Note: Descriptions are shown in the official language in which they were submitted.
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Human Tumor Necrosis Factor Receptor-Like 2
Background of the Invention
Field of the Invention
The present invention relates to novel members of the Tumor Necrosis
Factor (TNF) receptor family. More specifically, isolated nucleic acid
molecules
are provided encoding a human TNF receptor-related protein, referred to herein
as the TR2 receptor of FIG. IA-1B, having considerable homology to murine
CD40. Two different TR2 splice variants, referred to as TR2-SVI and TR2-SV2,
are also provided. TR2 polypeptides are also provided with homology to human
type 2 TNF receptor (TNF-RII). Further provided are vectors, host cells and
recombinant methods for producing the same. The invention also relates to both
the inhibition and enhancement of the activity of TR2 receptor polypeptides
and
diagnostic methods for detecting TR2 receptor gene expression.
Related Art
Human tumor necrosis factors a (TNF-a) and P (TNF-(3 or lymphotoxin)
are related members of a broad class of polypeptide mediators, which includes
the
interferons, interleukins and growth factors, collectively called cytokines
(Beutler,
B. and Cerami, A., Annu. Rev. Immunol., 7:625-655 (1989)).
Tumor necrosis factor (TNF-a and TNF-(3) was originally discovered as
a result of its anti-tumor activity, however, now it is recognized as a
pleiotropic
cytokine playing important roles in a host of biological processes and
pathologies.
To date, there are ten known members of the TNF-related cytokine family, TNF-
a, TNF-(3 (lymphotoxin-a), LT-P, TRAIL and ligands for the Fas receptor, CD30,
CD27, CD40, OX40 and 4-1BB receptors. These proteins have conserved C-
terminal sequences and variable N-terminal sequences which are often used as
membrane anchors, with the exception of TNF-(3. Both TNF-a and TNF-(3
function as homotrimers when they bind to TNF receptors.
TNF is produced by a number of cell types, including monocytes,
fibroblasts, T-cells, natural killer (NK) cells and predominately by activated
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macrophages. TNF-a has been reported to have a role in the rapid necrosis of
tumors, immunostimulation, autoimmune disease, graft rejection, producing an
anti-viral response, septic shock, cerebral malaria, cytotoxicity, protection
against
deleterious effects of ionizing radiation produced during a course of
chemotherapy, such as denaturation of enzymes, lipid peroxidation and DNA
damage (Nata et al., J. Immunol. 136(7):2483 (1987)), growth regulation,
vascular endothelium effects and metabolic effects. TNF-a also triggers
endothelial cells to secrete various factors, including PAI-1, IL-1, GM-CSF
and
IL-6 to promote cell proliferation. In addition, TNF-a up-regulates various
cell
adhesion molecules such as E-Selectin, ICAM-1 and VCAM-1. TNF-a and the
Fas ligand have also been shown to induce programmed cell death.
TNF-P has many activities, including induction of an antiviral state and
tumor necrosis, activation of polymorphonuclear leukocytes, induction of class
I
major histocompatibility complex antigens on endothelial cells, induction of
adhesion molecules on endothelium and growth hormone stimulation (Ruddle, N.
and Homer, R., Prog. Allergy 40:162-182 (1988)).
Both TNF-a and TNF-(3 are involved in growth regulation and interact
with hemopoietic cells at several stages of differentiation, inhibiting
proliferation
of various types of precursor cells, and inducing proliferation of immature
myelomonocytic cells. Porter, A., Tibtech 9:158-162 (1991).
Recent studies with "knockout" mice have shown that mice deficient in
TNF-P production show abnormal development of the peripheral lymphoid organs
and morphological changes in spleen architecture (reviewed in Aggarwal el al.,
Eur Cytokine Netw, 7(2):93-124 (1996)). With respect to the lymphoid organs,
the popliteal, inguinal, para-aortic, mesenteric, axillary and cervical lymph
nodes
failed to develop in TNF-(3 -/- mice. In addition, peripheral blood from TNF-P
-/-
mice contained a three fold reduction in white blood cells as compared to
normal
mice. Peripheral blood from TNF-P -/- mice, however, contained four fold more
B cells as compared to their normal counterparts. Further, TNF-P, in contrast
to
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TNF-a has been shown to induce proliferation of EBV-infected B cells. These
results indicate that TNF-(3 is involved in lymphocyte development.
The first step in the induction of the various cellular responses mediated
by TNF-a or TNF-P is their binding to specific cell surface or soluble
receptors.
Two distinct TNF receptors of approximately 55-KDa (TNF-RI) and 75-KDa
(TNF-RII) have been identified (Hohman et al., J. Biol. Chem., 264:14927-14934
(1989)), and human and mouse cDNAs corresponding to both receptor types have
been isolated and characterized (Loetscher et al., Cell, 61:351 (1990)). Both
TNF-Rs share the typical structure of cell surface receptors including
extracellular,
transmembrane and intracellular regions.
These molecules exist not only in cell bound forms, but also in soluble
forms, consisting of the cleaved extra-cellular domains of the intact
receptors
(Nophar et al., EMBO Journal, 9 (10):3269-76 (1990)) and otherwise intact
receptors wherein the transmembrane domain is lacking. The extracellular
domains of TNF-RI and TNF-RII share 28% identity and are characterized by four
repeated cysteine-rich motifs with significant intersubunit sequence homology.
The majority of cell types and tissues appear to express both TNF receptors
and
both receptors are active in signal transduction, however, they are able to
mediate
distinct cellular responses. Further, TNF-RII was shown to exclusively mediate
human T-cell proliferation by TNF as shown in PCT WO 94/09137.
TNF-RI dependent responses include accumulation of C-FOS, IL-6, and
manganese superoxide dismutase mRNA, prostaglandin E2 synthesis, IL-2
receptor and MHC class I and II cell surface antigen expression, growth
inhibition,
and cytotoxicity. TNF-RI also triggers second messenger systems such as
phospholipase A2, protein kinase C, phosphatidylcholine-specific phospholipase
C and sphingomyelinase (Pfefferk et al., Cell, 73:457-467 (1993)).
Several interferons and other agents have been shown to regulate the
expression of TNF receptors. Retinoic acid, for example, has been shown to
induce the production of TNF receptors in some cells type while down
regulating
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production in other cells. In addition, TNF-a has been shown effect the
localization of both types of receptor. TNF-a induces internalization of TNF-
RI
and secretion of TNF-RII (reviewed in Aggarwal et al., supra). Thus, the
production and localization of both TNF-Rs are regulated by a variety of
agents.
Both the yeast two hybrid system and co-precipitation and purification
have been used to identify ligands which associate with both types of the TNF-
Rs
(reviewed in Aggarwal et al., supra and Vandenabeele et al., Trends in Cell
Biol.
5:392-399 (1995)). Several proteins have been identified which interact with
the
cytoplasmic domain of a murine TNF-R. Two of these proteins appear to be
related to the baculovirus inhibitor of apoptosis, suggesting a direct role
for TNF-
R in the regulation of programmed cell death.
Summary of the Invention
An object of the present invention is to provide human tumor necrosis
factor receptor-like 2. In accordance with an aspect of the present invention,
there
is provided an isolated nucleic acid molecule comprising a polynucleotide
having
a nucleotide sequence at least 95% identical to a sequence selected from the
group
consisting of
(a) a nucleotide sequence encoding the TR2 receptor having
the amino acid sequence at positions from about -36 to about 247 in SEQ ID
NO:2;
(b) a nucleotide sequence encoding the TR2 receptor
polypeptide having the amino acid sequence at positions from about -3 5 to
about
247 in SEQ ID NO:2;
(c) a nucleotide sequence encoding the TR2 receptor
polypeptide having the amino acid sequence at positions from about I to about
247 in SEQ ID NO:2;
(d) a nucleotide sequence encoding the TR2 receptor having
the amino acid sequence encoded by the eDNA clone contained in ATCC Deposit
Number 97059,
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(e) a nucleotide sequence encoding the mature TR2 receptor
having the amino acid sequence encoded by the cDNA clone contained in ATCC
Deposit Number 97059;
(f) a nucleotide sequence encoding the TR2 extracellular
domain;
(g) a nucleotide sequence encoding the TR2 transmembrane
domain;
(h) a nucleotide sequence encoding the TR2 intracellular
domain;
(1) a nucleotide sequence encoding the TR2-SV I receptor
having the amino acid sequence at positions from about -36 to about 149 in SEQ
ID NO:5;
(j) a nucleotide sequence encoding the TR2-SV 1 receptor
having the amino acid sequence at positions from about -35 to about 149 in SEQ
ID NO:5;
(k) a nucleotide sequence encoding the TR2-SVI receptor
having the amino acid sequence at positions from about I to about 149 in SEQ
ID
NO:5;
(1) a nucleotide sequence encoding the TR2-SVI receptor
having the amino acid sequence encoded by the cDNA clone contained in ATCC
Deposit Number 97058;
(m) a nucleotide sequence encoding the mature TR2-SVI
receptor having the amino acid sequence encoded by the cDNA clone contained
in ATCC Deposit Number 97058,
(n) a nucleotide sequence encoding the TR2-SV2 receptor
having the amino acid sequence in SEQ ID NO:8;
(o) a nucleotide sequence encoding the TR2-SV2 receptor
having the amino acid sequence at positions from about 2 to about 13 6 in SEQ
ID
NO:8;
(p) a nucleotide sequence encoding the TR2-SV2 receptor
having the amino acid sequence encoded by the cDNA clone contained in ATCC
Deposit Number 97057; and
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(q) a nucleotide sequence complementary to any of the
nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k),
(1), (m), (n),
(o) or (p).
In accordance with another aspect of the invention, there is provided an
isolated nucleic acid molecule comprising a polynucleotide having a nucleotide
sequence in SEQ ID NO:1 wherein nucleotide 314 is either guanine or adenine,
nucleotide 386 is either thymine or cytosine, and nucleotide 627 is either
thymine
or cytosine.
In accordance with another aspect of the invention, there is provided an
isolated nucleic acid molecule comprising a polynucleotide having a nucleotide
sequence encoding the TR2 receptor having the amino acid sequence in SEQ ID
NO:2 wherein amino acid number -20 is either lysine or arginine and amino acid
number 5 is either serine or phenylalanine.
In accordance with another aspect of the invention, there is provided an
isolated TR2 polypeptide having an amino acid sequence at least 95% identical
to
a sequence selected from the group consisting of
(a) the amino acid sequence of the TR2 polypeptide having the
amino acid sequence at positions from about -36 to about-247 in SEQ ID NO:2,
(b) the amino acid sequence of the TR2 polypeptide having the
amino acid sequence at positions from about -35 to about 247 in SEQ ID N0:2;
(c) the amino acid sequence of the TR2 polypeptide having the
amino acid sequence at positions from about I to about 247 in SEQ ID NO:2;
(d) the amino acid sequence of the TR2 polypeptide having the
amino acid sequence encoded by the cDNA clone contained in ATCC Deposit
Number 97059;
(e) the amino acid sequence of the mature TR2 polypeptide
having the amino acid sequence encoded by the cDNA clone contained in ATCC
Deposit Number 97059;
(f) the amino acid sequence of the TR2 receptor extracellular
domain;
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(g) the amino acid sequence of the TR2 receptor
transmembrane domain;
(h) the amino acid sequence of the TR2 receptor intracellular
domain;
(i) the amino acid sequence encoding the TR2-SV1 receptor
having the amino acid sequence at positions from about-36 to about 149 in SEQ
ID NO:5;
(j) the amino acid sequence encoding the TR2-S V 1 receptor
having the amino acid sequence at positions from about -35 to about 149 in SEQ
ID NO:5;
(k) the amino acid sequence encoding the TR2-S V 1 receptor
having the amino acid sequence at positions from about 1 to about 149 in SEQ
ID
NO:5;
(1) the amino acid sequence encoding the TR2-S V 1 receptor
having the complete amino acid sequence encoded by the cDNA clone contained
in ATCC Deposit Number 97058;
(m) the amino acid sequence encoding the mature TR2-SVI
receptor having the amino acid sequence encoded by the cDNA clone contained
in ATCC Deposit Number 97058;
(n) the amino acid sequence encoding the TR2-SV2 receptor
having the amino acid sequence in SEQ ID NO:8;
(o) the amino acid sequence encoding the TR2-SV2 receptor
having the amino acid sequence at positions from about 2 to about 136 in SEQ
ID
NO:8,
(p) the amino acid sequence encoding the TR2-SV2 receptor
having the amino acid sequence encoded by the cDNA clone contained in ATCC
Deposit Number 97057; and
(q) the amino acid sequence of an epitope-bearing portion of
any one of the polypeptides of (a), (b), (c), (d), (e), (f), (g), (h), (i),
(j), (k), (1),
(m), (n), (o) or (p).
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In accordance with another aspect of the invention, there is provided an
isolated polypeptide comprising an epitope-bearing portion of a TR2 receptor
protein, wherein said portion is selected from the group consisting of. a
polypeptide comprising amino acid residues from about 3 to about 34 in SEQ
ID NO:2; a polypeptide comprising amino acid residues from about 70 to about
84 in SEQ ID NO:2, a polypeptide comprising amino acid residues from about
106 to about 153 in SEQ ID NO:2; a polypeptide comprising amino acid residues
from about 240 to about 247 in SEQ ID NO:2; a polypeptide comprising amino
acid residues from about 3 to about 34 in SEQ ID NO:5; a polypeptide
comprising
amino acid residues from about 63 to about 100 in SEQ ID NO:5; a polypeptide
comprising amino acid residues from about 135 to about 149 in SEQ ID NO:51
a polypeptide comprising amino acid residues from about 56 to about 68 in SEQ
ID NO:8; or a polypeptide comprising amino acid residues from about 93 to
about
136 in SEQ ID NO: S.
In accordance with another aspect of the invention, there is provided a
method of treating herpes simplex viral infection comprising introducing an
effective amount of a soluble fragment of a TR2 polypeptide into an individual
to
be treated in admixture with a pharmaceutically acceptable carrier.
In accordance with another aspect of the invention, there is provided a
method of treating a disease state associated with aberrant cell survival
comprising
introducing an effective amount of a TR2 protein, or agonist or antagonist
thereof,
into an individual to be treated in admixture with a pharmaceutically
acceptable
carrier.
In accordance with another aspect of the invention, there is provided a
method of screening for agonists and antagonists of TR2 activity comprising:
(a) contacting cells which express TR2 polypeptides with a
candidate compound,
(b) assaying a cellular response, and
(c) comparing the cellular response to a standard cellular
response made in absence of the candidate compound, whereby, an increased
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cellular response over the standard indicates that the compound is an agonist
and
a decreased cellular response over the standard indicates that the compound is
an
antagonist.
The present invention provides isolated nucleic acid molecules comprising
polynucleotides encoding a TR2 receptor and splice variants thereof having the
amino acid sequences shown in FIG. IA-1B (SEQ ID NO:2), FIG. 4A-4C (SEQ
ID NO:5) and FIG. 7A-7C (SEQ ID NO:8) or the amino acid sequence encoded
by the cDNA clone encoding the TR2 receptors deposited in bacterial hosts as
ATCC Deposit Numbers 97059, 97058 and 97057 on February 13, 1995, The
present invention also relates to recombinant vectors, which include the
isolated
nucleic acid molecules of the present invention, and to host cells containing
the
recombinant vectors, as well as to methods of making such vectors and host
cells
and for using them for production of TR2 polypeptides or peptides by
recombinant techniques.
The invention further provides isolated TR2 polypeptides having amino
acid sequences encoded by the polynucleotides described herein.
The present invention also provides a screening method for identifying
compounds capable of enhancing or inhibiting a cellular response induced by
TR2
receptors, which involves contacting cells which express TR2 receptors with
the
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candidate compound, assaying a cellular response, and comparing the cellular
response to a standard cellular response, the standard being assayed when
contact
is made in absence of the candidate compound; whereby, an increased cellular
response over the standard indicates that the compound is an agonist and a
decreased cellular response over the standard indicates that the compound is
an
antagonist.
In another aspect, a screening assay for agonists and antagonists is
provided which involves determining the effect a candidate compound has on the
binding of cellular ligands to TR2 receptors. In particular, the method
involves
contacting TR2 receptors with a ligand polypeptide and a candidate compound
and determining whether ligand binding to the TR2 receptors is increased or
decreased due to the presence of the candidate compound.
The invention further provides a diagnostic method useful during diagnosis
or prognosis of a disease states resulting from aberrant cell proliferation
due to
alterations in TR2 receptor expression.
An additional aspect of the invention is related to a method for treating an
individual in need of an increased level of a TR2 receptor activity in the
body
comprising administering to such an individual a composition comprising a
therapeutically effective amount of isolated TR2 polypeptides of the invention
or
an agonist thereof
A still further aspect of the invention is related to a method for treating an
individual in need of a decreased level of a TR2 receptor activity in the body
comprising, administering to such an individual a composition comprising a
therapeutically effective amount of a TR2 receptor antagonist.
The invention additionally provides soluble forms of the polypeptides of
the present invention. Soluble peptides are defined by amino acid sequences
wherein the sequence comprises the polypeptide sequences lacking a
transmembrane domain. Such soluble forms of the TR2 receptors are useful as
antagonists of the membrane bound forms of the receptors.
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Brief Description of the Figures
FIG. IA-IB shows the nucleotide (SEQ ID NO:1) and deduced amino
acid (SEQ ID NO:2) sequences of a TR2 receptor. The protein has a predicted
leader sequence of about 36 amino acid residues (underlined) (amino acid
residues
-36 to -1 in SEQ ID NO:2) and a deduced molecular weight of about 30,417 kDa.
It is further predicted that amino acid residues from about 37 to about 200
(amino
acid residues 1 to 164 in SEQ ID NO:2) constitute the extracellular domain;
from
about 201 to about 225 (amino acid residues 165 to 189 in SEQ ID NO:2) the
transmembrane domain (underlined); and from about 226 to about 283 (amino
acid residues 190 to 247 in SEQ ID NO:2) the intracellular domain. Two
potential asparagine-linked glycosylation sites are located at amino acid
positions
110 and 173 (amino acid residues 74 to 137 in SEQ ID NO:2).
FIG. 2 shows the regions of similarity between the amino acid sequences
of the TR2 receptor protein of FIG. IA-113 and a murine CD40 protein (SEQ ID
NO:3).
FIG. 3 shows an analysis of the TR2 receptor amino acid sequence of FIG.
IA-1B. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;
amphipathic regions; flexible regions; antigenic index and surface probability
are
shown. In the "Antigenic Index - Jameson-Wolf' graph, amino acid residues 39
to 70, 106 to 120, 142 to 189 and 276 to 283 in FIG. 1 A-IB (amino acid
residues
3 to 34, 70 to 84, 106 to 153 and 240 to 247 in SEQ ID NO:2) correspond to the
shown highly antigenic regions of the TR2 receptor protein.
FIG. 4A-4C shows the nucleotide (SEQ ID NO:4) and deduced amino
acid (SEQ ID NO:5) sequences of the TR2-SV1 receptor. The protein has a
predicted leader sequence of about 36 amino acid residues (underlined) (amino
acid residues -36 to -1 in SEQ ID NO:5) and a deduced molecular weight of
about
19.5 kDa.
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FIG. 5 shows the regions of similarity between the amino acid sequences
of the full-length TR2-SV1 receptor protein and a human type 2 TNF receptor
(SEQ ID NO:6).
FIG. 6 shows an analysis of the TR2-S V I receptor amino acid sequence.
Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;
amphipathic
regions, flexible regions; antigenic index and surface probability are shown.
In the
"Antigenic Index - Jameson-Wolf" graph, amino acid residues 39 to 70, 99 to
136
and 171 to 185 in FIG. 4A-4C (amino acid residues 3 to 34, 63 to 100 and 13 5
to
149 in SEQ ID NO:5) correspond to the shown highly antigenic regions of the
TR2-SV1 receptor protein.
FIG. 7A-7C shows the nucleotide (SEQ ID NO:7) and deduced amino
acid (SEQ ID NO:8) sequences of the TR2-SV2 receptor. This protein lacks a
putative leader sequence and has a deduced molecular weight of about 14 kDa.
FIG. 8 shows the regions of similarity between the amino acid sequences
of the TR2-SV2 receptor protein and a human type 2 TNF receptor (SEQ ID
NO:9).
FIG. 9 shows an analysis of the TR2-SV2 receptor amino acid sequence.
Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;
amphipathic
regions; flexible regions; antigenic index and surface probability are shown.
In the
"Antigenic Index - Jameson-Wolf' graph, amino acid residues 56 to 68 and 93 to
13 6 in FIG. 7A-7C (SEQ ID NO:8) correspond to the shown highly antigenic
regions of the TR2-SV2 receptor protein.
FIG. 10 shows the regions of similarity between the amino acid sequences
of the TR2 receptor protein of FIG. IA-lB and the TR2-SVI receptor protein of
FIG.4A-4C.
FIG. 11 shows the regions of similarity between the amino acid sequences
of the TR2 receptor protein of FIG. lA-1B and the TR2-SV2 receptor protein of
FIG. 7A-7C.
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FIG. 12 shows the regions of similarity between the amino acid sequences
of the TR2-SV I and the TR2-SV2 receptor proteins.
FIG. 13A-13C shows the regions of similarity between the nucleotide
sequences encoding the TR2 receptor protein of FIG. I A-1 B and the TR2-SV 1
receptor protein of FIG. 4A-4C.
FIG. 14A-14D shows the regions of similarity between the nucleotide
sequences encoding the TR2 receptor protein of FIG. IA-1B and the TR2-SV2
receptor protein of FIG. 7A-7C.
FIG. 15A-15E shows the regions of similarity between the nucleotide
sequences encoding the TR2-SVI and the TR2-SV2 receptor proteins
FIG. 16 shows an alignment of the amino acid sequence of the TR2
receptor of FIG. IA-IB (SEQ ID NO:2) with other TNFR family members. The
amino acid sequence of TR2 was aligned with those of TNFR-I (SEQ ID NO:10),
TNFR-II (SEQ ID NO:11), CD40 (SEQ ID NO:12) and 4-1BB (SEQ ID NO: 13)
on the basis of sequence homology and conserved cysteine residues.
Detailed Description of the Preferred Embodiments
The present invention provides isolated nucleic acid molecules comprising
polynucleotides encoding a TR2 polypeptide (FIG. lA-IB (SEQ ID NO:2)) and
splice variants thereof, TR2-SVI (FIG. 4A-4C (SEQ ID NO:5)) and TR2-SV2
(FIG. 7A-7C (SEQ ID NO:8)), the amino acid sequences of which were
determined by sequencing cloned cDNAs. The TR2 protein shown in FIG. IA-lB
shares sequence homology with the murine CD40 receptor (FIG. 2 (SEQ ID
NO:3)). On February 13, 1995 a deposit was made at the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, and given
accession number 97059. The nucleotide sequence shown in FIG. IA-1B (SEQ
ID NO: I) was obtained by sequencing a cDNA clone (Clone ID HLHAB49)
containing the same amino acid coding sequences as the clone in ATCC Accession
No. 97059 with minor deviation. The cDNA sequence shown in FIG. IA-1B
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(SEQ ID NO:1) differs from that of the ATCC deposit in the 5' and 3' noncoding
nucleotide sequences and three nucleotides.
The clone deposited in ATCC Accession No. 97059 contains 8 nucleotides
5' to the TR2 initiation codon and 21 nucleotides 3' to the TR2 stop codon. In
contrast, the TR2 cDNA sequence of HLHAB49, shown in FIG. 1 A-1 B (SEQ ID
NO: 1), contains considerably longer non-coding nucleotides sequence on both
the
5' and 3' ends of the TR2 coding sequences. Further, the TR2 receptor
nucleotide
sequence shown in FIG. IA-1B (SEQ ID NO:1) contains an adenine at nucleotide
314, a cytosine at nucleotide 386, and a cytosine at nucleotide 627. In
contrast,
the clone of ATCC Accession No. 97059 contains a guanine at nucleotide 314, a
thyrnine at nucleotide 386, and a thymine at nucleotide 627.
The TR2 receptors of the present invention include several allelic variants
containing alterations in at least these three nucleotides and two amino
acids.
Nucleotide sequence variants which have been identified include either guanine
or
adenine at nucleotide 314, thymine or cytosine at nucleotide 386, and thymine
or
cytosine at nucleotide 627 shown in FIG. IA-1B (SEQ ID NO: 1). While the
identified alteration at nucleotide 627 is silent, the alteration at
nucleotide 386
results in the codon at nucleotides 385 to 387 encoding either serine or
phenylalanine and the alteration at nucleotide 314 .results in the codon at
nucleotides 313 to 315 encoding either lysine or arginine.
The nucleotide sequences shown in FIG. 4A-4C (SEQ ID NO:4) and FIG.
7A-7C (SEQ ID NO:7) were also obtained by sequencing cDNA clones deposited
on February 13, 1995 at the American Type Culture Collection and given
accession numbers 97058 (TR2-SVI) and 97057 (TR2-SV2), respectively, The
deposited clones are contained in the pBluescript SK(-) plasmid (Stratagene,
LaJolla, CA).
As used herein the phrase "splice variant" refers to cDNA molecules
produced from a RNA molecules initially transcribed from the same genomic DNA
sequence which have undergone alternative RNA splicing. Alternative RNA
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splicing occurs when a primary RNA transcript undergoes splicing, generally
for
`
the removal of introns, which results in the production of more than one m-RNA
molecule each of which may encode different amino acid sequences. The term
"splice variant" also refers to the proteins encoded by the above cDNA
molecules.
