subject relative to treatment with the naturally
occurring form
15 of the STRIFEI or STRIFE2 protein.
In one embodiment, variants of a STRIFEI or STRIFE2 protein which function
as either STRIFE 1 or STRIFE2 agonists (mimetics) or as STRIFE 1 or STRIFE2
antagonists can be identified by screening combinatorial libraries of mutants,
e.g.,
truncation mutants, of a STRIFE1 or STRIFE2 protein for STRIFEI or STRIFE2
protein
20 agonist or antagonist activity. In one embodiment, a variegated library of
STRIFE1 or
STRIFE2 variants is generated by combinatorial mutagenesis at the nucleic acid
level
and is encoded by a variegated gene library. A variegated library of STRIFE1
or
STRIFE2 variants can be produced by, for example, enzymatically ligating a
mixture of
synthetic oligonucleotides into gene sequences such that a degenerate set of
potential
25 STRIFEI or STRIFE2 sequences is expressible as individual polypeptides, or
alternatively, as a set of larger fusion proteins (e.g., for phage display)
containing the set
of STRIFEi or STRIFE2 sequences therein. There are a variety of methods which
can
be used to produce libraries of potential STRIFE1 or STRIFE2 variants from a
degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene
sequence
30 can be performed in an automatic DNA synthesizer, and the synthetic gene
then ligated
into an appropriate expression vector. Use of a degenerate set of genes allows
for the
provision, in one mixture, of all of the sequences encoding the desired set of
potential
STRIFEI or STRIFE2 sequences. Methods for synthesizing degenerate
oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983)
Tetrahedron 39:3;
35 Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984)
Science
198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
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In addition, libraries of fragments of a STRIFE1 or STRIFE2 protein coding
sequence can be used to generate a variegated population of STRIFE1 or STRIFE2
fragments for screening and subsequent selection of variants of a STRIFE1 or
STRIFE2
protein. In one embodiment, a library of coding sequence fragments can be
generated by
S treating a double stranded PCR fragment of a STRIFE1 or STRIFE2 coding
sequence
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 S 1 nuclease, and
ligating
10 the resulting fragment library into an expression vector. By this method,
an expression
library can be derived which encodes N-terminal, C-terminal and internal
fragments of
various sizes of the STRIFE1 or STRIFE2 protein.
Several techniques are known in the art for screening gene products of
combinatorial libraries made by point mutations or truncation, and for
screening cDNA
15 libraries for gene products having a selected property. Such techniques are
adaptable for
rapid screening of the gene libraries generated by the combinatorial
mutagenesis of
STRIFE1 or STRIFE2 proteins. 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
20 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. Recrusive ensemble mutagenesis (REM), a new
technique which enhances the frequency of functional mutants in the libraries,
can be
used in combination with the screening assays to identify STRIFE1 or STRIFE2
variants
25 (Arkin and Yourvan {1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein
Engineering 6(3):327-331).
In one embodiment, cell based assays can be exploited to analyze a variegated
STRIFE 1 or STRIFE2 library. For example, a library of expression vectors can
be
transfected into a cell line which ordinarily responds to a particular ligand,
e.g., a
30 cytokine, in a STRIFE1 or STRIFE2-dependent manner. The transfected cells
are then
contacted with the ligand and the effect of expression of the mutant on
signaling by the
ligand can be detected, e.g., by measuring NF-xB activity or cell survival.
Plasmid
DNA can then be recovered from the cells which score for inhibition, or
alternatively,
potentiation of cytokine induction, and the individual clones further
characterized.
35 An isolated STRIFE1 or STRIFE2 protein, or a portion or fragment thereof,
can
be used as an immunogen to generate antibodies that bind STRIFE 1 or STRIFE2
using
standard techniques for polyclonal and monoclonal antibody preparation. A full-
Length
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-31 -
STRIFE 1 or STRIFE2 protein can be used or, alternatively, the invention
provides
antigenic peptide fragments of STRIFE1 or STRIFE2 for use as immunogens. The
antigenic peptide of STRIFE 1 or STRIFE2 comprises at least 8 amino acid
residues of
the amino acid sequence shown in SEQ ID N0:2 or SEQ ID N0:6 and encompasses an
5 epitope of STRIFE 1 or STRIFE2 such that an antibody raised against the
peptide forms
a specific immune complex with STRIFE 1 or STRIFE2. Preferably, the antigenic
peptide comprises at least 10 amino acid residues, more preferably at least 15
amino acid
residues, even more preferably at least 20 amino acid residues, and most
preferably at
least 30 amino acid residues.
Preferred epitopes encompassed by the antigenic peptide are regions of STRIFE
1
or STRIFE2 that are located on the surface of the protein, e.g., hydrophilic
regions.
A STRIFE1 or STRIFE2 immunogen typically is used to prepare antibodies by
immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal)
with the
immunogen. An appropriate immunogenic preparation can contain, for example,
15 recombinantly expressed STRIFE1 or STRIFE2 protein or a chemically
synthesized
STRIFE1 or STRIFE2 polypeptide. The preparation can further include an
adjuvant,
such as Freund's complete or incomplete adjuvant, or similar immunostimulatory
agent.
Immunization of a suitable subject with an immunogenic STRIFEI or STRIFE2
preparation induces a polyclonal anti-STRIFE I or STRIFE2 antibody response.
20 Accordingly, another aspect of the invention pertains to anti-STRIFEl or
STRIFE2 antibodies. The term "antibody" as used herein refers to
immunoglobulin
molecules and immunologically active portions of immunoglobulin molecules,
i.e.,
molecules that contain an antigen binding site which specifically binds
(immunoreacts
with) an antigen, such as STRIFE1 or STRIFE2. Examples of immunologically
active
25 portions of immunoglobulin molecules include Flab) and F(ab'~ fragments
which can
be generated by treating the antibody with an enzyme such as pepsin. The
invention
provides polyclonal and monoclonal antibodies that bind STRIFE1 or STRIFE2.
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
30 binding site capable of immunoreacting with a particular epitope of STRIFE1
or
STRIFE2. A monoclonal antibody composition thus typically displays a single
binding
affinity for a particular STRIFE1 or STRIFE2 protein with which it
immunoreacts.
Polyclonal anti-STRIFE 1 or STRIFE2 antibodies can be prepared as described
above by immunizing a suitable subject with a STRIFE1 or STRIFE2 immunogen.
The
35 anti-STRIFEI or STRIFE2 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 STRIFEI or STRIFE2. If desired, the antibody
molecules
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-32-
directed against STRIFE1 or STRIFE2 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 anti-STRIFE1 or STRIFE2 antibody titers are highest, antibody-
5 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) (see also, Brown et al. (
1981 ) J.
Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem .255:4980-83; Yeh et
al.
(1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the
more
10 recent 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. Liss, Inc., pp. 77-96) or trioma techniques. The technology
for
producing monoclonal antibody hybridomas is well known (see generally R. H.
Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses,
Plenum
15 Publishing Corp., New York, New York (1980); E. A. Lerner (1981) Yale J.
Biol. Med.,
54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:231-36). Briefly,
an
immortal cell line (typically a myeloma) is fused to lymphocytes (typically
splenocytes)
from a mammal immunized with a STRIFE1 or STRIFE2 immunogen as described
above, and the culture supernatants of the resulting hybridoma cells are
screened to
20 identify a hybridoma producing a monoclonal antibody that binds STRIFE1 or
STRIFE2.
Any of the many well known protocols used for fusing lymphocytes and
immortalized cell lines can be applied for the purpose of generating an anti-
STRIFE1 or
STRIFE2 monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature
266:55052;
25 Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med.,
cited supra;
Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled
worker
will appreciate that there are many variations of such methods which also
would be
useful. Typically, the immortal cell line (e.g., a myeloma cell line) is
derived from the
same mammalian species as the lymphocytes. For example, marine hybridomas can
be
30 made by fusing lymphocytes from a mouse immunized with an immunogenic
preparation of the present invention with an immortalized mouse cell line.
Preferred
immortal cell lines are mouse myeloma cell lines that are sensitive to culture
medium
containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a
number of myeloma cell lines can be used as a fusion partner according to
standard
35 techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma
lines.
These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse
myeloma cells are fused to mouse splenocytes using polyethylene glycol
("PEG").
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- 33 -
Hybridoma cells resulting from the fusion are then selected using HAT medium,
which
kills unfused and unproductively fused myeloma cells (unfused splenocytes die
after
several days because they are not transformed). Hybridoma cells producing a
monoclonal antibody of the invention are detected by screening the hybridoma
culture
S supernatants for antibodies that bind STRIFE1 or STRIFE2, e.g., using a
standard
ELISA assay.
Alternative to preparing monoclonal antibody-secreting hybridomas, a
monoclonal anti-STRIFE1 or STRIFE2 antibody can be identified and isolated by
screening a recombinant combinatorial immunoglobulin library (e.g., an
antibody phage
10 display library) with STRIFE1 or STRIFE2 to thereby isolate immunoglobulin
library
members that bind STRIFE 1 or STRIFE2. Kits for generating and screening phage
display libraries are commercially available (e.g., the Pharmacia Recombinant
Phage
Antibody System, Catalog No. 27-9400-O 1; and the Stratagene SurfZAPTM Phage
Display Kit, Catalog No. 240612). Additionally, examples of methods and
reagents
15 particularly amenable for use in generating and screening antibody display
library can be
found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al.
PCT
International Publication No. WO 92/18619; Dower et al. PCT International
Publication
No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791;
Markland
et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT
International
20 Publication WO 93/01288; McCafferty et al. PCT International Publication
No. WO
92/01047; Garrard et al. PCT International Publication No. WO 92/09690; Ladner
et al.
PCT International Publication No. WO 90/02809; Fuchs et al. (1991)
BiolTechnology
9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al.
(1989)
Science 246:1275-1281; Grifflths et al. (1993) EMBD J 12:725-734; Hawkins et
al.
25 (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-
628; Gram et
al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) BiolTechnology 9:1373-1377;
Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137; Barbas et al. (1991)
PNAS
88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.
Additionally, recombinant anti-STRIFE1 or STRIFE2 antibodies, such as
30 chimeric and humanized monoclonal antibodies, comprising both human and non-
human portions, which can be made using standard recombinant DNA techniques,
are
within the 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 Robinson et al. International Application No.
PCT/US86/02269;
35 Akira, et al. European Patent Application 184,187; Taniguchi, M., European
Patent
Application 171,496; Morrison et al. European Patent Application 173,494;
Neuberger
et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S.
Patent No.
CA 02318743 2000-07-24
WO 99137818 PCTNS99/01679
-34-
4,816,567; Cabilly et al. European Patent Application 125,023; Better et al.
(1988)
Science 240:1041-1043; Liu et al. (1987) PNAS 84:3439-3443; Liu et al. (1987)
J.
Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al.
(1987)
Canc. Res. 47:999-100; Wood et al. (1985) Nature 314:446-449; and Shaw et al.
5 (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science
229:1202-
1207; Oi et al. (1986) BioTechniques 4:214; Winter 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. Immunol. 141:4053-4060.
An anti-STRIFE1 or STRIFE2 antibody (e.g., monoclonal antibody) can be used
to isolate STRIFE1 or STRIFE2 by standard techniques, such as affinity
chromatography or immunoprecipitation. An anti-STRIFE1 or STRIFE2 antibody can
facilitate the purification of natural STRIFE1 or STRIFE2 from cells and of
recombinantly produced STRIFE1 or STRIFE2 expressed in host cells. Moreover,
an
anti-STRIFE1 or STRIFE2 antibody can be used to detect STRIFE1 or STRIFE2
protein
15 (e.g., in a cellular lysate or cell supernatant) in order to evaluate the
abundance and
pattern of expression of the STRIFE1 or STRIFE2 protein. Anti-STRIFE1 or
STRIFE2
antibodies can 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 (i.e., physically
linking) the
20 antibody to a detectable substance. Examples of detectable substances
include various
enzymes, prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent materials, and radioactive materials. Examples of suitable
enzymes
include horseradish peroxidase, alkaline phosphatase, ~i-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group complexes include
25 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
30 1251 131h 355 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 STRIFE1 or STRIFE2 (or a portion
thereof).
35 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
CA 02318743 2000-07-24
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-35-
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
S 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 are capable of directing
the
expression of genes to which they are operatively linked. Such vectors are
referred to
herein as "expression vectors". In general, expression vectors of utility in
recombinant
DNA techniques are often in the form of plasmids. In the present
specification,
"plasmid" and "vector" can be used interchangeably as the plasmid is the most
commonly used form of vector. 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, which
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
operatively 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 includes promoters, enhancers and other expression
control
elements (e.g., polyadenyladon 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 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 (e.g., STRIFE1 or STRIFE2 proteins, mutant forms of STRIFE1
or
STRIFE2, fusion proteins, etc.).
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The recombinant expression vectors of the invention can be designed for
expression of STRIFE1 or STRIFE2 in prokaryotic or eukaryotic cells. For
example,
STRIFE1 or STRIFE2 can be expressed in bacterial cells such as E. coli, insect
cells
(using baculovirus expression vectors) yeast cells or mammalian cells.
Suitable host
5 cells are discussed further in Goeddel, Gene Expression Technology: Methods
in
Enrymology 185, Academic Press, San Diego, CA (1990). Alternatively, the
recombinant expression vector can be transcribed and translated in vitro, for
example
using T7 promoter regulatory sequences and T7 polymerise.
Expression of proteins in prokaryotes is most often carried out in E. coli
with
10 vectors containing constitutive or inducible promotors 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
15 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.
20 Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;
Smith, D.B.
and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly,
MA)
and pRITS (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase
(GST),
maltose E binding protein, or protein A, respectively, to the target
recombinant protein.
Purified fusion proteins can be utilized in STRIFE1 or STRIFE2 activity
assays,
25 in STRIFE1 or STRIFE2 ligand binding (e.g., direct assays or competitive
assays
described in detail below), to generate antibodies specific for STRIFE1 or
STRIFE2
proteins, as examples. In a preferred embodiment, a STRIFE1 or STRIFE2 fusion
expressed in a retroviral expression vector of the present invention can be
utilized to
infect bone marrow cells which are subsequently transplanted into irradiated
recipients.
30 The pathology of the subject recipient is then examined after sufficient
time has passed
(e.g six (6) weeks).
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc (Amann et al., (1988) Gene 69:301-315) and pET 1 ld (Studier et al., Gene
Expression Technology: Methods in Enzymology 185, Academic Press, San Diego,
35 California (1990) 60-89). Target gene expression from the pTrc vector
relies on host
RNA polymerise transcription from a hybrid trp-lac fusion promoter. Target
gene
expression from the pET 11 d vector relies on transcription from a T7 gn 10-
lac fusion
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-37-
promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral
polymerase is supplied by host strains BL21 (DE3) or HMS 174(DE3) from a
resident ~.
prophage harboring a T7 gnl gene under the transcriptional control of the
lacUV 5
promoter.
