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CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
Antibodies that Imnaunospeci~ZCally Bind to TRAIL Receptors
Field of the Inyention
[0001] The present invention relates to antibodies and related molecules that
immunospecifically bind to TRAIL receptors. Such antibodies have uses, for
example, in
the prevention and treatment of cancers and other proliferative disorders. The
invention
also relates to nucleic acid molecules encoding anti-TRAIL receptor
antibodies, vectors
and host cells containing these nucleic acids, and methods for producing the
same. The
present invention relates to methods and compositions for preventing,
'detecting,
diagnosing, treating or ameliorating a disease or disorder, especially cancer
and other
hyperproliferative disorders, comprising administering to an animal,
preferably a human,
an effective amount of one or more antibodies or fragments or variants
thereof, or related
molecules, that immunospecifically bind to TRAIL receptor.
Background of the Invention
[0002] 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.
[0003] 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.
[0004] Among the ligands there are included TNF-a, lymphotoxin-oc (LT- oc,
also
known as TNF-(3), LT-(3 (found in complex heterotrimer LT-a2-(3), 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
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CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
protein, FAS antigen or APO-1, CD40, CD27, CD30, 4-1BB, OX40, low affinity p75
and
NGF-receptor (Meager, A., Biologicals, 22:291-295 (1994)).
[0005] 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).
[0006] 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, R., et
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, R.C. 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, K.F. et
al., Cell 69:737 (1992)).
[0007] TNF and LT-a 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-a,
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-oc are involved in the pathogenesis of a wide range of diseases,
including
endotoxic shock, cerebral malaria, tumors, autoimmune disease, AmS and graft-
host
rejection (Beutler, B. and Von Huffel, C., Science 264:667-668 (1994)).
Mutations in the
p55 Receptor cause increased susceptibility to microbial infection.
[0008] Moreover, an about 80 amino acid domain near the C-terminus of TNFRl
(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)).
[0009] Apoptosis, or programmed cell death, is a physiologic process essential
to
the normal development and homeostasis of multicellular organisms (H. 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 (C.B. Thompson, Science 267, 1456-1462 (1995)).
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CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
Recently, much attention has focused on the signal transduction and biological
function of
two cell surface death receptors, Fas/APO-1 and TNFR-1 (J.L. Cleveland, et
al., CeIL 81,
479-482 (1995); A. Fraser, et al., Cell 85, 781-784 (1996); S. Nagata, et al.,
Science 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 (C.A. Smith, et al.,
Science
248, 1019-23 (1990); M. Tewari, et al., in Modular Texts in Molecular and 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 Drosophila
suicide gene,
reaper (P. Golstein, et al., Cell 81, 185-6 (1995); K. 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 the death domain-containing adapter molecule FADD/MORT1
(A.M.
Chinnaiyan, et al., Cell 81, 505-12 (1995); M. P. Boldin, et al., J. Biol Chem
270, 7795-8
(1995); F.C. I~ischkel, 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 (M. Muzio et al., Cell 85, 817-827 (1996); M.P.
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-kB (L.A. Tartaglia, et al., Imnauf~ol Today 13, 151-3
(1992)).
Accordingly, TNFR-1 recruits the multivalent adapter molecule TRADD, which
like
FADD, also contains a death domain (H. Hsu, et al., Cell 81, 495-504 (1995);
H. 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-kB activation (H. Hsu, et al., Cell 84, 299-308 (1996); H. Hsu, et al.,
Immunity 4, 387-
396 (1996)).
[0010] One TNF-related apoptosis inducing ligand has been reported by several
groups and ghas been acribed the name Apoptosis Inducing Molecule I (AIM-I)
(Intenation Application No. WO 97/33899) and TNF-related apoptosis-inducing
ligand or
(TRAIL) (Whey, S.R. et al., Immunity 3:673-682 (1995)). Pitti, R.M. et al.,
refer to the
new molecule as Apo-2 ligand or ("Apo-2L"). For convenience, it will be
referred to
herein as TRAIL.
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[0011] Unlike FAS ligand whose transcripts appear to be largely restricted to
stimulated T-cells, significant levels of TRAIL are seen in many tissues, and
it is
constitutively transcribed by some cell lines. It has been shown that TRAIL
acts
independently from FAS ligand (Wiley, S.R., et al. (1995)), supra). Studies by
Marsters,
S.A. et al., have indicated that TRAIL activates apoptosis rapidly, within a
time frame that
is similar to death signalling by FAS/Apo-1L but much faster than TNF-induced
apoptosis
(Current Biology, 6:750-752 (1996)).
[0012] At least four TRAIL receptors have been identified, including TRAIL
receptor
1 (TRAIL-R1, also referred to as TR4, and death receptor 4 (DR4), Pan et al.,
Science
276:111-3 (1997), International Patent Application Nos. WO 98/32856,
WOOO/67793,
WO 99/37684, WO 2000/34355, WO 99/02653, SEQ ID NO:l); TRAIL receptor 2
(TRAIL-R2, also referred to as TR7, DRS, and FILLER, Pan et al., Science
277:815-8
(1997), Sheridan et al., Science 277:818-21 (1997), Chaudhury et al., Immunity
7:821-30
(1997), International Patent Application Nos. WO 98/46643, WO 99/09165, WO
99/11791, WO 98/41629, W000/66156, and WO 98/35986, SEQ ID N0:3); TRAIL
receptor 3 (TRAIL-R3, also referred to as TRS, decoy receptor 1 (DcRl) and
TRID)
(Degli-Esposti et al., J. Exp. Med. 186:1165-70 (1997), International Patent
Application
Nos. W098/30693, W00071150, WO 99100423, EP867509, WO 98/58062, SEQ ID
NO:2); and TRAIL Receptor 4 (TRAIL-R4, also referred to as TR10, DcR2, and
TRUNDD, Pan et al., FEBS Lett. 424:41-5 (1998), Degli-Eposti et al., Immunity
7:813-20
(1997), International Patent Application Nos. WO 98/54202, WO00/73321, WO
2000/08155, WO 99/03992, WO 2000/34355 and W09910484, SEQ ID N0:4). TRAIL
receptors 1 and 2 contain death domains in their cytoplasmic tails and the
triggering of
these receptors results in apoptosis. On the other hand TRAIL receptor 3 and 4
inhibit
apoptosis induced by the cytotoxic ligand TRAIL in part because of their
absent or
truncated cytoplasmic death domains, respectively. Each of the publications
and patents
cited above is hereby incorporated by reference in their entireties.
[0013] The effects of TNF family ligands and TNF family 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 compositions, such as antibodies, that influence the
biological activity
of TNF receptors, both normally and in disease states. In particular, there is
a need to
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WO 02/079377 PCT/USO1/42996
isolate and characterize antibodies that modulate the biological activities of
TRAIL
receptors.
Summary of the Invention
[0014] The present invention encompasses antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind to a TRAIL receptor polypeptide or polypeptide
fragment or
variant of a TRAIL receptor. In particular, the invention encompasses
antibodies
(including molecules comprising, or alternatively consisting of, antibody
fragments or
variants thereof) that immunospecifically bind to a polypeptide or polypeptide
fragment or
variant of human TRAIL receptors such as those of SEQ ID NOS:l-4.
[0015] The present invention relates to methods and compositions for
preventing,
treating or ameliorating a disease or disorder comprising administering to an
animal,
preferably a human, an effective amount of one or more antibodies or fragments
or
variants thereof, or related molecules, that immunospecifically bind to a
TRAIL receptor
or a fragment or variant thereof. In specific embodiments, the present
invention relates to
methods and compositions for preventing, treating or ameliorating a disease or
disorder
associated with TRAIL receptor function or TRAIL receptor ligand function or
aberrant
TRAIL receptor or TRAIL receptor ligand expression, comprising administering
to an
animal, preferably a human, an effective amount of one or more antibodies or
fragments or
variants thereof, or related molecules, that immunospecifically bind to a
TRAIL receptor
or a fragment or variant thereof. In highly preferred embodiments, the present
invention
relates to antibody-based methods and compositions for preventing, treating or
ameliorating cancers and other hyperproliferative disorders (e.g., leukemia,
carcinoma,
and lymphoma). Other diseases and disorders which can be treated, prevented or
ameliorated with the antibodies of the invention include, but are not limited
to,
neurodegenerative disorders (e.g., Parkinson's disease, Alzheimer's disease,
and
Huntington's disease), immune disorders (e.g., lupus, rheumatoid arthritis,
multiple
sclerosis, myasthenia gravis, Hashimoto's disease, and immunodeficiency
syndrome),
inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid
arthritis),
infectious diseases (e.g., AIDS, herpes viral infections, and other viral
infections) and
proliferative disorders.
CA 02426710 2003-04-23
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[0016] The present invention also encompasses methods and compositions for
detecting, diagnosing, or prognosing diseases or disorders comprising
administering to an
animal, preferably a human, an effective amount of one or more antibodies or
fragments or
variants thereof, or related molecules, that immunospecifically bind to TRAIL
receptor or
a fragment or variant thereof. In specific embodiments, the present invention
also
encompasses methods and compositions for detecting, diagnosing, or prognosing
diseases
or disorders associated with TRAIL receptor function or TRAIL receptor ligand
function
or aberrant TRAIL receptor or TRAIL receptor ligand expression, comprising
administering to an animal, preferably a human, an effective amount of one or
more
antibodies or fragments or variants thereof, or related molecules, that
irnrnunospecifically
bind to TRAIL receptor or a fragment or variant thereof. In highly preferred
embodiments, the present invention relates to antibody-based methods and
compositions
for detecting, diagnosing, or prognosing cancers and other hyperproliferative
disorders
(e.g., leukemia, carcinoma, and lymphoma). Other diseases and disorders which
can be
detected, diagnosed or prognosed with the antibodies of the invention include,
but are not
limited to, neurodegenerative disorders (e.g., Parkinson's disease,
Alzheimer's disease,
and Huntington's disease), immune disorders (e.g., lupus, rheumatoid
arthritis, multiple
sclerosis, myasthenia gravis, Hashimoto's disease, andv immunodeficiency
syndrome),
inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid
arthritis),
infectious diseases (e.g., AIDS, herpes viral infections, and other viral
infections), and
proliferative disorders.
[0017] Another embodiment of the present invention includes the use of the
antibodies
of the invention as a diagnostic tool to monitor the expression of TRAIL
receptor
expression on cells.
[0018] The present inventors have generated hybridoma cell lines that express
antibodies that imW unospecifically bind one or more TRAIL receptor
polypeptides (e.g.,
SEQ ID NOs:l-4). Thus, the invention encompases these cell lines, listed in
Table 1
below which were deposited with the American Type Culture Collection ("ATCC")
on
the dates listed in Table 1 and given the ATCC Deposit Numbers identified in
Table 1
The ATCC is located at 10801 University Boulevard, Manassas, VA 20110-2209,
USA.
The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the
international recognition of the deposit of microorganisms for purposes of ~
patent
procedure.
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[0019] Further, the present invention encompasses the polynucleotides encoding
the
antibodies expressed by these cell lines, as well as the amino acid sequences
encoding the
antibodies expressed by these cell lines. Molecules comprising, or
alternatively consisting
of, fragments or variants of these antibodies (e.g., heavy chains, VH domains,
VH CDRs,
light chains, VL domains, or VL CDRs having an amino acid sequence of any one
of those
expressed by one or more cell lines referred to in Table 1), that
imrnunospecifically bind
to one or more TRAIL receptors or fragments or variants thereof are also
encompassed by
the invention, as are nucleic acid molecules that encode these antibodies
and/or molecules.
In highly preferred embodiments, the present invention encompasses antibodies,
or
fragments or variants thereof, that bind to the extracellular regions/domains
of one or more
TRAIL receptors or fragments and variants thereof.
[0020] The present invention also provides antibodies that bind one or more
TRAIL
receptor polypeptides which are coupled to a detectable label, such as an
enzyme, a
fluorescent label, a luminescent label, or a bioluminescent label. The present
invention
also provides antibodies that bind one or more TRAIL receptor polypeptides
which are
coupled to a therapeutic or cytotoxic agent. The present invention also
provides antibodies
that bind one or more TRAIL receptor polypeptides which are coupled to a
radioactive
material.
[0021] The present invention also provides antibodies that bind one or more
TRAIL
receptor polypeptides that act as either TRAIL receptor agonists or TRAIL
receptor
antagonists. In specific embodiments, the antibodies of the invention
stimulate apoptosis
of TRAIL receptor expressing cells. In other specific embodiments, the
antibodies of the
invention inhibit TRAIL binding to a TRAIL receptor. In other specific
embodiments, the
antibodies of the invention upregulate TRAIL receptor expression.
[0022] The present invention also provides antibodies that inhibit apoptosis
of TRAIL
receptor expressing cells. In other specific embodiments, the antibodies of
the invention
downregulate TRAIL receptor expression.
[0023] In further embodiments, the antibodies of the invention have a
dissociation
constant (KD) of 10-7 M or less. In preferred embodiments, the antibodies of
the invention
have a dissociation constant (KD) of 10-9 M or less.
[0024] The present invention further provides antibodies that stimulate
apoptosis of
TRAIL receptor expressing cells better than an equal concentration of TRAIL
polypeptide
stimulates apoptosis of TRAIL receptor expressing cells.
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[0025] The present invention further provides antibodies that stimulate
apoptosis of
TRAIL receptor expressing cells equally well in the presence or absence of
antibody
cross-linking reagents; and/or stimulate apoptosis with equal or greater
potency as an
equal concentration of TRAIL in the absence of a cross-linking antibody or
other cross-
linking agent.
[0026] In further embodiments, antibodies of the invention have an off rate
(ko~) of 10-
3lsec or less. In preferred embodiments, antibodies of the invention have an
off rate (ko~)
of 10-4/sec or less. In other preferred embodiments, antibodies of the
invention have an off
rate (ko~) of 10-5/sec or less.
[0027] The present invention also provides for antibodies that preferentially
bind one
or more of the TRAIL receptors selected from the group of TR4, TRS, TR7, and
TR10.
[0028] In certain embodiments, properties of the antibodies of the present
invention, as
detailed in the Examples below, make the antibodies better therapeutic agents
than
previously described TRAIL receptor binding antibodies.
[0029] The present invention also provides panels of antibodies (including
molecules
comprising, or alternatively consisting of, antibody fragments or variants)
wherein the
panel members correspond to one, two, three, four, five, ten, fifteen, twenty,
or more
different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab')2
fragments, Fd
fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies,
and scFvs).
The present invention further provides mixtures of antibodies, wherein the
mixture
corresponds to one, two, three, four, five, ten, fifteen, twenty, or more
different antibodies
of the invention (e.g., whole antibodies, Fabs, F(ab')2 fragments, Fd
fragments, disulfide-
linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and scFvs)). The
present invention
also provides for compositions comprising, or alternatively consisting of,
one, two, three,
four, five, ten, fifteen, twenty, or more antibodies of the present invention
(including
molecules comprising, or alternatively consisting of, antibody fragments or
variants
thereof). A composition of the invention may comprise, or alternatively
consist of, one,
two, three, four, five, ten, fifteen, twenty, or more amino acid sequences of
one or more
antibodies or fragments or variants thereof. Alternatively, a composition of
the invention
may comprise, or alternatively consist of, nucleic acid molecules encoding one
or more
antibodies of the invention.
[0030] The present invention also provides for fusion proteins comprising an
antibody
(including molecules comprising, or alternatively consisting of, antibody
fragments or
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WO 02/079377 PCT/USO1/42996
variants thereof) of the invention, and a heterologous polypeptide (i.e., a
polypeptide
unrelated to an antibody or antibody domain). Nucleic acid molecules encoding
these
fusion proteins are also encompassed by the invention. A composition of the
present
invention may comprise, or alternatively consist of, one, two, three, four,
five, ten, fifteen,
twenty or more fusion proteins of the invention. Alternatively, a composition
of the
invention may comprise, or alternatively consist of, nucleic acid molecules
encoding one,
two, three, four, five, ten, fifteen, twenty or more fusion proteins of the
invention.
[0031] The present invention also provides for a nucleic acid molecule(s),
generally
isolated, encoding an antibody (including molecules, such as scFvs, VH
domains, or VL
domains, that comprise, or alternatively consist of, an antibody fragment or
variant
thereof) of the invention. The present invention also provides a host cell
transformed with
a nucleic acid molecule of the invention and progeny thereof. The present
invention also
provides a method for the production of an antibody (including a molecule
comprising, or
alternatively consisting of, an antibody fragment or variant thereof) of the
invention. The
present invention further provides a method of expressing an antibody
(including a
molecule comprising, or alternatively consisting of, an antibody fragment or
variant
thereof) of the invention from a nucleic acid molecule. These and other
aspects of the
invention are described in further detail below.
Brief Descriution of the Fi~nres
[0032] Figure 1: Flow cytometric staining of Hela, SW480 and HT1080 cells for
TR4 (TRAIL-R1) expression using monoclonal antibody 7.3. Cells were incubated
with 1
microgram/ml monoclonal antibody 7.3 for 45 minutes, washed and stained with
anti-
human IgG2-FITC detector antibody. Reactivity of the 7.3 monoclonal antibody
with the
cells is shown in the histogram with the dark line; isotype control staining
is shown in the
shaded histogram.
[0033] Figure 2: Sensitivity of HeLa cells to killing mediated by TRAIL (A),
monoclonal antibody 7.3 (B) or monoclonal antibody 7.12 (C). Sensitivity of
HeLa cells
to anti-TRAIL receptor monoclonal antibodies was tested in the presence of
cycloheximide either with or without an equivalent amount of secondary goat
anti-human
Ig Fc specific antibody. Use of an equivalent amount of secondary goat anti-
human Ig Fc
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CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
specific antibody means that the secondary goat anti-human Ig Fc specific
antibody
concentration was equal to the concentration of the test antibodies, 7.3 and
7.12.
[0034] Figure 3: Sensitivity of SW480 cells to killing mediated by TRAIL (A),
monoclonal antibody 7.3 (B) monoclonal antibody 7.12 (C). Sensitivity of SW480
cells
to monoclonal antibodies was tested in the presence of cycloheximide either
with or
without an equivalent amount of secondary goat anti-human Ig Fc specific
antibody.
[0035] Figure 4: Sensitivity of HT1080 cells to killing mediated by TRAIL.
(A),
monoclonal antibody 7.3 (B) or monoclonal antibody 7.12 (C). Sensitivity of
HT1080
cells to monoclonal antibodies was tested in the presence of cycloheximide
either with or
without an equivalent amount of secondary goat anti-human Ig Fc specific
antibody.
[0036] Figure 5: Sensitivity of HeLa, SW480 and HT1080 cells to killing
mediated
by anti- monoclonal antibody 7.12. Sensitivity of cells to monoclonal
antibodies were
tested in the absence of either cycloheximide or additional crosslinking with
a secondary
goat anti-human Ig Fc specific antibody.
[0037] Figure 6: Sensitivity of HeLa and SW480 to TRAIL-Receptor mediated
killing mediated induced by monoclonal antibody 7.12 in the presence of
TOPOTECAN.
A comparison is shown for sensitization of cells to TRAIL-R1 monoclonal
antibody
killing using either cycloheximide or topotecan.
[0038] Figure 7: Sensitivity of SW480 cells to killing mediated by anti-TRAIL
receptor monoclonal antibodies 7.3, 7.3.1, 7.3.2 or 7.3.3 (A) monoclonal
antibodies 7.12,
7.12.1, 7.12.2, or 7.12.3 (B), monoclonal antibodies 7.10, 7.10.1, 7.10.2, or
7.10.3, or
monoclonal antibodies 7.1.3, 7.2, 7.8, 8.3.1, or 8.3.2. Sensitivity of SW480
cells to
monoclonal antibodies was tested in the presence of cycloheximide
[0039] Figure 8: Effect of 7.12.2 treatment on tumor growth in Swiss nu/nu
mice.
[0040] Figure 9: Effect of 7.12.2 treatment on tumor growth in Swiss nu/nu
mice-II.
to
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Detailed Description of the Invention
Definitions
[0041] 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. As
such, the
term antibody encompasses not only whole antibody molecules, but also antibody
fragments as well as variants (including derivatives) of antibodies and
antibody fragments.
Examples of molecules which are described by the term "antibody" herein
include, but are
not limited to: single chain Fvs (scFvs), Fab fragments, Fab' fragments,
F(ab')2, disulfide
linked Fvs (sdFvs), Fvs, and fragments comprising or alternatively consisting
of, either a
VL or a VH domain. The term "single chain Fv" or "scFv" as used herein refers
to a
polypeptide comprising a VL domain of antibody linked to a VH domain of an
antibody.
Antibodies that immunospecifically bind to a TRAIL receptor may have cross-
reactivity
with other antigens. Preferably, antibodies that immunospecifically bind to a
TRAIL
receptor do not cross-react with other antigens (e.g., other TRAIL receptors
or other
members of the Tumor Necrosis Factor Receptor superfamily). Antibodies that
immunospecifically bind to a TRAIL receptor can be identified, for example, by
immunoassays or other techniques known to those of skill in the art, e.g., the
immunoassays described in the Examples below.
[0042] Antibodies of the invention include, but are not limited to,
monoclonal,
multispecific, human or chimeric antibodies, single chain antibodies, Fab
fragments,
F(ab') fragments, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-
Id antibodies to
antibodies of the invention), intracellularly-made antibodies (i.e.,
intrabodies), and
epitope-binding fragments of any of the above. 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 irnmunoglobulin molecule.
Preferably, an
antibody of the invention comprises, or alternatively consists of, a VH
domain, VH CDR,
VL domain, or VL CDR having an amino acid sequence of any one of those
referred to in
Table l, or a fragment or variant thereof. In a preferred embodiment, the
immunoglobulin
is an IgGl isotype. In another preferred embodiment, the immunoglobulin is an
IgG4
isotype. Immunoglobulins may have both a heavy and light chain. An array of
IgG, IgE,
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IgM, IgD, IgA, and IgY heavy chains may be paired with a light chain of the
kappa or
lambda forms.
[0043] The term "variant" as used herein refers to a polypeptide that
possesses a
similar or identical function as a TRAIL receptor polypeptide, a fragment of a
TRAIL
receptor polypeptide, an anti-TRAIL receptor antibody or antibody fragment
thereof, but
does not necessarily comprise a similar or identical amino acid sequence of a
TRAIL
receptor polypeptide, a fragment of a TRAIL receptor polypeptide, an anti-
TRAIL
receptor antibody or antibody fragment thereof, or possess a similar or
identical structure
of a TRAIL receptor polypeptide, a fragment of a TRAIL receptor polypeptide,
an anti-
TRAIL receptor antibody or antibody fragment thereof. A variant having a
similar amino
acid refers to a polypeptide that satisfies at least one of the following: (a)
a polypeptide
comprising, or alternatively consisting of, an amino acid sequence that is at
least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95% or at
least 99% identical to the amino acid sequence of a TRAIL receptor
polypeptide, a
fragment of , an anti-TRAIL receptor antibody or antibody fragment thereof
(including a
VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one
of those expressed by one or more cell lines referred to in Table 1) described
herein; (b) a
polypeptide encoded by a nucleotide sequence, the complementary sequence of
which
hybridizes under stringent conditions to a nucleotide sequence encoding a
TRAIL receptor
polypeptide (e.g., SEQ ID NO:1-4), a fragment of a TRAIL receptor polypeptide,
an anti-
TRAIL receptor antibody or antibody fragment thereof (including a VH domain,
VHCDR,
VL domain, or VLCDR having an amino acid sequence of any one of those referred
to in
Table 1), described herein, of at least 5 amino acid residues, at least 10
amino acid
residues, at least 15 amino acid residues, at least 20 amino acid residues, at
least 25 amino
acid residues, at least 30 amino acid residues, at least 40 amino acid
residues, at least 50
amino acid residues, at least 60 amino residues, at least 70 amino acid
residues, at least 80
amino acid residues, at least 90 amino acid residues, at least 100 amino acid
residues, at
least 125 amino acid residues, or at least 150 amino acid residues; and (c) a
polypeptide
encoded by a nucleotide sequence that is at least 30%, at least 35%, at least
40%, at least
45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at
least 80%, at least 85%, at least 90%, at least 95% or at least '99%,
identical to the
nucleotide sequence encoding a TRAIL receptor polypeptide, a fragment of a
TRAIL
12
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
receptor polypeptide, an anti-TRAIL receptor antibody or antibody fragment
thereof
(including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid
sequence of any one of those expressed by one or more cell lines referred to
in Table 1),
described herein. A polypeptide with similar structure to a TRAIL receptor
polypeptide, a
fragment of a TRAIL receptor polypeptide, an anti-TRAIL receptor antibody or
antibody
fragment thereof, described herein refers to a polypeptide that has a similar
secondary,
tertiary or quaternary structure of a TRAIL receptor polypeptide, a fragment
of a TRAIL
receptor polypeptide, an anti-TRAIL receptor antibody, or antibody fragment
thereof,
described herein. The structure of a polypeptide can determined by methods
known to
those skilled in the art, including but not limited to, X-ray crystallography,
nuclear
magnetic resonance, and crystallographic electron microscopy.
[0044] To determine the percent identity of two amino acid sequences or of two
nucleic acid sequences, the sequences are aligned for optimal comparison
purposes (e.g.,
gaps can be introduced in the sequence of a first amino acid or nucleic acid
sequence for
optimal alignment with a second amino acid or nucleic acid sequence). The
amino acid
residues or nucleotides at corresponding amino acid positions or nucleotide
positions are
then compared. When a position in the first sequence is occupied by the same
amino acid
residue or nucleotide at the corresponding position in the second sequence,
then the
molecules are identical at that position. The percent identity between the two
sequences is
a function of the number of identical positions shared by the sequences (i.e.,
°Io identity =
number of identical overlapping positions/total number of positions x
100°Io). In one
embodiment, the two sequences are the same length.
[0045] The determination of percent identity between two sequences can be
accomplished using a mathematical algorithm known to those of skill in the
art. An
example of a mathematical algorithm for comparing two sequences is the
algorithm of
Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-2268(1990), modified as
in
Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-5877(1993). The BLASTn
and
BLASTx programs of Altschul, et al. J. Mol. Biol. 215:403-410(1990) have
incorporated
such an alogrithm. BLAST nucleotide searches can be performed with the BLASTn
program (score = 100, wordlength = 12) to obtain nucleotide sequences
homologous to a
nucleic acid molecules of the invention. BLAST protein searches can be
performed with
the BLASTx program (score = 50, wordlength = 3) to obtain amino acid sequences
homologous to a protein molecules of the invention. To obtain gapped
alignments for
13
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
comparison purposes, Gapped BLAST can be utilized as described in Altschul et
al.
Nucleic Acids Res. 25:3589-3402(1997). Alternatively, PSI-BLAST can be used to
perform an iterated search which detects distant relationships between
molecules (Id.).
When utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the default
parameters of the respective programs (e.g., BLASTx and BLASTn) can be used.
(See
http://www.ncbi.nlm.nih.gov.)
[0046] Another example of a mathematical algorithm utilized for the comparison
of
sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN
program
(version 2.0) which is part of the GCG sequence alignment software package has
incorporated such an alogrithm. Other algorithms for sequence analysis known
in the art
include ADVANCE and ADAM as described in Torellis and Robotti Corraput. Appl.
Biosci., 10 :3-5(1994); and FASTA described in Pearson and Lipman Proc. Natl.
Acad.
Sci. 85:2444-8(1988). Within FASTA, ktup is a control option that sets the
sensitivity and
speed of the search.
[0047] The term "derivative" as used herein, refers to a variant polypeptide
of the
invention that comprises, or alternatively consists of, an amino acid sequence
of a TRAIL
receptor polypeptide, a fragment of a TRAIL receptor polypeptide, or an
antibody of the
invention that immunospecifically binds to a TRAIL receptor polypeptide, which
has been
altered by the introduction of amino acid residue substitutions, deletions or
additions. The
term "derivative" as used herein also refers to a TRAIL receptor polypeptide,
a fragment
of a TRAIL receptor polypeptide, an antibody that immunospecifically binds to
a TRAIL
receptor polypeptide which has been modified, e.g., by the covalent attachment
of any
type of molecule to the polypeptide. For example, but not by way of
limitation, a TRAIL
receptor polypeptide, a fragment of a TRAIL receptor polypeptide, or an anti-
TRAIL
receptor antibody, may be modified, e.g., by glycosylation, acetylation,
pegylation,
phosphorylation, amidation, derivatization by known protecting/blocking
groups,
proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A
derivative of a
TRAIL receptor polypeptide, a fragment of a TRAIL receptor polypeptide, or an
anti-
TRAIL receptor antibody, may be modified by chemical modifications using
techniques
known to those of skill in the art, including, but not limited to, specific
chemical cleavage,
acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a
derivative of
a TRAIL receptor polypeptide, a fragment of a TRAIL receptor polypeptide, or
an anti-
TRAIL receptor antibody, may contain one or more non-classical amino acids. A
14
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
polypeptide derivative possesses a similar or identical function as a TRAIL
receptor
polypeptide, a fragment of a TRAIL receptor polypeptide, or an anti-TRAIL
receptor
antibody, described herein.
[0048] The term "epitopes" as used herein refers to portions of TRAIL receptor
having
antigenic or irnmunogenic activity in an animal, preferably a mammal. An
epitope having
immunogenic activity is a portion of TRAIL receptor that elicits an antibody
response in
an animal. An eptiope having antigenic activity is a portion of TRAIL receptor
to which
an antibody immunospecifically binds as determined by any method known in the
art, for
example, by the immunoassays described herein. Antigenic epitopes need not
necessarily
be immunogenic.
[0049] The term "fragment" as used herein refers to a polypeptide comprising
an
amino acid sequence of at least 5 amino acid residues, at least 10 amino acid
residues, at
least 15 amino acid residues, at least 20 amino acid residues, at least 25
amino acid
residues, at least 30 amino acid residues, at least 35 amino acid residues, at
least 40 amino
acid residues, at least 45 amino acid residues, at least 50 amino acid
residues, at least 60
amino residues, at least 70 amino acid residues, at least 80 amino acid
residues, at least 90
amino acid residues, at least 100 amino acid residues, at least 125 amino acid
residues, at
least 150 amino acid residues, at least 175 amino acid residues, at least 200
amino acid
residues, or at least 250 amino acid residues, of the amino acid sequence of a
TRAIL
receptor, or an anti-TRAIL receptor antibody (including molecules such as
scFv's, that
comprise, or alternatively consist of, antibody fragments or variants thereof)
that
immunospecifically binds to TRAIL receptor.
[0050] The term "fusion protein" as used herein refers to a polypeptide that
comprises,
or alternatively consists of, an amino acid sequence of an anti-TRAIL receptor
antibody
of the invention and an amino acid sequence of a heterologous polypeptide
(i.e., a
polypeptide unrelated to an antibody or antibody domain).
[0051] The term "host cell" as used herein refers to the particular subject
cell
transfected with a nucleic acid molecule and the progeny or potential progeny
of such a
cell. Progeny may not be identical to the parent cell transfected with the
nucleic acid
molecule due to mutations or environmental influences that may occur in
succeeding
generations or integration of the nucleic acid molecule into the host cell
genome.
CA 02426710 2003-04-23
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Antibody Structure
[0052] The basic antibody structural unit is known to comprise a tetramer.
Each
tetramer is composed of two identical pairs of polypeptide chains, each pair
having one
"light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-
ternninal
portion of each chain includes a variable region of about 100 to 110 or more
amino acids
primarily responsible for antigen recognition. The carboxy-terminal portion of
each chain
defines a constant region primarily responsible for effector function. Human
light chains
are classified as kappa and lambda light chains. Heavy chains are classified
as mu, delta,
gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, lgG,
IgA, and
IgE, respectively. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed.,
2nd ed.
Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all
purposes). The
variable regions of each light/heavy chain pair form the antibody binding
site.
[0053] Thus, an intact IgG antibody has two binding sites. Except in
bifunctional or
bispecific antibodies, the two binding sites are the same.
[0054] The chains all exhibit the same general structure of relatively
conserved
framework regions (FR) joined by three hyper variable regions, also called
complementarity determining regions or CDRs. The CDRs from the heavy and the
ligt
chains of each pair are aligned by the framework regions, enabling binding to
a specific
epitope. From N-terminal to C-terminal, both light and heavy chains comprise
the domains
FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each
domain is in accordance with the definitions of Kabat Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and
1991)), or
Chothia & Lesk J Mol. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-
883
(1989).
[0055] A bispecific or bifunctional antibody is an artificial hybrid antibody
having two
different heavy/light chain pairs and two different binding sites. Bispecific
antibodies can
be produced by a variety of methods including fusion of hybridomas or linking
of Fab'
fragments. See, e.g., Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321
(1990),
Kostelny et al. J Immunol. 148:1547 1553 (1992). In addition, bispecific
antibodies may
be formed as "diabodies" (Holliger et al. "Diabodies': small bivalent and
bispecific
antibody fragments" PNAS USA 90:6444-6448 (1993)) or "Janusins" (Traunecker et
al.
"Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on
HIV
16
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
infected cells" EMBO J 10:3655-3659 (1991) and Traunecker et al. "Janusin: new
molecular design for bispecific reagents" Int J Cancer Suppl 7:51-52 (1992)).
[0056] Production of bispecific antibodies can be a relatively labor intensive
process
compared with production of conventional antibodies and yields and degree of
purity are
generally lower for bispecific antibodies. Bispecific antibodies do not exist
in the form of
fragments having a single binding site (e.g., Fab, Fab', and Fv).
Anti-TRAIL Receptor Antibodies
[0057] The present invention is directed to fully human antibodies, generally
isolated,
that immunospecifically bind one or more TRAIL receptor polypeptides.
Essentially,
XenoMouse lines of mice from Abgenix, Inc. (Fremont, CA) expressing human
antibodies
were immunized with TRAIL receptor polypeptides, lymphatic cells (such as B-
cells)
were recovered from the mice that had high titers of anti-TRAIL receptor
antibodies, and
such recovered cells were fused with a myeloid-type cell line to prepare
immortal
hybridoma cell lines. Hybridoma cell lines were screened to select and
identify
hybridoma cell Iines that produced antibodies specific to the immunogen. We
utilized
these techniques in accordance with the present invention for the preparation
of antibodies
specific to TRAIL receptor polypeptides. Herein, we describe the production of
multiple
hybridoma cell lines that produce antibodies specific to TRAIL receptor
polypeptides.
Further, we provide a characterization of the antibodies produced by such cell
lines.
[0058] The antibodies derived from hybridoma cell lines discussed herein are
listed in
Table 1. Preferred antibodies of the invention include, antibodies expressed
by the
following cell lines: 1.2, 1.3, 7.1, 7.3, 7.8, 7.10, 7.12, and 8.3 (including
the antibodies
expressed by each of the subclones of these Iines). XenoMouse strains of mice
from
Abgenix, Inc. express human kappa light chains with either human IgGl, IgG2,
or IgG4.
The IgG2 expressing strain was used to make the cell lines and antibodies of
the present
invention, thus each of the antibodies produced by cell lines are fully human
IgG2 heavy
chains with human kappa light chains. These hybridoma cell lines were
deposited with the
American Type Culture Collection ("ATCC") on the date listed in Table l, and
given
ATCC Deposit Numbers listed in Table 1. The ATCC is located at 10801
University
Boulevard, Manassas, VA 20110-2209, USA. The ATCC deposit was made pursuant to
the terms of the Budapest Treaty on the international recognition of the
deposit of
microorganisms for purposes of patent procedure.
17
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
[0059] As described in Example 1, hybridoma cell lines that have a numeric
designation that contains one period indicates a primary hybridoma isolate.
The number
preceding the period indicates the fusion panel a hybridoma came from and the
number
after the period designates the primary hybridoma isolate number. By "primary
hybridoma isolate" is meant a hybridoma obtained by fusing spleen cells of
immunized
mice with a fusion partner, plating the cells at limiting dilution in 96 well
plates, and
selecting a hybridoma cell line, that by visual inspection appeared to have
only a single
colony, i.e., that appeared to have grown up from a single cell. Such a
hybridoma cell line
is most likely a monoclonal cell line. Primary hybridoma isolates were also
subcloned. In
a subcloning procedure, cells corresponding to a single primary hybridoma
isolate are
plated out at limiting dilution, and hybridoma sublones, that by visual
inspection appeared
to have only a single colony, i.e., that appeared to have grown up from a
single cell, are
selected. In this application, hybridoma subclones have designations
containing two
periods. As above, the number preceding the first period indicates which
fusion panel a
hybridoma came from; the number immediately after the first period designates
the
primary hybridoma isolate number; and the number following the second period
indicates
the number of a particular subclone derived from the primary hybridoma isolate
with the
designation indicated by the number immediately following the first period.
Subcloned
cell lines are monoclonal and are typically more stable with respect to
antibody
expression.
[0060] The following hybridoma cell lines deposited at the American Type
Culture
Collection (ATCC) contain equal proportions of three sublones of a particular
hybridoma
isolate. Hybridomas 7.3.1, 7.3.2, and 7.3.3 were collectively deposited at the
ATCC on
November 16, 2000 and given ATCC Deposit Number PTA-2687. Hybridomas 7.8.1,
7.8.2, and 7.8.3 were collectively deposited at the ATCC on November 27, 2000
and given
ATCC Deposit Number PTA-2730. Hybridomas 7.10.1, 7.10.2, and 7.10.3 were
collectively deposited at the ATCC on November 27, 2000 and given ATCC Deposit
Number PTA-2729. Hybridomas 7.12.1, 7.12.2, and 7.12.3 were collectively
deposited at
the ATCC on November 27, 2000 and given ATCC Deposit Number PTA-2728.