As used herein, "TR2 proteins", 11TR2 receptors", "TR2 receptor proteins"
and "TR2 polypeptides" refer to all proteins resulting from the alternate
splicing
of the genomic DNA sequences encoding proteins having regions of amino acid
sequence identity and receptor activity which correspond to the proteins shown
in FIG. I A-113 (SEQ ID NO:2), FIG. 4A-4C (SEQ ID NO:5) or FIG. 7A-7C
(SEQ ID NO:8). The TR2 protein shown in FIG. IA-1B, the TR2-SVI protein
shown FIG. 4A-4C and the TR2-SV2 protein shown in FIG. 7A-7C are examples
of such receptor proteins.
Nucleic Acid Molecules
Unless otherwise indicated, all nucleotide sequences determined by
sequencing a DNA molecule herein were determined using an automated DNA
sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino
acid sequences of polypeptides encoded by DNA molecules determined herein
were predicted by translation of a DNA sequence determined as above.
Therefore, as is known in the art for any DNA sequence determined by this
automated approach, any nucleotide sequence determined herein may contain
some errors. Nucleotide sequences determined by automation are typically at
least
about 90% identical, more typically at least about 95% to at least about 99.9%
identical to the actual nucleotide sequence of the sequenced DNA molecule. The
actual sequence can be more precisely determined by other approaches including
manual DNA sequencing methods well known in the art. As is also known in the
art, a single insertion or deletion in a determined nucleotide sequence
compared
to the actual sequence will cause a frame shift in translation of the
nucleotide
sequence such that the predicted amino acid sequence encoded by a determined
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nucleotide sequence will be completely different from the amino acid sequence
actually encoded by the sequenced DNA molecule, beginning at the point of such
an insertion or deletion.
Using the information provided herein, such as the nucleotide sequence in
FIG. 1 A-1 B, FIG. 4A-4C or FIG. 7A-7C, nucleic acid molecules of the present
invention encoding TR2 polypeptides may be obtained using standard cloning and
screening procedures, such as those used for cloning cDNAs using mRNA as,
starting material. Illustrative of the invention, the nucleic acid molecule
described
in FIG. IA-1B (SEQ ID NO:1) was discovered in a cDNA library derived from
activated human T-lymphocytes. The nucleic acid molecules described in FIG.
4A-4C (SEQ ID NO:4) and FIG. 7A-7C (SEQ ID NO:7) were discovered in
cDNAs library derived from human fetal heart and human stimulated monocytes,
respectively.
As described in Example 6, TR2 mRNA was detected in numerous tissues
including lung, spleen and thymus and may be ubiquitously expressed in human
cells. TR2 RNA was also found to be expressed in B lymphocytes (CD 19+), both
CD4 ' (TH, and THZ clones) and CD8' T lymphocytes, monocytes and endothelial
cells.
As also noted in Example 6, the production of TR2 mRNA was inducible
in MG 63 cells by TNFa. Further, the accumulation of TR2 mRNA was observed
in HL60, U937 and THP 1 cells upon PMA or DMSO treatment. PMA. and
DMSO are agents known to induce differentiation of these three cell types.
The determined nucleotide sequence of the TR2 cDNA of FIG. 1 A-IB
(SEQ ID NO: 1) contains an open reading frame encoding a protein of about 283
amino acid residues, with a predicted leader sequence of about 36 amino acid
residues, and a deduced molecular weight of about 30,417 kDa. The amino acid
sequence of the predicted mature TR2 receptor is shown in FIG. l A- l B from
amino acid residue about 37 to residue about 283 (amino acid residues I to 247
in SEQ ID NO:2). As noted in Example 6, the location of the leader sequence
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cleavage site was confirmed for a TR2-Fc fusion protein and found to be
between
amino acids 36 and 37 shown in FIG. lA-lB (amino acid residues -1 to 1 in SEQ
ID NO:2). The TR2 protein shown in FIG. IA-1B (SEQ ID NO:2) is about 29%
identical and about 47% similar to the murine CD40 protein shown in SEQ ID
NO:3 (see FIG. 2).
Similarly, the determined cDNA nucleotide sequences of the TR2-SVI
splice variant of TR2 (FIG. 4A-4C (SEQ ID NO:4)) contains an open reading
frame encoding a protein of about 185 amino acid residues, with a predicted
leader sequence of about 36 amino acid residues, and a deduced molecular
weight
of about 19.5 kDa. The amino acid sequence of the predicted mature TR2-SV I
receptor is shown in FIG. 4A-4C (SEQ ID NO:5) from amino acid residue about
37 to residue about 185 (amino acid residues 1 to 149 in (SEQ ID NO:5). The
TR2-SVI protein shown in FIG. 4A-4C (SEQ ID NO:5) is about 25% identical
and about 48% similar to the human type 2 TNF receptor protein shown in SEQ
ID NO:6 (see FIG. 5).
The determined cDNA nucleotide sequences of the TR2-SV2 splice
variant of TR2 (FIG. 7A-7C (SEQ ID NO:7)) contains an open reading frame
encoding a protein of about 136 amino acid residues, without a predicted
leader
sequence, and a deduced molecular weight of about 14 kDa. The amino acid
sequence of the predicted TR2-SV2 receptor is shown in FIG. 7A-7C (SEQ ID
NO:8) from amino acid residue about I to residue about 136. The TR2-SV2
protein shown in FIG. 7A-7C (SEQ ID NO:8) is about 27% identical and about
45% similar to the human type 2 TNF receptor protein shown in SEQ ID NO: 9
(see FIG. 8).
A comparison of both the nucleotide and amino acid sequences of the
TR2, TR2-SVI and TR2-SV2 receptor proteins shown in FIG. IA-IB, FIG. 4A-
4C and FIG. 7A-7C shows several regions of near identity. While the amino acid
sequence of the TR2 receptor protein, shown in FIG. IA-lB (SEQ ID NO:2), is
about 60% identical and about 73% similar to the amino acid sequence of the
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TR2-SVl receptor protein, shown in FIG. 4A-4C (SEQ ID NO:5), in
approximately the first one hundred amino acids of their respective sequences
the
two proteins differ in one location (FIG. 10).
Similarly, the amino acid sequence of the TR2 receptor protein of FIG.
1A-1B (SEQ ID NO:2) is about 60% identical and about 71% similar to the amino
acid sequence of the TR2-SV2 receptor protein, shown in FIG. 7A-7C (SEQ ID
NO:8); however, the two proteins are almost identical over a 60 amino acid
=`? stretch in the central portion of the TR2-SV2 protein (FIG. 11).
In contrast, the TR2-SV1 and TR2-SV2 proteins are only about 20%
identical and about 38% similar at the amino acid level to each other. Unlike
the
comparisons of either of these proteins to the TR2 protein shown in FIG. 1 A-1
B
(SEQ ID NO:2), these proteins share their homology over the entire 136 amino
acid sequence of the TR2-SV2 protein (FIG. 12).
With respect to their nucleotide sequences of the cDNAs encoding the
disclosed TR2 proteins, a comparison of these sequences indicates that the TR2
cDNAs share large regions of near identity at the nucleic acid level (FIG, 13A-
13C, FIG. 14A-14D and FIG. 15A-15E). The cDNA sequences encoding the
TR2 and TR2-SV1 proteins, for example, share large regions of near identity in
their nucleotide sequences which encode both the N termini of the respective
proteins and their 5' and 3' noncoding regions (FIG. 13A-13C). Further, the
nucleotide sequences of the cDNAs encoding the TR2-SV1 and TR2-SV2
proteins share considerable homology but this identity is limited to their 3'
regions
well beyond their respective coding sequences (FIG. 15A-15E).
Such regions of near identity between two different cDNA sequences,
when maintained over an extended stretch of sequence, indicates to one skilled
in
the art that the respective molecules were originally transcribed from the
same
genon-iic DNA sequence. One skilled in the art would further recognize that,
since
more than one codon can encode the same amino acid, identity between two
proteins at the amino acid level does not necessarily mean that the DNA
sequences
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encoding those proteins will share similar regions of identity. The above data
indicates that the TR2 receptors of the present invention are transcribed from
a
single genomic DNA sequence and represent multiple splice variants of one
initial
RNA transcript.
Related proteins which are produced from alternately spliced RNA,
referred to as splice variants, are known in the art. The transcript of the
src gene,
for example, undergoes alternate RNA splicing to produce cell type specific
products. In most cells the Src protein consists of 533 amino acids while in
nerve
cells an additional short exon is included in the mRNA resulting in a protein
of 539
amino acids. See Alberts, B. et al., MOLECULAR BIOLOGY OF THE CELL (3rd
Edition, Garland Publishing, Inc., 1994), 455. Similarly, sex specific mRNA
transcripts have been identified in Drosophila where alternate mRNA splicing
results in a protein named Dsx which is approximately 550 amino acids in
length
in males and 430 amino acids in length in females. These two splice variant
proteins share a common core sequence of about 400 amino acids. See id at 457.
In the present instance, the TR2 receptor protein shown in FIG. lA-lB
(SEQ ID NO:2) is believed to be the full-length polypeptide encoded by the RNA
from which the TR2 receptor proteins are translated. The RNA encoding the
TR2-SVI splice variant shown in FIG. 4A-4B (SEQ ID NO:5) is believed to
contain an insertion in the region encoding amino acid residue 102 of the
amino
acid sequence shown in FIG. lA-lB and a deletion in the region encoding amino
acid residue 184 of the amino acid sequence shown in FIG. I A-1 B. The RNA
encoding the TR2-SV2 splice variant shown in FIG. 7A-7C is believed to begin
with the nucleotide sequence encoding amino acid residue 102 of the amino acid
sequence shown in FIG. IA-lB and contain insertions in the regions encoding
amino acid residues 184 and 243 of the amino acid sequence shown in FIG. 1 A-
1 B.
As indicated, the present invention also provides the mature forms of the
TR2 receptors of the present invention. According to the signal hypothesis,
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proteins secreted by mammalian cells have a signal or secretory leader
sequence
which is cleaved from the mature protein once export of the growing protein
chain
across the rough endoplasmic reticulum has been initiated. Most mammalian
cells
and even insect cells cleave secreted proteins with the same specificity.
However,
in some cases, cleavage of a secreted protein is not entirely uniform, which
results
in two or more mature species on the protein. Further, it has long been known
that the cleavage specificity of a
secreted protein is ultimately determined by the
primary structure of the complete protein, that is, it is inherent in the
amino acid
sequence of the polypeptide. Therefore, the present invention provides
nucleotide
sequences encoding mature TR2 polypeptides having the amino acid sequences
encoded by the cDNA clones contained in the host identified as ATCC Deposit
Numbers 97059 and 97058 and as shown in FIG. IA-1B (SEQ ID NO:2) and
FIG. 4A-4C (SEQ ID NO:5). By the mature TR2 polypeptides having the amino
acid sequences encoded by the cDNA clones contained in the host identified as
ATCC Deposit Numbers 97059 and 97058 is meant the mature form(s) of the
TR2 receptors produced by expression in a mammalian cell (e.g., COS cells, as
described below) of the complete open reading frame encoded by the human DNA
sequence of the clone contained in the vector in the deposited host.
The invention also provides nucleic acid sequences encoding the TR2-SV2
receptor protein of FIG. 7A-7C (SEQ ID NO:8), having the amino acid sequence
encoded by the cDNA clone contained in ATCC Deposit Number 97057, which
does not contain a secretory leader sequence.
Methods for predicting whether a protein has a secretory leader as well as
the cleavage point for that leader sequence are available. For instance, the
methods of McGeoch (Virus Res. 3:271-286 (1985)) and von Heinje (Nucleic
Acids Res. 14:4683-4690 (1986)) can be used. The accuracy of predicting the
cleavage points of known mammalian secretory proteins for each of these
methods
is in the range of 75-80%. von Heinje, supra. However, the two methods do not
always produce the same predicted cleavage point(s) for a given protein.
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In the present case, the predicted amino acid sequences of the complete
TR2 polypeptides shown in FIG. lA-lB (SEQ ID NO:2), FIG. 4A-4C (SEQ ID
NO:5) and FIG. 7A-7C (SEQ ID NO:8) were analyzed by a computer program
("PSORT") (K. Nakai and M. Kanehisa, Genomics 14:897-911 (1992)), which is
an expert system for predicting the cellular location of a protein based on
the
amino acid sequence. As part of this computational prediction of localization,
the
methods of McGeoch and von Heinje are incorporated. The analysis by the
PSORT program predicted the cleavage sites between amino acids -1 and 1 in
SEQ ID NO:2 and SEQ ID NO:5. Thereafter, the complete amino acid sequences
were further analyzed by visual inspection, applying a simple form of the (-1,-
3)
rule of von Heine. von Heinje, supra. Thus, the leader sequences for the TR2
protein shown in SEQ ID NO:2 and the TR2-SV1 protein are predicted to consist
of amino acid residues -36 to -1 in both SEQ ID NO:2 and SEQ ID NO:5, while
the predicted mature TR2 proteins consist of amino acid residues I to 247 for
the
TR2 protein shown in SEQ ID NO:2 and residues I to 149 for the TR2-S V I
protein shown in SEQ ID NO:5.
As noted in Example 6, the cleavage site of the leader sequence of a TR2-
Fc fusion protein was confirmed using amino acid analysis of the expressed
fusion
protein. This fusion protein was found to begin at amino acid 37, which
corresponds to amino acid I in SEQ ID NO:2 and SEQ ID NO:5, indicating that
the cleavage site of the leader sequence is between amino acids 36 and 37 in
this
protein (corresponding to amino acid residues -1 to I in SEQ ID NO:2 and SEQ
ID NO:5).
As one of ordinary skill would appreciate, however, due to the possibilities
of sequencing errors, as well as the variability of cleavage sites for leaders
in
different known proteins, the TR2 receptor polypeptide encoded by the cDNA of
ATCC Deposit Number 97059 comprises about 283 amino acids, but may be
anywhere in the range of 250 to 316 amino acids; and the leader sequence of
this
protein is about 36 amino acids, but may be anywhere in the range of about 30
to
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about 42 amino acids. Similarly, the TR2-SV1 receptor polypeptide encoded by
the cDNA of ATCC Deposit Number 97058 comprises about 185 amino acids,
but may be anywhere in the range of 163-207 amino acids; and the leader
sequence of this protein is about 36 amino acids, but may be anywhere in the
range of about 30 to about 42 amino acids. Further, the TR2-SV2 receptor
polypeptide encoded by the cDNA of ATCC Deposit Number 97057 comprises
about 136 amino acids, but may be anywhere in the range of 120-152 amino acids
As indicated, nucleic acid molecules of the present invention may be in the
form of RNA, such as mRNA, or in the form of DNA, including, for instance,
cDNA and genomic DNA obtained by cloning or produced synthetically. The
DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA
may be the coding strand, also known as the sense strand, or it may be the
non-coding strand, also referred to as the anti-sense strand.
By "isolated" nucleic acid molecule(s) is intended a nucleic acid molecule,
DNA or RNA, which has been removed from its native environment For example,
recombinant DNA molecules contained in a vector are considered isolated for
the
purposes of the present invention. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution. Isolated RNA
molecules include in vivo or in vitro RNA transcripts of the DNA molecules of
the
present invention. Isolated nucleic acid molecules according to the present
invention further include such molecules produced synthetically.
Isolated nucleic acid molecules of the present invention include DNA
molecules comprising an open reading frame (ORF) shown in FIG. lA-1B (SEQ
ID NO: 1); DNA molecules comprising the coding sequence for the mature TR2
receptor shown in FIG. lA-1B (SEQ IDNO:2) (last 247 amino acids); and DNA
molecules which comprise a sequence substantially different from those
described
above but which, due to the degeneracy of the genetic code, still encode the
TR2
receptor protein shown in FIG. IA-1B (SEQ ID NO:2). Of course, the genetic
CA 02270913 2001-01-04
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code is well known in the art. Thus, it would be routine for one skilled in
the art
to generate such degenerate variants.
Similarly, isolated nucleic acid molecules of the present invention include
DNA molecules comprising an open reading frame (ORF) shown in FIG. 4A-4C
(SEQ ID NO:4); DNA molecules comprising the coding sequence for the mature
TR2-SVI receptor shown in FIG. 4A-4C (SEQ ID NO:5) (last 149 amino acids);
and DNA molecules which comprise a sequence substantially different from those
described above but which, due to the degeneracy of the genetic code, still
encode
the TR2-SV1 receptor.
Further, isolated nucleic acid molecules of the present invention include
DNA molecules comprising an open reading frame (ORF) shown in FIG. 7A-7C
(SEQ ID NO:7) and DNA molecules which comprise a sequence substantially
different from those described above but which, due to the degeneracy of the
genetic code, still encode the TR2-SV2 receptor.
In another aspect, the invention provides isolated nucleic acid molecules
encoding the TR2, TR2-SV1 and TR2-SV2 polypeptides having the amino acid
sequences encoded by the cDNA clones contained in the plasmid deposited as
ATCC Deposit No. 97059, 97058 and 97057, respectively, on February 13, 1995.
In a further embodiment, these nucleic acid molecules will encode a mature
polypeptide or thefull-length polypeptide lacking the N-terminal methionine.
The
invention further provides isolated nucleic acid molecules having the
nucleotide
sequences shown in FIG. lA-1B (SEQ ID NO:1), FIG. 4A-4C (SEQ ID NO:4),
and FIG. 7A-7C (SEQ ID NO:7); the nucleotide sequences of the cDNAs
contained in the above-described deposited clones; or nucleic acid molecules
having a sequence complementary to one of the above sequences. Such isolated
molecules, particularly DNA molecules, are useful as probes for gene mapping,
by
in situ hybridization with chromosomes, and for detecting expression of the
TR2
receptor genes of the present invention in human tissue, for instance, by
Northern
blot analysis.
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I
The present invention is further directed to fragments of the isolated
nucleic acid molecules described herein. By a fragment of an isolated nucleic
acid
molecule having the nucleotide sequence of the deposited cDNAs or the
nucleotide sequence shown in FIG. lA-1B (SEQ ID NO:1), FIG. 4A-4C (SEQ ID
NO:4), or FIG. 7A-7C (SEQ ID NO:7) is intended fragments at least about 15 nt,
and more preferably at least about 20 nt, still more preferably at least about
30 nt,
and even more preferably, at least about 40 nt in length which are useful as
diagnostic probes and primers as discussed herein. Of course, larger fragments
50-400 nt in length are also useful according to the present invention as are
fragments corresponding to most, if not all, of the nucleotide sequences of
the
deposited cDNAs or as shown in FIG. IA-IB (SEQ ID NO: 1), FIG. 4A-4C (SEQ
ID NO:4), or FIG. 7A-7C (SEQ ID NO:7). By a fragment at least 20 nt in length,
for example, is intended fragments which include 20 or more contiguous bases
from the nucleotide sequences of the deposited cDNAs or the nucleotide
sequences as shown in FIG. 1A-1B (SEQ ID NO:1), FIG. 4A-4C (SEQ ID
NO:4), or FIG. 7A-7C (SEQ ID NO:7).
Preferred nucleic acid fragments of the present invention include nucleic
acid molecules encoding: a polypeptide comprising the TR2 receptor protein of
FIG. IA-113 (SEQ ID NO:2) extracellular domain (predicted to constitute amino
acid residues from about 37 to about 200 in FIG. 1A-lB (amino acid residues 1
to 164 in SEQ ID NO:2)); a polypeptide comprising the TR2 receptor
transmembrane domain (amino acid residues 201 to 225 in FIG. I A-IB (amino
=? acid residues 165 to 189 in SEQ ID NO:2)); a polypeptide comprising the TR2
receptor intracellular domain (predicted to constitute amino acid residues
from
about 226 to about 283 in FIG. IA-1B (amino acid residues 190 to 247 in SEQ
ID NO:2)); and a polypeptide comprising the TR2 receptor protein of FIG. lA-IB
(SEQ ID NO:2) extracellular and intracellular domains with all or part of the
transmembrane domain deleted.
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Preferred nucleic acid fragments of the present invention also include
nucleic acid molecules encoding polypeptides comprising the mature TR2-SV1
receptor (predicted to constitute amino acid residues from about 37 to about
185
in FIG. 4A-4C (amino acid residues I to 149 in SEQ ID NO:5)) and the complete
TR2-SV2 receptor (predicted to constitute amino acid residues from about I to
about 136 in FIG. 7A-7C (SEQ ID NO: 8)).
As above with the leader sequence, the amino acid residues constituting
the extracellular, transmembrane and intracellular domains have been predicted
by
computer analysis. Thus, as one of ordinary skill would appreciate, the amino
acid
residues constituting these domains may vary slightly (e.g., by about I to
about 15
amino acid residues) depending on the criteria used to define each domain.
Preferred nucleic acid fragments of the present invention also include
nucleic acid molecules encoding epitope-bearing portions of the TR2 receptor
proteins. In particular, such nucleic acid fragments of the present invention
include nucleic acid molecules encoding: a polypeptide comprising amino acid
residues from about 39 to about 70 in FIG. IA-1B (amino acid residues 3 to 34
in SEQ ID NO:2); a polypeptide comprising amino acid residues from about 106
to about 120 in FIG, I (amino acid residues 70 to 84 in SEQ ID NO:2); a
polypeptide comprising amino acid residues from about 142 to about 189 in FIG.
1 A- l B (amino acid residues 106 to 153 in SEQ ID NO:2); a polypeptide
comprising amino acid residues from about 276 to about 283 in FIG. 1A-1B
(amino acid residues 240 to 247 in SEQ ID NO:2); a polypeptide comprising
amino acid residues from about 39 to about 70 in FIG. 4A-4C (amino acid
residues 3 to 34 in SEQ ID NO:5); amino acid residues from about 99 to about
136 in FIG. 4A-4C (amino acid residues 63 to 100 in SEQ ID NO:5); amino acid
residues from about 171 to about 185 in FIG. 4A-4C (amino acid residues 135 to
149 in SEQ ID NO:5); amino acid residues from about 56 to about 68 in FIG. 7A-
7C (SEQ ID NO:8); amino acid residues from about 93 to about 136 in FIG. 7A-
7C (SEQ ID NO:8). The inventors have determined that the above polypeptide
CA 02270913 2001-01-04
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fragments are antigenic regions of the TR2 receptors. Methods for determining
other such epitope-bearing portions of the TR2 proteins are described in
detail
below.
In another aspect, the invention provides isolated nucleic acid molecules
comprising polynucleotides which hybridizes under stringent hybridization
conditions to a portion of the polynucleotide of one of the nucleic acid
molecules
of the invention described above, for instance, the cDNA clones contained in
ATCC Deposits 97059, 97058 and 97057. By "stringent hybridization conditions"
is intended overnight incubation at 42 C in a solution comprising: 50%
formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium
phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/ml
denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1x
SSC at about 65 C.
By a polynucleotide which hybridizes to a "portion" of a polynucleotide
is intended a polynucleotide (either DNA or RNA) hybridizing to at least about
15 nucleotides (nt), and more preferably at least about 20 nt, still more
preferably
at least about 30 nt, and even more preferably about 30-70 nt of the reference
polynucleotide. These are useful as diagnostic probes and primers as discussed
above and in more detail below.
By a portion of a polynucleotide of "at least 20 nt in length," for example,
is intended 20 or more contiguous nucleotides from the nucleotide sequence of
the
reference polynucleotide (e.g., the deposited cDNAs or the nucleotide
sequences
as shown in FIG. I A- 113 (SEQ ID NO: 1), FIG. 4A-4C (SEQ ID NO:4), or FIG.
7A-7C (SEQ ID NO:7)).
Of course, a polynucleotide which hybridizes only to a poly A sequence
(such as the 3' terminal poly(A) tract of the TR2 receptor cDNA sequences
shown
in FIG. I A-1B (SEQ ID NO:1), FIG. 4A-4C (SEQ ID NO:4), or FIG. 7A-7C
(SEQ ID NO:7)), or to a complementary stretch of T (or U) resides, would not
be included in a polynucleotide of the invention used to hybridize to a
portion of
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a nucleic acid of the invention, since such a polynucleotide would hybridize
to any
nucleic acid molecule containing a poly (A) stretch or the complement thereof
(e.g., practically any double-stranded cDNA clone).
As indicated, nucleic acid molecules of the present invention which encode
TR2 polypeptides may include, but are not limited to those encoding the amino
acid sequences of the mature polypeptides, by itself, the coding sequence for
the
mature polypeptides and additional sequences, such as those encoding the about
36 amino acid leader or secretory sequences, such as pre-, or pro- or prepro-
protein sequences; the coding sequence of the mature polypeptides, with or
without the aforementioned additional coding sequences, together with
additional,
non-coding sequences, including for example, but not limited to introns and
non-coding 5' and 3' sequences, such as the transcribed, non-translated
sequences
that play a role in transcription, mRNA processing, including splicing and
polyadenylation signals, for example - ribosome binding and stability of mRNA;
an additional coding sequence which codes for additional amino acids, such as
those which provide additional functionalities. Thus, the sequence encoding
the
polypeptides may be fused to a marker sequence, such as a sequence encoding a
peptide which facilitates purification of the fused polypeptide. In certain
preferred
embodiments of this aspect of the invention, the marker amino acid sequence is
a
hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen,
Inc.),
among others, many of which are commercially available. As described in Gentz
et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-
histidine
provides for convenient purification of the fusion protein. The "HA" tag is
another peptide useful for purification which corresponds to an epitope
derived
from the influenza hemagglutinin protein, which has been described by Wilson
et
al., Cell 37: 767 (1984). As discussed below, other such fusion proteins
include
the TR2 receptors fused to IgG-Fc at the N- or C-terminus.
The present invention further relates to variants of the nucleic acid
molecules of the present invention, which encode portions, analogs or
derivatives
CA 02270913 2001-01-04
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of the TR2 receptors. Variants may occur naturally, such as a natural allelic
variant. By an "allelic variant" is intended one of several alternate forms of
a gene
occupying a given locus on a chromosome of an organism. Genes 11, Lewin, B.,
ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may
be produced using art-known mutagenesis techniques.
