Note: Descriptions are shown in the official language in which they were submitted.
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HUMAN TUMOR NECROSIS FACTOR RECEPTOR TR9
Field of the Invention
The present invention relates to a novel member of the tumor necrosis factor
family of receptors. More specifically, isolated nucleic acid molecules are
provided
encoding a novel human tumor necrosis factor receptor, TR9 (also known as
Death
Domain Containing Receptor 6, or simply DR6). TR9 polypeptides are also
provided, as
are vectors, host cells and recombinant methods for producing the same. The
invention
also relates to both the inhibition and enhancement of the activities of TR9
receptor
polypeptides and diagnostic methods for detecting TR9 receptor gene
expression. The
invention further relates to screening methods for identifying agonists and
antagonists of
TR9 activity.
Background of the Invention
Many biological actions, for instance, response to certain stimuli and natural
biological processes, are controlled by factors, such as cytokines. Many
cytokines act
through receptors by engaging the receptor and producing an intra-cellular
response.
For example, tumor necrosis factors (TNF) alpha and beta are cytokines, which
act through TNF receptors to regulate numerous biological processes, including
protection against infection and induction of shock and inflammatory disease.
The TNF
molecules belong to the "TNF-ligand" superfamily, and act together with their
receptors
or counter-ligands, the "TNF-receptor" superfamily. So far, nine members of
the TNF
ligand superfamily have been identified and ten members of the TNF-receptor
superfamily
have been characterized.
Among the ligands there are included TNF-alpha, lymphotoxin-alpha (LT-alpha,
also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-
beta),
Fast, CD40L, CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor (NGF). The
superfamily of TNF receptors includes the p55TNF receptor, p75TNF receptor,
TNF
receptor-related protein, FAS antigen or APO-l, CD40, CD27, CD30, 4-1BB, OX40,
low affinity p75 and NGF-receptor (Meager, A., Biologicals 22:291-295 (1994)).
Many members of the TNF-ligand superfamily are expressed by activated T-cells,
implying that they are necessary for T-cell interactions with other cell types
which
underlie cell ontogeny and functions. (Meager, A., supra).
Considerable insight into the essential functions of several members of the
TNF
receptor family has been gained from the identification and creation of
mutants that
abolish the expression of these proteins. For example, naturally occurring
mutations in
the FAS antigen and its ligand cause lymphoproliferative disease (Watanabe-
Fukunaga. et
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al., Nature 356:314 ( 1992)), perhaps reflecting a failure of programmed cell
death.
Mutations of the CD40 ligand cause an X-linked immunodeficiency state
characterized by
high levels of immunoglobulin M and low levels of immunoglobulin G in plasma,
indicating faulty T-cell-dependent B-cell activation (Allen et al., Science
259:990 (1993)).
Targeted mutations of the low affinity nerve growth factor receptor cause a
disorder
characterized by faulty sensory innovation of peripheral structures (Lee et
al., Cell 69:737
( 1992)).
TNF and LT-alpha are capable of binding to two TNF receptors (the 55- and 75-
kd TNF receptors). A large number of biological effects elicited by TNF and LT-
alpha,
acting through their receptors, include hemorrhagic necrosis of transplanted
tumors,
cytotoxicity, a role in endotoxic shock, inflammation, immunoregulation,
proliferation
and anti-viral responses, as well as protection against the deleterious
effects of ionizing
radiation. TNF and LT-alpha are involved in the pathogenesis of a wide range
of
diseases, including endotoxic shock, cerebral malaria, tumors, autoimmune
disease,
AIDS and graft-host rejection (Beutler et al., ScieJZCe 264:667-668 (1994)).
Mutations in
the p55 receptor cause increased susceptibility to microbial infection.
Moreover, an about 80 amino acid domain near the C-terminus of TNFRI (p55)
and Fas was reported as the "death domain," which is responsible for
transducing signals
for programmed cell death (Tartaglia et al., Cell 74:845 ( 1993)).
Apoptosis, or programmed cell death, is a physiologic process essential to the
normal development and homeostasis of multicellular organisms (Steller,
Science
267:1445-1449 (1995)). Derangements of apoptosis contribute to the
pathogenesis of
several human diseases including cancer, neurodegenerative disorders, and
acquired
immune deficiency syndrome (Thompson C. B., Science 267:1456-1462 (1995)).
Recently, much attention has focused on the signal transduction and biological
function of
two cell surface death receptors, Fas/APO-1 and TNFR-1 (Cleveland et al., Cell
81:479-
482 (1995); Fraser et al., Cell 85:781-784 (1996); S. Nagata et al., Scie~zce
267:1449-56
(1995)). Both are members of the TNF receptor family, which also include TNFR-
2,
low affinity NGFR, CD40, and CD30, among others (Smith et al., Science
248:1019-23
( 1990); Tewari et al., in Modular- Texts in Molecular a~2d Cell Biology; M.
Purton,
Heldin, Carl, Ed. (Chapman and Hall, London, 1995). While family members are
defined by the presence of cysteine-rich repeats in their extracellular
domains, Fas/APO-1
and TNFR-1 also share a region of intracellular homology, appropriately
designated the
"death domain," which is distantly related to the Drosoplzila suicide gene,
reaper (Golstein
et al., Cell 81:185-6 ( 1995); White et al., Science 264:677-83 ( 1994)). This
shared death
domain suggests that both receptors interact with a related set of signal
transducing
molecules that, until recently, remained unidentified. Activation of Fas/APO-1
recruits
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WO 00/56862 PCT/US00/06831
-,
the death domain-containing adapter molecule FADD/MORT 1 (Chinnaiyan et cil.;
Cell
81:505-512 ( 1995); Boldin et al., J. Biol. Chem. 270:7795-8 (1995); Kischkel
et al.,
EMBO 14:5579-5588 ( 1995)), which in turn binds and presumably activates
FLICE/MACH1, a member of the ICE/CED-3 family of pro-apoptotic proteases
(Muzio et
al., Cell 85:817-827 ( 1996); Boldin et al., Cell 85:803-815 ( 1996)). While
the central
role of Fas/APO-1 is to trigger cell death, TNFR-1 can signal an array of
diverse
biological activities-many of which stem from its ability to activate NF-
kappaB (Tartaglia
et al., hnmurZOl Today 13:151-153 (1992)). Accordingly, TNFR-1 recruits the
multivalent adapter molecule TRADD, which like FADD, also contains a death
domain
(Hsu et al., Cell 81:495-504 (1995): Hsu et al., Cell 84:299-308 (1996)).
Through its
associations with a number of signaling molecules including FADD, TRAF2, and
RIP,
TRADD can signal both apoptosis and NF-kappaB activation (Hsu et al., Cell
84:299-308
(1996); Hsu et al., hnfnunity 4:387-396 (1996)).
The effects of TNF family ligands and receptors are varied and influence
numerous functions, both normal and abnormal, in the biological processes of
the
mammalian system. There is a clear need, therefore, for identification and
characterization of additional novel TNF receptors and ligands that influence
biological
activity, both normally and in disease states.
Summary of the Invention
The present invention provides isolated nucleic acid molecules, or
alternatively
consisting of, a polynucleotide encoding the TR9 receptor having the amino
acid sequence
shown in Figures lA-D (SEQ ID N0:2) or the amino acid sequence encoded by the
eDNA clone deposited as ATCC Deposit Number 209037 on May 15, 1997.
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 TR9 receptor polypeptides or peptides by
recombinant
techniques.
The invention further provides an isolated TR9 polypeptide having an amino
acid
sequence encoded by a polynucleotide described herein.
In certain embodiments, TR9 polypeptides of the invention, or agonists
thereof,
are administered, to treat, prevent, prognose and/or diagnose an
immunodeficiency (e.g.,
severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine
deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA),
Breton's
disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia,
acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset
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1
agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient
hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia,
agammaglobulinemia, common variable immunodeficiency (CVID) (acquired),
Wiskott-
Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IaM, non X-linked
immunodeficiency with hyper IgM, selective IaA deficiency, IgG subclass
deficiency
(with or without IaA 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.) or
conditions
associated with an immunodeficiency.
In a specific embodiment, TR9 polypeptides or polynucleotides of the
invention,
or agonists thereof, is administered to treat, prevent, prognose and/or
diagnose common
variable immunodeficiency.
In a specific embodiment, TR9 polypeptides or polynucleotides of the
invention,
or agonists thereof, is administered to treat, prevent, prognose and/or
diagnose X-linked
agammaglobulinemia.
In another specific embodiment, TR9 polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent, prognose
and/or diagnose
severe combined immunodeficiency (SCID).
In another specific embodiment, TR9 polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent, prognose
and/or' diagnose
Wiskott-Aldrich syndrome.
In another specific embodiment, TR9 polypeptides or polynucleotides of the
invention, or agonists thereof, is administered to treat, prevent, prognose
and/or diagnose
X-linked Ig deficiency with hyper IgM.
In another embodiment, TR9 antagonists (e.g., an anti-TR9 antibody), are
administered to treat, prevent, prognose and/or diagnose an autoimmune disease
(e.g.,
rheumatoid arthritis, systemic lupus erhythematosus, idiopathic
thrombocytopenia
purpura, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia,
autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis,
allergic
encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart
disease,
glomerulonephritis (e.g, IgA nephropathy), Multiple Sclerosis, Neuritis,
Uveitis
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Ophthahnia, 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,
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 , 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, and other inflammatory,
granulamatous, degenerative, and atrophic disorders) or conditions associated
with an
autoimmune disease. In a specific preferred embodiment, rheumatoid arthritis
is treated,
prevented, prognosed and/or diagnosed using anti-TR9 antibodies andlor other
antagonist
of the invention. In another specific preferred embodiment, systemic lupus
erythemosus
is treated, prevented, prognosed, and/or diagnosed using anti-TR9 antibodies
and/or other
antagonist of the invention. In another specific preferred embodiment,
idiopathic
thrombocytopenia purpura is treated, prevented, prognosed, and/or diagnosed
using anti-
TR9 antibodies and/or other antagonist of the invention. In another specific
preferred
embodiment IgA nephropathy is treated, prevented, prognosed and/or diagnosed
using
anti-TR9 antibodies and/or other antagonist of the invention. In a preferred
embodiment,
the autoimmune diseases and disorders and/or conditions associated with the
diseases and
disorders recited above are treated, prevented, prognosed and/or diagnosed
using anti-
TR9 antibodies.
The invention further provides compositions comprising a TR9 polynucleotide, a
TR9 polypeptide, and/or an anti-TR9 antibody, for administration to cells in
vitro, to cells
ex vivo, and to cells in vivo, or to a multicellular organism. In preferred
embodiments,
the compositions of the invention comprise a TR9 polynucleotide for expression
of a TR9
polypeptide in a host organism for treatment of disease. In a most preferred
embodiment,
the compositions of the invention comprise a TR9 polynucleotide for expression
of a TR9
polypeptide in a host organism for treatment of an immunodeficiency and/or
conditions
associated with an immunodeficiency. Particularly preferred in this regard is
expression
in a human patient for treatment of a dysfunction associated with aberrant
endogenous
activity of a TR9 gene (e.g., expression to enhance the normal B-cell function
by
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6
expanding B-cell numbers or increasing B cell lifespan; or expression to
enhance the
normal T cell function by expanding T cell numbers or increasing T cell
lifespan).
The present invention also provides a screening method for identifying
compounds capable of enhancing or inhibiting a cellular response induced by
the TR9
receptor. The method involves contacting cells which express the TR9 receptor
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 aaonists and antagonists is provided
which involves determining the effect a candidate compound has on the binding
of cellular
ligands to TR9 receptors. In particular, the method involves contacting TR9
receptors
with a ligand polypeptide and a candidate compound and determining whether
ligand
binding to the TR9 receptors is increased or decreased due to the presence of
the candidate
compound.
The invention further provides diagnostic assays such as quantitative and
diagnostic assays for detecting levels of TR9 receptor protein. Thus, for
instance, a
diagnostic assay in accordance with the invention for detecting over-
expression of TR9,
or soluble form thereof, compared to normal control tissue samples, may be
used to detect
the presence of tumors.
Tumor Necrosis Factor (TNF) family ligands are known to be among the most
pleiotropic cytokines, inducing a large number of cellular responses,
including
cytotoxicity, anti-viral activity, immunoregulatory activities, and the
transcriptional
regulation of several genes. Cellular response to TNF-family ligands include
not only
normal physiological responses, but also diseases associated with increased
apoptosis or
the inhibition of apoptosis. Apoptosis-programmed cell death-is a
physiological
mechanism involved in the deletion of peripheral T lymphocytes of the immune
system,
and its dysregulation can lead to a number of different pathogenic processes.
Diseases
associated with increased cell survival, or the inhibition of apoptosis,
include cancers,
autoimmune disorders, viral infections, inflammation, graft vs. host disease,
acute graft
rejection, and chronic graft rejection. Diseases associated with increased
apoptosis
include AIDS, neurodegenerative disorders, myelodysplastic syndromes, ischemic
injury,
toxin-induced liver disease, septic shock, cachexia, and anorexia.
Thus, the invention further provides a method for enhancing apoptosis induced
by
a TNF-family ligand, which involves administering to a cell which expresses
the TR9
polypeptide an effective amount of an agonist capable of increasing TR9
mediated
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7
signaling. Preferably, TR9 mediated signaling is increased to treat, prevent,
diagnose,
and/or detect a disease wherein decreased apoptosis is exhibited.
In a further aspect, the present invention is directed to a method for
inhibiting
apoptosis induced by a TNF-family ligand, which involves administering to a
cell which
expresses the TR9 polypeptide an effective amount of an antagonist capable of
decreasing
TR9 mediated signaling. Preferably. TR9 mediated signaling is decreased to
treat,
prevent, diagnose, and/or detect a disease wherein increased apoptosis is
exhibited.
Brief Descriptio~z of the Figures
Figures lA-D show the nucleotide sequence (SEQ ID NO:1 ) and deduced
amino acid sequence (SEQ ID N0:2) of the TR9 receptor. Analysis using the
computer
program PSORT reveals that the protein has a predicted leader sequence of
about 40
amino acid residues (underlined) and a deduced molecular weight of about 72
kDa. It is
further predicted that amino acid residues from about 41 to about 350
constitute the
extracellular domain (amino acid residues from about 1 to about 310 in SEQ ID
N0:2);
from about 351 to about 367 the transmembrane domain (amino acid residues from
about
311 to about 327 in SEQ ID N0:2); from about 368 to about 655 the
intracellular domain
(amino acid residues from about 328 to about 615 in SEQ ID N0:2); and from
about 429
to about 495 the death domain (amino acid residues from about 389 to about 455
in SEQ
ID N0:2).
Figure 2 shows the regions of similarity between the amino acid sequences of
the TR9 receptor (SEQ ID N0:2) and Fas (SEQ ID N0:3), NGFR p75 (SEQ ID N0:4),
and TNFR 1 (SEQ ID NO:S). Residues that match the consensus are shaded.
Figure 3 shows an analysis of the TR9 amino acid sequence. Alpha, beta, turn
and coil regions; hydrophilicity and hydrophobicity; amphipathic regions;
flexible regions;
antigenic index and surface probability are shown, as predicted for the amino
acid
sequence depicted in Figures 1 A-D using the default parameters of the recited
computer
programs. In the "Antigenic Index - Jameson-Wolf" Graph, amino acid residues
about 44
to about 121, about 156 to about 31 l, about 323 to about 348, about 376 to
about 412,
about 433 to about 474, about 485 to about 599, and about 611 to about 628 in
Figures
lA-D correspond to the shown highly antigenic regions of the TR9 protein.
These highly
antigenic fragments in Figures 1 A-D correspond to the following fragments,
respectively,
in SEQ ID N0:2: amino acid residues about 4 to about 81, about 116 to about
271, about
283 to about 308, about 336 to about 372, about 393 to about 434, about 445 to
about
559, and about 571 to about 588.
Figures 4A-C. Highlight the predicted amino acid sequence of TR9.
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8
Figure 4A: The open reading frame for TR9 defines a type I transmembrane
protein of
655 amino acids (SEQ ID N0:2). Application of a computer program other than
PSORT
has predicted the mature protein to start at amino acid 42 (Gln, indicated by
a black
triangle). The putative signal peptide and transmembrane domain are single and
double
underlined, respectively. Six potential N-glycosylation sites are indicated by
black dots.
The cytoplasmic death domain is boxed. An intracellular region containing a
potential
leucine-zipper motif overlapping with a proline rich sequence is underlined
with a thick
line. Figure 4B: Sequence alignment of extracellular cysteine-rich domains of
TR9
(SEQ ID N0:19) and osteoprotegerin (SEQ ID N0:20). Alignment was done with
Megalign (DNASTAR) software. Shading represents identical residues. Figure 4C:
Sequence comparison of death domains of TR9 (SEQ ID N0:21 ), CD95 (SEQ ID
N0:22), TNFR1 (SEQ ID N0:23), DR3 (SEQ ID N0:24), DR4 (SEQ ID N0:25), and
DRS (SEQ ID N0:26). Alignment was performed and represented in the same way as
in
Figure 4B. OPG; osteoprotegerin.
Figure 5. TR9 induces apoptosis in mammalian cells. Ectopic expression of
TR9 induces apoptosis in Hela cells, but not in MCF7 cells. Hela and MCF7
cells were
cotransfected with a empty vector, TR9, TR9 delta, or DR4, together with a
beta-
galactosidase-expressing reporter construct using a lipofectamine method
according to the
manufacturer's instructions (BRL). Nineteen hours after transfection, cells
were stained
with 5-bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside (X-Gal) and examined
as
described in Chinnaiyan et al., Cell 81:505-512 (1995). The data (mean ~ SD)
represent
the percentage of round, apoptotic cells as a function of total beta-
galactosidase-positive
cells (n=4).
Figure 6. TR9 mediates nuclear factor NF-kappaB activation. Cotransfection
of 293 cells was performed with the indicated expression constructs and a NF-
kappaB
luciferase reporter construct. After transfection (at 36 hours), cell extracts
were prepared
and luciferase activities determined as previously described (Chinnaiyan et
al., Science
274:990-992 (1996); and Pan et al., Science 276:1 I l-113 (1997)).
Transfection
efficiency was monitored by beta-galactosidase activity. A portion of the
transfected cells
was used to monitor expression of TR9 or TR9 delta. Cell lysates were prepared
and
immunoprecipitated with FLAG M2 affinity gel and the presence of TR9 or TR9
delta
detected by blotting with anti-FLAG.
Detailed Description of tlae Preferred Embodiments
The present invention provides isolated nucleic acid molecules, or
alternatively
consisting of, a polynucleotide encoding a TR9 receptor polypeptide having the
amino
acid sequence shown in Figures lA-C (SEQ ID N0:2), which was determined by
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9
sequencing a cloned cDNA. As shown in Figure 2, the TR9 receptor protein of
the
present invention shares sequence homology with Fas (SEQ ID N0: 3 ), NGFR p75
(SEQ
ID N0:4), and TNFR 1 (SEQ ID NO:S). The nucleotide sequence shown in SEQ ID
NO: l was obtained by sequencing a cDNA clone, which was deposited on May 15,
1997
at the American Type Culture Collection, 10801 University Boulevard. Manassas,
Virginia, 20110-2209, and given accession number 209037. The deposited clone
is
inserted in the pBluescript SK(-) plasmid (Stratagene, LaJolla, CA) using the
EcoRI and
XhoI restriction endonuclease cleavage sites.
Nucleic Acid Molecules
Unless otherwise indicated, all nucleotide sequences determined by sequencing
a
DNA molecule herein were determined using an automated DNA sequencer (such as
the
Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of
polypeptides
encoded by DNA molecules determined herein were predicted by translation of a
DNA
sequence determined as above. Therefore, as is known in the art for any DNA
sequence
determined by this automated approach, any nucleotide sequence determined
herein may
contain some errors. Nucleotide sequences determined by automation are
typically at least
about 90% identical, more typically at least about 95% to at least about 99.9%
identical to
the actual nucleotide sequence of the sequenced DNA molecule. The actual
sequence can
be more precisely determined by other approaches including manual DNA
sequencing
methods well known in the art. As is also known in the art, a single insertion
or deletion
in a determined nucleotide sequence compared to the actual sequence will cause
a frame
shift in translation of the nucleotide sequence such that the predicted amino
acid sequence
encoded by a determined nucleotide sequence will be completely different from
the amino
acid sequence actually encoded by the sequenced DNA molecule, beginning at the
point of
such an insertion or deletion.
Using the information provided herein, such as the nucleotide sequence in
Figures
IA-D (SEQ ID NO:I), a nucleic acid molecule of the present invention encoding
a TR9
polypeptide may be obtained using standard cloning and screening procedures,
such as
those for cloning cDNAs using mRNA as starting material. Illustrative of the
invention,
the nucleic acid molecule described in Figures lA-D (SEQ ID NO: l ) was
discovered in a
cDNA library derived from human microvascular endothelial cells. The gene was
also
identified in cDNA libraries from the following tissues: human placenta,
stromal cells,
human amygdala, human umbilical vein endothelial cells, kidney cancer, human
gall
bladder, snares adult brain, normal human liver, hepatocellular tumor,
keratinocytes,
bone marrow, macrophage, human synovial sarcoma, human hippocampus, and human
tonsils.
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The determined nucleotide sequence of the TR9 cDNA of Figures lA-D (SEQ ID
NO:1) contains an open reading frame encoding a protein of about 615 amino
acid
residues, with a predicted leader sequence of about 40 amino acid residues,
and a deduced
molecular weight of about 72 kDa. The amino acid sequence of the predicted
mature TR9
5 receptor is shown in Figures lA-D (SEQ ID N0:2) from amino acid residue
about 1 to
residue about 615. The TR9 protein shown in Figures 1 A-D (SEQ ID N0:2) is
about
24% identical and about 43% similar to NGFR (Figure 2).
As predicted by the sequence homology exhibited between TR9 and other death
domain containing receptors (see Figure 4C), TR9 induces apoptosis of
mammalian cells
10 (see Figure 6). It is expected that TR9-induced apoptosis will be
efficiently blocked by
inhibitors of death proteases including z-VAD-fmk, an irreversible broad
spectrum
caspase inhibitor and CrmA, a cowpox virus encoded serpin that preferentially
inhibits
apical caspases such as FLICE/MACH-1 (caspase-8).
As indicated, the present invention also provides the mature forms) of the TR9
receptor 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 a nucleotide sequence encoding the mature TR9 receptor polypeptides
having the
amino acid sequence encoded by the cDNA clone contained in the host identified
as
ATCC Deposit No. 209037 and as shown in Figures lA-D (SEQ ID N0:2). By the
mature TR9 protein having the amino acid sequence encoded by the cDNA clone
contained in the host identified as ATCC Deposit 209037 is meant the mature
forms) of
the TR9 receptor produced by expression in a mammalian cell (e.g., COS cells,
as
described below) of the complete open reading frame encoded by the human DNA
sequence of the clone contained in the vector in the deposited host. As
indicated below,
the mature TR9 receptor having the amino acid sequence encoded by the cDNA
clone
contained in ATCC Deposit No. 209037 may or may not differ from the predicted
"mature" TR9 receptor protein shown in SEQ ID N0:2 (amino acids from about 1
to
about 615) depending on the accuracy of the predicted cleavage site based on
computer
analysis.
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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 (Vircrs Res. 3:271-286 (1985)) and von Heinje (Nucleic Acids Res.
14:4683-
4690 ( 1986) ) can be used. The accuracy of predicting the cleavage points of
known
mammalian secretory proteins for each of these methods is in the range of 75-
80%. von
Heinje, supr~cs. However, the two methods do not always produce the same
predicted
cleavage points) for a given protein.
In the present case, the predicted amino acid sequence of the complete TR9
polypeptides of the present invention 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
site
between amino acid residues 40 and 41 in Figures lA-D (amino acid residues -1
and 1 in
SEQ ID N0:2). Thereafter, the complete amino acid sequences were further
analyzed by
visual inspection, applying a simple form of the (-l,-3) rule of von Heinje.
von Heinje,
supra. Thus, the leader sequence for the TR9 receptor protein is predicted to
consist of
amino acid residues from about 1 to 40 in Figures lA-D (amino acid residues -
40 to about
-1 in SEQ ID N0:2), while the mature TR9 protein is predicted to consist of
residues
from about 41 to 655 in Figures lA-D (about 1 to about 615 of SEQ ID N0:2).
Analysis
using a different computer program predicts that the mature protein of TR9
starts at amino
acid 42 (Gln) as depicted in Figures 1 A-D and 4A. The results of this
analysis are
presented in Figure 4A and described in Example 6.
As one of ordinary skill would appreciate, due to the possibility of
sequencing
errors, as well as the variability of cleavage sites for leaders in different
known proteins,
the predicted TR9 receptor polypeptide encoded by the deposited cDNA comprises
about
655 amino acids, but may be anywhere in the range of 645-665 amino acids; and
the
predicted leader sequence of this protein is about 40 amino acids, but may be
anywhere in
the range of about 30 to about 50 amino acids. It will further be appreciated
that, the
domains described herein have been predicted by computer analysis, and
accordingly, that
depending on the analytical criteria used for identifying various functional
domains, the
exact "address" of, for example, the extracelluar domain, intracelluar domain,
death
domain, cystein-rich motifs, and transmembrane domain of TR9 may differ
slightly. For
example, the exact location of the TR9 extracellular domain in Figures 1 A-D
(SEQ ID
N0:2) may vary slightly (e.g., the address may "shift" by about 1 to about 20
residues,
more likely about 1 to about 5 residues) depending on the criteria used to
define the
domain. In any event, as discussed further below, the invention further
provides
CA 02365255 2001-09-24
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1?
polypeptides having various residues deleted from the N-terminus and/or C-
terminus of
the complete TR9. including polypeptides lacking one or more amino acids from
the
N-termini of the extracellular domain described herein, which constitute
soluble forms of
the extracellular domain of the TR9 polypeptides.
As indicated, nucleic acid molecules of the present invention may be in the
form of
RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and
genomic DNA obtained by cloning or produced synthetically. The DNA may be
double-
stranded or single-stranded. Single-stranded DNA or RNA may be the coding
strand,
also known as the sense strand, or it may be the non-coding strand, also
referred to as the
anti-sense strand.
By "isolated" nucleic acid 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. However, a nucleic acid molecule
contained in
a clone that is a member of a mixed clone library (e.g., a genomic or cDNA
library) and
that has not been isolated from other clones of the library (e.g., in the form
of a
homogeneous solution containing the clone without other members of the
library) or a
chromosome isolated or removed from a cell or a cell lysate (e.g., a
"chromosome
spread", as in a karyotype), is not "isolated" for the purposes of this
invention. Isolated
RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules
of the
present invention. Isolated nucleic acid molecules according to the present
invention
further include such molecules produced synthetically.
Isolated nucleic acid molecules of the present invention include DNA
molecules,
or alternatively consisting of, an open reading frame (ORF) shown in Figures
lA-D (SEQ
ID NO:1); DNA molecules , or alternatively consisting of, the coding sequence
for the
mature TR9 protein; and DNA molecules which comprise a sequence substantially
different from those described above but which, due to the degeneracy of the
genetic
code, still encode the TR9 protein shown in Figures lA-D (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.
In addition, the invention provides nucleic acid molecules having nucleotide
sequences related to extensive portions of the nucleotide sequence in Figures
lA-D of
(SEQ ID NO:1 ), which have been determined from the following related cDNA
clones:
HIBEJ86R (SEQ ID N0:6), HL1AA79R (SEQ ID N0:7), HHFGD57R (SEQ ID N0:8),
HSABG38R (SEQ ID N0:9), and HHPDZ31R (SEQ ID NO:10).
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is
Further, the invention includes a polynucleotide. or alternatively consisting
of.
any portion of at least about 30 nucleotides, preferably at least about 50
nucleotides. of
the nucleotide sequence disclosed in Figures lA-D from nucleotides 655 to 907
(nucleotides 615 to 867 of SEQ ID NO:1 ) and/or the nucleotide sequence
disclosed in
Figures 1 A-D from nucleotides to 540 to 1020 (nucleotides 500 to 980 as
depicted in SEQ
ID NO:1).
In another aspect, the invention provides isolated nucleic acid molecules
encoding
the TR9 receptor polypeptide having an amino acid sequence as encoded by the
cDNA
clone contained in the plasmid deposited as ATCC Deposit No. 209037 on May 15.
1997. In a further embodiment, nucleic acid molecules are provided encoding
the mature
TR9 receptor polypeptide or the full-length TR9 receptor polypeptide lacking
the N-
terminal methionine. The invention also provides an isolated nucleic acid
molecule having
the nucleotide sequence shown in Figures 1 A-D (SEQ ID NO:1 ) or the
nucleotide
sequence of the TR9 cDNA contained in the above-described deposited clone, or
a nucleic
acid molecule having a sequence complementary to one of the above sequences.
Such
isolated molecules, particularly DNA molecules, have uses which include, but
are not
limited to, as probes for gene mapping, by irz .sitcr hybridization with
chromosomes, and
for detecting expression of the TR9 receptor gene in human tissue, for
instance, by
Northern blot analysis.
The present invention is further directed to fragments of the isolated nucleic
acid
molecules described herein. By a fragment of an isolated nucleic acid molecule
having the
nucleotide sequence of the deposited cDNA (the clone deposited as ATCC Deposit
No.
209037), or the nucleotide sequence shown in Figures 1 A-D (SEQ ID NO:1 ), or
the
complementary strand thereto, is intended fragments at least about 15 nt, and
more
preferably at least about 20 nt, still more preferably at least about 30 nt,
and even more
preferably, at least about 40, 50, 100, 150, 200, 250, 300, 400, or 500 nt in
length.
These fragments have numerous uses which include, but are not limited to,
diagnostic
probes and primers as discussed herein. Of course, larger fragments of 50-1500
nt or
501-1500 nt in length are also useful according to the present invention as
are fragments
corresponding to most, if not all, of the nucleotide sequence of the deposited
cDNA or as
shown in Figures lA-D (SEQ ID NO:1). By a fragment at least about 20 nt in
length, for
example, is intended fragments which include 20 or more contiguous bases from
the
nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown
in
Figures lA-D (SEQ ID NO:1). In this context "about" includes the particularly
recited
size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either
terminus or at
both termini.
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14
Representative examples of TR9 polynucleotide frayTments of the invention
include, for example, fragments that comprise, or alternatively, consist of, a
sequence
from about nucleotide 1-50, 51-100, 101-150. 151-200, 201-250, 251-300. 301-
350,
351-400, 401-450, 445-879, 451-500. 501-550, 551-600, 615-651, 651-700, 701-
750,
751-800, 800-850, 850-867, 851-900, 901-950. 951-1000, 1001-1050, 1051-1100,
1101-1150, 1151-1200. 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450,
1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800,
1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-3000, or 3001 to
the
end of SEQ ID NO:1, or the complementary DNA strand thereto, or the cDNA
contained
in the deposited clone. In this context "about" includes the particularly
recited ranges,
larger or smaller by several (5, 4, 3, 2, or 1 ) nucleotides, at either
terminus or at both
termini.
In specific embodiments, the polynucleotide fragments of the invention encode
a
polypeptide which demonstrates a TR9 functional activity. By a polypeptide
demonstrating a TR9 "functional activity" is meant, a polypeptide capable of
displaying
one or more known functional activities associated with a complete (full-
length) or mature
TR9 polypeptide. Such functional activities include, but are not limited to,
biological
activity (e.g., ability to induce apoptosis in cells expressing the
polypeptide (see e.g.,
Example 5), the ability to activate monocytes (e.g., induce TNF-alpha and/or
MCP-1
secretion from monocytes), and the ability to increase survival of monocytes
(see, e.g.,
Example 8), antigenicity [ability to bind (or compete with a TR9 polypeptide
for binding)
to an anti-TR9 antibody], immunogenicity (ability to generate antibody which
binds to a
TR9 polypeptide), ability to form multimers, and ability to bind to a receptor
or ligand for
a TR9 polypeptide (e.g., AIM-II and TR9 ligands expressed on the surface of
monocytes).
The functional activity of TR9 polypeptides, and fragments, variants
derivatives,
and analogs thereof, can be assayed by various methods.
For example, in one embodiment where one is assaying for the ability to bind
or
compete with full-length (complete) TR9 polypeptide for binding to anti-TR9
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, ifi situ immunoassays (using colloidal gold, enzyme or
radioisotope labels, for example), western blots, precipitation reactions,
agglutination
assays (e.g., gel agglutination assays. hemagglutination assays), complement
fixation
assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis
CA 02365255 2001-09-24
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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
5 detecting binding in an immunoassay and are within the scope of the present
invention.
In another embodiment, where a TR9 liaand is identified (e.g., AIM-II and TR9
ligands expressed on the surface of monocytes), 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-
10 reducing gel chromatography, protein affinity chromatography, and affinity
blotting. See
generally, Phizicky, et al., Microbiol. Rev. 59:94-123 ( 1995). In another
embodiment,
physiological correlates of TR9 binding to its substrates (signal
transduction) can be
assayed.
In addition, assays described herein (see Examples 5 and 8 and otherwise known
15 in the art may routinely be applied to measure the ability of TR9
polypeptides and
fragments, variants derivatives and analogs thereof to elicit TR9 related
biological activity
(e.g., ability to induce apoptosis in cells expressing the polypeptide (see
e.g., Example
6), the ability to activate monocytes, and the ability to increase survival of
monocytes
(see, e.g., Examples 8 and 9) in vitro or in vivo. For example, biological
activity can
routinely be measured using the cell death assays performed essentially as
previously
described (Chinnaiyan et al., Cell 81:505-512 (1995); Boldin et al., J. Biol.
Chen2.
270:7795-8(1995); Kischkel et al., EMBO 14:5579-5588 (1995); Chinnaiyan et
al., J.
Biol. Chem. 271:4961-4965 (1996)) and as set forth in Example 5 below. In one
embodiment involving MCF7 cells, plasmids encoding full-length TR9 or a
candidate
death domain containing receptor are co-transfected with the pLantern reporter
construct
encoding green fluorescent protein. Nuclei of cells transfected with TR9 will
exhibit
apoptotic morphology as assessed by DAPI staining.
Other methods will be known to the skilled artisan and are within the scope of
the
invention.
Preferred nucleic acid fragments of the present invention include a nucleic
acid
molecule encoding a member selected from the group: a polypeptide comprising
or
alternatively, consisting of, the TR9 receptor extracellular domain (predicted
to constitute
amino acid residues from about 1 to about 310 in SEQ ID N0:2); a polypeptide
comprising or alternatively, consisting of, the four TNFR-like cysteine rich
motifs of TR9
(amino acid residues 67 to 211 in Figures lA-D; amino acid residues 27 to 171
in SEQ ID
N0:2); a polypeptide comprising or alternatively, consisting of, the TR9
receptor
transmembrane domain (predicted to constitute amino acid residues from about
311 to
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16
about 327 in SEQ ID N0:2); a polypeptide comprising, or alternatively,
consisting of, a
fragment of the predicted mature TR9 polypeptide, wherein the fragment has a
TR9
functional activity (e.g., antigenic activity or biological acitivity); a
polypeptide
comprising or alternatively, consisting of, the TR9 receptor intracellular
domain
(predicted to constitute amino acid residues from about 328 to about 615 in
SEQ ID
N0:2); a polypeptide comprising or alternatively, consisting of, the TR9
receptor
extracellular and intracellular domains with all or part of the transmembrane
domain
deleted; a polypeptide comprising, or alternatively , consisting of, the TR9
receptor death
domain (predicted to constitute amino acid residues from about 389 to about
455 in SEQ
ID N0:2); and a polypeptide comprising, or alternatively, consisting of, one,
two, three,
four or more, epitope bearing portions of the TR9 receptor protein. In
additional
embodiments, the polynucleotide fragments of the invention encode a
polypeptide
comprising, or alternatively, consisting of, any combination of l, 2. 3, 4, 5,
6, 7, or all 8
of the above members. As above, with the leader sequence, the amino acid
residues
constituting the TR9 receptor extracellular, transmembrane and intracellular
domains have
been predicted by computer analysis. Thus, as one of ordinary skill would
appreciate, the
amino acid residues constituting these domains may vary slightly (e.g., by
about 1 to
about 15 amino acid residues) depending on the criteria used to define each
domain.
Polypeptides encoded by these nucleic acid molecules are also encompassed by
the
invention.
It is believed one or more of the four extracellular cysteine rich motifs of
TR9
disclosed in Figures lA-D and Figure 4B is important for interactions between
TR9 and
its (e.g., AIM-II and TR9 ligands expressed on the surface of monocytes).
Accordingly,
specific embodiments of the invention are directed to polynucleotides encoding
polypeptides which comprise, or alternatively consist of, the amino acid
sequence of
amino acid residues 27 to 65, 66 to 105, 106 to 145, and/or 146 to 171 of SEQ
ID N0:2,
as disclosed in Figures lA-D and Figure 4B. Additional embodiments of the
invention
are directed to polynucleotides encoding TR9 polypeptides which comprise, or
alternatively consist of, any combination of 1, 2, 3, or all 4, of the
extracellular cysteine-
rich motifs disclosed in Figures lA-D and Figure 4B. Polypeptides encoded by
these
polynucleotides are also encompassed by the invention.
In additional embodiments, the polynucleotides of the invention encode
functional
attributes of TR9. Preferred embodiments of the invention in this regard
include
fragments 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,
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l7
beta amphipathic regions, flexible regions, surface-forming regions and high
antigenic
index regions of TR9 polypeptides.
Certain preferred regions in this regard are set out in Figure 3 (Table 1).
The data
presented in Figure 3 and that presented in Table I, merely present a
different format of
the same results obtained when the amino acid sequence of TR9 as disclosed in
Figures
lA-D, is analyzed using the default parameters of the DNAxSTAR computer
algorithm.
The above-mentioned preferred regions set out in Figure 3 and in Table I
include,
but are not limited to, regions of the aforementioned types identified by
analysis of the
amino acid sequence set out in Figures lA-D. As set out in Figure 3 and in
Table I, such
preferred regions include Gamier-Robson alpha-regions, beta-regions, turn-
regions, and
coil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions, Kyte-
Doolittle
hydrophilic regions and hydrophobic regions, Eisenberg alpha- and beta-
amphipathic
regions, Karplus-Schulz flexible regions, Emini surface-forming regions and
Jameson-Wolf regions of high antigenic index. Among highly preferred
polynucleotides
in this regard are those that encode polypeptides comprise, or alternatively
consist of,
regions of TR9 that combine several structural features, such as several
(e.g., 1, 2, 3, or
4) of the same or different region features set out above.
Additionally, the data presented in columns VIII, IX, XIII, and XIV of Table I
can routinely be used to determine regions of TR9 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.
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18
Table
1
ResPositionI II IIIIV V VI Vll VIIIIX X XI X11Xlll X1V
Met1 . . . . T . . 0.180.2 . . . 0.3 0.71
Gly2 . . . . T . . 0.270.2 . . . 0.58 0.86
Thr3 . . . . . . C 0.360.16 . . . 0.66 0.9
Ser4 . . . . . T C 0.440.11 . . . 1.29 1.22
Pro5 . . . . . T C 0.52-0.11. . F 2.32 1.65
Ser6 . . . . T T . 0.53-0.06. . F 2.8 1.65
Ser7 . . . . . T C 0.07-0.04. . F 2.32 1.24
Ser8 . A . . . . C -0.210.26 . . F 0.89 0.66
Thr9 A A . . . . . -0.210.33 . . F 0.41 0.5
Ala10 A A . . . . . -0.670.33 . . F 0.13 0.5
LeuI1 A A . . . . . -0.670.51 * . . -0.6 0.2
Ala12 A . . . . T . -0.260.51 * * . -0.2 0.19
Ser13 A . . . . T . -0.840.03 * * . 0.1 0.36
Cys14 A . . . . T . -1.120.21 * . . 0.1 0.31
Ser15 A . . . . T . -0.420.03 * . . 0.1 0.31
Arg16 A A . . . . . 0.5 -0.47* * . 0.3 0.45
Ile17 A A . . . . . 0.5 -0.86* * . 0.75 1.63
Ala18 A A . . . . . 0.49-0.93* * . 0.75 1.23
Arg19 A A . . . . . 0.57-0.83* * . 0.6 0.91
Arg20 A A . . . . . 0.56-0.33* * F 0.6 1.31
Ala21 A A . . . . . -0.16-0.53* * F 0.9 1.87
Thr22 A A . . . . . -0.16-0.41* . . 0.3 0.94
Ala23 A A . . . . . -0.160.27 * . . -0.3 0.34
Thr24 A A . . . . . -0.610.77 . . . -0.6 0.34
Met25 A A . . . . . -1.020.7 . * . -0.6 0.23
Ile26 A A . . . . . -1.240.6 . . . -0.6 0.31
Ala27 A A . . . . . -1.740.79 . . . -0.6 0.18
Gly28 A A . . . . . -1.970.99 . . . -0.6 0.15
Ser29 A A . . . . . -2.471.06 . . . -0.6 0.17
Leu30 A A . . . . . -2.211.06 . . . -0.6 0.14
Leu31 A A . . . . . -2.020.99 . . . -0.6 0.14
Leu32 A A . . . . . -2.241.34 . . . -0.6 0.09
Leu33 A A . . . . . -2.21.64 . . . -0.6 0.09
Gly34 A . . B . . . -2.211.34 . . . -0.6 0.15
Phe35 . . B B . . . -1.711.14 . . . -0.6 0.26
Leu36 . . . B . . C -1.210.94 . . . -0.4 0.45
Ser37 . . . B . . C -0.990.74 . . F -0.250.66
Thr38 . . . B . . C -0.180.81 . . F -0.250.77
Thr39 . . . B . . C -0.040.43 . . F -0.1 1.62
Thr40 . . . B . . C 0.660.17 . . F 0.2 1.86
Ala41 . A . B . . C 1.47-0.21. * F 0.8 2.24
Gln42 A A . B . . . 1.81-0.3 . * F 0.6 2.68
Pro43 A A . B . . . 1.53-0.79. * F 0.9 3.72
Glu44 A A . . . . . 1.54-0.77. * F 0.9 3.72
Gln45 A A . . . . . 1.86-0.89. * F 0.9 2.88
Lys46 A A . . . . . 1.63-0.89* * F 0.9 2.99
Ala47 A . . . . T . 0.74-0.63* . F 1.3 1.43
Ser48 . . . . . T C 0.610.06 * . F 0.45 0.58
Asn49 . . . . . T C 0.3 0.09 * . F 0.45 0.29
Leu50 . . . . . T C 0.060.57 * * . 0 0.41
Ile51 . . . . T . . 0.120.83 * * . O.17 0.48
Gly52 . . . . T T . 0.680.44 * . . 0.54 0.58
Thr53 . . B . . T . 0.120.54 * . . 0.31 0.96
Tyr54 . . B . . T . 0.120.5 * * . 0.63 1.01
Arg55 . . B . . T . 1.04-0.19* * . 1.7 1.71
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19
Table
1
(continued)
ResPosition1 IIIIV V VI VlIVlll IX X XI XIIXIII XIV
II
His56 . B B . . . 1.34 -0.61* . 1.43 2.32
.
Val57 . . B . . C 1.38 -0.6 * * . 1.77 1.5
.
Asp58 . . B T . . 1.34 -0.87* * F 2.26 1.1
.
Ark59 . . . T . . 1.59 -0.44* * F 2.15 0.8
.
Ala60 . . . T T . 0.62 -0.54* * F 2.94 1.87
.
Thr61 . . . T T . -0.16-0.54* * F 3.1 0.83
.
Gly62 . . . T T . 0.39 0.14 * * F 1.89 0.35
.
Gln63 . . . T T . -0.280.63 * * F 1.28 0.5
.
Val64 . B . . . . -0.390.7 * * . 0.22 0.19
.
Leu65 . B . . . . 0.24 0.21 . . . 0.21 0.31
.
Thr66 . . . T T . -0.11-0.21. . . 1.1 0.36
.
Cys67 . . . T T . 0.02 -0.04. . . 1.41 0.26
.
Asp68 . . . T T . -0.57-0.26. . F 1.87 0.49
.
Lys69 . . . T T . -0.06-0.44. . F 2.18 0.34
.
Cys70 . . . . T C 0.44 -0.5 . . F 2.59 0.63
.
Pro71 . . . T T . 0.51 -0.59. . F 3.1 0.55
.
Ala72 . . . T T . 0.32 0.17 . . F 1.89 0.43
.
Gly73 . . . T T . 0.02 0.81 . . F 1.28 0.59
.
Thr74 . . B T . . -0.020.63 . . F 0.57 0.52
.
Tyr75 . B B . . . 0.61 0.2 . . . 0.01 0.88
.
Val76 . B B . . . 0.16 0.2 . . . -0.151.22
.
Ser77 . B B . . . 0.43 0.34 * . . -0.050.45
.
Glu78 . B . . . . 0.78 0.34 . . . 0.4 0.42
.
His79 . . . T . . 0.78 -0.01. . . 1.65 0.9
.
Cys80 . . . T T . 0.72 -0.17. . . 2.1 0.97
.
Thr81 . . . T T . 0.77 -0.17* * F 2.5 0.75
.
Asn82 . . . T T . 1.18 0.51 * * F 1.35 0.45
.
Thr83 . . . T T . 0.32 0.01 * * F 1.55 1.66
.
Ser84 . . B T . . -0.310.09 * * F 0.75 0.85
.
Leu85 . . B T . . 0.06 0.17 * * . 0.35 0.28
.
Ark86 . . B T . . 0.07 0.16 * * . 0.1 0.26
.
Val87 . . B T . . -0.6 0.06 * * . 0.1 0.26
.
Cys88 . . . T T . -0.5 0.24 . * . 0.5 0.17
.
Ser89 . . . T T . - -0.01. * . 1.1 0.14
. I
.06
Ser90 . . . T T . -0.590.63 . * . 0.2 0.14
.
Cys91 . . . . T C -1.010.41 . * . 0 0.25
.
Pro92 . . B T . . -0.860.33 . . . 0.1 0.27
.
Val93 . . B T . . -0.5 0.73 * . . -0.2 0.17
.
Gly94 . . B T . . -0.090.83 * . F -0.050.47
.
Thr95 . B B . . . 0.18 0.26 * . F -0.150.59
.
Phe96 . B B . . . 0.84 0.33 * . F 0 1.09
.
Thr97 A . B . . . 1.06 -0.31* . F 0.6 1.91
.
Ark98 A . B . . . 1.57 -0.34* . F 0.6 2.13
.
His99 A . . . T . 1.02 -0.4 * . F 1 2.43
.
Glu100 . . . . T C 1.33 -0.5 * . F 1.2 1.18
.
Asn101 . . . T T . 2.08 -0.99* . F 1.7 1.04
.
Gly102 . . . T T . 1.72 -0.99* * F 1.7 1.53
.
Ile103 A . . T . . 1.58 -0.91* . F 1.63 0.47
.
Glu104 A . . . . . 1.61 -0.41* . F 1.21 0.4
.
Lys105 . . . T . . 0.94 -0.81* . . 2.04 0.68
.
Cys106 . . . T . . 0.64 -0.67* . . 2.32 0.52
.
His107 . . . T T . 0.99 -0.97* . . 2.8 0.4
.
Asp108 . . . T T . 1.67 -0.57* . . 2.52 0.35
.
Cys109 . . . T T . 1 -0.14. . . 2.09 1
.
Ser110 . . . T T . 0.74 -0.14. . F 1.81 0.39
.
Gln111 . . . T . . 1.12 -0.21. . F 1.33 0.37
.
