Note: Descriptions are shown in the official language in which they were submitted.
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LA PRESENTE PARTIE I)E CETTE DEMANDE OU CE BREVETS
COMPRI~:ND PLUS D'UN TOME.
CECI EST ~.E TOME 1 DE 2
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
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JUMBO APPLICATIONS / PATENTS
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THAN ONE VOLUME.
THIS IS VOLUME 1 OF 2
NOTE: For additional vohxmes please contact the Canadian Patent Oi~ice.
CA 02365405 2001-09-21
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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. lA-1B, having considerable homology to murine
CD40. Two different TR2 splice variants, referred to as TR2-SV 1 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 functional activities of TR2 receptor
polypeptides and diagnostic methods for detecting TR2 receptor gene
expression.
Related Art
Human tumor necrosis factors a (TNF-a) and (3 (TNF-~3 or lymphotoxin)
are related members of a broad class ofpolypeptide mediators, which includes
the
interferons, interleukins and growth factors, collectively called cytokines
(Beutler,
B. and Cerami, A., Ar~mr. IZev. Imnn~r2ol. 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-(3, 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.
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TNF is produced by a number of cell types, including monocytes,
fibroblasts, T-cells, natural killer (NK) cells and predominately by activated
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., .~ 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-l, 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-(3 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., P~°og. 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-(3 production show abnormal development ofthe peripheral lymphoid organs
and morphological changes in spleen architecture (reviewed in Aggarwal et al.,
E2~r 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-
(3 -/-
mice contained a three fold reduction in white blood cells as compared to
normal
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mice. Peripheral blood from TNF-~3 -/- mice, however, contained four fold more
B cells as compared to their normal counterparts. Further, TNF-(3, in contrast
to
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-~3 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 a7., 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 .lournal, 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)).
. 22-05-2000 CA 02365405 2001-09-21 US 000007521
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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
production in other cells. In addition, TNF-a has been shown to affect 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 marine 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
The present invention provides isolated nucleic acid molecules
comprising, or alternatively consisting of, polynucleotides encoding TR2
receptors and splice variants thereof having the amino acid sequences shown in
SEQ 1D N0:26, FIG. lA-1B (SEQ lD N0:2), FIG. 4A-4C (SEQ ID N0:5) and
FIG. 7A-7C (SEQ B7 N0:8) or the amino acid sequence encoded by the cDNA
encoding the TR2 receptors deposited 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.
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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
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.
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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, or alternatively consists of, 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.
Brief Description of the Figures
FIG. lA-1B shows the nucleotide (SEQ ID NO:I) and deduced amino
acid (SEQ ID N0:2) sequences of a TRZ receptor. The protein has a predicted
leader sequence of about 36 amino acid residues (underlined) (amino acid
residues
-36 to -1 in SEQ ID N0:2) 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 I 64 in SEQ ID N0:2) constitute the extracellular domain;
from
about 201 to about 225 (amino acid residues 165 to 189 in SEQ ID N0:2) the
transmembrane domain (underlined); and from about 226 to about 283 (amino
acid residues 190 to 247 in SEQ ID N0:2) the intracellular domain. Two
potential asparagine-linked glycosylation sites are located at amino acid
positions
1 10 and 173 (amino acid residues 74 to 137 in SEQ ID N0:2).
FIG. 2 shows the regions of similarity between the amino acid sequences
of the TR2 receptor protein of FIG. lA-1B and a murine CD40 protein (SEQ ID
N0:3).
FIG. 3 shows an analysis ofthe TR2 receptor amino acid sequence ofFIG.
lA-IB. 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, I 06 to 120, 142 to 189 and 276 to 283 in FIG. I A-1 B (amino acid
residues
3 to 34, 70 to 84, 106 to 153 and 240 to 247 in SEQ ID N0:2) correspond to the
shown highly antigenic regions of the TR2 receptor protein.
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FIG. 4A-4C shows the nucleotide (SEQ ID N0:4) and deduced amino
acid (SEQ ID NO:S) 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 117 NO:S) and a deduced molecular weight of
about
19.5 kDa.
FIG. 5 shows the regions of similarity between the amino acid sequences
of the full-length TR2-S V 1 receptor protein and a human type 2 TNF receptor
(SEQ ID N0:6).
FIG. 6 shows an analysis of the TR2-SV 1 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 135 to 149 in SEQ B7 NO:S) correspond to the shown highly antigenic
1 S regions of the TR2-SV 1 receptor protein.
FIG. 7A-7C shows the nucleotide (SEQ 113 N0:7) and deduced amino
acid (SEQ ID N0:8) 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 Tlt2-SV2 receptor protein and a human type 2 TNF receptor (SEQ ID
N0:9).
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 136 in FIG. 7A-7C (SEQ ID N0:8) correspond to the shown
highly antigenic regions of the TR2-SV2 receptor protein.
AMENDED SHEET
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FIG.10 shows the regions of similarity between the amino acid sequences
of the TR2 receptor protein of FIG. lA-1B and the TR2-SVl 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.
FIG.12 shows the regions of similarity between the amino acid sequences
of the TR2-SV1 and the TR2-SV2 receptor proteins.
FIG. 13A-13D shows the regions of similarity between the nucleotide
sequences encoding the TR2 receptor protein of FIG. lA-1B and the TR2-SVl
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. lA-1B and the TR2-SV2
receptor protein of FIG. 7A-7C.
FIG. 15A-15F shows the regions of similarity between the nucleotide
sequences encoding the TR2-SV 1 and the TR2-SV2 receptor proteins.
FIG. 16 shows an alignment of the amino acid sequence of the TR2
receptor of FIG. lA-1B (SEQ ID N0:2) 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 lD NO:11), CD40 (SEQ lD N0:12) and 4-1BB (SEQ ID NO:l 3)
on the basis of sequence homology and conserved cysteine residues. Cysteine
repeat regions are defined by amino acid residues 5 to 40, 41 to 84, 85 to
127, and
128 to 166 in SEQ B7 N0:2, respectively referred to as cysteine repeat regions
A-D.
FIG. 17 shows the effect of TR2 on B cell in vitro proliferation. B
lymphocytes were purified from human tonsils by immunomagnetic selection.
Cells were cultured for 72 hours followed by a 24 hour 3H thymidine pulse in
RPMI1640 medium added with 10% FBS, 4 mM 1- glutamine, 5 X 10'5 M 2ME,
100 Ulml Penicillin, 100 ~.g/ml Streptomycin, and the indicated factors.
AMENDED SHEET
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Detailed Description of the Preferred Embodiments
The present invention provides isolated nucleic acid molecules
comprising, or alternatively consisting of, polynucleotides encoding a TR2
polypeptide (FIG. 1 A-1B (SEQ ID N0:2)) and splice variants thereof, TR2-SV 1
S (FIG. 4A-4C (SEQ ID N0:5)) and TIZ2-SV2 (FIG. 7A-7C (SEQ ID N0:8)), the
amino acid sequences of which were determined by sequencing cDNAs. The
TR2 protein shown in FIG. lA-1B shares sequence homology with the marine
CD40 receptor (FIG. 2 (SEQ )D N0:3)). On February 13, 1995 a deposit was
made at the American Type Culture Collection, 10801 University Blvd.,
Manassas, VA 20110-2209, USA, and given ATCC Accession No. 97059. The
nucleotide sequence shown in FIG. lA-1B (SEQ ID NO:1) was obtained by
sequencing a cDNA which is believed to contain the same amino acid coding
sequences as the cDNA contained in the deposited plasmid assigned ATCC
Accession No. 97059 (Clone ID HLHAB49).
The TR2 receptors of the present invention include several allelic variants
containing alterations in at least four nucleotides and two amino acids.
Nucleotide sequence variants which have been identified include either guanine
or adenine at nucleotide 314 and either thymine or cytosine at nucleotides
386,
624 and 627 shown in FIG. lA-1B (SEQ ID NO:1). While the identified
alteration at nucleotides 624 and 627 are silent, the alteration at nucleotide
386
results in the codon at nucleotides 38S 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 N0:4) and FIG.
7A-7C (SEQ D7 N0:7) were also obtained by sequencing cDNAs deposited on
February 13, 1995 at the American Type Culture Collection and given accession
numbers 97058 (TR2-SV 1) and 97057 (TR2-SV2), respectively. The deposited
cDNAs are contained in the pBluescript SK(-) plasmid (Stratagene, LaJolla,
CA).
AMENDED SHEET
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As used herein the ghrase "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,
splicing occurs when a primary RNA transcript undergoes splicing, generally
for
the removal of introns, which results in the production of more than one mRNA
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", "TR2 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 SEQ 1D N0:26, FIG. lA-1B (SEQ ID N0:2), FIG. 4A-4C (SEQ ID NO:S} or
FIG. 7A-7C (SEQ ID N0:8), as well as TR2 allellic variants. The TR2 proteins
shown in SEQ ID N0:26 and FIG.1 A-1 B, the TR2-SV 1 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
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manual DNA sequencing methods well known in the art. As is also known in the
art, a single insertion or deletion in a determined nucleotide sequence
compared
to the actual sequence will cause a frame shift in translation of the
nucleotide
sequence such that the predicted amino acid sequence encoded by a determined
nucleotide sequence will be completely different from the amino acid sequence
actually encoded by the sequenced DNA molecule, beginning at the point of such
an insertion or deletion.
Using the information provided herein, such as the nucleotide sequence
in SEQ >D N0:26, FIG. lA-1B, 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 1D 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 N0:4) and FIG. 7A-7C (SEQ ID
N0:7) 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 (CD19+), both
CD4+ (TH, and T~ clones) and CD8+ T lymphocytes, monocytes and endothelial
cells.
As also noted in Example 6, the production of TRZ mRNA was inducible
in MG 63 cells by TNFa. Further, the accumulation of TR2 mRNA was observed
in HL60, U937 and THP1 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. lA-1B
(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
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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. lA-1B from
amino acid residue about 37 to residue about 283 (amino acid residues 1 to 247
in SEQ D7 N0:2). In this context "about" includes the particularly recited
value
S and values larger or smaller by severai (5, 4, 3, 2, or 1 ) amino acids. As
noted in
Example 6, the location of the leader sequence cleavage site was confirmed for
a TR2-Fc fusion protein and found to be between amino acids 36 and 37 shown
in FIG. lA-1B (amino acid residues -1 to I in SEQ 113 NO:2). The TR2 protein
shown in FIG. IA-1B (SEQ ID N0:2) is about 29% identical and about 47%
similar to the marine CD40 protein shown in SEQ ID N0:3 (see FIG. 2).
Similarly, the determined cDNA nucleotide sequences of the TR2-SV 1
splice variant of TR2 (FIG. 4A-4C (SEQ ID N0:4)) contains an open reading
frame encoding a protein of about 185 amino acid residues, with a predicted
leader sequence of about 3 6 amino acid residues, and a deduced molecular
weight
of about 19.5 lcDa. The amino acid sequence of the predicted mature TR2-SV 1
receptor is shown in FIG. 4A-4C (SEQ ID NO:S) from amino acid residue about
37 to residue about 185 (amino acid residues 1 to 149 in (SEQ ID NO:S). In
this
context "about" includes the particularly recited value and values larger or
smaller
by several (5, 4, 3, 2, or 1 ) amino acids. The TR2-S V 1 protein shown in
FIG. 4A-
4C (SEQ >I7 NO:S) is about 25% identical and about 48% similar to the human
type 2 TNF receptor protein shown in SEQ ID N0:6 (see FIG. 5).
The determined cDNA nucleotide sequences of the Tlt2-SV2 splice
variant of TR2 (FIG. 7A-7C (SEQ 11'7 N0:7)) 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 Tlt2-SV2 receptor is shown in FIG. 7A-7C (SEQ ID
N0:8) from amino acid residue about 1 to residue about 136. In this context
"about" includes the particularly recited value and values larger or smaller
by
several (5, 4, 3, 2, or 1) amino acids. The TRZ-SV2 protein shown in FIG. 7A-
7C
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(SEQ ID N0:8) is about 27% identical and about 45% similar to the human type
2 TNF receptor protein shown in SEQ ID N0:9 (see FIG. 8).
A comparison of both the nucleotide and amino acid sequences of the
TR2, TR2-SV 1 and TR2-SV2 receptor proteins shown in FIG. lA-1B, FIG. 4A-
S 4C and FIG. 7A-7C shows several regions of near identity. While the amino
acid
sequence of the TR2 receptor protein, shown in FIG. lA-1B (SEQ ID N0:2), is
about 60% identical'and about 73% similar to the amino acid sequence of the
TRZ-SV 1 receptor protein, shown in FIG. 4A-4C (SEQ ID NO:S), 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.
1 A-1 B (SEQ ID N0:2) is about 60% identical and about 7I % similar to the
amino
acid sequence of the TR2-SV2 receptor protein, shown in FIG. 7A-7C (SEQ 1D
N0:8); 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-SV 1 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, lA-1B
(SEQ ID N0:2), 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-13D, FIG.14A-14D and FIG.15A-15F). The cDNA sequences encoding the
TR2 and TR2-SV 1 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-13D). Further, the
nucleotide sequences of the cDNAs encoding the TR2-SV 1 and TR2-SV2
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proteins share considerable homology but this identity is limited to their
3'regions
well beyond their respective coding sequences (FIG. 15A-15F).
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
genomic 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 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-1B
(SEQ ID N0:2) 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-SV 1 splice variant shown in FIG. 4A-4C (SEQ ID N0:5) is believed to
contain an insertion in the region encoding amino acid residue 102 of the
amino
acid sequence shown in FIG. lA-1B and a deletion in the region encoding amino
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acid residue I84 of the amino acid sequence shown in FIG. lA-1B. 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. I A-1 B and contain insertions in the regions encoding
amino acid residues 184 and 243 of the amino acid sequence shown in FIG.
lA-1B.
As indicated, the present invention also provides the mature forms of the
TR2 receptors of the present invention. According to the signal hypothesis,
proteins secreted by mammalian cells have a signal or secretory leader
sequence
which is cleaved from the mature protein once export of the growing protein
chain
across the rough endoplasmic reticulum has been initiated. Most mammalian
cells
and even insect cells cleave secreted proteins with the same specificity.
However,
in some cases, cleavage of a secreted protein is not entirely uniform, which
results
in two or more mature species on the protein. Further, it has long been known
that the cleavage specificity of a secreted protein is ultimately determined
by the
primary structure of the complete protein, that is, it is inherent in the
amino acid
sequence of the polypeptide. Therefore, the present invention provides
nucleotide
sequences encoding mature TR2 polypeptides having the amino acid sequences
encoded by the cDNAs contained in the deposits identified as ATCG Deposit
Numbers 97059 and 97058 and as shown in SEQ ID N0:26, FIG. IA-1B (SEQ
ID N0:2) and FIG. 4A-4C (SEQ ID NO:S). By the mature TR2 polypeptides
having the amino acid sequences encoded by the cDNAs contained in the deposits
identif ed as ATCC Deposit Numbers 97059 and 97058 is meant the mature
forms) of the Tlt2 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 cDNA contained in the deposited plasmids.
The invention also provides nucleic acid sequences encoding the TR2-SV2
receptor protein of FIG. 7A-7C (SEQ 1T3 N0:8), having the amino acid sequence
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encoded by the cDNA 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 ofthese methods
is in the range of 75-80%. von Heinje, supra. However, the two methods do not
always produce the same predicted cleavage points) for a given protein.
In the present case, the predicted amino acid sequences of the complete
TR2 polypeptides shown in FIG. lA-1B (SEQ ID N0:2), FIG. 4A-4C (SEQ ID
N0:5) and FIG. 7A-7C (SEQ ID N0:8) 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 117 N0:2 and SEQ ID N0:5. Thereafter, the complete amino acid sequences
were fiu~ther 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-SV 1 protein are predicted to consist
of amino acid residues -36 to -1 in both SEQ ID N0:2 and SEQ ID NO:S, while
the predicted mature TR2 proteins consist of amino acid residues 1 to 247 for
the
TR2 protein shown in SEQ ID N0:2 and residues 1 to 149 for the TR2-SV1
protein shown in SEQ ID NO:S.
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 1 in SEQ ID N0:2 and SEQ ID NO:S, indicating that
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the cleavage site of the leader sequence is between amino acids 36 and 37 in
this
protein (corresponding to amino acid residues -1 to 1 in SEQ ID N0:2 and SEQ
ID NO:S).
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 3 I 6 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. Similarly, the TR2-SV I 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-I 52 amino
acids.
In this context "about" includes the particularly recited value and values
larger or
smaller by several (5, 4, 3, 2, or 1 ) amino acids.
The leader sequences for the TR2 protein shown in SEQ ID N0:26 is
predicted to consist of amino acid residues -38 to -1 in SEQ ID N0:26, while
the
predicted mature TR2 protein consists of amino acid residues 1 to 245 in SEQ
ID
N0:26.
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.
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By "isolated" nucleic acid molecules) is intended a nucleic acid molecule,
DNA or RNA, which has been removed from its native environment For example,
recombinant DNA molecules contained in a vector are considered isolated for
the
purposes of the present invention. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution. Isolated RNA
molecules include in vzvo 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.
However, a nucleic acid contained in a clone that is a member of a library
(e.g., a genomic or cDNA library) that has not been isolated from other
members
of the library (e.g., in the form of a homogeneous solution containing the
clone
and other members of the library) or a chromosome isolated or removed from a
cell or a cell lysate (e.g., a "chromosome spread," as in a karyotype), is not
"isolated" for the purposes of the invention. As discussed further herein,
isolated
nucleic acid molecules according to the present invention may be produced
naturally, recombinantly, or synthetically.
Isolated nucleic acid molecules of the present invention include DNA
molecules comprising, or alternatively consisting of, an open reading frame
(ORF)
shown in SEQ ID N0:26 or FIG. lA-1B (SEQ ID NO:I); DNA molecules
comprising, or alternatively consisting of, the coding sequence for the mature
TRZ
receptor shown in SEQ ID N0:26 (last 245 amino acids) or FIG. 1 A-1B (SEQ ID
N0:2) (last 247 amino acids); and DNA molecules which comprise, or
alternatively consist of, a sequence substantially different from those
described
above but which, due to the degeneracy of the genetic code, still encode the
TR2
receptor protein shown in SEQ ID N0:26 or FIG. lA-1B (SEQ ID N0:2). Of
course, the genetic code is well known in the art. Thus, it would be routine
for
one skilled in the art to generate such degenerate variants.
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Similarly, isolated nucleic acid molecules of the present invention include
DNA molecules comprising, or alternatively consisting of, an open reading
frame
(ORF) shown in FIG. 4A-4C (SEQ ID N0:4); DNA molecules comprising, or
alternatively consisting of, the coding sequence for the mature TR2-SV 1
receptor
shown in FIG. 4A-4C (SEQ ID NO: S) (last 149 amino acids); and DNA molecules
which comprise, or alternatively consist of, a sequence substantially digerent
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, or alternatively consisting of, an open reading
frame
(ORF) shown in FIG. 7A-7C (SEQ ID N0:7) and DNA molecules which
comprise, or alternatively consist of, a sequence substantially different from
those
described above but which, due to the degeneracy of the genetic code, still
encode
the 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 cDNAs 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 the full-length polypeptide lacking the N-terminal methionine.
The
invention further provides isolated nucleic acid molecules having the
nucleotide
sequences shown in SEQ ID N0:25, FIG. lA-1B (SEQ )D NO:1), FIG. 4A-4C
(SEQ ID N0:4), and FIG. 7A-7C (SEQ ID N0:7); the nucleotide sequences of the
cDNAs contained in the above-described deposited cDNAs; or nucleic acid
molecules having a sequence complementaryto one ofthe above sequences. Such
isolated molecules, particularly DNA molecules, are useful, for example, 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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Further embodiments of the invention include isolated nucleic acid
molecules comprising, or alternatively consisting of, a polynucleotide having
a
nucleotide sequence at least 80% identical, and more preferably at Least 85%,
90%, 92%, 95%, 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence
encoding the TR2 polypeptide having the complete amino acid sequence shown
in SEQ ID N0:26, FIG.1 A-1 B (amino acid residues -3 6 to 247 in SEQ ID N0:2),
FIG. 4A-4C (amino acid residues -36 to 149 in SEQ ID NO:S), or FIG. 7A-7C
(amino acid residues 1 to 136 in SEQ ID N0:8); (b) a nucleotide encoding the
complete amino sequence shown in SEQ ID N0:26, FIG. lA-1B (amino acid
residues -35 to 247 in SEQ ID N0:2), FIG. 4A-4C (amino acid residues -35 to
149 in SEQ ID NO:S), 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 1 to
about 245 in SEQ ID N0:26, from about 37 to about 283 in FIG. lA-1B (amino
acid residues 1 to 247 in SEQ 1D N0:2) or the amino acid sequence at positions
from about 37 to about 185 in FIG. 4A-4C (amino acid residues 1 to 149 in SEQ
ID NO:S), or the amino acid sequence at positions from about 1 to about 136 in
FIG. 7A-7C (SEQ ID N0:8); {d) a nucleotide sequence encoding the TR2, TR2-
SV1 or TR2-SV2 polypeptides having the complete amino acid sequence
including the leader encoded by the cDNAs contained in ATCC Deposit Numbers
97059, 97058, and 97057, respectively; (e) a nucleotide sequence encoding the
mature TR2 or TR2-S V I receptors having the amino acid sequences encoded by
the cDNAs contained inATCC DepositNumbers 97059 and 97058, respectively;
(f) a nucleotide sequence encoding the TR2 or TIt2-SV1 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 TRZ receptor extracellular and
intracellular domains with all or part of the transmembrane domain deleted;
and
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(j) a nucleotide sequence complementary to any of the nucleotide sequences in
(a), (b), (c), (d), (e), (f), (g), (h)~ or (i). In this context "about"
includes the
particularly recited value and values larger or smaller by several (5, 4, 3,
2, or 1 )
amino acids.
By a polynucleotide having a nucleotide sequence at least, for example,
95% "identical" to a reference nucleotide sequence encoding a TR2 receptor
polypeptide is intended that the nucleotide sequence of the polynucleotide is
identical to the reference sequence except that the polynucleotide sequence
may
include up to five mismatches per each 100 nucleotides of the reference
nucleotide sequence encoding a TR2 receptor. 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 mismatches 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. The reference (query) sequence may be the entire TR2
receptor encoding nucleotide sequence shown in SEQ ID N0:25, FIG. lA-1B
(SEQ ID NO:1 ), FIG. 4A-4C (SEQ II7 N0:4), or FIG. 7A-7C (SEQ ID N0:7) or
any TR2 receptor polynucleotide fragment (e.g., a polynucleotide encoding the
amino acid sequence of any of the TIZ2 receptor N- and/or C- terminal
deletions
described herein), variant, derivative or analog, as described herein.
As a practical matter, whether any particular nucleic acid molecule is at
least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for
instance, the encoding nucleotide sequence shown in SEQ ID N0:25, FIG.1 A-1 B
(SEQ ID NO: l), FIG. 4A-4C (SEQ ID N0:4), or FIG. 7A-7C (SEQ ID N0:7), or
to the nucleotide sequence of the deposited cDNAs, can be determined
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conventionally using known computer programs such as the Bestfit program
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group, University Research Park, 575 Science Drive, Madison, WI 53711).
Bestfit uses the local homology algorithm of Smith and Waterman, Advances in
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.
In a specific embodiment, the identity between a reference (query)
sequence (a sequence of the present invention) and a subject sequence, also
referred to as a global sequence alignment, is determined using the FASTDB
computer program based on the algorithm of Brutlag el al. (Comp. App. Biosci.
6:237-245 ( 1990)). Preferred parameters used in a FASTDB alignment of DNA
sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4,
Mismatch
Penalty=1, Joining Penalty=30, Randomization Group Length=0, CutoffScore=1,
Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the
subject nucleotide sequence, whichever is shorter. According to this
embodiment,
if the subject sequence is shorter than the query sequence because of 5' or 3'
deletions, not because of internal deletions, a manual correction is made to
the
results to take into consideration the fact that the FASTDB program does not
account for 5' and 3' truncations ofthe subject sequence when calculating
percent
identity. For subject sequences truncated at the 5' or 3' ends, relative to
the query
sequence, the percent identity is corrected by calculating the number of bases
of
the query sequence that are 5' and 3' of the subject sequence, which are not
matched/aligned, as a percent of the total bases of the query sequence. A
determination of whether a nucleotide is matched/aligned is determined by
results
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of the FASTDB sequence alignment. This percentage is then subtracted from the
percent identity, calculated by the above FASTDB program using the specified
parameters, to arrive at a final percent identity score. This corrected score
is what
is used for the purposes of this embodiment. Only bases outside the S' and 3'
bases of the subject sequence, as displayed by the FASTDB alignment, which are
not matched/aligned with the query sequence, are calculated for the purposes
of
manually adjusting the percent identity score. For example, a 90 base subject
sequence is aligned to a 100 base query sequence to determine percent
identity.
The deletions occur at the 5' end of the subject sequence and therefore, the
FASTDB alignment does not show a matched/alignment of the first 10 bases at
S' end. The 10 unpaired bases represent 10% of the sequence (number of bases
at the S' and 3' ends not matched/total number of bases in the query sequence)
so
10% is subtracted from the percent identity score calculated by the FASTDB
program. If the remaining 90 bases were perfectly matched the final percent
identity would be 90%. In another example, a 90 base subject sequence is
compared with a 100 base query sequence. This time the deletions are internal
deletions so that there are no bases on the 5' or 3' of the subject sequence
which
are not matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only bases 5' and
3' ofthe subject sequence which are not matched/aligned with the query
sequence
are manually corrected for. No other manual corrections are made for the
purposes of this embodiment.
The present application is directed to nucleic acid molecules at least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid
sequences disclosed herein, (e.g., encoding a polypeptide having the amino
acid
sequence of an N and/or C terminal deletion disclosed herein, such as, for
example, a nucleic acid molecule encoding amino acids 50 to 283 of SEQ ID
N0:2), irrespective of whether they encode a polypeptide having a TRZ receptor
functional activity. This is because even where a particular nucleic acid
molecule
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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 polymerise 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 alai, ( 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 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
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid
sequence shown in SEQ 117 N0:25, FIG. lA-1B (SEQ ID NO:1), FIG. 4A-4C
(SEQ ID N0:4), or FIG. 7A-7C (SEQ ID N0:7) or to the nucleic acid sequence
of the deposited cDNAs which do, in fact, encode a polypeptide having TR2
receptor activity.
Preferably, the polynucleotide fragments of the invention encode a
polypeptide which demonstrates a TR2 functional 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 (e.g., complete (full-length) TR2 receptor polypeptides, mature TR2
receptor polypeptides, secreted TR2 receptor polypeptides, and soluble TR2
receptor polypeptides (e.g., having sequences contained in the extracellular
domain of a TR2 receptor ) as measured, for example, in a particular
immunoassay
or biological assay. For example, a TR2 receptor activity can routinely be
measured by determining the ability of a TR2 receptor polypeptide to bind a
TR2
receptor ligand (e.g., AIM II (International Publication No. WO 97/34911),
Lymphotoxin-a, and the Herpes virus protein HSV 1 gD). TR2 receptor activity
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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.
Other methods will be known to the skilled artisan and are within the
scope of the invention.
Of course, due to the degeneracy of the genetic code, one of ordinary skill
in the art will immediately recognize that a large number of the nucleic acid
molecules having a sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%
or 99% identical to the nucleic acid sequence of the deposited cDNAs or the
nucleic acid sequences shown in SEQ ID N0:25, FIG. lA-1B (SEQ ID NO:1),
FIG. 4A-4C (SEQ ID N0:4), or FIG. 7A-7C (SEQ ID N0:7) 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 TR.2 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 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.
The present invention is further directed to polynucleotides comprising, or
alternatively consisting of, fragments of the isolated nucleic acid molecules
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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
SEQ ID N0:25, FIG. lA-1B (SEQ ID NO:1), FIG. 4A-4C (SEQ ID N0:4), or
FIG. 7A-7C (SEQ D7 N0:7) is intended fragments at least about 15 nt, and more
S 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. In this context "about" includes the
particularly recited value and values larger or smaller by several (5, 4, 3,
2, or 1)
nucleotides.
Ofcourse, largerfragments 50, 75,100,125,150,175, 200, 225, 250, 275,
300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650,
675,
700, 725, 750, 775, 800, 825 or 848 nt in length are also useful according to
the
present invention as are fragments corresponding to most, if not all, of the
nucleotide sequences ofthe deposited cDNAs or as shown in SEQ ID N0:25, FIG.
lA-1B (SEQ 1D NO:I), FIG. 4A-4C (SEQ ID N0:4), or FIG. 7A-7C (SEQ ID
N0:7). 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 SEQ ID N0:25, FIG.
IA-1B (SEQ ID NO:1), FIG. 4A-4C (SEQ ID N0:4), or FIG. 7A-7C (SEQ ID
N0:7).
Preferred nucleic acid fragments of the present invention include nucleic
acid molecules encoding polypeptides comprising, or alternatively consisting
of,
the mature TR2-SV1 receptor (predicted to constitute amino acid residues from
about 37 to about 185 in FIG. 4A-4C (amino acid residues 1 to 149 in SEQ ID
NO:S)) and the complete TR2-SV2 receptor (predicted to constitute amino acid
residues from about 1 to about 136 in FIG. 7A-7C (SEQ )T7 N0:8)). In this
context "about" includes the particularly recited value and values larger or
smaller
by several (5, 4, 3, 2, or 1) amino acids.
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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 1 to
about
1 S amino acid residues) depending on the criteria used to define each domain.
In
this context "about" includes the particularly recited value and values larger
or
smaller by several (5, 4, 3, 2, or 1) amino acids.
Preferred nucleic acid fragments of the present invention also include
nucleic acid molecules encoding: a polypeptide comprising, or alternatively
consisting of, the TR2 receptor protein of FIG. lA-IB (SEQ ID N0:2)
extracellular domain (predicted to constitute amino acid residues from about
37
to about 200 in FIG. IA-1B (amino acid residues I to 164 in SEQ ID N0:2)); a
polypeptide comprising, or alternatively consisting of, the TR2 receptor
transmembrane domain (amino acid residues 201 to 225 in FIG. lA-1B (amino
acid residues 165 to 189 in SEQ ID N0:2)); a polypeptide comprising, or
alternatively consisting of, the TR2 receptor intracellular domain (predicted
to
constitute amino acid residues from about 226 to about 283 in FIG. 1 A-1 B
(amino
acid residues 190 to 247 in SEQ ID N0:2)); and a polypeptide comprising, or
alternatively consisting of, the TRZ receptor protein of FIG. lA-1B (SEQ ID
N0:2) extracellular and intracellular domains with all or part of the
transmembrane domain deleted. In this context "about" includes the
particularly
recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino
acids.
Preferred nucleic acid fragments of the present invention also include
nucleic acid molecules encoding amino acid residues the extracellular domain
of
the TR2 protein having the amino acid sequence set out in SEQ ID N0:26, both
with and without the associated leader sequence (amino acid residues -38 to
162
of SEQ ID N0:26 and amino acid residues I to 162 of SEQ ID N0:26,
respectively).
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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, or
alternatively consisting of, one, two, three, four, five or more amino acid
sequences selected from amino acid residues from about 39 to about 70 in FIG.
lA-1B (amino acid residues 3 to 34 in SEQ ID N0:2); apolypeptide comprising,
or alternatively consisting of, amino acid residues from about 106 to about
120
in FIG. 1 (amino acid residues 70 to 84 in SEQ ID N0:2); a polypeptide
comprising, or alternatively consisting of, amino acid residues from about 142
to
about 189 in FIG. IA-IB (amino acid residues 106 to 153 in SEQ ID N0:2); a
polypeptide comprising, or alternatively consisting of, amino acid residues
from
about 276 to about 283 in FIG. 1 A-1 B (amino acid residues 240 to 247 in SEQ
ID N0:2); a polypeptide comprising, or alternatively consisting of, amino acid
residues from about 39 to about 70 in FIG. 4A-4C (amino acid residues 3 to 34
in SEQ ID NO:S); amino acid residues from about 99 to about 136 in FIG. 4A-4C
(amino acid residues 63 to 100 in SEQ ID NO:S); amino acid residues from about
I7I to about 185 in FIG. 4A-4C (amino acid residues 135 to 149 in SEQ 117
NO: S); amino acid residues from about 56 to about 68 in FIG. 7A-7C (SEQ ID
N0:8); amino acid residues from about 93 to about 136 in FIG. 7A-7C (SEQ ID
N0:8). In this context "about" includes the particularly recited value and
values
larger or smaller by several (5, 4, 3, 2, or 1) amino acids. The inventors
have
determined that the above polypeptide 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. Polypeptides encoded by these
polynucleotides are also encompassed by the invention.
Representative examples of TR2 receptor polynucleodde fragments of the
invention include, for example, fragments that comprise, or alternatively,
consist
of, a sequence from about nucleotide 1 to 64, 65 to 100, l0I to 150, 151 to
200,
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201 to 250, 22S-265, 251 to 300, 301 to 350, 3S 1 to 372, 373 to 450, 4S 1 to
500,
SO1 to 550, 551 to 600, 601 to 650, 651 to 700, 701 to 750, 7S 1 to 800, 801
to
850, 8S 1 to 900, 901 to 950, 9S 1 to 1000, 1001 to l OSO, l OS 1 to 1100,
1070-1113, 1101 to 11 S0, 1151 to 1200, 1201 to 1250, 12S 1 to 1300, 1301 to
S 1350, 1351 to 1400, 1401 to 1450, 1451 to 1500, 1501 to 1550, 1SS1 to 1600,
or 1601 to 1670, of SEQ ID NO:1, the cDNA contained in the deposited
identified as ATCC Deposit No. 97059, or the complementary strand of any of
these fragments. In this context "about" includes the particularly recited
ranges,
larger or smaller by several (S, 4, 3, 2, or 1 ) nucleotides, at either
terminus or at
both termini. Polypeptides encoded by these polynucleotides are also
encompassed by the invention.
Further representative examples ofTR2 receptor polynucleotide fragments
of the invention include, for example, fragments that comprise, or
alternatively,
consist of, a sequence from about nucleotide 373 to 433, 373 to 450, 4S 1 to
500,
1S 501 to SSO, SS 1 to 600, 601 to 650, 651 to 700, 701 to 750, 7S 1 to 800,
801 to
850, 8S I to 900, or 901 to 927 of SEQ ID N0:4, from about nucleotide 247 to
300, 301 to 350, 351 to 372, 373 to 450, 451 to 500, SOl to SSO, SSl to 600,
or
601 to 6S4 of SEQ ID N0:7, or the cDNA contained in the deposited identified
as ATCC Deposit No. 97058 or 97057, or the complementary strand of any of
these polynucleotides. In this context "about" includes the particularly
recited
ranges, larger or smaller by several (S, 4, 3, 2, or 1 ) nucleotides, at
either terminus
or at both termini. Polypeptides encoded by these polynucleotides are also
encompassed by the invention.
It is believed one or more of the cysteine repeat regions of the TR2
2S receptor disclosed in FIG. 1 A-1 B are important for interactions between
the TR2
receptor and its ligands (e.g., AIM II (International Publication No. WO
97/34911 ), Lymphotoxin-a, and the Herpes virus protein HSV 1 glycoproteimD
(gD)). Accordingly, specific embodiments of the invention are directed to
polynucleotides encoding polypeptides which comprise, or alternatively consist
of,
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the amino acid sequence of cysteine repeat region A, B, C, or D disclosed in
FIG.
16 and described in Example 6. Additional embodiments of the invention are
directed to polynucleotides encoding TRZ receptor polypeptides which comprise,
or alternatively consist of, any combination of 1, 2, 3, or all 4 of cysteine
repeat
regions A-D disclosed in FIG. 16 and described in Example 6. Additional
preferred embodiments of the invention are directed to polypeptides which
comprise, or alternatively consist of, the TR2 receptor amino acid sequence of
cysteine repeat region A, B, C, or D disclosed in FIG. 16 and described in
Example 6. Additional embodiments ofthe invention are directed to TR2 receptor
polypeptides which comprise, or alternatively consist of, any combination of
1, 2,
3, or all 4 of cysteine repeat regions A-D disclosed in FIG. 16 and described
in
Example 6.
In certain embodiments, polynucleotides of the invention comprise, or
alternatively consist of, a polynucleotide sequence at least 80%, 85%, 90%,
92%,
95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding
one, two, or all three of the cysteine-rich motifs described above. The
present
invention also encompasses the above polynucleotide sequences fused to a
heterologous polynucleotide sequence. Polypeptides encoded by these nucleic
acids and/or polynucleotide sequences are also encompassed by the invention.
In another embodiment, the invention provides an isolated nucleic acid
molecule comprising, or alternatively consisting of, a polynucleotide which
hybridizes under stringent hybridization conditions to one, two, or all three
of the
cysteine-rich motifs described above polynucleotides of the invention
described
above, orthe complementary strand thereof. The meaning ofthe phrase "stringent
conditions" as used herein is described infra.
Preferably, the polynucleotide fragments of the invention encode a
polypeptide which demonstrates one or more TR2 receptor functional activities.
By a polypeptide demonstrating a TR2 receptor "functional activity" is meant,
a
polypeptide capable of displaying one or more known functional activities
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associated with a full-length (complete) TR2 receptor protein. Such functional
activities include, but are not limited to, biological activity (e.g.,
inhibition of B
cell proliferation), antigenicity, immunogenicity (ability to generate
antibodywhich
binds to a TR2 receptor polypeptide), the ability to bind (or compete with a
TRZ
receptor polypeptide for binding) to an anti-TR2 receptor antibody, the
ability to
form multimers with TR2 receptor polypeptides of the invention, and ability to
bind to a receptor or ligand for a TR2 receptor polypeptide (e.g., AIM II
(International Publication No. WO 97/34911 ), Lymphotoxin-a, and the Herpes
virus protein HSV1 glycoprotein D (gD)).
The functional activity of TR2 receptor polypeptides, and fragments,
variants derivatives, and analogs thereof, can be assayed by various methods.
For example, in one embodiment where one is assaying for the ability to
bind or compete with full-length TR2 receptor polypeptides for binding to anti-
TRZ receptor antibody, various immunoassays known in the art can be used,
including but not limited to, competitive and non-competitive assay systems
using
techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent
assay), "sandwich" immunoassays, immunoradiometric assays, gel difFusion
precipitation reactions, immunodiffusion assays, iy7 site immunoassays (using
colloidal gold, enzyme or radioisotope labels, for example), western blots,
precipitation reactions, agglutination assays (e.g., gel agglutination assays,
hemagglutination assays), complement fixation assays, immunofluorescence
assays, protein A assays, and immunoelectrophoresis assays, etc. In one
embodiment, antibody binding is detected by detecting a label on the primary
antibody. In another embodiment, the primary antibody is detected by detecting
binding of a secondary antibody or reagent to the primary antibody. In a
further
embodiment, the secondary antibody is labeled. Many means are known in the art
for detecting binding in an immunoassay and are within the scope of the
present
invention.
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In another embodiment, where a TR2 receptor ligand is identified (e.g.,
AIM II (International Publication No. WO 97/34911), Lymphotoxin-a, and the
Herpes virus protein HSV 1 gD), or the ability of a polypeptide fragment,
variant
or derivative of the invention to multimerize is being evaluated, binding can
be
assayed, e.g., by means well-known in the art, such as, for example, reducing
and
non-reducing gel chromatography, protein affinity chromatography, and affinity
blotting. See generally, Phizicky, E., et al., MicYObiol. Rev. 59:94-123
(1995).
In another embodiment, physiological correlates of TR2 receptor binding to its
substrates (signal transduction) can be assayed.
In addition, assays described herein (see, e.g., Examples 6 and 8 and
otherwise known in the art may routinely be applied to measure the ability of
TR2
receptor polypeptides and fragments, variants derivatives and analogs thereof
to
elicit TR2 receptor related biological activity (e.g., inhibition ofB cell
proliferation
ly7 vIIT(J O7" in vivo). Other methods will be known to the skilled artisan
and are
within the scope of the invention.
In another aspect, the invention provides isolated nucleic acid molecules
comprising, or alternatively consisting of, 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
complement of a polynucleotide frgament described herein, or the cDNAs
contained in ATCC Deposits 97059, 97058 and 97057. By "stringent
hybridization conditions" is intended overnight incubation at 42°C in a
solution
comprising, or alternatively consisting of: 50% formamide, Sx SSC (750 mM
NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), Sx
Denhardt's solution, 10% dextran sulfate, and 20 ~ g/ml denatured, sheared
salmon
sperm DNA, followed by washing the filters in O.lx 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
1 S nucleotides (nt), and more preferably at least about 20 nt, still more
preferably
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at least about 30 nt, and even more preferably about 30-70 nt of the reference
polynucleotide. In this context "about" includes the particularly recited
value and
values larger or smaller by several (5, 4, 3, 2, or 1) nucleotide. 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
ofthe
reference polynucleotide (e.g., the deposited cDNAs or the nucleotide
sequences
as shown in SEQ ID N0:25, FIG. lA-1B (SEQ ID NO:1), FIG. 4A-4C (SEQ ID
N0:4}, or FIG. 7A-7C (SEQ ID N0:7)).
Of course, a polynucleotide which hybridizes only to a poly A sequence
(such as the 3' terminal poly(A) tract of the TIt2 receptor cDNA sequences
shown
in SEQ ID N0:25, FIG. IA-1B (SEQ ID NO:1), FIG. 4A-4C (SEQ ID N0:4), or
FIG. 7A-7C (SEQ ID N0:7)), or to a complementary stretch of T (or In resides,
would not be included in a polynucleotide of the invention used to hybridize
to
a portion of 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
generated
from an oligo-dT primed cDNA library).
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 3 6 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;
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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., Pnoc. Nall. 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., C.'ell 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 ofthe present invention, which encode portions, analogs or
derivatives
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. Ger~e.s II, Lewin, B.,
ed., John Wiley & Sons, New York (1985).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques, which include, but are not limited to: oligonucleotide
mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed
mutagenesis (see, e.g., Carter et al., Nucl. Acids Res. 13:4331 (1986); and
Zoller
et al., Nucl. Acids Res. 10:6487 (1982)), cassette mutagenesis (see, e.g.,
Wells et
al., Gene 34:315 (1985)), restriction selection mutagenesis (see, e.g., Wells
etal.,
Philos. TYan.s. R. Soc. London See°.A 317:41 S ( 1986)).
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
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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
ofthe TR2
receptors or portions thereof. Also especially preferred in this regard are
conservative substitutions.
Vectors onrl 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 TRZ
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 irr 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, tip and tac 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
resistance for eukaryotic cell culture and tetracycline or ampicillin
resistance genes
for culturing in E co7i and other bacteria. Representative examples of
appropriate
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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 S~7 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, pNHBA, pNHl6a, pNHIBA, pNH46A, available from Stratagene; and
ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS available from Pharmacia.
Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl
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, DEAF-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 a7., 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
and to facilitate purification, among others, are familiar and routine
techniques in
the art. A preferred fusion protein comprises a heterologous region from
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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., ,lom°nal of Molecular Recognitior7, Vol. 8:52-58 (1995)
and K.
Johanson et al., The Journal of Biological ChemistJy, 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
("HI'LC") is employed for purification. Polypeptides of the present invention
include naturally purified products, products ofchemical synthetic procedures,
and
products produced by recombinant techniques from a prokaryotic or eukaryotic
host, including, for example, bacterial, yeast, higher plant, insect and
mammalian
cells. Depending upon the host employed in a recombinant production procedure,
the polypeptides of the present invention may be glycosylated or may be
non-glycosylated. In addition, polypeptides of the invention may also include
an
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initial modified methionine residue, in some cases as a result of host-
mediated
processes.
In addition to encompassing host cells containing the vector constructs
discussed herein, the invention also encompasses primary, secondary, and
immortalized host cells of vertebrate origin, particularly mammalian origin,
that
have been engineered to delete or replace endogenous genetic material (e.g.,
the
TR2 receptor coding sequence), and/or to include genetic material (e.g.,
heterologous polynucleotide sequences) that is operably associated with TRZ
receptor polynucleotides of the invention, and which activates, alters, andlor
amplifies endogenous TR2 receptor polynucleotides. For example, techniques
known in the art may be used to operably associate heterologous control
regions
(e.g., promoter and/or enhancer) and endogenous TR2 receptor polynucleotide
sequences via homologous recombination (see, e.g., U.S. Patent Number
5,641,670, issued June 24,1997; International PublicationNumber WO 96/29411,
1 S published September 26, 1996; International Publication Number WO
94/12650,
published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. U.S.A. 86:8932-
8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures
of
each of which are incorporated by reference in their entireties).
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
shown in SEQ ID N0:26, FIG. lA-1B (SEQ 117 N0:2), FIG. 4A-4C (SEQ ID
NO:S), or FIG. 7A-7C (SEQ ID N0:8), or a peptide or polypeptide comprising, or
alternatively consisting of, 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, or alternatively consists of, the polypeptide
sequence lacking the transmembrane domain. One example of such a soluble form
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of the TR2 receptor is the TR2-SV 1 splice variant which has a secretory
leader
sequence but lacks both the intracellular and transmembrane domains. Thus, the
TR2-SV 1 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 infra- 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. IA-1B (SEQ ID N0:2) 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.