Such variants include those produced by nucleotide substitutions, deletions
or additions, which may involve one or more nucleotides. The variants may be
altered in coding regions, non-coding regions, or both. Alterations in the
coding
regions may produce conservative or non-conservative amino acid substitutions,
deletions or additions. Especially preferred among these are silent
substitutions,
additions and deletions, which do not alter the properties and activities of
the TR2
receptors or portions thereof. Also especially preferred in this regard are
conservative substitutions.
Further embodiments of the invention include isolated nucleic acid
molecules comprising a polynucleotide having a nucleotide sequence at least
90%
identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to
(a) a nucleotide sequence encoding the TR2 polypeptide having the complete
amino acid sequence shown in FIG. IA-113 (amino acid residues -36 to 247 in
SEQ ID NO:2), FIG. 4A-4C (amino acid residues -36 to 149 in SEQ ID NO:5),
or FIG. 7A-7C (amino acid residues I to 136 in SEQ ID NO:8); (b) a nucleotide
encoding the complete amino sequence shown in FIG. 1A-lB (amino acid residues
-35 to 247 in SEQ ID NO:2), FIG. 4A-4C (amino acid residues -35 to 149 in SEQ
ID NO:5), or FIG. 7A-7C (amino acid residues 2 to 136 in SEQ ID NO:8) but
lacking the N-terminal methionine; (c) a nucleotide sequence encoding the
mature
TR2 receptors (full-length polypeptide with any attending leader sequence
removed) having the amino acid sequence at positions from about 37 to about
283
in FIG. IA-1B (amino acid residues 1 to 247 in SEQ ID NO:2) or the amino acid
sequence at positions from about 37 to about 185 in FIG. 4A-4C (amino acid
residues I to 149 in SEQ ID NO:5), or the amino acid sequence at positions
from
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about 1 to about 136 in FIG. 7A-7C (SEQ ID NO:8); (d) a nucleotide sequence
encoding the TR2, TR2-SVI or TR2-SV2 polypeptides having the complete
amino acid sequence including the leader encoded by the cDNA clones contained
in ATCC Deposit Numbers 97059, 97058, and 97057, respectively; (e) a
nucleotide sequence encoding the mature TR2 or TR2-SVI receptors having the
amino acid sequences encoded by the cDNA clones contained in ATCC Deposit
Numbers 97059 and 97058, respectively; (f) a nucleotide sequence encoding the
TR2 or TR2-SV 1 receptor extracellular domain; (g) a nucleotide sequence
encoding the TR2 receptor transmembrane domain ; (h) a nucleotide sequence
encoding the TR2 receptor intracellular domain; (i) a nucleotide sequence
encoding the TR2 receptor extracellular and intracellular domains with all or
part
of the transmembrane domain deleted; and (j) a nucleotide sequence
complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e),
(f), (g),
(h), or (i).
By a polynucleotide having a nucleotide sequence at least, for example,
95% "identical" to a reference nucleotide sequence encoding a TR2 polypeptide
is intended that the nucleotide sequence of the polynucleotide is identical to
the
reference sequence except that the polynucleotide sequence may include up to
five
point mutations per each 100 nucleotides of the reference nucleotide sequence
encoding the TR2 receptors. In other words, to obtain a polynucleotide having
a nucleotide sequence at least 95% identical to a reference nucleotide
sequence,
up to 5% of the nucleotides in the reference sequence may be deleted or
substituted with another nucleotide, or a number of nucleotides up to 5% of
the
total nucleotides in the reference sequence may be inserted into the reference
sequence. These mutations of the reference sequence may occur at the 5' or 3'
terminal positions of the reference nucleotide sequence or anywhere between
those terminal positions, interspersed either individually among nucleotides
in the
reference sequence or in one or more contiguous groups within the reference
sequence.
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As a practical matter, whether any particular nucleic acid molecule is at
least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the
nucleotide
sequence shown in FIG. 1A-IB (SEQ ID NO: 1) or to the nucleotides sequence
of the deposited cDNA clone encoding that protein can be determined
conventionally using known computer programs such as the BestfitTprogram
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group, University Research Park, 575 Science Drive, Madison, WI 53711.
Bestfit uses the local homology algorithm of Smith and Waterman, Advances in
Applied Mathematics 2: 482-489 (1981), to find the best segment of homology
between two sequences. When using Bestfit or any other sequence alignment
program to determine whether a particular sequence is, for instance, 95%
identical
to a reference sequence according to the present invention, the parameters are
set,
of course, such that the percentage of identity is calculated over the full
length of
the reference nucleotide sequence and that gaps in homology of up to 5% of the
total number of nucleotides in the reference sequence are allowed.
The present application is directed to nucleic acid molecules at least 90%,
95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences shown in
FIG. IA-1B (SEQ ID NO:1), FIG. 4A-4C (SEQ ID NO:4), or FIG. 7A-7C (SEQ
ID NO:7) or to the nucleic acid sequence of the deposited cDNAs, irrespective
of
whether they encode a polypeptide having TR2 receptor activity. This is
because
even where a particular nucleic acid molecule does not encode a polypeptide
having TR2 receptor activity, one of skill in the art would still know how to
use
the nucleic acid molecule, for instance, as a hybridization probe or a
polymerase
chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present
invention that do not encode a polypeptide having TR2 receptor activity
include,
inter alia, (1) isolating a TR2 receptor gene or allelic or splice variants
thereof in
a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase
chromosomal spreads to provide precise chromosomal location of a TR2 receptor
gene, as described in Verma et al., Human Chromosomes: A Manual of Basic
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Techniques, Pergamon Press, New York (1988), and (3) Northern Blot analysis
for detecting TR2 receptor mRNA expression in specific tissues.
Preferred, however, are nucleic acid molecules having sequences at least
90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown
in FIG. IA-1B (SEQ ID NO:1), FIG. 4A-4C (SEQ ID NO:4), or FIG. 7A-7C
(SEQ ID NO:7) or to the nucleic acid sequence of the deposited cDNAs which
do, in fact, encode a polypeptide having TR2 receptor activity. By "a
polypeptide
having TR2 receptor activity" is intended polypeptides exhibiting activity
similar,
but not necessarily identical, to an activity of the TR2 receptors of the
present
invention (either the full-length protein, the splice variants, or,
preferably, the
mature protein), as measured in a particular biological assay. For example,
TR2
receptor activity can be measured by determining the ability of a polypeptide-
Fc
fusion protein to inhibit lymphocyte proliferation as described below in
Example
6. TR2 receptor activity may also be measured by determining the ability of a
polypeptide, such as cognate ligand which is free or expressed on a cell
surface,
to confer proliferatory activity in intact cells expressing the receptor.
Of course, due to the degeneracy of the genetic code, one of ordinary skill
in the art will immediately recognize that a large number of the nucleic acid
molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99%
identical to the nucleic acid sequence of the deposited cDNAs or the nucleic
acid
sequences shown in FIG. IA-113 (SEQ ID NO:1), FIG.4A-4C (SEQ ID NO:4)
or FIG. 7A-7C (SEQ ID NO:7) will encode polypeptides "having TR2 receptor
activity." In fact, since degenerate variants of any of these nucleotide
sequences
all encode the same polypeptide, this will be clear to the skilled artisan
even
without performing the above described comparison assay. It will be further
recognized in the art that, for such nucleic acid molecules that are not
degenerate
variants, a reasonable number will also encode a polypeptide having TR2
protein
activity. This is because the skilled artisan is fully aware of amino acid
substitutions that are either less likely or not likely to significantly
effect protein
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function (e.g., replacing one aliphatic amino acid with a second aliphatic
amino
acid).
For example, guidance concerning how to make phenotypically silent
amino acid substitutions is provided in Bowie, J. U. et al., "Deciphering the
Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science
247:1306-1310 (1990), wherein the authors indicate that proteins are
surprisingly
tolerant of amino acid substitutions.
Vectors and Host Cells
The present invention also relates to vectors which include the isolated
DNA molecules of the present invention, host cells which are genetically
engineered with the recombinant vectors, and the production of TR2
polypeptides
or fragments thereof by recombinant techniques.
The polynucleotides may be joined to a vector containing a selectable
marker for propagation in a host. Generally, a plasmid vector is introduced in
a
precipitate, such as a calcium phosphate precipitate, or in a complex with a
charged lipid. If the vector is a virus, it may be packaged in vitro using an
appropriate packaging cell line and then transduced into host cells.
The DNA insert should be operatively linked to an appropriate promoter,
such as the phage lambda PL promoter, the E. coli lac, trp and lac promoters,
the
SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
Other suitable promoters will be known to the skilled artisan. The expression
constructs will further contain sites for transcription initiation,
termination and, in
the transcribed region, a ribosome binding site for translation. The coding
portion
of the mature transcripts expressed by the constructs will preferably include
a
translation initiating at the beginning and a termination codon (UAA, UGA or
UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one
selectable marker. Such markers include dihydrofolate reductase or neomycin
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resistance for eukaryotic cell culture and tetracycline or ampicillin
resistance genes
for culturing in E. coli and other bacteria. Representative examples of
appropriate
heterologous hosts include, but are not limited to, bacterial cells, such as
E. coli,
Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast
cells;
insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such
as
CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture
mediums and conditions for the above-described host cells are known in the
art.
Among vectors preferred for use in bacteria include pQE70, pQE60 and
pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript
vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and
ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1
and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL
available from Pharmacia. Other suitable vectors will be readily apparent to
the
skilled artisan.
Introduction of the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-dextran mediated transfection, cationic
lipid-mediated transfection, electroporation, transduction, infection or other
methods. Such methods are described in many standard laboratory manuals, such
as Davis et al., Basic Methods In Molecular Biology (1986).
The polypeptide may be expressed in a modified form, such as a fusion
protein, and may include not only secretion signals, but also additional
heterologous functional regions. For instance, a region of additional amino
acids,
particularly charged amino acids, may be added to the N-terminus of the
polypeptide to improve stability and persistence in the host cell, during
purification, or during subsequent handling and storage. Also, peptide
moieties
may be added to the polypeptide to facilitate purification. Such regions may
be
removed prior to final preparation of the polypeptide. The addition of peptide
moieties to polypeptides to engender secretion or excretion, to improve
stability
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and to facilitate purification, among others, are familiar and routine
techniques in
the art. A preferred fusion protein comprises a heterologous region from
immunoglobulin that is useful to solubilize proteins. For example, EP-A-O 464
533 (Canadian counterpart 2045869) discloses fusion proteins comprising
various
portions of constant region of immunoglobin molecules together with another
human protein or part thereof In many cases, the Fc part in a fusion protein
is
thoroughly advantageous for use in therapy and diagnosis and thus results, for
example, in improved pharmacokinetic properties (EP-A 0232 262). On the other
hand, for some uses it would be desirable to be able to delete the Fc part
after the
fusion protein has been expressed, detected and purified in the advantageous
manner described. This is the case when Fc portion proves to be a hindrance to
use in therapy and diagnosis, for example when the fusion protein is to be
used as
antigen for immunizations. In drug discovery, for example, human proteins,
such
as, human hIL-5 receptor has been fused with Fc portions for the purpose of
high-throughput screening assays to identify antagonists of hIL-5. See, D.
Bennett et al., Journal of Molecular Recognition, Vol. 8:52-58 (1995) and K.
Johanson et al., The Journal of Biological Chemistry, Vol. 270, No.
16:9459-9471 (1995).
TR2 receptors can be recovered and purified from recombinant cell
cultures by well-known methods including ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
affinity chromatography, hydroxylapatite chromatography and lectin
chromatography. Most preferably, high performance liquid chromatography
("HPLC") is employed for purification. Polypeptides of the present invention
include naturally purified products, products of chemical synthetic
procedures, and
products produced by recombinant techniques from a prokaryotic or eukaryotic
host, including, for example, bacterial, yeast, higher plant, insect and
mammalian
cells. Depending upon the host employed in a recombinant production procedure,
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the polypeptides of the present invention may be glycosylated or may be
non-glycosylated. In addition, polypeptides of the invention may also include
an
initial modified methionine residue, in some cases as a result of host-
mediated
processes.
TR2 Polypeptides and Fragments
The invention further provides isolated TR2 polypeptides having the amino
acid sequence encoded by the deposited cDNAs, or the amino acid sequence in
FIG. lA-lB (SEQ ID NO:2), FIG. 4A-4C (SEQ ID NO:5), or FIG. 7A-7C (SEQ
ID NO:8), or a peptide or poiypeptide comprising a portion of the above
polypeptides.
The polypeptides of this invention may be membrane bound or may be in
a soluble circulating form. Soluble peptides are defined by amino acid
sequence
wherein the sequence comprises the polypeptide sequence lacking the
transmembrane domain. One example of such a soluble form of the TR2 receptor
is the TR2-SV1 splice variant which has a secretory leader sequence but lacks
both the intracellular and transmembrane domains. Thus, the TR2-SVI receptor
protein appears to be secreted in a soluble form from cells which express this
protein.
The polypeptides of the present invention may exist as a membrane bound
receptor having a transmembrane region and an intra- and extracellular region
or
they may exist in soluble form wherein the transmembrane domain is lacking.
One
example of such a form of the TR2 receptor is the TR2 receptor shown in FIG.
1 A- l B (SEQ ID NO:2) which contains, in addition to a leader sequence,
{ transmembrane, intracellular and extracellular domains. Thus, this form of
the
TR2 receptor appears to be localized in the cytoplasmic membrane of cells
which
express this protein
It will be recognized in the art that some amino acid sequences of the TR2
receptors can be varied without significant effect to the structure or
function of
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the protein. If such differences in sequence are contemplated, it should be
remembered that there will be critical areas on the protein which determine
activity. Thus, the invention further includes variations of the TR2 receptors
which show substantial TR2 receptor activity or which include regions of TR2
proteins such as the protein portions discussed below, Such mutants include
deletions, insertions, inversions, repeats, and type substitutions. As
indicated
above, guidance concerning which amino acid changes are likely to be
phenotypically silent can be found in Bowie, J. U., et al., "Deciphering the
Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science
2-17. 1306-1310 (1990).
Thus, the fragment, derivative or analog of the polypeptides of FIG. I A-
1 B (SEQ ID NO:2), FIG. 4A-4C (SEQ ID NO:5), and FIG. 7A-7C (SEQ ID
NO:8), or that encoded by the deposited cDNAs, may be (i) one in which one or
more of the amino acid residues are substituted with a conserved or non-
conserved amino acid residue (preferably a conserved amino acid residue) and
such substituted amino acid residue may or may not be one encoded by the
genetic
code, or (ii) one in which one or more of the amino acid residues includes a
substituent group, or (iii) one in which the mature polypeptide is fused with
another compound, such as a compound to increase the half-life of the
polypeptide
(for example, polyethylene glycol), or (iv) one in which the additional amino
acids
are fused to the mature polypeptide, such as an IgG Fc fusion region peptide
or
leader or secretory sequence or a sequence which is employed for purification
of
the mature polypeptide or a proprotein sequence. Such fragments, derivatives
and
analogs are deemed to be within the scope of those skilled in the art from the
teachings herein.
Of particular interest are substitutions of charged amino acids with another
charged amino acid and with neutral or negatively charged amino acids. The
latter
results in proteins with reduced positive charge to improve the
characteristics of
the TR2 proteins. The prevention of aggregation is highly desirable.
Aggregation
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of proteins not only results in a loss of activity but can also be problematic
when
preparing pharmaceutical formulations, because they can be immunogenic.
(Pinckard et al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al.,
Diabetes
36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier systems
10:307-377 (1993)).
The replacement of amino acids can also change the selectivity of binding
to cell surface receptors. Ostade et al., Nature 361:266-268 (1993) describes
certain mutations resulting in selective binding of TNF-a to only one of the
two
known types of TNF receptors. Thus, the TR2 receptors of the present invention
may include one or more amino acid substitutions, deletions or additions,
either
from natural mutations or human manipulation.
As indicated, changes are preferably of a minor nature, such as
conservative amino acid substitutions that do not significantly affect the
folding
or activity of the protein (see Table 1).
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TABLE 1
CONSERVATIVE AMINO ACID SUBSTITUTIONS.
Aromatic Phenylalanine
Tryptophan
Tyrosine
Hydrophobic Leucine
Isoleucine
Valine
Polar Glutamine
Asparagine
Basic Arginine
Lysine
Histidine
Acidic Aspartic Acid
Glutamic Acid
Small Alanine
Serine
Threonine
Methionine
Glycine
Amino acids in the TR2 proteins of the present invention that are essential
for function can be identified by methods known in the art, such as site-
directed
mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science
244:1081-1085 (1989)). The latter procedure introduces single alanine
mutations
at every residue in the molecule. The resulting mutant molecules are then
tested
for biological activity such as receptor binding or in vitro, or in vitro
proliferative
activity. Sites that are critical for ligand-receptor binding can also be
determined
by structural analysis such as crystallization, nuclear magnetic resonance or
photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de
Vos
et al. Science 255:306-312 (1992)).
The polypeptides of the present invention are preferably provided in an
isolated form. By "isolated polypeptide", is intended a polypeptide removed
from
its native environment. Thus, a polypeptide produced and contained within a
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recombinant host cell would be considered "isolated" for purposes of the
present
invention. Also intended as an "isolated polypeptide" are polypeptides that
have
been purified, partially or substantially, from a recombinant host. For
example,
recombinantly produced versions of the TR2 receptors can be substantially
purified by the one-step method described in Smith and Johnson, Gene 67.31-40
(1988).
The polypeptides of the present invention also include the polypeptide
encoded by the deposited cDNAs including the leader; the polypeptide encoded
by the deposited the cDNAs minus the leader (i.e., the mature protein); the
polypeptides of FIG. lA-lB (SEQ ID NO:2) or FIG. 4A-4C (SEQ ID NO:5)
including the leader; the polypeptides of FIG. IA-1B (SEQ ID NO:2) or FIG. 4A
4C (SEQ ID NO:5) including the leader but minus the N-terminal methionine; the
polypeptides of FIG. I A-lB (SEQ ID NO:2) or FIG. 4A-4C (SEQ ID NO:5)
minus the leader; the polypeptide of FIG. 7A-7C (SEQ ID NO:8); the
extracellular
domain, the transmembrane domain, and the intracellular domain of the TR2
receptor shown in FIG. IA-1B (SEQ ID NO:2); and polypeptides which are at
least 80% identical, more preferably at least 90% or 95% identical, still more
preferably at least 96%, 97%, 98% or 99% identical to the polypeptides
described
above, and also include portions of such polypeptides with at least 30 amino
acids
and more preferably at least 50 amino acids.
By a polypeptide having an amino acid sequence at least, for example,
95% "identical" to a reference amino acid sequence of a TR2 polypeptide is
intended that the amino acid sequence of the polypeptide is identical to the
reference sequence except that the polypeptide sequence may include up to five
amino acid alterations per each 100 amino acids of the reference amino acid of
a
TR2 receptor. In other words, to obtain a polypeptide having an amino acid
sequence at least 95% identical to a reference amino acid sequence, up to 5%
of
the amino acid residues in the reference sequence may be deleted or
substituted
with another amino acid, or a number of amino acids up to 5% of the total
amino
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acid residues in the reference sequence may be inserted into the reference
sequence. These alterations of the reference sequence may occur at the amino
or
carboxy terminal positions of the reference amino acid sequence or anywhere
between those terminal positions, interspersed either individually among
residues
in the reference sequence or in one or more contiguous groups within the
reference sequence.
As a practical matter, whether any particular polypeptide is at least 90%,
95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence
shown in FIG. 1 A-1B (SEQ ID NO:2), FIG. 4A-4C (SEQ ID NO:5), or FIG. 7A-
7C (SEQ ID NO:8) or to the amino acid sequence encoded by one of the
deposited cDNTA clones can be determined conventionally using known computer
programs such the Bestfit program (Wisconsin Sequence Analysis Package,
Version 8 for Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, WI 53711). When using Bestfit or any other sequence
alignment program to determine whether a particular sequence is, for instance,
95% identical to a reference sequence according to the present invention, the
parameters are set, of course, such that the percentage of identity is
calculated
over the full length of the reference amino acid sequence and that gaps in
homology of up to 5% of the total number of amino acid residues in the
reference
sequence are allowed.
The polypeptides of the present invention could be used as a molecular
weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns
using methods well known to those of skill in the art.
In another aspect, the invention provides peptides or polypeptides
comprising epitope-bearing portions of the polypeptides of the invention. The
epitopes of these polypeptide portions are an immunogenic or antigenic
epitopes
of the polypeptides described herein. An "immunogenic epitope" is defined as a
part of a protein that elicits an antibody response when the whole protein is
the
immunogen. On the other hand, a region of a protein molecule to which an
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antibody can bind is defined as an "antigenic epitope." The number of
immunogenic epitopes of a protein generally is less than the number of
antigenic
epitopes. See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-
4002 (1983).
As to the selection of peptides or polypeptides bearing an antigenic epitope
(i.e., that contain a region of a protein molecule to which an antibody can
bind),
it is well known in that art that relatively short synthetic peptides that
mimic part
of a protein sequence are routinely capable of eliciting an antiserum that
reacts
with the partially mimicked protein. See, for instance, Sutcliffe, J. G.,
Shinnick,
T. M., Green, N. and Learner, R.A. (1983) Antibodies that react with
predetermined sites on proteins. Science 219:660-666. Peptides capable of
eliciting protein-reactive sera are frequently represented in the primary
sequence
of a protein, can be characterized by a set of simple chemical rules, and are
confined neither to immunodominant regions of intact proteins (i.e.,
immunogenic
epitopes) nor to the amino or carboxyl terminals.
Antigenic epitope-bearing peptides and polypeptides of the invention are
therefore useful to raise antibodies, including monoclonal antibodies, that
bind
specifically to a polypeptide of the invention. See, for instance, Wilson et
al., Cell
37:767-778 (1984) at 777. Antigenic epitope-bearing peptides and polypeptides
of the invention preferably contain a sequence of at least seven, more
preferably
at least nine and most preferably between at least about 15 to about 30 amino
acids contained within the amino acid sequence of a polypeptide of the
invention.
Non-limiting examples of antigenic polypeptides or peptides that can be
used to generate TR2 receptor-specific antibodies include: a polypeptide
comprising amino acid residues from about 39 to about 70 in FIG. I (amino acid
residues 3 to 34 in SEQ ID NO:2); a polypeptide comprising amino acid residues
from about 106 to about 120 in FIG. lA-1B (amino acid residues 70 to 84 in SEQ
ID NO:2); a polypeptide comprising amino acid residues from about 142 to about
189 in FIG. IA-1B (amino acid residues 106 to 153 in SEQ ID NO:2); a
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polypeptide comprising amino acid residues from about 276 to about 283 in
FIG. 1 (amino acid residues 240 to 247 in SEQ ID NO:2); a polypeptide
comprising amino acid residues from about 39 to about 70 in FIG. 4A-4C (amino
acid residues 3 to 34 in SEQ ID NO:5); a polypeptide comprising amino acid
residues from about 99 to about 136 in FIG. 4A-4C (amino acid residues 63 to
100 in SEQ ID NO:5); a polypeptide comprising amino acid residues from about
171 to about 185 in FIG. 4A-4C (amino acid residues 135 to 149 in SEQ ID
NO: 5)-, a polypeptide comprising amino acid residues from about 56 to about
68
in FIG. 7A-7C (SEQ ID NO:8); and a polypeptide comprising amino acid residues
from about 93 to about 136 in FIG. 7A-7C (SEQ ID NO:8). As indicated above,
the inventors have determined that the above polypeptide fragments are
antigenic
regions of the TR2 receptor proteins.
The epitope-bearing peptides and polypeptides of the invention may be
produced by any conventional means. Houghten, R. A. (1985) General method
for the rapid solid-phase synthesis of large numbers of peptides: specificity
of
antigen-antibody interaction at the level of individual amino acids. Proc.
Natl.
Acad. Sci. USA 82:5131-5135. This "Simultaneous Multiple Peptide Synthesis
(SMPS)" process is further described in U.S. Patent No. 4,631,211 to Houghten
et al. (1986).
As one of skill in the art will appreciate, TR2 polypeptides of the present
invention and the epitope-bearing fragments thereof described above can be
combined with parts of the constant domain of immunoglobulins (IgG), resulting
in chimeric polypeptides. These fusion proteins facilitate purification and
show
an increased half-life in vivo. This has been shown, e.g., for chimeric
proteins
consisting of the first two domains of the human CD4-polypeptide and various
domains of the constant regions of the heavy or light chains of mammalian
immunoglobulins (EPA 394,827; Traunecker et at., Nature 331:84-86 (1988)).
Fusion proteins that have a disulfide-linked dimeric structure due to the IgG
part
can also be more efficient in binding and neutralizing other molecules than
the
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monomeric TR2 receptor proteins or protein fragments alone (Fountoulakis et
al.,
J. Biochem 270:3958-3964 (1995)).
Detection of Disease States
The TNF-family ligands induce various cellular responses by binding to
TNF-family receptors, including the TR2 receptors of the present invention.
TNF-
P, a potent ligand of the TNF receptor proteins, is known to be involved in a
number of biological processes including lymphocyte development, tumor
necrosis, induction of an antiviral state, activation of polymorphonuclear
leukocytes, induction of class I major histocompatibility complex antigens on
endothelial cells, induction of adhesion molecules on endothelium and growth
hormone stimulation (Ruddle and Homer, Prog. Allergy, 40:162-182 (1988)).