One strategy to maximize recombinant protein expression in E. toll is to
express
the protein in a host bacteria with an impaired capacity to proteolytically
cleave the
recombinant protein (Gottesman, S., Gene Expression Technology: Methods in
Enzymolo~ 185, Academic Press, San Diego, Califonua {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. toll (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 STRIFE1 or STRIFE2 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 et al., (1987) Gene 54:113-123),
pYES2
(Invitrogen Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego,
CA).
Alternatively, STRIFE1 or STRIFE2 can be expressed in insect cells using
baculovirus expression vectors. 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 pCDM8 (Seed, B. (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, J.,
Fritsh, E. F.,
and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press,. Cold Spring Harbor,
NY,
1989.
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-
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WO 99/37818 PCT/US99/01679
-38-
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.
( I 987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
( I 988)
Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto
and
5 Baltimore (1989) EMBOJ. 8:729-733) and immunoglobulins (Banerji et al.
(1983) Cell
33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific
promoters
(e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 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
10 European Application Publication No. 264,166). Developmentally-regulated
promoters
are also encompassed, for example the marine hox promoters {Kessel and Grass {
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
1 S DNA molecule of the invention cloned into the expression vector in an
antisense
orientation. That is, the DNA molecule is operatively 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 STRIFE I or STRIFE2 mRNA. Regulatory
sequences operatively linked to a nucleic acid cloned in the antisense
orientation can be
20 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
25 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, H.
et al.,
Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in
Genetics,
Vol. 1(1) 1986.
30 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
35 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.
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WO 99/37818 PCT/US99/01679
-39-
A host cell can be any prokaryotic or eukaryotic cell. For example, STRIFE1 or
STRIFE2 protein can be expressed in bacterial cells such as E. coli, insect
cells, yeast or
mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
Other
suitable host cells are known to those skilled in the art.
5 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 (e.g., DNA) into a host cell,
including
calcium phosphate or calcium chloride co-precipitation, DEAF-dextran-mediated
10 transfection, lipofection, or electroporation. Suitable methods for
transforming or
transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the
15 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., 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, such as
6418,
20 hygromycin and methotrexate. Nucleic acid encoding a selectable marker can
be
introduced into a host cell on the same vector as that encoding STRIFE1 or
STRIFE2 or
can be introduced on a separate vector. 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).
25 A host cell of the invention, such as a prokaryotic or eukaryotic host cell
in
culture, can be used to produce (i.e., express) STRIFEl or STRIFE2 protein.
Accordingly, the invention further provides methods for producing STRIFE1 or
STRIFE2 protein using the host cells of the invention. In one embodiment, the
method
comprises culturing the host cell of invention (into which a recombinant
expression
30 vector encoding STRIFE1 or STRIFE2 has been introduced) in a suitable
medium such
that STRIFE1 or STR.IFE2 protein is produced. In another embodiment, the
method
further comprises isolating STRIFE1 or STRIFE2 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
35 oocyte or an embryonic stem cell into which STRIFE1 or STRIFE2-coding
sequences
have been introduced. Such host cells can then be used to create non-human
transgenic
animals in which exogenous STRIFE1 or STRIFE2 sequences have been introduced
into
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-40-
their genome or homologous recombinant animals in which endogenous STRIFE1 or
STRIFE2 sequences have been altered. Such animals are useful for studying the
function and/or activity of STRIFE1 or STRIFE2 and for identifying and/or
evaluating
modulators of STRIFE1 or STRIFE2 activity. As used herein, a "transgenic
animal" is a
5 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 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
10 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, a
"homologous recombinant animal" is a non-human animal, preferably a mammal,
more
preferably a mouse, in which an endogenous STRIFE 1 or STRIFE2 gene has been
altered by homologous recombination between the endogenous gene and an
exogenous
15 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 STRIFE1 or
STRIFE2-encoding nucleic acid into the male pronuclei of a fertilized oocyte,
e.g., by
microinjection, retroviral infection, and allowing the oocyte to develop in a
20 pseudopregnant female foster animal. The marine STRIFE1 or STRIFE2 cDNA
sequence of SEQ ID NO:1 or SEQ ID N0:4 can be introduced as a transgene into
the
genome of a non-human animal. Alternatively, a nonmurine homologue of a marine
STRIFE1 or STRIFE2 gene, such as a human STRIFE1 or STRIFE2 gene, can be
isolated based on hybridization to the marine STRIFE1 or STRIFE2 cDNA
(described
25 further in subsection I above) and used as a transgene. 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 STRIFE 1 or STRIFE2 transgene to direct expression of STRIFE 1
or
STRIFE2 protein to particular cells. Methods for generating transgenic animals
via
30 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, both by Leder et al., U.S. Patent No. 4,873,191 by
Wagner et
al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor
Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for
production of
35 other transgenic animals. A transgenic founder animal can be identified
based upon the
presence of the STRIFE1 or STRIFE2 transgene in its genome andlor expression
of
STRIFE 1 or STitIFE2 mRNA in tissues or cells of the animals. A transgenic
founder
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-41 -
animal can then be used to breed additional animals carrying the transgene.
Moreover,
transgenic animals carrying a transgene encoding STRIFE1 or STRIFE2 can
further be
bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains
5 at least a portion of a STRIFE1 or STRIFE2 gene into which a deletion,
addition or
substitution has been introduced to thereby alter, e.g., functionally disrupt,
the STRIFE1
or STRIFE2 gene. The STRIFE1 or STRIFE2 gene can be a marine gene (e.g., the
cDNA of SEQ ID N0:3 or SEQ ID N0:7), but can also be a non-marine homologue of
a
marine STRIFE1 or STRIFE2 gene. For example, a human STRIFE1 or STRIFE2 gene
10 can be used to construct a homologous recombination vector suitable for
altering an
endogenous STRIFE1 or STRIFE2 gene in the mouse genome. In a preferred
embodiment, the vector is designed such that, upon homologous recombination,
the
endogenous STRIFE1 or STRIFE2 gene is functionally disrupted (i.e., no longer
encodes a functional protein; also referred to as a "knock out" vector).
Alternatively, the
15 vector can be designed such that, upon homologous recombination, the
endogenous
STRIFE1 or STRIFE2 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 STRIFE1 or STRIFE2 protein). In the homologous
recombination vector, the altered portion of the STRIFEI or STRIFE2 gene is
flanked at
20 its 5' and 3' ends by additional nucleic acid of the STRIFE1 or STRIFE2
gene to allow
for homologous recombination to occur between the exogenous STRIFE 1 or
STRIFE2
gene carned by the vector and an endogenous STRIFE1 or STRIFE2 gene in an
embryonic stem cell. The additional flanking STRIFE1 or STRIFE2 nucleic acid
is of
sufficient length for successful homologous recombination with the endogenous
gene.
25 Typically, several kilobases of flanking DNA (both at the 5' and 3' ends)
are included in
the vector (see e.g., Thomas, K.R. and Capecchi, M. R. (1987) Cell 51:503 for
a
description of homologous recombination vectors). The vector is introduced
into an
embryonic stem cell line (e.g., by electroporation) and cells in which the
introduced
STRIFE1 or STRIFE2 gene has homologously recombined with the endogenous
30 STRIFE1 or STRIFE2 gene are selected (see e.g., Li, E. 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, A. in Teratocarcinomas and Embryonic
Stem
Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-
152}. A
chimeric embryo can then be implanted into a suitable pseudopregnant female
foster
35 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
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-42-
transgene. Methods for constructing homologous recombination vectors and
homologous recombinant animals are described further in Bradley, A. (1991)
Current
Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos.:
WO
90/11354 by Le Mouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by
5 Zijlstra et al.; and WO 93/04169 by Berns et al.
In another embodiment, transgenic non-humans animals can be produced which
contain selected systems which allow for regulated expression of the
transgene. One
example of such a system is the crelloxP recombinase system of bacteriophage
P1. For
a description of the crelloxP recombinase system, see, e.g., Lakso et al.
(1992) PNAS
10 89:6232-6236. Another example of a recombinase system is the FLP
recombinase
system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-
1355. If
a crelloxP 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
15 animals, e.g., by mating two 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, I. et al. (1997) Nature
385:810-
813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In
brief,
20 a cell, e.g., a somatic cell, from the transgenic animal can be isolated
and induced to exit
the growth cycle and enter Go phase. The quiescent cell can then be fused,
e.g., through
the use of electrical pulses, to an enucleated oocyte from an animal of the
same species
from which the quiescent cell is isolated. The recontructed oocyte is then
cultured such
that it develops to morula or blastocyte and then transferred to
pseudopregnant female
25 foster animal. The offspring borne of this female foster animal will be a
clone of the
animal from which the cell, e.g., the somatic cell, is isolated.
IV. Pharmaceutical Com~oeition8
The STRIFE1 or STRIFE2 nucleic acid molecules, STRIFE1 or STRIFE2
30 proteins, and anti-STRIFE1 or STRIFE2 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
35 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
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- 43 -
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.
A pharmaceutical composition of the invention is formulated to be compatible
5 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,
10 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 bisulfate; 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,
15 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
20 extemporaneous preparation of sterile injectable solutions or dispersion.
For
intravenous administration, suitable carriers include physiological saline,
bacteriostatic
water, Cremophor ELTM (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
25 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
30 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 manitol, sorbitol,
sodium
35 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.
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_4ø_
Sterile injectable solutions can be prepared by incorporating the active
compound (e.g., a STRIFE1 or STRIFE2 protein or anti-STRIFE1 or STRIFE2
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,
5 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 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
10 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
15 using a fluid carrier for use as a mouthwash, wherein the compound in the
fluid Garner 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
20 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
peppenmint,
methyl salicylate, or orange flavoring.
25 For administration by inhalation, the compounds are delivered in the form
of an
aerosol spray from pressured 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 transdermal administration, penetrants appropriate to the
barrier to be
30 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
35 the art.
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- 45 -
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 Garners that will
5 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.
Methods for preparation of such formulations will be apparent to those skilled
in the art.
10 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 Garners. 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.
15 It is especially advantageous to formulate oral or parenteral compositions
in
dosage unit form for ease of administration and uniformity of dosage. Dosage
unit 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
20 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 for the treatment of individuals.
Toxicity and therapeutic efficacy of such compounds can be determined by
25 standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., for
determining the LD50 (the dose lethal to 50% of the population) and the ED50
(the dose
therapeutically effective in 50% of the population). The dose ratio between
toxic and
therapeutic effects is the therapeutic index and it can be expressed as the
ratio
LD50/ED50. Compounds which exhibit large therapeutic indices are preferred.
While
30 compounds that exhibit toxic side effects may be used, care should be taken
to design a
delivery system that targets such compounds to the site of affected tissue in
order to
minimize potential damage to uninfected cells and, thereby, reduce side
effects.
The data obtained from the cell culture assays and animal studies can be used
in
formulating a range of dosage for use in humans. The dosage of such compounds
lies
35 preferably within a range of circulating concentrations that include the
ED50 with little
or no toxicity. The dosage may vary within this range depending upon the
dosage form
employed and the route of administration utilized. For any compound used in
the
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-46-
method of the invention, the therapeutically effective dose can be estimated
initially
from cell culture assays. A dose may be formulated in animal models to achieve
a
circulating plasma concentration range that includes the IC50 (i.e., the
concentration of
the test compound which achieves a half maximal inhibition of symptoms) as
5 determined in cell culture. Such information can be used to more accurately
determine
useful doses in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
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
10 example, intravenous injection, local administration (see U.S. Patent
5,328,470) or by
stereotactic injection {see e.g., Chen et al. (1994) PNAS 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. Alternatively, where the complete gene delivery
vector
15 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.
20
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) detecting assays (e.g., chromosome mapping, tissue typing, and
forensic
25 biology); c) predictive medicine (e.g., diagnostic assays, prognostic
assays, monitoring
clinical trials, and pharmacogenetics); and d) methods of treatment (e.g.,
therapeutic and
prophylactic methods as well as such methods in the context of
pharmacogenomics). As
described herein, a STRIFE1 protein of the invention has one or more of the
following
activities: (i) interaction of a STRIFE1 protein on the cell surface with a
second non-
30 STRIFE1 protein molecule on the surface of the same cell; (ii) interaction
of a STRIFE1
protein on the cell surface with a second non-STRIFE 1 protein molecule on the
surface
of a different cell; (iii) complex formation between a membrane-bound STRIFE1
protein
and a cytokine, e.g., TNF; (iv) interaction of a STRIFE1, protein with an
intracellular
protein including SH2 domain-containing proteins or cytoskeletal proteins; (v)
35 formation of a homogeneous multimeric signaling complex with like STRIFE1
proteins;
and (vi) formation of a heterogeneous multimeric signaling complex with other
TNFR
superfamily proteins. As described herein, STRIFE2 protein of the invention
has one or
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-47-
more of the following activities: (i) interaction of a STRIFE2 protein with a
membrane-
bound STRIFE2 receptor; (ii) interaction of a STRIFE2 protein with a soluble
form of a
STRIFE2 receptor; (iii) interaction of a STRIFE2 protein with an intracellular
protein
via a membrane-bound ~STRIFE2 receptor; (iv) complex formation between a
soluble
5 STRIFE2 protein and a second soluble STRIFE2 binding partner; (v) complex
formation
between a soluble STRIFE2 protein and a second soluble STRIFE2 binding
partner,
wherein the STRIFE2 binding partner is a non-STRIFE2 protein molecule; and
(vi)
complex formation between a soluble STRIFE2 protein and a second soluble
STRIFE2
binding partner, wherein the STRIFE2 binding partner is a second STRIFE2
protein
10 molecule. The STRIFE 1 and STRIFE2 proteins of the invention can can thus
be used
in, for example, ( 1 ) modulation of cellular signal transduction, either in
vitro or in vivo;
(2) regulation of gene transcription in a cell involved in development or
differentiation,
either in vitro or in vivo; (3) regulation of gene transcription in a cell
involved in in
development or differentiation, wherein at least one gene encodes a
differentiation-
15 specific protein; (4) regulation of gene transcription in a cell involved
in in development
or differentaition, wherein at least one gene encodes a second secreted
protein; (5)
regulation of gene transcription in a cell involved in development or
differentiation,
wherein at least one gene encodes a signal transduction molecule; and (6)
regulation of
cellular proliferation, either in vitro or in vivo. The isolated nucleic acid
molecules of
20 the invention can be used, for example, to express STRIFE1 or STRIFE2
protein (e.g.,
via a recombinant expression vector in a host cell in gene therapy
applications), to detect
STRIFEI or STRIFE2 mRNA (e.g., in a biological sample) or a genetic alteration
in a
STRIFE 1 or STRIFE2 gene, and to modulate STRIFE 1 or STRIFE2 activity, as
described further below. In addition, the STRIFE1 or STRIFE2 proteins can be
used to
25 screen drugs or compounds which modulate the STRIFEI or STRIFE2 activity as
well
as to treat disorders characterized by insufficient or excessive production of
STRIFE 1 or
STRIFE2 protein or production of STRIFE 1 or STRIFE2 protein forms which have
decreased or aberrant activity compared to STRIFE1 or STRIFE2 wild type
protein (e.g.,
developmental disorders or proliferative diseases such as cancer). Moreover,
the anti-
30 STRIFEI or STRIFE2 antibodies of the invention can be used to detect and
isolate
STRIFE1 or STRIFE2 proteins, regulate the bioavailability of STRIFE1 or
STRIFE2
proteins, and modulate STRIFE1 or STRIFE2 activity.