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 SEQ ID N0:26, FIG.1 A-1B (SEQ ID N0:2) or FIG. 4A-4C (SEQ
ID NO:S) including the leader; the polypeptides of SEQ ID N0:26, FIG. lA-1B
(SEQ ID N0:2) or FIG. 4A-4C (SEQ lI7 NO:S) including the leader but minus the
N-terminal methionine; the polypeptides of SEQ ID N0:26, FIG.1 A- I B (SEQ ID
N0:2) or FIG. 4A-4C (SEQ 1D NO:S) minus the leader; the polypeptide of FIG.
7A-7C (SEQ ID N0:8); the extracellular domain, the transmembrane domain, and
the intracellular domain of the TR2 receptor shown in SEQ ID N0:26 or FIG.1 A-
1 B (SEQ 11? N0:2); and polypeptides which are at least 80% identical, more
preferably at least 85%, 90%, 92% 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
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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
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 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the
amino acid sequence shown in SEQ 117 N0:26, FIG. lA-1B (SEQ 117 N0:2), FIG.
4A-4C (SEQ ID N0:5), or FIG. 7A-7C (SEQ m N0:8) or to the amino acid
sequence encoded by one of the deposited cDNAs can be determined
conventionally using known computer programs such the Bestfit program
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group, UniversityResearchPark, 575 Science Drive, Madison, WI53711). 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.
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In a specific embodiment, the identity between a reference (query)
sequence (a sequence of the present invention) and a subject sequence, also
referred to as a global sequence alignment, is determined using the FASTDB
computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci.
6:237-245 (1990)). Preferred parameters used in a FASTDB amino acid
alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining
Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window
Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500
or the length of the subject amino acid sequence, whichever is shorter.
According
to this embodiment, ifthe subject sequence is shorter than the query sequence
due
to N- or C-terminal deletions, not because of internal deletions, a manual
correction is made to the results to take into consideration the fact that the
FASTDB program does not account for N- and C-terminal truncations of the
subject sequence when calculating global percent identity. For subject
sequences
truncated at the N- and C-termini, relative to the query sequence, the percent
identity is corrected by calculating the number of residues of the query
sequence
that are N- and C-terminal of the subject sequence, which are not
matched/aligned
with a corresponding subject residue, as a percent of the total bases of the
query
sequence. A determination of whether a residue is matched/aligned is
determined
by results ofthe FASTDB sequence alignment. This percentage is then subtracted
from the percent identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score. This final
percent
identity score is what is used for the purposes of this embodiment. Only
residues
to the N- and C-termini of the subject sequence, which are not matched/aligned
with the query sequence, are considered for the purposes of manually adjusting
the
percent identity score. That is, only query residue positions outside the
farthest
N- and C-terminal residues ofthe subject sequence. For example, a 90 amino
acid
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residue subject sequence is aligned with a 100 residue query sequence to
determine percent identity. The deletion occurs at the N-terminus of the
subject
sequence and therefore, the FASTDB alignment does not show a
matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired
residues represent 10% of the sequence (number of residues at the N- and C-
termini not matched/total number of residues in the query sequence) so 10% is
subtracted from the percent identity score calculated by the FASTDB program.
If the remaining 90 residues were perfectly matched the final percent identity
would be 90%. In another example, a 90 residue subject sequence is compared
with a 100 residue query sequence. This time the deletions are internal
deletions
so there are no residues at the N- or C-termini of the subject sequence which
are
not matched/aligned with the query. In this case the percent identity
calculated
by FASTDB is not manually corrected. Once again, only residue positions
outside
the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB
alignment, which are not matched/aligned with the query sequence are manually
corrected for. No other manual corrections are made for the purposes of this
embodiment.
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
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. Guidance
concerning which amino acid changes are likely to be phenotypically silent can
be
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found in Bowie, J. U., et al., "Deciphering the Message in Protein Sequences:
Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990).
Thus, the fragment, derivative or analog of the polypeptides of SEQ ID
N0:26, FIG. lA-IB (SEQ 117 N0:2), FIG. 4A-4C (SEQ ID NO:S), and FIG. 7A-
7C (SEQ LD N0:8), 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. Polynucleotides encoding these fragments, derivatives or
analogs are also encompassed by the invention.
Ofparticular 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
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)).
AMENDED SHEET
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The replacement of amino acids can also change the selectivity of binding
to cell surface receptors. Ostade et al., Nafure 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 I).
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TABLE I
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 vita°o, or in
vita°o proliferative
activity. Sites that are critical for ligand-receptor binding can also be
determined
by structural analysis such as crystallization, nuclear magnetic resonance or
photoaffmity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de
Vos
et al. Science 255:3 06-312 ( 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 ofthe present invention have uses which include, but are
not limited to, as sources for generating antibodies that bind the
polypeptides of
the invention, and as molecular weight markers on SDS-PAGE gels or on
molecular sieve gel filtration columns using methods well known to those of
skill
in the art.
TR2 polypeptides of the invention can also inhibit mixed lymphocyte
reactions (MLRs). As discussed below in Example 6, TR2 polypeptides inhibit
three-way MLRs. An additional method for performing three-way MLRs is
discussed in Harrop et al., Jour. Immunol. 161:1786-1794 (1998), which
incorporated herein by reference.
The present application is also directed to proteins containing polypeptides
at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the TR2
receptor polypeptide sequence set forth herein as n'-m', n2-m2, n3-m~, n4-m4,
and/or n5-ms. In preferred embodiments, the application is directed to
proteins
containing polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or
99% identical to polypeptides having the amino acid sequence ofthe specific
TR2
receptor N- and C-terminal deletions recited herein. Polynucleotides encoding
these polypeptides are also encompassed by the invention.
In certain preferred embodiments, TR2 receptor proteins of the invention
comprise, or alternatively consist of, fusion proteins as described above
wherein
the TR2 receptor polypeptides are those described as n'-m', nz-m2, n3-m3, n4-
m'',
and/or ns-ms herein. In preferred embodiments, the application is directed to
nucleic acid molecules at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or
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99% identical to the nucleic acid sequences encoding polypeptides having the
amino acid sequence of the specific N- and C-terminal deletions recited
herein.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
As mentioned above, even if deletion of one or more amino acids from the
N-terminus of a protein results in modification of loss of one or more
biological
functions of the protein, other biological activities may still be retained.
Thus, the
ability of shortened TR2 muteins to induce and/or bind to antibodies which
recognize the complete or mature forms of the polypeptides generally will be
retained when less than the majority of the residues of the complete or mature
polypeptide are removed from the N-terminus. Whether a particular polypeptide
lacking N-terminal residues of a complete polypeptide retains such immunologic
activities can readily be determined by routine methods described herein and
otherwise known in the art. It is not unlikely that a TR2 mutein with a large
number of deleted N-terminal amino acid residues may retain some biological or
immunogenic activities. In fact, peptides composed of as few as six TR2 amino
acid residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having
one or more residues deleted from the amino terminus of the TR2 amino acid
sequence shown in FIG. lA-1B (i.e., SEQ ID N0:2), up to the glycine residue at
position number 278 and polynucleotides encoding such polypeptides. In
particular, the present invention provides polypeptides comprising, or
alternatively
consisting of, the amino acid sequence ofresidues n'-283 ofFIG. lA-1B (SEQ ID
N0:2), where n' is an integer in the range of 2 to 278. Polynucleotides
encoded
by these polypeptides are also encompassed by the invention.
More in particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, the amino acid
sequence
of residues of E-2 to H-283; P-3 to H-283; P-4 to H-283; G-5 to H-283; D-6 to
H-283; W-7 to H-283; G-8 to H-283; P-9 to H-283; P-10 to H-283; P-11 to
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H-283; W-12 to H-283; R-13 to H-283; S-14 to H-283; T-15 to H-283; P-16 to
H-283; K-17 to H-283; T-18 to H-283; D-19 to H-283; V-20 to H-283; L-21 to
H-283; R-22 to H-283; L-23 to H-283; V-24 to H-283; L-25 to H-283; Y-26 to
H-283; L-27 to H-283; T-28 to H-283; F-29 to H-283; L-30 to H-283; G-31 to
H-283; A-32 to H-283; P-33 to H-283; C-34 to H-283; Y-35 to H-283; A-36 to
H-283; P-37 to H-283; A-38 to H-283; L-39 to H-283; P-40 to H-283; S-41 to
H-283; C-42 to H-283; K-43 to H-283; E-44 to H-283; D-45 to H-283; E-46 to
H-283; Y-47 to H-283; P-48 to H-283; V-49 to H-283; G-50 to H-283; S-S1 to
H-283; E-52 to H-283; C-53 to H-283; C-54 to H-283; P-55 to H-283; K-56 to
H-283; C-57 to H-283; S-58 to H-283; P-59 to H-283; G-60 to H-283; Y-61 to
H-283; R-62 to H-283; V-63 to H-283; K-64 to H-283; E-65 to H-283; A-66 to
H-283; C-67 to H-283; G-68 to H-283; E-69 to H-283; L-70 to H-283; T-71 to
H-283; G-72 to H-283; T-73 to H-283; V-74 to H-283; C-75 to H-283; E-76 to
H-283; P-77 to H-283; C-78 to H-283; P-79 to H-283; P-80 to H-283; G-81 to
H-283; T-82 to H-283; Y-83 to H-283; I-84 to H-283; A-85 to H-283; H-86 to
H-283; L-87 to H-283; N-88 to H-283; G-89 to H-283; L-90 to H-283; S-91 to
H-283; K-92 to H-283; C-93 to H-283; L-94 to H-283; Q-95 to H-283; C-96 to
H-283; Q-97 to H-283; M-98 to H-283; C-99 to H-283; D-100 to H-283; P-101
to H-283; A-102 to H-283; M-103 to H-283; G-104 to H-283; L-105 to H-283;
R-106 to H-283; A-107 to H-283; S-108 to H-283; R-109 to H-283; N-I 10 to
H-283; C-I 11 to H-283; S-112 to H-283; R-113 to H-283; T-114 to H-283;
E-115 to H-283; N-116 to H-283; A-117 to H-283; V-118 to H-283; C-119 to
H-283; G-120 to H-283; C-121 to H-283; S-122 to H-283; P-123 to H-283;
G-124 to H-283; H-125 to H-283; F-126 to H-283; C-127 to H-283; I-128 to
H-283; V-129 to H-283; Q-130 to H-283; D-131 to H-283; G-132 to H-283;
D-133 to H-283; H-134 to H-283; C-135 to H-283; A-136 to H-283; A-137 to
H-283; C-138 to H-283; R-139 to H-283; A-140 to H-283; Y-141 to H-283;
A-142 to H-283; T-143 to H-283; S-144 to H-283; S-145 to H-283; P-146 to
H-283; G-147 to H-283; Q-148 to H-283; R-149 to H-283; V-150 to H-283;
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Q-151 to H-283; K-152 to H-283; G-153 to H-283; G-154 to H-283; T-155 to
H-283; E-156 to H-283; S-157 to H-283; Q-158 to H-283; D-159 to H-283;
T-160 to H-283; L-161 to H-283; C-162 to H-283; Q-163 to H-283; N-164 to
H-283; C-165 to H-283; P-166 to H-283; P-167 to H-283; G-168 to H-283;
T-169 to H-283; F-170 to H-283; S-171 to H-283; P-172 to H-283; N-173 to
H-283; G-174 to H-283; T-175 to H-283; L-176 to H-283; E-177 to H-283;
E-178 to H-283; C-179 to H-283; Q-180 to H-283; H-181 to H-283; Q-182 to
H-283; T-183 to H-283; K-184 to H-283; C-185 to H-283; S-186 to H-283;
W-187 to H-283; L-188 to H-283; V-189 to H-283; T-190 to H-283; K-191 to
H-283; A-192 to H-283; G-193 to H-283; A-194 to H-283; G-195 to H-283;
T-196 to H-283; S-197 to H-283; S-198 to H-283; S-199 to H-283; H-200 to
H-283; W-201 to H-283; V-202 to H-283; W-203 to H-283; W-204 to H-283;
F-205 to H-283; L-206 to H-283; S-207 to H-283; G-208 to H-283; S-209 to
H-283; L-210 to H-283; V-211 to H-283; I-212 to H-283; V-213 to H-283; I-214
to H-283; V-215 to H-283; C-216 to H-283; S-217 to H-283; T-218 to H-283;
V-219 to H-283; G-220 to H-283; L-221 to H-283; I-222 to H-283; I-223 to
H-283; C-224 to H-283; V-225 to H-283; K-226 to H-283; R-227 to H-283;
R-228 to H-283; K-229 to H-283; P-230 to H-283; R-231 to H-283; G-232 to
H-283; D-233 to H-283; V-234 to H-283; V-235 to H-283; K-236 to H-283;
V-237 to H-283; I-238 to H-283; V-239 to H-283; S-240 to H-283; V-241 to
H-283; Q-242 to H-283; R-243 to H-283; K-244 to H-283; R-245 to H-283;
Q-246 to H-283; E-247 to H-283; A-248 to H-283; E-249 to H-283; G-250 to
H-283; E-251 to H-283; A-252 to H-283; T-253 to H-283; V-254 to H-283;
I-255 to H-283; E-256 to H-283; A-257 to H-283; L-258 to H-283; Q-259 to
H-283; A-260 to H-283; P-261 to H-283; P-262 to H-283; D-263 to H-283;
V-264 to H-283; T-265 to H-283; T-266 to H-283; V-267 to H-283; A-268 to
H-283; V-269 to H-283; E-270 to H-283; E-271 to H-283; T-272 to H-283;
I-273 to H-283; P-274 to H-283; S-275 to H-283; F-276 to H-283; T-277 to
H-283; and G-278 to H-283 of the TRZ sequence shown in FIG. lA-1B. The
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present invention is also directed to nucleic acid molecules comprising, or
alternatively consisting of, a polynucleotide sequence at least 80%, 8S%, 90%,
92%, 9S%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences
encoding the polypeptides described above. The present invention also
S encompasses the above polynucleotide sequences fused to a heterologous
polynucleotide sequence. Polypeptides encoded by these polynucleotide
sequences are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids from
the C-terminus of a protein results in modification of loss of one or more
biological functions of the protein, other biological activities may still be
retained.
Thus, the ability of the shortened TR2 mutein to induce and/or bind to
antibodies
which recognize the complete or mature forms of the polypeptide generally will
be retained when less than the majority of the residues of the complete or
mature
polypeptide are removed from the C-terminus. Whether a particular polypeptide
1S lacking C-terminal residues of a complete polypeptide retains such
immunologic
activities can readily be determined by routine methods described herein and
otherwise known in the art. It is not unlikely that a TR2 mutein with a large
number of deleted C-terminal amino acid residues may retain some biological or
immunogenic activities. In fact, peptides composed of as few as six TR2 amino
acid residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having
one or more residues deleted from the carboxy terminus of the amino acid
sequence ofthe TR2 polypeptide shown in FIG. 1 A-1 B (SEQ ID N0:2), up to the
aspartic acid residue at position number 6, and polynucleotides encoding such
2S polypeptides. In particular, the present invention provides polypeptides
comprising, or alternatively consisting of, the amino acid sequence of
residues
1-m' ofFIG. lA-1B (i.e., SEQ ID N0:2), where m' is an integer in the range of
6 to 282. Polynucleotides encoded by these polypeptides are also encompassed
by the invention.
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More in particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, the amino acid
sequence
of residues M-I to N-282; M-1 to P-281; M-I to S-280; M-I to R-279; M-1 to
G-278; M-I to T-277; M-I to F-276; M-1 to S-275; M-1 to P-274; M-1 to I-273;
M-1 to T-272; M-1 to E-271; M-1 to E-270; M-1 to V-269; M-I to A-268; M-1
to V-267; M-1
to T-266; M-I
to T-265; M-I
to V-264; M-1
to D-263; M-I
to
P-262; M-1 to P-261; M-1 to A-260;to Q-259; to L-258;
M-1 M-I M-1 to
A-257; M-1 to E-256; M-I to I-255;to V-254; to T-253;
M-1 M-1 M-1 to
A-252; M-I to E-251; M-1 to G-250;to E-249; to A-248;
M-1 M-1 M-1 to
E-247; M-1 to Q-246; M-1 to R-245;to K-244; to R-243 ;
M-1 M-1 M-1 to
Q-242; M-1 to V-241; M-I to S-240; M-1 to V-239; M-1 to I-238; M-I to
V-237; M-1 to K-236; M-1 to V-235; M-I to V-234; M-1 to D-233; M-1 to
G-232; M-I to R-231; M-1 to P-230; M-1 to K-229; M-1 to R-228; M-1 to
R-227; M-I to K-226; M-1 to V-225; M-I to C-224; M-I to I-223; M-1 to I-222;
M-I to L-221; M-I to G-220; M-I to V-219; M-1 to T-218; M-1 to S-217; M-1
to C-216; M-1 to V-215; M-1 to I-214; M-1 to V-213; M-1 to I-212; M-1 to
V-211; M-I to L-210; M-1 to S-209; M-1 to G-208; M-I to S-207; M-1 to
L-206; M-1 to F-205; M-1 to W-204; M-1 to W-203; M-I to V-202; M-1 to
W-201; M-I to H-200; M-I to S-199; M-I to S-198; M-1 to S-197; M-I to
T-196; M-1 to G-195; M-1 to A-194; M-1 to G-193; M-I to A-192; M-1 to
K-191; M-1 to T-190; M-I to V-189; M-1 to L-188; M-1 to W-187; M-1 to
S-186; M-1 to C-185; M-1 to K-184; M-I to T-183; M-1 to Q-182; M-I to
H-181; M-1 to Q-180; M-1 to C-179; M-1 to E-178; M-I to E-177; M-1 to
L-176; M-1 to T-175; M-1 to G-174; M-1 to N-173; M-1 to P-172; M-1 to
S-171; M-1 to F-170; M-I to T-169; M-I to G-168; M-I to P-167; M-I to P-166;
M-1 to C-165; M-1 to N-164; M-1 to Q-163; M-1 to C-162; M-I to L-161; M-1
to T-160; M-1 to D-159; M-I to Q-I58; M-1 to S-157; M-1 to E-156; M-1 to
T-I55; M-1 to G-154; M-1 to G-153; M-1 to K-152; M-I to Q-151; M-1 to
V-150; M-1 to R-149; M-1 to Q-148; M-1 to G-147; M-1 to P-146; M-1 to
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S-145; M-1 to S-144; M-1 to T-143; M-1 to A-142; M-1 to Y-141; M-1 to
A-140; M-1 to R-139; M-1 to C-138; M-1 to A-137; M-1 to A-136; M-1 to
C-135; M-1 to H-134; M-1 to D-133; M-1 to G-132; M-1 to D-131; M-1 to
Q-130; M-1 to V-129; M-1 to I-128; M-1 to C-127; M-1 to F-126; M-1 to
H-125; M-I to G-124; M-1 to P-123; M-1 to S-122; M-1 to C-121; M-1 to
G-120; M-1 to C-119; M-I to V-118; M-1 to A-117; M-I to N-116; M-1 to
E-115; M-1 to T-114; M-1 to R-113; M-1 to S-112; M-1 to C-111; M-I to
N-110; M-1 to R-109; M-1 to S-108; M-I to A-107; M-1 to R-106; M-1 to
L-105; M-1 to G-104; M-I to M-103; M-1 to A-102; M-1 to P-101; M-1 to
D-100; M-I to C-99; M-1 to M-98; M-1 to Q-97; M-1 to C-96; M-1 to Q-95;
M-1 to L-94; M-1 to C-93 ; M-1 to K-92; M-1 to S-91; M- I to L-90; M-1 to
G-89; M-1 to N-88; M-1 to L-87; M-1 to H-86; M-I to A-85; M-1 to I-84; M-1
to Y-83; M-1 to T-82; M-1 to G-81; M-1 to P-80; M-1 to P-79; M-1 to C-78;
M-1 to P-77; M-1 to E-76; M-1 to C-75; M-1 to V-74; M-1 to T-73; M-1 to
G-72; M-1 to T-71; M-1 to L-70; M-1 to E-69; M-I to G-68; M-1 to C-67; M-I
to A-66; M-1 to E-65; M-1 to K-64; M-1 to V-63; M-1 to R-62; M-1 to Y-61;
M-I to G-60; M-1 to P-59; M-1 to S-58; M-1 to C-57; M-1 to K-56; M-1 to
P-55; M-1 to C-54; M-1 to C-53; M-1 to E-52; M-1 to S-51; M-I to G-50; M-1
to V-49; M-1 to P-48; M-1 to Y-47; M-1 to E-46; M-1 to D-45; M-1 to E-44;
M-1 to K-43; M-1 to C-42; M-1 to S-41; M-1 to P-40; M-1 to L-39; M-1 to
A-38; M-1 to P-37; M-1 to A-36; M-1 to Y-35; M-1 to C-34; M-1 to P-33; M-1
to A-32; M-1 to G-31; M-1 to L-30; M-1 to F-29; M-1 to T-28; M-1 to L-27;
M-1 to Y-26; M-1 to L-25; M-1 to V-24; M-1 to L-23; M-1 to R-22; M-1 to
L-21; M-I to V-20; M-1 to D-19; M-1 to T-18; M-1 to K-17; M-1 to P-16; M-1
to T-15; M-I to S-14; M-1 to R-13; M-1 to W-12; M-I to P-11; M-1 to P-10;
M-1 to P-9; M-I to G-8; M-1 to W-7; and M-1 to D-6 ofthe sequence ofthe TR2
sequence shown in FIG. lA-1B. The present invention is also directed to
nucleic
acid molecules comprising, or alternatively consisting of, a polynucleotide
sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical
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to the polynucleotide sequences encoding the polypepddes described above. The
present invention also encompasses the above polynucleotide sequences fused to
a heterologous polynucleotide sequence. Polypeptides encoded by these
polynucleotide sequences are also encompassed by the invention.
The invention also provides polypeptides having one or more amino acids
deleted from both the amino and the carboxyl termini of a TR2 polypeptide,
which
may be described generally as having residues n'-m' of FIG. 1 A-1 B (i. e. ,
SEQ ID
N0:2), where n' and m' are integers as described above. Polynucleotides
encoded
by these polypeptides are also encompassed by the invention.
Also mentioned above, even if deletion of one or more amino acids from
the N-terminus of a protein results in modification of loss of one or more
biological functions of the protein, other biological activities may still be
retained.
Thus, the ability of shortened TR2-SV1 muteins to induce and/or bind to
antibodies which recognize the complete or mature forms of the polypeptides
I S generally will be retained when less than the majority of the residues of
the
complete or mature polypeptide are removed from the N-terminus. Whether a
particular polypeptide lacking N-terminal residues of a complete polypeptide
retains such immunologic activities can readily be determined by routine
methods
described herein and otherwise known in the art. It is not unlikely that a TR2-
SV 1
mutein with a large number of deleted N-terminal amino acid residues may
retain
some biological or immunogenic activities. In fact, peptides composed of as
few
as six TR2-SV1 amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having
one or more residues deleted from the amino terminus of the TR2-SV 1 amino
acid
sequence shown in FIG. 4A-4C (i.e., SEQ ID NO:S), up to the threonine residue
at position number 180 and polynucleotides encoding such polypeptides. In
particular, the present invention provides polypeptides comprising, or
altenzatively
consisting of, the amino acid sequence ofresidues n2-185 ofFIG. 4A-4C (SEQ ID
AMENDED SHEET
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NO:S), where n2 is an integer in the range of 2 to 180, and 180 is the
position of
the first residue from the N-terminus of the complete TR2-SV1 polypeptide
believed to be required for at least immunogenic activity of the TR2-SV1
polypeptide. Polynucleotides encoded by these polypeptides are also
encompassed by the invention.
More in particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, the amino acid
sequence
of residues of E-2 to A-185; P-3 to A-185; P-4 to A-185; G-5 to A-185; D-6 to
A-185; W-7 to A-185; G-8 to A-185; P-9 to A-185; P-10 to A-185; P-I I to
A-185; W-12 to A-185; R-13 to A-185; S-14 to A-185; T-IS to A-185; P-16 to
A-185; R-17 to A-185; T-18 to A-185; D-19 to A-185; V-20 to A-185; L-21 to
A-185; R-22 to A-185; L-23 to A-185; V-24 to A-185; L-25 to A-185; Y-26 to
A-185; L-27 to A-185; T-28 to A-185; F-29 to A-185; L-30 to A-185; G-31 to
A-185; A-32 to A-185; P-33 to A-185; C-34 to A-185; Y-35 to A-185; A-36 to
IS A-185; P-37 to A-185; A-38 to A-185; L-39 to A-185; P-40 to A-185; S-41 to
A-185; C-42 to A-185; K-43 to A-185; E-44 to A-185; D-45 to A-185; E-46 to
A-185; Y-47 to A-185; P-48 to A-185; V-49 to A-185; G-50 to A-185; S-51 to
A-185; E-52 to A-185; C-53 to A-185; C-54 to A-185; P-55 to A-185; K-56 to
A-185; C-57 to A-185; S-58 to A-185; P-59 to A-185; G-60 to A-185; Y-61 to
A-185; R-62 to A-185; V-63 to A-185; K-64 to A-185; E-65 to A-185; A-66 to
A-185; C-67 to A-185; G-68 to A-185; E-69 to A-185; L-70 to A-185; T-71 to
A-185; G-72 to A-185; T-73 to A-185; V-74 to A-185; C-75 to A-185; E-76 to
A-185; P-77 to A-185; C-78 to A-185; P-79 to A-185; P-80 to A-185; G-81 to
A-185; T-82 to A-185; Y-83 to A-185; I-84 to A-185; A-85 to A-185; H-86 to
A-185; L-87 to A-185; N-88 to A-185; G-89 to A-185; L-90 to A-185; S-91 to
A-185; K-92 to A-185; C-93 to A-185; L-94 to A-185; Q-95 to A-185; C-96 to
A-185; Q-97 to A-185; M-98 to A-185; C-99 to A-185; D-100 to A-185; P-101
to A-185; D-102 to A-185; I-103 to A-185; G-104 to A-185; S-105 to A-185;
P-106 to A-185; C-107 to A-185; D-108 to A-185; L-109 to A-185; R-110 to
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-55-
A-185; G-I l I to A-185; R-112 to A-185; G-I13 to A-185; H-114 to A-185;
L-115 to A-185; E-116 to A-185; A-117 to A-185; G-118 to A-185; A-119 to
A-185; H-120 to A-185; L-121 to A-185; S-122 to A-185; P-123 to A-185; G-124
to A-185; R-125 to A-185; Q-126 to A-185; K-127 to A-185; G-128 to A-I85;
S E-129 to A-185; P-130 to A-185; D-13I to A-185; P-132 to A-185; E-133 to
A-I 85; V-134 to A-185; A-135 to A-185; F-136 to A-185; E-137 to A-185; S-138
to A-185; L-139 to A-185; S-140 to A-185; A-141 to A-185; E-142 to A-185;
P-143 to A-185; V-144 to A-185; H-145 to A-185; A-146 to A-185; A-147 to
A-185; N-148 to A-185; G-149 to A-185; S-150 to A-185; V-151 to A-185;
P-152 to A-185; L-153 to A-185; E-154 to A-I85; P-155 to A-185; H-156 to
A-185; A-157 to A-185; R-158 to A-185; L-I59 to A-185; S-160 to A-185;
M-161 to A-185; A-I62 to A-185; S-163 to A-185; A-164 to A-185; P-165 to
A-185; C-I66 to A-185; G-167 to A-185; Q-168 to A-185; A-169 to A-185;
G-170 to A-185; L-171 to A-185; H-172 to A-185; L-173 to A-185; R-174 to
A-185; D-175 to A-185; R-176 to A-185; A-177 to A-185; D-178 to A-185;
G-179 to A-185; and T-180 to A-185 of the TR2-SVl sequence shown in FIG.
4A-4.C. The present invention is also directed to nucleic acid molecules
comprising, or alternatively consisting of, a polynucleotide sequence at least
80%,
85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide
sequences encoding the polypeptides described above. The present invention
also
encompasses the above polynucleotide sequences fused to a heterologous
polynucleotide sequence. Polypeptides encoded bythesepolynucleotide sequences
are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids
from the C-terminus of a protein results in modification of loss of one or
more
biological functions ofthe protein, other biological activities may still be
retained.
Thus, the ability of the shortened TR2-SVl mutein to induce and/or bind to
antibodies which recognize the complete or mature forms of the polypeptide
generally will be retained when less than the majority of the residues of the
AMENDED SHEET
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-56-
complete or mature polypeptide are removed from the C-terminus. Whether a
particular polypeptide lacking C-terminal residues of a complete poiypeptide
retains such immunologic activities can readily be determined by routine
methods
described herein and otherwise known in the art. It is not uniWPi~ that a
TR2-SV 1 mutein with a large number of deleted C-terminal amino acid residues
may retain some biological or immunogenic activities. In fact, peptides
composed
of as few as six TR2-SV1 amino acid residues may often evoke an immune
response.
Accordingly, the present invention further provides polypeptides having
one or more residues deleted from the carboxy terminus of the amino acid
sequence ofthe TR2-SV1 polypeptide shown in FIG. 4A-4C (SEQ 1D NO:S), up
to the aspartic acid residue at position number 6, and polynucleotides
encoding
such polypeptides. In particular, the present invention provides polypeptides
comprising, or alternatively consisting of, the amino acid sequence of
residues
1-m2 of FIG. 4A-4C (i. e., SEQ ID NO: S), where m2 is an integer in the range
of
6 to 184. Polynucleotides encoded by these polypeptides are also encompassed
by the invention.
More in particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, the amino acid
sequence
ofresidues M-1 to R-184; M-1 to G-183; M-1 to G-182; M-1 to P-181; M-1 to
T-180; M-1 to G-179; M-1 to D-178; M-1 to A-177; M-1 to R-176; M-1 to
D-175; M-I to R-174; M-1 to L-173; M-1 to H-172; M-1 to L-171; M-1 to
G-170; M-I to A-169; M-1 to Q-168; M-1 to G-167; M-1 to C-166; M-I to
P-165; M-1 to A-164; M-1 to S-163; M-1 to A-162; M-1 to M-161; M-1 to
S-160; M-1 to L-159; M-I to R-158; M-1 to A-157; M-1 to H-156; M-1 to
P-155; M-1 to E-154; M-1 to L-153; M-1 to P-I52; M-1 to V-151; M-1 to
S-150; M-1 to G-149; M-1 to N-148; M-1 to A-147; M-1 to A-146; M-1 to
H-145; M-1 to V-I44; M-1 to P-143; M-1 to E-142; M-1 to A-141; M-1 to
S-140; M-1 to L-139; M-1 to S-138; M-1 to E-137; M-1 to F-136; M-1 to
AMENDED SHEET
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-57-
A-135; M-1 to V-134; M-1 to E-133; M-1 to P-132; M-1 to D-131; M-1 to
P-130; M-1 to E-129; M-1 to G-128; M-1 to K-127; M-1 to Q-126; M-1 to
R-125; M-1 to G-124; M-1 to P-123; M-1 to S-122; M-1 to L-121; M-1 to
H-120; M-1 to A-119; M-1 to G-118; M-1 to A-117; M-1 to E-116; M-1 to
L-115; M-1 to H-114; M-1 to G-l I3; M-1 to R-112; M-1 to G-111; M-1 to
R-110; M-1 to L-109; M-1 to D-108; M-1 to C-107; M-1 to P-106; M-1 to
S-105; M-1 to G-104; M-1 to I-103; M-1 to D-102; M-1 to P-101; M-1 to
D-100; M-1 to C-99; M-1 to M-98; M-1 to Q-97; M-1 to C-96; M-1 to Q-95;
M-1 to L-94; M-1 to C-93; M-1 to K-92; M-1 to S-91; M-1 to L-90; M-1 to
G-89; M-1 to N-88; M-1 to L-87; M-1 to H-86; M-1 to A-85; M-1 to I-84; M-1
to Y-83; M-1 to T-82; M-1 to G-81; M-1 to P-80; M-i to P-79; M-1 to C-78;
M-1 to P-77; M-1 to E-76; M-1 to C-75; M-1 to V-74; M-1 to T-73; M-1 to
G-72; M-1 to T-7I; M-1 to L-70; M-1 to E-69; M-1 to G 68; M-1 to C-67; M-1
to A-66; M-1 to E-65; M-1 to K-64; M-1 to V-63; M-1 to R-62; M-1 to Y-61;
M-1 to G-60; M-1 to P-59; M-1 to S-58; M-1 to C-57; M-1 to K-56; M-1 to
P-55; M-1 to C-54; M-1 to C-53; M-1 to E-52; M-1 to S-51; M-1 to G-50; M-1
to V-49; M-1 to P-48; M-1 to Y-47; M-1 to E-46; M-1 to D-45; M-1 to E-44;
M-I to K-43; M-1 to C-42; M-1 to S-41; M-I to P-40; M-1 to L-39; M-I to
A-38; M-1 to P-37; M-1 to A-36; M-1 to Y-35; M-1 to C-34; M-1 to P-33; M-1
to A-32; M-1 to G-31; M-1 to L-30; M-1 to F-29; M-1 to T-28; M-1 to L-27;
M-i to Y-26; M-1 to L-25; M-1 to V-24; M-1 to L-23; M-1 to R-22; M-1 to
L-21; M-1 to V-20; M-1 to D-19; M-1 to T-18; M-1 to R-17; M-1 to P-16; M-1
to T-15; M-1 to S-14; M-1 to R-13; M-1 to W-12; M-1 to P-11; M-1 to P-10;
M-1 to P-9; M-1 to G-8; M-I to W-7; and M-1 to D-6 of the sequence of the
TR2-S V 1 sequence shown in FIG. 4A-4C. The present invention is also directed
to nucleic acid molecules comprising, or alternatively consisting of, a
polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%,
or 99% identical to the polynucleotide sequences encoding the polypeptides
described above. The present invention also encompasses the above
AMENDED SHEET
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-58-
polynucleotide sequences fused to a heterologous polynucleotide sequence.
Polypeptides encoded by these polynucleotide sequences are also encompassed
by the invention.
The invention also provides polypeptides having one or more amino acids
deleted from both the amino and the carboxyl termini of a TR2-SVl polypeptide,
which may be described generally as having residues n2-rn2 of FIG. 4A-4C (i.
e.,
SEQ ID NO:S), where n2 and mz are integers as described above. Polynucleotides
encoded by these polypeptides are also encompassed by the invention.
Also mentioned above, even if deletion of one or more amino acids from
the N-terminus of a protein results in modification of loss of one or more
biological functions of the protein, other biological activities may still be
retained.
Thus, the ability of shortened TR2-SVZ muteins to induce and/or bind to
antibodies which recognize the complete or mature forms of the polypeptides
generally will be retained when less than the majority of the residues of the
complete or mature polypeptide are removed from the N-terminus. Whether a
particular polypeptide lacking N-terminal residues of a complete polypeptide
retains such immunologic activities can readily be determined by routine
methods
described herein and otherwise known in the art. It is not unlikely that a TR2-
SV2
mutein with a large number of deleted N-terminal amino acid residues may
retain
some biological or immunogenic activities. In fact, peptides composed of as
few
as six TRZ-SV2 amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having
one or more residues deleted from the amino terminus of the TR2-S V2 amino
acid
sequence shown in FIG. 7A-7C (i.e., SEQ ID N0:8), up to the serine residue at
position number 131 and polynucleotides encoding such polypeptides. In
particular, the present invention provides polypeptides comprising, or
alternatively
consisting of, the amino acid sequence of residues n3-136 of FIG. 7A-7C (i.e.,
SEQ ID N0:8), where n3 is an integer in the range of 2 to 131. Polynucleotides
encoded by these polypeptides are also encompassed by the invention.
AMENDED SHEET
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More in particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, the amino acid
sequence
of residues of L-2 to K-136; G-3 to K-136; T-4 to K-136; S-5 to K-136; G-6 to
K-136; H-7 to K-136; L-8 to K-136; V-9 to K-136; W-10 to K-136; L-11 to
K-136; S-12 to K-136; Q-13 to K-136; G-14 to K-136; F-15 to K-136; S-16 to
K-136; L-17 to K-136; A-18 to K-136; G-19 to K-136; R-20 to K-136; P-21 to
K-136; G-22 to K-136; S-23 to K-136; S-24 to K-136; P-25 to K-136; W-26 to
K-136; P-27 to K-136; V-28 to K-136; D-29 to K-136; A-30 to K-136; V-31 to
K-136; L-32 to K-136; A-33 to K-136; C-34 to K-136; G-35 to K-136; W-36 to
K-136; C-37 to K-136; P-38 to K-136; G-39 to K-136; L-40 to K-136; H-41 to
K-136; V-42 to K-136; P-43 to K-136; P-44 to K-136; L-45 to K-136; S-46 to
K-136; P-47 to K-136; S-48 to K-136; S-49 to K-136; W-50 to K-136; T-51 to
K-136; P-52 to K-136; A-53 to K-136; M-54 to K-136; G-55 to K-136; L-56 to
K-136; R-57 to K-136; A-58 to K-136; S-59 to K-136; R-60 to K-136; N-61 to
K-136; C-62 to K-136; S-63 to K-136; R-64 to K-136; T-65 to K-136; E-66 to
K-136; N-67 to K-136; A-68 to K-136; V-69 to K-136; C-70 to K-136; G-71 to
K-136; C-72 to K-136; S-73 to K-136; P-74 to K-136; G-75 to K-136; H-76 to
K-136; F-77 to K-136; C-78 to K-136; I-79 to K-136; V-80 to K-136; Q-81 to
K-136; D-82 to K-136; G-83 to K-136; D-84 to K-136; H-85 to K-136; C-86 to
K-136; A-87 to K-136; A-88 to K-136; C-89 to K-136; R-90 to K-136; A-91 to
K-136; Y-92 to K-136; A-93 to K-136; T-94 to K-136; S-95 to K-136; S-96 to
K-136; P-97 to K-136; G-98 to K-136; Q-99 to K-136; R-100 to K-136; V-101
to K-136; Q-102 to K-136; K-103 to K-136; G-104 to K-136; G-105 to K-136;
T-106 to K-136; E-107 to K-136; S-108 to K-136; Q-109 to K-136; D-I 10 to
K-136; T-111 to K-136; L-112 to K-136; C-113 to K-136; Q-114 to K-136;
N-115 to K-136; C-116 to K-136; P-117 to K-136; R-118 to K-136; G-119 to
K-136; P-120 to K-136; S-121 to K-136; L-122 to K-136; P-123 to K-136;
M-124 to K-136; G-125 to K-136; P-126 to K-136; W-127 to K-136; R-128 to
K-136; N-129 to K-136; V-130 to K-136; and S-131 to K-136 of the TR2-SV2
22-05-2000 CA 02365405 2001-09-21 US 000007521
-60-
sequence shown in FIG. 7A-7C. The present invention is also directed to
nucleic
acid molecules comprising, or alternatively consisting of, a polynucleotide
sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical
to the polynucleotide sequences encoding the polypeptides described above. The
present invention also encompasses the above polynucleotide sequences fused to
a heterologous polynucleotide sequence. Polypeptides encoded by these
polynucleotide sequences are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids
from the C-terminus of a protein results in modification of loss of one or
more
biological functions of the protein, other biological activities may still be
retained. Thus, the ability of the shortened TR2-SV2 mutein to induce and/or
bind to antibodies which recognize the complete or mature forms of the
polypeptide generally will be retained when less than the majority of the
residues
of the complete or mature polypeptide are removed from the C-terminus.
Whether a particular polypeptide lacking C-terminal residues of a complete
polypeptide retains such immunologic activities can readily be determined by
routine methods described herein and otherwise known in the art. It is not
unlikely that a TR2-SV2 mutein with a large number of deleted C-terminal amino
acid residues may retain some biological or immunogenic activities. In fact,
peptides composed of as few as six TR2-SV2 amino acid residues may often
evoke an immune response.
Accordingly, the present invention further provides polypeptides having
one or more residues deleted from the carboxy terminus of the amino acid
sequence of the TR2-SV2 polypeptide shown in FIG. 7A-7C (i. e., SEQ 1D N0:8),
up to the glycine residue at position number 6, and polynucleotides encoding
such
polypeptides. In particular, the present invention provides polypeptides
comprising, or alternatively consisting of, the amino acid sequence of
residues
1-m3 of FIG. 7A-7C (i.e., SEQ )D N0:8), where m3 is an integer in the range of
6 to 135. Polynucleotides encoded by these polypeptides are also encompassed
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by the invention.
More in particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, the amino acid
sequence
of residues M-1 to S-135; M-1 to P-134; M-1 to R-133; M-1 to T-132; M-1 to
S-131; M-1 to V-130; M-1 to N-129; M-1 to R-128; M-1 to W-127; M-1 to
P-126; M-1 to G-125; M-1 to M-124; M-1 to P-123; M-1 to L-122; M-1 to S-I21;
M-1 to P-120; M-1 to G-119; M-1 to R-118; M-1 to P-117; M-1 to C-116; M-1
to N-115; M-I to Q-1 I4; M-1 to C-113; M-1 to L-l I2; M-1 to T-111; M-1 to
D-110; M-1 to Q-109; M-1 to S-108; M-1 to E-107; M-1 to T-106; M-1 to G-105;
M-1 to G-104; M-1 to K-103; M-1 to Q-102; M-1 to V-101; M-1 to R-100; M-1
to Q-99; M-1 to G-98; M-1 to P-97; M-1 to S-96; M-1 to S-95; M-1 to T-94; M-1
to A-93; M-1 to Y-92; M-1 to A-91; M-1 to R-90; M-1 to C-89; M-I to A-88;
M-1 to A-87; M-1 to C-86; M-I to H-85; M-1 to D-84; M-1 to G-83; M-1 to
D-82; M-1 to Q-81; M-1 to V-80; M-1 to I-79; M-1 to C-78; M-1 to F-77; M-1
to H-76; M-1 to G-75; M-1 to P-74; M-1 to S-73; M-1 to C-72; M-1 to G-71; M-1
to C-70; M-1 to V-69; M-1 to A-68; M-1 to N-67; M-1 to E-66; M-1 to T-65;
M-1 to R-64; M-1 to S-63; M-1 to C-62; M-1 to N-61; M-1 to R-60; M-1 to S-59;
M-1 to A-58; M-1 to R-57; M-1 to L-56; M-1 to G-55; M-1 to M-54; M-1 to
A-53; M-1 to P-52; M-1 to T-51; M-1 to W-50; M-1 to S-49; M-1 to S-48; M-1
to P-47; M-1 to S-46; M-I to L-45; M-1 to P-44; M-1 to P-43; M-1 to V-42; M-1
to H-41; M-1 to L-40; M-1 to G-39; M-1 to P-38; M-1 to C-37; M-1 to W-36;
M-1 to G-35; M-1 to C-34; M-1 to A-33; M-1 to L-32; M-1 to V-31; M-1 to
A-30; M-1 to D-29; M-1 to V-28; M-I to P-27; M-1 to W-26; M-1 to P-25; M-1
to S-24; M-1 to S-23; M-1 to G-22; M-I to P-21; M-1 to R-20; M-I to G-19; M-1
to A-18; M-1 to L-17; M-1 to S-16; M-1 to F-15; M-1 to G-14; M-I to Q-13; M-1
to S-12; M-1 to L-11; M-1 to W-10; M-1 to V-9; M-1 to L-8; M-1 to H-7; and
M-1 to G-6 of the sequence of the TR2-S V2 sequence shown in FIG. 7A-7C. The
present invention is also directed to nucleic acid molecules comprising, or
alternatively consisting
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of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%,
98%, or 99% identical to the polynucleotide sequences encoding the
polypeptides
described above. The present invention also encompasses the above
polynucleotide sequences fused to a heterologous polynucleotide sequence.
Polypeptides encoded by these polynucleotide sequences are also encompassed
by the invention.
The invention also provides polypeptides having one or more amino acids
deleted from both the amino and the carboxyl termini of a TR2-S V2
polypeptide,
which may be described generally as having residues n3-m3 of FIG. 7A-7C (i.
e.,
SEQ TD N0:8), where n3 and m3 are integers as described above. Polynucleotides
encoded by these polypeptides are also encompassed by the invention.
In addition, the present invention further provides polypeptides having
one or more residues deleted from the amino terminus of the predicted
extracellular domain of the TR2 amino acid sequence shown in SEQ ID N0:2
I5 (FIG. lA-IB), up to the glycine residue at position number 159 and
polynucleotides encoding such polypeptides. In particular, the present
invention
provides polypeptides comprising, or alternatively consisting of, the amino
acid
sequence of residues n4-164 of SEQ ID N0:2, where n4 is an integer in the
range
of 1 to 159. Polynucleotides encoded by these polypeptides are also
encompassed by the invention.