CA 02365255 2001-09-24
- WO 00/56862 PCT/US00/06831
Table
1
(continued)
ResPosition1 ll IV V VI VII V111IX X XI Xll XIIIXIV
III
Pro112 . . . . T . 0.940.7 . . F 0.150.72
Cys113 . . . . T T . 0.3=10.56 . F 0.350.83
Pro114 . . . . T T . 0.120.79 . . . 0.2 0.47
Trp115 . . . . . T C 0.421.07 . . . 0 0.21
Pro116 A . . . . T C 0.470.64 . . . 0 0.69
Met117 A A . . . . . -0.130.07 * . . -0.30.9
Ile118 A A . . . . . 0.320.33 * . . -0.30.7
Glu119 A A . . . . . -0.13-0.16* . . 0.3 0.7
Lys120 A A . . . . -0.43-0.01* . . 0.3 0.38
Leu121 A A . . . . . -0.81-0.13* . . 0.3 0.55
Pro122 A A . . . . . -1.02-0.31* . . 0.3 0.32
Cys123 A A . . . . . -0.440.37 * . . -0.30.13
Ala124 A A . . . . . -0.440.86 . * . -0.60.23
Ala125 A A . . . . . -0.380.17 . * . -0.30.25
Leu126 A A . . . . . 0.43-0.26. . . 0.3 0.91
Thr127 A A . . . . . -0.02-0.83. . . 0.751.56
Asp128 . A . . T . . 0.33-0.76. . F 1.150.83
Arg129 . A . . T . . 0.26-0.77. . F 1.3 1.45
Glu130 . A . . T . . 0.63-0.89. . F 1.150.54
Cys131 . A . . T . . 1.23-0.94. . . 1 0.5
Thr132 . . . . T . . 1.2 -0.51. . . 1.2 0.39
Cys133 . . . . . . C 0.6 -0.09* . F 0.850.22
Pro134 . . . . . T C -0.210.53 * . F 0.150.42
Pro135 . . . . T T . -0.210.74 . . F 0.350.25
Gly136 . . . . T T 0.160.66 . . . 0.2 0.8
Met137 . . . . T T . 0.470.47 . * . 0.2 0.7
Phe138 . . . . T . . 0.540.44 . * . 0 0.73
Gln139 . . . . T T . 0.440.51 . * F 0.350.74
Ser140 . . . . T T . -0.010.57 . * F 0.5 1.08
Asn141 . . . . T T . -0.260.53 . * F 0.350.67
Ala142 . . . . T T . 0.130.24 . * F 0.650.39
Thr143 . . . . T . . 0.8 0.27 . * . 0.3 0.45
Cys144 . . . . T . . 0.490.39 . . . 0.3 0.38
Ala145 . . . . . T C -0.070.47 . . . 0 0.54
Pro146 . . . . T T . -0.730.61 . . . 0.2 0.28
His147 . . . . T T . -0.360.7 . . . 0.2 0.28
Thr148 . . B . T T . -0.90.56 . . . 0.2 0.43
Val149 . . B . . . . -0.580.7 . . . -0.40.21
Cys150 . . B . . T . -0.280.7 . . . -0.20.15
Pro151 . . . . T T . -0.411.11 . . . 0.2 0.11
Val152 . . . . T T . -1.231.06 * * . 0.2 0.15
Gly153 . . . . T T . -0.811.06 * * . 0.2 0.2
Trp154 . . . B T . . 0.090.49 * * . 0.1 0.25
Gly155 A . . B . . . 0.8 0.06 . * . 0.3 0.69
Val156 . . . B . . C 0.67-0.59. * . 1.851.39
Arg157 . . . B . . C 1.21-0.59* * F 2.3 1.31
Lys158 . . . . . T C 1.56-1.01. * F 3 1.9
Lys159 . . . . . T C 1.53-1.44. * F 2.7 4.44
Gly160 . . . . . T C 1.88-1.6 * * F 2.4 3.27
Thr161 . . . . . T C 2.73-1.6 * * F 2.1 2.83
Glu162 A A . . . . . 1.77-1.6 . * F 1.2 2.37
Thr163 A A . . . . . 1.83-0.96. * F 0.9 1.77
Glu164 A A . . . . . 1.12-1.39. * F 0.9 2.41
Asp165 A A . . . . . 1.51-1.3 . * F 0.750.75
Val166 A A . . . . . 1.82-1.3 . * F 0.9 1.03
Arj167 A A . . . . . 1.16-1.39. * . 0.751.03
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
21
Table
1
(continued)
Res PositionI II 111IV VI VIIVlll IX X XI XIIXI11XIV
V
Cys l68 A A . . . . . 0.88 -0.81. * . 0.880.33
Lys 169 A A . . . . . 0.99 -0.31. * . 0.860.45
Gln 170 A A . . . . . 0.64 -0.96. * . 1.4-10.45
Cys 171 . . . . T T . 1.19 -0.53* * . 2.520.83
Ala 172 . . . . T T . 0.38 -0.61* * . 2.8 0.6
Arg 173 . . . . T T . 0.74 0.17 * * F 1.770.3
Gly 174 . . . . T T . 0.7 0.16 * * F 1.490.75
Thr 175 . . . . T . . -0.16-0.41* * F 1.761.24
Phe 176 . . . . T . . 0.3 -0.27* * F 1.330.47
Ser 177 . . . . . . C 0.59 0.16 * * F 0.380.74
Asp 178 . . . . . . C 0.18 0.11 * * F 0.510.68
Val 179 . . . . . T C -0.330.01 * . F 0.991.06
Pro 180 . . . . T T . -0.62-0.13* . F 1.770.59
Ser 181 . . . . T T . 0.12 0.1 * . F 1.3 0.35
Ser 182 A . . . . T . -0.240.1 * * F 0.770.94
Val 183 A A . . . . . -0.2 0.03 . * . 0.090.32
Met 184 A A . . . . . 0.07 -0.4 . * . 0.560.48
Lys 185 A A . . . . . 0.03 -0.29. * . 0.430.36
Cys 186 A A . . . . . 0.02 0.09 . * . -0.30.77
Lys 187 A A . . . . . 0.32 -0.07. * . 0.451.12
Ala 188 A A . . . . . 0.51 -0.69. * . 0.6 0.94
Tyr 189 A . . . . T . 0.3 -0.11. * . 0.7 0.94
Thr 190 A . . . . T . -0.040 * * . 0.7 0.39
Asp 191 A . . . . T . 0.62 0.39 * * . 0.1 0.51
Cys 192 A . . . . T . 0.58 0.29 * * . 0.1 0.57
Leu 193 A . . . . . . 0.36 -0.07. . F 0.650.63
Ser 194 A . . . . T . -0.260.13 . . F 0.250.31
Gln 195 A . . . . T . -0.8 0.77 . . F -0.050.43
Asn 196 A . . . . T . -1.690.84 * . F -0.050.39
Leu 197 . . B . . T . -0.980.84 . . . -0.20.2
Val 198 . . B B . . . -0.380.46 . . . -0.60.23
Val 199 . . B B . . . -0.420.49 . . . -0.260.23
Ile 200 . . B B . . . -0.730.51 . * . 0.080.27
Lys 201 . . . . . T C -0.690.31 . . F 1.470.53
Pro 202 . . . . . T C 0.12 -0.33* . F 2.561.42
Gly 203 . . . . T T . 0.67 -0.97* * F 3.4 3.51
Thr 204 . . . . . T C 1.52 -1.17* . F 2.862.53
Lys 205 . . . . . . C 2.41 -1.17* * F 2.542.73
Glu 206 . . . . T . . 1.51 -1.2 * . F 2.624.44
Thr 207 . . . . T . . 1.06 -0.99* . F 2.5 2.28
Asp 208 . . . . T . . 1.06 -0.9 * . F 2.230.61
Asn 209 . . . . T T . 1.06 -0.47* . . 2.2 0.35
Val 210 . . . . T T . 0.2 0.01 * . . 1.380.35
Cys 211 . . . . T T . -0.010.21 . . . 1.160.17
Gly 212 . . . . T T . 0 0.64 * . . 0.640.17
Thr 213 . . B . . . . -0.7 0.63 * . F -0.030.3
Leu 214 . . . . . T C -I 0.77 . . F 0.150.48
Pro 215 . . . . . T C -0.440.59 . . F 0.150.66
Ser 216 . . . . T T . -0.080.54 . . F 0.350.61
Phe 217 . . . . T T . -0.040.44 . . F 0.350.99
Ser 218 . . . . T T . -0.030.24 . . F 0.850.92
Ser 219 . . . . T T . 0.57 0.2 . . F 1.050.92
Ser 220 . . . . T T . 0.48 0.24 . . F 1.4 1.65
Thr 221 . . . . . T C 0.57 -0.16. . F 2 1.65
Ser 222 . . . . . . C 0.92 -0.11. . F 2 1.9
Pro 223 . . . . . . C 0.91 -0.07. . F 1.8 1.41
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
Table 1 (continued)
Res Position 1 II III IV V VI VII VIII IX X XI XII XIII XIV
Ser 224 . . . . . T C 0.63 0.0 . . F 1.2 1.41
3
Pro 225 . . . . T T . 0.03 0.04 . . F 1.2 1.06
Gly 226 . . . . T T . -0.360.34 . . F 0.850.48
Thr 227 . . . . . T C -0.270.7 * . F 0.150.31
Ala 228 . . . . . . C 0.06 0.74 * . . 0.040.31
Ile 229 . . B . . . C 0.14 0.31 . * . 0.580.61
Phe 230 . . B . . T . 0.36 0.31 * . . 0.820.66
Pro 231 . . . . . T C 0.67 -0.17* . F 2.161.13
Arb 232 . . . . . T C 0.38 -0.17. . F 2.4 2.19
Pro 233 A . . . . T C 0.97 -0.24. . F 2.162.5
Glu 234 A A . . . . . 1.54 -1.03. * F 1.622.8
His 235 A A . . . . . 2.21 -0.97. . . 1.232.07
Met 236 A A . . . . . 2.42 -0.47. . . 0.691.82
Glu 237 A A . . . . . 1.46 -0.9 . . . 0.751.82
Thr 238 A A . . . . . 1.46 -0.26. . . 0.580.99
His 239 A A . . . . . 1.16 -0.33. . . 1.011.55
Glu 240 A A . . . . . 0.89 -0.56. . F 1.741.2
Val 241 A . . . . T . 1.18 -0.17. . F 2.12I.11
Pro 242 . . . . T T . 0.93 -0.17. . F 2.8 1.18
Ser 243 . . . . T T . 0.39 0.09 . . F 1.921.07
Ser 244 . . . . T T . 0.21 0.73 . . F 1.341.07
Thr 245 . . . . T . . 0.26 0.51 . . F 0.861.07
Tyr 246 . . B . . . . 0.77 0.09 . . F 0.481.59
Val 247 . . . . . T C 0.38 0.13 * . F 0.6 1.18
Pro 248 . . . . T T . 0.68 0.36 * . F 0.650.81
Lys 249 . . . . T T . 0.68 0.27 * . F 0.650.83
Gly 250 . . . . . T C 0.68 -0.1 * . F 1.2 1.5
Met 251 . . . . . . C 0.92 -0.26* . F 1.3 1.4
Asn 252 . . . . . . C 1.48 -0.69* . F 1.9 1.21
Ser 253 . . . . . T C 1.69 -0.3 . . F 2.1 1.64
Thr 254 . . . . . T C 1.34 -0.33. . F 2.4 2.66
Glu 255 . . . . . T C 1.39 -0.56. . F 3 2.22
Ser 256 . . . . . T C 1.4 -0.57. . F 2.7 2.22
Asn 257 . . . . T T . 1.1 -0.46. * F 2.3 1.55
Ser 258 . . . . . T C 0.54 -0.56. * F 2.1 1.2
Ser 259 . . . . . T C 0.97 0.09 . * F 0.750.67
Ala 260 . . . . . T C 0.76 -0.3 . * F 1.050.81
Ser 261 . . . B . . C 1.1 -0.27. * F 0.890.93
Val 262 A . . B . . . 0.24 -0.66. * F 1.381.4
Ark 263 . . B B . . . -0.27-0.4 . * F 1.321.03
Pro 264 A . . . . . . -0.27-0.21. * F 1.610.63
Lys 265 . . . . T . . 0.02 -0.21. * F 2.4 1.14
Val 266 . . . . . . C -0.57-0.47. * F 1.810.78
Leu 267 . . . . . . C 0.29 0.21 . * F 0.970.35
Ser 268 . . B . . . . 0.18 0.19 . * F 0.530.31
Ser 269 . . B . . . . 0.04 0.19 * * F 0.290.71
Ile 270 . . B . . . . -0.31-0.03* * F 0.650.86
Gln 271 . . B . . . . -0.31-0.23* . F 0.950.92
Glu 272 . . . . T . . 0.29 0.03 . . F 1.050.51
Gly 273 . . . . T . . 0.59 0.07 . . F 1.5 1.13
Thr 274 . . . . . . C 0.89 -0.61. . F 2.5 1.09
Val 275 . . . . . T C 1.47 -0.61. . F 3 1.01
Pro 276 . . . . . T C 1.17 -0.13. . F 2.4 1.47
Asp 277 . . . . T T . 0.87 -0.17. . F 2.3 1.37
Asn 278 . . . . . T C 0.62 -0.27. * F 2.1 2.47
Thr 279 . . . . . . C 1.04 -0.41. * F 1.9 1.61
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
Table
1
(continued)
Res Position1 II III V VI VIIVIII IX X XI XIIXIIIXIV
IV
Ser 280 . . . . . C 1.56 -0.84. * F 2.2 1.89
Ser 281 . . . . . T C 1.81 -0.41. F 2.4 1.16
Ala 282 . . . . . T C 1.81 -0.81. * F 3 1.61
Ark 283 A . . . . T . 1.81 -1.3 . * F 2.5 2.08
Gly 284 A . . . . T . 1.27 -1.69* * F 2.2 2.6
Lys 285 A . . . . . . 1.57 -1.43* * F 2.041.91
Glu 286 A . . . . . . 1.91 -1.53* * F 2.081.57
Asp 287 A . . . . T . 2.19 -1.53* * F 2.323.16
Val 288 A . . . . T . 1.27 -1.47* * F 2.662.28
Asn 289 . . . . T T . 1.4 -0.79* * F 3.4 1.09
Lys 290 . . . . T T . 1.36 -0.36* * F 2.761.01
Thr 291 . . . . . . C 0.54 0.04 * . F 1.422.18
Leu 292 . . . . . T C 0.54 0.09 * * F 1.281.12
Pro 293 A . . . . T . 0.54 0.09 * . F 0.590.97
Asn 294 A . . . T T . -0.310.73 . . . 0.2 0.5
Leu 295 A . . . . T . -0.360.89 . * . -0.20.45
Gln 296 . . B B . . . -0.080.6 . . . -0.60.47
Val 297 . . B B . . . 0.73 0.67 . . . -0.60.39
Val 298 . . B B . . . 0.94 0.67 . . . -0.60.83
Asn 299 . . . B . . C 0.6 0.39 . . . -0.10.83
His 300 . . . B T . . 1.2 0.41 . . F 0.1 1.11
Gln 301 . . . B T . . 1.17 0.2 . . F 0.642.3
Gln 302 . . . B . . C 1.99 0.06 . . F 0.681.95
Gly 303 . . . . . T C 2.96 0.16 . . F 1.321.95
Pro 304 . . . . . T C 2.92 -0.34. . F 2.162.2
His 305 . . . . . T C 2.07 -0.24. . F 2.4 1.73
His 306 A . . . . T . 1.26 0.04 * * . 1.211.23
Arg 307 A A . . . . . 1.3 0.3 * * . 0.420.65
His 308 A A . . . . . 0.83 -0.13* * . 0.780.96
Ile 309 A A . . . . . 0.23 0.06 * * . -0.060.58
Leu 310 A A . . . . . 0.06 0.24 * * . -0.30.25
Lys 311 A A . . . . . -0.210.67 * * . -0.60.28
Leu 312 . A . . . . C -0.920.56 * * . -0.40.53
Leu 313 . . . . . T C -0.890.49 * * F 0.150.64
Pro 314 . . . . . T C -0.59-0.2 * * F 1.050.55
Ser 315 A . . . . T . -0.090.3 . * F 0.250.68
Met 316 A . . . . T . -0.480.1 . * . 0.251.19
Glu 317 A . . . . . . -0.01-0.16. . F 0.950.76
Ala 318 A . . . . T . 0.8 -0.16. . F 1.450.56
Thr 319 A . . . . T . 1.06 -0.54. * F 2.050.98
Gly 320 A . . . . T . 1.06 -1.16. * F 2.5 1.13
Gly 321 . . . . . T C 1.36 -0.77. . F 3 1.5
Glu 322 A . . . . T . 1.04 -0.89. . F 2.5 1.4
Lys 323 . . . . . T C 1.42 -0.89* . F 2.4 2.04
Ser 324 . . . . T T . 0.84 -0.89* * F 2.3 3.18
Ser 325 . . . . . T C 1.23 -0.63* * F 1.8 1.29
Thr 326 . . . . . . C 1.23 -0.63* * F 1.3 1.29
Pro 327 . . . . . . C 1.02 -0.2 . * F 1.190.95
Ile 328 . . . . T . . 1.02 -0.16* * F 1.881.1
Lys 329 . . . . T . . 1.43 -0.54* * F 2.521.52
Gly 330 . . . . . T C 1.39 -1.03* * F 2.861.93
Pro 331 . . . . T T . 1.67 -1.03* * F 3.4 2.72
Lys 332 . . . . T T . 1.67 -1.21* * F 3.061.85
Arg 333 . . . . T T . 2.67 -0.79* * F 3 2.89
Gly 334 . . . . . . C 2.62 -1.21* * F 2.543.66
His 335 . . . . . T C 2.97 -1.24. * F 2.683.17
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
24
Table
1
(continued)
Res PositionI II IV V VI VIlVIII IX X XI XIIXIII XIV
IlI
Pro 336 . . . . . T C 2.37 -0.84. * F 2.62 2.6
Ark 337 . . . . T T . 2.29 -0.16. * F 2.8 2.17
Gln 338 A . . . . T . 2.22 _0.09. * F 2.12 2.17
Asn 339 A A . . . . . 2.53 -0.59* * F 1.74 2.81
Leu 340 A A . . . . . 1.87 -0.51* * F 1.46 1.95
His 341 A A . . . . . 2.08 0.27 * * . -0.020.97
Lys 342 . A . . . . C 1.08 -0.13. * . 0.65 1.01
His 343 . A . . . . C 1.08 0.16 * * . -0.1 0.86
Phe 344 . A . . . . C 1.08 -0.13. * . 0.65 1.02
Asp 345 A A . . . . . 1.86 -0.63* * . 0.6 0.88
Ile 346 A A . . . . . 1.08 -0.13. * . 0.3 0.88
Asn 347 A A . . . . . 0.82 0.06 . * . -0.3 0.84
Glu 348 A A . . . . . 0.57 -0.3 . * . 0.3 0.78
His 349 A A . . . . . 0.67 0.61 * * . -0.451.16
Leu 350 . . . B . . C -0.220.54 . * . -0.4 0.72
Pro 351 A . . B . . . -0.190.83 . * . -0.6 0.29
Trp 352 A . . B . . . -1 1.47 . . . -0.6 0.16
Met 353 A . . B . . . -1.7 1.66 . . . -0.6 0.16
Ile 354 A . . B . . . -2.481.76 . . . -0.6 0.09
Val 355 A . . B . . . -2.482.01 . . . -0.6 0.07
Leu 356 A . . B . . . -3.081.79 . . . -0.6 0.06
Phe 357 A . . B . . . -3.641.86 . . . -0.6 0.07
Leu 358 A . . B . . . -3.861.81 . . . -0.6 0.07
Leu 359 A . . B . . . -3.821.86 . . . -0.6 0.07
Leu 360 A . . B . . . -3.821.81 . . . -0.6 0.06
Val 361 A . . B . . . -3.9 1.67 . . . -0.6 0.05
Leu 362 A . . B . . . -4.061.67 . . . -0.6 0.04
Val 363 A . . B . . . -4.1 1.63 . . . -0.6 0.04
Va1 364 A . . B . . . -3.961.59 . . . -0.6 0.04
Ile 365 A . . B . . . -3.441.51 . . . -0.6 0.03
Val 366 . . B B . . . -3.481.21 . * . -0.6 0.05
Val 367 . . B B . . . -2.561.26 * . . -0.6 0.04
Cys 368 . . B B . . . -1.660.61 * . . -0.6 0.12
Ser 369 A . . B . . . -1.1 -0.07. . . 0.64 0.34
Ile 370 . . . B T . . -0.51-0.33* * . 1.38 0.61
Arg 371 . . . B T . . 0.46 -0.59* * F 2.32 1.51
Lys 372 . . . B T . . 1 -1.16* * F 2.66 2.21
Ser 373 . . . . T T . 0.86 -1.06* * F 3.4 4.56
Ser 374 . . . . T T . 1.2 -1.06* * F 3.06 1.92
Arg 375 . . . . T T . 2.13 -1.06* * F 2.72 1.92
Thr 376 . . . . T T . 1.68 -1.06* . F 2.38 2.86
Leu 377 . . . . T . . 1.42 -1.01* . F 1.84 2.12
Lys 378 . . . . T . . 1.83 -0.97* . F 1.84 1.67
Lys 379 . . . . T . . 2.13 -0.97* . F 2.18 2.27
Gly 380 . . . . . T C 2.02 -1.06* * F 2.52 4.76
Pro 381 . . . . . T C 2.12 -1.74* * F 2.86 3.97
Arg 382 . . . . T T . 2.63 -1.31* * F 3.4 3.07
Gln 383 . . . . . T C 2 -0.93* . F 2.86 4.16
Asp 384 . . . . . T C 1.07 -0.86* . F 2.52 2.72
Pro 385 . . . . . T C 0.56 -0.6 * * F 2.03 0.97
Ser 386 . . . . . T C 0.77 0.04 . * F 0.79 0.42
Ala 387 A . . . . T . 0.7 -0.36* * . 0.7 0.43
Ile 388 A A . B . . . 0.11 -0.36* . . 0.3 0.56
Val 389 A A . B . . . -0.23-0.29. . . 0.3 0.42
Glu 390 A A . B . . . -0.83-0.24. * . 0.3 0.41
Lys 391 A A . . . . . -0.49-0.06. . F 0.45 0.49
CA 02365255 2001-09-24
i~VO 00/56862 PCT/US00/06831
Table
1
(continued)
ResPosition1 II IV V VI VII VIIIIX X XI XllXlll XIV
III
Ala392 A A . . . . . 0.14-0.7-4. . F 0.9 1.31
Gly393 A A . . . . . 0.73-1.39. * F 0.9 1.51
Leu394 A A . . . . . 0.99-1 . . F 0.9 1.01
Lys395 A A . . . . . 0.68-0.39. * F 0.45 0.99
Lys396 A A . . . . . 0.42-0.4 . * F 0.6 1.45
Ser397 . . . . T . . 0.7 -0.4 . . F 1.2 2.71
Met398 . . . . . . C 1.04-0.6 * . F 1.6 1.96
Thr399 . . . . . T C 1.86-0.2 * * F 1.8 1.7
Pro400 . . . . . T C 1.920.2 . . F 1.5 2.03
Thr401 . . . . . T C 1.88-0.19. * F 2.4 4.03
Gln402 . . . . . T C 2,22-0.8 . * F 3 4.83
Asn403 . A . . . . C 2.53-1.29. . F 2.3 6.25
Arg404 . A . . T . . 1.96-0.8 . * F 2.2 4.55
Glu405 . A . . T . . 1.92-0.6 . * F 1.9 1.84
Lys406 . A . . T . . 1.99-0.24. * F 1.3 1.8
Trp407 . A . . T . . 1.320.11 . * . 0.25 1.44
Ile408 . . . . T . . 1.320.69 . * . 0 0.44
Tyr409 . . . . T . . 0.871.09 . * . 0 0.36
Tyr410 . . . . T . . 0.831.51 . . . 0 0.34
Cys411 . . . . T . . 0.441.1 . . . 0 0.65
Asn412 . . . . T T . -0.160.84 . . . 0.2 0.41
Gly413 . . . . T T . 0.730.77 . . . 0.2 0.18
His414 . . . . T T . 0.090.01 . . . 0.5 0.58
Gly415 . . . . . T C -0.480.13 . . . 0.3 0.25
Ile416 A . . B . . . 0.230.41 . . . -0.6 0.21
Asp417 A . . B . . . -0.58-0.01* . . 0.3 0.31
Ile418 A . . B . . . -1.090.17 * . . -0.3 0.26
Leu419 A . . B . . . -1.640.39 * . . -0.3 0.27
Lys420 A . . B . . . -1.890.2 * . . -0.3 0.16
Leu421 A . . B . . . -1 0.7 * . . -0.6 0.24
Val422 A . . B . . . -1.860.41 * . . -0.6 0.5
Ala423 A . . B . . . -1.310.37 * . . -0.3 0.18
Ala424 A . . B . . . -0.80.8 . * . -0.6 0.22
Gln425 A . . B . . . -0.840.5 . * . -0.6 0.4
Val426 A . . B . . . -0.320.26 . * . -0.020.68
Gly427 A . . . . T . 0.580.67 * * F 0.51 0.71
Ser428 . . . . . T C 1.170.17 * * F 1.29 0.82
Gln429 . . . . T T . 0.87-0.23* * F 2.52 1.85
Trp430 . . . . T T . 0.62-0.19* * F 2.8 1.31
Lys431 . . . B T . . 1.480.14 * * F 1.52 1.53
Asp432 . . . B T . . 1.120.16 * . . 1.09 1.53
Ile433 . . B B . . . 0.610.54 * . . 0.11 1.26
Tyr434 . . B B . . . -0.060.31 * . . -0.020.52
Gln435 . . B B . . . 0.230.89 * . . -0.6 0.17
Phe436 . . . B T . . -0.41.29 * . . -0.2 0.38
Leu437 . . . B . . C -0.71.1 * . . -0.4 0.25
Cys438 . . . B T . . 0.190.73 * * . -0.2 0.19
Asn439 . . . . . T C 0.540.33 * * . 0.3 0.38
Ala440 . . . . . T C 0.54-0.46* * F 1.05 0.91
Ser441 A . . . . T . 0.39-1.14. . F 1.3 2.93
Glu442 A . . . . T . 0.61-1.07* . F 1.3 1.35
Arg443 A A . . . . . 0.69-0.97* . F 0.9 1.35
Glu444 A A . . . . . -0.01-0.97* . . 0.75 1.02
Val445 A A . . . . . 0.2 -0.5 * . . 0. 0.51
8 7 6
Ala446 A A . . . . . 0.58-0.19* . . 0.3 0.35
Ala447 A A . . . . . 0.230.21 * . . -0.3 0.32
CA 02365255 2001-09-24
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26
Table
1
(continued)
ResPosition1 II I V VI VII VIIIIX X XI XII XIIIXIV
II IV
Phe448 A . . . T . -0.120.64 * . . -0.20.43
.
Ser449 A . . . T . -0.430.76 . . F -0.20.67
.
Asn450 . . . T T . -0.170.74 . . F 0.350.96
.
Gly451 . . . T T . 0.420.74 . . F 0.5 1.12
.
Tyr452 . . . . . C 0.98-0.04. . F 1 1.39
.
Thr453 . A . . . C 1.680.07 . . . 0.051.18
.
Ala454 A A . . . . 2.09-0.33. . . 0.452.06
.
Asp455 A A . . . . 1.5 -0.76. . . 0.752.58
.
His456 A A . . . . 1.6 -1.01. . . 0.751.8
.
Glu457 A A . . . . 1.26-0.74. . . 0.752.8
.
Arg458 A A . . . . 0.98-0.74. * . 0.751.69
.
Ala459 A A . . . . 0.76-0.24. * . 0.451.26
.
Tyr460 A A . . . . 0.76-0.06* * . 0.3 0.6
.
Ala461 A A . . . . 0.760.34 * * . -0.30.53
.
Ala462 A A . . . . 0.470.84 * * . -0.60.71
.
Leu463 A . B . . . 0.041.26 . * . -0.60.48
.
Gln464 A . B . . . -0.260.99 . * . -0.60.68
.
His465 A . B . . . 0.1 1.17 . * . -0.60.47
.
Trp466 . . B T . . 0.340.67 . * . -0.051.13
.
Thr467 . . B . . C 0.720.41 * * . -0.10.64
.
Ile468 . . B . . C 1.530.44 * * F 0.350.73
.
Arg469 . . B . . C 0.94-0.06. * F 1.7 1.2
.
Gly470 . . . . T C 0.68-0.47. * F 2.250.84
.
Pro471 . . . . T C 0.16-0.57. * F 3 1.61
.
Glu472 . . . . T C -0.12-0.57* * F 2.550.68
.
Ala473 A . . . T . 0.77-0.07* * F 1.750.69
.
Ser474 A A . . . . -0.16-0.1 * . . 0.9 0.78
.
Leu475 A A B . . . -0.70.16 . . . 0 0.37
.
Ala476 A A B . . . -0.790.84 . . . -0.60.26
.
Gln477 A A B . . . -1.380.73 * . . -0.60.26
.
Leu478 A A B . . . -1.60.84 * * . -0.60.31
.
Ile479 A A B . . . -1.190.84 * . . -0.60.26
.
Ser480 A A . . . . -0.380.34 * . . -0.30.29
.
Ala481 A A . . . . 0.180.34 * . . -0.30.61
.
Leu482 A A . . . . 0.290.16 * . . 0.191.18
.
Arg483 A A . . . . 1.21-0.53* . . 1.431.73
.
Gln484 A A . . . . 2.1 -0.91* . F 1.923.35
.
His485 . . . T T . 2.4 -1.01* * F 3.066.53
.
Arg486 . . . T T . 2.13-1.7 * . F 3.4 5.57
.
Arg487 . . . T T . 2.09-1.06. * F 3.062.39
.
Asn488 . . . T T . 1.98-0.81* . F 2.721.3
.
Asp489 . . . T . . 2.02-1.31* * F 2.181.15
.
Val490 A . . . . . 1.17-1.31. * F 1.441.17
.
Val491 A . . . . . 1.17-0.63. * F 0.950.51
.
Glu492 A . . . . . 0.71-1.03* * F 0.950.6
.
Lys493 A . . . . . -0.1-0.6 * * F 0.950.8
.
Ile494 A . . . . . -0.7-0.56* * F 0.950.89
.
Arg495 A A . . . . 0.16-0.59* * F 0.750.51
.
Gly496 A A . . . . 1.01-0.59* * . 0.6 0.44
.
Leu497 A A . . . . 0.7 -0.59* * . 0.751.05
.
Met498 A A . . . . 0.34-0.79* * . 0.6 0.77
.
Glu499 A A . . . . 1.23-0.3 * * F 0.6 1.13
.
Asp500 A . . . T . 0.31-0.33* * F 1 2.37
.
Thr501 A . . . T . 0.66-0.33. . F 1 1.97
.
Thr502 A . . . T . 1.16-0.94* * F 1.3 1.97
.
Gln503 A . . . T . 1.76-0.46. * F I 1.7
.
CA 02365255 2001-09-24
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?7
Table 1 (continued)
Res Position 1 11 III IV V VI VII VIII IX X XI XI1 XIII XIV
Leu 504 A A . . . . . 1.8 -0.46 . F 0.6 1.97
.
Glu 505 A A . . . . . 0.99-0.94 . F 0.9 2.73
.
Thr 506 A A . . . . . 0.71-0.74 . F 0.9 1.3
.
Asp 507 A A . . . . . 0.21-0.64 . F 0.9 1.59
.
Lys 508 A A . . . . . 0 -0.64 * F 0.75 0.76
.
Leu 509 A A . . . . . 0.21-0.21 * . 0.3 0.81
.
Ala 510 A A . . . . . -0.09-0.09 * . 0.3 0.48
.
Leu 511 . A . . . . C 0.010.3 . * . -0.1 0.32
Pro 512 . A . . T . . -0.290.73 . * . -0.2 0.61
Met 513 . . . . T . . -0.540.43 . * . 0 0.8
Ser 514 . . . . . T C -0.540.36 . . F 0.6 1.51
Pro 515 . . . . . T C -0.260.36 . . F 0.45 0.8
Ser 516 . . . . . T C 0.340.31 . . F 0.6 1.09
Pro 517 . . . . T T . 0.260.13 . . F 0.8 1.26
Leu 518 . . . . T . . 0.640.13 . . F 0.6 1.09
Ser 519 . . . . . T C 0.060.13 . . F 0.6 1.26
Pro 520 . . . . . T C 0.060.43 . . F 0.15 0.57
Ser 521 . . . . . T C 0.060.43 . . F 0.3 1.07
Pro 522 . . . . . T C 0.060.13 . . F 0.6 1.07
Ile 523 . . . . . . C 0.870.17 . . F 0.4 1.07
Pro 524 . . . . . . C 0.580.14 . . F 0.4 1.28
Ser 525 . . . . . T C 0.830.26 . * F 0.45 0.84
Pro 526 . . . . . T C 0.32-0.17 * F 1.2 2.39
.
Asn 527 . . . . . T C 0.53-0.17 * F 1.2 1.27
.
Ala 528 A . . . . T . 1.42-0.6 . * F 1.3 1.64
Lys 529 A A . . . . . 1.33-0.59 * F 0.9 1.71
.
Leu 530 A A . . . . . 1.04-0.63 * F 0.9 1.43
.
Glu 531 A A . . . . . 0.44-0.53 * F 0.9 1.43
.
Asn 532 A A . . . . . -0.37-0.34 * F 0.45 0.59
.
Ser 533 A . . B . . . -0.090.34 . * F -0.150.59
Ala 534 A . . B . . . -0.990.14 * * . -0.3 0.49
Leu 535 A . . B . . . -0.180.79 . * . -0.6 0.23
Leu 536 A . . B . . . -0.390.39 . * . -0.3 0.29
Thr 537 A . . B . . . -0.690.43 . * . -0.6 0.45
Val 538 . . B B . . . -0.60.31 . * . -0.3 0.73
Glu 539 . . B . . T . -0.010.06 . * F 0.4 1.36
Pro 540 . . . . . T C 0.8 -0.23 * F 1.54 1.64
.
Ser 541 . . . . . T C 1.66-0.71 * F 2.18 3.68
.
Pro 542 A . . . . T . 1.97-1.36 * F 2.32 4.25
.
Gln 543 . . . . T . . 2.87-0.96 * F 2.86 4.42
.
Asp 544 . . . . T T . 2.52-1.39 * F 3.4 6.59
.
Lys 545 . . . . T T . 2.03-1.34 * F 3.06 4.22
.
Asn 546 . . . . T T . 1.63-0.99 . F 2.72 2.11
.
Lys 547 . . . . T T . 0.99-0.6 . . F 2.38 1.09
Gly 548 . . . B . . C 0.990.04 . . F 0.39 0.41
Phe 549 . . B B . . . 0.990.04 . . . -0.3 0.42
Phe 550 . . B B . . . 0.64-0.36 . . 0.3 0.37
.
Val 551 A . . B . . . 0.640.03 . . . -0.3 0.49
Asp 552 A . . B . . . 0.39-0.4 . . F 0.45 0.99
Glu 553 A A . . . . . -0.08-0.76 . F 0.9 1.77
.
Ser 554 A A . . . . . -0.19-0.86 * F 0.9 1.96
*
Glu 555 A A . . . . . 0.62-0.81 * F 0.75 0.97
*
Pro 556 A A . . . . . 0.81-0.81 * F 0.9 1.1
*
Leu 557 A A . . . . . 0.81-0.24 * F 0.45 0.44
*
Leu 558 A A . . . . . 0.51-0.63 * . 0.91 0.42
.
Arg 559 A A . . . . . 0.5 -0.24 * . 0.92 0.37
*
CA 02365255 2001-09-24
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?8
Table
1
(continued)
Res PositionI II IV V VI VIIVIII IX X XI XllXlll XIV
III
Cys 560 A . . . . T . 0.2 -0.19* * F 1.78 0.64
Asp 561 . . . . T T . 0.11 -0.49. * F 2.64 1.04
Ser 562 . . . . T T . 0.58 -0.79. * F 3.1 0.71
Thr 563 . . . . T T . 1.09 -0.36. * F 2.64 1.32
Ser 564 . . . . . T C 0.68 -0.54. * F 2.43 1.06
Ser 565 . . . . . T C 0.76 -0.16. . F 1.82 1.06
Gly 566 . . . . T T . -0.06-0.04. . F 1.56 0.74
Ser 567 . . . . . T C -0.060.16 * . F 0.45 0.45
Ser 568 . . . . . . C 0.37 0.16 * . F 0.53 0.45
Ala 569 . . . . . . C 0.67 -0.23* . F 1.41 0.9
Leu 570 . . . . . . C 0.62 -0.26* . F 1.84 1.08
Ser 571 . . . . . T C 0.67 -0.21* . F 2.17 0.8
Arg 572 . . . . T T . 0.27 -0.21* . F 2.8 1.06
Asn 573 . . . . T T . -0.320.07 * . F 1.92 1.11
Gly 574 . . . . T T . -0.040.07 * . F 1.49 0.58
Ser 575 . . . B . . C 0.81 0.17 * . F 0.61 0.43
Phe 576 A . . B . . . 1.11 0.17 * . F 0.13 0.53
Ile 577 A . . B . . . 1.04 -0.23* . . 0.3 0.93
Thr 578 A . . B . . . 1.09 -0.66. . F 0.9 1.39
Lys 579 A . . B . . . 1.43 -1.04* . F 0.9 3.21
Glu 580 A . . . . . . 1.42 -1.83. . F 1.1 7.65
Lys 581 A . . . . T . 1.27 -2.03. . F 1.3 7.65
Lys 582 A . . . . T . 1.34 -1.87* * F 1.3 2.84
Asp 583 A . . . . T . 1.77 -1.19* * F 1.3 1.35
Thr 584 A . . . . T . 1.72 -1.19* . F 1.3 1.32
Val 585 A . . B . . . 0.87 -0.79* . . 0.75 1.15
Leu 586 A . . B . . . 0.93 -0.14* * . 0.3 0.51
Arg 587 A . . B . . . 0.08 -0.14* * . 0.3 0.69
Gln 588 . . B B . . . 0.08 0.06 * * . -0.3 0.77
Val 589 . . B B . . . 0.18 -0.59* * . 1.06 1.56
Arg 590 . . . B T . . 0.37 -0.84* * . 1.77 1.23
Leu 591 . . . B T . . 1.18 -0.27* . . 1.63 0.38
Asp 592 . . . . . T C 0.26 -0.67* * F 2.59 0.86
Pro 593 . . . . T T . 0.26 -0.63. * F 3.1 0.36
Cys 594 . . . . T T . 0.9 -0.23* * . 2.34 0.76
Asp 595 . . . . T T . -0.1 -0.49* * . 2.03 0.7
Leu 596 . . . . . . C 0.01 0.2 . * . 0.72 0.32
Gln 597 . . B . . . . 0.01 0.56 * . . -0.090.51
Pro 598 A . . . . . . 0.22 -0.01* * . 0.5 0.51
Ile 599 A A . . . . . 0.29 -0.01* * . 0.45 1.04
Phe 600 A A . . . . . -0.52-0.09* * . 0.3 0.59
Asp 601 A A . . . . . 0.26 0.2 * * . -0.3 0.32
Asp 602 A A . . . . . -0.440.27 * . . -0.3 0.61
Met 603 A A . . . . . -1.040.37 * . . -0.3 0.61
Leu 604 A A . . . . . -0.160.27 * . . -0.3 0.3
His 605 A A . . . . . 0.33 0.67 * . . -0.6 0.29
Phe 606 A A . . . . . 0.33 1.1 . . . -0.6 0.46
Leu 607 . A . . . . C 0.33 0.49 . . . -0.4 0.96
Asn 608 A . . . . T . 0.12 -0.2 . * F 1 1.22
Pro 609 A . . . . T . 1.04 -0.01. * F 1 1.16
Glu 610 A . . . . T . 0.22 -0.8 . * F 1.3 2.76
Glu 611 A . . . . T . 0.03 -0.84* . F 1.3 1.27
Leu 612 A A . B . . . 0.84 -0.56* * . 0.6 0.58
Arg 613 A A . B . . 0.84 -0.99* * . 0.6 0.58
Val 614 A A . B . . . 0.17 -0.99* * . 0.6 0.58
Ile 615 A A . B . . . -0.04-0.3 * * . 0.3 0.49
CA 02365255 2001-09-24
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?9
Table
1
(continued)
ResPosition1 11 IV V VI VII VIII IX X XI XIlXI11 XIV
III
Glu616 A A . B . . . -0.04 -0.56* * . 0.6 0.39
Glu617 A A . . . . . 0.18 -0.16* * F 0.45 0.9
Ile618 A A . . . . . 0.07 -0.3 * * F 0.6 1.3
Pro619 A A . . . . . 0.92 -0.99* . F 0.9 1.3
Gln620 A A . . . . . 1.86 -0.99* * F 0.9 1.26
Ala621 A A . . . . . 1.04 -0.99* * F 0.9 3.59
Glu622 A A . . . . . 1.04 -0.99* . F 0.9 1.91
Asp623 A A . . . . . 2.04 -1.41* . F 0.9 1.84
Lys624 A A . . . . . 1.44 -1.81* * F 0.9 3.58
Leu625 A A . . . . . 0.74 -1.63* * F 0.9 1.7
Asp626 A A . . . . . 1.33 -0.84* * F 0.75 0.88
Arg627 A A . . . . . 0.44 -0.84* . F 0.75 0.76
Leu628 A A . B . . . -0.44 -0.16* . . 0.3 0.65
Phe629 A A . B . . . -0.83 -0.16* . . 0.3 0.27
Glu630 A A . B . . . -0.88 0.27 * . . -0.3 0.14
Ile631 A A . B . . . -0.83 0.91 * . . -0.6 0.12
Ile632 A A . B . . . -1.24 0.23 * * . -0.3 0.29
Gly633 A A . B . . . -0.43 -0.17. . . 0.3 0.22
Val634 A A . B . . . 0.27 0.23 . . F -0.150.55
Lys635 A A . B . . . -0.32 -0.46. . F 0.6 1.35
Ser636 . A . . . . C 0.27 -0.64. . F 1.1 1.38
Gln637 A A . . . . . 1.16 -0.69. * F 0.9 2.49
Glu638 A A . . . . . 1.19 -0.93. . F 0.9 2.16
Ala639 A A . B . . . 1.23 -0.44. . F 0.6 2.33
Ser640 A A . B . . . 0.3 8 -0.14* . F 0.6 1.1
I
Gln641 A A . B . . . 0.68 0.14 * . F -0.150.53
Thr642 A A . B . . . 0.38 0.14 * . F -0.150.87
Leu643 A A . B . . . -0.48 0.03 * . F -0.150.87
Leu644 A A . B . . . -0.13 0.29 * . F -0.150.37
Asp645 . A . B T . . -0.13 0.64 * . F -0.050.41
Ser646 . A . B T . . -0.17 0.54 * . . -0.2 0.66
Val647 . . B B . . . -0.67 0.36 * . . -0.151.09
Tyr648 . . . B T . . -0.07 0.36 * . . 0.1 0.54
Ser649 . . . . T . . 0.74 0.79 * . . 0 0.62
His650 . A . . . . C -0.07 0.4 * . . 0.05 1.39
Leu651 . A . . . . C -0.58 0.44 * . . -0.4 0.73
Pro652 A A . . . . . -0.11 0.37 * . F -0.150.45
Asp653 A A . . . . . -0.26 0.41 * . . -0.6 0.42
Leu654 A A . . . . . -0.34 0.34 * . . -0.3 0.66
Leu655 A A . . . . . -0.7 0.09 * . . -0.3 0.54
CA 02365255 2001-09-24
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3O
Preferred nucleic acid fragments of the present invention also include nucleic
acid
molecules encoding polypeptides comprising. or alternatively consisting of,
one, two,
three, four, five. or more epitope-bearing portions of the TR9 receptor
protein. In
particular, such nucleic acid fragments of the present invention include
nucleic acid
molecules encoding: a polypeptide comprising or alternatively, consisting of,
amino acid
residues from about 4 to about 81 in SEQ ID N0:2; a polypeptide comprising or
alternatively, consisting of, amino acid residues from about 116 to about 271
in SEQ ID
N0:2; a polypeptide comprising or alternatively, consisting of, amino acid
residues from
about 283 to about 308 in SEQ ID N0:2; a polypeptide comprising or
alternatively,
consisting of, amino acid residues from about 336 to about 372 in SEQ ID N0:2;
a
polypeptide comprising or alternatively, consisting of, amino acid residues
from about
393 to about 434 in SEQ ID N0:2; a polypeptide comprising or alternatively,
consisting
of, amino acid residues from about 445 to about 559 in SEQ ID N0:2; and a
polypeptide
comprising or alternatively, consisting of, amino acid residues from about 571
to about
588 in SEQ ID N0:2. In this context "about" includes the particularly recited
range,
larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either
terminus or at both
termini. The inventors have determined that the above polypeptide fragments
are
antigenic regions of the TR9 receptor. Methods for determining other such
epitope-
bearing portions of the TR9 protein are described in detail below.
In specific embodiments, the polynucleotides of the invention are less than
100000 kb, 50000 kb, 10000 kb, 1000 kb, 500 kb, 400 kb, 350 kb, 300 kb, 250
kb,
200 kb, 175 kb, 150 kb, 125 kb, 100 kb, 75 kb, 50 kb, 40 kb, 30 kb, 25 kb, 20
kb, 15
kb, 10 kb, 7.5 kb, or 5 kb in length.
In further embodiments, polynucleotides of the invention comprise, or
alternatively consist of, at least 15, at least 30, at least 50, at least 100,
or at least 250, at
least 500, or at least 1000 contiguous nucleotides of TR9 coding sequence, but
consist of
less than or equal to 1000 kb, 500 kb, 250 kb, 200 kb, 150 kb, 100 kb, 75 kb,
50 kb, 30
kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kb of genomic DNA that flanks the 5' or
3' coding
nucleotide set forth in Figures lA-D (SEQ ID NO:1). In further embodiments,
polynucleotides of the invention comprise, or alternatively consist of, at
least 15, at least
30, at least 50, at least 100, or at least 250, at least 500, or at least 1000
contiguous
nucleotides of TR9 coding sequence, but do not comprise all or a portion of
any TR9
intron. In another embodiment, the nucleic acid comprising, or alternatively
consisting
of, TR9 coding sequence does not contain coding sequences of a genomic
flanking gene
(i.e., 5' or 3' to the TR9 gene in the genome). In other embodiments, the
polynucleotides
of the invention do not contain the coding sequence of more than 1000, 500,
250, 100,
50, 25, 20, 15, I0, 5, 4, 3, 2, or 1 genomic flanking gene(s).
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31
In another embodiment. the invention provides an isolated nucleic acid
molecule
comprising, or alternatively consisting of, a polynucleotide which hybridizes,
preferably
under stringent hybridization conditions, to a portion of the polynucleotide
sequence of a
polynucleotide of the invention such as, for instance, the sequence
complementary to the
coding and/or noncoding (i.e., transcribed, untranslated) sequence depicted in
Figures
lA-D, the sequence of the cDNA clone contained in ATCC Deposit 209037 and the
sequence encoding a TR9 domain or a polynucleotide fragment as described
herein. By
"stringent hybridization conditions" is intended overnight incubation at
42°C in a solution
comprising: 50% formamide, 5x SSC (750 mM NaCI, 75mM trisodium citrate), 50 mM
sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20
g/ml
denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx
SSC at
about65°C.
By a polynucleotide which hybridizes to a "portion" of a polynucleotide is
intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15
nucleotides (nt), and more preferably at least about 20 nt, still more
preferably at least
about 30 nt, and even more preferably about 30-70, or 80-150 nt, or the entire
length of
the reference polynucleotide. By a portion of a polynucleotide of "at least
about 20 nt in
length," for example, is intended 20 or more contiguous nucleotides from the
nucleotide
sequence of the reference polynucleotide (e.g., the deposited cDNA or the
nucleotide
sequence as shown in Figures lA-D (SEQ ID NO:1). In this context "about"
includes the
particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1)
nucleotides, at either
terminus or at both termini. These have uses, which include, but are not
limited to, as
diagnostic probes and primers as discussed above and in more detail below.
In specific embodiments, polynucleotides of the invention hybridize to a
complementary strand of a polynucleotide encoding amino acid residues 40-152,
40-48,
40-51, 51-66, 66-73, 73-83, 83-104, 104-110, 110-128, 128-146, and/or 146-152
as
depicted in SEQ ID N0:2.
Of course, a polynucleotide which hybridizes only to a poly A sequence (such
as
the 3' terminal poly tract of the TR9 receptor cDNA shown in SEQ ID NO:1 ), or
to a
complementary stretch of T (or U) residues, 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
using oligo dT as a primer).
As indicated, nucleic acid molecules of the present invention which encode a
TR9
receptor polypeptide may include, but are not limited to, those encoding the
amino acid
sequence of the mature polypeptide, by itself; the coding sequence for the
mature
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37
polypeptide and additional sequences. such as those encoding the about amino
acid leader
or secretory sequence, such as a pre-, or pro- or prepro- protein sequence;
the coding
sequence of the mature polypeptide, with or without the aforementioned
additional coding
sequences, together with additional, non-coding sequences, including for
example, but
not limited to introns and non-coding 5' and 3' sequences, such as the
transcribed, non-
translated sequences that play a role in transcription, mRNA processing,
including
splicing and polyadenylation signals, for example - ribosome binding and
stability of
mRNA; an additional coding sequence which codes for additional amino acids,
such as
those which provide additional functionalities. Thus, the sequence encoding
the
polypeptide 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., Pr-oc. Natl. Acad.
Sci. USA
86:821-824 (1989), for instance, hexa-histidine provides for convenient
purification of
the fusion protein. The "HA" tag is another peptide useful for purification
which
corresponds to an epitope derived from the influenza hemagglutinin protein,
which has
been described by Wilson et al., Cell 37:767-778 ( 1984). As discussed below,
other
such fusion proteins include the TR9 receptor fused to Fc at the N- or C-
terminus.
The present invention further relates to variants of the nucleic acid
molecules of
the present invention, which encode portions, analogs or derivatives of the
TR9 receptor.
Variants may occur naturally, such as a natural allelic variant. By an
"allelic variant" is
intended one of several alternate forms of a gene occupying a given locus on a
chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New
York
(1985). Non-naturally occurring variants may be produced using art-known
mutagenesis
techniques, 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 er al., Philos. Trans. R. Soc. London SerA
317:415
( 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 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 functional activities of the
TR9 receptor or
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~i ~
J J
portions thereof. Also especially preferred in this regard are conservative
substitutions.
Further embodiments of the invention include isolated nucleic acid molecules
comprising, or alternatively consisting of, a polynucleotide having a
nucleotide sequence
at least 90% identical, and more preferably at least 80%, 85%, 95%, 96%, 97%,
98% or
99% identical to (a) a nucleotide sequence encoding the polypeptide having the
amino acid
sequence shown in Figures lA-D (SEQ ID N0:2); (b) a nucleotide sequence
encoding the
polypeptide having the amino acid sequence shown in Figures lA-D (SEQ ID
N0:2), but
lacking the N-terminal methionine; (c) a nucleotide sequence encoding the
predicted
mature TR9 polypeptide (full-length polypeptide with any attending leader
sequence
removed) comprising, or alternatively consisting of, the amino acid sequence
at positions
from about 1 to about 615 in SEQ ID N0:2; (d) a nucleotide sequence encoding
the TR9
polypeptide having the amino acid sequence encoded by the cDNA clone contained
in
ATCC Deposit No. 209037; (e) a nucleotide sequence encoding the mature TR9
polypeptide having the amino acid sequence encoded by the cDNA clone contained
in
ATCC Deposit No. 209037; (f) a nucleotide sequence encoding the TR9 receptor
extracellular domain; (g) a nucleotide sequence encoding one, two, three or
all four
TNFR-like cysteine rich motifs of TR9 (amino acid residues 67 to 211 in
Figures lA-D;
amino acid residues 27 to 171 in SEQ ID N0:2); (h) a nucleotide sequence
encoding the
TR9 receptor transmembrane domain; (i) a nucleotide sequence encoding the TR9
receptor intracellular domain; (j) a nucleotide sequence encoding the TR9
receptor
extracellular and intracellular domains with all or part of the transmembrane
domain
deleted; (k) a nucleotide sequence encoding the TR9 receptor death domain; (1)
a
nucleotide sequence encoding the TR9 leucine zipper; (m) a fragment of the
polpeptide of
(c) having TR9 functional activity (e.a., antigenic or biological activity);
and (n) a
nucleotide sequence complementary to any of the nucleotide sequences in (a),
(b), (c),
(d), (e), (f), (g), (h), (i), (j), (k), (1), or (m). Polypeptides encoded by
these
polynucleotides are also encompassed by the invention.
By a polynucleotide having a nucleotide sequence at least, for example, 95%
"identical" to a reference nucleotide sequence encoding a TR9 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 the TR9
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
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34
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 TR9 nucleotide sequence shown
in
Figures lA-D (SEQ ID NO: I) or any fragment (e.a., a polynucleotide encoding
the amino
acid sequence of a TR9 N and/or C terminal deletion described herein) as
described
herein.
As a practical matter, whether any particular nucleic acid molecule is at
least 80%,
85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance. the nucleotide
sequence shown in Figures lA-D (SEQ ID NO:1 ) or to the nucleotides sequence
of the
deposited cDNA clone can be determined conventionally using known computer
programs such as the Bestfit program (Wisconsin Sequence Analysis Package,
Version 8
for Unix, Genetics Computer Group, University Research Park, 575 Science
Drive,
Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and
Waterman,
Advances iiZ Applied Mat7~enzatics 2:482-489 (1981), to find the best segment
of
homology between two sequences. When using Bestfit or any other sequence
alignment
program to determine whether a particular sequence is, for instance, 95%
identical to a
reference sequence according to the present invention, the parameters are set,
of course,
such that the percentage of identity is calculated over the full length of the
reference
nucleotide sequence and that gaps in homology of up to 5% of the total number
of
nucleotides in the reference sequence are allowed.
In a specific embodiment, the identity between a reference (query) sequence (a
sequence of the present invention) and a subject sequence, also referred to as
a global
sequence alignment, is determined using the FASTDB computer program based on
the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 ( 1990)). Preferred
parameters
used in a FASTDB alignment of DNA sequences to calculate percent identity are:
Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining Penalty=30,
Randomization
Group Length=0, Cutoff Score=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 of the subject sequence when calculating
percent
identity. For subject sequences truncated at the 5' or 3' ends, relative to
the query
sequence, the percent identity is corrected by calculating the number of bases
of the query
sequence that are 5' and 3' of the subject sequence, which are not
matched/aligned, as a
percent of the total bases of the query sequence. A determination of whether a
nucleotide
CA 02365255 2001-09-24
w0 00/56862 PCT/US00/06831
~J
is matched/aligned is determined by results of the FASTDB sequence alignment.
This
percentage is then subtracted from the percent identity, calculated by the
above FASTDB
program using the specified parameters, to arrive at a final percent identity
score. This
corrected score is what is used for the proposes of this embodiment. Only
bases outside
the 5' and 3' bases of the subject sequence, as displayed by the FASTDB
alignment,
which are not matched/aligned with the query sequence. are calculated for the
purposes of
manually adjusting the percent identity score. For example, a 90 base subject
sequence is
aligned to a 100 base query sequence to determine percent identity. The
deletions occur at
the 5' end of the subject sequence and therefore, the FASTDB alignment does
not show a
matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases
represent 10%
of the sequence (number of bases at the 5' and 3' ends not matched/total
number of bases
in the query sequence) so 10% is subtracted from the percent identity score
calculated by
the FASTDB program. If the remaining 90 bases were perfectly matched the final
percent
identity would be 90%. In another example, a 90 base subject sequence is
compared with
a 100 base query sequence. This time the deletions are internal deletions so
that there are
no bases on the 5' or 3' of the subject sequence which are not matched/aligned
with the
query. In this case the percent identity calculated by FASTDB is not manually
corrected.
Once again, only bases 5' and 3' of the 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%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in
Figures lA-D (SEQ ID NO:1) or to the nucleic acid sequence of the deposited
cDNA,
irrespective of whether they encode a polypeptide having TR9 receptor
functional activity.
Polypeptides encoded by these polynucleotides are also encompassed by the
invention.
This is because even where a particular nucleic acid molecule does not encode
a
polypeptide having TR9 receptor functional activity, one of skill in the art
would still
know how to use the nucleic acid molecule, for instance, as a hybridization
probe or a
polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of
the
present invention that do not encode a polypeptide having TR9 receptor
functional activity
include, inter alia, (1) isolating the TR9 receptor gene or allelic variants
thereof in a cDNA
library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal
spreads to
provide precise chromosomal location of the TR9 receptor gene, as described in
Verma et
al., Human Chrof~iosonves: A Mar2atal of Basic Techniques, Pergamon Press,
N.Y.
(1988); and (3) Northern Blot analysis for detecting TR9 receptor mRNA
expression in
specific tissues.
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36
Preferred, however, are nucleic acid molecules having sequences at least 80%,
85%, 90%. 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence
shown
in Figures 1 A-D (SEQ ID NO:1 ), the nucleic acid sequence of the deposited
cDNA, or
fragments thereof, which do, in fact, encode a polypeptide having TR9 receptor
S functional activity. Polypeptides encoded by these polynucleotides are also
encompassed
by the invention. By "a polypeptide having TR9 receptor functional activity"
is intended
polypeptides exhibiting activity similar, but not necessarily identical, to a
functional
activity of the TR9 receptor of the invention (e.g., the full-length (i.e.,
complete) protein
or, preferably, the mature protein), as measured in a particular functional
assay (e.g.,
immunoassay and/or biological assay). For example, TR9 polypeptide functional
activity
can be measured by the ability of a polypeptide sequence described herein to
form
multimers (e.g., homodimers and homotrimers) with complete TR9, and to bind a
TR9
ligand (e.g., TR9 ligand expressed on the surface of monocytes). TR9
polypeptide
functional activity can be also be measured, for example, by determining the
ability of a
polypeptide of the invention to activate monocytes, increase cell survival of
monocytes or
to induce apoptosis in cells expressing the polypeptide. These functional
assays can be
routinely performed using techniques described herein and otherwise known in
the art.
For example, TR9 receptor functional activity (e.g., biological activity) can
routinely be measured using the cell death assays performed essentially as
previously
described (Chinnaiyan et al., Cell 81:505-512 (1995); Boldin et al., J. Biol.
Chem.
270:7795-8(1995); Kischkel et al., EMBO 14:5579-5588 (1995); Chinnaiyan et
al., J.
Biol. Chem. 271:4961-4965 ( 1996)) and as set forth in Example 5 below. In
MCF7
cells, plasmids encoding full-length TR9 or a candidate death domain
containing receptor
are co-transfected with the pLantern reporter construct encoding green
fluorescent protein.
Nuclei of cells transfected with TR9 will exhibit apoptotic morphology as
assessed by
DAPI staining. It is expected that like TNFR-1 and Fas/APO-1 (Muzio et al.,
Cell
85:817-827 ( 1996); Boldin et al., Cell 85:803-815 ( 1996); Tewari et al., J.
Biol. Chem.
270:3255-60 ( 1995)), TR9-induced apoptosis will be blocked by the inhibitors
of ICE-
like proteases, CrmA and z-VAD-fmk. In addition, it is expected that apoptosis
induced
by TR9 will be blocked by dominant negative versions of FADD (FADD-DN) or
FLICE
(FLICE-DN/MACHa 1 C360S).
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%, 95%, 96%, 97%, 98%, or 99% identical to, for
example, the nucleic acid sequence of the deposited cDNA or the nucleic acid
sequence
shown in Figures 1 A-D (SEQ ID NO:1 ), or fragments thereof, will encode a
polypeptide
"having TR9 receptor functional activity." In fact, since degenerate variants
of these
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
37
nucleotide sequences all encode the same polypeptide, in many instances, this
will be
clear to the skilled artisan even without performing the above described
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 TR9
receptor
functional activity. This is because the skilled artisan is fully aware of
amino acid
substitutions that are either less likely or not likely to significantly
effect protein function
(e.g., replacing one aliphatic amino acid with a second aliphatic amino acid),
as further
described below.
For example, guidance concerning how to make phenotypically silent amino acid
substitutions is provided in J.U. Bowie 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.
Polynucleotide assays
This invention is also related to the use of TR9 polynucleotides to detect
complementary polynucleotides such as, for example, as a diagnostic reagent.
Detection
of a mutated form of TR9 associated with a dysfunction will provide a
diagnostic tool that
can add or define a diagnosis of a disease or susceptibility to a disease
which results from
under-expression, over-expression, or altered expression of TR9 or a soluble
form
thereof, such as, for example, tumors or autoimmune diseases.
Individuals carrying mutations in the TR9 gene may be detected at the DNA
level
by a variety of techniques. Nucleic acids for diagnosis may be obtained from a
patient's
cells, such as from blood, urine, saliva, tissue biopsy and autopsy material.
The genomic
DNA may be used directly for detection or may be amplified enzymatically by
using PCR
prior to analysis. (Saiki et al., Nature 324:163-166 ( 1986)). RNA or cDNA may
also be
used in the same ways. As an example, PCR primers complementary to the nucleic
acid
encoding TR9 can be used to identify and analyze TR9 expression and mutations.
For
example, deletions and insertions can be detected by a change in size of the
amplified
product in comparison to the normal genotype. Point mutations can be
identified by
hybridizing amplified DNA to radiolabeled TR9 RNA or alternatively,
radiolabeled TR9
antisense DNA sequences. Perfectly matched sequences can be distinguished from
mismatched duplexes by RNase A digestion or by differences in melting
temperatures.