Hybridomas 8.3.1 and 8.3.2 were collectively deposited at the ATCC on November
27,
2000 and given ATCC Deposit Number PTA-2731.
18
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WO 02/079377 PCT/USO1/42996
[0061] Individual hybridoma 7.1.3 was deposited at the ATCC on March 02, 2001
and
given ATCC Deposit Number PTA-3149. Individual hybridoma 7.3.3 was deposited
at
the ATCC on May 11, 2001 and given ATCC Deposit Number PTA-3368. Individual
hybridoma 7.12.2 was deposited at the ATCC on May 11, 2001 and given ATCC
Deposit
Number PTA-3369.
[0062] The ATCC is located at 10801 University Boulevard, Manassas, VA 20110-
2209, USA. Each of the ATCC deposits described herein was made pursuant to the
terms
of the Budapest Treaty on the international recognition of the deposit of
microorganisms
for purposes of patent procedure. The ATCC Deposit Numbers and the hybridoma
designations are also presented in Table 1.
19
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
.-, ooo ~
0 0 0 0
0
0 0 0 0
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O ~ ~ O
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c
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H H H
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V
M 7
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N N
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CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
0 0 0 0 0 0 p o
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000 000 000 0 00
O O O O O O N O
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N N N N N N ~ N
N N N N
w w w w w w H w
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21
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
O ~ N c'~d'~n~O
a\ a>~ a>~ a>a>
22
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
[0063] If an antibody expressed by one (or two) of the sublcones has a
property
that is distinct from the remaining sublone(s) of a given primary hybridoma
isolate, a cell
line expressing the antibody with that property can be retrieved from the
pooled ATCC
deposit using methods that are routine in the art. Briefly, retrieval of such
a clone would
require plating cells of the ATCC deposit at limiting dilution, growing the
cells in culture,
selecting monoclonal cell lines, and testing the antibodies expressed by the
monoclonal
cell lines for the presence or absence of the property using methods that are
routine in the
art. By way of non-limiting example, if one of the subclones expressed an
antibody with a
superior affinity for a TRAIL receptor polypeptide of the invention, one would
test the
affinities of the antibodies expressed by the monoclonal cell Iines derived
from the ATCC
deposit. A monoclonal cell line that expressed an antibody with an affinity
matching or
resembling the desired affinity (making allowances for experimental variations
in
determinations of affinity) would be equivalent to the specific subclone from
the ATCC
deposit sought after.
[0064] As a matter of convenience, reference to an antibody herein by the
primary
hybridoma designation references not only to the primary hybridoma isolate but
also each
of its subclones deposited at the ATCC. For example reference to hybridoma
cell line 7.3,
references hybridoma cell lines 7.3, 7.3.1, 7.3.2, and 7.3.3. The only
exceptions to this
policy are the recitation of hybridoma designations in the Figures, Figure
Legends,
Examples, and Claims. In those portions of the application, reference to a
particular
hybridoma designation references only the hybridoma defined by that
designation.
Additionally, the antibody secreted by a hybridoma cell lines has the same
designation as
the cell line itself. Thus the term 7.3 may also reference the antibody
expressed by
hybridoma cell Iines 7.3, 7.3.1, 7.3.2, and 7.3.3.
[0065] The present invention encompasses antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind to a TRAIL receptor polypeptide or a fragment,
variant, or fusion
protein thereof. A TRAIL receptor polypeptide includes, but is not limited to,
TR4 (SEQ
ID NO:1) or the polypeptide encoded by the cDNA in clone HCUDS60 contained in
ATCC Deposit 97853 deposited Jan 21, 1997; TR5 (SEQ ID N0:2) or the
polypeptide
encoded by the cDNA in clone HPRCB54 contained in ATCC Deposit 97798 deposited
Nov. 22, 1996; TR7 (SEQ ID N0:3) or the polypeptide encoded by the cDNA in
clone
HLYBX88 contained in ATCC Deposit 97920 deposited Mar. 7, 1997, andlor TR10
(SEQ
23
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
ID N0:4) or the polypeptide encoded by the cDNA in clone HI~AB035 contained in
ATCC Deposit 209040 deposited May 15, 1997. TRAIL receptors may be produced
through recombinant expression of nucleic acids encoding the polypeptides of
SEQ ID
NOS:1-4, (e.g., the cDNAs in the ATCC Deposit Numbers 97853, 97798, 97920, or
209040).
[0066] In one embodiment, the antibodies of the invention preferentially bind
TR4
(SEQ ID N0:1), or fragments, variants, or fusion proteins thereof (e.g., the
extracellular
region of TR4 fused to an Fc domain) relative to their ability to bind TRS,
TR7, or TR10
(SEQ ID NOS:2-4) or fragments, variants, or fusion proteins thereof. In
another preferred
embodiment, antibodies of the invention preferentially bind TR7, fragments,
variants, or
fusion proteins thereof (e.g., the extracellular region of TR7 fused to an Fc
domain)
relative to their ability to bind TR4, TRS, or TR10 (SEQ Ip NOS:1, 2, and 4)
or
fragments, variants, or fusion proteins thereof. In other preferred
embodiments, the
antibodies of the invention preferentially bind to TR4 and TR7 (SEQ ID NOS:1
and 3), or
fragments and variants thereof relative to their ability to bind TR5 or TR10
(SEQ ID
NOS:2 and 4) or fragments, variants, or fusion proteins thereof. In other
preferred
embodiments, the antibodies of the invention preferentially bind to TR5 and
TR10 (SEQ
ID NOS:2 and 4), or fragments and variants thereof relative to their ability
to bind TR4 or
TR7 (SEQ ID NOS:l and 3) or fragments, variants, or fusion proteins thereof.
In other
preferred embodiments, the antibodies of the invention bind TR4, TRS, TR7 and
TR10
(SEQ ID NOS:l-4). In another embodiment, antibodies of the invention
preferentially
bind TR5 (SEQ ID N0:2), or fragments and variants thereof relative to their
ability to bind
TR4, TR7 or TR10 (SEQ ID NOS:l, 2, and 3). In another embodiment, antibodies
of the
invention preferentially bind TR10 (SEQ ID N0:4), or fragments and variants
thereof
relative to their ability to bind TR4, TRS, or TR7 (SEQ ID NOS:1-3). An
antibody's
ability to preferentially bind one antigen compared to another antigen may be
determined
using any method known in the art.
TRAIL Receptor Polypeptides
TR4
[0067] In certain embodiments of the present invention, the antibodies of the
present
invention bind TR4 polypeptide, or fragments or variants thereof. The
following section
describes the TR4 polypeptides, fragments and variants that may be bound by
the
24
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
antibodies of the invention in more detail. The TR4 polypeptides, fragments
and variants
which may be bound by the antibodies of the invention are also described in
International
Publication Numbers, for example, W098/32856 and WO00/67793 which are herein
incorporated by reference in their entireties.
[0068] In certain embodiments, the antibodies of the present invention
immunospecifically bind TR4 polypeptide. An antibody that immunospecifically
binds
TR4 may, in some embodiments, bind fragments, variants (including species
orthologs of
TR4), multimers or modified forms of TR4. For example, an antibody
immunospecific for
TR4 may bind the TR4 moiety of a fusion protein comprising all or a portion of
TR4.
[0069] TR4 proteins may be found as monomers or multimers (i.e., dimers,
trimers,
tetramers, and higher multimers). Accordingly, the present invention relates
to antibodies
that bind TR4 proteins found as monomers or as part of multimers. In specific
embodiments, antibodies of the invention bind TR4 monomers, dirners, trimers
or
tetramers. In additional embodiments, antibodies of the invention bind at
least dimers, at
least trimers, or at least tetramers containing one or more TR4 polypeptides.
[0070] Antibodies of the invention may bind TR4 homomers or heteromers. As
used
herein, the term hornomer, refers to a multimer containing only TR4 proteins
of the
invention (including TR4 fragments, variants, and fusion proteins, as
described herein).
These homomers may contain TR4 proteins having identical or different
polypeptide
sequences. In a specific embodiment, a homomer of the invention is a multimer
containing only TR4 proteins having an identical polypeptide sequence. In
another
specific embodiment, antibodies of the invention bind TR4 homomers containing
TR4
proteins having different polypeptide sequences. In specific embodiments,
antibodies of
the invention bind a TR4 homodimer (e.g., containing TR4 proteins having
identical or
different polypeptide sequences) or a homotrimer (e.g., containing TR4
proteins having
identical or different polypeptide sequences). In additional embodiments,
antibodies of
the invention bind at least a homodimer, at least a homotrimer, or at least a
homotetramer
of TR4.
[0071] As used herein, the term heteromer refers to a multimer containing
heterologous proteins (i.e., proteins containing polypeptide sequences that do
not
correspond to a polypeptide sequences encoded by the TR4 gene) in addition to
the TR4
proteins of the invention. In a specific embodiment, antibodies of the
invention bind a
heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments,
the
CA 02426710 2003-04-23
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antibodies of the invention bind at least a homodimer, at least a homotrimer,
or at least a
homotetramer containing one or more TR4 polypeptides.
[0072] Multimers bound by one or more antibodies of the invention may be the
result
of hydrophobic, hydrophilic, ionic and/or covalent associations andlor may be
indirectly
linked, by for example, liposome formation. Thus, in one embodiment, multimers
bound
by one or more antibodies of the invention, such as, for example, homodimers
or
homotrimers, are formed when TR4 proteins contact one another in solution. In
another
embodiment, heteromultimers bound by one or more antibodies of the invention,
such as,
for example, heterotrimers or heterotetramers, are formed when proteins of the
invention
contact antibodies to the TR4 polypeptides (including antibodies to the
heterologous
polypeptide sequence in a fusion protein) in solution. In other embodiments,
multimers
bound by one or more antibodies of the invention are formed by covalent
associations with
and/or between the TR4 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 NO:l or the polypeptide
encoded by the
deposited cDNA clone of ATCC Deposit 97853). 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
occurnng)
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 TR4 fusion protein. In one example, covalent associations are
between the
heterologous sequence contained in a fusion protein (see, e.g., US Patent
Number
5,478,925). In a specific example, the covalent associations are between the
heterologous
sequence contained in a TR4-Fc fusion protein (as described herein). In
another specific
example, covalent associations of fusion proteins are between heterologous
polypeptide
sequences from another TNF family ligand/receptor member that is capable of
forming
covalently associated multimers, such as for example, oseteoprotegerin (see,
e.g.,
International Publication No. WO 98/49305, the contents of which are herein
incorporated
by reference in its entirety).
[0073] The multimers that may be bound by one or more antibodies 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
26
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
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 that may be bound by one or more
antibodies 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 that may be bound by one or more antibodies 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 that may be
bound by one
or more antibodies of the invention (see, e.g., US Patent Number 5,478,925,
which is
herein incorporated by reference in its entirety).
[0074] Alternatively, multimers that may be bound by one or more antibodies of
the
invention may be generated using genetic engineering techniques known in the
art. In one
embodiment, proteins contained in multimers that may be bound by one or more
antibodies 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 that may be bound by one or more
antibodies of
the invention are generated by ligating a polynucleotide sequence encoding a
TR4
polypeptide 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 TR4 polypeptides 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). In another embodiment, two or more
TR4
27
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
polypeptides are joined through synthetic linkers (e.g., peptide, carbohydrate
or soluble
polymer linkers). Examples include those peptide linkers described in U.S.
Pat. No.
5,073,627 (hereby incorporated by reference). Proteins comprising multiple TR4
polypeptides separated by peptide linkers may be produced using conventional
recombinant DNA technology. In specific embodiments, antibodies of the
invention bind
proteins comprising multiple TR4 polypeptides separated by peptide linkers.
[0075] Another method for preparing multimer TR4 polypeptides involves use of
TR4
polypeptides fused to a leucine zipper or isoleucine polypeptide sequence.
Leucine zipper
domains and isoleucine zipper domains are polypeptides that promote
multimerization of
the proteins in which they are found. Leucine zippers were originally
identified in several
DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have
since been
found in a variety of different proteins. Among the known leucine zippers are
naturally
occurnng peptides and derivatives thereof that dimerize or trimerize. Examples
of leucine
zipper domains suitable for producing soluble multimeric TR4 proteins are
those described
in PCT application WO 94/10308, hereby incorporated by reference. Recombinant
fusion
proteins comprising a soluble TR4 polypeptide fused to a peptide that
dimerizes or
trimerizes in solution are expressed in suitable host cells, and the resulting
soluble
multimeric TR4 is recovered from the culture supernatant using techniques
known in the
art. In specific embodiments, antibodies of the invention bind TR4-leucine
zipper fusion
protein monomers and/or TR4-leucine zipper fusion protein multimers.
[0076] Certain members of the TNF family of proteins are believed to exist in
trimeric
form (Beutler and Huffel, Science 264:667, 1994; Banner et al., Cell 73:431,
1993). Thus,
trimeric TR4 may offer the advantage of enhanced biological activity.
Preferred leucine
zipper moieties are those that preferentially form trimers. One example is a
leucine zipper
derived from lung surfactant protein D (SPD), as described in Hoppe et al.
(FEBS Letters
344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby
incorporated
by reference. In specific embodiments, antibodies of the invention bind TR4-
leucine
zipper fusion protein trimers.
[0077] Other peptides derived from naturally occurring trimeric proteins may
be
employed in preparing trimeric TR4. In specific embodiments, antibodies of the
invention
bind TR4- fusion protein monomers and/or TR4 fusion protein trimers.
[0078] Antibodies that bind TR4 polypeptides are preferably provided in an
isolated
form, and preferably are substantially purified. By "isolated polypeptide" is
intended a
28
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
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. Antibodies of the present invention may bind TR4 polypeptide
fragments
comprising or alternatively, consisting of, an amino acid sequence contained
in SEQ ll~
NO:1, encoded by the cDNA contained in ATCC deposit Number 97853, or encoded
by
nucleic acids which hybridize (e.g., under stringent hybridization conditions)
to the
nucleotide sequence contained in ATCC deposit Number 97853, 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. Antibodies of the present invention may bind polypeptide
fragments,
including, for example, fragments that comprise or alternatively, consist of
from about
amino acid residues: 1 to 23, 24 to 43, 44 to 63, 64 to 83, 84 to 103, 104 to
123, 124 to
143, 144 to 163, 164 to 183, 184 to 203, 204 to 223, 224 to 238, 239 to 264,
265 to 284,
285 to 304, 305 to 324, 325 to 345, 346 to 366, 367 to 387, 388 to 418, 419 to
439, and/or
440 to 468 of SEQ ID NO:1. 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. Moreover, polypeptide fragments bound by the antibodies of the
invention
can be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,
140, 150, 175
or 200 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.
[0079) Preferably, antibodies of the present invention bind polypeptide
fragments
selected from the group: a polypeptide comprising or alternatively, consisting
of, the TR4
receptor extracellular domain (predicted to constitute amino acid residues
from about 24 to
about 238 in SEQ ID NO:1); a polypeptide comprising or alternatively,
consisting of, both
TR4 cysteine rich domains (both of which may be found in the protein fragment
consisting
of amino acid residues from about 131 to about 229 in SEQ ID NO:1); a
polypeptide
comprising or alternatively, consisting of, the TR4 cysteine rich domain
consisting of
amino acid residues from about 131 to about 183 in SEQ ID NO:1); a polypeptide
comprising or alternatively, consisting of, the TR4 cysteine rich domain
consisting of
amino acid residues from about 184 to about 229 in SEQ ID N0:1); a polypeptide
comprising or alternatively, consisting of, the TR4 receptor transmembrane
domain
(predicted to constitute amino acid residues from about 239 to about 264 in
SEQ ID
29
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
NO:1); a polypeptide comprising or alternatively, consisting of, fragment of
the predicted
mature TR4 polypeptide, wherein the fragment has a TR4 functional activity
(e.g.,
antigenic activity or biological acitivity); a polypeptide comprising or
alternatively,
consisting of, the TR4 receptor intracellular domain (predicted to constitute
amino acid
residues from about 265 to about 468 in SEQ ID NO:1); a polypeptide comprising
or
alternatively, consisting of, the TR4 receptor extracellular and intracellular
domains with
all or part of the transmembrane domain deleted; a polypeptide comprising, or
alternatively consisting of, the TR4 receptor death domain (predicted to
constitute amino
acid residues from about 379 to about 422 in SEQ ID NO:l); and a polypeptide
comprising, or alternatively, consisting of, one, two, three, four or more,
epitope bearing
portions of the TR4 receptor protein. In additional embodiments, the
polypeptide
fragments of the invention comprise, or alternatively, consist of, any
combination of 1, 2,
3, 4, 5, 6, 7, or all 8 of the above members. The amino acid residues
constituting the TR4
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 I5 amino
acid residues) depending on the criteria used to define each domain.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
[0080] It is believed that one or both of the extracellular cysteine rich
motifs of TR4 is
important for interactions between TR4 and its ligands (e.g., TRAIL).
Accordingly, in
highly preferred embodiments, antibodies of the present invention bind TR4
polypeptide
fragments comprising, or alternatively consisting of amino acid residues 131
to 183,
and/or 184 to 229 of SEQ ll~ NO:1. In another highly preferred embodiment,
antibodies
of the present invention bind TR4 polypeptides comprising, or alternatively
consisting of
both of the extracellular cysteine rich motifs (amino acid residues 131 to 229
of SEQ ID
N0:1.) In another preferred embodiment, antibodies of the present invention
bind TR4
polypeptides comprising, or alternatively consisting the extracellular soluble
domain of
TR4 (amino acid residues 24-238 of SEQ ID NO:1.) In highly preferred
embodiments, the
antibodies of the invention that bind all or a portion of the extracellular
soluble domain of
TR4 (e.g., one or both cysteine rich domains) prevent TRAIL Iigand from
binding to TR4.
In other highly preferred embodiments, the antibodies of the invention that
bind all or a
portion of the extracellular soluble domain of TR4 (e.g., one or both cysteine
rich
domains) agonize the TR4 receptor. Tn other highly preferred embodiments, the
antibodies
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
of the invention that bind all or a portion of the extracellular soluble
domain of TR4 (e.g.,
one or both cysteine rich domains) induce cell death of the cell expressing
the TR4
receptor.
[0081] Antibodies of the invention may also bind fragments comprising, or
alternatively, consisting of structural or functional attributes of TR4. 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"),
hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic
regions, surface forming regions, and high antigenic index regions (i.e.,
containing four or
more contiguous amino acids having an antigenic index of greater than or equal
to 1.5, as
identified using the default parameters of the Jameson-Wolf program) of
complete (i.e.,
full-length) TR4. Certain preferred regions are those set out in Table 2 and
include, but are
not limited to, regions of the aforementioned types identified by analysis of
the amino acid
sequence depicted in (SEQ ID N0:1), such preferred regions include; Gamier-
Robson
predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-
Fasman
predicted alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle
predicted
hydrophilic 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.
[0082] The data representing the structural or functional attributes of TR4
set forth in
Table 2, as described above, was generated using the various modules and
algorithms of
the DNA*STAR set on default parameters. Column I represents the results of a
Garnier-
Robson analysis of alpha helical regions; Column II represents the results of
a Chou-
Fasman analysis of alpha helical regions; Column III represents the results of
a Gamier
Robson analysis of beta sheet regions; Column IV represents the results of a
Chou-
Fasman analysis of beta sheet regions; Column V represents the results of a
Gamier
Robson analysis of turn regions; Column VI represents the results of a Chou-
Fasman
analysis of turn regions; Column VII represents the results of a Gamier Robson
analysis of
coil regions; Column VIII represents a Kyte-Doolittle hydrophilicity plot;
Column;
Column 1X represents the results of an Eisenberg analysis of alpha amphipathic
regions;
Column X represents the results of an Eisenberg analysis of beta amphipathic
regions;
Column XI represents the results of a Karplus-Schultz analysis of flexible
regions;
31
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Column XII represents the Jameson-Wolf antigenic index score; and Column XIII
represents the Emini surface probability plot.
[0083] In a preferred embodiment, the data presented in columns VIII, XII, and
XIII
of Table 2 can ~ be used to determine regions of TR4 which exhibit a high
degree of
potential for antigenicity. Regions of high antigenicity are determined from
the data
presented in columns VIII, XII, and/or XIII 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.
[0084] The above-mentioned preferred regions set out in Table 2 include, but
are not
limited to, regions of the aforementioned types identified by analysis of the
amino acid
sequence set out in SEQ ID NO:1. As set out in Table 2, such preferred regions
include
Gamier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions,
Chou-Fasman
alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic
regions,
Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible
regions,
Jameson-Wolf regions of high antigenic index and Emini surface-forming
regions. Among
preferred polypeptide fragmnents bound by one or more antibodies of the
invention are
those that comprise regions of TR4 that combine several structural features,
such as
several (e.g., I, 2, 3 , or 4) of the same or different region features set
out above and in
Table 2.
32
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WO 02/079377 PCT/USO1/42996
Table 2
ResPositionI II III IV V VI VII VIII IX X XI XII XIII
Met1 . . B . . . . 0.12 . . . -0.100.90
Ala2 . . . . . . C -0.08* * . 0.25 1.08
Pro3 . . . . . . C 0.42 * * . 0.10 0.86
Pro4 . . . . . T C -0.04* * . 1.05 1.69
Pro5 A . . . . T . 0.31 . * F 1.00 1.24
Ala6 A . . . . T . 0.10 . * F 1.00 1.10
Arg7 A . . . . T . 0.34 . * . 0.10 0.58
Val8 . . B B . . . -0.03. * . -0.300.37
His9 . . B B . . . -0.52. * . -0.300.37
Leu10 . . B B . . . -1.12. * . -0.600.17
Gly11 . . B B . . . -2.22. * . -0.600.28
Ala12 . . B B . . . -2.09. * . -0.600.14
Phe13 . . B B . . . -1.54. * . -0.600.12
Leu14 . . B B . . . -1.72. . . -0.600.18
Ala15 . . B B . . . -0.91. . . -0.600.27
Val16 . . B B . . . -0.78. . . -0.600.51
Thr17 . . B B . . . -0.53. . F -0.450.95
Pro18 . . . B . . C -0.13. . F 0.05 0.93
Asn19 . . . . . T C 0.09 . . F 0.60 1.69
Pro20 . . . . . T C 0.09 . . F 0.60 1.18
Gly21 . . . . T T . 0.64 . . F 0.65 0.77
Ser22 . . . . . T C 0.61 . . F 0.45 0_64
Ala23 . . . . . . C 0,51 . . F ~ 0.41
0.25
Ala24 . . . . . T C 0.51 . . F 0.45 0.60
Ser25 ~ . . B . . T . 0.13 . . F 0.85 0.78
Gly26 A . . . . T . -0.11. . F 0.85 0.78
Thr27 A . . . . T . -0.40. . F 0.85 0.78
Glu28 A A . . . . . -0.40. . F 0.45 0.58
Ala29 A A . . . . . -0.12. . . 0.30 0.60
Ala30 A A . . . . . -0.03. . . 0.30 0.60
Ala31 A A . . . . . 0.01 . . . 0.30 0.53
Ala32 A A . . . . . 0.37 . . . -0.300.71
Thr33 A . . . . T . -0.49* . F 1.00 1.40
Pro34 A . . . . T . -0.19. . F 1.00 1.03
Ser35 . . B . . T . 0.06 . . F 0.40 1.07
Lys36 . . B . . T . 0.34 . . F 0.25 0.73
Val37 . . B B . . . 0.63 . . F -0.150.64
Trp38 . . B B . . . 0.36 . . F -0.150.64
Gly39 . . B B . . . 0.22 * * F -0.150.32
Ser40 . . . . . . C 0.63 * * F -0.050.43
Ser41 . . . . . T C -0.30* * F 0.45 0.80
Ala42 . . . . . T C 0.56 * * F 1.05 0.57
Gly43 . . . . . T C 0.63 * * F 1.35 0.73
Arg44 . . B . . T . 1.09 * * F 1.49 0.84
Ile45 . . B . . . . 2.04 * * F 1.78 1.63
Glu46 . . B . . . . 1.00 * * F 2.12 1.63
Pro47 . . B . . T . 1.24 * * F 2.51 0.83
Arg48 . . . . T T . 1.70 * * F 3.40 1.17
Gly49 . . . . T T . 1.24 * * F 3.06 1.32
Gly50 . . . . T T . 1.54 * * F 2.57 0.84
33
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Table 2 (continued)
ResPositionI II III IV VI VII VIII IX X XI XII XIII
V.
Gly51 . . . . . T C 0.73 * * F 2.03 0.44
Arg52 . . . . . T C 0.73 * * F 1.39 0.36
Gly53 . . B . . T . 0.31 * * F 0.85 0.57
A1a54 . . B . . T . 0.36 . * F 0.85 0.83
Leu55 . . B . . . . 0.10 . * F 0.65 0.57
Pro56 . . B . . . . 0.10 . * F -0.25 0.57
Thr57 . . B . . . . -0.01. * F -0.25 0.55
Ser58 . . B . . T . 0.30 . . F 0.10 1.16
Met59 . . B . . T . 0.54 . . F 0.40 1.02
Gly60 . . B . . T . 1.14 . . F 0.25 0.70
Gln61 . . . . T T . 1.06 . . F 0.65 0.81
His62 . . . . . . C 0.78 . * F 0.40 1.10
Gly63 . . . . . T C 1.19 . * F 0.60 1.12
Pro64 . . . . . T C 1.20 . * F 1.20 1.27
Ser65 . . . . . T C 1.66 . * F 1.05 0.94
Ala66 . . B . . T . 1.07 . * F 1.30 1.86
Arg67 . . B . . . . 0.76 * * . 1.29 1.22
Ala68 . . B . . . . 1.21 * * . 1.48 0.90
Arg69 . . B . . T . 0.83 . * . 2.17 1.74
Ala70 . . B . . T . 0.92 . * F 2.51 0.90
Gly71 . . . . T T . 1.17 . * F 3.40 1.37
Arg72 . . . . . T C 0.84 . * F 2.71 0.69
Ala73 . . . . . T C 1.54 * . F 2.48 1.06
Pro74 . . . . . T C 1.22 * . F 2.70 2.10
G1y75 . . . . . T C 1.22 * . F 2.62 1.66
Pro76 . . . . . T C 1.68 * * F 2.24 1.66
Arg77 . . . . . C 1.57 * . F 2.60 2.10
Pro78 . A B . . . . 1.57 * . F 1.94 3.68
Ala79 . A B . . . . 1.48 * . F 1.68 2.40
Arg80 . A B . . . . 1.61 * * F 1.42 1.64
Glu81 . A B . . . . 1.93 * * F 1.16 1.64
Ala82 A A . . . . . 1.01 * * F 0.90 3.19
Ser83 A . . . . T . 1.33 * * F 1.30 1.34
Pro84 A . . . . T . 1.07 * * F 1.30 1.52
Arg85 A . . . . T . 0.92 * * F 1.00 1.12
Leu86 A . . . . T . 0.97 . * . 0.85 2.13
Arg87 A . . B . . . 1.24 . * . 0.75 1.46
Val88 A . . B . . . 0.84 * * . 0.75 1.08
His89 A . . B . . . 1.10 . * . -0.15 1.13
Lys90 A . . B . . . 0.29 * * F 0.90 1.16
Thr91 . . B B . . . 0.24 * * F 0.00 1.35
Phe92 . . B B . . . -0.72* * . -0.30 0.74
Lys93 . . B B . . . -0.72* * . -0.30 0.27
Phe94 . . B B . . . -1.03* . . -0.60 0.14
Val95 . . B B . . . -1.93* . . -0.60 0.16
Val96 . . B B . . . -2.43. * . -0.60 0.06
Val97 . . B B . . . -2.54. * . -0.60 0.06
Gly98 . . B B . . . -2.59. * . -0.60 0.06
Val99 . . B B . . . -2.74. . . -0.60 0.15
Leu100 . . B B . . . -2.74* . . -0.60 0.15
34
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Table 2 (continued)
ResPositionI II III IV V VI VII VIII IX X XI XII XIII
Leu101 . . B B . . . -2.10* . . -0.60 0.11
Gln102 . . B B . . . -1.54* . . -0.60 0.23
Val103 . . B B . . . -1.50. . . -0.60 0.37
Val104 . . B . . T . -1.23. . . -0.20 0.61
Pro105 . . B . . T . -1.01* . F 0.25 0.35
Ser106 A . . . . T . -0.51* . F -0.05 0.48
Ser107 A . . . . T . -1.40* * F 0.25 0.94
Ala108 A . . . . . . -0.50. * F 0.05 0.43
Ala109 A . . . . . . -0.46. * . 0.50 0.63
Thr110 A . . . . . . -0.28. * . -0.10 0.39
Ile111 A . . . . . . 0.02 . * . -0.10 0.53
Lys112 . . B . . . . 0.32 . * . 0.50 0,87
Leu113 . . B . . . . 0.61 . * F 1.05 1.04
His114 . . B . . . . 0.31 . * F 1.30 1.99
Asp17.5 . . . . . T C 0.28 * * F 1.80 0.70
Gln126 . . . . T T . 0.86 . * F 1.65 0.84
Ser117 . . . . T T . 0.81 . . F 2.50 0.89
Ile118 . . . . T T . 1.62 . . F 2.25 0.92
Gly119 . . . . . . C 1.37 . . F 1.00 0.92
Thr120 . . . . . . C 1.37 . . F 0.45 0.72
Gln121 . . B . . . C 1.33 . F 0.65 1.79
Gln122 . . B . . . . 1.33 . . F 0.20 2.46
Trp123 . . B . . . . 2.01 . . . 0.05 2.28
Glu124 . . . . . . C 1.54 . . . 0.25 2.04
His125 . . . . . . C 1.51 . . . 0.10 0.97
Ser126 . . . . . T C 2.51 . . F 0.45 0.91
Pro127 . . . . T T . 0.70 . . F 1.55 0.91
Leu128 . . . . T T . 0.32 . . F 0.65 0.55
Gly129 . . . . T T . 0.11 . . F 0.65 0.22
Glu130 . . . . T . . -0.07. . F 0.45 0.22
Leu131 . . B . . . . -0.11* . . 0.18 0.42
Cys132 . . B . . . . -0.20* . F 1.21 0.42
,
Pro133 . . B . . T . 0.58 * * F 1.69 0.32
Pro134 . . . . T T . 1.03 . * F 1.47 0.53
Gly135 . . . . T T . 0.73 . * F 2.80 1.94
Ser136 . . . . . T C 1.54 * . F 2,32 1.68
His137 . . . . . . C 2.32 * . F 2,48 1.88
Arg138 . . B . . . . 2.32 * . F 2.34 3.72
Ser139 . . B . . . . 2.19 * . F 2.40 4.29
Glu140 . . . . T . . 1.94 * . F 2.86 3.12
Arg141 . . . . T T . 1.58 * . F 3.40 1.61
Pro142 . . . . T T . 1.61 . * F 2.91 0.64
Gly143 . . . . T T . 1.61 . * F 2.57 0.60
Ala144 . . . . T T . 1.24 . * . 2.08 0.60
Cys7.45 . . . . T . . 0.93 . * . 1.41 0.21
Asn146 . . B . . . . 0.82 . * . 0.84 0.30
Arg7.47 . . B . . . . 0.69 * . . 1.01 0.52
Cys148 . . B . . T . 0.18 * . F 1.83 0.96
Thr7.49 . . B . . T . 0.42 * . F 1.70 0.44
Glu150 . . B . . T . 0.84 * . F 1.53 0.22
CA 02426710 2003-04-23
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Table 2 (continued)
ResPositionI II III IV V VI VTI VIII IX X XI XII XIIT
Gly151 . . B . . T . 0.53 * . F 0.76 0.65
Val152 . . B B . . . 0.42 . * F 0.19 0.65
Gly153 . . B B . . . 0.50 . . . -0.13 0.61
Tyr154 . . B B . . . 0.51 . . . -0.60 0.62
Thr155 . . B B . . . 0.51 . . F -0.30 1.12
Asn156 . . . B . . C 0.86 . . F 0.20 1.81
Ala157 . . . . T T . 0.90 . . F 0.80 1.86
Ser158 . . . . T T . 0.54 . . F 0.80 1.06
Asn159 . . . . T T . 0.20 . . F 0.35 0.57
Asn160 . . . . T T . -0.16* . F 0.35 0.57
Leu161 . A B . . . . -0.97* . . -0.60 0.23
Phe162 . A B . . . . -0.59. . . -0.60 0.12
Ala163 . A B . . . . -0:96. . . -0.60 0.11
Cys164 . A B . . . . -1.27* . . -0.60 0.07
Leu165 . . B . . T . -1.86. . . -0.20 0.12
Pro166 . . B . . T . -1.71* . . -0.20 0.12
Cys167 . . . . T T . -0.97* . . 0.20 0.12
Thr168 A . . . . T . -0.68. . . 0.10 0.30
Ala169 A . . . . . . -0.01. . . 0.50 0.26
Cys170 A . . . . T . 0.80 . . . 0.70 0.80
Lys171 A . . . . T . 1.01 . . F 1.15 0,.96
Ser172 A . . . . T . 1.68 . * F 1.30 1.65
Asp173 A . . . . T . 2.10 . * F 1.30 5.33
Glu174 A A . . . . . 2.39 . * F 0.90 5.22
Glu175 A A . . . . . 2.84 . * F 1.24 5.22
Glu176 A A . . . . . 2.13 . * F 1.58 4.83
Arg177 . A . . T _ . 2.12 . . F 2.32 1.50
Ser178 . . . . . T C 1.81 . . F 2.86 1.25
Pro179 . . . . T T . 1.50 * . F 3.40 1.04
Cys180 . . . . T T . 1.61 * . F 2.61 0.77
Thr181 . . . . T T . 1.61 * . F 2.67 1.12
Thr182 . . . . T . . 1.19 * * F 2.38 1.16
Thr183 . . . . T T . 0.90 . . F 2.49 3.13
Arg184 . . . . T T . 0.44 . . F 2.40 2.19
Asn185 . . . . T T . 1.11 . . F 2.50 0.81
Thr186 . . . . T T . 0.76 * . F 2.25 0.98
Ala187 _ . . . T . . 1.11 * . . 1.65 0.27
Cys188 . . . . T . . 1.21 * . . 1.40 0.33
Gln189 . . B . . . . 0.76 * . . 0.75 0.36
Cys190 . . B . . . . 0.44 . . . 0.50 0.35
Lys191 . . B . . T . 0.06 . * F 0.85 0.94
Pro192 . . . . T T . 0.76 . . F 0:65 0.47
Gly193 . . . . T T . 1.42 . * F 1.74 1.72
Thr194 . . B . . T . 1.42 . * F 1.68 1.38
Phe195 . . B . . . . 2.09 . * F 1.82 1.49
Arg196 . . . . T . . 1.74 . * F 2.56 2.42
Asn197 . . . . T T . 1.37 . * F 3.40 2.25
Asp198 . . . . T T . 1.71 . * F 3.06 2.63
Asn199 . . . . . T C 1.42 . * F 2.52 2.32
Ser200 A . . . . T . 1.46 . * F 1.98 1.43
36
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Table 2 (continued)
ResPositionI II III IV V VI VII VIII IX X XI XII XIII
A1a201 A . . . , . . 1.46 . * . 1.14 0.46
Glu202 A . . . . . . 2.50 * . . 0.80 0.56
Met203 A . . . . . . 0.83 * . . 1.11 0.83
Cys204 A . . . . T . 0.53 * . . 1.62 0.44
Arg205 . . . T T . 0.52 * . . 2.33 0.34
Lys206 . . . . T T . 0.77 * . F 2.49 0.50
Cys207 . . . . T T . 0.10 * . F 3.10 0.92
Ser208 . . . . T , . 0.49 * * F 2.59 0.25
Thr209 . . . . T . . 1.27 * * F 1.98 0.19
Gly210 . . . . T . . 0.81 * . F 1.67 0.71
Cys211 . . B . . T . 0.17 * * F 1.16 0.53
Pro212 . . . . T T . -0.02* * F 1.25 0.36
Arg213 . . . . T T . 0.32 * * F 0.65 0.27
Gly214 . . B . . T . -0.22* * . 0.85 1.01
Met215 . . B B . . . 0.17 * * . 0.30 0.48
Val216 . . B B . . . 0.83 * * _ 0.79 0.49
Lys217 . . B B . . . 0.38 * * . 0.98 0.83
Val218 . . B B . . . -0.04* * F 1.32 0.45
Lys219 . . B B . . . 0.09 . * F 1.51 0.88
Asp220 . . B . . . . 0.40 . * F 1.90 0.68
Cys221 . . B . . . . 0.96 . * F 0.81 0.96
Thr222 . . . . . T C 0.91 . * F 1.62 0.65
Pro223 . . . . T T . 0.88 . * F 1.63 0.65
Trp224 . . . . T T . 0.83 . * F 0.54 0.84
Ser225 A . . . . T . 0.17 . . F 1.00 1.01
Asp226 A A . . . . . -0.02. . F 0.45 0.35
Ile227 A A . . . . . 0.26 * . . -0.30 0.25
Glu228 A A . . . . . 0.51 * . . 0.30 0.25
Cys229 . A B . . . . 0.80 * . . 0.60 0.30
Val230 A A . . . . . 0.80 * * . 0.60 0.74
His231 A A . . . . . 0.46 * * . 0.60 0.58
Lys232 A A . . . . . 1.34 * . F 0.60 1.06
Glu233 . A . . T . . 1.00 * . F 1.30 2.30
Ser234 . . . , T T . 1.63 * . F 1.70 1.68
Gly235 . . . . T T . 2.49 * . F 1.70 1.14
Asn236 . . . , T T . 1.63 * . F 1.40 1.06
Gly237 . . . . . T C 1.30 * . F 0.45 0.55
His238 . . . B . . C 0.44 . . . -0.40 0.59
Asn239 . . . B . . C -0.14. . . -0.40 0.27
Ile240 . . B B . . . -0.61. . . -0.60 0.19
Trp241 . . B B . . . -1.47. . . -0.60 0.12
Val242 . . B B . . . -1.98. . . -0.60 0.05
Ile243 . . B B . . . -2.26. . . -0.60 0.06
Leu244 . . B B . . . -3.07. . . -0.60 0.08
Val245 . . B B . . . -3.03. . . -0.60 0.09
Val246 . . B B . . . -3.60. . . -0.60 0.09
Thr247 . . B B . . . -2.96. . . -0.60 0.08
Leu248 . . B B . . . -2.88. . . -0.60 0.17
Val249 . . B B . . . -2.88. * . -0.60 0.19
Val250 . . B B . . . -2.83. . . -0.60 0.11
37
CA 02426710 2003-04-23
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Table 2 (continued)
ResPositionI II III IV V VI VII VIII IX X XI XII XIII
Pro251 . . B B . . . -2.83. . . -0.600.11
Leu252 . . B B . . . -3.11. . . -0.600.11
Leu253 A . . B . . . -3.16. . . -0.600.15
Leu254 A . . B . . . -3.11. . . -0.600.07
Val255 A . . B . . . -3.14. . . -0.600.07
Ala256 A . . B . . . -3.79. . . -0.600.06
Val257 . . B B . . . -3.64. . . -0.600.05
Leu258 , . B B . . . -3.50. . . -0.600.04
Ile259 . . B B . . . -3.36. . . -0.600.02
Val260 , . B B . . . -3.39. . . -0.600.02
Cys261 . . B B . . . -3.14. . . -0.600.01
Cys262 . . B B . . . -2.59. . . -0.600.02
Cys263 . . B B . . . -2.12. . . -0.600.03
Tle264 . . B B . . . -1.90. . . -0.600.06
Gly265 . . . . T T . -1.39. . F 0.35 0.06
Ser266 . . . . T T . -1.07. . F 0.35 0.11
G1y267 . . . . T T . -0.40. . F 0.65 0.16
Cys268 . . . . T T . 0.06 . . F 1.25 0.27
Gly269 . . . . T . . 0.99 . * F 1.39 0.31
Gly270 . . . . T . . 0.67 _ . F 2.03 0.62
Asp271 . . . . . T C 0.37 . . F 2.37 0.62
Pro272 . . . . T T . 0.71 * * F 2.91 0.62
Lys273 . . . . T T . 1.49 * * F 3.40 1.05
Cys274 . . B . . T . 0.98 * * . 2.51 1.23
Met275 . . B B . . . 0.66 * * . 1.62 0.59
Asp276 . . B B . . . -0.04* * . 1.28 0.16
Arg277 . . B B . . . -0.12. * . 0.04 0.26
Val278 . . B B . . . -0.06. * . -0.600.27
Cys279 . . B B . . . -0.20. . . 0.30 0.32
Phe280 . . B B . . . 0.06 . * . -0.600.13
Trp281 . . B B . . . -0.76. . . -0.600.18
Arg282 . . B B . . . -1.68. . . -0.600.28
Leu283 . . B B . . . -0.71. . . -0.600.26
G1y284 . . . B T . . -0.39. * . -0.200.49
Leu285 . . . B . . C 0.10 . * . 0.50 0.25
Leu286 . . . B . . C 0.04 . * . 0.20 0.46
Arg287 . . . B . . C -0.66. . F 0.65 0.46
Gly288 . . , . . T C 0.16 . . F 1.35 0.57
Pro289 . . . . . T C 0.50 . * F 2.70 1.19
Gly290 . . . . . T C 1.31 * * F 3.00 1.01
Ala291 A . . . . T . 1.53 . * F 2.50 1.65
Glu292 A . . . . . . 1.39 . . F 2.00 1.08
Asp293 A . . . . . . 1.73 . . F 1.70 1.48
Asn294 A . . . . T . 2.94 . * . 1.45 2.36
Ala295 A . . . . T . 1.40 . . . 1.15 2.36
His296 A . . . . T . 1.18 * . . 1.00 0.99
Asn297 A . . . . T . 0.88 . . . 0.10 0.51
Glu298 A . . . . . . 0.88 * . . -0.200.67
Ile299 A . . . . . . 0.29 * * . -0.100.80
Leu300 A . . . . . . 0.88 * * . -0.100.50
38
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Table 2 (continued)
ResPositionI II III IV V VI VII VIII IX X XI XII XTII
Ser301 A . . . . . . 0.61 * . F 0.65 0.48
Asn302 A . . . . T . -0.20* . F 0.25 0.92
Ala303 A . . . . T . -0.50* . F 0.25 0.92
Asp304 A . . . . T . 0.08 * . F 0.85 0.92
Ser305 . . . . . T C 0.19 * . F 1.05 0.83
Leu306 . . . B . . C -0.37* . F 0.05 0.71
Ser307 . . B B . . . -0.67* . F -0.150.31
Thr308 . . B B . . . -0.08* . . -0.600.31
Phe309 . . B B . . . -0.08* . . -0.300.66
Val310 A . . B . . . 0.22 . . F -0.250.85
Ser311 A A . . . . . 0.43 . . F 0.00 1.03
.