More in particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, the amino acid
sequence
of residues of P-1 to H-164; A-2 to H-164; L-3 to H-164; P-4 to H-164; S-5 to
H-164; C-6 to H-164; K-7 to H-I64; E-8 to H-164; D-9 to H-164; E-10 to H-164;
Y-11 to H-164; P-12 to H-164; V-13 to H-164; G-14 to H-164; S-15 to H-164;
E-16 to H-164; C-17 to H-164; C-18 to H-,I64; P-19 to H-164; K-20 to H-164;
C-21 to H-164; S-22 to H-164; P-23 to H-164; G-24 to H-164; Y-25 to H-164;
R-26 to H-164; V-27 to H-164; K-28 to H-164; E-29 to H-164; A-30 to H-164;
C-31 to H-164; G-32 to H-164; E-33 to H-164; L-34 to H-164; T-35 to
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H-164; G-3 6 to H-164; T-3 7 to H-164; V-3 8 to H-164; C-3 9 to H-164; E-40 to
H-164; P-41 to H-164; C-42 to H-164; P-43 to H-164; P-44 to H-164; G-45 to
H-164; T-46 to H-164; Y-47 to H-164; I-48 to H-164; A-49 to H-164; H-SO to
H-164; L-51 to H-164; N-52 to H-164; G-53 to H-164; L-54 to H-164; S-55 to
H-164; K-56 to H-164; C-57 to H-164; L-58 to H-164; Q-59 to H-164; C-60 to
H-164; Q-61 to H-164; M-62 to H-164; C-63 to H-164; D-64 to H- I 64; P-65 to
H-164; A-66 to H-164; M-67 to H-164; G-68 to H-164; L-69 to H-164; R-70 to
H-164; A-71 to H-164; S-72 to H-164; R-73 to H-164; N-74 to H-164; C-75 to
H-164; S-76 to H-164; R-77 to H-164; T-78 to H-164; E-79 to H-164; N-80 to
H-164; A-81 to H-164; V-82 to H-164; C-83 to H-164; G-84 to H-164; C-85 to
H-164; S-86 to H-164; P-87 to H-164; G-88 to H-164; H-89 to H-164; F-90 to
H-164; C-91 to H-164; I-92 to H-164; V-93 to H-164; Q-94 to H-164; D-95 to
H-164; G-96 to H-164; D-97 to H-164; H-98 to H-164; C-99 to H-164; A-100
to H-164; A-101 to H-164; C-102 to H-164; R-103 to H-164; A-104 to H-164;
Y-105 to H-164; A-106 to H-164; T-107 to H-164; S-108 to H-164; S-109 to
H-164; P-110 to H-164; G-111 to H-164; Q-112 to H-164; R-I 13 to H-164;
V-114 to H-164; Q-11 S to H-164; K-116 to H-164; G-117 to H-164; G-118 to
H- I 64; T- I 19 to H-164; E-120 to H-164; S-121 to H-164; Q-122 to H-164;
D-123 to H-164; T-124 to H-164; L-125 to H-164; C-126 to H-164; Q-127 to
H-164; N-128 to H-164; C-129 to H-164; P-130 to H-164; P-131 to H-164;
G-132 to H-164; T-133 to H-164; F-134 to H-164; S-135 to H-164; P-136 to
H-164; N-137 to H-164; G-138 to H-164; T-139 to H-164; L-140 to H-164;
E-141 to H-164; E-142 to H-164; C- I 43 to H-164; Q-144 to H-164; H- I 45 to
H-164; Q-146 to H-164; T-147 to H-164; K-148 to H-164; C-149 to H-164;
S-150 to H-164; W-151 to H-164; L-152 to H-164; V-153 to H-164; T-154 to
H-164; K-155 to H-164; A-156 to H-164; G-157 to H-164; A-158 to H-164; and
G-159 to H-164 ofthe TRZ amino acid sequence shown in SEQ ID N0:2 (which
is identical to that shown in FIG. lA-IB, with the exception that the amino
acid
residues in FIG. lA-1B are numbered consecutively from 1 through 283 from the
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N-terminus to the C-terminus, while the amino acid residues in SEQ ID N0:2 are
numbered consecutively from -36 through 247 to reflect the position of the
predicted signal peptide). The present invention is also directed to nucleic
acid
molecules comprising, or alternatively consisting of, a polynucleotide
sequence at
least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the
polynucleotide sequences encoding the polypeptides described above. The
present
invention also encompasses the above polynucleotide sequences fused to a
heterologous polynucleotide sequence. Polypeptides encoded by these
polynucleotide sequences are also encompassed by the invention.
The present invention further provides polypeptides having one or more
residues deleted from the carboxy terminus of the predicted extracellular
domain
of the amino acid sequence of the TR2 shown in SEQ ID N0:2 (FIG. 1 A-1 B), up
to the cysteine residue at position number 6 in SEQ ID N0:2, and
polynucleotides
encoding such polypeptides. In particular, the present invention provides
polypeptides comprising, or alternatively consisting of, the amino acid
sequence
of residues 1-m4 of SEQ ID N0:2 (FIG. IA-IB), where m4 is an integer in the
range of 6 to 164. Polynucleotides encoded by these polypeptides are also
encompassed by the invention.
More in particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, the amino acid
sequence
ofresidues P-1 to H-164; P-1 to S-163; P-1 to S-162; P-1 to S-161; P-1 to T-
160;
P-1 to G-159; P-1 to A-158; P-1 to G-157; P-1 to A-156; P-1 to K-155; P-1 to
T-1 _54; P-1 to V-153; P-1 to L-152; P-1 to W-151; P-1 to S-150; P-I to C-149;
P-1 to K-148; P-1 to T-147; P-1 to Q-146; P-1 to H-145; P-1 to Q-144; P-1 to
C-143; P-1 to E-142; P-1 to E-141; P-1 to L-140; P-I to T-139; P-1 to G-138;
P-1 to N-137; P-1 to P-136; P-1 to S-135; P-1 to F-134; P-I to T-133; P-1 to
G-132; P-1 to P-131; P-1 to P-130; P-1 to C-129; P-1 to N-128; P-1 to Q-127;
P-1 to C-126; P-1 to L-125; P-1 to T-124; P-1 to D-123; P-1 to Q-122; P-1 to
S-121; P-1 to E-120; P-1 to T-11-9; P-1 to G-118; P-1 to G-117; P-1 to K-116;
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P-1 to Q-1 I5; P-I to V-114; P-1 to R-I 13; P-1 to Q-112; P-1 to G-1 I I; P-1
to
P-110; P-1 to S-109; P-1 to S-108; P-I to T-107; P-1 to A-106; P-1 to Y-105;
P-I to A-104; P-1 to R-103; P-I to C-102; P-1 to A-101; P-1 to A-100; P-1 to
C-99; P-1 to H-98; P-1 to D-97; P-1 to G-96; P-1 to D-95; P-1 to Q-94; P-1 to
V-93; P-1 to I-92; P-1 to C-91; P-1 to F-90; P-1 to H-89; P-1 to G-88; P-I to
P-87; P-I to S-86; P-1 to C-85; P-1 to G-84; P-1 to C-83; P-1 to V-82; P-1 to
A-81; P-1 to N-80; P-I to E-79; P-I to T-78; P-1 to R-77; P-1 to S-76; P-1 to
C-75; P-I to N-74; P-1 to R-73; P-I to S-72; P-1 to A-71; P-1 to R-70; P-1 to
L-69; P-1 to G-68; P-1 to M-67; P-1 to A-66; P-1 to P-65; P-1 to D-64; P-1 to
C-63; P-1 to M-62; P-1 to Q-61; P-1 to C-60; P-I to Q-59; P-1 to L-58; P-1 to
C-57; P-I to K-56; P-1 to S-55; P-1 to L-54; P-1 to G-53; P-1 to N-52; P-1 to
L-51; P-1 to H-S0; P-I to A-49; P-I to I-48; P-1 to Y-47; P-1 to T-46; P-I to
G-45; P-I to P-44; P-1 to P-43; P-1 to C-42; P-I to P-41; P-1 to E-40; P-I to
C-39; P-I to V-38; P-1 to T-37; P-1 to G-36; P-1 to T-35; P-I to L-34; P-1 to
E-33; P-1 to G-32; P-1 to C-31; P-1 to A-30; P-1 to E-29; P-1 to K-28; P-1 to
V-27; P-1 to R-26; P-I to Y-25; P-1 to G-24; P-I to P-23; P-1 to S-22; P-1 to
C-21; P-I to K-20; P-1 to P-19; P-I to C-18; P-I to C-17; P-1 to E-16; P-I to
S-15; P-1 to G-14; P-1 to V-13; P-I to P-12; P-1 to Y-11; P-I to E-10; P-1 to
D-9; P-I to E-8; P-1 to K-7; and P-1 to C-6 of the sequence of the TR2
sequence
shown in SEQ ID N0:2 (which is identical to the sequence shown as FIG. 1 A-1
B,
with the exception that the amino acid residues in FIG. lA-1B are numbered
consecutively from I through 283 from the N-terminus to the C-terminus, while
the amino acid residues in SEQ ID N0:2 are numbered consecutively from -36
through 247 to reflect the position of the predicted signal peptide). The
present
invention is also directed to nucleic acid molecules comprising, or
alternatively
consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%,
96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the
polypeptides described above. The present invention also encompasses the above
polynucleotide sequences fused to a heterologous polynucleotide sequence.
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Polypeptides encoded by these polynucleotide sequences are also encompassed
by the invention.
The invention also provides polypeptides having one or more amino acids
deleted from both the amino and the carboxyl termini of a soluble TR2
polypeptide, which may be described generally as having residues n'-m4 of SEQ
1D N0:2 (FIG. 1 A-1 B), where n4 and m4 are integers as described above.
Polynucleotides encoded by these polypeptides are also encompassed by the
invention.
In addition, the present invention further provides polypeptides having one
or more residues deleted from the amino terminus of the predicted
extracellular
domain of the TR2-SV 1 amino acid sequence shown in SEQ 117 NO:S (FIG.
4A-4C), up to the threonine residue at position number 144 and polynucleotides
encoding such polypeptides. In particular, the present invention provides
polypeptides comprising, or alternatively consisting of, the amino acid
sequence
of residues ns-149 of SEQ ID N0:5, where ns is an integer in the range of 1 to
144. Polynucleotides encoded by these polypeptides are also encompassed by the
invention.
More in particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, the amino acid
sequence
of residues of P-1 to A-149; A-2 to A-149; L-3 to A-149; P-4 to A-149; S-5 to
A-149; C-6 to A-I49; K-7 to A-149; E-8 to A-149; D-9 to A-149; E-10 to A-149;
Y-11 to A-149; P-12 to A-149; V-13 to A-149; G-14 to A-149; S-15 to A-149;
E-16 to A-149; C-17 to A-149; C-18 to A-149; P-19 to A-149; K-20 to A-149;
C-21 to A-149; S-22 to A-149; P-23 to A-149; G-24 to A-149; Y-25 to A-149;
R-26 to A-149; V-27 to A-149; K-28 to A-149; E-29 to A-149; A-30 to A-149;
C-31 to A-149; G-32 to A-149; E-33 to A-149; L-34 to A-149; T-35 to A-149;
G-36 to A-149; T-37 to A-149; V-38 to A-149; C-39 to A-149; E-40 to A-149;
P-41 to A-149; C-42 to A-149; P-43 to A-149; P-44 to A-149; G-45 to A-149;
T-46 to A-149; Y-47 to A-149; I-48 to A-149; A-49 to A-149; H-50 to A-149;
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L-51 to A-149; N-52 to A-149; G-53 to A-149; L-54 to A-149; S-55 to A-149;
K-56 to A-149; C-57 to A-149; L-58 to A-149; Q-59 to A-149; C-60 to A-149;
Q-61 to A-149; M-62 to A-149; C-63 to A-149; D-64 to A-149; P-65 to A-149;
D-66 to A-149; I-67 to A-149; G-68 to A-149; S-69 to A-149; P-70 to A-149;
S C-7I to A-149; D-72 to A-149; L-73 to A-149; R-74 to A-149; G-75 to A-149;
R-76 to A-149; G-77 to A-149; H-78 to A-149; L-79 to A-149; E-80 to A-149;
A-81 to A-149; G-82 to A-149; A-83 to A-149; H-84 to A-149; L-85 to A-149;
S-86 to A-.149; P-87 to A-I49; G-88 to A-149; R-89 to A-149; Q-90 to A-149;
K-91 to A-149; G-92 to A-149; E-93 to A-I49; P-94 to A-149; D-95 to A-149;
P-96 to A-149; E-97 to A-149; V-98 to A-149; A-99 to A-149; F-100 to A-149;
E-101 to A-149; S-102 to A-I49; L-103 to A-149; S-104 to A-149; A-105 to
A-149; E-106 to A-149; P-107 to A-149; V-108 to A-149; H-109 to A-149;
A-110 to A-149; A-111 to A-149; N-112 to A-149; G-113 to A-149; S-114 to
A-149; V-115 to A-149; P-116 to A-149; L-117 to A-149; E-118 to A-149; P-1 I9
to A-149; H-I20 to A-149; A-121 to A-149; R-122 to A-149; L-123 to A-149;
S-124 to A-149; M-125 to A-149; A-126 to A-149; S-127 to A-149; A-I28 to
A-149; P-129 to A-149; C-130 to A-149; G-131 to A-149; Q-132 to A-149;
A-133 to A-149; G-134 to A-149; L-135 to A-149; H-136 to A-149; L-137 to
A-I49; R-138 to A-149; D-139 to A-149; R-140 to A-149; A-141 to A-149;
D-142 to A-149; G-143 to A-149; and T-144 to A-149 of the TR2-SVl amino
acid sequence shown in SEQ ID NO:S (which is identical to that shown in FIG.
4A-4.C, with the exception that the amino acid residues in FIG. 4A-4C are
numbered consecutively from 1 through 185 from the N-terminus to the
C-terminus, while the amino acid residues in SEQ ID NO:S are numbered
consecutively from -36 through 149 to reflect the position of the predicted
signal
peptide). The present invention is also directed to nucleic acid molecules
comprising, or alternatively consisting of, a polynucleotide sequence at least
80%,
85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide
sequences encoding the polypeptides described above. The present invention
also
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encompasses the above polynucleotide sequences fused to a heterologous
polynucleotide sequence. Polypeptides encoded by these polynucleotide
sequences are also encompassed by the invention.
The present invention flu ther provides polypeptides having one or more
residues deleted from the carboxy terminus of the predicted extracellular
domain
of the amino acid sequence of the TR2-SV 1 shown in SEQ ID NO:S (FIG.
4A-4C), up to the cysteine residue at position number 6 in SEQ ID NO:S, and
polynucleotides encoding such polypeptides. In particular, the present
invention
provides polypeptides comprising, or alternatively consisting of, the amino
acid
sequence of residues 1-ms of SEQ ID NO:S (FIG. 4A-4C), where ms is an integer
in the range of 6 to 149. Polynucleotides encoded by these polypeptides are
also
encompassed by the invention.
More in particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, the amino acid
sequence
of residues P-1 to A-149; P-1 to R-148; P-1 to G-147; P-1 to G-146; P-1 to
P-145; P-1 to T-144; P-1 to G-143; P-1 to D-142; P-1 to A-141; P-1 to R-140;
P-1 to D-139; P-1 to R-138; P-1 to L-137; P-1 to H-136; P-1 to L-135; P-1 to
G-134; P-1 to A-133; P-1 to Q-132; P-1 to G-131; P-1 to C-130; P-1 to P-129;
P-1 to A-128; P-1 to S-127; P-1 to A-126; P-1 to M-125; P-1 to S-124; P-1 to
L-I23; P-1 to R-122; P-1 to A-121; P-1 to H-120; P-1 to P-119; P-1 to E-118;
P-1 to L-117; P-1 to P-116; P-1 to V-115; P-1 to S-114; P-1 to G-113; P-1 to
N-112; P-1 to A-I 11; P-1 to A-110; P-1 to H-109; P-1 to V-108; P-1 to P-107;
P-1 to E-106; P-1 to A-105; P-1 to S-104; P-1 to L-103; P-1 to S-102; P-1 to
E-101; P-1 to F-100; P-1 to A-99; P-1 to V-98; P-1 to E-97; P-1 to P-96; P-1
to
D-95; P-1 to P-94; P-1 to E-93; P-1 to G-92; P-I to K-9I; P-1 to Q-90; P-1 to
R-89; P-1 to G-88; P-1 to P-87; P-1 to S-86; P-1 to L-85; P-1 to H-84; P-1 to
A-83; P-1 to G-82; P-1 to A-81; P-1 to E-80; P-1 to L-79; P-1 to H-78; P-1 to
G-77; P-1 to R-76; P-1 to G-75; P-1 to R-74; P-1 to L-73; P-1 to D-72; P-1 to
C-71; P-1 to P-70; P-1 to S-69; P-1 to G-68; P-1 to I-67; P-1 to D-66; P-1 to
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P-65; P-1 to D-64; P-1 to C-63; P-1 to M-62; P-1 to Q-61; P-1 to C-60; P-1 to
Q-59; P-1 to L-58; P-1 to C-57; P-1 to K-56; P-1 to S-55; P-1 to L-54; P-1 to
G-53; P-1 to N-52; P-1 to L-SI; P-1 to H-50; P-1 to A-49; P-I to I-48; P-1 to
Y-47; P-I to T-46; P-1 to G-45; P-1 to P-44; P-1 to P-43; P-1 to C-42; P-I to
P-41; P-1 to E-40; P-1 to C-39; P-1 to V-38; P-1 to T-37; P-1 to G-36; P-1 to
T-35; P-1 to L-34; P-1 to E-33; P-1 to G-32; P-I to C-31; P-1 to A-30; P-1 to
E-29; P-1 to K-28; P-1 to V-27; P-1 to R-26; P-1 to Y-25; P-1 to G-24; P-1 to
P-23; P-1 to S-22; P-1 to C-21; P-1 to K-20; P-1 to P-19; P-I to C-18; P-1 to
C-I7; P-1 to E-16; P-1 to S-15; P-1 to G-14; P-1 to V-13; P-1 to P-12; P-1 to
Y-I1; P-1 to E-10; P-1 to D-9; P-1 to E-8; P-1 to K-7; and P-I to C-6 of the
sequence of the TR2-SV 1 sequence shown in SEQ ID NO:S (which is identical
to the sequence shown as FIG. 4A-4C, with the exception that the amino acid
residues in FIG. 4A-4C are numbered consecutively from 1 through 185 from the
N-terminus to the C-terminus, while the amino acid residues in SEQ ID NO:S are
numbered consecutively from -36 through 149 to reflect the position of the
predicted signal peptide). The present invention is also directed to nucleic
acid
molecules comprising, or alternatively consisting of, a polynucleotide
sequence
at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the
polynucleotide sequences encoding the polypeptides described above. The
present invention also encompasses the above polynucleotide sequences fused to
a heterologous polynucleotide sequence. Polypeptides encoded by these
polynucleotide sequences are also encompassed by the invention.
The invention also provides polypeptides having one or more amino acids
deleted from both the amino and the carboxyl termini of a soluble TR2-SV1
polypeptide, which may be described generally as having residues ns-m5 of SEQ
ID NO:S (FIG. 4A-4C), where ns and ms are integers as described above.
Polynucleotides encoded by these polypeptides are also encompassed by the
invention.
Additionally, one or more of the amino acid residues of the polypeptides
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of the invention (e.g., arginine and lysine residues) may be deleted or
substituted
with another residue to eliminate undesired processing by proteases such as,
for
example, furins or kexins.
The invention further provides for the proteins containing polypeptide
sequences encoded by the polynucleotides of the invention.
Among the especially preferred fragments of the invention are fragments
characterized by structural or functional attributes of TR2 receptors of the
invention. Such fragments include amino acid residues that comprise, or
alternatively consist of, alpha-helix and alpha-helix forming regions ("alpha-
regions"), beta-sheet and beta-sheet-forming regions ("beta-regions"), turn
and
turn-forming regions ("turn-regions"), coil and coil-forming regions ("coil-
regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic
regions,
beta amphipathic regions, surface forming regions, and high antigenic index
regions (i.e., containing four or more contiguous amino acids having an
antigenic
index of greater than or equal to 1.5, as identified using the default
parameters of
the Jameson-Wolf program) of complete (i. e., full-length) TR2 receptor (SEQ
ID
N0:2). Certain preferred regions are those set out in FIG. 3 and include, but
are
not limited to, regions of the aforementioned types identified by analysis of
the
amino acid sequence depicted in FIG. lA-1B (SEQ ID N0:2), such preferred
regions include; Gamier-Robson predicted alpha-regions, beta-regions, turn-
regions, and coil-regions; Chou-Fasman predicted alpha-regions, beta-regions,
turn-regions, and coil-regions; Kyte-Doolittle predicted hydrophilic and
hydrophobic regions; Eisenberg alpha and beta amphipathic regions; Emini
surface-forming regions; and Jameson-Wolf high antigenic index regions, as
predicted using the default parameters of these computer programs.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
In additional embodiments, the polynucleotides of the invention encode
functional attributes of TR2 receptors. Preferred embodiments of the invention
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in this regard include fragments that comprise, or alternatively consist of,
one,
two, three, four or more of one or more of the following functional domains:
alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and
beta-sheet forming regions ("beta-regions"), turn and turn-forming regions
("turn-regions"), coil and coil-forming regions ("coil-regions"), hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic
regions, flexible regions, surface-forming regions and high antigenic index
regions
of TR2 receptors.
The data representing the structural or functional attributes of the TR2
receptors set forth in FIGS. 3, 6 and 9 and Tables II, II and IX were
generated
using the various identified modules and algorithms of the DNA*STAR set on
default parameters. In a preferred embodiment, the data presented in columns
VIII, IX, XIII, and XIV of Table II can be used to determine regions of TR2
receptors which exhibit a high degree of potential for antigenicity. Regions
of
high antigenicity are determined from the data presented in columns VIII, IX,
XIII, and/or IV by choosing values which represent regions of the polypeptide
which are likely to be exposed on the surface of the polypeptide in an
environment
in which antigen recognition may occur in the process of initiation of an
immune
response.
Certain preferred regions in these regards are set out in FIGS. 3, 6 and 9,
but may, as shown in Tables II, III and IV, respectively, be represented or
identified by using tabular representations of the data presented in FIGS. 3,
6 and
9. The DNA*STAR computer algorithm used to generate FIGS. 3, 6 and 9 (set
on the original default parameters) was used to present the data in FIGS. 3, 6
and
9 in a tabular format. (See Tables II, III and IV, respectively).
The above-mentioned preferred regions set out in FIGS. 3, 6 and 9 and in
Tables II, III and IV include, but are not limited to, regions of the
aforementioned
types identified by analysis of the amino acid sequence set out in FIGs. l, 4
and
7. As set out in FIGs. 3, 6 and 9, and in Tables II, III and IV, such
preferred
CA 02365405 2001-09-21
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regions include Gamier-Robson alpha-regions, beta-regions, turn-regions, and
coil-regions (columns I, III, V, and VII in Tables II, III and IV), Chou-
Fasman
alpha-regions, beta-regions, and turn-regions (columns II, IV, and VI in
Tables II,
III and IV), Kyte-Doolittle hydrophilic regions (column VIII in Tables II, III
and
IV), Hopp-Woods hydrophobic regions (column IX in Tables II, III and IV),
Eisenberg alpha- and beta-amphipathic regions (columns X and XI in Tables II,
III and IV), Karplus-Schulz flexible regions (column XII in Tables II, III and
IV),
Jameson-Wolf regions of high antigenic index (column XIII in Tables II, III
and
IV), and Emini surface-forming regions (column XIV in Tables II, III and IV).
CA 02365405 2001-09-21
WO 00/56405 PCT/US00/07521
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N N N N N M M M M M M M
~rl cW 1 rl ~-i rl r1 r-i r-I .-i r-i r-I rl
N
O
N ~ O f~ b~ s~ ~-I ~ ~ is O S-I u~
N .-I~IS-i~t~r~N.~~~N
P4 C~ w E-~ r~C rC ~ ~ N ~C w u7 a
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Among highly preferred fragments in this regard are those that comprise,
or alternatively consist of, regions of TR2 receptors that combine several
structural features, such as several of the features set out above.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
As one of skill in the art will appreciate, TR2 polypeptides of the present
invention and epitope-bearing fragments thereof can be combined with
heterologous polypeptide sequences. For example, the polypeptides ofthe
present
invention may be fused with the constant domain of immunoglobulins (IgA, IgE,
IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof,
including both entire domains and portions thereof), resulting in chimeric
polypeptides. These fusion proteins facilitate purification and show an
increased
half life.
The present invention is further directed to isolated polypeptides
comprising, or alternatively consisting of, fragments of TR2, TR2-SVl, and
TR2-SV2. In particular, the invention provides isolated polypeptides
comprising,
or alternatively consisting of, the amino acid sequences of a member selected
from
the group consisting of amino acids -36 to 24, -26 to 34, -16 to 44, -6 to 54,
1 to
60, 1 I to 70, 21 to 80, 31 to 90, 41 to 100, 51 to I 10, 61 to 120, 71 to
130; 81
to 140, 91 to 150, 101 to 160, 111 to 170, 121 to 180, 131 to 190, 141 to 200,
1 S 1 to 210, I 61 to 220, 171 to 230, 181 to 240, and 191 to 247 of SEQ ID
N0:2,
as well as isolated polynucleotides which encode these polypeptides. The
invention further provides isolated polypeptides comprising, or alternatively
consisting of, the amino acid sequences of a member selected from the group
consisting of amino acids -36 to 24, -26 to 34, -16 to 44, -6 to 54, 1 to 60,
11 to
70, 21 to 80, 31 to 90, 41 to 100, 51 to 110, 61 to 120, 71 to 130, 81 to 140,
and
91 to 149 of SEQ ID NO:S, as well as isolated polynucleotides which encode
these polypeptides. The invention also provides isolated polypeptides
comprising,
or alternatively consisting of, the amino acid sequences of a member selected
from
the group consisting of amino acids 1 to 60, I 1 to 70, 21 to 80, 31 to 90, 41
to
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100, 51 to 110, 61 to 120, 71 to 130, and 81 to 136 of SEQ ID N0:8, as well as
isolated polynucleotides which encode these polypeptides.
The present invention is also directed to isolated polypeptides comprising,
or alternatively consisting of, domains of TR2, TR2-SVl, and TR2-SV2. In
particular, the invention provides polypeptides comprising, or alternatively
consisting of, beta-sheet regions of TR2, TR2-SV1, and TR2-SV2 set out in
Tables II, III and IV. These polypeptides include polypeptides comprising, or
alternatively consisting of, amino acid sequences of a member selected from
the
group consisting of amino acid residues from about -19 to about -5, amino acid
residues from about -18 to about -6, amino acid residues from about -2 to
about
4, amino acid residues from about 25 to about 31, amino acid residues from
about
46 to about 51, amino acid residues from about 57 to about 71, amino acid
residues from about 99 to about 104, amino acid residues from about 151 to
about
156, amino acid residues from about 175 to about 191, amino acid residues from
1 S about 174 to about I 90, amino acid residues from about I 97 to about 206,
amino
acid residues from about 197 to about 208, amino acid residues from about 215
to about 220, amino acid residues from about 228 to about 238, and amino acid
residues from about 229 to about 241 of SEQ ID N0:2; amino acid residues from
about -19 to about -5, amino acid residues from about -18 to about -6, amino
acid
residues from about -2 to about 3, amino acid residues from about 26 to about
31,
amino acid residues from about 34 to about 40, amino acid residues from about
46 to about 50, amino acid residues from about 57 to about 64, amino acid
residues from about 69 to about 74, amino acid residues from about 122 to
about
128, and amino acid residues from about 132 to about 140 of SEQ ID NO:S; and
amino acid residues from about 6 to about 13, amino acid residues from about
26
to about 33, amino acid residues from about 50 to about 58, and amino acid
residues from about 86 to about 93 of SEQ ID N0:8. The invention is further
directed to isolated polynucleotides comprising, or alternatively consisting
of,
nucleic acid molecules which encode the beta-sheet regions set out in Tables
II,
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III and IV, and isolated polypeptides comprising, or alternatively consisting
of,
amino acid sequences at least 80% identical, and more preferably at least 85%,
90%, 92%, 95%, 96%, 97%, 98% or 99% identical to nucleic acid molecules
encoding beta-sheet regions of the TR2, TR2-SV 1, and TR2-SV2 proteins.
The TR2 receptor proteins of the invention may be in monomers or
multimers (i.e., dimers, trimers, tetramers, and higher multimers).
Accordingly,
the present invention relates to monomers and multimers of the TR2 receptor
proteins of the invention, their preparation, and compositions (preferably,
pharmaceutical compositions) containing them. In specific embodiments, the
polypeptides of the invention are monomers, dimers, trimers or tetramers. In
additional embodiments, the multimers of the invention are at least dimers, at
least
trimers, or at least tetramers.
Multimers encompassed by the invention may be homomers or heteromers.
As used herein, the term homomer, refers to a multimer containing only TRZ
receptor proteins of the invention (including TR2 receptor fragments,
variants,
and fusion proteins, as described herein). These homomers may contain TR2
receptor proteins having identical or different polypeptide sequences. In a
specific
embodiment, a homomer of the invention is a multimer containing only TR2
receptor proteins having an identical polypeptide sequence. In another
specific
embodiment, a homomer of the invention is a multimer containing TR2 receptor
proteins having different polypeptide sequences. In specific embodiments, the
multimer of the invention is a homodimer (e.g., containing TR2 receptor
proteins
having identical or different polypeptide sequences) or a homotrimer (e.g.,
containing TR2 receptor proteins having identical or different polypeptide
sequences). In additional embodiments, the homomeric multimer ofthe invention
is at least a homodimer, at least a homotrimer, or at least a homotetramer.
As used herein, the term heteromer refers to a multimer containing
heterologous proteins (i.e., proteins containing only polypeptide sequences
that
do not correspond to a polypeptide sequences encoded by the TR2 receptor gene)
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in addition to the TR2 receptor proteins of the invention. In a specific
embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or
a
heterotetramer. In additional embodiments, the heteromeric multimer of the
invention is at least a heterodimer, at least a heterotrimer, or at least a
heterotetramer.
Multimers of the invention may be the result of hydrophobic, hydrophilic,
ionic and/or covalent associations and/or may be indirectly linked, by for
example,
liposome formation. Thus, in one embodiment, multimers of the invention, such
as, for example, homodimers or homotrimers, are formed when proteins of the
invention contact one another in solution. In another embodiment,
heteromultimers of the invention, such as, for example, heterotrimers or
heterotetramers, are formed when proteins of the invention contact antibodies
to
the polypeptides of the invention (including antibodies to the heterologous
polypeptide sequence in a fusion protein of the invention) in solution. In
other
embodiments, multimers of the invention are formed by covalent associations
with
and/or between the TR2 receptor proteins of the invention. Such covalent
associations may involve one or more amino acid residues contained in the
polypeptide sequence ofthe TR2 receptor proteins (e.g. , the polypeptide
sequence
recited in SEQ ID N0:2, SEQ ID NO:S, SEQ ID N0:8, or SEQ ID N0:26, or the
polypeptides encoded by the cDNAs contained in ATCC Deposit Numbers 97059,
97058, or 97057). In one instance, the covalent associations are cross-linking
between cysteine residues located within the polypeptide sequences ofthe
proteins
which interact in the native (i.e., naturally occurring) polypeptide. In
another
instance, the covalent associations are the consequence of chemical or
recombinant manipulation. Alternatively, such covalent associations may
involve
one or more amino acid residues contained in the heterologous polypeptide
sequence in a TR2 receptor fusion protein. In one example, covalent
associations
are between the heterologous sequence contained in a fusion protein of the
invention (see, e.g., U.S. Patent No. 5,478,925). In a specific example, the
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covalent associations are between the heterologous sequence contained in a TR2
receptor-Fc fusion protein of the invention (as described herein). In another
specific example, covalent associations of fusion proteins of the invention
are
between heterologous polypeptide sequences from another TNF family
ligand/receptor member that is capable of forming covalently associated
multimers, such as for example, oseteoprotegerin (see, e.g., International
Publication No. WO 98/49305, the contents of which are herein incorporated by
reference in its entirety). In another embodiment, two or more TR2
polypeptides
of the invention are joined through synthetic linkers (e.g., peptide,
carbohydrate
or soluble polymer linkers). Examples include, but are not limited to, those
peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by
reference). Proteins comprising multiple TRZ polypeptides separated by peptide
linkers may be produced using conventional recombinant DNA technology.
Another method for preparing multimer TR2 polypeptides of the invention
1 S involves use of TR2 polypeptides fused to a leucine zipper polypeptide
sequence.
Leucine zipper domains are polypeptides that promote multimerization of the
proteins in which they are found. Leucine zippers were originally identified
in
several DNA-binding proteins (Landschulz et al., Science 240:1759, ( 1988)),
and
have since been found in a variety of different proteins. Among the known
leucine
zippers are naturally occurring peptides and derivatives thereof that dimerize
or
trimerize. Examples of leucine zipper domains suitable for producing soluble
multimeric TR2 proteins are those described in PCT application WO 94/10308,
hereby incorporated by reference. Recombinant fusion proteins comprising a
soluble TR2 polypeptide fused to a peptide that dimerizes or trimerizes in
solution
are expressed in suitable host cells, and the resulting soluble multimeric TR2
is
recovered from the culture supernatant using techniques known in the art.
Certain members of the TNF family of proteins are believed to exist in
trimeric form (Beutler and Huffel, Science 264:667, 1994; Banner et al., Cell
73:431, 1993). Thus, trimeric TR2 may offer the advantage of enhanced
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biological activity. Preferred leucine zipper moieties are those that
preferentially
form trimers. One example is a leucine zipper derived from lung surfactant
protein D (SPD), as described in Hoppe et al. (FEBSLetters 344:191, (1994))
and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by
reference. Other peptides derived from naturally occurring trimeric proteins
may
be employed in preparing trimeric TR2.
In another example, proteins ofthe invention are associated by interactions
between Flag~ polypeptide sequence contained in Flag~-TR2 or Flag~-TR2
fusion proteins of the invention. In a further embodiment, associations
proteins
ofthe invention are associated by interactions between heterologous
polypeptide
sequence contained in Flag~-TR2 or Flag~-TR2 fusion proteins of the invention
and anti-Flag~ antibody.
The multimers of the invention may be generated using chemical
techniques known in the art. For example, proteins desired to be contained in
the
multimers of the invention may be chemically cross-linked using linker
molecules
and linker molecule length optimization techniques known in the art (.see,
e.g.,
U.S. Patent Number 5,478,925, which is herein incorporated by reference in its
entirety). Additionally, multimers of the invention may be generated using
techniques known in the art to form one or more inter-molecule cross-links
between the cysteine residues located within the polypeptide sequence of the
proteins desired to be contained in the multimer (.see, e.g., U.S. Patent
Number
5,478,925, which is herein incorporated by reference in its entirety).
Further,
proteins of the invention may be routinely modified by the addition of
cysteine or
biotin to the C-terminus or N-terminus ofthe polypeptide sequence ofthe
protein
and techniques known in the art may be applied to generate multimers
containing
one or more ofthese modified proteins (see, e.g., U.S. Patent Number
5,478,925,
which is herein incorporated by reference in its entirety). Additionally,
techniques
known in the art may be applied to generate liposomes containing the protein
components desired to be contained in the multimer of the invention (.see,
e.g.,
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U.S. Patent Number 5,478,925, which is herein incorporated by reference in its
entirety).
Alternatively, multimers of the invention may be generated using genetic
engineering techniques known in the art. In one embodiment, proteins contained
S in multimers of the invention are produced recombinantly using fusion
protein
technology described herein or otherwise known in the art (see, e.g., U.S.
Patent
Number 5,478,925, which is herein incorporated by reference in its entirety).
In
a specific embodiment, polynucleotides coding for a homodimer of the invention
are generated by ligating a polynucleotide sequence encoding a polypeptide
ofthe
invention to a sequence encoding a linker polypeptide and then further to a
synthetic polynucleotide encoding the translated product of the polypeptide in
the
reverse orientation from the original C-terminus to the N-terminus (lacking
the
leader sequence) (see, e.g., U.S. Patent Number 5,478,925, which is herein
incorporated by reference in its entirety). In another embodiment, recombinant
techniques described herein or otherwise known in the art are applied to
generate
recombinant polypeptides ofthe invention which contain a transmembrane domain
and which can be incorporated by membrane reconstitution techniques into
liposomes (see, e.g., U. S. Patent Number 5,478,925, which is herein
incorporated
by reference in its entirety).
The present invention encompasses polypeptides comprising, or
alternatively consisting of, an epitope of the polypeptide having an amino
acid
sequence of SEQ ID N0:2, SEQ ID NO:S, SEQ ID N0:8, or SEQ ID N0:26, or
an epitope of the polypeptide sequence encoded by a polynucleotide sequence
contained in the deposited cDNA identified as ATCC Accession No. 97059,
97058 or 97057 or encoded by a polynucleotide that hybridizes to the
complement
ofthe polynucleotide sequence of SEQ ID NO: l, SEQ ID N0:4, SEQ ID N0:7,
or SEQ ID N0:25, or contained in the deposited cDNA identified as ATCC
Accession No. 97059, 97058 or 97057 under stringent hybridization conditions
or lower stringency hybridization conditions as defined herein. The present
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invention further encompasses polynucleotide sequences encoding an epitope of
a polypeptide sequence of the invention (such as, for example, the sequence
disclosed in SEQ ID N0:2, SEQ ID NO:S, SEQ ID N0:8, or SEQ ID N0:26),
polynucleotide sequences of the complementary strand of a polynucleotide
sequence encoding an epitope of the invention, and polynucleotide sequences
which hybridize to the complementary strand under stringent hybridization
conditions or lower stringency hybridization conditions defined herein.
The term "epitopes," as used herein, refers to portions of a polypeptide
having antigenic or immunogenic activity in an animal, preferably a mammal,
and
most preferably in a human. In a preferred embodiment, the present invention
encompasses a polypeptide comprising an epitope, as well as the polynucleotide
encoding this polypeptide. An "immunogenic epitope," as used herein, is
defined
as a portion of a protein that elicits an antibody response in an animal, as
determined by any method known in the art, for example, by the methods for
1 S generating antibodies described infi°a. (See, for example, Geysen
et al., Proc.
Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term "antigenic epitope," as
used herein, is defined as a portion of a protein to which an antibody can
immunospecifically bind its antigen as determined by any method well known in
the art, for example, by the immunoassays described herein. Immunospecific
binding excludes non-specific binding but does not necessarily exclude
cross-reactivity with other antigens. Antigenic epitopes need not necessarily
be
immunogenic.
As to the selection ofpeptides 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
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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 epitopes of the invention preferably contain a sequence of at
least 4, at least 5, at least 6, at least 7, more preferably at least 8, at
least 9, at least
10, at least I 5, at least 20, at least 25, and, most preferably, between
about I S to
about 30 amino acids. In this context "about" includes the particularly
recited
value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids.
Preferred polypeptides comprising immunogenic or antigenic epitopes are at
least
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100
amino
acid residues in length. Antigenic epitopes are useful, for example, to raise
antibodies, including monoclonal antibodies, that specifically bind the
epitope.
Antigenic epitopes can be used as the target molecules in immunoassays. (See,
for instance, Wilson et al., Cell 37:767-778 (1984); Sutclif~e el al., Science
219:660-666 (1983)).
Non-limiting examples of antigenic polypeptides or peptides that can be
used to generate TR2 receptor-specific antibodies include: a polypeptide
comprising, or alternatively consisting of, amino acid residues from about 39
to
about 70 in FIG. 1 (amino acid residues 3 to 34 in SEQ ID N0:2); a polypeptide
comprising, or alternatively consisting of, amino acid residues from about 106
to
about 120 in FIG. lA-1B (amino acid residues 70 to 84 in SEQ ID N0:2); a
polypeptide comprising, or alternatively consisting of, amino acid residues
from
about 142 to about 189 in FIG. lA-1B (amino acid residues 106 to 153 in SEQ
ID N0:2); a polypeptide comprising, or alternatively consisting of, amino acid
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residues from about 276 to about 283 in FIG. 1 (amino acid residues 240 to 247
in SEQ ID N0:2); a polypeptide comprising, or alternatively consisting of,
amino
acid residues from about 39 to about 70 in FIG. 4A-4C (amino acid residues 3
to
34 in SEQ ID NO:S); a polypeptide comprising, or alternatively consisting of,
S amino acid residues from about 99 to about 136 in FIG. 4A-4C (amino acid
residues 63 to 100 in SEQ 1D NO:S); a polypeptide comprising, or alternatively
consisting of, amino acid residues from about 171 to about 18S in FIG. 4A-4C
(amino acid residues 13S to 149 in SEQ 117 NO:S); a polypeptide comprising, or
alternatively consisting of, amino acid residues from about S6 to about 68 in
FIG.
7A-7C (SEQ ID N0:8); and a polypeptide comprising, or alternatively consisting
of, amino acid residues from about 93 to about 136 in FIG. 7A-7C (SEQ ID
N0:8). In this context "about" includes the particularly recited value and
values
larger or smaller by several (S, 4, 3, 2, or 1) amino acids. As indicated
above, the
inventors have determined that the above polypeptide fragments are antigenic
1 S 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-S13S. This "Simultaneous Multiple Peptide Synthesis
(SMPS)" process is further described in U.S. Patent No. 4,631,211 to Houghten
et al. (1986).
Similarly, immunogenic epitopes can be used, for example, to induce
antibodies according to methods well known in the art. (See, for instance,
Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad.
Sci.
USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). A
preferred immunogenic epitope includes the secreted protein. The polypeptides
comprising one or more immunogenic epitopes may be presented for eliciting an
antibody response together with a carnerprotein, such as an albumin, to an
animal
AMENDED SHEET
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system (such as, for example, rabbit or mouse), or, if the polypeptide is of
sufl;icient length (at least about 25 amino acids), the polypeptide may be
presented
without a carrier. However, immunogenic epitopes comprising as few as 8 to 10
amino acids have been shown to be sufficient to raise antibodies capable
ofbinding
to, at the very least, linear epitopes in a denatured polypeptide (e.g., in
Western
blotting).
Epitope-bearing polypeptides of the present invention may be used to
induce antibodies according to methods well known in the art including, but
not
limited to, in vivo immunization, in vitro immunization, and phage display
methods. See, e.gl., Sutcliffe et al., supra; Wilson et al., s?rpna, and
Bittle et al.,
J. Gen. Virol., 66:2347-23 54 ( 1985). If in vivo immunization is used,
animals may
be immunized with free peptide; however, anti-peptide antibody titer may be
boosted by coupling the peptide to a macromolecular carrier, such as keyhole
limpet hemacyanin (KL,H) or tetanus toxoid. For instance, peptides containing
cysteine residues may be coupled to a carrier using a linker such as
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may
be coupled to carriers using a more general linking agent such as
glutaraldehyde.
Animals such as, for example, rabbits, rats, and mice are immunized with
either
free or carrier-coupled peptides, for instance, by intraperitoneal and/or
intradermal
injection of emulsions containing about 100 micrograms of peptide or carrier
protein and Freund's adjuvant or any other adjuvant known for stimulating an
immune response. Several booster injections may be needed, for instance, at
intervals of about two weeks, to provide a useful titer of anti-peptide
antibody that
can be detected, for example, by ELISA assay using free peptide adsorbed to a
solid surface. The titer of anti-peptide antibodies in serum from an immunized
animal may be increased by selection of anti-peptide antibodies, for instance,
by
adsorption to the peptide on a solid support and elution ofthe selected
antibodies
according to methods well known in the art.
As one of skill in the art will appreciate, and as discussed above, the
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polypeptides of the present invention comprising an immunogenic or antigenic
epitope can be fused to other polypeptide sequences. For example, the
polypeptides of the present invention may be fused with the constant domain of
immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or
S any combination thereof and portions thereof) resulting in chimeric
polypeptides.
Such fusion proteins may facilitate purification and may increase half life in
vivo.
This has been shown 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. See, e.g., EP 394,827;
Traunecker e1 al., Nat~ne, 331:84-86 (1988). IgG Fusion proteins that have a
disulfide-linked dimeric structure due to the IgG portion disulfide bonds have
also
been found to be more effcient in binding and neutralizing other molecules
than
monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et
al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above
epitopes can also be recombined with a gene of interest as an epitope tag
(e.g., the
hemagglutinin ("HA") tag or flag tag) to aid in detection and purification of
the
expressed polypeptide. For example, a system described by Janknecht et al.
allows for the ready purification of non-denatured fusion proteins expressed
in
human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA
88:8972-897). In this system, the gene of interest is subcloned into a
vaccinia
recombination plasmid such that the open reading frame of the gene is
translationally fused to an amino-terminal tag consisting of six histidine
residues.
The tag serves as a matrix-binding domain for the fusion protein. Extracts
from
cells infected with the recombinant vaccinia virus are loaded onto Ni2+
nitriloacetic
acid-agarose column and histidine-tagged proteins can be selectively eluted
with
imidazole-containing bui~ers.