TNF-a, also a ligand of the TNF receptor proteins, has been reported to have a
role in the rapid necrosis of tumors, immunostimulation, autoimmune disease,
graft rejection, producing an anti-viral response, septic shock, cerebral
malaria,
cytotoxicity, protection against deleterious effects of ionizing radiation
produced
during a course of chemotherapy, such as denaturation of enzymes, lipid
peroxidation and DNA damage (Nata et al, J. Immunol. 136(7):2483 (1987);
Porter, Tibtech 9:158-162 (1991)), growth regulation, vascular endothelium
effects and metabolic effects. TNF-a also triggers endothelial cells to
secrete
various factors, including PAI-1, IL-1, GM-CSF and IL-6 to promote cell
proliferation. In addition, TNF-a up-regulates various cell adhesion molecules
such as E-Selectin, ICAM-1 and VCAM-1. TNF-a and the Fas ligand have also
been shown to induce programmed cell death.
Cells which express the TR2 polypeptides and are believed to have a
potent cellular response to TR2 receptor ligands include B lymphocytes (CD
19'),
both CD4+ and CD8+ T lymphocytes, monocytes, endothelial cells and other cell
types shown in Tables 2 and 3. By "a cellular response to a TNF-family ligand"
is intended any genotypic, phenotypic, and/or morphologic change to a cell,
cell
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line, tissue, tissue culture or patient that is induced by a TNF-family
ligand. As
indicated, such cellular responses include not only normal physiological
responses
to TNF-family ligands, but also diseases associated with increased cell
proliferation or the inhibition of increased cell proliferation, such as by
the
inhibition of apoptosis. Apoptosis-programmed cell death-is a physiological
mechanism involved in the deletion of peripheral T lymphocytes of the immune
system, and its dysregulation can lead to a number of different pathogenic
processes (Ameisen, J.C., AIDS 8:1197-1213 (1994); Krammer, P.H. et al., Curr.
Opin. Immunol. 6:279-289 (1994)).
It is believed that certain tissues in mammals with specific disease states
associated with aberrant cell survival express significantly altered levels of
the TR2
receptor protein and mRNA encoding the TR2 receptor protein when compared
to a corresponding "standard" mammal, i.e., a mammal of the same species not
having the disease state. Further, since some forms of this protein are
secreted,
it is believed that enhanced levels of the TR2 receptor protein can be
detected in
certain body fluids (e.g., sera, plasma, urine, and spinal fluid) from mammals
with
the disease state when compared to sera from mammals of the same species not
having the disease state. Thus, the invention provides a diagnostic method
useful
during diagnosis of disease states, which involves assaying the expression
level of
the gene encoding the TR2 receptor protein in mammalian cells or body fluid
and
comparing the gene expression level with a standard TR2 receptor gene
expression level, whereby an increase or decrease in the gene expression level
over
the standard is indicative of certain disease states associated with aberrant
cell
survival.
Where diagnosis of a disease state involving the TR2 receptors of the
present invention has already been made according to conventional methods, the
present invention is useful as a prognostic indicator, whereby patients
exhibiting
significantly aberrant TR2 receptor gene expression will experience a worse
clinical outcome relative to patients expressing the gene at a lower level.
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By "assaying the expression level of the gene encoding the TR2 receptor
protein" is intended qualitatively or quantitatively measuring or estimating
the
level of the TR2 receptor protein or the level of the mRNA encoding the TR2
receptor protein in a first biological sample either directly (e.g., by
determining or
estimating absolute protein level or mRNA level) or relatively (e.g., by
comparing
to the TR2 receptor protein level or mRNA level in a second biological
sample).
Preferably, the TR2 receptor protein level or mRNA level in the first
biological sample is measured or estimated and compared to a standard TR2
receptor protein level or mRNA level, the standard being taken from a second
biological sample obtained from an individual not having the disease state. As
will
be appreciated in the art, once a standard TR2 receptor protein level or mRNA
level is known, it can be used repeatedly as a standard for comparison.
By "biological sample" is intended any biological sample obtained from an
individual, cell line, tissue culture, or other source which contains TR2
receptor
protein or mRNA. Biological samples include mammalian body fluids (such as
sera, plasma, urine, synovial fluid and spinal fluid) which contain secreted
mature
TR2 receptor protein, and thymus, prostate, heart, placenta, muscle, liver,
spleen,
lung, kidney and other tissues. Methods for obtaining tissue biopsies and body
fluids from mammals are well known in the art. Where the biological sample is
to
include mRNA, a tissue biopsy is the preferred source.
Diseases associated with increased cell survival, or the inhibition of
apoptosis, include cancers (such as follicular lymphomas, carcinomas with p53
mutations, and hormone-dependent tumors); autoimmune disorders (such as
systemic lupus erythematosus and immune-related glomerulonephritis rheumatoid
arthritis) and viral infections (such as herpes viruses, pox viruses and
adenoviruses), information graft v. host disease, acute graft rejection, and
chronic
graft rejection. Diseases associated with decreased cell survival, or
increased
apoptosis, include AIDS; neurodegenerative disorders (such as Alzheimer's
disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis
pigmentosa,
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Cerebellar degeneration); myelodysplastic syndromes (such as aplastic anemia),
ischemic injury (such as that caused by myocardial infarction, stroke and
reperfusion injury), toxin-induced liver disease (such as that caused by
alcohol),
septic shock, cachexia and anorexia.
Assays available to detect levels of soluble receptors are well known to
those of skill in the art, for example, radioimmunoassays, competitive-binding
assays, Western blot analysis, and preferably an ELISA assay may be employed.
TR2 receptor-protein specific antibodies can be raised against the intact
TR2 receptor protein or an antigenic polypeptide fragment thereof, which may
presented together with a carrier protein, such as an albumin, to an animal
system
(such as rabbit or mouse) or, if it is long enough (at least about 25 amino
acids),
without a carrier.
As used herein, the term "antibody" (Ab) or "monoclonal antibody" (mAb)
is meant to include intact molecules as well as antibody fragments (such as,
for
example, Fab and F(ab')2 fragments) which are capable of specifically binding
to
TR2 receptor protein. Fab and F(ab')2 fragments lack the Fc fragment of intact
antibody, clear more rapidly from the circulation, and may have less non-
specific
tissue binding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325
(1983)). Thus, these fragments are preferred.
The antibodies of the present invention may be prepared by any of a
variety of methods. For example, cells expressing the TR2 receptor protein or
an
antigenic fragment thereof can be administered to an animal in order to induce
the
production of sera containing polyclonal antibodies. In a preferred method, a
preparation of TR2 receptor protein is prepared and purified to render it
substantially free of natural contaminants. Such a preparation is then
introduced
into an animal in order to produce polyclonal antisera of greater specific
activity.
In the most preferred method, the antibodies of the present invention are
monoclonal antibodies (or TR2 receptor protein binding fragments thereof).
Such
monoclonal antibodies can be prepared using hybridoma technology (Kohler et
al.,
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Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et
al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., In: Monoclonal
Antibodies and T-Cell Hybridomas, Elsevier, N.Y., (1981) pp. 563-68 1). In
general, such procedures involve immunizing an animal (preferably a mouse)
with
a TR2 receptor protein antigen or, more preferably, with a TR2 receptor
protein-expressing cell. Suitable cells can be recognized by their capacity to
bind
anti-TR2 receptor protein antibody. Such cells may be cultured in any suitable
tissue culture medium; however, it is preferable to culture cells in Earle's
modified
Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about
56 C), and supplemented with about 10 g/l of nonessential amino acids, about
1,000 U/ml of penicillin, and about 100 g/ml of streptomycin. The splenocytes
of such mice are extracted and fused with a suitable myeloma cell line. Any
suitable myeloma cell line may be employed in accordance with the present
invention; however, it is preferable to employ the parent myeloma cell line
(SP2O),
available from the American Type Culture Collection, Rockville, Maryland.
After
fusion, the resulting hybridoma cells are selectively maintained in HAT
medium,
and then cloned by limiting dilution as described by Wands el al.
(Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained through
such a selection are then assayed to identify clones which secrete antibodies
capable of binding the TR2 receptor protein antigen.
Agonists and Antagonists of TR2 Receptor Function
In one aspect, the present invention is directed to a method for inhibiting
an activity of TR2 induced by a TNF-family ligand (e.g., cell proliferation,
hematopoietic development), which involves administering to a cell which
expresses a TR2 polypeptide an effective amount of a TR2 receptor ligand,
analog
or an antagonist capable of decreasing TR2, receptor mediated signaling.
Preferably, TR2 receptor mediated signaling is increased to treat a disease
wherein
increased cell proliferation is exhibited. An antagonist can include soluble
forms
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of the TR2 receptors and antibodies directed against the TR2 polypeptides
which
block TR2 receptor mediated signaling. Preferably, TR2 receptor mediated
signaling is decreased to treat a disease.
In a further aspect, the present invention is directed to a method for
increasing cell proliferation induced by a TNF-family ligand, which involves
administering to a cell which expresses a TR2 polypeptide an effective amount
of
an agonist capable of increasing TR2 receptor mediated signaling. Preferably,
TR2 receptor mediated signaling is increased to treat a disease wherein
decreased
cell proliferation is exhibited. Agonists of the present invention include
monoclonal antibodies directed against the TR2 polypeptides which stimulate
TR2
receptor mediated signaling. Preferably, TR2 receptor mediated signaling is
increased to treat a disease.
By "agonist" is intended naturally occurring and synthetic compounds
capable of enhancing cell proliferation and differentiation mediated by TR2
polypeptides. Such agonists include agents which increase expression of TR2
receptors or increase the sensitivity of the expressed receptor. By
"antagonist" is
intended naturally occurring and synthetic compounds capable of inhibiting TR2
mediated cell proliferation and differentiation. Such antagonists include
agents
which decrease expression of TR2 receptors or decrease the sensitivity of the
expressed receptor. Whether any candidate "agonist" or "antagonist" of the
present invention can enhance or inhibit cell proliferation and
differentiation can
be determined using art-known TNF-family ligand/receptor cellular response
assays, including those described in more detail below.
One such screening technique involves the use of cells which express the
receptor (for example, transfected CHO cells) in a system which measures
extracellular pH changes caused by receptor activation, for example, as
described
in Science 246:181-296 (October 1989). For example, compounds may be
contacted with a cell which expresses the receptor polypeptide of the present
invention and a second messenger response, e.g., signal transduction or pH
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changes, may be measured to determine whether the potential compound activates
or inhibits the receptor.
Another such screening technique involves introducing RNA encoding the
receptor into Xenopus oocytes to transiently express the receptor. The
receptor
oocytes may then be contacted with the receptor ligand and a compound to be
screened, followed by detection of inhibition or activation of a calcium
signal in
the case of screening for compounds which are thought to inhibit activation of
the
receptor.
Another method involves screening for compounds which inhibit activation
of the receptor polypeptide of the present invention antagonists by
determining
inhibition of binding of labeled ligand to cells which have the receptor on
the
surface thereof. Such a method involves transfecting a eukaryotic cell with
DNA
encoding the receptor such that the cell expresses the receptor on its surface
and
contacting the cell with a compound in the presence of a labeled form of a
known
ligand. The ligand can be labeled, e.g., by radioactivity. The amount of
labeled
ligand bound to the receptors is measured, e.g., by measuring radioactivity of
the
receptors. If the compound binds to the receptor as determined by a reduction
of
labeled ligand which binds to the receptors, the binding of labeled ligand to
the
receptor is inhibited.
Soluble forms of the polypeptides of the present invention may be utilized
in the ligand binding assay described above. These forms of the TR2 receptors
are
contacted with ligands in the extracellular medium after they are secreted. A
determination is then made as to whether the secreted protein will bind to TR2
receptor ligands.
Further screening assays for agonist and antagonist of the present
invention are described in Tartaglia, L.A., and Goeddel, D.V., J. Biol. Chem.
267(7) :4304-4307(1992).
Thus, in a further aspect, a screening method is provided for determining
whether a candidate agonist or antagonist is capable of enhancing or
inhibiting a
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cellular response to a TNF-family ligand. The method involves contacting cells
which express TR2 polypeptides with a candidate compound and a TNF-family
ligand, assaying a cellular response, and comparing the cellular response to a
standard cellular response, the standard being assayed when contact is made
with
the ligand in absence of the candidate compound, whereby an increased cellular
response over the standard indicates that the candidate compound is an agonist
of
the ligand/receptor signaling pathway and a decreased cellular response
compared
to the standard indicates that'the candidate compound is an antagonist of the
ligand/receptor signaling pathway. By "assaying a cellular response" is
intended
qualitatively or quantitatively measuring a cellular response to a candidate
compound and/or a TNF-family ligand (e.g., determining or estimating an
increase
or decrease in T cell proliferation or tritiated thymidine labeling). By the
invention, a cell expressing a TR2 polypeptide can be contacted with either an
endogenous or exogenously administered TNF-family ligand.
In an additional aspect, a thymocyte proliferation assay may be employed
to identify both ligands and potential drug candidates. For example, thymus
cells
are disaggregated from tissue and grown in culture medium. Incorporation of
DNA precursors such as 3H-thymidine or 5-bromo-2'-deoxyuridine (BrdU) is
monitored as a parameter for DNA synthesis and cellular proliferation. Cells
which have incorporated BrdU into DNA can be detected using a monoclonal
antibody against BrdU and measured by an enzyme or fluorochrome-conjugated
second antibody. The reaction is quantitated by fluorimetry or by
spectrophotometry. Two control wells and an experimental well are set up as
above and TNF-(3 or cognate ligand is added to all wells while soluble
receptor
polypeptides of the present invention are added individually to the second
control
wells, with the experimental well containing a compound to be screened. The
ability of the compound to be screened to stimulate or inhibit the above
interaction
may then be quantified.
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Agonists according to the present invention include compounds such as,
for example, TNF-family ligand peptide fragments, transforming growth factor
P,
and neurotransmitters (such as glutamate, dopamine, N-methyl-D-aspartate).
Preferred agonist include polyclonal and monoclonal antibodies raised against
TR2
polypeptide, or a fragment thereof. Such agonist antibodies raised against a
TNF-
family receptor are disclosed in Tartaglia, L.A., et al., Proc. Natl. Acad.
Sc!.
USA 88:9292-9296 (1991); and Tartaglia, L.A., and Goeddel, D.V., J. Biol.
Chem. 267 (7):4304-4307 (1992). See, also, PCT Application WO 94/09137.
Further preferred agonists include chemotherapeutic drugs such as, for
example,
cisplatin, doxorubicin, bleomycin, cytosine arabinoside, nitrogen mustard,
methotrexate and vincristine. Others include ethanol and P-amyloid peptide.
(.Science 267:1457-1458 (1995)).
Antagonist according to the present invention include soluble forms of the
TR2 receptors (e.g., fragments of the TR2 receptor shown in FIG. IA-IB that
include the ligand binding domain from the extracellular region of the full
length
receptor). Such soluble forms of the receptor, which may be naturally
occurring
or synthetic, antagonize TR2, TR2-SVI or TR2-SV2 mediated signaling by
competing with the cell surface bound forms of the receptor for binding to TNF-
family ligands.. Antagonists of the present invention also include antibodies
specific for TNF-family ligands and TR2-Fc fusion proteins such as the one
described below in Examples 5 and 6.
By a "TNF-family ligand" is intended naturally occurring, recombinant, and
synthetic ligands that are capable of binding to a member of the TNF receptor
family and inducing the ligand/receptor signaling pathway. Members of the TNF
ligand family include, but are not limited to, TNF-a, lymphotoxin-a (LT-a,
also
known as TNF-P), LT-(3 (found in complex heterotrimer LT-a2-p), FasL,
CD40L, CD27L, CD30L, 4-IBBL, OX40L and nerve growth factor (NGF).
The experiments set forth in Example 6 demonstrate that the TR2
receptors of the present invention are capable of inducing the proliferation
of
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lymphocytes. Further, such proliferation can be inhibited by a TR2 protein
fragment fused to an Fc antibody fragment.
TNFa has been shown to protect mice from infection with herpes simplex
virus type 1 (HSV-1). Rossol-Voth, R. et al., J. Gen. Virol. 72:143-147
(1991).
The mechanism of the protective effect of TNFa is unknown but appears to
involve neither interferons not NK cell killing. One member of the TNFR family
has been shown to mediate HSV-1 entry into cells. Montgomery, R. et al., Eur.
C:ytokine Newt. 7:159 (1996). Further, antibodies specific for the
extracellular
domain of this TNFR block HSV-1 entry into cells. Thus, TR2 receptors of the
present invention include both TR2 amino acid sequences and antibodies capable
of preventing TNFR mediated viral entry into cells. Such sequences and
antibodies can function by either competing with cell surface localized TNFR
for
binding to virus or by directly blocking binding of virus to cell surface
receptors.
Antibodies according to the present invention may be prepared by any of
a variety of methods using TR2 receptor immunogens of the present invention.
Such TR2 receptor immunogens include the TR2 receptor protein shown in FIG.
1A-1B (SEQ ID NO:2) and the TR2-SV1 (FIG. 4A-4C (SEQ ID NO:5)) and
TR2-SV2 (FIG. 7A-7C(SEQ ID NO:8)) polypeptides (any of which may or may
not include a leader sequence) and polypeptide fragments of the receptors
comprising the ligand binding, extracellular, transmembrane, the intracellular
domains of the TR2 receptors, or any combination thereof.
Polyclonal and monoclonal antibody agonist or antagonist according to the
present invention can be raised according to the methods disclosed in
Tartaglia
and Goeddel, I. Biol. Chem. 267(7):4304-4307(1992)); Tartaglia et al., Cell
73:213-216 (1993)), and PCT Application WO 94/09137. The term "antibody"
(Ab) or "monoclonal antibody" (mAb) as used herein is meant to include intact
molecules as well as fragments thereof (such as, for example, Fab and F(ab')2
fragments) which are capable of binding an antigen. Fab and F(ab')Z fragments
lack the Fc fragment of intact antibody, clear more rapidly from the
circulation,
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and may have less non-specific tissue binding of an intact antibody (Wahl et
al.,
J. Nucl. Med. 24:316-325 (1983)).
In a preferred method, antibodies according to the present invention are
mAbs. Such mAbs can be prepared using hybridoma technology (Kohler and
Millstein, Nature 256:495-497 (1975) and U.S. Patent No. 4,376,110; Harlow et
al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY, 1988; Monoclonal Antibodies and Hybridomas: A New
Dimension in Biological Analyses, Plenum Press, New York, NY, 1980;
Campbell, "Monoclonal Antibody Technology," In: Laboratory Techniques in
Biochemistry and Molecular Biology, Volume 13 (Burdon et al., eds.), Elsevier,
Amsterdam (1984)).
Proteins and other compounds which bind the TR2 receptor domains are
also candidate agonist and antagonist according to the present invention. Such
binding compounds can be "captured" using the yeast two-hybrid system (Fields
and Song, Nature 340:245-246 (1989)). A modified version of the yeast two-
hybrid system has been described by Roger Brent and his colleagues (Gyuris, J.
et al., Cell 75:791-803 (1993); Zervos, A.S. et al., Cell 72:223-232 (1993)).
Preferably, the yeast two-hybrid system is used according to the present
invention
to capture compounds which bind to the ligand binding, extracellular,
intracellular,
and transmembrane domains of the TR2 receptors. Such compounds are good
candidate agonist and antagonist of the present invention.
Using the two-hybrid assay described above, the intracellular domain of
the TR2 receptor, or a portion thereof, may be used to identify cellular
proteins
which interact with the receptor in vivo. Such an assay may also be used to
identify ligands with potential agonistic or antagonistic activity of TR2
receptor
function. This screening assay has previously been used to identify protein
which
interact with the cytoplasmic domain of the murine TNF-RII and led to the
identification of two receptor associated proteins. Rothe, M. et al., Cell
78:681
(1994). Such proteins and amino acid sequences which bind to the cytoplasmic
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domain of the TR2 receptors are good candidate agonist and antagonist of the
present invention.
Other screening techniques include the use of cells which express the
polypeptide of the present invention (for example, transfected CHO cells) in a
system which measures extracellular pH changes caused by receptor activation,
for example, as described in Science, 246:181-296 (1989). In another example,
potential agonists or antagonists may be contacted with a cell which expresses
the
polypeptide of the present invention and a second messenger response, e.g.,
signal
transduction may be measured to determine whether the potential antagonist or
agonist is effective.
The TR2 receptor agonists may be employed to stimulate ligand activities,
such as inhibition of tumor growth and necrosis of certain transplantable
tumors.
The agonists may also be employed to stimulate cellular differentiation, for
example, T-cell, fibroblasts and hemopoietic cell differentiation. Agonists to
the
TR2 receptor may also augment TR2's role in the host's defense against
microorganisms and prevent related diseases (infections such as that from
Listeria
monocytogenes) and Chlamidiae. The agonists may also be employed to protect
against the deleterious effects of ionizing radiation produced during a course
of
radiotherapy, such as denaturation of enzymes, lipid peroxidation, and DNA
damage.
Agonists to the receptor polypeptides of the present invention may be used
to augment TNFs role in host defenses against microorganisms and prevent
related diseases. The agonists may also be employed to protect against the
deleterious effects of ionizing radiation produced during a course of
radiotherapy,
such as denaturation of enzymes, lipid peroxidation, and DNA damage.
The agonists may also be employed to mediate an anti-viral response, to
regulate growth, to mediate the immune response and to treat
immunodeficiencies
related to diseases such as HIV by increasing the rate of lymphocyte
proliferation
and differentiation.
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The antagonists to the polypeptides of the present invention may be
employed to inhibit ligand activities, such as stimulation of tumor growth and
necrosis of certain transplantable tumors. The antagonists may also be
employed
to inhibit cellular differentiation, for example, T-cell, fibroblasts and
hemopoietic
cell differentiation. Antagonists may also be employed to treat autoimmune
diseases, for example, graft versus host rejection and allograft rejection,
and T-cell
mediated autoimmune diseases such as AIDS. It has been shown that T-cell
proliferation is stimulated via a type 2 TNF receptor. Accordingly,
antagonizing
the receptor may prevent the proliferation of T-cells and treat T-cell
mediated
autoimmune diseases.
The state of immunodeficiency that defines AIDS is secondary to a
decrease in the number and function of CD4+ T-lymphocytes. Recent reports
estimate the daily loss of CD4+ T cells to be between 3.5 X 107 and 2 X 109
cells
(Wei X., et al., Nature 373:117-122 (1995)). One cause of CD4+ T cell
depletion
in the setting of HIV infection is believed to be HIV-induced apoptosis.
Indeed,
HIV-induced apoptotic cell death has been demonstrated not only in vitro but
also, more importantly, in infected individuals (Ameisen, J.C., AIDS 8:1197-
1213
(1994) ; Finkel, T.H., and Banda, N.K., Curr. Opin. Immunol. 6:605-615(1995);
Muro-Cacho, C.A. et al., J. Immunol. 154:5555-5566 (1995)). Furthermore,
apoptosis and CD4+ T-lymphocyte depletion is tightly correlated in different
animal models of AIDS (Brunner, T., et al., Nature 373:441-444 (1995);
Gougeon, M.L., et al., AIDS Res. Hum. Retroviruses 9:553-563 (1993)) and,
apoptosis is not observed in those animal models in which viral replication
does
not result in AIDS (Gougeon, M.L. et al., AIDS Res. Hum. Retroviruses 9:553-
563 (1993)). Further data indicates that uninfected but primed or activated T
lymphocytes from HIV-infected individuals undergo apoptosis after encountering
the TNF-family ligand FasL. Using monocytic cell lines that result in death
following HIV infection, it has been demonstrated that infection of U937 cells
with HIV results in the de novo expression of FasL and that FasL mediates HIV-
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induced apoptosis (Badley, A.D. et al., J. Virol. 70:199-206 (1996)). Further
the
TNF-family ligand was detectable in uninfected macrophages and its expression
was upregulated following HIV infection resulting in selective killing of
uninfected
CD4+ T-lymphocytes (Badley, A.D et al., J. Virol. 70:199-206 (1996)).
As shown in Example 6, the TR2 receptor shown in FIG. ]A-lB is
expressed in CD4` T-lymphocytes and is capable of inducing lymphocyte
proliferation. Thus, by the invention, a method for treating HIV+ individuals
is
provided which involves administering an agonist of the present invention to
increase the rate of proliferation and differentiation of CD4+ T-lymphocytes.
Such
agonists include agents capable of inducing the expression of TR2 receptors
(e.g.,
TNFa, PMA and DMSO) or enhancing the signal of such receptors which induces
lymphocyte proliferation and differentiation. Modes of administration and
dosages
are discussed in detail below.
In rejection of an allograft, the immune system of the recipient animal has
not previously been primed to respond because the immune system for the most
part is only primed by environmental antigens. Tissues from other members of
the
same species have not been presented in the same way that, for example,
viruses
and bacteria have been presented. In the case of allograft rejection,
immunosuppressive regimens are designed to prevent the immune system from
reaching the effector stage. However, the immune profile of xenograft
rejection
may resemble disease recurrence more that allograft rejection. In the case of
disease recurrence, the immune system has already been activated, as evidenced
by destruction of the native islet cells. Therefore, in disease recurrence the
immune system is already at the effector stage. Antagonists of the present
invention are able to suppress the immune response to both allografts and
xenografts by decreasing the rate of TR2 mediated lymphocyte proliferation and
differentiation. Such antagonists include the TR2-Fc fusion protein described
in
Examples 5 and 6. Thus, the present invention further provides a method for
suppression of immune responses.
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In addition, TNFa has been shown to prevent diabetes in strains of animals
which are prone to this affliction resulting from autoimmunity. See Porter,
A.,
Tibtech 9:158-162 (1991). Thus, agonists and antagonists of the present
invention may be useful in the treatment of autoimmune diseases such as type 1
diabetes.
In addition, the role played by the TR2 receptors in cell proliferation and
differentiation indicates that agonist or antagonist of the present invention
may be
used to treat disease states involving aberrant cellular expression of these
receptors. TR2 receptors may in some circumstances induce an inflammatory
response, and antagonists may be useful reagents for blocking this response.
Thus
TR2 receptor antagonists (e.g., soluble forms of the TR2 receptors;
neutralizing
antibodies) may be useful for treating inflammatory diseases, such as
rheumatoid
arthritis, osteoarthritis, psoriasis, septicemia, and inflammatory bowel
disease.