Glu312 A A . . . . . 0.73 . . F 0.00 1.17
Gln313 A A . . . . . 0.74 . . F 0.90 2.73
Gln314 A A . . . . . 1.44 . . F 0.90 2.73
Met315 A A . . . . . 2.30 . . F 0.90 2.73
Glu316 A A . . . . . 2.39 . . F 0.90 2.73
Ser317 A A . . . . . 2.80 . * F 0.90 2.44
Gln318 A A . . . . . 1.80 . * F 0.90 2.49
Glu319 A A . . . . . 0.99 . * F 0.90 2.40
Pro320 A A . .- . . . 1.28 . * F 0.90 1.48
Ala321 A A . . . . . 0.93 . . F 0.60 1.23
Asp322 A A . B . . . 0.38 . . F 0.45 0.70
Leu323 A A . B . . . 0.07 . . F -0.150.34
Thr324 . A B B . . . -0.79. . F -0.150.48
G1y325 . A B B . . . -0.58. . . -0.300.21
Val326 . . B B . . . -0.29. . . -0.600.45
Thr327 . . B B . . . -0.50. . . -0.600.42
Val328 . . B B . . . -0.03. * F -0.170.65
Gln329 . . B B . . . 0.28 , * F 0.11 0.87
Sex330 . . . . . T C 0.03 . * F 2.04 1.05
Pro331 . . . . . T C 0.89 . * F 2.32 1.42
Gly332 . . . . T T . 0.53 . * F 2.80 1.42
Glu333 A . . . . T . 0.58 . * F 1.97 0.57
Ala334 . . B . . . . -0.23. * . 0.74 0.30
Gln335 . . B . . . . -0.28. . . 0.46 0.25
Cys336 . . B , . . . -0.28. . . 0.18 0.14
Leu337 . . B . . . . -0.52. * . -0.400.22
Leu338 . . B . . . . -0.52. * . -0.400.13
Gly339 . A . . . . C -0.52. * F 0.05 0.42
Pro340 A A . . . . . -0.52. * F -0.150.51
Ala341 A A . . . . . -0.20. * F 0.60 1.07
Glu342 A A . . . . . 0.31 . * F 0.90 1.07
Ala343 A A . . . . . 1.12 * * F 0.75 0.93
Glu344 A A . . . . . 1.58 . * F 0.90 1.60
G1y345 A A . . . . . 1.90 . * F 0.90 1.80
Ser346 A . . . . T . 2.60 . * F 2.30 3.50
Gln347 A . . . . T . 1.79 . * F 1.30 3.96
Arg348 A . . . . T . 1.57 . * F 1.30 3.30
Arg349 . . B . . T . 0.71 . * F 1.30 2.03
Arg350 . . B B . . . 0.84 . * F 0.75 0.87
39
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Table Z (continued)
ResPositionI II III IV V VI VTI VIII IX X XI XIT XIII
Leu351 . . B B . . . 0.56 . * . 0.60 0.69
Leu352 . . B B . . . 0.56 . * . 0.30 0.35
Val353 . . B B . . . 0.10 * * . -0.300.29
Pro354 . . B . . T , -0.60* . . -0,200.35
Ala355 . . . . T T . -0.71. * . 0.50 0.43
Asn356 . . . . . T C -0.11. . F 1.65 0.96
Gly357 . . . . . T C 0.39 . . F 1.95 0.96
Ala358 . . . . . . C 1.24 . . F 2.20 1.37
Asp359 . . . . . T C 1.14 . . F 3.00 1.48
Pro360 A . . . . T . 0.92 * . F 2.50 2.16
Thr361 A . . . . T . 0.32 . . F 1.90 1.76
Glu362 A . . . . T . -0.14. . F 1.60 1.04
Thr363 A . . B . . . -0.26. . F 0.15 0.56
Leu364 A . . B . . . -0.96* . . -0.600.33
Met365 A . . B . . . -0.74* . . -0.600.17
Leu366 A . . B . . . -0.39* . . -0.600.19
~
Phe367 A . . B . . . -1.09* . . -0.600.47
Phe368 A . . B . . . -1.37* . . -0.600.41
Asp369 A . . B . . . -0.56* . . -0.600.50
Lys370 A A . . . . . -0.84* . . -0.300.93
Phe371 A A . B . . . -0.89* . . -0.300.75
Ala372 A A . B . . . -0.40* . . -0.300.34
Asn373 . A B B . . . -0.40* . . -0.600.26
Ile374 . A B B . . . -0.40* . . -0.600.26
VaI375 . A B B . . . -0.74. . . -0.600.43
Pro376 . A . B . . C -0.33. . . -0.100.36
Phe377 . . . . T T . 0.26 . . . 0.20 0.54
Asp378 . . . . T T . 0.26 . . F 0.80 1.21
Ser379 . . . . T T . 0.33 . . F 1.40 2.35
Trp380 A . . . . T . 0.59 * * F 0.40 1.29
Asp381 A A . . . . . 0.91 * . F -0.15Ø76
Gln382 A A . . . . . 1.61 * . . -0.151.11
Leu383 A A . . . . . 0.80 * . . -0.151.84
Met384 A A . . , . . 1.10 * . . 0.30 0.91
Arg385 A A . . . . . 0.58 * . . 0.30 0.87
Gln386 A A . . . . . 0.27 * . . -0.300.87
Leu387 A A . . . . . 0.31 * . . 0.45 1.27
Asp388 A A . . . . . 1.12 * . . 0.75 1.30
Leu389 A A . . . . . 1.72 * . F 0.60 2.21
Thr390 A . . . . T . 0.72 * . F 1.30 2.54
Lys391 A . . . . T . 0.72 . * F 1.30 1.07
Asn392 A . . . . T . 0.68 * * F 1.30 2.16
Glu393 A . . . . T . -0.18* . F 1.30 1.11
Ile394 . . B B . . . 0.74 * . F 0.75 0.41
Asp395 . . B B . . . 0.47 * * . 0.60 0.50
Val396 , . B B . . . 0.08 * * . 0.60 0.29
Val397 . . B B . . . -0.23. . . 0.51 0.41
Arg398 . . B . . T . -0.82* . . 1.12 0.36
Ala399 . . B , . T . -0.28* . . 0.73 0.49
Gly400 . . . . T T . -0.49* . F 2.09 0.65
CA 02426710 2003-04-23
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Table 2 (continued)
ResPositionI II III IV V VI VII VIII IX X XI XII XIII
'
Thr401 . . . . . T C 0.02 * * F 2.10 0.51
Ala402 . . . . . . C 0.88 * * F 1.09 0.50
Gly403 . . . . . T C 0.18 * * F 1.68 0.85
Pro404 . . . . . T C -0.04. . F 1.47 0.59
Gly405 . . . . . T C 0.06 . . F 1.26 0.48
Asp406 A . . . . T . -0.22. . F 0.25 0.76
Ala407 A A . . . . . -0.23. . . -0.300.50
Leu408 A A . . . . . -0.70. . . -0.600.50
Tyr409 A A . . . . . -1.09* . . -0.600.25
Ala410 A A . . . . . -0.70* . . -0.600.24
Met411 A A . . . . . -0.99* . . -0.600.59
Leu412 A A . . . . . -1.26* . . -0.600.39
Met413 A A . . . . . -0.44* . . -0.600.29
Lys414 A A . B . . . -0.16* . . -0.600.47
Trp415 A A . B . . . 0.12 * . . 0.15 1.14
Val416 A A . B . . . 0.38 * * . 0.45 1.66
Asn417 A . . . . T . 1.30 * . F 1.75 0.82
Lys418 A . . . . T . 1.90 * . F 2.20 1.53
~
Thr419 . . . . . T C 1.27 * . F 3.00 3.32
Gly420 . . . . . T C 1.26 * . F 2.70 2.08
Arg421 . . . . T . . 1.22 * . F 2.40 1.40
Asn422 . . . . . T C 1.19 * . F 1.65 0.68
Ala423 . . B . . T . 0.83 . . . 1.00 0.93
Ser424 . . B . . T . 0.33 . . . 0.70 0.69
Ile425 . . B . . T . -0.13. * . -0.200.35
His426 . A B . . . -0.24. * . -0.600.29
Thr427 . A B . . . . -0.83* * . -0.600.36
Leu428 A A . . . . . -1.06* * . -0.600.52
Leu429 A A . . . . . -0.76* * . -0.600.31
Asp430 A A . . . . . 0.24 * * . -0.300.38
Ala431 A A . . . . . -0.32* * . 0.30 0.89
.
Leu432 A A . . . . . -0.02* * . 0.75 1.07
Glu433 A A . . . . . 0.80 * * . 0.75 1.11
Arg434 A A . . . . 1.72 * * F 0.90 1.90
Met435 A A . . . . . 1.69 * * F 0.90 4.52
Glu436 A A . . . . . 1.69 * * F 0.90 3.55
Glu437 A A . . . . . 2.54 * . F 0.90 1.83
Arg438 A A . . . . . 2.54 * * F 0.90 3.70
His439 A A . . . . . 2.48 * * F 0.90 3.70
Ala440 A A . . . . . 2.19 * * F 0.90 4.28
Lys441 A A . . . . . 2.19 * * F 0.90 7..53
Glu442 A A . . . . . 2.19 * . F 0.90 1.95
Lys443 A A . . . . . 1.27 * * F 0.90 3.22
Ile444 A A . . . . . 0.49 * * F 0.90 1.33
Gln445 A A . . . . . 0.22 * * F 0.75 0.63
Asp446 A A . . . . . 0.18 * * F -0.150.23
Leu447 A A . . . . . -0.12* . . -0.300.56
Leu448 A A . . . . . -0.51* . . 0.55 0.43
Val449 A A . . . . . 0.42 * . F 0.95 0:26
Asp450 A . . . . T . -0.28* . F 1.60 0_62
41
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WO 02/079377 PCT/USO1/42996
Table 2 (continued)
ResPosition II III V VI VII VIII IX XI XI2 XIII
I IV X
Ser451 . . . . T T . -1.17 * F 2.25 0.65
.
Gly452 . . . . T T . -0.60 * F 2.50 0.62
.
Lys453 . . B . . T . -0.60 . F 1.25 0.58
.
Phe454 . A B . . . . 0.26 . . . 0.15 0.36
Ile455 . A B . . . , 0.26 . . . 0.20 0.62
Tyr456 . A B . . . . 0.21 . . . 0.55 0.52
Leu457 . A B . . . . 0.24 . . . -0.030.59
Glu458 . A B . . . . -0.14 . F 0.54 1.22
.
Asp459 . A . . T . . 0.26 . . F 1.66 0.77
Gly460 . . . . T T . 0.56 . . F 2.78 1.26
Thr461 . . . . . T C -0.06 * F 2.70 0.73
.
Gly462 . . . . . T C 0.46 * . F 2.13 0.33
Ser463 . . . . . T C -0.36 '. F 1.26 0.44
.
Ala464 A . . . . . . -0.36 . , 0.14 0.25
.
Val465 . . B . . . . -0.40 . . 0.17 0.44
.
Ser466 . . B . . . . -0.48 . , -0.100.42
.
Leu467 . . B . . . . -0,52 . . -0.100.53
.
Glu468 A . . . . . . -0.61 . . 0.50 0.92
.
42
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
[0085] In another aspect, the invention provides an antibody that binds a
peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide described
herein. The
epitope of this polypeptide portion is an immunogenic or antigenic epitope of
a
polypeptide of the invention. 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).
[0086] As to the selection of peptides or polypeptides bearing an antigenic
epitope
(i.e., that contain a region of a protein molecule to which an antibody can
bind), it is well
known in that art that relatively short synthetic peptides that mimic part of
a protein
sequence are routinely capable of eliciting an antiserum that reacts with the
partially
mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green,
N. and
Learner, R.A. (1983) Antibodies that react with predetermined sites on
proteins. Scieface
219:660-666. Peptides capable of eliciting protein-reactive sera are
frequently represented
in the primary sequence of a protein, can be characterized by a set of simple
chemical
rules, and are confined neither to immunodominant regions of intact proteins
(i.e.,
immunogenic epitopes) nor to the amino or carboxyl terminals.
[0087] Antigenic epitope-bearing peptides and polypeptides are therefore
useful to
raise antibodies, including monoclonal antibodies, that bind to a TR4
polypeptide of the
invention. See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777.
Antigenic
epitope-bearing peptides and polypeptides 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 SEQ ll~ NO:1.
[0088] Antibodies of the invention may bind one or more antigenic TR4
polypeptides
or peptides including, but not limited to: a polypeptide comprising amino acid
residues
from about 35 to about 92 of SEQ m NO:1; a polypeptide comprising amino acid
residues
from about 114 to about 160 of SEQ m N0:1; a polypeptide comprising amino acid
residues from about 169 to about 240 of SEQ m N0:1; a polypeptide comprising
amino
acid residues from about 267 to about 298 of SEQ ID NO:I; a polypeptide
comprising
amino acid residues from about 330 to about 364 of SEQ m NO:1; a polypeptide
comprising amino acid residues from about 391 to about 404 of SEQ m NO:l;
and%or a
43
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
polypeptide comprising amino acid residues from about 418 to about 465 of SEQ
ID
NO:1. In this context "about" includes the particularly recited range, larger
or smaller by
several (5, 4, 3, 2, or 1) amino acids, at either terminus or at both termini.
As indicated
above, the inventors have determined that the above polypeptide fragments are
antigenic
regions of the TR4 protein. Epitope-bearing TR4 peptides and polypeptides may
be
produced by any conventional means. Houghten, R.A., "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. 4,631,21 I to Houghten et al. (1986).
[0089] As one of skill in the art will appreciate, TR4 polypeptides and the
epitope-bearing fragments thereof described herein (e.g., corresponding to a
portion of the
extracellular domain such as, for example, amino acid residues 1 to 240 of SEQ
ID NO:1
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 TR4 protein or protein fragment alone
(Fountoulakis et al.,
J Bioclaerra 270:3958-3964 (1995)). Thus, antibodies of the invention may bind
fusion
proteins that comprise all or a portion of a TRAIL receptor polypeptide such
as TR4.
[0090] 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 yiel°ds and show better solubility than the corresponding
natural polypeptide, at
least under certain purification and storage conditions. Antibodies of the
present invention
may also bind such modified TR4 polypeptides or TR4 polypeptide fragments or
variants.
[009] For instance, 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
44
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
without substantial loss of biological function, or Ioss of the ability to be
bound by a
specific antibody. For instance, Ron et al., J. Biol. Chem., 26:2984-2988
(1993) reported
modified KGF proteins that had heparin binding activity even if 3, 8, or 27
amino-ternninal
amino acid residues were missing. In the present case, since TR4 is a member
of the death
domain containing receptor (DDCR) polypeptide family, deletions of N-terminal
amino
acids up to the cysteine residue at position 109 in SEQ ID NO:1 may retain
some
biological activity such as the ability to induce apoptosis. Polypeptides
having further N-
tenninal deletions including the cysteine residue at position 109 (C-109) in
SEQ ID N0:1
would not be expected to retain such biological activities because this
residue is conserved
among family members and may be required for forming a disulfide bridge to
provide
structural stability which is needed for ligand binding.
[0092] However, 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
TR4 ligand (e.g., TRAIL)) may still be retained. For example, the ability of
shortened
TR4 polypeptides to induce and/or bind to antibodies which recognize the
complete or
mature forms of the TR4 polypeptides generally will be retained when less than
the
majority of the residues of the complete or mature polypeptide are removed
from the
N-terminus. Whether a particular polypeptide lacking N-terminal residues of a
complete
polypeptide retains such immunologic activities can readily be determined by
routine
methods described herein and otherwise known in the art. It is not unlikely
that a TR4
polypeptide 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
TR4 amino acid residues may often evoke an immune response.
[0093] Accordingly, the present invention further provides antibodies that
bind
polypeptides having one or more residues deleted from the amino terminus of
the TR4
amino acid sequence of SEQ ID NO:l up to the serine residue at position number
463 and
polynucleotides encoding such polypeptides. In particular, the present
invention provides
antibodies that bind polypeptides comprising the amino acid sequence of
residues n1-468
of SEQ ID NO:I, where n1 is an integer from 2 to 463 corresponding to the
position of the
amino acid residue in SEQ ID NO:1.
[0094] More in particular, the invention provides antibodies that bind
polypeptides
comprising, or alternatively consisting of, the amino acid sequence of
residues of A-2 to
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
E-468; P-3 to E-468; P-4 to E-468; P-5 to E-468; A-6 to E-468; R-7 to E-468; V-
8 to E-
468; H-9 to E-468; L-10 to E-468; G-11 to E-468; A-I2 to E-468; F-13 to E-468;
L-14 to
E-468; A-15 to E-468; V-16 to E-468; T-17 to E-468; P-18 to E-468; N-I9 to E-
468; P-20
to E-468; G-21 to E-468; S-22 to E-468; A-23 to E-468; A-24 to E-468; S-25 to
E-468; G-
26 to E-468; T-27 to E-468; E-28 to E-468; A-29 to E-468; A-30 to E-468; A-31
to E-468;
A-32 to E-468; T-33 to E-468; P-34 to E-468; S-35 to E-468; K-36 to E-468; V-
37 to E-
468; W-38 to E-468; G-39 to E-468; S-40 to E-468; S-41 to E-468; A-42 to E-
468; G-43
to E-468; R-44 to E-468; I-45 to E-468; E-46 to E-468; P-47 to E-468; R-48 to
E-468; G-
49 to E-468; G-50 to E-468; G-51 to E-468; R-52 to E-468; G-53 to E-468; A-54
to E-
468; L-55 to E-468; P-56 to E-468; T-57 to E-468; S-58 to E-468; M-59 to E-
468; G-60 to
E-468; Q-61 to E-468; H-62 to E-468; G-63 to E-468; P-64 to E-468; S-65 to E-
468; A-66
to E-468; R-67 to E-468; A-68 to E-468; R-69 to E-468; A-70 to E-468; G-71 to
E-468; R-
72 to E-468; A-73 to E-468; P-74 to E-468; G-75 to E-468; P-76 to E-468; R-77
to E-468;
P-78 to E-468; A-79 to E-468; R-80 to E-468; E-81 to E-468; A-82 to E-468; S-
83 to E-
468; P-84 to E-468; R-85 to E-468; L-86 to E-468; R-87 to E-468; V-88 to E-
468; H-89 to
E-468; K-90 to E-468; T-9I to E-468; F-92 to E-468; K-93 to E-468; F-94 to E-
468; V-95
to E-468; V-96 to E-468; V-97 to E-468; G-98 to E-468; V-99 to E-468; L-100 to
E-468;
L-l0I to E-468; Q-102 to E-468; V-103 to E-468; V-104 to E-468; P-105 to E-
468; S-106
to E-468; S-107 to E-468; A-108 to E-468; A-109 to E-468; T-110 to E-468; I-
111 to E-
468; K-112 to E-468; L-113 to E-468; H-114 to E-468; D-I15 to E-468; Q-116 to
E-468;
S-117 to E-468; I-I18 to E-468; G-119 to E-468; T-120 to E-468; Q-12I to E-
468; Q-122
to E-468; W-123 to E-468; E-124 to E-468; H-125 to E-468; S-126 to E-468; P-
127 to E-
468; L-128 to E-468; G-129 to E-468; E-130 to E-468; L-131 to E-468; C-132 to
E-468;
P-133 to E-468; P-I34 to E-468; G-135 to E-468; S-136 to E-468; H-137 to E-
468; R-138
to E-468; S-139 to E-468; E-140 to E-468; R-14I to E-468; P-142 to E-468; G-
143 to E-
468; A-144 to E-468; C-145 to E-468; N-146 to E-468; R-147 to E-468; C-I48 to
E-468;
T-149 to E-468; E-150 to E-468; G-151 to E-468; V-152 to E-468; G-153 to E-
468; Y-154
to E-468; T-155 to E-468; N-156 to E-468; A-157 to E-468; S-158 to E-468; N-
159 to E-
468; N-160 to E-468; L-161 to E-468; F-162 to E-468; A-163 to E-468; C-164 to
E-468;
L-165 to E-468; P-166 to E-468; C-167 to E-468; T-168 to E-468; A-169 to E-
468; C-170
to E-468; K-171 to E-468; S-172 to E-468; D-173 to E-468; E-174 to E-468; E-
175 to E-
468; E-176 to E-468; R-177 to E-468; S-178 to E-468; P-179 to E-468; C-180 to
E-468;
T-181 to E-468; T-182 to E-468; T-183 to E-468; R-184 to E-468; N-185 to E-
468; T-186
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CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
to E-468; A-187 to E-468; C-188 to E-468; Q-189 to E-468; C-190 to E-468; K-
191 to E-
468; P-192 to E-468; G-193 to E-468; T-194 to E-468; F-195 to E-468; R-196 to
E-468;
N-197 to E-468; D-198 to E-468; N-199 to E-468; S-200 to E-468; A-201 to E-
468; E-202
to E-468; M-203 to E-468; C-204 to E-468; R-205 to E-468; K-206 to E-468; C-
207 to E-
468; S-208 to E-468; T-209 to E-468; G-210 to E-468; C-211 to E-468; P-212 to
E-468;
R-213 to E-468; G-214 to E-468; M-215 to E-468; V-2I6 to E-468; K-217 to E-
468; V-
218 to E-468; K-219 to E-468; D-220 to E-468; C-221 to E-468; T-222 to E-468;
P-223 to
E-468; W-224 to E-468; S-225 to E-468; D-226 to E-468; I-227 to E-468; E-228
to E-468;
C-229 to E-468; V-230 to E-468; H-231 to E-468; K-232 to E-468; E-233 to E-
468; S-234
to E-468; G-235 to E-468; N-236 to E-468; G-237 to E-468; H-238 to E-468; N-
239 to E-
468; I-240 to E-468; W-241 to E-468; V-242 to E-468; I-243 to E-468; L-244 to
E-468;
V-245 to E-468; V-246 to E-468; T-247 to E-468; L-248 to E-468; V-249 to E-
468; V-250
to E-468; P-251 to E-468; L-252 to E-468; L-253 to E-468; L-254 to E-468; V-
255 to E-
468; A-256 to E-468; V-257 to E-468; L-258 to E-468; I-259 to E-468; V-260 to
E-468;
C-261 to E-468; C-262 to E-468; C-263 to E-468; I-264 to E-468; G-265 to E-
468; S-266
to E-468; G-267 to E-468; C-268 to E-468; G-269 to E-468; G-270 to E-468; D-
271 to E-
468; P-272 to E-468; K-273 to E-468; C-274 to E-468; M-275 to E-468; D-276 to
E-468;
R-277 to E-468; V-278 to E-468; C-279 to E-468; F-280 to E-468; W-281 to E-
468; 8-
282 to E-468; L-283 to E-468; G-284 to E-468; L-285 to E-468; L-286 to E-468;
R-287 to
E-468; G-288 to E-468; P-289 to E-468; G-290 to E-468; A-291 to E-468; E-292
to E-
468; D-293 to E-468; N-294 to E-468; A-295 to E-468; H-296 to E-468; N-297 to
E-468;
E-298 to E-468; I-299 to E-468; L-300 to E-468; S-301 to E-468; N-302 to E-
468; A-303
to E-468; D-304 to E-468; S-305 to E-468; L-306 to E-468; S-307 to E-468; T-
308 to E-
468; F-309 to E-468; V-3I0 to E-468; S-311 to E-468; E-312 to E-468; Q-313 to
E-468;
Q-314 to E-468; M-315 to E-468; E-316 to E-468; S-317 to E-468; Q-318 to E-
468; E-319
to E-468; P-320 to E-468; A-321 to E-468; D-322 to E-468; L-323 to E-468; T-
324 to E-
468; G-325 to E-468; V-326 to E-468; T-327 to E-468; V-328 to E-468; Q-329 to
E-468;
S-330 to E-468; P-331 to E-468; G-332 to E-468; E-333 to E-468; A-334 to E-
468; Q-335
to E-468; C-336 to E-468; L-337 to E-468; L-338 to E-468; G-339 to E-468; P-
340 to E-
468; A-341 to E-468; E-342 to E-468; A-343 to E-468; E-344 to E-468; G-345 to
E-468;
S-346 to E-468; Q-347 to E-468; R-348 to E-468; R-349 to E-468; R-350 to E-
468; L-351
to E-468; L-352 to E-468; V-353 to E-468; P-354 to E-468; A-355 to E-468; N-
356 to E-
468; G-357 to E-468; A-358 to E-468; D-359 to E-468; P-360 to E-468; T-361 to
E-468;
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E-362 to E-468; T-363 to E-468; L-364 to E-468; M-365 to E-468; L-366 to E-
468; F-367
to E-468; F-368 to E-468; D-369 to E-468; K-370 to E-468; F-371 to E-468; A-
372 to E-
468; N-373 to E-468; I-374 to E-468; V-375 to E-468; P-376 to E-468; F-377 to
E-468; D-
378 to E-468; S-379 to E-468; W-380 to E-468; D-381 to E-468; Q-382 to E-468;
L-383
to E-468; M-384 to E-468; R-385 to E-468; Q-386 to E-468; L-387 to E-468; D-
388 to E-
468; L-389 to E-468; T-390 to E-468; K-391 to E-468; N-392 to E-468; E-393 to
E-468; I-
394 to E-468; D-395 to E-468; V-396 to E-468; V-397 to E-468; R-398 to E-468;
A-399
to E-468; G-400 to E-468; T-401 to E-468; A-402 to E-468; G-403 to E-468; P-
404 to E-
468; G-405 to E-468; D-406 to E-468; A-407 to E-468; L-408 to E-468; Y-409 to
E-468;
A-410 to E-468; M-411 to E-468; L-412 to E-468; M-413 to E-468; K-414 to E-
468; W-
415 to E-468; V-416 to E-468; N-417 to E-468; K-418 to E-468; T-419 to E-468;
G-420
to E-468; R-421 to E-468; N-422 to E-468; A-423 to E-468; S-424 to~E-468; I-
425 to E-
468; H-426 to E-468; T-427 to E-468; L-428 to E-468; L-429 to E-468; D-430 to
E-468;
A-431 to E-468; L-432 to E-468; E-433 to E-468; R-434 to E-468; M-435 to E-
468; E-436
to E-468; E-437 to E-468; R-438 to E-468; H-439 to E-468; A-440 to E-468; K-
441 to E-
468; E-442 to E-468; K-443 to E-468; I-444 to E-468; Q-445 to E-468; D-446 to
E-468;
L-447 to E-468; L-448 to E-468; V-449 to E-468; D-450 to E-468; S-451 to E-
468; G-452
to E-468; K-453 to E-468; F-454 to E-468; I-455 to E-468; Y-456 to E-468; L-
457 to E-
468; E-458 to E-468; D-459 to E-468; G-460 to E-468; T-461 to E-468; G-462 to
E-468;
and/or S-463 to E-468 of the TR4 sequence of SEQ ~ NO:1.
[0095] In another embodiment, N-terminal deletions of the TR4 polypeptide can
be
described by the general formula n2 to 238 where n2 is a number from 2 to 238
corresponding to the amino acid sequence identified of SEQ ID N0:1. In
specific
embodiments, antibodies of the invention bind N terminal deletions of the TR4
comprising, or alternatively consisting of, the amino acid sequence of
residues: A-2 to H-
238; P-3 to H-238; P-4 to H-238; P-5 to H-238; A-6 to H-238; R-7 to H-238; V-8
to H-
238; H-9 to H-238; L-10 to H-238; G-11 to H-238; A-12 to H-238; F-13 to H-238;
L-14 to
H-238; A-15 to H-238; V-16 to H-238; T-I7 to H-238; P-18 to H-238; N-19 to H-
238; P-
20 to H-238; G-21 to H-238; S-22 to H-238; A-23 to H-238; A-24 to H-238; S-25
to H-
238; G-26 to H-238; T-27 to H-238; E-28 to H-238; A-29 to H-238; A-30 to H-
238; A-31
to H-238; A-32 to H-238; T-33 to H-238; P-34 to H-238; S-35 to H-238; K-36 to
H-238;
V-37 to H-238; W-38 to H-238; G-39 to H-238; S-40 to H-238; S-41 to H-238; A-
42 to H-
238; G-43 to H-238; R-44 to H-238; I-45 to H-238; E-46 to H-238; P-47 to H-
238; R-48 to
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H-238; G-49 to H-238; G-50 to H-238; G-51 to H-238; R-52 to H-238; G-53 to H-
238; A-
54 to H-238; L-55 to H-238; P-56 to H-238; T-57 to H-238; S-58 to H-238; M-59
to H-
238; G-60 to H-238; Q-61 to H-238; H-62 to H-238; G-63 to H-238; P-64 to H-
238; S-65
to H-238; A-66 to H-238; R-67 to H-238; A-68 to H-238; R-69 to H-238; A-70 to
H-238;
G-71 to H-238; R-72 to H-238; A-73 to H-238; P-74 to H-238; G-75 to H-238; P-
76 to H-
238; R-77 to H-238; P-78 to H-238; A-79 to H-238; R-80 to H-238; E-81 to H-
238; A-82
to H-238; S-83 to H-238; P-84 to H-238; R-85 to H-238; L-86 to H-238; R-87 to
H-238;
V-88 to H-238; H-89 to H-238; K-90 to H-238; T-91 to H-238; F-92 to H-238; K-
93 to H-
238; F-94 to H-238; V-95 to H-238; V-96 to H-238; V-97 to H-238; G-98 to H-
238; V-99
to H-238; L-100 to H-238; L-101 to H-238; Q-102 to H-238; V-103 to H-238; V-
104 to
H-238; P-105 to H-238; S-106 to H-238; S-107 to H-238; A-108 to H-238; A-109
to H-
238; T-110 to H-238; I-111 to H-238; K-112 to H-238; L-113 to H-238; H-114 to
H-238;
D-115 to H-238; Q-116 to H-238; S-117 to H-238; I-118 to H-238; G-119 to H-
238; T-
120 to H-238; Q-121 to H-238; Q-122 to H-238; W-123 to H-238; E-124 to H-238;
H-125
to H-238; S-126 to H-238; P-127 to H-238; L-128 to H-238; G-129 to H-238; E-
130 to H-
238; L-131 to H-238; C-132 to H-238; P-133 to H-238; P-I34 to H-238; G-135 to
H-238;
S-136 to H-238; H-137 to H-238; R-138 to H-238; S-139 to H-238; E-140 to H-
238; 8-
141 to H-238; P-142 to H-238; G-143 to H-238; A-144 to H-238; C-145 to H-238;
N-146
to H-238; R-147 to H-238; C-148 to H-238; T-149 to H-238; E-150 to H-238; G-
151 to H-
238; V-152 to H-238; G-153 to H-238; Y-154 to H-238; T-155 to H-238; N-156 to
H-238;
A-I57 to H-238; S-158 to H-238; N-159 to H-238; N-160 to H-238; L-161 to H-
238; F-
162 to H-238; A-163 to H-238; C-164 to H-238; L-165 to H-238; P-166 to H-238;
C-167
to H-238; T-168 to H-238; A-169 to H-238; C-170 to H-238; K-17I to H-238; ~S-
I72 to H-
238; D-173 to H-238; E-174 to H-238; E-175 to H-238; E-176 to H-238; R-177 to
H-238;
S-178 to H-238; P-179 to H-238; C-180 to H-238; T-181 to H-238; T-182 to H-
238; T-183
to H-238; R-184 to H-238; N-185 to H-238; T-186 to H-238; A-187 to H-238; C-
188 to
H-238; Q-189 to H-238; C-190 to H-238; K-191 to H-238; P-192 to H-238; G-193
to H-
238; T-194 to H-238; F-195 to H-238; R-196 to H-238; N-197 to H-238; D-198 to
H-238;
N-199 to H-238; S-200 to H-238; A-201 to H-238; E-202 to H-238; M-203 to H-
238; C-
204 to H-238; R-205 to H-238; K-206 to H-238; C-207 to H-238; S-208 to H-238;
T-209
to H-238; G-210 to H-238; C-211 to H-238; P-212 to H-238; R-213 to H-238; G-
214 to H-
238; M-215 to H-238; V-216 to H-238; K-217 to H-238; V-218 to H-238; K-219 to
H-
238; D-220 to H-238; C-221 to H-238; T-222 to H-238; P-223 to H-238; W-224 to
H-238;
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S-225 to H-238; D-226 to H-238; I-227 to H-238; E-228 to H-238; C-229 to H-
238; V-230
to H-238; H-231 to H-238; K-232 to H-238; andlor E-233 to H-238; of the TR4
extracellular domain sequence of SEQ ID NO:1.