Sequence differences between a reference gene and genes having mutations also
may be revealed by direct DNA sequencing. In addition, cloned DNA segments may
be
employed as probes to detect specific DNA segments. The sensitivity of such
methods
can be greatly enhanced by appropriate use of PCR or another amplification
method. For
CA 02365255 2001-09-24
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p8
example, a sequencing primer is used with double-stranded PCR product or a
single-
stranded template molecule generated by a modified PCR. The sequence
determination is
performed by conventional procedures with radiolabeled nucleotide or by
automatic
sequencing procedures with fluorescent-tags.
Genetic testing based on DNA sequence differences may be achieved by detection
of alteration in electrophoretic mobility of DNA fragments in gels, with or
without
denaturing agents. Small sequence deletions and insertions can be visualized
by high
resolution gel electrophoresis. DNA fragments of different sequences may be
distinguished on denaturing formamide gradient gels in which the mobilities of
different
DNA fragments are retarded in the gel at different positions according to
their specific
melting or partial melting temperatures (see, e.g., Myers et al., Scie~zce
230:1242
( 1985)).
Sequence changes at specific locations also may be revealed by nuclease
protection assays, such as RNase and Sl protection or the chemical cleavage
method
(e.g., Cotton et al., Pr-oc. Natl. Acad. Sci. USA 85:4397-4401 ( 1985)).
Thus, the detection of a specific DNA sequence may be achieved by methods such
as hybridization, RNase protection, chemical cleavage, direct DNA sequencing
or the use
of restriction enzymes, (e.g., restriction fragment length polymorphisms
("RFLP") and
Southern blotting of genomic DNA.
In addition to more conventional gel-electrophoresis and DNA sequencing,
mutations also can be detected by in situ analysis.
Vectors and Host Cells
The present invention also relates to vectors which include the isolated DNA
molecules of the present invention, host cells which are genetically
engineered with the
recombinant vectors, or which are otherwise engineered to produce the
polypeptides of
the invention, and the production of TR9 receptor polypeptides, or fragments
thereof, by
recombinant or synthetic techniques.
The polynucleotides may be joined to a vector containing a selectable marker
for
propagation in a host. Generally, a plasmid vector is introduced in a
precipitate, such as a
calcium phosphate precipitate, or in a complex with a charged lipid. If the
vector is a
virus, it may be packaged in vitro using an appropriate packaging cell line
and then
transduced into host cells.
In one embodiment, the DNA of the invention is operatively associated with an
appropriate heterologous regulatory element (e.g., promoter or enhancer), such
as, the
phage lambda PL promoter, the E. coli lac, trp, and tac promoters, the SV40
early and
CA 02365255 2001-09-24
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39
late promoters and promoters of retroviral LTRs, to name a few. Other suitable
promoters will be known to the skilled artisan.
In embodiments in which vectors contain expression constructs, these
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.
coli and other bacteria. Representative examples of appropriate hosts include,
but are not
limited to, bacterial cells, such as E. coli, Streptomyces and Salmo~zella
typhiwuriurn
cells; fungal cells, such as yeast cells (e.g., Saccharornvces cerevisiae or
Pichia pastoris
(ATCC Accession No. 201178)); insect cells such as Drosophila S2 and
Spodoptera Sf9
cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant
cells.
Appropriate culture mediums and conditions for the above-described host cells
are known
in the art.
Among vectors preferred for use in bacteria include pHE4 (ATCC Accession
Number 209645, deposited February 25, 1998), pQE70, pQE60 and pQE-9, available
from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNHBA,
pNHl6a,
pNHl8A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3,
pDR540, pRITS available from Pharmacia. Among preferred eukaryotic vectors are
pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene; and pSVK3,
pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for
use
in yeast systems include, but are not limited to, pYES2, pYDI, pTEFI/Zeo,
pYES2/GS,
pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K,
pPIC9K, and PA0815 (all available from Invitrogen, Carlsbad, CA). Other
suitable
vectors will be readily apparent to the skilled artisan.
Selection of appropriate vectors and promoters for expression in a host cell
is a
well known procedure and the requisite techniques for expression vector
construction,
introduction of the vector into the host and expression in the host are
routine skills in the
art.
The present invention also relates to host cells containing the vector
constructs
discussed herein, and additionally encompasses host cells containing
nucleotide
sequences of the invention that are operably associated with one or more
heterologous
control regions (e.g., promoter and/or enhancer) using techniques known of in
the art.
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The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g.,
a human
derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host
cell can be a
prokaryotic cell, such as a bacterial cell. The host strain may be chosen
which modulates
the expression of the inserted gene sequences, or modifies and processes the
gene product
in the specific fashion desired. Expression from certain promoters can be
elevated in the
presence of certain inducers; thus expression of the genetically engineered
polypeptide
may be controlled. Furthermore, different host cells have characteristics and
specific
mechanisms for the translational and post-translational processing and
modification (e.g.,
phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen
to ensure the
10 desired modifications and processing of the foreign protein expressed.
Introduction of the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-
mediated
transfection, electroporation, transduction, infection or other methods. Such
methods are
described in many standard laboratory manuals, such as Davis et al., Basic
Methods In
15 Molecular Biology (1986).
TR9 polypeptides can be recovered and purified from recombinant cell cultures
by
well-known methods including ammonium sulfate or ethanol precipitation, acid
extraction, anion or canon exchange chromatography, phosphocellulose
chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite
20 chromatography and lectin chromatography. Most preferably, high performance
liquid
chromatography ("HPLC") is employed for purification.
TR9 polypeptides, and preferably the secreted form, can also be recovered
from:
products purified from natural sources, including bodily fluids, tissues and
cells, whether
directly isolated or cultured; products of chemical synthetic procedures; and
products
25 produced by recombinant techniques from a prokaryotic or eukaryotic host,
including, for
example, bacterial, yeast, higher plant, insect, and mammalian cells.
In one embodiment, the yeast Pichia pastoris is used to express TR9 protein in
a
eukaryotic system. Pichia pastoris is a methylotrophic yeast which can
metabolize
methanol as its sole carbon source. A main step in the methanol metabolization
pathway
30 is the oxidation of methanol to formaldehyde using O~. This reaction is
catalyzed by the
enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon
source, Pichia
pastoris must generate high levels of alcohol oxidase due, in part, to the
relatively low
affinity of alcohol oxidase for O,. Consequently, in a growth medium depending
on
methanol as a main carbon source, the promoter region of one of the two
alcohol oxidase
35 genes (AOXI ) is highly active. In the presence of methanol, alcohol
oxidase produced
from the AOXI gene comprises up to approximately 30% of the total soluble
protein in
Pichia pastoris. See, Ellis, S.B., et al., Mol. Cell. Biol. 5:1111-21 ( 1985);
Koutz, P.J,
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
41
et crl., Yectst 5:167-77 ( 1989); Tschopp, J.F., et crl., Ncicl. Acids Res. 1
x:3859-76
( 1987). Thus, a heterologous coding sequence, such as, for example, a TR9
polynucleotide of the present invention, under the transcriptional regulation
of all or part
of the AOXI regulatory sequence is expressed at exceptionally high levels in
Pichica yeast
grown in the presence of methanol.
In one example, the plasmid vector pPIC9K is used to express DNA encoding a
TR9 polypeptide of the invention, as set forth herein, in a Pichea yeast
system essentially
as described in "Pichicc Protocols: Methods in Molecular Biology," D.R.
Higgins and J.
Cregg, eds. The Humana Press, Totowa, NJ, 1998. This expression vector allows
expression and secretion of a TR9 protein of the invention by virtue of the
strong AOXI
promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory
signal peptide
(i.e., leader) located upstream of a multiple cloning site.
Many other yeast vectors could be used in place of pPIC9K, such as, pYES2,
pYDI, pTEFI/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-
D2, pHIL-S1, pPIC3.5K, and PA0815, as one skilled in the art would readily
appreciate, as long as the proposed expression construct provides
appropriately located
signals for transcription, translation. secretion (if desired), and the like,
including an in-
frame AUG as required.
In another embodiment, high-level expression of a heterologous coding
sequence,
such as, for example, a TR9 polynucleotide of the present invention, may be
achieved by
cloning the heterologous polynucleotide of the invention into an expression
vector such
as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the
absence of
methanol.
Depending upon the host employed in a recombinant production procedure, the
TR9 polypeptides may be glycosylated or may be non-glycosylated. In addition,
TR9
polypeptides may also include an initial modified methionine residue, in some
cases as a
result of host-mediated processes. Thus, it is well known in the art that the
N-terminal
methionine encoded by the translation initiation codon generally is removed
with high
efficiency from any protein after translation in all eukaryotic cells. While
the N-terminal
methionine on most proteins also is efficiently removed in most prokaryotes,
for some
proteins, this prokaryotic removal process is inefficient, depending on the
nature of the
amino acid to which the N-terminal methionine is covalently linked.
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., TR9 coding sequence), and/or to
include
genetic material (e.g., heterologous polynucleotide sequences) that is
operably associated
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WO 00/56862 PCT/US00/06831
42
with TR9 polynucleotides of the invention, and which activates, alters, and/or
amplifies
endogenous TR9 polynucleotides. For example, techniques known in the art may
be
used to operably associate heterologous control regions (e.g., promoter and/or
enhances)
and endogenous TR9 polynucleotide sequences via homologous recombination (see,
e.g.,
US Patent Number 5,641,670, issued June 24, 1997; International Publication
Number
WO 96/2941 l, published September 26, 1996; International Publication Number
WO
94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA
86:8932-
8935 ( 1989); and Zijlstra et al., Nature 342:435-438 ( 1989), the disclosures
of each of
which are incorporated by reference in their entireties).
The polypeptide may be expressed or synthesized in a modified form, such as a
fusion protein (comprising, or alternatively consisting of, the polypeptide
joined via a
peptide bond to a heterologous protein sequence (of a different protein)), and
may include
not only secretion signals, but also additional heterologous functional
regions. Such a
fusion protein can be made by ligating polynucleotides of the invention and
the desired
nucleic acid sequence encoding the desired amino acid sequence to each other,
by
methods known in the art, in the proper reading frame, and expressing the
fusion protein
product by methods known in the art. Alternatively, such a fusion protein can
be made
by protein synthetic techniques, e.g., by use of a peptide synthesizer. Thus,
for instance,
a region of additional amino acids, particularly charged amino acids, may be
added to the
N-terminus of the polypeptide to improve stability and persistence in the host
cell, during
purification, or during subsequent handling and storage. Additionally, 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.
For example,
in one embodiment, polynucleotides encoding TR9 polypeptides of the invention
may be
fused to the pelB pectate lyase signal sequence to increase the efficiency to
expression and
purification of such polypeptides in Gram-negative bacteria. See, US Patent
Nos.
5,576,195 and 5,846,818, the contents of which are herein incorporated by
reference in
their entireties.
A preferred fusion protein comprises a heterologous region from immunoglobulin
that is useful to solubilize proteins. For example, EP-A-O 464 533 (Canadian
counterpart
2045869) discloses fusion proteins comprising various portions of constant
region of
immunoglobin molecules together with another human protein or part thereof. In
many
cases, the Fc part in a fusion protein is thoroughly advantageous for use in
therapy and
diagnosis and thus results, for example, in improved pharmacokinetic
properties (EPA 0
232 262). Thus, in a specific embodiment, fusion proteins of the invention
comprise, or
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WO 00/56862 PCT/US00/06831
43
alternatively. consist of, amino acid residues 1 to 310 of SEQ ID N0:2 fused
to an Fc
polypepitde sequence. On the other hand, for some uses it would be desirable
to be able
to delete the Fc part after the fusion protein has been expressed, detected
and purified in
the advantageous manner described. This is the case when the Fc portion proves
to be a
hindrance to use in therapy and diagnosis, for example when the fusion protein
is to be
used as an antigen for immunizations. In drug discovery, for example, human
proteins,
such as the hILS-receptor, have been fused with Fc portions for the propose of
high-
throughput screening assays to identify antagonists of hIL-5. See, Bennett et
al., J. of
Moles. Reco~IrZitiora 8:52-58 (1995) and Johanson et al., J. Biol. Client.
270:9459-9471
(1995).
TR9 polypeptides of the present invention include naturally purified products,
products of chemical synthetic procedures, and products produced by
recombinant
techniques from a prokaryotic or eukaryotic host, including, for example,
bacterial, yeast,
higher plant. insect and mammalian cells. Depending upon the host employed in
a
recombinant production procedure, the polypeptides of the present invention
may be
glycosylated or may be non-glycosylated. In addition, polypeptides of the
invention may
also include an initial modified methionine residue or alternatively, may be
missing the N-
terminal methionine, in some cases as a result of host-mediated processes.
In addition, polypeptides 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.,
Nature
310:105-111 ( 1984)). For example, a peptide corresponding to a fragment of
the TR9
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 TR9 polypeptide sequence.
Non-classical
amino acids include, but are not limited to, to the D-isomers of the common
amino acids,
2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-
amino
butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric
acid,
3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline,
sarcosine,
citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,
phenylglycine,
cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as
b-methyl
amino acids. Ca-methyl amino acids. Na-methyl amino acids, and amino acid
analogs in
general. Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
Non-naturally occurring variants may be produced using art-known mutagenesis
techniques, which include, but are not limited to oligonucleotide mediated
mutagenesis,
alanine scanning, PCR mutagenesis, site directed mutagenesis (see, e.g.,
Carter et al.,
Nucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487
(1982)),
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WO 00/56862 PCT/US00/06831
44
cassette mutagenesis (see, e.,g., Wells et crl., Gene 34:315 ( 1985)),
restriction selection
mutagenesis (see, e.g., Wells et al., Plrilos. Tnczns. R. Soc. London SerA
317:415
( 1986)).
The invention additionally, encompasses TR9 proteins 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, NaBH~, acetylation, formylation, oxidation, reduction, metabolic
synthesis in
the presence of tunicamycin; etc.
Additional post-translational modifications encompassed by the invention
include,
for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-
terminal or
C-terminal ends), attachment of chemical moieties to the amino acid backbone,
chemical
modifications of N-linked or O-linked carbohydrate chains, and addition or
deletion of an
N-terminal methionine residue as a result of procaryotic host cell expression.
The
polypeptides may also be modified with a detectable label, such as an
enzymatic,
fluorescent, isotopic or affinity label to allow for detection and isolation
of the protein.
Also provided by the invention are chemically modified derivatives of TR9
which
may provide additional advantages such as increased solubility, stability and
circulating
time of the polypeptide, or decreased immunogenicity (see US 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,
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WO 00/56862 PCT/US00/06831
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.
5 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. Biotechnnl. 56:59-72 ( 1996);
Vorobjev et
al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al.,
Bioconjug. Chenz.
10:638-646 ( 1999), the disclosures of each of which are incorporated herein
by reference.
10 The polyethylene glycol molecules (or other chemical moieties) should be
attached
to the protein with consideration of effects on functional or antigenic
domains of the
protein. There are a number of attachment methods available to those skilled
in the art,
e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF),
see also
Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-
CSF
15 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
20 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
25 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
30 (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and
combinations thereof) of
the protein.
One may specifically desire polypeptides 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.),
35 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
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
46
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 polypeptides chemically
modified at
the N-terminus modification may be accomplished by reductive alkylation which
exploits
differential reactivity of different types of primary amino groups (lysine
versus the
N-terminal) available for derivatization in a particular protein. Under the
appropriate
reaction conditions, substantially selective derivatization of the protein at
the N-terminus
with a carbonyl group containing polymer is achieved.
As indicated above, pegylation of the proteins of the invention may be
accomplished by any number of means. For example, polyethylene glycol may be
attached to the protein either directly or by an intervening linker.
Linkerless systems for
attaching polyethylene glycol to proteins are described in Delgado et al.,
Crit. Rev. Tlzercr.
Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol.
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 of which are incorporated herein by
reference.
One system for attaching polyethylene glycol directly to amino acid residues
of
proteins without an intervening linker employs tresylated MPEG, which is
produced by
the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride
(C1SO,CH,CF;). 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 produced by reacting proteins of the invention
with a
polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
Polyethylene glycol can also be attached to proteins using a number of
different
intervening linkers. For example, U.S. Patent No. 5,612,460, the entire
disclosure 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 l, 2, 3, 4, 5, 6, 7,
8, 9, 10, 12,
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47
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-
1 l, 10-12.
11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol
moieties
per protein molecule. Methods for determining the degree of substitution are
discussed.
for example, in Delgado et ccl., Crit. Rev. Thera. Drag Carrier Sys. 9:249-304
( 1992 ).
The TR9 can be recovered and purified by standard methods which include, but
are not limited to, ammonium sulfate or ethanol precipitation, acid
extraction, anion or
canon exchange chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography, hydroxylapatite
chromatography
and lectin chromatography. Most preferably, high performance liquid
chromatography
("HPLC") is employed for purification.
TR9 receptor polynucleotides and polypeptides may be used in accordance with
the present invention for a variety of applications, particularly those that
make use of the
chemical and biological properties of TR9. Among these are applications in
treatment,
prevention, diagnosis, and/or detection of tumors, resistance to parasites,
bacteria and
viruses, to induce proliferation of T-cells, endothelial cells and certain
hematopoietic cells,
to treat, prevent, diagnose, and/or detect restenosis, graft vs. host disease,
to regulate
anti-viral responses and to prevent certain autoimmune diseases after
stimulation of TR9
by an agonist. Additional applications relate to diagnosis and to treatment,
prevention,
diagnosis, and/or detection of disorders of cells, tissues and organisms.
These aspects of
the invention are discussed further below.
Transgenics and "knock-outs "
The TR9 polypeptides of the invention can also be expressed in transgenic
animals. Animals of any species, including, but not limited to, mice, rats,
rabbits,
hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human
primates,
e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic
animals.
In a specific embodiment, techniques described herein or otherwise known in
the art, are
used to express polypeptides of the invention in humans, as part of a gene
therapy
protocol.
Any technique known in the art may be used to introduce the transgene (i.e.,
nucleic acids of the invention) into animals to produce the founder lines of
transgenic
animals. Such techniques include, but are not limited to, pronuclear
microinjection
(Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et
al.,
Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY)
9:830-834
(1991); and Hoppe et al., US Patent Number 4,873,191 (1989)); retrovirus
mediated
gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci.,
USA
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48
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., Cell
57:717-723
( 1989); ete. For a review of such techniques, see Gordon, "Transgenic
Animals," Intl.
Rev. Cytol. 11:171-229 (1989), which is incorporated by reference herein 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. (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
targeting
is preferred. Briefly, when such a technique is to be utilized, vectors
containing some
nucleotide sequences homologous to the endogenous gene axe designed for the
purpose of
integrating, via homologous recombination with chromosomal sequences, into and
disrupting the function of the nucleotide sequence of the endogenous gene. The
transgene
may also be selectively introduced into a particular cell type, thus
inactivating the
endogenous gene in only that cell type, by following, for example, the
teaching of Gu et
al. (Science 265:103-106 ( 1994)). The regulatory sequences required for such
a cell-type
specific inactivation will depend upon the particular cell type of interest,
and will be
apparent to those of skill in the art. The contents of each of the documents
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
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WO 00/56862 PCT/US00/06831
49
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. i~z sitic 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 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
TR9 polypeptides, studying conditions and/or disorders associated with
aberrant TR9
expression, and in screening for compounds effective in ameliorating such
conditions
and/or disorders.
In further embodiments of the invention, cells that are genetically engineered
to
express the proteins of the invention, or alternatively, that are genetically
engineered not
to express the proteins of the invention (e.g., knockouts) are administered to
a patient in
vivo. Such cells may be obtained from the patient (i.e., animal, including
human) or an
MHC compatible donor and can include, but are not limited to fibroblasts, bone
marrow
cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial
cells, etc.
The cells are genetically engineered in vitro using recombinant DNA techniques
to
introduce the coding sequence of polypeptides of the invention into the cells,
or
alternatively, to disrupt the coding sequence and/or endogenous regulatory
sequence
associated with the polypeptides of the invention, e.g., by transduction
(using viral
vectors, and preferably vectors that integrate the transgene into the cell
genome) or
transfection procedures, including, but not limited to, the use of plasmids,
cosmids,
YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the
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>0
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 of the 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 implanted in the body, e.g., genetically engineered
fibroblasts can be
implanted as part of a skin graft; genetically engineered endothelial cells
can be implanted
as part of a lymphatic or vascular graft. (See, for example, Anderson et al.,
US Patent
Number 5,399.349; and Mulligan & Wilson, US Patent Number 5,460,959, each of
which is incorporated by reference herein in its entirety).
When the cells to be administered are non-autologous or non-MHC compatible
cells, they can be administered using well known techniques which prevent the
development of a host immune response against the introduced cells. For
example, the
cells may be introduced in an encapsulated form which, while allowing for an
exchange
of components with the immediate extracellular environment, does not allow the
introduced cells to be recognized by the host immune system.
TR9 Receptor Proteins and Fragments
The invention further provides for proteins containing polypeptide sequences
encoded by polynucleotides of the invention.
The TR9 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 TR9 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 TR9
proteins of the
invention (including TR9 fragments, variants, and fusion proteins, as
described herein).
These homomers may contain TR9 proteins having identical or different
polypeptide
sequences. In a specific embodiment, a homomer of the invention is a multimer
containing only TR9 proteins having an identical polypeptide sequence. In
another
specific embodiment, a homomer of the invention is a multimer containing TR9
proteins
having different polypeptide sequences. In specific embodiments, the multimer
of the
invention is a homodimer (e.g., containing TR9 proteins having identical or
different
polypeptide sequences) or a homotrimer (e.g., containing TR9 proteins having
identical
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Jl
or different polypeptide sequences). In additional embodiments, the homomeric
multimer
of the invention is at least a homodimer, at least a homotrimer, or at least a
homotetramer.
As used herein, the term heteromer refers to a multimer containing
heterologous
proteins (i.e., proteins containing only polypeptide sequences that do not
correspond to a
polypeptide sequences encoded by the TR9 gene) in addition to the TR9 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 homomeric
multimer of
the invention is at least a homodimer, at least a homotrimer, or at least a
homotetramer.
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 TR9 proteins of the invention. Such covalent associations
may
involve one or more amino acid residues contained in the polypeptide sequence
of the
protein ( e.g., the polypeptide sequence recited in SEQ ID N0:2 or contained
in the
polypeptide encoded by the deposited cDNA clone. In one instance, the covalent
associations are cross-linking between cysteine residues located within the
polypeptide
sequences of the proteins which interact in the native (i.e., naturally
occurring)
polypeptide. In another instance, the covalent associations are the
consequence of
chemical or recombinant manipulation. Alternatively, such covalent
associations may
involve one or more amino acid residues contained in the heterologous
polypeptide
sequence in a TR9 fusion protein. In one example, covalent associations are
between the
heterologous sequence contained in a fusion protein of the invention (see,
e.g., US Patent
Number 5,478,925). In a specific example, the covalent associations are
between the
heterologous sequence contained in a TR9-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 ligandlreceptor 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).
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57
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., US Patent Number
5,478,925,
which is herein incorporated by reference in its entirety). Additionally,
multimers of the
invention may be generated using techniques known in the art to form one or
more inter-
molecule cross-links between the cysteine residues located within the
polypeptide
sequence of the proteins desired to be contained in the multimer (see, e.g.,
US Patent
Number 5,478,925, which is herein incorporated by reference in its entirety).
Further,
proteins of the invention may be routinely modified by the addition of
cysteine or biotin to
the C terminus or N-terminus of the polypeptide sequence of the protein and
techniques
known in the art may be applied to generate multimers containing one or more
of these
modified proteins (see, e.g., US Patent Number 5,478,925, which is herein
incorporated
by reference in its entirety). Additionally, techniques known in the art may
be applied to
generate liposomes containing the protein components desired to be contained
in the
multimer of the invention (see, e.g., US Patent Number 5,478,925, which is
herein
incorporated by reference in its entirety).
Alternatively, multimers of the invention may be generated using genetic
engineering techniques known in the art. In one embodiment, proteins contained
in
multimers of the invention are produced recombinantly using fusion protein
technology
described herein or otherwise known in the art (see, e.g., US Patent Number
5,478,925,
which is herein incorporated by reference in its entirety). In a specific
embodiment,
polynucleotides coding for a homodimer of the invention are generated by
ligating a
polynucleotide sequence encoding a polypeptide of the invention to a sequence
encoding a
linker polypeptide and then further to a synthetic polynucleotide encoding the
translated
product of the polypeptide in the reverse orientation from the original C-
terminus to the
N-terminus (lacking the leader sequence) (see, e.g., US Patent Number
5,478,925,
which is herein incorporated by reference in its entirety). In another
embodiment,
recombinant techniques described herein or otherwise known in the art are
applied to
generate recombinant polypeptides of the invention which contain a
transmembrane
domain and which can be incorporated by membrane reconstitution techniques
into
liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated
by
reference in its entirety).
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j3
In one embodiment, the invention provides an isolated TR9 protein having the
amino acid sequence encoded by the deposited cDNA, or the amino acid sequence
in
Figures lA-D (SEQ ID N0:2), or a polypeptide comprising, or alternatively
consisting
of, a portion (i.e., fragment) of the above polypeptides.
Polypeptide fragments of the present invention include polypeptides
comprising,
or alternatively, consisting of, an amino acid sequence contained in SEQ ID
N0:2,
encoded by the cDNA contained in the deposited clone, or encoded by nucleic
acids
which hybridize (e.g., under stringent hybridization conditions) to the
nucleotide
sequence contained in the deposited clone, or shown in Figures lA-D (SEQ ID
NO:1) or
the complementary strand thereto. Protein fragments may be "free-standing." or
comprised within a larger polypeptide of which the fragment forms a part or
region, most
preferably as a single continuous region. Representative examples of
polypeptide
fragments of the invention, include, for example, fragments that comprise or
alternatively,
consist of, from about amino acid residues -40 to 1, 1 to 20, 21 to 40. 41 to
60, 61 to 83,
84 to 100, 101 to 120, 121 to 140, 141 to 160, 160-167, 161 to 180, 181 to
200, 201 to
220, 221 to 240, 241 to 260, 261 to 280, 281 to 310, 311 to 350, 3~ 1 to 400,
401 to
450, 451 to 500. 551 to 600, or 601 to the end of the coding region of SEQ ID
N0:2.
Polynucleotides encoding these polypeptides are also encompassed by the
invention. In
this context "about" includes the particularly recited ranges, larger or
smaller by several
(5, 4, 3, 2, or 1 ) amino acids, at either extreme or at both extremes.
Moreover,
polypeptide fragments can be at least about 20, 30, 40, 50, 60, 70, 80, 90,
100, 110,
120, 130, 140, or 150 amino acids in length. In this context "about" includes
the
particularly recited value, larger or smaller by several (5, 4, 3, 2, or 1 )
amino acids, at
either extreme or at both extremes.
In specific embodiments, polypeptide fragments of the invention comprise, or
alternatively, consist of, amino acid residues: 40 to 48, 40 to 51, 51 to 66,
66 to 73, 73 to
83, 83 to 104, 104 to 110, 110 to 128, 128 to 146, 146 to 152, 40 to 152,
and/or 28 to
171 in SEQ ID N0:2. Polynucleotides encoding these polypeptides are also
encompassed
by the invention.
Preferred polypeptide fragments of the present invention include a member
selected from the group: a polypeptide comprising or alternatively, consisting
of, the TR9
receptor extracellular domain (predicted to constitute amino acid residues
from about 1 to
about 310 in SEQ ID N0:2); a polypeptide comprising or alternatively,
consisting of, the
four TNFR-like cysteine rich motifs of TR9 (amino acid residues 67 to 211 in
Figures
lA-D; amino acid residues 27 to 171 in SEQ ID N0:2); a polypeptide comprising
or
alternatively, consisting of, the TR9 receptor transmembrane domain (predicted
to
constitute amino acid residues from about 311 to about 327 in SEQ ID N0:2); a
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J4
polypeptide comprising or alternatively, consisting of, fragment of the
predicted mature
TR9 polypeptide. wherein the fragment has a TR9 functional activity (e.g.,
antigenic
activity or biological acitivity); a polypeptide comprising or alternatively,
consisting of,
the TR9 receptor intracellular domain (predicted to constitute amino acid
residues from
about 328 to about 615 in SEQ ID N0:2); a polypeptide comprising or
alternatively,
consisting of, the TR9 receptor extracellular and intracellular domains with
all or part of
the transmembrane domain deleted; a polypeptide comprising, or alternatively
consisting
of, the TR9 receptor death domain (predicted to constitute amino acid residues
from about
389 to about 455 in SEQ ID N0:2); and a polypeptide comprising, or
alternatively,
consisting of, one. two, three, four or more, epitope bearing portions of the
TR9 receptor
protein. In additional embodiments, the polypeptide fragments of the invention
comprise,
or alternatively. consist of, any combination of l, 2, 3, 4, 5, 6, 7, or all 8
of the above
members. As above, with the leader sequence, the amino acid residues
constituting the
TR9 receptor extracellular, transmembrane and intracellular domains have been
predicted
by computer analysis. Thus, as one of ordinary skill would appreciate, the
amino acid
residues constituting these domains may vary slightly (e.g., by about 1 to
about 15 amino
acid residues) depending on the criteria used to define each domain.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
As discussed above, it is believed that one or more of the four extracellular
cysteine-rich motifs of TR9 is important for interactions between TR9 and its
ligands.
Accordingly, in preferred embodiments, polypeptide fragments of the invention
comprise,
or alternatively consist of amino acid residues 27 to 65, 66 to 105, 106 to
145, and/or 146
to 171 of SEQ ID N0:2. Polynucleotides encoding these polypeptides are also
encompassed by the invention. Additional embodiments of the invention are
directed to
polypeptides which comprise, or alternatively consist of, any combination of
l, 2, 3, or
all 4 of the extracellular cysteine-rich motifs disclosed in Figures lA-D and
Figure 4B.
Among the especially preferred fragments of the invention are fragments
comprising, or alternatively, consisting of structural or functional
attributes of TR9. Such
fragments include amino acid residues that comprise alpha-helix and alpha-
helix forming
regions ("alpha-regions"), beta-sheet and beta-sheet-forming regions ("beta-
regions"),
turn and turn-forming regions ("turn-regions"), coil and coil-forming regions
("coil-
regions"), hydrophillic regions, hydrophobic regions, alpha amphipathic
regions, beta
amphipathic regions, surface forming regions, and high antigenic index regions
(i.e.,
regions of polypeptides consisting of amino acid residues having an antigenic
index of or
equal to greater than 1.5, as identified using the default parameters of the
Jameson-Wolf
program) of TR9. Certain preferred regions are those disclosed in Figure 3 and
Table I
and include, but are not limited to, regions of the aforementioned types
identified by
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J
analysis of the amino acid sequence depicted in Figures 1 A-D, such prefewed
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 another' aspect, the invention provides a peptide or polypeptide
comprising, or
alternatively, consisting of, one, two, three, four, five or more, epitope-
bearing portions
of a polypeptide of the invention. The epitope of this polypeptide portion is
an
immunogenic or antigenic epitope of a polypeptide described herein. An
"immunogenic
epitope" is defined as a part of a protein that elicits an antibody response
when the whole
protein is the immunogen. On the other hand. a region of a protein molecule to
which an
antibody can bind is defined as an "antigenic epitope." The number of
immunogenic
epitopes of a protein generally is less than the number of antigenic epitopes.
See, for
instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998- 4002 ( 1983).
As to the selection of peptides or polypeptides bearing an antigenic epitope
(i.e.,
that contain a region of a protein molecule to which an antibody can bind), it
is well
known in that art that relatively short synthetic peptides that mimic part of
a protein
sequence are routinely capable of eliciting an antiserum that reacts with the
partially
mimicked protein. See, for instance, J.G. Sutcliffe et al., "Antibodies That
React With
Predetermined Sites on Proteins," Sciei2ce 219:660-666 (1983). Peptides
capable of
eliciting protein-reactive sera are frequently represented in the primary
sequence of a
protein, can be characterized by a set of simple chemical rules, and are
confined neither to
immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to
the amino
or carboxyl terminals.
Antigenic epitope-bearing peptides and polypeptides of the invention are
therefore
useful to raise antibodies, including monoclonal antibodies, that bind
specifically to a
polypeptide of the invention. See, for instance, Wilson et al., Cell 37:767-
778 ( 1984) at
777. Antigenic epitope-bearing peptides and polypeptides of the invention
preferably
contain a sequence of at least seven, more preferably at least nine and most
preferably
between at least about 15 to about 30 amino acids contained within the amino
acid
sequence of a polypeptide of the invention.
Non-limiting examples of antigenic polypeptides or peptides that can be used
to
generate TR9 receptor-specific antibodies include: a polypeptide comprising,
or
alternatively consisting of, amino acid residues from about 4 to about 81 in
SEQ ID
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>6
N0:2, about 1 16 to about 271 in SEQ ID N0:2, about 283 to about 308 in SEQ ID
N0:2, about 336 to about 372 in SEQ ID N0:2, about 393 to about 4 34 in SEQ ID
N0:2,
about 445 to about 559 in SEQ ID N0:2, and about 571 to about 588 in SEQ ID
N0:2. In
this context "about" includes the particularly recited ranges, larger or
smaller- by several
(5, 4, 3, 2, or 1 ) nucleotides, at either terminus or at both termini. As
indicated above,
the inventors have determined that the above polypeptide fragments are
antigenic regions
of the TR9 receptor protein. Polynucleotides encoding these polypeptides are
also
encompassed by the invention.
The epitope-bearing peptides and polypeptides of the invention may be produced
by any conventional means. R.A. Houghten, "General Method for the Rapid Solid-
Phase
Synthesis of Large Numbers of Peptides: Specificity of Antigen-Antibody
Interaction at
the Level of Individual Amino Acids." Proc. Natl. Acad. Sci. USA 82:5131-5135
(1985). This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is
further
described in U.S. Patent No. 4,631,211 to Houghten et al. (1986).
As one of skill in the art will appreciate, TR9 receptor polypeptides of the
present
invention and the epitope-bearing fragments thereof described above can be
combined
with parts of the constant domain of immunoglobulins (IgG), resulting in
chimeric
polypeptides. These fusion proteins facilitate purification and show an
increased half-life
in vivo. This has been shown, e.g., for chimeric proteins consisting of the
first two
domains of the human CD4-polypeptide and various domains of the constant
regions of
the heavy or light chains of mammalian immunoglobulins (EPA 394,827;
Traunecker et
al., Nature 331:84-86 ( 1988)). Fusion proteins that have a disulfide-linked
dimeric
structure due to the IgG part can also be more efficient in binding and
neutralizing other
molecules than the monomeric TR9 protein or protein fragment alone
(Fountoulakis et al.,
J. Biochem. 270:3958-3964 (1995)).
To improve or alter the characteristics of TR9 polypeptides, protein
engineering
may be employed. Recombinant DNA technology known to those skilled in the art
can be
used to create novel mutant proteins or "muteins including single or multiple
amino acid
substitutions, deletions, additions or fusion proteins. Such modified
polypeptides can
show, e.g., enhanced activity or increased stability. In addition, they may be
purified in
higher yields and show better solubility than the corresponding natural
polypeptide, at
least under certain purification and storage conditions. For many proteins,
including the
extracellular domain of a membrane associated protein or the mature forms) of
a secreted
protein, it is known in the art that one or more amino acids may be deleted
from the N-
terminus or C-terminus without substantial loss of biological function.
However, even if
deletion of one or more amino acids from the N-terminus or C-terminus of a
protein
results in modification or loss of one or more biological functions of the
protein, other
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57
TR9 functional activities may still be retained. For example, in many
instances, the ability
of the shortened protein to induce and/or bind to antibodies which recognize
TR9
(preferably antibodies that bind specifically to TR9) will retained
irrespective of the size or
location of the deletion. In fact, polypeptides composed of as few as six TR9
amino acid
residues may often evoke an immune response. Whether a particular polypeptide
lacking
N-terminal and/or C-terminal residues of a complete protein retains such
immunologic
activities can readily be determined by routine methods described herein and
otherwise
known in the art.
As mentioned above, even if deletion of one or more amino acids from the
N-terminus of a protein results in modification or loss of one or more
biological
functions of the protein, other functional activities (e.g., biological
activities, ability to
multimerize, ability to bind TR9 ligand) may still be retained. For- example,
the ability of
shortened TR9 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 an TR9
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 TR9
amino acid residues may often evoke an immune response.
Accordingly, in one embodiment, the present invention further provides
polypeptides having one or more residues deleted from the amino terminus of
the amino
acid sequence of the TR9 polypeptide depicted in Figures lA-D (SEQ ID N0:2) or
encoded by the cDNA of the deposited clone. Particularly, in one embodiment, N-
terminal deletions of the TR9 polypeptide can be described by the general
formula m to
615, where m is a number from -39 to 614 corresponding to the position of
amino acid
identified in SEQ ID N0:2 and preferably, corresponds to one of the N-terminal
amino
acid residues identified in the N-terminal deletions specified herein. In
specific
embodiments, N-terminal deletions of the TR9 polypeptide of the invention
comprise, or
alternatively consist of, amino acid residues: Q-2 to L-615; P-3 to L-615; E-4
to L-615;
Q-5 to L-615; K-6 to L-615; A-7 to L-615; S-8 to L-615; N-9 to L-615; L-10 to
L-615; I-
11 to L-615; G-12 to L-615; T-13 to L-615; Y-14 to L-615; R-15 to L-615; H-16
to L-
615; V-17 to L-615; D-18 to L-615; R-19 to L-615; A-20 to L-615; T-21 to L-
615; G-22
to L-615; Q-23 to L-615; V-24 to L-615; L-25 to L-615; T-26 to L-615; C-27 to
L-615;
D-28 to L-615; K-29 to L-615; C-30 to L-615; P-31 to L-615; A-32 to L-615: G-
33 to L-
615; T-34 to L-615; Y-35 to L-615; V-36 to L-615; S-37 to L-615; E-38 to L-
615; H-39
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58
to L-615; C-40 to L-615: T-41 to L-615: N-42 to L-615; T-43 to L-615: S-44 to
L-615;
L-45 to L-615: R-46 to L-615; V-47 to L-615; C-48 to L-615; S-49 to L-615; S-
50 to L-
615; C-51 to L-615; P-52 to L-615; V-53 to L-615; G-54 to L-615; T-55 to L-
615; F-56
to L-615; T-57 to L-615; R-58 to L-615; H-59 to L-615; E-60 to L-615; N-61 to
L-615;
G-62 to L-615: I-63 to L-615; E-64 to L-615; K-65 to L-615; C-66 to L-615; H-
67 to L-
615; D-68 to L-615; C-69 to L-615; S-70 to L-615; Q-71 to L-615; P-72 to L-
615; C-73
to L-615; P-74 to L-615; W-75 to L-615; P-76 to L-615; M-77 to L-615; I-78 to
L-615;
E-79 to L-615: K-80 to L-615; L-81 to L-615; P-82 to L-615; C-83 to L-615; A-
84 to L-
615; A-85 to L-615; L-86 to L-615; T-87 to L-615; D-88 to L-615; R-89 to L-
615; E-90
to L-615; C-91 to L-615; T-92 to L-615; C-93 to L-615; P-94 to L-615; P-95 to
L-615;
G-96 to L-615; M-97 to L-615; F-98 to L-615; Q-99 to L-615; S-100 to L-61_5; N-
101 to
L-615 : A-102 to L-615 ; T-103 to L-615 ; C-104 to L-615 ; A-105 to L-615 ; P-
106 to L-
615; H-107 to L-615; T-108 to L-615; V-109 to L-615; C-110 to L-615; P-111 to
L-615;
V-112 to L-615: G-113 to L-615; W-114 to L-615; G-115 to L-615; V-116 to L-
615; R-
117 to L-615; K-118 to L-615; K-119 to L-615; G-120 to L-615; T-121 to L-615;
E-122
to L-615; T-123 to L-615; E-124 to L-615; D-125 to L-615; V-126 to L-615; R-
127 to L-
615; C-128 to L-615; K-129 to L-615; Q-130 to L-615; C-131 to L-615; A-132 to
L-615;
R-133 to L-615; G-134 to L-615; T-135 to L-615; F-136 to L-615; S-137 to L-
615; D-
138 to L-615; V-139 to L-615; P-140 to L-615; S-141 to L-615; S-142 to L-615;
V-143
to L-615; M-144 to L-615; K-145 to L-615; C-146 to L-615; K-147 to L-615; A-
148 to
L-615; Y-149 to L-615; T-150 to L-615; D-151 to L-615; C-152 to L-615; L-153
to L-
615; S-154 to L-615; Q-155 to L-615; N-156 to L-615; L-157 to L-615; V-158 to
L-615;
V-159 to L-615: I-160 to L-615; K-161 to L-615; P-162 to L-615; G-163 to L-
615; T-
164 to L-615 ; K-165 to L-615 ; E-166 to L-615 ; T-167 to L-615 ; D-168 to L-
615 ; N-169
to L-615; V-170 to L-615; C-171 to L-615; G-172 to L-615; T-173 to L-615; L-
174 to L-
615; P-175 to L-615; S-176 to L-615; F-177 to L-615; S-178 to L-615; S-179 to
L-615;
S-180 to L-615; T-181 to L-615; S-182 to L-615; P-183 to L-615; S-184 to L-
615; P-185
to L-615; G-186 to L-615; T-187 to L-615; A-188 to L-615; I-189 to L-615; F-
190 to L-
615; P-191 to L-615; R-192 to L-615; P-193 to L-615; E-194 to L-615; H-195 to
L-615;
M-196 to L-615; E-197 to L-615; T-198 to L-615; H-199 to L-615; E-200 to L-
615; V-
201 to L-615; P-202 to L-615; S-203 to L-615; S-204 to L-615; T-205 to L-615;
Y-206
to L-615; V-207 to L-615; P-208 to L-615; K-209 to L-615; G-210 to L-615; M-
211 to
L-615; N-212 to L-615; S-213 to L-615; T-214 to L-615; E-215 to L-615; S-216
to L-
615; N-217 to L-615; S-218 to L-615; S-219 to L-615; A-220 to L-615; S-221 to
L-615;
V-222 to L-615; R-223 to L-615; P-224 to L-615; K-225 to L-615; V-226 to L-
615; L-
227 to L-615; S-228 to L-615; S-229 to L-615; I-230 to L-615; Q-231 to L-615;
E-232 to
L-615; G-233 to L-615; T-234 to L-615; V-235 to L-615; P-236 to L-615; D-237
to L-
CA 02365255 2001-09-24
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59
615; N-238 to L-615; T-239 to L-615; S-240 to L-615; S-241 to L-615: A-242 to
L-615;
R-243 to L-615; G-244 to L-615; K-245 to L-615; E-246 to L-615; D-247 to L-
615; V-
248 to L-615; N-249 to L-615; K-250 to L-615; T-251 to L-615; L-252 to L-615:
P-253
to L-615; N-254 to L-615; L-255 to L-615: Q-256 to L-615; V-257 to L-615; V-
258 to L-
615; N-259 to L-615; H-260 to L-615; Q-261 to L-615; Q-262 to L-615; G-263 to
L-615;
P-264 to L-615; H-265 to L-615; H-266 to L-615; R-267 to L-615; H-268 to L-
615: I-
269 to L-615; L-270 to L-615; K-271 to L-615; L-272 to L-615; L-273 to L-615:
P-274
to L-615; S-275 to L-615; M-276 to L-615: E-277 to L-615: A-278 to L-615; T-
279 to L-
615; G-280 to L-615; G-281 to L-615; E-282 to L-615; K-283 to L-615; S-284 to
L-615;
S-285 to L-615; T-286 to L-615; P-287 to L-615; I-288 to L-615; K-289 to L-
615: G-290
to L-615; P-291 to L-615; K-292 to L-615: R-293 to L-615; G-294 to L-615; H-
295 to L-
615; P-296 to L-615; R-297 to L-615; Q-298 to L-615; N-299 to L-615; L-300 to
L-615;
H-301 to L-615; K-302 to L-615; H-303 to L-615; F-304 to L-615; D-305 to L-
615; I-
306 to L-615; N-307 to L-615; E-308 to L-615; H-309 to L-615; L-310 to L-615;
P-311
to L-615; W-312 to L-615; M-313 to L-615; I-314 to L-615; V-315 to L-615; L-
316 to L-
615; F-317 to L-615; L-318 to L-615; L-319 to L-615; L-320 to L-615; V-321 to
L-615;
L-322 to L-615; V-323 to L-615; V-324 to L-615; I-325 to L-615; V-326 to L-
615; V-327
to L-615; C-328 to L-615; S-329 to L-615; I-330 to L-615; R-331 to L-615; K-
332 to L-
615; S-333 to L-615; S-334 to L-615; R-335 to L-615; T-336 to L-615; L-337 to
L-615;
K-338 to L-615; K-339 to L-615; G-340 to L-615; P-341 to L-615; R-342 to L-
615; Q-
343 to L-615; D-344 to L-615; P-345 to L-615; S-346 to L-615; A-347 to L-615:
I-348 to
L-615; V-349 to L-615; E-350 to L-615; K-351 to L-615: A-352 to L-615; G-353
to L-
615; L-354 to L-615; K-355 to L-615; K-356 to L-615; S-357 to L-615: M-358 to
L-615;
T-359 to L-615; P-360 to L-615; T-361 to L-615; Q-362 to L-615; N-363 to L-
615; R-
364 to L-615; E-365 to L-615; K-366 to L-615; W-367 to L-615; I-368 to L-615;
Y-369
to L-615; Y-370 to L-615; C-371 to L-615; N-372 to L-615; G-373 to L-615; H-
374 to
L-615; G-375 to L-615; I-376 to L-615; D-377 to L-615; I-378 to L-615; L-379
to L-615;
K-380 to L-615; L-381 to L-615; V-382 to L-615; A-383 to L-615; A-384 to L-
615; Q
385 to L-615; V-386 to L-615; G-387 to L-615; S-388 to L-615; Q-389 to L-615;
W-390
to L-615; K-391 to L-615; D-392 to L-615; I-393 to L-615; Y-394 to L-615; Q-
395 to L-
615: F-396 to L-615; L-397 to L-615; C-398 to L-615; N-399 to L-615; A-400 to
L-615;
S-401 to L-615; E-402 to L-615; R-403 to L-615; E-404 to L-615; V-405 to L-
615; A-
406 to L-615; A-407 to L-615; F-408 to L-615; S-409 to L-615; N-410 to L-615;
G-411
to L-615; Y-412 to L-615; T-413 to L-615; A-414 to L-615; D-415 to L-615; H-
416 to L-
615; E-417 to L-615; R-418 to L-615; A-419 to L-615; Y-420 to L-615; A-421 to
L-615;
A-422 to L-615; L-423 to L-615; Q-424 to L-615; H-425 to L-615; W-426 to L-
615; T-
427 to L-615; I-428 to L-615; R-429 to L-615; G-430 to L-615; P-431 to L-615;
E-432 to
CA 02365255 2001-09-24
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L-615; A-433 to L-615; S-434 to L-615; L-435 to L-615; A-436 to L-615; Q-437
to L-
615; L-438 to L-615; I-439 to L-615: S-440 to L-615; A-441 to L-615: L-442 to
L-615;
R-443 to L-615; Q-444 to L-615; H-445 to L-615; R-446 to L-615; R-447 to L-
615; N-
448 to L-615; D-449 to L-615; V-450 to L-615; V-451 to L-615; E-452 to L-615:
K-453
5 to L-615; I-454 to L-615; R-455 to L-615; G-456 to L-615; L-457 to L-615; M-
458 to L-
615; E-459 to L-615; D-460 to L-615; T-461 to L-615; T-462 to L-615; Q-463 to
L-615;
L-464 to L-615; E-465 to L-615; T-466 to L-615; D-467 to L-615; K-468 to L-
615; L-
469 to L-615; A-470 to L-615; L-471 to L-615; P-472 to L-615; M-473 to L-615;
S-474
to L-615; P-475 to L-615; S-476 to L-615; P-477 to L-615; L-478 to L-615; S-
479 to L-
10 615; P-480 to L-615; S-481 to L-615; P-482 to L-615; I-483 to L-615: P-484
to L-615;
S-485 to L-615; P-486 to L-615; N-487 to L-615; A-488 to L-615; K-489 to L-
615; L-
490 to L-615; E-491 to L-615; N-492 to L-615; S-493 to L-615; A-494 to L-615;
L-495
to L-615; L-496 to L-615; T-497 to L-615; V-498 to L-615; E-499 to L-615; P-
500 to L-
615; S-501 to L-615; P-502 to L-615; Q-503 to L-615; D-504 to L-615; K-505 to
L-615;
15 N-506 to L-615; K-507 to L-615; G-508 to L-615; F-509 to L-615; F-510 to L-
615; V-
511 to L-615; D-512 to L-615; E-513 to L-615; S-514 to L-615; E-515 to L-615;
P-516
to L-615; L-517 to L-615; L-518 to L-615; R-519 to L-615; C-520 to L-615; D-
521 to L-
615; S-522 to L-615; T-523 to L-615; S-524 to L-615; S-525 to L-615; G-526 to
L-615;
S-527 to L-615; S-528 to L-615; A-529 to L-615; L-530 to L-615; S-531 to L-
615; R-
20 532 to L-615; N-533 to L-615; G-534 to L-615; S-535 to L-615; F-536 to L-
615; I-537
to L-615; T-538 to L-615; K-539 to L-615; E-540 to L-615; K-541 to L-615; K-
542 to L-
615; D-543 to L-615; T-544 to L-615; V-545 to L-615; L-546 to L-615; R-547 to
L-615;
Q-548 to L-615; V-549 to L-615; R-550 to L-615; L-551 to L-615; D-552 to L-
615; P-
553 to L-615; C-554 to L-615; D-555 to L-615; L-556 to L-615; Q-557 to L-615;
P-558
25 to L-615; I-559 to L-615; F-560 to L-615; D-561 to L-615; D-562 to L-615; M-
563 to L-
615; L-564 to L-615; H-565 to L-615; F-566 to L-615; L-567 to L-615; N-568 to
L-615;
P-569 to L-615; E-570 to L-615; E-571 to L-615; L-572 to L-615; R-573 to L-
615; V-
574 to L-615; I-575 to L-615; E-576 to L-615; E-577 to L-615; I-578 to L-615;
P-579 to
L-615; Q-580 to L-615; A-581 to L-615; E-582 to L-615; D-583 to L-615; K-584
to L-
30 615; L-585 to L-615; D-586 to L-615; R-587 to L-615; L-588 to L-615; F-589
to L-615;
E-590 to L-615; I-591 to L-615; I-592 to L-615; G-593 to L-615; V-594 to L-
615; K-595
to L-615; S-596 to L-615; Q-597 to L-615; E-598 to L-615; A-599 to L-615; S-
600 to L-
615; Q-601 to L-615; T-602 to L-615; L-603 to L-615; L-604 to L-615; D-605 to
L-615;
S-606 to L-615; V-607 to L-615; Y-608 to L-615; S-609 to L-615; and/or H-610
to L-
35 615 of SEQ ID N0:2. Polynucleotides encoding these polypeptides are also
encompassed by the invention. The present invention is also directed to
nucleic acid
molecules comprising, or alternatively, consisting of, a polynucleotide
sequence at least
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61
80%, 85%, 90%, 92%, 95%, 96C/c, 97%, 98% or 99% identical to the
polynucleotide
sequences encoding the TR9 polypeptides described above, and the polypeptides
encoded
thereby. The present invention also encompasses the above polynucleotide
sequences
fused to a heterologous polynucleotide sequence, and the polypeptides encoded
thereby.