Antagonists to the TR2 receptor may also be employed to treat and/or
prevent septic shock, which remains a critical clinical condition. Septic
shock
results from an exaggerated host response, mediated by protein factors such as
TNF and IL-1, rather than from a pathogen directly. For example,
lipopolysaccharides have been shown to elicit the release of TNF leading to a
strong and transient increase of its serum concentration. TNF causes shock and
tissue injury when administered in excessive amounts. Accordingly, it is
believed
that antagonists to the TR2 receptor will block the actions of TNF and
treat/prevent septic shock. These antagonists may also be employed to treat
meningococcemia in children which correlates with high serum levels of TNF.
Among other disorders which may be treated by the antagonists to TR2
receptors, there are included, inflammation which is mediated by TNF receptor
ligands, and the bacterial infections cachexia and cerebral malaria. The TR2
receptor antagonists may also be employed to treat inflammation mediated by
ligands to the receptor such as TNF.
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Modes of administration
The agonist or antagonists described herein can be administered in vitro,
ex vivo, or in vivo to cells which express the receptor of the present
invention. By
administration of an "effective amount" of an agonist or antagonist is
intended an
amount of the compound that is sufficient to enhance or inhibit a cellular
response
to a TNF-family ligand and include polypeptides. In particular, by
administration
of an "effective amount" of an agonist or antagonists is intended an amount
effective to enhance or inhibit TR2 receptor mediated activity. Of course,
where
cell proliferation and/or differentiation is to be enhanced, an agonist
according to
the present invention can be co-administered with a TNF-family ligand. One of
ordinary skill will appreciate that effective amounts of an agonist or
antagonist can
be determined empirically and may be employed in pure form or in
pharmaceutically acceptable salt, ester or pro-drug form. The agonist or
antagonist may be administered in compositions in combination with one or more
pharmaceutically acceptable excipients.
It will be understood that, when administered to a human patient, the total
daily usage of the compounds and compositions of the present invention will be
decided by the attending physician within the scope of sound medical
judgement.
The specific therapeutically effective dose level for any particular patient
will
depend upon factors well known in the medical arts.
As a general proposition, the total pharmaceutically effective amount of
a TR2 polypeptide administered parenterally per dose will be in the range of
about
1 gg/kg/day to 10 mg/kg/day of patient body weight, although, as noted above,
this will be subject to therapeutic discretion. More preferably, this dose is
at least
0.01 mg/kg/day, and most preferably for humans between about 0.01 and I
mg/kg/day for the hormone. If given continuously, the TR2 polypeptide is
typically administered at a dose rate of about I g/kg/hour to about 50
gg/kg/hour, either by 1-4 injections per day or by continuous subcutaneous
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infusions, for example, using a mini-pump. An intravenous bag solution may
also
be employed.
Pharmaceutical compositions containing the TR2 receptor polypeptides of
the invention may be administered orally, rectally, parenterally,
intracistemally,
intravaginally, intraperitoneally, topically (as by powders, ointments, drops
or
transdermal patch), bucally, or as an oral or nasal spray. By
"pharmaceutically
acceptable carrier" is meant a non-toxic solid, semisolid or liquid filler,
diluent,
encapsulating material or formulation auxiliary of any type. The term
"parenteral"
as used herein refers to modes of administration which include intravenous,
intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular
injection and infusion.
Having generally described the invention, the same will be more readily
understood by reference to the following examples, which are provided by way
of
illustration and are not intended as limiting.
Example 1
Expression and Purification of TR2 in E. coli
The bacterial expression vector pQE60 is used for bacterial expression in
this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311).
pQE60 encodes ampicillin antibiotic resistance ("Amp"') and contains a
bacterial
origin of replication ("ori"), an IPTG inducible promoter, a ribosome binding
site
("RBS"), six codons encoding histidine residues that allow affinity
purification
using nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin sold by QIAGEN,
Inc., supra, and suitable single restriction enzyme cleavage sites. These
elements
are arranged such that a DNA fragment encoding a polypeptide may be inserted
in such as way as to produce that polypeptide with the six His residues (i.e.,
a "6
X His tag") covalently linked to the carboxyl terminus of that polypeptide.
However, in this example, the polypeptide coding sequence is inserted such
that
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translation of the six His codons is prevented and, therefore, the polypeptide
is
produced with no 6 X His tag.
The DNA sequence encoding the desired portion of the TR2 protein
lacking the hydrophobic leader sequence is amplified from the deposited cDNA
clone using PCR oligonucleotide primers which anneal to the amino terminal
sequences of the desired portion of the TR2 protein and to sequences in the
deposited construct 3' to the cDNA coding sequence. Additional nucleotides
containing restriction sites to facilitate cloning in the pQE60 vector are
added to
the 5' and 3' sequences, respectively.
For cloning the mature protein, the 5' primer has the sequence:
5' CGCCCATGGCCCCAGCTCTGCCGTCCT 3' (SEQ ID NO:14) containing
the underlined NcoI restriction site followed by 18 nucleotides complementary
to
the amino terminal coding sequence of the mature TR2 sequence in FIG. I A-1 B.
One of ordinary skill in the art would appreciate, of course, that the point
in the
protein coding sequence where the 5' primer begins may be varied to amplify a
desired portion of the complete protein shorter or longer than the mature
form.
The 3' primer has the sequence:
5' CGCAAGCTTATTGTGGGAGCTGCTGGTCCC 3' (SEQ ID NO:15)
containing the underlined HindIIl restriction site followed by 18 nucleotides
complementary to the 3' end of the nucleotide sequence shown in FIG. IA-1B
(SEQ ID NO:1) encoding the extracellular domain of the TR2 receptor.
The amplified TR2 DNA fragments and the vector pQE60 are digested
with NcoI and HindIII and the digested DNAs are then ligated together.
Insertion
of the TR2 DNA into the restricted pQE60 vector places the TR2 protein coding
region including its associated stop codon downstream from the IPTG-inducible
promoter and in-frame with an initiating AUG. The associated stop codon
prevents translation of the six histidine codons downstream of the insertion
point.
The ligation mixture is transformed into competent E. coli cells using
standard procedures such as those described in Sambrook et al., Molecular
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Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY (1989). E. coli strain M15/rep4, containing multiple
copies of the plasmid pREP4, which expresses the lac repressor and confers
kanamycin resistance ("Kan""), is used in carrying out the illustrative
example
described herein. This strain, which is only one of many that are suitable for
expressing TR2 protein, is available commercially from QIAGEN, Inc., supra.
Transformants are identified by their ability to grow on LB plates in the
presence
of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies
and
the identity of the cloned DNA confirmed by restriction analysis, PCR and DNA
sequencing.
Clones containing the desired constructs are grown overnight ("O/N") in
liquid culture in LB media supplemented with both ampicillin (100 .tg/ml) and
kanamycin (25 pg/ml). The ON culture is used to inoculate a large culture, at
a
dilution of approximately 1:25 to 1:250. The cells are grown to an optical
density
at 600 nm ("OD600") of between 0.4 and 0.6. Isopropyl-b-D-
thiogalactopyranoside ("IPTG") is then added to a final concentration of 1 mM
to
induce transcription from the lac repressor sensitive promoter, by
inactivating the
lacl repressor. Cells subsequently are incubated further for 3 to 4 hours.
Cells
then are harvested by centrifugation.
The cells are then stirred for 3-4 hours at 4 C in 6 M guanidine-HCI, pH
8. The cell debris is removed by centrifugation, and the supernatant
containing the
TR2 is dialyzed against 50 mM Na-acetate buffer pH 6, supplemented with 200
mM NaCl. Alternatively, the protein can be successfully refolded by dialyzing
it
against 500 mM NaCl, 20% glycerol, 25 mM Tris/HCI pH 7.4, containing
protease inhibitors. After renaturation the protein can be purified by ion
exchange, hydrophobic interaction and size exclusion chromatography.
Alternatively, an affinity chromatography step such as an antibody column can
be
used to obtain pure TR2 protein. The purified protein is stored at 4 C or
frozen
at -80 C.
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Example 2
Example 2(a): Cloning and Expression of a Soluble Fragment of TR2 Protein
in a Baculovirus Expression System
In this example, the plasmid shuttle vector pA2 GP was used to insert the
cloned DNA encoding the mature extracellular domain of the TR2 receptor
protein shown in FIG. IA-1B, lacking its naturally associated secretory signal
(leader) sequence, into a baculovirus. This protein was expressed using a
baculovirus leader and standard methods as described in Summers et al., A
Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures,
Texas Agricultural Experimental Station Bulletin No. 1555 (1987). This
expression vector contains the strong polyhedrin promoter of the Autographa
californica nuclear polyhedrosis virus (AcMNPV) followed by the secretory
signal
peptide (leader) of the baculovirus gp67 protein and convenient restriction
sites
such as BamFH, Xbal and Asp7l8. The polyadenylation site of the simian virus
40
("SV40") is used for efficient polyadenylation. For easy selection of
recombinant
virus, the plasmid contains the beta-galactosidase gene from E. coli under
control
of a weak Drosophila promoter in the same orientation, followed by the
polyadenylation signal of the polyhedrin gene. The inserted genes are flanked
on
both sides by viral sequences for cell-mediated homologous recombination with
wild-type viral DNA to generate viable virus that expresses the cloned
polynucleotide.
Many other baculovirus vectors could be used in place of the vector above,
such as pAc373, pVL941 and pAcIMl, as one skilled in the art would readily
appreciate, as long as the construct provides appropriately located signals
for
transcription, translation, secretion and the like, including a signal peptide
and an
in-frame AUG as required. Such vectors are described, for instance, in Luckow
et al., Virology 170:31-39.
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The cDNA sequence encoding essentially the mature extracellular domain
(amino acids 37 to 200 shown in FIG. IA-IB) of the TR2 receptor protein in the
deposited clone (ATCC Deposit Number 97059) was amplified using PCR
oligonucleotide primers corresponding to the relevant 5' and 3' sequences of
the
gene. The 5' primer for each of the above has the sequence:
5' CGCGGATCCCGGAGCCCCCTGCTAC 3' (SEQ ID NO: 16) containing the
underlined BamHI restriction enzyme site, an efficient signal for initiation
of
translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol.
196:947-
950 (1987), followed by 15 bases of the coding sequence of the TR2 protein
shown in FIG. lA-lB, beginning with the nucleotide 354. The 3' primer has the
sequence:
5' CGC GG TACCATTGTGGGAGCTGCTGGTCCC 3' (SEQ ID NO:17)
containing the underlined, Asp718 restriction sites followed by 17 nucleotides
complementary to the coding sequences in FIG. lA-1B.
The amplified fragment was isolated from a 1% agarose gel using a
commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The
fragment was then digested with BamHI and Asp718 and purified on a 1%
agarose gel. This fragment is designated herein "Fl".
The plasmid was digested with the restriction enzymes BamHI and Asp718
dephosphorylated using calf intestinal phosphatase. The DNA was then isolated
from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101
Inc., La Jolla, Ca.). This vector DNA was designated herein "V Ill.
Fragment F 1 and the dephosphorylated plasmid V 1 were ligated together
with T4 DNA ligase. E. coli HB 101 cells were transformed with the ligation
mixture and spread on culture plates. Other suitable E. coli hosts such as XL-
I
Blue (Stratagene Cloning Systems, La Jolla, CA) may also be used. Bacteria
were
identified that contain the plasmid with the human TR2 sequences using the PCR
method, in which one of the primers that was used to amplify the gene and the
second primer was from well within the vector so that only those bacterial
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colonies containing TR2 gene fragments show amplification of the DNA. The
sequence of the cloned fragment was confirmed by DNA sequencing. The plasmid
was designated herein pBacTR2-T.
Five gg of pBacTR2-T was co-transfected with 1.0 pg of a commercially
available linearized baculovirus DNA ("BaculoGoldTM baculovirus DNA",
Pharmingen, San Diego, CA.), using the lipofection method described by Feigner
et al., Proc. Natl. Acad Sci. USA 84:7413-7417 (1987). 1 g of BaculoGoldTM
virus DNA and 5 pg of plasmid pBacTR2-T were mixed in a sterile well of a
microtiter plate containing 50 l of serum-free Grace's medium (Life
Technologies
Inc., Gaithersburg, MD). Afterwards, 10 pl Lipofectin plus 90 gl Grace's
medium
were added, mixed and incubated for 15 minutes at room temperature. Then the
transfection mixture was added drop-wise to Sf9 insect cells (ATCC CRL 1711)
seeded in a 35 mm tissue culture plate with I ml Grace's medium without serum.
The plate was rocked back and forth to mix the newly added solution. The plate
was then incubated for 5 hours at 27'C. After 5 hours the transfection
solution
was removed from the plate and 1 ml of Grace's insect medium supplemented with
10% fetal calf serum was added. The plate was put back into an incubator and
cultivation was continued at 27 C for four days.
After four days the supernatant was collected and a plaque assay was
performed, as described by Summers and Smith, supra. An agarose gel with
"Blue Gal" (Life Technologies Inc., Gaithersburg) was used to allow easy
identification and isolation of gal-expressing clones, which produce blue-
stained
plaques. (A detailed description of a "plaque assay" of this type can also be
found
in the user's guide for insect cell culture and baculovirology distributed by
Life
Technologies Inc., Gaithersburg, page 9-10). After appropriate incubation,
blue
stained plaques were picked with the tip of a micropipettor (e.g., Eppendorf).
The
agar containing the recombinant viruses was then resuspended in a
microcentrifuge tube containing 200 l of Grace's medium and the suspension
containing the recombinant baculovirus was used to infect Sf9 cells seeded in
35
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mm dishes. Four days later the supernatants of these culture dishes were
harvested and then they were stored at 4 C. The recombinant virus is called V-
TR2-T.
To verify the expression of the gene used, Sf cells were grown in Grace's
medium supplemented with 10% heat inactivated FBS. The cells were infected
with the recombinant baculovirus V-TR2-T at a multiplicity of infection
("MOI")
of about 2. Six hours later the medium was removed and replaced with SF900 II
medium minus methionine and cysteine (available from Life Technologies Inc.,
Rockville, MD). Forty-two hours later, 5 Ci of 35S-methionine and 5 Ci 35S-
cysteine (available from Amersham) were added to radiolabel proteins. The
cells
were further incubated for 16 hours and then they were harvested by
centrifugation. The proteins in the supernatant as well as the intracellular
proteins
were analyzed by SDS-PAGE followed by autoradiography. Microsequencing of
the amino acid sequence of the amino terminus of purified protein was used to
determine the amino terminal sequence of the mature protein and thus the
cleavage point and length of the secretory signal peptide.
Example 2(b): Cloning and Expression of TR2 Protein in a Baculovirus
Expression System
Similarly to the cloning and expression of the truncated version of the TR2
receptor described in Example 2(a), recombinant baculoviruses were generated
which express the full length TR2 receptor protein shown in FIG. 1 A-1 B (SEQ
ID NO: 2).
In this example, the plasmid shuttle vector pA2 was used to insert the
cloned DNA encoding the complete protein, including its naturally associated
secretary signal (leader) sequence, into a baculovirus to express the mature
TR2
protein. Other attributes of the pA2 vector are as described for the pA2 GP
vector used in Example 2(a).
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The cDNA sequence encoding the full length TR2 protein in the deposited
clone, including the AUG initiation codon and the naturally associated leader
sequence shown in FIG. IA-113 (SEQ ID NO:2), was amplified using PCR
oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
The
5' primer has the sequence:
5' GCGCGGATCCACCATGGAGCCTCCTGGAGACTGG 3' (SEQ IDNO:18)
containing the underlined Bam1U restriction enzyme site, an efficient signal
for
initiation of translation in eukaryotic cells, as described by Kozak, M., J.
Mol.
Biol. 196:947-950 (1987), followed by 21 bases of the sequence of the complete
TR2 protein shown in FIG. IA-1B, beginning with the AUG initiation codon. The
3' primer has the sequence:
5' GCGCGGTACCTCTACCCCAGCAGGGGCGCCA 3' (SEQ ID NO: 19)
containing the underlined, Asp718 restriction site followed by 21 nucleotides
complementary to the 3' noncoding sequence in FIG. IA-1B.
The amplified fragment was isolated and digested with restriction enzymes
as described in Example 2(a) to produce plasmid pBacTR2
5 g of pBacTR2 was co-transfected with 1 gg of BaculoGoldTM
(Pharmingen) viral DNA and 10 l of LipofectinTM (Life Technologies, Inc.) in
a
total volume of 200 l serum free media. The primary viruses were harvested at
4-5 days post-infection (pi), and used in plaque assays. Plaque purified
viruses
were subsequently amplified and frozen, as described in Example 2(a).
For radiolabeling of expressed proteins, Sf9 cells were seeded in 12 well
dishes with 2.0 ml of a cell suspension containing 0.5 x 10' cells/ml and
allowed
to attach for 4 hours. Recombinant baculoviruses were used to infect the cells
at
an MOI of 1-2. After 4 hours, the media was replaced with 1.0 ml of serum free
media depleted for methionine and cysteine (-Met/-Cys). At 3 days pi, the
culture
media was replaced with 0.5 ml -Met/-Cys containing 2 gCi each [35S]-Met and
[35S]-Cys. Cells were labeled for 16 hours after which the culture media was
removed and clarified by centrifugation (Supernatant). The cells were lysed in
the
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dish by addition of 0.2 ml lysis buffer (20 mM HEPES, pH 7.9; 130 mM NaCI; 0.2
mM EDTA; 0.5 mM DTT and 0.5% vol/vol NP-40) and then diluted up to 1.0 ml
with dH2O (Cell Extract). 30 l of each supernatant and cell extract were
resolved by 15% SDS-PAGE. Protein gels were stained, destained, amplified,
dried and autoradiographed. Labeled bands corresponding to the recombinant
proteins were visible after 16-72 hours exposure.
Example 3
Cloning and Expression of TR2 in Mammalian Cells
A typical mammalian expression vector contains the promoter element,
which mediates the initiation of transcription of mRNA, the protein coding
sequence, and signals required for the termination of transcription and
polyadenylation of the transcript. Additional elements include enhancers,
Kozak
sequences and intervening sequences flanked by donor and acceptor sites for
RNA
splicing. Highly efficient transcription can be achieved with the early and
late
promoters from SV40, the long terminal repeats (LTRS) from Retroviruses, e.g.,
RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).
However, cellular elements can also be used (e.g., the human actin promoter).
Suitable expression vectors for use in practicing the present invention
include, for
example, vectors such as PSVL and PMSG (Pharmacia, Uppsala, Sweden),
pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC
67109). Mammalian host cells that could be used include, human HeLa 293, H9
and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail
QC 1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
Alternatively, the gene can be expressed in stable cell lines that contain the
gene integrated into a chromosome. The co-transfection with a selectable
marker
such as dhfr, gpt, neomycin, or hygromycin allows the identification and
isolation
of the transfected cells.
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The transfected gene can also be amplified to express large amounts of the
encoded protein. The DHFR (dihydrofolate reductase) marker is useful to
develop cell lines that carry several hundred or even several thousand copies
of the
gene of interest. Another useful selection marker is the enzyme glutamine
synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington el
al.,
Bio/Technology 10:169-175 (1992)). Using these markers, the mammalian cells
are grown in selective medium and the cells with the highest resistance are
selected. These cell lines contain the amplified gene(s) integrated into a
chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the
production of proteins.
The expression vectors pC1 and pC4 contain the strong promoter (LTR)
of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-
447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell
41:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme
cleavage sites Barn}, Xbal and Asp7l8, facilitate the cloning of the gene of
interest. The vectors contain in addition the 3' intron, the polyadenylation
and
termination signal of the rat preproinsulin gene.
Example 3(a): Cloning and Expression in COS Cells
The expression plasmid, pTR2 HA, is made by cloning a cDNA encoding
TR2 into the expression vector pcDNAI/Amp or pcDNAIII (which can be
obtained from Invitrogen, Inc.).
The expression vector pcDNAI/amp contains: (1) an E. coli origin of
replication effective for propagation in E. coli and other prokaryotic cells;
(2) an
ampicillin resistance gene for selection of plasmid-containing prokaryotic
cells; (3)
an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV
promoter, a polylinker, an SV40 intron; (5) several codons encoding a
hemagglutinin fragment (i.e., an "HA" tag to facilitate purification) followed
by
a termination codon and polyadenylation signal arranged so that a cDNA can be
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conveniently placed under expression control of the CMV promoter and operably
linked to the SV40 intron and the polyadenylation signal by means of
restriction
sites in the polylinker. The HA tag corresponds to an epitope derived from the
influenza hemagglutinin protein described by Wilson et al., Cell 37:767
(1984).
The fusion of the HA tag to the target protein allows easy detection and
recovery
of the recombinant protein with an antibody that recognizes the HA epitope.
pcDNAIII contains, in addition, the selectable neomycin marker.
A DNA fragment encoding a TR2 is cloned into the polylinker region of
the vector so that recombinant protein expression is directed by the CMV
promoter. The plasmid construction strategy is as follows. The TR2 cDNA of the
deposited clone is amplified using primers that contain convenient restriction
sites,
much as described above for construction of vectors for expression of TR2 in
E.
coll. Suitable primers include the following, which are used in this example.
The
5' primer, containing the underlined Baml-II site, a Kozak sequence, an AUG
start
codon and 6 additional codons of the 5' coding region of the complete TR2 has
the following sequence:
5' GCGCGGATCCACCATGGAGCCTCCTGGAGACTGG 3' (SEQ ID NO:20).
The 3' primer, containing the underlined XbaI site, a stop codon, HA tag, and
19
bp of 3' coding sequence has the following sequence (at the 3' end):
5' GCGCTCTAGATCAAGCGTAGTCTGGGACGTCGT
ATGGGTAGTGGTTTGGGCTCCTCCC 3' (SEQ ID NO:21).
The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are
digested with BamHI and Xbal and then ligated. The ligation mixture is
transformed into E. coil strain SURE (available from Stratagene Cloning
Systems,
11099 North Torrey Pines Road, La Jolla, CA 92037), and the transformed
culture is plated on ampicillin media plates which then are incubated to allow
growth of ampicillin resistant colonies. Plasmid DNA is isolated from
resistant
colonies and examined by restriction analysis or other means for the presence
of
the TR2-encoding fragment.
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For expression of recombinant TR2, COS cells are transfected with an
expression vector, as described above, using DEAE-DEXTRAN, as described, for
instance, in Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold
Spring Laboratory Press, Cold Spring Harbor, New York (1989). Cells are
incubated under conditions for expression of TR2 by the vector.
Expression of the TR2-HA fusion protein is detected by radiolabeling and
immunoprecipitation, using methods described in, for example Harlow et all,
Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York (1988). To this end, two days after
transfection, the cells are labeled by incubation in media containing 35S-
cysteine
for 8 hours. The cells and the media are collected, and the cells are washed
and
lysed with detergent-containing RIPA buffer: 150 mM NaCI, 1% NP-40, 0.1%
SDS, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited
above. Proteins are precipitated from the cell lysate and from the culture
media
using an HA-specific monoclonal antibody. The precipitated proteins then are
analyzed by SDS-PAGE and autoradiography. An expression product of the
expected size is seen in the cell lysate, which is not seen in negative
controls.
Example 3(b): Cloning and Expression in CHO Cells
The vector pC4 is used for the expression of TR2 protein. Plasmid pC4
is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). The
plasmid contains the mouse DHFR gene under control of the SV40 early
promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity
that are transfected with these plasmids can be selected by growing the cells
in a
selective medium (alpha minus MEM, Life Technologies) supplemented with the
chemotherapeutic agent methotrexate. The amplification of the DHFR genes in
cells resistant to methotrexate (MTX) has been well documented (see, e.g.,
Alt,
F. W., Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, JBiol. Chem.
253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,
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1097:107-143, Page, M. J. and Sydenham, M.A. 1991, Biotechnology 9:64-68).
Cells grown in increasing concentrations of MTX develop resistance to the drug
by overproducing the target enzyme, DHFR, as a result of amplification of the
DHFR gene. If a second gene is linked to the DHFR gene, it is usually co-
amplified and over-expressed. It is known in the art that this approach may be
used to develop cell lines carrying more than 1,000 copies of the amplified
gene(s). Subsequently, when the methotrexate is withdrawn, cell lines are
obtained which contain the amplified gene integrated into one or more
chromosome(s) of the host cell.
Plasmid pC4 contains for expressing the gene of interest the strong
promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen,
et al., Molecular and Cellular Biology, March 1985:438-447) plus a fragment
isolated from the enhancer of the immediate early gene of human
cytomegalovirus
(CMV) (Boshart et al., Cell 41:521-530 (1985)). Downstream of the promoter
are BamHI, XbaI, and Asp718 restriction enzyme cleavage sites that allow
integration of the genes. Behind these cloning sites the plasmid contains the
3'
intron and polyadenylation site of the rat preproinsulin gene. Other high
efficiency
promoters can also be used for the expression, e.g., the human R-actin
promoter,
the S V40 early or late promoters or the long terminal repeats from other
retroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On gene
expression systems and similar systems can be used to express the TR2 protein
in
a regulated way in mammalian cells (Gossen, M., & Bujard, H. 1992, Proc. Natl.
Acad. Sci. USA 89: 5547-5551). For the polyadenylation of the mRNA other
signals, e.g., from the human growth hormone or globin genes can be used as
well.
Stable cell lines carrying a gene of interest integrated into the chromosomes
can
also be selected upon co-transfection with a selectable marker such as gpt,
G418
or hygromycin. It is advantageous to use more than one selectable marker in
the
beginning, e.g., G418 plus methotrexate.