[0096] As mentioned above, even if deletion of one or more amino acids from
the
C-terminus of a protein results in modification of loss of one or more
biological functions
of the protein, other functional activities (e.g., biological activities,
ability to multimerize,
ability to bind DR4 ligand (e.g., TRAIL)) may still be retained. For example
the ability of
the shortened TR4 polypeptide to induce and/or bind to antibodies which
recognize the
complete or mature forms of the TR4 polypeptide generally will be retained
when less
than the majority of the residues of the complete or mature polypeptide are
removed from
the C-terminus. Whether a particular polypeptide lacking C-terminal residues
of a
complete polypeptide retains such immunologic activities can readily be
determined by
routine methods described herein and otherwise known in the art. It is not
unlikely that a
TR4 polypeptide with a large number of deleted C-terminal amino acid residues
may
retain some biological or immunogenic activities. In fact, peptides composed
of as few as
six TR4 amino acid residues may often evoke an immune response.
[0097] Accordingly, the present invention further provides antibodies that
bind
polypeptides having one or more residues deleted from the carboxy terminus of
the amino
acid sequence of the TR4 polypeptide sequence of SEQ ID NO:1 up to the alanine
residue
at position number 30, and polynucleotides encoding such polypeptides. In
particular, the
present invention provides antibodies that bind polypeptides comprising the
amino acid
sequence of residues 24-ml of SEQ ID NO:1, where ml is an integer from 30 to
467
corresponding to the position of the amino acid residue in SEQ ID NO:l.
[009] More in particular, the invention provides antibodies that bind
polypeptides
comprising, or alternatively consisting of, the amino acid sequence of
residues A-24 to L-
467; A-24 to S-466; A-24 to V-465; A-24 to A-464; A-24 to S-463; A-24 to G-
462; A-24
to T-461; A-24 to G-460; A-24 to D-459; A-24 to E-458; A-24 to L-457; A-24 to
Y-456;
A-24 to I-455; A-24 to F-454; A-24 to K-453; A-24 to G-452; A-24 to S-451; A-
24 to D-
450; A-24 to V-449; A-24 to L-448; A-24 to L-447; A-24 to D-446; A-24 to Q-
445; A-24
to I-444; A-24 to K-443; A-24 to E-442; A-24 to K-441; A-24 to A-440; A-24 to
H-439;
A-24 to R-438; A-24 to E-437; A-24 to E-436; A-24 to M-435; A-24 to R-434; A-
24 to E-
433; A-24 to L-432; A-24 to A-431; A-24 to D-430; A-24 to L-429; A-24 to L-
428; A-24
to T-427; A-24 to H-426; A-24 to I-425; A-24 to S-424; A-24 to A-423; A-24 to
N-422;
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A-24 to R-421; A-24 to G-420; A-24 to T-419; A-24 to K-418; A-24 to N-417; A-
24 to V-
416; A-24 to W-415; A-24 to K-414; A-24 to M-413; A-24 to L-412; A-24 to M-
411; A-
24 to A-410; A-24 to Y-409; A-24 to L-408; A-24 to A-407; A-24 to D-406; A-24
to 6-
405; A-24 to P-404; A-24 to G-403; A-24 to A-402; A-24 to T-401; A-24 to G-
400; A-24
to A-399; A-24 to R-398; A-24 to V-397; A-24 to V-396; A-24 to D-395; A-24 to
I-394;
A-24 to E-393; A-24 to N-392; A-24 to K-391; A-24 to T-390; A-24 to L-389; A-
24 to D-
388; A-24 to L-387; A-24 to Q-386; A-24 to R-385; A-24 to M-384; A-24 to L-
383; A-24
to Q-382; A-24 to D-381; A-24 to W-380; A-24 to S-379; A-24 to D-378; A-24 to
F-377;
A-24 to P-376; A-24 to V-375; A-24 to I-374; A-24 to N-373; A-24 to A-372; A-
24 to F-
371; A-24 to K-370; A-24 to D-369; A-24 to F-368; A-24 to F-367; A-24 to L-
366; A-24
to M-365; A-24 to L-364; A-24 to T-363; A-24 to E-362; A-24 to T-361; A-24 to
P-360;
A-24 to D-359; A-24 to A-358; A-24 to G-357; A-24 to N-356; A-24 to A-355; A-
24 to P-
354; A-24 to V-353; A-24 to L-352; A-24 to L-351; A-24 to R-350; A-24 to R-
349; A-24
to R-348; A-24 to Q-347; A-24 to S-346; A-24 to G-345; A-24 to E-344; A-24 to
A-343;
A-24 to E-342; A-24 to A-341; A-24 to P-340; A-24 to G-339; A-24 to L-338; A-
24 to L-
337; A-24 to C-336; A-24 to Q-335; A-24 to A-334; A-24 to E-333; A-24 to G-
332; A-24
to P-331; A-24 to S-330; A-24 to Q-329; A-24 to V-328; A-24 to T-327; A-24 to
V-326;
A-24 to G-325; A-24 to T-324; A-24 to L-323; A-24 to D-322; A-24 to A-321; A-
24 to P-
320; A-24 to E-319; A-24 to Q-318; A-24 to S-317; A-24 to E-316; A-24 to M-
315; A-24
to Q-314; A-24 to Q-313; A-24 to E-312; A-24 to S-311; A-24 to V-310; A-24 to
F-309;
A-24 to T-308; A-24 to S-307; A-24 to L-306; A-24 to S-305; A-24 to D-304; A-
24 to A-
303; A-24 to N-302; A-24 to S-301; A-24 to L-300; A-24 to I-299; A-24 to E-
298; A-24 to
N-297; A-24 to H-296; A-24 to A-295; A-24 to N-294; A-24 to D-293; A-24 to E-
292; A-
24 to A-291; A-24 to G-290; A-24 to P-289; A-24 to G-288; A-24 to R-287; A-24
to L-
286; A-24 to L-285; A-24 to G-284; A-24 to L-283; A-24 to R-282; A-24 to W-
281; A-24
to F-280; A-24 to C-279; A-24 to V-278; A-24 to R-277; A-24 to D-276; A-24 to
M-275;
A-24 to C-274; A-24 to K-273; A-24 to P-272; A-24 to D-271; A-24 to G-270; A-
24 to 6-
269; A-24 to C-268; A-24 to G-267; A-24 to S-266; A-24 to G-265; A-24 to I-
264; A-24
to C-263; A-24 to C-262; A-24 to C-261; A-24 to V-260; A-24 to I-259; A-24 to
L-258;
A-24 to V-257; A-24 to A-256; A-24 to V-255; A-24 to L-254; A-24 to L-253; A-
24 to L-
252; A-24 to P-251; A-24 to V-250; A-24 to V-249; A-24 to L-248; A-24 to T-
247; A-24
to V-246; A-24 to V-245; A-24 to L-244; A-24 to I-243; A-24 to V-242; A-24 to
W-241;
A-24 to I-240; A-24 to N-239; A-24 to H-238; A-24 to G-237; A-24 to N-236; A-
24 to G-
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235; A-24 to S-234; A-24 to E-233; A-24 to K-232; A-24 to H-231; A-24 to V-
230; A-24
to C-229; A-24 to E-228; A-24 to I-227; A-24 to D-226; A-24 to S-225; A-24 to
W-224;
A-24 to P-223; A-24 to T-222; A-24 to C-221; A-24 to D-220; A-24 to K-219; A-
24 to V-
218; A-24 to K-217; A-24 to V-216; A-24 to M-215; A-24 to G-214; A-24 to R-
213; A-24
to P-212; A-24 to C-211; A-24 to G-210; A-24 to T-209; A-24 to S-208; A-24 to
C-207;
A-24 to K-206; A-24 to R-205; A-24 to C-204; A-24 to M-203; A-24 to E-202; A-
24 to A-
ZOI; A-24 to S-200; A-24 to N-199; A-24 to D-198; A-24 to N-197; A-24 to R-
196; A-24
to F-195; A-24 to T-194; A-24 to G-193; A-24 to P-192; A-24 to K-191; A-24 to
C-190;
A-24 to Q-189; A-24 to C-188; A-24 to A-187; A-24 to T-186; A-24 to N-185; A-
24 to 8-
184; A-24 to T-183; A-24 to T-182; A-24 to T-181; A-24 to C-180; A-24 to P-
179; A-24
to S-178; A-24 to R-177; A-24 to E-176; A-24 to E-175; A-24 to E-I74; A-24 to
D-173;
A-24 to S-172; A-24 to K-171; A-24 to C-170; A-24 to A-169; A-24 to T-168; A-
24 to C-
167; A-24 to P-166; A-24 to L-165; A-24 to C-164; A-24 to A-163; A-24 to F-
162; A-24
to L-161; A-24 to N-160; A-24 to N-159; A-24 to S-158; A-24 to A-157; A-24 to
N-156;
A-24 to T-155; A-24 to Y-154; A-24 to G-153; A-24 to V-152; A-24 to G-151; A-
24 to E-
150; A-24 to T-149; A-24 to C-148; A-24 to R-147; A-24 to N-146; A-24 to C-
145; A-24
to A-144; A-24 to G-143; A-24 to P-142; A-24 to R-141; A-24 to E-140; A-24 to
S-139;
A-24 to R-138; A-24 to H-137; A-24 to S-136; A-24 to G-135; A-24 to P-134; A-
24 to P-
133; A-24 to C-132; A-24 to L-131; A-24 to E-130; A-24 to G-129; A-24 to L-
128; A-24
to P-127; A-24 to S-126; A-24 to H-125; A-24 to E-124; A-24 to W-123; A-24 to
Q-122;
A-24 to Q- I2I; A-24 to T-120; A-24 to G-119; A-24 to I-118; A-24 to S-I17; A-
24 to Q-
116; A-24 to D-115; A-24 to H-114; A-24 to L-113; A-24 to K-112; A-24 to I-
111; A-24
to T-110; A-24 to A-109; A-24 to A-108; A-24 to S-107; A-24 to S-106; A-24 to
P-105;
A-24 to V-104; A-24 to V-103; A-24 to Q-102; A-24 to L-101; A-24 to L-100; A-
24 to V-
99; A-24 to G-98; A-24 to V-97; A-24 to V-96; A-24 to V-95; A-24 to F-94; A-24
to K-
93; A-24 to F-92; A-24 to T-91; A-24 to K-90; A-24 to H-89; A-24 to V-88; A-24
to R-
87; A-24 to L-86; A-24 to R-85; A-24 to P-84; A-24 to S-83; A-24 to A-82; A-24
to E-81;
A-24 to R-80; A-24 to A-79; A-24 to P-78; A-24 to R-77; A-24 to P-76; A-24 to
G-75; A-
24 to P-74; A-24 to A-73; A-24 to R-72; A-24 to G-71; A-24 to A-70; A-24 to R-
69; A-24
to A-68; A-24 to R-67; A-24 to A-66; A-24 to S-65; A-24 to P-64; A-24 to G-63;
A-24- to
H-62; A-24 to Q-61; A-24 to G-60; A-24 to M-59; A-24 to S-58; A-24 to T-57; A-
24 to P-
56; A-24 to L-55; A-24 to A-54; A-24 to G-53; A-24 to R-52; A-24 to G-51; A-24
to G-
50; A-24 to G-49; A-24 to R-48; A-24 to P-47; A-24 to E-46; A-24 to I-45; A-24
to R-44;
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A-24 to G-43; A-24 to A-42; A-24 to S-41; A-24 to S-40; A-24 to G-39; A-24 to
W-38;
A-24 to V-37; A-24 to K-36; A-24 to S-35; A-24 to P-34; A-24 to T-33; A-24 to
A-32; A-
24 to A-31; and/or A-24 to A-30 of the TR4 sequence of SEQ ID NO:1.
[0099 In another embodiment, antibodies of the invention bind C-terminal
deletions
of the TR4 polypeptide that can be described by the general formula 24-m2
where m2 is a
number from 30 to 238 corresponding to the amino acid sequence identified of
SEQ ID
NO:I. In specific embodiments, the invention provides antibodies that bind TR4
polypeptides comprising, or alternatively consisting of, the amino acid
sequence of
residues: A-24 to G-237; A-24 to N-236; A-24 to G-235; A-24 to S-234; A-24 to
E-233;
A-24 to K-232; A-24 to H-231; A-24 to V-230; A-24 to C-229; A-24 to E-228; A-
24 to I-
227; A-24 to D-226; A-24 to S-225; A-24 to W-224; A-24 to P-223; A-24 to T-
222; A-24
to C-221; A-24 to D-220; A-24 to K-219; A-24 to V-218; A-24 to K-217; A-24 to
V-216;
A-24 to M-2I5; A-24 to G-2I4; A-24 to R-213; A-24 to P-212; A-24 to C-211; A-
24 to 6-
210; A-24 to T-209; A-24 to S-208; A-24 to C-207; A-24 to K-206; A-24 to R-
205; A-24
to C-204; A-24 to M-203; A-24 to E-202; A-24 to A-201; A-24 to S-200; A-24 to
N-I99;
A-24 to D-198; A-24 to N-I97; A-24 to R-196; A-24 to F-195; A-24 to T-194; A-
24 to 6-
193; A-24 to P-I92; A-24 to K-19I; A-24 to C-190; A-24 to Q-189; A-24 to C-
I88; A-24
to A-187; A-24 to T-186; A-24 to N-185; A-24 to R-184; A-24 to T-183; A-24 to
T-182;
A-24 to T-181; A-24 to C-180; A-24 to P-179; A-24 to S-178; A-24 to R-177; A-
24 to E-
176; A-24 to E-175; A-24 to E-I74; A-24 to D-173; A-24 to S-172; A-24 to K-
171; A-24
to C-170; A-24 to A-169; A-24 to T-168; A-24 to C-167; A-24 to P-166; A-24 to
L-165;
A-24 to C-164; A-24 to A-163; A-24 to F-162; A-24 to L-161; A-24 to N-160; A-
24 to N-
159; A-24 to S-158; A-24 to A-157; A-24 to N-156; A-24 to T-155; A-24 to Y-
154; A-24
to G-153; A-24 to V-152; A-24 to G-151; A-24 to E-150; A-24 to T-149; A-24 to
C-148;
A-24 to R-147; A-24 to N-146; A-24 to C-145; A-24 to A-144; A-24 to G-143; A-
24 to P-
142; A-24 to R-141; A-24 to E-I40; A-24 to S-139; A-24 to R-138; A-24 to H-
137; A-24
to S-136; A-24 to G-135; A-24 to P-134; A-24 to P-133; A-24 to C-132; A-24 to
L-131;
A-24 to E-130; A-24 to G-129; A-24 to L-128; A-24 to P-227; A-24 to S-126; A-
24 to H-
125; A-24 to E-124; A-24 to W-123; A-24 to Q-122; A-24 to Q-121; A-24 to T-
120; A-24
to G-1I9; A-24 to I-I18; A-24 to S-1I7; A-24 to Q-116; A-24 to D-115; A-24 to
H-114;
A-24 to L-113; A-24 to K-112; A-24 to I-111; A-24 to T-110; A-24 to A-109; A-
24 to A-
108; A-24 to S-107; A-24 to S-106; A-24 to P-105; A-24 to V-104; A-24 to V-
103; A-24
to Q-102; A-24 to L-101; A-24 to L-100; A-24 to V-99; A-24 to G-98; A-24 to V-
97; A-
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24 to V-96; A-24 to V-95; A-24 to F-94; A-24 to K-93; A-24 to F-92; A-24 to T-
91; A-24
to K-90; A-24 to H-89; A-24 to V-88; A-24 to R-87; A-24 to L-86; A-24 to R-85;
A-24 to
P-84; A-24 to S-83; A-24 to A-82; A-24 to E-81; A-24 to R-80; A-24 to A-79; A-
24 to P-
78; A-24 to R-77; A-24 to P-76; A-24 to G-75; A-24 to P-74; A-24 to A-73; A-24
to R-72;
A-24 to G-71; A-24 to A-70; A-24 to R-69; A-24 to A-68; A-24 to R-67; A-24 to
A-66;
A-24 to S-65; A-24 to P-64; A-24 to G-63; A-24 to H-62; A-24 to Q-61; A-24 to
G-60; A-
24 to M-59; A-24 to S-58; A-24 to T-57; A-24 to P-56; A-24 to L-55; A-24 to A-
54; A-24
to G-53; A-24 to R-52; A-24 to G-51; A-24 to G-50; A-24 to G-49; A-24 to R-48;
A-24 to
P-47; A-24 to E-46; A-24 to I-45; A-24 to R-44; A-24 to G-43; A-24 to A-42; A-
24 to S-
41; A-24 to S-40; A-24 to G-39; A-24 to W-38; A-24 to V-37; A-24 to K-36; A-24
to S-
35; A-24 to P-34; A-24 to T-33; A-24 to A-32; A-24 to A-31; and/or A-24 to A-
30; of the
TR4 extracellular domain sequence of SEQ ID N0:1.
[0100] The present invention further provides antibodies that bind
polypeptides having
one or more residues from the carboxy terminus of the amino acid sequence of
the TR4
polypeptide of SEQ ID NO:1, up to C-221 of SEQ ID NO:1. In particular, the
present
invention provides antibodies that bind polypeptides having the amino acid
sequence of
residues 1-m9 of the amino acid sequence in SEQ ID N0:1, where m9 is any
integer in the
range of 221-468 and residue C-221 is the position of the first residue from
the C-
terminus of the complete TR4 polypeptide (shown in SEQ ID NO:1) believed to be
required for receptor binding activity of the TR4 protein.
[0101] The invention also provides antibodies that bind polypeptides having
one or
more amino acids deleted from both the amino and the carboxyl termini of a TR4
polypeptide, which may be described generally as having residues n1- ml and/or
n2- m2 of
SEQ ID NO:1, where n1, n2, ml, and m2 are integers as described above.
[0102] Also included are antibodies that bind a polypeptide consisting of a
portion of
the complete TR4 amino acid sequence encoded by the cDNA clone contained in
ATCC
Deposit No. 97853, where this portion excludes from 1 to about 108 amino acids
from the
amino terminus of the complete amino acid sequence encoded by the cDNA clone
contained in ATCC Deposit No. 97853, or from 1 to about 247 amino acids from
the
carboxy terminus, or any combination of the above amino terminal and carboxy
terminal
deletions, of the complete amino acid sequence encoded by the cDNA clone
contained in
ATCC Deposit No. 97853.
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[0103] Preferably, antibodies of the present invention bind fragments of TR4
comprising a portion of the extracellular domain; i.e., within residues 24-238
of SEQ ID
NO:1, since any portion therein is expected to be soluble.
[0104] It will be recognized in the art that some amino acid sequence of TR4
can be
varied without significant effect of the structure or function of the protein.
If such
differences in sequence are contemplated, it should be remembered that there
will be
critical areas on the protein which determine activity. Such areas will
usually comprise
residues which make up the ligand binding site or the death domain, or which
form tertiary
structures which affect these domains.
[0105] Thus, the invention further includes antibodies that bind variations of
the TR4
protein which show substantial TR4 protein activity or which include regions
of TR4 such
as the protein fragments discussed below. Such mutants include deletions,
insertions,
inversions, repeats, and type substitution. Guidance concerning which amino
acid changes
are likely to be phenotypically silent can be found in Bowie, J.IJ. et al.,
Science
247:1306-1310 (1990).
[0106] Thus, antibodies of the present invention may bind a fragment,
derivative, or
analog of the polypeptide of SEQ ID NO:1, or that encoded by the cDNA in ATCC
deposit 97853. Such fragments, variants or derivatives may be (i) one in which
at least
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
residue may or may not be one encoded by the genetic code, or (ii) one in
which one or
more of the amino acid residues includes a substituent group, or (iii) one in
which the
mature polypeptide is fused with another compound, such as a compound to
increase the
half life of the polypeptide (for example, polyethylene glycol), or (iv) one
in which the
additional amino acids are fused to the mature polypeptide, such as an IgG Fc
fusion
region peptide or leader or secretory sequence or a sequence which is employed
for
purification of the mature polypeptide or a proprotein sequence. Such
fragments,
derivatives and analogs are deemed to be within the scope of those skilled in
the art from
the teachings herein.
[0107] 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
TR4 protein.
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
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., Clir2 Exp.
Immunol.
2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al.
Crit. Rev.
Therapeutic Drug Carrier Systefn,s 10:307-377 (1993)).
[0108] The replacement of amino acids can also change the selectivity of
binding to
cell surface receptors. Ostade et al., Nature 361:266-268 (I993) describes
certain
mutations resulting in selective binding of TNF-alpha to only one of the two
known types
of TNF receptors. Thus, the antibodies of the present invention may bind a TR4
receptor
that contains one or more amino acid substitutions, deletions or additions,
either from
natural mutations or,human manipulation.
[0109] 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 3).
TABLE 3. Conservative Amino Acid Substitutions.
Aromatic Phenylalanine
Tryptophan
Tyrosine
Hydrophobic Leucine
Isoleucine
. Valine
Polar ~ Glutamine
Asparagine
Basic Arginine
Lysine
Histidine
Acidic ~ Aspartic Acid
Glutamic Acid
Small Alanine
Serine
Threonine
Methionine
Glycine
56
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
[0110] In specific embodiments, the number of substitutions, additions or
deletions in
the amino acid sequence of SEQ ID N0:1 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, I5, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 30-20, 20-15, 20-10,
15-10, IO-1, 5-10,
1-5, 1-3 or 1-2.
[0111] In specific embodiments, the antibodies of the invention bind TR4
polypeptides
or fragments or variants thereof (especially a fragment comprising or
alternatively
consisting of, the extracellular soluble domain of TR4), that contains any one
or more of
the following conservative mutations in TR4: Ml replaced with A, G, I, L, S,
T, or V; A2
replaced with G, I, L, S, T, M, or V; A6 replaced with G, I, L, S, T, M, or V;
R7 replaced
with H, or K; V8 replaced with A, G, I, L, S, T, or M; H9 replaced with K, or
R; LIO
replaced with A, G, I, S, T, M, or V; G11 replaced with A, I, L, S, T, M, or
V; A12
replaced with G, I, L, S, T, M, or V; F13 replaced with W, or Y; L14 replaced
with A, G,
I, S, T, M, or V; A15 replaced with G, I, L, S, T, M, or V; V16 replaced with
A, G, I, L, S,
T, or M; T27 replaced with A, G, I, L, S, M, or V; N19 replaced with Q; G21
replaced
with A, I, L, S, T, M, or V; S22 replaced with A, G, I, L, T, M, or V; A23
replaced with G,
I, L, S, T, M, or V; A24 replaced with G, I, L, S, T, M, or V; S25 replaced
with A, G, I, L,
T, M, or V; G26 replaced with A, I, L, S, T, M, or V; T27 replaced with A, G,
I, L, S, M,
or V; E28 replaced with D; A29 replaced with G, I, L, S, T, M, or V; A30
replaced with G,
I, L, S, T, M, or V; A31 replaced with G, I, L, S, T, M, or V; A32 replaced
with G, I, L, S,
T, M, or V; T33 replaced with A, G, I, L, S, M, or V; S35 replaced with A, G,
I, L, T, M,
or V; K36 replaced with H, or R; V37 replaced with A, G, I, L, S, T, or M; W38
replaced
with F, or Y; G39 replaced with A, I, L, S, T, M, or V; S40 replaced with A,
G, I, L, T, M,
or V; S41 replaced with A, G, I, L, T, M, or VA42 replaced with G, I, L, S, T,
M, or V;
G43 replaced with A, I, L, S, T, M, or V; R44 replaced with H, or K; I45
replaced with A,
G, L, S, T, M, or V; E46 replaced with D; R48 replaced with H, or K; G49
replaced with
A, I, L, S, T, M, or V; G50 replaced with A, I, L, S, T, M, or V; G51 replaced
with A, I, L,
S, T, M, or V; R52 replaced with H, or K; G53 replaced with A, I, L, S, T, M,
or V; A54
replaced with G, I, L, S, T, M, or V; L55 replaced with A, G, I, S, T, M, or
V; T57
replaced with A, G, I, L, S, M, or V; S58 replaced with A, G, I, L, T, M, or
V; M59
replaced with A, G, I, L, S, T, or V; G60 replaced with A, I, L, S, T, M, or
V; Q61 A
replaced with N; H62 replaced with K, or R; G63 replaced with A, I, L, S, T,
M, or V; S65
57
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
replaced with A, G, I, L, T, M, or V; A66 replaced with G, I, L, S, T, M, or
V; R67
replaced with H, or K; A68 replaced with G, I, L, S, T, M, or V; R69 replaced
with H, or
K; A70 replaced with G, I, L, S, T, M, or V; G71 replaced with A, I, L, S, T,
M, or V; R72
replaced with H, or K; A73 replaced with G, I, L, S, T, M, or V; G75 replaced
with A, I,
L, S, T, M, or V; R77 replaced with H, or K; A79 replaced with G, I, L, S, T,
M, or V;
R80 replaced with H, or K; E81 replaced with D; A82 replaced with G, I, L, S,
T, M, or V;
S83 replaced with A, G, I, L, T, M, or V; R85 replaced with H, or K; L86
replaced with A,
G, I, S, T, M, or V; R87 replaced with H, or K; V88 replaced with A, G, I, L,
S, T, or M;
H89 replaced with K, or R; K90 replaced with H, or R; T91 replaced with A, G,
I, L, S, M,
or V; F92 replaced with W, or Y; K93 replaced with H, or R; F94 replaced with
W, or Y;
V95 replaced with A, G, I, L, S, T, or M; V96 replaced with A, G, I, L, S, T,
or M; V97
replaced with A, G, I, L, S, T, or M; G98 replaced with A, I, L, S, T, M, or
V; V99
replaced with A, G, I, L, S, T, or M; L100 replaced with A, G, I, S, T, M, or
V; L101
replaced with A, G, I, S, T, M, or V; Q102 replaced with N; V103 replaced with
A, G, I,
L, S, T, or M; V 104 replaced with A, G, I, L, S, T, or M; S 106 replaced with
A, G, I, L, T,
M, or V; 5107 replaced with A, G, I, L, T, M, or V; A108 replaced with G, I,
L, S, T, M,
or V; A109 replaced with G, I, L, S, T, M, or V; T110 replaced with A, G, I,
L, S, M, or
V; I111 replaced with A, G, L, S, T, M, or V; K112 replaced with H, or R; L113
replaced
with A, G, I, S, T, M, or V; H114 replaced with K, or R; D115 replaced with E;
Q116
replaced with N; S 117 replaced with A, G, I, L, T, M, or V; I118 replaced
with A, G, L, S,
T, M, or V; 6119 replaced with A, I, L, S, T, M, or V; T120 replaced with A,
G, I, L, S,
M, or V; Q121 replaced with N; Q122 replaced with N; W123 replaced with F, or
Y; E124
replaced with D; H125 replaced with K, or R; S 126 replaced with A, G, I, L,
T, M, or V;
L128 replaced with A, G, I, S, T, M, or V; 6129 replaced with A, I, L, S, T,
M, or V;
E130 replaced with D; L131 replaced with A, G, I, S, T, M, or V; 6135 replaced
with A, I,
L, S, T, M, or V; S136 replaced with A, G, I, L, T, M, or V; H137 replaced
with K, or R;
8138 replaced with H, or K; 5139 replaced with A, G, I, L, T, M, or V; E140
replaced
with D; 8141 replaced with H, or K; 6143 replaced with A, I, L, S, T, M, or V;
A144
replaced with G, I, L, S, T, M, or V; N146 replaced with Q; 8147 replaced with
H, or K;
T149 replaced with A, G, I, L, S, M, or V; E150 replaced with D; 6151 replaced
with A, I,
L, S, T, M, or V; V152 replaced with A, G, I, L, S, T, or M; 6153 replaced
with A, I, L, S,
T, M, or V; Y154 replaced with F, or W; T155 replaced with A, G, I, L, S, M,
or V; N156
replaced with Q; A157 replaced with G, I, L, S, T, M, or V; 5158 replaced with
A, G, I, L,
58
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
T, M, or V; N159 replaced with Q; N160 replaced with Q; L161 replaced with A,
G, I, S,
T, M, or V; F162 replaced with W, or Y; A163 replaced with G, I, L, S, T, M,
or V; L165
replaced with A, G, I, S, T, M, or V; T168 replaced with A, G, I, L, S, M, or
V; A169
replaced with G, I, L, S, T, M, or V; K171 replaced with H, or R; S 172
replaced with A,
G, I, L, T, M, or V; D173 replaced with E; E174 replaced with D; E175 replaced
with D;
E176 replaced with D; 8177 replaced with H, or K; S 178 replaced with A, G, I,
L, T, M,
or V; T181 replaced with A, G, I, L, S, M, or V; T182 replaced with A, G, I,
L, S, M, or
V; T183 replaced with A, G, I, L, S, M, or V; 8184 replaced with H, or K; N185
replaced
with Q; T186 replaced with A, G, I, L, S, M, or V; A187 replaced with G, I, L,
S, T, M, or
V; Q189 replaced with N; K191 replaced with H, or R; 6193 replaced with A, I,
L, S, T,
M, or V; T194 replaced with A, G, I, L, S, M, or V; F195 replaced with W, or
Y; 8196
replaced with H, or K; N197 replaced with Q;,D198 replaced with E; N199
replaced with
Q; 5200 replaced with A, G, I, L, T, M, or V; A201 replaced with G, I, L, S,
T, M, or V;
E202 replaced with D; M203 replaced with A, G, I, L, S, T, or V; 8205 replaced
with H,
or K; K206 replaced with H, or R; 5208 replaced with A, G, I, L, T, M, or V;
T209
replaced with A, G, I, L, S, M, or V; 6210 replaced with A, I, L, S, T, M, or
V; 8213
replaced with H, or K; 6214 replaced with A, I, L, S, T, M, or V; M215
replaced with A,
G, I, L, S, T, or V; V216 replaced with A, G, I, L, S, T, or M; K217 replaced
with H, or R;
V218 replaced with A, G, I, L, S, T, or M; K219 replaced with H, or R; D220
replaced
with E; T222 replaced with A, G, I, L, S, M, or V; W224 replaced with F, or Y;
5225
replaced with A, G, I, L, T, M, or V; D226 replaced with E; I227 replaced with
A, G, L, S,
T, M, or V; E228 replaced with D; V230 replaced with A, G, I, L, S, T, or M;
H231
replaced with K, or R; K232 replaced with H, or R; E233 replaced with D; S234
replaced
with A, G, I, L, T, M, or V; 6235 replaced with A, I, L, S, T, M, or V; N236
replaced with
Q; 6237 replaced with A, I, L, S, T, M, or V; H238 replaced with K, or R; N239
replaced
with Q; I240 replaced with A, G, L, S, T, M, or V; W241 replaced with F, or Y;
V242
replaced with A, G, I, L, S, T, or M; I243 replaced with A, G, L, S, T, M, or
V; L244
replaced with A, G, I, S, T, M, or V; V245 replaced with A, G, I, L, S, T, or
M; V246
replaced with A, G, I, L, S, T, or M; T247 replaced with A, G, I, L, S, M, or
V; L248
replaced with A, G, I, S, T, M, or V; V249 replaced with A, G, I, L, S, T, or
M; V250
replaced with A, G, I, L, S, T, or M; L252 replaced with A, G, I, S, T, M, or
V; L253
replaced with A, G, I, S, T, M, or V; L254 replaced with A, G, I, S, T, M, or
V; V255
replaced with A, G, I, L, S, T, or M; A256 replaced with G, I, L, S, T, M, or
V; V257
59
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
replaced with A, G, I, L, S, T, or M; L258 replaced with A, G, I, S, T, M, or
V; I259
replaced with A, G, L, S, T, M, or V; V260 replaced with A, G, I, L, S, T, or
M; I264
replaced with A, G, L, S, T, M, or V; 6265 replaced with A, I, L, S, T, M, or
V; 5266
replaced with A, G, I, L, T, M, or V; 6267 replaced with A, I, L, S, T, M, or
V; 6269
replaced with A, I, L, S, T, M, or V; 6270 replaced with A, I, L, S, T, M, or
V; D271
replaced with E; K273 replaced with H, or R; M275 replaced with A, G, I, L, S,
T, or V;
D276 replaced with E; 8277 replaced with H, or K; V278 replaced with A, G, I,
L, S, T,
or M; F280 replaced with W, or Y; W281 °replaced with F, or Y; 8282
replaced with H, or
K; L283 replaced with A, G, I, S, T, M, or V; 6284 replaced with A, I, L, S,
T, M, or V;
L285 replaced with A, G, I, S, T, M, or V; L286 replaced with A, G, I, S, T,
M, or V;
8287 replaced with H, or K; 6288 replaced with A, I, L, S, T, M, or V; 6290
replaced
with A, I, L, S, T, M, or V; A291 replaced with G, I, L, S, T, M, or V; E292
replaced with
D; D293 replaced with E; N294 replaced with Q; A295 replaced with G, I, L, S,
T, M, or
V; H296 replaced with K, or R; N297 replaced with Q; E298 replaced with D;
I299
replaced with A, G, L, S, T, M, or V; L300 replaced with A, G, I, S, T, M, or
V; 5301
replaced with A, G, I, L, T, M, or V; N302 replaced with Q; A303 replaced with
G, I, L, S,
T, M, or V; D304 replaced with E; 5305 replaced with A, G, I, L, T, M, or V;
L306
replaced with A, G, I, S, T, M, or V; 5307 replaced with A, G, I, L, T, M, or
V; T308
replaced with A, G, I, L, S, M, or V; F309 replaced with W, or Y; V310
replaced with A,
G, I, L, S, T, or M; 5311 replaced with A, G, I, L, T, M, or V; E312 replaced
with D;
Q313 replaced with N; Q314 replaced with N; M315 replaced with A, G, I, L, S,
T, or V;
E316 replaced with D; S317 replaced with A, G, I, L, T, M, or V; Q318 replaced
with N;
E319 replaced with D; A321 replaced with G, I, L, S, T, M, or V; D322 replaced
with E;
L323 replaced with A, G, I, S, T, M, or V; T324 replaced with A, G, I, L, S,
M, or V;
6325 replaced with A, I, L, S, T, M, or V; V326 replaced with A, G, I, L, S,
T, or M;
T327 replaced with A, G, I, L, S, M, or V; V328 replaced with A, G, I, L, S,
T, or M;
Q329 replaced with N; 5330 replaced with A, G, I, L, T, M, or V; 6332 replaced
with A,
I, L, S, T, M, or V; E333 replaced with D; A334 replaced with G, I, L, S, T,
M, or V;
Q335 replaced with N; L337 replaced with A, G, I, S, T, M, or V; L338 replaced
with A,
G, I, S, T, M, or V; 6339 replaced with A, I, L, S, T, M, or V; A341 replaced
with G, I, L,
S, T, M, or V; E342 replaced with D; A343 replaced with G, I, L, S, T, M, or
V; E344
replaced with D; 6345 replaced with A, I, L, S, T, M, or V; 5346 replaced with
A, G, I, L,
T, M, or V; Q347 replaced with N; 8348 replaced with H, or K; 8349 replaced
with H, or
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
K; 8350 replaced with H, or K; L351 replaced with A, G, I, S, T, M, or V; L352
replaced
with A, G, I, S, T, M, or V; V353 replaced with A, G, I, L, S, T, or M; A355
replaced with
G, I, L, S, T, M, or V; N356 replaced with Q; 6357 replaced with A, I, L, S,
T, M, or V;
A358 replaced with G, I, L, S, T, M, or V; D359 replaced with E; T361 replaced
with A,
G, I, L, S, M, or V; E362 replaced with D; T363 replaced with A, G, I, L, S,
M, or V;
L364 replaced with A, G, I, S, T, M, or V; M365 replaced with A, G, I, L, S,
T, or V;
L366 replaced with A, G, I, S, T, M, or V; F367 replaced with W, or Y; F368
replaced
with W, or Y; D369 replaced with E; K370 replaced with H, or R; F371 replaced
with W,
or Y; A372 replaced with G, I, L, S, T, M, or V; N373 replaced with Q; I374
replaced with
A, G, L, S, T, M, or V; V375 replaced with A, G, I, L, S, T, or M; F377
replaced with W,
or Y; D378 replaced with E; 5379 replaced with A, G, I, L, T, M, or V; W380
replaced
with F, or Y; D381 replaced with E; Q382 replaced with N; L383 replaced with
A, G, I, S,
T, M, or V; M384 replaced with A, G, I, L, S, T, or V; 8385 replaced with H,
or K; Q386
replaced with N; L387 replaced with A, G, I, S, T, M, or V; D388 replaced with
E; L389
replaced with A, G, I, S, T, M, or V; T390 replaced with A, G, I, L, S, M, or
V; K391
replaced with H, or R; N392 replaced with Q; E393 replaced with D; I394
replaced with
A, G, L, S, T, M, or V; D395 replaced with E; V396 replaced with A, G, I, L,
S, T, or M;
V397 replaced with A, G, I, L, S, T, or M; 8398 replaced with H, or K; A399
replaced
with G, I, L, S, T, M, or V; 6400 replaced with A, I, L, S, T, M, or V; T40I
replaced with
A, G, I, L, S, M, or V; A402 replaced with G, I, L, S, T, M, or V; 6403
replaced with A, I,
L, S, T, M, or V; 6405 replaced with A, I, L, S, T, M, or V; D406 replaced
with E; A407
replaced with G, I, L, S, T, M, or V; L408 replaced with A, G, I, S, T, M, or
V; Y409
replaced with F, or W; A410 replaced with G, I, L, S, T, M, or V; M411
replaced with A,
G, I, L, S, T, or V; L412 replaced with A, G, I, S, T, M, or V; M413 replaced
with A, G, I,
L, S, T, or V; K414 replaced with H, or R; W415 replaced with F, or Y; V416
replaced
with A, G, I, L, S, T, or M; N417 replaced with Q; K418 replaced with H, or R;
T419
replaced with A, G, I, L, S, M, or V; 6420 replaced with A, I, L, S, T, M, or
V; 8421
replaced with H, or K; N422 replaced with Q; A423 replaced with G, I, L, S, T,
M, or V;
5424 replaced with A, G, I, L, T, M, or V; I425 replaced with A, G, L, S, T,
M, or V;
H426 replaced with K, or R; T427 replaced with A, G, I, L, S, M, or V; L428
replaced
with A, G, I, S, T, M, or V; LA.29 replaced with A, G, I, S, T, M, or V; D430
replaced with
E; A431 replaced with G, I, L, S, T, M, or V; I~32 replaced With A, G, I, S,
T, M, or V;
E433 replaced with D; 8434 replaced with H, or K; M435 replaced with A, G, I,
L, S, T,
61
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
or V; E436 replaced with D; E437 replaced with D; 8438 replaced with H, or K;
H439
replaced with K, or R; A440 replaced with G, I, L, S, T, M, or V; K441
replaced with H,
or R; E442 replaced with D; K443 replaced with H, or R; I444 replaced with A,
G, L, S, T,
M, or V; Q445 replaced with N; D446 replaced with E; L447 replaced with A, G,
I, S, T,
M, or V; L448 replaced with A, G, I, S, T, M, or V; V449 replaced with A, G,
I, L, S, T,
or M; D450 replaced with E; 5451 replaced with A, G, I, L, T, M, or V; 6452
replaced
with A, I, L, S, T, M, or V; K453 replaced with H, or R; F454 replaced with W,
or Y; I455
replaced with A, G, L, S, T, M, or V; Y456 replaced with F, or W; L457
replaced with A,
G, I, S, T, M, or V; E458 replaced with D; D459 replaced with E; 6460 replaced
with A,
I, L, S, T, M, or V; T461 replaced with A, G, I, L, S, M, or V; 6462 replaced
with A, I, L,
S, T, M, or V; S463 replaced with A, G, I, L, T, M, or V; A464 replaced with
G, I, L, S, T,
M, or V; V465 replaced with A, G, I, L, S, T, or M; 5466 replaced with A, G,
I, L, T, M,
or V; L467 replaced with A, G, I, S, T, M, or V; and/or E468 replaced with D
of SEQ ID
N0:1.