A. Screening Assavs:
35 The invention provides a method (also referred to herein as a "screening
assay")
for identifying modulators, i.e., candidate or test compounds or agents (e.g.,
peptides,
peptidomimetics, small molecules or other drugs) which bind to STRIFE1 or
STRIFE2
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- 48 -
proteins or have a stimulatory or inhibitory effect on, for example, STRIFE1
or
STRIFE2 expression or STRIFE1 or STRIFE2 activity.
There are assays that can be used to identify candidate or test compounds or
agents which have a stimulatory or inhibitory effect on, for example, STRIFE 1
or
5 STRIFE2 expression or STRIFE1 or STRIFE2 activity. For example, assays based
on
the effects of TNF on some cells can be used to evaluate the modulatory
activity of test
compounds on STRIFEl or STRIFE2 expression or STRIFE1 or STRIFE2 activity.
Known effects of TNF on fibroblast cells include effects on mitogenesis, IL-6
secretion
and HLA class II antigen induction. Known effects of TNF on monocytes include
10 effects on secretion of cytokines such as GM-CSF, IL-6, and IL-8. TNF is
known to be
cytotoxic to some cells, such as WEHI-164 marine fibrosarcoma cells (described
in
Espevik et al. (1986) J. Immunol. Methods 95:99-105). TNF is also known to
have
effects on cytokine secretion by endothelial cells, as well as effect
induction of adhesion
molecules such as ICAM-1, E-selectin, VCAM, and tissue factor production in
15 endothelial cells. Thus, these cells and the detectable phenotypic changes
resulting from
the effect of TNF in the presence or absence of a test compound can be used to
evaluate
the modulatory activity of the test compound on STRIFE1 or STRIFE2 expression
or
STRIFE1 or STRIFE2 activity. Furthermore, TNF is known to modulate neutrophil
responses. Comparisons can be made between TNF effects on neutrophils in the
20 presence or absence of a test compound using cellular activation, priming,
degranulation, and/or superoxide production as detectable endpoints for
evaluation of
STRIFE1 or STRIFE2 modulatory activity. These and other related assays are
well
known to those having ordinary skill in the art.
In one embodiment, the invention provides assays for screening candidate or
test
25 compounds which bind to or modulate the activity of a STRIFE1 or STRIFE2
protein or
polypeptide or biologically active portion thereof. In another embodiment, the
invention
provides assays for screening candidate or test compounds which bind to or
modulate
the activity of a STRIFE1 receptor. The test compounds of the present
invention can be
obtained using any of the numerous approaches in combinatorial library methods
known
30 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
35 oligomer or small molecule libraries of compounds (Lam, K.S. (1997)
Anticancer Drug
Des. 12:145).
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WO 99/37818 PCTNS99/01679
-49-
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. U.S.A.
90:6909; 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.
5 Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.
33:2061; and in
Gallop et al. (1994) J. Med. Chem. 37:1233.
Libraries of compounds may be presented in solution (e.g., Houghten (1992)
Biotechniques 13 :412-421 ), or on beads (Lam ( 1991 ) Nature 3 54:82-84),
chips {Fodor
(1993) Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner
USP
10 '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or
on phage
(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-
406);
(Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J.
Mol. Biol.
222:301-310); (Ladnersupra.).
In one embodiment, an assay is a cell-based assay in which a cell which
1 S expresses a STRIFE 1 or STRIFE2 receptor on the cell surface is contacted
with a test
compound and the ability of the test compound to bind to a STRIFEi or STRIFE2
receptor is determined. The cell preferably expresses a human STRIFE1 or
STRIFE2
receptor, e.g., the human receptor encoded by clone AX92 3 contained in ATCC
Deposit Number 98101 (described in PCT application number WO 98/01554,
published
20 on January 15, 1998) or the human OAF065 receptor (described in PCT
application
number WO 98/38304, published on September 3, I998). The cell, for example,
can be
of mammalian origin or a yeast cell. Determining the ability of the test
compound to
bind to a STRIFEI or STRIFE2 receptor can be accomplished, for example, by
coupling
the test compound with a radioisotope or enzymatic label such that binding of
the test
25 compound to the STRIFE1 or STRIFE2 receptor can be determined by detecting
the
labeled compound in a complex. For example, test compounds can be labeled with
~zSI,
355 ~4C~ 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 labeled with, for example, horseradish peroxidase,
alkaline
30 phosphatase, or luciferase, and the enzymatic label detected by
determination of
conversion of an appropriate substrate to product.
It is also within the scope of this invention to determine the ability of a
test
compound to interact with a STRIFE 1 or STRIFE2 receptor without the labeling
of any
of the interactants. For example, a microphysiometer can be used to detect the
35 interaction of a test compound with a STRIFE1 or STRIFE2 receptor without
the
labeling of either the test compound or the receptor. McConnell, H. M. et al.
( 1992)
Science 257:1906-1912. As used herein, a "microphysiometer" (e.g.,
CytosensorT"") is
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an analytical instrument that measures the rate at which a cell acidifies its
environment
using a light-addressable potentiometric sensor (LAPS). Changes in this
acidification
rate can be used as an indicator of the interaction between ligand and
receptor.
In a preferred embodiment, the assay comprises contacting a cell which
5 expresses a STRIFE1 or STRIFE2 receptor on the cell surface with a STRIFE1
or
STRIFE2 protein or biologically-active portion thereof, to form an assay
mixture,
contacting the assay mixture with a test compound, and determining the ability
of the
test compound to interact with a STRIFE1 or STRIFE2 receptor, wherein
determining
the ability of the test compound to interact with a STRIFE1 or STRIFE2
receptor
10 comprises determining the ability of the test compound to preferentially
bind to the
STRIFE1 or STRIFE2 receptor as compared to the ability of STRIFE1 or STRIFE2,
or a
biologically active portion thereof, to bind to the receptor.
In another embodiment, an assay is a cell-based assay comprising contacting a
cell which expresses a STRIFE1 or STRIFE2 target molecule with a test compound
and
15 determining the ability of the test compound to modulate the activity of
the STRIFE1 or
STRIFE2 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 an appropriate substrate, detecting the induction of a reporter gene
(comprising a
20 STRIFE1 or STRIFE2-responsive regulatory element operatively linked to a
nucleic
acid encoding a detectable marker, e.g., luciferase), or detecting a cellular
response, for
example, development, differentiation or rate of proliferation.
In yet another embodiment, an assay of the present invention is a cell-free
assay
in which a STRIFE1 or STRIFE2 protein or biologically active portion thereof
is
25 contacted with a test compound and the ability of the test compound to bind
to the
STRIFE1 or STRIFE2 protein or biologically active portion thereof is
determined.
Binding of the test compound to the STRIFE 1 or STRIFE2 protein can be
determined
either directly or indirectly as described above. In a preferred embodiment,
the assay
includes contacting the STRIFE1 or STRIFE2 protein or biologically active
portion
30 thereof with a known compound which binds STRIFE1 or STRIFE2 to form an
assay
mixture, contacting the assay mixture with a test compound, and determining
the ability
of the test compound to interact with a STRIFE1 or STRIFE2 protein, wherein
determining the ability of the test compound to interact with a STRIFE 1 or
STRIFE2
protein comprises determining the ability of the test compound to
preferentially bind to
35 STRIFE1 or STRIFE2 or biologically active portion thereof as compared to
the known
compound.
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In another embodiment, the assay is a cell-free assay in which a STRIFE1 or
STRIFE2 protein or biologically active portion thereof is contacted with a
test
compound and the ability of the test compound to modulate (e.g., stimulate or
inhibit)
the activity of the STRIFE 1 or STRIFE2 protein or biologically active portion
thereof is
5 determined. Determining the ability of the test compound to modulate the
activity of a
STRIFE1 or STRIFE2 protein can be accomplished, for example, by determining
the
ability of the STRIFE1 or STRIFE2 protein to bind to a STRIFE1 or STRIFE2
target
molecule by one of the methods described above for determining direct binding.
Determining the ability of the STRIFE1 or STRIFE2 protein to bind to a STRIFE1
or
10 STRIFE2 target molecule can also be accomplished using a technology such as
real-time
Biomolocular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C.
(1991)
Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol.
5:699-705.
As used herein, "BIA" is a technology for studying biospecific interactions in
real time,
without labeling any of the interactants (e.g., BIAcoreT""). Changes in the
optical
15 phenomenon surface plasmon resonance (SPR) can be used as an indication of
real-time
reactions between biological molecules.
In an alternative embodiment, determining the ability of the test compound to
modulate the activity of a STRIFEl or STRIFE2 protein can be accomplished by
determining the ability of the STRIFE1 or STRIFE2 protein to further modulate
the
20 activity of a STRIFE1 or STRIFE2 target molecule. For example, the
catalytic/enzymatic activity of the target molecule on an appropriate
substrate can be
determined as previously described.
In yet another embodiment, the cell-free assay involves contacting a STRIFE 1
or
STRIFE2 protein or biologically active portion thereof with a known compound
which
25 binds the STRIFE 1 or STRIFE2 protein 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 STRIFE1 or STRIFE2 protein, wherein determining the ability
of the
test compound to interact with the STRIFE 1 or STRIFE2 protein comprises
determining
the ability of the STRIFE1 or STRIFE2 protein to preferentially bind to or
modulate the
30 activity of a STRIFE1 or STRIFE2 target molecule.
The cell-free assays of the present invention are amenable to use of both
soluble
and/or membrane-bound forms of isolated proteins (e.g., STRIFE1 or STRIFE2
proteins
or biologically active portions thereof or STRIFE1 or STRIFE2 target
molecules). In
the case of cell-free assays in which a membrane-bound form an isolated
protein is used
35 (e.g., a STRIFE2 target molecule or receptor) it may be desirable to
utilize a solubilizing
agent such that the membrane-bound form of the isolated protein is maintained
in
solution. Examples of such solubilizing agents include non-ionic detergents
such as n-
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octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-
methylglucamide,
decanoyl-N-methylglucamide, Triton~ X-100, Triton~ X-114, Thesit~,
Isotridecypoly(ethylene glycol ether), 3-[(3-cholamidopropyl)dimethylamminio)-
1-
propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio)-2-hydroxy-1-
5 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 STRIFE1 or STRIFE2 or its
target
molecule to facilitate separation of complexed from uncomplexed forms of one
or both
10 of the proteins, as well as to accommodate automation of the assay. Binding
of a test
compound to a STRIFE1 or STRIFE2 protein, or interaction of a STRIFE1 or
STRIFE2
protein with a target molecule in the presence and absence of a candidate
compound, can
be accomplished in any vessel suitable for containing the reactants. Examples
of such w
vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In
one
I 5 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/
STRIFE 1 or STRIFE2 fusion proteins or glutathione-S-transferase/target 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
20 compound or the test compound and either the non-adsorbed target protein or
STRIFE1
or STRIFE2 protein, 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,
the
matrix immobilized in the case of beads, complex determined either directly or
25 indirectly, for example, as described above. Alternatively, the complexes
can be
dissociated from the matrix, and the level of STRIFE 1 or STRIFE2 binding or
activity
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 a STRIFE1 or STRIFE2
protein
30 or a STRIFE1 or STRIFE2 target molecule can be immobilized utilizing
conjugation of
biotin and streptavidin. Biotinylated STRIFE1 or STRIFE2 protein 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).
35 Alternatively, antibodies reactive with STRIFE1 or STRIFE2 protein or
target molecules
but which do not interfere with binding of the STRIFE1 or STRIFE2 protein to
its target
molecule can be derivatized to the wells of the plate, and unbound target or
STRIFE 1 or
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STRIFE2 protein 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
STRiFEI or STRIFE2 protein or target molecule, as well as enzyme-linked assays
5 which rely on detecting an enzymatic activity associated with the STRIFE1 or
STRIFE2
protein or target molecule.
In another embodiment, modulators of STRIFE1 or STRIFE2 expression are
identified in a method wherein a cell is contacted with a candidate compound
and the
expression of STRIFE 1 or STRIFE2 mRNA or protein in the cell is determined.
The
10 level of expression of STRIFE1 or STRIFE2 mRNA or protein in the presence
of the
candidate compound is compared to the level of expression of STRIFE 1 or
STRIFE2
mRNA or protein in the absence of the candidate compound. The candidate
compound
can then be identified as a modulator of STRIFE1 or STRIFE2 expression based
on this
comparison. For example, when expression of STRIFE1 or STRIFE2 nIRNA or
protein
15 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
STRIFE1 or
STRIFE2 mRNA or protein expression. Alternatively, when expression of STRIFE1
or
STRIFE2 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
20 inhibitor of STRIFE1 or STRIFE2 mRNA or protein expression. The level of
STRIFE1
or STRIFE2 mRNA or protein expression in the cells can be determined by
methods
described herein for detecting STRIFE 1 or STRIFE2 mRNA or protein.
In yet another aspect of the invention, the STRIFE1 or STRIFE2 proteins can be
used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see,
e.g., U.S. Patent
25 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) Biotechniques 14:920-924; Iwabuchi
et al.
(1993) Oncogene 8:1693-1696; and Brent W094/10300), to identify other
proteins,
which bind to or interact with STRIFE1 or STRIFE2 ("STRIFE1- or STRIFE2-
binding
proteins" or "STRIFE1 or STRIFE2-by") and modulate STRIFE1 or STRIFE2
activity.