In another embodiment, N-terminal deletions of the TR9 polypeptide can be
described by the general formula m to 310 where m is a number from -40 to 309
corresponding to the amino acid sequence identified in SEQ ID N0:2. In
specific
embodiments, N terminal deletions of the TR9 of the invention comprise, or
alternatively,
consist of, amino acid residues: Q-2 to L-310; P-3 to L-310; E-4 to L-310; Q-5
to L-310;
K-6 to L-310: A-7 to L-310; S-8 to L-310; N-9 to L-310; L-10 to L-310; I-11 to
L-310;
G-12 to L-310; T-13 to L-310; Y-14 to L-310; R-15 to L-310; H-16 to L-310; V-
17 to L-
310; D-18 to L-310; R-19 to L-310; A-20 to L-310; T-21 to L-310; G-22 to L-
310; Q-23
to L-310; V-24 to L-310; L-25 to L-310; T-26 to L-310; C-27 to L-310; D-28 to
L-310;
K-29 to L-310; C-30 to L-310; P-31 to L-310; A-32 to L-310; G-33 to L-310; T-
34 to L-
310; Y-35 to L-310; V-36 to L-310; S-37 to L-310; E-38 to L-310; H-39 to L-
310; C-40
to L-310; T-41 to L-310; N-42 to L-310; T-43 to L-310; S-44 to L-310; L-45 to
L-310;
R-46 to L-310; V-47 to L-310; C-48 to L-310; S-49 to L-310; S-50 to L-310; C-
51 to L-
310; P-52 to L-310; V-53 to L-310; G-54 to L-310; T-55 to L-310; F-56 to L-
310; T-57
to L-310; R-58 to L-310; H-59 to L-310; E-60 to L-310; N-61 to L-310; G-62 to
L-310;
I-63 to L-310: E-64 to L-310; K-65 to L-310; C-66 to L-310; H-67 to L-310; D-
68 to L-
310; C-69 to L-310; S-70 to L-310; Q-71 to L-310; P-72 to L-310; C-73 to L-
310; P-74
to L-310; W-75 to L-310; P-76 to L-310; M-77 to L-310; I-78 to L-310; E-79 to
L-310;
K-80 to L-310; L-81 to L-310; P-82 to L-310; C-83 to L-310; A-84 to L-310; A-
85 to L-
310; L-86 to L-310; T-87 to L-310; D-88 to L-310; R-89 to L-310; E-90 to L-
310; C-91
to L-310; T-92 to L-310; C-93 to L-310; P-94 to L-310; P-95 to L-310; G-96 to
L-310;
M-97 to L-310; F-98 to L-310; Q-99 to L-310; S-100 to L-310; N-101 to L-310; A-
102 to
L-310; T-103 to L-310; C-104 to L-310; A-105 to L-310; P-106 to L-310; H-107
to L-
310; T-108 to L-310; V-109 to L-310; C-110 to L-310; P-111 to L-310; V-112 to
L-310;
G-113 to L-310; W-114 to L-310; G-115 to L-310; V-116 to L-310; R-117 to L-
310; K-
118 to L-310; K-119 to L-310; G-120 to L-310; T-121 to L-310; E-122 to L-310;
T-123
to L-310; E-124 to L-310; D-125 to L-310; V-126 to L-310; R-127 to L-310; C-
128 to L-
310; K-129 to L-310; Q-130 to L-310; C-131 to L-310; A-132 to L-310; R-133 to
L-310;
G-134 to L-310; T-135 to L-310; F-136 to L-310; S-137 to L-310; D-138 to L-
310; V-
139 to L-310; P-140 to L-310; S-141 to L-310; S-142 to L-310; V-143 to L-310;
M-144
to L-310; K-145 to L-310; C-146 to L-310; K-147 to L-310; A-148 to L-310; Y-
149 to L-
310; T-150 to L-310; D-151 to L-310; C-152 to L-310; L-153 to L-310; S-154 to
L-310;
Q-155 to L-310; N-156 to L-310; L-157 to L-310; V-158 to L-310; V-159 to L-
310; I-
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62
160 to L-310; K-161 to L-310; P-162 to L-310; G-163 to L-310; T-164 to L-310;
K-165
to L-310; E- I 66 to L-310; T-167 to L-310; D-168 to L-310; N-169 to L-310; V-
170 to L-
310; C-171 to L-310: G-172 to L-310; T-173 to L-310; L-174 to L-310; P-175 to
L-310;
S-176 to L-310; F-177 to L-310; S-178 to L-310; S-179 to L-310; S-180 to L-
310; T-181
to L-310; S- I 82 to L-310; P-183 to L-310: S-184 to L-310; P-185 to L-310; G-
186 to L-
310; T-187 to L-310; A-188 to L-310; I-189 to L-310; F-190 to L-310; P-191 to
L-310;
R-192 to L-310; P-193 to L-310; E-194 to L-310: H-195 to L-310; M-196 to L-
310; E-
197 to L-310; T-198 to L-310; H-199 to L-310; E-200 to L-310; V-201 to L-310;
P-202
to L-310; S-203 to L-310; S-204 to L-310; T-205 to L-310; Y-206 to L-310; V-
207 to L-
310; P-208 to L-310; K-209 to L-310; G-210 to L-310; M-211 to L-310; N-212 to
L-310;
S-213 to L-310; T-214 to L-310; E-215 to L-310; S-216 to L-310; N-217 to L-
310; S-
218 to L-310; S-219 to L-310; A-220 to L-310; S-221 to L-310; V-222 to L-310;
R-223
to L-310; P-224 to L-310; K-225 to L-310; V-226 to L-310; L-227 to L-310; S-
228 to L-
310; S-229 to L-310; I-230 to L-310; Q-231 to L-310; E-232 to L-310; G-233 to
L-310;
T-234 to L-310; V-235 to L-310; P-236 to L-310; D-237 to L-310; N-238 to L-
310; T-
239 to L-310; S-240 to L-310; S-241 to L-310; A-242 to L-310; R-243 to L-310;
G-244
to L-310; K-245 to L-310; E-246 to L-310; D-247 to L-310; V-248 to L-310; N-
249 to L-
310; K-250 to L-310; T-251 to L-310; L-252 to L-310; P-253 to L-310; N-254 to
L-310;
L-255 to L-310; Q-256 to L-310; V-257 to L-310; V-258 to L-310; N-259 to L-
310; H-
260 to L-310; Q-261 to L-310; Q-262 to L-310; G-263 to L-310; P-264 to L-310;
H-265
to L-310; H-266 to L-310; R-267 to L-310; H-268 to L-310; I-269 to L-310; L-
270 to L-
310; K-271 to L-310; L-272 to L-310; L-273 to L-310; P-274 to L-310; S-275 to
L-310;
M-276 to L-310; E-277 to L-310; A-278 to L-310; T-279 to L-310; G-280 to L-
310; 6-
281 to L-310; E-282 to L-310; K-283 to L-310; S-284 to L-310; S-285 to L-310;
T-286
to L-310; P-287 to L-310; I-288 to L-310; K-289 to L-310; G-290 to L-310; P-
291 to L-
310; K-292 to L-310; R-293 to L-310; G-294 to L-310; H-295 to L-310; P-296 to
L-310;
R-297 to L-310; Q-298 to L-310; N-299 to L-310; L-300 to L-310; H-301 to L-
310; K-
302 to L-310; H-303 to L-310; F-304 to L-310; and/or D-305 to L-310 of SEQ ID
N0:2.
Polynucleotides encoding these polypeptides are also encompassed by the
invention. 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 TR9
polypeptides described above, and the polypeptides encoded thereby. The
present
invention also encompasses the above polynucleotide sequences fused to a
heterologous
polynucleotide sequence, and the polypeptides encoded thereby.
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
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63
functions of the protein, other functional activities (e.g., biological
activities, ability to
multimerize, ability to bind TR9 ligand) may still be retained. For example
the ability of
the shortened TR9 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 an TR9
mutein with a large number of deleted C-terminal amino acid residues may
retain some
biological or immunogenic activities. In fact, as discussed above, peptides
composed of
as few as six TR9 amino acid residues may often evoke an immune response.
Accordingly, further embodiments of the invention are directed to C-terminal
deletions of the TR9 polypeptide described by the general formula 1 to n,
where n is a
number from 2 to 614 corresponding to the position of amino acid residue
identified in
SEQ ID N0:2 and preferably, corresponds to one of the C-terminal amino acid
residues
identified in the C-terminal deletions specified herein. In specific
embodiments, C
terminal deletions of the TR9 polypeptide of the invention comprise, or
alternatively,
consist of, amino acid residues: A-1 to L-614; A-1 to D-613; A-1 to P-612; A-1
to L-61 l;
A-1 to H-610; A-1 to S-609; A-1 to Y-608: A-1 to V-607; A-1 to S-606; A-1 to D-
605; A-
1 to L-604; A-1 to L-603 ; A-1 to T-602; A-1 to Q-601; A-1 to S-600; A-1 to A-
599; A-1
to E-598; A-1 to Q-597; A-1 to S-596; A-1 to K-595; A-1 to V-594; A-1 to G-
593; A-1 to
I-592; A-1 to I-591; A-1 to E-590; A-1 to F-589; A-1 to L-588; A-1 to R-587; A-
1 to D-
586; A-1 to L-585; A-1 to K-584; A-1 to D-583; A-1 to E-582; A-1 to A-581; A-1
to Q-
580; A-1 to P-579; A-1 to I-578; A-1 to E-577; A-1 to E-576; A-1 to I-575; A-1
to V-574;
A-1 to R-573; A-1 to L-572; A-1 to E-571; A-1 to E-570; A-1 to P-569; A-1 to N-
568; A-
1 to L-567; A-1 to F-566; A-1 to H-565; A-1 to L-564; A-1 to M-563; A-1 to D-
562; A-1
to D-561; A-1 to F-560; A-1 to I-559; A-1 to P-558; A-1 to Q-557; A-1 to L-
556; A-1 to
D-555; A-1 to C-554; A-1 to P-553; A-1 to D-552; A-1 to L-551; A-1 to R-550; A-
1 to V-
549; A-1 to Q-548; A-1 to R-547; A-1 to L-546; A-1 to V-545; A-1 to T-544; A-1
to D-
543; A-1 to K-542; A-1 to K-541; A-1 to E-540; A-1 to K-539; A-1 to T-538; A-1
to I-
537; A-1 to F-536; A-1 to S-535; A-1 to G-534; A-1 to N-533; A-1 to R-532; A-1
to 5-
531; A-1 to L-530; A-1 to A-529; A-1 to S-528; A-1 to S-527; A-1 to G-526; A-1
to S-
525; A-1 to S-524; A-1 to T-523; A-1 to S-522; A-1 to D-521; A-1 to C-520; A-1
to 8-
519; A-1 to L-518; A-1 to L-517; A-1 to P-516; A-1 to E-515; A-1 to S-514; A-1
to E-
513; A-1 to D-512; A-1 to V-511; A-1 to F-510; A-1 to F-509; A-1 to G-508; A-1
to K-
507; A-1 to N-506; A-1 to K-505; A-1 to D-504; A-1 to Q-503; A-1 to P-502; A-1
to S-
501; A-1 to P-500; A-1 to E-499; A-1 to V-498; A-1 to T-497; A-1 to L-496; A-1
to L-
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64
495; A-1 to A-494; A-1 to S-493 ; A-1 to N-492; A-1 to E-491; A-1 to L-490;
A=1 to K-
489; A-1 to A-488; A-1 to N-487; A-1 to P-486; A-1 to S-485; A-1 to P-484; A-1
to I-
483; A-1 to P-482; A-1 to S-481; A-1 to P-480: A-1 to S-479; A-1 to L-478; A-1
to P-
477; A-1 to S-476; A-1 to P-475; A-1 to S-474; A-1 to M-473; A-1 to P-472; A-1
to L-
471; A-1 to A-470; A-1 to L-469; A-1 to K-468; A-1 to D-467; A-1 to T-466; A-1
to E-
465; A-1 to L-464; A-1 to Q-463 ; A-1 to T-462; A-1 to T-461; A-1 to D-460; A-
1 to E-
459; A-1 to M-458; A-1 to L-457; A-1 to G-456; A-1 to R-455; A-1 to I-454; A-1
to K-
453; A-1 to E-452; A-1 to V-451; A-1 to V-450; A-1 to D-449; A-1 to N-448; A-1
to R-
447; A-1 to R-446; A-1 to H-445; A-1 to Q-444; A-1 to R-443; A-1 to L-442; A-1
to A-
441; A-1 to S-440; A-1 to I-439; A-1 to L-438; A-1 to Q-437; A-1 to A-436; A-1
to L-
435; A-1 to S-434; A-1 to A-433; A-1 to E-432; A-1 to P-431; A-1 to G-430; A-1
to 8-
429; A-1 to I-428; A-1 to T-427; A-1 to W-426; A-1 to H-425; A-1 to Q-424; A-I
to L-
423; A-1 to A-422; A-1 to A-421; A-1 to Y-420; A-1 to A-419; A-1 to R-418; A-1
to E-
417; A-1 to H-416; A-1 to D-415; A-1 to A-414; A-1 to T-4 I 3; A-1 to Y-412; A-
1 to G-
411; A-1 to N-410; A-1 to S-409; A-1 to F-408; A-1 to A-407; A-1 to A-406; A-1
to V-
405; A-1 to E-404; A-1 to R-403 ; A-1 to E-402; A-1 to S-401; A-1 to A-400; A-
1 to N-
399; A-1 to C-398; A-1 to L-397; A-1 to F-396; A-1 to Q-395; A-1 to Y-394; A-1
to I-
393; A-1 to D-392; A-1 to K-391; A-1 to W-390; A-1 to Q-389; A-1 to S-388; A-1
to 6-
387; A-1 to V-386; A-1 to Q-385; A-1 to A-384; A-1 to A-383; A-1 to V-382; A-1
to L-
381; A-1 to K-380; A-1 to L-379; A-1 to I-378; A-1 to D-377; A-1 to I-376; A-1
to 6-
375; A-1 to H-374; A-1 to G-373; A-1 to N-372; A-1 to C-371; A-1 to Y-370; A-1
to Y-
369; A-1 to I-368; A-1 to W-367; A-1 to K-366; A-1 to E-365; A-1 to R-364; A-1
to N-
363; A-1 to Q-362; A-1 to T-361; A-1 to P-360; A-1 to T-359; A-1 to M-358; A-1
to S-
357; A-1 to K-356; A-1 to K-355; A-1 to L-354; A-1 to G-353; A-1 to A-352; A-1
to K-
351; A-1 to E-350; A-1 to V-349; A-1 to I-348; A-1 to A-347; A-1 to S-346; A-1
to P-
345; A-1 to D-344; A-1 to Q-343 ; A-1 to R-342; A-1 to P-341; A-1 to G-340; A-
1 to K-
339; A-1 to K-338; A-1 to L-337; A-1 to T-336; A-1 to R-335; A-1 to S-334; A-1
to S-
333; A-1 to K-332; A-1 to R-331; A-1 to I-330; A-1 to S-329; A-1 to C-328; A-1
to V-
327; A-1 to V-326; A-1 to I-325; A-1 to V-324; A-1 to V-323; A-1 to L-322; A-1
to V-
321; A-1 to L-320; A-1 to L-319; A-1 to L-318; A-1 to F-317; A-1 to L-316; A-1
to V-
315; A-1 to I-314; A-1 to M-313; A-1 to W-312; A-1 to P-311; A-1 to L-310; A-1
to H-
309; A-1 to E-308; A-1 to N-307; A-1 to I-306; A-1 to D-305; A-1 to F-304; A-1
to H-
303; A-1 to K-302; A-1 to H-301; A-1 to L-300; A-1 to N-299; A-1 to Q-298; A-1
to 8-
297; A-I to P-296; A-1 to H-295; A-1 to G-294; A-1 to R-293; A-1 to K-292; A-1
to P-
291; A-1 to G-290; A-1 to K-289; A-1 to I-288; A-1 to P-287; A-1 to T-286; A-1
to S-
285; A-1 to S-284; A-1 to K-283; A-1 to E-282; A-1 to G-281; A-1 to G-280; A-1
to T-
279; A-1 to A-278; A-1 to E-277; A-1 to M-276; A-1 to S-275; A-1 to P-274; A-1
to L-
CA 02365255 2001-09-24
WO 00!56862 PCT/US00/06831
273; A-1 to L-272; A-1 to K-271; A-1 to L-270; A-1 to I-269: A-1 to H-268; A-I
to R-
267; A-1 to H-266; A-1 to H-265; A-1 to P-264; A-1 to G-263; A-1 to Q-262; A-1
to Q-
261; A-1 to H-260; A-1 to N-259; A-1 to V-258; A-1 to V-257: A-I to Q-256; A-1
to L-
255; A-1 to N-254; A-1 to P-253; A-1 to L-252; A-1 to T-251; A-I to K-250; A-1
to N-
5 249; A-I to V-248; A-1 to D-247; A-1 to E-246; A-1 to K-245; A-1 to G-244; A-
1 to 8-
243; A-1 to A-242; A-1 to S-241; A-1 to S-240; A-1 to T-239: A-1 to N-238; A-1
to D-
237; A-1 to P-236; A-1 to V-235; A-1 to T-234; A-1 to G-233; A-1 to E-232; A-1
to Q-
231; A-1 to I-230; A-1 to S-229; A-1 to S-228; A-1 to L-227: A-I to V-226; A-1
to K-
225; A-I to P-224; A-I to R-223; A-1 to V-222; A-1 to S-221; A-1 to A-220; A-1
to S-
10 219; A-1 to S-218; A-1 to N-217; A-1 to S-216; A-1 to E-215; A-1 to T-214;
A-1 to S-
213; A-1 to N-212; A-1 to M-211; A-I to G-210; A-1 to K-209; A-1 to P-208; A-1
to V-
207; A-1 to Y-206; A-1 to T-205; A-1 to S-204; A-1 to S-203; A-1 to P-202; A-1
to V-
201; A- I to E-200; A-1 to H-199; A-1 to T-198 ; A- I to E-197 ; A-1 to M-196;
A-1 to H-
195; A-1 to E-194; A-1 to P-193; A-1 to R-192; A-1 to P-191; A-1 to F-190; A-1
to I-
I5 189; A-I to A-188; A-1 to T-187; A-I to G-186; A-1 to P-185; A-1 to S-184;
A-I to P-
183; A-1 to S-182; A-1 to T-181; A-1 to S-180; A-1 to S-179; A-1 to S-178; A-1
to F-
177; A-1 to S-176; A-1 to P-175; A-1 to L-174; A-I to T-173; A-1 to G-172; A-1
to C-
171; A-1 to V-170; A-I to N-169; A-1 to D-168; A-1 to T-167; A-1 to E-166; A-1
to K-
165; A-1 to T-164; A-1 to G-163; A-I to P-162; A-I to K-161; A-1 to I-160; A-1
to V-
20 159; A-1 to V-158; A-1 to L-157; A-1 to N-156; A-I to Q-155; A-1 to S-154;
A-I to L-
153; A-1 to C-152; A-1 to D-151; A-1 to T-150; A-1 to Y-149; A-1 to A-148; A-1
to K-
147; A-1 to C-146; A-1 to K-145; A-1 to M-144; A-1 to V-143; A-1 to S-142; A-1
to S-
141; A-1 to P-140; A-1 to V-139; A-1 to D-138; A-1 to S-137; A-1 to F-136; A-1
to T-
135; A-1 to G-134; A-I to R-133; A-1 to A-132; A-1 to C-131; A-1 to Q-130; A-I
to K-
25 129; A-1 to C-128; A-1 to R-127; A-I to V-126; A-1 to D-125; A-1 to E-124;
A-1 to T-
123; A-1 to E-122; A-1 to T-121; A-I to G-120; A-1 to K-I 19; A-1 to K-118; A-
1 to 8-
117; A-1 to V-116; A-1 to G-115; A-1 to W-114; A-1 to G-113; A-1 to V-112; A-1
to P-
111; A-I to C-110; A-1 to V-109; A-1 to T-108; A-1 to H-107; A-1 to P-106; A-I
to A-
105; A-1 to C-104; A-1 to T-103; A-1 to A-102; A-1 to N-101; A-1 to S-100; A-1
to Q-
30 99; A-1 to F-98; A-1 to M-97; A-I to G-96; A-1 to P-95; A-1 to P-94; A-1 to
C-93; A-1
to T-92; A-1 to C-91: A-1 to -90; A-1 to R-89; A-1 to D-88; A-1 to T-87; A-1
to L-86; A-
1 to A-85; A-1 to A-84; A-1 to C-83; A-1 to P-82; A-I to L-81; A-1 to K-80; A-
1 to E-79;
A-1 to I-78; A-1 to M-77; A-1 to P-76; A-1 to W-75; A-1 to P-74; A-1 to C-73;
A-1 to P-
72; A-I to Q-71; A-1 to S-70; A-1 to C-69; A-1 to D-68; A-1 to H-67; A-1 to C-
66; A-1
35 to K-65; A-1 to E-64; A-1 to I-63; A-1 to G-62; A-1 to N-61; A-1 to E-60; A-
1 to H-59;
A-1 to R-58; A-1 to T-57; A-1 to F-56; A-1 to T-55; A-1 to G-54; A-1 to V-53;
A-1 to P-
52; A-1 to C-51; A-1 to S-50; A-1 to S-49; A-1 to C-48; A-1 to V-47; A-1 to R-
46; A-1 to
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66
L-45; A-I to S-44; A-1 to T-43; A-I to N-42; A-1 to T-41; A-1 to C-40; A-1 to
H-39; A-I
to E-38; A-I to S-37; A-1 to V-36; A-I to Y-35; A-1 to T-34; A-1 to G-33; A-1
to A-32;
A-1 to P-31; A-1 to C-30; A-I to K-29; A-1 to D-28; A-I to C-27; A-1 to T-26;
A-I to L-
25; A-1 to V-24: A-I to Q-23; A-1 to G-22; A-1 to T-21; A-1 to A-20: A-1 to R-
19; A-1
to D-18; A-1 to V-17; A-1 to H-16; A-I to R-15; A-1 to Y-14; A-1 to T-13; A-1
to G-12;
A-1 to I-11; A-1 to L-10; A-I to N-9; A-1 to S-8; A-1 to A-7; and/or A-1 to K-
6 of SEQ
ID N0:2. Polynucleotides encoding these polypeptides are also encompassed by
the
invention. 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
TR9 polypeptides described above, and the polypeptides encoded thereby. The
present
invention also encompasses the above polynucleotide sequences fused to a
heterologous
polynucleotide sequence, and the polypeptides encoded thereby.
Further embodiments of the invention are directed to polypeptide fragments
comprising, or alternatively, consisting of, amino acids described by the
general formula
m to n, where m and n correspond to any one of the amino acid residues
specified above
for these symbols, respectively. Polynucleotides encoding these polypeptides
are also
encompassed by the invention.
However, many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are related
to SEQ ID NO: I and may have been publicly available prior to conception of
the present
invention. Preferably, such related polynucleotides are specifically excluded
from the
scope of the present invention. To list every related sequence would be
cumbersome.
Similarly, preferably excluded from the present invention are one or more
polynucleotides
comprising a nucleotide sequence described by the general formula of a'-b',
where a' is
any integer between 1 to 3437 of SEQ ID NO: I, b' is an integer of 15 to 3452,
where
both a' and b' correspond to the positions of nucleotide residues shown in SEQ
ID NO:1,
and where the b' is greater than or equal to a' + 14.
In specific embodiments, the polynucleotides of the invention are less than
300
kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, or 7.5 kb in length. In a further
embodiment,
polynucleotides of the invention comprise at least 15 contiguous nucleotides
of TR9
coding sequence, but do not comprise all or a portion of any TR9 intron. In
another
embodiment, the nucleic acid comprising TR9 coding sequence does not contain
coding
sequences of a genomic flanking gene (i.e., 5' or 3' to the TR9 gene in the
genome).
In specific embodiments, the polynucleotides of the invention are less than
100,000 kb, 50,000 kb, 10,000 kb, 1,000 kb, 500 kb, 400 kb, 350 kb, 300 kb,
250 kb,
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67
200 kb, 175 kb. 150 kb, 125 kb, 100 kb, 75 kb, 50 kb. 40 kb, 30 kb. 25 kb, 20
kb, 15
kb, 10 kb, 7.5 kb, or 5 kb in length.
In further embodiments, polynucleotides of the invention comprise at least 15,
at
least 30, at least 50, at least 100, or at least 250, at least 500, or at
least 1000 contiguous
nucleotides of TR9 coding sequence. but consist of less than or equal to 1000
kb, 500 kb,
250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10
kb, or 5
kb of genomic DNA that flanks the 5' or 3' coding nucleotide sequences set
forth in
Figures lA-D (SEQ ID NO:1). In further embodiments, polynucleotides of the
invention
comprise at least 15, at least 30, at least 50, at least 100, or at least 250.
at least 500, or at
least 1000 contiguous nucleotides of TR9 coding sequence, but do not comprise
all or a
portion of any TR9 intron. In another embodiment, the nucleic acid comprising
TR9
coding sequence does not contain coding sequences of a genomic flanking gene
(i.e., 5'
or 3' to the TR9 gene in the genome). In other embodiments, the
polynucleotides of the
invention do not contain the coding sequence of more than 1000, 500, 250, 100,
50, 25,
20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
The invention further provides isolated TR9 polypeptides having the amino acid
sequence encoded by the deposited cDNAs, or the amino acid sequences in
Figures lA-D
(SEQ ID N0:2) or a peptide or polypeptide comprising a portion of the above
polypeptides.
The polypeptides of the 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.
The polypeptides of the present invention may exist as a membrane bound
receptor having a transmembrane region and an intra- and extracellular region
or they may
exist in soluble form wherein the transmembrane domain is lacking. One example
of such
a form of the TR9 receptor is the TR9 receptor shown in Figures 1 A-D (SEQ ID
N0:2)
which contains, in addition to a leader sequence, transmembrane, intracellular
and
extracellular domains. Thus, this form of the TR9 receptor appears to be
localized in the
cytoplasmic membrane of cells which express this protein.
In specific embodiments, the polypeptide fragments of the invention (i.e.,
those
described herein) are not larger than 610, 600, 580, 570, 550, 525, 500, 475,
450, 400,
425, 390, 380, 375, 350, 336, 334, 331, 305, 300, 295, 290, 285, 280, 275,
260, 250,
225, 200, 185, 175, 170, 165, 160. 155, 150, 145, 140, 135, 130. 125, 120,
115, 110,
105, 100, 90, 80, 75, 60, 50, 40, 30, or 25 amino acid residues in length.
It will be recognized in the art that some amino acid sequences of the TR9
receptor
can be varied without significant effect on the structure or function of the
protein. If such
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68
differences in sequence are contemplated, it should be remembered that there
will be
critical areas on the protein which detemnine activity. Thus, the invention
further includes
variations of the TR9 receptor which show substantial TR9 receptor functional
activity
(e.g., biological activity) or which include regions of TR9 receptor
polypeptide such as
the protein portions discussed herein. Such mutants include deletions,
insertions,
inversions, repeats, and type substitutions. As indicated above, guidance
concerning
which amino acid changes are likely to be phenotypically silent can be found
in Bowie et
al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid
Substitutions," Science 247:1306-1310 (1990).
Of special interest are substitutions of charged amino acids with other
charged or
neutral amino acids which may produce proteins with highly desirable improved
characteristics, such as less aggregation. Aggregation may not only reduce
activity but
also be problematic when preparing pharmaceutical formulations, because
aggregates can
be immunogenic (Pinckard et al., Cliti. Exp. hnmunol. 2:331-340 (1967);
Robbins et al.,
Diabetes 36: 838-845 ( 1987); Cleland et al., Crit. Rev. Therapeutic Df-ug
Carrier Systems
10:307-377 (1993).
Thus, the fragment, derivative or analog of the polypeptide of Figures lA-D
(SEQ
ID N0:2), or that encoded by the deposited cDNA, 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(s), and more preferably at
least one
but less than ten conserved amino acid residues), and such substituted amino
acid
residues) 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 Fe
fusion
region peptide or leader or secretory sequence or a sequence which is employed
for
purification of the mature polypeptide or a proprotein sequence. Such
fragments,
derivatives and analogs are deemed to be within the scope of those skilled in
the art from
the teachings herein.
Of particular interest are substitutions of charged amino acids with another
charged amino acid and with neutral or negatively charged amino acids. The
latter results
in proteins with reduced positive charge to improve the characteristics of the
TR9
receptor. 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
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69
al., Crit. Rev. Therapeutic Drrry Carrier S~~stem,s 10:307-377 ( 1993)).
The replacement of amino acids can also change the selectivity of binding to
cell
surface receptors. Ostade et al., Ncrtrrre 361:266-268 (1993), describes
certain mutations
resulting in selective binding of TNF-alpha to only one of the two known types
of TNF
receptors. Thus, the TR9 receptor of the present invention may include one or
more
amino acid substitutions, deletions, or additions, either from natural
mutations or human
manipulation.
As indicated, changes are preferably of a minor nature, such as conservative
amino acid substitutions that do not significantly affect the folding or
activity of the
protein (see Table II).
TABLE II
Conservative Amino Acid Substitutions
Phenylalani
Tryptophan
Tyrosine
Hydrophobic Leucine
Isoleucine
Valine
Polar ~ Glutamine
Asparagine
Basic Arginine
Lysine
Histidine
Acidic ~ Aspartic Acid
Glutamic Acid
Small Alanine
Serine
Threonine
Methionine
Glycine
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In specific embodiments, the number of substitutions, additions or deletions
in the
amino acid sequence of Figures lA-D and/or any of the polypeptide fragments
described
herein (e.g., the extracellular domain or intracellular domain) is 75, 70, 60,
50, 40, 35,
30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3. 2, 1 or 30-20, 20-15, 20-10, 15-10,
10-l, 5-10,
5 1-5, 1-3 or 1-2.
Amino acids in the TR9 protein of the present invention that are essential for
function can be identified by methods known in the art, such as site-directed
mutagenesis
or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085
(1989)). The latter procedure introduces single alanine mutations at every
residue in the
10 molecule. The resulting mutant molecules are then tested for biological
activity such as,
receptor/ligand binding or in vitro proliferative, and/or activation activity
upon
monocytes. Sites that are critical for ligand-receptor binding can also be
determined by
structural analysis such as crystallization, nuclear magnetic resonance or
photoaffinity
labeling (Smith et al., J. Mol. Biol. 224:899-904 ( 1992) and de Vos et al.
Scie~zce
15 255:306-312 ( 1992)).
Additionally, protein engineering may be employed to improve or alter the
characteristics of TR9 polypeptides. Recombinant DNA technology known to those
skilled in the art can be used to create novel mutant proteins or muteins
including single or
multiple amino acid substitutions, deletions, additions or fusion proteins.
Such modified
20 polypeptides can show, e.g., enhanced activity or increased stability. In
addition, they
may be purified in higher yields and show better solubility than the
corresponding natural
polypeptide, at least under certain purification and storage conditions.
Non-naturally occurring variants may be produced using art-known mutagenesis
techniques, which include, but are not limited to oligonucleotide mediated
mutagenesis,
25 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 et al., Philos. Traps. R. Soc. Lor2don SerA
317:415
( 1986)).
30 Thus, the invention also encompasses TR9 derivatives and analogs that have
one
or more amino acid residues deleted, added, or substituted to generate TR9
polypeptides
that are better suited for expression, scale up, etc., in the host cells
chosen. For example,
cysteine residues can be deleted or substituted with another amino acid
residue in order to
eliminate disulfide bridges; N-linked glycosylation sites can be altered or
eliminated to
35 achieve, for example, expression of a homogeneous product that is more
easily recovered
and purified from yeast hosts which are known to hyperglycosylate N-linked
sites. To
this end, a variety of amino acid substitutions at one or both of the first or
third amino
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WO 00/56862 PCT/US00/06831
71
acid positions on any one or- more of the glycosylation recognitions sequences
in the TR9
polypeptides of the invention, and/or an amino acid deletion at the second
position of any
one or more such recognition sequences will prevent glycosylation of the TR9
at the
modified tripeptide sequence (see, e.g., Miyajimo et al., EMBO J 5(6):1193-
1197).
Additionally, 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 TR9 thereby effectively generating agonists and
antagonists of
TR9. See genercrlh~, U.S. Patent Nos. 5.605,793, 5,811,238, 5,830.721,
5,834.252,
and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33
(1997);
Harayama, S. Treads 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 of these patents and publications are hereby incorporated by
reference).
In one embodiment, alteration of TR9 polynucleotides and corresponding
polypeptides
may be achieved by DNA shuffling. DNA shuffling involves the assembly of two
or
more DNA segments into a desired TR9 molecule by homologous, or site-specific,
recombination. In another embodiment, TR9 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 TR9 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,
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
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, TR 10 (International Publication No. WO 98/54202), 31 X2
(International Publication No. WO 98/06842), TR11, TR11SV1, TR11SV2, TR12, and
TNF-Rl, TRAMP/DR3/APO-3/WSL/LARD, TRAIL-R1/DR4/APO-2, TRAIL-R2/DRS,
DcRI/TRAIL-R3/TRID/LIT, DcR2/TRAIL-R4, CAD, TRAIL, TRAMP, v-FLIP.
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
7?
In further preferred embodiments, the heterologous molecules are any member of
the TNF family.
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/or contained within a
recombinant host
cell is considered isolated for purposes of the present invention. Also
intended as an
"isolated polypeptide" are polypeptides that have been purified, partially or
substantially,
from a recombinant host cell. For example, a recombinantly produced version of
the
TR9 receptor can be substantially purified by the one-step method described in
Smith and
Johnson, Gene 67:31-40 (1988).
The polypeptides of the present invention include a polypeptide comprising, or
alternatively, consisting of, the polypeptide encoded by the deposited cDNA
including the
leader; a polypeptide comprising, or alternatively, consisting of, the mature
polypeptide
encoded by the deposited cDNA minus the leader (i.e., the mature protein); a
polypeptide
comprising, or alternatively, consisting of, amino acids about -40 to about
615 in SEQ ID
N0:2; a polypeptide comprising, or alternatively, consisting of, amino acids
about -39 to
about 615 in SEQ ID N0:2; a polypeptide comprising, or alternatively.
consisting of,
amino acids about 1 to about 615 in SEQ ID N0:2; a polypeptide comprising, or
alternatively, consisting of, the extracellular domain; a polypeptide
comprising, or
alternatively, consisting of, the four TNFR-like cysteine rich motifs of TR9
(amino acid
residues 67 to 211 in Figures lA-D; amino acid residues 27-171 in SEQ ID
N0:2); a
polypeptide comprising, or alternatively, consisting of, the transmembrane
domain; a
polypeptide comprising, or alternatively, consisting of, the intracellular
domain; a
polypeptide comprising, or alternatively, consisting of, the extracellular and
intracellular
domains with all or part of the transmembrane domain deleted; a polypeptide
comprising,
or alternatively. consisting of, the death domain (amino acid residues 429-495
as depicted
in Figures lA-D; amino acid residues 389-455 in SEQ ID N0:2); and/or a
polypeptide
comprising, or alternatively, consisting of, the TR9 leucine zipper (amino
acid residues
497-518 of Figures lA-D; amino acid residues 457-478 of SEQ ID N0:2); as well
as
polypeptides which are at least 80% identical, more preferably at least 85%,
90% or 95%
identical, still more preferably at least 96%, 97%, 98% or 99% identical to
the
polypeptides described above, and also include portions of such polypeptides
with at least
30 amino acids and more preferably at least 50 amino acids. Polynucleotides
encoding
these polypeptides are also encompassed by the invention.
By a polypeptide having an amino acid sequence at least, for example, 95%
°'identical" to a reference amino acid sequence of a TR9 receptor
polypeptide is intended
that the amino acid sequence of the polypeptide is identical to the reference
sequence
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73
except that the polypeptide sequence may include up to five amino acid
alterations per
each 100 amino acids of the reference amino acid of the TR9 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%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid
sequence
shown in Figures lA-D (SEQ ID N0:2), the amino acid sequence encoded by
deposited
eDNA clone, or fragments thereof, can be determined conventionally using known
computer programs such the Bestfit program (Wisconsin Sequence Analysis
Package,
Version 8 for Unix, Genetics Computer Group, University Research Park, 575
Science
Drive, Madison, WI 53711). When using Bestfit or any other sequence alignment
program to determine whether a particular sequence is, for instance, 95%
identical to a
reference sequence according to the present invention, the parameters are set,
of course,
such that the percentage of identity is calculated over the full length of the
reference amino
acid sequence and that gaps in homology of up to 5% of the total number of
amino acid
residues in the reference sequence are allowed.
In a specific embodiment, the identity between a reference (query) sequence (a
sequence of the present invention) and a subject sequence, also referred to as
a global
sequence alignment, is determined using the FASTDB computer program based on
the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred
parameters
used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch
Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l,
Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window
Size=500 or the length of the subject amino acid sequence, whichever is
shorter.
According to this embodiment, if the subject sequence is shorter than the
query sequence
due to N- or C-terminal deletions, not because of internal deletions, a manual
correction is
made to the results to take into consideration the fact that the FASTDB
program does not
account for N- and C-terminal truncations of the subject sequence when
calculating global
percent identity. For subject sequences truncated at the N- and C-termini,
relative to the
query sequence, the percent identity is corrected by calculating the number of
residues of
the query sequence that are N- and C-terminal of the subject sequence, which
are not
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
74
matched/aligned with a corresponding subject residue, as a percent of the
total bases of
the query sequence. A determination of whether a residue is matched/aligned is
determined by results of the FASTDB sequence alignment. This percentage is
then
subtracted from the percent identity, calculated by the above FASTDB program
using the
specified parameters, to arrive at a final percent identity score. This final
percent identity
score is what is used for the purposes of this embodiment. Only residues to
the N- and
C-termini of the subject sequence, which are not matched/aligned with the
query
sequence, are considered for the purposes of manually adjusting the percent
identity
score. That is, only query residue positions outside the farthest N- and C-
terminal
residues of the subject sequence. For example, a 90 amino acid residue subject
sequence
is aligned with a 100 residue query sequence to determine percent identity.
The deletion
occurs at the N-terminus of the subject sequence and therefore, the FASTDB
alignment
does not show a matching/alignment of the first 10 residues at the N-terminus.
The 10
unpaired residues represent 10% of the sequence (number of residues at the N-
and C-
termini not matched/total number of residues in the query sequence) so 10% is
subtracted
from the percent identity score calculated by the FASTDB program. If the
remaining 90
residues were perfectly matched the final percent identity would be 90%. In
another
example, a 90 residue subject sequence is compared with a 100 residue query
sequence.
This time the deletions are internal deletions so there are no residues at the
N- or C-termini
of the subject sequence which are not matched/aligned with the query. In this
case the
percent identity calculated by FASTDB is not manually corrected. Once again,
only
residue positions outside the N- and C-terminal ends of the subject sequence,
as displayed
in the FASTDB alignment, which are not matched/aligned with the query sequence
are
manually corrected for. No other manual corrections are made for the purposes
of this
embodiment.
The polypeptides of the present invention have uses which include, but are not
limited to, molecular weight marker on SDS-PAGE gels or on molecular sieve gel
filtration columns and as a source for generating antibodies that bind the
polypeptides of
the invention, using methods well known to those of skill in the art.
The present application is also directed to proteins cotaining polypeptides at
least
80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the TR9 polypeptide
sequences set forth herein as m-n. In preferred embodiments, the application
is directed
to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%
or
99% identical to polypeptides having the amino acid sequence of the a specific
TR9 N-
and/or C-terminal deletion recited herein. Polynucleotides encoding these
polypeptides
are also encompassed by the invention.
In certain preferred embodiments, TR9 proteins of the invention comprise, or
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
alternatively consist of, fusion proteins. as described above, wherein the TR9
palypeptide
component of the fusion protein is one of the polypeptide sequences set forth
herein as m-
n. In preferred embodiments, the polypeptide sequence component of the fusion
protein
that is homologous to the TR9 polypeptides of the invention is at least 80%,
85%, 90%,
95%, 96%, 97%, 98% or 99% identical to the polypeptide acid sequence of a
specific N-
and/or C-terminal deletion recited herein. Polynucleotides encoding these
polypeptides
are also encompassed by the invention.
The present invention encompasses polypeptides comprising, or alternatively
consisting of, an epitope of the polypeptide having an amino acid sequence of
SEQ ID
10 N0:2, or an epitope of the polypeptide sequence encoded by a polynucleotide
sequence
contained in ATCC deposit No. 209037 or encoded by a polynucleotide that
hybridizes to
the complement of the sequence of SEQ ID NO: l or contained in ATCC deposit
No.
209037 under stringent hybridization conditions or lower stringency
hybridization
conditions as defined supra. The present invention further encompasses
polynucleotide
15 sequences encoding an epitope of a polypeptide sequence of the invention
(such as, for
example, the sequence disclosed in SEQ ID NO:1 ), 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
20 supra.
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
25 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 generating antibodies
described infra.
(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
30 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.
Fragments which function as epitopes may be produced by any conventional
3~ means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135
(1985), further
described in U.S. Patent No. 4,631,211).
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76
In the present invention, antigenic epitopes preferably contain a sequence of
at
least 4, at least 5. at least 6, at least 7, more preferably at least 8, at
least 9, at least 10, at
least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at
least 25, at least 30,
at least 40, at least 50, and, most preferably, between about 15 to about 30
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. Additional non-exclusive preferred antigenic epitopes
include the
antigenic epitopes disclosed herein, as well as portions thereof. Antigenic
epitopes are
useful, for example, to raise antibodies, including monoclonal antibodies,
that specifically
bind the epitope. Preferred antigenic epitopes include the antigenic epitopes
disclosed
herein, as well as any combination of two, three, four, five or more of these
antigenic
epitopes. Antigenic epitopes can be used as the target molecules in
immunoassays. (See,
for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science
219:660-666
( 1983)).
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). Preferred immunogenic epitopes
include the
immunogenic epitopes disclosed herein, as well as any combination of two,
three, four,
five or more of these immunogenic epitopes. The polypeptides comprising one or
more
immunogenic epitopes may be presented for eliciting an antibody response
together with a
carrier protein, such as an albumin, to an animal system (such as rabbit or
mouse), or, if
the polypeptide is of sufficient length (at least about 25 amino acids), the
polypeptide may
be presented without a carrier. However, immunogenic epitopes comprising as
few as 8
to 10 amino acids have been shown to be sufficient to raise antibodies capable
of binding
to, at the very least, linear epitopes in a denatured polypeptide (e.g., in
Western blotting).
Epitope-bearing polypeptides of the present invention may be used to induce
antibodies according to methods well known in the art including, but not
limited to, in
vivo immunization, in vitro immunization, and phage display methods. See,
e.g.,
Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-2354
( 1985). If in vivo immunization is used, animals may be immunized with free
peptide;
however, anti-peptide antibody titer may be boosted by coupling the peptide to
a
macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus
toxoid.
For instance, peptides containing cysteine residues may be coupled to a
carrier using a
linker such as maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carriers using a more general linking agent such
aglutaraldehyde. Animals such as rabbits, rats and mice are immunized with
either free or
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77
carrier- coupled peptides, for instance, by intraperitoneal and/or intradermal
injection of
emulsions containing about 100 pg 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 which can be detected, for example, by
ELISA assay
using free peptide adsorbed to a solid surface. The titer of anti-peptide
antibodies in
serum from an immunized animal may be increased by selection of anti-peptide
antibodies, for instance, by adsorption to the peptide on a solid support and
elution of the
selected antibodies according to methods well known in the art.
As one of skill in the art will appreciate, and as discussed above, the
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 (CH 1, CH2, CH3, or 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 et al., Nature, 331:84-86 ( 1988). Enhanced delivery of an antigen
across the
epithelial barrier to the immune system has been demonstrated for antigens
(e.g., insulin)
conjugated to an FcRn binding partner such as IgG or Fe fragments (see, e.g.,
PCT
Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that have a
disulfide-linked dimeric structure due to the IgG portion desulfide bonds have
also been
found to be more efficient 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 Ni'+ nitriloacetic acid-
agarose column
and histidine-tagged proteins can be selectively eluted with imidazole-
containing buffers.
CA 02365255 2001-09-24
WO 00!56862 PCT/US00/06831
78
In another- embodiment, the TR9 polypeptides of the present invention arid the
epitope-bearing fragments thereof are fused with a heterologous antigen (e.g.,
polypeptide, carbohydrate, phospholipid, or nucleic acid). In specific
embodiments, the
heterologous antigen is an immunogen.
In a more specific embodiment, the heterologous antigen is the gp 120 protein
of
HIV, or a fragment thereof. Polynucleotides encoding these polypeptides are
also
encompassed by the invention.
In another embodiment, the TR9 polypeptides of the present invention and the
epitope-bearing fragments thereof are fused with polypeptide sequences of
another TNF
family member (or biologically active fragments or variants thereof). In a
specific
embodiment, the TR9 polypeptides of the present invention are fused with a
CD40L
polypeptide sequence. In a preferred embodiment, the CD40L polypeptide
sequence is
soluble.
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 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 of polynucleotides corresponding to SEQ ID NO:1,
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 encoding 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.
In a preferred embodiments, TR9 polypeptides of the invention (inlcuding
biologically active fragments or variants thereof), are fusedwith soluble
CD40L
polypeptides, or biologically acitve fragments or variants thereof.
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79
Antibodies
Further polypeptides of the invention relate to antibodies and T-cell antigen
receptors (TCR) which immunospecifically bind a polypeptide, polypeptide
fragment, or
variant of SEQ ID N0:2, and/or an epitope, of the present invention (as
determined by
immunoassays well known in the art for assaying specific antibody-antigen
binding).
Antibodies of the invention include. but are not limited to, polyclonal,
monoclonal.
multispeeific, human, humanized or chimeric antibodies, single chain
antibodies, Fab
fragments, F(ab') fragments, fragments produced by a Fab expression library,
anti-
idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to
antibodies of the
invention), and epitope-binding fragments of any of the above. The term
"antibody," as
used herein, refers to immunoglobulin molecules and immunologically active
portions of
immunoglobulin molecules, i.e., molecules that contain an antigen binding site
that
immunospecifically binds an antigen. The immunoglobulin molecules of the
invention
can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGI,
IgG2,
IgG3, IgG4, IgAI and IgA2) or subclass of immunoglobulin molecule.
Immunoglobulins may have both a heavy and light chain. An array of IgG, IgE,
IgM,
IgD, IgA, and IgY heavy chains may be paired with a light chain of the kappa
or lambda
forms.
Most preferably the antibodies are human antigen-binding antibody fragments of
the
present invention and include, but are not limited to, Fab, Fab' and F(ab')2,
Fd, single-chain
Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments
comprising
either a VL or VH domain. Antigen-binding antibody fragments, including single-
chain
antibodies, may comprise the variable regions) alone or in combination with
the entirety or a
portion of the following: hinge region, CH1, CH2, and CH3 domains. Also
included in the
invention are antigen-binding fragments also comprising any combination of
variable regions)
with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the
invention may be
from any animal origin including birds and mammals. Preferably, the antibodies
are human,
murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel,
horse, or chicken.
As used herein, "human" antibodies include antibodies having the amino acid
sequence of a
human immunoglobulin and include antibodies isolated from human immunoglobulin
libraries
or from animals transgenic for one or more human immunoglobulin and that do
not express
endogenous immunoglobulins, as described infra and, for example in, U.S.
Patent No.
5,939,598 by Kucherlapati et al.
The antibodies of the present invention may be monospecific, bispecific,
trispecific or
of greater multispecificity. Multispecific antibodies may be specific for
different epitopes of a
polypeptide of the present invention or may be specific for both a polypeptide
of the present
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
invention as well as for a heterologous epitope, such as a heterologous
polypeptide or solid
support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO
91/00360;
WO 92/05793: Tutt, et al., J. Immunol. 147:60-69 ( 1991 ); U.S. Patent Nos.
4,474,893;
4.714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.
148:1547-1553
5 ( 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 they
recognize or
specifically bind. The epitope(s) or polypeptide portions) may be specified as
described
herein, e.g., by N-terminal and C-terminal positions, by size in contiguous
amino acid
10 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
15 cross-reactivity. Antibodies that do not bind any other analog, ortholog,
or homolog of a
polypeptide of the present invention are included. Antibodies that bind
polypeptides with at
least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least
70%, at least 65%, at
least 60%, at least 55%, and at least 50% identity (as calculated using
methods known in the art
and described herein) to a polypeptide of the present invention are also
included in the present
20 invention. In specific embodiments, antibodies of the present invention
cross-react with
murine, rat and/or rabbit homologs of human proteins and the corresponding
epitopes thereof.
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)
25 to a polypeptide of the present invention are also included in the present
invention. In a
specific embodiment, the above-described cross-reactivity is with respect to
any single specific
antigenic or immunogenic polypeptide, or combinations) of 2, 3, 4, 5, or more
of the specific
antigenic and/or immunogenic polypeptides disclosed herein. Further included
in the present
invention are antibodies which bind polypeptides encoded by polynucleotides
which hybridize
30 to a polynucleotide of the present invention under stringent hybridization
conditions (as
described herein). Antibodies of the present invention may also be described
or specified in
terms of their binding affinity to a polypeptide of the invention. Preferred
binding affinities
include those with a dissociation constant or Kd less than 5 X 10-' M, 10~' M,
5 X 10-~ M, 10-~
M, 5 X 10-'' M. 10-'' M, 5 X 105 M, 10-s M, 5 X 10-6 M, 10-6M, 5 X 10-' M, 10'
M, 5 X 10-~
35 M, 10-g M, 5 X 10-9 M, 10-~ M, 5 X 10~'° M, 10-'° M, 5 X 10-"
M, 10-" M, 5 X 10~''- M, '°-'-
M, 5 X 10-" M. 10~'~ M, 5 X 10-''' M, 10-''' M, 5 X 10-'5 M, or 10-'' M.
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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 95%, at least
90%, at least 85 %, at least 80%, at least 75%, 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. Preferrably, antibodies of the present invention bind an
antigenic epitope
disclosed herein. or a portion thereof. The invention features both receptor-
specific antibodies
and ligand-specific antibodies. The invention also features receptor-specific
antibodies which
do not prevent ligand binding but prevent receptor activation. Receptor
activation (i.e.,
signaling) may be determined by techniques described herein or otherwise known
in the art.
For example, receptor activation can be determined by detecting the
phosphorylation (e.g.,
tyrosine or serine/threonine) of the receptor or its substrate by
immunoprecipitation followed by
western blot analysis (for example, as described supra). In specific
embodiments, antibodies
are provided that inhibit ligand activity or receptor activity by at least
95%, at least 90%, at least
85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%
of the activity in
absence of the antibody.
The invention also features receptor-specific antibodies which both prevent
ligand
binding and receptor activation as well as antibodies that recognize the
receptor-ligand complex,
and, preferably. do not specifically recognize the unbound receptor or the
unbound ligand.
Likewise, included in the invention are neutralizing antibodies which bind the
ligand and
prevent binding of the ligand to the receptor, as well as antibodies which
bind the ligand,
thereby preventing receptor activation, but do not prevent the ligand from
binding the receptor.
Further included in the invention are antibodies which activate the receptor.
These antibodies
may act as receptor agonists, i.e., potentiate or activate either all or a
subset of the biological
activities of the ligand-mediated receptor activation, for example, by
inducing dimerization of
the receptor. 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., J.
Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214
(1998); Yoon
et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci.
111(Pt2):237-247
(1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et
al., Cytokine
9(4):233-241 ( 1997); Carlson et al., J. Biol. Chem. 272( 17):11295-11301 (
1997); Taryman et
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82
al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1 167
(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 may be used, for example. but not limited
to, to
purify, detect, and target the polypeptides of the present invention,
including both in vitro and
in vivo diagnostic and therapeutic methods. For example, the antibodies have
use in
immunoassays for qualitatively and quantitatively measuring levels of the
polypeptides of the
present invention in biological samples. See, e.a., Harlow et al., Antibodies:
A Laboratory
Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by
reference
herein in its entirety).
As discussed in more detail below, the antibodies of the present invention may
be used
either alone or in combination with other compositions. The antibodies may
further be
recombinantly fused to a heterologous polypeptide at the N- or C-terminus or
chemically
conjugated (including covalently and non-covalently conjugations) to
polypeptides or other
compositions. For example, antibodies of the present invention may be
recombinantly fused
or conjugated to molecules useful as labels in detection assays and effector
molecules such as
heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT
publications WO
92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.
The antibodies of the invention include derivatives that are modified, i.e, by
the
covalent attachment of any type of molecule to the antibody such that covalent
attachment does
not prevent the antibody from generating an anti-idiotypic response. For
example, but not by
way of limitation, the antibody derivatives include antibodies that have been
modified, e.g., by
glycosylation, acetylation, pegylation, phosphylation, 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 of tunicamycin, etc. Additionally, the derivative may contain one or
more non-
classical amino acids.
The antibodies of the present invention may be generated by any suitable
method
known in the art. Polyclonal antibodies to an antigen-of- interest can be
produced by various
procedures well known in the art. For example, a polypeptide of the invention
can be
administered to various host animals including, but not limited to, rabbits,
mice, rats, etc. to
induce the production of sera containing polyclonal antibodies specific for
the antigen. Various
adjuvants may be used to increase the immunological response, depending on the
host species,
and include but are not limited to, Freund's (complete and incomplete),
mineral gels such as
aluminum hydroxide, surface active substances such as lysolecithin, pluronic
polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and
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potentially useful human adjuvants such as BCG (bacille Cahnette-Guerin) and
corynebacterium parvum. 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
technologies, or a
combination thereof. For example, monoclonal antibodies can be produced using
hybridoma
techniques including those known in the art and taught, for example, in Harlow
et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988);
Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier,
N.Y., 1981 ) (said references incorporated by reference in their entireties).
The term
"monoclonal antibody" as used herein is not limited to antibodies produced
through hybridoma
technology. The term "monoclonal antibody" refers to an antibody that is
derived from a single
clone, including any eukaryotic, prokaryotic, or phage clone, and not the
method by which it
is produced.
A "monoclonal antibody" may comprise, or alternatively consist of, two
proteins,
i.e., a heavy and a light chain.
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 the Examples
(e.g., Example 11). In a non-limiting example, mice can be immunized with a
polypeptide of
the invention or a cell expressing such peptide. Once an immune response is
detected, e.g.,
antibodies specific for the antigen are detected in the mouse serum, the mouse
spleen is
harvested and splenocytes isolated. The splenocytes are then fused by well-
known techniques
to any suitable myeloma cells, for example cells from cell line SP20 available
from the ATCC.
Hybridomas are selected and cloned by limited dilution. The hybridoma clones
are then
assayed by methods known in the art for cells that secrete antibodies capable
of binding a
polypeptide of the invention. Ascites fluid, which generally contains high
levels of antibodies,
can be generated by immunizing mice with positive hybridoma 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 solated 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 which recognize specific epitopes may be generated by known
techniques. For example, Fab and F(ab')2 fragments of the invention may be
produced by
proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain
(to produce
Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments
contain the
variable region, the light chain constant region and the CH 1 domain of the
heavy chain.
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For example, the antibodies of the present invention can also be generated
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 embodiment, such phage can be
utilized to display
antigen binding domains expressed from a repertoire or combinatorial antibody
library (e.g.,
human or murine). Phage expressing an antigen binding domain that binds the
antigen of
interest can be selected or identified with antigen, e.g., using labeled
antigen or antigen bound
or captured to a solid surface or bead. Phage used in these methods are
typically filamentous
phage including fd and M13 binding domains expressed from phage with Fab, Fv
or disulfide
stabilized Fv antibody domains recombinantly fused to either the phage gene
III or gene VIII
protein. Examples of phage display methods that can be used to make the
antibodies of the
present invention include those disclosed in Brinkman et al., J. Immunol.
Methods 182:41-50
(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et
al., Eur. J.
Immunol. 24:92-958 ( 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 (
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).
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Examples of techniques which can be used to produce single-chain Fvs and
antibodies
include those described in U.S. Patents 4,946,778 and 5,258,498; Huston et
al., Methods in
Enzymology 203:46-88 ( 1991 ); Shu et al., PNAS 90:7995-7999 ( 1993); and
Skerra et al.,
Science 240:1038-1040 (1988). For some uses, including in vivo use of
antibodies in
5 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
10 229:1202 ( 1985); Oi et al., BioTechniques 4:214 ( 1986); Gillies et al., (
1989) J. Immunol.
Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397,
which are
incorporated herein by reference in their entirety. 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
15 regions from a human immunoglobulin molecule. Often, framework residues in
the human
framework regions will be substituted with the corresponding residue from the
CDR donor
antibody to alter, preferably improve, antigen binding. These framework
substitutions are
identified by methods well known in the art, e.g., by modeling of the
interactions of the CDR
and framework residues to identify framework residues important for antigen
binding and
20 sequence comparison to identify unusual framework residues at particular
positions. (See,
e.g., Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature
332:323 ( 1988),
which are incorporated herein by reference in their entireties.) Antibodies
can be humanized
using a variety of techniques known in the art including, for example, CDR-
grafting (EP
239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101;
and
25 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan,
Molecular
Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering
7(6):805-814
(1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S.
Patent No.
5,565,332).
Completely human antibodies are particularly desirable for therapeutic
treatment,
30 detection, and/or prevention in human patients. Human antibodies can be
made by a variety of
methods known in the art including phage display methods described above using
antibody
libraries derived from human immunoglobulin sequences. See also, U.S. Patent
Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO
98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of
which is
35 incorporated herein by reference in its entirety.