Additional fusion proteins of the invention may be generated through the
techniques of gene-shuffling, motif shuffling, exon-shuffling, and/or
codon-shuffling (collectively referred to as "DNA shuffling"). DNA shuffling
may
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be employed to modulate the activities of polypeptides of the invention, such
methods can be used to generate polypeptides with altered activity, as well as
agonists and antagonists of the polypeptides. See, generally, U.S. Patent Nos.
5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al.,
Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol.
16(2):76-82 ( 1998); Hansson et al., J. Mol. Biol. 287:265-76 ( 1999); and
Lorenzo
and Blasco, BioTechniques 24(2):308-13 (1998) (each of these patents and
publications are hereby incorporated by reference in its entirety). In one
embodiment, alteration ofpolynucleotides corresponding to SEQ ID NO: I and the
polypeptides encoded by these polynucleotides may be achieved by DNA
shuffling. DNA shuffling involves the assembly of two or more DNA segments
by homologous or site-specific recombination to generate variation in the
polynucleotide sequence. In another embodiment, polynucleotides of the
invention, or the encoded polypeptides, may be altered by being subjected to
random mutagenesis by error-prone PCR, random nucleotide insertion or other
methods prior to recombination. In another embodiment, one or more
components, motifs, sections, parts, domains, fragments, etc., of a
polynucleotide
coding a polypeptide of the invention may be recombined with one or more
components, motifs, sections, parts, domains, fragments, etc. of one or more
heterologous molecules.
Proteins of the invention can be chemically synthesized using techniques
known in the art (e.g., .see Creighton, 1983, Proteins: Structures and
Molecular
Principles, W.H. Freeman & Co., N.Y., and Hunkapiller, M., et al.,
Natm°e
310:1 OS-111 ( I 984)). For example, a peptide corresponding to a fragment of
the
TR2 receptor polypeptides of the invention can be synthesized by use of a
peptide
synthesizer. Furthermore, if desired, nonclassical amino acids or chemical
amino
acid analogs can be introduced as a substitution or addition into the TR2
receptor
polypeptide sequence. Non-classical amino acids include, but are not limited
to,
to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino
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isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, E-Ahx,
6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,
ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,
homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, (3-alanine, fluoro-amino acids, designer amino acids such
as ~3-
methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino
acid analogs in general. Furthermore, the amino acid can be D (dextrorotary)
or
L (levorotary).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques, which include, but are not limited to oligonucleotide
mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed
mutagenesis (see, e.g., Carter et al., Nucl. AcidsRe.s. 13:4331 (1986); and
Zoller
et al., Nucl. AcidsRes. 10:6487 (1982)), cassette mutagenesis (.see, e.g.,
Wells et
al., Gene 34:315 (1985)), restriction selection mutagenesis (see, e.g., Wells
etal.,
Philos. Traps. R. Soc. London SetA 317:415 (1986)).
The invention additionally, encompasses TR2 receptor polypeptides which
are differentially modified during or after translation, e.g., by
glycosylation,
acetylation, phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to an antibody
molecule
or other cellular ligand, etc. Any of numerous chemical modifications may be
carried out by known techniques, including but not limited to, specific
chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease,
NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in
the
presence of tunicamycin; etc.
Additional post-translational modifications encompassed by the invention
include, for example, e.g., N-linked or O-linked carbohydrate chains,
processing
of N-terminal or C-terminal ends), attachment of chemical moieties to the
amino
acid backbone, chemical modifications of N-linked or O-linked carbohydrate
chains, and addition or deletion of an N-terminal methionine residue as a
result of
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procaryotic host cell expression. The polypeptides may also be modified with a
detectable label, such as an enzymatic, fluorescent, isotopic or affinity
label to
allow for detection and isolation of the protein.
Also provided by the invention are chemically modified derivatives of TR2,
TR2-SV1 and TRZ-SV2 receptor polypeptides which may provide additional
advantages such as increased solubility, stability and circulating time of the
polypeptides, or decreased immunogenicity (see U. S. Patent No. 4,179,337).
The
chemical moieties for derivitization may be selected from water soluble
polymers
such as polyethylene glycol, ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The
polypeptides
may be modified at random positions within the molecule, or at predetermined
positions within the molecule and may include one, two, three or more attached
chemical moieties.
The polymer may be of any molecular weight, and may be branched or
unbranched. For polyethylene glycol, the preferred molecular weight is between
about 1 kDa and about 100 kDa (the term "about" indicating that in
preparations
of polyethylene glycol, some molecules will weigh more, some less, than the
stated
molecular weight) for ease in handling and manufacturing. Other sizes may be
used, depending on the desired therapeutic profile (e.g., the duration of
sustained
release desired, the effects, if any on biological activity, the ease in
handling, the
degree or lack of antigenicity and other known effects of the polyethylene
glycol
to a therapeutic protein or analog). For example, the polyethylene glycol may
have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500,
3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000,
9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000,
14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500,
19,000;
19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000,
65,000,
70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
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As noted above, the polyethylene glycol may have a branched structure.
Branched polyethylene glycols are described, for example, in U.S. Patent No.
5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996);
Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et
al., Bioconjug. Cherrz. 10:638-646 (1999), the disclosures of each of which
are
incorporated herein by reference.
The polyethylene glycol molecules (or other chemical moieties) should be
attached to the protein with consideration of effects on functional or
antigenic
domains of the protein. There are a number of attachment methods available to
those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference
(coupling PEG to G-CSF), see also Malik et al., Exp. Hen~alol. 20:1028-1035
(1992) (reporting pegylation of GM-CSF using tresyl chloride). For example,
polyethylene glycol may be covalently bound through amino acid residues via a
reactive group, such as, a free amino or carboxyl group. Reactive groups are
those to which an activated polyethylene glycol molecule may be bound. The
amino acid residues having a free amino group may include lysine residues and
the
N-terminal amino acid residues; those having a free carboxyl group may include
aspartic acid residues, glutamic acid residues and the C-terminal amino acid
residue. Sulfhydryl groups may also be used as a reactive group for attaching
the
polyethylene glycol molecules. Preferred for therapeutic purposes is
attachment
at an amino group, such as attachment at the N-terminus or lysine group.
As suggested above, polyethylene glycol may be attached to proteins via
linkage to any of a number of amino acid residues. For example, polyethylene
glycol can be linked to a proteins via covalent bonds to lysine, histidine,
aspartic
acid, glutamic acid, or cysteine residues. One or more reaction chemistries
may
be employed to attach polyethylene glycol to specific amino acid residues
(e.g.,
lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein
or to more
than one type of amino acid residue (e.g., lysine, histidine, aspartic acid,
glutamic
acid, cysteine and combinations thereof) of the protein.
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One may specifically desire proteins chemically modified at the N-
terminus. Using polyethylene glycol as an illustration of the present
composition,
one may select from a variety of polyethylene glycol molecules (by molecular
weight, branching, etc.), the proportion of polyethylene glycol molecules to
protein (or peptide) molecules in the reaction mix, the type of pegylation
reaction
to be performed, and the method of obtaining the selected N-terminally
pegylated
protein. The method of obtaining the N-terminally pegylated preparation (i.e.,
separating this moiety from other monopegylated moieties if necessary) may be
by purification of the N-terminally pegylated material from a population of
pegylated protein molecules. Selective proteins chemically modified at the N-
terminus modification may be accomplished by reductive alkylation which
exploits
differential reactivity of different types of primary amino groups (lysine
versus the
N-terminal) available for derivatization in a particular protein. Under the
appropriate reaction conditions, substantially selective derivatization ofthe
protein
at the N-terminus with a carbonyl group containing polymer is achieved.
As indicated above, pegylation of the proteins of the invention may be
accomplished by any number of means. For example, polyethylene glycol may be
attached to the protein either directly or by an intervening linker.
Linkerless
systems for attaching polyethylene glycol to proteins are described in Delgado
et
al., C~°it. Rev. They°a. Drug Cap°~°ier S'ys.
9:249-304 (1992); Francis et al., Interr2.
.I. of Hemafol. 68:1-18 (1998); U.S. Patent No. 4,002,531; U.S. Patent No.
5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each ofwhich are
incorporated herein by reference.
One system for attaching polyethylene glycol directly to amino acid
residues of proteins without an intervening linker employs tresylated MPEG,
which is produced by the modification of monmethoxy polyethylene glycol
(MPEG) using tresylchloride (C1SOZCHZCF3). Upon reaction of protein with
tresylated MPEG, polyethylene glycol is directly attached to amine groups of
the
protein. Thus, the invention includes protein-polyethylene glycol conjugates
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produced by reacting proteins ofthe invention with a polyethylene glycol
molecule
having a 2,2,2-trifluoreothane sulphonyl group.
Polyethylene glycol can also be attached to proteins using a number of
different intervening linkers. For example, U. S. Patent No. 5,612,460, the
entire
disclosure of which is incorporated herein by reference, discloses urethane
linkers
for connecting polyethylene glycol to proteins. Protein-polyethylene glycol
conjugates wherein the polyethylene glycol is attached to the protein by a
linker
can also be produced by reaction of proteins with compounds such as MPEG-
succinimidylsuccinate, MPEG activated with l, l'-carbonyldiimidazole, MPEG-
2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various
MPEG-succinate derivatives. A number additional polyethylene glycol
derivatives
and reaction chemistries for attaching polyethylene glycol to proteins are
described
in WO 98/32466, the entire disclosure of which is incorporated herein by
reference. Pegylated protein products produced using the reaction chemistries
set
out herein are included within the scope of the invention.
The number of polyethylene glycol moieties attached to each protein of the
invention (i.e., the degree of substitution) may also vary. For example, the
pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4,
5, 6,
7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly,
the
average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7,
6-8,
7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-IS, 14-16, 15-17, 16-18, 17-19, or 18-
20 polyethylene glycol moieties per protein molecule. Methods for determining
the degree of substitution are discussed, for example, in Delgado et al.,
C~°it. Rev.
Thera. Drug Cagier Sys. 9:249-304 (1992).
Antibodies
The present invention further relates to antibodies and T-cell antigen
receptors (TCR) which specifically bind the polypeptides of the present
invention.
The antibodies of the present invention include IgG (including IgGl, IgG2,
IgG3,
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and IgG4), IgA (including IgAl and IgA2), IgD, IgE, or IgM, and IgY. As used
herein, the term "antibody" (Ab) is meant to include whole antibodies,
including
single-chain whole antibodies, and antigen-binding fragments thereof. Most
preferably the antibodies are human antigen binding antibody fragments of the
present invention include, but are not limited to, Fab, Fab' and F(ab')2, Fd,
single-
chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and
fragments comprising either a VL or VH domain. The antibodies may be from any
animal origin including birds and mammals. Preferably, the antibodies are
human,
murine, rabbit, goat, guinea pig, camel, horse, or chicken. As used herein,
"human" antibodies include antibodies having the amino acid seduence of a
human
immunoglobulin and include antibodies isolated from human immunoglobulin
libraries or from animals transgenic for one or more human immunoglobulin and
that do not express endogenous immunoglobulins, as described infi°a
and, for
example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
Antigen-binding antibody fragments, including single-chain antibodies, may
comprise the variable regions) alone or in combination with the entire or
partial
of the following: hinge region, CH1, CH2, and CH3 domains. Also included in
the invention are any combinations of variable regions) and hinge region, CHI,
CH2, and CH3 domains. The present invention further includes monoclonal,
polyclonal, chimeric, humanized, and human monoclonal and polyclonal
antibodies
which specifically bind the polypeptides of the present invention. The present
invention further includes antibodies which are anti-idiotypic to the
antibodies of
the present invention.
The antibodies of the present invention may be monospecific, bispecific,
trispecific or of greater multispecificity. Multispecific antibodies may be
specific
for different epitopes of a polypeptide of the present invention or may be
specific
for both a polypeptide of the present invention as well as for heterologous
compositions, such as a heterologous polypeptide or solid support material.
See,
e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al.,
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.~ Inamunod. 147:60-69 (1991); U.S. Patent Numbers 5,573,920, 4,474,893,
5,601,819, 4,714,681, 4,925,648; Kostelny, S.A. et al, J. Immunol.
148:1547-1553 (1992).
Antibodies of the present invention may be described or specified in terms
of the epitope(s) or portions) of a polypeptide of the present invention which
are
recognized or specifically bound by the antibody. The epitope(s) or
polypeptide
portions) may be specified as described herein, e.g., by N-terminal and C-
terminal
positions, by size in contiguous amino acid residues, or listed in the Tables
and
Figures. Antibodies which specifically bind any epitope or polypeptide of the
present invention may also be excluded. Therefore, the present invention
includes
antibodies that specifically bind polypeptides of the present invention, and
allows
for the exclusion of the same.
Antibodies of the present invention may also be described or specified in
terms of their cross-reactivity. Antibodies that do not bind any other analog,
ortholog, or homolog of the polypeptides of the present invention are
included.
Antibodies that do not bind polypeptides with less than 95%, less than 90%,
less
than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less
than
60%, less than 55%, and less than 50% identity (as calculated using methods
known in the art and described herein) to a polypeptide of the present
invention
are also included in the present invention. Further included in the present
invention are antibodies which only bind polypeptides encoded by
polynucleotides
which hybridize to a polynucleotide of the present invention under stringent
hybridization conditions (as described herein). Antibodies ofthe present
invention
may also be described or specified in terms of their binding affinity.
Preferred
binding affinities include those with a dissociation constant or Kd less than
SX10-~M, 10-~M, SX10-'M, 10-'M, SX10-~M, 10-~M, SX10-9M, 10-9M,
5X10''°M,
10-'"M, SX10-"M, 10-"M, SX10-'zM, 10-'ZM, SX10-'3M, 10-'3M, SX10-'4M,
10-'4M, SX10-'SM, and 10-'SM.
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The invention also provides antibodies that competitively inhibit binding
of an antibody to an epitope of the invention as determined by any method
known
in the art for determining competitive binding, for example, the immunoassays
described herein. In preferred embodiments, the antibody competitively
inhibits
binding to the epitope by at least 90%, at least 80%, at least 70%, at least
60%,
or at least 50%.
Antibodies of the present invention may act as agonists or antagonists of
the polypeptides of the present invention. For example, the present invention
includes antibodies which disrupt the receptor/ligand interactions with the
polypeptides of the invention either partially or fully. The invention
features both
receptor-specific antibodies and ligand-specific antibodies. The invention
also
features receptor-specific antibodies which do not prevent ligand binding but
prevent receptor activation. Receptor activation (i.e., signaling) may be
determined by technigues described herein or otherwise known in the art. For
example, receptor activation can be determined by detecting the
phosphorylation
(e.gJ., tyrosine or serine/threonine) of the receptor or its substrate by
immunoprecipitation followed by western blot analysis (for example, as
described
.supra). In specific embodiments, antibodies are provided that inhibit ligand
or
receptor activity by at least 90%, at least 80%, at least 70%, at least 60%,
or at
least 50% of the activity in absence of the antibody.
The invention also features receptor-specific antibodies which both prevent
ligand binding and receptor activation as well as antibodies that recognize
the
receptor-ligand complex, and, preferably, do not specifically recognize the
unbound receptor or the unbound ligand. Likewise, included in the invention
are
neutralizing antibodies which bind the ligand and prevent binding of the
ligand to
the receptor, as well as antibodies which bind the ligand, thereby preventing
receptor activation, but do not prevent the ligand from binding the receptor.
Further included in the invention are antibodies which activate the receptor.
These antibodies may act as receptor agonists, i.e., potentiate or activate
either all
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or a subset of the biological activities of the ligand-mediated receptor
activation.
The antibodies may be specified as agonists, antagonists or inverse agonists
for
biological activities comprising the specific biological activities of the
peptides of
the invention disclosed herein. The above antibody agonists can be made using
methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent
No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer
Res. 58(16):3668-3678 (1998); Harrop et al., .I. Immunol. 161(4):1786-1794
(1998); Zhu et al., Cancer Res. 58(IS):3209-3214 (1998); Yoon et al., J.
Immunol. 160(7):3170-3179 (1998); Prat et al., .~ Cell. Sei. Ill(Pt2):237-247
(1998); Pitard etal.,.~ Immunol. Methods205(2):177-190 (1997); Liautardetal.,
Cytokine 9(4):233-241 (1997); Carlson et al., .I. Biol. Chem.
272(17):11295-11301 (1997); Taryman et al., Neuron 14(-1):755-762 (1995);
Muller et al., Sty°ucture 6(9):1153-1167 (1998); Bartunek et al.,
Cytokine
8(1):14-20 (1996) (which are all incorporated by reference herein in their
entireties).
Antibodies of the present invention have uses that include, but are not
limited to, methods known in the art to purify, detect, and target the
polypeptides
of the present invention including both in vitro and in vivo diagnostic and
therapeutic methods. For example, the antibodies have use in immunoassays for
qualitatively and quantitatively measuring levels ofthe polypeptides ofthe
present
invention in biological samples. See, e.g., Harlow et al., ANTIBODIES: A
LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988) (incorporated by reference in the entirety).
The antibodies of the present invention may be used either alone or in
combination with other compositions. The antibodies may further be
recombinantly fused to a heterologous polypeptide at the N- or C-terminus or
chemically conjugated (including covalently and non-covalently conjugations)
to
polypeptides or other compositions. For example, antibodies of the present
invention may be recombinantly fused or conjugated to molecules useful as
labels
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in detection assays and effector molecules such as heterologous polypeptides,
drugs, or toxins. See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S.
Patent Number 5,314,995; and EP 0 396 387.
The antibodies of the invention include derivatives that are modified, i. e.,
by the covalent attachment of any type of molecule to the antibody such that
covalent attachment does not prevent the antibody from generating an
anti-idiotypic response. For example, but not by way of limitation, the
antibody
derivatives include antibodies that have been modified, e.g., by
glycosylation,
acetylation, pegylation, phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand
or
other protein, etc. Any of numerous chemical modifications may be carried out
by known techniques, including, but not limited to specific chemical cleavage,
acetylation, formylation, metabolic synthesis oftunicamycin, etc.
Additionally, the
derivative may contain one or more non-classical amino acids.
The antibodies of the present invention may be prepared by any suitable
method known in the art. Polyclonal antibodies to an antigen-of interest can
be
produced by various procedures well known in the art. For example, a
polypeptide ofthe invention can be administered to various host animals
including,
but not limited to, rabbits, mice, rats, etc. to induce the production of sera
containing polyclonal antibodies specific for the antigen. Various adjuvants
may
be used to increase the immunological response, depending on the host species,
and include but are not limited to, Freund's (complete and incomplete),
mineral
gels such as aluminum hydroxide, surface active substances such as
lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and Coiyrrebacteriun~ parvnm. Such adjuvants are also well
known in the art.
Monoclonal antibodies can be prepared using a wide variety of techniques
known in the art including the use of hybridoma, recombinant, and phage
display
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technologies, or a combination thereof. For example, monoclonal antibodies can
be produced using hybridoma techniques including those known in the art and
taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold
Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in:
Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)
(said references incorporated by reference in their entireties). The term
"monoclonal antibody" is not limited to antibodies produced through hybridoma
technology. The term "monoclonal antibody" refers to an antibody that is
derived
from a single clone, including any eukaryotic, prokaryotic, or phage clone,
and not
the method by which it is produced. Monoclonal antibodies can be prepared
using
a wide variety of techniques known in the art including the use of hybridoma,
recombinant and phage display technology.
Hybridoma techniques include those known in the art and taught in
Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring
1 S Harbor Laboratory Press, 2nd ed. 1988); and Hammerling, et al., in:
MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference in their
entireties).
Fab and F(ab')2 fragments may be produced by proteolytic cleavage, using
enzymes such as papain (to produce Fab fragments) or pepsin (to produce
F(ab')2
fragments).
Alternatively, antibodies ofthe present invention can be produced through
the application of recombinant DNA and phage display technology or through
synthetic chemistry using methods known in the art. For example, the
antibodies
of the present invention can be prepared using various phage display methods
known in the art. In phage display methods, functional antibody domains are
displayed on the surface of a phage particle which carries polynucleotide
sequences encoding them. Phage with a desired binding property are selected
from a repertoire or combinatorial antibody library (e.g. human or murine) by
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selecting directly with antigen, typically antigen bound or captured to a
solid
surface or bead. Phage used in these methods are typically filamentous phage
including fd and M 13 with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII protein.
Examples
of phage display methods that can be used to make the antibodies of the
present
invention include those disclosed in Brinkman U. et al., .I. Immunol. Methods
182:41-50 (1995); Ames, R.S. et al., J. Immunol. Methods 184:177-186 (1995);
Kettleborough, C.A. etal., Eur. .~ Immunol. 24:952-958 (1994); Persic, L. et
al.,
Gene 187:9-I 8 ( 1997); Burton, D.R. et al., Advances in Immunology ~ 7: I 91-
280
(1994); PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO
92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Patent
Numbers 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753,
5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743
(said references incorporated by reference in their entireties).
As described in the above references, after phage selection, the antibody
coding regions from the phage can be isolated and used to generate whole
antibodies, including human antibodies, or any other desired antigen binding
fragment, and expressed in any desired host including mammalian cells, insect
cells, plant cells, yeast, and bacteria. For example, techniques to
recombinantly
produce Fab, Fab' and F(ab')2 fragments can also be employed using methods
known in the art such as those disclosed in WO 92/22324; Mullinax, R.L. et
al.,
BioTechniques 12:864-869 (1992); and Sawai, H. et al. A.IRI 34:26-34 (1995);
and Better, M. et al., Science 240: I 041-1043 ( I 988) (said references
incorporated
by reference in their entireties).
Examples of techniques which can be used to produce single-chain Fvs and
antibodies include those described in U.S. Patent Numbers 4,946,778 and
5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu, L. et
al., PNAS 90:7995-7999 (1993); and Skerra, A. et al., Science 240:1038-1040
( 1988). For some uses, including in viva use of antibodies in humans and in
vitro
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detection assays, it may be preferable to use chimeric, humanized, or human
antibodies. Methods for producing chimeric antibodies are known in the art.
See
e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214
(1986);
Gillies, S.D. et al., J Immunol. Methods 125:191-202 (1989); and U.S. Patent
Number 5,807,715. Antibodies can be humanized using a variety of techniques
including CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Patent Numbers
5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519
596; Padlan E.A., Molecular Immunology 28(4/5):489-498 (1991); Studnicka
G.M. et ad., Protein Engineering 7:805-814 (1994); Roguska M.A. et al., PNAS
91:969-973 ( 1994)), and chain shuffling (U. S. Patent Number 5,565,332).
Human
antibodies can be made by a variety of methods known in the art including
phage
display methods described above. S'ee also, U.S. Patent Numbers 4,444,887,
4,716,111, 5,545,806, and 5,814,318; and WO 98/46645, WO 98/50433, WO
98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741 (said
references incorporated by reference in their entireties).
Further included in the present invention are antibodies recombinantly
fused or chemically conjugated (including both covalently and non-covalently
conjugations) to a polypeptide of the present invention. The antibodies may be
specific for antigens other than polypeptides of the present invention. For
example, antibodies may be used to target the polypeptides of the present
invention to particular cell types, either irr vitro or in vivo, by fusing or
conjugating the polypeptides of the present invention to antibodies specific
for
particular cell surface receptors. Antibodies fused or conjugated to the
polypeptides of the present invention may also be used in irr vitro
immunoassays
and purification methods using methods known in the art. See e.g., Harbor et
al.,
supra and WO 93/21232; EP 0 439 095; Naramura, M. et al., Immufiol. Lett.
39:91-99 (1994); U.S. Patent 5,474,981; Gillies, S.O. etal., PNAS89:1428-1432
(1992); Fell, H.P. et al., J. Immunol. 146:2446-2452 (1991) (said references
incorporated by reference in their entireties).
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The present invention further includes compositions comprising the
polypeptides of the present invention fused or conjugated to antibody domains
other than the variable regions. For example, the polypeptides of the present
invention may be fused or conjugated to an antibody Fc region, or portion
thereof.
The antibody portion fused to a polypeptide ofthe present invention may
comprise
the hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination
of whole domains or portions thereof. The polypeptides of the present
invention
may be fused or conjugated to the above antibody portions to increase the in
vivo
half life of the polypeptides or for use in immunoassays using methods known
in
the art. The polypeptides may also be fused or conjugated to the above
antibody
portions to form multimers. For example, Fc portions fused to the polypeptides
of the present invention can form dimers through disulfide bonding between the
Fc portions. Higher multimeric forms can be made by fusing the polypeptides to
portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of
I S the present invention to antibody portions are known in the art. See e.g.,
U. S.
Patent Numbers 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851,
5,112,946; EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570;
Ashkenazi, A. et al. (1991) PNAS 88:10535-10539; Zheng, X.X. et al. (1995) J.
Immunol. 154:5590-5600; and Vil, H. et al. (1992) PNAS 89:11337-11341 (said
references incorporated by reference in their entireties).
The invention further relates to antibodies which act as agonists or
antagonists of the polypeptides of the present invention. Antibodies which act
as
agonists or antagonists of the polypeptides of the present invention include,
for
example, antibodies which disrupt receptor/ligand interactions with the
polypeptides of the invention either partially or fully. For example, the
present
invention includes antibodies which disrupt the ability of the proteins of the
invention to multimerize. In another example, the present invention includes
antibodies which allow the proteins of the invention to multimerize, but
disrupts
the ability of the proteins of the invention to bind one or more TRZ
receptors) or
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ligand(s) (e.g., AIM II (International Publication No. WO 97/34911),
Lymphotoxin-a, and the Herpes virus protein HSV 1 gD). In yet another example,
the present invention includes antibodies which allow the proteins of the
invention
to multimerize, and bind TR2 receptors) or ligand(s) (e.g., AIM II
(International
Publication No. WO 97/3491 I), Lymphotoxin-a, and the Herpes virus protein
HSV 1 gD), but blocks biological activity associated with the TR2
receptor/ligand
complex.
Antibodies which act as agonists or antagonists of the polypeptides of the
present invention also include, both receptor-specific antibodies and ligand
specific antibodies. Included are receptor-specific antibodies which do not
prevent ligand binding but prevent receptor activation. Receptor activation
(i. e. ,
signaling) may be determined by techniques described herein or otherwise known
in the art. Also included are receptor-specific antibodies which both prevent
ligand binding and receptor activation. Likewise, included are neutralizing
I 5 antibodies which bind the ligand and prevent binding of the ligand to the
receptor,
as well as antibodies which bind the ligand, thereby preventing receptor
activation,
but do not prevent the ligand from binding the receptor. Further included are
antibodies which activate the receptor. These antibodies may act as agonists
for
either all or less than all of the biological activities affected by ligand-
mediated
receptor activation. The antibodies may be specified as agonists or
antagonists for
biological activities comprising specific activities disclosed herein. The
above
antibody agonists can be made using methods known in the art. See e.g., WO
96/40281; U.S. Patent Number 5,811,097; Deng, B. et al., Blood 92:1981-1988
(1998); Chen, Z. et al., Cancer Res. 58:3668-3678 (1998); Harrop, J.A. et al.,
.I.
Immur~ol. 161:1786-1794 (1998); Zhu, Z. et al., Cancer Res. 58:3209-3214
(1998); Yoon, D.Y. et al., .l. Immunod. 160:3170-3179 (1998); Prat, M. et al.,
J.
Cell. Sci. Ill(I't2):237-247 (1998); Pitard, V. et al., .l. Immunol. Methods
205:177-190 (1997); Liautard, J. et al., Cytokine 9(4):233-241 (1997);
Carlson,
N.G. et al., J Biol. Chem. 272:11295-11301 (1997); Taryman, R.E. et al.,
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Neuron 14:755-762 (1995); Muller, Y.A. et al., Structure 6:1153-1167 (1998);
Bartunek, P. et al., Cytokine 8:14-20 (1996) (said references incorporated by
reference in their entireties).
Methods for producing and screening for specific antibodies using
hybridoma technology are routine and well-known in the art and are discussed
in
detail in Example 17. Briefly, mice can be immunized with a polypeptide of the
invention or a cell expressing such peptide. Once an immune response is
detected,
e.g., antibodies specific for the antigen are detected in the mouse serum, the
mouse spleen is harvested and splenocytes isolated. The splenocytes are then
fused by well-known techniques to any suitable myeloma cells, for example
cells
from cell line SP20 available from the ATCC. Hybridomas are selected and
cloned by limited dilution. The hybridoma clones are then assayed by methods
known in the art for cells that secrete antibodies capable of binding a
polypeptide
of the invention. Ascites fluid, which generally contains high levels of
antibodies,
i 5 can be generated by immunizing mice with positive hybridoma clones.
Accordingly, the present invention provides methods of generating
monoclonal antibodies as well as antibodies produced by the method comprising
culturing a hybridoma cell secreting an antibody of the invention wherein,
preferably, the hybridoma is generated by fusing splenocytes isolated from a
mouse immunized with an antigen of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma clones that
secrete an antibody able to bind a polypeptide of the invention.
Antibody fragments that recognize specific epitopes may be generated by
known techniques. For example, Fab and F(ab')2 fragments of the invention may
be produced by proteolytic cleavage ofimmunoglobulin molecules, using enzymes
such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2
fragments). F(ab')2 fragments contain the variable region, the light chain
constant
region and the CH 1 domain of the heavy chain.
For example, the antibodies of the present invention can also be generated
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using various phage display methods known in the art. In phage display
methods,
functional antibody domains are displayed on the surface of phage particles
which
carry the polynucleotide sequences encoding them. In a particular, such phage
can
be utilized to display antigen-binding domains expressed from a repertoire or
S combinatorial antibody library (e.g., human or murine). Phage expressing an
antigen binding domain that binds the antigen of interest can be selected or
identified with antigen, e.g., using labeled antigen or antigen bound or
captured
to a solid surface or bead. Phage used in these methods are typically
filamentous
phage including fd and M 13 binding domains expressed from phage with Fab, Fv
or disulfide stabilized Fv antibody domains recombinantly fused to either the
phage gene III or gene VIII protein. Examples of phage display methods that
can
be used to make the antibodies ofthe present invention include those disclosed
in
Brinkman etal., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.
Methods 184:177-186 ( 1995); Kettleborough et al., Eur. J. Immunol. 24:952-958
(1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in
Immunology 57:191-280 (1994); PCT application No. PCT/GB91/Ol 134; PCT
publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO
93/11236; WO 95/15982; WO 95/20401; and U.S. Patent Nos. 5,698,426;
5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;
5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of
which is incorporated herein by reference in its entirety.
As described in the above references, after phage selection, the antibody
coding regions from the phage can be isolated and used to generate whole
antibodies, including human antibodies, or any other desired antigen binding
fragment, and expressed in any desired host, including mammalian cells, insect
cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments
can also be employed using methods known in the art such as those disclosed in
PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869
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(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science
240:1041-1043 (1988) (said references incorporated by reference in their
entireties).
Examples oftechniques which can be used to produce single-chain Fvs and
S antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS
90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For
some uses, including in vivo use of antibodies in humans and in vitro
detection
assays, it may be preferable to use chimeric, humanized, or human antibodies.
A
chimeric antibody is a molecule in which different portions of the antibody
are
derived from different animal species, such as antibodies having a variable
region
derived from a murine monoclonal antibody and a human immunoglobulin
constant region. Methods for producing chimeric antibodies are known in the
art.
See, e.g., Morrison, Science 229:1202 (1985); Oi et ad., BioTechniyues =1:214
(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Patent
Nos.
5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference
in their entireties. Humanized antibodies are antibody molecules from non-
human
species antibody that binds the desired antigen having one or more
complementarity determining regions (CDRs) from the non-human species and
framework regions from a human immunoglobulin molecule. Often, framework
residues in the human framework regions will be substituted with the
corresponding residue from the CDR donor antibody to alter, preferably
improve,
antigen binding. These framework substitutions are identified by methods well
known in the art, e.g., by modeling ofthe interactions ofthe CDR and framework
residues to identify framework residues important for antigen binding and
sequence comparison to identify unusual framework residues at particular
positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089; Riechmann et
al.,
Nature 332:323 (1988), which are incorporated herein by reference in their
entireties.) Antibodies can be humanized using a variety of techniques known
in
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the art including, for example, CDR-grafting (EP 239,400; PCT publication WO
91/09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or
resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology
28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814
S (1994); Roguska. e1 al., PNAS 91:969-973 (1994)), and chain shuffling (U.S.
Patent No. 5,565,332).
Completely human antibodies are particularly desirable for therapeutic
treatment of human patients. Human antibodies can be made by a variety of
methods known in the art including phage display methods described above using
antibody libraries derived from human immunoglobulin sequences. See also, U.S.
Patent Nos. 4,444,887 and 4,716,11 I; and PCT publications WO 98/46645, WO
98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO
91/10741; each of which is incorporated herein by reference in its entirety.
Human antibodies can also be produced using transgenic mice which are
I 5 incapable of expressing functional endogenous immunoglobulins, but which
can
express human immunoglobulin genes. For example, the human heavy and light
chain immunoglobulin gene complexes may be introduced randomly or by
homologous recombination into mouse embryonic stem cells. Alternatively, the
human variable region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and light chain
genes. The mouse heavy and light chain immunoglobulin genes may be rendered
non-functional separately or simultaneously with the introduction of human
immunoglobulin loci by homologous recombination. In particular, homozygous
deletion of the JH region prevents endogenous antibody production. The
modified embryonic stem cells are expanded and microinjected into blastocysts
to
produce chimeric mice. The chimeric mice are then bred to produce homozygous
offspring that express human antibodies. The transgenic mice are immunized in
the normal fashion with a selected antigen, e.g., all or a portion of a
polypeptide
of the invention. Monoclonal antibodies directed against the antigen can be
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obtained from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the transgenic
mice rearrange during B cell differentiation, and subsequently undergo class
switching and somatic mutation. Thus, using such a technique, it is possible
to
produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an
overview of this technology for producing human antibodies, see Lonberg and
Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this
technology for producing human antibodies and human monoclonal antibodies and
protocols for producing such antibodies, see, e.g., PCT publications WO
98/24893; WO 96/34096; WO 96/33735; U.S. PatentNos. 5,413,923; 5,625,126;
5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, which
are
incorporated by reference herein in their entirety. In addition, companies
such as
Abgenix, Inc. (Freemont, CA) and GenPharm (San Jose, CA) can be engaged to
provide human antibodies directed against a selected antigen using technology
similar to that described above.
Completely human antibodies which recognize a selected epitope can be
generated using a technique referred to as "guided selection." In this
approach a
selected non-human monoclonal antibody, e.g., a mouse antibody, is used to
guide
the selection of a completely human antibody recognizing the same epitope.
(Jespers et al., Bioltechnology 12:899-903 (1988)).
As discussed above, antibodies to the TR2 receptor proteins of the
invention can, in turn, be utilized to generate anti-idiotype antibodies that
"mimic"
TRZ receptors using techniques well known to those skilled in the art. (See,
e.g.,
Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinofh, J. Immunol.
147(8):2429-243 8 ( 1991 )). For example, antibodies which bind to TR2
receptors
and competitively inhibit TR2 receptor multimerization and/or binding to
ligand
can be used to generate anti-idiotypes that "mimic" TR2 receptor
multimerization
and/or binding domain and, as a consequence, bind to and neutralize TR2
receptors and/or their ligand(s). Such neutralizing anti-idiotypes or Fab
fragments
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of such anti-idiotypes can be used in therapeutic regimens to neutralize TR2
receptor ligand(s). For example, such anti-idiotypic antibodies can be used to
bind
TR2 receptors, or to bind TRZ receptors or ligands, and thereby block TR2
receptor mediated inhibition of apoptosis.
A. Polynucleotides Encoding Antibodies
The invention further provides polynucleotides comprising a nucleotide
sequence encoding an antibody of the invention and fragments thereof. The
invention also encompasses polynucleotides that hybridize under stringent or
lower stringency hybridization conditions, e.g., as defined herein, to
polynucleotides that encode an antibody, preferably, that specifically binds
to a
polypeptide of the invention, preferably, an antibody that binds to a
polypeptide
having the amino acid sequence of SEQ ID N0:2, SEQ ID NO:S, SEQ ID N0:8,
or SEQ ID N0:26.
The polynucleotides may be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. For example, ifthe
nucleotide sequence of the antibody is known, a polynucleotide encoding the
antibody may be assembled from chemically synthesized oligonucleotides (e.g.,
as
described in Kutmeier et al., BioTechnigue.s 17:242 (1994)), which, briefly,
involves the synthesis of overlapping oligonucleotides containing portions of
the
sequence encoding the antibody, annealing and ligation of those
oligonucleotides,
and then amplification of the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an antibody may be generated
from nucleic acid from a suitable source. If a clone containing a nucleic acid
encoding a particular antibody is not available, but the sequence of the
antibody
molecule is known, a nucleic acid encoding the immunoglobulin may be obtained
from a suitable source (e.g., an antibody cDNA library, or a cDNA library
generated from, or nucleic acid, preferably polyA+ RNA, isolated from, any
tissue
or cells expressing the antibody, such as hybridoma cells selected to express
an
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antibody of the invention) by PCR amplification using synthetic primers
hybridizable to the 3' and 5' ends of the sequence or by cloning using an
oligonucleotide probe specific for the particular gene sequence to identify,
e.g.,
a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic
acids generated by PCR may then be cloned into replicable cloning vectors
using
any method well known in the art.
Once the nucleotide sequence and corresponding amino acid sequence of
the antibody is determined, the nucleotide sequence of the antibody may be
manipulated using methods well known in the art for the manipulation of
nucleotide sequences, e.g., recombinant DNA techniques, site directed
mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook
et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998,
Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both
incorporated by reference herein in their entireties), to generate antibodies
having
a different amino acid sequence, for example to create amino acid
substitutions,
deletions, and/or insertions.
In a specific embodiment, the amino acid sequence of the heavy and/or
light chain variable domains may be inspected to identify the sequences of the
complementarity determining regions (CDRs) by methods that are well know in
the art, e.g., by comparison to known amino acid sequences of other heavy and
light chain variable regions to determine the regions of sequence
hypervariability.
Using routine recombinant DNA techniques, one or more of the CDRs may be
inserted within framework regions, e.g., into human framework regions to
humanize a non-human antibody, as described supra. The framework regions may
be naturally occurring or consensus framework regions, and preferably human
framework regions (.see, e.g., Chothia et al., J Mol. Biol. 278:457-479 (1998)
for
a listing of human framework regions). Preferably, the polynucleotide
generated
by the combination of the framework regions and CDRs encodes an antibody that
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specifically binds a polypeptide of the invention. Preferably, as discussed
supra,
one or more amino acid substitutions may be made within the framework regions,
and, preferably, the amino acid substitutions improve binding of the antibody
to
its antigen. Additionally, such methods may be used to make amino acid
substitutions or deletions of one or more variable region cysteine residues
participating in an intrachain disulfide bond to generate antibody molecules
lacking
one or more intrachain disulfide bonds. Other alterations to the
polynucleotide are
encompassed by the present invention and within the skill of the art.
In addition, techniques developed for the production of "chimeric
antibodies" (Morrisonetal., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger
et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by
splicing genes from a mouse antibody molecule of appropriate antigen
specificity
together with genes from a human antibody molecule of appropriate biological
activity can be used. As described supra, a chimeric antibody is a molecule in
1 S which difFerent portions are derived from dif~'erent animal species, such
as those
having a variable region derived from a murine mAb and a human
immunoglobulin constant region, e.g., humanized antibodies.
Alternatively, techniques described for the production of single chain
antibodies (U. S. Patent No. 4,694,778; Bird, 1988, Science 242:423- 42;
Huston
et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989,
Nature 334:544-54) can be adapted to produce single chain antibodies. Single
chain antibodies are formed by linking the heavy and light chain fragments of
the
Fv region via an amino acid bridge, resulting in a single chain polypeptide.
Techniques for the assembly of functional Fv fragments in E. coli may also be
used (Skerra et al., 1988, Science 242:1038-1041).
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B. Methods of Producing Antibodies
The antibodies of the invention can be produced by any method known in
the art for the synthesis of antibodies, in particular, by chemical synthesis
or
preferably, by recombinant expression techniques.
S Recombinant expression of an antibody of the invention, or fragment,
derivative or analog thereof, e.g., a heavy or light chain of an antibody of
the
invention, requires construction of an expression vector containing a
polynucleotide that encodes the antibody. Once a polynucleotide encoding an
antibody molecule or a heavy or light chain of an antibody, or portion thereof
(preferably containing the heavy or light chain variable domain), of the
invention
has been obtained, the vector for the production of the antibody molecule may
be
produced by recombinant DNA technology using techniques well known in the
art. Thus, methods for preparing a protein by expressing a polynucleotide
containing an antibody encoding nucleotide sequence are described herein.
1 S Methods which are well known to those skilled in the art can be used to
construct
expression vectors containing antibody coding sequences and appropriate
transcriptional and translational control signals. These methods include, for
example, in vitro recombinant DNA techniques, synthetic techniques, and irmivo
genetic recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding an antibody molecule ofthe
invention,
or a heavy or light chain thereof, or a heavy or light chain variable domain,
operably linked to a promoter. Such vectors may include the nucleotide
sequence
encoding the constant region ofthe antibody molecule (see, e.g., PCT
Publication
WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464)
2S and the variable domain of the antibody may be cloned into such a vector
for
expression of the entire heavy or light chain.
The expression vector is transferred to a host cell by conventional
techniques and the transfected cells are then cultured by conventional
techniques
to produce an antibody of the invention. Thus, the invention includes host
cells
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containing a polynucleotide encoding an antibody of the invention, or a heavy
or
light chain thereof, operably linked to a heterologous promoter. In preferred
embodiments for the expression of double-chained antibodies, vectors encoding
both the heavy and light chains may be co-expressed in the host cell for
expression
of the entire immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the
antibody molecules of the invention. Such host-expression systems represent
vehicles by which the coding sequences of interest may be produced and
subsequently purified, but also represent cells which may, when transformed or
transfected with the appropriate nucleotide coding sequences, express an
antibody
molecule of the invention in situ. These include but are not limited to
microorganisms such as bacteria (e.g., E. co7i, B. sublili.s) transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing antibody coding sequences; yeast (e.g., Saccharomyces,
Yichia) transformed with recombinant yeast expression vectors containing
antibody coding sequences; insect cell systems infected with recombinant virus
expression vectors (e.g. , baculovirus) containing antibody coding sequences;
plant cell systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed
with recombinant plasmid expression vectors (e.g., Ti plasmid) containing
antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK,
293, 3 T3 cells) harboring recombinant expression constructs containing
promoters
derived from the genome of mammalian cells (e.g., metallothionein promoter) or
from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus
7.SK promoter). Preferably, bacterial cells such as Escherichia coli, and more
preferably, eukaryotic cells, especially for the expression of whole
recombinant
antibody molecule, are used for the expression of a recombinant antibody
molecule. For example, mammalian cells such as Chinese hamster ovary cells
(CHO), in conjunction with a vector such as the major intermediate early gene
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promoter element from human cytomegalovirus is an effective expression system
for antibodies (Foecking et al., 1986, Gene 45:101; Cockett et al., 1990,
Bio/Technology 8:2).
In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the antibody
molecule being expressed. For example, when a large quantity of such a protein
is to be produced, for the generation of pharmaceutical compositions of an
antibody molecule, vectors which direct the expression of high levels of
fusion
protein products that are readily purified may be desirable. Such vectors
include,
but are not limited, to the E. coli expression vector pUR278 (Ruther et al.,
1983,
EMBO J. 2:1791 ), in which the antibody coding sequence may be ligated
individually into the vector in frame with the lac Z coding region so that a
fusion
protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.
13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and
I S the like. pGEX vectors may also be used to express foreign polypeptides as
fusion
proteins with glutathione S-transferase (GST). In general, such fusion
proteins
are soluble and can easily be purified from lysed cells by adsorption and
binding
to a matrix glutathione-agarose beads followed by elution in the presence of
free
glutathione. The pGEX vectors are designed to include thrombin or factor Xa
protease cleavage sites so that the cloned target gene product can be released
from the GST moiety.
In an insect system, Autog~°apha californica nuclear polyhedrosis
virus
(AcNPV) is used as a vector to express foreign genes. The virus grows in
Spodoptera frugiperda cells. The antibody coding sequence may be cloned
individually into non-essential regions (for example the polyhedrin gene) of
the
virus and placed under control of an AcNPV promoter (for example the
polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may
be utilized. In cases where an adenovirus is used as an expression vector, the
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antibody coding sequence of interest may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter and
tripartite
leader sequence. This chimeric gene may then be inserted in the adenovirus
genome by iro vitro or in vivo recombination. Insertion in a non- essential
region
of the viral genome (e.g., region El or E3) will result in a recombinant virus
that
is viable and capable of expressing the antibody molecule in infected hosts.
(e.g.,
see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific
initiation signals may also be required for efficient translation of inserted
antibody
coding sequences. These signals include the ATG initiation codon and adjacent
sequences. Furthermore, the initiation codon must be in phase with the reading
frame of the desired coding sequence to ensure translation of the entire
insert.