30 Such STRIFE1-binding proteins are also likely to be involved in the
propagation of
signals by the STRIFE 1 proteins as, for example, downstream elements of a
STRIFE 1-
mediated signaling pathway. Alternatively, such STRIFE2-binding proteins are
likely to
be cell-surface molecules associated with non-STRIFE2-expressing cells,
wherein such
STRIFE2-binding proteins are involved in signal transduction.
35 The two-hybrid system is based on the modular nature of most transcription
factors, which consist of separable DNA-binding and activation domains.
Briefly, the
assay utilizes two different DNA constructs. In one construct, the gene that
codes for a
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STRIFE1 or STRIFE2 protein is fused to a gene encoding the DNA binding domain
of a
known transcription factor (e.g., GAL-4). In the other construct, a DNA
sequence, from
a library of DNA sequences, that encodes an unidentified protein ("prey" or
"sample") is
fused to a gene that codes for the activation domain of the known
transcription factor. If
5 the "bait" and the "prey" proteins are able to interact, in vivo, forming an
STRIFE1 or
STRIFE2-dependent complex, the DNA-binding and activation domains of the
. transcription factor are brought into close proximity. This proximity allows
transcription of a reporter gene (e.g., LacZ) which is operably linked to a
transcriptional
regulatory site responsive to the transcription factor. Expression of the
reporter gene can
10 be detected and cell colonies containing the functional transcription
factor can be
isolated and used to obtain the cloned gene which encodes the protein which
interacts
with the STRIFE 1 or STRIFE2 protein.
This invention further pertains to novel agents identified by the above-
described
screening assays. Accordingly, it is within the scope of this invention to
further use an
1 S agent identified as described herein in an appropriate animal model. For
example, an
agent identified as described herein (e.g., a STRIFE1 or STRIFE2 modulating
agent, an
antisense STRIFE1 or STRIFE2 nucleic acid molecule, a STRIFE1 or STRIFE2-
specific
antibody, or a STRIFE1 or STRIFE2-binding partner) can be used in an animal
model to
determine the efficacy, toxicity, or side effects of treatment with such an
agent.
20 Alternatively, an agent identified as described herein can be used in an
animal model to
determine the mechanism of action of such an agent. Furthermore, this
invention
pertains to uses of novel agents identif ed by the above-described screening
assays for
treatments as described herein.
In the screening assays described herein, either the marine STRIFE 1 or
STRIFE2
25 receptors could be used, or preferably, a human STRIFE1 or STRIFE2
receptor, e.g., the
human receptor encoded by clone AX92 3 contained in ATCC Deposit Number 981 O
1
(described in PCT application number WO 98/01554, published on January 15,
1998) or
the human OAF065 receptor (described in PCT application number WO 98/38304,
published on September 3, 1998), may be used.
30
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
35 respective genes on a chromosome; and, thus, locate gene regions associated
with
genetic disease; (ii) identify an individual from a minute biological sample
(tissue
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typing); and (iii) aid in forensic identification of a biological sample.
These applications
are described in the subsections below.
1. Chromosome Mapping
5 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. This
process is
called chromosome mapping. Accordingly, portions or fragments of the STRIFE1
or
STRIFE2 nucleotide sequences, described herein, can be used to map the
location of the
STRIFE 1 or STRIFE2 genes on a chromosome. The mapping of the STRIFE 1 or
10 STRIFE2 sequences to chromosomes is an important first step in correlating
these
sequences with genes associated with disease.
Briefly, STRIFE1 or STRIFE2 genes can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 by in length) from the STRIFE1 or
STRIFE2
nucleotide sequences. Computer analysis of the STRIFE1 or STRIFE2 sequences
can
15 be used to predict 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 STRIFE 1 or
STRIFE2
sequences will yield an amplified fragment.
20 Somatic cell hybrids are prepared by fusing somatic cells from different
mammals (e.g., human and mouse cells). As hybrids of human and mouse cells
grow
and divide, they gradually lose human chromosomes in random order, but retain
the
mouse chromosomes. By using media in which mouse cells cannot grow, because
they
lack a particular enzyme, but human cells can, the one human chromosome that
contains
25 the gene encoding the needed enzyme, will be retained. By using various
media, panels
of hybrid cell lines can be established. Each cell line in a panel contains
either a single
human chromosome or a small number of human chromosomes, and a full set of
mouse
chromosomes, allowing easy mapping of individual genes to specific human
chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924). Somatic cell
hybrids
30 containing only fragments of human chromosomes can also be produced by
using
human chromosomes with translocations and deletions.
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 STRIFE1 or STRIFE2
35 nucleotide sequences 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 STRIFE 1 or STRIFE2 sequence to its chromosome
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include in situ hybridization (described in Fan, Y. et al. ( 1990) PNAS,
87:6223-27), pre-
screening with labeled flow-sorted chromosomes, and pre-selection by
hybridization to
chromosome specific cDNA libraries.
Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase
5 chromosomal spread can further be used to provide a precise chromosomal
location in
one step. Chromosome spreads can be made using cells whose division has been
blocked in metaphase by a chemical such as colcemid that disrupts the mitotic
spindle.
The chromosomes can be treated briefly with trypsin, and then stained with
Giemsa. A
pattern of light and dark bands develops on each chromosome, so that the
chromosomes
10 can be identified individually. The FISH technique can be used with a DNA
sequence
as short as 500 or 600 bases. However, clones larger than 1,000 bases have a
higher
likelihood of binding to a unique chromosomal location with sufficient signal
intensity
for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases
will
since to get good results at a reasonable amount of time. For a review of this
15 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
20 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
25 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 a gene and a disease, mapped to the same chromosomal
region, can
then be identified through linkage analysis (co-inheritance of physically
adjacent genes),
described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.
30 Moreover, differences in the DNA sequences between individuals affected and
unaffected with a disease associated with the STRIFE 1 or STRIFE2 gene, 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
35 first looking for structural alterations in the 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
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individuals can be performed to confirm the presence of a mutation and to
distinguish
mutations from polymorphisms.
2. Tissue Tvuing
S The STRIFE1 or STRIFE2 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
10 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
15 alternative technique which determines the actual base-by-base DNA sequence
of
selected portions of an individual's genome. Thus, the STRIFE1 or STRIFE2
nucleotide
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.
20 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 be used to obtain such identification sequences from
individuals
and from tissue. The STRIFE 1 or STRIFE2 nucleotide sequences of the invention
25 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
30 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 SEQ ID NO:S, can comfortably provide
positive individual identification with a panel of perhaps.10 to 1,000 primers
which each
yield a noncoding amplified sequence of 100 bases. If predicted coding
sequences, such
35 as those in SEQ ID N0:3 or SEQ ID N0:7 are used, a more appropriate number
of
primers for positive individual identification would be 500-2,000.
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If a panel of reagents from STRIFE1 or STRIFE2 nucleotide 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,
5 can be made from extremely small tissue samples.
3. Use of Partial STRIFEl or STRIFE2 Seauences in Forensic Biology
DNA-based identification techniques can also be used in forensic biology.
Forensic biology is a scientific field employing genetic typing of biological
evidence
10 found at a crime scene as a means for positively identifying, for example,
a perpetrator
of a crime. To 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
15 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
20 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 of SEQ ID NOs:l
or
SEQ ID NO:S are particularly appropriate for this use as greater numbers of
polymorphisms occur in the noncoding regions, making it easier to
differentiate
25 individuals using this technique. Examples of polynucleotide reagents
include the
STRIFE1 or STRIFE2 nucleotide sequences or portions thereof, e.g., fragments
derived
from the noncoding regions of SEQ ID NO: l or SEQ ID NO:S, having a length of
at
least 20 bases, preferably at least 30 bases.
The STRIFE1 or STRIFE2 nucleotide sequences described herein can further be
30 used to pmvide 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 or lung tissue. This can be very useful in cases where a forensic
pathologist is
presented with a tissue of unknown origin. Panels of such STRIFEl or STRIFE2
probes
can be used to identify tissue by species and/or by organ type.
35 In a similar fashion, these reagents, e.g., STRIFE1 or STRIFE2 primers or
probes can be used to screen tissue culture for contamination (i.e. screen for
the presence
of a mixture of different types of cells in a culture).
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C. Predictive Medicine:
The present invention also pertains to the field of predictive medicine in
which
diagnostic assays, prognostic assays, and monitoring clinical trails are used
for
5 prognostic (predictive) purposes to thereby treat an individual
prophylactically.
Accordingly, one aspect of the present invention relates to diagnostic assays
for
determining STRIFE 1 or STRIFE2 protein and/or nucleic acid expression as well
as
STRIFE 1 or STRIFE2 activity, 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
10 disorder, or is at risk of developing a disorder, associated with aberrant
STRIFE 1 or
STRIFE2 expression or activity. The invention also provides for prognostic (or
predictive) assays for determining whether an individual is at risk of
developing a
disorder associated with STRIFE 1 or STRIFE2 protein, nucleic acid expression
or
activity. For example, mutations in a STRIFE1 or STRIFE2 gene can be assayed
in a
15 biological sample. Such assays can be used for prognostic or predictive
purpose to
thereby phophylactically treat an individual prior to the onset of a disorder
characterized
by or associated with STRIFEI or STRIFE2 protein, nucleic acid expression or
activity.
Another aspect of the invention pertains to monitoring the influence of agents
{e.g., drugs, compounds) on the expression or activity of STRIFE1 or STRIFE2
in
20 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 STRIFE 1 or
25 STRIFE2 protein or nucleic acid 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 STRIFE 1 or STRIFE2 protein or nucleic acid (e.g.,
mRNA,
genomic DNA) that encodes STRIFE1 or STRIFE2 protein such that the presence of
STRIFE 1 or STRIFE2 protein or nucleic acid is detected in the biological
sample. A
30 preferred agent for detecting STRIFE1 or STRIFE2 mRNA or genomic DNA is a
labeled nucleic acid probe capable of hybridizing to STRIFE1 or STRIFE2 mRNA
or
genomic DNA. The nucleic acid probe can be, for example, a full-length STRIFE
1 or
STRIFE2 nucleic acid, such as the nucleic acid of SEQ ID NO: 1 or SEQ ID NO:S,
or a
portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250
or 500
35 nucleotides in length and sufficient to specifically hybridize under
stringent conditions
to STRIFE 1 or STRIFE2 mRNA or genomic DNA. Other suitable probes for use in
the
diagnostic assays of the invention are described herein.
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A preferred agent for detecting STRIFE1 or STRIFE2 protein is an antibody
capable of binding to STRIFE1 or STRIFE2 protein; preferably an antibody with
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
5 "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
10 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
STRIFE1 or STRIFE2 mRNA, protein, or genomic DNA in a biological sample in
vitro
15 as well as in vivo. For example, in vitro techniques for detection of
STRIFE1 or
STRIFE2 mRNA include Northern hybridizations and in situ hybridizations. In
vitro
techniques for detection of STRIFE1 or STRIFE2 protein include enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations and
immunofluorescence. In vitro techniques for detection of STRIFE 1 or STRIFE2
20 genomic DNA include Southern hybridizations. Furthermore, in vivo
techniques for
detection of STRIFE1 or STRIFE2 protein include introducing into a subject a
labeled
anti-STRIFE1 or STRIFE2 antibody. 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.
25 In one embodiment, the biological sample contains protein molecules from
the
test subject: Alternatively, 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 serum sample isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control
30 biological sample from a control subject, contacting the control sample
with a
compound or agent capable of detecting STRIFE1 or STRIFE2 protein, mRNA, or
genomic DNA, such that the presence of STRIFE 1 or STRIFE2 protein, mRNA or
genomic DNA is detected in the biological sample, and comparing the presence
of
STRIFE1 or STRIFE2 protein, mRNA or genomic DNA in the control sample with the
35 presence of STRIFE1 or STRIFE2 protein, mRNA or genomic DNA in the test
sample.
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The invention also encompasses kits for detecting the presence of STRIFE1 or
STRIFE2 in a biological sample. For example, the kit can comprise a labeled
compound
or agent capable of detecting STRIFE 1 or STRIFE2 protein or mRNA in a
biological
sample; means for determining the amount of STRIFE1 or STRIFE2 in the sample;
and
5 means for comparing the amount of STRIFE1 or STRIFE2 in the sample with a
standard. The compound or agent can be packaged in a suitable container. The
kit can
further comprise instructions for using the kit to detect STRIFE 1 or STRIFE2
protein or
nucleic acid.
10 2. Prognostic Assts
The diagnostic methods described herein can furthermore be utilized to
identify
subjects having or at risk of developing a disease or disorder associated with
aberrant
STRIFE1 or STRIFE2 expression or activity. For example, the assays described
herein,
such as the preceding diagnostic assays or the following assays, can be
utilized to
15 identify a subject having or at risk of developing a disorder associated
with STRIFE1 or
STRIFE2 protein, nucleic acid expression or activity such as a TNF-associated
disorder,
e.g., inflammatory, immune, or neoplastic disorder. Thus, the present
invention
provides a method for identifying a disease or disorder associated with
aberrant
STRIFE1 or STRIFE2 expression or activity in which a test sample is obtained
from a
20 subject and STRIFEI or STRIFE2 protein or nucleic acid (e.g, mRNA, genomic
DNA)
is detected, wherein the presence of STRIFEi or STRIFE2 protein or nucleic
acid is
diagnostic for a subject having or at risk of developing a disease or disorder
associated
with aberrant STRIFE 1 or STRIFE2 expression or activity. As used herein, a
"test
sample" refers to a biological sample obtained from a subject of interest. For
example, a
25 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 STRIFE1 or STRIFE2
expression
30 or activity. For example, such methods can be used to determine whether a
subject can
be effectively treated with an agent for a disorder, such as a proliferative
disorder, a
differentiative or developmental disorder, or a hematopoietic disorder.
Alternatively,
such methods can be used to determine whether a subject can be effectively
treated with
an agent for a differentiative or proliferative disease (e.g., cancer). Thus,
the present
35 invention provides methods for determining whether a subject can be
effectively treated
with an agent for a disorder associated with aberrant STRIFE1 or STRIFE2
expression
or activity in which a test sample is obtained and STRIFE 1 or STRIFE2 protein
or
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nucleic acid expression or activity is detected (e.g., wherein the abundance
of STRIFE1
or STRIFE2 protein or nucleic acid expression or activity is diagnostic for a
subject that
can be administered the agent to heat a disorder associated with aberrant
STRIFE1 or
STRIFE2 expression or activity.)