Human antibodies can also be produced using transgenic mice which are
incapable of
expressing functional endogenous immunoglobulins, but which can express human
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86
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 which express human antibodies. The transgenic
mice are
immunized in the normal fashion with a selected antigen, e.g., all or a
portion of a polypeptide
of the invention. Monoclonal antibodies directed against the antigen can be
obtained from the
immunized, transgenic mice using conventional hybridoma technology. The human
immunoglobulin transgenes harbored by the 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, Int. Rev. Immunol. 13:65-93 (1995). 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 92/01047; WO
96/34096; WO
96/33735; European Patent No. 0 598 877; U.S. Patent Nos. 5,413,923;
5,625,126;
5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771;
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., Biotechnology
12:899-903
( 1988)).
Further, antibodies to the polypeptides of the invention can, in turn, be
utilized to
generate anti-idiotype antibodies that "mimic" polypeptides of the invention
using techniques
well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J.
7(5):437-444;
(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,
antibodies
which bind to and competitively inhibit polypeptide multimerization and/or
binding of a
polypeptide of the invention to a ligand can be used to generate anti-
idiotypes that "mimic" the
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87
polypeptide multimerization and/or binding domain and, as a consequence, bind
to and
neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or
Fab fragments of
such anti-idiotypes can be used in therapeutic regimens to neutralize
polypeptide ligand. For
example, such anti-idiotypic antibodies can be used to bind a polypeptide of
the invention
and/or to bind its ligands/receptors, and thereby block its biological
activity.
Polyrzcccleotides EncodifZg Antibodies
The invention further provides polynucleotides comprising a nucleotide
sequence
encoding an antibody of the invention and fragments thereof. The invention
also
encompasses polynucleotides that hybridize under stringent or lower stringency
hybridization conditions, e.g., as defined supra, to polynucleotides that
encode an
antibody, preferably, that specifically binds to a polypeptide of the
invention, preferably,
an antibody that binds to a polypeptide having the amino acid sequence of SEQ
ID N0:2.
The polynucleotides may be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. For example, if
the
nucleotide sequence of the antibody is known, a polynucleotide encoding the
antibody
may be assembled from chemically synthesized oligonucleotides (e.g., as
described in
Kutmeier et al., BioTechniques 17:242 ( 1994)), which, briefly, involves the
synthesis of
overlapping oligonucleotides containing portions of the sequence encoding the
antibody,
annealing and ligating 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 chemically synthesized or
obtained
from a suitable source (e.g., an antibody cDNA library, or a cDNA library
generated
from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or
cells
expressing the antibody, such as hybridoma cells selected to express an
antibody of the
invention) by PCR amplification using synthetic primers hybridizable to the 3'
and 5'
ends of the sequence or by cloning using an oligonucleotide probe specific for
the
particular gene sequence to identify, e.g., 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
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88
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 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"
(Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al.,
Nature
312:604-608 ( 1984); Takeda et al., Nature 314:452-454 ( 1985)) by splicing
genes from a
mouse antibody molecule of appropriate antigen specificity together with genes
from a
human antibody molecule of appropriate biological activity can be used. As
described
supra, a chimeric antibody is a molecule in which different portions are
derived from
different 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. 4,946,778; Bird, Science 242:423- 42 (1988); Huston et al.,
Proc.
Natl. Acad. Sci. USA 85:5879-5883 ( 1988); and Ward et al., Nature 334:544-54
( 1989))
can be adapted to produce single chain antibodies. Single chain antibodies are
formed by
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89
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., Science 242:1038- 1041 (
1988)).
Methods of'Producing Aiztibodies
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.
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 or a single
chain antibody of the invention), requires construction of an expression
vector containing
a polynucleotide that encodes the antibody. Once a polynucleotide encoding an
antibody
molecule or a heavy or light chain of an antibody, or portion thereof
(preferably
containing the heavy or light chain variable domain), of the invention has
been obtained,
the vector for the production of the antibody molecule may be produced by
recombinant
DNA technology using techniques well known in the art. Thus, methods for
preparing a
protein by expressing a polynucleotide containing an antibody encoding
nucleotide
sequence are described herein. Methods which are well known to those skilled
in the art
can be used to construct expression vectors containing antibody coding
sequences and
appropriate transcriptional and translational control signals. These methods
include, for
example, in vitro recombinant DNA techniques, synthetic techniques, and in
vivo genetic
recombination. The invention, thus, provides replicable vectors comprising a
nucleotide
sequence encoding an antibody molecule of the invention, or a heavy or light
chain
thereof, or a heavy or light chain variable domain, operably linked to a
promoter. Such
vectors may include the nucleotide sequence encoding the constant region of
the antibody
molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036;
and
U.S. Patent No. 5,122,464) and the variable domain of the antibody may be
cloned into
such a vector for expression of the entire heavy or light chain.
The expression vector is transferred to a host cell by conventional techniques
and
the transfected cells are then cultured by conventional techniques to produce
an antibody
of the invention. Thus, the invention includes host cells containing a
polynucleotide
encoding an antibody of the invention, or a heavy or light chain thereof, or a
single chain
antibody of the invention, operably linked to a heterologous promoter. In
preferred
embodiments for the expression of double-chained antibodies, vectors encoding
both the
heavy and light chains may be co-expressed in the host cell for expression of
the entire
immunoglobulin molecule, as detailed below.
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A variety of host-expression vector systems may be utilized to express the
antibody molecules of the invention. Such host-expression systems represent
vehicles by
which the coding sequences of interest may be produced and subsequently
purified, but
also represent cells which may, when transformed or transfected with the
appropriate
5 nucleotide coding sequences, express an antibody molecule of the invention
in situ.
These include but are not limited to microorganisms such as bacteria (e.g., E.
coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or
cosmid
DNA expression vectors containing antibody coding sequences; yeast (e.g.,
Saccharomyces, Pichia) transformed with recombinant yeast expression vectors
10 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
15 mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring
recombinant expression constructs containing promoters derived from the genome
of
mammalian cells (e.g., metallothionein promoter) or from mammalian viruses
(e.g., the
adenovirus late promoter; the vaccinia virus 7.SK promoter). Preferably,
bacterial cells
such as Escherichia coli, and more preferably, eukaryotic cells, especially
for the
20 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
promoter element from human cytomegalovirus is an effective expression system
for
antibodies (Foecking et al., Gene 45:101 ( 1986); Cockett et al.,
Bio/Technology 8:2
25 ( 1990)).
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
30 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., EMBO J. 2:1791 ( 1983)), 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, Nucleic Acids Res. 13:3101-3109
(1985); Van
3~ Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX
vectors
may also be used to express foreign polypeptides as fusion proteins with
glutathione S-
transferase (GST). In general, such fusion proteins are soluble and can easily
be purified
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from lysed cells by adsorption and binding to matrix glutathione-agarose beads
followed
by elution in the presence of free glutathione. The pGEX vectors are designed
to include
thrombin or factor Xa protease cleavage sites so that the cloned target gene
product can be
released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV)
is used as a vector to express foreign genes. The virus grows in
Spodoptercc,fi-ccgiperdcc
cells. The antibody coding sequence may be cloned individually into non-
essential
regions (for example the polyhedrin gene) of the virus and placed under
control of an
AcNPV promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may be
utilized. In cases where an adenovirus is used as an expression vector, the
antibody
coding sequence of interest may be ligated to an adenovirus
transcription/translation
control complex, e.g., the late promoter and tripartite leader sequence. This
chimeric
gene may then be inserted in the adenovirus genome by in vitro or in vivo
recombination.
Insertion in a non- essential region of the viral genome (e.g., region 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, Proc. Natl. Acad. Sci. USA 81:355-
359
( 1984)). 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., Methods in Enzymol. 153:51-544 (1987)).
In addition, a host cell strain may be chosen which modulates the expression
of
the inserted sequences, or modifies and processes the gene product in the
specific fashion
desired. Such modifications (e.g., glycosylation) and processing (e.g.,
cleavage) of
protein products may be important for the function of the protein. Different
host cells
have characteristic and specific mechanisms for the post-translational
processing and
modification of proteins and gene products. Appropriate cell lines or host
systems can be
chosen to ensure the correct modification and processing of the foreign
protein expressed.
To this end, eukaryotic host cells which possess the cellular machinery for
proper
processing of the primary transcript, glycosylation, and phosphorylation of
the gene
product may be used. Such mammalian host cells include but are not limited to
CHO,
VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer
cell
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lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal
mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
For long-term. high-yield production of recombinant proteins. stable
expression is
preferred. For example, cell lines which stably express the antibody molecule
may be
engineered. Rather than using expression vectors which contain viral origins
of
replication, host cells can be transformed with DNA controlled by appropriate
expression
control elements (e.g., promoter, enhances, sequences, transcription
terminators,
polyadenylation sites, ete.), 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 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., Cell 11:223 ( 1977)),
hypoxanthine-
guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad.
Sci. USA
48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell
22:817 (1980))
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., Natl. Acad. Sci. USA 77:357
(1980);
O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA
78:2072
(1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical
Pharmacy
12:488-505; Wu and Wu, Biotherapy 3:87-95 ( 1991 ); Tolstoshev, Ann. Rev.
Pharmacol.
Toxicol. 32:573-596 ( 1993); Mulligan, Science 260:926-932 ( 1993); and Morgan
and
Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-
215); and hygro, which confers resistance to hygromycin (Santerre et al., Gene
30:147
( 1984)). Methods commonly known in the art of recombinant DNA technology may
be
routinely applied to select the desired recombinant clone, and such methods
are described,
for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology,
John
Wiley & Sons, NY ( 1993); Kriegler, Gene Transfer and Expression, A Laboratory
Manual, Stockton Press, NY ( 1990); and in Chapters 12 and 13, Dracopoli et
al. (eds),
Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre
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93
Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by
reference herein in
their entireties.
The expression levels of an antibody molecule can be increased by vector
amplification (for a review, see Bebbington and Hentschel, The use of vectors
based on
gene amplification for the expression of cloned genes in mammalian cells in
DNA
cloning, Vol.3. {Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of inhibitor
present in
culture of host cell will increase the number of copies of the marker gene.
Since the
amplified region is associated with the antibody gene, production of the
antibody will also
increase (Grouse et al., Mol. Cell. Biol. 3:257 (1983)).
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, and is capable of
expressing,
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,
Nature 322:52 ( 1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 ( 1980)).
The
coding sequences for the heavy and light chains may comprise cDNA or genomic
DNA.
Once an antibody molecule of the invention has been produced by an animal,
chemically synthesized, or 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. In addition,
the antibodies of the present invention or fragments thereof can be fused to
heterologous
polypeptide sequences described herein or otherwise known in the art, to
facilitate
purification.
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, 30, 40, 50, 60, 70, 80, 90 or
100 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, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of
the
polypeptide) of the present invention. For example, antibodies may be used to
target the
polypeptides of the present invention to particular cell types, either in
vitro or in vivo, by
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fusing or conjugating the polypeptides of the present invention to antibodies
specific for
particular cell surface receptors. Antibodies fused or conjugated to the
polypeptides of the
present invention may also be used in in vitro immunoassays and purification
methods
using methods known in the art. See e.g., Harbor et al., supra, and PCT
publication WO
93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S.
Patent
5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.
146:2446-2452( 1991), which are incorporated by reference in their entireties.
The present invention further includes compositions comprising the
polypeptides
of the present invention fused or conjugated to antibody domains other than
the variable
regions. For example, the polypeptides of the present invention may be fused
or
conjugated to an antibody Fc region, or portion thereof. The antibody portion
fused to a
polypeptide of the present invention may comprise the constant region, hinge
region,
CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or
portions thereof. The polypeptides may also be fused or conjugated to the
above antibody
portions to form multimers. For example, Fc portions fused to the polypeptides
of the
present invention can form dimers through disulfide bonding between the Fc
portions.
Higher multimeric forms can be made by fusing the polypeptides to portions of
IgA and
IgM. Methods for fusing or conjugating the polypeptides 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 corresponding to a polypeptide,
polypeptide fragment, or a variant of SEQ ID N0:2, 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
corresponding
to SEQ ID N0:2 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
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9~
diagnosis, and thus can result in, for example, improved pharmacokinetic
properties. (EP
A 232,262). Alternatively, deleting the Fc part after the fusion protein has
been
expressed, detected, and purified, would be desired. For example, the Fc
portion may
hinder therapy and diagnosis if the fusion protein is used as an antigen for
immunizations.
In drug discovery, for example, human proteins, such as hIL-5, have been fused
with Fc
portions for the purpose of high-throughput screening assays to identify
antagonists of
hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58 ( 1995);
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 facilitate purification. In
preferred
embodiments, the marker amino acid sequence is a hexa-histidine peptide, such
as the tag
provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA,
91311 ),
among others, many of which are commercially available. As described in Gentz
et al.,
Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine
provides for
convenient purification of the fusion protein. Other peptide tags useful for
purification
include, but are not limited to, the "HA" tag, which corresponds to an epitope
derived
from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 ( 1984))
and the
"flag" tag.
The present invention further encompasses antibodies or fragments thereof
conjugated to a diagnostic or therapeutic agent. The antibodies can be used
diagnostically
to, for example, monitor the development or progression of a tumor as part of
a clinical
testing procedure to, e.g., determine the efficacy of a given treatment
regimen. Detection
can be facilitated by coupling the antibody to a detectable substance.
Examples of
detectable substances include various enzymes, prosthetic groups, fluorescent
materials,
luminescent materials, bioluminescent materials, radioactive materials,
positron emitting
metals using various positron emission tomographies, and nonradioactive
paramagnetic
metal ions. The detectable substance may be coupled or conjugated either
directly to the
antibody (or fragment thereof) or indirectly, through an intermediate (such
as, for
example, a linker known in the art) using techniques known in the art. See,
for example,
U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies
for use
as diagnostics according to the present invention. Examples of suitable
enzymes include
horseradish peroxidase, alkaline phosphatase, 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;
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examples of bioluminescent materials include luciferase, luciferin, and
aequorin; and
examples of suitable radioactive material include''~I,'''I, "'In or ''''Tc.
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, e.g., alpha-emitters such as, for example. 213Bi. A
cytotoxin or
cytotoxic agent includes any agent that is detrimental to cells. Examples
include
paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,
mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,
propranolol, and
puromycin and analogs or homologs thereof. Therapeutic agents include, but are
not
limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-
thioguanine,
cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and
cis-
dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g.,
daunorubicin
(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic
agents
(e.g., vincristine and vinblastine).
The conjugates of the invention can be used for modifying a given biological
response, the therapeutic agent or drug moiety is not to be construed as
limited to classical
chemical therapeutic agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins may
include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria
toxin; a
protein such as tumor necrosis factor, alpha-interferon, beta-interferon,
nerve growth
factor, platelet derived growth factor, tissue plasminogen activator, an
apoptotic agent,
e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO
97/33899),
AIM II (See, International Publication No. WO 97/34911 ), Fas Ligand
(Takahashi et al.,
Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.
WO
99/23105), CD40 Ligand, 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 macrophage 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
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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 et 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 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 Of
Antibody-Toxin Conjugates", Immunol. Rev. 62:119-58 (1982).
Alternatively, an antibody can be conjugated to a second antibody to form an
antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980,
which is
incorporated herein by reference in its entirety.
An antibody, with or without a therapeutic moiety conjugated to it,
administered
alone or in combination with cytotoxic factors) and/or cytokine(s) can be used
as a
therapeutic.
Immunophenotyping
The antibodies of the invention may be utilized for immunophenotyping of cell
lines and biological samples. The translation product of the gene of the
present invention
may be useful as a cell specific marker, or more specifically as a cellular
marker that is
differentially expressed at various stages of differentiation and/or
maturation of particular
cell types. Monoclonal antibodies directed against a specific epitope, or
combination of
epitopes, will allow for the screening of cellular' populations expressing the
marker.
Various techniques can be utilized using monoclonal antibodies to screen for
cellular
populations expressing the marker(s), and include magnetic separation using
antibody-
coated magnetic beads, "panning" with antibody attached to a solid matrix
(i.e., plate),
and flow cytometry (See, e.g., U.S. Patent 5,985,660: and Morrison et al.,
Cell, 96:737-
49 ( 1999)).
These techniques allow for the screening of particular populations of cells,
such as
might be found with hematological malignancies (i.e. minimal residual disease
(MRD) in
acute leukemic patients) and "non-self' cells in transplantations to prevent
Graft-versus-
Host Disease (GVHD). Alternatively, these techniques allow for the screening
of
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hematopoietic stem and progenitor cells capable of undergoing proliferation
and/or
differentiation, as might be found in human umbilical cord blood.
As.scays For Antibody Bir2ding
The antibodies of the invention may be assayed for immunospecific binding by
any method known in the art. The immunoassays which can be used include but
are not
limited to competitive and non-competitive assay systems using techniques such
as
western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel
diffusion precipitin reactions, immunodiffusion assays, agglutination assays,
complement-fixation assays, immunoradiometric assays, fluorescent
immunoassays,
protein A immunoassays, to name but a few. Such assays are routine and well
known in
the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular
Biology, Vol.
1, John Wiley & Sons, Inc., New York, which is incorporated by reference
herein in its
entirety). Exemplary immunoassays are described briefly below (but are not
intended by
way of limitation).
Immunoprecipitation protocols generally comprise lysing a population of cells
in a
lysis buffer such as RIPA buffer ( I % NP-40 or Triton X-100, 1 % sodium
deoxycholate,
0.1 % SDS, 0.15 M NaCI, 0.01 M sodium phosphate at pH 7.2, 1 % Trasylol)
supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA,
PMSF,
aprotinin, sodium vanadate), adding the antibody of interest to the cell
lysate, incubating
for a period of time (e.g., I-4 hours) at 4° C, adding protein A and/or
protein G sepharose
beads to the cell lysate, incubating for about an hour or more at 4° C,
washing the beads
in lysis buffer and resuspending the beads in SDS/sample buffer. The ability
of the
antibody of interest to immunoprecipitate a particular antigen can be assessed
by, e.g.,
western blot analysis. One of skill in the art would be knowledgeable as to
the parameters
that can be modified to increase the binding of the antibody to an antigen and
decrease the
background (e.g., pre-clearing the cell lysate with sepharose beads). For
further
discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al,
eds, 1994,
Current Protocols in Molecular Biology, Vol. l, John Wiley & Sons, Inc., New
York at
10.16.1.
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
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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., ''P or''~I) diluted in blocking buffer, washing the membrane
in wash
buffer, and detecting the presence of the antigen. One of skill in the art
would be
knowledgeable as to the parameters that can be modified to increase the signal
detected
and to reduce the background noise. For further discussion regarding western
blot
protocols see, e.g., Ausubel et al. eds, 1994. Current Protocols in Molecular
Biology,
Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96 well microtiter
plate
with the antigen, adding the antibody of interest conjugated to a detectable
compound
such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline
phosphatase) to
the well and incubating for a period of time, and detecting the presence of
the antigen. In
ELISAs the antibody of interest does not have to be conjugated to a detectable
compound;
instead, a second antibody (which recognizes the antibody of interest)
conjugated to a
detectable compound may be added to the well. Further, instead of coating the
well with
the antigen, the antibody may be coated to the well. In this case, a second
antibody
conjugated to a detectable compound may be added following the addition of the
antigen
of interest to the coated well. One of skill in the art would be knowledgeable
as to the
parameters that can be modified to increase 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 affinity of an antibody to an antigen and the off-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 125I) 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
off rates can be determined from the data by scatchard plot analysis.
Competition with a
second antibody can also be determined using radioimmunoassays. In this case,
the
antigen is incubated with antibody of interest conjugated to a labeled
compound (e.g., ;H
or''SI) in the presence of increasing amounts of an unlabeled second antibody.
Therapeutic Uses
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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, detecting, and/or preventing
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 of the
invention (including fragments, analogs and derivatives thereof and anti-
idiotypic
antibodies as described herein). The antibodies of the invention can be used
to treat,
diagnose, 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,
any one or more of the diseases, disorders, or conditions described herein
(e.g.,
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, rheumatic heart disease,
glomerulonephritis (e.g,
IgA nephropathy), 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, and other inflammatory, granulamatous, degenerative,
and
atrophic disorders).
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. The
treatment,
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detection, and/or prevention of diseases, disorders, or conditions 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. disorders or conditions.
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 of treatments (e.g., radiation therapy, chemotherapy, hormonal
therapy,
immunotherapy and anti-tumor agents, antibiotics, and immunoglobulin).
Generally,
administration of products of a species origin or species reactivity (in the
case of
antibodies) that is the same species as that of the patient is preferred.
Thus, in a preferred
embodiment, human antibodies, fragments derivatives, analogs, or nucleic
acids, are
administered to a human patient for therapy or prophylaxis.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or
neutralizing antibodies against polypeptides or polynucleotides of the present
invention,
fragments or regions thereof, for both immunoassays directed to and therapy of
disorders
related to polynucleotides or polypeptides, including fragments thereof, of
the present
invention. Such antibodies, fragments, or regions, will preferably have an
affinity for
polynucleotides or polypeptides of the invention, including fragments thereof.
Preferred
binding affinities include those with a dissociation constant or Kd less than
5 X 10~' M,
10-' M, 5 X 10-~ M, 10~~ M, 5 X 10-~ M, 10-~ M, 5 X 105 M, 105 M, 5 X 10-6 M,
10-6 M,
5 X 10-' M, 10-' M, 5 X 10-~ M, 10-g M, 5 X 10-9 M, 10-~ M, 5 X 10-'°
M, 10~'° M, 5 X
10-" M, 10-" M, 5 X 10-" M, 10-" M, 5 X 10~' ~ M, 10-' ~ M, 5 X 10-' ~ M, 10-'
~ M, 5 X
10''5 M, and 10-'5 M.
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Gene Tlter~cr~y
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.,
Clinical
Pharmacy 12:488-505 ( 1993); Wu and Wu, Biotherapy 3:87-95 ( 1991 );
Tolstoshev,
Ann. Rev. Pharmacol. Toxicol. 32:573-596 ( 1993); Mulligan, Science 260:926-
932
(1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
TIBTECH 11 (5):155-215 ( 1993). Methods commonly known in the art of
recombinant
DNA technology which can be used are described in Ausubel et al. (eds.),
Current
Protocols in Molecular Biology, John Wiley & Sons, NY ( 1993); and Kriegler,
Gene
Transfer and Expression, A Laboratory Manual, Stockton Press, NY ( 1990).
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 encoding nucleic
acids (Koller
and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al.,
Nature
342:435-438 (1989). 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 in vivo or
ex vivo
gene therapy.
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In a specific embodiment, the nucleic acid sequences are directly administered
in
vivo, where it is expressed to produce the encoded product. This can be
accomplished
by any of numerous methods known in the art, e.g., by constructing them as
part of an
appropriate nucleic acid expression vector and administering it so that they
become
intracellular, e.g., by infection using defective or attenuated retrovirals or
other viral
vectors (see U.S. Patent No. 4,980,286). or by direct injection of naked DNA,
or by use
of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating
with lipids
or cell-surface receptors or transfecting agents, encapsulation in liposomes,
microparticles, or microcapsules, or by administering them in linkage to a
peptide which
is known to enter the nucleus, by administering it in linkage to a ligand
subject to
receptor-mediated endocytosis (see, e.g., Wu and Wu, J: Biol. Chem. 262:4429-
4432
( 1987)) (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; WO 92/22635; W092/20316; W093/14188,
WO 93/20221 ). Alternatively, the nucleic acid can be introduced
intracellularly and
incorporated within host cell DNA for expression, by homologous recombination
(Koller
and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 ( 1989); Zijlstra et
al., Nature
342:435-438 (1989)).
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., Meth. Enzymol. 217:581-599 ( 1993)). These retroviral
vectors
contain the components necessary for the correct packaging of the viral genome
and
integration into the 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.,
Biotherapy 6:291-302 ( 1994), 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., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood
83:1467-1473
{1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and
Grossman
and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
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
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disease. Other targets for adenovirus-based delivery systems are liver. the
central
nervous system, endothelial cells, and muscle. Adenoviruses have the advantage
of being
capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion
in
Genetics and Development 3:499-503 ( 1993) present a review of adenovirus-
based gene
therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) 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.,
Science 252:431-434 ( 1991 ); Rosenfeld et al., Cell 68:143- 155 ( 1992):
Mastrangeli et
al., J. Clin. Invest. 91:225-234 (1993); PCT Publication W094/12649: and Wang,
et al.,
Gene Therapy 2:775-783 ( 1995). In a preferred embodiment, adenovirus vectors
are
used.
Adeno-associated virus (AAV) has also been proposed for use in gene therapy
(Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No.
5,436,146).
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 selection to
isolate those
cells that have taken up and are expressing the transferred gene. Those cells
are then
delivered to a patient.
In this embodiment, the nucleic acid is introduced into a cell prior to
administration in vivo of the resulting recombinant cell. Such introduction
can be carried
out by any method known in the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or bacteriophage
vector containing
the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer,
microcell-
mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known
in the
art for the introduction of foreign genes into cells (see, e.g., Loeffler and
Behr, Meth.
Enzymol. 217:599-618 ( 1993); Cohen et al., Meth. Enzymol. 217:618-644 (
1993);
Cline, Pharmac. Ther. 29:69-92m (1985) 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 of
the 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
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depends on the desired effect, patient state, etc., and can be determined by
one skilled in
the art.
Cells into which a nucleic acid can be introduced for purposes of gene therapy
encompass any desired, available cell type, and include but are not limited to
epithelial
cells, endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells
such as 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.
In a preferred embodiment, the cell used for gene therapy is autologous to the
patient.
In an embodiment in which recombinant cells are used in gene therapy, nucleic
acid sequences encoding an antibody are introduced into the cells such that
they are
expressible by the cells or their progeny, and the recombinant cells are then
administered
in vivo for therapeutic effect. In a specific embodiment, stem or progenitor
cells are used.
Any stem and/or progenitor cells which can be isolated and maintained in vitro
can
potentially be used in accordance with this embodiment of the present
invention (see e.g.
PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 ( 1992);
Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo
Clinic Proc.
61:771 ( 1986)).
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.
Demonstration of Therapeutic or- Prophylactic Activity
The compounds or pharmaceutical compositions of the invention are preferably
tested in vitro, and then in vivo for the desired therapeutic or prophylactic
activity, prior to
use in humans. For example, in vitro assays to demonstrate the therapeutic or
prophylactic utility of a compound or pharmaceutical composition include, the
effect of a
compound on a cell line or a patient tissue sample. The effect of the compound
or
composition on the cell line and/or tissue sample can be determined utilizing
techniques
known to those of skill in the art including, but not limited to, rosette
formation assays
and cell lysis assays. In accordance with the invention, in vitro assays which
can be used
to determine whether administration of a specific compound is indicated,
include in vitro
cell culture assays in which a patient tissue sample is grown in culture, and
exposed to or
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otherwise administered a compound, and the effect of such compound upon the
tissue
sample is observed.
TherapeuticlProphylactic Administration and Composition
The invention provides methods of treatment, inhibition and prophylaxis by
administration to a subject of an effective amount of a compound or
pharmaceutical
composition of the invention, preferably an antibody of the invention. In a
preferred
aspect, the compound is substantially purified (e.g., substantially free from
substances
that limit its effect or produce undesired side-effects). The subject is
preferably an
animal, including but not limited to animals such as cows, pigs, horses,
chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably human.
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
1 S described herein below.
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, J. Biol. Chem. 262:4429-4432 ( 1987)), 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 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,
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said implant being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. Preferably, when
administering a
protein, including an antibody, of the invention, care must be taken to use
materials to
which the protein does not absorb.
In another embodiment, the compound or composition can be delivered in a
vesicle, in particular a liposome (see Langer, 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. 353- 365 ( 1989); Lopez-Berestein, ibid.,
pp. 317-
327; see generally ibid.)
In yet another embodiment, the compound or composition can be delivered in a
controlled release system. In one embodiment, a pump may be used (see Langer,
supra;
Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery
88:507
( 1980); Saudek et al., N. Engl. J. Med. 321:574 ( 1989)). In another
embodiment,
polymeric materials can be used (see Medical Applications of Controlled
Release, Langer
and Wise (eds.), CRC Pres., Boca Raton, Florida ( 1974); Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.),
Wiley,
New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem.
23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al.,
Ann. Neurol.
25:351 ( 1989); Howard et al., J.Neurosurg. 71:105 ( 1989)). In yet another
embodiment,
a controlled release system can be placed in proximity of the therapeutic
target, i.e., the
brain, thus requiring only a fraction of the systemic dose (see, e.g.,
Goodson, in Medical
Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
Other controlled release systems are discussed in the review by Langer
(Science
249:1527-1533 (1990)).
In a specific embodiment where the compound of the invention is a nucleic acid
encoding a protein, the nucleic acid can be administered in vivo to promote
expression of
its encoded protein, by constructing it as part of an appropriate nucleic acid
expression
vector and administering it so that it becomes intracellular, e.g., by use of
a retroviral
vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of
microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or
cell-surface
receptors or transfecting agents, or by administering it in linkage to a
homeobox- like
peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc.
Natl. Acad. Sci.
USA 88:1864-1868 ( 1991 )), 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
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108
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 carriers such as pharmaceutical grades of mannitol, lactose, starch,
magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of
suitable
pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences"
by E.W.
Martin. Such compositions will contain a therapeutically effective amount of
the
compound, preferably in purified form, together with a suitable amount of
carrier so as to
provide the form for proper administration to the patient. The formulation
should suit the
mode of administration.
In a preferred embodiment, the composition is formulated in accordance with
routine procedures as a pharmaceutical composition adapted for intravenous
administration to human beings. Typically, compositions for intravenous
administration
are solutions in sterile isotonic aqueous buffer. Where necessary, the
composition may
also include a solubilizing agent and a local anesthetic such as lignocaine to
ease pain at
the site of the injection. Generally, the ingredients are supplied either
separately or mixed
together in unit dosage form, for example, as a dry lyophilized powder or
water free
concentrate in a hermetically sealed container such as an ampoule or sachette
indicating the
quantity of active agent. Where the composition is to be administered by
infusion, it can
be dispensed with an infusion bottle containing sterile pharmaceutical grade
water or
saline. Where the composition is administered by injection, an ampoule of
sterile water
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for injection or saline can be provided so that the ingredients may be mixed
prior to
administration.
The compounds of the invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with anions such as
those derived
from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those
formed with
cations such as those derived from sodium, potassium, ammonium, calcium,
ferric
hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
The amount of the compound of the invention which will be effective in the
treatment, inhibition and prevention of a disease or disorder associated with
aberrant
expression and/or activity of a polypeptide of the invention can be determined
by standard
clinical techniques. In addition, in vitro assays may optionally be employed
to help
identify optimal dosage ranges. The precise dose to be employed in the
formulation will
also depend on the route of administration, and the seriousness of the disease
or disorder,
and should be decided according to the judgment of the practitioner and each
patient's
circumstances. Effective doses may be extrapolated from dose-response curves
derived
from in vitro or animal model test systems.
For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to
100
mg/kg of the patient's body weight. Preferably, the dosage administered to a
patient is
between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1
mg/kg
to 10 mg/kg of the patient's body weight. Generally, human antibodies have a
longer
half life within the human body than antibodies from other species due to the
immune
response to the foreign polypeptides. Thus, lower dosages of human antibodies
and less
frequent administration is often possible. Further, the dosage and frequency
of
administration of antibodies of the invention may be reduced by enhancing
uptake and
tissue penetration (e.g., into the brain) of the antibodies by modifications
such as, for
example, lipidation.
The invention also provides a pharmaceutical pack or kit comprising one or
more
containers filled with one or more of the ingredients of the pharmaceutical
compositions
of the invention. Optionally associated with such containers) can be a notice
in the form
prescribed by a governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects approval by the
agency of
manufacture, use or sale for human administration.
Diagnosis and Imaging
Labeled antibodies, and derivatives and analogs thereof, which specifically
bind to
a polypeptide of interest can be used for diagnostic purposes to detect,
diagnose, or
monitor diseases and/or disorders associated with the aberrant expression
and/or activity
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of a polypeptide of the invention. The invention provides for the detection of
aberrant
expression of a polypeptide of interest. comprising (a) assaying the
expression of the
polypeptide of interest in cells or body fluid of an individual using one or
more antibodies
specific to the polypeptide interest and (b) comparing the level of gene
expression with a
standard gene expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard expression level is
indicative
of aberrant expression.
The invention provides a diagnostic assay for diagnosing a disorder,
comprising
(a) assaying the expression of the polypeptide of interest in cells or body
fluid of an
individual using one or more antibodies specific to the polypeptide interest
and (b)
comparing the level of gene expression with a standard gene expression level,
whereby an
increase or decrease in the assayed polypeptide gene expression level compared
to the
standard expression level is indicative of a particular disorder. With respect
to cancer,
the presence of a relatively high amount of transcript in biopsied tissue from
an individual
may indicate a predisposition for the development of the disease, or may
provide a means
for detecting the disease prior to the appearance of actual clinical symptoms.
A more
definitive diagnosis of this type may allow health professionals to employ
preventative
measures or aggressive treatment earlier thereby preventing the development or
further
progression of the cancer.
Antibodies of the invention can be used to assay protein levels in a
biological
sample using classical immunohistological methods known to those of skill in
the art
(e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et
al., J. Cell.
Biol. 105:3087-3096 ( 1987)). Other antibody-based methods useful for
detecting protein
gene expression include immunoassays, such as the enzyme linked immunosorbent
assay
(ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are
known in
the art and include enzyme labels, such as, glucose oxidase; radioisotopes,
such as iodine
('3'I, "SI,''~I,'''I), carbon ('4C), sulfur (;5S), tritium (;H), indium
("SmIn, "~mln, "ZIn,
"'In), and technetium (~~Tc, 99mTc), thallium ('°'Ti), gallium (6gGa,
6'Ga), palladium
('°~Pd), molybdenum (99Mo), xenon ('~;Xe), fluorine ('gF), 'S~Sm, "'Lu,
'S9Gd, '49Pm,
'4°La "5Yb '66Ho ~°Y '"Sc '86Re 'BgRe '~ZPr '°SRh 9'Rw
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).
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One aspect of the invention is the detection and diagnosis of a disease or
disorder
associated with aberrant expression of a polypeptide of interest in an animal,
preferably a
mammal and most preferably a human. In one embodiment, diagnosis comprises: a)
administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a subject
an effective amount of a labeled molecule which specifically binds to the
polypeptide of
interest; b) waiting for a time interval following the administering for
permitting the
labeled molecule to preferentially concentrate at sites in the subject where
the polypeptide
is expressed (and for unbound labeled molecule to be cleared to background
level); c)
determining background level; and d) detecting the labeled molecule in the
subject, such
that detection of labeled molecule above the background level indicates that
the subject has
a particular disease or disorder associated with aberrant expression of the
polypeptide of
interest. Background level can be determined by various methods including,
comparing
the amount of labeled molecule detected to a standard value previously
determined for a
particular system.
As described herein, specific embodiments of the invention are directed to the
use
of the antibodies of the invention to quantitate or qualitate concentrations
of cells of B cell
lineage or cells of monocytic lineage.
Also as described herein, antibodies of the invention may be used to treat,
diagnose, or prognose an individual having an immunodeficiency. In a specific
embodiment, antibodies of the invention are used to treat, diagnose, and/or
prognose an
individual having common variable immunodeficiency disease (CVID) or a subset
of this
disease. In another embodiment, antibodies of the invention are used to
diagnose,
prognose, treat or prevent a disorder characterized by deficient serium
immunoglobulin
production, recurrent infections, and/or immune system dysfunction.
Also as described herein, antibodies of the invention may be used to treat,
diagnose, or prognose an individual having an autoimmune disease or disorder.
In a
specific embodiment, antibodies of the invention are used to treat, diagnose,
and/or
prognose an individual having systemic lupus erythematosus, or a subset of the
disease.
In another specific embodiment, antibodies of the invention are used to treat,
diagnose
and/or prognose an individual having rheumatoid arthritis, or a subset of this
disease.
It will be understood in the art that the size of the subject and the imaging
system
used will determine the quantity of imaging moiety needed to produce
diagnostic images.
In the case of a radioisotope moiety, for a human subject, the quantity of
radioactivity
injected will normally range from about 5 to 20 millicuries of 99mTc. The
labeled antibody
or antibody fragment will then preferentially accumulate at the location of
cells which
contain the specific protein. In vivo tumor imaging is described in S.W.
Burchiel et al.,
"Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments."
(Chapter
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13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and
B. A.
Rhodes, eds., Masson Publishing Inc. ( 1982).
Depending on several variables, including the type of label used and the mode
of
administration, the time interval following the administration for permitting
the labeled
molecule to preferentially concentrate at sites in the subject and for unbound
labeled
molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours
or 6 to 12
hours. In another embodiment the time interval following administration is 5
to 20 days
or 5 to 10 days.
In an embodiment, monitoring of the disease or disorder is carried out by
repeating the method for diagnosing the disease or disease, for example, one
month after
initial diagnosis, six months after initial diagnosis, one year after initial
diagnosis, etc.
Presence of the labeled molecule can be detected in the patient using methods
known in the art for in vivo scanning. These methods depend upon the type of
label
used. Skilled artisans will be able to determine the appropriate method for
detecting a
particular label. Methods and devices that may be used in the diagnostic
methods of the
invention include, but are not limited to, computed tomography (CT), whole
body scan
such as position emission tomography (PET), magnetic resonance imaging (MRI),
and
sonography.
In a specific embodiment, the molecule is labeled with a radioisotope and is
detected in the patient using a radiation responsive surgical instrument
(Thurston et al.,
U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled
with a
fluorescent compound and is detected in the patient using a fluorescence
responsive
scanning instrument. In another embodiment, the molecule is labeled with a
positron
emitting metal and is detected in the patent using positron emission-
tomography. In yet
another embodiment, the molecule is labeled with a paramagnetic label and is
detected in a
patient using magnetic resonance imaging (MRI).
Kits
The present invention provides kits that can be used in the above methods. In
one
embodiment, a kit comprises an antibody of the invention, preferably a
purified antibody,
in one or more containers. In a specific embodiment, the kits of the present
invention
contain a substantially isolated polypeptide comprising an epitope which is
specifically
immunoreactive with an antibody included in the kit. Preferably, the kits of
the present
invention further comprise a control antibody which does not react with the
polypeptide of
interest. In another specific embodiment, the kits of the present invention
comprise two
or more antibodies (monoclonal and/or polyclonal) that recognize the same
and/or
different sequences or regions of the polypeptide of the invention. In another
specific
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embodiment, the kits of the present invention contain a means for detecting
the binding of
an antibody to a polypeptide of interest (e.g., the antibody may be conjugated
to a
detectable substrate such as a fluorescent compound, an enzymatic substrate, a
radioactive
compound or a luminescent compound, or a second antibody which recognizes the
first
antibody may be conjugated to a detectable substrate).
In another specific embodiment of the present invention, the kit is a
diagnostic kit
for use in screening serum containing antibodies specific against
proliferative and/or
cancerous polynucleotides and polypeptides. Such a kit may include a control
antibody
that does not react with the polypeptide of interest. Such a kit may include a
substantially
isolated polypeptide antigen comprising an epitope which is specifically
immunoreactive
with at least one anti-polypeptide antigen antibody. Further, such a kit
includes means for
detecting the binding of said antibody to the antigen (e.g., the antibody may
be conjugated
to a fluorescent compound such as fluorescein or rhodamine which can be
detected by
flow cytometry). In specific embodiments, the kit may include a recombinantly
produced
or chemically synthesized polypeptide antigen. The polypeptide antigen of the
kit may
also be attached to a solid support.
In a more specific embodiment the detecting means of the above-described kit
includes a solid support to which said polypeptide antigen is attached. Such a
kit may also
include a non-attached reporter-labeled anti-human antibody. In this
embodiment, binding
of the antibody to the polypeptide antigen can be detected by binding of the
said reporter-
labeled antibody.
In an additional embodiment, the invention includes a diagnostic kit for use
in
screening serum containing antigens of the polypeptide of the invention. The
diagnostic
kit includes a substantially isolated antibody specifically immunoreactive
with polypeptide
or polynucleotide antigens, and means for detecting the binding of the
polynucleotide or
polypeptide antigen to the antibody. In one embodiment, the antibody is
attached to a
solid support. In a specific embodiment, the antibody may be a monoclonal
antibody. The
detecting means of the kit may include a second, labeled monoclonal antibody.
Alternatively, or in addition, the detecting means may include a labeled,
competing
antigen.
In one diagnostic configuration, test serum is reacted with a solid phase
reagent
having a surface-bound antigen obtained by the methods of the present
invention. After
binding with specific antigen antibody to the reagent and removing unbound
serum
components by washing, the reagent is reacted with reporter-labeled anti-human
antibody
to bind reporter to the reagent in proportion to the amount of bound anti-
antigen antibody
on the solid support. The reagent is again washed to remove unbound labeled
antibody,
and the amount of reporter associated with the reagent is determined.
Typically, the
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reporter is an enzyme which is detected by incubating the solid phase in the
presence of a
suitable fluorometric, luminescent or colorimetric substrate (Sigma, St.
Louis, MO).
The solid surface reagent in the above assay is prepared by known techniques
for
attaching protein material to solid support material, such as polymeric beads,
dip sticks,
96-well plate or filter material. These attachment methods generally include
non-specific
adsorption of the protein to the support or covalent attachment of the
protein, typically
through a free amine group, to a chemically reactive group on the solid
support, such as
an activated carboxyl, hydroxyl, or aldehyde group. Alternatively,
streptavidin coated
plates can be used in conjunction with biotinylated antigen(s).
Thus, the invention provides an assay system or kit for carrying out this
diagnostic method. The kit generally includes a support with surface- bound
recombinant
antigens, and a reporter-labeled anti-human antibody for detecting surface-
bound anti-
antigen antibody.
The invention further relates to antibodies that act as agonists or
antagonists of the
polypeptides of the present invention. For example, the present invention
includes
antibodies that disrupt the receptor/ligand interactions with the polypeptides
of the
invention either partially or fully. Included are both receptor-specific
antibodies and
ligand-specific antibodies. Included are receptor-specific antibodies that 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 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 that 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.
Further included
are antibodies that bind to TR9 irrespective of whether TR9 is bound to a TR9
ligand.
These antibodies act as TR9 agonists as reflected in an increase in cellular
proliferation in
response to binding of TR9 to a TR9 ligand in the presence of these
antibodies. The
above antibody agonists can be made using methods known in the art. See e.g.,
WO
96/40281; US Patent 5,811,097; Deng, B. et al., Blood 92(6):1981-1988 (1998);
Chen,
Z. et al., Cancer Res. 58( 16):3668-3678 ( 1998); Harrop, J.A. et al., J.
Immunol.
161(4):1786-1794 (1998); Zhu, Z. et al., Cancer Res. 58(15):3209-3214 (1998);
Yoon,
D.Y. et al., J. Immunol. 160(7):3170-3179 ( 1998); Prat, M. et al., J. Cell.
Sci.
111(Pt2):237-247 (1998); Pitard, V. et al., J. Immunol. Methods 205(2):177-190
(1997);
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Liautard, J. et al., Cytokinde 9(4):233-241 (1997); Carlson, N.G. et al., J.
Biol: Chem.
272(17):11295-11301 (1997); Taryman, R.E. et al., Neuron 14(4):755-762 (1995);
Muller, Y.A. et al., Structure 6(9):1153-1167 (1998); Bartunek, P. et al.,
Cytokine
8( 1 ):14-20 ( 1996) (said references incorporated by reference in their
entireties).
The invention encompasses antibodies that inhibit or reduce the ability of TR9
to
bind TR9 ligand in vitro and/or in vivo. In a specific embodiment, antibodies
of the
invention inhibit or reduce the ability of TR9 to bind TR9 ligand in vitro. In
another
nonexclusive specific embodiment, antibodies of the invention inhibit or
reduce the ability
of TR9 to bind bind TR9 ligand in vivo. Such inhibition can be assayed using
techniques
described herein or otherwise known in the art.
The invention also encompasses, antibodies that bind specifically to TR9, but
do
not inhibit the ability of TR9 to bind TR9 ligand in vitro and/or in vivo. In
a specific
embodiment, antibodies of the invention do not inhibit or reduce the ability
of TR9 to bind
TR9 ligand in vitro. In another nonexclusive specific embodiment, antibodies
of the
invention do not inhibit or reduce the ability of TR9 to bind TR9 ligand in
vivo.
As described above, the invention encompasses antibodies that inhibit or
reduce a
TR9-mediated biological activity in vitro and/or in vivo. In a specific
embodiment,
antibodies of the invention inhibit or reduce TR9-mediated B or T cell
proliferation in
vitro. Such inhibition can be assayed by routinely modifying B or T cell
proliferation
assays described herein or otherwise known in the art. In another nonexclusive
specific
embodiment, antibodies of the invention inhibit or reduce TR9-mediated B or T
cell
proliferation in vivo.
Alternatively, the invention also encompasses, antibodies that bind
specifically to
a TR9, but do not inhibit or reduce a TR9-mediated biological activity in
vitro and/or in
vivo (e.g., stimulation of B or T cell proliferation). In a specific
embodiment, antibodies
of the invention do not inhibit or reduce a TR9-mediated biological activity
in vitro. In
another non-exclusive embodiment, antibodies of the invention do not inhibit
or reduce a
TR9-mediated biological activity in vivo.
As described above, the invention encompasses antibodies that specifically
bind to
the same epitope as at least one of the antibodies specifically referred to
herein, in vitro
and/or in vivo.
In a specific embodiment, the specific antibodies described above are
humanized
using techniques described herein or otherwise known in the art and then used
as
therapeutics as described herein.
In another specific embodiment, any of the antibodies listed above are used in
a
soluble form.
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In another specific embodiment, any of the antibodies listed above are
conjugated
to a toxin or a label (as described infra). Such conjugated antibodies are
used to kill a
particular population of cells or to quantitate a particular population of
cells. In a
preferred embodiment, such conjugated antibodies are used to kill B cells
expressing TR9
on their surface. In another prefewed embodiment, such conjugated antibodies
are used
to quantitate B cells expressing TR9 on their surface. In a preferred
embodiment, such
conjugated antibodies are used to kill T cells expressing TR9 on their
surface. In another
preferred embodiment, such conjugated antibodies are used to quantitate T
cells
expressing TR9 on their surface.
In another specific embodiment, any of the antibodies listed above are
conjugated
to a toxin or a label (as described infra). Such conjugated antibodies are
used to kill a
particular population of cells or to quantitate a particular population of
cells.
The antibodies of the invention also have uses as therapeutics and/or
prophylactics
which include, but are not limited to, inactivating lymphocytes or blocking
lymphocyte
activation and/or killing lymphocyte lineages that express TR9 on their cell
surfaces (e.g.,
to treat, prevent, and/or diagnose myeloid leukemias, lymphocyte based
leukemias and
lymphomas, lymphocytosis, lymphocytopenia, rheumatoid arthritis, and other
diseases or
conditions associated with activated lymphocytes). In a specific embodiment,
the
antibodies of the invention fix complement. In other specific embodiments, as
further
described herein, the antibodies of the invention (or fragments thereof) are
associated with
heterologous polypeptides or nucleic acids (e.g. toxins, such as, compounds
that bind
and activate endogenous cytotoxic effecter systems, and radioisotopes; and
cytotoxic
prodrugs).
In another embodiment, one or more monoclonal antibodies are produced wherein
they recognize or bind TR9 and/or a mutein thereof, but do not recognize or
bind TR9
and/or a mutein thereof. In a related embodiment, one or more monoclonal
antibodies are
produced wherein they recognize or bind TR9 and/or a mutein thereof, but do
not
recognize or bind TR9 and/or a mutein thereof.
As discussed above, antibodies to the TR9 polypeptides of the invention can,
in
turn, be utilized to generate anti-idiotype antibodies that "mimic" the TR9,
using
techniques well known to those skilled in the art. (See, e.g., Greenspan &
Bona, FASEB
J. 7(5):437-444 (1989), and Nissinoff, J. In2rnunol. 147(8):2429-2438 (1991)).
For
example, antibodies which bind to TR9 and competitively inhibit TR9
multimerization
and/or binding to ligand can be used to generate anti-idiotypes that "mimic"
the TR9 TNF
mutimerization and/or binding domain and, as a consequence, bind to and
neutralize TR9
and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such
anti-idiotypes
can be used in therapeutic regimens to neutralize TR9 ligand. For example,
such anti-
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idiotypic antibodies can be used to bind TR9 on the surface of cells of B or T
cell lineage,
and thereby block TR9-mediated B or T cell activation, proliferation, and/or
differentiation.
Polypeptide Assays
The present invention also relates to diagnostic assays such as quantitative
and
diagnostic assays for detecting levels of TR9 receptor protein, or the soluble
form thereof,
in cells and tissues, including determination of normal and abnormal levels.
Thus, for
instance, a diagnostic assay in accordance with the invention for detecting
over-
expression of TR9, or soluble form thereof, compared to normal control tissue
samples
may be used to detect the presence of tumors, for example. Assay techniques
that can be
used to determine levels of a protein, such as a TR9 protein of the present
invention, or a
soluble form thereof, in a sample derived from a host are well-known to those
of skill in
the art. Such assay methods include radioimmunoassays, competitive-binding
assays,
Western Blot analysis and ELISA assays.
Assaying TR9 protein levels in a biological sample can occur using any art-
known
method. By "biological sample" is intended any biological sample obtained from
an
individual, cell line, tissue culture, or other source which contains TR9
receptor protein or
mRNA. Preferred for assaying TR9 protein levels in a biological sample are
antibody-
based techniques. For example, TR9 protein expression in tissues can be
studied with
classical immunohistological methods. (Jalkanen et al., J. Cell. Biol. 101:976-
985
( 1985); Jalkanen et al., J. Cell. Biol. 105: 3087-3096 ( 1987)). Other
antibody-based
methods useful for detecting TR9 receptor gene expression include
immunoassays, such
as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay
(RIA).
Suitable labels are known in the art and include enzyme labels, such as,
glucose
oxidase, and radioisotopes, such as iodine ("5I,'2'I), carbon ('''C), sulphur
(ASS), tritium
(3H), indium ("'In), and technetium (99mTc), and fluorescent labels, such as
fluorescein
and rhodamine, and biotin.
Therapeutics
The tumor necrosis factor (TNF) family ligands are known to be among the most
pleiotropic cytokines, inducing a large number of cellular responses,
including
cytotoxicity, anti-viral activity, immunoregulatory activities, and the
transcriptional
regulation of several genes (D.V. Goeddel et al., "Tumor Necrosis Factors:
Gene
Structure and Biological Activities," SyYnp. Quarzt. Biol. 51:597- 609 (1986),
Cold
Spring Harbor; B. Beutler and A. Cerami, Annu. Rev. Biochenz. 57:505-518
(1988);
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L.J. Old, Sci. Ana. 258:59-75 ( 1988); W. Fiers, FEBS Lett. 285:199-224 ( 1991
)). The
TNF-family ligands induce such various cellular responses by binding to TNF-
family
receptors, including the TR9 of the present invention. Cells which express the
TR9
polypeptide and are believed to have a potent cellular response to TR9 ligands
include
fetal liver, PBL, lung, kidney, small intestine, colon, keratinocytes,
endothelial cells, and
monocyte activated tissue. By "a cellular response to a TNF-family ligand" is
intended
any genotypic, phenotypic, and/or morphologic change to a cell, cell line,
tissue, tissue
culture or patient that is induced by a TNF-family ligand. As indicated, such
cellular
responses include not only normal physiological responses to TNF-family
ligands, but
also diseases associated with increased apoptosis or the inhibition of
apoptosis.
Apoptosis-programmed cell death-is a physiological mechanism involved in the
deletion
of peripheral T lymphocytes of the immune system, and its dysregulation can
lead to a
number of different pathogenic processes (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,
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, prostrate cancer, Kaposi's sarcoma and ovarian cancer);
autoimmune
disorders (such as multiple sclerosis, Sjogren's syndrome, Hashimoto's
thyroiditis,
biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic
lupus
erythematosus and immune-related glomerulonephritis rheumatoid arthritis);
viral
infections (such as herpes viruses, pox viruses and adenoviruses);
inflammation; graft vs.
host disease; acute graft rejection and chronic graft rejection. In preferred
embodiments,
TNFR polynucleotides, polypeptides, and/or antagonists of the invention are
used to
inhibit growth, progression, andlor metasis of cancers, in particular those
listed above
and in the paragraph that follows.
Additional diseases or conditions associated with increased cell survival
include,
but are not limited to, progression, and/or metastases of malignancies and
related
disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic
leukemia,
acute myelocytic leukemia (including myeloblastic, promyelocytic,
myelomonocytic,
monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic
myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera,
lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple
myeloma,
Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors
including, but
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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 include AIDS; neurodegenerative
disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic
lateral
sclerosis, Retinitis pigmentosa, Cerebellar degeneration); and brain tumor or
prior
associated disease); autoimmune disorders (such as, multiple sclerosis,
Sjogren's
syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease,
Crohn's disease,
polymyositis, systemic lupus erythematosus and immune-related
glomerulonephritis and
rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia),
graft v. host
disease, ischemic injury (such as that caused by myocardial infarction, stroke
and
reperfusion injury), liver injury (e.g., hepatitis related liver injury,
ischemialreperfusion
injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver
disease (such as
that caused by alcohol), septic shock, cachexia and anorexia. In preferred
embodiments,
TNFR polynucleotides, polypeptides and/or agonists are used to treat, prevent,
diagnose,
and/or detect the diseases and disorders listed above.
Immunodeficiencies that may be treated, prevented, diagnosed, and/or prognosed
with TR9 polynucleotides or polypeptides or TR9 agonists or antagonists (e.g.,
anti-TR9
antibodies) of the invention, include, but are not limited to one or more
immunodeficiencies selected from: severe combined immunodeftciency (SCID)-X
linked,
SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked
agammaglobulinemia (XLA), Breton'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), Wiskott-Aldrich Syndrome (WAS), X-linked immunodeficiency
with
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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.
Autoimmune diseases or disorders that may be treated, diagnosed, or prognosed
using TR9 polynucleotides or polypeptides or TR9 agonists or antagonists
(e.g., anti-
TR9 antibodies) of the invention include, but are not limited to, one or more
of the
following: 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 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 dnig
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, and
other
inflammatory, granulamatous, degenerative, and atrophic disorders.
TR9 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 or disorders, or conditions associated therewith: primary
immuodeficiencies,
immune-mediated thrombocytopenia, Kawasaki syndrome, bone marrow transplant
(e.g.,
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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, TR9 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 B 19, patients with
stable
mutliple myeloma who 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.