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The plasmid pC4 is digested with the restriction enzymes Bami and
Asp718 and then dephosphorylated using calf intestinal phosphatase by
procedures
known in the art. The vector is then isolated from a 1% agarose gel.
The DNA sequence encoding the complete TR2 protein including its
leader sequence is amplified using PCR oligonucleotide primers corresponding
to
the 5' and 3' sequences of the gene. The 5' primer has the sequence:
5' GCGC TCCACCATGGAGCCTCCTGGAGACTGG 3' (SEQ ID NO:22)
containing the underlined BamHI restriction enzyme site followed by an
efficient
signal for initiation of translation in eukaryotes, as described by Kozak, M.,
J.
Mol. Biol. 196:947-950 (1987), and 21 bases of the coding sequence of TR2
protein shown in FIG. lA-1B (SEQ ID NO: 1). The 3' primer has the sequence:
5' GCGCGGTACCTCTACCCCAGCAGGGGCGCCA 3' (SEQ ID NO: 19)
containing the underlined Asp718 restriction site followed by 21 nucleotides
complementary to the non-translated region of the TR2 gene shown in FIG. I A-
lB (SEQ ID NO:1).
The amplified fragment is digested with the endonucleases BamHI and
Asp718 and then purified again on a 1% agarose gel. The isolated fragment and
the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB
101
or XL-1 Blue cells are then transformed and bacteria are identified that
contain the
fragment inserted into plasmid pC4 using, for instance, restriction enzyme
analysis.
Chinese hamster ovary cells lacking an active DHFR gene are used for
transfection. 5 pg of the expression plasmid pC4 is cotransfected with 0.5 g
of
the plasmid pSV2-neo using lipofectin (Feigner et al., supra). The plasmid
pSV2-
neo contains a dominant selectable marker, the neo gene from Tn5 encoding an
enzyme that confers resistance to a group of antibiotics including G418. The
cells
are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,
the cells are trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of
metothrexate plus I mg/ml G418. After about 10-14 days single clones are
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trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using
different
concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).
Clones growing at the highest concentrations of methotrexate are then
transferred
to new 6-well plates containing even higher concentrations of methotrexate
(1 M, 2 M, 5 M, 10 mM, 20 mM). The same procedure is repeated until
clones are obtained which grow at a concentration of 100 - 200 M. Expression
of the desired gene product is analyzed, for instance, by SDS-PAGE and Western
blot or by reverse phase HPLC analysis.
Example 4
Tissue distribution of TR2 mRNA expression
Northern blot analysis is carried out to examine TR2 gene expression in
human tissues, using methods described by, among others, Sambrook et al.,
cited
above. A cDNA probe containing the entire nucleotide sequence of the TR2
protein (SEQ ID NO: 1) is labeled with 32P using the rediprimeTM DNA labeling
system (Amersham Life Science), according to manufacturer's instructions.
After
labeling, the probe is purified using a CHROMA SPIN- I OOTM column (Clontech
Laboratories, Inc.), according to manufacturer's protocol number PT 1200-1.
The
purified labeled probe is then used to examine various human tissues for TR2
mRNA.
Multiple Tissue Northern (MTN) blots containing various human tissues
(H) or human immune system tissues (IM) are obtained from Clontech and are
examined with the labeled probe using ExpressHybTM hybridization solution
(Clontech) according to manufacturer's protocol number PT1190-1. Following
hybridization and washing, the blots are mounted and exposed to film at -70 C
overnight, and films developed according to standard procedures.
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Example 5
Example 5(a): Expression and Purification of TR2-Fc(TR2-Ig Fusion
Protein) and Cleaved TR2
The putative transmembrane domain of translated TR2 receptor was
determined by hydrophobicity using the method of Goldman et at. (Ann. Rev. of
Biophys. Biophys. Chem. 15:321-353 (1986)) for identifying nonpolar
transbilayer
helices. The region upstream of this transmembrane domain, encoding the
putative leader peptide and extracellular domain, was chosen for the
production
of an Fc fusion protein. Primers were designed to PCR the corresponding coding
region from HTXBS40 with the addition of a BgIII site (single underlined), a
Factor Xa protease site and an Asp7181 site (double underlined) at the 3' end.
PCR with this primer pair (forward 35-mer:
5' CAGGAATTCGCAGCCATGGAGCCTCCTGGAGACTG 3' (SEQ ID NO:23),
and reverse primer 53-mer:
5 ' CCATACCCAGGTACCCCTTCCCTCGATA ATCT
TGCCTTCGTCACCAGCCAGC 3' (SEQ ID NO:24)), which contains 18
nucleotides of the TR2 coding sequence, resulted in one band of the expected
size.
This was cloned into COSFclink to give the TR2-Fclink plasmid. The PCR
product was digested with EcoRI and Asp718I and ligated into the COSFclink
plasmid (Johansen, et al., J. Biol. Chem. 270:9459-9471 (1995)) to produce TR2-
Fclink.
COS cells were transiently transfected with TR2-Fclink and the resulting
supernatant was immunoprecipitated with protein A agarose. Western blot
analysis of the immunoprecipitate using goat anti-human Fc antibodies revealed
a strong band consistent with the expected size for glycosylated TR2-Fc
(greater
than 47.5 kD). A 15L transient COS transfection was performed and the
resulting
supernatant was purified (see below). The purified protein was used to
immunize
mice following DNA injection for the production of mAbs.
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CHO cells were transfected with TR2-Fclink to produce stable cell lines.
Five lines were chosen by dot blot analysis for expansion and were adapted to
shaker flasks. The line with the highest level of TR2-Fc protein expression
was
identified by Western blot analysis. TR2-Fc protein purified from the
supernatant
of this line was used for cell binding studies by flow cytometry, either as
intact
protein or after factor Xa cleavage and biotinylation (see below).
Clone HTXBS40 is an allelic variant of TR2 which differs from the
sequence shown in FIG. ]A-1B (SEQ ID NO:1) in that HTXBS40 contains
guanine at nucleotide 314, thymine at nucleotide 386 and cytosine at
nucleotide
627.
A plasmid suitable for expression of the extracellular domain of TR2 was
constructed as follows to immunize mice for the production of anti-TR2 mAbs.
The Fc fragment was removed from TR2-Fclink by a BglII/XbaI digestion,
Klenow was used to fill in the overhangs, and the blunt ends of the plasmid
were
religated. The resulting frame shift introduced a stop codon immediately
following the amino acids which had originally been introduced into TR2-Fclink
by the addition of the BglH site. Thus, the C terminus of the extracellular
domain
of TR2 is followed by only 2 amino acids (RS) in this constructed (TR2exlink).
Example 5(b): Purification of TR2-Fc from CHO EJA Conditioned Media
Followed by Cleavage and Biotinylation of TR2.
Assays
Product purity through the purification was monitored on 15% Laemmli
SDS-PAGE gels run under reducing and non-reducing conditions. Protein
concentration was monitored by A280 assuming an extinction coefficient of 0.7
for
the receptor and 1.28 for the chimera, both calculated from the sequence.
Extinction coefficients were confirmed by AAA.
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Protein G Chromatography of the TR2-Fc Fusion Protein
All steps described below were carried out at 4 C. 15L of CHO
conditioned media (CM) (0.2 filtered following harvest in cell culture) was
applied to a 5 X 10 cm column of Protein G at a linear flow rate of 199 cm/h.
The
column had been washed with 100 mM glycine, pH 2.5 and equilibrated in 20 mm
sodium phosphate, 150 mM sodium chloride, pH 7 prior to sample application.
After the CM was loaded the column was washed with 5 column volumes of 20
mM sodium phosphate, 150 mM sodium chloride, pH 7 and eluted with 100 mM
glycine, pH 2.5. 435 ml of eluate was immediately neutralized with 3 M Tris,
pH
8.5 and 0.2 g filtered. Based on A280, extinction coefficient 1.28, 65 mg of
protein
was recovered at 0.15 mg/ml.
Concentration/Dialysis
385 ml of Protein G eluate was concentrated in an Amicon stirred cell
fitted with a 30K membrane to 34 ml at a final concentration of 1.7. The
concentrate was dialyzed against buffer.
Factor Xa Cleavage and Purification to Generate Free Receptor
Six ml (10.2 mg) of TR2-Fc was added to 50 g of Factor Xa resulting in
a 1:200, e:s ratio. The mixture was incubated overnight at 4 C.
Protein G Chromatography of the Free TR2 receptor
A 1 nil column of Protein G was equilibrated in 20 mM sodium phosphate,
150 mM sodium chloride, pH 6.5 in a disposable column using gravity flow. The
cleaved receptor was passed over the column 3 times after which the column was
washed with 20 mM sodium phosphate, 150 mM sodium chloride, pH 6.5 until no
A280 absorbance was seen. The column was eluted with 2.5 ml of 100 mM
glycine, pH 2.5 neutralized with 83 i of 3 M Tris, pH 8.5. TR2 eluted in the
nonbound fraction.
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Concentration
The nonbound fraction from the Protein G column, about 12 ml, was
TM
concentrated in a Centricon IOK cell (Amicon) to about I ml to a final
concentration of 3.5 mg/ml estimated by A280, extinction coefficient 0.7.
Mono S Chromatography
The concentrated sample was diluted to 5 ml with 20 mM sodium
phosphate, pH 6 and applied to a 0.5 X 5 cm Mono S column equilibrated in 20
mM sodium phosphate, pH 6 at a linear flow rate of 300 cm/h. The column was
washed with 20 mM sodium phosphate, pH 6 and eluted with a 20 column volume
linear gradient of 20 mM sodium phosphate, pH 6 to 20 mM sodium phosphate,
1 M sodium chloride, pH 6. TR2 protein eluted in the nonbound fraction.
Concentration/Dialysis
TM
The 3 ml nonbound fraction from the Mono S column was concentrated
to I ml as above using a Centricon 10K cell and dialyze against 20 mM sodium
phosphate, 150 mM sodium chloride, pH 7. The concentration following dialysis
was 2.1 mg/ml.
Biotinylation
0.5 mg of TR2 at 2.1 mg/ml was dialyzed against 100 mM borate, pH 8.5.
A 20-fold molar excess of NHS-LC Biotin was added and the mixture was left on
a rotator overnight at 4 C. The biotinylated TR2 was dialyzed against. 20 mM
sodium phosphate, 150 mM sodium chloride, pH 7, sterile filtered and stored at
-70 C. Biotinylation was demonstrated on a Western blot probed with
strepavidin
HRP and subsequently developed with ECL reagent.
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Example 6
The Membrane Bound Form of the TR2 Receptor is a TNFR which Induces
Lymphocytes Proliferation and Differentiation
The members of the tumor necrosis factor (TNFR)/nerve growth factor
receptor (NGFR) superfamily are characterized by the presence of three to six
repeats of a cysteine-rich motif that consists of approximately 30 to 40 amino
acids in the extracellular part of the molecule (Mallett, S. and Barclay, AN,
Immunol. Today 12:220 (1991)). The crystal structure of TNFR-I showed that
the cysteine-rich motif (TNFR domain) was composed of three elongated strands
of residues held together by a twisted ladder of disulfide bonds (Banner, D.W.
et
al., Cell 73:431 (1993). These receptors contain a hinge-like region
immediately
adjacent to the transmembrane domain, characterized by a lack of cysteine
residues and a high proportion of serine, threonine, and proline, which are
likely
to be glycosylated with O-linked sugars. A cytoplasmic part of these molecules
shows limited sequence similarities - a finding which may be the basis for
diverse
cellular signaling. At present, the members identified from human cells
include
CD40 (Stamenkovic, I. et al., F.MBO J. 8:1403 (1989)), 4-1BB (Kwon, B.S. and
Weissman, S.M., Proc. Natl. Acad. Sci. USA 86:1963 (1989)), OX-40 (Mallett,
S. et al., EMBO J. 9:1063 (1990)), TNFR-I (Loetscher, H. et al., (.'ell 61:351
(1990); Schall, T.J. el al., Cell 61:361 (1990)), TNFR-II (Smith, C.A. et al.,
Science 248:1019 (1990)), CD27 (Van Lier, R.A. et al., J. Immunol. 139:1589
(1987)), Fas (Itoh, N. etal., Cell 66:233 (1991)), NGFR (Johnson, D. el al.,
Cell
47:545 (1986)), CD30 (Durkop, H. et al., Cell 68:421 (1992)) and LTBR (Baens,
M. et al., Genomics 16:214 (1993)). Viral open reading frames encoding soluble
TNFRs have also been identified, such as SFV-T2 (Smith, C.A. et al., Science
248:1019 (1990)), Va53 (Howard, S.T. et al., Virology 180:633 (1991)), G4RG
(Hu, F.-Q, et al., Virology 204:343 (1994)) and crmB (Smith, G.L.,,1. Gen.
Viol.
74:1725 (1993)).
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Recent intensive studies have shown that these molecules are involved in
diverse biological activities such as immunoregulation (Armitage, R.J., Curr.
Opin. Immunol. 6:407 (1994); Smith, C.A. et al., Cell 75:959 (1994)), by
regulating cell proliferation (Banchereau, J. et al., Science 251:70 (1991);
Pollok,
K.E. et al., J. Immunol. 150:771 (1993); Baum, P.R. et al., EMBO J. 13:3992
(1994)), cell survival (Grass, H.-J. et al., Blood 83:2045 (1994); Torcia, M.
et al.,
('ell 85:345-356 (1996)), and cell death (Tartaglia, L.A. et al., Cell 74:845
(1993); Gillette-Ferguson, I. and Sidman, C.L., Eur. J. Immunol. 24:1181
(1994);
Krammer, P.H. etal., (..'urr. Opin. Immunol. 6:279 (1994)).
Because of their biological significance and the diverse membership of this
superfamily, we predicted that there would be further members of the
superfamily.
By searching an EST-data base, we have identified a new member of the TNFR
superfamily. We report here the initial characterization of the molecule
called
TR2.
Material and Methods
Identification and Cloning of New Members of the TNFR Superfamily
An expressed sequence tag (EST) cDNA data base, obtained over 500
different cDNA libraries (Adams, M.D. et al., Science 252:1651 (1991); Adams,
M.D. et al., Nature 355:632 (1992)), was screened for sequence similarity with
cysteine-rich motif of the TNFR superfamily, using the blastn and tblastn
algorithms (Altschul, S.F. et al., J. Mol. Biol. 215:403 (1990)). One EST
(HTISB52 - ATCC Accession No. 97059) was identified in a human T cell line
library which showed significant identity to TNFR-II at the amino acid level.
This
sequence was used to clone the missing 5' end by RACE (rapid amplification of
cDNA ends) using a 5'-RACE-ready cDNA of human leukocytes (Clontech,
PT1155-1. Cat. #7301-1). This sequence matched four further ESTs (HTOBH42,
HTOAU65, HLHA49 and HTXBS40). Complete sequencing of these and other
cDNAs indicated that they contained an identical open reading frame homologous
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to the TNFR superfamily and was named TR2. Analysis of several other ESTs
and cDNAs indicated that some cDNAs had additional sequences inserted in the
open reading frame identified above, and might represent various partially-
spliced
mRNAs.
Cells
The myeloid and B-cell lines studied represent cell types at different stages
of the differentiation pathway. KG1a and PLB 985 (Koeffler, H. el al., Blood
56:265 (1980); Tucker, K. et al., Blood 70:372 (1987)) were obtained from
Phillip Koeffler (UCLA School of Medicine), BJA-B was from Z. Jonak
(SmithKline Beecham), and TF 274, a stromal cell line exhibiting osteoblastic
features, was generated from the bone marrow of a healthy male donor (Tan &
Jonak, unpublished). All of the other cell lines were obtained from the
American
Type Culture Collection (Rockville, MD). Monocytes were prepared by
differential centrifugation of peripheral blood mononuclear cells (PBMC) and
adhesion to tissue culture dish. CD19', CD4' and CD8' were isolated from
PBMC by immunomagnetic beads (Dynal, Lake Success, NY). Endothelial cells
from human coronary artery were purchased from clonetics (Clonetics, CA).
RNA and DNA Blot Hybridization
Total RNA of adult tissues was purchases from Clontech (Palo Alto, CA),
or extracted from primary cells and cell lines with TriReagent (Molecular
Research Center, Inc., Cincinnati, OH). 5 to 7.5 jig of total RNA was
fractionated in a 1% agarose gel containing formaldehyde, as described
(Sambrook el al., Molecular Cloning, Cold Springs Harbor (1989)) and
transferred quantitatively to Zeta-probe nylon membrane (Biorad, Hercules, CA)
by vacuum-blotting. The blots were prehybridized, hybridized with 32P-labeled
XhoI/EcoRI fragment of TR2 or OX-40 probe, washed under stringent conditions
and exposed to X-ray films.
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High molecular weight human DNA was digested with various restriction
enzymes and fractionated in 0.8% agarose gel. The DNA was denatured,
neutralized and transferred to nylon membrane and hybridized to 32P-labeled TR-
2
or its variant cDNA.
In Situ Hybridization and FISH Detection
The in situ hybridization and FISH detection of TR2 location in human
chromosome were performed as previously described (Heng, H.H.Q. et al., Proc.
Natl. Acad. Sci. USA 89:9509 (1992); Heng, H.H.Q. el al., Human Molecular
Genetics 3:61 (1994)). FISH signals and the DAPI banding pattern were recorded
separately by taking photographs, and the assignment of the FISH mapping data
with chromosomal bands was achieved by superimposing FISH signals with DAPI
banded chromosome (Heng, H.H.Q. and Tsui, L.-C., Chromosoma. 102:325
(1993)).
Production of Recombination TR2-Fc Fusion Proteins
The 5' portion of the TR2 containing the entire putative open reading
frame of extracellular domain was amplified by polymerase chain reaction
(Saiki,
R.K. et al., Science 239:487 (1988)). For correctly oriented cloning, a
Hindlll
site on the 5' end of the forward primer and a BgIII site on the 5' end of the
reverse primer were created. The Fc portion of human IgG, was PCR-amplified
from ARH-77 (ATCC) cell RNA and cloned in Smal site of pGem7 vector
(Promega). The Fc fragment including hinge, CH2, and CH3 domain sequences
contained a BglII site at its 5' end and an Xhol site at its 3' end. The
HindlIl-
Bg1II fragment of TR2 cDNA was inserted into the upstream of human IgG,Fc
and an in frame fusion was confirmed by sequencing. The TR2-Fc fragment was
released by digesting the plasmid with HindIII-XhoI and cloned it into pcDNA3
expression plasmid.
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The TR2-Fc plasmid, linearized with Pvul, was transferred into NIH 3T3
by the calcium phosphate co-precipitation method. After selection in 400 g/ml
G418, neomycin-resistant colonies were picked and expanded. ELISA with anti-
human IgG, and Northern analysis with 32P-labeled TR2 probe were used to
select
clones that produce high levels of TR2-Fc in the supernatant. In some
experiments, a slightly different engineered TR2-Fc produced in Chinese
hamster
ovary (CHO) cells was used. The TR2-Fc was purified by protein G
chromatography, and the amino acid sequence of N-terminus of the TR2-Fc fusion
protein was determined by automatic peptide sequencer (ABI). TR2-Fc was used
to produce polyclonal rabbit anti-TR2 antibodies.
Blocking MLR-Mediated PBMC Proliferation
TM
PBMC were isolated from three healthy adult volunteers by Ficoll gradient
centrifugation at 400 x g for 30 minutes. PBMCs were recovered, washed in
RPMI 1640 (GIBCO-BRL) supplemented with 10% FBS, 300 g/ml L-glutamine
and 50 .tg/ml genetomycin, and adjusted to l x 106 cells/ml for two donors and
to 2 x 1 OS cells/ml for the third donor.
Fifty 1 of each cell suspension was added to 96-well (round bottom)
plates (Falcon, Franklin Lakes, NS) together with 50 l of TR2-Fc, IL-5R-Fc,
anti-CD4 mAb or control mAb. Plates were incubated at 37 C in 5% CO2 for
96 hours. One iCi of [3H]-methylthymidine (ICN Biomedicals, Costa Mesa, CA)
was then added for an additional 16 hours. Cells were harvested and
radioactivity
was counted.
Results and Discussion
TR2 is a New Member of the TNFR Superfamily
FIG. IA-113 (SEQ ID NO:2) shows the amino acid sequence of TR2
deduced from the longest open reading frame of one of the isolated cDNAs
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(HLHAB49). Comparison with other sequenced cDNAs and ESTs in the
database indicated potential allelic variants which resulted in amino acid
changes
at positions 17 (either Arg or Lys) and 41 (either Ser or Phe) of the protein
sequence shown in FIG. lA-lB (amino acid residues -20 and 5 in SEQ ID NO:2).
The open reading frame encodes 283 amino acids with a calculated
molecular weight of 30,417. The TR2 protein was expected to be a receptor.
Therefore, the potential signal sequence and transmembrane domain were sought.
A hydrophobic stretch of 23 amino acids towards the C terminus (amino acids
201-225) (FIG. lA-1B) was assigned as a transmembrane domain because it made
a potentially single helical span, but the signal sequence was less obvious.
The
potential ectodomain TR2 was expressed in NIH 3T3 and CHO cells as a
Fc-fusion protein, and the N-terminal amino acid sequence of the recombinant
TR2-Fc protein was determined in both cases. The N-terminal sequence of the
processed mature TR2 started from amino acid 37, indicating that the first 36
amino acids constituted the signal sequence (FIG. IA-IB).
Using a polyclonal rabbit antibody raised to TR2, the molecular size of
natural TR2 was determined to be 38 kD by Western analysis. Since the protein
backbone of processed TR2 would be composed of 247 amino acids with an Mr
of 26,000, the protein must be modified post-translationally. Two potential
asparagine-linked glycosylation sites are located at amino acid positions 110
and
173 (FIG. IA-113). Along with the other members of the TNFR family, TR2
contains the characteristic cysteine-rich motifs which have been shown by X-
ray
crystallography (Banner et al., Cell 73:431 (1993)) to represent a repetitive
structural unit (Banner, D.W. et al., Cell 73:431 (1993)). FIG. 16 shows the
potential TNFR domain aligned among TR2 (SEQ ID NO:2), TNFR-I (SEQ ID
NO: 10), TNFR-II (SEQ ID NO: 11), CD40 (SEQ ID NO: 12) and 4-1BB (SEQ
ID NO: 13). TR2 contained two perfect TNFR domain and two imperfect ones.
The TR2 cytoplasmic tail (TR2 cy) appears to be more closely related to
those ofCD40cy and 4-1BBcy, and does not contain the death domain seen in the
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Fas and TNFR-I intracellular domains. Although the homology is moderate, the
Thr" of TR2 is aligned with Thr233 of 4-1 BB and Thr254 of CD40. This may be
significant because Inui et al., (Inui, S. et al., Eur. J. Immunol. 20:1747
(1990))
found that Thr254 was essential for CD40 signal transduction and when the
Thr254
of CD40 was mutated, the CD40 bd did not bind to the CD40cy (Hu, H.M. el al.,
J. Biol. Chem. 269:30069 (1994)). Signals through 4-1BB and CD40 have been
shown to be costimulatory to T cells and B cells respectively (Banchereau, J.
and
Rousset, F., Nature 353:678 (1991); Hurtaldo, J. et al., J Immunol. 155:3360
(1995)).
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TABLE 2
GENE EXPRESSION OF TR2 AND OX40 IN TISSUES AND CELLS
GENE
SOURCE TR2 OX-40
TISSUES (adult)
Brain +/- -
Heart + -
Lung + -
Thymus ++ -
Spleen +-r+ -
Liver + -
Kidneyy + -
Small Intestine +++ -
Prostate ++ -
Skeletal Muscle +/- -
Ovary +
Pancreas +
Colon +
Thyroid +
Spinal Cord +
"i rachea +
Adrenal Gland +
Lymph Node +++
PRIMARY CELLS
PBL, CD 19+ ++ -
PBL,CD8+ ++ -
PBL, CD8+ (activated) ++ ++
PBL +++
PBL, CD4+ (activated) ++ ++
Bone Marrow + -
Monocle ++ -
Endothelial + -
HEMATOPOIETIC CELL LINES
Erythroid
K562
HEL +
Myeloid
KG 1 a romyeloblast) + +
KGI Myeloblast) t t
PLB985 ate myeloblast)
HL60 romyelocyte)
U937 Promonoc e) t
THP-1 onocyte)~ + -
B-Lymphocyte
REH -preB f
BJA-B arty B + -
Rai azure'B; I +
IM 9 -Nature B, LgG) - -
T-Lymphocyte
Sup-TI CD4+
Molt-3 CD4+ f -
H9 CI)4+ +
Jurkat CD4+ + +
no en+try = not tested, - = not detected
to = increasing amounts of RNA detected
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TR2 RNA Expression
A human tissue RNA blot was used to determine tissue distribution of TR2
RNA expression. TR2 RNA was detected in several tissues with a relatively high
level in the lung, spleen and thymus (Table 2) but was not detected by this
method
in the brain, liver or skeletal muscle (Table 2). TR-2 was also expressed in
monocytes, CD19+ B cells, and resting or PMA plus PHA-treated CD4+ or CD8"
T cells. It was only weakly expressed in bone marrow and endothelial cells
(Tables 2 and 3), although expression was observed in the hematopoietic cell
line
KGla (Table 2). For comparison, the tissue distribution of OX-40, another
member of the TNFR superfamily, was examined (Table 2). Unlike TR2, OX-40
was not detected in any tissues examined, and was detected only in activated T-
cells and KGIa. Several cell lines were negative for TR2 expression, including
TF
274 (bone marrow stromal), MG 63 and TE 85 (osteosarcoma), RL 95-2
(endometrial sarcoma), MCF-7 and T-47D (breast cancer cells), BE, HT 29
(colon cancer cells), HTB-1I and MM-32 (neuroblastoma), although TR2 was
found in the rhabdosarcoma HTB-82 (data not shown),
Several cell lines were examined for inducible TR2 expression. HL60,
U937 and THP1, which belong to the myelomonocytic lineage, all increased TR2
expression in response to the differentiation agents PMA or DMSO. Increases in
expression in response to these agents were observed in KGIa and Jurkat cells.