These exogenous translational control signals and initiation codons can be of
a
variety of origins, both natural and synthetic. The efficiency of expression
may be
enhanced by the inclusion of appropriate transcription enhancer elements,
transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol.
153:51-544).
In addition, a host cell strain may be chosen which modulates the
expression of the inserted sequences, or modifies and processes the gene
product
in the specific fashion desired. Such modifications (e.g., glycosylation) and
processing (e.g., cleavage) of protein products may be important for the
function
ofthe protein. Different host cells have characteristic and specific
mechanisms for
the post-translational processing and modification of proteins and gene
products.
Appropriate cell lines or host systems can be chosen to ensure the correct
modification and processing of the foreign protein expressed. To this end,
eukaryotic.host cells which possess the cellular machinery for proper
processing
of the primary transcript, glycosylation, and phosphorylation of the gene
product
may be used. Such mammalian host cells include but are not limited to CHO,
VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast
cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D,
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and normal mammary gland cell line such as, for example, CRL7030 and
Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable
expression is preferred. For example, cell lines which stably express the
antibody
molecule may be engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with DNA
controlled by
appropriate expression control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a selectable
marker.
Following the introduction of the foreign DNA, engineered cells may be allowed
to grow for 1-2 days in an enriched media, and then are switched to a
selective
media. The selectable marker in the recombinant plasmid confers resistance to
the
selection and allows cells to stably integrate the plasmid into their
chromosomes
and grow to form foci which in turn can be cloned and expanded into cell
lines.
This method may advantageously be used to engineer cell lines which express
the
1 S antibody molecule. Such engineered cell lines may be particularly useful
in
screening and evaluation of compounds that interact directly or indirectly
with the
antibody molecule.
A number of selection systems may be used, including but not limited to
the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223),
hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192,
Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase
(Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt-
cells, respectively. Also, antimetabolite resistance can be used as the basis
of
selection for the following genes: dhfr, which confers resistance to
methotrexate
(Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc.
Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic
acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which
confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505;
Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
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Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and
Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH
11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et
al., 1984, Gene 30:147). Methods commonly known in the art of recombinant
DNA technology which can be used are described in Ausubel et al. (eds.), 1993,
Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990,
Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and
in Chapters 12 and 13, Dracopoli ef al. (eds), 1994, Current Protocols in
Human
Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol.
150:1, which are incorporated by reference herein in their entireties.
The expression levels of an antibody molecule can be increased by vector
amplification (for a review, see Bebbington and Hentschel, The use of vectors
based on gene amplification for the expression of cloned genes in mammalian
cells
in DNA cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in
the vector system expressing antibody is amplifiable, increase in the level of
inhibitor present in culture of host cell will increase the number of copies
of the
marker gene. Since the amplified region is associated with the antibody gene,
production ofthe antibody will also increase (Grouse et al., 1983, Mol. Cell.
Biol.
3:257).
The host cell may be co-transfected with two expression vectors of the
invention, the first vector encoding a heavy chain derived polypeptide and the
second vector encoding a light chain derived polypeptide. The two vectors may
contain identical selectable markers which enable equal expression of heavy
and
light chain polypeptides. Alternatively, a single vector may be used which
encodes
both heavy and light chain polypeptides. In such situations, the light chain
should
be placed before the heavy chain to avoid an excess of toxic free heavy chain
(Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA
77:2197). The coding sequences for the heavy and light chains may comprise
cDNA or genomic DNA.
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Once an antibody molecule of the invention has been recombinantly
expressed, it may be purified by any method known in the art for purification
of
an immunoglobulin molecule, for example, by chromatography (e.g., ion
exchange, affinity, particularly by affinity for the specific antigen after
Protein A,
and sizing column chromatography), centrifugation, differential solubility, or
by
any other standard technique for the purification of proteins.
G Antibody conjugates
The present invention encompasses antibodies recombinantly fused or
chemically conjugated (including both covalently and non-covalently
conjugations)
to a polypeptide (or portion thereof, preferably at least 10, 20 or 50 amino
acids
of the polypeptide) of the present invention to generate fusion proteins. The
fusion does not necessarily need to be direct, but may occur through linker
sequences. The antibodies may be specific for antigens other than polypeptides
(or portion thereof, preferably at least 10, 20 or 50 amino acids of the
polypeptide) of the present invention. For example, antibodies may be used to
target the polypeptides of the present invention to particular cell types,
either in
vitro or in viva, by fusing or conjugating the polypeptides of the present
invention
to antibodies specific for particular cell surface receptors. Antibodies fused
or
conjugated to the polypeptides of the present invention may also be used in in
vitro immunoassays and purification methods using methods known in the art.
See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095;
Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Patent 5,474,981;
Gillies
et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.
146:2446-2452( 1991 ), which are incorporated by reference in their
entireties.
The present invention further includes compositions comprising the
polypeptides of the present invention fused or conjugated to antibody domains
other than the variable regions. For example, the polypeptides of the present
invention may be fused or conjugated to an antibody Fc region, or portion
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thereof. The antibody portion fused to a polypeptide of the present invention
may
comprise the constant region, hinge region, CHl domain, CH2 domain, and CH3
domain or any combination of whole domains or portions thereof. The
polypeptides may also be fused or conjugated to the above antibody portions to
form multimers. For example, Fc portions fused to the polypeptides ofthe
present
invention can form dimers through disulfide bonding between the Fc portions.
Higher multimeric forms can be made by fusing the polypeptides to portions of
IgA and IgM. Methods for fusing or conjugating the polypeptides of the present
invention to antibody portions are known in the art. See, e.g., U.S. Patent
Nos.
5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434;
EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al.,
Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.
154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-
11341 (1992) (said references incorporated by reference in their entireties).
As discussed, .supra, the polypeptides of the present invention may be
fused or conjugated to the above antibody portions to increase the in vivo
half life
of the polypeptides or for use in immunoassays using methods known in the art.
Further, the polypeptides of the present invention may be fused or conjugated
to
the above antibody portions to facilitate purification. One reported example
describes chimeric proteins consisting of the first two domains of the human
CD4-polypeptide and various domains of the constant regions of the heavy or
light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al.,
Nature 331:84-86 (1988). The polypeptides of the present invention fused or
conjugated to an antibody having disulfide- linked dimeric structures (due to
the
IgG) may also be more efficient in binding and neutralizing other molecules,
than
the monomeric secreted protein or protein fragment alone. (Fountoulakis et
al.,
J. Biochem. 270:3958-3964 (1995)). In many cases, the Fc part in a fusion
protein is beneficial in therapy and diagnosis, and thus can result in, for
example,
improved pharmacokinetic properties. (EP A 232,262). Alternatively, deleting
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the Fc part after the fusion protein has been expressed, detected, and
purified,
would be desired. For example, the Fc portion may hinder therapy and diagnosis
if the fusion protein is used as an antigen for immunizations. In drug
discovery,
for example, human proteins, such as hIL-5 receptor, have been fused with Fc
portions for the purpose of high-throughput screening assays to identify
antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-
58
(1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
Moreover, the antibodies or fragments thereof of the present invention can
be fused to marker sequences, such as a peptide to facilitates their
purification.
In preferred embodiments, the marker amino acid sequence is a hexa-histidine
peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton
Avenue, Chatsworth, CA, 91311 ), among others, many of which are commercially
available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824
(1989), for instance, hexa-histidine provides for convenient purification of
the
fusion protein. Other peptide tags useful for purification include, but are
not
limited to, the "HA" tag, which corresponds to an epitope derived from the
influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the
"flag"
tag.
The present invention further encompasses antibodies or fragments thereof
conjugated to a diagnostic or therapeutic agent. The antibodies can be used
diagnostically to, for example, monitor the development or progression of a
tumor
as part of a clinical testing procedure to, e.g., determine the e~cacy of a
given
treatment regimen. Detection can be facilitated by coupling the antibody to a
detectable substance. Examples ofdetectable substances include various
enzymes,
prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent
materials, radioactive materials, positron emitting metals using various
positron
emission tomographies, and nonradioactive paramagnetic metal ions. See, for
example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to
antibodies for use as diagnostics according to the present invention. Examples
of
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suitable enzymes include horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic
group
complexes include streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride
or
phycoerythrin; an example of a luminescent material includes luminol; examples
of bioluminescent materials include luciferase, luciferin, and aequorin; and
examples of suitable radioactive material include'ZSI, '3'I, 1"In or 99Tc.
Further, an antibody or fragment thereof may be conjugated to a
therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent,
a
therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent
includes any agent that is detrimental to cells. Examples include paclitaxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
I S dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,
I-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,
propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents
include, but are not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine),
alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine
platinum
(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin)
and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),
bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
The conjugates of the invention can be used for modifying a given
biological response, the therapeutic agent or drug moiety is not to be
construed
as limited to classical chemical therapeutic agents. For example, the drug
moiety
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may be a protein or polypeptide possessing a desired biological activity. Such
proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas
exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-
interferon,
13-interferon, nerve growth factor, platelet derived growth factor, tissue
S plasminogen activator, a thrombotic agent or an anti- angiogenic agent,
e.g.,
angiostatin or endostatin; or, biological response modifiers such as, for
example,
lymphokines, interleukin-1 ("IL-1 "), interleukin-2 ("IL-2"), interleukin-6
("IL-6"),
granulocyte macrophase colony stimulating factor ("GM-CSF"), granulocyte
colony stimulating factor ("G-CSF"), or other growth factors.
Antibodies may also be attached to solid supports, which are particularly
useful for immunoassays or purification of the target antigen. Such solid
supports
include, but are not limited to, glass, cellulose, polyacrylamide, nylon,
polystyrene,
polyvinyl chloride or polypropylene.
Techniques for conjugating such therapeutic moiety to antibodies are well
known, see, e.g., Arnon el al., "Monoclonal Antibodies For Immunotargeting Of
Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy,
Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et
al.,
"Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed. ),
Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody
Carriers
2fl Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies
'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506
(1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of
Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer
Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press
1985),
and Thorpe et al., "The Preparation And Cytotoxic Properties OfAntibody-Toxin
Conjugates", Immunol. Rev. 62:119-58 (1982).
Alternatively, an antibody can be conjugated to a second antibody to form
an antibody heteroconjugate as described by Segal in U. S. Patent No.
4,676,980,
which is incorporated herein by reference in its entirety.
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An antibody, with or without a therapeutic moiety conjugated to it,
administered alone or in combination with cytotoxic factors) and/or
cytokine(s)
can be used as a therapeutic.
D. Assays For Antibody Binding
The antibodies ofthe invention may be assayed for immunospecific binding
by any method known in the art. The immunoassays which can be used include
but are not limited to competitive and non-competitive assay systems using
technidues such as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays,
precipitin reactions, gel diffusion precipitin reactions, immunodiffusion
assays,
agglutination assays, complement-fixation assays, immunoradiometric assays,
fluorescent immunoassays, protein A immunoassays, to name but a few. Such
assays are routine and well known in the art (see, e.g., Ausubel et al., eds,
1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New
York, which is incorporated by reference herein in its entirety). Exemplary
immunoassays are described briefly below (but are not intended by way of
limitation).
Immunoprecipitation protocols generally comprise lysing a population of
cells in a lysis buffer such as RIPA buffer ( 1 % NP-40 or Triton X- 100, I
sodium deoxycholate, 0. I % SDS, 0. I 5 M NaCI, 0.01 M sodium phosphate at pH
7.2, I% Trasylol) supplemented with protein phosphatase and/or protease
inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody
of interest to the cell lysate, incubating for a period of time (e.g., I -4
hours) at 4°
C, adding protein A and/or protein G sepharose beads to the cell lysate,
incubating
for about an hour or more at 4° C, washing the beads in lysis buffer
and
resuspending the beads in SDS/sample buffer. The ability of the antibody of
interest to immunoprecipitate a particular antigen can be assessed by, e.g.,
western
blot analysis. One of skill in the art would be knowledgeable as to the
parameters
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that can be modified to increase the binding of the antibody to an antigen and
decrease the background (e.g., pre-clearing the cell lysate with sepharose
beads).
For further discussion regardingimmunoprecipitationprotocols see, e.g.,
Ausubel
et al., eds, 1994, Current Protocols in Molecular Biology, Vol. l, John Wiley
&
Sons, Inc., New York at 10.16.1.
Western blot analysis generally comprises preparing protein samples,
electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-PAGE depending on the molecular weight of the antigen), transferring the
protein sample from the polyacrylamide gel to a membrane such as
nitrocellulose,
PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3%
BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween
20), blocking the membrane with primary antibody (the antibody of interest)
diluted in blocking buffer, washing the membrane in washing buffer, blocking
the
membrane with a secondary antibody (which recognizes the primary antibody,
e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g.,
horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
3ZP
or 'ZSI) diluted in blocking buffer, washing the membrane in wash buffer, and
detecting the presence of the antigen. One of skill in the art would be
knowledgeable as to the parameters that can be modified to increase the signal
detected and to reduce the background noise. For further discussion regarding
western blot protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols
in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96 well
microtiter plate with the antigen, adding the antibody of interest conjugated
to a
detectable compound such as an enzymatic substrate (e.g., horseradish
peroxidase
or alkaline phosphatase) to the well and incubating for a period of time, and
detecting the presence of the antigen. In ELISAs the antibody of interest does
not
have to be conjugated to a detectable compound; instead, a second antibody
(which recognizes the antibody of interest) conjugated to a detectable
compound
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may be added to the well. Further, instead of coating the well with the
antigen,
the antibody may be coated to the well. In this case, a second antibody
conjugated to a detectable compound may be added following the addition of the
antigen of interest to the coated well. One of skill in the art would be
knowledgeable as to the parameters that can be modified to increase the signal
detected as well as other variations of ELISAs known in the art. For further
discussion regarding ELISAs see, e.g., Ausubel et al., eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
11.2.1.
The binding af~'mity of an antibody to an antigen and the ofd rate of an
antibody-antigen interaction can be determined by competitive binding assays.
One example of a competitive binding assay is a radioimmunoassay comprising
the
incubation of labeled antigen (e.g., 3H or'25I) with the antibody of interest
in the
presence of increasing amounts of unlabeled antigen, and the detection of the
antibody bound to the labeled antigen. The affinity of the antibody of
interest for
a particular antigen and the binding ofd rates can be determined from the data
by
scatchard plot analysis. Competition with a second antibody can also be
determined using radioimmunoassays. In this case, the antigen is incubated
with
antibody of interest is conjugated to a labeled compound (e.g., ~H or'ZSI) in
the
presence of increasing amounts of an unlabeled second antibody.
E. Therapeutic Uses
The present invention is further directed to antibody-based therapies which
involve administering antibodies of the invention to an animal, preferably a
mammal, and most preferably a human, patient for treating one or more of the
disclosed diseases, disorders, or conditions. Therapeutic compounds of the
invention include, but are not limited to, antibodies of the invention
(including
fragments, analogs and derivatives thereof as described herein) and nucleic
acids
encoding antibodies ofthe invention (including fragments, analogs and
derivatives
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thereof and anti-idiotypic antibodies as described herein). The antibodies of
the
invention can be used to treat, inhibit or prevent diseases, disorders or
conditions
associated with aberrant expression and/or activity of a polypeptide of the
invention, including, but not limited to, autoimmune diseases, disorders, or
conditions associated with such diseases or disorders, including, but not
limited
to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia,
idiopathic thrombocytopenia purpura, autoimmunocytopenia, hemolytic anemia,
antiphospholipid syndrome, dermatitis, allergic encephalomyelitis,
myocarditis,
relapsing polychondritis, ulcerative colitis, dense deposit disease, rheumatic
heart
disease, glomerulonephritis (e.g., IgA nephropathy), pemphigus vulgaris,
discoid
lupus, Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies,
Purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff Man
Syndrome, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome,
insulin dependent diabetes mellitis, and autoimmune inflammatory eye,
autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis),
systemic
lupus erhythematosus, Goodpasture's syndrome, Pemphigus, Receptor
autoimmunities such as, for example, (a) Graves' Disease , (b) Myasthenia
Gravis,
and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune
thrombocytopenic purpura , rheumatoid arthritis, schleroderma with anti-
collagen
antibodies, mixed connective tissue disease, polymyositis/dermatomyositis,
pernicious anemia, idiopathic Addison's disease, infertility,
glomerulonephritis
such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid,
Sjogren's syndrome, diabetes millitus, and adrenergic drug resistance
(including
adrenergic drug resistance with asthma or cystic fibrosis), chronic active
hepatitis,
primary biliary cirrhosis, other endocrine gland failure, vitiligo,
vasculitis, post-MI,
cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory
myopathies, graft v. host diseases (GVI~) and other inflammatory,
granulamatous, degenerative, and atrophic disorders).
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In a specific embodiment, antibodies of the invention are be used to treat,
inhibit, prognose, diagnose or prevent rheumatoid arthritis.
In another specific embodiment, antibodies of the invention are used to
treat, inhibit, prognose, diagnose or prevent systemic lupus erythematosis.
S Additionally, the antibodies of the invention can be used to treat, inhibit
or prevent diseases, disorders or conditions associated with
immunodeficiencies
including, but not limited to, severe combined immunodeficiency (SCID)-X
linked,
SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked
agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia,
X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult
onset agammaglobulinemia, late-onset agammaglobulinemia,
dysgammaglobulinemia, hypogammaglobulinemia, transient
hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia,
agammaglobulinemia, common variable immunodeficiency (CVID) (acquired),
I 5 Wiskott-Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IgM,
non X-linked imrnunodeficiency with hyper IgM, selective IgA deficiency, IgG
subclass deficiency (with or without IgA deficiency), antibody deficiency with
normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain
deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD),
selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type),
reticular dysgenesis, neonatal neutropenia, autoimmune neutropenia, severe
congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with
immunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linked
lymphoproliferative syndrome (XLP), Nezelof syndrome-combined
immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP),
MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined
immunodeficiency.
Antibodies of the invention are used to prevent graft rejection and
inflammation and for the treatment of arthritis.
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The treatment and/or prevention of diseases and disorders associated with
aberrant expression and/or activity of a polypeptide of the invention
includes, but
is not limited to, alleviating symptoms associated with those diseases and
disorders. Antibodies of the invention may be provided in pharmaceutically
acceptable compositions as known in the art or as described herein.
A summary of the ways in which the antibodies of the present invention
may be used therapeutically includes binding polynucleotides or polypeptides
of
the present invention locally or systemically in the body or by direct
cytotoxicity
of the antibody, e.g., as mediated by complement (CDC) or by effector cells
(ADCC). Some of these approaches are described in more detail below. Armed
with the teachings provided herein, one of ordinary skill in the art will know
how
to use the antibodies of the present invention for diagnostic, monitoring or
therapeutic purposes without undue experimentation.
The antibodies of this invention may be advantageously utilized in
combination with other monoclonal or chimeric antibodies, or with lymphokines
or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for
example,
which serve to increase the number or activity of effector cells which
interact with
the antibodies.
The antibodies of the invention may be administered alone or in
combination with other types oftreatments (e.g., radiation therapy,
chemotherapy,
hormonal therapy, immunotherapy and anti-tumor agents). Generally,
administration of products of a species origin or species reactivity (in the
case of
antibodies) that is the same species as that of the patient is preferred.
Thus, in a
preferred embodiment, human antibodies, fragments derivatives, analogs, or
nucleic acids, are administered to a human patient for therapy or prophylaxis.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or
neutralizing antibodies against polypeptides or polynucleotides of the present
invention, fragments or regions thereof, for both immunoassays directed to and
therapy of disorders related to polynucleotides or polypeptides, including
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fragments thereof, of the present invention. Such antibodies, fragments, or
regions, will preferably have an amity for polynucleotides or polypeptides,
including fragments thereof. Preferred binding amities include those with a
dissociation constant orKdlessthan5X10~6M, 10-6M, SX10~'M, 10-'M, SX10-8M,
S 10-gM, 5 X 10-9M, 10-9M, 5 X 10-' °M, I 0-' °M, 5 X 10~"M, 10-
"M, 5 X 10-' zM, 10-' zM,
SX10-'3M, 10-'3M, SX10-'4M, 10-'4M, SX10-'SM, and 10~'SM.
Transgenic Non-Human Animals
The proteins of the invention can also be expressed in transgenic animals.
Animals of any species, including, but not limited to, mice, rats, rabbits,
hamsters,
guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates,
e.g.,
baboons, monkeys, and chimpanzees may be used to generate transgenic animals.
In a specific embodiment, techniques described herein or otherwise known in
the
art, are used to express polypeptides of the invention in humans, as part of a
gene
therapy protocol.
Any technique known in the art may be used to introduce the transgene
(i.e., nucleic acids of the invention) into animals to produce the founder
lines of
transgenic animals. Such techniques include, but are not limited to,
pronuclear
microinjection (Paterson et al., Appl. Microbfiol. Biotechraol. 40:691-698
(1994);
Carver et al., Biolechrzology (NY) 11:1263-1270 (1993); Wright et al.,
Biotechnology (N3') 9:830-834 (1991); and Hoppe et al., U.S. Patent Number
4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der
Putten et al., P~°oc. Natl. Acad. Sci., USA 82:6148-6152 (1985)),
blastocysts or
embryos; gene targeting in embryonic stem cells (Thompson et al., Cell
56:313-321 (1989)); electroporation of cells or embryos (Lo, Mol Cell. Biol.
3:1803-1814 (1983)); introduction of the polynucleotides of the invention
using
a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing
nucleic
acid constructs into embryonic pleuripotent stem cells and transferring the
stem
cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et
al.,
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Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon,
"Transgenic Animals," Irrtl. Rev. Cytol. 115:171-229 (1989), which is
incorporated by reference herein in its entirety. See, also, U.S. Patent No.
5,464,764 (Capecchi, et al., Positive-Negative Selection Methods and Vectors);
U.S. Patent No. 5,631,153 (Capecchi, et al., Cells and Non-Human Organisms
Containing Predetermined Genomic Modifications and Positive-Negative
Selection Methods and Vectors for Making Same); U.S. Patent No. 4,736,866
(Leder, et al., Transgenic Non-Human Animals); and U. S. Patent No. 4,873,191
(Wagner, et al., Genetic Transformation of Zygotes); each of which is hereby
incorporated by reference in its entirety. Further, the contents of each of
the
documents recited in this paragraph is herein incorporated by reference in its
entirety.
Any technique known in the art may be used to produce transgenic clones
containing polynucleotides of the invention, for example, nuclear transfer
into
enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells
induced
to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature
385:810-813 (1997)), each of which is herein incorporated by reference in its
entirety).
The present invention provides for transgenic animals that carry the
transgene in all their cells, as well as animals which carry the transgene in
some,
but not all their cells, i. e., mosaic animals or chimeric animals. The
transgene may
be integrated as a single transgene or as multiple copies such as in
concatamers,
e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be
selectively introduced into and activated in a particular cell type by
following, for
example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci.
USA
89:6232-6236 (1992)). The regulatory sequences required for such a cell-type
specific activation will depend upon the particular cell type of interest, and
will be
apparent to those of skill in the art. When it is desired that the
polynucleotide
transgene be integrated into the chromosomal site of the endogenous gene, gene
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targeting is preferred. Briefly, when such a technique is to be utilized,
vectors
containing some nucleotide sequences homologous to the endogenous gene are
designed for the purpose of integrating, via homologous recombination with
chromosomal sequences, into and disrupting the function of the nucleotide
sequence of the endogenous gene. The transgene may also be selectively
introduced into a particular cell type, thus inactivating the endogenous gene
in
only that cell type, by following, for example, the teaching of Gu et al. (Gu
et al.,
Science 265:103-106 (1994)). The regulatory sequences required for such a cell-
type specific inactivation will depend upon the particular cell type of
interest, and
will be apparent to those of skill in the art. The contents of each of the
documents
recited in this paragraph is herein incorporated by reference in its entirety.
Once transgenic animals have been generated, the expression of the
recombinant gene may be assayed utilizing standard techniques. Initial
screening
may be accomplished by Southern blot analysis or PCR techniques to analyze
animal tissues to verify that integration of the transgene has taken place.
The level
of mRNA expression of the transgene in the tissues of the transgenic animals
may
also be assessed using techniques which include, but are not limited to,
Northern
blot analysis of tissue samples obtained from the animal, ire .situ
hybridization
analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-
expressing tissue may also be evaluated immunocytochemically or
immunohistochemically using antibodies specific for the transgene product.
Once the founder animals are produced, they may be bred, inbred, outbred,
or crossbred to produce colonies of the particular animal. Examples of such
breeding strategies include, but are not limited to: outbreeding of founder
animals
with more than one integration site in order to establish separate lines;
inbreeding
of separate lines in order to produce compound transgenics that express the
transgene at higher levels because of the effects of additive expression of
each
transgene; crossing of heterozygous transgenic animals to produce animals
homozygous for a given integration site in order to both augment expression
and
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eliminate the need for screening of animals by DNA analysis; crossing of
separate
homozygous lines to produce compound heterozygous or homozygous lines; and
breeding to place the transgene on a distinct background that is appropriate
for an
experimental model of interest.
Transgenic and "knock-out" animals of the invention have uses which
include, but are not limited to, animal model systems useful in elaborating
the
biological function of TRZ receptor polypeptides, studying conditions and/or
disorders associated with aberrant TR2 receptor expression, and in screening
for
compounds effective in ameliorating such conditions and/or disorders.
In further embodiments of the invention, cells that are genetically
engineered to express the proteins of the invention, or alternatively, that
are
genetically engineered not to express the proteins of the invention (e.g.,
knockouts) are administered to a patient irr vivo. Such cells may be obtained
from
the patient (i.e., animal, including human) or an MHC compatible donor and can
1 S include, but are not limited to fibroblasts, bone marrow cells, blood
cells (e.g.,
lymphocytes), adipocytes, muscle cells, endothelial cells, etc. The cells are
genetically engineered in vib°o using recombinant DNA techniques to
introduce
the coding sequence of polypeptides of the invention into the cells, or
alternatively, to disrupt the coding sequence and/or endogenous regulatory
sequence associated with the polypeptides of the invention, e.g., by
transduction
(using viral vectors, and preferably vectors that integrate the transgene into
the
cell genome) or transfection procedures, including, but not limited to, the
use of
plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The
coding sequence of the polypeptides of the invention can be placed under the
control of a strong constitutive or inducible promoter or promoter/enhancer to
achieve expression, and preferably secretion, of the polypeptides ofthe
invention.
The engineered cells which express and preferably secrete the polypeptides of
the
invention can be introduced into the patient systemically, e.g., in the
circulation,
or intraperitoneally. Alternatively, the cells can be incorporated into a
matrix and
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implanted in the body, e.g., genetically engineered fibroblasts can be
implanted as
part of a skin graft; genetically engineered endothelial cells can be
implanted as
part of a lymphatic or vascular graft. (See, e.g., Anderson et al. U. S.
Patent
Number 5,399,349; and Mulligan & Wilson, U.S. Patent Number 5,460,959, each
of which is incorporated by reference herein in its entirety).
When the cells to be administered are non-autologous or non-MHC
compatible cells, they can be administered using well known techniques which
prevent the development of a host immune response against the introduced
cells.
For example, the cells may be introduced in an encapsulated form which, while
allowing for an exchange of components with the immediate extracellular
environment, does not allow the introduced cells to be recognized by the host
immune system.
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-~3, 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, Pnog. Alleyy, 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 Immuhol. 136(7):2483 (1987);
Porter, Tibtech 9:158-162 (1991)), growth regulation, vascular endothelium
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effects and metabolic effects. TNF-a also triggers endothelial cells to
secrete
various factors, including PAI-l, 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 CD4T and CD8+ T lymphocytes, monocytes, endothelial cells and other cell
types shown in Tables V and VI. By "a cellular response to a TNF-family
ligand"
is intended any genotypic, phenotypic, and/or morphologic change to a cell,
cell
line, tissue, tissue culture or patient that is induced by a TNF-family
ligand. As
indicated, such cellular responses include not only normal physiological
responses
to TNF-family ligands, but also diseases associated with increased 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., AIDSB:1197-1213 (1994); Krammer, P.H. et al., Curr.
Opirz. Inznzunol. 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
TR2
receptor protein and mRNA encoding 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 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
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the gene encoding 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.
By "assaying the expression level of the gene encoding TR2 receptor
protein" is intended qualitatively or quantitatively measuring or estimating
the
level of TR2, TR2-SV 1 and/or TR2-SV2 receptor protein or the level of the
mRNA encoding TR2, TR2-SVl and/or TR2-SV2 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 TR2, TR2-SVl
and/or TR2-SV2 receptor protein level or mRNA level in a second biological
sample).
Preferably, 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,
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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, including, but not limited to, colon
cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,
glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach
cancer,
neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma,
osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate
cancer, Kaposi's sarcoma and ovarian cancer); 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
1 S 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,
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.
In preferred embodiments TR2, TR2-SV 1 and/or TR2-SV2
polynucleotides or polypeptides of the invention are used to treat or prevent
autoimmune diseases and/or inhibit the growth, progression, and/or metastasis
of
cancers, including, but not limited to, those cancers disclosed herein, such
as, for
example, lymphocytic leukemias (including, for example, MLL and chronic
lymphocytic leukemia (CLL)) and follicular lymphomas. In another embodiment
TR2, TR2-SV 1 and/or TR2-SV2 polynucleotides or polypeptides ofthe invention
are used to activate, differentiate or proliferate cancerous cells or tissue
(e.g., B
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cell lineage related cancers (e.g., CLL and MLL), lymphocytic leukemia, or
lymphoma) and thereby render the cells more vulnerable to cancer therapy
(e.g.,
chemotherapy or radiation therapy).
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 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')z 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 TR2, TR2-SV 1 and/or
TR2-SV2 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, TR2-SV1 and/or
TR2-SV2 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
etal.,
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NatuJ~e 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler
et
al., Em°. J. Immunol. 6:292 (1976); Hammerling et al., In: Monoclonal
Antibodies and T Cell Hybridomas, Elsevier, N.Y., (1981) pp. 563-681). In
general, such procedures involve immunizing an animal (preferably a mouse)
with
S 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/1 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
(SPzO),
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 et al.
(Gash°oenterology 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
a TR2 activity induced by a TNF-family ligand (e.g., cell proliferation,
hematopoietic development), which involves administering to a cell which
expresses a TRZ 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,
TRZ 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, TRZ 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 TRZ 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
1 S 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
ofthe
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 ofthe 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 TRZ
receptor ligands.
Further screening assays for agonist and antagonist of the present
invention are described in Tartaglia, L.A., and Goeddel, D.V., .I. 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 ~H-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.
Agonists according to the present invention include compounds such as,
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for example, TNF-family ligand peptide fragments, transforming growth factor
Vii,
and neurotransmitters (such as glutamate, dopamine, N methyl-D-aspartate).
Preferred agonist include polyclonal and monoclonal antibodies raised against
a
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.
Sci. USA 88:9292-9296 ( 1991 ); and Tartaglia, L.A., and Goeddel, D. V., J.
Biol.
Chem. 267 (7):4304-4307 (1992). See, also, PCT Application WO 94/09137.
Further preferred agonists include chemotherapeutic drugs such as, for
example,
cisplatin, doxorubicin, bleomycin, cytosine arabinoside, nitrogen mustard,
methotrexate and vincristine. Others include ethanol and (3-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. lA-1B 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-SVl 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-(3), LT-(3 (found in complex heterotrimer LT-a2-~3), FasL,
CD40L, CD27L, CD30L, 4-1BBL, 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
lymphocytes. Further, such proliferation can be inhibited by a TR2 protein
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fragment fused to an Fc antibody fragment. Thus, specifically included within
the scope of the invention are TR2 receptor/Fc fusion proteins, and nucleic
acid
molecules which encode such proteins. These fusion proteins include those
having amino acid sequences of the extracellular domains of the TR2 proteins
of
the invention. Examples of portions of TR2 extracellular domains which are
useful in the preparation of TR2 receptor/Fc fusion proteins include amino
acids
1 to 192, 37 to 192, 50 to 192 and 100 to 192 in SEQ ID N0:2.
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).
IO 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.
Cytokine Newt. 7:159 (1996). Further, antibodies specific for the
extracellular
domain of this TNFR block HSV-I 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.
Similarly, antibodies specific for the extracellular domain of the TR2
receptors of the invention, as well as other TR2 antagonists, can also block
HSV-1 entry into cells. These antagonists are thus useful in the treatment and
prevention of Herpes simplex infections.
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 SEQ
ID N0:26, FIG. lA-1B (SEQ ID N0:2) and the TR2-SV 1 (FIG. 4A-4C (SEQ ID
N0:5)) and TR2-SV2 (FIG. 7A-7C (SEQ ID N0:8)) polypeptides (any of which
may or may not include a leader sequence) and polypeptide fragments of the
receptors comprising, or alternatively consisting of, the ligand binding,
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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, J. 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,
and may have less non-specific tissue binding of an intact antibody (Wahl et
al.,
.~. 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, Natm°e 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;MonoclonalAntibodies acrdHybridomas: ANew
Dimension ire Biological Analyses, Plenum Press, New York, NY, 1980;
Campbell, "Monoclonal Antibody Technology," In: Laboratory Techniques in
Biochen~i.stry andMolecular 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, Natm°e 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,
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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
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 ofthe present invention and a second messenger response, e.g.,
signal
transduction may be measured to determine whether the potential antagonist or
agonist is elective.
The TRZ 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
fromListeria
monocytogene.s) 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
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damage.
Agonists to the receptor polypeptides ofthe present invention may be used
to augment TNF's role in host defenses against microorganisms and prevent
related diseases. The agonists may also be employed to protect against the
deleterious effects ofionizing radiation produced during a course
ofradiotherapy,
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.
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 10' and 2 X 109
cells
(Wei X., et al., Natm°e 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 vitYO but
also, more importantly, in infected individuals (Ameisen, J.C., AIDS 8:1197-
1213
(1994) ; Finkel, T.H., and Banda, N.K., Curs. Opin. Immunol. 6:605-615(1995);
Muro-Cacho, C.A. et al., J. Imnzunol. 154:5555-5566 (1995)). Furthermore,
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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. Relroviruses 9:553-563 (1993)) and,
apoptosis is not observed in those animal models in which viral replication
does
S not result in AIDS (Gougeon, M.L. et al., AIDS Res. Hum. Relroviruses 9:553-
563 (1993)). Further data indicates that uninfected but primed or activated T
lymphocytes from HIV-infected individuals undergo apoptosis after encountering
the TNF-family ligand Fast. Using monocytic cell lines that result in death
following HIV infection, it has been demonstrated that infection of U937 cells
with HIV results in the de novo expression of Fast and that Fast mediates HIV-
induced apoptosis (Badley, A.D. et al., .I Tirol. 70:199-206 (1996)). Further
the
TNF-family ligand was detectable in uninfected macrophages and its expression
was upregulated following HIV infection resulting in selective killing
ofuninfected
CD4+ T-lymphocytes (Badley, A.D et al., J virol. 70:199-206 (1996)).
As shown in Example 6, the TRZ receptor shown in FIG. lA-1B 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 ofadministration 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
ofthe
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
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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.
In addition, TNF-a 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
1 S 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-l, 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
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tissue inj ury 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 T'R2
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.
In specific embodiments, antagonists according to the present invention
are nucleic acids corresponding to the sequences contained in SEQ ID N0:25,
FIG. lA-IB (SEQ 117 NO:1), FIG. 4A-4C (SEQ ID N0:4) or FIG. 7A-7C (SEQ
ID N0:7) or the complementary strand thereof, and/or to the deposited
nucleotide
sequences of ATCC Deposit Numbers 97059, 97058 or 97057. In one
embodiment, antisense sequence is generated internally by the organism, in
another embodiment, the antisense sequence is separately administered (see,
e.g,
O'Connor, J. Neurochem. 56:560 (1991), and Oligodeoxynucleotides as Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (i988). Antisense
technology can be used to control gene expression through antisense DNA or
RNA, ox through triple-helix formation. Antisense techniques are discussed for
example, in Okano, J. Neurochem. X6:560 (199I); Oligodeoxynucleotides as
Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).
Triple helix formation is discussed in, for instance, Lee et al., Nucleic
Acids
Research 6:3073 {1979); Cvoney et al., Science 241:456 (1988); and Dervan et
al., Science 251:1300 (1991). The methods are based on binding of a
polynucleotide to a complementary DNA or RNA.
For example, the 5' coding portion of a polynucleotide that encodes the
mature polypeptide of the present invention may be used to design an antisense
RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA
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oligonucleotide is designed to be complementary to a region of the gene
involved
in transcription thereby preventing transcription and the production of the
receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and
blocks translation of the mRNA molecule into receptor polypeptide.
In one embodiment, the TR2 receptor antisense nucleic acid of the
invention is produced intracellularly by transcription from an exogenous
sequence.
For example, a vector or a portion thereof, is transcribed, producing an
antisense
nucleic acid (RNA) of the invention. Such a vector would contain a sequence
encoding the TRZ receptor antisense nucleic acid. Such a vector can remain
episomal or become chromosomally integrated, as long as it can be transcribed
to
produce the desired antisense RNA. Such vectors can be constructed by
recombinant DNA technology methods standard in the art. Vectors can be
plasmid, viral, or others know in the art, used for replication and expression
in
vertebrate cells. Expression of the sequence encoding a TR2 receptor, or
fragments thereof, can be by any promoter known in the art to act in
vertebrate,
preferably human cells. Such promoters can be inducible or constitutive. Such
promoters include, but are not limited to, the SV40 early promoter region
(Bernoist and Chambon, Nature 29:304-310 ( 1981 ), the promoter contained in
the
3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-
797
(1980), the Herpes thymidine promoter (Wagner et al., Pnoc. Natl. Acad. Sci.
U. S.A. 78:1441-1445 ( 1981 ), the regulatory sequences ofthe metallothionein
gene
(Brinster, et al., Nature 296:39-42 (1982)), etc.
The antisense nucleic acids of the invention comprise a sequence
complementary to at least a portion of an RNA transcript of a TR2 receptor
gene.
However, absolute complementarity, although preferred, is not required. A
sequence "complementary to at least a portion of an RNA," referred to herein,
means a sequence having sufficient complementarity to be able to hybridize
with
the RNA, forming a stable duplex; in the case of double stranded TR2 receptor
antisense nucleic acids, a single strand of the duplex DNA may thus be tested,
or
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triplex formation may be assayed. The ability to hybridize will depend on both
the
degree of complementarity and the length of the antisense nucleic acid
Generally,
the larger the hybridizing nucleic acid, the more base mismatches with a TR2
receptor RNA it may contain and still form a stable duplex (or triplex as the
case
may be). One skilled in the art can ascertain a tolerable degree of mismatch
by use
of standard procedures to determine the melting point of the hybridized
complex.
Oligonucleotides that are complementaryto the 5' end ofthe message, e.g.,
the S' untranslated sequence up to and including the AUG initiation codon,
should
work most efficiently at inhibiting translation. However, sequences
complementary to the 3' untranslated sequences of mRNAs have been shown to
be effective at inhibiting translation of mRNAs as well. See generally,
Wagner,
R., Nature 372:333-335 (1994). Thus, oligonucleotides complementary to either
the 5'- or 3'- non- translated, non-coding regions of the TRZ receptor shown
in
SEQ ID N0:25, FIG. 1 A-1 B (SEQ ID NO:1 ), FIG. 4A-4C (SEQ II3 N0:4) or FIG.
7A-7C (SEQ ID N0:7) could be used in an antisense approach to inhibit
translation of endogenous TR2 receptor mRNA. Oligonucleotides complementary
to the 5' untranslated region of the mRNA should include the complement of the
AUG start codon. Antisense oligonucleotides complementary to mRNA coding
regions are less efficient inhibitors of translation but could be used in
accordance
with the invention. Whether designed to hybridize to the 5'-, 3'- or coding
region
of TR2 receptor mRNA, antisense nucleic acids should be at least six
nucleotides
in length, and are preferably oligonucleotides ranging from b to about 50
nucleotides in length. In specific aspects the oligonucleotide is at least 10
nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50
nucleotides.
The polynucleotides of the invention can be DNA or RNA or chimeric
mixtures or derivatives or modified versions thereof, single-stranded or
double-
stranded. The oligonucleotide can be modified at the base moiety, sugar
moiety,
or phosphate backbone, for example, to improve stability of the molecule,
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hybridization, etc. The oligonucleotide may include other appended groups such
as peptides (e.g., for targeting host cell receptors in vivo), or agents
facilitating
transport across the cell membrane (.see, e.g., Letsinger et al., Proc. Nail.
Acad.
,S'ci. U.S.A. 86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci.
84:648-
652 (1987); PCT Publication No. W088/09810, published December 15, 1988)
or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134,
published
April 25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et
al.,
BioTechniqZres 6:958-976 (1988)) or intercalating agents. (See, e.g., Zon,
Pha~°nz
Re.s. 5:539-549 (1988)). To this end, the oligonucleotide may be conjugated to
another molecule, e.g., a peptide, hybridization triggered cross-linking
agent,
transport agent, hybridization-triggered cleavage agent, etc.
The antisense oligonucleotide may comprise at least one modified base
moiety which is selected from the group including, but not limited to,
S-fluorouracil, 5-bromouracil, S-chlorouracil, 5-iodouracil, hypoxanthine,
xantine,
4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, S-carboxymethylaminomethyl-
2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-
galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine,
1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,
3-methylcytosine, 5-methyl cytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-
D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,
pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil,
4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-
oxyacetic
acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil,
(acp3)w,
and 2,6-diaminopurine.
The antisense oligonucleotide may also comprise at least one modified
sugar moiety selected from the group including, but not limited to, arabinose,
2-fluoroarabinose, xylulose, and hexose.
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In yet another embodiment, the antisense oligonucleotide comprises at
least one modified phosphate backbone selected from the group including, but
not
limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate,
a
phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl
phosphotriester, and a formacetal or analog thereof.
In yet another embodiment, the antisense oligonucleotide is an a.-anomeric
oligonucleotide. An a,-anomeric oligonucleotide forms specific double-stranded
hybrids with complementary RNA in which, contrary to the usual [3-units, the
strands run parallel to each other (Gautier et al., Nucl. Acids Re.s. 1:6625-
6641
(1987)). The oligonucleotide is a 2'-0-methylribonucleotide (moue et ad.,
Nucl.
Acids Res. 15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (moue ei
al., FEBSLett. 215:327-330 (1987)).
Polynucleotides of the invention may be synthesized by standard methods
known in the art, e.g. by use of an automated DNA synthesizer (such as are
commercially available from Biosearch, Applied Biosystems, etc.). As examples,
phosphorothioate oligonucleotides may be synthesized by the method of Stein et
a1. (Nucl. Acids Res. 16:3209 (1988)), methylphosphonate oligonucleotides can
be prepared by use of controlled pore glass polymer supports (Sarin el al.,
Pnoc.
Natl. Acad Sci. U.S.A. 85:7448-7451 (1988)), etc.
While antisense nucleotides complementary to TR2 receptor coding region
sequences could be used, those complementary to the transcribed untranslated
region are most preferred.
Potential antagonists according to the invention also include catalytic
RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364,
published October 4, 1990; Sarver et al, Science 247:1222-1225 (1990). While
ribozymes that cleave mRNA at site specific recognition sequences can be used
to destroy TRZ receptor mRNAs, the use of hammerhead ribozymes is preferred.
Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions
that form complementary base pairs with the target mRNA. The sole requirement
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is that the target mRNA have the following sequence of two bases: f-UG-3'. The
construction and production of hammerhead ribozymes is well known in the art
and is described more fully in Haseloff and Gerlach, Nature 334:585-591
(1988).
There are numerous potential hammerhead ribozyme cleavage sites within the
nucleotide sequence of the TR2 receptors (SEQ ID N0:25, FIG. 1 A-1 B (SEQ 1D
NO:1), FIG. 4A-4C (SEQ B7 N0:4) and FIG. 7A-7C (SEQ ID N0:7}). Preferably,
the ribozyme is engineered so that the cleavage recognition site is located
near the
S' end of the subj ect TR2 receptor mRNA; i. e. , to increase efficiency and
minimize
the intracellular accumulation of non-functional mRNA transcripts.
As in the antisense approach, the ribozymes of the invention can be
composed of modified oligonucleotides (e.g. for improved stability, targeting,
etc.)
and should be delivered to cells which express TR2 receptors in vivo. DNA
constructs encoding the ribozyme may be introduced into the cell in the same
manner as described above for the introduction of antisense encoding DNA. A
preferred method of delivery involves using a DNA construct "encoding" the
ribozyme under the control of a strong constitutive promoter, such as, for
example,
pot III or pot II promoter, so that transfected cells will produce sufficient
quantities
of the ribozyme to destroy endogenous TR2 receptor messages and inhibit
translation. Since ribozymes unlike antisense molecules, are catalytic, a
lower
intracellular concentration is required for eff ciency.