Administration to an animal (e.g., mouse, rat, rabbit, hamster, guinea pig,
pigs,
micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human
primate, and
human, most preferably human) to boost the immune system to produce increased
quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce
higher
affinity antibody production (e.g., IgG, IgA, IgM, and IgE), and/or to
increase an
immune response. In a specific nonexclusive embodiment, TR9 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, TR9
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, TR9 polypeptides of the invention and/or agonists thereof, are
administered
to boost the immune system to produce increased quantities of IgM.
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.
Thus, in one aspect, the present invention is directed to a method for
enhancing
apoptosis induced by a TNF-family ligand, which involves administering to a
cell which
expresses the TR9 polypeptide, an effective amount of TR9 ligand, analog or an
agonist
capable of increasing TR9 mediated signaling. Preferably, TR9 mediated
signaling is
increased to treat, prevent, diagnose, and/or detect a disease wherein
decreased apoptosis
or decreased cytokine and adhesion molecule expression is exhibited. Agonists
include.
but are not limited to, soluble forms of TR9 and antibodies (preferably
monoclonal)
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1?2
directed against the TR9 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 TR9 polypeptide an effective amount of an antagonist capable of
decreasing
TR9 mediated signaling. Preferably, TR9 mediated signaling is decreased to
treat,
prevent, diagnose, and/or detect a disease «~herein increased apoptosis, NFkB
expression
and/or JNK expression is exhibited. Antagonists include, but are not limited
to, soluble
forms of TR9 polypeptide and antibodies (preferably monoclonal) directed
against the
TR9 polypeptide.
As disclosed in Example 8, a TR9-Fc fusion protein containing a polypeptide
sequence located in the extracellular domain of TR9 activates monocytes and
increases
monocyte survival. Accordingly, in specific embodiments, the invention
encompasses
methods of stimulating an inflammatory response, which includes administering
to a cell
(e.g., monocytes in vitro or in vivo) a polynucleotide, polypeptide, aaonist,
or antagonist
of the invention.
Additionally, the invention encompasses a method of enhancing macrophage
activity which involves administering to a cell (e.g., monocytes in vitro or
in vivo) a
polynucleotide, polypeptide, agonist, or antagonist of the invention. In a
specific
embodiment, a polynucleotide, polypeptide, agonist, or antagonist of the
invention is
administered as a prophylactic or therapeutic agent to generate resistance to
pathogens.
In another embodiment, a polynucleotide, polypeptide, agonist, or antagonist
of
the invention, which demonstrates anti-inflammatory activity and/or which acts
as an
antagonist to a TR9-Fc polypeptide, is administered to treat, prevent,
diagnose, and/or
detect an inflammatory disease. In another embodiment a polynucleotide,
polypeptide,
agonist, or antagonist of the invention, which demonstrates anti-inflammatory
activity
and/or which acts as an antagonist to a TR9-Fc polypeptide, is administered to
treat,
prevent, diagnose, and/or detect a THl-associated condition. In a specific
embodiment, a
polynucleotide, polypeptide, agonist, or antagonist of the invention, which
demonstrate
anti-inflammatory activity and/or which acts as an antagonist to a TR9-Fc
polypeptide is
administered to treat, prevent, diagnose, and/or detect autoimmune disorders,
such as
those disclosed herein.
By "agonist" is intended naturally occurring and synthetic compounds capable
of
enhancing or potentiating apoptosis. By "antagonist" is intended naturally
occurring and
synthetic compounds capable of inhibiting apoptosis. Whether any candidate
"agonist" or
'°antagonist" of the present invention can enhance or inhibit apoptosis
can be determined
using art-known TNF-family ligand/receptor cellular response assays, including
those
described in more detail below.
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One such screening procedure involves the use of melanophores which are
transfected to express the receptor of the present invention. Such a screening
technique is
described in PCT WO 92/01810, published February 6, 1992. Such an assay may be
employed, for example, for screening for a compound which inhibits (or
enhances)
activation of the receptor polypeptide of the present invention by contacting
the
melanophore cells which encode the receptor with both a TNF-family ligand and
the
candidate antagonist (or agonist). Inhibition or enhancement of the signal
generated by
the ligand indicates that the compound is an antagonist or agonist of the
ligand/receptor
signaling pathway.
Other screening techniques include the use of cells which express the receptor
(for
example, transfected CHO cells) in a system which measures extracellular pH
changes
caused by receptor activation, for example, as described in Science 246:181-
296 ( 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 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 Xer2opus oocytes to transiently express the receptor. The
receptor oocytes
may then be contacted with the receptor ligand and a compound to be screened,
followed
by detection of inhibition or activation of a calcium signal in the case of
screening for
compounds which are thought to inhibit activation of the receptor.
Another screening technique well known in the art involves expressing in cells
a
construct wherein the receptor is linked to a phospholipase C or D. Exemplary
cells
include endothelial cells, smooth muscle cells, embryonic kidney cells, etc.
The
screening may be accomplished as hereinabove described by detecting activation
of the
receptor or inhibition of activation of the receptor from the phospholipase
signal.
Another method involves screening for compounds which inhibit activation of
the
receptor polypeptide of the present invention antagonists by determining
inhibition of
binding of labeled ligand to cells which have the receptor on the surface
thereof. Such a
method involves transfecting a eukaryotic cell with DNA encoding the receptor
such that
the cell expresses the receptor on its surface and contacting the cell with a
compound in
the presence of a labeled form of a known ligand. The ligand can be labeled,
e.g., by
radioactivity. The amount of labeled ligand bound to the receptors is
measured, e.g., by
measuring radioactivity of the receptors. If the compound binds to the
receptor as
determined by a reduction of labeled ligand which binds to the receptors, the
binding of
labeled ligand to the receptor is inhibited.
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Soluble forms of the polypeptides of the present invention may be utilized in
the
ligand binding assay described above. These forms of the TR9 receptors are
contacted
with ligands in the extracellularmedium after they are secreted. A
determination is then
made as to whether the secreted protein will bind to TR9 receptor ligands.
Further screening assays for agonists and antagonists of the present invention
are
described in Tartaglia et al., J. Biol. Cheat. 267:4304-4307( 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
cellular response
to a TNF-family ligand. The method involves contacting cells which express the
TR9
polypeptide with a candidate compound and a TNF-family ligand, assaying a
cellular
response, and comparing the cellular response to a standard cellular response,
the
standard being assayed when contact is made with the ligand in absence of the
candidate
compound, whereby an increased cellular response over the standard indicates
that the
candidate compound is an agonist of the ligand/receptor signaling pathway and
a
decreased cellular response compared to the standard indicates that the
candidate
compound is an antagonist of the ligand/receptor signaling pathway. By
"assaying a
cellular response" is intended qualitatively or quantitatively measuring a
cellular response
to a candidate compound and/or a TNF-family ligand (e.g., determining or
estimating an
increase or decrease in T cell proliferation or tritiated thymidine labeling).
By the
invention, a cell expressing the TR9 polypeptide can be contacted with either
an
endogenous or exogenously administered TNF-family ligand.
Agonist according to the present invention include naturally occurring and
synthetic compounds such as, for example, TNF family ligand peptide fragments,
transforming growth factor, neurotransmitters (such as glutamate, dopamine, N-
methyl-
D-aspartate), tumor suppressors (p53), cytolytic T cells and antimetabolites.
Preferred
agonists include chemotherapeutic drugs such as, for example, cisplatin,
doxorubicin,
bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate and
vincristine. Others
include ethanol and -amyloid peptide. (Science 267:1457-1458 (1995)). Further
preferred agonists include polyclonal and monoclonal antibodies raised against
the TR9
polypeptides of the invention, or a fragment thereof. Such agonist antibodies
raised
against a TNF-family receptor are disclosed in Tartaglia et al., Proc. Natl.
Acad. Sci.
USA 88:9292-9296 ( 1991 ); and Tartaglia et al., J. Biol. Chem. 267:4304-
4307( 1992).
See, also, PCT Application WO 94/09137.
Antagonists according to the present invention include naturally occurring and
synthetic compounds such as, for example, the CD40 ligand, neutral amino
acids, zinc,
estrogen, androgens, viral genes (such as Adenovirus EIB, Baculovirus p35 and
IAP,
Cowpox virus crmA, Epstein-Barr virus BHRFl, LMP-I, African swine fever virus
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125
LMWS-HL, and Herpesvirus yl 34.5), calpain inhibitors, cysteine protease
inhibitors,
and tumor promoters (such as PMA, Phenobarbital, and = Hexachlorocyclohexane).
In specific embodiments, antagonists according to the present invention are
nucleic acids corresponding to the sequences contained in Figures lA-D, or the
complementary strand thereof, and/or to nucleotide sequences contained in the
deposited
clone. In one embodiment, antisense sequence is generated internally by the
organism, in
another embodiment, the antisense sequence is separately administered (see,
for example,
O'Connor, J., Neurocllettt. 56:560 (1991); and Oligodeoxyttttcleotides
asAntisense
Inhibitors of Gerte Expression, CRC Press, Boca Raton, FL ( 1988). Antisense
technology can be used to control gene expression through antisense DNA or
RNA, or
through triple-helix formation. Antisense techniques are discussed for
example, in
Okano, J., Neurochem. 56:560 ( 1991 ); OligodeoxytZCtcleotides as Antisense
Inhibitors of
Gene Expression, CRC Press, Boca Raton, FL (1988). Triple helix formation is
discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073 (1979);
Cooney et
al., Science 241:456 ( 1988); and Dervan et al., Science 251:1300 ( 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
polynucleotide of from about 10 to 40 base pairs in length. A DNA
polynucleotide 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
polypeptide hybridizes to the mRNA itz vivo and blocks translation of the mRNA
molecule into receptor polypeptide. The polynucleotides described herein can
also be
delivered to cells such that the antisense RNA or DNA may be expressed in vivo
to inhibit
production of the TR9 receptor.
In one embodiment, the TR9 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 TR9 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 TR9, 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
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126
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., Proc. Natl.
Aced. Sci.
U.S.A. 78:1441-1445 (1981), the regulatory sequences of the 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 TR9 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 TR9 antisense nucleic acids, a single strand of the
duplex DNA
may thus be tested, or triplex formation may be assayed. The ability to
hybridize will
depend on both the degree of complementarity and the length of the antisense
nucleic acid
Generally, the larger the hybridizing nucleic acid, the more base mismatches
with a TR9
RNA it may contain and still form a stable duplex (or triplex as the case may
be). One
skilled in the art can ascertain a tolerable degree of mismatch by use of
standard
procedures to determine the melting point of the hybridized complex.
Oligonucleotides that are complementary to the 5' end of the message, e.g.,
the 5'
untranslated sequence up to and including the AUG initiation codon, should
work most
efficiently at inhibiting translation. However, sequences complementary to the
3'
untranslated sequences of mRNAs have been shown to be effective at inhibiting
translation of mRNAs as well. See generally, Wagner, R., 1994, Nature 372:333-
335.
Thus, oligonucleotides complementary to either the 5'- or 3'- non- translated,
non-coding
regions of the TR9 shown in Figures lA-D could be used in an antisense
approach to
inhibit translation of endogenous TR9 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 TR9 mRNA, antisense
nucleic
acids should be at least six nucleotides in length, and are preferably
oligonucleotides
ranging from 6 to about 50 nucleotides in length. In specific aspects the
oligonucleotide
is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides
or at least 50
nucleotides.
The polynucleotides of the invention can be DNA or RNA or chimeric mixtures or
derivatives or modified versions thereof, single-stranded or double-stranded.
The
oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate
backbone,
for example, to improve stability of the molecule, hybridization, etc. The
oligonucleotide
may include other appended groups such as peptides (e.g., for targeting host
cell
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127
receptors in vivo), or agents facilitating transport across the cell membrane
(see, e.g.,
Letsinger et al., Proc. Natl. Acad. Sci. 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., BioTechniques 6:958-976 ( 1988)) or intercalating agents.
(See, e.g.,
Zon, Pharm. Res. 5:539-549 ( 1988)). To this end, the oligonucleotide rnay 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, 5-
fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-
acetylcytosine,
5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine,
inosine,
N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-
adenine,
7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-
D-mannosylqueosine, 5= methoxycarboxymethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,
pseudouracil, queosine, 2-thiocytosine, 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.
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 - anomeric
oligonucleotide. An alpha-anomeric oligonucleotide forms specific double-
stranded
hybrids with complementary RNA in which, contrary to the usual beta-units, the
strands
run parallel to each other (Gautier et al., Nucl. Acids Res. 15:6625-6641 (
1987)). The
oligonucleotide is a 2_-0-methylribonucleotide (moue et al., Nucl. Acids Res.
15:6131-
6148 (1987)), or a chimeric RNA-DNA analogue (moue et al., FEBS Lett. 215:327-
330
( 1987)).
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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 al. (Nucl. Acids
Res.
16:3209 ( 1988 ) ). methylphosphonate oligonucleotides can be prepared by use
of
controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci.
U.S.A.
85:7448-7451 ( 1988)), etc.
While antisense nucleotides complementary to the TR9 coding region sequence
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 TR9 mRl'~lAs,
the use of
hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at
locations
dictated by flanking regions that form complementary base pairs with the
target mRNA.
The sole requirement is that the target mRNA have the following sequence of
two bases:
5'-UG-3'. The construction and production of hammerhead ribozymes is well
known in
the art and is described more fully in Haseloff and Gerlach, Nature 334:585-
591 ( 1988).
There are numerous potential hammerhead ribozyme cleavage sites within the
nucleotide
sequence of TR9 (Figures lA-D). Preferably, the ribozyme is engineered so that
the
cleavage recognition site is located near the 5' end of the TR9 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 TR9 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, pol III or pol II promoter, so that transfected cells
will produce
sufficient quantities of the ribozyme to destroy endogenous TR9 messages and
inhibit
translation. Since ribozymes, unlike antisense molecules are catalytic. a
lower
intracellular concentration is required for efficiency.
Endogenous gene expression can also be reduced by inactivating or "knocking
out" the TR9 gene and/or its promoter using targeted homologous recombination.
(E.g.,
see Smithies et al., Nature 317:230-234 ( 1985); Thomas & Capecchi, Cell
51:503-512
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129
(1987); Thompson et al., Cell 5:313-321 (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
negative selectable
marker, to transfect cells that express polypeptides of the invention in 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 (e.g., see Thomas & Capecchi 1987 and Thompson 1989, 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 in 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.
Further antagonists according to the present invention include soluble forms
of
TR9, (e.g., fragments of the TR9 receptor sequence depicted in Figures lA-D
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
TR9 mediated signaling by competing with the cell surface TR9 for binding to
TNF-
family ligands. Thus, soluble forms of the receptor that include the ligand
binding
domain are novel cytokines capable of inhibiting apoptosis induced by TNF-
family
ligands. These are preferably expressed as dimers or trimers, since these have
been
shown to be superior to monomeric forms of soluble receptor as antagonists,
e.g.,
IgGFc-TNF receptor family fusions. Other such cytokines are known in the art
and
include Fas B (a soluble form of the mouse Fas receptor) that acts
physiologically to limit
apoptosis induced by Fas ligand (Hughes and Crispe, J. Exp. Med. 182:1395-1401
(1995)). 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 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-b), LT-b (found in complex heterotrimer LT-a2-b), Fast, 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),
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neutrokine-alpha (International Publication No. WO 98/18921), CD40L, CD27L;
CD30L, 4-1BBL, OX40L and nerve growth factor (NGF). The experiments set forth
in
Example 5 and 6, indicate that the TR9 receptor, like other homologous
proteins, is a
death domain-containing molecule capable of triggering apoptosis, which is
important in
the regulation of the immune system. In addition, the experiments set forth
below suggest
that TR9-induced apoptosis will be blocked by the inhibitors of ICE-like
proteases, CrmA
and z-VAD-fmk. Importantly, it is also expected that apoptosis induced by TR9
will be
blocked by dominant negative versions of FADD (FADD-DN) or FLICE (FLICE-
DN/MACHa1C360S), which were previously shown to inhibit death signaling by
Fas/APO-1 and TNFR-1. Thus, inhibitors of ICE-like proteases, FADD-DN and
FLICE-
DN/MACHa1C360S could also be used as antagonists for TR9 activity.
Antagonists of the present invention also include antibodies specific for TNF-
family ligands or the TR9 polypeptides of the invention. The term "antibody"
(Ab) or
"monoclonal antibody" (mAb) as used herein is meant to include intact
molecules as well
as fragments thereof (such as, e.g., Fab and F(ab'), fragments) which are
capable of
binding an antigen. Fab and F(ab')~ fragments lack the Fc fragment of intact
antibody,
clear more rapidly from the circulation, and may have less non-specific tissue
binding of
an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).
Antibodies according to the present invention may be prepared by any of a
variety
of standard methods using TR9 immunogens of the present invention. As
indicated, such
TR9 immunogens include the full-length (complete) TR9 polypeptide depicted in
Figures
lA-D (SEQ ID N0:2) (which may or may not include the leader sequence) and TR9
polypeptide fragments comprising, or alternatively consisting of, for example,
the ligand
binding domain, extracellular domain, transmembrane domain, intracellular
domain, death
domain, incomplete death domain, 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(7):4304-4307( 1992)); Tartaglia et al., Cell 73:213-216 (
1993)), and
PCT Application WO 94/09137 (the contents of each of these three publications
are herein
incorporated by reference in their entireties), and are preferably specific to
(i.e., bind
uniquely to polypeptides of the invention having the amino acid sequence of
SEQ ID
N0:2.
In a preferred method, antibodies according to the present invention are mAbs.
Such mAbs can be prepared using hybridoma technology (Kohler and Millstein,
Nature
256:495-497 (1975) and U.S. Patent No. 4,376,110; Harlow et al., Af~tibodies:
A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
131
1988; Monoclonal Antibodies and Hybridomas: A New Dimension in Biological
Analyses, Plenum Press, New York, NY, 1980; Campbell, "Monoclonal Antibody
Technology," In: Laboratory Tech~2iyares in Biochen~istm aml
MoleczrlcrrBiology,
Volume 13 (Burdon et al., eds.), Elsevier, Amsterdam (1984)).
Proteins and other compounds which bind the TR9 domains are also candidate
agonists and antagonists according to the present invention. Such binding
compounds
can be "captured" using the yeast two-hybrid system (Fields and Song, Nature
340:245-
246 (1989)). A modified version of the yeast two- hybrid system has been
described by
Roger Brent and his colleagues (Gyuris, Cel175:791-803 (1993); Zervos 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
domain,
extracellular, intracellular, transmembrane. and death domain of the TR9. Such
compounds are good candidate agonists and antagonists of the present
invention.
Using the two-hybrid assay described above, the intracellular domain of the
TR9
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 TR9 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 et
al., Cell 78:681 ( 1994). Such proteins and amino acid sequences which bind to
the
cytoplasmic domain of the TR9 receptors are good candidate agonist and
antagonist of the
presentinvention.
Other screening techniques include the use of cells which express the
polypeptide
of the present invention (for example, transfected CHO cells) in a system
which measures
extracellular pH changes caused by receptor activation, for example, as
described in
Science, 246:181-296 (1989). In another example, potential agonists or
antagonists may
be contacted with a cell which expresses the polypeptide of the present
invention and a
second messenger response, e.g., signal transduction may be measured to
determine
whether the potential antagonist or agonist is effective.
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 TR9 ligands including TRAIL, TNF-alpha,
lymphotoxin-
alpha (LT-alpha. also known as TNF-beta), LT-beta (found in complex
heterotrimer LT-
alpha2-beta), Fast, CD40, CD27, CD30, 4-1BB, OX40, and nerve growth factor
(NGF)
and other TNF ligand family members described herein.
Representative therapeutic applications of the present invention are discussed
in-
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132
more detail below. 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 10' cells (Wei et
al., Nature
373:117-122 (1995)). One cause of CD4+ T cell depletion in the setting of HIV
infection
is believed to be HIV-induced apoptosis. Indeed, HIV-induced apoptotic cell
death has
been demonstrated not only irZ vitro but also. more importantly, in infected
individuals
(Ameisen, J.C., AIDS 8:1197-1213 ( 1994): Finkel and Banda, Cetrr. Opin.
Immunol.
6:605-615(1995); Muro-Cacho et al., J. Innnmaol. 154:5555-5566 (1995)).
Furthermore, apoptosis and CD4+ T-lymphocyte depletion is tightly correlated
in different
animal models of AIDS (Brunner et al., Nature 373:441-444 (1995): Gougeon et
al.,
AIDS Res. Hum. Retrovirccses 9:553-563 (1993)) and, apoptosis is not observed
in those
animal models in which viral replication does not result in AIDS. Id. 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 et al., J. Virol. 70:199-206
(1996)).
Further, the TNF-family ligand was detectable in uninfected macrophages and
its
expression was upregulated following HIV infection resulting in selective
killing of
uninfected CD4 T-lymphocytes. Id. Thus, by the invention, a method for
treating,
preventing, diagnosing, and/or detecting HIV+ individuals is provided which
involves
administering an antagonist of the present invention to reduce selective
killing of CD4 T-
lymphocytes. Modes of administration and dosages are discussed in detail
below.
In rejection of an allograft, the immune system of the recipient animal has
not
previously been primed to respond because the immune system for the most part
is only
primed by environmental antigens. Tissues from other members of the same
species have
not been presented in the same way than, for example, viruses and bacteria
have been
presented. In the case of allograft rejection, immunosuppressive regimens are
designed to
prevent the immune system from reaching the effector stage. However, the
immune
profile of xenograft rejection may resemble disease recurrence more than
allograft
rejection. In the case of disease recurrence, the immune system has already
been
activated, as evidenced by destruction of the native islet cells. Therefore,
in disease
recurrence, the immune system is already at the effector stage. Agonists of
the present
invention are able to suppress the immune response to both allografts and
xenografts
because lymphocytes activated and differentiated into effector cells will
express the TR9
polypeptide, and thereby are susceptible to compounds which enhance apoptosis.
Thus,
the present invention further provides a method for creating immune privileged
tissues.
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133
TR9 antagonists of the invention can further be used in the treatment,
prevention,
diagnosis, and/or detection of inflammatory diseases and stress response
related diseases,
such as inflammatory bowel disease, rheumatoid arthritis, osteoarthritis,
psoriasis, and
septicemia.
In addition, due to lymphoblast expression of TR9, soluble TR9 agonist or
antagonist antibodies (e.g., mABs) may be used to treat, prevent, diagnose,
and/or detect
this form of cancer. Further. soluble TR9 or neutralizing mABs may be used to
treat,
prevent, diagnose, and/or detect various chronic and acute forms of
inflammation such as
rheumatoid arthritis, osteoarthritis, psoriasis, septicemia, and inflammatory
bowel
disease.
Polynucleotides and/or polypeptides of the invention, and/or agonists and/or
antagonists thereof, are useful in the diagnosis and treatment, prevention.
diagnosis,
and/or detection 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 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.), Helicobacter
pylori
infection, invasive Staphylococcia, etc.), parasitic infection, nephritis,
bone disease (e.g.,
osteoporosis), atherosclerosis, pain, cardiovascular disorders (e.g.,
neovascularization,
hypovascularization or reduced circulation (e.g., ischemic disease (e.g.,
myocardial
infarction, stroke, etc.)), AIDS, allergy, inflammation, neurodegenerative
disease (e.g.,
Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,
pigmentary
retinitis, cerebellar degeneration, etc.), graft rejection (acute and
chronic), graft vs. host
disease, diseases due to osteomyelodysplasia (e.g., aplastic anemia, etc.),
joint tissue
destruction in rheumatism, liver disease (e.g., acute and chronic hepatitis,
liver injury,
and cirrhosis), autoimmune disease (e.g., multiple sclerosis, rheumatoid
arthritis,
systemic lupus erythematosus, immune complex glomerulonephritis, 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.
CA 02365255 2001-09-24
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1 34
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).
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.
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,
prevention,
diagnosis, and/or detection of autoimmune disorders. In specific embodiments,
polynucleotides and/or polypeptides of the invention are used to treat,
prevent, diagnose,
and/or detect chronic inflammatory, allergic or autoimmune conditions, such as
those
described herein or are otherwise known in the art.
Modes of Admitzistratiora
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 TR9
mediated apoptosis. Of course, where it is desired for apoptosis to be
enhanced, an
agonist according to the present invention can be co-administered with a TNF-
family
ligand. One of ordinary skill will appreciate that effective amounts of an
agonist or
antagonist can be determined empirically and may be employed in pure form or
in
pharmaceutically acceptable salt, ester or prodrug form. The agonist or
antagonist may be
administered in compositions in combination with one or more pharmaceutically
acceptable excipients (i.e., carriers).
It will be understood that, when administered to a human patient, the total
daily
usage of the compounds and compositions of the present invention will be
decided by the
attending physician within the scope of sound medical judgment. The specific
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135
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 TR9
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 above, this will be
subject to
therapeutic discretion. More preferably, this dose is at least 0.01 mg/ka/day,
and most
preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If
given
continuously, the TR9 polypeptide is typically administered at a dose rate of
about 1
~,g/kg/hour to about 50 ~tg/kg/hour, either by 1-4 injections per day or by
continuous
subcutaneous infusions, for example, using a mini-pump. An intravenous bag
solution
may also be employed.
Dosaging may also be arranged in a patient specific manner to provide a
predetermined concentration of an agonist or antagonist in the blood, as
determined by the
RIA technique. Thus patient dosaging may be adjusted to achieve regular on-
going
trough blood levels, as measured by RIA, on the order of from 50 to 1000
ng/ml,
preferably 150 to 500 ng/ml.
Pharmaceutical compositions are provided comprising an agonist (including TR9
receptor polynucleotides, polypeptides or antibodies of the invention) or
agonist (e.g.,
TR9 polynucleotides, polypeptides of the invention or antibodies thereto) of
TR9 and a
pharmaceutically acceptable carrier or excipient, which may be administered
orally,
rectally, parenterally, intracistemally, intravaginally, intraperitoneally,
topically (as by
powders, ointments, drops or transdermal patch), bucally, or as an oral or
nasal spray,
In one embodiment "pharmaceutically acceptable carrier" means a non-toxic
solid,
semisolid or liquid filler, diluent, encapsulating material or formulation
auxiliary of any
type. In a specific embodiment, "pharmaceutically acceptable" means approved
by a
regulatory agency of the federal or a state government or listed in the U.S.
Pharmacopeia
or other generally recognized pharmacopeia for use in animals, and more
particularly
humans. Nonlimiting examples of suitable pharmaceutical carriers according to
this
embodiment are provided in "Remington's Pharmaceutical Sciences" by E.W.
Martin,
and include sterile liquids, such as water and oils, including those of
petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and
the like. Water is a preferred carrier when the pharmaceutical composition is
administered
intravenously. Saline solutions and aqueous dextrose and glycerol solutions
can be
employed as liquid carriers, particularly for injectable solutions.
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136
The term "parenteral" as used herein refers to modes of administration which
include intravenous, intramuscular, intraperitoneal, intrasternal,
subcutaneous and
intraarticular injection and infusion.
Pharmaceutical compositions of the present invention for parenteral injection
can
comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions as well as sterile powders for
reconstitution into
sterile injectable solutions or dispersions just prior to use.
In addition to soluble TR9 polypeptides, TR9 polypeptides containing the
transmembrane region can also be used when appropriately solubilized by
including
detergents, such as CHAPS or NP-40, with buffer.
The compositions of the invention may be administered alone or in combination
with other therapeutic agents. Therapeutic agents that may be administered in
combination with the compositions of the invention, include but not limited
to, other
members of the TNF family, chemotherapeutic agents, antibiotics, steroidal and
non-
steroidal anti-inflammatories, conventional immunotherapeutic agents,
cytokines and/or
growth factors. Combinations may be administered either concomitantly, e.g.,
as an
admixture, separately but simultaneously or concurrently; or sequentially.
This includes
presentations in which the combined agents are administered together as a
therapeutic
mixture, and also procedures in which the combined agents are administered
separately
but simultaneously, e.g., as through separate intravenous lines into the same
individual.
Administration "in combination" further includes the separate administration
of one of the
compounds or agents given first, followed by the second.
In one embodiment, the compositions of the invention are administered in
combination with other members of the TNF family. TNF, TNF-related or TNF-like
molecules that may be administered with the compositions of the invention
include, but
are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha,
also
known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta),
OPGL,
Fast, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International
Publication No. WO 96/14328), AIM-I (International Publication No. WO
97/33899),
AIM-II ((International Publication No. WO 97/34911), endokine-alpha
(International
Publication No. WO 98/07880), TR6 (International Publication No. WO 98/30694),
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,
TR2 (International Publication No. WO 96/34095), DR3 (International
Publication No.
WO 97/33904), DR4 (International Publication No. WO 98/32856), TRS
(International
Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694),
TR7
(International Publication No. WO 98/41629), TRANK, TR9 (International
Publication
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137
No. WO 98/56892), TR10 (International Publication No. WO 98/54202). 31X2
(International Publication No. WO 98/06842 ), and TR 11. TR 11 S V 1. TR 11 S
V2, TR 12.
and soluble forms CD 154, CD70, and CD 1 ~3.
In a preferred embodiment, compositions of the invention are administered in
combination with endokine-alpha.
In a preferred embodiment, the compositions of the invention are administered
in
combination with CD401igand (CD40L), a soluble form of CD40L (e.g.,
AVRENDT"").
bioloigically 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).
Conventional nonspecific immunosuppressive agents, that may be administered
in combination with the compositions of the invention include, but are not
limited to.
steroids, cyclosporine, cyclosporine analogs, cyclophosphamide
methylprednisone,
prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other
immunosuppressive
agents that act by suppressing the function of responding T cells.
In a further embodiment, the compositions of the invention are administered in
combination with an antibiotic agent. Antibiotic agents that may be
administered with the
compositions of the invention include, but are not limited to, tetracycline,
metronidazole,
amoxicillin, beta-lactamases, aminoglycosides, macrolides, quinolones,
fluoroquinolones, cephalosporins, erythromycin, ciprofloxacin, and
streptomycin.
In certain embodiments, compositions of the invention are administered in
combination with antiretroviral agents, nucleoside reverse transcriptase
inhibitors, non-
nucleoside reverse transcriptase inhibitors, and/or protease inhibitors.
Nucleoside reverse
transcriptase inhibitors that may be administered in combination with the
compositions of
the invention, include, but are not limited to, RETROVIRT"~ (zidovudine/AZT),
VIDEXT"~
(didanosine/ddI), HIVIDT"" (zalcitabine/ddC), ZERITT"" (stavudine/d4T),
EPIVIRT""
(lamivudine/3TC), and 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, VIRAMUNET"~ (nevirapine),
RESCRIPTORTM (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
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138
agents, nucleoside reverse transcriptase inhibitors. non-nucleoside reverse
transcciptase
inhibitors, and/or protease inhibitors may be used in any combination with
compositions
of the invention to treat AIDS and/or to prevent or treat HIV infection.
In other embodiments, compositions of the 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-SLLFAMETHOXAZOLET"", DAPSONET"~,
PENTAMIDINET"~, ATOVAQUONET"", ISONIAZIDT"~, RIFAMPINT"~
PYRAZINAMIDET"~, ETHAMBUTOLT"~, RIFABUTINT"~, CLARITHROMYCINT"~,
AZITHROMYCINTM, GANCICLOVIRT"", FOSCARNETTM, CIDOFOVIRT"~,
FLUCONAZOLET"~, ITRACONAZOLET"", KETOCONAZOLET"~, ACYCLOVIRT"~,
FAMCICOLVIRT"~, PYRIMETHAMINET"", LEUCOVORINT"", NEUPOGENTM
(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"", PENTAMIDINET"",
and/or ATOVAQUONET"" to prophylactically treat or prevent an opportunistic
Pneumocystis carinii pneumonia infection. In another specific embodiment,
compositions
of the invention are used in any combination with ISONIAZIDT"~, RIFAMPINT"",
PYRAZINAMIDETM, and/or ETHAMBUTOLT"" to prophylactically treat or prevent an
opportunistic Mycobacterium aviurn complex infection. In another specific
embodiment,
compositions of the invention are used in any combination with RIFABUTINT"~,
CLARITHROMYCINT"", and/or AZITHROMYCINTM to prophylactically treat or prevent
an opportunistic Mycobacterium tuberculosis infection. In another specific
embodiment,
compositions of the invention are used in any combination with GANCICLOVIRT"~,
FOSCARNETT"", and/or CIDOFOVIRT"~ to prophylactically treat or prevent 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 or prevent
an
opportunistic fungal infection. In another specific embodiment, compositions
of the
CA 02365255 2001-09-24
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I p9
invention are used in any combination with ACYCLOVIRT"" and/or FAMCICOLVIRT""
to
prophylactically treat or prevent an opportunistic herpes simplex virus type I
and/or type
II infection. In another specific embodiment, compositions of the invention
are used in
any combination with PYRIMETHAMINET"~ and/or LEUCOVORINT"" to prophylactically
treat or prevent an opportunistic Toxoplnsmn go~zdii infection. In another
specific
embodiment. compositions of the invention are used in any combination with
LEUCOVORINr"~ and/or NEUPOGENT"" to prophylactically treat or prevent an
opportunistic bacterial infection.
In a further embodiment, the compositions of the invention are administered in
combination with an antiviral agent. Antiviral agents that may be administered
with the
compositions of the invention include. but are not limited to, acyclovir,
ribavirin,
amantadine, and remantidine.
In a further embodiment, the compositions of the invention are administered in
combination with an antibiotic agent. Antibiotic agents that may be
administered with the
compositions of the invention include, but are not limited to, amoxicillin,
aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin,
chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin,
fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,
rifampin,
streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-
sulfamthoxazole,
and vancomycin.
Conventional nonspecific immunosuppressive agents, that may be administered in
combination with the compositions of the invention include, but are not
limited to,
steroids, cyclosporine, cyclosporine analogs, cyclophosphamide
methylprednisone,
prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other
immunosuppressive
agents that act by suppressing the function of responding T cells.
Additionally, immunosuppressants preparations that may be administered with
the
compositions of the invention include, but are not limited to, ORTHOCLONET"~
(OKT3),
SANI~IMMUNET"~/NEORALT"~/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,
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140
prednisone, and methylprednisolone (e.g., IV methylprednisolone). In a
specific
embodiment, compositions of the invention are administered in combination with
prednisone. In a further specific embodiment, the compositions of the
invention are
administered in combination with prednisone and an immunosuppressive agent.
Immunosuppressive agents that may be administered with the compositions of the
invention and prednisone are those described herein. and include, but are not
limited to,
azathioprine, cylophosphamide, and cyclophosphamide IV. In a another specific
embodiment, compositions of the invention are administered in combination with
methylprednisolone. In a further specific embodiment, the compositions of the
invention
are administered in combination with methylprednisolone and an
immunosuppressive
agent. Immunosuppressive agents that may be administered with the compositions
of the
invention and methylprednisolone are those described herein, and include, but
are not
limited to, azathioprine, cylophosphamide, and cyclophosphamide IV.
In 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 (Dong Wha),
darbufelone
mesylate (Warner-Lambert), soluble TNF receptor 1 (synergen; Amgen), IPR-6001
(Institute for Pharmaceutical Research), trocade (Hoffman-La Roche), EF-5
(Scotia
Pharmaceuticals), BIIL-284 (Boehringer Ingelheim), BIIF-1149 (Boehringer
Ingelheim),
LeukoVax (Inflammatics), MK-663 (Merck), ST-1482 (Sigma-Tau), and butixocort
propionate (WarnerLambert).
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),
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1=11
eyclophosphamide, chlorambucil, gold. ENBRELT"" (Etanercept), anti-TNF
antibody, and
prednisolone.
In a more preferred embodiment, the compositions of the invention we
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 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 ENBRELTM, methotrexate and suflasalazine. In other embodiments, one or
mere
antimalarials is combined with one of the above-recited combinations. In a
specfic
embodiment, the compositions of the invention are administered in combination
with an
antimalarial (e.g., hydroxychloroquine), ENBRELT"", methotrexate and
suflasalazine. In
another specfic 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, GAMMART"~,
IVEEGAMT"~,
SANDOGLOBULINT"~, GAMMAGARD S/DT"", and GAMIMUNETM. In a specific
embodiment, compositions of the invention are administered in combination with
intravenous immune globulin preparations in transplantation therapy (e.g.,
bone marrow
transplant).
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1 X12
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 an additional embodiment, the compositions of the invention are
administered
alone or in combination with an anti-inflammatory agent. Anti-inflammatory
agents that
may be administered with the compositions of the invention include, but are
not limited
to, glucocorticoids and the nonsteroidal anti-inflammatories,
aminoarylcarboxylic acid
derivatives, arylacetic acid derivatives, arylbutyric acid derivatives,
arylcarboxylic acids,
arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid
derivatives,
thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-
hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome,
difenpiramide,
ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol,
paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.
In another embodiment, compostions of the invention are administered in
combination with a chemotherapeutic agent. Chemotherapeutic agents that may be
administered with the compositions of the invention include, but are not
limited to,
antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and
dactinomycin);
antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU,
methotrexate,
floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine,
and 6-
thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU,
cytosine
arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine,
mitomycin,
busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,
medroxyprogesterone,
estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol
acetate,
methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and
testolactone);
nitrogen mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine
(nitrogen
mustard) and thiotepa); steroids and combinations (e.g., bethamethasone sodium
phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine
sulfate,
vinblastine sulfate, and etoposide).
In 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, IL2, IL3, IL4,
ILS, IL6,
IL7, IL10, IL12, IL13, ILLS, anti-CD40, CD40L, IFN-gamma and TNF-alpha.
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
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143
Growth Factor (GDGF), as disclosed in European Patent Number EP-399816;
Platelet
Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-
682110; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European
Patent
Number EP-282317; Placental Growth Factor (P1GF), as disclosed in
International
Publication Number WO 92/06194; Placental Growth Factor-2 (PIGF-2), as
disclosed in
Hauser et al., Gorwth Factors, 4:259-268 ( 1993); Vascular Endothelial Growth
Factor
(VEGF), as disclosed in International Publication Number WO 90/13649; Vascular
Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number
EP-
506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in
International
Publication Number WO 96/39515; Vascular Endothelial Growth Factor B-186 (VEGF-
B 186), 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 DE 19639601. 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 tha
may be
administered with the compositions of the invention include, but are not
limited to, FGF-
l, 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.
Chromosome Assays
The nucleic acid molecules of the present invention are also valuable for
chromosome identification. The sequence is specifically targeted to and can
hybridize
with a particular location on an individual human chromosome. The mapping of
DNAs to
chromosomes according to the present invention is an important first step in
correlating
those sequences with genes associated with disease.
In certain preferred embodiments in this regard, the cDNA herein disclosed is
used to clone genomic DNA of a TR9 receptor gene. This can be accomplished
using a
variety of well known techniques and libraries, which generally are available
commercially. The genomic DNA then is used for in situ chromosome mapping
using
well known techniques for this purpose.
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144
In addition, in some cases, sequences can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis
of the
3. untranslated region of the gene is used to rapidly select primers that do
not span more
than one exon in the genomic DNA, thus complicating the amplification process.
These
primers are then used for PCR screening of somatic cell hybrids containing
individual
human chromosomes.
Fluorescence in .sitic hybridization ("FISH") of a cDNA clone to a metaphase
chromosomal spread can be used to provide a precise chromosomal location in
one step.
This technique can be used with probes from the cDNA as short as 50 or 60 bp.
For a
review of this technique, see Verma et al., Human Chromosomes: A Manccal Of
Basic
Techniques, Pergamon Press, N.Y. ( 1988).
Once a sequence has been mapped to a precise chromosomal location, the
physical
position of the sequence on the chromosome can be correlated with genetic map
data.
Such data are found, for example, in V. McKusick, Mendelian Inheritance In
Man,
available on-line through Johns Hopkins University, Welch Medical Library. The
relationship between genes and diseases that have been mapped to the same
chromosomal
region are then identified through linkage analysis (coinheritance of
physically adjacent
genes).
Next, it is necessary to determine the differences in the cDNA or genomic
sequence between affected and unaffected individuals. If a mutation is
observed in some
or all of the affected individuals but not in any normal individuals, then the
mutation is
likely to be the causative agent of the disease.
Having generally described the invention, the same will be more readily
understood by reference to the following examples, which are provided by way
of
illustration and are not intended as limiting.
Examples
Example l: Expression and Purification of the TR9 Receptor in E. coli
The bacterial expression vector pQE60 is used for bacterial expression in this
example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311 ). pQE60
encodes ampieillin antibiotic resistance ("Amp"') and contains a bacterial
origin of
replication ("ori"), an IPTG inducible promoter, a ribosome binding site
("RBS"), six
codons encoding histidine residues that allow affinity purification using
nickel-nitrilo-tri-
acetic acid ("Ni-NTA") affinity resin sold by QIAGEN, Inc., supra, and
suitable single
restriction enzyme cleavage sites. These elements are arranged such that a DNA
fragment
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145
encoding a polypeptide may be inserted in such as way as to produce that
polypeptide
with the six His residues (i.e.. a "6 X His tag") covalently linked to the
carboxyl terminus
of that polypeptide. However, in this example, the polypeptide coding sequence
is
inserted such that translation of the six His codons is prevented and,
therefore, the
polypeptide is produced with no 6 X His tag.
The DNA sequence encoding the desired portion of the TR9 receptor protein
lacking the hydrophobic leader sequence is amplified from the deposited cDNA
clone
using PCR oligonucleotide primers which anneal to the amino terminal sequences
of the
desired portion of the TR9 receptor protein and to sequences in the deposited
construct 3'
to the cDNA coding sequence. Additional nucleotides containing restriction
sites to
facilitate cloning in the pQE60 vector are added to the 5' and 3' sequences,
respectively.
For cloning the mature protein, the 5' primer has the sequence:
5'-CGC CCA TGG CTC AGC CAG AAC AGA AG-3' (SEQ ID NO:11) containing the
underlined NcoI restriction site followed by 17 nucleotides complementary to
the amino
terminal coding sequence of the mature TR9 receptor sequence in Figures lA-D.
One of
ordinary skill in the art would appreciate, of course, that the point in the
protein coding
sequence where the 5' primer begins may be varied to amplify a desired portion
of the
complete protein shorter or longer than the mature form. The 3' primer has the
sequence:
5'-CGC AAG CTT TTA GGG CAA ATG CTC ATT G-3' (SEQ ID N0:12) containing
the underlined HindIII restriction site followed by 19 nucleotides
complementary to the 3'
end of the non-coding sequence in the TR9 receptor DNA sequence in Figures 1 A-
D.
The amplified TR9 receptor DNA fragments and the vector pQE60 are digested
with NcoI and HindIII, and the digested DNAs are then ligated together.
Insertion of the
TR9 receptor DNA into the restricted pQE60 vector places the TR9 receptor
protein
coding region including its associated stop codon downstream from the IPTG-
inducible
promoter and in-frame with an initiating AUG. The associated stop codon
prevents
translation of the six histidine codons downstream of the insertion point.
The ligation mixture is transformed into competent E. coli cells using
standard
procedures such as those described in Sambrook et al., Molecular Cloning: a
Laboratory
Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989). E. coli strain M15/rep4, containing multiple copies of the plasmid
pREP4, which
expresses the lac repressor and confers kanamycin resistance ("Kan"'), is used
in carrying
out the illustrative example described herein. This strain, which is only one
of many that
are suitable for expressing TR9 receptor protein, is available commercially
from
~5 QIAGEN, Inc., supra. Transformants are identified by their ability to grow
on LB plates
in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from
resistant
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146
colonies and the identity of the cloned DNA confirmed by restl-iction
analysis, PCR and
DNA sequencing.
Clones containing the desired constructs are grown overnight ("O/N") in liquid
culture in LB media supplemented with both ampicillin (100 ~g/ml) and
kanamycin (25
p,g/ml). The O~ culture is used to inoculate a large culture, at a dilution of
approximately
1:25 to 1:250. The cells are grown to an optical density at 600 nm ("OD600")
of between
0.4 and 0.6. Isopropyl-b-D-thiogalactopyranoside ("IPTG") is then added to a
final
concentration of 1 mM to induce transcription from the lac repressor sensitive
promoter,
by inactivating the lacI repressor. Cells subsequently are incubated further
for 3 to 4
hours. Cells then are harvested by centrifugation.
The cells are then stirred for 3-4 hours at 4°C in 6M guanidine-HCI,
pHB. The
cell debris is removed by centrifugation, and the supernatant containing the
TR9 receptor
is dialyzed against 50 mM Na-acetate buffer pH6, supplemented with 200 mM
NaCI.
Alternatively, the protein can be successfully refolded by dialyzing it
against 500 mM
NaCI, 20% glycerol, 25 mM Tris/HCl pH7.4, containing protease inhibitors.
After
renaturation the protein can be purified by ion exchange, hydrophobic
interaction and size
exclusion chromatography. Alternatively, an affinity chromatography step such
as an
antibody column can be used to obtain pure TR9 receptor protein. The purified
protein is
stored at 4°C or frozen at -80°C .
Example 2: Cloning and Expression of the TR9 Receptor Protein in a
Baculovirus Expression System
In this illustrative example, the plasmid shuttle vector pA2 is used to insert
the
cloned DNA encoding the complete protein, including its naturally associated
secretary
signal (leader) sequence, into a baculovirus to express the mature TR9
receptor protein,
using standard methods as described in Summers et al., A Manual of Methods for
Baculovirus Vectors and Insect Cell Cultc~re Procedures, Texas Agricultural
Experimental
Station Bulletin No. 1555 ( 1987). This expression vector contains the strong
polyhedrin
promoter of the Autographa califorizica nuclear polyhedrosis virus (AcMNPV)
followed
by convenient restriction sites such as BawHI and Asp718. The polyadenylation
site of
the simian virus 40 ("SV40") is used for efficient polyadenylation. For easy
selection of
recombinant virus, the plasmid contains the beta-galactosidase gene from E.
coli under
control of a weak Drosophila promoter in the same orientation, followed by the
polyadenylation signal of the polyhedrin gene. The inserted genes are flanked
on both
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147
sides by viral sequences for cell-mediated homologous recombination with wild-
type viral
DNA to generate viable virus that express the cloned polynucleotide.
Many other baculovirus vectors could be used in place of the vector above,
such
as pAc373, pVL941 and pAcIMI, as one skilled in the art would readily
appreciate, as
long as the construct provides appropriately located signals for
transcription, translation,
secretion and the like, including a signal peptide and an in-frame AUG as
required. Such
vectors are described, for instance, in Luckow et al., Virology 170:31-39
(1989).
The cDNA sequence encoding the full length TR9 protein in the deposited clone,
including the AUG initiation codon and the naturally associated leader
sequence shown in
Figures lA-D (SEQ ID N0:2), is amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' sequences of the gene. The 5' primer has the
sequence:
5'-CGC CCC GGG GCC ATC ATG GGG ACC TCT CCG AGC-3' (SEQ ID N0:13)
containing the underlined SmaI restriction enzyme site, an efficient signal
for initiation of
translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol.
196:947-950
( 1987), followed by a number of bases of the sequence of the complete TR9
receptor
protein shown in Figures 1 A-D, beginning with the AUG initiation codon.
The 3' primer (for cloning the soluble form) has the sequence: 5'-CGC GGT
ACC TTA GGG CAA ATG CTC ATT G-3' (SEQ ID N0:14) containing the underlined
Asp718 restriction site followed by nucleotides complementary to the 3'
noncoding
sequence in Figures lA-D.
The amplified fragment is isolated from a 1 % agarose gel using a commercially
available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment then is
digested
with SmaI and Asp718 and again is purified on a 1 % agarose gel. This fragment
is
designated herein "F 1 ".
The plasmid is digested with the restriction enzymes SmaI and Asp718 and
optionally, can be dephosphorylated using calf intestinal phosphatase, using
routine
procedures known in the art. The DNA is then isolated from a 1 % agarose gel
using a
commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This
vector DNA is
designated herein "V 1 ".
Fragment Fl and the dephosphorylated plasmid V 1 are ligated together with T4
DNA ligase. E. coli HB 101 or other suitable E. coli hosts such as XL-1 Blue
(Stratagene
Cloning Systems, La Jolla, CA) cells are transformed with the ligation mixture
and spread
on culture plates. Bacteria are identified that contain the plasmid with the
human TR9
receptor gene using the PCR method, in which one of the primers that is used
to amplify
the gene and the second primer is from well within the vector so that only
those bacterial
colonies containing the TR9 receptor gene fragment will show amplification of
the DNA.
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The sequence of the cloned fragment is confirmed by DNA sequencing. This
plasmid is
designated herein pBacTR9.
Five ~g of the plasmid pBacTR9 are co-transfected with 1.0 ~tg of a
commercially
available linearized baculovirus DNA ("BaculoGoIdTM baculovirus DNA",
Pharmingen,
San Diego, CA.), using the lipofection method described by Felgner et al.,
Proc. Natl.
Acnd. Sci. USA 84:7413-7417 (1987). One ~g of BaculoGoldTM virus DNA and 5 ~g
of
the plasmid pBacTR9 are mixed in a sterile well of a microtiter plate
containing 50 ~1 of
serum-free Grace's medium (Life Technologies Inc., Rockville, MD). Afterwards,
10 ~l
Lipofectin plus 90 ~1 Grace's medium are added, mixed and incubated for 15
minutes at
room temperature. Then the transfection mixture is added drop-wise to Sf9
insect cells
(ATCC CRL 1711 ) seeded in a 35 mm tissue culture plate with 1 ml Grace's
medium
without serum. The plate is rocked back and forth to mix the newly added
solution. The
plate is then incubated for 5 hours at 27°C. After 5 hours the
transfection solution is
removed from the plate and 1 ml of Grace's insect medium supplemented with 10%
fetal
calf serum is added. The plate is put back into an incubator and cultivation
is continued at
27°C for four days.
After four days the supernatant is collected and a plaque assay is performed,
as
described by Summers and Smith, supra. An agarose gel with "Blue Gal" (Life
Technologies Inc., Rockville, MD.) is used to allow easy identification and
isolation of
gal-expressing clones, which produce blue-stained plaques. (A detailed
description of a
"plaque assay" of this type can also be found in the user's guide for insect
cell culture and
baculovirology distributed by Life Technologies Inc., Rockville, MD., page 9-
10). After
appropriate incubation, blue stained plaques are picked with the tip of a
micropipettor
(e.g., Eppendorf). The agar containing the recombinant viruses is then
resuspended in a
microcentrifuge tube containing 200 ~tl of Grace's medium and the suspension
containing
the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm
dishes. Four
days later the supernatants of these culture dishes are harvested and then
they are stored at
4°C . The recombinant virus is called V-TR9.
To verify the expression of the V-TR9 gene, Sf9 cells are grown in Grace's
medium supplemented with 10% heat inactivated FBS. The cells are infected with
the
recombinant baculovirus V-TR9 at a multiplicity of infection ("MOI") of about
2. Six
hours later the medium is removed and is replaced with SF900 II medium minus
methionine and cysteine (available from Life Technologies Inc., Rockville,
MD). If
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149
radiolabeled proteins are desired, 42 hours later, 5 ~tCi of 'SS-methionine
and 5 ~tCi ';S-
cysteine (available from Amersham) are added. The cells are further incubated
for 16
hours and then they are harvested by centrifugation. The proteins in the
supernatant as
well as the intracellular proteins are analyzed by SDS-PAGE followed by
autoradiography
(if radiolabeled). Microsequencing of the amino acid sequence of the amino
terminus of
purified protein may be used to determine the amino terminal sequence of the
mature
protein and thus the cleavage point and length of the secretory signal
peptide.
Example 3: Clozzing artd Expression of TR9 in Mammalian Cells
A typical mammalian expression vector contains the promoter element, which
mediates the initiation of transcription of mRNA, the protein coding sequence,
and signals
required for the termination of transcription and polyadenylation of the
transcript.
Additional elements include enhancers, Kozak sequences and intervening
sequences
flanked by donor and acceptor sites for RNA splicing. Highly efficient
transcription can
be achieved with the early and late promoters from SV40, the long terminal
repeats
(LTRS) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the
cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the
human
actin promoter). Suitable expression vectors for use in practicing the present
invention
include, for example, vectors such as PSVL and PMSG (Pharmacia, Uppsala,
Sweden),
pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBCI2MI (ATCC 67109).
Mammalian host cells that could be used include, human Hela 293, H9 and Jurkat
cells,
mouse NIH3T3 and C127 cells, Cos l, Cos 7 and CV 1, quail QC1-3 cells, mouse L
cells and Chinese hamster ovary (CHO) cells.
Alternatively, the gene can be expressed in stable cell lines that contain the
gene
integrated into a chromosome. The co-transfection with a selectable marker
such as dhfr,
gpt, neomycin, or hygromycin allows the identification and isolation of the
transfected
cells.
The transfected gene can also be amplified to express large amounts of the
encoded protein. The dihydrofolate reductase (DHFR) marker is useful to
develop cell
lines that carry several hundred or even several thousand copies of the gene
of interest.
Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy
et al.,
Biochem J. 227:277-279 ( 1991 ); Bebbington et al., BiolTechnology 10:169-175
( 1992)).
Using these markers, the mammalian cells are grown in selective medium and the
cells
with the highest resistance are selected. These cell lines contain the
amplified genes)
integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are
often
used for the production of proteins.
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150
The expression vectors pCl and pC4 contain the strong promoter (LTR) of the
Rous Sarcoma Virus (Cullen et al., Molec. Cell. Binl. 5:438-447 ( 1985)) plus
a fragment
of the CMV-enhances (Boshart et al., Cel141:521-530 (1985)). Multiple cloning
sites,
e.g., with the restriction enzyme cleavage sites BafnHI, XbaI and Asp718,
facilitate the
cloning of the gene of interest. The vectors contain in addition the 3'
intron, the
polyadenylation and termination signal of the rat preproinsulin gene.
Example 3(a): Cloning and Expression in COS Cells
The expression plasmid, pTR9-HA, is made by cloning a cDNA encoding TR9
into the expression vector pcDNAI/Amp or pcDNAIII (which can be obtained from
Invitrogen, Inc.).
The expression vector pcDNAI/Amp contains: (1) an E. coli origin of
replication
effective for propagation in E. coli and other prokaryotic cells; (2) an
ampicillin resistance
gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin
of
replication for propagation in eukaryotic cells; (4) a CMV promoter, a
polylinker, an
SV40 intron; (5) several codons encoding a hemagglutinin fragment (i.e., an
"HA" tag to
facilitate purification) followed by a termination codon and polyadenylation
signal
arranged so that a cDNA can be conveniently placed under expression control of
the CMV
promoter and operably linked to the SV40 intron and the polyadenylation signal
by means
of restriction sites in the polylinker. The HA tag corresponds to an epitope
derived from
the influenza hemagglutinin protein described by Wilson et al., Cell 37: 767-
778 ( 1984).