5 The methods of the invention can also be used to detect genetic alterations
in an
STRIFE1 or STRIFE2 gene, thereby determining if a subject with the altered
gene is at
risk for a disorder characterized by aberrant development, aberrant cellular
differentiation, aberrant cellular proliferation or an aberrant hematopoietic
response. In
preferred embodiments, the methods include detecting, in a sample of cells
from the
10 subject, the presence or absence of a genetic alteration characterized by
at least one of an
alteration affecting the integrity of ~a gene encoding a STRIFE 1 or STRIFE2-
protein, or
the mis-expression of the STRIFEI or STRIFE2 gene. For example, such genetic
alterations can be detected by ascertaining the existence of at least one of 1
) a deletion of
one or more nucleotides from an STRIFE1 or STRIFE2 gene; 2) an addition of one
or
15 more nucleotides to a STRIFE1 or STRIFE2 gene; 3) a substitution of one or
more
nucleotides of a STRIFE1 or STRIFE2 gene, 4) a chromosomal rearrangement of a
STRIFE1 or STRIFE2 gene; 5) an alteration in the level of a messenger RNA
transcript
of a STRIFE1 or STRIFE2 gene, 6) aberrant modification of a STRIFE1 or STRIFE2
gene, such as of the methylation pattern of the genomic DNA, 7) the presence
of a non-
20 wild type splicing pattern of a messenger RNA transcript of a STRIFE1 or
STRIFE2
gene, 8) a non-wild type level of a STRIFE1 or STRIFE2-protein, 9) allelic
loss of a
STRIFE1 or STRIFE2 gene, and 10) inappropriate post-translational modification
of a
STRIFE 1 or STRIFE2-protein. As described herein, there are a large number of
assay
techniques known in the art which can be used for detecting alterations in a
STRIFE 1 or
25 STRIFE2 gene. A preferred biological sample is a tissue or serum sample
isolated by
conventional means from a subject.
In certain embodiments, detection of the alteration involves the use of a
probe/primer in a polymerise chain reaction (PCR) (see, e.g., U.S. Patent Nos.
4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively,
in a
30 ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science
241:1077-1080;
and Nakazawa et al. (1994) PNAS 91:360-364), the latter of which can be
particularly
useful for detecting point mutations in the STRIFE1 or STRIFE2-gene (see
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
35 or both) from the cells of the sample, contacting the nucleic acid sample
with one or
more primers which specifically hybridize to a STRIFE1 or STRIFE2 gene under
conditions such that hybridization and amplification of the STRIFE1 or STRIFE2-
gene
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(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
5 described herein.
Alternative amplification methods include: self sustained sequence replication
(Guatelli, J.C. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878),
transcriptional
amplification system (Kwoh, D.Y. et al., 1989, Proc. Natl. Acad. Sci. USA
86:1173-
1177), Q-Beta Replicase (Lizardi, P.M. et all, 1988, Bio/Technology 6:1197),
or any
10 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 STRIFE 1 or STRIFE2 gene from a
15 sample cell can be identified by alterations in restriction enzyme cleavage
patterns. For
example, sample and control DNA is isolated, amplified (optionally), digested
with one
or more restriction endonucleases, 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
20 specific ribozymes (see, for example, 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 in STRIFE 1 or STRIFE2 can be
identified by hybridizing a sample and control nucleic acids, e.g., DNA or
RNA, to high
25 density arrays containing hundreds or thousands of oligonucleotides probes
(Cronin,
M.T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature
Medicine 2: 753-759). For example, genetic mutations in STRIFE1 or STRIFE2 can
be
identified in two dimensional arrays containing light-generated DNA probes as
described in Cronin, M.T. et al. supra. Briefly, a first hybridization array
of probes can
30 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 ovelapping
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.
35 Each mutation array is composed of parallel probe sets, one complementary
to the wild-
type gene and the other complementary to the mutant gene.
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In yet another embodiment, any of a variety of sequencing reactions known in
the art can be used to directly sequence the STRIFE1 or STRIFE2 gene and
detect
mutations by comparing the sequence of the sample STRIFE1 or STRIFE2 with the
corresponding wild-type (control) sequence. Examples of sequencing reactions
include
5 those based on techniques developed by Maxim and Gilbert ((1977) PNAS
74:560) or
Sanger ((1977) PNAS 74:5463). It is also contemplated that any of a variety of
automated sequencing procedures can be utilized when performing the diagnostic
assays
((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see,
e.g.,
PCT International Publication No. WO 94/16101; Cohen et al. '(1996) Adv.
Chromatogr.
10 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-
159).
Other methods for detecting mutations in the STRIFE1 or STRIFE2 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 art technique of "mismatch cleavage" starts by providing
heteroduplexes of
15 formed by hybridizing (labeled) RNA or DNA containing the wild-type STRIFE1
or
STRIFE2 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. For instance, RNA/DNA duplexes can be
20 treated with RNase and DNA/DNA hybrids treated with S 1 nuclease to
enzymatically
digesting the 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
25 gels to determine the site of mutation. See, for example, Cotton et al.
(1988) Proc. Natl
Acad Sci 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
30 "DNA mismatch repair" enzymes) in defined systems for detecting and mapping
point
mutations in STRIFE 1 or STRIFE2 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
35 on an STRIFE1 or STRIFE2 sequence, e.g., a wild-type STRIFE1 or STRIFE2
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
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detected from electrophoresis protocols or the like. See, for example, U.S.
Patent No.
5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to
identify mutations in STRIFE1 or STRIFE2 genes. For example, single strand
5 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; and
Hayashi
( 1992) Genet Anal Tech Appl 9:73-79). Single-stranded DNA fragments of sample
and
control STRIFE1 or STRIFE2 nucleic acids will be denatured and allowed to
renature.
10 The secondary structure of single-stranded nucleic acids varies according
to sequence,
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 using RNA (rather than DNA), in
which the
secondary structure is more sensitive to a change in sequence. In a preferred
15 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
20 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
25 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
30 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.
35 Alternatively, allele specific amplification technology which depends on
selective PCR amplification may be used in conjunction with the instant
invention.
Oligonucleotides used as primers for specific amplification may carry the
mutation of
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interest in the center of the molecule (so that amplification depends on
differential
hybridization) (Gibbs et al. (1989) Nucleic Acids Res. i 7:2437-2448) 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
5 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
Iigase for
amplification (Barany (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
10 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
15 patients exhibiting symptoms or family history of a disease or illness
involving a
STRIFE1 or STRIFE2 gene.
Furthermore, any cell type or tissue in which STRIFE1 or STRIFE2 is expressed
may be utilized in the prognostic assays described herein.
20 3. Monitoring of Effects Durine Clinical Trials
Monitoring the influence of agents (e.g., drugs, compounds) on the expression
or
activity of STRIFE1 or STRIFE2 (e.g., modulation of cellular signal
transduction,
regulation of gene transcription in a cell involved in development or
differentiation,
regulation of cellular proliferation) can be applied not only in basic drug
screening, but
25 also in clinical trials. For example, the effectiveness of an agent
determined by a
screening assay as described herein to increase STRIFE 1 or STRIFE2 gene
expression,
protein levels, or upregulate STRIFE1 or STRIFE2 activity, can be monitored in
clinical
trails of subjects exhibiting decreased STRIFE1 or STRIFE2 gene expression,
protein
levels, or downregulated STRIFE1 or STRIFE2 activity. Alternatively, the
effectiveness
30 of an agent determined by a screening assay to decrease STRIFE1 or STRIFE2
gene
expression, protein levels, or downregulate STRIFE1 or STRIFE2 activity, can
be
monitored in clinical trails of subjects exhibiting increased STRIFE1 or
STRIFE2 gene
expression, protein levels, or upregulated STRIFE1 or STRIFE2 activity. In
such
clinical trials, the expression or activity of STRIFE1 or.STRIFE2 and,
preferably, other
35 genes that have been implicated in, for example, a proliferative disorder
can be used as a
"read out" or markers of the phenotype of a particular cell.
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-67-
For example, and not by way of limitation, genes, including STRIFE1 or
STRIFE2, that are modulated in cells by treatment with an agent (e.g.,
compound, drug
or small molecule) which modulates STRIFEI or STRIFE2 activity (e.g.,
identified in a
screening assay as described herein) can be identified. Thus, to study the
effect of
5 agents on proliferative disorders, developmental or differentiative
disorder, or
hematopoietic disorder, for example, in a clinical trial, cells can be
isolated and RNA
prepared and analyzed for the levels of expression of STRIFE1 or STRIFE2 and
other
genes implicated in the proliferative disorder, developmental or
differentiative disorder,
or hematopoietic disorder, respectively. The levels of gene expression (i.e.,
a gene
10 expression pattern) can be quantified by Northern blot analysis or RT-PCR,
as described
herein, or alten!latively by measuring the amount of protein produced, by one
of the
methods as described herein, or by measuring the levels of activity of STRIFE1
or
STRIFE2 or other genes. 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
1 S 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
20 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 expression of a STRIFE1 or STRIFE2 protein,
mRNA,
or genomic DNA in the preadministration sample; (iii) obtaining one or more
post-
administration samples from the subject; (iv) detecting the level of
expression or activity
25 of the STRIFE1 or STRIFE2 protein, mRNA, or genomic DNA in the post-
administration samples; (v) comparing the level of expression or activity of
the
STRIFE 1 or STRIFE2 protein, mRNA, or genomic DNA in the pre-administration
sample with the STRIFE1 or STRIFE2 protein, mRNA, or genomic DNA in the post
administration sample or samples; and (vi) altering the administration of the
agent to the
30 subject accordingly. For example, increased administration of the agent may
be
desirable to increase the expression or activity of STRIFE1 or STRIFE2 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
STRIFE1 or STRIFE2 to lower levels than detected, i.e. to decrease the
effectiveness of
35 the agent. According to such an embodiment, STRIFE1 or STRIFE2 expression
or
activity may be used as an indicator of the effectiveness of an agent, even in
the absence
of an observable phenotypic response.
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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
5 with aberrant STRIFEI or STRIFE2 expression or activity. With regards to
both
prophylactic and therapeutic methods of treatment, such treatments may be
specifically
tailored or modified, based on knowledge obtained from the field of
pharmacogenomics:
"Pharmacogenomics", as used herein, refers to the application of genomics
technologies
such as gene sequencing, statistical genetics, and gene expression analysis to
drugs in
10 clinical development and on the market. More specifically, the term refers
to the study
of how a patient's genes determine his or her response to a drug (e.g., a
patient's "drug
response phenotype", or "drug response genotype"). Thus, another aspect of the
invention provides methods for tailoring an individual's prophylactic or
therapeutic
treatment with either the STRIFEl or STRIFE2 molecules of the present
invention or
15 STRIFEI or STRIFE2 modulators according to that individual's drug response
genotype.
Pharmacogenomics allows a clinician or physician to target prophylactic or
therapeutic
treatments to patients who will most benefit from the treatment and to avoid
treatment of
patients who will experience toxic drug-related side effects.
20 1. Prophylactic Methods
In one aspect, the invention provides a method for preventing in a subject, a
disease or condition associated with an aberrant STRIFE1 or STRIFE2 expression
or
activity, by administering to the subject an agent which modulates STRIFE1 or
STRIFE2 expression or at least one STRIFEI or STRIFE2 activity. Subjects at
risk for
25 a disease which is caused or contributed to by aberrant STRIFE1 or STRIFE2
expression or activity 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 the manifestation of symptoms characteristic of the
STRIFE 1 or
STRIFE2 aberrancy, such that a disease or disorder is prevented or,
alternatively,
30 delayed in its progression. Depending on the type of STRIFE1 or STRIFE2
aberrancy,
for example, an STRIFE1 or STRIFE2 agonist or STRIFE1 or STRIFE2 antagonist
agent can be used for treating the subject. The appropriate agent can be
determined
based on screening assays described herein. The prophylactic methods of the
present
invention are fixrther discussed in the following subsections.
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2. Therapeutic Methods
Another aspect of the invention pertains to methods of modulating STRIFE 1 or
STRIFE2 expression or activity for therapeutic purposes. The modulatory method
of the
invention involves contacting a cell with an agent that modulates one or more
of the
5 activities of STRIFE1 or STRIFE2 protein activity associated with the cell.
An agent
that modulates STRIFE1 or STRIFE2 protein activity can be an agent as
described
herein, such as a nucleic acid or a protein, a naturally-occurring target
molecule of a
STRIFE 1 or STRIFE2 protein, a peptide, a STRIFE 1 or STRIFE2 peptidomimetic,
or
other small molecule. In one embodiment, the agent stimulates one or more
STRIFE1 or
10 STRIFE2, protein activity. Examples of such stimulatory agents include
active STRIFE 1
or STRIFE2 protein and a nucleic acid molecule encoding STRIFE1 or STRIFE2
that
has been introduced into the cell. In another embodiment, the agent inhibits
one or more
STRIFE 1 or STRIFE2 protein activity. Examples of such inhibitory agents
include
antisense STRIFE 1 or STRIFE2 nucleic acid molecules and anti-STRIFE 1 or
STRIFE2
15 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
STRIFEI or STRIFE2 protein or nucleic acid molecule. In one embodiment, the
20 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) STRIFE1 or STRIFE2 expression or activity. In another
embodiment,
the method involves administering a STRIFE1 or STRIFE2 protein or nucleic acid
molecule as therapy to compensate for reduced or aberrant STRIFE1 or STRIFE2
25 expression or activity.
Stimulation of STRIFE 1 or STRIFE2 activity is desirable in situations in
which
STRIFE 1 or STRIFE2 is abnormally downregulated and/or in which increased
STRIFE 1
or STRIFE2 activity is likely to have a beneficial effect. Likewise,
inhibition of
STRIFE1 or STRIFE2 activity is desirable in situations in which STRIFE1 or
STRIFE2
30 is abnormally upregulated and/or in which decreased STRIFE 1 or STRIFE2
activity is
likely to have a beneficial effect. One example of such a situation is where a
subject has
a TNF-associated disorder, e.g., an inflammatory, immune, or neoplastic
disorder.
3. Pharmacogenomics
35 The STRIFE1 or STRIFE2 molecules of the present invention, as well as
agents,
or modulators which have a stimulatory or inhibitory effect on STRIFE 1 or
STRIFE2
activity (e.g., STRIFEI or STRIFE2 gene expression) as identified by a
screening assay
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described herein can be administered to individuals to treat (prophylactically
or
therapeutically) disorders (e.g, TNF-associated disorders) associated with
aberrant
STRIFE1 or STRIFE2 activity. In conjunction with such treatment,
phaimacogenomics
(i.e., the study of the relationship between an individual's genotype and that
individual's
5 response to a foreign compound or drug) 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, a
physician or clinician may consider applying knowledge obtained in relevant
pharmacogenomics studies in determining whether to administer an STRIFE 1 or
10 STRIFE2 molecule or STRIFE1 or STRIFE2 modulator as well as tailoring the
dosage
and/or therapeutic regimen of treatment with an STRIFE1 or STRIFE2 molecule or
STRIFE1 or STRIFE2 modulator.