The compounds or pharmaceutical compositions of the invention are
preferably tested in vitro, and then in vivo for the desired therapeutic or
prophylactic activity, prior to use in humans. For example, in vitro assays to
demonstrate the therapeutic or prophylactic utility of a compound or
pharmaceutical composition include, the effect of a compound on a cell line or
a
patient tissue sample. The effect of the compound or composition on the cell
line
and/or tissue sample can be determined utilizing techniques known to those of
skill
in the art including, but not limited to, inhibition or stimulation of
proliferation.
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In accordance with the invention, in vitYO assays which can be used to
determine
whether administration of a specific compound is indicated, include in
vita°o cell
culture assays in which a patient tissue sample is grown in culture, and
exposed
to or otherwise administered a compound, and the efFect of such compound upon
the tissue sample is observed.
Endogenous gene expression can also be reduced by inactivating or
"knocking out" the TR2 receptor gene and/or its promoter using targeted
homologous recombination. (See, e.g., Smithies et al., Nature 317:230-234
(1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell
x:313-321 (1989); each of which is incorporated by reference herein in its
entirety). For example, a mutant, non-functional polynucleotide of the
invention
(or a completely unrelated DNA sequence) flanked by DNA homologous to the
endogenous polynucleotide sequence (either the coding regions or regulatory
regions of the gene) can be used, with or without a selectable marker and/or a
I S negative selectable marker, to transfect cells that express polypeptides
of the
invention i~ vivo. In another embodiment, techniques known in the art are used
to generate knockouts in cells that contain, but do not express the gene of
interest.
Insertion of the DNA construct, via targeted homologous recombination, results
in inactivation of the targeted gene. Such approaches are particularly suited
in
research and agricultural fields where modifications to embryonic stem cells
can
be used to generate animal offspring with an inactive targeted gene (see,
e.g.,
Thomas & Capecchi 1987 and Thompson I 989, ,supra). However this approach
can be routinely adapted for use in humans provided the recombinant DNA
constructs are directly administered or targeted to the required site ire vivo
using
appropriate viral vectors that will be apparent to those of skill in the art.
The
contents of each of the documents recited in this paragraph is herein
incorporated
by reference in its entirety.
In other embodiments, antagonists according to the present invention
include soluble forms of TRZ receptor (e.g., fragments of the TR2 receptors
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shown in SEQ ID N0:26, FIG. lA-1B (SEQ ID N0:2), FIG. 4A-4C (SEQ ID
NO:S) or FIG. 7A-7C (SEQ ID N0:8)) that include the ligand binding domain
from the extracellular region of the full length receptor). Such soluble forms
of
the TR2 receptor, which may be naturally occurring or synthetic, antagonize
TR2
receptor mediated signaling by competing with the cell surface bound forms
ofthe
receptor for binding to TNF-family ligands. Antagonists of the present
invention
also include antibodies specific for TNF-family ligands and TR2 receptor-Fc
fusion proteins.
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 and/or blocking the ligandlreceptor signaling pathway.
Members of the TNF ligand family include, but are not limited to, TNF-a,
lymphotoxin-a (LT-a, also known as TNF-(3), LT-~i (found in complex
heterotrimer LT-a 2-Vii), FasL, VEGI (International Publication No. WO
96114328), AIM I (International Publication No. WO 97/33899), AIM B
(International Publication No. WO 97/34911), APRIL (J. Exp. Med.
188(6):1185-1190), endokine-a (International Publication No. WO 98/07880),
neutrokine-a (International Publication No. WO 98/18921), CD40L, CD27L,
CD30L, 4-1BBL, OX40L and nerve growth factor (NGF).
TNF-a has been shown to protect mice from infection with Herpes
simplex virus type 1 (HSV-1). Rossol-Voth et al., J.Gen. Yirol. 72:143-147
( 1991 ). The mechanism of the protective effect of TNF-a is unknown but
appears
to involve neither interferons nor NK cell killing. One member of the family
has
been shown to mediate HSV-1 entry into cells. Montgomery et al., Eur. Cytokine
Newt. 7:159 (1996). Further, antibodies specifc for the extracellular domain
of
this block HSV-1 entry into cells. Thus, TR2 receptor antagonists of the
present
invention include both TR2 receptor amino acid sequences and antibodies
capable
ofpreventing mediated viral entry into cells. Such sequences and antibodies
can
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function by either competing with cell surface localized 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 standard methods using TR2 receptor immunogens of the present
invention. Such TR2 receptor immunogens include the TR2 receptor proteins
shown in SEQ ID N0:26, FIG, lA-1B (SEQ m N0:2}, FIG. 4A-4.C (SEQ 1D
NO:S) and FIG. 7A-7C (SEQ 1D N0:8) (which may or may not include a leader
sequence) and polypeptide fragments of the receptor comprising the ligand
binding, extracellular (e.g., one or more of the cysteine repeat regions),
transmembrane, the intracellular domains of TR2 receptor, or any combination
thereof.
Polyclonal and monoclonal antibody agonists or antagonists according to
the present invention can be raised according to the methods disclosed herein
and/or known in the art, such as, for example, those methods described in
Tartaglia and Goeddel, J. Biol. Chem. 267:4304-4307(1992)); Tartaglia et al.,
Cell 73:213-216 (1993)), and PCT Application WO 94/09137 (the contents of
each of these three applications are herein incorporated by reference in their
entireties), and are preferably specific to polypeptides of the invention
having the
amino acid sequence of SEQ 1D N0:2, SEQ >D NO:S, SEQ ID N0:8, or SEQ )D
N0:26.
The techniques of gene-shuffling, motif shuffling, exon-shuffling, and/or
codon-shuffling (collectively referred to as "DNA shuffling") may be employed
to modulate the activities of TR2 thereby effectively generating agonists and
antagonists of TR2. See generally, U.S. Patent Nos. 5,605,793, 5,811,238,
5,830,721, 5,834,252, and 5,837,458, and Patters, P. A., et al., Curr. Opinion
Biotechnol. 8:724-33 (1997); Harayama, S., Trends Biotechnol. 16(2):76-82
(1998); Hansson, L. O. et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo,
M.
M. and Blasco, R., BioTechniques 24(2):308-13 (1998) (each ofthese patents and
publications are hereby incorporated by reference). In one embodiment,
alteration
AMENDED SHEET
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of TR2 polynucleotides and corresponding polypeptides may be achieved by DNA
shuffling. DNA shuffling involves the assembly of two or more DNA segments
into a desired TR2 molecule by homologous, or site-specific, recombination. In
another embodiment, TR2 polynucleotides and corresponding polypeptides may
be altered by being subjected to random mutagenesis by error-prone PCR, random
nucleotide insertion or other methods prior to recombination. In another
embodiment, one or more components, motifs, sections, parts, domains,
fragments, etc., of TR2 may be recombined with one or more components, motifs,
sections, parts, domains, fragments, etc. of one or more heterologous
molecules.
In preferred embodiments, the heterologous molecules are, for example, TNF-
alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found
in complex heterotrimer LT-alpha2-beta), OPGL, Fast, CD27L, CD30L, CD40L,
4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO
96/14328), AIM I (International Publication No. WO 97/33899), AIM II
(International Publication No. WO 97/34911 ), APRIL (J Exp. Med. 188(6):1185-
1190), endokine-alpha (International Publication No. WO 98/07880), Neutrokine-
alpha (International Publication No. WO 98/18921), OPG, and neutrokine-alpha
(International Publication No. WO 98/18921, OX40, and nerve growth factor
(NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, DR3
(International Publication No. WO 97/33904), DR4 (International PublicationNo.
WO 98/32856), TRS (International Publication No. WO 98/30693), TR6
(International Publication No. WO 98/30694),TR7 (International Publication No.
WO 98/41629), TRANK, TR9 (International Publication No. WO
98/56892),TR10 (International Publication No. WO 98/54202),31X2
(International Publication No. WO 98/06842), TR12, and TNF-R1,
TRAMP/DR3/APO-3/WSL/LARD, TRAIL-R1lDR4/APO-2, TRAIL-R2/DRS,
DcRl/TRAIL-R3/TRID/LIT, DcR2/TRAIL-R4, CAD, TRAIL, TRAMP, v-FLIP.
In further preferred embodiments, the heterologous molecules are any
member of the TNF family.
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Therapeutic antl Other Uses
The Tumor Necrosis Factor (TNF) family ligands are known to be among
the most pleiotropic cytokines, inducing a large number of cellular responses,
including cytotoxicity, anti-viral activity, immunoregulatory activities, and
the
transcriptional regulation of several genes (Goeddel, D.V., et al., "Tumor
Necrosis Factors: Gene Structure and Biological Activities," Symp. Ouant.
Biol.
~ 1:597-609, Cold Spring Harbor ( 1986); Beutler, B., and Cerami, A., Ann?.r.
Rev.
Biochem. 57:505-518 (1988); Old, L.J., Sci. Am. 258:59-75 (1988); Fiers, W.,
FEBS Left. 28~ :199-224 ( 1991 )). The TNF-family ligands induce such various
cellular responses by binding to TNF-family receptors.
TR2 polynucleotides or polypeptides, or agonists of TR2, can be used in
the treatment of infectious agents. For example, by increasing the immune
response, particularly increasing the proliferation and difFerentiation of B
cells,
infectious diseases may be treated. The immune response may be increased by
either enhancing an existing immune response, or by initiating a new immune
response. Alternatively, TR2 polynucleotides or polypeptides, or agonists or
antagonists of TR2, may also directly inhibit the infectious agent, without
necessarily eliciting an immune response.
As noted above, TR2 polynucleotides and polypeptides, and anti-TR2
antibodies, are useful for diagnosis of conditions involving abnormally high
or low
expression of TR2, TR2-SV1 and/or TR2-SV2 and/or TR2, TR2-SV1 and/or
TR2-SV2 activities. Given the cells and tissues where TR2, TR2-SV1 and/or
TR2-SV2 is expressed as well as the activities modulated by TR2, TR2-SV1
and/or TR2-SV2, it is readily apparent that a substantially altered (increased
or
decreased) level of expression of TR2, TR2-SV 1 and/or TR2-SV2 in an
individual
compared to the standard or "normal" level produces pathological conditions
related to the bodily systems) in which TR2, TR2-SVl and/or TR2-SV2 is
expressed and/or is active.
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It will also be appreciated by one of ordinary skill that, since the TRZ
polypeptides of the invention are members of the TNF family, the extracellular
domains of the respective proteins may be released in soluble form from the
cells
which express TR2, TR2-SV1 and/or TR2-SV2 by proteolytic cleavage and
therefore, when TR2, TR2-SV1 and/or TR2-SV2 polypeptide (particularly a
soluble form of the respective extracellular domains) is added from an
exogenous
source to cells, tissues or the body of an individual, the polypeptide will
exert its
modulating activities on any of its target cells of that individual. Also,
cells
expressing this type II transmembrane protein may be added to cells, tissues
or the
body of an individual whereby the added cells will bind to cells expressing
receptor for TR2, TR2-SV 1 and/or TR2-SV2 whereby the cells expressing TR2,
TR2-SV1 and/or TR2-SV2 can cause actions (e.g., reduced proliferation or
cytotoxicity) on the receptor-bearing target cells.
In one embodiment, the invention provides a method of delivering
I S compositions containing the polypeptides of the invention (e.g.,
compositions
containing TR2, TR2-SVl and/or TR2-SV2 polypeptides or anti-TR2,
anti-TR2-SV1 and/or anti-TR2-SV2 antibodies associated with heterologous
polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted
cells,
such as, for example, B cells expressing a TR2, TR2-SVl and/or TR2-SV2
receptor, or monocytes expressing the cell surface bound form of TR2, TR2-SV 1
and/or TR2-SV2. TR2, TR2-SVl and/or TR2-SV2 polypeptides or anti-TR2,
anti-TR2-SV I and/or anti-TR2-SV2 antibodies ofthe invention may be associated
with heterologous polypeptides, heterologous nucleic acids, toxins, or
prodrugs
via hydrophobic, hydrophilic, ionic and/or covalent interactions.
In one embodiment, the invention provides a method for the specific
delivery of compositions of the invention to cells by administering
polypeptides
ofthe invention (e.g., TR2, TR2-SV 1 and/or TR2-SV2 polypeptides or anti-TR2,
anti-TR2-SVl and/or anti-TR2-SV2 antibodies) that are associated with
heterologous polypeptides or nucleic acids. In one example, the invention
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provides a method for delivering a therapeutic protein into the targeted cell.
In
another example, the invention provides a method for delivering a single
stranded
nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid
(e.g.,
DNA that can integrate into the cell's genome or replicate episomally and that
can
be transcribed) into the targeted cell.
In another embodiment, the invention provides a method for the specific
destruction of cells (e.g., the destruction of tumor cells) by administering
polypeptides ofthe invention (e.g., TR2, TR2-SV I and/or TR2-SV2 polypeptides
or anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies) in association with
toxins or cytotoxic prodrugs.
In a specific embodiment, the invention provides a method for the specific
destruction of cells of B cell lineage (e.g., B cell related leukemias or
lymphomas)
by administering TR2, TR2-SV1 and/or TR2-SV2 polypeptides and/or anti-TR2
antibodies in association with toxins or cytotoxic prodrugs.
By "toxin" is meant compounds that bind and activate endogenous
cytotoxic effector systems, radioisotopes, holotoxins, modified toxins,
catalytic
subunits oftoxins, cytotoxins (cytotoxic agents), or any molecules or enzymes
not
normally present in or on the surface of a cell that under defined conditions
cause
the cell's death. Toxins that may be used according to the methods of the
invention include, but are not limited to, radioisotopes known in the art,
compounds such as, for example, antibodies (or complement fixing containing
portions thereof) that bind an inherent or induced endogenous cytotoxic
effector
system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin,
Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin,
pokeweed antiviral protein, alpha-sarcin and cholera toxin. "Toxin" also
includes
a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal
ion, e.g.,
alpha-emitters such as, for example, 213Bi, or other radioisotopes such as,
for
example, 1113pd' 133Xe' 131I' 6RGe' S7CO' GSzn' SSsr' 32P' 35S' 90Y' l~3sm'
153Gd' 169p
~lCr' S4Mn' 75se' 113Sn' 9llYttrlum, 117T1n, 186~enlLlm, l6~Holmium, and
IRBRhenium;
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luminescent labels, such as luminol; and fluorescent labels, such as
fluorescein and
rhodamine, and biotin.
Techniques known in the art may be applied to label antibodies of the
invention. Such techniques include, but are not limited to, the use of
bifunctional
conjugating agents (see e.g., U.S. Patent Nos. 5,756,065; 5,714,631;
5,696,239;
5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119;
4,994,560; and 5,808,003; the contents of each of which are hereby
incorporated
by reference in its entirety). A cytotoxin or cytotoxic agent includes any
agent
that is detrimental to cells. Examples include paclitaxol, cytochalasin B,
gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin
dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,
glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin
and
analogs or homologs thereof. Therapeutic agents include, but are not limited
to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,
cytarabine,
5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C,
cis-dichlorodiamine platinum (II) (DDP), cisplatin, anthracyclines (e.g.,
daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g.,
dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
By "cytotoxic prodrug" is meant a non-toxic compound that is converted
by an enzyme, normally present in the cell, into a cytotoxic compound.
Cytotoxic
prodrugs that may be used according to the methods of the invention include,
but
are not limited to, glutamyl derivatives of benzoic acid mustard alkylating
agent,
phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,
daunorubisin, and phenoxyacetamide derivatives of doxorubicin.
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It will be appreciated that conditions caused by a decrease in the standard
or normal level of TR2, TR2-SV 1 and/or TR2-SV2 activity in an individual,
particularly disorders of the immune system, can be treated by administration
of
TR2, TR2-SV 1 and/or TR2-SV2 polypeptide (in the form of soluble extracellular
domain or cells expressing the complete protein) or agonist. Thus, the
invention
also provides a method of treatment of an individual in need of an increased
level
of TR2, TR2-SV1 and/or TR2-SV2 activity comprising administering to such an
individual a pharmaceutical composition comprising an amount of an isolated
TR2, TR2-SV 1 and/or TR2-SV2 polypeptide ofthe invention, or agonist thereof,
effective to increase the TR2, TR2-SV1 and/or TR2-SV2 activity level in such
an
individual.
It will also be appreciated that conditions caused by a increase in the
standard or normal level of TR2, TR2-SVI and/or TR2-SV2 activity in an
individual, particularly disorders of the immune system, can be treated by
IS administration of TR2, TR2-SVI and/or TR2-SV2 polypeptides (in the form of
soluble extracellular domain or cells expressing the complete protein) or
antagonist (e.g., an anti-TR2 antibody). Thus, the invention also provides a
method of treatment of an individual in need of an decreased level of TR2,
TR2-SV I and/or TR2-SV2 activity comprising administering to such an
individual
a pharmaceutical composition comprising an amount of an isolated TR2,
TR2-SVI and/or TR2-SV2 polypeptide of the invention, or antagonist thereof,
effective to decrease the TR2, TR2-SV1 and/or TR2-SV2 activity level in such
an individual.
TR2 polynucleotides or polypeptides of the invention, or agonists or
antagonists of TR2 can be used in the treatment of infectious agents. For
example, by increasing the immune response, particularly increasing the
proliferation and dif~'erentiation of B cells, infectious diseases may be
treated. The
immune response may be increased by either enhancing an existing immune
response, or by initiating a new immune response. Alternatively, TR2
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polynucleotides or polypeptides, or agonists of TR2 may also directly inhibit
the
infectious agent, without necessarily eliciting an immune response.
Viruses are one example of an infectious agent that can cause disease or
symptoms that can be treated by TR2, TR2-S V 1 and/or TR2-SV2 polynucleotides
or polypeptides, or agonists of TR2, TR2-SV1 and/or TR2-SV2. Examples of
viruses, include, but are not limited to the following DNA and RNA viruses and
viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus,
Birnaviridae,
Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV,
Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as,
Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g.,
Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g.,
Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae,
Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia),
Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and
Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause
a
variety of diseases or symptoms, including, but not limited to: arthritis,
bronchiollitis, respiratory syncytial virus, encephalitis, eye infections
(e.g.,
conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E,
Chronic
Active, Delta), Japanese B encephalitis. Junin, Chikungunya, Rift Valley
fever,
yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia,
Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,
Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually
transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. TR2,
TR2-SVl and/or TR2-SV2 polynucleotides or polypeptides, or agonists or
antagonists of TR2, TR2-SVl and/or TR2-SV2, can be used to treat, prevent,
diagnose, and/or detect any of these symptoms or diseases. In specific
embodiments, TR2, TR2-SV 1 and/or TR2-SV2 polynucleotides, polypeptides, or
agonists are used to treat, prevent, and/or diagnose: meningitis, Dengue, EBV,
and/or hepatitis (e.g., hepatitis B). In an additional specific embodiment
TR2,
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TR2-SV 1 and/or TR2-SV2 polynucleotides, polypeptides, or agonists are used to
treat patients nonresponsive to one or more other commercially available
hepatitis
vaccines. In a further specific embodiment, TR2, TR2-SVI and/or TR2-SV2
polynucleotides, polypeptides, or agonists are used to treat, prevent, and/or
diagnose AIDS. In an additional specific embodiment TR2, TR2-SV1 and/or
TR2-S V2 receptor polynucleotides, polypeptides, agonists, and/or antagonists
are
used to treat, prevent, and/or diagnose patients with cryptosporidiosis.
Similarly, bacterial or fungal agents that can cause disease or symptoms
and that can be treated by TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or
polypeptides, or agonists or antagonists of TR2, TR2-SVI and/or TR2-SV2,
include, but not limited to, the following Gram-Negative and Gram-positive
bacteria and bacterial families and fungi: Actinomycetales (e.~ ,
Corynebacteriunr,
Mycobacterium, Nnrcardia), Cryplococcus neofornrans, Aspergillosis,
Bacillaceae (e.g., Anthr°ax, Clostridium), Bacteroidaceae,
Blaslonrycosis,
I S Bordetella, Borrelia (e.g., Borrelia burgdorferi, Brucellosi.s,
Candidiasis,
C.'crnrpylobacter, Coccidioidonrycosis, Cryptococcosis, Dermatocycose.s, E.
tote
(e.g., EnterotoxigenicE. tote and EnterohemorrhagicE. tote),
Errterobacter°iaceae
(Klebsiella, Salmonella (e.g., Salmonella yphi and Salmonella paratyphi),
S'er°ratia, Yer.sinia), Erysipelolhrix, Helicobacter, Legionello.sis,
Leplo.spir°osis,
Li.steria (e.g., Listeria monocyfogenes), Mycoplasmatales, Mycobacterium
leprae, T~ibr°io cholerae, Neisseriaceae (e.g., Acinetobacter,
Gonorrhea,
Menigococcal), Meisseria meningitides, Pasteurellacea Infections (e.g.,
Actinobacillrrs, Heanrophilu.s (e.g., Heanrophilus ir~uenza type B),
Pasterrrella),
Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp.,
Staphylococcal, Meningiococcal, Pneunrococcal and Str°eptococcal
(e.g.,
Streptococcus pnerrnroniae and Group B Streptococcus). These bacterial or
fungal families can cause the following diseases or symptoms, including, but
not
limited to: bacteremia, endocarditis, eye infections (conjunctivitis,
tuberculosis,
uveitis), gingivitis, opportunistic infections (e.g., AIDS related
infections),
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paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract
infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,
Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid,
pneumonia, Gonorrhea, meningitis (e.g., mengitis types A and B), Chlamydia,
Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus,
Botulism,
gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually
transmitted
diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary
tract
infections, wound infections. TR2, TR2-SV1 and/or TR2-SV2 polynucleotides
or polypeptides, or agonists or antagonists of TR2, TR2-SV1 and/or TR2-SV2,
can be used to treat, prevent, diagnose, and/or detect any of these symptoms
or
diseases. In specific embodiments, TRZ polynucleotides, polypeptides, or
agonists
thereof are used to treat, prevent, and/or diagnose: tetanus, Diphtheria,
botulism,
and/or meningitis type B.
Moreover, parasitic agents causing disease or symptoms that can be
treated by TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides, or
agonists of TR2, TR2-SV1 and/or TR2-SV2, include, but not limited to, the
following families or class: Amebiasis, Babesiosis, Coccidiosis,
Cryptosporidiosis,
Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis,
Leishmaniasis,
Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans
(e.g., Plasmodium virax, Plasmodium falciparium, Plasn2odiuna malariae and
Plasmodium ovate). These parasites can cause a variety of diseases or
symptoms,
including, but not limited to: Scabies, Trombiculiasis, eye infections,
intestinal
disease (e.g., dysentery, giardiasis), liver disease, lung disease,
opportunistic
infections (e.g., AIDS related), malaria, pregnancy complications, and
toxoplasmosis. TR2, TR2-SV I and/or TR2-SV2 polynucleotides or polypeptides,
or agonists or antagonists of TR2, TR2-SV1 and/or TR2-SV2, can be used to
treat, prevent, diagnose, and/or detect any of these symptoms or diseases. In
specific embodiments, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides,
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polypeptides, or agonists thereof are used to treat, prevent, and/or diagnose
malaria.
TR2 receptor polynucleotides, polypeptides, agonists or antagonists ofthe
invention may be used in developing treatments for any disorder mediated
(directly
or indirectly) by defective, or insufficient amounts ofTR2. TR2, TR2-SV I
and/or
TR2-SV2 receptor polypeptides, agonists or antagonists may be administered to
a patient (e.g., mammal, preferably human) afflicted with such a disorder.
Alternatively, a gene therapy approach may be applied to treat such disorders.
Disclosure herein of TR2 receptor nucleotide sequences permits the detection
of
defective TR2 receptor genes, and the replacement thereof with normal TR2
receptor-encoding genes. Defective genes may be detected in in vitro
diagnostic
assays, and by comparison of the TR2 receptor nucleotide sequence disclosed
herein with that of a TR2 receptor gene derived from a patient suspected of
harboring a defect in this gene.
In another embodiment, the polypeptides of the present invention are used
as a research tool for studying the biological effects that result from
inhibiting
AIM II/TRZ receptor and/or lymphotoxin-a/TR2 receptor interactions on
different
cell types. TR2 receptor polypeptides also may be employed in in vita°o
assays for
detecting AIM II, lymphotoxin-a or TRZ receptor or the interactions thereof.
In another embodiment, a purified TR2 receptor polypeptide or antagonist
is used to inhibit binding of AIM-II or lymphotoxin-a to endogenous cell
surface
AIM II and/or lymphotoxin-a receptors. Certain ligands of the TNF family (of
which AIM II and lymphotoxin-a are members) have been reported to bind to
more than one distinct cell surface receptor protein. AIM II and lymphotoxin-a
likewise are believed to bind multiple cell surface proteins. By binding AIM
II
and/or lymphotoxin-a, soluble TR2 receptor polypeptides of the present
invention
may be employed to inhibit the binding of AIM II and/or lymphotoxin-a not only
to cell surface TR2 receptor, but also to AIM II and/or lymphotoxin-a receptor
proteins that are distinct from TR2 receptor. Thus, in another embodiment, TR2
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receptor polynucleotides, polypeptides, agonists or antagonists are used to
inhibit
a biological activity of AIM II and/or lymphotoxin-a, in in vitro or in vivo
procedures. By inhibiting binding of AIM II and/or lymphotoxin-a to cell
surface
receptors, TR2 receptor polynucleotides, polypeptides, agonists or antagonists
also inhibit biological effects that result from the binding of AIM II and/or
lymphotoxin-a to endogenous receptors. Various forms of TR2 receptor may be
employed, including, for example, the above-described TR2 receptor fragments,
derivatives, and variants that are capable ofbinding AIM II and/or lymphotoxin-
a.
In one preferred embodiment, a soluble TR2 receptor polypeptide is employed to
I 0 inhibit a biological activity of AIM-II (e.g.. to inhibit AIM II-mediated
apoptosis
of cells susceptible to such apoptosis). In another preferred embodiment, a
soluble TR2 receptor polypeptide is employed to inhibit a biological activity
of
lymphotoxin-a (e.g., induction of inflammation and immune responses,
maintenance of lymphoid tissues, induction of B cell proliferation).
In a further embodiment, a TR2 receptor polynucleotide, polypeptide,
agonist or antagonist is administered to a mammal (e.g., a human) to treat a
AIM II-mediated and/or lymphotoxin-a mediated disorder. Such
AIM II-mediated and/or lymphotoxin-a mediated disorders include conditions
caused (directly or indirectly) or exacerbated by AIM II and/or lymphotoxin-a.
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, including, but not limited to colon
cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,
glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach
cancer,
neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma,
osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate
cancer, Karposi's sarcoma and ovarian cancer); autoimmune disorders (such as,
multiple sclerosis, Sjogren's syndrome, Grave's disease, Hashimoto's
thyroiditis,
autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn's disease,
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polymyositis, systemic lupus erythematosus and immune-related
glomerulonephritis, autoimmune gastritis, autoimmune thrombocytopenic purpura,
and rheumatoid arthritis) and viral infections (such as herpes viruses, pox
viruses
and adenoviruses), inflammation, graft vs. host disease (acute and/or
chronic),
S acute graft rejection, and chronic graft rejection. In preferred
embodiments, TR2
receptor polynucleotides, polypeptides, agonists, or antagonists of the
invention
are used to inhibit growth, progression, and/or metastasis of cancers, in
particular
those listed above or in the paragraph that follows.
Additional diseases or conditions associated with increased cell survival
include, but are not limited to, progression, and/or metastases ofmalignancies
and
related disorders such as leukemia (including acute leukemias (e.g., acute
lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic,
promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic
leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic
lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease
and non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, heavy chain disease, and solid tumors including, but not
limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate
cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat
gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic
carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma,
seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular
tumor,
lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
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ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis include AIDS;
neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease,
S Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration
and
brain tumor or prior associated disease); autoimmune disorders (such as,
multiple
sclerosis, Sjogren's syndrome, Grave's disease Hashimoto's thyroiditis,
autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn's disease,
polymyositis, systemic lupus erythematosus, immune-related glomerulonephritis,
autoimmune gastritis, thrombocytopenic purpura, and rheumatoid arthritis)
myelodysplastic syndromes (such as aplastic anemia), graft vs. host disease
(acute
and/or chronic), ischemic injury (such as that caused by myocardial
infarction,
stroke and reperfusion injury), liver injury or disease (e.g., hepatitis
related liver
injury, cirrhosis, ischemia/reperfusion injury, cholestosis (bile duct injury)
and liver
cancer); toxin-induced liver disease (such as that caused by alcohol), septic
shock,
ulcerative colitis, cachexia and anorexia. In preferred embodiments, TR2
receptor
polynucleotides, polypeptides, agonists, and/or antagonists are used to treat
the
diseases and disorders listed above.
Many of the pathologies associated with HIV are mediated by apoptosis,
including HIV-induced nephropathy and HIV encephalitis. Thus, in additional
preferred embodiments, TR2 receptor polynucleotides, polypeptides, agonists or
antagonists of the invention are used to treat AIDS and pathologies associated
with AIDS.
Another embodiment of the present invention is directed to the use of TR2
receptor polynucleotides, polypeptides, agonists or antagonists to reduce
AIM II-mediated death of T cells in HIV-infected patients. The role of T cell
apoptosis in the development of AIDS has been the subject of a number of
studies
(see, for example, Meyaard et al., Science 257:217-219 (1992); Groux et al.,
J.
Exp. Med. 175:331 (1992); and Oyaizu et al., in "Cell Activation andApoptosis
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in HIU Infection, " Andrieu and Lu, eds., Plenum Press, New York, pp.
101-114(1995). Fas-mediated apoptosis has been implicated in the loss of T
cells
in HIV individuals (Katsikis et al., J. Exp. Med. 181:2029-2036 (1995). It is
also
likely that T cell apoptosis occurs through multiple mechanisms. For example,
at
least some of the T cell death seen in HIV patients is likely to be mediated
by
AIM II.
Activated human T cells are induced to undergo programmed cell death
(apoptosis) upon triggering through the CD3/T cell receptor complex, a process
termed activated-induced cell death (AICD). AICD of CD4 T cells isolated from
HIV-Infected asymptomatic individuals has been reported (Groux et al.,
sups°a).
Thus, AICD may play a role in the depletion of CD4+ T cells and the
progression
to AIDS in HIV-infected individuals. Thus, the present invention provides a
method of inhibiting AIM II-mediated T cell death in HIV patients, comprising
administering a TR2 receptor polynucleotides, polypeptides, agonists or
antagonists of the invention (preferably, a soluble TR2 receptor polypeptide)
to
the patients. In one embodiment, the patient is asymptomatic when treatment
with
TR2 receptor polynucleotides, polypeptides, agonists or antagonists commences.
If desired, prior to treatment, peripheral blood T cells may be extracted from
an
HIV patient, and tested for susceptibility to AIM II-mediated cell death by
procedures known in the art. In one embodiment, a patient's blood or plasma is
contacted with TR2 receptor polypeptides of the invention ex vivo. The TR2
receptor polypeptides may be bound to a suitable chromatography matrix by
procedures known in the art. The patient's blood or plasma flows through a
chromatography column containing TR2 receptor polypeptide bound to the
matrix, before being returned to the patient. The immobilized TR2 receptor
polypeptide binds AIM II, thus removing AIM-II protein from the patient's
blood.
In additional embodiments a TR2 receptor polynucleotide, polypeptide,
agonist or antagonist of the invention is administered in combination with
other
inhibitors of T cell apoptosis. For example, as discussed above, Fas-mediated
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apoptosis also has been implicated in loss of T cells in HIV individuals
(Katsikis
et al., .I Exp. Med. 181:2029-2036 (1995). Thus, a patient susceptible to both
Fas ligand mediated and AIM II mediated T cell death may be treated with both
an agent that blocks AIM II/AIM II receptor interactions and an agent that
blocks
S Fas-ligand/Fas interactions. Suitable agents for blocking binding of Fas-
ligand to
Fas include, but are not limited to, soluble Fas polypeptides; multimeric
forms of
soluble Fas polypeptides (e.g., dimers of sFas/Fc); anti-Fas antibodies that
bind
Fas without transducing the biological signal that results in apoptosis;
anti-Fas-ligand antibodies that block binding of Fas-ligand to Fas; and
muteins of
Fas-ligand that bind Fas but do not transduce the biological signal that
results in
apoptosis. Preferably, the antibodies employed according to this method are
monoclonal antibodies. Examples of suitable agents for blocking Fas-ligand/Fas
interactions, including blocking anti-Fas monoclonal antibodies, are described
in
WO 95/10540, hereby incorporated by reference.
In another example, agents which block binding of TRAIL to a TRAIL
receptor are administered with the TR2 receptor polynucleotides, polypeptides,
agonists or antagonists of the invention. Such agents include, but are not
limited
to, soluble TRAIL receptor polypeptides (e.g., a soluble form of OPG, DR4 (WO
98/32856); TRS (WO 98/30693); DRS (WO 98/41629); and TR10 (WO
98/54202)); multimeric forms of soluble TRAIL receptor polypeptides; and
TRAIL receptor antibodies that bind the TRAIL receptor without transducing the
biological signal that results in apoptosis, anti-TRAIL antibodies that block
binding of TRAIL to one or more TRAIL receptors, and muteins of TRAIL that
bind TRAIL receptors but do not transduce the biological signal that results
in
apoptosis. Preferably, the antibodies employed according to this method are
monoclonal antibodies.
Another embodiment of the present invention is directed to the use of TR2
as a regulator of B cell proliferation and differentiation. The assays and
experiments described herein clearly provide the scientific rational for the
use of
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TR2 as a regulator of B cell proliferation and dif~'erentiation. The possible
uses
of the soluble or membrane bound TR2, its native ligand and various ligand
antagonists are diverse and include treatment of autoimmune disorders and
immunodeficiencies resulting from infection, anti-neoplastic therapy and/or
inherited disorders. Moreover, many of the pre-neoplastic monoclonal
gammopathies and neoplastic B cell diseases such as multiple myeloma may
utilize
TR2 or its ligand as either inducing or progressing factors.
Accordingly, TR2 or derived, functional agonists (including anti-TR2
antibodies, soluble forms having amino acids sequences contained in the
extracellular domain of TRZ (e.g., TR2-Fc) and TR2 ligands), may find
application as the following:
As an agent to direct an individuals immune system towards development
of a humoral response (i.e., TH2) as opposed to a THl cellular response.
As an antigen for the generation of antibodies to inhibit or enhance TRZ
1 S mediated responses.
As a means of activating T cells.
As a means of regulating secreted cytokines that are elicited by TR2.
Antagonists of TR2 include binding and/or inhibitory antibodies, antisense
nucleic acids, ribozymes, soluble forms of TR2 (e.g., TR2-fc) and TR2
ligand(s).
These would be expected to reverse many of the activities of the receptor
described above as well as find clinical or practical application as:
Antagonists of TRZ activities can also be used to treat or prevent Herpes
viral infections. Such antagonists include full-length and mature TR2
polypeptides
of the invention, TR2 fragments (e.g., soluble fragments), and antibodies
having
specificity for TR2 polypeptides. While not wishing to be limited to a
specific
mechanism, TR2 antagonist are believed to function in the treatment or
prevention
Herpes viral infections by blocking Herpes viral entry into cells.
An additional condition, disease or symptom that can be treated by TR2
polynucleotides or polypeptides, or agonists of TR2, is osteomyelitis.
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Preferably, treatment using TR2 polynucleotides or polypeptides, or
agonists of TR2, could either be by administering an ef~'ective amount of TR2
polypeptide to the patient, or by removing cells from the patient, supplying
the
cells with TR2 polynucleotide, and returning the engineered cells to the
patient (ex
vivo therapy). Moreover, as further discussed herein, the TR2 polypeptides or
polynucleotides can be used as an adjuvant in a vaccine to raise an immune
response against infectious disease.
In another embodiment, TR2, TRZ-SV I and/or TR2-SV2 polynucleotides
or polypeptides of the invention and/or agonists and/or antagonists thereof,
are
used to treat, prevent, and/or diagnose inner ear infection (such as, for
example,
otitis media), as well as other infections characterized by infection with
Stneplococcz~s pnezrmoniae and other pathogenic organisms.
In a specific embodiment, TR2, TR2-SV 1 and/or TR2-SV2
polynucleotides or polypeptides, or agonists or antagonists thereof (e.g. ,
anti-TR2,
anti-TR2-SV I and/or anti-TR2-SV2 antibodies) are used to treat or prevent a
disorder characterized by deficient serum immunoglobulin production, recurrent
infections, and/or immune system dysfunction. Moreover, TR2, TR2-S V I and/or
TR2-SV2 polynucleotides or polypeptides, or agonists or antagonists thereof
(e.g., anti-TR2, anti-TR2-SV 1 and/or anti-TR2-SV2 antibodies) may be used to
treat or prevent infections of the joints, bones, skin, and/or parotid glands,
blood-borne infections (e.g., sepsis, meningitis, septic arthritis, and/or
osteomyelitis), autoimmune diseases (e.g., those disclosed herein),
inflammatory
disorders, and malignancies, and/or any disease or disorder or condition
associated
with these infections, diseases, disorders and/or malignancies) including, but
not
limited to, CVID, other primary immune deficiencies, HIV disease, CLL,
recurrent
bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis,
meningitis,
herpes zoster (e.g., severe herpes zoster), and/or pheumocystis carnii.
TR2, TR2-SVl and/or TR2-SV2 polynucleotides or polypeptides ofthe
invention, or agonists or antagonists thereof, may be used to diagnose,
prognose,
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treat or prevent one or more of the following diseases or disorders, or
conditions
associated therewith: primary immuodeficiencies, immune-mediated
thrombocytopenia, Kawasaki syndrome, bone marrow transplant (e.g., recent
bone marrow transplant in adults or children), chronic B-cell lymphocytic
leukemia, HIV infection (e.g., adult or pediatric HIV infection), chronic
inflammatory demyelinating polyneuropathy, and post-transfusion purpura.
Additionally, TR2, TR2-SV 1 and/or TR2-SV2 polynucleotides or
polypeptides of the invention, or agonists or antagonists thereof, may be used
to
diagnose, prognose, treat or prevent one or more of the following diseases,
disorders, or conditions associated therewith, Guillain-Barre syndrome, anemia
(e.g., anemia associated with parvovirus B19, patients with stable mutliple
myelomawho are at high risk for infection (e.g., recurrent infection),
autoimmune
hemolytic anemia (e.g., warm-type autoimmune hemolytic anemia),
thrombocytopenia (e.g., neonatal thrombocytopenia), and immune-mediated
neutropenia), transplantation (e.g., cytamegalovirus (CMV)-negative recipients
of CMV-positive organs), hypogammaglobulinemia (e.g.,
hypogammaglobulinemic neonates with risk factor for infection or morbidity),
epilepsy (e.g., intractable epilepsy), systemic vasculitic syndromes,
myasthenia
gravis (e.g., decompensation in myasthenia gravis), dermatomyositis, and
polymyositis.
Additional preferred embodiments of the invention include, but are not
limited to, the use of TR2, TR2-SV 1 and/or TR2-SV2 polypeptides, TR2,
TR2-SV I and/or TR2-SV2 polynucleotides, and functional agonists thereof, in
the
following applications:
Administration to an animal (e.g., mouse, rat, rabbit, hamster, guinea pig,
pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human
primate, and human, most preferably human) to boost the immune system to
produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgM,
and
IgE), to induce higher affinity antibody production (e.g., IgG, IgA, IgM, and
IgE),
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and/or to increase an immune response. In a specific nonexclusive embodiment,
TR2, TR2-SVl and/or TR2-SV2 polypeptides of the invention, and/or agonists
thereof, are administered to boost the immune system to produce increased
quantities of IgG. In another specific nonexclusive embodiment, TR2, TR2-SV 1
and/or TR2-SV2 polypeptides of the invention and/or agonists thereof, are
administered to boost the immune system to produce increased quantities of
IgA.
In another specific nonexclusive embodiment, TR2, TR2-SVl and/or TR2-SV2
polypeptides of the invention and/or agonists thereof, are administered to
boost
the immune system to produce increased quantities of IgM.
Administration to an animal (including, but not limited to, those listed
above, and also including transgenic animals) incapable of producing
functional
endogenous antibody molecules or having an otherwise compromised endogenous
immune system, but which is capable of producing human immunoglobulin
molecules by means of a reconstituted or partially reconstituted immune system
from another animal (see, e.g., published PCT Application Nos. W098/24893,
WO/9634096, WO/9633735, and WO/9110741).
A vaccine adjuvant that enhances immune responsiveness to specific
antigen. In a specific embodiment, the vaccine adjuvant is a TR2, TR2-SVl
and/or TR2-SV2 polypeptide described herein. In another specific embodiment,
the vaccine adjuvant is a TR2, TR2-SV 1 and/or TR2-SV2 polynucleotide
described herein (i.e., the TR2, TR2-SVl and/or TR2-SV2 polynucleotide is a
genetic vaccine adjuvant). As discussed herein, TR2, TR2-SV 1 and/or TR2-SV2
polynucleotides may be administered using techniques known in the art,
including
but not limited to, liposomal delivery, recombinant vector delivery, injection
of
naked DNA, and gene gun delivery.
An adjuvant to enhance tumor-specific immune responses.
An adjuvant to enhance anti-viral immune responses. Anti-viral immune
responses that may be enhanced using the compositions of the invention as an
adjuvant, include, but are not limited to, virus and virus associated diseases
or
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symptoms described herein or otherwise known in the art. In specific
embodiments, the compositions of the invention are used as an adjuvant to
enhance an immune response to a virus, disease, or symptom selected from the
group consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g.,
hepatitis
B). In another specific embodiment, the compositions of the invention are used
as an adjuvant to enhance an immune response to a virus, disease, or symptom
selected from the group consisting of HIV/AIDS, Respiratory syncytial virus,
Dengue, Rotavirus, Japanese B encephalitis, Influenza A and B, Parainfluenza,
Measles, Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever,
Herpes
simplex, and yellow fever. In another specific embodiment, the compositions of
the invention are used as an adjuvant to enhance an immune response to the HIV
gp I 20 antigen.
An adjuvant to enhance anti-bacterial or anti-fungal immune responses.
Anti-bacterial or anti-fungal immune responses that may be enhanced using the
compositions of the invention as an adjuvant, include bacteria or fungus and
bacteria or fungus associated diseases or symptoms described herein or
otherwise
known in the art. In specific embodiments, the compositions of the invention
are
used as an adjuvant to enhance an immune response to a bacteria or fungus,
disease, or symptom selected from the group consisting of: tetanus,
Diphthe~°ia,
botulism, and meningitis type B. In another specific embodiment, the
compositions of the invention are used as an adjuvant to enhance an immune
response to a bacteria or fungus, disease, or symptom selected from the group
consisting o~ vibrio cholerae, Mycohacterinn~ leprae, Salmonella typhi,
Salmonellaparatyphi, Meisseriameningitidis, Streptococcuspneumoniae, Group
B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli,
Enlerohemorrhagic E. coli, Borrelia burgdo~feri, and Plasmodium (malaria).
An adjuvant to enhance anti-parasitic immune responses. Anti-parasitic
immune responses that may be enhanced using the compositions of the invention
as an adjuvant, include parasite and parasite associated diseases or symptoms
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described herein or otherwise known in the art. In specific embodiments, the
compositions of the invention are used as an adjuvant to enhance an immune
response to a parasite. In another specific embodiment, the compositions of
the
invention are used as an adjuvant to enhance an immune response to Plasmodium
(malaria).
As a stimulator of B cell responsiveness to pathogens.
As an agent that elevates the immune status of an individual prior to their
receipt of immunosuppressive therapies.
As an agent to induce higher affinity antibodies.
As an agent to increase serum immunoglobulin concentrations.
As an agent to accelerate recovery of immunocompromised individuals.
As an agent to boost immunoresponsiveness among aged populations.
As an immune system enhancer prior to, during, or after bone marrow
transplant and/or other transplants (e.g., allogeneic or xenogeneic organ
transplantation). With respect to transplantation, compositions of the
invention
may be administered prior to, concomitant with, and/or after transplantation.
In
a specific embodiment, compositions of the invention are administered after
transplantation, prior to the beginning of recovery of T-cell populations. In
another specific embodiment, compositions ofthe invention are first
administered
after transplantation after the beginning of recovery of T cell populations,
but
prior to full recovery of B cell populations.
As an agent to boost immunoresponsiveness among B cell
immunodeficient individuals, such as, for example, an individual who has
undergone a partial or complete splenectomy. B cell immunodeficiencies that
may
be ameliorated or treated by administering the TR2, TR2-SV1 and/or TR2-SV2
polypeptides or polynucleotides of the invention, or agonists thereof,
include, but
are not limited to, severe combined immunodeficiency (SCID)-X linked,
SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked
agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia,
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X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult
onset agammaglobulinemia, late-onset agammaglobulinemia,
dysgammaglobulinemia, hypogammaglobulinemia, transient
hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia,
agammaglobulinemia, common variable immunodeficiency (CVID) (acquired),
Wiskott-Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IgM,
non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG
subclass deficiency (with or without IgA deficiency), antibody deficiency with
normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain
deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD),
selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type),
reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia,
thymic
alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-
telangiectasia,
short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof
syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase
deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and
severe combined immunodeficiency.