The fusion of the HA tag to the target protein allows easy detection and
recovery of the
recombinant protein with an antibody that recognizes the HA epitope. pcDNAIII
contains,
in addition, the selectable neomycin marker.
A DNA fragment encoding the TR9 is cloned into the polylinker region of the
vector so that recombinant protein expression is directed by the CMV promoter.
The
plasmid construction strategy is as follows. The TR9 cDNA of the deposited
clone is
amplified using primers that contain convenient restriction sites, much as
described above
for construction of vectors for expression of TR9 in E. coli. Suitable primers
include the
following, which are used in this example.
The 5' primer, containing the underlined SmaI site, a Kozak sequence, an AUG
start codon and codons of the 5' coding region of the complete TR9 receptor
has the
following sequence: 5'-CGC CCC GGG GCC ATC ATG GGG ACC TCT CCG AGC-
3' (SEQ ID N0:13).
The 3' primer, containing the underlined XbaI site, a stop codon, and
nucleotides
of the 3' coding sequence, has the following sequence (at the 3' end): 5'-CGC
TCT AGA
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TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA GGG CAA ATG CTC ATT G-
3' (SEQ ID N0:15).
The PCR amplified DNA fragment and the vector. pcDNAI/Amp. are digested
with SmaI and XbaI and then ligated. The ligation mixture is transformed into
E. coli
strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey
Pines
Road, La Jolla, CA 92037), and the transformed culture is plated on ampicillin
media
plates which then are incubated to allow growth of ampicillin resistant
colonies. Plasmid
DNA is isolated from resistant colonies and examined by restriction analysis
or other
means for the presence of the TR9-encoding fragment.
For expression of recombinant TR9, COS cells are transfected with an
expression
vector, as described above, using DEAE-DEXTRAN, as described, for instance, in
Sambrook et ul., Molecular- Cloning: a Laboratory Manual, Cold Spring
Laboratory
Press, Cold Spring Harbor, N.Y. (1989). Cells are incubated under conditions
for
expression of TR9 by the vector.
Expression of the TR9-HA fusion protein is detected by radiolabeling and
immunoprecipitation, using methods described in, for example Harlow et al.,
Antibodies:
A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. ( 1988). To this end, two days after transfection, the cells are
labeled by
incubation in media containing ~5S-cysteine for 8 hours. The cells and the
media are
collected, and the cells are washed and lysed with detergent-containing RIPA
buffer: 150
mM NaCI, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH 7.5. as described by
Wilson et al. cited above. Proteins are precipitated from the cell lysate and
from the
culture media using an HA-specific monoclonal antibody. The precipitated
proteins then
are analyzed by SDS-PAGE and autoradiography. An expression product of the
expected
size is seen in the cell lysate, which is not seen in negative controls.
Example 3(b): Cloning and Expression in CHO Cells
The vector pC4 is used for the expression of TR9 protein. Plasmid pC4 is a
derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). The plasmid
contains the mouse DHFR gene under control of the SV40 early promoter. Chinese
hamster ovary- or other cells lacking dihydrofolate activity that are
transfected with these
plasmids can be selected by growing the cells in a selective medium (alpha
minus MEM,
Life Technologies, Rockville, MD) supplemented with the chemotherapeutic agent
methotrexate. The amplification of the DHFR genes in cells resistant to
methotrexate
(MTX) has been well documented (see, e.g., Alt et al., J. Biol. C72em.
253:1357-1370
(1978); Hamlin et. al., Biochem. et Biophys. Acta, 1097:107-143 (1990); and
Page et.
al., Biotechnology 9:64-68 ( 1991 )). Cells grown in increasing concentrations
of MTX
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develop resistance to the drug by overproducing the target enzyme, DHFR, as a
result of
amplification of the DHFR gene. If a second gene is linked to the DHFR gene,
it is
usually co-amplified and over-expressed. It is known in the art that this
approach may be
used to develop cell lines carrying more than 1,000 copies of the amplified
gene(s).
Subsequently, when the methotrexate is withdrawn, cell lines are obtained
which contain
the amplified gene integrated into one or more chromosomes) of the host cell.
Plasmid pC4 contains for expressing the gene of interest the strong promoter
of
the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen et al.,
Molec. Cell.
Biol. 5:438-447 ( 1985)) plus a fragment isolated from the enhancer of the
immediate early
gene of human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-X30 ( 1985)).
Downstream of the promoter are BamHI, XbaI, and Asp718 restriction enzyme
cleavage
sites that allow integration of the genes. Behind these cloning sites the
plasmid contains
the 3' intron and polyadenylation site of the rat preproinsulin gene. Other
high efficiency
promoters can also be used for the expression, e.g., the human (3-actin
promoter, the
SV40 early or late promoters or the long terminal repeats from other
retroviruses, e.g.,
HIV and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems and
similar
systems can be used to express the TR9 in a regulated way in mammalian cells
(Gossen
et. al., Proc. Natl. Acad. Sci. USA 89:5547-5551 ( 1992). For the
polyadenylation of the
mRNA other signals, e.g., from the human growth hormone or globin genes can be
used
as well. Stable cell lines carrying a gene of interest integrated into the
chromosomes can
also be selected upon co-transfection with a selectable marker such as gpt,
6418 or
hygromycin. It is advantageous to use more than one selectable marker in the
beginning,
e.g., G418 plus methotrexate.
The plasmid pC4 is digested with the restriction enzymes SmaI and Asp718 and
then dephosphorylated using calf intestinal phosphatase by procedures known in
the art.
The vector is then isolated from a 1 % agarose gel.
The DNA sequence encoding the complete TR9 protein including its leader
sequence is amplified using PCR oligonucleotide primers corresponding to the
5' and 3'
sequences of the gene.
The 5' primer has the sequence: 5'-CGC CCC GGG GCC ATC ATG GGG ACC
TCT CCG AGC-3' (SEQ ID N0:13) restriction enzyme site, an efficient signal for
initiation of translation in eukaryotic cells, as described by Kozak, M., J.
Mol. Biol.
196:947-950 ( 1987), followed by a number of bases of the coding sequence of
the TR9
receptor protein shown in Figures 1 A-D (SEQ ID NO:1 ).
The 3' primer (for cloning the soluble form) has the sequence: 5'-CGC GGT
ACC TTA GGG CAA ATG CTC ATT G-3' (SEQ ID N0:14) containing the underlined
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Asp718 restriction site followed by nucleotides complementary to the non-
translated
region of the TR9 receptor gene shown in Figures 1 A-D (SEQ ID NO:1 ).
The amplified fragment is digested with the endonucleases SmaI and then
purified
again on a 1 % agarose gel. The isolated fragment and the dephosphorylated
vector are
then ligated with T4 DNA ligase. E. coli HB 101 or XL-1 Blue cells are then
transformed
and bacteria are identified that contain the fragment inserted into plasmid
pC4 using, for
instance, restriction enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR gene are used for
transfection. Five ~g of the expression plasmid pC4 is cotransfected with 0.5
~tg of the
plasmid pSV2-neo using lipofectin (Felgner et al., secpc-cr). The plasmid
pSV2neo
contains a dominant selectable marker, the neo gene from Tn5 encoding an
enzyme that
confers resistance to a group of antibiotics including 6418. The cells are
seeded in alpha
minus MEM supplemented with 1 mg/ml 6418. After 2 days, the cells are
trypsinized
and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM
supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml 6418. After
about
10-14 days single clones are trypsinized and then seeded in 6-well petri
dishes or 10 ml
flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM,
400 nM,
800 nM). Clones growing at the highest concentrations of methotrexate are then
transferred to new 6-well plates containing even higher concentrations of
methotrexate (1
pM, 2 ~M, 5 pM, 10 ~M, 20 pM). The same procedure is repeated until clones are
obtained which grow at a concentration of 100-200 ~M. Expression of the
desired gene
product is analyzed, for instance, by SDS-PAGE and Western blot or by reverse
phase
HPLC analysis.
Example 4: Tissue Distribution of TR9 mRNA Expression
Northern blot analysis is carried out to examine TR9 gene expression in human
tissues, using methods described by, among others, Sambrook et cal., supra. A
cDNA
probe containing the entire nucleotide sequence of the TR9 protein (SEQ ID NO:
1 ) is
labeled with ;'P using the rediprimeT'~' DNA labeling system (Amersham Life
Science),
according to manufacturer's instructions. After labeling, the probe was
purified using a
CHROMA SPIN-100TM column (Clontech Laboratories, Inc.), according to
manufacturer's protocol number PT1200-1. The purified labeled probe is then
used to
examine various human tissues for TR9 mRNA.
Multiple Tissue Northern (MTN) blots containing various human tissues (H) or
human immune system tissues (IM) are obtained from Clontech and are examined
with
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the labeled probe using ExpressHyb~'''f hybridization solution (Clontech)
according to
manufacturer's protocol number PTl 190-1. Following hybridization and washing,
the
blots are mounted and exposed to film at -70°C overnight, and films
developed according
to standard procedures.
Example 5: TR9 Irzduced Apoptosis
Overexpression of Fas/APO-1 and TNFR-1 in mammalian cells mimics receptor
activation (M. Muzio et al., Cell 85:817-827 (1996); M. P. Boldin et al., Cell
85:803-815
( 1996)). Thus, this system is utilized to study the functional role of TR9.
Transient
expression of TR9 in MCF7 breast carcinoma cells and 293 human embryonic
kidney
cells is investigated for induction of apoptosis.
Experifnental Desigfi
Cell death assays are performed essentially as previously described (A.M.
Chinnaiyan et al., Cell 81:505-512 (1995); M.P. Boldin et al., J. Biol. Chem.
270: 7795-
8 (1995); F.C. Kischkel et al., EMBO 14:5579-5588 (1995); A.M. Chinnaiyan et
al., J.
Biol. Chefs. 271:4961-4965 (1996)). Briefly, MCF-7 human breast carcinoma
clonal
cell lines stably transfected with either vector alone, a CrmA expression
construct (M.
Tewari et al., J. Biol. Chem. 270:3255-60 ( 1995)), or FADD-DN expression
construct
(A.M. Chinnaiyan et al., J. Biol. Chem. 271:4961-4965 (1996)) are transiently
transfected with pCMV- TR9- galatosidase in the presence of a ten-fold excess
of
pcDNA3 expression constructs encoding the indicated proteins using
lipofectamine
(GIBCO-BRL). 293 cells are likewise transfected using the CaP04 method. The
ICE
family inhibitor z-VAD-fmk (Enzyme Systems Products, Dublin, CA) is added to
the
cells at a concentration of lOuM, 5 hrs after transfection. 32 hours following
transfection, cells are fixed and stained with X-Gal as previously described
(A.M.
Chinnaiyan et al., Cell 81:505-12 ( 1995); M.P. Boldin et al., J. Biol. Chem.
270:7795-8
(1995); F.C. Kischkel et al., EMBO 14:5579-5588 (1995)).
Results
The affected cells will display morphological alterations typical of cells
undergoing
apoptosis, becoming rounded, condensed, and detaching from the dish. Similar
to
TNFR-1 and Fas/APO-1 (M. Muzio et al., Cell 85:817-827 (1996); M. P. Boldin et
al.,
Cell 85:803-815 (1996); M. Tewari et al., J. Biol. Chem. 270:3255-60 (1995)),
TR9-
induced apoptosis is blocked by the inhibitors of ICE-like proteases, CrmA and
z-VAD-
fmk.
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Example 6: Characterization of TR9
Members of the TNF receptor family are crucial modulators of inflammatory and
cellular immune responses, and mediate a variety of biological functions,
ranging from
cell proliferation, differentiation and apoptosis to cell survival (Nagata,
S., Cell 88:355-
365 (1997); Armitage, R. J., Ccrc-r. Opicc. Immuno. 6:407-413 (1994);
Golstein, P.,
Curc-. Biol. 7:8750-8753 (1997); Baichwal et al., Cccrr. Biol. 7:894-896.
(1997): Smith
et al., Cell 76: 959-962 ( 1994); Anderson et al., Nuture 390:175-179 ( 1997);
and
Cleveland et al., Cell 81:479-482 ( 1995)). This family of receptors is
characterized by
several extracellular, cysteine-rich motifs that compose the ligand binding
domain
(Armitage, R. J., Cccrr. Opin. Immccno. 6:407-413 ( 1994); and Smith et al.,
Cell 76: 959-
962 ( 1994)). Upon ligation by their cognate ligands, these receptors engage a
number of
signal transduction pathways, including apoptosis, activation of NF B and JNK
pathways that modulate the expression of genes involved in the immune and
stress
response (Smith et al., Cell 76: 959-962 ( 1994)).
Within the TNF receptor family, six members have emerged as a distinct
subgroup termed death receptors; they contain a cytoplasmic death domain and
activation
of these receptors leads to engagement of components of the cell death pathway
(Nagata,
S., Cell 88:355-365 ( 1997); and Golstein, P., Cccrr. Biol. 7:8750-8753 (
1997)).
Transmission of the death signal is mediated by a series of homophilic protein-
protein
interactions involving the death domain and death effector domain that was
originally
defined as being present in the adaptor molecule FADD/MORT 1 and the death
protease
caspase-8 (Chinnaiyan et al., Semmin. ImnZUnol. 9:66-67 ( 1997)). For example,
when
the death receptor CD95/Fas is ligated by cognate ligand or agonist antibody,
the adaptor
molecule FADD and the death protease caspase-8 are recruited to the signalling
complex
through interactions involving death and death effector domains, respectively
(Chinnaiyan
et al., Semcnin. Immunol. 9:66-67 ( 1997); Muzio et al., Cell 85: 817-827 (
1996); and
Boldin et al., Cell 85:803-815 ( 1996)). On approximation, caspase-8 undergoes
an
autoactivation, initiating activation of the downstream caspases, cleavage of
death
substrates and demise of the cell (Muzio et al, J. Biol. Chew. 273:2952-2956
(1997);
Barinaga, M., Science 280:32-34 ( 1998); Salvesen et al., Cell 91:443-446 (
1997): and
Martin et al., Cell 82: 349-352 ( 1995)). In contrast to CD-95 that directly
engages the
FADD-caspase-8 pathway (Muzio et al., Cell 85: 817-827 (1996); Boldin et al.,
Cell
85:803-815 (1996); Chinnaiyan et al., Cell 81:505-512 (1995); and Boldin et
al., J. Biol.
Chem. 270:7795-7789 (1995)), both TNFR1 and DR3 utilize a primary adaptor
molecule
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termed TRADD, around which assembles the FADD-caspase-8 pathway, an NF B
activating pathway involving the death domain-containing Ser/Thr kinase RIP
and a JNK
activating pathway that is mediated by the adaptor molecule TRAF2 (Hsu et al.,
Cell
81:495-504 (1995); Hsu et al., Immerjiih~ 4:387-396 (1996); Chinnaiyan et al.,
Science
274:990-992 ( 1996); Kitson et al., Natcrc-e 384:372-375 ( 1996); Yeh et al.,
Inioncnaity
7:715-725 (1997); Lee et al., Immunity 7:703-713 (1997); and Kelliher et al.,
Immunity
8:297-303 ( 1998). Finally, there exists a subsidiary death pathway involving
the death
domain-containing adaptor RAIDD that binds to caspase-2 and has been shown to
be part
of the TNFR1 receptor complex, although the exact physiologic relevance of
this
redundant pathway remains unclear (Duan et al., Nature 385:86-89 ( 1997); and
Ahmad et
al., Cancer Res. 57:615-619 ( 1997).
Here, we report the identification and initial characterization of TR9, a new
member of the TNF receptor family possessing a cytoplasmic death domain. TR9
induced
apoptosis in mammalian cells and was capable of engaging the NF B and JNK
pathways.
Materials and methods
Expression Constructs -- TR9 (amino acid residues 42-655 as dipicted in
Figures lA-D;
amino acid residues 2-615 as presented in SEQ ID N0:2) and TR9 delta (amino
acid
residues 42-460 as dipicted in Figures lA-D; amino acid residues 2-420 as
presented in
SEQ ID N0:2) were cloned into pCMV 1FLAG (IBI-Kodak) as in frame fusions to a
TR9-terminal Preprotrypsin leader sequence and FLAG tag encoded by the vector.
cDNAs were obtained by polymerase chain reaction using DNA oligo primers for
TR9:
5'-GGA AGA TCT GCC AGA ACA GAA GGC CTC GAA T-3' (SEQ ID N0:16) and
5'-CCA TCT TCC TGA CCT GCT GTA GTC TAG AGC C-3' (SEQ ID N0:17) and for
TR9 delta: 5'-GGA AGA TCT GCC AGA ACA GAA GGC CTC GAA T-3' (SEQ ID
N0:16) and 5'-GCC GAC CAC GAG CGG GCC TAG TCT AGA GCC-3' (SEQ ID
N0:18). Constructs encoding DR4, FADD, CD95, DR3, TRADD, ICH1-pro, RAIDD
and RIP have been described previously (Chinnaiyan et al., Cell 81:505-512 (
1995); Hsu
et al., Cell 81:495-504 ( 1995); Hsu et al., Immuni y 4:387-396 ( 1996);
Chinnaiyan et al.,
Science 274:990-992 ( 1996); Kelliher et al., hnmunity 8:297-303 ( 1998); and
Pan et al.,
Science 276:111-113 (1997)).
Apoptosis Assao -- Cell death assays were performed as previously described
(Chinnaiyan et al., Cell 81:505-512 (1995); and Pan et al., Science 276:111-
113 (1997)).
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Both Hela and MCF7 cells were transfected using the lipofectamine procedure
(Life
Technologies, Inc.) according to the manufacturer's instructions.
Co-imnuzioprecipitation Assay -- hz vivo interaction assays have been
described
elsewhere (Chinnaiyan et al., Cell 81:05-512 (1995); and Pan et al., Science
276:111-
113 ( 1997)). 293 cells were co-transfected with FLAG-TR9, FLAG-TR9 delta.
FLAG-
CD95, FLAG-DR3, FLAG-TNFR1. and ICH-lpro-FLAG, expression constructs using
standard calcium phosphate precipitation. After transfection (at 38-40 hours),
cell lysates
were prepared and the FLAG-tagged expressed proteins were immunoprecipitated
with
FLAG M2 affinity gel (IBI-Kodak) and the presence of FADD, myc-tagged TRADD
and
RIP (myc-TRADD and myc-RIP), or RAIDD detected by immunoblotting with
polyclonal
antibody to FADD horseradish peroxidase (HRP)-conjugated antibody to myc
(BMB), or
polyclonal antibody to RAIDD.
NF- _B Luciferase Assay -- NF B luciferase assays were done as described
elsewhere
(Chinnaiyan et al., Cell 81:505-512 (1995); and Pan et al., Science 276:111-
113 (1997)).
JNKActivatiozz Assay -- 293 cells were cultured in MEM containing 10% FBS.
Cells
were plated in 6-well plates and transfected with TR9 expressing plasmid or
vector alone
at 60-70% confluency by the lipofectamine method according to the
manufacturer's
instructions. Forty hours post transfection, cell extracts were prepared in
lysis buffer
containing 20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCI, 0.1 % NP-40, 2
micrograms/ml leupeptin, 2 micrograms/ml aprotinin, 1 mM PMSF, 0.5
micrograms/ml
benzamide, 1 mM DTT and 1mM orthovanadate. The C-jun kinase assay was
performed
by a modified method as described (Haridas et al., Iznzzzuzzol. 160:3152-3162
( 1998)).
Briefly, cell extracts (70 micrograms) were subjected to immunoprecipitation
with 0.03
_g anti-JNK antibody for 30 min at 4°C. Immuno-complexes were collected
by
incubation with protein A/G-sepharose beads for 30 min at 4°C. The
beads were
extensively washed with lysis buffer (4 X 400 microliters) and kinase buffer
(2 X 400
microliters: 20 mM HEPES, pH 7.4, 1 mM DTT, 25 mM NaCI) and the kinase
reaction
allowed to proceed for 15 min at 30 C with 2micrograms GST-Jun (1-79) in
2microliters
containing 20 mM HEPES, pH 7.4, 10 mM MgCh 1 mM DTT and 10 microcuries
[gamma~'P]ATP. Reactions were stopped by the addition of 15 microliters SDS-
sample
buffer and resolved by SDS-polyacrylamide gel electrophoresis. GST-Jun (1-79)
was
visualized by staining with Coomassie Blue and the dried gel visualized
following
Phosphorimager analysis (Molecular Dynamics; Sunyvale, CA) and quantitation by
ImageQuant Software (Molecular Dynamics). A specific assay for JNK activity
involved
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the co-transfection of 3 X l Oh 293 cells with vector, or the CD40, TR9, or
TR9 delta
expression constructs (6.4 micrograms) together with 2.4 micrograms of a JNK-
myc
expression plasmid using the calcium phosphate precipitation method. After
transfection
(approximately 36 hours), cell extracts were prepared by lysis in NP 40 buffer
(20 mM
Tris-Cl, pH 8Ø 137 mM NaCI, 10% Glycerol, 2 mM EDTA, 5 mM Na,VO~, 0.5 mM
PMSF and 1 % NP40) plus protease inhibitor cocktail (BMB). Immunoprecipitation
of
JNK-myc was performed using monoclonal anti-myc antibody ( 10 micrograms,
Babco)
and immunocomplexes precipitated with 20 microliters protein G-sepharose (50%
slurry,
Sigma) and detected by blotting with anti-myc-HRP. FLAG tagged CD40, TR9, and
TR9 delta were immunoprecipitated with anti-FLAG M2 affinity gel and detected
by
blotting with anti-FLAG antibody. The kinase assay utilized 2 micrograms GST
Jun(1-
79) as substrate, 50 mM ATP and 5 microcuries [gammaj'P]ATP in 30 microliters
kinase
buffer (30 mM HEPES, pH 7.4, 7 mM Mn Cl" 5 mM MgCI~ and 1 mM DTT).
Results and discussion
TR9 has a putative signal sequence (amino acid residues 1-41 as depicted in
Figures lA-D and 4A; amino acid residues -40 to 1 in SEQ ID N0:2), with the
mature
form predicted to start at amino acids 42 (Gln) as depicted in Figures lA-D
and 4A
(Nielson et al., Protein. Eng. 10:1-6 (1997)). The extracellular portion
(amino acid
residues 42-350 as depicted in Figures lA-D and 4A; amino acid residues 2-310
in SEQ
ID N0:2) contains four TNFR-like cysteine-rich motifs of TR9 (amino acid
residues 67-
211 as depicted in Figures lA-D; amino acid residues 27-171 in SEQ ID N0:2)
that are
most related to those of osteoprotegerin (OPG) and TNFR2 with 36% and 42%
amino
acid identities, respectively (Figure.4B; data not shown). A transmembrane
domain
(amino acids 351 to 370 as depicted in Figures lA-D and 4A; residues 311 to
330 of SEQ
ID N0:2) is followed by a 285-amino acid long cytoplasmic portion of the
molecule that
contains a death domain related to those of all known death receptors (Figure
4C), being
most related to the death domain of TNFR1 (27.2%) and least like that of DRS
(19.7%).
Curiously, unlike other death receptors that have death domains present in
their COOH-
terminus, the death domain in TR9 was located adjacent to the transmembrane
domain
followed by a 150 amino acid tail. Interestingly, following the death domain
was a
putative leucine zipper sequence overlapping with a proline-rich region
reminescent of a
SH3 domain-binding motif (Figure 4A) (Pawson et al., Science 278:2075-2080 (
1997)).
TR9 mRNA expression in human tissues and cancer cell lines -- A 4-kb TR9
transcript
was found in most human adult tissue, immune tissue, and cancer cell lines
represented
on Northern blots (Clontech) that were probed with TR9 cDNA according to the
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manufacturers instructions (data not shown). The transcript was abundant in
heairt. brain,
placenta, pancreas, lymph node, thymus and prostate. Lower levels were
detected in
lung, skeletal muscle, kidney, testis, uterus, small intestine, colon, spleen,
bone marrow,
and fetal liver. However, adult liver and peripheral blood leukocytes
expressed little TR9
mRNA. Additionally, smaller transcripts of 3.1 and 2.4 kb were observed in the
testis
and fetal liver, respectively.
Among human cancer cell lines, abundant levels of 4-kb transcript was detected
in
several nonlymphoid tumor cells, including cervical carcinoma Hela S3.
colorectal
adenocarcinoma SW480, lung carcinoma A549, and melanoma 6361 cells.
Significantly,
less or no expression was observed in lines of hematopoietic origin (e.g.,
Raji, K562.
and HL-60; data not shown).
TR9 indatces apoptosis in mammalian cells -- Since ectopic expression of death
receptors
can induce cell death in a ligand-independent manner (Chinnaiyan et al., Cell
81:50-512
(1995); Boldin et al., J. Biol. Chem. 270:7795-7789 (1995); Chinnaiyan et al.,
Sciet2ce
274:990-992 (1996); Kitson et al., Natecre 384:372-375 (1996); and Pan et al.,
Sciecice
276:111-113 (1997)), we tested if TR9 could induce apoptosis upon
overexpression.
When Hela S3 cervical carcinoma cells were transfected with a TR9-expressing
construct,
43% of the transfected cells underwent morphological changes characteristic of
apoptosis
(Figure 5). As expected, deletion of the putative death domain (TR9 delta)
abolished its
killing activity. Significantly, TR9 was unable to induce cell death in human
breast
carcinoma MCF7 cells although they were very sensitive to DR4 killing (Figure
5 and not
shown), suggesting that the cell death pathway engaged by TR9 may be distinct
from that
engaged by other death receptors. Alternatively, the apoptotic activity of TR9
may be
modulated by other signaling pathways it activates (see below) or ligand
binding may be
required to unveil its full killing capacity.
Interaction of TR9 with adaptor molecules in vivo -- Death receptors utilize
the adaptor
molecules FADD (for CD95) or both TRADD and FADD (for TNFR1 and DR3) to
transmit the death signal (Chinnaiyan et al., Cell 81:505-512 (1995); Boldin
et al., J.
Biol. Chem. 270:7795-7789 (1995); Chinnaiyan et al., Science 274:990-992
(1996); and
Kitson et al., Nature 384:372-375 (1996)). We thus determined if TR9 could
bind any of
these adaptor molecules in human embryonic kidney 293 cells. TR9 did not
interact with
FADD, although the association between CD95 and FADD was readily detected
under
similar conditions (data not shown). Interestingly, TR9 was found to associate
with
TRADD, although the interaction was weaker than that between DR3 and TRADD
(data
not shown). This observation is consistent with the observation that TR9 has a
weaker
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killing ability. Alternatively, TR9 may use a TRADD-related molecule as an
adaptor, or
the observed association might be bridged by another adaptor protein.
Interaction was not
detectable between TR9 and RAIDD or RIP, two other adaptor molecules known to
be
recruited to the TNFR1 and DR3 signalling complexes (data not shown).
TR9 activates fiarclecar factor--kappaB -- Both TNFR1 and DR3 can engage a
signal
transduction pathway that leads to the activation of NF-kappaB (Smith et al.,
Cell 76:
959-962 ( 1994): Chinnaiyan et al., Science 274:990-992 ( 1996); Kitson et
al., Nature
384:372-375 (1996); and Baker et al., Oncogejae 12:1-9 (1996)). The ability of
TR9 to
activate NF-kappaB was tested in a luciferase reporter assay and was found to
induce NF-
kappaB activation in a dose-dependent manner (Figure 6). Presumably
overexpressing
the receptor allowed it to achieve an active configuration that was competent
to signal the
NF-kappaB system. Interestingly, the cytoplasmic deletion of TR9 that
abolished its
apoptotic activity similarly abrogated its ability to activate NF-kappaB (data
not shown),
suggesting that these two signaling pathways may be mediated by a common
receptor-
proximal adapter molecule.
Ectopic expressioh of TR9 induces JNK activatiofi -- JNK activation is known
to
be induced by several TNF receptors including TNFR1 and CD40 (Smith et al.,
Cell76:
959-962 ( 1994); Yeh et al., ImrnurZi y 7:715-725 ( 1997); Lee et al.,
Immunity 7:703-713
(1997); and Baker et al., Oncogene 12:1-9 (1996)). We next determined whether
overexpression of TR9 could lead to JNK activation using an in vitro kinase
assay. TR9
was found to induce JNK activation in a dose-dependent manner (data not
shown). The
cytoplasmic truncation that attenuated cell death or NF-kappaB activation had
surprisingly
little effect on JNK activation (data not shown). This would be consistent
with the notion
that JNK activation is mediated by a cytoplasmic segment different from that
responsible
for apoptosis and NF-kappaB induction. It is noteworthy that two potential
TRAF-
binding motifs are present adjacent to the transmembrane domain PRQDP (amino
acid
residues 381-385 as depicted in Figures lA-D; amino acid residues 341-345 as
presented
in SEQ ID N0:2), and PTQNR (amino acid residues 400-404 as depicted in Figures
lA-
D; amino acid residues 360-364 as presented in SEQ ID N0:2) (Gedrich et al.,
J. Biol.
Chem. 271:12852-12858 (1996) and Boucher et al., Biochena. and Biophy. Res.
Communi. 233:592-600 ( 1997).
In conclusion, we have identified a novel death domain-containing TNF receptor
designated TR9. TR9 engages a cell death pathway different from those
initiated by the
CD95, TNFR1 or TRAIL/Apo2L receptors. In addition, TR9 also activates NF-
kappaB
and JNK, two signaling pathways shared by TNFR 1. Thus, it is likely that like
the other
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members of the TNF receptor family. TR9 plays a role in inflammatory responses
and
immune regulation.
Example 7: Gene Therapy Using Endogenoz~s TR9 GefZe
Another method of gene therapy according to the present invention involves
operably associating the endogenous TR9 sequence with a promoter via
homologous
recombination as described, for example, in US Patent Number 5,641,670, issued
June
24, 1997; International Publication Number WO 96/29411, published September
26,
1996; International Publication Number WO 94/12650, published August 4, 1994;
Koller
et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 ( 1989); and Zijlstra et al.,
Nature
342:435-438 ( 1989). This method involves the activation of a gene which is
present in
the target cells. but which is not expressed in the cells, or is expressed at
a lower level
than desired. Polynucleotide constructs are made which contain a promoter and
targeting
sequences, which are homologous to the ~' non-coding sequence of endogenous
TR9,
flanking the promoter. The targeting sequence will be sufficiently near the 5'
end of TR9
so the promoter will be operably linked to the endogenous sequence upon
homologous
recombination. The promoter and the targeting sequences can be amplified using
PCR.
Preferably, the amplified promoter contains distinct restriction enzyme sites
on the 5' and
3' ends. Preferably, the 3' end of the first targeting sequence contains the
same
restriction enzyme site as the 5' end of the amplified promoter and the 5' end
of the
second targeting sequence contains the same restriction site as the 3' end of
the amplified
promoter.
The amplified promoter and the amplified targeting sequences are digested with
the appropriate restriction enzymes and subsequently treated with calf
intestinal
phosphatase. The digested promoter and digested targeting sequences are added
together
in the presence of T4 DNA ligase. The resulting mixture is maintained under
conditions
appropriate for ligation of the two fragments. The construct is size
fractionated on an
agarose gel then purified by phenol extraction and ethanol precipitation.
In this Example, the polynucleotide constructs are administered as naked
polynucleotides via electroporation. However, the polynucleotide constructs
may also be
administered with transfection-facilitating agents, such as liposomes, viral
sequences,
viral particles, precipitating agents, etc. Such methods of delivery are known
in the art.
Once the cells are transfected, homologous recombination will take place which
results in the promoter being operably linked to the endogenous TR9 sequence.
This
results in the expression of TR9 in the cell. Expression may be detected by
immunological
staining, or any other method known in the art.
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Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue
is
placed in DMEM + 10% fetal calf serum. Exponentially growing or early
stationary phase
fibroblasts are trypsinized and rinsed from the plastic surface with nutrient
medium. An
aliquot of the cell suspension is removed for counting, and the remaining
cells are
subjected to centrifugation. The supernatant is aspirated and the pellet is
resuspended in 5
ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCI, ~ mM KCI, 0.7
mM Na2 HP04, 6 mM dextrose). The cells are recentrifuged, the supernatant
aspirated,
and the cells resuspended in electroporation buffer containing 1 mg/ml
acetylated bovine
serum albumin. The final cell suspension contains approximately 3X 106
cells/ml.
Electroporation should be performed immediately following resuspension.
Plasmid DNA is prepared according to standard techniques. For example, to
construct a plasmid for targeting to the TR9 locus, plasmid pUC 18 (MBI
Fermentas,
Amherst, NY) is digested with HindIII. The CMV promoter is amplified by PCR
with
an XbaI site on the 5' end and a BamHI site on the 3'end. Two TR9 non-coding
sequences are amplified via PCR: one TR9 non-coding sequence (TR9 fragment 1)
is
amplified with a HindIII site at the 5' end and an Xba site at the 3'end; the
other TR9 non-
coding sequence (TR9 fragment 2) is amplified with a BamHI site at the 5'end
and a
HindIII site at the 3'end. The CMV promoter and TR9 fragments are digested
with the
appropriate enzymes (CMV promoter - XbaI and BamHI; TR9 fragment 1 - XbaI; TR9
fragment 2 - BamHI) and ligated together. The resulting ligation product is
digested with
HindIII, and ligated with the HindIII-digested pUC 18 plasmid.
Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio-
Rad).
The final DNA concentration is generally at least 120 pg/ml. 0.5 ml of the
cell suspension
(containing approximately 1.5.X106 cells) is then added to the cuvette, and
the cell
suspension and DNA solutions are gently mixed. Electroporation is performed
with a
Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960 pF and
250-300
V, respectively. As voltage increases, cell survival decreases, but the
percentage of
surviving cells that stably incorporate the introduced DNA into their genome
increases
dramatically. Given these parameters, a pulse time of approximately 14-20 mSec
should
be observed.
Electroporated cells are maintained at room temperature for approximately 5
min,
and the contents of the cuvette are then gently removed with a sterile
transfer pipette. The
cells are added directly to 10 ml of prewarmed nutrient media (DMEM with 15%
calf
serum) in a 10 cm dish and incubated at 37°C. The following day, the
media is aspirated
and replaced with 10 ml of fresh media and incubated for a further 16-24
hours.
The engineered fibroblasts are then injected into the host, either alone or
after
having been grown to confluence on cytodex 3 microcarrier beads. The
fibroblasts now
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produce the protein product. The fibroblasts can then be introduced into a
patient as
described above
Example: 8: TR9 Activates Monocytes and Increases Mouocyte Survival
The effects of TR9-Fc (containing amino acids residues Ml to L350 of the
sequence depicted in Figures 1 A-D fused to an ISG 1 fc fusion protein) on
monocytes
were evaluated using monocyte survival and TNF-alpha release functional
assays.
~ Methods:
Monocyte Survival
Monocytes were cultured for 48 hours in polypropilene tubes: in serum-free
medium (positive control); in the presence of 100 ng/ml TNF-alpha (negative
control);
and in the presence of 2ug/ml and 20 ug/ml of TR9-Fc. The cultured cells were
stained
with Annexin V and propidium iodide to determine the number of apoptotic and
dead
cells.
TNF-alpha release
Monocytes (Sx 105) were incubated for 1 day on immobilized TR9-Fc ( 10 ug/ml).
Conditioned media were collected and analyzed in ELISA for TNF-alpha content
using
R&D Systems kits.
MCP-1 release
Monocytes (5x105) were incubated for 1 day on wells coated with TR9-Fc (10
ug/ml). Conditioned media were collected and analyzed for MCP-1 content by
ELISA
using R&D system kits.
~ Results:
The effects of TR9-Fc on monocyte activation were examined using the above-
described monocyte survival and TNF-alpha release assays.
In the monocyte survival assay, monocytes were cultured in serum free media
and serum free media containing TR9-Fc (at concentrations of 2ug/ml or 20
ug/ml), or
TNF-alpha ( 100 nglml). After 48 hours the percentage of apoptotic or dead
cells was
determined by staining with Annexin V and propidium iodide. The results
revealed that
81% of untreated cells were apoptotic or dead, while only 25% of the cells
treated with
TNF-alpha were killed. Monocytes treated with 20 ug/ml of TR9-Fc were 48%
apoptotic, indicating that TR9-Fc enhances monocte survival.
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In the TNF-alpha release assay, as described above, monocytes were incubated
alone or on immobilized TR9-Fc (10 ug/ml) and the production of TNF-alpha was
measured by a standard ELISA assay. The concentration of TNF-alpha release of
the
TR9-Fc treated monocytes was in all of the four donor lots tested Qrecrten
than S,f~ld than
that observed in the absence of TR9-Fc (see Table III). Interestingly,
additional
experiments conducted as described above, but in the presence or absence of
Interferon-
gamma (5 ng/ml; Peprotech) revealed that the combination of TR9-Fc and
Interferon-
gamma results in a synergistic release of TNF-alpha (see Table III, Donor 4).
Table III
TR9-Fc-induced TNF-alpha secretion from monocytes
Treatment TNF-alpha
(pg/ml)
Donor 1
None 0
TR9-Fc 331
Donor 2
None 0
TR9-Fc 460
Donor 3
None 60
TR9-Fc 5290
Donor 4
None 0
TR9-Fc 5
IFN- 25
TR9-Fc + IFN- 85
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16s
Monocytes (5x105) were incubated for 1 day on wells coated with TR9-Fc (10
_g/ml).
Conditioned media were collected and analyzed for TNF-alpha content by ELISA.
In the MCP-1 release assay. as described above, monocytes were incubated alone
or on immobilized TR9-Fc and the production of MCP-1 was measured by a
standard
ELISA assay. The concentration of MCP-1 release of the TR9-Fc treated
monocytes was
greater than that observed in the absence of TR9-Fc (see Table IV).
Table IV
TR9-Fc-induced MCP-1 secretion from monocytes
Treatment MCP-1
(Pg/~)
None 0
TR9-Fc 351
Monocytes (Sx 105) were incubated for 1 day on wells coated with TR9-Fc ( 10
_g/ml).
Conditioned media were collected and analyzed for MCP-1 content by ELISA.
Example: 9 Assays for Monocyte Activation and/or Increased Survival
Assays for molecules that activate (or alternatively, inactivate) monocytes
and/or
increase monocyte survival (or alternatively, decrease monocyte survival) are
known in
the art and may routinely be applied to determine whether a molecule of the
invention
functions as a TR9 agonists (or alternatively, a TR9 antagonist). Three of
such assays are
described below.
~ Methods:
Monocyte survival Assay
Monocytes are cultured for 48 hours in polypropylene tubes in serum-free
medium (positive control), in the presence of 100 ng/ml TNF-alpha (negative
control),
and in the presence of varying concentrations of the compound to be tested. In
assays for
antagonists, the assays include varying concentrations of the composition to
be tested in
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the presence of 20 ug/ml of TR9-Fc. Monocyte survival is assayed by staining
the cells
with Annexin V and propidium iodide and determining the number of apoptotic
and dead
cells.
As exemplified in Example 8. compounds that function as TR9 agonists will
enhance monocyte survival when compared to the media alone (i.e., the positive
control).
In contrast, compounds that function as TR9 antagonists will decrease monocyte
survival when compared to that observed when the cells are contacted with the
TR9-Fc
protein alone.
TNF-alpha release
To identify TR9-Fc agonists, monocytes (Sx 105) are incubated for 1 day with
varying concentrations of the compound to be tested. Culture media are
collected and
analyzed in ELISA for TNF-alpha content using R&D Systems kits. TR9-Fc
agonists will
induce a greater concentration of TNF-alpha release when compared to that
observed
when monocyte cells are incubated for 1 day under the same conditions, but in
the
absence of the test compound.
To identify TR9-Fc antagonists, monocytes (5x 105) are incubated for 1 day
with
varying concentrations of the compound to be tested in the presence and
absence of
immobilized TR9-Fc ( 10 ug/ml). Culture media are collected and analyzed in
ELISA for
TNF-alpha content using R&D Systems kits. TR9-Fc antagonists elicit a reduced
TNF-
alpha release from the monocytes when compared to that observed when monocytes
cells
are incubated in the presence of immobilized TR9-Fc, but in the absence of the
test
compound.
MCP-1 release
To identify TR9-Fc agonists, monocytes (Sx 105) are incubated for 1 day with
varying concentrations of the compound to be tested. Culture media are
collected and
analyzed in ELISA for MCP-1 content using R&D Systems kits. TR9-Fc agonists
will
induce a greater concentration of MCP-1 release when compared to that observed
when
monocyte cells are incubated for 1 day under the same conditions, but in the
absence of
the test compound.
To identify TR9-Fc antagonists, monocytes (5x105) are incubated for 1 day with
varying concentrations of the compound to be tested in the presence and
absence of
immobilized TR9-Fc ( 10 ug/ml). Culture media are collected and analyzed in
ELISA for
MCP-1 content using R&D Systems kits. TR9-Fc antagonists elicit a reduced MCP-
1
release from the monocytes when compared to that observed when monocytes cells
are
incubated in the presence of immobilized TR9-Fc, but in the absence of the
test
compound.
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Example 10: Protein Fusions of TR9
TR9 polypeptides of the invention are optionally fused to other proteins.
These
fusion proteins can be used for a variety of applications. For example. fusion
of TR9
polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding
protein
facilitates purification. (See EP A 394,827; Traunecker, et al., Nature 331:84-
86 ( 1988).)
Similarly, fusion to IgG- l, IgG-3, and albumin increases the halflife time in
vivo.
Nuclear localization signals fused to TR9 polypeptides can target the protein
to a specific
subcellular localization, while covalent heterodimer or homodimers can
increase or
decrease the activity of a fusion protein. Fusion proteins can also create
chimeric
molecules having more than one function. Finally, fusion proteins can increase
solubility
and/or stability of the fused protein compared to the non-fused protein. All
of the types of
fusion proteins described above can be made using techniques known in the art
or by
using or routinely modifying the following protocol, which outlines the fusion
of a
polypeptide to an IgG molecule.
Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using
primers that span the 5' and 3' ends of the sequence described below. These
primers also
preferably contain convenient restriction enzyme sites that will facilitate
cloning into an
expression vector, preferably a mammalian expression vector.
For example, if the pC4 (Accession No. 209646) expression vector is used, the
human Fc portion can be ligated into the BamHI cloning site. Note that the 3'
BamHI site
should be destroyed. Next, the vector containing the human Fc portion is re-
restricted
with BamHI, linearizing the vector, and TR9 polynucleotide, isolated by the
PCR
protocol described in Example 1, is ligated into this BamHI site. Note that
the
polynucleotide is cloned without a stop codon, otherwise a fusion protein will
not be
produced.
If the naturally occurring signal sequence is used to produce the secreted
protein,
pC4 does not need a second signal peptide. Alternatively, if the naturally
occurring signal
sequence is not used, the vector can be modified to include a heteroloaous
signal
sequence. (See, e.g., WO 96/34891.)
Human IgG Fc region:
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCC
AGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGA
CGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG
GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG
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TACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCA
AGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAA
AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG
CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGG
TCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA
GCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
TTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAG
AAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGA
GGAT (SEQ ID N0:27).
Example ll . Production of an Antibody.
(a) Hybridoma Technoloay
The antibodies of the present invention can be prepared by a variety of
methods.
(See, Current Protocols, Chapter 2.) As one example of such methods. cells
expressing
polypeptide(s) of the invention are administered to an animal to induce the
production of
sera containing polyclonal antibodies. In a preferred method, a preparation of
polypeptide(s) of the invention is prepared and purified to render it
substantially free of
natural contaminants. Such a preparation is then introduced into an animal in
order to
produce polyclonal antisera of greater specific activity.
Monoclonal antibodies specific for polypeptide(s) of the invention are
prepared
using hybridoma technology. (Kohler et al., Nature 256:495 ( 1975); Kohler et
al., Eur.
J. Immunol. 6:511 ( 1976); Kohler et al., Eur. J. Immunol. 6:292 ( 1976);
Hammerling et
al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-
681
(1981)). In general, an animal (preferably a mouse) is immunized with
polypeptide(s) of
the invention or, more preferably, with a secreted polypeptide-expressing
cell. Such
polypeptide-expressing cells are cultured in any suitable tissue culture
medium, preferably
in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum
(inactivated at about 56°C), and supplemented with about 10 g/1 of
nonessential amino
acids, about 1,000 U/ml of penicillin, and about 100 pg/ml of streptomycin.
The splenocytes of such mice are extracted and fused with a suitable myeloma
cell
line. Any suitable myeloma cell line may be employed in accordance with the
present
invention; however, it is preferable to employ the parent myeloma cell line
(SP20),
available from the ATCC. After fusion, the resulting hybridoma cells are
selectively
maintained in HAT medium, and then cloned by limiting dilution as described by
Wands
et al. (Gastroenterology 80:225-232 ( 1981 )). The hybridoma cells obtained
through
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such a selection are then assayed to identify clones which secrete antibodies
capable of
binding the polypeptide(s) of the invention.
Alternatively, additional antibodies capable of binding to polypeptide(s) of
the
invention can be produced in a two-step procedure using anti-idiotypic
antibodies. Such a
method makes use of the fact that antibodies are themselves antigens, and
therefore, it is
possible to obtain an antibody which binds to a second antibody. In accordance
with this
method, protein specific antibodies are used to immunize an animal, preferably
a mouse.
The splenocytes of such an animal are then used to produce hybridoma cells,
and the
hybridoma cells are screened to identify clones which produce an antibody
whose ability
to bind to the protein-specific antibody can be blocked by polypeptide(s) of
the invention.
Such antibodies comprise anti-idiotypic antibodies to the protein-specific
antibody and are
used to immunize an animal to induce formation of further protein-specific
antibodies.
For in vivo use of antibodies in humans, an antibody is "humanized". Such
antibodies can be produced using genetic constructs derived from hybridoma
cells
producing the monoclonal antibodies described above. Methods for producing
chimeric
and humanized antibodies are known in the art and are discussed herein. (See,
for
review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214
(1986);
Cabilly et al., U.S. Patent No. 4,816.567; Taniguchi et al., EP 171496;
Morrison et al.,
EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;
Boulianne et
al., Nature 312:643 ( 1984); Neuberger et al., Nature 314:268 ( 1985).)
(b) Isolation Of Antibody Fragments Directed Against Polypeptide(sl From A
Library Of scFvs
Naturally occurring V-genes isolated from human PBLs are constructed into a
library of antibody fragments which contain reactivities against
polypeptide(s) of the
invention to which the donor may or may not have been exposed (see e.g., U.S.
Patent
5,885,793 incorporated herein by reference in its entirety).
Rescue of tlTe Library.
A library of scFvs is constructed from the RNA of human PBLs as described in
PCT publication WO 92/01047. To rescue phage displaying antibody fragments,
approximately 109 E. coli harboring the phagemid are used to inoculate 50 ml
of 2xTY
containing 1 % glucose and 100 pg/ml of ampicillin (2xTY-AMP-GLU) and grown to
an
O.D. of 0.8 with shaking. Five ml of this culture is used to innoculate 50 ml
of 2xTY-
AMP-GLU, 2 x 108 TU of delta gene 3 helper (M 13 delta gene III, see PCT
publication
WO 92/01047) are added and the culture incubated at 37°C for 45 minutes
without
shaking and then at 37°C for 45 minutes with shaking. The culture is
centrifuged at 4000
r.p.m. for 10 min. and the pellet resuspended in 2 liters of 2xTY containing
100 pg/ml
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ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are prepared as
described in PCT publication WO 92/01047.
M 13 delta gene III is prepared as follows: M 13 delta gene III helper phage
does
not encode gene III protein, hence the phage(mid) displaying antibody
fragments have a
greater avidity of binding to antigen. Infectious M 13 delta gene III
particles are made by
growing the helper phage in cells harboring a pUCl9 derivative supplying the
wild type
gene III protein during phage morphogenesis. The culture is incubated for 1
hour at 37°
C without shaking and then for a further hour at 37°C with shaking.
Cells are spun down
(IEC-Centra 8,400 r.p.m. for 10 min >, resuspended in 300 ml 2xTY broth
containing 100
pg ampicillin/ml and 25 yg kanamycin/ml (2xTY-AMP-KAN) and grown overnight,
shaking at 37°C. Phage particles are purified and concentrated from the
culture medium
by two PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS and
passed
through a 0.45 pm filter (Minisart NML; Sartorius) to give a final
concentration of
approximately 1013 transducing units/ml (ampicillin-resistant clones).
Panning of the Library.
Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 qg/ml
or 10 pglml of a polypeptide of the present invention. Tubes are blocked with
2%
Marvel-PBS for 2 hours at 37°C and then washed 3 times in PBS.
Approximately 1013
TU of phage is applied to the tube and incubated for 30 minutes at room
temperature
tumbling on an over and under turntable and then left to stand for another 1.5
hours.
Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage
are
eluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes on an
under and
over turntable after which the solution is immediately neutralized with 0.5 ml
of 1.OM
Tris-HCI, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1
by
incubating eluted phage with bacteria for 30 minutes at 37°C. The E.
coli are then plated
on TYE plates containing 1 % glucose and 100 ~g/ml ampicillin. The resulting
bacterial
library is then rescued with delta gene 3 helper phage as described above to
prepare phage
for a subsequent round of selection. This process is then repeated for a total
of 4 rounds
of affinity purification with tube-washing increased to 20 times with PBS, 0.1
% Tween-
20 and 20 times with PBS for rounds 3 and 4.
Characterization of Binders.
Eluted phage from the 3rd and 4th rounds of selection are used to infect E.
coli
HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single
colonies for
assay. ELISAs are performed with microtitre plates coated with either 10 pg/ml
of the
polypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clones
positive in
ELISA are further characterized by PCR fingerprinting (see, e.g., PCT
publication WO
92/01047) and then by sequencing. These ELISA positive clones may also be
further
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171
characterized by techniques known in the art, such as, for example, epitope
mapping.
binding affinity. receptor signal transduction, ability to block or
competitively inhibit
antibody/antigen binding, and competitive agonistic or antagonistic activity.
Example 12: Method of Determining Alteratiofzs in the TR9 Gene
RNA is isolated from entire families or individual patients presenting with a
phenotype of interest (such as a disease). cDNA is then generated from these
RNA
samples using protocols known in the art. (See, Sambrook.) The cDNA is then
used as
a template for PCR, employing primers surrounding regions of interest in SEQ
ID NO:1.
Suggested PCR conditions consist of 35 cycles at 95° C for 30 seconds;
60-120 seconds
at 52-58° C; and 60-120 seconds at 70° C, using buffer solutions
described in Sidransky,
D., et al., Science 252:706 ( 1991 ).
PCR products are then sequenced using primers labeled at their 5' end with T4
polynucleotide kinase, employing SequiTherm Polymerise. (Epicentre
Technologies).
The intron-exon borders of selected exons of TR9 are also determined and
genomic PCR
products analyzed to confirm the results. PCR products harboring suspected
mutations in
TR9 is then cloned and sequenced to validate the results of the direct
sequencing.
PCR products of TR9 are cloned into T-tailed vectors as described in Holton,
T.A. and Graham, M.W., Nucleic Acids Research, 19:1156 (1991) and sequenced
with
T7 polymerise (United States Biochemical). Affected individuals are identified
by
mutations in TR9 not present in unaffected individuals.
Genomic rearrangements are also observed as a method of determining
alterations
in the TR9 gene. Genomic clones isolated using techniques known in the art are
nick-
translated with digoxigenindeoxy-uridine 5'-triphosphate (Boehringer Manheim),
and
FISH performed as described in Johnson, Cg. et al., Methods Cell Biol. 35:73-
99
(1991). Hybridization with the labeled probe is carried out using a vast
excess of human
cot-1 DNA for specific hybridization to the TR9 genomic locus.
Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium
iodide, producing a combination of C- and R-bands. Aligned images for precise
mapping
are obtained using a triple-band filter set (Chroma Technology, Brattleboro,
VT) in
combination with a cooled charge-coupled device camera (Photometrics, Tucson,
AZ) and
variable excitation wavelength filters. (Johnson, Cv. et al., Genet. Anal.
Tech. Appl.,
8:75 (1991).) Image collection, analysis and chromosomal fractional length
measurements are performed using the ISee Graphical Program System. (Inovision
Corporation, Durham, NC.) Chromosome alterations of the genomic region of TR9
(hybridized by the probe) are identified as insertions, deletions, and
translocations. These
TR9 alterations are used as a diagnostic marker for an associated disease.
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Example 13: Method of Detecting Abttorfnal Levels of TR9 in a Biological
Sample
TR9 polypeptides can be detected in a biological sample, and if an increased
or
decreased level of TR9 is detected. this polypeptide is a marker for a
particular phenotype.
Methods of detection are numerous, and thus, it is understood that one skilled
in the art
can modify the following assay to fit their particular needs.
For example, antibody-sandwich ELISAs are used to detect TR9 in a sample,
preferably a biological sample. Wells of a microtiter plate are coated with
specific
antibodies to TR9, at a final concentration of 0.2 to 10 ug/ml. The antibodies
are either
monoclonal or polyclonal and are produced using technique known in the art.
The wells
are blocked so that non-specific binding of TR9 to the well is reduced.
The coated wells are then incubated for > 2 hours at RT with a sample
containing
TR9. Preferably, serial dilutions of the sample should be used to validate
results. The
plates are then washed three times with deionized or distilled water to remove
unbounded
TR9.
Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at a
concentration
of 25-400 ng, is added and incubated for 2 hours at room temperature. The
plates are
again washed three times with deionized or distilled water to remove unbounded
conjugate.
75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate
(NPP) substrate solution is then added to each well and incubated 1 hour at
room
temperature to allow cleavage of the substrate and flourescence. The
flourescence is
measured by a microtiter plate reader. A standard curve is preparded using the
experimental results from serial dilutions of a control sample with the sample
concentration plotted on the X-axis (log scale) and fluorescence or absorbance
on the Y-
axis (linear scale). The TR9 polypeptide concentration in a sample is then
interpolated
using the standard curve based on the measured flourescence of that sample.
Examplel4: Method of Treating Decreased Levels of TR9
The present invention relates to a method for treating an individual in need
of a
decreased level of TR9 biological activity in the body comprising,
administering to such
an individual a composition comprising a therapeutically effective amount of
TR9
antagonist. Preferred antagonists for use in the present invention are TR9-
specific
antibodies.
Moreover, it will be appreciated that conditions caused by a decrease in the
standard or normal expression level of TR9 in an individual can be treated by
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173
administering TR9, preferably in a soluble and/or secreted form. Thus, the
invention also
provides a method of treatment of an individual in need of an increased level
of TR9
polypeptide comprising administering to such an individual a pharmaceutical
composition
comprising an amount of TR9 to increase the biological activity level of TR9
in such an
individual.