Pharmacogenomics deals with clinically significant hereditary variations in
the
response to drugs due to altered drug disposition and abnormal action in
affected
15 persons. See e.g., Eichelbaum, M., Clin Exp Pharmacol Physiol, 1996, 23(10-
11) :983-
985 and Linder, M.W., Clin Chem, 1997, 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 (altered drug action) or
genetic
conditions transmitted as single factors altering the way the body acts on
drugs (altered
20 drug metabolism). These pharmacogenetic conditions can occur either as rare
genetic
defects or as naturally-occurring polymorphisms. For example, glucose-6-
phosphate
dehydrogenase deficiency (G6PD) is a common inherited enrymopathy in which the
main clinical complication is haemolysis after ingestion of oxidant drugs
(anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
25 One pharmacogenomics approach to identifying genes that predict drug
response, known as "a genome-wide association", relies primarily on a high-
resolution
map of the human genome consisting of already known gene-related markers
(e.g., a "bi-
allelic" gene marker map which consists of 60,000-100,000 polymorphic or
variable
sites on the human genome, each of which has two variants). Such a high-
resolution
30 genetic map can be compared to a map of the genome of each of a
statistically
significant number of patients taking part in a Phase II/III drug trial to
identify markers
associated with a particular observed drug response or side ei~'ect.
Alternatively, such a
high resolution map can be generated from a combination of some ten-million
known
single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a
35 "SNP" is a common alteration that occurs in a single nucleotide base in a
stretch of
DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may
be involved in a disease process, however, the vast majority may not be
disease-
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associated. Given a genetic map based on the occurrence of such SNPs,
individuals can
be grouped into genetic categories depending on a particular pattern of SNPs
in their
individual genome. In such a manner, treatment regimens can be tailored to
groups of
genetically similar individuals, taking into account traits that may be common
among
5 such genetically similar individuals.
Alternatively, a method termed the "candidate gene approach", can be utilized
to
identify genes that predict drug response. According to this method, if a gene
that
encodes a drug target is known (e.g., a STRIFE1 or STRIFE2 protein or a
STRIFE1
receptor of the present invention), all common variants of that gene can be
identified in
10 the population and a particular drug response can be associated with one or
more genes.
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 CYP2C 19) has provided an
explanation
15 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
20 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, PM show no therapeutic
response, as
demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed
25 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.
Alternatively, a method termed the "gene expression profiling", can be
utilized to
identify genes that predict drug response. For example, the gene expression of
an
30 animal dosed with a drug (e.g., a STRIFE1 or STRIFE2 molecule or STRIFE1 or
STRIFE2 modulator of the present invention) can give an indication whether
gene
pathways related to toxicity have been turned on.
Information generated from more than one of the above phanmacogenomics
approaches can be used to determine appropriate dosage and treatment regimens
for
35 prophylactic or therapeutic treatment an individual. 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
STRIFE1
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or STRIFE2 molecule or STRIFE1 or STRIFE2 modulator, such as a modulator
identified by one of the exemplary screening assays described herein.
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 incorporated herein by
reference.
EXAMPLES
EXAMPLE 1: IDENTIFICATION OF MURINE STRIFEl AND STRIFE2 cDNA
In this example, the isolation and characterization of the cDNA encoding
marine
10 STRIFE1 and STRIFE2 is described. STRIFE is a mouse gene which encodes a
protein
belonging to the TNFR family. Two splice forms have been identified, one that
is
predicted to be membrane bound (STRIFEI) and one that is secreted (STRIFE2).
STRIFE was identified as a TNFR homologue by a computer-based search of the
public EST databases. More specifically, the marine STRIFE1 and STRIFE2 cDNA
15 were identified by searching against a copy of the GenBank nucleotide
database using
the BLASTNT"" program (BLASTN 1.3MP: Altschul et al., J. Mol. Bio. 215:403,
1990).
Numerous clones that consisted mostly of 3' reads and some that were 5' reads
within the
3' untranslated region were found by this search. The sequences were analyzed
against a
non-redundant protein database with the BLASTXT"" program, which translates a
nucleic
20 acid sequence in all six frames and compares it against available protein
databases
{BLASTX I .3MP:Altschul et al., supra). This protein database is a combination
of the
Swiss-Prot, PIR, and NCBI GenPept protein databases. Two clones (Accession
Numbers AA036247 and AA003356) were obtained from the IMAGE consortium, and
fully sequenced. The additional sequencing of AA036247 (T 127a; STRIFE 1 )
extended
25 the original EST by 623 nucleotides (see SEQ ID NO:1 ) and the further
sequencing of
AA003356 (T127b; STRIFE2) extended the original EST by 254 nucleotides (see
SEQ
ID NO:S).
A BLASTNTM search of the EST database revealed the following ESTs having
significant homology to clone Accession Number AA036247:
30
EST Database hits S ecies Base Pairs % Coding?
Covered Identi
Accession # AA495217 zebrafish 602-711 82 yes
A BLASTNTM search of the EST and nucleotide database revealed the following
ESTs and nucleotides having significant homology to clone Accession Number
AA003356:
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EST Database hits S ecies Base Pairs % Coding?
Covered Identi
Accession # AA686080rat 297-367 64 yes
Accession # AA209382human 150-210 67 yes
Accession # AA409240mouse 284-319 80 yes
Accession # N91779mouse 519-489 83 yes
EXAMPLE 2: TISSUE EXPRESSION OF THE STRIFEl AND STRIFE2 GENE
Human I and mouse multiple tissue northern (MTN) blots (Clontech, Palo Alto,
5 CA) containing 2 p,g of poly A+ RNA per lane were probed with a 750bp
EcoRl/Notl
fragment of the mouse STRIFE1 cDNA. The filters were prehybridized in 10 ml of
Express Hyb hybridization solution (Clontech, Palo Alto, CA) at 68°C
for 1 hour, after
which 100 ng of 32P labeled probe was added. The probe was generated using the
Stratagene Prime-It kit, Catalog Number 300392 (Clontech, Palo Alto, CA).
10 Hybridization was allowed to proceed at 68°C for approximately 2
hours. The filters
were washed in a 0.05% SDS/2X SSC solution for 15 minutes at room temperature
and
then twice with a 0.1% SDS/O.1X SSC solution for 20 minutes at SO°C and
then
exposed to autoradiography film overnight at -80°C with one screen. The
mouse tissues
tested included: heart, brain, spleen, lung, liver, skeletal muscle, kidney,
and testis. The
15 human tissues tested included: heart, placenta, lung, liver, skeletal
muscle, kidney, and
pancreas.
There was a strong hybridization to both mouse and human heart, brain, and
lung
indicating that the approximately 4.4 kb STRIFE1 and STRIFE2 gene transcript
is
expressed in these tissues.
20
EXAMPLE 3: EXPRESSION OF RECOMBINANT STRIFE1 AND STRIFE2
PROTEIN IN BACTERIAL CELLS
In this example, STRIFE1 or STRIFE2 is expressed as a recombinant
glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion
polypeptide
25 is isolated and characterized. Specifically, STRIFE1 or STRIFE2 is fused to
GST and
this fusion polypeptide is expressed in E. coli, e.g., strain PEB 199. As the
marine
STRIFE1 and STRIFE2 proteins are predicted to be approximately 23.55 kDa and
16.72
kDa, respectively, and GST is predicted to be 26 kDa, the fusion polypeptides
are
predicted to be approximately 49.55 kDa and 42.72 kDa, respectively, in
molecular
30 weight. Expression of the GST-STRIFE 1 or STRIFE2 fusion protein in PEB 199
is
induced with IPTG. The recombinant fusion polypeptide is purified from crude
bacterial
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lysates of the induced PEB 199 strain by affinity chromatography on
glutathione beads.
Using polyacrylamide gel electrophoretic analysis of the polypeptide purified
from the
bacterial lysates, the molecular weight of the resultant fusion polypeptide is
determined.
5 EXAMPLE 4: EXPRESSION OF RECOMBINANT STRIFEl AND
STRIFE2 PROTEIN IN COS CELLS
To express the STRIFE1 or STRIFE2 gene in COS cells, the pcDNA/Amp
vector by Invitrogen Corporation (San Diego, CA) is used. This vector contains
an
SV40 origin of replication, an ampicillin resistance gene, an E. coli
replication origin, a
10 CMV promoter followed by a polylinker region, and an SV40 intron and
polyadenylation site. A DNA fragment encoding the entire STRIFE1 or STRIFE2
protein and a HA tag (Wilson et al. (1984) Cell 37:767) fused in-frame to its
3' end of
the fragment is cloned into the polylinker region of the vector, thereby
placing the
expression of the recombinant protein under the control of the CMV promoter.
1 S To construct the plasmid, the STRIFE1 or STRIFE2 DNA sequence is amplified
by PCR using two primers. The 5' primer contains the restriction site of
interest
followed by approximately twenty nucleotides of the STRIFE1 or STRIFE2 coding
sequence starting from the initiation codon; the 3' end sequence contains
complementary
sequences to the other restriction site of interest, a translation stop codon,
the HA tag
20 and the last 20 nucleotides of the STRIFE1 or STRIFE2 coding sequence. The
PCR
amplified fragment and the pCDNA/Amp vector are digested with the appropriate
restriction enzymes and the vector is dephosphorylated using the CIAP enzyme
(New
England Biolabs, Beverly, MA). Preferably the two restriction sites chosen are
different
so that the STRIFE1 or STRIFE2 gene is inserted in the correct orientation.
The ligation
25 mixture is transformed into E. coli cells (strains HB101, DHSa, SURE,
available from
Stratagene Cloning Systems, La Jolla, CA, can be used), the transformed
culture is
plated on ampicillin media plates, and resistant colonies are selected.
Plasmid DNA is
isolated from transformants and examined by restriction analysis for the
presence of the
correct fragment.
30 COS cells are subsequently transfected with the STRIFE1 or STRIFE2-
pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-
precipitation methods, DEAF-dextran-mediated transfection, lipofection, or
electroporation. Other suitable methods for transfecting host cells can be
found in
Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory
Manual.
35 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press, Cold
Spring Harbor, NY, 1989. The expression of the STRIFE l and STRIFE2
polypeptide is
detected by radiolabelling (35S-methionine or 35S-cysteine available from NEN,
Boston,
CA 02318743 2000-07-24
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MA, can be used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies:
A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
1988) using an HA specific monoclonal antibody. Briefly, the cells are
labelled for 8
hours with 35S-methioiiine (or 35S-cysteine). The culture media are then
collected and
5 the cells are lysed using detergents (RIPA buffer, 150 mM NaCI, 1% NP-40,
0.1% SDS,
0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are
precipitated with an HA specific monoclonal antibody. Precipitated
polypeptides are
then analyzed by SDS-PAGE.
Alternatively, DNA containing the STRIFE1 or STRIFE2 coding sequence is
10 cloned directly into the polylinker of the pCDNA/Amp vector using the
appropriate
restriction sites. The resulting plasmid is transfected into COS cells in the
manner
described above, and the expression of the STRIFE1 or STRIFE2 polypeptide is
detected by radiolabelling and immunoprecipitation using an STRIFE1 or STRIFE2
specific monoclonal antibody.
15
EXAMPLE 5: CHARACTERIZATION OF THE MURINE STRIFE! AND
STRIFE2 PROTEINS
STRIFE 1 is approximately 981 nucleotides in length and has an open reading
frame of 645 nucleotides that is predicted to encode a protein of 214 amino
acids.
20 STRIFE2 is approximately 655 nucleotides long with an open reading frame of
453
nucleotides predicted to encode a protein of 150 amino acids. Both clones have
been
subcloned into a variety of expression vectors including those for retroviral
delivery and
for expression in bacterial, yeast and mammalian cells.
BlastX searching of the protein database confirms the homology of this clone
to
25 various members of the TNFR family. The extracellular domains of STRIFE1
and
STRIFE2 are approximately 40% identical to OX40. Importantly, a number of
cysteine
residues within the extracellular domains of STRIFE1 and STRIFE2 match the
cysteine-
rich domain signature of the TNFR/NGFR family (Prosite Accession PDOC00561).
The program SignalP {Nielsen et al, 1997) predicts a 30 amino acid signal
peptide at the
30 very N-terminus of both STRIFE1 and STRIFE2 (i.e., as 1-29 of SEQ ID NOs:I
and 5).
The predicted molecular weight for STRIFE1 is approximately 23.55 lcDa with
the
signal peptide and 20.34 lcDa without the signal peptide which is presumed to
be cleaved
in the mature protein. There are no obvious motifs in the small intracellular
domain of
STRIFE1. STRIFE2 is predicted to be 16.72 lcDa with the signal peptide and
13.51 kDa
35 without the signal peptide.
A FASTA search (described in Pearson W.R. & Lipman D.J. {1988) PNAS
85:2444-2448, score matrix: PAM120) using the STRIFE1 protein sequence as a
query,
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WO 99137818 PCTNS99l01679
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indicates that STRIFEI is 85.7% identical to the human OAF065 receptor
(Accession
number W70387; described in PCT application number WO 98/38304, published on
September 3, 1998) over amino acid residues 1-203. The results from this
search are
shown in Figure 4.
5 A FASTA search (described in Pearson W.R. & Lipman D.J. (1988) PNAS
85:2444-2448, score matrix: PAM120) using the STRIFEI nucleotide sequence as a
query, indicates that STRIFE1 is 70.6% identical to the nucleic acid molecule
encoding
the human OAF065 receptor (Accession number V33362; described in PCT
application
number WO 98/38304, published on September 3, 1998) over nucleotide residues
65-
981. The results from this search are shown in Figure 5.
Structure of the STRIFE! and STRIFE2 Famil~r proteins
An alignment of the amino acid sequences of marine STRIFE1, STRIFE2, and
marine OX40 (Accesssion Number P47741) is shown in Figure 3. Amino acid
residues
I S which are conserved between marine STRIFE 1 and STRIFE2 family members are
highlighted. The percent identity was calculated using the alignment generated
using
MegAlignT''" sequence alignment software. The initial pairwise alignment step
was
performed using a Wilbur-Lipmann algorithm with a K-tuple of 2, a GAP penalty
of 5, a
window of 4, and diagonals saved set to = 4. The multiple alignment step was
20 performed using the Clustal algorithm with a PAM 250 residue weight Table,
a GAP
penalty of 10, and a GAP length penalty of 10.
Retroviral Delivery of STRIFE! and STRIFE2 into mice
The entire open reading frame of STRIFE1 or STRIFE2 is subcloned into the
25 retroviral vector MSCVneo, described in Hawley et al.(1994) Gene Therapy
1:136-138.