In contrast, PMA did not induce TR2 expression in MG63, but unexpectedly
TNF-a did.
In almost all cases, the predominant mRNA was approximately 1.7 kb in
size, although several higher molecular weight species could be detected in
some
tissues. While many cDNAs and ESTs which were sequenced contained insertions
in the coding region indicative of partial splicing, we only detected one
major
protein by Western blot, suggesting that if these encode alternate proteins
they are
not evident in the cells we examined. The abundance of higher MW mRNAs
raises the possibility that TR2 may in part be regulated at the level of mRNA
maturation.
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TABLE 3
RELATIVE ABUNDANCE (RA) OF TR2 RNA IN
VARIOUS TISSUE AND CELL TYPES
Tissue or Cell Type RA Tissue or Cell Type RA
Activated Macrophage (LPS) 22 Fetal Heart 1
Breast Lymph Node 5 Fetal Lung 2
B Cell Lymphoma 5 Gliobiastoma 1
Activated Monocytes 2 Hypothalamus, Schizophrenia 1
Activated T Cells 3 Infant Brain 2
Activated Neutrophil 2 Lung 2
Tonsils 5 Osteosarcoma I
Thymus 3 Pancreas Tumor 1
Anergic T-cell 1 Placenta 2
Jurkat T-Cell 3 Small Intestine 1
Raji Cells (Cycloheximide Treated) 3 Smooth Muscle 1
Atrophic Endometrium I Stomach 2
Bone Marrow 1 T-Cell Lymphoma 1
Brain I T-Cells 1
Breast 1 Testes 3
CD34 Depleted Buffy Coat (Cord Blood) I Testes Tumor 2
Cerebellum I Tongue I
Corpus Colosum 1 Umbilical Vein Endothelial Cells 2
Caco-2 Cells (adenocarcinoma, colon) 1 White Fat 3
Fetal Dura Mater I
TR2 Maps at 1P36.2-P36.3
The FISH mapping procedure was applied to localize the TR2 gene to a
specific human chromosomal region. The assignment of a hybridization signal to
the short arm of chromosome I was obtained with the aid of DAPI banding. A
total of 10 metatic figures. were photographed which indicated that the TR2
gene
is located on the chromosome 1 region p36.2-p36.3. The TR2 position is in
close
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proximity with CD30 (Smith, C.A. et aL, Cell 73:1349-1360 (1993), 4-IBB
(Kwon, B.S. et al., J. ImmunoL 152:2256-2262 (1994); Goodwin, R.G. et aL,
Eur. J. ImmunoL 23:2631-2641 (1993), OX-40 (Birkeland, M.L. et al., Eur. J.
Immunol. 25:926-930 (1995), and TNFR-II (Baker, E. et al., Cytogenet. & Cell
Genet. 57:117-118 (1991), suggesting that it evolved through a localized gene
duplication event. Interestingly, all of these receptors have stimulatory
phenotypes in T cells in response to cognate ligand binding, in contrast to
Fas and
TNFR-I which stimulate apoptosis. This prompted us to test if TR2 might be
involved in lymphocyte stimulation.
TR2-Fc Interfaces with MLR-Mediated Proliferation of PBMC
To determine the possible involvement of cell surface TR2 with its ligand
in lymphocyte proliferation, we examined allogeneic MLR proliferative
responses.
When TR-2-Fc was added to the culture, a significant reduction of maximal
responses was observed (p <0.05). The addition of TR2-Fc at 100 4g/ml
inhibited
the proliferation up to 53%. No significant inhibition of proliferation was
observed with the control IL-5R-Fc. Surprisingly, at high concentrations (10-
100
gg/ml) IL-5R-Fc was shown to enhance proliferation. An anti-CD4 mAb assayed
simultaneously inhibited MLR-mediated proliferation up to 60%, whereas a
control anti-IL-S mAb failed to inhibit the proliferation. It is well known
that a
major component of the MLR proliferative response is T cell-dependent; hence,
it would appear that inhibiting the interaction of TR2 with its ligand
prevents
optimal T lymphocyte activation and proliferation. The inhibition of MLR
proliferation by TR2-Fc at concentrations of 1-100 g/ml compares favorably
with
biological effects seen with other TNFR-Fc superfamily members such as CD40-
Fc (unpublished results, Jeremy Harrop).
Hence, we have identified an additional member of the TNF receptor
superfamily which either plays a direct role in T cell stimulation or binds to
a
ligand which can stimulate T cell proliferation through one or more receptors
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which may include TR2. Consistent with a direct role for TR2 is the similarity
of
the cytoplasmic domain with CD40 and 4-1BB. We are currently trying to
identify this ligand to which TR2 binds in order to clarify its role.
It will be clear that the invention may be practiced otherwise than as
particularly described in the foregoing description and examples.
Numerous modifications and variations of the present invention are
possible in light of the above teachings and, therefore, are within the scope
of the
appended claims.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANTS: Human Genome Sciences, Inc.
9410 Key West Avenue
Rockville, MD 20850
United States of America
Smithkline Beecham Corporation
709 Swedeland Road
King of Prussia, PA 19406
United States of America
APPLICANTS/INVENTORS: Ni, Jian
Rosen, Craig A.
Gentz, Reiner L.
Lyn, Sally Doreen Patricia
Hurle, Mark Robert
(ii) TITLE OF INVENTION: Human Tumor Necrosis Factor
Receptor-Like 2
(iii) NUMBER OF SEQUENCES: 24
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Sterne, Kessler, Goldstein & Fox, P.L.L.C.
(B) STREET: 1100 New York Ave, Suite 600
(C) CITY: Washington
(D) STATE: DC
(E) COUNTRY: USA
(F) ZIP: 20005-3934
(v) COMPUTER READABLE FORM:
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(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: TBA
(B) FILING DATE: Herewith
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Goldstein, Jorge A.
(B) REGISTRATION NUMBER: 29,021
(C) REFERENCE/DOCKET NUMBER: 1488.077PC04/JAG/EKS/SGW
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 202-271-2600
(B) TELEFAX: 202-271-2540
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(A) LENGTH: 1704 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 265..1113
(ix) FEATURE:
(A) NAME/KEY: sigpeptide
(B) LOCATION: 265..372
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 373..1113
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GCACGAGCTG CCTCCCGCAG GCGCCACCTG TGTCCCCCAG CGCCGCTCCA CCCAGCAGGC 60
CTGAGCCCCT CTCTGCTGCC AGACACCCCC TGCTGCCCAC TCTCCTGCTG CTCGGGTTCT 120
GAGGCACAGC TTGTCACACC GAGGCGGATT CTCTTTCTCT TTCTCTTTCT CTTCTGGCCC 180
ACAGCCGCAG CAATGGCGCT GAGTTCCTCT GCTGGAGTTC ATCCTGCTAG CTGGGTTCCC 240
GAGCTGCCGG TCTGAGCCTG AGGC ATG GAG CCT CCT GGA GAC TGG GGG CCT 291
Met Glu Pro Pro Gly Asp Trp Gly Pro
-36 -35 -30
CCT CCC TGG AGA TCC ACC CCC AAA ACC GAC GTC TTG AGG CTG GTG CTG 339
Pro Pro Trp Arg Ser Thr Pro Lys Thr Asp Val Leu Arg Leu Val Leu
-25 -20 -15
TAT CTC ACC TTC CTG GGA GCC CCC TGC TAC GCC CCA GCT CTG CCG TCC 387
Tyr Leu Thr Phe Leu Gly Ala Pro Cys Tyr Ala Pro Ala Leu Pro Ser
-10 -5 1 5
TGC AAG GAG GAC GAG TAC CCA GTG GGC TCC GAG TGC TGC CCC AAG TGC 435
Cys Lys Glu Asp Glu Tyr Pro Val Gly Ser Glu Cys Cys Pro Lys Cys
15 20
AGT CCA GGT TAT CGT GTG AAG GAG GCC TGC GGG GAG CTG ACG GGC ACA 483
Ser Pro Gly Tyr Arg Val Lys Glu Ala Cys Gly Glu Leu Thr Gly Thr
25 30 35
GTG TGT GAA CCC TGC CCT CCA GGC ACC TAC ATT GCC CAC CTC AAT GGC 531
Val Cys Glu Pro Cys Pro Pro Gly Thr Tyr Ile Ala His Leu Asn Gly
40 45 50
CTA AGC AAG TGT CTG CAG TGC CAA ATG TGT GAC CCA GCC ATG GGC CTG 579
Leu Ser Lys Cys Leu Gln Cys Gln Met Cys Asp Pro Ala Met Gly Leu
55 60 65
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-87-
CGC GCG AGC CGG AAC TGC TCC AGG ACA GAG AAC GCC GTG TGT GGT TGC 627
Arg Ala Ser Arg Asn Cys Ser Arg Thr Glu Asn Ala Val Cys Gly Cys
70 75 80 85
AGC CCA GGC CAC TTC TGC ATC GTC CAG GAC GGG GAC CAC TGC GCC GCG 675
Ser Pro Gly His Phe Cys Ile Val Gln Asp Gly Asp His Cys Ala Ala
90 95 100
TGC CGC GCT TAC GCC ACC TCC AGC CCG GGC CAG AGG GTG CAG AAG GGA 723
Cys Arg Ala Tyr Ala Thr Ser Ser Pro Gly Gln Arg Val Gln Lys Gly
105 110 115
GGC ACC GAG AGT CAG GAC ACC CTG TGT CAG AAC TGC CCC CCG GGG ACC 771
Gly Thr Glu Ser Gln Asp Thr Leu Cys Gln Asn Cys Pro Pro Gly Thr
120 125 130
TTC TCT CCC AAT GGG ACC CTG GAG GAA TGT CAG CAC CAG ACC AAG TGC 819
Phe Ser Pro Asn Gly Thr Leu Glu Glu Cys Gln His Gln Thr Lys Cys
135 140 145
AGC TGG CTG GTG ACG AAG GCC GGA GCT GGG ACC AGC AGC TCC CAC TGG 867
Ser Trp Leu Val Thr Lys Ala Gly Ala Gly Thr Ser Ser Ser His Trp
150 155 160 165
GTA TGG TGG TTT CTC TCA GGG AGC CTC GTC ATC GTC ATT GTT TGC TCC 915
Val Trp Trp Phe Leu Ser Gly Ser Leu Val Ile Val Ile Val Cys Ser
170 175 180
ACA GTT GGC CTA ATC ATA TGT GTG AAA AGA AGA AAG CCA AGG GGT GAT 963
Thr Val Gly Leu Ile Ile Cys Val Lys Arg Arg Lys Pro Arg Gly Asp
185 190 195
GTA GTC AAG GTG ATC GTC TCC GTC CAG CGG AAA AGA CAG GAG GCA GAA 1011
Val Val Lys Val Ile Val Ser Val Gln Arg Lys Arg Gln Glu Ala Glu
200 205 210
GGT GAG GCC ACA GTC ATT GAG GCC CTG CAG GCC CCT CCG GAC GTC ACC 1059
Gly Glu Ala Thr Val Ile Glu Ala Leu Gln Ala Pro Pro Asp Val Thr
215 220 225
ACG GTG GCC GTG GAG GAG ACA ATA CCC TCA TTC ACG GGG AGG AGC CCA 1107
Thr Val Ala Val Glu Glu Thr Ile Pro Ser Phe Thr Gly Arg Ser Pro
230 235 240 245
AAC CAC TGACCCACAG ACTCTGCACC CCGACGCCAG AGATACCTGG AGCGACGGCT 1163
Asn His
GAATGAAAGA GGCTGTCCAC CTGGCGGAAC CACCGGAGCC CGGAGGCTTG GGGGCTCCAC 1223
CCTGGACTGG CTTCCGTCTC CTCCAGTGGA GGGAGAGGTG GCGCCCCTGC TGGGGTAGAG 1283
CTGGGGACGC CACGTGCCAT TCCCATGGGC CAGTGAGGGC CTGGGGCCTC TGTTCTGCTG 1343
TGGCCTGAGC TCCCCAGAGT CCTGAGGAGG AGCGCCAGTT GCCCCTCGCT CACAGACCAC 1403
ACACCCAGCC CTCCTGGGCC AACCCAGAGG GCCTTCAGAC CCCAGCTGTG TGCGCGTCTG 1463
ACTCTTGTGG CCTCAGCAGG ACAGGCCCCG GGCACTGCCT CACAGCCAAG GCTGGACTGG 1523
CA 02270913 1999-04-30
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-88-
GTTGGCTGCA GTGTGGTGTT TAGTGGATAC CACATCGGAA GTGATTTTCT AAATTGGATT 1583
TGAATTCGGC TCCTGTTTTC TATTTGTCAT GAAACAGTGT ATTTGGGGAG ATGCTGTGGG 1643
AGGATGTAAA TATCTTGTTT CTCCTCAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1703
A 1704
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 283 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg Ser Thr Pro
-36 -35 -30 -25
Lys Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr Phe Leu Gly Ala
-20 -15 -10 -5
Pro Cys Tyr Ala Pro Ala Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro
1 5 10
Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys
15 20 25
Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro
30 35 40
Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys
45 50 55 60
Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser
65 70 75
Arg Thr Glu Asn Ala Val Cys Gly Cys Ser Pro Gly His Phe Cys Ile
80 85 90
Val Gln Asp Gly Asp His Cys Ala Ala Cys Arg Ala Tyr Ala Thr Ser
95 100 105
Ser Pro Gly Gln Arg Val Gln Lys Gly Gly Thr Glu Ser Gln Asp Thr
110 115 120
Leu Cys Gln Asn Cys Pro Pro Gly Thr Phe Ser Pro Asn Gly Thr Leu
125 130 135 140
Glu Glu Cys Gln His Gln Thr Lys Cys Ser Trp Leu Val Thr Lys Ala
145 150 155
Gly Ala Gly Thr Ser Ser Ser His Trp Val Trp Trp Phe Leu Ser Gly
160 165 170
CA 02270913 1999-04-30
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-89-
Ser Leu Val Ile Val Ile Val Cys Ser Thr Val Gly Leu Ile Ile Cys
175 180 185
Val Lys Arg Arg Lys Pro Arg Gly Asp Val Val Lys Val Ile Val Ser
190 195 200
Val Gln Arg Lys Arg Gln Glu Ala Glu Gly Glu Ala Thr Val Ile Glu
205 210 215 220
Ala Leu Gln Ala Pro Pro Asp Val Thr Thr Val Ala Val Glu Glu Thr
225 230 235
Ile Pro Ser Phe Thr Gly Arg Ser Pro Asn His
240 245
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 281 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Val Ser Leu Pro Arg Leu Cys Ala Leu Trp Gly Cys Leu Leu Thr
1 5 10 15
Ala Val His Leu Gly Gln Cys Val Thr Cys Ser Asp Lys Gln Tyr Leu
20 25 30
His Asp Gly Gln Cys Cys Asp Leu Cys Gln Pro Gly Ser Arg Leu Thr
35 40 45
Ser His Cys Thr Ala Leu Glu Lys Thr Gln Cys His Pro Cys Asp Ser
50 55 60
Gly Glu Phe Ser Ala Gln Trp Asn Arg Glu Ile Arg Cys His Gin His
65 70 75 80
Arg His Cys Glu Pro Asn Gln Gly Leu Arg Val Lys Lys Glu Gly Thr
85 90 95
Ala Glu Ser Asp Thr Val Cys Thr Cys Lys Glu Gly Gln His Cys Thr
100 105 110
Ser Lys Asp Cys Glu Ala Cys Ala Gln His Thr Pro Cys Ile Pro Gly
115 120 125
Phe Gly Val Met Glu Met Ala Thr Glu Thr Thr Asp Thr Val Cys His
130 135 140
Pro Cys Pro Val Gly Phe Phe Ser Asn Gln Ser Ser Leu Phe Glu Lys
145 150 155 160
CA 02270913 1999-04-30
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-90-
Cys Tyr Pro Trp Thr Ser Cys Glu Asp Lys Asn Leu Glu Val Leu Gln
165 170 175
Lys Gly Thr Ser Gln Thr Asn Val Ile Cys Gly Leu Lys Ser Arg Met
180 185 190
Arg Ala Leu Leu Val Ile Pro Val Val Met Gly Ile Leu Ile Thr Ile
195 200 205
Phe Gly Val Phe Leu Tyr Ile Lys Lys Val Val Lys Lys Pro Lys Asp
210 215 220
Asn Glu Met Leu Pro Pro Ala Ala Arg Arg Gln Asp Pro Gln Glu Met
225 230 235 240
Glu Asp Tyr Pro Gly His Asn Thr Ala Ala Pro Val Gin Glu Thr Leu
245 250 255
His Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile
260 265 270
Ser Val Gln Glu Arg Gln Val Thr Asp
275 280
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2692 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 373..927
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION: 373..480
(ix) FEATURE:
(A) NAME/KEY: mat peptide
(B) LOCATION: 481..927
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CCCCCTTCTA CAGGAAACCC GGAGTGGACT GGAACGGTGC AGGGGGAGAA CTCGCCCCTC 60
CCATCGGGCG CCTCCTTCAT ACCGGCCCTT CCCCTCGGCT TTGCCTGGAC AGCTCCTGCC 120
TCAGGCAGCG CCACCTGTGT CGCCCAGCGC CGCTCCACCC AGCAGGCCTG AGCCCCTCTC 180
TGCTGCCAGA CACCCCCTGC TGCCCACTAC TCCTGCTGCT CGGGTTCTGA GGCACAGCTT 240
GTCACACCGA GGCGGATTCT CTTTCTCTTT CTCTTTCTCT TCTGGCCCAC AGCCGCAGCA 300
CA 02270913 1999-04-30
WO 98/18824 PCT/US96/18540
-91-
ATGGCGCTGA GTTCCTCTGC TGGAGTTCAT CCTGCTAGCT GGGTTCCCGA GCTGCCGGTC 360
TGAGCCTGAG TC ATG GAG CCT CCT GGA GAC TGG GGG CCT CCT CCC TGG 408
Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp
-36 -35 -30 -25
AGA TCC ACC CCC AGA ACC GAC GTC TTG AGG CTG GTG CTG TAT CTC ACC 456
Arg Ser Thr Pro Arg Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr
-20 -15 -10
TTC CTG GGA GCC CCC TGC TAC GCC CCA GCT CTG CCG TCC TGC AAG GAG 504
Phe Leu Gly Ala Pro Cys Tyr Ala Pro Ala Leu Pro Ser Cys Lys Glu
-5 1 5
GAC GAG TAC CCA GTG GGC TCC GAG TGC TGC CCC AAG TGC AGT CCA GGT 552
Asp Glu Tyr Pro Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly
15 20
TAT CGT GTG AAG GAG GCC TGC GGG GAG CTG ACG GGC ACA GTG TGT GAA 600
Tyr Arg Val Lys Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu
25 30 35 40
CCC TGC CCT CCA GGC ACC TAC ATT GCC CAC CTC AAT GGC CTA AGC AAG 648
Pro Cys Pro Pro Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys
45 50 55
TGT CTG CAG TGC CAA ATG TGT GAC CCA GAT ATT GGT TCC CCC TGT GAC 696
Cys Leu Gln Cys Gln Met Cys Asp Pro Asp Ile Gly Ser Pro Cys Asp
60 65 70
CTC AGG GGA AGA GGT CAC CTG GAG GCT GGT GCC CAC CTG AGT CCA GGC 744
Leu Arg Gly Arg Gly His Leu Glu Ala Gly Ala His Leu Ser Pro Gly
75 80 85
AGA CAG AAA GGG GAA CCA GAC CCA GAG GTG GCC TTT GAG TCA CTG AGC 792
Arg Gln Lys Gly Glu Pro Asp Pro Glu Val Ala Phe Glu Ser Leu Ser
90 95 100
GCA GAG CCT GTC CAT GCG GCC AAC GGC TCT GTC CCC TTG GAG CCT CAT 840
Ala Glu Pro Val His Ala Ala Asn Gly Ser Val Pro Leu Glu Pro His
105 110 115 120
GCC AGG CTC AGC ATG GCC AGT GCT CCC TGC GGC CAG GCA GGA CTG CAC 888
Ala Arg Leu Ser Met Ala Ser Ala Pro Cys Gly Gln Ala Gly Leu His
125 130 135
CTG CGG GAC AGG GCT GAC GGC ACA CCT GGG GGC AGG GCC TGAGCCTACA 937
Leu Arg Asp Arg Ala Asp Gly Thr Pro Gly Gly Arg Ala
140 145
GGGAGGCACA GGGCAGGTGG GCTAGCCATG AACAGAAGAG GAAGCTGGAG TGCTTTGGGG 997
GTTCATGCAT GTAGGCTGGG ATTTGGGGCT CACACCTCAA CCTGCATGCC CAGTTCCATG 1057
CCCCTCCCCT CTTGTGAAAG CACCTGTCTA CTTGGGCTGA GGATGTGGGG GCACAGGTGG 1117
CAGGTGAGGC TGCCCTCAGG AGGGGCCCAG GCCCAGCTTG TACCCCACCT CCACCAGTAC 1177
CTGAAGAAGT GGGGCTCTCA CCCTACCTGC CTCTGCCATT GGAATGGCCT GGTTTGCACA 1237
CA 02270913 1999-04-30
WO 98/18824 PCTIUS96/18540
-92-
GATGGGAAAC CCGTTTGAGG GGTGGGTGTC TGGGTGGGCA CGTGGGGCGA GGACCTGCCT 1297
GAGGGACCCT GCCCTGGAAC TGACAGTGCA AGCTCGGCGT CCTGCCCATC TGGGCAGAAG 1357
GCTGGTTTCT CCCATCAACG AAGCCCTCCC AGGACCTTCC TGCAAGCCCT CGTCCCACAC 1417
GCAGCTCTGC CGTCCCTTGG TGTCCCTCCC GGCCTCAGGT CCTCCATGCT GGGTACCTCT 1477
GGGCACCTCG TTTGGCTGAG CCAGGGGTTC AGCCTGGCAG GGCGCCCTGG CAGCAGTCCT 1537
TGGCCTGTGG ATGCTGTCCT GGCCTGTGGA TGGTGTCCCG CCCTCCACGT ACCCCTCTCA 1597
CCCCCTCCTC TTGGACTCCA GCCATGGGCC TGCGCGCGAG CCGGAACTGC TCCAGGACAG 1657
AGAACGCCGT GTGTGGCTGC AGCCCAGGCC ACTTCTGCAT CGTCCAGGAC GGGGACCACT 1717
GCGCCGCGTG CCGCGCTTAC GCCACCTCCA GCCCGGGCCA GAGGGTGCAG AAGGGAGGCA 1777
CCGAGAGTCA GGACACCCTG TGTCAGAACT GCCCCCCGGG GACCTTCTCT CCCAATGGGA 1837
CCCTGGAGGA ATGTCAGCAC CAGACCAATT GGCCTAATCA TATGTGTGAA AAGAAGAAAG 1897
CCAAGGGGTG AGCACACGGT GGCCCCATCA GGGTTCATGT CCCCAGCCGT CACCTCTTGG 1957
AGCTCTGTCA CCCCAGGCGT GGGAGGTGGC CCCAGAGCTT TTCCAGGATC CGCGGCTCCT 2017
CCCAGGGCAG CCACTGCAGG CTGGGGCAGG TGTATGTAGT CAAGGTGATC GTCTCCGTCC 2077
AGCGGTAAAA GACAGGAGGC AGAAGGTGAG GCCACAGTCA TTGAGCCCTG CAGGCCCCTC 2137
CGGACGTCAC CACGGTGGCC GTGGAGGAGA CAATACCCTC ATTCACGGGG AGGAGCCCAA 2197
ACCACTGACC CACAGACTCT GCACCCCGAC GCCAGAGATA CCTGGAGAGA CGGCTGCTGA 2257
TAGAGGCTGT CCACCTGGCG AAACCACCGG AGCCCGGAGG CTTGGGGGCT CCGCCCTGGG 2317
CTGGTTTCCG TCTCCTCCAG TGGAGGGAGA GGTGGTGCCC CTGCTGGTGG TAGAGCTGGG 2377
GACGCCACGT GCCATTCCCA TGGTTCAGTG AGGGGCTGGT GGCCTCTGTT CTGCTGTGGC 2437
CTGAGCTCCC CAGAGTCCTG AGGAGGAGCC CCAGTTGCCC CTCGCTCACA GACCACACAC 2497
CCAGCCCTCC TGGGCCAACC CAGAGGCCCC TTCAGACCCC AGCTGTCTGC GCGTCTGACT 2557
CTTGTGGCCT CAGCAGGACA GGCCCCGGGC ACTGCCTCAC AGCCAAGGCT GGAATGGGTT 2617
GGCTGCAGTG TGGTGTTTAG TGGATACCAC ATCGGAAGTG ATTTTCTAAA AATTGGATTT 2677
GAATTCGGAA AAAAA 2692
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 185 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
CA 02270913 1999-04-30
WO 98/18824 PCTIUS96/18540
-93-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg Ser Thr Pro
-36 -35 -30 -25
Arg Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr Phe Leu Gly Ala
-20 -15 -10 -5
Pro Cys Tyr Ala Pro Ala Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro
1 5 10
Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys
15 20 25
Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro
30 35 40
Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys
45 50 55 60
Gln Met Cys Asp Pro Asp Ile Gly Ser Pro Cys Asp Leu Arg Gly Arg
65 70 75
Gly His Leu Glu Ala Gly Ala His Leu Ser Pro Gly Arg Gln Lys Gly
80 85 90
Glu Pro Asp Pro Glu Val Ala Phe Glu Ser Leu Ser Ala Glu Pro Val
95 100 105
His Ala Ala Asn Gly Ser Val Pro Leu Glu Pro His Ala Arg Leu Ser
110 115 120
Met Ala Ser Ala Pro Cys Gly Gln Ala Gly Leu His Leu Arg Asp Arg
125 130 135 140
Ala Asp Gly Thr Pro Gly Gly Arg Ala
145
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 248 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu
1 5 10 15
Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr
20 25 30
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WO 98/18824 PCTIUS96/18540
-94-
Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln
35 40 45
Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys
50 55 60
Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp
65 70 75 80
Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys
85 90 95
Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg
100 105 110
Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu
115 120 125
Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg
130 135 140
Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val
145 150 155 160
Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr
165 170 175
Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly
180 185 190
Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser
195 200 205
Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser
210 215 220
Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser
225 230 235 240
Phe Leu Leu Pro Met Gly Pro Ser
245
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2637 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 247..654
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CA 02270913 1999-04-30
WO 98/18824 PCTIUS96/18540
-95-
AAAGCTCGGG CTCCACCGGG GACGACCGCT CCTAGAAACT GAGTGGTATC CCCCGGGCCT 60
GCAGGAATTC CAACCTGCCT GAAGGGACCC TGCCCTGGAA CTGACAGTGC AAGCTCGGCG 120
TCCTGCCCAT CTGGGAAGAA GGCTGGTTTC TCCCATCAAC GAAGCCCTCC CAGGACCTTC 180
CTGCAAGCCC TCGTCCCACA CGCAGCTCTG CCGTCCCTTG GTGTCCCTCC CGGCCTCAGG 240
TCCTCC ATG CTG GGT ACC TCT GGG CAC CTC GTT TGG CTG AGC CAG GGG 288
Met Leu Gly Thr Ser Gly His Leu Val Trp Leu Ser Gln Gly
150 155 160
TTC AGC CTG GCA GGG CGC CCT GGC AGC AGT CCT TGG CCT GTG GAT GCT 336
Phe Ser Leu Ala Gly Arg Pro Gly Ser Ser Pro Trp Pro Val Asp Ala
165 170 175
GTC CTG GCC TGT GGA TGG TGT CCC GGC CTC CAC GTA CCC CCT CTC AGC 384
Val Leu Ala Cys Gly Trp Cys Pro Gly Leu His Val Pro Pro Leu Ser
180 185 190 195
CCC TCC TCT TGG ACT CCA GCC ATG GGC CTG CGC GCG AGC CGG AAC TGC 432
Pro Ser Ser Trp Thr Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys
200 205 210
TCC AGG ACA GAG AAC GCC GTG TGT GGC TGC AGC CCA GGC CAC TTC TGC 480
Ser Arg Thr Glu Asn Ala Val Cys Gly Cys Ser Pro Gly His Phe Cys
215 220 225
ATC GTC CAG GAC GGG GAC CAC TGC GCC GCG TGC CGC GCT TAC GCC ACC 528
Ile Val Gln Asp Gly Asp His Cys Ala Ala Cys Arg Ala Tyr Ala Thr
230 235 240
TCC AGC CCG GGC CAG AGG GTG CAG AAG GGA GGC ACC GAG AGT CAG GAC 576
Ser Ser Pro Gly Gln Arg Val Gln Lys Gly Gly Thr Glu Ser Gln Asp
245 250 255
ACC CTG TGT CAG AAC TGC CCC CGG GGA CCT TCT CTC CCA ATG GGA CCC 624
Thr Leu Cys Gln Asn Cys Pro Arg Gly Pro Ser Leu Pro Met Gly Pro
260 265 270 275
TGG AGG AAT GTC AGC ACC AGA CCA AGT AAG TGAACCCGGG GGAGGCCAGC 674
Trp Arg Asn Val Ser Thr Arg Pro Ser Lys
280 285
TCTGTGCCCT GGGGAGGGGG CTCCACGTTG CTTCCCTGGG AGATGACCGT CTTCTCCAGC 734
AGAAAGGTTG AAGGTCCCAC CCTGAGCGGC ACCCTGGTCA CATGCCTGCG TCCAGGAGAG 794
CTGCAGGGTG AAGCCTGTGT GCCCCAGATA ACCCCTTCCA TGGGCCCAGA CAAAGCCTCA 854
TCAGATCTGA GCTTCCTGGA GGCTCAGGAT GGGCCTTCCC AGAAGCAGGC CCAGAGGGAG 914
GCTGCCTCCA GATCCCCTGT CCCCTGGGGC TGTGGGTGTC CCTGAATGTC AGGGCCATGG 974
GAGGGCCCCT GGGCTTCAGG GGTTGGGGAA AGTGAACACT CTGCTCTTTG TCCACCTTCG 1034
GGAGGACAAC CTTCAAATGC TGACCCTGGG CCCCTAACTG ACCTGAGACT TCAGAGCTTC 1094
TTGGGAGGAG CTGGGGTCCC CCAGCGGAGC CTGGGATGGA GCAGGGATGG CTGCCCCAGG 1154
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-96-
GAGGGGGCGG TGGGGCCTTC CATCCTGCTC TGCCCTCCTC GTCCTCTGGC CCCAGCTCAG 1214
TCCTGTCCAT CTCCAGCTCT AACCATTTGT GGCCCGACAC TGGCTCTCCC TCTACCTTCT 1274
GTCCTTGTCT GACACTGGTC TCCCGTGCTC TGGGGTCTCT GCACTGATGG CTGCCTCCCG 1334
CTTCTCTCCC CTCTCCCTCT GCCGTCCTGT CTCCTGTGGC CAGTCTCTCC TTGTTTCTCT 1394
TCTCCTCCTT CCTTCTCTCC ACCTCCCCAT AGCCGAGCTT GGAAAAGTCA GACAGACCTC 1454
TGAGGTCTCA TCCTGGAGCT GCCACCAGCC CAGCCTCCCT GGGACCTGTC TTCACTGCCT 1514