As an agent to boost immunoresponsiveness among individuals having an
acquired loss of B cell function. Conditions resulting in an acquired loss of
B cell
function that may be ameliorated or treated by administering the TR2, TR2-SV 1
and/or TR2-SV2 polypeptides or polynucleotides of the invention, or agonists
thereof, include, but are not limited to, HIV Infection, AIDS, bone marrow
transplant, and B cell chronic lymphocytic leukemia (CLL).
As an agent to boost immunoresponsiveness among individuals having a
temporary immune deficiency. Conditions resulting in a temporary immune
deficiency that may be ameliorated or treated by administering the TR2, TR2-SV
1
and/or TRZ-SV2 polypeptides or polynucleotides of the invention, or agonists
thereof, include, but are not limited to, recovery from viral infections
(e.g.,
influenza), conditions associated with malnutrition, recovery from infectious
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mononucleosis, or conditions associated with stress, recovery from measles,
recovery from blood transfusion, recovery from surgery.
As a regulator of antigen presentation by monocytes, dendritic cells, and/or
B-cells. In one embodiment, TR2, TR2-SVl and/or TR2-SV2 polypeptides (in
soluble, membrane-bound or transmembrane forms) or polynucleotides enhance
antigen presentation or antagonize antigen presentation in vitro or in vivo.
Moreover, in related embodiments, this enhancement or antagonization of
antigen
presentation may be useful in anti-tumor treatment or to modulate the immune
system.
As a mediator of mucosal immune responses. The expression of TR2 by
monocytes and the responsiveness ofB cells to this factor suggests that it may
be
involved in exchange of signals between B cells and monocytes or their
differentiated progeny. This activity is in many ways analogous to the
CD40-CD154 signaling between B cells and T cells. TR2 may therefore be an
important regulator of T cell independent immune responses to environmental
pathogens. In particular, the unconventional B cell populations (CDS+) that
are
associated with mucosal sites and responsible for much of the innate immunity
in
humans may respond to TR2 thereby enhancing an individual's protective immune
status.
As a means to induce tumor proliferation and thus make it more
susceptible to anti-neoplastic agents. For example, multiple myeloma is a
slowly
dividing disease and is thus refractory to virtually all anti-neoplastic
regimens. If
these cells were forced to proliferate more rapidly their susceptibility
profile would
likely change.
As a B cell specific binding protein to which specific activators or
inhibitors of cell growth may be attached. The result would be to focus the
activity of such activators or inhibitors onto normal, diseased, or neoplastic
B cell
populations.
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As a means of detecting B-lineage cells by virtue of its specificity. This
application may require labeling the protein with biotin or other agents
(e.g., as
described herein) to afford a means of detection.
As a stimulator of B cell production in pathologies such as AIDS, chronic
lymphocyte disorder and/or Common Variable Immunodificiency.
As part of a B cell selection device the function of which is to isolate B
cells from a heterogenous mixture of cell types. TR2 could be coupled to a
solid
support to which B cells would then specifically bind. Unbound cells would be
washed out and the bound cells subsequently eluted. A nonlimiting use of this
selection would be to allow purging of tumor cells from, for example, bone
marrow or peripheral blood prior to transplant.
As a therapy for generation and/or regeneration of lymphoid tissues
following surgery, trauma or genetic defect.
As a gene-based therapy for genetically inherited disorders resulting in
immuno-incompetence such as observed among SCID patients.
As an antigen for the generation of antibodies to inhibit or enhance TR2,
TR2-SV1 and/or TR2-SV2 mediated responses.
As a means of activating monocytes/macrophages to defend against
parasitic diseases that effect monocytes such as Le.shmania.
As pretreatment of bone marrow samples prior to transplant. Such
treatment would increase B cell representation and thus accelerate recover.
As a means of regulating secreted cytokines that are elicited by TR2,
TR2-SV1 and/or TR2-SV2.
TR2, TR2-SVI and/or TRZ-SV2 polypeptides or polynucleotides of the
invention, or agonists may be used to modulate IgE concentrations in vitro or
in
vwo.
Additionally, TR2, TRZ-SVl and/or TR2-SV2 polypeptides or
polynucleotides of the invention, or agonists thereof, may be used to treat,
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prevent, and/or diagnose IgE-mediated allergic reactions. Such allergic
reactions
include, but are not limited to, asthma, rhinitis, and eczema.
In a specific embodiment, TR2, TR2-SV1 and/or TR2-SV2 polypeptides
or polynucleotides of the invention, or agonists thereof, is administered to
treat,
prevent, diagnose, and/or ameliorate selective IgA deficiency.
In another specific embodiment, TR2, TR2-SV 1 and/or TR2-SV2
polypeptides or polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, diagnose, and/or ameliorate ataxia-
telangiectasia.
In another specific embodiment, TR2, TR2-SV1 and/or TR2-SV2
polypeptides or polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, diagnose, and/or ameliorate common variable
immunodeficiency.
In another specific embodiment, TR2, TR2-SVl and/or TR2-SV2
polypeptides or polynucleotides of the invention, or agonists thereof, is
1 S administered to treat, prevent, diagnose, and/or ameliorate X-linked
agammaglobulinemia.
In another specific embodiment, TR2, TR2-SV 1 and/or TR2-SV2
polypeptides or polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, diagnose, and/or ameliorate severe combined
immunodeficiency (SCID).
In another specific embodiment, TR2, TR2-SVl and/or TR2-SV2
polypeptides or polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, diagnose, and/or ameliorate Wiskott-Aldrich
syndrome.
In another specific embodiment, TR2, TR2-SVl and/or TR2-SV2
polypeptides or polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, diagnose, and/or ameliorate X-linked Ig
deficiency
with hyper IgM.
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In another specific embodiment, TR2, TR2-SV1 and/or TR2-SV2
polypeptides or polynucleotides ofthe invention, or agonists or antagonists
(e.g.,
anti-TR2 antibodies) thereof, is administered to treat, prevent, and/or
diagnose
chronic myelogenous leukemia, acute myelogenous leukemia, leukemia,
S hystiocytic leukemia, monocytic leukemia (e.g., acute monocytic leukemia),
leukemic reticulosis, Shilling Type monocytic leukemia, and/or other leukemias
derived from monocytes and/or monocytic cells and/or tissues.
In another specific embodiment, TR2, TR2-SV1 and/or TR2-SV2
polypeptides or polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, diagnose, and/or ameliorate monocytic
leukemoid
reaction, as seen, for example, with tuberculosis.
In another specific embodiment, TR2, TR2-SV1 and/or TR2-SV2
polypeptides or polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, diagnose, and/or ameliorate monocytic
leukocytosis, monocytic leukopenia, monocytopenia, and/or monocytosis.
In a specific embodiment, TR2, TR2-SVI and/or TR2-SV2
polynucleotides or polypeptides of the invention, and/or anti-TR2 antibodies
and/or agonists or antagonists thereof, are used to treat, prevent, detect,
and/or
diagnose primary B lymphocyte disorders and/or diseases, and/or conditions
associated therewith. In one embodiment, such primary B lymphocyte disorders,
diseases, and/or conditions are characterized by a complete or partial loss of
humoral immunity. Primary B lymphocyte disorders, diseases, and/or conditions
associated therewith that are characterized by a complete or partial loss of
humoral immunity and that may be prevented, treated, detected and/or diagnosed
with compositions of the invention include, but are not limited to, X-Linked
Agammaglobulinemia (XL,A), severe combined immunodeficiency disease (SCID),
and selective IgA deficiency.
In a preferred embodiment, TR2, TR2-SV1 and/or TR2-SV2
polynucleotides, polypeptides, and/or agonists and/or antagonists thereof are
used
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to treat, prevent, and/or diagnose diseases or disorders affecting or
conditions
associated with any one or more of the various mucous membranes of the body.
Such diseases or disorders include, but are not limited to, for example,
mucositis,
mucoclasis, mucocolitis, mucocutaneous leishmaniasis (such as, for example,
American leishmaniasis, leishmaniasis americana, nasopharyngeal leishmaniasis,
and New World leishmaniasis), mucocutaneous lymph node syndrome (for
example, Kawasaki disease), mucoenteritis, mucoepidermoid carcinoma,
mucoepidermoid tumor, mucoepithelial dysplasia, mucoid adenocarcinoma,
mucoid degeneration, myxoid degeneration; myxomatous degeneration;
myxomatosis, mucoid medial degeneration (for example, cystic medial necrosis),
mucolipidosis (including, for example, mucolipidosis I, mucolipidosis II,
mucolipidosis III, and mucolipidosis IV), mucolysis disorders, mucomembranous
enteritis, mucoenteritis, mucopolysaccharidosis (such as, for example, type I
mucopolysaccharidosis (i.e., Hurler's syndrome), type IS mucopolysaccharidosis
(i.e., Scheie's syndrome or type V mucopolysaccharidosis), type II
mucopolysaccharidosis (i.e., Hunter's syndrome), type III
mucopolysaccharidosis
(i.e., Sanfilippo's syndrome), type IV mucopolysaccharidosis (i.e., Morquio's
syndrome), type VI mucopolysaccharidosis (i.e., Maroteaux-Lamy syndrome),
type VII mucopolysaccharidosis (i.e, mucopolysaccharidosis due to
beta-glucuronidase deficiency), and mucosulfatidosis), mucopolysacchariduria,
mucopurulent conjunctivitis, mucopus, mucormycosis (i.e., zygomycosis),
mucosal disease (i. e., bovine virus diarrhea), mucous colitis (such as, for
example,
mucocolitis and myxomembranous colitis), and mucoviscidosis (such as, for
example, cystic fibrosis, cystic fibrosis ofthe pancreas, Clarke-Hadfield
syndrome,
fibrocystic disease of the pancreas, mucoviscidosis, and viscidosis). In a
highly
preferred embodiment, TR2, TR2-SVI and/or TR2-SV2 polynucleotides,
polypeptides, and/or agonists and/or antagonists thereof are used to treat,
prevent,
and/or diagnose mucositis, especially as associated with chemotherapy.
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In a preferred embodiment, TR2, TR2-SV1 and/or TR2-SV2
polynucleotides, polypeptides, and/or agonists and/or antagonists
thereofareused
to treat, prevent, and/or diagnose diseases or disorders ai~'ecting or
conditions
associated with sinusitis.
S An additional condition, disease or symptom that can be treated,
prevented, and/or diagnosed by TR2, TR2-SV 1 and/or TR2-SV2 polynucleotides
or polypeptides, or agonists of TR2, TRZ-SV I and/or TR2-SV2, is
osteomyelitis.
An additional condition, disease or symptom that can be treated,
prevented, and/or diagnosed by TR2, TR2-SV 1 and/or TR2-SV2 polynucleotides
or polypeptides, or agonists of TR2, TR2-SV 1 and/or TRZ-SV2, is endocarditis.
Antagonists of TR2, TRZ-SV1 and/or TRZ-SV2 include binding and/or
inhibitory antibodies, antisense nucleic acids, ribozymes, and TR2, TRZ-SV I
and/or TR2-SV2 polypeptides of the invention. These would be expected to
reverse many of the activities of the ligand described above as well as find
clinical
or practical application as:
A means of blocking various aspects of immune responses to foreign
agents or self. Examples include autoimmune disorders such as lupus, and
arthritis, as well as immunoresponsiveness to skin allergies, inflammation,
bowel
disease, injury and pathogens. Although our current data speaks directly to
the
potential role of TR2 in B cell and monocyte related pathologies, it remains
possible that other cell types may gain expression or responsiveness to TR2.
Thus, TR2 may, like CD40 and its ligand, be regulated by the status of the
immune system and the microenvironment in which the cell is located.
A therapy for preventing the B cell proliferation and immunoglobin
secretion associated with autoimmune diseases such as idiopathic
thrombocytopenic purpura, systemic lupus erythematosus and MS.
An inhibitor of graft versus host disease or transplant rejection.
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A therapy for B cell malignancies such as ALL, Hodgkin's disease,
non-Hodgkin lymphoma, Chronic lymphocyte leukemia, plasmacytomas, multiple
myeloma, Burkitt's lymphoma, and EBV-transformed diseases.
A therapy for chronic hypergammaglobulinemeia evident in such diseases
as monoclonalgammopathy ofundetermined significance (MGUS), Waldenstrom's
disease, related idiopathic monoclonalgammopathies, and plasmacytomas.
A therapy for decreasing cellular proliferation of Large B-cell Lymphomas.
A means of decreasing the involvement of B cells and immunoglobin
associated with Chronic Myelogenous Leukemia.
I O An immunosuppressive agent(s).
TR2, TR2-SV 1 and/or TRZ-SV2 polypeptides or polynucleotides of the
invention, or antagonists may be used to modulate IgE concentrations in vitro
or
III VIVO.
In another embodiment, administration of TR2, TR2-SV 1 and/or
I 5 TR2-SV2 polypeptides or polynucleotides ofthe invention; or antagonists
thereof,
may be used to treat, prevent, and/or diagnose IgE-mediated allergic reactions
including, but not limited to, asthma, rhinitis, and eczema.
An inhibitor of signaling pathways involving ERK 1, COX2 and Cyclin D2
which have been associated with TRZ induced B cell activation.
20 The above-recited applications have uses in a wide variety of hosts. Such
hosts include, but are not limited to, human, murine, rabbit, goat, guinea
pig,
camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep,
dog,
cat, non-human primate, and human. In specific embodiments, the host is a
mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or
cat. In
25 preferred embodiments, the host is a mammal. In most preferred embodiments,
the host is a human.
The agonists and antagonists may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as described herein.
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The antagonists may be employed for instance to inhibit TR2-mediated,
TR2-SVl-mediated and/or TR2-SV2-mediated chemotaxis and activation of
macrophages and their precursors, and of neutrophils, basophils, B lymphocytes
and some T-cell subsets, e.g., activated and CD8 cytotoxic T cells and natural
killer cells, in certain auto-immune and chronic inflammatory and infective
diseases. Examples of auto-immune diseases include multiple sclerosis, and
insulin-dependent diabetes. The antagonists may also be employed to treat,
prevent, and/or diagnose infectious diseases including silicosis, sarcoidosis,
idiopathic pulmonary fibrosis by preventing the recruitment and activation of
mononuclear phagocytes. They may also be employed to treat, prevent, and/or
diagnose idiopathic hyper-eosinophilic syndrome by preventing eosinophil
production and migration. Endotoxic shock may also be treated by the
antagonists by preventing the migration of macrophages and their production of
the TR2, TR2-SV 1 and/or TR2-SV2 polypeptides of the present invention. The
antagonists may also be employed for treating atherosclerosis, by preventing
monocyte infiltration in the artery wall. The antagonists may also be employed
to
treat, prevent, and/or diagnose histamine-mediated allergic reactions and
immunological disorders including late phase allergic reactions, chronic
urticaria,
and atopic dermatitis by inhibiting chemokine-induced mast cell and basophil
degranulation and release of histamine. IgE-mediated allergic reactions such
as
allergic asthma, rhinitis, and eczema may also be treated. The antagonists may
also be employed to treat, prevent, and/or diagnose chronic and acute
inflammation by preventing the attraction of monocytes to a wound area. They
may also be employed to regulate normal pulmonary macrophage populations,
since chronic and acute inflammatory pulmonary diseases are associated with
sequestration of mononuclear phagocytes in the lung. Antagonists may also be
employed to treat, prevent, and/or diagnose rheumatoid arthritis by preventing
the
attraction of monocytes into synovial fluid in the joints of patients.
Monocyte
influx and activation plays a significant role in the pathogenesis of both
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degenerative and inflammatory arthropathies. The antagonists may be employed
to interfere with the deleterious cascades attributed primarily to IL-1 and
TNF,
which prevents the biosynthesis of other inflammatory cytokines. In this way,
the
antagonists may be employed to prevent inflammation. The antagonists may also
S be employed to inhibit prostaglandin-independent fever induced by TR2,
TR2-SVl and/or TR2-SV2. The antagonists may also be employed to treat,
prevent, and/or diagnose cases of bone marrow failure, for example, aplastic
anemia and myelodysplastic syndrome. The antagonists may also be employed to
treat, prevent, and/or diagnose asthma and allergy by preventing eosinophil
accumulation in the lung. The antagonists may also be employed to treat,
prevent,
and/or diagnose subepithelial basement membrane fibrosis which is a prominent
feature of the asthmatic lung. The antagonists may also be employed to treat,
prevent, and/or diagnose lymphomas (e.g., one or more of the extensive, but
not
limiting, list of lymphomas provided herein).
I 5 TR2, TR2-SV 1 and/or TR2-SV2 polynucleotides or polypeptides of the
invention and/or agonists and/or antagonists thereof, may be used to treat,
prevent, and/or diagnose various immune system-related disorders and/or
conditions associated with these disorders, in mammals, preferably humans.
Many
autoimmune disorders result from inappropriate recognition of self as foreign
material by immune cells. This inappropriate recognition results in an immune
response leading to the destruction of the host tissue. Therefore, the
administration of TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or
polypeptides of the invention and/or agonists and/or antagonists thereof that
can
inhibit an immune response, particularly the proliferation of B cells and/or
the
production of immunoglobulins, may be an effective therapy in treating and/or
preventing autoimmune disorders. Thus, in preferred embodiments, TR2,
TR2-SV 1 and/or TR2-SV2 antagonists of the invention (e.g., polypeptide
fragments of TR2, TR2-SV 1 and/or TR2-SV2 and anti-TR2 antibodies) are used
to treat, prevent, and/or diagnose an autoimmune disorder.
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Autoimmune disorders and conditions associated with these disorders that
may be treated, prevented, and/or diagnosed with the TR2, TR2-SVl and/or
TR2-SV2 polynucleotides, polypeptides, and/or antagonist of the invention
(e.g.,
anti-TR2 antibodies), include, but are not limited to, autoimmune hemolytic
anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia
purpura, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,
dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis,
rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), Multiple
Sclerosis, Neuritis, Uveitis Ophthalmic, Polyendocrinopathies, Purpura (e.g.,
Henloch-Scoenlein purpura), Reiter's Disease, Stiff Man Syndrome, Autoimmune
Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes
mellitis, and autoimmune inflammatory eye disease.
Additional autoimmune disorders (that are highly probable) that may be
treated, prevented, and/or diagnosed with the compositions of the invention
include, but are not limited to, autoimmune thyroiditis, hypothyroidism (i.e.,
Hashimoto's thyroiditis) (often characterized, e.g., by cell-mediated and
humoral
thyroid cytotoxicity), systemic lupus erhythematosus (often characterized,
e.g., by
circulating and locally generated immune complexes), Goodpasture's syndrome
(often characterized, e.g., by anti-basement membrane antibodies), Pemphigus
(often characterized, e.g., by epidermal acantholytic antibodies), Receptor
autoimmunities such as, for example, (a) Graves' Disease (often characterized,
e.g., by TSH receptor antibodies), (b) Myasthenia Gravis (often characterized,
e.g., by acetylcholine receptor antibodies), and (c) insulin resistance (often
characterized, e.g., by insulin receptor antibodies), autoimmune hemolytic
anemia
(often characterized, e.g., by phagocytosis of antibody-sensitized RBCs),
autoimmune thrombocytopenic purpura (often characterized, e.g., byphagocytosis
of antibody-sensitized platelets.
Additional autoimmune disorders (that are probable) that may be treated,
prevented, and/or diagnosed with the compositions of the invention include,
but
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are not limited to, rheumatoid arthritis (often characterized, e.g., by immune
complexes in joints), schleroderma with anti-collagen antibodies (often
characterized, e.g., by nucleolar and other nuclear antibodies), mixed
connective
tissue disease (often characterized, e.g., by antibodies to extractable
nuclear
antigens (e.g., ribonucleoprotein)), polymyositis/dermatomyositis (often
characterized, e.g., by nonhistone ANA), pernicious anemia (often
characterized,
e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies),
idiopathic
Addison's disease (often characterized, e.g., by humoral and cell-mediated
adrenal
cytotoxicity, infertility (often characterized, e.g., by antispermatozoal
antibodies),
glomerulonephritis (often characterized, e.g., by glomerular basement membrane
antibodies or immune complexes) such as primary glomerulonephritis and 1gA
nephropathy, bullous pemphigoid (often characterized, e.g., by IgG and
complement in basement membrane), Sjogren's syndrome (often characterized,
e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)),
diabetes millitus (often characterized, e.g., by cell-mediated and humoral
islet cell
antibodies), and adrenergic drug resistance (including adrenergic drug
resistance
with asthma or cystic fibrosis) (often characterized, e.g., by beta-adrenergic
receptor antibodies).
Additional autoimmune disorders (that are possible) that may be treated,
prevented, and/or diagnosed with the compositions of the invention include,
but
are not limited to, chronic active hepatitis (often characterized, e.g., by
smooth
muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by
mitchondrial antibodies), other endocrine gland failure (often characterized,
e.g.,
by specific tissue antibodies in some cases), vitiligo (often characterized,
e.g., by
melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and
complement
in vessel walls and/or low serum complement), post-MI (often characterized,
e.g.,
by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by
myocardial antibodies), urticaria (often characterized, e.g., by IgG and IgM
antibodies to IgE), atopic dermatitis (often characterized, e.g., by IgG and
IgM
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antibodies to IgE), asthma (often characterized, e.g., by IgG and IgM
antibodies
to IgE), inflammatory myopathies, and many other inflammatory, granulamatous,
degenerative, and atrophic disorders.
In a preferred embodiment, the autoimmune diseases and disorders and/or
conditions associated with the diseases and disorders recited above are
treated,
prevented, and/or diagnosed using anti-TR2, anti-TR2-SV 1 and/or anti-TR2-SV2
antibodies.
In a specific preferred embodiment, rheumatoid arthritis is treated,
prevented, and/or diagnosed using anti-TR2, anti-TR2-SV 1 and/or anti-TR2-SV2
antibodies and/or other antagonist of the invention.
In a specific preferred embodiment, lupus is treated, prevented, and/or
diagnosed using anti-TR2, anti-TR2-SV 1 and/or anti-TR2-SV2 antibodies and/or
other antagonist of the invention.
In a specific preferred embodiment, nephritis associated with lupus is
treated, prevented, and/or diagnosed using anti-TR2, anti-TR2-SV1 and/or
anti-TR2-SV2 antibodies and/or other antagonist of the invention.
In a specific embodiment, TR2, TR2-SV1 and/or TR2-SV2
polynucleotides or polypeptides, or antagonists thereof (e.g., anti-TR2,
anti-TR2-SVl and/or anti-TR2-SV2 antibodies) are used to treat or prevent
systemic lupus erythematosus and/or diseases, disorders or conditions
associated
therewith. Lupus-associated diseases, disorders, or conditions that may be
treated
or prevented with TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or
polypeptides, or antagonists of the invention, include, but are not limited
to,
hematologic disorders (e.g., hemolytic anemia, leukopenia, lymphopenia, and
thrombocytopenia), immunologic disorders (e.g., anti-DNA antibodies, and
anti-Sm antibodies), rashes, photosensitivity, oral ulcers, arthritis, fever,
fatigue,
weight loss, serositis (e.g., pleuritus (pleuricy)), renal disorders (e.g.,
nephritis),
neurological disorders (e.g., seizures, peripheral neuropathy, CNS related
disorders), gastroinstestinal disorders, Raynaud phenomenon, and pericarditis.
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In a preferred embodiment, the TR2, TR2-SV 1 and/or TR2-SV2 polynucleotides
or polypeptides, or antagonists thereof (e.g., anti-TR2, anti-TR2-SVI and/or
anti-TR2-SV2 antibodies) are used to treat or prevent renal disorders
associated
with systemic lupus erythematosus. In a most preferred embodiment, TR2,
TR2-SVI and/or TR2-SV2 polynucleotides or polypeptides, or antagonists
thereof (e.g., anti-TR2, anti-TR2-SV 1 and/or anti-TR2-SV2 antibodies) are
used
to treat or prevent nephritis associated with systemic lupus erythematosus.
Similarly, allergic reactions and conditions, such as asthma (particularly
allergic asthma) or other respiratory problems, may also be treated by TR2,
TR2-SVI and/or TR2-SV2 polynucleotides or polypeptides of the invention
and/or agonists and/or antagonists thereof. Moreover, these molecules can be
used to treat, prevent, and/or diagnose anaphylaxis, hypersensitivity to an
antigenic molecule, or blood group incompatibility.
TR2, TR2-SVI and/or TR2-SV2 polynucleotides or polypeptides of the
invention and/or agonists and/or antagonists thereof, may also be used to
treat,
prevent, and/or diagnose organ rejection or graft-versus-host disease (GVHD)
and/or conditions associated therewith. Organ rejection occurs by host immune
cell destruction of the transplanted tissue through an immune response.
Similarly,
an immune response is also involved in GVHD, but, in this case, the foreign
transplanted immune cells destroy the host tissues. The administration of TR2,
TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention
and/or agonists and/or antagonists thereof, that inhibits an immune response,
particularly the proliferation, differentiation, or chemotaxis of T-cells, may
be an
effective therapy in preventing organ rejection or GVI~.
Similarly, TR2, TR2-SVI and/or TR2-SV2 polynucleotides or
polypeptides of the invention and/or agonists and/or antagonists thereof, may
also
be used to modulate inflammation. For example, TR2, TR2-SV1 and/or
TR2-SVZ polynucleotides or polypeptides of the invention and/or agonists
and/or
antagonists thereof, may inhibit the proliferation and differentiation of
cells
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involved in an inflammatory response. These molecules can be used to treat,
prevent, and/or diagnose inflammatory conditions, both chronic and acute
conditions, including chronic prostatitis, granulomatous prostatitis and
malacoplakia, inflammation associated with infection (e.g., septic shock,
sepsis,
or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion
injury,
endotoxin lethality, arthritis, complement-mediated hyperacute rejection,
nephritis,
cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's
disease, or resulting from over production of cytokines (e.g., TNF or IL-1).
In a specific embodiment, anti-TR2, anti-TR2-SV 1 and/or anti-TR2-SV2
antibodies of the invention are used to treat, prevent, modulate, detect,
and/or
diagnose inflammation.
In a specific embodiment, anti-TR2, anti-TR2-SV I and/or anti-TR2-SV2
antibodies of the invention are used to treat, prevent, modulate, detect,
and/or
diagnose inflammatory disorders.
I S In another specific embodiment, anti-TR2, anti-TR2-SV 1 and/or
anti-TR2-SV2 antibodies of the invention are used to treat, prevent, modulate,
detect, and/or diagnose allergy and/or hypersensitivity.
Antibodies against TR2, TR2-SV 1 and/or TR2-SV2 may be employed to
bind to and inhibit TR2, TR2-SVI and/or TR2-SV2 activity to treat, prevent,
and/or diagnose ARDS, by preventing infiltration ofneutrophils into the lung
after
injury. The agonists and antagonists of the instant may be employed in a
composition with a pharmaceutically acceptable carrier, e.g., as described
hereinafter.
TR2, TR2-SV 1 and/or TR2-SV2 and/or TR2 receptor polynucleotides or
polypeptides of the invention and/or agonists and/or antagonists thereof, are
used
to treat, prevent, and/or diagnose diseases and disorders of the pulmonary
system
(e.g., bronchi such as, for example, sinopulmonary and bronchial infections
and
conditions associated with such diseases and disorders and other respiratory
diseases and disorders. In specific embodiments, such diseases and disorders
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include, but are not limited to, bronchial adenoma, bronchial asthma,
pneumonia
(such as, e.g., bronchial pneumonia, bronchopneumonia, and tuberculous
bronchopneumonia), chronic obstructive pulmonary disease (COPD), bronchial
polyps, bronchiectasia (such as, e.g., bronchiectasia sicca, cylindrical
bronchiectasis, and saccular bronchiectasis), bronchiolar adenocarcinoma,
bronchiolar carcinoma, bronchiolitis (such as, e.g., exudative bronchiolitis,
bronchiolitis fibrosa obliterans, and proliferativebronchiolitis), bronchiolo-
alveolar
carcinoma, bronchitic asthma, bronchitis (such as, e.g., asthmatic bronchitis,
Castellani's bronchitis, chronic bronchitis, croupous bronchitis, fibrinous
bronchitis, hemorrhagic bronchitis, infectious avian bronchitis, obliterative
bronchitis, plastic bronchitis, pseudomembranousbronchitis, putrid bronchitis,
and
verminous bronchitis), bronchocentric granulomatosis, bronchoedema,
bronchoesophageal fistula, bronchogenic carcinoma, bronchogenic cyst,
broncholithiasis, bronchomalacia, bronchomycosis (such as, e.g.,
bronchopulmonary aspergillosis), bronchopulmonary spirochetosis, hemorrhagic
bronchitis, bronchorrhea, bronchospasm, bronchostaxis, bronchostenosis, Biot's
respiration, bronchial respiration, Kussmaul respiration, Kussmaul-Kien
respiration, respiratory acidosis, respiratory alkalosis, respiratory distress
syndrome of the newborn, respiratory insufficiency, respiratory scleroma,
respiratory syncytial virus, and the like.
In a specific embodiment, TR2, TR2-SV 1 and/or TR2-SV2
polynucleotides or polypeptides of the invention and/or agonists and/or
antagonists thereof, are used to treat, prevent, and/or diagnose chronic
obstructive
pulmonary disease (COPD).
In another embodiment, TR2, TR2-SV 1 and/or TR2-SV2 polynucleotides
or polypeptides of the invention and/or agonists and/or antagonists thereof,
are
used to treat, prevent, and/or diagnose fibroses and conditions associated
with
fibroses, such as, for example, but not limited to, cystic fibrosis (including
such
fibroses as cystic fibrosis of the pancreas, Clarke-Hadfield syndrome,
fibrocystic
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disease of the pancreas, mucoviscidosis, and viscidosis), endomyocardial
fibrosis,
idiopathic retroperitoneal fibrosis, leptomeningeal fibrosis, mediastinal
fibrosis,
nodular subepidermal fibrosis, pericentral fibrosis, perimuscular fibrosis,
pipestem
fibrosis, replacement fibrosis, subadventitial fibrosis, and Symmers' clay
pipestem
fibrosis.
The TNF family ligands are known to be among the most pleiotropic
cytokines, inducing a large number of cellular responses, including
cytotoxicity,
anti-viral activity, immunoregulatory activities, and the transcriptional
regulation
of several genes (D.V. Goeddel et al., "Tumor Necrosis Factors: Gene Structure
and Biological Activities," Symp. Quant. BioJ. 51:597- 609 (1986), Cold Spring
Harbor; B. Beutler and A. Cerami, Anrru. Rev. Biochen~. 57:505-518 (1988);
L.J.
Old, Sci. Arrz. 258:59-75 (1988); W. Fiers, FEBSLett. 28:199-224 (1991)). The
TNF-family ligands, including TR2, TR2-SV 1 and/or TR2-SV2 polypeptides of
the present invention, induce such various cellular responses by binding to
TNF-family receptors. TR2, TR2-SV1 and/or TR2-SV2 polypeptides are
believed to elicit a potent cellular response including any genotypic,
phenotypic,
and/or morphologic change to the cell, cell line, tissue, tissue culture or
patient.
As indicated, such cellular responses include not only normal physiological
responses to TNF-family ligands, but also diseases associated with increased
apoptosis or the inhibition of apoptosis. Apoptosis-programmed cell death-is a
physiological mechanism involved in the deletion of peripheral B and/or T
lymphocytes of the immune system, and its disregulation can lead to a number
of
different pathogenic processes (J.C. Ameisen, AIDS 8:1197-1213 (1994); P.H.
Krammer et al., Curr. Opin. Immunol. 6:279-289 (1994)).
Diseases associated with increased cell survival, or the inhibition of
apoptosis that may be diagnosed, treated, or prevented with the TR2, TR2-SV1
and/or TRZ-SV2 polynucleotides or polypeptides of the invention, and agonists
and antagonists thereof, include cancers (such as follicular lymphomas,
carcinomas
with p53 mutations, and hormone-dependent tumors, including, but not limited
to,
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colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,
glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach
cancer,
neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma,
osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate
cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as
systemic lupus erythematosus and immune-related glomerulonephritis rheumatoid
arthritis); viral infections (such as herpes viruses, pox viruses and
adenoviruses);
inflammation; graft vs. host disease; acute graft rejection and chronic graft
rejection. Thus, in preferred embodiments TR2, TR2-SVI and/or TRZ-SV2
polynucleotides or polypeptides of the invention and/or agonists or
antagonists
thereof are used to treat, prevent, and/or diagnose autoimmune diseases and/or
inhibit the growth, progression, and/or metastasis of cancers, including, but
not
limited to, those cancers disclosed herein, such as, for example, lymphocytic
leukemias (including, for example, MLL and chronic lymphocytic leukemia
(CLL)) and follicular lymphomas. In another embodiment TR2, TR2-SV 1 and/or
TR2-SV2 polynucleotides or polypeptides of the invention are used to activate,
differentiate or proliferate cancerous cells or tissue (e.g., B cell lineage
related
cancers (e.g., CLL and MLL), lymphocytic leukemia, or lymphoma) and thereby
render the cells more vulnerable to cancer therapy (e.g., chemotherapy or
radiation therapy).
Moreover, in other embodiments, TR2, TRZ-SVl and/or TR2-SV2
polynucleotides or polypeptides of the invention or agonists or antagonists
thereof, are used to inhibit the growth, progression, and/or metastases of
malignancies and related disorders such as leukemia (including acute leukemias
(e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including
myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia))
and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and
chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's
disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's
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macroglobulinemia, heavy chain disease, and solid tumors including, but not
limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate
cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat
gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic
carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma,
seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular
tumor,
lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis apoptosis that may be
diagnosed, treated, or prevented with the TR2, TR2-SV1 and/or TR2-SV2
polynucleotides or polypeptides of the invention, and agonists and antagonists
thereof, include AIDS; neurodegenerative disorders (such as Alzheimer's
disease,
Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa,
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. Thus, in preferred embodiments TR2, TR2-SV1 and/or
TR2-SV2 polynucleotides or polypeptides of the invention and/or agonists or
antagonists thereof, are used to treat, prevent, and/or diagnose the diseases
and
disorders listed above.
In preferred embodiments, TR2, TR2-SV 1 and/or TR2-SV2 polypeptides
of the invention and/or agonists or antagonists thereof (e.g., anti-TR2
antibodies)
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inhibit the growth of human histiocytic lymphoma U-93 7 cells in a dose-
dependent
manner. In additional preferred embodiments, TR2, TR2-SVI and/or TR2-SV2
polypeptides of the invention and/or agonists or antagonists thereof (e.g.,
anti-TR2 antibodies) inhibit the growth of PC-3 cells, HT-29 cells, HeLa
cells,
MCF-7 cells, and A293 cells. In highly preferred embodiments, TR2, TR2-SV 1
and/or TR2-SV2 polynucleotides or polypeptides ofthe invention and/or agonists
or antagonists thereof (e.g., anti-TR2 antibodies) are used to inhibit growth,
progression, and/or metastasis of prostate cancer, colon cancer, cervical
carcinoma, and breast carcinoma.
Thus, in additional preferred embodiments, the present invention is
directed to a method for enhancing apoptosis induced by a TNF-family ligand,
which involves administering to a cell which expresses a TR2, TR2-SVl and/or
TR2-SV2 receptor an efFective amount ofTR2, TR2-SV1 and/or TR2-SV2, or
an agonist or antagonist thereof, capable of increasing or decreasing TR2,
TR2-SV1 and/or TR2-SV2 mediated signaling. Preferably, TR2, TR2-SVI
and/or TR2-SV2 mediated signaling is increased or decreased to treat, prevent,
and/or diagnose a disease wherein decreased apoptosis or decreased cytokine
and
adhesion molecule expression is exhibited. An agonist or antagonist can
include
soluble forms of TR2, TR2-SVl and/or TR2-SV2 and monoclonal antibodies
directed against the TR2, TR2-SV1 and/or TR2-SV2 polypeptide.
In a further aspect, the present invention is directed to a method for
inhibiting apoptosis induced by a TNF-family ligand, which involves
administering
to a cell which expresses the TR2, TR2-SV1 and/or TR2-SV2 receptor an
effective amount of an agonist or antagonist capable of increasing or
decreasing
TR2, TR2-SV 1 and/or TR2-SV2 mediated signaling. Preferably, TR2, TR2-SV1
and/or TR2-SV2 mediated signaling is increased or decreased to treat, prevent,
and/or diagnose a disease wherein increased apoptosis or NF-kappaB expression
is exhibited. An agonist or antagonist can include soluble forms of TR2,
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TR2-SV 1 and/or TR2-SV2 and monoclonal antibodies directed against the TR2,
TR2-SVl and/or TR2-SV2 polypeptide.
Because TR2, TRZ-SV 1 and TR2-SV2 belong to the TNF superfamily, the
polypeptides should also modulate angiogenesis. In addition, since TR2,
TR2-SVl and TR2-SV2 inhibit immune cell functions, the polypeptides will have
a wide range of anti-inflammatory activities. TR2, TR2-SV1 and/or TR2-SV2
may be employed as an anti-neovascularizing agent to treat, prevent, and/or
diagnose solid tumors by stimulating the invasion and activation of host
defense
cells, e.g., cytotoxic T cells and macrophages and by inhibiting the
angiogenesis
of tumors. Those of skill in the art will recognize other non-cancer
indications
where blood vessel proliferation is not wanted. They may also be employed to
enhance host defenses against resistant chronic and acute infections, for
example,
mycobacterial infections via the attraction and activation of microbicidal
leukocytes. TR2, TR2-SVl and/or TR2-SV2 may also be employed to inhibit
1 S T-cell proliferation by the inhibition of IL-2 biosynthesis for the
treatment of
T-cell mediated auto-immune diseases and lymphocytic leukemias (including, for
example, chronic lymphocytic leukemia (CLL)). TR2, TR2-SV 1 and/or TR2-SV2
may also be employed to stimulate wound healing, both via the recruitment of
debris clearing and connective tissue promoting inflammatory cells. In this
same
manner, TR2, TR2-SV 1 and/or TR2-SV2 may also be employed to treat, prevent,
and/or diagnose other fibrotic disorders, including liver cirrhosis,
osteoarthritis
and pulmonary fibrosis. TR2, TR2-SVl and/or TR2-SV2 also increases the
presence of eosinophils that have the distinctive function of killing the
larvae of
parasites that invade tissues, as in schistosomiasis, trichinosis and
ascariasis. It
may also be employed to regulate hematopoiesis, by regulating the activation
and
differentiation of various hematopoietic progenitor cells, for example, to
release
mature leukocytes from the bone marrow following chemotherapy, i.e., in stem
cell mobilization. TR2, TR2-SV 1 and/or TR2-SV2 may also be employed to treat,
prevent, and/or diagnose sepsis.
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Polynucleotides and/or polypeptides of the invention and/or agonists
and/or antagonists thereof are useful in promoting angiogenesis, wound healing
(e.g., wounds, burns, and bone fractures) and regulating hematopoiesis.
Polynucleotides and/or polypeptides of the invention and/or agonists and/or
antagonists thereof are also useful as an adjuvant to enhance immune
responsiveness to specific antigen, anti-viral immune responses.
More generally, polynucleotides and/or polypeptides of the invention
and/or agonists and/or antagonists thereof are useful in regulating (i.e.,
elevating
or reducing) immune response. For example, polynucleotides and/or polypeptides
of the invention may be useful in preparation or recovery from surgery,
trauma,
radiation therapy, chemotherapy, and transplantation, or may be used to boost
immune response and/or recovery in the elderly and immunocompromised
individuals. Alternatively, polynucleotides and/or polypeptides of the
invention
and/or agonists and/or antagonists thereof are useful as immunosuppressive
agents, for example in the treatment or prevention of autoimmune disorders. In
specific embodiments, polynucleotides and/or polypeptides of the invention are
used to treat or prevent chronic inflammatory, allergic or autoimmune
conditions,
such as those described herein or are otherwise known in the art.
Preferably, treatment using TR2, TR2-SV1 and/or TR2-SV2
polynucleotides or polypeptides, and/or agonists or antagonists of TR2, TR2-SV
1
and/or TR2-SV2 (e.g., anti-TRZ antibody), could either be by administering an
ef~'ective amount of TR2, TR2-SV 1 and/or TRZ-SV2 polypeptide ofthe invention
,or agonist or antagonist thereof, to the patient, or by removing cells from
the
patient, supplying the cells with TR2, TRZ-SV 1 and/or TR2-S V2
polynucleotide,
and returning the engineered cells to the patient (ex vivo therapy). Moreover,
as
further discussed herein, the TR2, TR2-SV1 and/or TRZ-SV2 polypeptide or
polynucleotide can be used as an adjuvant in a vaccine to raise an immune
response against infectious disease.
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The agonists and antagonists may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as described hererin.
All ofthe above described applications may be used in veterinary medicine,
as well as in human treatment regimens.
The above-recited applications have uses in a wide variety of hosts. Such
hosts include, but are not limited to, human, murine, rabbit, goat, guinea
pig,
camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep,
dog,
cat, non-human primate, and human. In specific embodiments, the host is a
mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or
cat. In
preferred embodiments, the host is a mammal. In most preferred embodiments,
the host is a human.
Cardio>>r~sculur Disorders
TR2 polynucleotides, polypeptides, agonists or antagonists of the
invention may be used to treat cardiovascular disorders, including peripheral
artery
disease, such as limb ischemia.
Cardiovascular disorders include cardiovascular abnormalities, such as
arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous
malformations,
congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital
heart defects include aortic coarctation, cortriatriatum, coronaryvessel
anomalies,
crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly,
Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of
fallot, transposition of great vessels, double outlet right ventricle,
tricuspid atresia,
persistent truncus arteriosus, and heart septal defects, such as
aortopulmonary
septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of
Fallot, ventricular heart septal defects.
Cardiovascular disorders also include heart disease, such as arrhythmias,
carcinoid heart disease, high cardiac output, low cardiac output, cardiac
tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest,
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congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea,
cardiac
edema, heart hypertrophy, congestive cardiomyopathy, left ventricular
hypertrophy, right ventricular hypertrophy, post-infarction heart rupture,
ventricular septal rupture, heart valve diseases, myocardial diseases,
myocardial
ischemia, pericardial effusion, pericarditis (including constrictive and
tuberculous),
pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease,
rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular
pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and
cardiovascular tuberculosis.
Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter,
bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block,
sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine
Syndrome, Mahaim-type pre-excitation syndrome, Wolfs=Parkinson-White
syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation.
Tachycardias include paroxysmal tachycardia, supraventricular tachycardia,
accelerated idioventricular rhythm, atrioventricular nodal reentry
tachycardia,
ectopic atrial tachycardia, ectopic functional tachycardia, sinoatrial nodal
reentry
tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular
tachycardia.
Heart valve disease include aortic valve insu~ciency, aortic valve stenosis,
hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve
prolapse, mural valve insufficiency, mitral valve stenosis, pulmonary atresia,
pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia,
tricuspid valve insufficiency, and tricuspid valve stenosis.
Myocardial diseases include alcoholic cardiomyopathy, congestive
cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis,
pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas
cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns
Syndrome, myocardial reperfusion injury, and myocarditis.
Myocardial ischemias include coronary disease, such as angina pectoris,
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coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary
vasospasm, myocardial infarction and myocardial stunning.
Cardiovascular diseases also include vascular diseases such as aneurysms,
angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease,
Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic
edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome,
arterial
occlusive diseases, arteritis, enarteritis, polyarteritis nodosa,
cerebrovascular
disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis,
erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension,
hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-
occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion,
Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia
telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose
veins,
varicose ulcer, vasculitis, and venous insu~ciency.
Aneurysms include dissecting aneurysms, false aneurysms, infected
aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary
aneurysms, heart aneurysms, and iliac aneurysms.
Arterial occlusive diseases include arteriosclerosis, intermittent
claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular
occlusion, Moyamoya disease, renal artery obstruction, retinal artery
occlusion,
and thromboangiitis obliterans.
Cerebrovascular disorders include carotid artery diseases, cerebral amyloid
angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis,
cerebral
arteriovenous malformation, cerebral artery diseases, cerebral embolism and
thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's
syndrome,
cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid
hemorrhage, cerebral infarction, cerebral ischemia (including transient),
subclavian
steal syndrome, periventricular leukomalacia, vascular headache, cluster
headache,
migraine, and vertebrobasilar insufficiency.
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Embolisms include air embolisms, amniotic fluid embolisms, cholesterol
embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and
thromoboembolisms. Thrombosis include coronary thrombosis, hepatic vein
thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus
thrombosis,
Wallenberg's syndrome, and thrombophlebitis.
Ischemia includes cerebral ischemia, ischemic colitis, compartment
syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion
injuries, and peripheral limb ischemia. Vasculitis includes aortitis,
arteritis,
Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node
syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-
Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
In one embodiment, a TR2 receptor polynucleotide, polypeptide, agonist,
or antagonist of the invention is used to treat thrombotic microangiopathies.