[0112] In specific embodiments, the antibodies of the invention bind TR4
polypeptides
or fragments or variants thereof (especially a fragment comprising or
alternatively
consisting of, the extracellular soluble domain of TR4), that contains any one
or more of
the following non-conservative mutations in TR4: M1 replaced with D, E, H, K,
R, N, Q,
F, W, Y, P, or C; A2 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P3
replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P4 replaced with
D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P5 replaced with D, E, H, K, R,
A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or C; A6 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; R7
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V8
replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; H9 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, P, or C; L10 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G11
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; A12 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; F13 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
L14 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A15 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; V16 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T17
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; P18 replaced with D, E, H, K, R, A, G, I,
L, S, T, M,
V, N, Q, F, W, Y, or C; N19 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y,
P, or C; P20 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, or C; G21
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S22 replaced with D, E,
H, K, R, N,
62
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
Q, F, W, Y, P, or C; A23 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A24
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S25 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; G26 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
T27
replaced with D, E, H, K, R, N, Q, F, W, Y,'P, or C; E28 replaced with H, K,
R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; A29 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; A30 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A31 replaced with
D, E, H,
K, R; N, Q, F, W, Y, P, or C; A32 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
T33 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P34 replaced with D,
E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S35 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; K36 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; V37
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W38 replaced with D, E,
H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; G39 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; S40 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S41 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; A42 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; G43
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R44 replaced with D, E,
A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; I45 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
E46 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P47
replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R48 replaced
with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G49 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; G50 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G51
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; R52 replaced with D, E, A, G, I,
L, S, T, M, V,
N, Q, F, W, Y, P, or C; G53 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; A54
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L55 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; P56 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, or C; T57 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S58 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; M59 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
G60 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q61 replaced with D,
E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; H62 replaced with D, E, A, G, I, L,
S, T, M, V, N,
Q, F, W, Y, P, or C; G63 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
P64
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S65
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A66 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; R67 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; A68
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R69 replaced with D, E,
A, G, I, L, S,
63
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
T, M, V, N, Q, F, W, Y, P, or C; A70 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
G71 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R72 replaced with D,
E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; A73 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; P74 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; G75
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P76 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R77 replaced with D, E, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, P, or C; P78 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, or C; A79 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R80
replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E81 replaced with H, K,
R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; A82 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; S83 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P84 replaced with
D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R85 replaced with D, E, A, G,
I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; L86 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; R87
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V88
replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; H89 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, P, or C; K90 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; T91
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F92 replaced with D, E,
H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; K93 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F,
W, Y, P, or C; F94 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,
P, or C; V95
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V96 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; V97 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
G98
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V99 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; L100 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L101
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q102 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; V103 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; V104 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P105 replaced
with D, E,
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S 106 replaced with D,
E, H, K, R, N,
Q, F, W, Y, P, or C; 5107 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A108
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A109 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; T110 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
Il l l
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K112 replaced with D, E,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; L113 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; H114 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
D115 replaced
64
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q 116 replaced
with D, E, H,
K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S 117 replaced with D, E, H,
K, R, N, Q, F,
W, Y, P, or C; Il 18 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6119
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; T120 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; Q121 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C;
Q122 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;
W123 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; E124 replaced with
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H125 replaced with D, E, A, G, I,
L, S, T, M,
V, N, Q, F, W, Y, P, or C; 5126 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; P127
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L128
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6129 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; E130 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
L131 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 0132 replaced with
D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; P133 replaced with D, E, H, K,
R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or C; P134 replaced with D, E, H, K, R, A, G, I,
L, S, T, M,
V, N, Q, F, W, Y, or C; 6135 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; 5136
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H137 replaced with D, E,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; 8138 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, P, or C; 5139 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
E140 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 8141 replaced
with D, E, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P142 replaced with D, E, H, K, R,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; 6143 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or
C; A144 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C145 replaced
with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; N146 replaced with D, E, H,
K, R, A, G,
I, L, S, T, M, V, F, W, Y, P, or C; 8147 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, P, or C; C148 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y,
or P; T149 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E150 replaced
with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 6151 replaced with D, E, H,
K, R, N, Q,
F, W, Y, P, or C; V 152 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
6153
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y154 replaced with D, E,
H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; T155 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; N156 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;
A157
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S 158 replaced with D, E,
H, K, R, N,
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
Q, F, W, Y, P, or C; N159 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P,
or C; N160 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; L161
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F162 replaced with D, E,
H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; A163 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; C164 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or
P; L165
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P166 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; C167 replaced with D, E, H, K, R, A,
G, I, L, S,
T, M, V, N, Q, F, W, Y, or P; T168 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
A169 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C170 replaced with
D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; K171 replaced with D, E, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; S 172 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
D173 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
E174 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E175 replaced
with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E176 replaced with H, K, R, A,
G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; 8177 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, P, or C; 5178 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P179
replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; C180 replaced
with D, E,
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; T181 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; T182 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
T183
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 8184 replaced with D, E,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; N185 replaced with D, E, H, K, R, A, G, I,
L, S, T, M,
V, F, W, Y, P, or C; T186 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A187
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C188 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Q189 replaced with D, E, H, K, R, A,
G, I, L, S,
T, M, V, F, W, Y, P, or C; C190 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, or P; K191 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
P192 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
6193
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T194 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; F195 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or
C; 8196 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
N197 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; D 198 replaced
with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N199 replaced with D, E, H, K,
R, A, G, I,
L, S, T, M, V, F, W, Y, P, or C; 5200 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
66
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
A201 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E202 replaced with
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; M203 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; C204 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or P;
8205 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K206
replaced
t~ith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C207 replaced with
D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; 5208 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; T209 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6210
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; C211 replaced with D, E, H, K, R,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or P; P212 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, or C; 8213 replaced With D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or
C; 6214 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M215 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; V216 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
K217 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V218
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; K219 replaced with D, E, A, G, I,
L, S, T, M,
V, N, Q, F, W, Y, P, or C; D220 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, P, or C; 0221 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or P;
T222 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P223 replaced with
D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; W224 replaced with D, E, H, K,
R, N, Q,
A, G, I, L, S, T, M, V, P, or C; 5225 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
D226 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
I227 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E228 replaced with H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; C229 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, or P; V230 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
H231
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K232
replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E233 replaced with H, K, R,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; 5234 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; 6235 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N236 replaced
with D, E, H,
K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; 6237 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; H238 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
N239 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;
I240 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; W241 replaced with D, E, H, K, R,
N, Q, A, G,
I, L, S, T, M, V, P, or C; V242 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; I243
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L244 replaced with D, E,
H, K, R, N,
67
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
Q, F, W, Y, P, or C; V245 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V246
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T247 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; L248 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V249
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V250 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; P251 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, or C; L252 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L253
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; L254 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; V255 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A256 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; V257 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
L258 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I259 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; V260 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; C261
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; C262
replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; 0263 replaced
with D, E,
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; I264 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; 6265 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
5266
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6267 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; C268 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, or P; 6269 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6270
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; D271 replaced with H, K, R, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, P, or C; P272 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, or C; K273 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; 0274
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; M275
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; D276 replaced with H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; 8277 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, P, or C; V278 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 0279
replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; F280 replaced with
D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; W281 replaced with D, E, H, K, R, N,
Q, A, G, I,
L, S, T, M, V, P, or C; 8282 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; L283 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6284 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L285 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
L286 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 8287 replaced with
D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 6288 replaced with D, E, H, K, R, N,
Q, F, W, Y,
P, or C; P289 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, or C;
68
CA 02426710 2003-04-23
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6290 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A291 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; E292 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, P, or C; D293 replaced with H, K, R, A, G, I, L, S, T,.M, V, N, Q, F, W, Y,
P, or C;
N294 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;
A295 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; H296 replaced with D, E, A, G, I,
L, S, T, M,
V, N, Q, F, W, Y, P, or C; N297 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W,
Y, P, or C; E298 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
I299 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L300 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; 5301 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; N302
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A303
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; D304 replaced with H, K, R, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, P, or C; 5305 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; L306
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 5307 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; T308 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
F309
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; V310
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; 5311 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; E312 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
Q313
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; Q314
replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; M315 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; E316 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y,
P, or C; S317 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q318
replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E319 replaced with H, K,
R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; P320 replaced with D, E, H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, or C; A321 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C;
D322 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
L323 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; T324 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; 6325 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V326
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; T327 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; V328 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q329 replaced
with D, E,
H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; 5330 replaced with D, E, H,
K, R, N, Q,
F, W, Y, P, or C; P331 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y,
or C; 6332 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E333 replaced
with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A334 replaced with D, E, H,
K, R, N, Q,
69
CA 02426710 2003-04-23
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F, W, Y, P, or C; Q335 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or
C; C336 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or
P; L337
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L338 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; 6339 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
P340
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A341
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E342 replaced with H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; A343 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
E344 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
6345 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; 5346 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; Q347 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C;
8348 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 8349
replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 8350 replaced with
D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L351 replaced with D, E, H, K, R, N,
Q, F, W, Y,
P, or C; L352 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V353
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; P354 replaced with D, E, H, K, R, A, G, I,
L, S, T, M,
V, N, Q, F, W, Y, or C; A355 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; N356
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; 6357
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; A358 replaced with D, E, H, K, R, N, Q,
F, W, Y,
P, or C; D359 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; P360
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; T361
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E362 replaced with H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; T363 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
L364 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M365 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; L366 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; F367
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F368
replaced with D,
E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D369 replaced with H, K, R,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; K370 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, P, or C; F371 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,
V, P, or C;
A372 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N373 replaced with
D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; I374 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; V375 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P376
replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; F377 replaced with
D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; D378 replaced with H, K, R, A, G, I,
L, S, T, M,
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
V, N, Q, F, W, Y, P, or C; 5379 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C;
W380 replaced with D, E, H, K, R, N; Q, A, G, I, L, S, T, M, V, P, or C; D381
replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q382 replaced
with D, E, H,
K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L383 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; M384 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 8385
replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q386 replaced with
D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L387 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; D388 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
L389 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T390 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; K391 replaced with D, E, A, G, I, L, S, T, M, V,~N,
Q, F, W, Y,
P, or C; N392 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,
or C; E393
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I394
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; D395 replaced with H, K, R, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, P, or C; V396 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; V397
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 8398 replaced with D, E,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; A399 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; 6400 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T401 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; A402 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; a
6403 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P404 replaced with
D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; 6405 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; D406 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C;
A407 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L408 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; Y409 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V,
P, or C; A410 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M411
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; L412 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; M413 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K414 replaced
with D, E, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; W4I5 replaced with D, E, H, K, R,
N, Q, A, G,
I, L, S, T, M, V, P, or C; V416 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; N417
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K418
replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T419 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; 6420 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
8421
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N422
replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A423 replaced with D, E,
H, K, R, N,
7i
CA 02426710 2003-04-23
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Q, F, W, Y, P, or C; S424 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
I425
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H426 replaced with D, E,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; T427 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; L428 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I~1-29 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; D430 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, P, or C; A431 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L432
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E433 replaced with H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; 8434 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, P, or C; M435 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E436
replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E437 replaced with H,
K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 8438 replaced with D, E, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, P, or C; H439 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; A440 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K441 replaced
with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E442 replaced with H, K, R, A,
G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; K443 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, P, or C; I444 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q445
replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; D446 replaced
with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L447 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; L448 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V449
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; D450 replaced with H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; 5451 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
6452 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K453 replaced with
D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F454 replaced with D, E, H, K, R, N,
Q, A, G, I,
L, S, T, M, V, P, or C; I455 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; Y456
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; I~.57
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; E458 replaced with H, K, R, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, P, or C; D459 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; 6460 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T461 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; 6462 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or
C; 5463 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A464 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; V465 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
5466 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L467 replaced with
D, E, H, K,
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R, N, Q, F, W, Y, P, or C; and/or E468 replaced with H, I~, R, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, P, or C of SEQ ID N0:1.
[0113] Amino acids in the TR4 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
molecule. The resulting mutant molecules are then tested for biological
activity such as
receptor binding or irz vitro, or in vitro proliferative activity. Sites that
are critical for
ligand-receptor binding can also be determined by structural analysis such as
crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith
et al., J. Mol.
Biol. 224:899-904 (1992) and de Vos et al. Scief2ce 255:306-312 (1992)). In
preferred
embodiments, antibodies of the present invention bind regions of TR4 that are
essential for
TR4 function. In other preferred embodiments, antibodies of the present
invention bind
regions of TR4 that are essential for TR4 function and inhibit or abolish TR4
function. In
other preferred embodiments, antibodies of the present invention bind regions
of TR4 that
are essential for TR4 function and enhance TR4 function.
[0114] Additionally, protein engineering may be employed to improve or alter
the
characteristics of TR4 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
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.
Antibodies of the
present invention may bind such modified TR4 polypeptides.
[0115] Non-naturally occurring variants of TR4 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 holler 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. Trans. R. Soc. London
SerA 317:415
(1986)).
[0116] Thus, the invention also encompasses antibodies that bind TR4
derivatives and
analogs that have one or more amino acid residues deleted, added, or
substituted to
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generate TR4 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 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 acid positions on any one or more of the
glycosylation
recognition sequences in the TR4 polypeptides and/or an amino acid deletion at
the second
position of any one or more such recognition sequences will prevent
glycosylation of the
TR4 at the modified tripeptide sequence (see, e.g., Miyajimo et al., EMBO J
5(6):1193-
1197). Additionally, one or more of the amino acid residues of TR4
polypeptides (e.g.,
arginine and lysine residues) may be deleted or substituted with another
residue to
eliminate undesired processing by proteases such as, for example, furins or
kexins. .
[0117] The antibodies of the present invention also include antibodies that
bind a
polypeptide comprising, or alternatively, consisting of the polypeptide
encoded by the
deposited cDNA (the deposit having ATCC Accession Number 97853) including the
leader; a polypeptide comprising, or alternatively, consisting of the mature
polypeptide
encoded by the deposited the cDNA minus the leader (i.e., the mature protein);
a
polypeptide comprising, or alternatively, consisting of the polypeptide of SEQ
ID NO:1
including the leader; a polypeptide comprising, or alternatively, consisting
of the
polypeptide of SEQ ID NO:1 minus the amino terminal methionine; a polypeptide
comprising, or alternatively, consisting of the polypeptide of SEQ ID NO:1
minus the
leader; a polypeptide comprising, or alternatively, consisting of the TR4
extracellular
domain; a polypeptide comprising, or alternatively, consisting of the TR4
cysteine rich
domain; a polypeptide comprising, or alternatively, consisting of the TR4
transmembrane
domain; a polypeptide comprising, or alternatively, consisting of the TR4
intracellular
domain; a polypeptide comprising, or alternatively, consisting of the TR4
death domain; a
polypeptide comprising, or alternatively, consisting of soluble polypeptides
comprising all
or part of the extracellular and intracelluar domains but lacking the
transmembrane
domain; as well as polypeptides which are at least 80% identical, more
preferably at least
90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99%
identical to
the polypeptides described above (e.g., the polypeptide encoded by the
deposited cDNA
clone (the deposit having ATCC Accession Number 97853), the polypeptide of SEQ
ID
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N0:1, and portions of such polypeptides with at least 30 amino acids and more
preferably
at least 50 amino acids.
[0118] By a polypeptide having an amino acid sequence at least, for example,
95%
"identical" to a reference amino acid sequence of a TR4 polypeptide is
intended that the
amino acid sequence of the polypeptide is identical to the reference sequence
except that
the polypeptide sequence may include up to five amino acid alterations per
each 100
amino acids of the reference amino acid of the TR4 polypeptide. 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.
[0119] As a practical matter, whether any particular polypeptide is at least
90%, 95%,
96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown
in SEQ
ID NO:1 or to the amino acid sequence encoded by deposited cDNA clones can be
determined conventionally using known computer programs such the Bestfit
program
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group,
University Research Park, 575 Science Drive, Madison, WI 53711. When using
Bestfit or
any other sequence alignment program to determine whether a particular
sequence is, for
instance, 95% identical to a reference sequence according to the present
invention, the
parameters are set, of course, such that the percentage of identity is
calculated over the full
length of the reference amino acid sequence and that gaps in homology of up to
5% of the
total number of amino acid residues in the reference sequence are allowed.
[0120] In a specific embodiment, the identity between a reference (query)
sequence (a
sequence of the present invention) and a subject sequence, also referred to as
a global
sequence alignment, is determined using the FASTDB computer program based on
the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred
parameters
used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch
Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l,
Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window
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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
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 matchedlaligned 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.
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[0121] The present application is also directed to antibodies that bind
proteins
containing polypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to
the TR4
polypeptide sequence set forth herein as nl-ml, and/or n2-m2. In preferred
embodiments,
the application is directed to antibodies that bind proteins containing
polypeptides at least
90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid
sequence of the specific TR4 N- and C-terminal deletions recited herein.
[0122] In certain preferred embodiments, antibodies of the invention bind TR4
fusion
proteins as described above wherein the TR4 portion of the fusion protein are
those
described as nl-ml, and/or nz-m2 herein.
TRS
[0123] In certain embodiments of the present invention, the antibodies of the
present
invention bind TR5 polypeptide, or fragments or variants thereof. The
following section
describes the TR5 polypeptides, fragments and variants that may be bound by
the
antibodies of the invention in more detail. The TR5 polypeptides, fragments
and variants
which may be bound by the antibodies of the invention are also described in
International
Publication Numbers, for example, W098/30693 and W~00/71150 which are herein
incorporated by reference in their entireties. Amino acids 41-299 of SEQ ID
NO:2 are
identical to the TR5 protein disclosed in W098/30693 and WO00/71150.
[0124] In certain embodiments, the antibodies of the present invention
immunospecifically bind TR5 polypeptide. An antibody that immunospecifically
binds
TR5 may, in some embodiments, bind fragments, variants (including species
orthologs of
TR5), multimers or modified forms of TRS. For example, an antibody
immunospecific for
TR5 may bind the TR5 moiety of a fusion protein comprising all or a portion of
TRS.
[0125] TR5 proteins may be found as monomers or multimers (i.e., dimers,
trimers,
tetramers, and higher multimers). Accordingly, the present invention relates
to antibodies
that bind TR5 proteins found as monomers or as part of multimers. In specific
embodiments, antibodies of the invention bind TR5 monomers, dimers, trimers or
tetramers. In additional embodiments, antibodies of the invention bind at
least dimers, at
least trimers, or at least tetramers containing one or more TR5 polypeptides.
[0126] Antibodies of the invention may bind TR5 homomers or heteromers. As
used
herein, the term homomer, refers to a multimer containing only TR5 proteins of
the
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invention (including TR5 fragments, variants, and fusion proteins, as
described herein).
These homomers may contain TR5 proteins having identical or different
polypeptide
sequences. In a specific embodiment, a homomer of the invention is a multimer
containing only TR5 proteins having an identical polypeptide sequence. In
another
specific embodiment, antibodies of the invention bind TR5 homomers containing
TR5
proteins having different polypeptide sequences. In specific embodiments,
antibodies of
the invention bind a TR5 homodimer (e.g., containing TR5 proteins having
identical or
different polypeptide sequences) or a homotrimer (e.g., containing TR5
proteins having
identical or different polypeptide sequences). In additional embodiments,
antibodies of
the invention bind at least a homodimer, at least a homotrimer, or at least a
homotetramer
of TRS.
[0127] As used herein, the term heteromer refers to a multimer containing
heterologous proteins (i.e., proteins containing polypeptide sequences that do
not
correspond to a polypeptide sequences encoded by the TR5 gene) in addition to
the TR5
proteins of the invention. In a specific embodiment, antibodies of the
invention bind a
heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments,
the
antibodies of the invention bind at least a homodimer, at least a homotrimer,
or at least a
homotetramer containing one or more TR5 polypeptides.
[0128] Multimers bound by one or more antibodies 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
bound
by one or more antibodies of the invention, such as, for example, homodimers
or
homotrimers, are formed when TR5 proteins contact one another in solution. In
another
embodiment, heteromultimers bound by one or more antibodies of the invention,
such as,
for example, heterotrimers or heterotetramers, are formed when TR5 proteins
contact
antibodies to the polypeptides of the invention (including antibodies to the
heterologous
polypeptide sequence in a fusion protein) in solution. In other embodiments,
multimers
bound by one or more antibodies of the invention are formed by covalent
associations with
and/or between the TR5 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 the polypeptide
encoded by the
deposited cDNA clone of ATCC Deposit 97798). In one instance, the covalent
associations are cross-linking between cysteine residues located within the
polypeptide
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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 TR5 fusion protein. In one example, covalent associations are
between the
heterologous sequence contained in a fusion protein (see, e.g., US Patent
Number
5,478,925). In a specific example, the covalent associations are between the
heterologous
sequence contained in a TR5-Fc fusion protein (as described herein). In
another specific
example, covalent associations of fusion proteins are between heterologous
polypeptide
sequences from another TNF family ligand/receptor member that is capable of
forming
covalently associated multimers, such as for example, oseteoprotegerin (see,
e.g.,
International Publication No. WO 98/49305, the contents of which are herein
incorporated
by reference in its entirety).
[0129] The multimers that may be bound by one or more antibodies 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 that may be bound by one or more
antibodies 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 that may be bound by one or more antibodies 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 that may be
bound by one
or more antibodies of the invention (see, e.g., US Patent Number 5,478,925,
which is
herein incorporated by reference in its entirety).
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[0130] Alternatively, multimers that may be bound by one or more antibodies of
the
invention may be generated using genetic engineering techniques known in the
art. In one
embodiment, proteins contained in multimers that may be bound by one or more
antibodies 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 that may be bound by one or more
antibodies of
the invention are generated by ligating a polynucleotide sequence encoding a
TR5
polypeptide 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 TR5 polypeptides 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). In another embodiment, two or more
TR5
polypeptides of the invention are joined through synthetic linkers (e.g.,
peptide,
carbohydrate or soluble polymer linkers). Examples include those peptide
linkers
described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference).
Proteins
comprising multiple TR5 polypeptides separated by peptide linkers may be
produced
using conventional recombinant DNA technology. In specific embodiments,
antibodies of
the invention bind proteins comprising multiple TR5 polypeptides separated by
peptide
linkers.
[0131] Another method for preparing multimer TR5 polypeptides of the invention
involves use of TRS polypeptides fused to a leucine zipper or isoleucine
polypeptide
sequence. Leucine zipper domains and isoleucine zipper domains are
polypeptides that
promote multimerization of the proteins in which they are found. Leucine
zippers were
originally identified in several DNA-binding proteins (Landschulz et al.,
Science
240:1759, (1988)), and have since been found in a variety of different
proteins. Among
the known leucine zippers are naturally occurring peptides and derivatives
thereof that
dimerize or trimerize. Examples of leucine zipper domains suitable for
producing soluble
multimeric TR5 proteins are those described in PCT application WO 94/10308,
hereby
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incorporated by reference. Recombinant fusion proteins comprising a soluble
TR5
polypeptide fused to a peptide that dimerizes or trimerizes in solution are
expressed in
suitable host cells, and the resulting soluble multimeric TR5 is recovered
from the culture
supernatant using techniques known in the art. In specific embodiments,
antibodies of the
invention bind TR5-leucine zipper fusion protein monomers and/or TR5-leucine
zipper
fusion protein multirners.
[0132] Certain members of the TNF family of proteins are believed to exist in
trimeric
form (Beutler and Huffel, Science 264:667, 1994; Banner et al., Cell 73:431,
1993). Thus,
trimeric TR5 may offer the advantage of enhanced biological activity.
Preferred leucine
zipper moieties are those that preferentially form trimers. One example is a
leucine zipper
derived from lung surfactant protein D (SPD), as described in Hoppe et al.
(FEBS Letters
344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby
incorporated
by reference. In specific embodiments, antibodies of the invention bind TR5-
leucine
zipper fusion protein trimers.
[0133] Other peptides derived from naturally occurring trimeric proteins may
be
employed in preparing trimeric TRS. In specific embodiments, antibodies of the
invention
bind TR5- fusion protein monomers and/or TR5 fusion protein trimers.
[0134] The TR5 polypeptides are preferably provided in an isolated form, and
preferably are substantially purified. 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 TR5 polypeptide is substantially purified by the one-
step method
described in Smith and Johnson, Gene 67:31-40 (1988).
[0135] Antibodies of the present invention may bind TR5 polypeptides or
polypeptide
fragments including polypeptides comprising or alternatively, consisting of,
an amino acid
sequence contained in SEQ ID N0:2, encoded by the cDNA contained in ATCC
deposit
Number 97798, or encoded by nucleic acids which hybridize (e.g., under
stringent
hybridization conditions) to the cDNA contained in ATCC deposit Number 97798
or to
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. Antibodies of the
present invention
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may bind polypeptide fragments, including, for example, fragments that
comprise or
alternatively, consist of from about amino acid residues: 1 to 40, 41 to 66,
67 to 90, 91 to
140, 191 to 240, 241 to 280, andlor 281 to 299, of SEQ ID N0:2. Moreover,
polypeptide
fragments bound by the antibodies of the invention can be at least 10, 20, 30,
40, 50, 60,
70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids in length.
[0136] . In specific embodiments, antibodies of the present invention bind
polypeptide
fragments comprising, or alternatively consisting of, amino acid residues: 41-
299, 67-299,
67-280, 93-193, and/or 281-299, of TR5 (SEQ ID N0:2). Polynucleotides encoding
these
polypeptides are also encompassed by the invention.
[0137] In additional embodiments, antibodies of the present invention bind TR5
polypeptide fragments comprising, or alternatively consisting, of one or more
TR5
domains. Preferably, antibodies of the present invention bind TR5 polypeptides
or
polypeptide fragments selected from the group consisting of: (a) a polypeptide
comprising
or alternatively, consisting of, the TR5 transmembrane domain (predicted to
constitute
amino acid residues from about 281 to about 299 of SEQ ID NO:2); (b) a
polypeptide
comprising or alternatively, consisting of, the TR5 receptor extracellular
domain
(predicted to constitute amino acid residues from about 67 to about 280 of SEQ
ID N0:2);
(c)a polypeptide comprising or alternatively, consisting of, both TR5 cysteine
rich
domains (both of which may be found in the protein fragment consisting of
amino acid
residues from about 93 to about 193 in SEQ ID NO:2); (d) a polypeptide
comprising or
alternatively, consisting of, the TR5 cysteine rich domain consisting of amino
acid
residues from about 93-150 in SEQ ID N0:2); (e) a polypeptide comprising or
alternatively, consisting of, the TR5 cysteine rich domain consisting of amino
acid
residues from about 151 to about 193 in SEQ ID NO:2); (f) a polypeptide
comprising or
alternatively, consisting of, fragment of the predicted mature TR5
polypeptide, wherein
the fragment has a TR5 functional activity (e.g., antigenic activity or
biological acitivity);
or (g) any combination of polypeptides (a)-(g). As discussed above, it is
believed that one
or both of the extracellular cysteine rich motifs of TR5 is important for
interactions
between TR5 and its ligands (e.g., TRAIL). Accordingly, in highly preferred
embodiments, antibodies of the present invention bind TR5 polypeptide
fragments
comprising, or alternatively consisting of, amino acid residues 93 to 150
and/or 151-193
of SEQ ID N0:2. In another highly preferred embodiment, antibodies of the
present
invention bind TR5 polypeptides comprising, or alternatively consisting of,
both of the
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extracellular cysteine rich motifs (amino acid residues 93 to 193 SEQ ID
N0:2.) In
another preferred embodiment, antibodies of the present invention bind TR5
polypeptides
comprising, or alternatively consisting of the extracellular soluble domain of
TR5 (amino
acid residues 67-280 of SEQ ID N0:2.)
[0138] Antibodies of the invention may also bind fragments comprising, or
alternatively, consisting of structural or functional attributes of TRS. 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"),
hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic
regions, surface forming regions, and high antigenic index regions (i.e.,
containing four or
more contiguous amino acids having an antigenic index of greater than or equal
to 1.5, as
identified using the default parameters of the Jameson-Wolf program) of
complete (i.e.,
full-length) TRS. Certain preferred regions are those set out in Table 4 and
include, but are
not limited to, regions of the aforementioned types identified by analysis of
the amino acid
sequence depicted in (SEQ ID N0:2), such preferred regions include; Gamier-
Robson
predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-
Fasman
predicted alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle
predicted
hydrophilic 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.
[0139] The data representing the structural or functional attributes of TR5
set forth in
Table 4, as described above, was generated using the various modules and
algorithms of
the DNA*STAR set on default parameters. Column I represents the results of a
Garnier-
Robson analysis of alpha helical regions; Column II represents the results of
a Chou-
Fasman analysis of alpha helical regions; Column III represents the results of
a Gamier
Robson analysis of beta sheet regions; Column IV represents the results of a
Chou-
Fasman analysis of beta sheet regions; Column V represents the results of a
Gamier
Robson analysis of turn regions; Column VI represents the results of a Chou-
Fasman
analysis of turn regions; Column VII represents the results of a Gamier Robson
analysis of
coil regions; Column VIII represents a Kyte-Doolittle hydrophilicity plot;
Column IX
represents a Hopp-Woods hydrophobicity plot; Column X represents the results
of an
Eisenberg analysis of alpha amphipathic regions; Column XI represents the
results of an
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Eisenberg analysis of beta amphipathic regions; Column XII represents the
results of a
Karplus-Schultz analysis of flexible.regions; Column XIII represents the
Jameson-Wolf
antigenic index score; and Column XIV represents the Emini surface probability
plot.
[0140] In a preferred embodiment, the data presented in columns VIII, IX,
XIII, and
XIV of Table 4 can be used to determine regions of TR5 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 XIV 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.
[0141] The above-mentioned preferred regions set out in Table 4 include, but
are not
limited to, regions of the aforementioned types identified by analysis of the
amino acid
sequence set out in SEQ ID N0:2. As set out in Table 4, such preferred regions
include
Gamier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions,
Chou-Fasman
alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic
regions,
Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible
regions,
Jameson-Wolf regions of high antigenic index and Emini surface-forming
regions.
Preferably, antibodies of the present invention bind TR5 polypeptides or TR5
polypeptide
fragments and variants comprising regions of TR5 that combine several
structural features,
such as several (e.g., l, 2, 3 , or 4) of the same or different region
features set out above
and in Table 4.