GGGGCCCTGG GAGCCAGGGA GGCTCCCTGA GGCTGAGTGA ACACTGGGCG CTGCACCTGC 1574
CTCTCCCACG TCCTCGGCCC CACTCCCGCA GGTGCAGCTG GCTGGTGACG AAGCCCGGAG 1634
CTGGGACCAG CAGCTCCCAC TGGGTATGGT GGTTTCTCTC AGGGAGCCTC GTCATCGTCA 1694
TTGTTTGCTC CACAGTTGGC CTAATCATAT GTGTGAAAAG AAGAAAGCCA AGGGGTGATG 1754
TAGTCAAGGT GATCGTCTCC GTCCAGGTAT TGATCCTCCT CCCCCTCTCC CTCCCCCCTC 1814
CACCTTCCCA CCTCCCCTCT CCCCGCTGGG GCTGGTGTTT CTGGTGTACA TGGTGGGGGC 1874
TCCCAGTTCT CTGAGGGTCC TGAGTCTTTC AAGTACAGCC ACGGTAGCTC AGGAAAGAAC 1934
CCACCCCCTC AAACTGAAAG CAGTAAAATG AACCCGAGAA CCTGGAGTCC CAGGGGGGCC 1994
TGAGCAGGCA GGGTCTCCAC GATTCGTGTG CTCACAGCGG GAAAAGACAG GAGGCAGAAG 2054
GTGAGGCCAC AGTCATTGAG GCCCTGCAGG CCCCTCCGGA CGTCACCACG GTGGCCGTGG 2114
AGGAGACAAT ACCCTCATTC ACGGGGGAGG AGCCCAAACC ACTGACCCAC AGACTCTGCA 2174
CCCCGACGCC AGAGATACCT GGAGCGACGG CTGCTGAAAG AGGCTGTCCA CCTGGCGAAA 2234
CCACCGGAGC CCGGAGGTTT GGGGGCTCCG CCCTGGGCTG GTTTCCGTCT CCTCCAGTGG 2294
AGGGAGAGGT GGGGCCCCTG CTGGGGTAGA GCTGGGGACG CCACGTGCCA TTCCCATGGG 2354
CCAGTGAGGG CCTGGGGCCT CTGTTCTGCT GTGGCCTGAG CTCCCCAGAG TCCTGAGGAG 2414
GAGCGCCAGT TGCCCCTCGC TCACAGACCA CACACCCAGC CCTCCTGGGT CCAGCCCAGA 2474
GGGCCCTTCA GACCCCAGCT GTCTGCGCGT CTGACTCTTG TGGCCTCAGC AGGACAGGCC 2534
CCGGGCACTG CCTTCAAGCC AAGGCTGGAC TGGGTTGGCT GCAGTGTGGT GTTTAGTGGA 2594
TACCACATCG GAAGTGATTT TCTAAATTGG ATTTGAAAAA AAA 2637
(2) INFORMATION FOR SEQ ID NO:B:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 136 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
CA 02270913 1999-04-30
WO 98/18824 PCTIUS96/18540
-97-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Leu Gly Thr Ser Gly His Leu Val Trp Leu Ser Gln Gly Phe Ser
1 5 10 15
Leu Ala Gly Arg Pro Gly Ser Ser Pro Trp Pro Val Asp Ala Val Leu
20 25 30
Ala Cys Gly Trp Cys Pro Gly Leu His Val Pro Pro Leu Ser Pro Ser
35 40 45
Ser Trp Thr Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser Arg.
50 55 60
Thr Glu Asn Ala Val Cys Gly Cys Ser Pro Gly His Phe Cys Ile Val
65 70 75 80
Gln Asp Gly Asp His Cys Ala Ala Cys Arg Ala Tyr Ala Thr Ser Ser
85 90 95
Pro Gly Gln Arg Val Gln Lys Gly Gly Thr Glu Ser Gln Asp Thr Leu
100 105 110
Cys Gln Asn Cys Pro Arg Gly Pro Ser Leu Pro Met Gly Pro Trp Arg
115 120 125
Asn Val Ser Thr Arg Pro Ser Lys
130 135
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 198 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu
1 5 10 15
Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr
20 25 30
Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln
35 40 45
Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys
50 55 60
Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp
65 70 75 80
CA 02270913 1999-04-30
WO 98/18824 PCTIUS96/18540
-98-
Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys
85 90 95
Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg
100 105 110
Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu
115 120 125
Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg
130 135 140
Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val
145 150 155 160
Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr
165 170 175
Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly
180 185 190
Asn Ala Ser Met Asp Ala
195
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 154 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Val Cys Pro Gln Gly Lys Tyr Ile His Pro Gln Asn Asn Ser Ile Cys
1 5 10 15
Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly
20 25 30
Pro Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr
35 40 45
Ala Ser Glu Asn His Leu Arg His Cys Leu Ser Cys Ser Lys Cys Arg
50 55 60
Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cys Thr Val Asp Arg Asp
65 70 75 80
Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu
85 90 95
Asn Leu Phe Gln Cys Phe Asn Cys Ser Leu Cys Leu Asn Gly Thr Val
100 105 110
CA 02270913 1999-04-30
WO 98/18824 PCT/US96/18540
-99-
His Leu Ser Cys Gln Glu Lys Gln Asn Thr Val Cys Thr Cys His Ala
115 120 125
Gly Phe Phe Leu Arg Glu Asn Glu Cys Val Ser Cys Ser Asn Cys Lys
130 135 140
Lys Ser Leu Glu Cys Thr Lys Leu Cys Leu
145 150
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 163 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys
1 10 15
Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr
20 25 30
Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu
35 40 45
Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser
50 55 60
Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys
65 70 75 80
Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys
85 90 95
Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala
100 105 110
Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro
115 120 125
Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His
130 135 140
Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala
145 150 155 160
Val Cys Thr
(2) INFORMATION FOR SEQ ID NO:12:
CA 02270913 1999-04-30
WO 98/18824 PCT/US96/18540
-100-
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 163 amino acids
(B) TYPE; amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln Cys Cys Ser Leu
1 5 10 15
Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr Glu Pro Thr Glu
20 25 30
Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu Asp Thr Trp Asn
35 40 45
Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp Pro Asn Leu Gly
50 55 60
Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp Thr Ile Cys Thr
65 70 75 80
Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys Glu Ser Cys Val
85 90 95
Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys Gln Ile Ala Thr
100 105 110
Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val Gly Phe Phe Ser
115 120 125
Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp Thr Ser Cys Glu
130 135 140
Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn Lys Thr Asp Val
145 150 155 160
Val Cys Gly
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 132 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
CA 02270913 1999-04-30
WO 98/18824 PCTIUS96/18540
-101-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln
1 5 10 15
Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln
20 25 30
Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg
35 40 45
Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly
50 55 60
Phe His Cys Leu Gly Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys
65 70 75 80
Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly
85 90 95
Thr Phe Asn Lys Gln Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys
100 105 110
Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp
115 120 125
Val Val Cys Gly
130
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
CGCCCATGGC CCCAGCTCTG CCGTCCT 27
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
CA 02270913 1999-04-30
WO 98/18824 PCT/US96/18540
-102-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CGCAAGCTTA TTGTGGGAGC TGCTGGTCCC 30
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
CGCGGATCCC GGAGCCCCCT GCTAC 25
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
CGCGGTACCA TTGTGGGAGC TGCTGGTCCC 30
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GCGCGGATCC ACCATGGAGC CTCCTGGAGA CTGG 34
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
CA 02270913 1999-04-30
WO 98/18824 PCTIUS96/18540
-103-
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
GCGCGGTACC TCTACCCCAG CAGGGGCGCC A 31
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GCGCGGATCC ACCATGGAGC CTCCTGGAGA CTGG 34
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
GCGCTCTAGA TCAAGCGTAG TCTGGGACGT CGTATGGGTA GTGGTTTGGG CTCCTCCC 58
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
CA 02270913 1999-04-30
WO 98/18824 PCTIUS96/18540
-104-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
GCGCGGATCC ACCATGGAGC CTCCTGGAGA CTGG 34
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CAGGAATTCG CAGCCATGGA GCCTCCTGGA GACTG 35
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
CCATACCCAG GTACCCCTTC CCTCGATAGA TCTTGCCTTC GTCACCAGCC AGC 53
CA 02270913 1999-04-30
WO 98/18824 PCT/US96/18540
= 105-
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule l3bis)
A. The indications made below relate to the microorganism referred to in the
description
on page 4 = line ],8
D. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional
sheet E
Name of depositary institution
American Culture Collection
Address of depositary institution (iaclrtiwa portal eadera d cowry)
12301 Parkiawn Drive
Rockville, Maryland 20852
United States of krerica
Date of deposit Accession Number
February 13, 1995 ATOC 97057
C. ADDITIONAL INDICATIONS 0 love Usak i f am aprliaW4 This infatuation is
continued en au additional shit 0
Di3A Plasmid, 244,087
in respect of those designations in which a European Patent is sought a sample
of the deposited microorganism will be made available until the publication of
the mention of-the grant of the European patent or until the date on which the
application, has been refused or withdrawn or is deemed to be withdrawn, only
by
the issue of such a -san Ile to an expert nominated by the person requesting
the
le (Rule' 28 (4 EPC).
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (itrbeiadis-ionrareoo~~o-
aplai~aatdSfatet)
E. SEPARATE FURNISHING OF INDICATIONS (Tacos bfaek i/noe applicrblej
TLeindieaticuslistedbebwvwillbesubmittedtotheIttamationalBureaulater(rpeabterar
erl meefdwia/ialiear õ*Aezsniei
Number ofDeposiI
For receiving Office use only For International Bureau use only
This sheet was received with the international application [] This sheet was
received by the International Bureau on:
Authorized officer Authorized officer
PD
Verne PCt'fl O11 4 (July 19')2)
CA 02270913 1999-04-30
WO 98/1824 Pff/U396i18S4o
-106-
(DNA PLASMID, 244,087)
FINLAND
The applicant hereby requests that, until the application has been laid open
to public inspection (by the
National Board of Patents and Registration), or has been finally decided upon
by the National Board of
Patents and Registration without having been laid open to public inspection,
the furnishing of a sample
shell only be effected to an expert in the art.
DENMARK
Ile applicant hereby request that, until the application has been laid open to
public inspection (by the
Danish Patent Office), or has been finally decided upon by the Danish Patent
Office without having been
laid open to public inspection, the furnishing of a sample shall only be
effected to an expert in the art.
The request to this effect shall be filed by the applicant with the Danish
Patent Office not later than at
the time when the application is made available to the public under Sections
22 and 33(3) of the Danish
Patents Act. If such a request has been filed by the applicant, any request
made by a third party for the
furnishing of a sample shall indicate the expert to be used. That expert may
be any person entered on
a list of recognized experts drawn up by the Danish Patent office or any
person approved by the applicant
in the individual case.
SWEDEN
The applicant hereby request that, until the application has been laid open to
public inspection (by the
Swedish Patent Office), or has been finally decided upon by the Swedish Patent
Office without having
been laid open to public inspection, the furnishing of a sample shall only be
effected to an expert in the
art. The request to this effect shall be filed by the applicant with the
International Bureau before the
expiration of 16 months from the priority date (preferably on the Form
PCT/RO/134 reproduced in annex
Z of Volume I of the PCT Applicant's Guide). If such a request has been filed
by the applicant, any
request made by a third party for the furnishing of a sample shall indicate
the expert to be used. That
expert may be any person entered on a list of recognized experts drawn up by
the Swedish Patent office
or any person approved by the applicant in the individual can.
UNffED KINGDOM
The applicant hereby request that the furnishing of a sample of a
microorganism shall only be made
available to an expert. The request to this effect must be filed by the
applicant with the International
Bureau before the completion of the technical preparations for international
publication of the
application.
NETHERLANDS
The applicant hereby request that until the date of a grant of a Netherlands
patent or until the date on
which the application is refused or withdrawn or lapsed, the microorganism
shall be made available as
provided in Rule 31 F(1) of the Patent Rules only by the issue of a sample to
an expert. The request to
this effect must be furnished by the applicant with the Netherlands Industrial
Property Office before the
date on which the application is made available to the public under Section
22C or Section 25 of the
Patents Act of the Kingdom of the Netherlands, whichever of the two dates
occurs earlier.
CA 02270913 1999-04-30
WO 98/18824 PCTIUS96/IS540
-107-
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 13bis)
A. The indications made below relate to the microorganism referred to in the
description
on page 4 , line 18
B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional
:beet
Name of depositary institution
American 1~= Chili-i"_ C5n11P,-_j-_jcm
Address of depositary institution (including postal cola and country)
12301 Parklawn Drive
Rockville, Maryland 20852
United States of America
ate of deposit Accession Number
February 13, 1995 ATCC 97058
C. ADDITIONAL INDICATIONS (lewrbladt if ant a *mbk) This information is
continued on an additional sheet 0
DNA Plaslnid, 245,598
In respect of those designations in which a European Patent is sought a sample
of the deposited microorganism will be made available until the publication of
the mention of-the grant of the European patent or until the date on which the
application has been refused or withdrawn or is deemed to be withdrawn, only
by
the issue of such a sample to an expert rxmtinated by the person requesting
the
sample (Rule"28(4) EPC).
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE
(ifthewdlnao.r.nesoe/err.fllaigsrteiSt ter)
E. SEPARATE FURNISHING OF INDICATIONS (leans bleak f sot eppliwb
TheiodintiooslinedbelawvwllbesubmittedtodtebttbmationalBateaulater(spars
thegraodaetareaf1Miadariaareg., =Aoosaion
Nanrber of Deposit)
For receiving Office use only For International Bureau use only
This sheet was received with the international application 13 This sheet was
received by the International Bureau on:
Authorized officer NWMHM Authorized officer
Fnriti PGI'M0/134 (July 1 502)
CA 02270913 1999-04-30
W0 98/18824 PCT/US96/18540
(DNA PLASMID, 245,598) '108'
FINLAND
The applicant hereby requests that, until the application has been laid open
to public inspection (by the
National Board of Patents and Registration), or has been finally decided upon
by the National Board of
Patents and Registration without having been laid open to public inspection,
the furnishing of a sample
shall only be effected to an expert in the art.
DENMARK
The applicant hereby request that, until the application has been laid open to
public inspection (by the
Danish Patent Office), or has been finally decided upon by the Danish Patent
Office without having been
laid open to public inspection, the furnishing of a sample shall only be
effected to an expert in the art.
The request to this effect shall be filed by the applicant with the Danish
Patent Office not later than at
the time when the application is made available to the public under Sections
22 and 33(3) of the Danish
Patents Act. If such a request has been filed by the applicant, any request
made by a third party for the
furnishing of a sample shall indicate the expert to be used. That expert may
be any person entered on
a list of recognized experts drawn up by the Danish Patent office or any
person approved by the applicant
in the individual case.
SWEDEN
The applicant hereby request that, until the application has been laid open to
public inspection (by the
Swedish Patent Office), or has been finally decided upon by the Swedish Patent
Office without having
been laid open to public inspection, the furnishing of a sample shall only be
effected to an expert in the
art. The request to this effect shall be filed by the applicant with the
International Bureau before the
expiration of 16 months from the priority date (preferably on the Form
PCT/RO/l 34 reproduced in annex
Z of Volume I of the PCT Applicant's Guide). If such a request has been filed
by the applicant, any
request made by a third party for the furnishing of a sample shall indicate
the expert to be used. That
expert maybe any person entered on a list of recognized experts drawn up by
the Swedish Patent office
or any person approved by the applicant in the individual case.
UNITED KINGDOM
The applicant hereby request that the furnishing of a sample of a
microorganism shall only be made
available to an expert. The request to this effect must be filed by the
applicant with the International
Bureau before the completion of the technical preparations for international
publication of the
application.
NETHERLANDS
The applicant hereby request that until the date of a grant of a Netherlands
patent or until the date on
which the application is refused or withdrawn or lapsed, the microorganism
shall be made available as
provided in Rule 3 IF(l) of the Patent Rules only by the issue of a sample to
an expert. The request to
this effect must be furnished by the applicant with the Netherlands Industrial
Property Office before the
date on which the application is made available to the public under Section
22C or Section 25 of the
Patents Act of the Kingdom of the Netherlands, whichever of the two dates
occurs earlier.
CA 02270913 1999-04-30
WO 98/18824 PCT/US96I18540
-109-
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 13bis)
A. The indications made below relate to the microorganism referred to in the
description
on page . tine 18
B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional
sheet 0
Name of depositary institution
Address of depositary institution (iadudisa postal code and cow Wy)
12301 Parkiaiwn Drive
Rockville, Naryland 20852
United States of America
Date of deposit Accession Number
February 13, 1995 N1 97059
C. ADDITIONAL INDICATIONS (lawe blank i(aoI applicable) This information is
continued on an additional cbeer D
DM Plasmid, 60041
In respect of those designations in which a European Patent is sought a sample
of the deposited microorganism will be made available until the publication of
the mention of-the grant of the European patent or until the date on which the
application-has been refused or withdrawn or is deemed to be withdrawn, only
by
the issue of such a sample to an expert nominated by the person requesting'
the
seap3e (Rule:'28(4) EPC).
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE if also us&aliarr are ant
f.r eff &m VarredSates)
B. SEPARATE FURNISHING OF INDICATIONS (latrblaak if eet applicable)
TbobW&etimlWabdowwilibesubmittedtodolaftmationaI Boma
,N-b- -f D-pajitj
For receiving Office use only For International Bureau use only
This sheet was received with the international application [J his sheet was
received by the International Bureau on:
Authorized officer NlV (SSfl O~ Authorized officer
ilr C k*- ___'
l orui PCf/lt(1I34 (IMIy 1992)
CA 02270913 1999-04-30
WO 98/18824 PCT/US96/18540
-110-
(DNA PLASMID, 60041)
FINLAND
The applicant hereby requests that, until the application has been laid open
to public inspection (by the
National Board of Patents and Registration), or has been finally decided upon
by the National Board of
Patents and Registration without having been laid open to public inspection,
the furnishing of a sample
shall only be effected to an expert in the art.
DENMARK
The applicant hereby request that, until the application has been laid open to
public inspection (by the
Danish Patent Office), or has been finally decided upon by the Danish Patent
Office without having been
laid open to public inspection, the furnishing of a sample shall only be
effected to an expert in the art.
The request to this effect shall be filed by the applicant with the Danish
Patent Office not later than at
the time when the application is made available to the public under Sections
22 and 33(3) of the Danish
Patents Act. If such a request has been filed by the applicant, any request
made by a third party for the
furnishing of a sample shall indicate the expert to be used. That expert may
be any person entered on
a list of recognized experts drawn up by the Danish Patent office or any
person approved by the applicant
in the individual case.
SWEDEN
The applicant hereby request that, until the application has been laid open to
public inspection (by the
Swedish Patent Office), or has been finally decided upon by the Swedish Patent
Office without having
been laid open to public inspection, the furnishing of a sample shall only be
effected to an expert in the
art. The request to this effect shall be filed by the applicant with the
International Bureau before the
expiration of 16 months from the priority date (preferably on the Form
PCT/RO/134 reproduced in annex
Z of Volume I of the PCT Applicant's Guide). If such a request has been filed
by the applicant, any
request made by a third party for the furnishing of a sample shall indicate
the expert to be used. That
expert may be any person entered on a list of recognized experts drawn up by
the Swedish Patent office
or any person approved by the applicant in the individual case.
UNITED KINGDOM
The applicant hereby request that the furnishing of a sample of a
microorganism shall only be made
available to an expert. The request to this effect must be filed by the
applicant with the International
Bureau before the completion of the technical preparations for international
publication of the
application.
NETHERLANDS
The applicant hereby request that until the date of a grant of a Netherlands
patent or until the date on
which the application is refused or withdrawn or lapsed, the microorganism
shall be made available as
provided in Rule 31 F(1) of the Patent Rules only by the issue of a sample to
an expert. The request to
this effect must be furnished by the applicant with the Netherlands Industrial
Property Office before the
date on which the application is made available to the public under Section
22C or Section 25 of the
Patents Act of the Kingdom of the Netherlands, whichever of the two dates
occurs earlier.