Cells (293Ebna, Invitrogen) are then transiently transfected with the STRIFE1
or
STRIFE2 construct and with constructs containing viral regulatory elements, to
produce
high titre retrovirus containing the STRIFE! or STRIFE2 gene. This virus is
then used
to transfect mice. These mice are then tested for any gross pathology and for
changes in
30 their immune response using standard assays.
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
35 described herein. Such equivalents are intended to be encompassed by the
following
claims.
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(1) GENERAL INFORMATION:
( i ) APPLICANT':
(A) NAME: MILLENNIUM BIOTHERAPEUTICS, INC. et al.
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APPLICATION
DATA:
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APPLICATION
DATA:
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(B) FILING DATE: 27 JANUARY 1998
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INFORMATION:
'4S (A) NAME: MANDRAGOURAS, AMY E.
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(ix) TELECOMMUNICATION
INFORMATION:
SO (A) TELEPHONE: (617)227-7400 '
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CA 02318743 2000-07-24
WO 99!37818 PCTNS99/01679
-2-
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 981 base pairs
$ (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
10
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 107..748
1S
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID
NO:
l:
GAATTCGGCA CGAGGGCCGG CACCCCCGCG CTCAAACTGC 60
CCACCCCAGC AGTCCGGCGC
20
CGCGGGGCAG GACAAGGGGA AGGAATAAAC AGAGCC 115
ACGTTTGGTG ATG
GCA
CTC
Met
Ala
Leu
1
2S AAG GTCCTACCT CTA CAC AGG ACG TTC GCTGCCATT CTC TTC 163
GTG CTC
Lys ValLeuPro Leu His Arg Thr Phe AlaAlaIle Leu Phe
Val Leu
5 10 15
CTA CTCCACCTG GCA TGT AAA GTG GAA ACCGGAGAT TGC AGG 211
AGT TGC
30 Leu LeuHisLeu Ala Cys Lys Val Glu ThrGlyAsp Cys Arg
Ser Cys
20 25 30 35
CAG CAGGAATTC AAG GAT CGA TCT TGT GTCCTCTGC AAA CAG
GGA AAC
Gln GlnGluPhe Lys Asp Arg Ser Cys ValLeuCys Lys Gln
Gly Asn
3$ 40 45 50
TGC GGACCTGGC ATG GAG TTG TCC TGT GGCTTCGGC TAT GGG 307
AAG GAA
Cys GlyProGly Met Glu Leu Ser Cys GlyPheGly Tyr Gly
Lys Glu
S5 60 6S
40
GAG GATGCACAG TGT GTG CCC TGC CAC CGGTTCAAG GAA GAC 355
AGG CCG
Glu AspAlaGln Cys Val Pro Cys His ArgPheLys Glu Asp
Arg Pro
70 75 80
4S TGG GGTTTCCAG AAG TGT AAG CCA GAC TGTGCGCTG GTG AAC 403
TGT GCG
Trp GlyPheGln Lys Cys Lys Pro Asp CysAlaLeu Val Asn
Cys Ala
85 90 95
CGC TTTCAGAGG GCC AAC TGC TCA AGT GATGCTGTC TGC GGG 451
CAC ACC
S0 Arg PheGlnArg Ala Asn Cys Ser Ser'AspAlaVal Cys Gly
His Thr
100 105 110 115
GAC TGCCTGCCA GGA TTT TAC CGG AAA CTGGTTGGT TTT CAA 499
AAG ACC
Asp CysLeuPro Gly Phe Tyr Arg Lys LeuValGly Phe Gln
Lys Thr
$$ 120 125 130
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GACATG GAGTGT GTGCCCTGC GGAGACCCACCT CCTCCCTAC 547
GAA
CCA
AspMet GluCys ValProCys GlyAspProPro ProProTyr GluPro
135 140 145
S CACTGT ACCAGC AAGGTGAAC CTTGTGAAGATC TCCTCCACC GTCTCC
595
HisCys ThrSer LysValAsa LeuValLysIle SerSerThr ValSer
150 155 160
AGCCCT CGGGAC ACGGCGCTG GCTGCCGTCATC TGCAGTGCT CTGGCC 643
1~ SerPro ArgAsp ThrAlaLeu AlaAlaValIle CysSerAla LeuAla
165 170 175
ACGGTG CTGCTC GCCCTGCTC ATCCTGTGTGTC ATCTACTGC AAGAGG 6SI
IS ThrVal LeuLeu AlaLeuLeu IleLeuCysVal IleTyrCys LysArg
180 185 190 195
CAGTTC ATGGAG AAGAAACCC AGCTGTAAGCTC CCATCC.CTC TGTCTC 739
GlnPhe MetGlu LysLysPro SerCysLysLeu ProSerLeu CysLeu
2~ 200 205 210
ACTGTG AAGTGAGCTTGTT CAAGAGTT CTCAAGACAC 7gg
AGCATTGTCA
CC
ThrVal Lys
2S
CTGGCTGAGA CCTAAGACCT TTAGAGCATC AACAGCTACT TAGAATACAA GATGCAGGAA 848
AACGAGCCTC TTCAGGAATC TCAGGGCCTC CTAGGGATGC TGGCAAGGCT GTGATGTCTC 908
3O AAGGCTACCA GGAAAAAP,TA AAAGTTGTCT ATACCCTAAA AAAAAAAAAA p~ApppApAl~A 968
AACATGCGGC CGC 981
3S (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 214 amino acids
(B) TYPE: amino acid
4~ (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
4S
Met Ala Leu Lys Val Leu Pro Leu His Arg Thr Val Leu Phe Ala Ala
1 5 10 15
Ile Leu Phe Leu Leu His Leu Ala Cys Lys Val Ser Cys Glu Thr Gly
S~ 20 25 ~ 30
Asp Cys Arg Gln Gln Glu Phe Lys Asp Arg Ser Gly Asn Cys Val Leu
35 40 45
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-4-
Cys Lys Gln Cys Gly Pro Gly Met Glu Leu Ser Lys Glu Cys Gly Phe
50 55 60
Gly Tyr Gly Glu Asp Ala Gln Cys Val Pro Cys Arg Pro His Arg Phe
65 70 75
80
Lys Glu Asp Trp Gly Phe Gln Lys Cys Lys Pro Cys Ala Asp Cys Ala
85 90 95
10 Leu Val Asn Arg Phe Gln Arg Ala Asn Cys Ser His Thr Ser Asp Ala
100 105 110
Val Cys Gly Asp Cys Leu Pro Gly Phe Tyr Arg Lys Thr Lys Leu Val
115 120 125
15
Gly Phe Gln Asp Met Glu Cys Val Pro Cys Gly Asp Pro Pro Pro Pro
130 135 140
Tyr Glu Pro His Cys Thr Ser Lys Val Asn Leu Val Lys Ile Ser Ser
20 145 150 155 160
Thr Val Ser Ser Pro Arg Asp Thr Ala Leu Ala Ala Val Ile Cys Ser
165 170 175
25 Ala Leu Ala Thr Val Leu Leu Ala Leu Leu Ile Leu Cys Val Ile Tyr
180 185 190
Cys Lys Arg Gln Phe Met Glu Lys Lys Pro Ser Cys Lys Leu Pro Ser
195 200 205
30
Leu Cys Leu Thr Val Lys
210
3S (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 642 base pairs
(B) TYPE: nucleic acid
40 (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
45
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..642
50
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
ATG GCA CTC AAG GTC CTA CCT CTA CAC AGG ACG GTG CTC TTC GCT GCC
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-$-
Met Ala Leu Lys Val Leu Pro Leu His Arg Thr Val Leu Phe Ala Ala
1 5 10 15
ATT 96
CTC
TTC
CTA
CTC
CAC
CTG
GCA
TGT
AAA
GTG
AGT
TGC
GAA
ACC
GGA
$ Ile
Leu
Phe
Leu
Leu
His
Leu
Ala
Cys
Lys
Val
Ser
Cys
Glu
Thr
Gly
~
20 25 30
GAT 144
TGC
AGG
CAG
CAG
GAA
TTC
AAG
GAT
CGA
TCT
GGA
AAC
TGT
GTC
CTC
Asp Cys Arg Gln
Gln Glu Phe
Lys Asp Arg
Ser Gly Asn
Cys Val Leu
35 40 45
TGC AAA CAG TGC TTG TCC AAG GAA TGT GGC 192
GGA CCT GGC TTC
ATG GAG
Cys Lys Gln Cys Leu Ser Lys Glu Cys Gly
Gly Pro Gly Phe
Met Glu
50 55 60
1$
GGC TAT GGG GAG GCA CAG TGT CCC TGC AGG CCG CAC CGG 240
GAT GTG TTC
Gly Tyr Gly Glu Ala Gln Cys Pro Cys Arg Pro His Arg
Asp Val Phe
65 70 75 80
ZO AAG GAA GAC TGG TTC CAG AAG AAG CCA TGT GCG GAC TGT 288
GGT TGT GCG
Lys Glu Asp Trp Prs Gln Lys Lys Pro Cys Ala Asp Cys
Gly Cys Ala
85 90 95
CTG GTG AAC CGC CAG AGG GCC TGC TCA CAC ACC AGT GAT 336
TTT AAC GCT
2$ Leu Val Asn Arg Gln Arg Ala Cys Ser His Thr Ser Asp
Phe Asn Ala
100 105 110
GTC TGC GGG GAC CTG CCA GGA TAC CGG AAG ACC AAA CTG 384
TGC TTT GTT
Val Cys Gly Asp Leu Pro Gly Tyr Arg Lys Thr Lys Leu
Cys Phe Val
3~ 115 120 125
GGT TTT CAA GAC GAG TGT GTG TGC GGA GAC CCA CCT CCT 432
ATG CCC CCC
Gly Phe Gln Asp Glu Cys Val Cys Gly Asp Pro Pro Pro
Met Pro Pro
130 135 140
3$
TAC GAA CCA CAC ACC AGC AAG AAC CTT GTG AAG ATC TCC 480
TGT GTG TCC
Tyr Glu Pro His Thr Ser Lys Asn Leu Val Lys Ile Ser
Cys Val Ser
145 150 155 160
4O ACC GTC TCC AGC CGG GAC ACG CTG GCT GCC GTC ATC TGC 528
CCT GCG AGT
Thr Val Ser Ser Arg Asp Thr Leu Ala Ala Val Ile Cys
Pro Ala Ser
165 170 175
GCT CTG GCC ACG CTG CTC GCC CTC ATC CTG TGT GTC ATC 576
GTG CTG TAC
4$ Ala Leu Ala Thr Leu Leu Ala Leu Ile Leu Cys Val Ile
Val Leu Tyr
180 185 190
TGC AAG AGG CAG ATG GAG AAG CCC AGC TGT AAG CTC CCA 624
TTC AAA TCC
Cys Lys Arg Gln Met Glu Lys Pro Ser Cys Lys Leu Pro
Phe Lys Ser
$~ 195 200 ' 205
CTC TGT CTC ACT AAG 642
GTG
_ Leu Cys Leu Thr Lys
Val
210
$$
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(2) INFORMATION FOR SEQ ID N0:4:
(
i
)
SEQUENCE
CH...P.ACTERISTICS
(A) LENGTH: pairs
555 base
S (B) TYPE: nucleic
acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE cDNA
TYPE:
10
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..555
1S
(xi) SEQUENCE IPTION: SEQ ID
DESCR N0:4:
GAA ACCGGA GAT TGC CAGCAGGAA TTCAAGGAT CGATCTGGAAAC 8
AGG
20 Glu ThrGly Asp Cys GlnGlnGlu PheLysAsp ArgSerGlyAsn
Arg
1 5 10 15
TGT GTCCTC TGC AAA TGCGGACCT GGCATGGAG TTGTCCAAGGAA 96
CAG
Cys ValLeu Cys Lys CysGlyPro GlyMetGlu LeuSerLysGlu
Gln
2S 20 25 30
TGT GGCTTC GGC TAT GAGGATGCA CAGTGTGTG CCCTGCAGGCCG 144
GGG
Cys GlyPhe Gly Tyr GluAspAla GlnCysVal ProCysArgPro
Gly
35 40 45
30
CAC CGGTTC AAG GAA TGGGGTTTC CAGAAGTGT AAGCCATGTGCG 192
GAC
His ArgPhe Lys Glu TrpGlyPhe GlnLysCys LysProCysAla
Asp
50 55 60
3S GAC TGTGCG CTG GTG CGCTTTCAG AGGGCCAAC TGCTCACACACC 240
AAC
Asp CysAla Leu Val ArgPheGln ArgAlaAsn CysSerHisThr
Asn
65 70 75 g0
AGT GATGCT GTC TGC C~.CTGCCTG CCAGGATTT TACCGGAAGACC 288
GGG
40 Ser AspAla Val Cys AspCysLeu ProGlyPhe TyrArgLysThr
Gly
85 90 95
AAA CTGGTT GGT TTT GACATGGAG TGTGTGCCC TGCGGAGACCCA 336
CAA
Lys LeuVal Gly Phe AspMetGlu CysValPro CysGlyAspPro
Gln
4S loo l05 llo
CCT CCTCCC TAC GAA CACTGTACC AGCAAGGTG AACCTTGTGAAG 384
CCA
Pro ProPro Tyr Glu HisCysThr SerLysVal AsnLeuValLys
Pro
115 120 125
S0
ATC TCCTCC ACC GTC AGC~CTCGG GACACGGCG CTGGCTGCCGTC 432
TCC
Ile SerSer Thr Val SerProArg AspThrAla LeuAlaAlaVal
Ser
130 135 140
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ATC TGC AGT GCT CTG GCC ACG GTG CTG CTC GCC CTG CTC ATC CTG TGT 480
Ile Cys Ser Ala Leu Ala Thr Val Leu Leu Ala Leu Leu Ile Leu Cys
145 150 155 160
S GTC ATC TAC TGC AAG AGG CAG TTC ATG GAG AAG AAA CCC AGC TGT AAG 528
Val Ile Tyr Cys Lys Arg Gln Phe Met Glu Lys Lys Pro Ser Cys Lys
165 170 175
CTC CCA TCC CTC TGT CTC ACT GTG AAG 555
10 Leu Pro Ser Leu Cys Leu Thr Val Lys
180 185
(2) INFORMATION FOR SEQ ID N0:5:
IS (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 655 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
20
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
2S (A) NAME/KEY: CDS
(B) LOCATION: 110..559
(xi) SEQID
SEQUENCE N0:5:
DESCRIPTION:
30
GAATTCGGCA CGAGGGCGTT GCTGCGGAAA 60
TGGCGCGGAA GCGTGAGTCT
GTGCTACCAA
GGAGCACAGC ACTGGCGAGT GTGAGAGCC 115
AGCAGGAATA ATG
AACACGTTTG GCA
3S Met
Ala
1
CTC AAGGTCCTA CCTCTACAC AGGACGGTGCTC TTCGCTGCCATT CTC 163
Leu LysValLeu ProLeuHis ArgThrValLeu PheAiaAlaIle Leu
5 10 15
40
TTC CTACTCCAC CTGGCATGT AAAGTGAGTTGC GAAACCGGAGAT TGC 211