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Table 4
ResPosition II III IV V VI VII IX X XI XII XTII XIV
I VIII
Met1 . . . . . . C 0.19 0.37 * . . 0.10 0.47
Gln2 . . . . . . C 0.58 -0.06 * . 0.70 0.74
G1y3 . A . . . . C 2.08 -0.49* * . 0.50 1.00
Val4 . A . . . . C 0.77 -0.91* * . 0.95 1.98
Lys5 . A . . . . C 0.34 -0.74* * F 0.95 0.99
Glu6 . A B . . . . 0.73 -0.46* * F 0.45 0.82
Arg7 . A B . . . . -0.08-0.46* * F 0.60 1.72
Phe8 . . B . . . . -0.08-0.41* * . 0.50 0.71
Leu9 . . B . . T . 0.78 0.01 * * . 0.10 0.40
Pro10 . . . . . T C 0.43 0.41 * * . 0.00 0.33
Leu11 . . . . T T . 0.09 0.80 * . F 0.69 0.51
Gly12 . . . . T T . -0.020.44 * * F 1.03 0.62
Asn13 . . . . T T . 0.79 -0.24* . F 2.27 0.67
Ser14 . . . . . T C 1.01 -0.67* * F 2.86 1.58
Gly15 . . . . T T . 1.01 -0.86* . F 3.40 1.62
Asp16 . . . . T T . 1.93 -0.86* . F 3.06 1.55
Arg17 . . . . . . C 2.07 -1.26* . F 2.32 2.27
Ala18 . . . . . . C 1.86 -1.21* . F 2.32 3.55
Pro19 . . . . . . C 2.16 -1.21* . F 2.32 3.28
Arg20 . . . . . . C 2.16 -1.21* * F 2.32 2.80
Pro21 . . . . . T C 2.27 -0.79* * F 2.86 2.74
Pro22 . . . . T T . 1.81 -1.29* * F 3.40 3.47
Asp23 . . . . T T . 2.51 -1.29* * F 3.06 1.76
Gly24 . . . . T T . 1.87 -1.29. * F 2.72 2.22
Arg25 . . . B T . . 1.87 -1.07. * F 1.98 1.07
Gly26 . . . B T . . 1.87 -1.50. * F 1.64 1.25
Arg27 . . . B T . . 2.19 -1.07. * F 1.30 1.96
Val28 . _ B B . . . 1.88 -1.50. * F 1.24 1.96
Arg29 . . B B . . . 2.22 -1.01. * F 1.58 2.85
Pro30 . . B . . . . 2.11 -1.04. * F 2.12 2.52
Arg31 . . . . T . . 2.11 -1.04* * F 2.86 5.68
Thr32 . . . . T T . 1.14 -1.26* * F 3.40 2.87
Gln33 . . . . T T . 1.66 -0.61. * F 3.06 1.38
Asp34 . . . . T T . 1.54 -0.61. * F 2.57 0.70
Gly35 . . . . T T . 1.72 -0.21* * F 1.93 0.77
Val36 . . . . . . C 1.30 -0.20* * F 1.19 0.61
Gly37 . . . . . . C 1.01 -0.11. . F 0.85 0.53
Asn38 . A . . . . C 0.42 0.50 . * . -0.400.53
His39 . A . . . . C 0.53 0.57 * * . -0.400.72
Thr40 . A . . . . C -0.01-0.07* * . 0.65 1.42
Met41 . A B . . . . 0.63 0.19 * * . -0.300.62
Ala42 . A B . . . . 1.02 0.21 * . . 0.00 0.70
Arg43 . A B . . . . 0'.71-0.29* * . 0.90 0.97
Ile44 . . B . . . . -0.07-0.29* * . 1.55 1.42
Pro45 . . . . . . C 0.29 -0.21* * F 2.20 1.16
Lys46 . . . . T . . 0.19 -0.71* * F 3.00 1.18
Thr47 . . . B . . C -0.080.07 * * F 1.40 1.46
Leu48 . . . B . . C -1.040.03 * * F 0.95 0.70
Lys49 . . B B . . . -1.010.24 * * . 0.30 0.26
Phe50 . . B B . . . -1.690.89 * . . -0.300.13
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Table 4 (continued)
Res Position II III IV V VI VIT IX X XI XII XIII XIV
I VIII
Val 51 . . B B . . -2.591.09 * * . -0.600.11
Val 52 . . B B . . . -2.871.04 . * . -0.600.04
Val 53 . . B B . . . -2.911.54 * * . -0.600.05
Ile 54 . . B B . . . -3.771.40 * * . -0.600.05
Val 55 . . B B . . . -3.881.44 . . . -0.600.05
Ala 56 . . B B . . . -3.231.49 . . . -0.600.06
Val 57 . . B B . . . -3.231.27 . . . -0.600.13
Leu 58 . . B B . . . -3.191.23 . . . -0.600.13
Leu 59 . . B B . . . -2.891.27 * . . -0.600.11
Pro 60 . . B B . . . -2.281.27 . . . -0.600.15
Val 61 . . B B . . . -1.991.39 . . . -0.600.28
Leu 62 . . B B . . . -1.721.09 . . . -0.600.46
Ala 63 . . B B . . . -1.220.90 . . . -0.600.30
Tyr 64 . . B B . . . -0.720.96 . . . -0.600.58
Ser 65 . . . B . . C -1.100.80 * * . -0.251.02
Ala 66 . . . B . . C -0.130.61 * . . -0.251.02
Thr 67 . . . B . . C 0.68 0.11 . * F 0.20 1.28
Thr 68 . A . B . . C 1.27 -0.24. . F 0.80 1.65
Ala 69 . A . B . . C 1.51 -0.63. . F 1.10 2.83
Arg 70 . A . B . . C 0.96 -1.13. . F 1.10 3.40
Gln 71 . A . B . . C 1.33 -0.97. . F 1.10 1.75
Glu 72 . A . B . . C 1.64 -1.03. . F 1.10 2.68
Glu 73 . A . . . . C 1.96 -1.13. . F 1.10 2.37
Val 74 . A . . . . C 2.23 -0.73* . F 1.10 2.37
Pro 75 . A . . T . . 1.27 -0.64* . F 1.30 1.97
Gln 76 . . . B T . . 0.68 0.00 . . F 0.85 0.85
Gln 77 . . . B . . C 0.47 0.50 . . F -0.101.15
Thr 78 . . . B . . C 0.47 0.29 . . F 0.20 1.15
Va1 79 . . . B . . C 1.32 0.26 . . F 0.20 1.15
Ala 80 . . . B . . C 1.53 0.26 * . F 0.20 1.15
Pro 81 . . . B . . C 1.64 0.26 . . F 0.20 1.38
Gln 82 . . . . T . . 1.61 -0.23* . F 1.54 3.64
Gln 83 . . . . . . C 1.62 -0.37. * F 1.68 4.91
Gln 84 . . . . T T . 1.78 -0.49* * F 2.42 4.25
Arg 85 . . . . T T . 2.41 -0.13* * F 2.76 2.13
His 86 . . . . T T . 2.28 -0.53* * F 3.40 2.46
Ser 87 . . . . . T C 2.28 -0.50* * . 2.71 1.40
Phe 88 . A . . T . . 2.28 -0.90* * F 2.32 1.24
Lys 89 . A . . T . . 1.61 -0.90* * F 1.98 1.58
Gly 90 . A . . T . . 1.29 -0.83* * F 1.80 0.63
Glu 91 . A . . T . . 0.73 -0.79. * F 1.92 1.13
Glu 92 . A . . . . C 0.69 -1.07. * F 1.88 0.57
Cys 93 . . . . . T C 1.09 -0.64. * F 2.59 0.57
Pro 94 . . . . T T . 1.01 -0.69* * F 3.10 0.44
Ala 95 . . . . T T . 1.47 -0.19* . F 2.49 0.35
Gly 96 . . . . . T C 1.17 -0.19. * F 2.13 1.27
Ser 97 . . . . . . C 1.17 -0.37. * F 1.62 1.10
His 98 . . . . . C 1.80 -0.80. * F 1.95 1.88
Arg 99 . . . . T . . 1.70 -0.80. * F 2.18 2.59
Ser 100 . . . . T . . 1.94 -0.74. * F 2.52 2.78
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Table 4 (continued)
ResPosition III IV V VI VIIVIII IX X XI XIT XIII XIV
I II
Glu101 . . . . T . . 1.70 -0.70. * F 2.86 2.03
His102 . . . . T T . 1.33 -0.70. * F 3.40 1.04
Thr103 . . . . T T . 1.37 -0.13* * F 2.61 0.42
Gly104 . . . . T T . 1.04 -0.11* * . 2.12 0.39
Ala105 . . . . T T . 0.68 0.31 . . . 1.18 0.44
Cys106 . . . . T . . 0.37 0.39 . . . 0.64 0.16
Asn107 . . . . . T C 0.40 0.39 . . . 0.30 0.24
Pro108 . . . . T T . 0.37 -0.04* . F 1.53 0.41
Cys109 . . . . T T . -0.14-0.11* . F 1.81 0.76
Thr110 . . . . T T . 0.44 -0.04* . F 2.09 0.35
Glu111 . . B . . . . 0.87 -0.44* . F 1.77 0.38
Gly112 . . . . T T . 0.56 -0.11* . F 2.80 1.10
Val113 . . B . . T . 0.77 -0.20. . F 2.12 1.10
Asp114 . . B . . T . 0.84 -0.29. . . 1.69 1.02
Tyr115 . . . . . T C 0.86 0.21 . . . 1.01 1.04
Thr116 . . . . . . C 0.86 0.17 . . F 0.68 1.88
Asn117 . . . . . . C 1.20 -0.07. . F 1.34 1.81
Ala118 . . . . . T C 2.06 0.33 . . F 1.28 1.86
Ser119 . . . . T T . 1.84 -0.43. . F 2.42 2.24
Asn120 . . . . T T . 1.79 -0.49. . F 2.76 2.15
Asn121 . . . . T T . 1.43 -0.50* . F 3.40 2.85
Glu122 . . . . . T C 0.73 -0.43* . F 2.56 1.14
Pro123 . . . . T T . 1.11 -0.03. . F 2.27 0.61
Ser124 . . . . T T . 0.74 0.00 . . F 1.93 0.59
Cys125 . . . . T T . 0.43 0.17 * . . 0.84 0.18
Phe126 . . B . . T . -0.420.66 . . . -0.200.17
Pro127 . . . . T T . -1.090.87 * . . 0.20 0.09
Cys128 . . . . T T . -0.831.06 * . . 0.20 0.09
Thr129 . . . T T . -0.830.49 . . . 0.54 0.22
Val130 . . . . T . . -0.170.09 * . . 0.98 0.19
Cys131 . . . . T T . 0.53 -0.34* * . 2.12 0.59
Lys132 . . . . T T . 0.79 -0.51* . F 2.91 0.71
Ser133 . . . . T T . 1.42 -1.00. * F 3.40 1.90
Asp134 . . . . T T . 1.78 -1.14* * F 3.06 4.84
G1n135 . . . . T . . 2.33 -1.71* * F 2.83 4.84
Lys136 . . . . T . . 2.70 -1.33. * F 2.80 4.84
His137 . . . . T T . 1.99 -1.33. * F 2.97 3.88
Lys138 . . . . T T . 1.98 -0.76. * F 2.94 1.20
Ser139 . . . . T T . 1.38 -0.67. * F 3.10 0.87
Ser140 . . . . T T . 1.07 -0.06* * F 2.49 0.63
Cys141 . . . B T . . 1.13 -0.07* * F 1.78 0.45
Thr142 . . . B T . . 1.17 -0.07* . . 1.32 0.66
Met143 . . . B T . . 0.81 -0.46* * . 1.01 0.83
Thr144 . . . . T T . 0.26 -0.36. . F 1.40 2.23
Arg145 . . . . T T . -0.11-0.29. . F 1.40 1.15
Asp146 . . . . T T . 0.56 -0.20* . F 1.25 0.62
Thr147 . . . . T T . 0.20 -0.41* . . 1.10 0.75
Val148 . . . B T . . 0.84 -0.33* . . 1.04 0.20
Cys149 . . B T . . 1.16 -0.33* . . 1.38 0.24
Gln150 . . . B T . . 0.70 -0.33* . . 1.72 0.29
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Table 4 (continued)
ResPosition II III IV V VI VII IX X XT XII XIII XIV
I VIII
Cys151 . . . . T T . 0.39 -0.39. . . 2.46 0.39
Lys152 . . . . T T . 0.00 -0.54* * F 3.40 1.05
Glu153 . . . . T T . 0.97 -0.33* * F 2.61 0.53
Gly154 . . . . T T . 1.63 -0.73. * F 3.06 1.92
Thr155 . . . . T . . 1.63 -0.90* * F 2.86 1.55
Phe156 . . . . . . C 2.30 -0.90. * F 2.66 1.55
Arg157 . . . . T . . 1.96 -0.50. * F 2.86 2.51
Asn158 . . . . T T . 1.74 -0.54* * F 3.40 2.33
Glu159 . . . . T T . 2.09 -0.60. * F 3.06 4.17
Asn160 . . . . . T C 1.80 -1.39. * F 2.52 3.68
Ser161 . . . . . T C 1.83 -0.77. * F 2.18 2.27
Pro162 . . . . T . . 1..83-0.60* * F 1.69 0.70
Glu163 . . . . T . . 1.88 -0.60* . F 1.66 0.85
Met164 . . . . T . . 1.21 -1_00* . . 1.97 1.28
Cys165 . . . . T T . 0.91 -0.81* . . 2.33 0.44
Arg166 . . . . T T . 1.32 -0.86* * . 2.64 0.34
Lys167 . . . . T T . 0.87 -0.86* * F 3.10 0.68
Cys168 . . . . T T . 0.66 -0.90* * F 2.79 0.68
Ser169 . . . . T . . 0.96 -1.04* * F 2.59 0.53
Arg170 . . . . T . . 1.28 -0.66* * F 2.59 0.36
Cys171 . . . . . T C 1.17 -0.23* * F 2.29 0.66
Pro172 . . . . T T . 0.27 -0.80* . F 2.79 0.85
Ser173 . . . . T T . 0.93 -0.54* * F 3.10 0.32
Gly174 . . . . T T . 0.38 -0.14* * F 2.64 1.05
Glu175 . . . B T . . -0.03-0.07* * F 1.78 0.50
Val176 . . B B . . . 0.63 -0.11. * F 1.07 0.50
Gln177 . . B B . . . 0.18 -0.10. * . 0.61 0.82
Val178 . . B . . T . 0.17 0.04 . * . 0.10 0.25
Ser179 . . . . T T . 0.21 0.53 . * . 0.20 0.49
Asn180 . . . . T T . -0.080.27 . * F 0.65 0.38
Cys181 . . . . T T . 0.78 0.79 . * F 0.63 0.54
Thr182 . . . . T . . 0.78 0.14 . . F 1.01 0.67
Ser183 . . . . T . . 0.74 -0.24. . F 1.89 0.70
Trp184 . . . . T T . 1.04 0.04 . . F 1.77 0.91
Asp185 . . . . T T . 0.38 -0.13. . F 2.80 1.09
Asp186 . . . . T T . 0.19 -0.04* . F 2.37 0.44
Ile187 . . . . . T C 0.50 0.21 * . . 1.14 0.31
Gln188 . A B . . . . 0.80 -0.70* . . 1.16 0.32
Cys189 . A B . . . . 0.39 -0.70* . . 0.88 0.33
Val190 . A B . . . . 0.04 0.09 * * . -0.300.41
Glu191 . A . . . . C -0.54-0.17* * . 0.50 0.23
Glu192 . A . . T . . 0.34 -0.07* * . 0.70 0.44
Phe193 . A . . T . . -0.24-0.24* * . 0.88 0.96
Gly194 . . . . T T . 0.11 -0.39. * . 1.46 0.56
Ala195 . . . . . T C 0.11 0.10 . * . 0.84 0.47
Asn196 . . . . . T C 0.11 0.74 . * . 0.72 0.40
Ala197 . . . . . T C -0.20-0.04. * . 1.80 0.70
Thr198 . A . . . . C 0.29 0.01 . * . 0.62 1.00
Val199 . A . . . . C 0.04 -0.06. * F 1.19 0.96
Glu200 . A . . . . C 0.04 0.04 . * F 0.41 0.96
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Table 4 (continued)
ResPositionI II III IV V VI VII IX X XI XII XITI XIV
VIII
Thra 201 . A . . . . C 0.04 0.04 . * F 0.23 0.67
Pro202 . A . . . . C 0.63 -0.44 . F 0.80 1.57
*
Ala203 A A . . . . . 0.63 -1.09 . F 0.90 1.57
.
A1a204 A A . . . . . 0.89 -0.60 . F 0.90 1.57
*
Glu205 A A . . . . . 0.89 -0.47 . F 0.60 1.00
.
Glu206 A A . . . . . 0.89 -0.50 . F 0.90 1.60
.
Thr207 . A . . T . . 0.80 -0.51 . F 1.30 2.28
.
Met208 . A . . T . . 1.18 -0.63 . F 1.30 1.76
.
Asn209 . A . . T . . 1.42 -0.20 . F 1.00 1.57
.
Thr210 . A . . . . C 1.11 0.23 . . F 0.20 1.08
Ser211 . . . . . T C 0.90 0.23 . . F 0.60 1.57
Pro212 . . . . T T . 0.62 0.04 . . F 0.80 1.51
Gly213 . . . . . T C 1.01 0.14 . . F 0.60 1.06
Thr214 . . . . . T C 0.42 0.09 . . F 0.60 1.22
Pro215 . . . . . . C 0.14 0.20 . . F 0.25 0.80
Ala216 . A . . . .' C 0.44 0.27 . . F 0.05 0.82
Pro217 . A . . . . C 0.66 -0.16 . . 0.50 0.98
.
Ala218 . A . . . . C 0.69 -0.64 . . 0.95 1.10
.
Ala219 A A . . . . . 0.40 -0.59 * . 0.75 1.57
.
Glu220 A A . . . . . 0.61 -0.47 * F 0.60 1.00
.
Glu221 A A . . . . . 0.89 -0.50 * F 0.90 1.60
.
Thr222 . A . . T . . 0.80 -0.51 . F 1.30 2.28
.
Met223 . A . . T . . 1.18 -0.63 . F 1.30 1.76
.
Asn224 . A . . T . . 1.42 -0.20 . F 1.00 1.57
.
Thr225 . A . . . . C 1.11 0.23 . . F 0.20 1.08
Ser226 . . . . . T C 0.90 0.23 . . F 0.60 1.57
Pro227 . . . . T T . 0.62 0.04 . . F 0.80 1.51
Gly228 . . . . . T C 1.01 0.14 . . F 0.60 1.06
Thr229 . . . . . T C 0.42 0.09 . . F 0.60 1.22
Pro230 . . . . . . C 0.14 0.20 . . F 0.25 0.80
Ala231 . A . . . . C 0.44 0.27 . . F 0.05 0.82
Pro232 . A . . . . C 0.66 -0.16 . . 0.50 0.98
.
Ala233 . A . . . . C 0.69 -0.64 . . 0.95 1.10
.
Ala234 A A . . . . . 0.40 -0.59 . . 0.75 1.57
.
Glu235 A A . . . . . 0.30 -0.47 . F 0.60 1.00
.
Glu236 A A . . . . . 0.58 -0.41 . F 0.60 1.43
.
Thr237 . A B . . . . 0.49 -0.43 . F 0.60 2.05
.
Met238 . A . . T . . 0.87 -0.54 . F 1.30 1.58
.
Thr239 . A . . T . . 1.11 -0.11 . F 1.00 1.41
.
Thr240 . A . . . . C 0.80 0.31 . . F 0.05 0.97
Ser241 . . . . . T C 0.59 0.31 . . F 0.60 1.41
Pro242 . . . . T T . 0.31 0.13 . . F 0.80 1.51
Gly243 . . . . . T C 0.70 0.14 . . F 0.60 1.06
Thr244 . . . . . T C 0.42 0.09 . . F 0.60 1.22
Pro245 . . . . . . C 0.14 0.20 . . F 0.25 0.80
Ala246 . A . . . . C 0.44 0.27 . . F 0.05 0.82
Pro247 . A . . . . C 0.66 -0.16 . . 0.50 0.98
.
Ala248 . A . . . . C 0.69 -0.64 . . 0.95 1.10
.
Ala249 A A . . . . . 0.40 -0.59 . . 0.75 1.57
.
Glu250 A A . . . . . 0.30 -0.47 . F 0.60 1.00
.
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Table 4 (continued)
ResPositionI II III IV V VI VII IX X XI XII XIII XIV
VIII
Glu251 A A . . . . . 0.58 -0.41 . F 0.60 1.43
.
'~hr252 . A B . . . . 0.49 -0.43 . F 0.60 2.05
.
Met253 . A . . T . . 0.87 -0.54 . F 1.30 1.58
.
Thr254 . A . . T . . 1.11 -0.11 . F 1.00 1.41
.
Thr255 . A . . . . C 0.80 0.31 . . F 0.05 0.97
Ser256 . . . . . T C 0.59 0.31 . . F 0.60 1.41
Pro257 . . . . T T . 0.31 0.13 . . F 0.80 1.51
Gly258 . . . . . T C 0.70 0.14 . . F 0.60 1.06
Thr259 . . . . . T C 0.42 0.09 . . F 0.60 1.22
Pro260 . . . . . . C 0.14 0.20 . . F 0.25 0.80
Ala261 . A . . . . C 0.44 0.27 . . F 0.05 0.82
Pro262 . A . . . . C 0.66 -0.16 . . 0.50 0.98
.
Ala263 . A . . . . C 0.69 -0.64 . . 0.95 1.10
.
A1a264 A A . . . . . 0.40 -0.59 . . 0.75 1.57
.
Glu265 A A . . . . . 0.30 -0.47 . F 0.60 1.00
.
Glu266 A A . . . . . 0.58 -0.41 . F 0.60 1.43
.
Thr267 . A B . . . . 0.49 -0.43 . F 0.60 2.05
.
Met268 . A . . T . . 0.87 -0.54 . F 1.30 1.58
.
Thr269 . A . . T . . 1.11 -0.11 . F 1.00 1.41
.
Thr270 . A . . . . C 0.80 0.31 . . F 0.05 0.97
Ser271 . . . . . T C 0.59 0.31 . . F 0.60 1.41
Pro272 . . . . . T C 0.31 0.13 . . F 0.60 1.51
Gly273 . . . . . T C 0.61 0.14 . . F 0.60 1.06
Thr274 . . . . . T C 0.62 0.04 . . F 0.60 1.06
Pro275 . . . . . . C 0.90 0.04 . . F 0.25 0.92
Ala276 . . . . T . . 0.96 0.11 . . F 0.60 1.26
Ser277 . . . . T T . 0.36 0.44 . . F 0.50 1.37
Ser278 . . . . T T . 0.40 0.64 . . . 0.20 0.73
His279 . . . . T T . 0.04 0.60 . . . 0.20 0.97
Tyr280 . . . . T T . -0.060.67 . * . 0.20 0.39
Leu281 . . . B T . . -0.360.77 . . . -0.200.42
Ser282 . . . B T . . -0.911.07 . . . -0.200.22
Cys283 . . B B . . . -0.961.21 . . . -0.600.10
Thr284 . . B B . . . -1.810.89 * . . -0.600.12
Ile285 . . B B . . . -2.460.89 . . . -0.600.06
Val286 . . B B . . . -2.501.19 . . . -0.600.08
Gly287 . . B B . . . -3.011.26 . . . -0.600.04
Ile288 . . B B . . . -3.231.46 * . . -0.600.05
Ile289 . . B B . . . -3.781.46 . . . -0.600.05
Val290 . . B B . . . -3.701.46 . . . -0.600.04
Leu291 . . B B . . . -3.661.71 . . . -0.600.04
Ile292 . . B B . . . -4.201.71 . . . -0.600.05
Val293 . . B B . . . -4.171.71 . . . -0.600.05
~
Leu294 . . B B . . . -3.981.71 . . . -0.600.04
Leu295 . . B B . . . -3.981.81 . . . -0.600.05
Ile296 . . B B . . . -3.561.77 . . . -0.600.05
Val297 . . B B . . . -3.061.56 . . . -0.600.08
Phe298 . . B B . . . -2.591.30 . . . -0.600.13
Val299 . . B B . . . -2.171.04 . . . -0.600.23
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[0142] In another aspect, the invention provides an antibody that binds a
peptide or
polypeptide comprising, or alternatively, consisting of, one, two, three,
four, five or more,
epitope-bearing portions of TRS. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide of the invention. 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. LISA 81:3998- 4002 (1983).
[0143] As to the selection of peptides or polypeptides bearing an antigenic
epitope
(i.e., that contain a region of a protein molecule to which an antibody can
bind), it is well
known in that art that relatively short synthetic peptides that mimic part of
a protein
sequence are routinely capable of eliciting an antiserum that reacts with the
partially
mimicked protein. See, for instance, Sutcliffe, J. G., et al., "Antibodies
That React With
Predetermined Sites on Proteins," Science, 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.
[0144] Antigenic epitope-bearing peptides and polypeptides of the invention
are
therefore useful to raise antibodies, including monoclonal antibodies, that
bind to a TR5
polypeptide. See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777.
Antigenic
epitope-bearing peptides and polypeptides preferably contain a sequence of at
least
seven, more preferably at least nine and most preferably between about 15 to
about 30
amino acids contained within the amino acid sequence of a polypeptide of the
invention.
[0145] Antibodies of the invention may bind one or more antigenic TR5
polypeptides
or peptides including, but not limited to: a polypeptide comprising amino acid
residues
from about Gln-82 to about Glu-92 SEQ ID N0:2; a polypeptide comprising amino
acid
residues from about His-98 to about Cys-106 in SEQ ID N0:2; a polypeptide
comprising
amino acid residues from about Gln-82 to about Thr-Cys-106 in SEQ ID NO:2; a
polypeptide comprising amino acid residues from about Pro-108 to about Thr-116
in SEQ
ID N0:2; a polypeptide comprising amino acid residues from about Ser-119 to
about Cys-
125 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about
Cys-131
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to about Thr-142 in SEQ ID N0:2; a polypeptide comprising amino acid residues
from
about Gln-150 to about Pro-162 in SEQ m N0:2; a polypeptide comprising amino
acid
residues from about Arg-166 to about Val-176 in SEQ >D N0:2; a polypeptide
comprising
amino acid residues from about Gln-150 to about Val-176 in SEQ m N0:2; and a
polypeptide comprising amino acid residues from about Thr-182 to about Gln-188
in SEQ
m N0:2) As indicated above, the inventors have determined that the above
polypeptide
fragments are antigenic regions of the TR5 protein. 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 TR5 receptor
protein.
[0146] The epitope-bearing TR5 peptides and polypeptides may be produced by
any
conventional means (See, e.g., Houghten, R. A. "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)).
[0147] Epitope-bearing peptides and polypeptides of the invention are used to
induce
antibodies according to methods well known in the art. See, for instance,
Sutcliffe et al.,
supra; Wilson et al., supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA
82:910-914; and
Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985). Immunogenic epitope-
bearing
peptides of the invention, i.e., those parts of a protein that elicit an
antibody response when
the whole protein is the immunogen, are identified according to methods known
in the art.
See, for instance, Geysen et al., supra. Further still, U.S. Patent No.
5,194,392 to Geysen
(1990) describes a general method of detecting or determining the sequence of
monomers
(amino acids or other compounds) which is a topological equivalent of the
epitope (i.e., a
"mimotope") which is complementary to a particular paratope (antigen binding
site) of an
antibody of interest. More generally, U.S. Patent No. 4,433,092 to Geysen
(1989)
describes a method of detecting or determining a sequence of monomers which is
a
topographical equivalent of a ligand which is complementary to the ligand
binding site of
a particular receptor of interest. Similarly, U.S. Patent No. 5,480,971 to
Houghten, R. A.
et al. (1996) on Peralkylated Oligopeptide Mixtures discloses linear C1-C7-
alkyl
peralkylated oligopeptides and sets and libraries of such peptides, as well as
methods for
using such oligopeptide sets and libraries for determining the sequence of a
peralkylated
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oligopeptide that preferentially binds to an acceptor molecule of interest.
Thus,
non-peptide analogs of the epitope-bearing peptides of the invention also can
be made
routinely by these methods.
[0148] As one of skill in the art will appreciate, TR5 receptor polypeptides
of the
present invention and the epitope-bearing fragments thereof described herein
above (e.g.,
corresponding to a portion of the extracellular domain, such as, for example,
polypeptide
sequence comprising, or alternatively, consisting of, amino acid residues 41
to 280, 67 to
280, 70 to 280, 75 to 280, 80 to 280 and 90 to 280 of SEQ ID N0:2) 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 (EP A 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 TR5 protein or protein fragment
alone~(Fountoulakis et al.,
J. Biochem. 270:3958-3964 (1995)). Thus, antibodies of the invention may bind
fusion
proteins that comprise all or a portion of a TRAIL receptor polypeptide such
as TRS.
[0149] ' 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. Antibodies of the
present invention
may also bind such modified TR5 polypeptides or TR5 polypeptide fragments or
variants.
[0150] For instance, 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 or loss of the ability to be
bound by a
specific antibody. For instance, Ron et al., J. Biol. Chetra., 268:2984-2988
(1993) reported
modified KGF proteins that had heparin binding activity even if 3, 8, or 27
amino-terminal
amino acid residues were missing. In the present case, since the proteins of
the invention
are members of the TNFR polypeptide family, deletions of N-terminal amino
acids up to
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the cysteine at position C-93 of SEQ ID N0:2 may retain some biological
activity such as
regulation of proliferation and apoptosis of lymphoid cells. Polypeptides
having further
N-terminal deletions including the C-93 residue of SEQ ID N0:2, would not be
expected
to retain such biological activities because it is known that these residues
in a TR5-related
polypeptide are required for forming a disulfide bridge to provide structural
stability
which is needed for ligand binding. Thus, in specific embodiments, antibodies
of the
present invention bind N-terminally deleted protein fragments which retain
biological
activity.
[0151] 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 TRAIL ligand) may still be retained. For example, the ability
of the
shortened protein to induce and/or bind to antibodies which recognize the
complete or
mature form of the TR5 protein generally will be retained when less than the
majority of
the residues of the complete protein or extracellular domain are removed from
the
N-terminus. Whether a particular polypeptide lacking N-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. It is not unlikely that a TR5
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 TR5 amino
acid
residues may often evoke an immune response.
[0152] Accordingly, the present invention further provides antibodies that
bind TR5
polypeptides having one or more residues deleted from the amino terminus of
TR5 (SEQ
ID N0:2), up to the cysteine residue in each which is at position number 93,
and
polynucleotides encoding such polypeptides. In particular, the present
invention provides
antibodies that bind TR5 polypeptides comprising the amino acid sequence of
residues
n3-299 of SEQ ID N0:2 where n3 is an integer in the range of 1-93 where 93 is
the
position of the first cysteine residue from the N-terminus of the complete TR5
polypeptide
believed to be required for activity of the TR5 protein.
[0153] More in particular, the invention provides antibodies that bind
polypeptides
having the amino acid sequence of residues: Q-2 to V-299; G-3 to V-299; V-4 to
V-299;
K-5 to V-299; E-6 to V-299; R-7 to V-299; F-8 to V-299; L-9 to V-299; P-10 to
V-299; L-
11 to V-299; G-12 to V-299; N-13 to V-299; S-14 to V-299; G-15 to V-299; D-16
to V-
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299; R-17 to V-299; A-18 to V-299; P-19 to V-299; R-20 to V-299; P-21 to V-
299; P-22
to V-299; D-23 to V-299; G-24 to V-299; R-25 to V-299; G-26 to V-299; R-27 to
V-299;
V-28 to V-299; R-29 to V-299; P-30 to V-299; R-31 to V-299; T-32 to V-299; Q-
33 to V-
299; D-34 to V-299; G-35 to V-299; V-36 to V-299; G-37 to V-299; N-38 to V-
299; H-39
to V-299; T-40 to V-299; M-41 to V-299; A-42 to V-299; R-43 to V-299; I-44 to
V-299;
P-45 to V-299; K-46 to V-299; T-47 to V-299; L-48 to V-299; K-49 to V-299; F-
50 to V-
299; V-51 to V-299; V-52 to V-299; V-53 to V-299; I-54 to V-299; V-55 to V-
299; A-56
to V-299; V-57 to V-299; L-58 to V-299; L-59 to V-299; P-60 to V-299; V-61 to
V-299;
L-62 to V-299; A-63 to V-299; Y-64 to V-299; S-65 to V-299; A-66 to V-299; T-
67 to V-
299; T-68 to V-299; A-69 to V-299; R-70 to V-299; Q-71 to V-299; E-72 to V-
299; E-73
to V-299; V-74 to V-299; P-75 to V-299; Q-76 to V-299; Q-77 to V-299; T-78 to
V-299;
V-79 to V-299; A-80 to V-299; P-81 to V-299; Q-82 to V-299; Q-83 to V-299; Q-
84 to V-
299; R-85 to V-299; H-86 to V-299; S-87 to V-299; F-88 to V-299; K-89 to V-
299; G-90
to V-299; E-91 to V-299; E-92 to V-299; and/or C-93 to V-299 of SEQ ID N0:2.
[0154] Similarly, the present invention further provides antibodies that bind
polypeptides having one or more residues deleted from the amino terminus of
the TR5
amino acid sequence shown in SEQ ID N0:2, up to the leucine residue at
position number
295. In particular, the present invention provides antibodies that bind
polypeptides
comprising the amino acid sequence of residues n4-299 of SEQ 117 N0:2, where
n4 is an
integer from 2 to 294 corresponding to the position of the amino acid residue
in SEQ ID
NO:2.
[0155] More in particular, the invention provides antibodies that bind
polypeptides
comprising, or alternatively consisting of, the amino acid sequence of
residues: Q-2 to V-
299; G-3 to V-299; V-4 to V-299; K-5 to V-299; E-6 to V-299; R-7 to V-299; F-8
to V-
299; L-9 to V-299; P-10 to V-299; L-11 to V-299; G-12 to V-299; N-13 to V-299;
S-14 to
V-299; G-15 to V-299; D-16 to V-299; R-17 to V-299; A-18 to V-299; P-19 to V-
299; R-
20 to V-299; P-21 to V-299; P-22 to V-299; D-23 to V-299; G-24 to V-299; R-25
to V-
299; G-26 to V-299; R-27 to V-299; V-28 to V-299; R-29 to V-299; P-30 to V-
299; R-31
to V-299; T-32 to V-299; Q-33 to V-299; D-34 to V-299; G-35 to V-299; V-36 to
V-299;
G-37 to V-299; N-38 to V-299; H-39 to V-299; T-40 to V-299; M-41 to V-299; A-
42 to
V-299; R-43 to V-299; I-44 to V-299; P-45 to V-299; K-46 to V-299; T-47 to V-
299; L-48
to V-299; K-49 to V-299; F-50 to V-299; V-51 to V-299; V-52 to V-299; V-53 to
V-299;
I-54 to V-299; V-55 to V-299; A-56 to V-299; V-57 to V-299; L-58 to V-299; L-
59 to V-
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299; P-60 to V-299; V-61 to V-299; L-62 to V-299; A-63 to V-299; Y-64 to V-
299; S-65
to V-299; A-66 to V-299; T-67 to V-299; T-68 to V-299; A-69 to V-299; R-70 to
V-299;
Q-71 to V-299; E-72 to V-299; E-73 to V-299; V-74 to V-299; P-75 to V-299; Q-
76 to V
299; Q-77 to V-299; T-78 to V-299; V-79 to V-299; A-80 to V-299; P-81 to V-
299; Q-82
to V-299; Q-83 to V-299; Q-84 to V-299; R-85 to V-299; H-86 to V-299; S-87 to
V-299;
F-88 to V-299; K-89 to V-299; G-90 to V-299; E-91 to V-299; E-92 to V-299; C-
93 to V
299; P-94 to V-299; A-95 to V-299; G-96 to V-299; S-97 to V-299; H-98 to V-
299; R-99
to V-299; S-100 to V-299; E-101 to V-299; H-102 to V-299; T-103 to V-299; G-
104 to V
299; A-105 to V-299; C-106 to V-299; N-107 to V-299; P-108 to V-299; C-109 to
V-299;
T-110 to V-299; E-111 to V-299; G-112 to V-299; V-113 to V-299; D-114 to V-
299; Y
115 to V-299; T-116 to V-299; N-117 to V-299; A-118 to V-299; S-119 to V-299;
N-120
to V-299; N-121 to V-299; E-122 to V-299; P-123 to V-299; S-124 to V-299; C-
125 to V
299; F-126 to V-299; P-127 to V-299; C-128 to V-299; T-129 to V-299; V-130 to
V-299;
C-131 to V-299; K-132 to V-299; S-133 to V-299; D-134 to V-299; Q-135 to V-
299; K
136 to V-299; H-137 to V-299; K-138 to V-299; S-139 to V-299; S-140 to V-299;
C-141
to V-299; T-142 to V-299; M-143 to V-299; T-144 to V-299; R-145 to V-299; D-
146 to
V-299; T-147 to V-299; V-148 to V-299; C-149 to V-299; Q-150 to V-299; C-151
to V
299; K-152 to V-299; E-153 to V-299; G-154 to V-299; T-155 to V-299; F-156 to
V-299;
R-157 to V-299; N-158 to V-299; E-159 to V-299; N-160 to V-299; S-161 to V-
299; P
162 to V-299; E-163 to V-299; M-164 to V-299; C-165 to V-299; R-166 to V-299;
K-167
to V-299; C-168 to V-299; S-169 to V-299; R-170 to V-299; C-171 to V-299; P-
172 to V
299; S-173 to V-299; G-174 to V-299; E-175 to V-299; V-176 to V-299; Q-177 to
V-299;
V-178 to V-299; S-179 to V-299; N-180 to V-299; C-181 to V-299;.T-182 to V-
299; 5
183 to V-299; W-184 to V-299; D-185 to V-299; D-186 to V-299; I-187 to V-299;
Q-188
to V-299; C-189 to V-299; V-190 to V-299; E-191 to V-299; E-192 to V-299; F-
193 to V
299; G-194 to V-299; A-195 to V-299; N-196 to V-299; A-197 to V-299; T-198 to
V-299;
V-199 to V-299; E-200 to V-299; T-201 to V-299; P-202 to V-299; A-203 to V-
299; A
204 to V-299; E-205 to V-299; E-206 to V-299; T-207 to V-299; M-208 to V-299;
N-209
to V-299; T-210 to V-299; S-211 to V-299; P-212 to V-299; G-213 to V-299; T-
214 to V
299; P-215 to V-299; A-216 to V-299; P-217 to V-299; A-218 to V-299; A-219 to
V-299;
E-220 to V-299; E-221 to V-299; T-222 to V-299; M-223 to V-299; N-224 to V-
299; T
225 to V-299; S-226 to V-299; P-227 to V-299; G-228 to V-299; T-229 to V-299;
P-230
to V-299; A-231 to V-299; P-232 to V-299; A-233 to V-299; A-234 to V-299; E-
235 to V
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299; E-236 to V-299; T-237 to V-299; M-238 to V-299; T-239 to V-299; T-240 to
V-299;
S-241 to V-299; P-242 to V-299; G-243 to V-299; T-244 to V-299; P-245 to V-
299; A-
246 to V-299; P-247 to V-299; A-248 to V-299; A-249 to V-299; E-250 to V-299;
E-251
to V-299; T-252 to V-299; M-253 to V-299; T-254 to V-299; T-255 to V-299; S-
256 to V-
299; P-257 to V-299; G-258 to V-299; T-259 to V-299; P-260 to V-299; A-261 to
V-299;
P-262 to V-299; A-263 to V-299; A-264 to V-299; E-265 to V-299; E-266 to V-
299; T-
267 to V-299; M-268 to V-299; T-269 to V-299; T-270 to V-299; S-271 to V-299;
P-272
to V-299; G-273 to V-299; T-274 to V-299; P-275 to V-299; A-276 to V-299; S-
277 to V-
299; S-278 to V-299; H-279 to V-299; Y-280 to V-299; L-281 to V-299; S-282 to
V-299;
C-283 to V-299; T-284 to V-299; I-285 to V-299; V-286 to V-299; G-287 to V-
299; I-288
to V-299; I-289 to V-299; V-290 to V-299; L-291 to V-299; I-292 to V-299; V-
293 to V-
299; and/or L-294 to V-299 of the TR5 sequence shown in SEQ ID N0:2.
[0156] Similarly, many examples of biologically functional C-terminal deletion
muteins are known. For instance, interferon gamma shows up to ten times higher
activities by deleting 8-10 amino acid residues from the carboxy terminus of
the protein
(Dobeli et al., J. Biotechnology 7:199-216 (I988)). In the present case, since
the protein
of the invention is a member of the TNFR polypeptide family, deletions of C-
terminal
amino acids up to the cysteine at position 189 of SEQ ID N0:2, may retain some
biological activity such as regulation of proliferation and apoptosis of
lymphoid cells.
Polypeptides having further C-terminal deletions including the cysteine at
position 189 of
SEQ ID N0:2 would not be expected to retain such biological activities because
it is
known that this residue in TNF receptor-related polypeptides is required for
forming a
disulfide bridge to provide structural stability which is needed for ligand
binding.
[0157] Also as mentioned above, even if deletion of one or more amino acids
from the
C-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 TR5 ligand (e.g., TRAIL)) may still be retained. Thus, the
ability of the
shortened protein to induce and/or bind to antibodies which recognize the
complete or
mature form of the protein generally will be retained when less than the
majority of the
residues of the complete or mature form protein are removed from the C-
terminus.
Whether a particular polypeptide lacking 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. It is not unlikely that a TR5
polypeptide with a
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large number of deleted C-terminal amino acid residues may retain some
biological or
immunogenic activities. In fact, peptides composed of as few as six TR5 amino
acid
residues may often evoke an immune response.
[0158] ~ Accordingly, the present invention further provides antibodies that
bind
polypeptides having one or more residues from the carboxy terminus of the
amino acid
sequence of TR5 shown in SEQ 1D N0:2 up to the cysteine at position 189 of SEQ
ID
N0:2. In particular, the present invention provides antibodies that bind
polypeptides
having the amino acid sequence of residues 1-x of the amino acid sequence in
SEQ ID
N0:2, where x is any integer in the range of 189-299.