For example, a patient with decreased levels of TR9 polypeptide receives a
daily
dose 0.1-100 ug/ka of the polypeptide for six consecutive days. Preferably,
the
polypeptide is in a soluble and/or secreted form.
Example I5: Method of Treating Ifzcreased Levels of TR9
The present invention also relates to a method for treating an individual in
need of
an increased level of TR9 biological activity in the body comprising
administering to such
an individual a composition comprising a therapeutically effective amount of
TR9 or an
agonist thereof.
Antisense technology is used to inhibit production of TR9. This technology is
one example of a method of decreasing levels of TR9 polypeptide, preferably a
soluble
and/or secreted form, due to a variety of etiologies, such as cancer.
For example, a patient diagnosed with abnormally increased levels of TR9 is
administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and
3.0 mg/kg
day for 21 days. This treatment is repeated after a 7-day rest period if the
is determined to
be well tolerated.
Example 16: Method of Treatment Usi~zg Gene Therapy - Ex Vivo
One method of gene therapy transplants fibroblasts, which are capable of
expressing soluble and/or mature TR9 polypeptides, onto a patient. Generally,
fibroblasts are obtained from a subject by skin biopsy. The resulting tissue
is placed in
tissue-culture medium and separated into small pieces. Small chunks of the
tissue are
placed on a wet surface of a tissue culture flask, approximately ten pieces
are placed in
each flask. The flask is turned upside down, closed tight and left at room
temperature
over night. After 24 hours at room temperature, the flask is inverted and the
chunks of
tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's
F12 media,
with 10% FBS, penicillin and streptomycin) is added. The flasks are then
incubated at 37
degree C for approximately one week.
At this time, fresh media is added and subsequently changed every several
days.
After an additional two weeks in culture, a monolayer of fibroblasts emerge.
The
monolayer is trypsinized and scaled into larger flasks.
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174
pMV-7 (Kirschmeier, P.T. et al., DNA, 7:219-25 (1988)), flanked by the long
terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI
and
HindIII and subsequently treated with calf intestinal phosphatase. The linear
vector is
fractionated on agarose gel and purified. using glass beads.
The cDNA encoding TR9 can be amplified using PCR primers which correspond
to the 5' and 3' end encoding sequences respectively. Preferably, the 5'
primer contains
an EcoRI site and the 3' primer includes a HindIII site. Equal quantities of
the Moloney
murine sarcoma virus linear backbone and the amplified EcoRI and HindIII
fragment are
added together, in the presence of T4 DNA ligase. The resulting mixture is
maintained
under conditions appropriate for ligation of the two fragments. The liaation
mixture is
then used to transform E. coli HB 101, which are then plated onto agar
containing
kanamycin for the purpose of confirming that the vector contains properly
inserted TR9.
The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture
to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf
serum (CS), penicillin and streptomycin. The MSV vector containing the TR9
gene is
then added to the media and the packaging cells transduced with the vector.
The
packaging cells now produce infectious viral particles containing the TR9
gene(the
packaging cells are now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the
media is harvested from a 10 cm plate of confluent producer cells. The spent
media,
containing the infectious viral particles, is filtered through a millipore
filter to remove
detached producer cells and this media is then used to infect fibroblast
cells. Media is
removed from a sub-confluent plate of fibroblasts and quickly replaced with
the media
from the producer cells. This media is removed and replaced with fresh media.
If the
titer of virus is high, then virtually all fibroblasts will be infected and no
selection is
required. If the titer is very low, then it is necessary to use a retroviral
vector that has a
selectable marker, such as neo or his. Once the fibroblasts have been
efficiently infected,
the fibroblasts are analyzed to determine whether TR9 protein is produced.
The engineered fibroblasts are then transplanted onto the host, either alone
or after
having been grown to confluence on cytodex 3 microcarrier beads.
Example 17: Method of Treatment Using Gene Therapy - In Vivo
Another aspect of the present invention is using in vivo gene therapy methods
to
treat disorders, diseases and conditions. The gene therapy method relates to
the
introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) TR9
sequences into an animal to increase or decrease the expression of the TR9
polypeptide.
The TR9 polynucleotide may be operatively linked to a promoter or any other
genetic
CA 02365255 2001-09-24
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17~
elements necessary for the expression of the TR9 polypeptide by the target
tissue. Such
gene therapy and delivery techniques and methods are known in the art, see,
for example,
W090/11092, W098/1 1779; U.S. Patent NO. 5693622, 5705151, ~~80859; Tabata H.
et al., Cardiovcese. Res. 35:470-479 (1997); Chao J. et al., Phcarncacol. Re
s. 35:517-522
(1997); Wolff J.A. Neoronucscul. Disorcl. 7:314-318 (1997); Schwartz B. et
crl., Gene
Ther. 3:405-411 ( 1996); Tsurumi Y. et al., Circcclcztion 94:3281-3290 ( 1996)
(incorporated herein by reference).
The TR9 polynucleotide constructs may be delivered by any method that delivers
injectable materials to the cells of an animal, such as, injection into the
interstitial space of
tissues (heart, muscle, skin, lung, liver, intestine and the like). The TR9
polynucleotide
constructs can be delivered in a pharmaceutically acceptable liquid or aqueous
carrier.
The term "naked" polynucleotide, DNA or RNA, refers to sequences that are free
from any delivery vehicle that acts to assist, promote, or facilitate entry
into the cell.
including viral sequences, viral particles, liposome formulations, lipofectin
or
precipitating agents and the like. However, the TR9 polynucleotides may also
be
delivered in liposome formulations (such as those taught in Felgner P.L. et
al. (1995)
Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol. Cell
85(1):1-7)
which can be prepared by methods well known to those skilled in the art.
The TR9 polynucleotide vector constructs used in the gene therapy method are
preferably constructs that will not integrate into the host genome nor will
they contain
sequences that allow for replication. Any strong promoter known to those
skilled in the
art can be used for driving the expression of DNA. Unlike other gene therapies
techniques, one major advantage of introducing naked nucleic acid sequences
into target
cells is the transitory nature of the polynucleotide synthesis in the cells.
Studies have
shown that non-replicating DNA sequences can be introduced into cells to
provide
production of the desired polypeptide for periods of up to six months.
The TR9 polynucleotide construct can be delivered to the interstitial space of
tissues within the an animal, including of muscle, skin, brain, lung, liver,
spleen, bone
marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
bladder,
stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,
and
connective tissue. Interstitial space of the tissues comprises the
intercellular fluid,
mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic
fibers in the
walls of vessels or chambers, collagen fibers of fibrous tissues, or that same
matrix
within connective tissue ensheathing muscle cells or in the lacunae of bone.
It is similarly
the space occupied by the plasma of the circulation and the lymph fluid of the
lymphatic
channels. Delivery to the interstitial space of muscle tissue is preferred for
the reasons
discussed below. They may be conveniently delivered by injection into the
tissues
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176
comprising these cells. They are preferably delivered to and expressed in
persistent, non-
dividing cells which are differentiated. although delivery and expression may
be achieved
in non-differentiated or less completely differentiated cells, such as, for
example, stem
cells of blood or skin fibroblasts. In viva muscle cells are particularly
competent in their
ability to take up and express polynucleotides.
For the naked TR9 polynucleotide injection, an effective dosage amount of DNA
or RNA will be in the range of from about 0.05 g/kg body weight to about 50
mg/kg
body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20
mg/kg
and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the
artisan
of ordinary skill will appreciate, this dosage will vary according to the
tissue site of
injection. The appropriate and effective dosage of nucleic acid sequence can
readily be
determined by those of ordinary skill in the art and may depend on the
condition being
treated and the route of administration. The preferred route of administration
is by the
parenteral route of injection into the interstitial space of tissues. However,
other
parenteral routes may also be used, such as, inhalation of an aerosol
formulation
particularly for delivery to lungs or bronchial tissues, throat or mucous
membranes of the
nose. In addition, naked TR9 polynucleotide constructs can be delivered to
arteries
during angioplasty by the catheter used in the procedure.
The dose response effects of injected TR9 polynucleotide in muscle in viva is
determined as follows. Suitable TR9 template DNA for production of rnRNA
coding for
TR9 polypeptide is prepared in accordance with a standard recombinant DNA
methodology. The template DNA, which may be either circular or linear, is
either used as
naked DNA or complexed with liposomes. The quadriceps muscles of mice are then
injected with various amounts of the template DNA.
Five to six week old female and male Balb/C mice are anesthetized by
intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is
made on the
anterior thigh, and the quadriceps muscle is directly visualized. The TR9
template DNA
is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle
over one
minute, approximately 0.5 cm from the distal insertion site of the muscle into
the knee and
about 0.2 cm deep. A suture is placed over the injection site for future
localization, and
the skin is closed with stainless steel clips.
After an appropriate incubation time (e.g., 7 days) muscle extracts are
prepared by
excising the entire quadriceps. Every fifth 15 um cross-section of the
individual
quadriceps muscles is histochemically stained for TR9 protein expression. A
time course
for TR9 protein expression may be done in a similar fashion except that
quadriceps from
different mice are harvested at different times. Persistence of TR9 DNA in
muscle
following injection may be determined by Southern blot analysis after
preparing total
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cellular DNA and HIRT supernatants from injected and control mice. The results
of the
above experimentation in mice can be use to extrapolate proper dosages and
other
treatment parameters in humans and other animals using TR9 naked DNA.
Example 18: Effect of TR9 on the Expressiozz of MHC Class II,
Costimulatory azzd Adhesiozz Molecules and Cell Differentiation of
~norzocytes and Mozzocyte-Derived Huznazz Dezzdritic Cells
Dendritic cells are generated by the expansion of proliferating precursors
found in
the peripheral blood: adherent PBMC or elutriated monocytic fractions are
cultured for 7
10 days with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells have
the
characteristic phenotype of immature cells (expression of CD 1, CD80, CD86,
CD40 and
MHC class II antigens). Treatment with activating factors, such as TNF-a,
causes a
rapid change in surface phenotype (increased expression of MHC class I and II,
costimulatory and adhesion molecules, downregulation of FCyRII, upregulation
of
CD83). These changes correlate with increased antigen-presenting capacity and
with
functional maturation of the dendritic cells.
FACS analysis of surface antigens is performed as follows. Cells are treated 1-
3
days with increasing concentrations of TR9 or LPS (positive control), washed
with PBS
containing 1 % BSA and 0.02 mM sodium azide, and then incubated with 1:20
dilution of
appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at
4°C. After an
additional wash, the labeled cells are analyzed by flow cytometry on a FACScan
(Becton
Dickinson).
Effect on the production of cytokines.
Cytokines generated by dendritic cells, in particular IL-12, are important in
the
2~ initiation of T-cell dependent immune responses. IL-12 strongly influences
the
development of Thl helper T-cell immune response, and induces cytotoxic T and
NK cell
function. An ELISA is used to measure the IL-12 release as follows. Dendritic
cells
( 106/ml) are treated with increasing concentrations of TR9 for 24 hours. LPS
( 100 ng/ml)
is added to the cell culture as positive control. Supernatants from the cell
cultures are then
collected and analyzed for IL-12 content using commercial ELISA kit (e.g., R &
D
Systems (Minneapolis, MN)). The standard protocols provided with the kits are
used.
Effect on the expression of MHC Class II, costimulatory and adhesion
molecules.
Three major families of cell surface antigens can be identified on monocytes:
adhesion molecules, molecules involved in antigen presentation, and Fc
receptor.
Modulation of the expression of MHC class II antigens and other costimulatory
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178
molecules, such as B7 and ICAM-l, may result in changes in the antigen
presenting
capacity of monocytes and ability to induce T cell activation. Increase
expression of Fc
receptors may correlate with improved monocyte cytotoxic activity, cvtokine
release and
phagocytosis.
FACS analysis is used to examine the surface antigens as follows. Monocytes
are
treated 1-5 days with increasing concentrations of TR9 or LPS (positive
control), washed
with PBS containing 1 % BSA and 0.02 mM sodium azide, and then incubated with
1:20
dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30
minutes at 4°C.
After an additional wash, the labeled cells are analyzed by flow cytometry on
a FACScan
(Becton Dickinson).
Morzocyte activation uradlor increased setwival.
Assays for molecules that activate (or alternatively, inactivate) monocytes
and/or
increase monocyte survival (or alternatively, decrease monocyte survival) are
known in
the art and may routinely be applied to determine whether a molecule of the
invention
functions as an inhibitor or activator of monocytes. TR9, agonists, or
antagonists of TR9
can be screened using the three assays described below. For each of these
assays,
Peripheral blood mononuclear cells (PBMC) are purified from single donor
leukopacks
(American Red Cross, Baltimore, MD) by centrifugation through a Histopaque
gradient
(Sigma). Monocytes are isolated from PBMC by counterflow centrifugal
elutriation.
1. Monocyte Survival Assay. Human peripheral blood monocytes
progressively lose viability when cultured in absence of serum or other
stimuli. Their
death results from internally regulated process (apoptosis). Addition to the
culture of
activating factors, such as TNF-alpha dramatically improves cell survival and
prevents
DNA fragmentation. Propidium iodide (PI) staining is used to measure apoptosis
as
follows. Monocytes are cultured for 48 hours in polypropylene tubes in serum-
free
medium (positive control), in the presence of 100 ng/ml TNF-alpha (negative
control),
and in the presence of varying concentrations of the compound to be tested.
Cells are
suspended at a concentration of 2 x 106/ml in PBS containing PI at a final
concentration of
S ~.g/ml, and then incubated at room temperature for 5 minutes before FAC Scan
analysis.
PI uptake has been demonstrated to correlate with DNA fragmentation in this
experimental
paradigm.
2. Effect on cytokine release. An important function of
monocytes/macrophages is their regulatory activity on other cellular
populations of the
immune system through the release of cytokines after stimulation. An ELISA to
measure
cytokine release is performed as follows. Human monocytes are incubated at a
density of
Sx 10' cells/ml with increasing concentrations of TR9 and under the same
conditions, but
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179
in the absence of TR9. For IL-12 production, the cells are primed overnight
with IFN-_
(100 U/ml) in presence of TR9. LPS ( 10 ng/ml) is then added. Conditioned
media are
collected after 24h and kept frozen until use. Measurement of TNF-_, IL-10,
MCP-1 and
IL-8 is then performed using a commercially available ELISA kit (e.g., R & D
Systems
(Minneapolis, MN)) applying the standard protocols provided with the kit.
3. Oxidative burst. Purified monocytes are plated in 96-well plate at 2-
1x105 cell/well. Increasing concentrations of TR9 ai-e added to the wells in a
total volume
of 0.2 ml culture medium (RPMI 1640 + 10% FCS, glutamine and antibiotics).
After 3
days incubation, the plates are centrifuged and the medium is removed from the
wells. To
the macrophage monolayers, 0.2 ml per well of phenol red solution ( 140 n~IVI
NaCI, 10
mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and
19
U/ml of HRPO) is added, together with the stimulant (200 nM PMA). The plates
are
incubated at 37°C for 2 hours and the reaction is stopped by adding 20
pl 1N NaOH per
well. The absorbance is read at 610 nm. To calculate the amount of H,O,
produced by the
macrophages, a standard curve of a H,O, solution of known molarity is
performed for
each experiment.
The studies described in this example tested activity in TR9 protein. However,
one skilled in the art could easily modify the exemplified studies to test the
activity of TR9
polynucleotides (e.g., gene therapy), agonists, and/or antagonists of TR9.
It will be clear that the invention may be practiced otherwise than as
particularly
described in the foregoing description and examples.
Numerous modifications and variations of the present invention are possible in
light of the above teachings and, therefore, are within the scope of the
appended claims.
The entire disclosure of all publications (including patents, patent
applications,
journal articles, laboratory manuals, books, or other documents) cited herein
are hereby
incorporated by reference.
Further, the Sequence Listing submitted herewith, and the Sequence Listing
submitted with U.S. Provisional Application Serial No. 60/126,019, filed on
March 24,
1999; and U.S. Provisional Application Serial No. 60/134,220, filed on May 14,
1999,
in both computer and paper forms, are each hereby incorporated by reference in
its
entirety.
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180
I~-DICATIONS RELATI\G TO A DEPOSTTED i~IICROORGAIVIS>\'I
(PCT Rule l3bi.s)
A. The indications made below
relate to the microorganism referred
to in the description
on page 3 , line 24
B. H)ENTIFICATIO\OFDEPOSIT Further
deposits areidentifiedonanadditionalsheet
Nameofdepositaryinstitution American
Type Culture Collection
Address of depositay institution
lincltrdig postal code and cotw/
10801 University Boulevard
Mantissas, Virginia 20110-2209
United States of America
Date ofdeposit Accession Number
15 May 1997 209037
C. ADDITIONAL IN DICATIONS Ileaae
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information is continued on an
additional sheet
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (iJthe indications
arenot/orall designated Stated
Europe
In respect to those designations
in which a European Patent is
sought a sample of the deposited
microorganism will be made available
until the publication of the
mention of the grant of the European
patent or until the date on which
application has been refused
or withdrawn or is deemed to
be withdrawn,
only by the issue of such a sample
to an expert nominated by the
person requesting the sample
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EPC).
E. SEPARATE FURNISHING OFINDICATIONS(/eaveb/anki/notapplicabJe~
The indications listed below will
be submitted to the International
Bureau later (specythegenerolnanrreo/theindicariome.g.,
'Accession
Number of Deposit' j
For receiving Office use only For International Bureau use only
This sheet was received with the international application ~ This sheet
wasreceivedbythelntemationalBureauon:
1 s MAR 2000
Authorized officer Authorized officer
t:;r
Form PCT/RO/134 (July 199?)
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
181
ATCC Deposit No.: 209037
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The applicant requests that, until either a Canadian patent has been issued on
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The applicant hereby requests that the application has been laid open to
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the applicant with
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The applicant hereby gives notice that the furnishing of a sample of a
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FINLAND
The applicant hereby requests that, until the application has been laid open
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CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
182
ATCC Deposit No.: 209037
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SWEDEN
The applicant hereby requests that, until the application has been laid open
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Netherlands, whichever of the two dates occurs earlier.
CA 02365255 2001-09-24
- WO 00/56862 PCT/US00/06831
SEQUENCE LISTING
<110> Human Genome Sciences, Inc.
<120> Human Tumor Necrosis Factor Receptor TR9
<130> PF375PCTZ
<140> Unassigned
<141> 2000-03-16
<150> 60/126,019
<151> 1999-03-24
<150> 60/134,220
<151> 1999-05-14
<160> 27
<170> PatentIn Ver. 2.1
<210> 1
<211> 3474
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (247)..(2211)
<220>
<221> sig_peptide
<222> (247)..(366)
<220>
<221> mat~eptide
<222> (367)..(2211)
<400> 1
gcgggctgca gtcgcggcgg cttctccccg cctgggcggc cgcgccgctg ggcaggtgct 60
gagcgcccct agagcctccc ttgccgcctc cctcctctgc ccggccgcag cagtgcacat 120
ggggtgttgg aggtagatgg gctcccggcc cgggaggcgg cggtggatgc ggcgctgggc 180
agaagcagcc gccgattcca gctgccccgc gcgccccggg cgcccctgcg agtccccggt 240
tcagcc atg ggg acc tct ccg agc agc agc acc gcc ctc gcc tcc tgc 288
Met Gly Thr Ser Pro Ser Ser Ser Thr Ala Leu Ala Ser Cys
-40 -35 -30
agc cgc atc gcc cgc cga gcc aca gcc acg atg atc gcg ggc tcc ctt 336
Ser Arg Ile Ala Arg Arg Ala Thr Ala Thr Met Ile Ala Gly Ser Leu
-25 -20 -15
ctc ctg ctt gga ttc ctt agc acc acc aca get cag cca gaa cag aag 384
Leu Leu Leu Gly Phe Leu Ser Thr Thr Thr Ala Gln Pro Glu Gln Lys
-10 -5 -1 1 5
CA 02365255 2001-09-24
- WO 00/56862 PCT/US00/06831
2
gcc tcg aat ctc att ggc aca tac cgc cat gtt gac cgt gcc acc ggc 432
Ala Ser Asn Leu Ile Gly Thr Tyr Arg His Val Asp Arg Ala Thr Gly
15 20
cag gtg cta acc tgt gac aag tgt cca gca gga acc tat gtc tct gag 480
Gln Val Leu Thr Cys Asp Lys Cys Pro Ala Gly Thr Tyr Val Ser Glu
25 30 35
cat tgt acc aac aca agc ctg cgc gtc tgc agc agt tgc cct gtg ggg 528
His Cys Thr Asn Thr Ser Leu Arg Val Cys Ser Ser Cys Pro Val Gly
40 45 50
acc ttt acc agg cat gag aat ggc ata gag aaa tgc cat gac tgt agt 576
Thr Phe Thr Arg His Glu Asn Gly Ile Glu Lys Cys His Asp Cys Ser
55 60 65 70
cag cca tgc cca tgg cca atg att gag aaa tta cct tgt get gcc ttg 624
Gln Pro Cys Pro Trp Pro Met Ile Glu Lys Leu Pro Cys Ala Ala Leu
75 80 85
act gac cga gaa tgc act tgc cca cct ggc atg ttc cag tct aac get 672
Thr Asp Arg Glu Cys Thr Cys Pro Pro Gly Met Phe Gln Ser Asn Ala
90 g5 100
acc tgt gcc ccc cat acg gtg tgt cct gtg ggt tgg ggt gtg cgg aag 720
Thr Cys Ala Pro His Thr Val Cys Pro Val Gly Trp Gly Val Arg Lys
105 110 115
aaa ggg aca gag act gag gat gtg cgg tgt aag cag tgt get cgg ggt 768
Lys Gly Thr Glu Thr Glu Asp Val Arg Cys Lys Gln Cys Ala Arg Gly
120 125 130
acc ttc tca gat gtg cct tct agt gtg atg aaa tgc aaa gca tac aca 816
Thr Phe Ser Asp Val Pro Ser Ser Val Met Lys Cys Lys Ala Tyr Thr
135 140 145 150
gac tgt ctg agt cag aac ctg gtg gtg atc aag ccg ggg acc aag gag 864
Asp Cys Leu Ser Gln Asn Leu Val Val Ile Lys Pro Gly Thr Lys Glu
155 160 165
aca gac aac gtc tgt ggc aca ctc ccg tcc ttc tcc agc tcc acc tca 912
Thr Asp Asn Val Cys Gly Thr Leu Pro Ser Phe Ser Ser Ser Thr Ser
170 175 180
cct tcc cct ggc aca gcc atc ttt cca cgc cct gag cac atg gaa acc 960
Pro Ser Pro Gly Thr Ala Ile Phe Pro Arg Pro Glu His Met Glu Thr '
185 190 195
cat gaa gtc cct tcc tcc act tat gtt ccc aaa ggc atg aac tca aca 1008
His Glu Val Pro Ser Ser Thr Tyr Val Pro Lys Gly Met Asn Ser Thr
200 205 210
gaa tcc aac tct tct gcc tct gtt aga cca aag gta ctg agt agc atc 1056
Glu Ser Asn Ser Ser Ala Ser Val Arg Pro Lys Val Leu Ser Ser Ile
215 220 225 230
cag gaa ggg aca gtc cct gac aac aca agc tca gca agg ggg aag gaa 1104
Gln Glu Gly Thr Val Pro Asp Asn Thr Ser Ser Ala Arg Gly Lys Glu
235 240 245
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
3
gacgtgaac aagaccctccca aaccttcag gtagtcaaccac cagcaa 1152
AspValAsn LysThrLeuPro AsnLeuGln ValValAsnHis GlnGln
250 255 260
ggcccccac cacagacacatc ctgaagctg ctgccgtccatg gaggcc 1200
GlyProHis HisArgHisIle LeuLysLeu LeuProSerMet GluAla
265 270 275
actgggggc gagaagtccagc acgcccatc aagggccccaag agggga 1248
ThrGlyGly GluLysSerSer ThrProIle LysGlyProLys ArgGly
280 285 290
catcctaga cagaacctacac aagcatttt gacatcaatgag catttg 1296
HisProArg GlnAsnLeuHis LysHisPhe AspIleAsnGlu HisLeu
295 300 305 310
ccctggatg attgtgcttttc ctgctgctg gtgcttgtggtg attgtg 1344
ProTrpMet IleValLeuPhe LeuLeuLeu ValLeuValVal IleVal
315 320 325
gtgtgcagt atccggaaaagc tcgaggact ctgaaaaagggg ccccgg 1392
ValCysSer IleArgLysSer SerArgThr LeuLysLysGly ProArg
330 335 340
caggatccc agtgccattgtg gaaaaggca gggctgaagaaa tccatg 1440
GlnAspPro SerAlaIleVal GluLysAla GlyLeuLysLys SerMet
345 350 355
actccaacc cagaaccgggag aaatggatc tactactgcaat ggccat 1488
ThrProThr GlnAsnArgGlu LysTrpIle TyrTyrCysAsn GlyHis
360 365 370
ggtatcgat atcctgaagctt gtagcagcc caagtgggaagc cagtgg 1536
GlyIleAsp IleLeuLysLeu ValAlaAla GlnValGlySer GlnTrp
375 380 385 390
aaagatatc tatcagtttctt tgcaatgcc agtgagagggag gttget 1584
LysAspIle TyrGlnPheLeu CysAsnAla SerGluArgGlu ValAla
395 400 405
getttctcc aatgggtacaca gccgaccac gagcgggcctac gcaget 1632
AlaPheSer AsnGlyTyrThr AlaAspHis GluArgAlaTyr AlaAla
410 415 420
ctgcagcac tggaccatccgg ggccccgag gccagcctcgcc cagcta 1680
LeuGlnHis TrpThrIleArg GlyProGlu AlaSerLeuAla GlnLeu
425 430 435
attagcgcc ctgcgccagcac cggagaaac gatgttgtggag aagatt 1728
IleSerAla LeuArgGlnHis ArgArgAsn AspValValGlu LysIle
440 445 450
cgtgggctg atggaagacacc acccagctg gaaactgacaaa ctaget 1776
ArgGlyLeu MetGluAspThr ThrGlnLeu GluThrAspLys LeuAla
455 460 465 470
ctcccgatg agccccagcccg cttagcccg agccccatcccc agcccc 1824
LeuProMet SerProSerPro LeuSerPro SerProIlePro SerPro
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
4
475 480 485
aacgcgaaacttgag aattcc getctcctgacg gtggagccttcc cca 1872
AsnAlaLysLeuGlu AsnSer AlaLeuLeuThr ValGluProSer Pro
490 495 500
caggacaagaacaag ggcttc ttcgtggatgag tcggagcccctt ctc 1920
GlnAspLysAsnLys GlyPhe PheValAspGlu SerGluProLeu Leu
505 510 515
cgctgtgactctaca tccagc ggctcctccgcg ctgagcaggaac ggt 1968
ArgCysAspSerThr SerSer GlySerSerAla LeuSerArgAsn Gly
520 525 530
tcctttattaccaaa gaaaag aaggacacagtg ttgcggcaggta cgc 2016
SerPheIleThrLys GluLys LysAspThrVal LeuArgGlnVal Arg
535 540 545 550
ctggacccctgtgac ttgcag cctatctttgat gacatgctccac ttt 2064
LeuAspProCysAsp LeuGln ProIlePheAsp AspMetLeuHis Phe
555 560 565
ctaaatcctgaggag ctgcgg gtgattgaagag attccccagget gag 2112
LeuAsnProGluGlu LeuArg ValIleGluGlu IleProGlnAla Glu
570 575 580
gacaaactagaccgg ctattc gaaattattgga gtcaagagccag gaa 2160
AspLysLeuAspArg LeuPhe GluIleIleGly ValLysSerGln Glu
585 590 595
gccagccagaccctc ctggac tctgtttatagc catcttcctgac ctg 2208
AlaSerGlnThrLeu LeuAsp SerValTyrSer HisLeuProAsp Leu
600 605 610
ctgtagaacatag ggatactgca tactcaattt agtggcaggg
2261
ttctggaaat
Leu
615
tggtttttta attttcttct gtttctgatt tttgttgttt ggggtgtgtg tgtgtgtttg 2321
tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tttaacagag aatatggcca 2381
gtgcttgagt tctttctcct tctctctctc tttttttttt aaataactct tctgggaagt 2441
tggtttataa gcctttgcca ggtgtaactg ttgtgaaata cccaccacta aagtttttta 2501
agttccatat tttctccatt ttgccttctt atgtattttc gagattattc tgtgcacttt 2561
aaatttactt aacttaccat aaatgcagtg tgacttttcc cacacactgg attgtgaggc 2621
tcttaacttc ttaaaagtat aatggcatct tgtgaatcct ataagcagtc tttatgtctc 2681
ttaacattca cacctacttt ttaaaaacaa atattattac tatttttatt attgtttgtc 2741
ctttataaat tttcttaaag attaagaaaa tttaagaccc cattgagtta ctgtaatgca 2801
attcaacttt gagttatctt ttaaatatgt cttgtatagt tcatattcat ggctgaaact 2861
tgaccacact attgctgatt gtatggtttt cacctggaca ccgtgtagaa tgcttgatta 2921
CA 02365255 2001-09-24
- WO 00/56862 PCTNS00/06831
cttgtactct tcttatgcta atatgctctg ggctggagaa atgaaatcct caagccatca 2981
ggatttgcta tttaagtggc ttgacaactg ggccaccaaa gaacttgaac ttcacctttt 3041
aggatttgag ctgttctgga acacattgct gcactttgga aagtcaaaat caagtgccag 3101
tggcgccctt tccatagaga atttgcccag ctttgcttta aaagatgtct tgttttttat 3161
atacacataa tcaataggtc caatctgctc tcaaggcctt ggtcctggtg ggattccttc 3221
accaattact ttaattaaaa atggctgcaa ctgtaagaac ccttgtctga tatatttgca 3281
actatgctcc catttacaaa tgtaccttct aatgctcagt tgccaggttc caatgcaaag 3341
gtggcgtgga ctccctttgt gtgggtgggg tttgtgggta gtggtgaagg accgatatca 3401
gaaaaatgcc ttcaagtgta ctaatttatt aataaacatt aggtgtttgt taaaaaaaaa 3461
aaaaaaaaaa aaa 3474
<210> 2
<211> 655
<212> PRT
<213> Homo sapiens
<400> 2
Met Gly Thr Ser Pro Ser Ser Ser Thr Ala Leu Ala Ser Cys Ser Arg
-40 -35 -30 -25
Ile Ala Arg Arg Ala Thr Ala Thr Met Ile Ala Gly Ser Leu Leu Leu
-20 -15 -10
Leu Gly Phe Leu Ser Thr Thr Thr Ala Gln Pro Glu Gln Lys Ala Ser
_5 _1 1 5
Asn Leu Ile Gly Thr Tyr Arg His Val Asp Arg Ala Thr Gly Gln Val
15 20
Leu Thr Cys Asp Lys Cys Pro Ala Gly Thr Tyr Val Ser Glu His Cys
25 30 35 40
Thr Asn Thr Ser Leu Arg Val Cys Ser Ser Cys Pro Val Gly Thr Phe
45 50 55
Thr Arg His Glu Asn Gly Ile Glu Lys Cys His Asp Cys Ser Gln Pro
60 65 70
Cys Pro Trp Pro Met Ile Glu Lys Leu Pro Cys Ala Ala Leu Thr Asp
75 80 85
Arg Glu Cys Thr Cys Pro Pro Gly Met Phe Gln Ser Asn Ala Thr Cys
g0 g5 100
Ala Pro His Thr Val Cys Pro Val Gly Trp Gly Val Arg Lys Lys Gly
105 110 115 120
Thr Glu Thr Glu Asp Val Arg Cys Lys Gln Cys Ala Arg Gly Thr Phe
CA 02365255 2001-09-24
- WO 00/56862 PCT/US00/06831
6
125 130 135
Ser Asp Val Pro Ser Ser Val Met Lys Cys Lys Ala Tyr Thr Asp Cys
140 145 150
Leu Ser Gln Asn Leu Val Val Ile Lys Pro Gly Thr Lys Glu Thr Asp
155 160 165
Asn Val Cys Gly Thr Leu Pro Ser Phe Ser Ser Ser Thr Ser Pro Ser
170 175 180
Pro Gly Thr Ala Ile Phe Pro Arg Pro Glu His Met Glu Thr His Glu
185 190 195 200
Val Pro Ser Ser Thr Tyr Val Pro Lys Gly Met Asn Ser Thr Glu Ser
205 210 215
Asn Ser Ser Ala Ser Val Arg Pro Lys Val Leu Ser Ser Ile Gln Glu
220 225 230
Gly Thr Val Pro Asp Asn Thr Ser Ser Ala Arg Gly Lys Glu Asp Val
235 240 245
Asn Lys Thr Leu Pro Asn Leu Gln Val Val Asn His Gln Gln Gly Pro
250 255 260
His His Arg His Ile Leu Lys Leu Leu Pro Ser Met Glu Ala Thr Gly
265 270 275 280
Gly Glu Lys Ser Ser Thr Pro Ile Lys Gly Pro Lys Arg Gly His Pro
285 290 295
Arg Gln Asn Leu His Lys His Phe Asp Ile Asn Glu His Leu Pro Trp
300 305 310
Met Ile Val Leu Phe Leu Leu Leu Val Leu Val Val Ile Val Val Cys
315 320 325
Ser Ile Arg Lys Ser Ser Arg Thr Leu Lys Lys Gly Pro Arg Gln Asp
330 335 340
Pro Ser Ala Ile Val Glu Lys Ala Gly Leu Lys Lys Ser Met Thr Pro
345 350 355 360
Thr Gln Asn Arg Glu Lys Trp Ile Tyr Tyr Cys Asn Gly His Gly Ile
365 370 375
Asp Ile Leu Lys Leu Val Ala Ala Gln Val Gly Ser Gln Trp Lys Asp
380 385 390
Ile Tyr Gln Phe Leu Cys Asn Ala Ser Glu Arg Glu Val Ala Ala Phe
395 400 405
Ser Asn Gly Tyr Thr Ala Asp His Glu Arg Ala Tyr Ala Ala Leu Gln
410 415 420
His Trp Thr Ile Arg Gly Pro Glu Ala Ser Leu Ala Gln Leu Ile Ser
425 430 435 440
CA 02365255 2001-09-24
- WO 00/56862 PCT/US00/06831
7
Ala Leu Arg Gln His Arg Arg Asn Asp Val Val Glu Lys Ile Arg Gly
445 450 455
Leu Met Glu Asp Thr Thr Gln Leu Glu Thr Asp Lys Leu Ala Leu Pro
460 465 470
Met Ser Pro Ser Pro Leu Ser Pro Ser Pro Ile Pro Ser Pro Asn Ala
475 480 485
Lys Leu Glu Asn Ser Ala Leu Leu Thr Val Glu Pro Ser Pro Gln Asp
490 495 500
Lys Asn Lys Gly Phe Phe Val Asp Glu Ser Glu Pro Leu Leu Arg Cys
505 510 515 520
Asp Ser Thr Ser Ser Gly Ser Ser Ala Leu Ser Arg Asn Gly Ser Phe
525 530 535
Ile Thr Lys Glu Lys Lys Asp Thr Val Leu Arg Gln Val Arg Leu Asp
540 545 550
Pro Cys Asp Leu Gln Pro Ile Phe Asp Asp Met Leu His Phe Leu Asn
555 560 565
Pro Glu Glu Leu Arg Val Ile Glu Glu Ile Pro Gln Ala Glu Asp Lys
570 575 580
Leu Asp Arg Leu Phe Glu Ile Ile Gly Val Lys Ser Gln Glu Ala Ser
585 590 595 600
Gln Thr Leu Leu Asp Ser Val Tyr Ser His Leu Pro Asp Leu Leu
605 610 615
<210> 3
<211> 281
<212> PRT
<213> Homo sapiens
<400> 3
Met Leu Gly Ile Trp Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala
1 5 10 15
Arg Leu Ser Ser Lys Ser Val Asn Ala Gln Val Thr Asp Ile Asn Ser
20 25 30
Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Va1 Glu Thr Gln Asn
35 40 45
Leu Glu Gly Leu His His Asp Gly Gln Phe Cys His Lys Pro Cys Pro
50 55 60
Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro
65 70 75 80
Asp Cys Val Pro Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His
85 90 95
CA 02365255 2001-09-24
- WO 00/56862 PCT/US00/06831
8
Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly
100 105 110
Leu Glu Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg
115 120 125
Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp
130 135 140
Pro Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr
145 150 155 160
Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn Leu Gly Trp
165 170 175
Leu Cys Leu Leu Leu Leu Pro Ile Pro Leu Ile Val Trp Val Lys Arg
180 185 190
Lys Glu Val Gln Lys Thr Cys Arg Lys His Arg Lys Glu Asn Gln Gly
195 200 205
Ser His Glu Ser Pro Thr Leu Asn Pro Glu Thr Val Ala Ile Asn Leu
210 215 220
Ser Asp Val Asp Leu Ser Lys Tyr Ile Thr Thr Ile Ala Gly Val Met
225 230 235 240
Thr Leu Ser Gln Val Lys Ala Asn Leu Cys Thr Leu Ala Glu Lys Ile
245 250 255
Gln Thr Ile Ile Leu Lys Asp Ile Thr Ser Asp Ser Glu Asn Ser Asn
260 265 270
Phe Arg Asn Glu Ile Gln Ser Leu Val
275 280
<210> 4
<211> 455
<212> PRT
<213> Homo Sapiens
<400> 4
Met Gly Ala Gly Ala Thr Gly Arg Ala Met Asp Gly Pro Arg Leu Leu
1 5 10 15
Leu Leu Leu Leu Leu Gly Val Ser Leu Gly Gly Ala Lys Glu Ala Cys
20 25 30
Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys Lys Ala Cys Asn
35 40 45
Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn Gln Thr Val Cys
50 55 60
Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val Val Ser Ala Thr
65 70 75 80
Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu Gln Ser Met Ser
CA 02365255 2001-09-24
- WO 00/56862 PCT/US00/06831
9
85 90 95
Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg Cys Ala Tyr Gly
100 105 110
Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala Cys Arg Val Cys
115 120 125
Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp Lys Gln Asn Thr
130 135 140
Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp Glu Ala Asn His
145 150 155 160
Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp Thr Glu Arg Gln
165 170 175
Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys Glu Glu Ile Pro
180 185 190
Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu Gly Ser Asp Ser Thr
195 200 205
Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu Gln Asp Leu Ile
210 215 220
Ala Ser Thr Val Ala Gly Val Val Thr Thr Val Met Gly Ser Ser Gln
225 230 235 240
Pro Val Val Thr Arg Gly Thr Thr Asp Asn Leu Ile Pro Val Tyr Cys
245 250 255
Ser Ile Leu Ala Ala Val Val Val Gly Leu Val Ala Tyr Ile Ala Phe
260 265 270
Lys Arg Trp Asn Ser Cys Lys Gln Asn Lys Gln Gly Ala Asn Ser Arg
275 280 285
Pro Val Asn Gln Thr Pro Pro Pro Glu Gly Glu Lys Leu His Ser Asp
290 295 300
Ser Gly Ile Ser Val Asp Ser Gln Ser Leu His Asp Gln Gln Pro His
305 310 315 320
Thr Gln Thr Ala Ser Gly Gln Ala Leu Lys Gly Asp Gly Gly Leu Tyr
325 330 335
Ser Ser Leu Pro Pro Ala Lys Arg Glu Glu Val Glu Lys Leu Leu Asn
340 345 350
Gly Ser Ala Gly Asp Thr Trp Arg His Leu Ala Gly Glu Leu Gly Tyr
355 360 365
Gln Pro Glu His Ile Asp Ser Phe Thr His Glu Ala Cys Pro Val Arg
370 375 380
Ala Leu Leu Ala Ser Trp Ala Thr Gln Asp Ser Ala Thr Leu Pro Arg
385 390 395 400
CA 02365255 2001-09-24
- WO 00/56862 PCT/US00/06831
Arg Glu Val Ala Pro Pro Tyr Gln Gly Ala Asp Pro Ile Leu Ala Thr
405 410 415
Ala Leu Ala Ser Asp Pro Ile Pro Asn Pro Leu Gln Lys Trp Glu Asp
420 425 430
Ser Ala His Lys Pro Gln Ser Leu Asp Thr Asp Asp Pro Cys Ser Glu
435 440 445
Ser Thr Ala Thr Ser Pro Val
450 455
<210> 5
<211> 455
<212> PRT
<213> Homo Sapiens
<400> 5
Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu
1 5 10 15
Glu Leu Leu Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro
25 30
His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys
35 40 45
Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys
50 55 60
Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp
65 70 75 80
Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu
85 90 95
Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val
100 105 110
Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg
115 120 125
Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe
130 135 140
Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu
145 150 155 160
Lys Gln Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu
165 170 175
Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr
180 185 190
Lys Leu Cys Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser
195 200 205
Gly Thr Thr Val Leu Leu Pro Leu Val Ile Phe Phe Gly Leu Cys Leu
CA 02365255 2001-09-24
- WO 00/56862 PCT/US00/06831
11
210 215 220
Leu Ser Leu Leu Phe Ile Gly Leu Met Tyr Arg Tyr Gln Arg Trp Lys
225 230 235 240
Ser Lys Leu Tyr Ser Ile Val Cys Gly Lys Ser Thr Pro Glu Lys Glu
245 250 255
Gly Glu Leu Glu Gly Thr Thr Thr Lys Pro Leu Ala Pro Asn Pro Ser
260 265 270
Phe Ser Pro Thr Pro Gly Phe Thr Pro Thr Leu Gly Phe Ser Pro Val
275 280 285
Pro Ser Ser Thr Phe Thr Ser Ser Ser Thr Tyr Thr Pro Gly Asp Cys
290 295 300
Pro Asn Phe Ala Ala Pro Arg Arg Glu Val Ala Pro Pro Tyr Gln Gly
305 310 315 320
Ala Asp Pro Ile Leu Ala Thr Ala Leu Ala Ser Asp Pro Ile Pro Asn
325 330 335
Pro Leu Gln Lys Trp Glu Asp Ser Ala His Lys Pro Gln Ser Leu Asp
340 345 350
Thr Asp Asp Pro Ala Thr Leu Tyr Ala Val Val Glu Asn Val Pro Pro
355 360 365
Leu Arg Trp Lys Glu Phe Val Arg Arg Leu Gly Leu Ser Asp His Glu
370 375 380
Ile Asp Arg Leu Glu Leu Gln Asn Gly Arg Cys Leu Arg Glu Ala Gln
385 390 395 400
Tyr Ser Met Leu Ala Thr Trp Arg Arg Arg Thr Pro Arg Arg Glu Ala
405 410 415
Thr Leu Glu Leu Leu Gly Arg Val Leu Arg Asp Met Asp Leu Leu Gly
420 425 430
Cys Leu Glu Asp Ile Glu Glu Ala Leu Cys Gly Pro Ala Ala Leu Pro
435 440 445
Pro Ala Pro Ser Leu Leu Arg
450 455
<210> 6
<211> 365
<212> DNA
<213> Homo sapiens
<400> 6
tcggctgtgt acccattgga gaaagcagca acctccctct cactggcatt gcaaagaaac 60
tgatagatat ctttccactg gcttcccact tgggctgcta caagcttcag gatatcgata 120
ccatggccat tgcagtagta gatccatttt cccggttctg ggttggagtc atggattttt 180
cagccctgcc ttttccacaa tggcactggg atcctgccgg ggcccctttt tagagtcctc 240
gagcttttcc ggatactgca caccacaatc accacaagca ccagcagcag gaaaagcaca 300
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
12
atcatccagg gcaaatgctc attgatgtca aaatgcttgt gtaggttctg tctaggatgt 360
cccct 365
<210> 7
<211> 378
<212> DNA
<213> Homo Sapiens
<400> 7
agaaacgatg ttgtggagaa gattcgtggg ctgatggaag acaccaccca gctggaaact 60
gacaaactag ctctcccgat gagccccagc ccgcttagcc cgagccccat ccccagcccc 120
aacgcgaaac ttgagaattc cgctctcctg acggtggagc cttccccaca ggacaagaac 180
aagggcttct tcgtggatga gtcggagccc cttctccgct gtactctaca tccagcggct 240
cctccgcgct gagcaggaac ggttccttta ttaccaaaga aaagaaggac acagtgttgc 300
ggcaggtacg cctggacccc tgtaaatttg cagcctatct ttgattgaca tgttccactt 360
tctaaatcct gaggagtt 378
<210> 8
<211> 345
<212> DNA
<213> Homo sapiens
<400> 8
ggcagaggca caaggtaatt tctcaatcat tggccatggg catggctgac tacagtcatg 60
gcatttctct atgccattct catgcctggt aaaggtcccc acagggcaac tgctgacaga 120
cgcgcggctt gtgttggtac atgctcagag acataggttc ctgctggaca cttgtcacag 180
gttagcacct agccggtggc acggtcaaca tggcggtatg tgccaatgag attcgaggcc 240
ttctgttctg gctgagctgt ggtggtgcta aggaatccaa gcggagaagg gagcccagat 300
catcgtggct gtggctggcg ggcgatgcgg ttcaggaggc cgagg 345
<210> 9
<211> 316
<212> DNA
<213> Homo Sapiens
<400> 9
gctaattagc gccctgccag accggagaaa cgatgtttgg agaagattcg tgggctgatg 60
gaagacacca cccagctgga aactgacaaa ctagctctcc cgatgagccc cagcccgctt 120
agcccgagcc ccatccccag ccccaacgcg aaacttgaga attccgctct cctgacggtg 180
gagcctttcc cacaggacaa gaacaagggc ttcttcgtgg atgagtcgga gccccttctc 240
cgctgtactc tacatccagc ggctcctccg gctgagcagg aacggttcct ttattaccaa 300
gaaaagaagg acacag 316
<210> 10
<211> 489
<212> DNA
<213> Homo Sapiens
<400> 10
aattcggcac gaggaatcct ataagcagtc tttatgtctc ttaacattca cacctacttt 60
ttaaaaacaa atattattac tatttttatt attgtttgtc ctttataaat tttcttaaag 120
attaagaaaa tttaagaccc cattgagtta ctgtaatgca attcaacttt gagttatctt 180
ttaaatatgt cttgtatagt tcatattcat ggctgaaact tgaccacact attgctgatt 240
gtatggttca cctggcaccg tgtagatgct tgattacttg tactctctta tgtaaatgct 300
ctgggctggg gaatgaatcc caggctcagg tttccctatt aaggggttca ctggccccaa 360
CA 02365255 2001-09-24
- WO 00/56862 PCT/US00/06831
13
gactgactcc cttggggttg ggtttggaca atgtcttggg agaaaagccg gggcttccag 420
ggttcccctt gtaagggttt taaaaaaaag ccattctgag ctcgccgggg tcccatttaa 480
489
aagggcccg
<210> 11
<211> 26
<212> DNA
<213> Homo Sapiens
<400> 11
cgcccatggc tcagccagaa cagaag 26
<210> 12
<211> 28
<212> DNA
<213> Homo Sapiens
<400> 12
cgcaagcttt. tagggcaaat gctcattg 28
<210> 13
<211> 33
<212> DNA
<213> Homo Sapiens
<400> 13
cgccccgggg ccatcatggg gacctctccg agc 33
<210> 14
<211> 28
<212> DNA
<213> Homo sapiens
<400> 14
cgcggtacct tagggcaaat gctcattg 28
<210> 15
<211> 55
<212> DNA
<213> Homo Sapiens
<400> 15
cgctctagat caagcgtagt ctgggacgtc gtatgggtag ggcaaatgct cattg 55
<210> 16
<211> 31
<212> DNA
<213> Homo Sapiens
<400> 16
ggaagatctg ccagaacaga aggcctcgaa t 31
CA 02365255 2001-09-24
- WO 00/56862 PCT/US00/06831
14
<210> 17
<211> 31
<212> DNA
<213> Homo Sapiens
<400> 17
ccatcttcct gacctgctgt agtctagagc c 31
<210> 18
<211> 30
<212> DNA
<213> Homo Sapiens
<400> 18
gccgaccacg agcgggccta gtctagagcc 30
<210> 19 .
<211> 147
<212> PRT
<213> Homo Sapiens
<400> 19
Thr Cys Asp Lys Cys Pro Ala Gly Thr Tyr Val Ser Glu His Cys Thr
1 5 10 15
Asn Thr Ser Leu Arg Val Cys Ser Ser Cys Pro Val Gly Thr Phe Thr
20 25 30
Arg His Glu Asn Gly Ile Glu Lys Cys His Asp Cys Ser Gln Pro Cys
35 40 45
Pro Trp Pro Met Ile Glu Lys Leu Pro Cys Ala Ala Leu Thr Asp Arg
50 55 60
Glu Cys Thr Cys Pro Pro Gly Met Phe Gln Ser Asn Ala Thr Cys Ala
65 70 75 80
Pro His Thr Val Cys Pro Val Gly Trp Gly Val Arg Lys Lys Gly Thr
85 90 95
Glu Thr Glu Asp Val Arg Cys Lys Gln Cys Ala Arg Gly Thr Phe Ser
100 105 110
Asp Val Pro Ser Ser Val Met Pro Cys Lys Ala Tyr Thr Asp Cys Leu
115 120 125
Ser Gln Asn Leu Val Val Ile Lys Pro Gly Thr Lys Glu Thr Asp Asn
130 135 140
Val Cys Gly
145
<210> 20
<211> 147
<212> PRT
<213> Homo sapiens
CA 02365255 2001-09-24
- WO 00/56862 PCT/US00/06831
<400> 20
Leu Cys Asp Lys Cys Pro Pro Gly Thr Tyr Leu Lys Gln His Cys Thr
1 5 10 15
Ala Lys Trp Lys Thr Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr
25 30
Asp Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys
35 40 45
Lys Glu Leu Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg
50 55 60
Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu
65 70 75 80
Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr
85 90 95
Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe Ser
100 105 110
Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn Cys Ser
115 120 125
Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr His Asp Asn
130 135 140
Ile Cys Ser
145
<210>
21
<211>
67
<212>
PRT
<213>
Homo
Sapiens
<400>
21
Gln Trp Asp Tyr PheLeuCysAsn AlaSerGlu ArgGlu
Lys Ile Gln
1 5 10 15
Val Ala Phe Asn TyrThrAlaAsp HisGluArg AlaTyr
Ala Ser Gly
20 25 30
Ala Ala Gln Trp IleArgGlyPro GluAlaSer LeuAla
Leu His Thr
35 40 45
Gln Leu Ser Leu GlnHisArgArg AsnAspVal ValGlu
Ile Ala Arg
50 55 60
Lys Ile
Arg
65
<210> 22
<211> 68
<212> PRT
<213> Homo Sapiens
a
CA 02365255 2001-09-24
- WO 00/56862 PCT/US00/06831
16
<400> 22
Gln Val Lys Gly Phe Val Arg Lys Asn Gly Val Asn Glu Ala Lys Ile
1 5 10 15
Asp Glu Ile Lys Asn Asp Asn Val Gln Asp Thr Ala Glu Gln Lys Val
20 25 30
Gln Leu Leu Arg Asn Trp His Gln Leu His Gly Lys Lys Glu Ala Tyr
35 40 45
Asp Thr Leu Ile Lys Asp Leu Lys Lys Ala Asn Leu Cys Thr Leu Ala
50 55 60
Glu Lys Ile Gln
<210> 23
<211> 68
<212> PRT
<213> Homo Sapiens
<400> 23
Arg Trp Lys Glu Phe Val Arg Arg Leu Gly Leu Ser Asp His Glu Ile
1 5 10 15
Asp Arg Leu Glu Leu Gln Asn Gly Arg Cys Leu Arg Glu Ala Gln Tyr
20 25 30
Ser Met Leu Ala Thr Trp Arg Arg Arg Thr Arg Arg Glu Ala Thr Leu
35 40 45
Glu Leu Leu Gly Arg Val Leu Arg Asp Met Asp Leu Leu Gly Cys Leu
50 55 60
Glu Asp Ile Glu
<210> 24
<211> 65
<212> PRT
<213> Homo Sapiens
<400> 24
Arg Trp Lys Glu Phe Val Arg Thr Leu Gly Leu Arg Glu Ala Glu Ile
5 10 15
Glu Ala Val Glu Val Glu Ile Gly Arg Phe Arg Asp Gln Gln Tyr Glu
20 25 30
Met Leu Lys Arg Trp Arg Gln Gln Gln Pro Ala Gly Leu Gly Ala Val
35 40 45
Tyr Ala Ala Leu Glu Arg Met Gly Leu Asp Gly Cys Val Glu Asp Leu
50 55 60
Arg
CA 02365255 2001-09-24
- WO 00/56862 PCT/US00/06831
17
65
<210>
25
<211>
67
<212>
PRT
<213> Sapiens
Homo
<400>
25
Ser Trp Gln LeuMetArg GlnLeuAsp LeuThrLysAsn GluIle
Asp
1 5 10 15
Asp Val Arg AlaGlyThr AlaGlyPro GlyAspAlaLeu TyrAla
Val
20 25 30
Met Leu Lys TrpValAsn LysThrGly ArgAsnAlaSer IleHis
Met
35 40 45
Thr Leu Asp AlaLeuGlu ArgMetGlu GluArgHisAla LysGlu
Leu
50 55 60
Lys Ile
Gln
65
<210> 26
<211> 67
<212> PRT
<213> Homo
Sapiens
<400> 26
Ser Trp Glu LeuMetArgLys LeuGlyLeu MetAspAsn GluIle
Pro
1 5 10 15
Lys Val Ala AlaGluAlaAla GlyHisArg AspThrLeu TyrThr
Lys
20 25 30
Met Leu Ile TrpValAsnLys ThrGlyArg AspAlaSer ValHis
Lys
35 40 45
Thr Leu Leu AlaLeuGluThr LeuGlyGlu ArgLeuAla LysGln
Asp
50 55 60
Lys Ile Glu
65
<210> 27
<211> 733
<212> DNA
<213> Homo
Sapiens
<400> 27
gggatccgga gcccaaatct tctgacaaaa ctcacacatg cccaccgtgc ccagcacctg 60
aattcgaggg tgcaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga 120
tctcccggac tcctgaggtc acatgcgtgg tggtggacgt aagccacgaa gaccctgagg 180
tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg 240
aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact 300
ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca acccccatcg 360
CA 02365255 2001-09-24
WO 00/56862 PCT/US00/06831
18
agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc 420
catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct 480
atccaagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga 540
ccacgcctcc cgtgctggac tccgacggct cettcttcct ctacagcaag ctcaccgtgg 600
acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc 660
acaaccacta cacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccgc 720
gactctagag gat 733