Phe LeuLeuHis LeuAlaCys LysValSerCys GluThrGlyAsp Cys
20 25 30
4S AGG CAGCAGGAA TTCAAGGAT CGATCTGGAAAC TGTGTCCTCTGC AAA 259
Arg GlnGlnGlu PheLysAsp ArgSerGlyAsn CysValLeuCys Lys
35 40 45 50
CAG TGCGGACCT GGCATGGAG TTGTCCAAGGAA TGTGGCTTCGGC TAT 307
SO Gln CysGlyPro GlyMetGlu LeuSerLysGlu CysGlyPheGly Tyr
55 60 65
GGG GAGGATGCA CAGTGTGTG CCCTGCAGGCCG CACCGGTTCAAG GAA 355
Gly GluAspAla GlnCysVal ProCysArgPro HisArgPheLys Glu
SS 70 75 80
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_g_
GAC TGG GGTTTCCAG TGT CCA GCG GAC TGT CTG GTG 403
AAG AAG TGT GCG
Asp Trp GlyPheGln CysLys Pro Ala Asp Cys Leu Val
Lys Cys Ala
85 90 95
$
AAC CGC TTTCAGAGG AACTGC TCA ACC AGT GAT GTC TGC 451
GCC CAC GCT
Asn Arg PheGlnArg AsnCys Ser Thr Ser Asp Val Cys
Ala His Ala
100 105 110
IO GGG GAC TGCCTGCCA TTTTAC CGG ACC AAA CTG GGT TTT 499
GGA AAG GTT
Gly Asp CysLeuPro PheTyr Arg Thr Lys Leu Gly Phe
Gly Lys Val
115 120 125 130
CAA GAC ATGGAGTGT CCCTGC GGA CCA CCT CCT TAC GAA 547
GTG GAC CCC
1$ Gln Asp MetGluCys ProCys Gly Pro Pro Pro Tyr Glu
Val Asp Pro
135 140 145
CCA CAC TGTGAGTGATGTGCCA 599
AGTGGCAGCA
GACCTTTAAA
AAAAAAAGAA
Pro His CysGlu
20 150
AAAAAAACAA i4AAAAAAAAAAAAAAAAA AAATTTCCGC 655
ACAAAAACAA GGCCGC
p~~
2$ (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 150 amino acids
(B) TYPE: amino acid
30 (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
3$
Met Ala Leu Lys Val Leu Pro Leu His Arg Thr Val Leu Phe Ala F.la
1 5 10 15
Ile Leu Phe Leu Leu His Leu Ala Cys Lys Val Ser Cys Glu Thr Gly
40 20 25 30
Asp Cys Arg Gln Gln Glu Phe Lys Asp Arg Ser Gly Asn Cys Val Leu
35 40 45
4$ Cys Lys Gln Cys Gly Pro Gly Met Glu Leu Ser Lys Glu Cys Gly Phe
50 55 60
Gly Tyr Gly Glu Asp Ala Gln Cys Val Pro Cys Arg Pro His Arg Phe
65 70 75 80
$0
Lys Glu Asp Trp Gly Phe Gln Lys Cys Lys Pry Cys Ala Asp Cys Ala
85 90 95
Leu Val Asn Arg Phe Gln Arg Ala Asn Cys Ser His Thr Ser Asp Ala
$$ 100 105 110
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Val Cys Gly Asp Cys Leu Pro Gly Phe Tyr Arg Lys Thr Lys Leu Val
115 120 125
S Gly Phe Gln Asp Met Glu Cys Val Pro Cys Gly Asp Pro Pro Pro Pro
130 ~ 135 140
Tyr Glu Pro His Cys Glu
145 150
lO
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
IS (A) LENGTH: 450 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
ZO (ii) MOLECULE TYPE: cDNA
(ix)
FEATURE:
(A) CDS
NAME/KEY:
2S (B) 1..450
LOCATION:
(xi) ID N0:7:
SEQUENCE
DESCRIPTION:
SEQ
3OATG GCA AAGGTC CCT CTA AGG ACG CTC TTCGCT GCC 48
CTC CTA CAC GTG
Met Ala LysVal Pro Leu Arg Thr Leu PheAla Ala
Leu Leu His Val
1 5 10 15
ATT CTC CTACTC CTG GCA AAA GTG TGC GAAACC GGA 96
TTC CAC TGT AGT
3SIle Leu LeuLeu Leu Ala Lys Val Cys GluThr Gly
Phe His Cys Ser
20 25 30
GAT TGC CAGCAG TTC AAG CGA TCT AAC TGTGTC CTC 144
AGG GAA GAT GGA
Asp Cys GlnGln Phe Lys Arg Ser Asn CysVal Leu
Arg Glu Asp Gly
40 35 40 45
TGC AAA TGCGGA GGC ATG TTG TCC GAA TGTGGC TTC 192
CAG CCT GAG AAG
Cys Lys CysGly Gly Met Leu Ser Glu CysGly Phe
Gln Pro Glu Lys
50 55 60
4S
GGC TAT GAGGAT CAG TGT CCC TGC CCG CACCGG TTC 240
GGG GCA GTG AGG
Gly Tyr GluAsp Gln Cys Pro Cys Pro HisArg Phe
Gly Ala Val Arg
65 70 75 80
SOAAG GAA TGGGGT CAG AAG AAG CCA GCG GACTGT GCG 288
GAC TTC TGT TGT
Lys Glu TrpGly Gln Lys Lys Pro Ala AspCys Ala
Asp Phe Cys Cys
85 90 95
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CTG GTG AAC CGC TTT CAG AGG GCC AAC TGC TCA CAC ACC AGT GAT GCT 336
Leu Val Asn Arg Phe Gln Arg Ala Asn Cys Ser His Thr Ser Asp Ala
100 105 110
S GTC TGC GGG GAC TGC CTG CCA GGA TTT TAC CGG AAG ACC AAA CTG GTT 384
Val Cys Gly Asp Cys' Leu Pro Gly Phe Tyr Arg Lys Thr Lys Leu Val
115 120 125
GGT TTT CAA GAC ATG GAG TGT GTG CCC TGC GGA GAC CCA CCT CCT CCC 432
10 Gly Phe Gln Asp Met Glu Cys Val Pro Cys Gly Asp Pro Pro Pro Pro
130 135 140
TAC
GAA
CCA
CAC
TGT
GAG
450
Tyr Glu Pro His Cys Glu
IS 145 150
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
20 (A) LENGTH: 363 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
zS (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
30 (B) LOCATION: 1..363
(xi) SEQUENCE DESCRIPTION: SEQ
ID N0:8:
3S GAA ACC GGA GAT TGC AGG CAG CAG GAA GAT TCT GGA AAC 48
TTC AAG CGA
Glu Thr Gly Asp Cys Arg Gln Gln Glu Asp Ser Gly Asn
Phe Lys Arg
1 5 10 15
TGT GTC CTC TGC AAA CAG TGC GGA CCT GAG TCC AAG GAA 96
GGC ATG TTG
40 Cys Val Leu Cys Lys Gln Cys Gly Pro Glu Ser Lys Glu
Gly Met Leu
20 25 30
TGT GGC TTC GGC TAT GGG GAG GAT GCA GTG TGC AGG CCG 144
CAG TGT CCC
Cys Gly Phe Gly Tyr Gly Glu Asp Ala Val Cys Arg Pro
Gln Cys Pro
4S 35 40 45
CAC CGG TTC AAG GAA GAC TGG GGT TTC TGT CCA TGT GCG 192
CAG AAG AAG
His Arg Phe Lys Glu Asp Trp Gly Phe Cys Pro Cys Ala
Gln Lys Lys
50 55 60
$~
GAC TGT GCG CTG GTG AAC CGC TTT CAG AAC TCA CAC ACC 240
AGG GCC TGC
Asp Cys Ala Leu Val Asn Arg Phe Gln Asn Ser His Thr
Arg Ala Cys
65 70 75 80
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AGT GAT GCT GTC TGC GGG GAC TGC CTG CCA GGA TTT TAC CGG AAG ACC 288
Ser Asp Ala Val Cys Gly Asp Cys Leu Pro Gly Phe Tyr Arg Lys Thr
85 90 95
S AAA CTG GTT GGT TTT CAA GAC ATG GAG TGT GTG CCC TGC GGA GAC CCA 336
Lys Leu Val Gly Phe Gln Asp Met Glu Cys Val Pro Cys Gly Asp Pro
100 105 110
CCT CCT CCC TAC GAA CCA CAC TGT GAG 363
10 Pro Pro Pro Tyr Glu Pro His Cys Glu
115 120
(2) INFORMATION FOR SEQ ID N0:9:
IS (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
20 (ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
2S
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Met Ala Leu Lys Val Leu Pro Leu His Arg Thr Val Leu Phe Ala Ala
1 5 10 15
30
Ile Leu Phe Leu Leu His Leu Ala Cys Lys Val Ser Cys
20 25
(2) INFORMATION FOR SEQ ID NO:10:
3S
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
40
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
4S
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ala Leu Ala Ala Val Ile Cys Ser Ala Leu Ala Thr Val Leu Leu Ala
S0 1 5 10 ~ 15
Leu Leu Ile Leu Cys Val Ile Tyr Cys
20 25
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(2) INFORMATION FOR SEQ ID NO:lI:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
1~ (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ
ID NO:11:
i5
Cys Arg Gln Gln Glu Phe Lys Asp Gly Asn Cys Val Leu
Arg Ser Cys
1 5 10 15
Lys Gln Cys Gly Pro Gly Met Glu Lys Glu Cys Gly Phe
Leu Ser Gly
2~ 20 25 30
Tyr Gly Glu Asp Ala Gln Cys
35
2S (2) INFORMATION
FOR
SEQ
ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 amino acids
(B) TYPE: amino acid
3~ (D) TOPOLOGY: linear
(ii) MOLECULE TYpE: protein
(v) FRAGMENT TYPE: internal
35
(xi) SEQUENCE DESCRIPTION: SEQ
ID N0:12:
40 Cys Arg Pro His Arg Phe Lys Glu Gly Phe Gln Lys Cys
Asp Trp Lys
1 5 10 15
Pro Cys Ala Asp Cys Ala Leu Val Phe Gln Arg Ala Asn
Asn Arg Cys
20 25 30
45
Ser His Thr Ser Asp Ala Val Cys
35 40
(2) INFORMATION
FOR
SEQ
ID N0:13:
$~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(8) TYPE: amino acid
(D) TOPOLOGY: linear
$5
CA 02318743 2000-07-24
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-13-
(ii) MOLECULE TYPE: protein
(v) FRAGMEi~~ ~L~YPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ
ID N0:13:
Met Ala Leu Lys Val Leu Pro Arg Thr Val Leu Ala
Leu His Phe Ala
1 s
l0 15
Ile Leu Phe Leu Leu His Leu Lys Val Ser Cys
Ala Cys
20 25
IS (2) INFORMATION
FOR
SEQ
ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39.amino acids
(B) TYPE: amino acid
2~ (D) TOPOLOGY: linear
(ii) MOLECULE TYpE: protein
(v) FRAGMENT TYPE: internal
25
(xi) SEQUENCE DESCRIPTION: SEQ 14:
ID N0:
30 Cys Arg Gln Gln Glu Phe Lys Ser Gly Asn Cys Leu
Asp Arg Val Cys
1 5 10 15
Lys Gln Cys Gly Pro Gly Met Ser Lys Glu Cys Phe
Glu Leu Gly Gly
20 25 30
3$
Tyr Gly Glu Asp Ala Gln Cys
35
(2) INFORMATION
FOR
3EQ
ID N0:15:
40
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
45
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
$0
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Cys Arg Pro His Arg Phe Lys Glu Asp Trp Gly Phe Gln Lys Cys Lys
$$ 1 5 10 15
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Pro Cys Ala Asp Cys Ala Leu Val Asn Arg Phe Gln Arg Ala Asn Cys
20 25 30
$ Ser His Thr Ser Asp Ala Val Cys
35 ~ 40
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
1$ (ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Prctein
(B) LOCATION: 2
(D) OTHER INFORMATION: /note= "Xaa is four or six amino
acids."
(ix) FEATURE:
(A) NAME/KEY: protein
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "Xaa is 5 or 10 amino
acids."
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: B
3$ (D) OTHER INFORMATION: /note= "Xaa is 0 or 2 amino acids."
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: i'v
40 (D) OTHER INFORMATION: /note= "Xaa is 7 or 11 amino
acids."
(ix) FEATURE:
(A) NAME/KEy; protein
45 (B) LOCATION: 12
(D) OTHER INFORMATION: /note= "Xaa is 4 or 6 amino acids."
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
$0
Cys Xaa Phe Tyr His Xa.. Cys Xaa Cys Xaa Cys Xaa Asp Asn Glu Gln
1 5 10 15
Ser.Lys Pro Xaa Xaa Cys
$$ 20
CA 02318743 2000-07-24
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- 1S -
S (2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
1~ (D) TOPOLOGY: linear
(ii) MOLECULE TYpE: protein
(v) FRAGMENT TYPE: N-terminal
1S
(ix) FEATURE:
{A) NAME/KEY: Protein
(B) LOCATION: 2
(D) OTHER INFORMATION: /note= °Xaa is between four and
fourteen
amino acids.°
(ix) FEATURE:
zS (A) NAME/KEY: protein
(B) LOCATION: 4
(D) OTHER INFORMATION: /note= ~~Xaa is between 0 and 2 amino
acids.°
3~ (ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "Xaa is between 2 and 4 amino
acids . ~~
3S
(ix) FEATURE:
{A) NAME/KEY: protein
(B) LOCATION: 8
(D) OTHER INFORMATION: /note= "Xaa is between 6 and 12 amino
40 acids."
(ix) FEATURE:
(A) NAME/KEy: protein
(B) LOCATION: 10
4S (D) OTHER INFORMATION: /note= ~~Xaa is between 6 and 10 amino
acids . ~~
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
$~
Cys Xaa Cys Xaa Cys Xaa Cys Xaa Cys Xaa Cys