[0159] The present invention further provides antibodies that bind
polypeptides having
one or more residues deleted from the carboxy terminus of the amino acid
sequence of the
TR5 polypeptide shown in SEQ ID N0:2 up to the glutaxnine residue at position
number at
position number 6. In particular, the present invention provides antibodies
that bind
polypeptides comprising the amino acid sequence of residues 1-m3 of SEQ ID
N0:3,
where m3 is an integer from 6 to 298 corresponding to the position of the
amino acid
residue in SEQ ID NO:3.
[0160] More in particular, the invention provides antibodies that bind
polypeptides
comprising, or alternatively consisting of, the amino acid sequence of
residues: M-1 to F-
298; M-1 to V-297; M-1 to I-296; M-1 to L-295; M-1 to L-294; M-1 to V-293; M-1
to I-
292; M-1 to L-291; M-1 to V-290; M-1 to I-289; M-1 to I-288; M-1 to G-287; M-1
to V-
286; M-1 to I-285; M-1 to T-284; M-1 to C-283; M-1 to S-282; M-1 to L-281; M-1
to Y-
280; M-1 to H-279; M-1 to S-278; M-1 to S-277; M-1 to A-276; M-1 to P-275; M-1
to T-
274; M-1 to G-273; M-1 to P-272; M-1 to S-27I; M-1 to T-270; M-1 to T-269; M-1
to M-
268; M-1 to T-267; M-1 to E-266; M-1 to E-265; M-1 to A-264; M-1 to A-263; M-1
to P-
262; M-1 to A-261; M-1 to P-260; M-1 to T-259; M-1 to G-258; M-1 to P-257; M-1
to 5-
256; M-1 to T-255; M-1 to T-254; M-1 to M-253; M-1 to T-252; M-1 to E-251; M-1
to E-
250; M-1 to A-249; M-1 to A-248; M-1 to P-247; M-1 to A-246; M-1 to P-245; M-1
to T-
244; M-1 to G-243; M-1 to P-242; M-1 to S-241; M-1 to T-240; M-1 to T-239; M-1
to M-
238; M-1 to T-237; M-1 to E-236; M-1 to E-235; M-1 to A-234; M-1 to A-233; M-1
to P-
232; M-1 to A-231; M-1 to P-230; M-1 to T-229; M-1 to G-228; M-1 to P-227; M-1
to 5-
226; M-1 to T-225; M-1 to N-224; M-1 to M-223; M-1 to T-222; M-1 to E-221; M-1
to E-
220; M-1 to A-219; M-1 to A-218; M-1 to P-217; M-1 to A-216; M-1 to P-215; M-1
to T-
214; M-1 to G-213; M-1 to P-212; M-1 to S-211; M-1 to T-210; M-1 to N-209; M-1
to M-
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208; M-1 to T-207; M-1 to E-206; M-I to E-205; M-1 to A-204; M-1 to A-203; M-1
to P-
202; M-1 to T-201; M-1 to E-200; M-1 to V-199; M-1 to T-198; M-1 to A-197; M-1
to N-
196; M-1 to A-195; M-1 to G-194; M-1 to F-193; M-1 to E-192; M-1 to E-19I; M-1
to V-
190; M-1 to C-189; M-1 to Q-188; M-1 to I-187; M-1 to D-186; M-1 to D-185; M-1
to W-
184; M-1 to S-183; M-1 to T-182; M-1 to C-181; M-1 to N-180; M-1 to S-179; M-1
to V-
178; M-1 to Q-177; M-1 to V-176; M-1 to E-175; M-1 to G-174; M-1 to S-173; M-1
to P-
172; M-1 to C-171; M-I to R-170; M-1 to S-169; M-1 to C-168; M-1 to K-167; M-1
to 8-
166; M-1 to C-165; M-1 to M-164; M-1 to E-163; M-1 to P-162; M-1 to S-161; M-1
to N-
160; M-1 to E-159; M-1 to N-158; M-1 to R-157; M-1 to F-156; M-1 to T-155; M-1
to 6-
254; M-1 to E-153; M-I to K-152; M-I to C-151; M-1 to Q-150; M-1 to C-149; M-1
to V-
148; M-1 to T-147; M-1 to D-146; M-1 to R-145; M-1 to T-144; M-1 to M-143; M-1
to T-
142; M-1 to C-141; M-1 to S-140; M-1 to S-139; M-1 to K-138; M-1 to H-137; M-1
to K-
136; M-1 to Q-235; M-1 to D-134; M-1 to S-133; M-1 to K-132; M-1 to C-131; M-1
to V-
130; M-1 to T-129; M-1 to C-128; M-1 to P-127; M-1 to F-126; M-1 to C-125; M-1
to S-
124; M-1 to P-123; M-1 to E-122; M-1 to N-121; M-1 to N-120; M-1 to S-119; M-1
to A-
I18; M-1 to N-I I7; M-1 to T-I I6; M-1 to Y-115; M-1 to D-114; M-1 to V-I13; M-
I to 6-
112; M-1 to E-111; M-1 to T-110; M-1 to C-109; M-1 to P-108; M-1 to N-107; M-1
to C-
106; M-1 to A-105; M-1 to G-104; M-1 to T-103; M-1 to H-102; M-1 to E-101; M-1
to S-
100; M-1 to R-99; M-1 to H-98; M-1 to S-97; M-1 to G-96; M-1 to A-95; M-1 to P-
94; M-
1 to C-93; M-1 to E-92; M-1 to E-91; M-1 to G-90; M-1 to K-89; M-1 to F-88; M-
1 to S-
87; M-1 to H-86; M-1 to R-85; M-1 to Q-84; M-1 to Q-83; M-1 to Q-82; M-1 to P-
81; M-
1 to A-80; M-1 to V-79; M-1 to T-78; M-1 to Q-77; M-1 to Q-76; M-1 to P-75; M-
1 to V-
74; M-1 to E-73; M-1 to E-72; M-1 to Q-71; M-1 to R-70; M-1 to A-69; M-1 to T-
68; M-1
to T-67; M-1 to A-66; M-1 to S-65; M-1 to Y-64; M-1 to A-63; M-1 to L-62; M-1
to V-
61; M-1 to P-60; M-1 to L-59; M-1 to L-58; M-1 to V-57; M-1 to A-56; M-1 to V-
55; M-1
to I-54; M-1 to V-53; M-1 to V-52; M-1 to V-51; M-1 to F-50; M-1 to K-49; M-1
to L-48;
M-1 to T-47; M-1 to K-46; M-1 to P-45; M-1 to I-44; M-1 to R-43; M-1 to A-42;
M-I to
M-41; M-I to T-40; M-1 to H-39; M-1 to N-38; M-1 to G-37; M-1 to V-36; M-1 to
G-35;
M-1 to D-34; M-1 to Q-33; M-1 to T-32; M-1 to R-31; M-1 to P-30; M-1 to R-29;
M-1 to
V-28; M-1 to R-27; M-1 to G-26; M-1 to R-25; M-1 to G-24; M-1 to D-23; M-1 to
P-22;
M-1 to P-21; M-1 to R-20; M-1 to P-19; M-1 to A-18; M-1 to R-17; M-1 to D-16;
M-1 to
G-15; M-1 to S-14; M-I to N-13; M-1 to G-12; M-1 to L-11; M-1 to P-10; M-1 to
L-9; M-
1 to F-8; andlor M-1 to R-7 of the TR5 sequence shown in SEQ ID N0:2.
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[0161] In another embodiment, antibodies of the invention bind C-terminal
deletions
of the extracellular domain of the TR5 polypeptide that can be described by
the general
formula 67-m4 where m4 is a number from 73 to 299 corresponding to the amino
acid
sequence identified in SEQ ID N0:2. In specific embodiments, antibodies of the
invention bind C terminal deletions of the TR5 polypeptide comprising, or
alternatively,
consisting of, amino acid residues: T-67 to F-298; T-67 to V-297; T-67 to I-
296; T-67 to
L-295; T-67 to L-294; T-67 to V-293; T-67 to I-292; T-67 to L-291; T-67 to V-
290; T-67
to I-289; T-67 to I-288; T-67 to G-287; T-67 to V-286; T-67 to I-285; T-67 to
T-284; T-67
to C-283; T-67 to S-282; T-67 to L-281; T-67 to Y-280; T-67 to H-279; T-67 to
S-278; T-
67 to S-277; T-67 to A-276; T-67 to P-275; T-67 to T-274; T-67 to G-273; T-67
to P-272;
T-67 to S-271; T-67 to T-270; T-67 to T-269; T-67 to M-268; T-67 to T-267; T-
67 to E-
266; T-67 to E-265; T-67 to A-264; T-67 to A-263; T-67 to P-262; T-67 to A-
261; T-67 to
P-260; T-67 to T-259; T-67 to G-258; T-67 to P-257; T-67 to S-256; T-67 to T-
255; T-67
to T-254; T-67 to M-253; T-67 to T-252; T-67 to E-251; T-67 to E-250; T-67 to
A-249; T-
67 to A-248; T-67 to P-247; T-67 to A-246; T-67 to P-245; T-67 to T-244; T-67
to G-243;
T-67 to P-242; T-67 to S-241; T-67 to T-240; T-67 to T-239; T-67 to M-238; T-
67 to T-
237; T-67 to E-236; T-67 to E-235; T-67 to A-234; T-67 to A-233; T-67 to P-
232; T-67 to
A-231; T-67 to P-230; T-67 to T-229; T-67 to G-228; T-67 to P-227; T-67 to S-
226; T-67
to T-225; T-67 to N-224; T-67 to M-223; T-67 to T-222; T-67 to E-221; T-67 to
E-220; T-
67 to A-219; T-67 to A-218; T-67 to P-217; T-67 to A-216; T-67 to P-215; T-67
to T-214;
T-67 to G-213; T-67 to P-212; T-67 to S-211; T-67 to T-210; T-67 to N-209; T-
67 to M-
208; T-67 to T-207; T-67 to E-206; T-67 to E-205; T-67 to A-204; T-67 to A-
203; T-67 to
P-202; T-67 to T-201; T-67 to E-200; T-67 to V-199; T-67 to T-198; T-67 to A-
197; T-67
to N-196; T-67 to A-195; T-67 to G-194; T-67 to F-193; T-67 to E-192; T-67 to
E-19I; T-
67 to V-190; T-67 to C-189; T-67 to Q-I88; T-67 to I-187; T-67 to D-186; T-67
to D-185;
T-67 to W-184; T-67 to S-183; T-67 to T-182; T-67 to C-181; T-67 to N-180; T-
67 to 5-
179; T-67 to V-178; T-67 to Q-177; T-67 to V-176; T-67 to E-175; T-67 to G-
174; T-67 to
S-173; T-67 to P-172; T-67 to C-171; T-67 to R-170; T-67 to S-169; T-67 to C-
168; T-67
to K-167; T-67 to R-166; T-67 to C-165; T-67 to M-164; T-67 to E-163; T-67 to
P-162; T-
67 to S-161; T-67 to N-160; T-67 to E-159; T-67 to N-158; T-67 to R-157; T-67
to F-156;
T-67 to T-155; T-67 to G-154; T-67 to E-153; T-67 to K-152; T-67 to C-151; T-
67 to Q-
150; T-67 to C-149; T-67 to V-148; T-67 to T-147; T-67 to D-146; T-67 to R-
145; T-67 to
T-144; T-67 to M-143; T-67 to T-142; T-67 to C-141; T-67 to S-140; T-67 to S-
139; T-67
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CA 02426710 2003-04-23
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to K-138; T-67 to H-137; T-67 to K-136; T-67 to Q-135; T-67 to D-134; T-67 to
S-133; T-
67 to K-132; T-67 to C-131; T-67 to V-130; T-67 to T-129; T-67 to C-128; T-67
to P-127;
T-67 to F-126; T-67 to C-125; T-67 to S-124; T-67 to P-123; T-67 to E-122; T-
67 to N-
121; T-67 to N-120; T-67 to S-119; T-67 to A-118; T-67 to N-117; T-67 to T-
116; T-67 to
Y-115; T-67 to D-114; T-67 to V-113; T-67 to G-112; T-67 to E-111; T-67 to T-
110; T-67
to C-109; T-67 to P-108; T-67 to N-107; T-67 to C-106; T-67 to A-105; T-67 to
G-104; T-
67 to T-103; T-67 to H-102; T-67 to E-101; T-67 to S-100; T-67 to R-99; T-67
to H-98; T-
67 to S-97; T-67 to G-96; T-67 to A-95; T-67 to P-94; T-67 to C-93; T-67 to E-
92; T-67 to
E-91; T-67 to G-90; T-67 to K-89; T-67 to F-88; T-67 to S-87; T-67 to H-86; T-
67 to R-
85; T-67 to Q-84; T-67 to Q-83; T-67 to Q-82; T-67 to P-81; T-67 to A-80; T-67
to V-79;
T-67 to T-78; T-67 to Q-77; T-67 to Q-76; T-67 to P-75; T-67 to V-74; and/or T-
67 to E-
73 of the TR5 extracellular domain sequence shown in SEQ ID N0:2.
[0162] The invention also provides antibodies that bind polypeptides having
one or
more amino acids deleted from both the amino and the carboxyl termini, which
may be
described generally as having residues n4 to m3 of SEQ ID N0:2, where n4 and
m3 are
integers as described above.
[0163] Also included are antibodies that bind a polypeptide consisting of a
portion of
the complete TR5 amino acid sequence encoded by a cDNA clone contained in ATCC
Deposit No. 97798, where this portion excludes from 1 to about 89 amino acids
from the
amino terminus of the complete amino acid sequence encoded by the cDNA clone
contained in ATCC Deposit No. 97798, or from 1 to about 110 amino acids from
the
carboxy terminus of the complete amino acid sequence encoded by the cDNA clone
contained in ATCC Deposit No. 97798, or any combination of the above amino
terminal
and carboxy terminal deletions, of the complete amino acid sequence encoded by
the
cDNA clone contained in ATCC Deposit No. 97798.
[0164] In addition to terminal deletion forms of the protein discussed above,
it will
also be recognized by one of ordinary skill in the art that some amino acid
sequences of
the TR5 polypeptide can be varied without significant effect on the structure
or function of
the proteins. If such differences in sequence are contemplated, it should be
remembered
that there will be critical areas on the protein which determine activity.
Thus, the invention
further includes variations of the TR5 polypeptide, which show substantial TR5
polypeptide activity or which include regions of TR5 protein such as the
protein portions
discussed below. Such mutants include deletions, insertions, inversions,
repeats, and type
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substitutions Guidance concerning which amino acid changes are likely to be
phenotypically silent can be found in Bowie, J. U. et al., "Deciphering the
Message in
Protein Sequences: Tolerance to Amino Acid Substitutions," Scietzce 247:1306-
1310
(1990).
[0165] Thus, antibodies of the present invention may bind a fragment,
derivative, or
analog of the polypeptide of SEQ 1D N0:2, or that encoded by the cDNA in ATCC
deposit 97798. Such fragments, variants or derivatives 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 residue(s)), 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 or soluble extracellular polypeptide is fused with another compound,
such as a
compound to increase the half life of the polypeptide (for example,
polyethylene glycol);
or (iv) one in which the additional amino acids are fused to the mature
polypeptide, such
as an IgG Fc fusion region peptide or leader or secretory sequence or a
sequence which is
employed fox 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.
[0166] Of particular interest are substitutions of charged amino acids with
another
charged amino acids and with neutral or negatively charged amino acids. The
latter results
in proteins with reduced positive charge to improve the characteristics of the
TR5 protein.
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.
Ifnrnuftol.
2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al.,
Crit. Rev.
Therapeutic Drug CaYt-ier Systeytts 10:307-377 (1993)).
[0167] The replacement of amino acids can also change the selectivity of
binding of a'
ligand to cell surface receptors. For example, Ostade et al., Nature 361:266-
268 (1993),
describes certain mutations resulting in selective binding of TNF-a to only
one of the two
known types of TNF receptors. 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.,
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Science 255:306-312 (1992)). Thus, the antibodies of the present invention may
bind a
TR5 receptor that contains one or more amino acid substitutions, deletions or
additions,
either from natural mutations or human manipulation.
[0168] 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 3 above).
[0169] In specific embodiments, the number of substitutions, additions or
deletions in
the amino acid sequence of SEQ ID N0:2 and/or any of the polypeptide fragments
described herein (e.g., the extracellular 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, 1-5,
1-3 or 1-2.
[0170] In specific embodiments, the antibodies of the invention bind TR5
polypeptides
or fragments or variants thereof (especially a fragment comprising or
alternatively
consisting of, the extracellular soluble domain of TR5), that contains any one
or more of
the following conservative mutations in TRS: M1 replaced with A, G, I, L, S,
T, or V; Q2
replaced with N; G3 replaced with A, I, L, S, T, M, or V; V4 replaced with A,
G, I, L, S,
T, or M; K5 replaced with H, or R; E6 replaced with D; R7 replaced with H, or
K; F8
replaced with W, or Y; L9 replaced with A, G, I, S, T, M, or V; L11 replaced
with A, G, I,
S, T, M, or V; G12 replaced with A, I, L, S, T, M, or V; N13 replaced with Q;
S14
replaced with A, G, I, L, T, M, or V; G15 replaced with A, I, L, S, T, M, or
V; D16
replaced with E; R17 replaced with H, or K; A18 replaced with G, I, L, S, T,
M, or V; R20
replaced with H, or K; D23 replaced with E; G24 replaced with A, I, L, S, T,
M, or V; R25
replaced with H, or K; G26 replaced with A, I, L, S, T, M, or V; R27 replaced
with H, or
K; V28 replaced with A, G, I, L, S, T, or M; R29 replaced with H, or K; R31
replaced with
H, or K; T32 replaced with A, G, I, L, S, M, or V; Q33 replaced with N; D34
replaced
with E; G35 replaced with A, I, L, S, T, M, or V; V36 replaced with A, G, I,
L, S, T, or M;
G37 replaced with A, I, L, S, T, M, or V; N38 replaced with Q; H39 replaced
with K, or
R; T40 replaced with A, G, I, L, S, M, or V; M41 replaced with A, G, I, L, S,
T, or V; A42
replaced with G, I, L, S, T, M, or V; R43 replaced with H, or K; I44 replaced
with A, G, L,
S, T, M, or V; K46 replaced with H, or R; T47 replaced with A, G, I, L, S, M,
or V; I~8
replaced with A, G, I, S, T, M, or V; K49 replaced with H, or R; F50 replaced
with W, or
Y; V51 replaced with A, G, I, L, S, T, or M; V52 replaced with A, G, I, L, S,
T, or M; V53
replaced with A, G, I, L, S, T, or M; I54 replaced with A, G, L, S, T, M, or
V; V55
replaced with A, G, I, L, S, T, or M; A56 replaced with G, I, L, S, T, M, or
V; V57
103
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replaced with A, G, I, L, S, T, or M; L58 replaced with A, G, I, S, T, M, or
V; L59
replaced with A, G, I, S, T, M, or V; V61 replaced with A, G, I, L, S, T, or
M; L62
replaced with A, G, I, S, T, M, or V; A63 replaced with G, I, L, S, T, M, or
V; Y64
replaced with F, or W; S65 replaced with A, G, I, L, T, M, or V; A66 replaced
with G, I,
L, S, T, M, or V; T67 replaced with A, G, I, L, S, M, or V; T68 replaced with
A, G, I, L, S,
M, or V; A69 replaced with G, I, L, S, T, M, or V; R70 replaced with H, or K;
Q71
replaced with N; E72 replaced with D; E73 replaced with D; V74 replaced with
A, G, I, L,
S, T, or M; Q76 replaced with N; Q77 replaced with N; T78 replaced with A, G,
I, L, S,
M, or V; V79 replaced with A, G, I, L, S, T, or M; A80 replaced with G, I, L,
S, T, M, or
V; Q82 replaced with N; Q83 replaced with N; Q84 replaced with N; R85 replaced
with
H, or K; H86 replaced with K, or R; S87 replaced with A, G, I, L, T, M, or V;
F88
replaced with W, or Y; K89 replaced with H, or R; G90 replaced with A, I, L,
S, T, M, or
V; E91 replaced with D; E92 replaced with D; A95 replaced with G, I, L, S, T,
M, or V;
G96 replaced with A, I, L, S, T, M, or V; S97 replaced with A, G, I, L, T, M,
or V; H98
replaced with K, or R; R99 replaced with H, or K; S 100 replaced with A, G, I,
L, T, M, or
V; E101 replaced with D; H102 replaced with K, or R; T103 replaced with A, G,
I, L, S,
M, or V; 6104 replaced with A, I, L, S, T, M, or V; A105 replaced with G, I,
L, S, T, M,
or V; N107 replaced with Q; T110 replaced with A, G, I, L, S, M, or V; E111
replaced
with D; 6112 replaced with A, I, L, S, T, M, or V; V113 replaced with A, G, I,
L, S, T, or
M; D114 replaced with E; Y115 replaced with F, or W; T116 replaced with A, G,
I, L, S,
M, or V; N117 replaced with Q; A118 replaced with G, I, L, S, T, M, or V; 5119
replaced
with A, G, I, L, T, M, or V; N120 replaced with Q; N121 replaced with Q; E122
replaced
with D; S 124 replaced with A, G, I, L, T, M, or V; F126 replaced with W, or
Y; T129
replaced with A, G, I, L, S, M, or V; V130 replaced with A, G, I, L, S, T, or
M; K132
replaced with H, or R; S 133 replaced with A, G, I, L, T, M, or V; D 134
replaced with E;
Q135 replaced with N; K136 replaced with H, or R; H137 replaced with K, or R;
K138
replaced with H, or R; S 139 replaced with A, G, I, L, T, M, or V; S 140
replaced with A,
G, I, L, T, M, or V; T142 replaced with A, G, I, L, S, M, or V; M143 replaced
with A, G,
I, L, S, T, or V; T144 replaced with A, G, I, L, S, M, or V; 8145 replaced
with H, or K;
D146 replaced with E; T147 replaced with A, G, I, L, S, M, or V; V148 replaced
with A,
G, I, L, S, T, or M; Q150 replaced with N; K152 replaced with H, or R; E153
replaced
with D; 6154 replaced with A, I, L, S, T, M, or V; T155 replaced with A, G, I,
L, S, M, or
V; F156 replaced with W, or Y; 8157 replaced with H, or K; N158 replaced with
Q; E159
104
CA 02426710 2003-04-23
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replaced with D; N160 replaced with Q; 5161 replaced with A, G, I, L, T, M, or
V; E163
replaced with D; M164 replaced with A, G, I, L, S, T, or V; 8166 replaced with
H, or I~;
K167 replaced with H, or R; S 169 replaced with A, G, I, L, T, M, or V; 8170
replaced
with H, or I~; S 173 replaced with A, G, I, L, T, M, or V; 6174 replaced with
A, I, L, S, T,
M, or V; E175 replaced with D; V176 replaced with A, G, I, L, S, T, or M; Q177
replaced
with N; V 178 replaced with A, G, I, L, S, T, or M; S 179 replaced with A, G,
I, L, T, M, or
V; N180 replaced with Q; T182 replaced with A, G, I, L, S, M, or V; 5183
replaced with
A, G, I, L, T, M, or V; W184 replaced with F, or Y; D185 replaced with E; D186
replaced
with E; I187 replaced with A, G, L, S, T, M, or V; Q188 replaced with N; V190
replaced
with A, G, I, L, S, T, or M; E191 replaced with D; E192 replaced with D; F193
replaced
with W, or Y; 6194 replaced with A, I, L, S, T, M, or V; A195 replaced with G,
I, L, S, T,
M, or V; N196 replaced with Q; A197 replaced with G, I, L, S, T, M, or V; T198
replaced
with A, G, I, L, S, M, or V; V199 replaced with A, G, I, L, S, T, or M; E200
replaced with
D; T201 replaced with A, G, I, L, S, M, or V; A203 replaced with G, I, L, S,
T, M, or V;
A204 replaced with G, I, L, S, T, M, or V; E205 replaced with D; E206 replaced
with D;
T207 replaced with A, G, I, L, S, M, or V; M208 replaced with A, G, I, L, S,
T, or V;
N209 replaced with Q; T210 replaced with A, G, I, L, S, M, or V; 5211 replaced
with A,
G, I, L, T, M, or V; 6213 replaced with A, I, L, S, T, M, or V; T214 replaced
with A, G, I,
L, S, M, or V; A216 replaced with G, I, L, S, T, M, or V; A218 replaced with
G, I, L, S, T,
M, or V; A219 replaced with G, I, L, S, T, M, or V; E220 replaced with D; E221
replaced
with D; T222 replaced with A, G, I, L, S, M, or V; M223 replaced with A, G, I,
L, S, T, or
V; N224 replaced with Q; T225 replaced with A, G, I, L, S, M, or V; 5226
replaced with
A, G, I, L, T, M, or V; 6228 replaced with A, I, L, S, T, M, or V; T229
replaced with A,
G, I, L, S, M, or V; A231 replaced with G, I, L, S, T, M, or V; A233 replaced
with G, I, L,
S, T, M, or V; A234 replaced with G, I, L, S, T, M, or V; E235 replaced with
D; E236
replaced with D; T237 replaced with A, G, I, L, S, M, or V; M238 replaced With
A, G, I,
L, S, T, or V; T239 replaced with A, G, I, L, S, M, or V; T240 replaced with
A, G, I, L, S,
M, or V; 5241 replaced with A, G, I, L, T, M, or V; 6243 replaced with A, I,
L, S, T, M,
or V; T244 replaced with A, G, I, L, S, M, or V; A246 replaced with G, I, L,
S, T, M, or
V; A248 replaced with G, I, L, S, T, M, or V; A249 replaced with G, I, L, S,
T, M, or V;
E250 replaced with D; E251 replaced with D; T252 replaced with A, G, I, L, S,
M, or V;
M253 replaced with A, G, I, L, S, T, or V; T254 replaced with A, G, I, L, S,
M, or V;
T255 replaced with A, G, I, L, S, M, or V; 5256 replaced with A, G, I, L, T,
M, or V;
105
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
6258 replaced with A, I, L, S, T, M, or V; T259 replaced with A, G, I, L, S,
M, or V;
A261 replaced with G, I, L, S, T, M, or V; A263 replaced with G, T, L, S, T,
M, or V;
A264 replaced with G, I, L, S, T, M, or V; E265 replaced with D; E266 replaced
with D;
T267 replaced with A, G, I, L, S, M, or V; M268 replaced with A, G, I, L, S,
T, or V;
T269 replaced with A, G, I, L, S, M, or V; T270 replaced with A, G, I, L, S,
M, or V;
5271 replaced with A, G, I, L, T, M, or V; 6273 replaced with A, I, L, S, T,
M, or V;
T274 replaced with A, G, I, L, S, M, or V; A276 replaced with G, I, L, S, T,
M, or V;
5277 replaced with A, G, I, L, T, M, or V; 5278 replaced with A, G, I, L, T,
M, or V;
H279 replaced with K, or R; Y280 replaced with F, or W; L281 replaced with A,
G, I, S,
T, M, or V; 5282 replaced with A, G, I, L, T, M, or V; T284 replaced with A,
G, I, L, S,
M, or V; I285 replaced with A, G, L, S, T, M, or V; V286 replaced with A, G,
I, L, S, T,
or M; 6287 replaced with A, I, L, S, T, M, or V; I288 replaced with A, G, L,
S, T, M, or
V; I289 replaced with A, G, L, S, T, M, or V; V290 replaced with A, G, I, L,
S, T, or M;
L291 replaced with A, G, I, S, T, M, or V; I292 replaced with A, G, L, S, T,
M, or V;
V293 replaced with A, G, I, L, S, T, or M; L294 replaced with A, G, I, S, T,
M, or V;
L295 replaced with A, G, I, S, T, M, or V; I296 replaced with A, G, L, S, T,
M, or V;
V297 replaced with A, G, I, L, S, T, or M; and/or F298 replaced with W, or Y;
V299
replaced with A, G, I, L, S, T, or M.
[0171] In specific embodiments, the antibodies of the invention bind TR5
polypeptides
or fragments or variants thereof (especially a fragment comprising or
alternatively
consisting of, the extracellular soluble domain of TR5), that contains any one
or more of
the following non-conservative mutations in TRS: MI replaced with D, E, H, K,
R, N, Q,
F, W, Y, P, or C; Q2 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C;
G3 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V4 replaced with D, E,
H, K, R,
N, Q, F, W, Y, P, or C; K5 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or
C; E6 replaced with H, K,.R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
R7 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F8 replaced with D,
E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; L9 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or
C; P10 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or
C; Ll l
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; GI2 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; N13 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P,
or C; S14 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G15 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; D16 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F,
106
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W, Y, P, or C; R17 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; A18
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P19 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R20 replaced with D, E, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, P, or C; P21 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, or C; P22 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C;
D23 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G24
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; R25 replaced with D, E, A, G, I,
L, S, T, M, V,
N, Q, F, W, Y, P, or C; G26 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; R27
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V28
replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; R29 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, P, or C; P30 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or
C; R31 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T32
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q33 replaced with D, E, H, K, R,
A, G, I, L, S,
T, M, V, F, W, Y, P, or C; D34 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, P, or C; G35 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V36
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; G37 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; N38 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;
H39 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T40 replaced with
D, E, H, K, R,
N, Q, F, W, Y, P, or C; M41 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; A42
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R43 replaced with D, E,
A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; I44 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
P45 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
K46 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T47 replaced with
D, E, H, K, R,
N, Q, F, W, Y, P, or C; L48 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; K49
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F50
replaced with D, E,
H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; V5I replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; V52 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V53
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; I54 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; V55 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A56
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; V57 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; L58 replaced with D, E, H, K, R,. N, Q, F, W, Y, P, or C; L59 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; P60 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, or C; V61 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L62
replaced with
107
CA 02426710 2003-04-23
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D, E, H, K, R, N, Q, F, W, Y, P, or C; A63 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; Y64 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
S65 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A66 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; T67 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T68
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; A69 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; R70 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q71
replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E72 replaced
with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E73 replaced with H, K, R, A, G,
I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; V74 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; P75
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; Q76
replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; Q77 replaced
with D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T78 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; V79 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A80
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; P81 replaced with D, E, H, K, R, A, G, I,
L, S, T, M,
V, N, Q, F, W, Y, or C; Q82 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y,
P, or C; Q83 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,
or C; Q84
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R85
replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H86 replaced with D, E, A,
G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; S87 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
F88 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K89
replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G90 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; E91 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; E92 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; C93
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; P94
replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A95 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; G96 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; S97
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H98 replaced with D, E,
A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; R99 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, P, or C; S 100 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E101
replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H102 replaced
with D, E, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T103 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; 6104 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A105
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; C106 replaced with D, E, H, K, R, A, G,
I, L, S, T,
108
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
M, V, N, Q, F, W, Y, or P; N107 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F,
W, Y, P, or C; P108 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or
C; C109 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or
P; T110
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E111 replaced with H, K,
R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; 6112 replaced with D, E, H, K, R, N, Q,
F, W, Y,
P, or C; V113 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D114
replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Y115 replaced with D, E,
H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; T116 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; N117 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;
A118
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S 119 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; N120 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P,
or C; N121 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; E122
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P123
replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S 124 replaced
with D, E, H, K,
R, N, Q, F, W, Y, P, or C; CI25 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, or P; F126 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,
P, or C;
P127 replaced with D, E, H, K, R, A, G, I, L; S, T, M, V, N, Q, F, W, Y, or C;
C128
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; T129
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; V 130 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; C I31 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or P;
K132 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S 133
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; D134 replaced with H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; Q135 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F,
W, Y, P, or C; K136 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
H137 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K138
replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S 139 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; S 140 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; C 141
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; T142
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; M143 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; T144 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 8145
replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D146 replaced with H, K,
R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; T147 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; V148 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C149 replaced
with D, E,
109
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Q150 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; C 151 replaced with D, E, H, K, R, A,
G, I, L, S, T,
M, V, N, Q, F, W, Y, or P; K152 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W,
Y, P, or C; E153 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
6154 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T155 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; F156 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V,
P, or C; 8157 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; N158
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y; P, or C; E159
replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N160 replaced with D,
E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; S 161 replaced with D, E, H, K, R,
N, Q, F, W, Y,
P, or C; P162 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, or C;
E163 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
M164 replaced
With D, E, H, K, R, N, Q, F, W, Y, P, or C; C165 replaced with D, E, H, K, R,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or P; 8166 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, P, or C; K167 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
C 168 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or
P; S 169
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 8170 replaced with D, E,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; C171 replaced with D, E, H, K, R, A, G, I,
L, S, T, M,
V, N, Q, F, W, Y, or P; P172 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, or C; 5173 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6174
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; E175 replaced with H, K, R, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, P, or C; V176 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; Q177
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V 178
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; S 179 replaced with D, E, H, K, R, N,
Q, F, W, Y, P,
or C; N280 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; C181
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; T182
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S 183 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; W184 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; D185
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D 186
replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I187 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; Q188 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y,
P, or C; C189 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, or P;
V190 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E191 replaced with
H, K, R, A,
110
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E192 replaced with H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; F193 replaced with D, E, H, K, R, N, Q, A, G, I,
L, S, T, M,
V, P, or C; 6194 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A195
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; N196 replaced with D, E, H, K, R, A, G,
I, L, S, T,
M, V, F, W, Y, P, or C; A197 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; T198
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V 199 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; E200 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; T201 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P202 replaced
with D, E,
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A203 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; A204 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
E205
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E206
replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T207 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; M208 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; N209
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T210
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; S211 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; P212 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; 6213
replaced with D; E, H, K, R, N, Q, F, W, Y, P, or C; T214 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; P215 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, or C; A216 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P217
replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A218 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; A219 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; E220
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E221
replaced with
H, K, R, A, G~ I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T222 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; M223 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; N224
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T225
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; 5226 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; P227 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; 6228
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T229 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; P230 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, or C; A231 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P232
replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A233 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; A234 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; E235
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E236
replaced with
111
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T237 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; M238 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; T239
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T240 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; 5241 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
P242
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; 6243
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; T244 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; P245 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C;
A246 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P247 replaced with
D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A248 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; A249 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E250
replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E251 replaced
with H, K, R,
A, G, I, L, S, T, M, V, ,N, Q, F, W, Y, P, or C; T252 replaced with D, E, H,
K, R, N, Q, F,
W, Y, P, or C; M253 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T254
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; T255 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; 5256 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P257
replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; 6258 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; T259 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; P260
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A261
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; P262 replaced with D, E, H, K, R,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; A263 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or
C; A264 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E265 replaced
with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E266 replaced with H, K, R, A,
G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; T267 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or
C; M268 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T269 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; T270 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
5271 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P272 replaced with
D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; 6273 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; T274 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P275
replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A276 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; 5277 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
5278 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H279 replaced with
D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Y280 replaced with D, E, H, K, R, N,
Q, A, G, I,
L, S, T, M, V, P, or C; L281 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; 5282
112
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C283 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or P; T284 replaced with D, E, H, K, R, N,
Q, F, W, Y,
P, or C; I285 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V286
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; 6287 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; I288 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I289 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; V290 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
L291 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I292 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; V293 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; L294
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L295 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; I296 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V297
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F298 replaced with D, E,
H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; and/or V299 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C.
[0172] Amino acids in the TR5 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
molecule. The resulting mutant molecules are then tested for biological
activity such as
receptor binding or in vitro proliferative activity. Sites that are critical
for ligand-receptor
binding can also be determined by structural analysis such as crystallization,
nuclear
magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol.
224:899-904
(1992) and de Vos et al. Science 255:306-312 (1992)). In preferred
embodiments,
antibodies of the present invention bind regions of TR5 that are essential for
TR5 function.
In other preferred embodiments, antibodies of the present invention bind
regions of TR5
that are essential for TR5 function and inhibit or abolish TR5 function. In
other preferred
embodiments, antibodies of the present invention bind regions of TR5 that are
essential for
TR5 function and enhance TR5 function.
[0173] Additionally, protein engineering may be employed to improve or alter
the
characteristics of TR5 polypeptides. Recombinant DNA technology known to those
skilled in the art can be used to create novel mutant proteins or polypeptides
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
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natural polypeptide, at least under certain purification and storage
conditions. Antibodies
of the present invention may bind such modified TR5 polypeptides.
[0174] TR5 occurnng variants that may be bound by the antibodies of the
invention
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 et al.,
Philos. Traps. R.
Soc. London SerA 317:415 (1986)).
[0175] In preferred embodiments, the antibodies of the invention bind a 259
amino
acid TR5 polypeptide (corresponding to amino acids residues 41-299 of SEQ ID
N0:2)
exhibiting two main structural domains. First, the extracellular TRAIL ligand
binding
domain was identified within residues from about 67 to about 280 in SEQ ID
N0:2.
Second, the transmembrane domain was identified within residues from about 281
to
about 299 in SEQ ID N0:2. As mentioned above, however, TRS, lacks a putative
intracellular signalling domain, thus, TR5 is also sometimes referred to as
"TRID"
(TRAIL Receptor Without an Intracellular Domain").
[0176] The antibodies of the present invention also include antibodies that
bind a
polypeptide comprising, or alternatively, consisting of the polypeptide
encoded by the
deposited cDNA including the leader; the mature polypeptide encoded by the
deposited
the cDNA minus the leader (i.e., the mature protein); a polypeptide comprising
amino
acids about 1 to about 299 in SEQ ID NO:2; a polypeptide comprising amino
acids about
41 to about 299 in SEQ ID N0:2; a polypeptide comprising amino acids about 42
to about
299 in SEQ ID NO:2 as well as polypeptides which are at least 80% identical,
more
preferably at least 90% or 95% identical, still more preferably at least 96%,
97%, 98%, or
99% identical to the polypeptides described above, and also include portions
of such
polypeptides with at least 30 amino acids and more preferably at least 50
amino acids.