One
such disorder is thrombotic thrombocytopenic purpura (TTP) (Kwaan, H.C.,
Semin. Hematnl. 24:71 (1987); Thompson et al., Blood 80:1890 (1992)).
Increasing TTP-associated mortality rates have been reported by the U. S.
Centers
for Disease Control (Torok et ad., Am. J. Hematol. 50:84 (1995)). Plasma from
patients afflicted with TTP (including HIV+ and HIV- patients) induces
apoptosis
of human endothelial cells of dermal microvascular origin, but not large
vessel
origin (Lawrence et al., Blood 87:3245 (1996)). Plasma of TTP patients thus is
thought to contain one or more factors that directly or indirectly induce
apoptosis.
Another thrombotic microangiopathy is hemolytic-uremic syndrome (HOTS)
(Moake, J.L., Lancet, 343:393 (1994); Melnyk et al., As°ch. Intern.
Med.
155:2077 (1995); Thompson et al., supra). Thus, in one embodiment, the
invention is directed to use of TR2 receptor to treat the condition that is
often
referred to as "adult HUS" (even though it can strike children as well). A
disorder
known as childhood/diarrhea-associated HUS differs in etiology from adult HUS.
In another embodiment, conditions characterized by clotting of small blood
vessels
may be treated using TR2 receptor. Such conditions include, but are not
limited
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to, those described herein. For example, cardiac problems seen in about 5-10%
of pediatric AIDS patients are believed to involve clotting of small blood
vessels.
Breakdown of the microvasculature in the heart has been reported in multiple
sclerosis patients. As a further example, treatment of systemic lupus
erythematosus (SLE) is contemplated. In one embodiment, a patient's blood or
plasma is contacted with TR2 receptor polypeptides ofthe invention ex vivo.
The
TR2 receptor polypeptides of the invention may be bound to a suitable
chromatography matrix by procedures known in the art. According to this
embodiment, the patient's blood or plasma flows through a chromatography
column containing TR2 receptor polynucleotides and/or polypeptides of the
invention bound to the matrix, before being returned to the patient. The
immobilized TR2 receptor binds AIM II, thus removing AIM II protein from the
patient's blood. Alternatively, TR2 receptor polynucleotides, polypeptides,
agonists or antagonists of the invention may be administered in vivo to a
patient
afflicted with a thrombotic microangiopathy. In one embodiment, a soluble form
of TR2 receptor polypeptide of the invention is administered to the patient.
Thus,
the present invention provides a method for treating a thrombotic
microangiopathy, involving use of an effective amount of TR2 receptor
polynucleotide, polypeptide, agonist or antagonist. A TR2 receptor polypeptide
may be employed in in vivo or ex vivo procedures, to inhibit AiM II-mediated
damage to (e.g., apoptosis of) microvascular endothelial cells.
While not intending to be bound by theory, cells which express TR2 are
believed to interact with cells that express AIM II.
TR2 receptorpolynucleotides, polypeptides, agonists or antagonists ofthe
invention may be employed in combination with other agents useful in treating
a
particular disorder. For example, in an in vitro study reported by Laurence et
al.,
Blood 87:3245 (1996), some reduction of TTP plasma-mediated apoptosis of
microvascular endothelial cells was achieved by using an anti-Fas blocking
antibody, aurintricarboxylic acid, or normal plasma depleted of
cryoprecipitate.
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Thus, a patient may be treated with a polynucleotide and/or polypeptide of the
invention in combination with an agent that inhibits Fas-ligand-mediated
apoptosis
of endothelial cells, such as, for example, an agent described above. In one
embodiment, a TR2 receptor polynucleotide, polypeptide, agonist or antagonist,
and an anti-FAS blocking antibody are both administered to a patient afflicted
with
a disorder characterized by thrombotic microanglopathy, such as TTP or HL1S.
Examples ofblocking monoclonal antibodies directed against Fas antigen (CD95)
are described in WO 95/10540, hereby incorporated by reference.
The naturally occurring balance between endogenous stimulators and
inhibitors of angiogenesis is one in which inhibitory influences predominate.
Rastinejad et al., Cell SG:345-355 (1989). In those rare instances in which
neovascularization occurs under normal physiological conditions, such as wound
healing, organ regeneration, embryonic development, and female reproductive
processes, angiogenesis is stringently regulated and spatially and temporally
delimited. Under conditions of pathological angiogenesis such as that
characterizing solid tumor growth, these regulatory controls fail.
Unregulated angiogenesis becomes pathologic and sustains progression of
many neoplastic and non-neoplastic diseases. A number of serious diseases are
dominated by abnormal neovascularization including solid tumor growth and
metastases, arthritis, some types ofeye disorders, and psoriasis. See, e.g.,
reviews
by Moses et al., Biotech. 9:630-634 (1991); Folkman et al., N. Engl. .I. Med.
333:1757-1763 (1995); Auerbach et al., J. Microvasc. Res. 29:401-411 (1985);
Folkman, Advances in Cancer Research, Klein and Weinhouse, eds., Academic
Press, New York, pp. 175-203 (1985); Patz, Am. .I. Opthalmol. 94:715-743
( 1982); and Folkman et al. , Science 221:719-725 ( 1983 ). In a number of
pathological conditions, the process of angiogenesis contributes to the
disease
state. For example, significant data have accumulated which suggest that the
growth of solid tumors is dependent on angiogenesis (Folkman and Klagsbrun,
Science 235:442-447 (1987)).
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The present invention provides for treatment of diseases or disorders
associated with neovascularization by administration of the TR2 receptor
polynucleotides and/or polypeptides of the invention (including TR2 receptor
agonists and/or antagonists). Malignant and metastatic conditions which can be
treated with the polynucleotides and polypeptides ofthe invention include, but
are
not limited to those malignancies, solid tumors, and cancers described herein
and
otherwise known in the art (for a review of such disorders, see Fishman et
al.,
Medicine, 2nd Ed., J. B. Lippincott Co., Philadelphia (1985)).
Additionally, ocular disorders associated with neovascularization which
can be treated with the TR2 receptor polynucleotides and polypeptides of the
present invention (including TR2 receptor agonists and TR2 receptor
antagonists)
include, but are not limited to: neovascular glaucoma, diabetic retinopathy,
retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity
macular
degeneration, corneal graft neovascularization, as well as other eye
inflammatory
diseases, ocular tumors and diseases associated with choroidal or iris
neovascularization. See, e.g. , reviews by Waltman et al., Anz .I. Ophthal.
85:704-
710 (1978) and Gartner et a7., Surv. Ophtha7. 22:291-312 (1978).
Additionally, disorders which can be treated with the TR2 receptor
polynucleotides and polypeptides ofthe present invention (including TRZ
receptor
agonists and TR2 receptor antagonists) include, but are not limited to,
hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques,
delayed
wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion
fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma,
and vascular adhesions.
Polynucleotides and/or polypeptides of the invention, and/or agonists
and/or antagonists thereof, are useful in the diagnosis and treatment or
prevention
of a wide range of diseases and/or conditions. Such diseases and conditions
include, but are not limited to, cancer (e.g., immune cell related cancers,
breast
cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer
associated
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with mutation or alteration of p53, brain tumor, bladder cancer, uterocervical
cancer, colon cancer, colorectal cancer, non-small cell carcinoma of the lung,
small cell carcinoma of the lung, stomach cancer, etc.), lymphoproliferative
disorders (e.g., lymphadenopathy), microbial (e.g., viral, bacterial, etc.)
infection
(e.g., HIV-1 infection, HIV-2 infection, herpesvirus infection (including, but
not
limited to, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus
infection, poxvirus infection, human papilloma virus infection, hepatitis
infection
(e.g., HAV, HBV, HCV, etc.), Helicobacte~° pylori infection, invasive
Staphylococcia, etc.), parasitic infection, nephritis, bone disease (e.g.,
osteoporosis) and bone formation (e.g., regulator of osteoclast
differentiation),
atherosclerosis, pain, cardiovascular disorders (e.g., neovascularization,
hypovascularization or reduced circulation (e.g., ischemic disease (e.g.,
myocardial infarction, stroke, etc.)), AIDS, allergy, inflammation,
neurodegenerative disease (e.g., Alzheimer's disease, Parkinson's disease,
amyotrophic lateral sclerosis, pigmentary retinitis, cerebellar degeneration,
etc.),
graft rejection (acute and chronic), graft vs. host disease, diseases due to
osteomyelodysplasia (e.gJ., aplastic anemia, etc.), joint tissue destruction
in
rheumatism, liver disease (e.g., acute and chronic hepatitis, liver injury,
and
cirrhosis), autoimmune disease (e.g., multiple sclerosis, rheumatoid
arthritis,
systemic lupus erythematosus, immune complex glomerulonephritis, myasthenia
gravis, autoimmune diabetes, autoimmune thrombocytopenic purpura, Grave's
disease, Hashimoto's thyroiditis, etc.), cardiomyopathy (e.g., dilated
cardiomyopathy), diabetes, diabetic complications (e.g., diabetic nephropathy,
diabetic neuropathy, diabetic retinopathy), influenza, asthma, psoriasis,
glomerulonephritis, septic shock, and ulcerative colitis.
Polynucleotides and/or polypeptides of the invention and/or agonists
and/or antagonists thereof are also useful as an adjuvant to enhance immune
responsiveness to specific antigen and/or anti-viral immune responses.
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More generally, polynucleotides and/or polypeptides of the invention
and/or agonists and/or antagonists thereof are useful in regulating (i. e.,
elevating
or reducing) immune response. For example, polynucleotides and/or polypeptides
of the invention may be useful in preparation or recovery from surgery,
trauma,
radiation therapy, chemotherapy, and transplantation, or may be used to boost
immune response and/or recovery in the elderly and immunocompromised
individuals. Alternatively, polynucleotides and/or polypeptides of the
invention
and/or agonists and/or antagonists thereof are useful as immunosuppressive
agents, for example in the treatment or prevention of autoimmune disorders. In
specific embodiments, polynucleotides and/or polypeptides of the invention are
used to treat or prevent chronic inflammatory, allergic or autoimmune
conditions,
such as those described herein or are otherwise known in the art.
All ofthe above described applications may be used in veterinary medicine,
as well as in human treatment regimens.
Gene Therapy
In a specific embodiment, nucleic acids comprising sequences encoding
antibodies or functional derivatives thereof, are administered to treat,
inhibit or
prevent a disease or disorder associated with aberrant expression and/or
activity
of a polypeptide of the invention, by way of gene therapy. Gene therapy refers
to
therapy performed by the administration to a subject of an expressed or
expressible nucleic acid. In this embodiment of the invention, the nucleic
acids
produce their encoded protein that mediates a therapeutic effect.
Any of the methods for gene therapy available in the art can be used
according to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al.,
1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;
Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993,
Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.
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62:191-217; May, 1993,TIBTECH11(5):155-215). Methods commonly known
in the art of recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John
Wiley
& Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory
S Manual, Stockton Press, NY.
In a preferred aspect, the compound comprises nucleic acid sequences
encoding an antibody, said nucleic acid sequences being part of expression
vectors that express the antibody or fragments or chimeric proteins or heavy
or
light chains thereof in a suitable host. In particular, such nucleic acid
sequences
have promoters operably linked to the antibody coding region, said promoter
being inducible or constitutive, and, optionally, tissue-specific. In another
particular embodiment, nucleic acid molecules are used in which the antibody
coding sequences and any other desired sequences are flanked by regions that
promote homologous recombination at a desired site in the genome, thus
providing for intrachromosomal expression of the antibody nucleic acids
(Koller
and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al.,
1989, Nature 342:435-438). In specific embodiments, the expressed antibody
molecule is a single chain antibody; alternatively, the nucleic acid sequences
include sequences encoding both the heavy and light chains, or fragments
thereof,
of the antibody.
Delivery of the nucleic acids into a patient may be either direct, in which
case the patient is directly exposed to the nucleic acid or nucleic acid-
carrying
vectors, or indirect, in which case, cells are first transformed with the
nucleic acids
in vitro, then transplanted into the patient. These two approaches are known,
respectively, as irr vivo or ex vivo gene therapy.
In a specific embodiment, the nucleic acid sequences are directly
administered in vivo, where it is expressed to produce the encoded product.
This
can be accomplished by any of numerous methods known in the art, e.g., by
constructing them as part of an appropriate nucleic acid expression vector and
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administering it so that they become intracellular, e.g., by infection using
defective
or attenuated retrovirals or other viral vectors (see U.S. Patent No.
4,980,286),
or by direct injection of naked DNA, or by use of microparticle bombardment
(e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface
receptors or transfecting agents, encapsulation in liposomes, microparticles,
or
microcapsules, or by administering them in linkage to a peptide which is known
to enter the nucleus, by administering it in linkage to a ligand subject to
receptor-
mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chena. 262:4429-
4432) (which can be used to target cell types specifically expressing the
receptors), etc. In another embodiment, nucleic acid-ligand complexes can be
formed in which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet
another embodiment, the nucleic acid can be targeted in vivo for cell specific
uptake and expression, by targeting a specific receptor (.see, e.g., PCT
Publications WO 92/06180 dated April 16, I 992 (Wu et a7. ); WO 92/22635 dated
December 23, 1992 (Wilson et al.); W092/20316 dated November 26, 1992
(Findeis et al.); W093/14188 dated July 22, 1993 (Clarke el al.), WO 93/20221
dated October 14, 1993 (Young)). Alternatively, the nucleic acid can be
introduced intracellularly and incorporated within host cell DNA for
expression,
by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci.
USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
In a specific embodiment, viral vectors that contains nucleic acid sequences
encoding an antibody of the invention are used. For example, a retroviral
vector
can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). These
retroviral vectors have been to delete retroviral sequences that are not
necessary
for packaging of the viral genome and integration into host cell DNA. The
nucleic
acid sequences encoding the antibody to be used in gene therapy are cloned
into
one or more vectors, which facilitates delivery of the gene into a patient.
More
detail about retroviral vectors can be found in Boesen et al., 1994,
Biotherapy
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6:291-302, which describes the use of a retroviral vector to deliver the mdrl
gene
to hematopoietic stem cells in order to make the stem cells more resistant to
chemotherapy. Other references illustrating the use of retroviral vectors in
gene
therapy are: Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al.,
1994,
Blood 83 :1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-
141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-
114.
Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses are especially attractive vehicles for delivering genes to
respiratory
epithelia. Adenoviruses naturally infect respiratory epithelia where they
cause a
mild disease. Other targets for adenovirus-based delivery systems are liver,
the
central nervous system, endothelial cells, and muscle. Adenoviruses have the
advantage ofbeing capable ofinfecting non-dividing cells. Kozarsky and Wilson,
1993, Current Opinion in Genetics and Development 3:499-503 present a review
of adenovirus-based gene therapy. Bout el al., I 994, Human Gene Therapy 5:3-
10 demonstrated the use of adenovirus vectors to transfer genes to the
respiratory
epithelia of rhesus monkeys. Other instances of the use of adenoviruses in
gene
therapy can be found in Rosenfeld et al., 1991, Science 252:431-434; Rosenfeld
e1 crl., 1992, Cell 68:143- 155; Mastrangeli et al., 1993, J. Clin. Invest.
91:225-
234; PCT Publication W094/12649; and Wang et al., 1995, Gene Therapy 2:775-
783. In a preferred embodiment, adenovirus vectors are used.
Adeno-associated virus (AAV) has also been proposed for use in gene
therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; U. S.
Patent
No. 5,43 6,146).
Another approach to gene therapy involves transferring a gene to cells in
tissue culture by such methods as electroporation, lipofection, calcium
phosphate
mediated transfection, or viral infection. Usually, the method of transfer
includes
the transfer of a selectable marker to the cells. The cells are then placed
under
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selection to isolate those cells that have taken up and are expressing the
transferred gene. Those cells are then delivered to a patient.
In this embodiment, the nucleic acid is introduced into a cell prior to
administration in vivo ofthe resulting recombinant cell. Such introduction can
be
carried out by any method known in the art, including but not limited to
transfection, electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell fusion,
chromosome-mediated gene transfer, microcell-mediated gene transfer,
spheroplast fusion, etc. Numerous techniques are known in the art for the
introduction offoreign genes into cells (.see, e.g., Loef~ler and Behr, 1993,
Meth.
Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Cline,
1985, Pharmac. Ther. 29:69-92) and may be used in accordance with the present
invention, provided that the necessary developmental and physiological
functions
of the recipient cells are not disrupted. The technique should provide for the
stable transfer ofthe nucleic acid to the cell, so that the nucleic acid is
expressible
by the cell and preferably heritable and expressible by its cell progeny.
The resulting recombinant cells can be delivered to a patient by various
methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or
progenitor cells) are preferably administered intravenously. The amount of
cells
envisioned for use depends on the desired ef~'ect, patient state, etc., and
can be
determined by one skilled in the art.
Cells into which a nucleic acid can be introduced for purposes of gene
therapy encompass any desired, available cell type, and include but are not
limited
to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle
cells,
hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes,
macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various
stem or progenitor cells, in particular hematopoietic stem or progenitor
cells, e.g.,
as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal
liver,
etc.
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In a preferred embodiment, the cell used for gene therapy is autologous to
the patient.
In an embodiment in which recombinant cells are used in gene therapy,
nucleic acid sequences encoding an antibody are introduced into the cells such
that
they are expressible by the cells or their progeny, and the recombinant cells
are
then administered in vivo for therapeutic effect. In a specific embodiment,
stem
or progenitor cells are used. Any stem and/or progenitor cells which can be
isolated and maintained ire vitro can potentially be used in accordance with
this
embodiment of the present invention (see, e.g., PCT Publication WO 94/08598,
dated April 28, 1994; Stemple and Anderson, I 992, Cell 71:973-985; Rheinwald,
1980, Meth. Cell Bio. 21 A:229; and Pittelkow and Scott, 1986, Mayo Clinic
Proc.
61:771).
In a specific embodiment, the nucleic acid to be introduced for purposes
of gene therapy comprises an inducible promoter operably linked to the coding
region, such that expression of the nucleic acid is controllable by
controlling the
presence or absence of the appropriate inducer of transcription.
Moles 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
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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.
The compositions of the invention may be administered alone or in
combination with other adjuvants. Adjuvants that may be administered with the
compositions of the invention include, but are not limited to, alum, alum plus
deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.),
BCG, and MPL. In a specific embodiment. compositions of the invention are
administered in combination with alum. In another specific embodiment,
compositions of the invention are administered in combination with QS-21.
Further adjuvants that may be administered with the compositions ofthe
invention
include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax
100a, QS-21, QS-I 8, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant
technology. Vaccines that may be administered with the compositions of the
invention include, but are not limited to, vaccines directed toward protection
against MMR (measles, mumps, rubella), polio, varicella, tetanus/diphtheria,
hepatitis A, hepatitis B, Haenaophilus inf72renzae B, whooping cough,
pneumonia,
influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese
encephalitis,
poliomyelitis, rabies, typhoid fever, and pertussis, and/or PNEUMOVAX-23T"".
Combinations may be administered either concomitantly, e.g., as an admixture,
separately but simultaneously or concurrently; or sequentially. This includes
presentations in which the combined agents are administered together as a
therapeutic mixture, and also procedures in which the combined agents are
administered separately but simultaneously, e.g., as through separate
intravenous
lines into the same individual. Administration "in combination" further
includes
the separate administration of one of the compounds or agents given first,
followed by the second.
In another specific embodiment, compositions of the invention are used in
combination with PNEUMOVAX-23T"~ to treat, prevent, and/or diagnose infection
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and/or any disease, disorder, and/or condition associated therewith. In one
embodiment, compositions of the invention are used in combination with
PNELJMOVAX-23T"~ to treat, prevent, and/or diagnose any Gram positive
bacterial infection and/or any disease, disorder, and/or condition associated
therewith. In another embodiment, compositions of the invention are used in
combination with PNELTMOVAX-23T"" to treat, prevent, and/or diagnose infection
and/or any disease, disorder, and/or condition associated with one or more
members of the genus E~terococcus and/or the genus Stneplococcus. In another
embodiment, compositions of the invention are used in any combination with
PNELJMOVAX-23T"" to treat, prevent, and/or diagnose infection and/or any
disease, disorder, and/or condition associated with one or more members of the
Group B streptococci. In another embodiment, compositions of the invention are
used in combination with PNELJMOVAX-23T"~ to treat, prevent, and/or diagnose
infection and/or any disease, disorder, and/or condition associated with
Streplococcu.s pneumoniae.
The compositions of the invention may be administered alone or in
combination with other therapeutic agents, including but not limited to, TNFs,
TNF blocking agents (e.g., antibodies which bind specifically to TNFs, such as
TNF-a, TNF-(3, or TNF-y), chemotherapeutic agents, antibiotics, antivirals,
steroidal and non-steroidal anti-inflammatories, conventional
immunotherapeutic
agents and cytokines. Combinations may be administered either concomitantly,
e.g., as an admixture, separately but simultaneously or concurrently; or
sequentially. This includes presentations in which the combined agents are
administered together as a therapeutic mixture, and also procedures in which
the
combined agents are administered separately but simultaneously, e.g., as
through
separate intravenous lines into the same individual. Administration "in
combination" further includes the separate administration ofone ofthe
compounds
or agents given first, followed by the second.
In one embodiment, the compositions of the invention are administered in
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combination with other members of the TNF family. TNF, TNF-related or
TNF-like molecules that may be administered with the compositions of the
invention include, but are not limited to, soluble forms of TNF, lymphotoxin
(LT,
also known as TNF-beta), LT-beta (found in complex heterotrimer LT 2-beta),
OPGL, Fast, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma
(International Publication No. WO 96/14328), AIM I (International Publication
No. WO 97/33899), AIM II (International Publication No. WO 97/34911),
APRIL (J. Exp. Med. 188(6):1185-1190), endokine (International Publication No.
WO 98/07880), TR6 (International Publication No. WO 98/30694), OPG
(International Publication No. WO 98/18921, OX40, and nerve growth factor
(NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2
(International Publication No. WO 96/34095), DR3 (International PublicationNo.
WO 97/33904), DR4 (International Publication No. WO 98/32856), TRS
(International Publication No. WO 98/30693), TR6 (International Publication
No.
WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK,
TR9 (International Publication No. WO 98/56892), TRIO (International
Publication No. WO 98/54202), 31X2 (International Publication No. WO
98/06842), TR12, and soluble forms CD I 54, CD70 and CD I 53.
In another embodiment, the compositions ofthe invention are administered
in combination with one or more TNF blocking agents. TNF blocking agents are
believed to be useful in the treatment of arthritis (e.g., rheumatoid
arthritis).
In a preferred embodiment, the compositions of the invention are
administered in combination with CD40 ligand (CD40L), a soluble form of
CD40L (e.g., AVRENDT""), biologically active fragments, variants, or
derivatives
of CD40L, anti-CD40L antibodies (e.g., agonistic or antagonistic antibodies),
and/or anti-CD40 antibodies (e.g., agonistic or antagonistic antibodies).
In certain embodiments, compositions ofthe invention are administered in
combination with antiretroviral agents, nucleoside reverse transcriptase
inhibitors,
non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors.
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Nucleoside reverse transcriptase inhibitors that may be administered in
combination with the compositions of the invention, include, but are not
limited
to, RETROVIRT"" (zidovudine/AZT), VIDEXT"" (didanosine/ddI), HIVIDT"~
(zalcitabine/ddC), ZERITT"~ (stavudine/d4T), EPIVIRT"~ (lamivudine/3TC), and
COMBIVIRT"" (zidovudine/lamivudine). Non-nucleoside reverse transcriptase
inhibitors that may be administered in combination with the compositions of
the
invention, include, but are not limited to, VIR.~~MUNETM (nevirapine),
RESCRIPTORT"" (delavirdine), and SUSTIVAT"" (efavirenz). Protease inhibitors
that may be administered in combination with the compositions of the
invention,
include, but are not limited to, CRIXIVANT"" (indinavir), NORVIRT""
(ritonavir),
INVIRASET"~ (saquinavir), and VIRACEPTT"" (nelfinavir). In a specific
embodiment, antiretroviral agents, nucleoside reverse transcriptase
inhibitors,
non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors
may be
used in any combination with compositions of the invention to treat, prevent,
and/or diagnose AIDS and/or to treat, prevent, and/or diagnose HIV infection.
In other embodiments, compositions ofthe invention may be administered
in combination with anti-opportunistic infection agents. Anti-opportunistic
agents
that may be administered in combination with the compositions of the
invention,
include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLET"~,
DAPSONETM, PENTAMIDINET"~, ATOVAQUONET"", ISONIAZIDT"~,
RIFAMPINT"~, PYRAZINAMIDET"", ETHAMBUTOLT"~, RIFABUTINT"~,
CLARITHROMYCINT"", AZITHROMYCINT"", GANCICLOVIRT"~,
FOSCARNETT"~, CIDOFOVIRT"", FLUCONAZOLET"", ITRACONAZOLET"",
KETOCONAZOLET"", ACYCLOVIRT~~, FAMCICOLVIRT"~,
PYRIMETHAMINET"~, LEUCOVORINT"", NEUPOGENT"" (filgrastim/G-CSF),
and LEUKINET~~ (sargramostim/GM-CSF). In a specific embodiment,
compositions of the invention are used in any combination with
TRIMETHOPRIM-SULFAMETHOXAZOLET"", DAPSONET"~,
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PENTAMIDINET"", and/or ATOVAQUONET"" to prophylactically treat, prevent,
and/or diagnose an opportunistic Pneumocysti.s carinii pneumonia infection. In
another specific embodiment, compositions of the invention are used in any
combination with ISONIAZIDT"", RIFAMPINT"", PYRAZINAMIDET"", and/or
ETHAMBUTOLT"~ to prophylactically treat, prevent, and/or diagnose an
opportunistic Mycobacterium avium complex infection. In another specific
embodiment, compositions of the invention are used in any combination with
RIFABUTINT"", CLARITHROMYC1NTM, and/or AZITHROMYCINT"~ to
prophylactically treat, prevent, and/or diagnose an
opportunisticMycobacteriuna
t7fberculo.si.s infection. In another specific embodiment, compositions of the
invention are used in any combination with GANCICLOVIRT"", FOSCARNETTM,
and/or CIDOFOVIRT"" to prophylactically treat, prevent, and/or diagnose an
opportunistic cytomegalovirus infection. In another specific embodiment,
compositions of the invention are used in any combination with
FLUCONAZOLET"", ITRACONAZOLET"", and/or KETOCONAZOLET"~ to
prophylactically treat, prevent, and/or diagnose an opportunistic fungal
infection.
In another specific embodiment, compositions of the invention are used in any
combination with ACYCLOVIRT"~ and/or FAMCICOLVIRT"" to prophylactically
treat, prevent, and/or diagnose an opportunistic herpes simplex virus type I
and/or
type II infection. In another specific embodiment, compositions of the
invention
are used in any combination with PYRINN>ETHAMINET"" and/or LEUCOVORINr"~
to prophylactically treat, prevent, and/or diagnose an opportunistic
Toxoplasma
gor~dii infection. In another specific embodiment, compositions of the
invention
are used in any combination with LEUCOVORINT"~ and/or NEUPOGENT"" to
prophylactically treat, prevent, and/or diagnose an opportunistic bacterial
infection.
In a further embodiment, the compositions of the invention are
administered in combination with an antiviral agent. Antiviral agents that may
be
administered with the compositions of the invention include, but are not
limited
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to, acyclovir, ribavirin, amantadine, and remantidine.
In a further embodiment, the compositions of the invention are
administered in combination with an antibiotic agent. Antibiotic agents that
may
be administered with the compositions ofthe invention include, but are not
limited
to, amoxicillin, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases,
Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin,
erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins,
quinolones, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim,
trimethoprim-sulfamthoxazole, and vancomycin.
Conventional nonspecific immunosuppressive agents, that may be
administered in combination with the compositions of the invention include,
but
are not limited to, steroids, cyclosporine, cyclosporine analogs,
cyclophosphamide, cyclophosphamide IV, methylprednisolone, prednisolone,
azathioprine, FK-506, I S-deoxyspergualin, and other immunosuppressive agents
that act by suppressing the function of responding T cells.
In specific embodiments, compositions of the invention are administered
in combination with immunosuppressants. Immunosuppressants preparations that
may be administered with the compositions of the invention include, but are
not
limited to, ORTHOCLONET"~ (OKT3), SANDIMMUNETM/NEORALTM~
SANGDYAT"" (cyclosporin), PROGRAFT"~ (tacrolimus), CELLCEPTT""
(mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNET"~
(sirolimus). In a specific embodiment, immunosuppressants may be used to
prevent rejection of organ or bone marrow transplantation.
In a preferred embodiment, the compositions of the invention are
administered in combination with steroid therapy. Steroids that may be
administered in combination with the compositions of the invention, include,
but
are not limited to, oral corticosteroids, prednisone, and methylprednisolone
(e.g.,
IV methylprednisolone). In a specific embodiment, compositions ofthe invention
are administered in combination with prednisone. In a further specific
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embodiment, the compositions of the invention are administered in combination
with prednisone and an immunosuppressive agent. Immunosuppressive agents
that may be administered with the compositions of the invention and prednisone
are those described herein, and include, but are not limited to, azathioprine,
cylophosphamide, and cyclophosphamide IV. In a another specific embodiment,
compositions of the invention are administered in combination with
methylprednisolone. In a further specific embodiment, the compositions of the
invention are administered in combination with methylprednisolone and an
immunosuppressive agent. Immunosuppressive agents that may be administered
with the compositions ofthe invention and methylprednisolone are those
described
herein, and include, but are not limited to, azathioprine, cylophosphamide,
and
cyclophosphamide IV.
In a preferred embodiment, the compositions of the invention are
administered in combination with an antimalarial. Antimalarials that may be
administered with the compositions of the invention include, but are not
limited
to, hydroxychloroquine, chloroquine, and/or quinacrine.
In a preferred embodiment, the compositions of the invention are
administered in combination with an NSAID.
In a nonexclusive embodiment, the compositions of the invention are
administered in combination with one, two, three, four, five, ten, or more of
the
following drugs: NRD-101 (Hoechst Marion Roussel), diclofenac (Dimethaid),
oxaprozin potassium (Monsanto), mecasermin (Chiron), T-614 (Toyama),
pemetrexed disodium (Eli Lilly), atreleuton (Abbott), valdecoxib (Monsanto),
eltenac (Byk Gulden), campath, AGM-1470 (Takeda), CDP-571 (Celltech
Chiroscience), CM-101 (CarboMed), ML-3000 (Merckle), CB-2431 (KS
Biomedix), CBF-BS2 (KS Biomedix), IL-1Ra gene therapy (Valentis), JTE-522
(Japan Tobacco), paclitaxel (Angiotech), DW-166HC (Doug Wha), darbufelone
mesylate (Warner-Lambent), soluble TNF receptor 1 (synergen; Amgen),
IPR-6001 (Institute for Pharmaceutical Research), trocade (Ho~man-La Roche),
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EF-5 (Scotia Pharmaceuticals), BIIL-284 (Boehringer Ingelheim), BIIF-1149
(Boehringer Ingelheim), LeukoVax (Inflammatics), MK-663 (Merck), ST-1482
(Sigma-Tau), and butixocort propionate (Warner-Lambert).
In a preferred embodiment, the compositions of the invention are
administered in combination with one, two, three, four, five or more of the
following drugs: methotrexate, sulfasalazine, sodium aurothiomalate,
auranofin,
cyclosporine, penicillamine, azathioprine, an antimalarial drug (e.g., as
described
herein), cyclophosphamide, chlorambucil, gold, ENBRELT"~ (Etanercept),
anti-TNF antibody, and prednisolone.
In a more preferred embodiment, the compositions of the invention are
administered in combination with an antimalarial, methotrexate, anti-TNF
antibody, ENBRELT"~ and/or suflasalazine. In one embodiment, the compositions
of the invention are administered in combination with methotrexate. In another
embodiment, the compositions of the invention are administered in combination
I S with anti-TNF antibody. In another embodiment, the compositions of the
invention are administered in combination with methotrexate and anti-TNF
antibody. In another embodiment, the compositions of the invention are
administered in combination with suflasalazine. In another specific
embodiment,
the compositions of the invention are administered in combination with
methotrexate, anti-TNF antibody, and suflasalazine. In another embodiment, the
compositions of the invention are administered in combination ENBRELT"'. In
another embodiment, the compositions of the invention are administered in
combination with ENBRELT"~ and methotrexate. In another embodiment, the
compositions of the invention are administered in combination with ENBRELT"~,
methotrexate and suflasalazine. In another embodiment, the compositions of the
invention are administered in combination with ENBRELT"", methotrexate and
suflasalazine. In other embodiments, one or more antimalarial is combined with
one of the above-recited combinations. In a specific embodiment, the
compositions ofthe invention are administered in combination with an
antimalarial
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(e.g., hydroxychloroquine), ENBRELT"", methotrexate and suflasalazine. In
another specific embodiment, the compositions of the invention are
administered
in combination with an antimalarial (e.g., hydroxychloroquine), sulfasalazine,
anti-TNF antibody, and methotrexate.
In an additional embodiment, compositions of the invention are
administered alone or in combination with one or more intravenous immune
globulin preparations. Intravenous immune globulin preparations that may be
administered with the compositions of the invention include, but not limited
to,
GAMMARTM, IVEEGAMT~~, SANDOGLOBULINT"~, GAMMAGARD S/DT"~,
and GAMIMUNET"~. In a specific embodiment, compositions of the invention are
administered in combination with intravenous immune globulin preparations in
transplantation therapy (e.g., bone marrow transplant).
In an additional embodiment, the compositions of the invention are
administered alone or in combination with an anti-inflammatory agent.
Anti-inflammatory agents that may be administered with the compositions ofthe
invention include, but are not limited to, glucocorticoids and the
nonsteroidal
anti-inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid
derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic
acid
derivatives, pyrazoles, pyrazolones, salicylic acid derivatives,
thiazinecarboxamides, E-acetamidocaproic acid, S-adenosylmethionine,
3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome,
difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide,
orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole,
and
tenidap.
In another embodiment, compositions of the invention are administered
in combination with a chemotherapeutic agent. Chemotherapeutic agents that
may be administered with the compositions of the invention include, but are
not
limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin,
daunorubicin, and
dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g.,
fluorouracil,
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5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid,
plicamycin,
mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU,
lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine,
hydroxyurea, procarbazine, mitomycin C, busulfan, cis-platin, and vincristine
sulfate); hormones (e.g., medroxyprogesterone, estramustine phosphate sodium,
ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone,
diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen
mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogen
mustard) and thiotepa); steroids and combinations (e.g., bethamethasone sodium
phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine
sulfate, vinblastine sulfate, and etoposide).
In a specific embodiment, compositions of the invention are administered
in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and
prednisone) or any combination of the components of CHOP. In another
embodiment, compositions ofthe invention are administered in combination with
Rituximab. In a further embodiment, compositions of the invention are
administered with Rituxmab and CHOP, or Rituxmab and any combination ofthe
components of CHOP.
In an additional embodiment, the compositions of the invention are
administered in combination with cytokines. Cytokines that may be administered
with the compositions of the invention include, but are not limited to, GM-
CSF,
G-CSF, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15,
anti-CD40, CD40L, IFN, IFN-beta, IFN-gamma, TNF, and TNF-beta. In another
embodiment, compositions of the invention may be administered with any
interleukin, including, but not limited to, IL-lalpha, IL-lbeta, IL-2, IL-3,
IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,
IL-17, IL-18, IL-19, IL-20, IL-21, and IL-22. In preferred embodiments, the
compositions of the invention are administered in combination with IL-4 and
IL-10. Both IL-4 and IL-10 have been observed by the inventors to enhance TR2
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mediated B cell proliferation.
In an additional embodiment, the compositions of the invention are
administered with a chemokine. In another embodiment, the compositions of the
invention are administered with chemokine beta-8, chemokine beta-1, and/or
S macrophage inflammatory protein-4. In a preferred embodiment, the
compositions of the invention are administered with chemokine beta-8.
In an additional embodiment, the compositions of the invention are
administered in combination with an IL-4 antagonist. IL-4 antagonists that may
be administered with the compositions ofthe invention include, but are not
limited
to: soluble IL-4 receptor polypeptides, multimeric forms of soluble IL-4
receptor
polypeptides; anti-IL-4 receptor antibodies that bind the IL-4 receptor
without
transducing the biological signal elicited by IL-4, anti-IL4 antibodies that
block
binding of IL-4 to one or more IL-4 receptors, and muteins of IL-4 that bind
IL-4
receptors but do not transduce the biological signal elicited by IL-4.
Preferably,
1 S the antibodies employed according to this method are monoclonal antibodies
(including antibody fragments, such as, for example, those described herein).
In an additional embodiment, the compositions of the invention are
administered in combination with hematopoietic growth factors. Hematopoietic
growth factors that may be administered with the compositions of the invention
include, but are not limited to, LELJKINET"~ (SARGRAMOSTIMT"~) and
NEUPOGENT"" (FILGRASTIMT"").
In an additional embodiment, the compositions of the invention are
administered in combination with angiogenic proteins. Angiogenic proteins that
may be administered with the compositions of the invention include, but are
not
limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European
Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as
disclosed in European Patent Number EP-682110; Platelet Derived Growth
Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317;
Placental Growth Factor (P1GF), as disclosed in International Publication
Number
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WO 92/06194; Placental Growth Factor-2 (P1GF-2), as disclosed in Hauser et
al.,
Gorwth Factors, 4:259-268 ( I 993); Vascular Endothelial Growth Factor (VEGF),
as disclosed in International Publication Number WO 90/13649; Vascular
Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent
Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as
disclosed in International Publication Number WO 96/39515; Vascular
Endothelial
Growth Factor B-186 (VEGF-B186), as disclosed in International Publication
Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as
disclosed in International Publication Number WO 98/02543; Vascular
Endothelial
Growth Factor-D (VEGF-D), as disclosed in International Publication Number
WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as
disclosed in German Patent Number DE19639601. The above mentioned
references are incorporated herein by reference herein.
In an additional embodiment, the compositions of the invention are
administered in combination with Fibroblast Growth Factors. Fibroblast Growth
Factors that may be administered with the compositions of the invention
include,
but are not limited to, FGF-I, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7,
FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
In additional embodiments, the compositions of the invention are
administered in combination with other therapeutic or prophylactic regimens,
such as, for example, radiation therapy. Such therapy may be administered
ssequentially and/or concomitantly
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 pg/kg/day to 10 mg/kg/day of patient body weight, although, as noted
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above, this will be subject to therapeutic discretion. More preferably, this
dose
is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01
and 1 mg/kg/day for the hormone. If given continuously, the TR2 polypeptide
is typically administered at a dose rate of about 1 pg/kg/hour to about 50
~g/kg/hour, either by 1-4 injections per day or by continuous subcutaneous
infusions, for example, using a mini-pump. An intravenous bag solution may
also
be employed.
The invention provides methods of treatment, inhibition and prophylaxis
by administration to a subject of an effective amount of a compound or
pharmaceutical composition ofthe invention (e.g., an antibody ofthe
invention).
In a preferred aspect, the compound is substantially purified (e.g.,
substantially
free from substances that limit its effect or produce undesired side-effects).
The
subject is preferably an animal, including but not limited to animals such as
cows,
pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most
preferably human.
Formulations and methods of administration that can be employed when
the compound comprises a nucleic acid or an immunoglobulin are described
above; additional appropriate formulations and routes of administration can be
selected from among those described herein below.
Pharmaceutical compositions containing the TR2 receptor polypeptides
ofthe 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.
The composition, if desired, can also contain minor amounts of wetting
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or emulsifying agents, or pH buffering agents. These compositions can take the
form of solutions, suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like.
TR2 compositions of the invention are also suitably administered by
sustained-release systems. Suitable examples of sustained-release compositions
include suitable polymeric materials (such as, for example, semi-permeable
polymer matrices in the form of shaped articles, e.g., films, or
mirocapsules),
suitable hydrophobic materials (for example as an emulsion in an acceptable
oil)
or ion exchange resins, and sparingly soluble derivatives (such as, for
example,
a sparingly soluble salt).
Various delivery systems are known and can be used to administer a
compound of the invention, e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant cells capable of expressing the compound, receptor-
mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-
1 S 4432), construction of a nucleic acid as part of a retroviral or other
vector, etc.
Methods of introduction include but are not limited to intradermal,
intramuscular,
intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral
routes.
The compounds or compositions may be administered by any convenient route,
for example by infusion or bolus injection, by absorption through epithelial
or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.)
and
may be administered together with other biologically active agents.
Administration can be systemic or local. In addition, it may be desirable to
introduce the pharmaceutical compounds or compositions of the invention into
the central nervous system by any suitable route, including intraventricular
and
intrathecal injection; intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir, such as an
Ommaya
reservoir. Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the
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pharmaceutical compounds or compositions of the invention locally to the area
in need of treatment; this may be achieved by, for example, and not by way of
limitation, local infusion during surgery, topical application, e.g., in
conjunction
with a wound dressing after surgery, by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant being of a
porous, non-porous, or gelatinous material, including membranes, such as
sialastic membranes, or fibers. Preferably, when administering a protein,
including an antibody, of the invention, care must be taken to use materials
to
which the protein does not absorb.
In another embodiment, the compound or composition can be delivered
in a vesicle, in particular a liposome (see Larger, 1990, Science 249:1527-
1533;
Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer,
Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-
Berestein, ibid., pp. 317-327; see generally ibid.)
I S In yet another embodiment, the compound or composition can be
delivered in a controlled release system. In one embodiment, a pump may be
used (see Larger, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201;
Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med.
321:574). In another embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, Larger and Wise (eds.), CRC Pres., Boca
Raton, Florida ( 1974); Controlled Drug Bioavailability, Drug Product Design
and
Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and
Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy
et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351;
Howard
et al., 1989, J. Neurosurg. 71:105). In yet another embodiment, a controlled
release system can be placed in proximity of the therapeutic target, i. e. ,
the brain,
thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in
Medical
Applications of Controlled Release, supra, vol. 2, pp. 115-13 8 ( 1984)).
Sustained-release matrices include polylactides (U. S. Pat. No. 3,773,919,
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EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate
(Sidman, U. et al., Biopolymers 22:547-556 (1983)), poly (2- hydroxyethyl
methacrylate) (R. Langer et al., J. Biomed. Mater. Res. I5: I 67-277 ( 1981 ),
and
R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et
al., Id.) or poly-D- (-)-3-hydroxybutyric acid (EP 133,988).
Sustained-release compositions also include liposomally entrapped
compositions of the invention (see generally, Langer, Science 249:1527-1533
(1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and
Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317 -327 and
353-365 (1989)).. Liposomes containing TR2 polypeptide my be prepared by
methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci.
(USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA)
77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP
142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and
4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about
200-800 Angstroms) unilamellar type in which the lipid content is greater than
about 30 mol. percent cholesterol, the selected proportion being adjusted for
the
optimal TR2 polypeptide therapy.
In yet an additional embodiment, the compositions of the invention are
delivered by way of a pump (see Langer, supra; Sefton, CRC C~°it. Ref.
Biomed
Errg. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N.
Engd. J Med 321:574 ( 1989)).
Other controlled release systems are discussed in the review by Langer
(Science 29:1527-1533 (1990)).
In a specific embodiment where the compound of the invention is a
nucleic acid encoding a protein, the nucleic acid can be administered in vivo
to
promote expression of its encoded protein, by constructing it as part of an
appropriate nucleic acid expression vector and administering it so that it
becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Patent No.
4,980,286),
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or by direct injection, or by use of microparticle bombardment (e.g., a gene
gun;
Biolistic, Dupont), or coating with lipids or cell-surface receptors or
transfecting
agents, or by administering it in linkage to a homeobox- like peptide which is
known to enter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad.
Sci.
USA 88:1864-1868), etc. Alternatively, a nucleic acid can be introduced
intracellularly and incorporated within host cell DNA for expression, by
homologous recombination.
The present invention also provides pharmaceutical compositions. Such
compositions comprise a therapeutically effective amount of a compound, and a
pharmaceutically acceptable carrier. In a specific embodiment, the term
"pharmaceutically acceptable" means approved by a regulatory agency of the
Federal or a state government or listed in the U. S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more particularly in
humans. The term "carrier" refers to a diluent, adjuvant, excipient, or
vehicle
with which the therapeutic is administered. Such pharmaceutical carriers can
be
sterile liquids, such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame
oil and the like. Water is a preferred carrier when the pharmaceutical
composition is administered intravenously. Saline solutions and aqueous
dextrose
and glycerol solutions can also be employed as liquid carriers, particularly
for
injectable solutions. Suitable pharmaceutical excipients include starch,
glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium
stearate,
glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,
propylene, glycol, water, ethanol and the like. The composition, if desired,
can
also contain minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions, suspensions,
emulsion, tablets, pills, capsules, powders, sustained-release formulations
and the
like. The composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can include
standard
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