[0177] By a polypeptide having an amino acid sequence at least, for example,
95%
"identical" to a reference amino acid sequence of a TR5 polypeptide is
intended that the
amino acid sequence of the polypeptide is identical to the reference sequence
except that
the polypeptide sequence may include up to five amino acid alterations per
each 100
amino acids of the reference amino acid of the TR5 polypeptide. In other
words, to obtain
a polypeptide having an amino acid sequence at least 95% identical to a
reference amino
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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.
[0178] As a practical matter, whether any particular polypeptide is at least
90%, 95%,
96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown
in SEQ
ID N0:2, or to the amino acid sequence encoded by the deposited cDNA clone,
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.
[0179] In a specific embodiment, the identity between a reference (query)
sequence (a
sequence of the present invention) and a subject sequence, also referred to as
a global
sequence alignment, is determined using the FASTDB computer program based on
the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred
parameters
used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch
Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1,
Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window
Size=500 or the length of the subject amino acid sequence, whichever is
shorter.
According to this embodiment, 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
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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.
[0180] The polypeptide of the present invention have uses which include, but
are not
limited to, as sources for generating antibodies that bind the polypeptides of
the invention,
and as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel
filtration
columns using methods well known to those of skill in the art.
[0181] The present application is also directed to proteins cotaining
polypeptides at
least 90%, 95%, 96%, 97%, 98% or 99% identical to the TR5 polypeptide sequence
set
forth herein as nl-mr. In preferred embodiments, the application is directed
to proteins
containing polypeptides at least 90%, 95%, 96°70, 97%, 98% or 99%
identical to
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polypeptides having the amino acid sequence of the specific TR5 N- and C-
terminal
deletions recited herein.
[0182] In certain preferred embodiments, TR5 proteins of the invention
comprise
fusion proteins as described above wherein the TR5 polypeptides are those
described as
n4-m3 herein. In preferred embodiments, the application is directed to nucleic
acid
molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic
acid
sequences encoding polypeptides having the amino acid sequence of the specific
N- and
C-terminal deletions recited herein. Polynucleotides encoding these
polypeptides are also
encompassed by the invention.
TR7
[0183] In certain embodiments of the present invention, the antibodies of the
present
invention bind TR7 polypeptide, or fragments or variants thereof. The
following section
describes the TR7 polypeptides, fragments and variants that may be bound by
the
antibodies of the invention in more detail. The TR7 polypeptides, fragments
and variants
which may be bound by the antibodies of the invention are also described in,
for example,
International Publication Numbers W098/41629, WO00/66156, and W098/35986 which
are herein incorporated by reference in their entireties.
[0184] In certain embodiments, the antibodies of the present invention
immunospecifically bind TR7 polypeptide. An antibody that immunospecifically
binds
TR7 may, in some embodiments, bind fragments, variants (including species
orthologs of
TR7), multimers or modified forms of TR7. For example, an antibody
immunospecific for
TR7 may bind the TR7 moiety of a fusion protein comprising all or a portion of
TR7.
[0185] TR7 proteins may be found as monomers or multimers (i.e., dimers,
trimers,
tetramers, and higher multimers). Accordingly, the present invention relates
to antibodies
that bind TR7 proteins found as monomers or as part of multimers. In specific
embodiments, the TR7 polypeptides 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.
[0186] Antibodies of the invention may bind TR7 homomers or heteromers. As
used
herein, the term homomer, refers to a multimer containing only TR7 proteins of
the
invention (including TR7 fragments, variants, and fusion proteins, as
described herein).
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These homomers may contain TR7 proteins having identical or different
polypeptide
sequences. In a specific embodiment, a homomer of the invention is a multimer
containing only TR7 proteins having an identical polypeptide sequence. In
another
specific embodiment, antibodies of the invention bind TR7 homomers containing
TR7
proteins having different polypeptide sequences. In specific embodiments,
antibodies of
the invention bind a TR7 homodimer (e.g., containing TR7 proteins having
identical or
different polypeptide sequences) or a homotrimer (e.g., containing TR7
proteins having
identical or different polypeptide sequences). In additional embodiments,
antibodies of
the invention bind at least a homodimer, at least a homotrimer, or at least a
homotetramer
of TR7.
[0187] As used herein, the term heteromer refers to a multimer containing
heterologous proteins (i.e., proteins containing polypeptide sequences that do
not
correspond to a polypeptide sequences encoded by the TR7 gene) in addition to
the TR7
proteins of the invention. In a specific embodiment, antibodies of the
invention bind a
heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments,
the
antibodies of the invention bind at least a homodimer, at least a homotrimer,
or at least a
homotetramer containing one or more TR7 polypeptides.
[0188] Multimers bound by one or more antibodies 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
bound
by one or more antibodies of the invention, such as, for example, homodimers
or
homotrimers, are formed when TR7 proteins contact one another in solution. In
another
embodiment, heteromultimers bound by one or more antibodies of the invention,
such as,
for example, heterotrimers or heterotetramers, are formed when TR7 proteins
contact
antibodies to the TR7 polypeptides (including antibodies to the heterologous
polypeptide
sequence in a fusion protein) in solution. In other embodiments, multimers
bound by one
or more antibodies of the invention are formed by covalent associations with
and/or
between the TR7 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 N0:3 or the polypeptide encoded by the
deposited cDNA clone of ATCC Deposit 97920). 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)
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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 TR7 fusion protein. In one example, covalent associations are
between the
heterologous sequence contained in a fusion protein (see, e.g., US Patent
Number
5,478,925). In a specific example, the covalent associations are between the
heterologous
sequence contained in a TR7-Fc fusion protein (as described herein). In
another specific
example, covalent associations of fusion proteins 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).
[0189] The multimers that may be bound by one or more antibodies 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 that may be bound by one or more
antibodies 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 that may be bound by one or more antibodies 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 that may be
bound by one
or more antibodies of the invention (see, e.g., US Patent Number 5,478,925,
which is
herein incorporated by reference in its entirety).
[0190] Alternatively, multimers that may be bound by one or more antibodies of
the
invention may be generated using genetic engineering techniques known in the
art. In one
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embodiment, proteins contained in multimers that may be bound by one or more
antibodies 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 that may be bound by one or more
antibodies of
the invention are generated by ligating a polynucleotide sequence encoding a
TR7
polypeptide 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 TR7 polypeptides 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). In another embodiment, two or more
TR7
polypeptides axe joined through synthetic linkers (e.g., peptide, carbohydrate
or soluble
polymer linkers). Examples include those peptide linkers described in U.S.
Pat. No.
5,073,627 (hereby incorporated by reference). Proteins comprising multiple TR7
polypeptides separated by peptide linkers may be produced using conventional
recombinant DNA technology. In specific embodiments, antibodies of the
invention bind
proteins comprising multiple TR7 polypeptides separated by peptide linkers.
[0191] Another method for preparing multimer TR7 polypeptides involves use of
TR7
polypeptides fused to a leucine zipper or isoleucine polypeptide sequence.
Leucine zipper
domains and isoleucine zipper domains are polypeptides that promote
multimerization of
the proteins in which they are found. Leucine zippers were originally
identified in several
DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have
since been
found in a variety of different proteins. Among the known leucine zippers are
naturally
occurring peptides and derivatives thereof that dimerize or trimerize.
Examples of leucine
zipper domains suitable for producing soluble multimeric TR7 proteins are
those described
in PCT application WO 94/10308, hereby incorporated by reference. Recombinant
fusion
proteins comprising a soluble TR7 polypeptide fused to a peptide that
dimerizes or
trimerizes in solution are expressed in suitable host cells, and the resulting
soluble
multimeric TR7 is recovered from the culture supernatant using techniques
known in the
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art. In specific embodiments, antibodies of the invention bind TR7-leucine
zipper fusion
protein monomers and/or TR7-leucine zipper fusion pxotein multimers.
[0192] Ceutain members of the TNF family of proteins are believed to exist in
trimeric
form (Beutler and Huffel, Science 264:667, 1994; Banner et al., Cell 73:431,
1993). Thus,
trimeric TR7 may offer the advantage of enhanced biological activity.
Preferred leucine
zipper moieties are those that preferentially form trimers. One example is a
leucine zipper
derived from lung surfactant protein D (SPD), as described in Hoppe et al.
(FEBS Letters
344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby
incorporated
by reference. In specific embodiments, antibodies of the invention bind TR7-
leucine
zipper fusion protein trimers.
[0193] Other peptides derived from naturally occurring trimeric proteins may
be
employed in preparing trimeric TR7. In specific embodiments, antibodies of the
invention
bind TR7- fusion protein monomers and/or TR7 fusion protein trimers.
[0194] The TR7 polypeptides are preferably provided in an isolated form, and
preferably are substantially purified. 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 TR7 polypeptide is substantially purified by the one-
step method
described in Smith and Johnson, GeyZe 67:31-40 (1988).
[0195] Antibodies of the present invention may bind TR7 polypeptides or
polypeptide
fragments including polypeptides comprising or alternatively, consisting of,
an amino acid
sequence contained in SEQ 1D N0:3, encoded by the cDNA contained in ATCC
deposit
Number 97920, or encoded by nucleic acids which hybridize (e.g., under
stringent
hybridization conditions) to the nucleotide sequence contained in the ATCC
deposit
Number 97920, 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. Antibodies of the
present invention
may bind polypeptide fragments, including, fox example, fragments that
comprise or
alternatively, consist of from about amino acid residues: 1 to 51, 52 to 78,
79 to 91, 92 to
111, 112 to 134, 135 to 151, 152 to 178, 179 to 180, 181 to 208, 209 to 218,
219 to 231,
232 to 251, 252 to 271, 272 to 291, 292 to 311, 312 to 323, 324 to 361, 362 to
391, 392 to
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411 of SEQ ID N0:3. 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 10, 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.
[0196] Preferred polypeptide fragments of the present invention include a
member
selected from the group: a polypeptide comprising or alternatively, consisting
of, the TR7
receptor extracellular domain (predicted to constitute amino acid residues
from about 52 to
about 184 in SEQ ID N0:3); a polypeptide comprising or alternatively,
consisting of, both
TR7 cysteine rich domains (both of which may be found in the protein fragment
consisting
of amino acid residues from about 84 to about 179 in SEQ ID N0:3); a
polypeptide
comprising or alternatively, consisting of, the TR7 cysteine rich domain
consisting of
amino acid residues from about 84 to about 131 in SEQ ID N0:3); a polypeptide
comprising or alternatively, consisting of, the TR7 cysteine rich domain
consisting of
amino acid residues from about 132 to about 179 in SEQ ID NO:3); a polypeptide
comprising or alternatively, consisting of, the TR7 receptor transmembrane
domain
(predicted to constitute amino acid residues from about 185 to about 208 in
SEQ ID
N0:3); a polypeptide comprising or alternatively, consisting of, fragment of
the predicted
mature TR7 polypeptide, wherein the fragment has a TR7 functional activity
(e.g.,
antigenic activity or biological acitivity); a polypeptide comprising or
alternatively,
consisting of, the TR7 receptor intracellular domain (predicted to constitute
amino acid
residues from about 209 to about 411 in SEQ ~ N0:3); a polypeptide comprising
or
alternatively, consisting of, the TR7 receptor extracellular and intracellular
domains with
all or part of the transmembrane domain deleted; a polypeptide comprising, or
alternatively consisting of, the TR7 receptor death domain (predicted to
constitute amino
acid residues from about 324 to about 391 in SEQ ID NO:3); and a polypeptide
comprising, or alternatively, consisting of, one, two, three, four or more,
epitope bearing
portions of the TR7 receptor protein. In additional embodiments, the
polypeptide
fragments of the invention comprise, or alternatively, consist of, any
combination of 1, 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 TR7 receptor extracellular, transmembrane and
intracellular
domains have been predicted by computer analysis. Thus, as one of ordinary
skill would
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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.
[0197] As discussed above, it is believed that one or both of the
extracellular cysteine
rich motifs of TR7 is important for interactions between TR7 and its ligands
(e.g.,
TRAIL). Accordingly, in highly preferred embodiments, antibodies of the
present
invention bind TR7 polypeptide fragments comprising, or alternatively
consisting of,
amino acid residues 84 to 131, and/or 132 to 179 of SEQ ID N0:3. In another
highly
preferred embodiment, antibodies of the present invention bind TR7
polypeptides
comprising, or alternatively consisting of, both of the extracellular cysteine
rich motifs
(amino acid residues 84 to 179 of SEQ ID N0:3.) In another preferred
embodiment,
antibodies of the present invention bind TR7 polypeptides comprising, or
alternatively
consisting the extracellular soluble domain of TR7 (amino acid residues 52 to
184 of SEQ
ID N0:2.) In other highly preferred embodiments, the antibodies of the
invention that bind
all or a portion of the extracellular soluble domain of TR7 (e.g., one or both
cysteine rich
domains) agonize the TR7 receptor.
[0198] In other highly preferred embodiments, the antibodies of the invention
that
bind all or a portion of the extracellular soluble domain of TR7 (e.g., one or
both cysteine
rich domains) induce cell death of the cell expressing the TR7 receptor.
[0199] Antibodies of the invention may also bind fragments comprising, or
alternatively, consisting of structural or functional attributes of TR7. 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 TR7. Certain preferred regions are those disclosed in Table 5 and include,
but are not
limited to, regions of the aforementioned types identified by analysis of the
amino acid
sequence of SEQ ID N0:3, such preferred regions include; Gamier-Robson
predicted
alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman
predicted alpha-
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regions, beta-regions, and turn-regions; Kyte-Doolittle predicted hydrophilic
regions and
Hopp-Woods predicted 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.
[0200] The data representing the structural or functional attributes of TR7
set forth in
Table 5, as described above, was generated using the various modules and
algorithms of
the DNA*STAR set on default parameters. Column I represents the results of a
Garnier-
Robson analysis of alpha helical regions; Column II represents the results of
a Chou-
Fasman analysis of alpha helical regions; Column III represents the results of
a Gamier
Robson analysis of beta sheet regions; Column IV represents the results of a
Chou-
Fasman analysis of beta sheet regions; Column V represents the results of a
Gamier
Robson analysis of turn regions; Column VI represents the results of a Chou-
Fasman
analysis of turn regions; Column VII represents the results of a Gamier Robson
analysis of
coil regions; Column VIII represents a Kyte-Doolittle hydrophilicity plot;
Column IX
represents a Hopp-Woods hydrophobicity plot; Column X represents the results
of an
Eisenberg analysis of alpha amphipathic regions; Column XI represents the
results of an
Eisenberg analysis of beta amphipathic regions; Column XII represents the
results of a
Karplus-Schultz analysis of flexible regions; Column XIII represents the
Jameson-Wolf
antigenic index score; and Column XIV represents the Emini surface probability
plot.
[0201] In a preferred embodiment, the data presented in columns VIII, IX,
XIII, and
XIV of Table 5 can be used to determine regions of TR7 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 XIV 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. The columns in Table 5 present the result of different
analysees of the
TR7 protein sequence.
[0202] The above-mentioned preferred regions set out in Table 5 include, but
are not
limited to, regions of the aforementioned types identified by analysis of the
amino acid
sequence set out in SEQ ID N0:3. As set out in Table 5, such preferred regions
include
Gamier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions,
Chou-Fasman
alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic
regions,
Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible
regions,
124
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Jameson-Wolf regions of high antigenic index and Emini surface-forming
regions.
Preferably, antibodies of the present invention bind TR7 polypeptides or TR7
polypeptide
fragments and variants comprising regions of TR7 that combine several
structural features,
such as several (e.g., l, 2, 3 , or 4) of the same or different region
features set out above
and in Table 5.
125
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Table 5
ResPositionI II III IV V VI VTI VIII IX X XII XIII XIV
XI
Met1 A . . . . . . 1.11 -0.70. . 1.29 2.18
*
Glu2 A . . . . . 1.50 -0.70. . 1.63 1.69
*
Gln3 A . . . . T . 1.89 -0.73. . 2.17 2.28
*
Arg4 . . . . T T . 1.69 -0.76. . 2.91 3.71
*
Gly5 . . . . T T . 1.87 -0.87. F 3.40 2.17
*
Gln6 . . . . T T . 1.88 -0.44. F 2.76 1.93
*
Asn7 . . . . . . C 1.29 -0.34. F 1.87 1.00
*
Ala8 . . . . . . C 0.99 0.16 . F 1.08 1.02
.
Pro9 . . . . . . C 0.53 0.11 . . 0.44 0.79
*
Ala10 A . . . . . ~ 0.29 0.14 . . -0.100.48
. *
Ala11 A . . . . T . 0.40 0.24 . . 0.10 0.48
.
Ser12 A . . . . T . 0.44 -0.26. F 0.85 0.61
*
Gly13 A . . . . T . 1.14 -0.69. F 1.30 1.22
*
Ala14 A . . . . T . 1.32 -1.19. F 1.30 2.36
*
Arg15 A . . . T . . 1.57 -1.19. F 1.50 2.39
*
Lys16 . . . . T . . 1.94 -1.14. F 1.50 2.39
.
Arg17 . . . . T . . 1.90 -1.14. F 1.80 3.66
*
His18 . . . . . . C 2.03 -1.21* F 1.90 1.85
*
Gly19 . . . . . T C 2.73 -0.79* F 2.40 1.43
*
Pro20 . . . . . T C 2.62 -0.79* F 2.70 1.43
*
Gly21 . . . . . T C 1.99 -0.79* F 3.00 1.82
.
Pro22 . . . . . T C 1.99 -0.79. F 2.70 1.86
*
Arg23 . A . . . . C 1.68 -1.21* F 2.30 2.35
.
G1u24 . A B . . . . 1.43 -1.21* F 2.10 2.35
.
Ala25 . A . . T . . 1.76 -1.14* F 2.50 1.54
.
Arg26 . A . . T . . 1.89 -1.57* F 2.50 1.54
.
Gly27 . . . . T . . 1.76 -1.14* F 3.00 1.37
.
Ala28 . . . . T . C 1.43 -0.71* F 2.70 1.35
*
Arg29 . . . . . T C 1.54 -0.79* F 2.66 1.06
*
Pro30 . . . . . T C 1.28 -0.79* F 2.62 2.10
*
Gly31 . . . . . T C 0.96 -0.57* F 2.58 1.54
*
Pro32 . . . . . T C 1.34 -0.64* F 2.54 1.22
*
Arg33 . . . . . . C 1.62 -0.64* F 2.60 1.58
*
Val34 . . . . . . C 0.70 -0.59* F 2.34 2.30
*
Pro35 . . B . . . . 0.06 -0.33* F 1.58 1.23
*
Lys36 . . B B . . . -0.41-0.11* F 0.97 0.46
.
Thr37 . _ B B . . . -1.060.57 * F -0.190.52
*
Leu38 . . B B . . . -2.020.57 * . -0.600.25
*
Val39 . . B B . . . -1.760.79 . . -0.600.09
.
Leu40 A . . B . . . -2.131.29 . . -0.600.06
.
Val41 A . . B . . . -3.031.30 . . -0.600.08
.
Val42 A . . B . . . -3.531.26 . . -0.600.08
.
Ala43 A . . B . . . -3.531.30 . . -0.600.08
.
Ala44 A . . B . . . -3.491.30 . . -0.600.09
.
Val45 A . . B . . . -3.531.34 . . -0.600.10
.
Leu46 A . . B . . . -2.981.34 . . -0.600.07
.
Leu47 A . . B . . . -2.711.23 . . -0.600.09
.
Leu48 A . . B . . . -2.121.23 . . -0.600.13
.
Val49 A . . B . . . -1.830.59 . . -0.600.27
.
Ser50 A . . B . . . -1.570.29 . . -0.300.44
*
126
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Table 5 (continued)
ResPositionI II III IV V VI VII VIII IX XI XII XIII XIV
X
Ala51 A A . . . . . -1.570.10 . . -0.300.54
.
Glu52 A A . . . . . -1.640.10 . . -0.300.60
.
Ser53 A A . B . . . -1.140.14 . . -0.300.31
.
Ala54 A A . B . . . -0.290.24 . . -0.300.45
.
Leu55 A A . B . . . 0.01 0.14 . . -0.300.45
.
Ile56 A A . B . . . 0.60 0.54 . . -0.600.58
.
Thr57 A A . B . . . -0.210.16 . F -0.150.96
.
Gln58 A A . B . . . -0.500.34 . F -0.150.96
.
Gln59 A A . B . . . -0.120.16 . F 0.00 1.38
.
Asp60 . A . B T . . 0.69 -0.10 . F 1.00 1.48
.
Leu61 . A . . . . C 1.58 -0.19 * F 0.80 1.48
.
Ala62 . A . . . . C 2.00 -0.19 * F 0.80 1.48
.
Pro63 . A . . . . C 1.41 -0.59 * F 1.10 1.73
.
Gln64 . A . . T . . 0.82 -0.09 * F 1.00 2.13
.
Gln65 A A . . . . . 0.61 -0.27 * F 0.60 2.13
.
Arg66 A A . . . . . 1.42 -0.34 * F 0.60 2.13
.
Ala67 A A . . . . . 2.01 -0.37 * F 0.94 2.13
.
Ala68 A A . . . . . 2.27 -0.37 * F 1.28 2.13
*
Pro69 A A . . . . . 2.38 -0.77 * F 1.92 2.17
*
Gln70 . A . . T . . 2.08 -0.77 . F 2.66 4.21
*
Gln71 . . . . T T . 1.67 -0.89 * F 3.40 5.58
*
Lys72 . . . . T T . 2.04 -1.00 . F 3.06 4.84
.
Arg73 . . . . T T . 2.33 -1.00 . F 2.97 4.32
.
Ser74 . . . . . T C 2.54 -1.01 . F 2.68 3.34
.
Ser75 . . . . . T C 2.20 -1.41 . F 2.59 2.89
.
Pro76 . . . . T T . 1.39 -0.99 . F 2.70 1.46
.
Ser77. . . . . T T . 0.68 -0.30 . F 2.50 0.90
.
Glu78 . . . . T T . 0.36 -0.11 * F 2.25 0.36
.
Gly79 . . . . T . . 0.44 -0.07 . F 1.80 0.36
.
Leu80 . . . . T . . 0.40 -0.07 . F 1.55 0.42
.
Cys81 . . . . . . C 0.58 -0.03 . . 0.95 0.24
.
Pro82 . . . . . T C 0.84 0.47 . F 0.15 0.33
*
Pro83 . . . . T T . -0.040.54 . F 0.35 0.54
*
Gly84 . . . . T T . 0.00 0.54 . . 0.20 0.70
*
His85 . . . . . T C 0.81 0.36 . . 0.30 0.61
*
His86 . . . . . . C 1.48 -0.07 . . 0.70 0.68
*
Ile87 . . . . . . C 1.34 -0.50 * . 1.19 1.15
*
Ser88 . . . . . . C 1.67 -0.50 * F 1.53 0.84
*
Glu89 . . . . T . . 2.01 -1.00 * F 2.52 1.21
*
Asp90 . . . . T . . 1.38 -1.50 * F 2.86 2.88
*
Gly91 . . . . T T . 0.52 -1.61 * F 3.40 1.15
*
Arg92 . . . . T T . 1.11 -1.31 * F 2.91 0.47
*
Asp93 . . . . T T 0.74 -0.93 * F 2.57 0.37
.
Cys94 . . . . T T . 0.79 -0.36 * . 1.78 0.20
.
Ile95 . . . . T . . 0.54 -0.79 * . 1.54 0.21
.
Ser96 . . . . T . . 0.54 -0.03 * . 1.18 0.19
.
Cys97 . . . . T T . 0.43 0.40 * . 0.76 0.36
..
Lys98 . . . . T T . 0.43 0.23 . . 1.34 0.88
.
Tyr99 . . . . T T . 0.86 -0.46 * F 2.52 1.10
.
Gly100 . . . . T T . 1.44 -0.09 * F 2.80 3.22
.
127
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Table 5
ResPositionI II III IV V VI VII VIII IX XI XII XIII XIV
X
Gln101 . . . . T T . 1.43 -0.27 . F 2.52 2.16
*
Asp102 . . . . T T . 2.07 0.21 * F 1.64 1.99
*
Tyr103 . . . . T T . 1.73 -0.04 * F 1.96 2.73
*
Ser104 . . . ' . T T . 1.98 0.44 . F 0.78 1.66
*
Thr105 . . . . T . . 2.32 0.44 . F 0.30 1.60
*
His106 . . . . T . . 1.51 0.44 . . 0.15 1.70
*
Trp107 . . . . T T . 0.70 0.37 . . 0.65 1.05
*
Asn108 . . . . T T . 0.24 0.67 . . 0.20 0.60
.
Asp109 . . . . T T . -0.12 0.97 . . 0.20 0.38
*
Leu110 A . . . . T . -0.62 1.04 * . -0.200.19
*
Leu111 . . . B T . . -0.48 0.81 * . -0.200.10
*
Phe112 . . . B T . . -0.86 0.41 * . -0.200.12
*
Cys113 . . . B T . . -1.17 0.99 * . -0.200.08
*
Leu114 . . . B T . . -1.06 0.79 * . -0.200.13
.
Arg115 . . . B T . . -0.91 0.10 * . 0.10 0.30
.
Cys116 . . . B T . . -0.10 -0.11 . . 0.70 0.30
.
Thr117 . . . B T . . 0.30 -0.69 * . 1.00 0.61
.
Arg118 . . . B T . . 0.62 -0.99 . F 1.49 0.42
.
Cys119 . . . . T T . 1.43 -0.56 . F 2.23 0.77
*
Asp120 . . . . T T . 0.47 -1.13 . F 2.57 0.92
*
Ser121 . . . . T T . 1.13 -0.97 * F 2.91 0.35
.
Gly122 . . . . T T . 0.63 -0.97 * F 3.40 1.13
.
Glu123 . A . . T . . 0.22 -0.86 * F 2.51 0.56
.
Val124 A A . . . . . 0.68 -0.47 * F 1.47 0.56
.
Glu125 . A . . T . . 0.01 -0.43 * . 1.38 0.87
.
Leu126 . A . . T . . 0.00 -0.29 * . 1.04 0.27
.
Ser127 . . . . . T C 0.03 0.20 * F 0.45 0.52
.
Pro128 . . . . T T . -0.28 0.04 * F 0.93 0.44
.
Cys129 . . . . T T . 0.69 0.53 * F 0.91 0.77
.
Thr130 . . . . T T . 0.69 -0.16 * F 2.24 1.12
.
Thr131 . . . . T . . 1.19 -0.14 * F 2.32 1.16
.
Thr132 . . . . T T . 0.63 -0.09 * F 2.80 3.13
.
Arg133 . . . . T T . 0.18 -0.01 . F 2.52 1.61
.
Asn134 . . . . T T . 0.84 0.07 . F 1.49 0.60
.
Thr135 . . . . T T . 0.49 -0.01 . F 1.51 0.72
.
Val136 . . . . T . C 0.80 0.07 . . 0.58 0.20
*
Cys137 . A . . T . . 1.11 0.07 . . 0.10 0.21
*
Gln138 . A B . . . . 0.66 -0.33 . . 0.30 0.25
*
Cys139 . A . . T . . 0.34 -0.39 . . 0.70 0.34
.
Glu140 A A . . . . . -0.04 -0.54 * F 0.75 0.91
*
Glu141 A A . . . . , 0.92 -0.33 * F 0.45 0.46
*
Gly142 . A . . T . . 1.59 -0.73 * F 1.30 1.67
.
Thr143 A A . . . . . 1.59 -1.30 * F 0.90 1.67
.
Phe144 A A . . . . . 2.26 -1.30 * F 0.90 1.67
.
Arg145 A A . . . . . 1.96 -1.30 * F 0.90 2.81
.
Glu146 A A . . . . . 1.74 -1.34 * F 0.90 2.61
.
Glu147 A A . . . . . 2.09 -1.40 * F 0.90 4.66
.
Asp148 A A . . . . . 1.80 -2.19 * F 0.90 4.12
.
Ser149 A . . . . T . 1.83 -1.57 * F 1.30 2.35
.
Pro150 A . . . . T . 1.83 -1.00 . F 1.15 0.73
.
128
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Table 5 (continued)
ResPositionI II TII IV V VI VII VIII IX X XII XIIIXIV
XT
G1u151 A . . . . T . 1.88 -1.00* F 1.150.85
.
Met152 A . . . . T . 1.21 -1.00* . 1.491.28
*
Cys153 A . . . . T . 1.32 -0.81* . 1.680.44
*
Arg154 A . . . . T . 1.31 -1.24* . 2.020.50
.
Lys155 . . . . T T . 1.18 -0.76* F 2.910.73
*
Cys156 . . . . T T . 0.51 -0.94* F 3.401.35
.
Arg157 . . . . T . . 0.90 -0.94* F 2.710.37
.
Thr158 . . . . T . . 1.68 -0.51* F 2.370.28
.
Gly159 . . . . T . . 1.22 -0.51* F 2.431.04
.
Cys160 . . . . . T C 0.58 -0.66. F 2.190.53
*
Pro161 . . . . T T . 0.39 -0.04. F 2.000.36
*
Arg162 . . . . T T . 0.32 0.11. F 1.650.27
*
Gly163 . . . . T T . -0.22 -0.31* . 2.501.01
*
Met164 . . B B . . . -0.22 -0.24* . 1.300.48
*
Va1165 . . B B . . . 0.44 -0.24* . 1.300.24
*
Lys166 . . B B . . . -0.01 -0.24* . 1.300.41
*
Val167 . . B . . T . -0.43 -0.10* F 1.850.22
*
Gly168 . . . . T T . -0.30 -0.23. F 2.250.44
.
Asp169 . . . . T T . 0.01 -0.44. F 2.500.34
.
Cys170 . . . . T T . 0.57 0.47. F 1.350.48
*
Thr171 . . . . . T C 0.52 0.21. F 1.200.65
*
Pro172 . . . . T T . 0.49 -0.21. F 1.750.65
*
Trp173 . . . . T T . 0.83 0.47. F 0.600.84
*
Ser174 A . . . . T . 0.17 -0.10. F 1.001.01
*
Asp175 A A . . . . . -0.02 -0.01. F 0.450.35
.
Ile176 A A . . . . . 0.26 0.20* . -0.300.25
*
Glu177 A A . . . . . 0.51 -0.21* . 0.300.25
.
Cys178 A A . . . . . 0.80 -0.60* . 0.600.30
.
Val179 A A . . . . . 0.80 -0.60* . 0.600.74
*
His180 A A . . . . . 0.46 -0.90. . 0.600.58
*
Lys181 A A . . . . . 0.46 -0.47* F 0.601.06
.
Glu182 A . . . . T . -0.43 -0.36* F 1.001.00
.
Ser183 A . . . . T . -0.66 -0.31. F 0.850.52
.
Gly184 A . . . T T . -0.14 -0.13. F 1.250.18
.
Ile185 A . . . . T . -0.97 0.30. . 0.100.10
.
I1e186 . . B B . . . -1.32 0.94* . -0.600.06
Ile187 . . B B . . . -2.18 1.04. . -0.600.08
.
Gly188 . . B B . . . -2.47 1.26. . -0.600.09
*
Val189 . . B B . . . -2.71 1.07. . -0.600.13
.
Thr190 A . . B . . . -2.68 0.89. . -0.600.18
*
Val191 A . . B . . . -2.64 0.84. . -0.600.14
.
Ala192 A . . B . . . -2.57 1.06. . -0.600.14
*
Ala193 A . . B . . . -3.11 1.10. . -0.600.08
~ .
Val194 A . . B . . . -3.11 1.30. . -0.600.07
.
Val195 A . . B . . . -3.39 1.30. . -0.600.05
.
Leu196 A . . B . . . -3.39 1.30. . -0.600.05
.
Ile197 A . . B . . . -3.50 1.44. . -0.600.05
.
Val198 A . . B . . . -3.77 1.59. . -0.600.06
.
Ala199 A . . B . . . -3.58 1.59. . -0.600.06
.
Val200 A . . B . . . -2.68 1.47. . -0.600.04
.
129
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Table 5 (continued)
ResPositionI II III IV V VI VII VIII IX X XII XIII XIV
XI
Phe201 A . . B . . . -2.170.79 . . -0.600.12
.
Val202 A . . B . . . -2.090.53 . . -0.600.16
.
Cys203 A . . . . T . -2.040.71 . . -0.200.17
.
Lys204 A . . . . T . -1.740.76 . . -0.200.17
.
Ser205 A . . . . T . -0.840.89 . . -0.200.24
.
Leu206 A . . . . T . -0.100.24 . . 0.10 0.88
.
Leu207 A A . . . . . -0.10-0.33. . 0.30 0.88
.
Trp208 A A . . . . . -0.240.31 . . -0.300.49
.
Lys209 A A . . . . . -0.500.61 . . -0.600.49
.
Lys2l0 A A . . . . . -0.440.36 * . -0.300.91
.
Val211 A A . . . . . -0.440.43 * . -0.451.36
*
Leu212 . A B . . . . 0.41 0.20 * . -0.300.56
*
Pro213 . A B . . . . 0.36 0.20 * . -0.300.56
.
Tyr214 . . . B T . . -0.580.63 * . -0.200.75
.
Leu215 . . . B T . . -1.290.67 * . -0.200.64
*
Lys216 . . . B T . . -0.730.56 * . -0.200.22
.
Gly217 . . B B . . . -0.270.51 * . -0.600.19
.
Ile218 . . B B . . . -0.400.19 * . -0.300.23
.
Cys219 . . B . . T . -0.50-0.07* . 0.70 0.11
.
Ser220 . . . . T T . -0.030.36 . F 0.65 0.11
*
Gly221 . . . . T T . -0.080.36 . F 0.65 0.16
.
Gly222 . . . . T T . 0.06 -0.33. F 1.25 0.49
.
Gly223 . . . . . . C 0.94 -0.47. F 0.85 0.57
.
Gly224 . . . . . . C 1.72 -0.86* F 1.15 0.99
.
Asp225 . . . . . T C 1.17 -1.29. F 1.50 1.97
*
Pro226 . . . . . T C 1.51 -1.07* F 1.84 1.47
.
Glu227 . . B . . T . 1.97 -1.50* F 1.98 2.49
.
Arg228 . . B . . T . 2.01 -1.93* F 2.32 2.92
.
Val229 . . . . T . . 2.06 -1.54* F 2.86 2.53
.
Asp230 . . . . T T . 2.06 -1.59* F 3.40 1.96
.
Arg231 . . . . T T . 2.38 -1.19* F 3.06 1.73
*
Ser232 . . . . T T . 2.17 -1.19* F 2.72 4.57
.
Ser233 . . . . T T . 1.71 -1.40* F 2.72 4.23
*
Gln234 . . . . . . C 1.98 -0.97* F 2.32 2.14
*
Arg235 . . . . . T C 1.98 -0.47* F 2.22 1.61
*
Pro236 . . . . . T C 1.87 -0.86* F 2.86 2.08
*
Gly237 . . . . T T . 2.17 -1.24. F 3.40 2.01
*
Ala238 . . . . . T C 1.61 -1.24. F 2.86 1.65
*
G1u239 A'. . . . . . 0.80 -0.60. F 1.97 0.79
*
Asp240 A . . . . . . 0.69 -0.34. F 1.33 0.66
*
Asn241 A . . . . . . 0.90 -0.37* . 0.99 1.05
.
Val242 A . . . . . . 0.36 -0.87* . 0.95 1.05
.
Leu243 A . . . . . . 0.09 -0.19* . 0.50 0.44
.
Asn244 A . . B . . . -0.210.46 * . -0.600.20
.
Glu245 A . . B . . . -1.100.44 * . -0.600.37
.
Ile246 A . . B . . . -1.910.49 * . -0.600.31
.
Val247 A . . B . . . -1.060.49 * . -0.600.16
.
Ser248 . . B B . . . -0.460.49 * . -0.600.16
~ .
Ile249 . . B B . . . -0.770.91 * . -0.600.35
.
Leu250 . . . B . . C -0.770.71 . . -0.400.69
.
130
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Table 5
(continued)
Res Position I II III IV V VI VII VIII IX XI XII XIIIXIV
X
Gln 251 . . . . . T C -0.730.47 . F 0.150.89
.
Pro 252 . . . . . T C -0.090.73 . F 0.150.94
.
Thr 253 . . . . . T C 0.21 0.47 . F 0.301.76
.
Gln 254 . . . . . T C 1.10 -0.21 . F 1.201.76
.
Val 255 . A . . . . C 1.91 -0.21 . F 0.801.97
.
Pro 256 . A . . . . C 1.31 -0.64 . F 1.102.37
.
Glu 257 A A . . . . . 1.52 -0.51 * F 0.901.35
.
Gln 258 A A . . . . . 0.98 -0.91 * F 0.903.16
.
Glu 259 A A . . . . . 0.98 -0.91 * F 0.901.51
.
Met 260 A A . . . . . 1.83 -0.94 * F 0.901.51
.
Glu 261 A A . . . . . 1.83 -0.94 * . 0.751.51
.
Val 262 A A . . . . . 1.24 -0.91 * F 0.901.35
.
Gln 263 A A . . . . . 1.24 -0.41 * F 0.601.38
.
Glu 264 A A . . . . . 1.03 -1.03 * F 0.901.38
.
Pro 265 A A . . . . . 1.32 -0.60 * F 1.182.88
.
Ala 266 A A . . . . . 0.98 -0.76 * F 1.462.40
.
Glu 267 A . . . . T . 0.98 -0.73 * F 2.141.37
.
Pro 268 A . . . . T . 0.98 -0.09 . F 1.970.66
.
Thr 269 . . . . T T . 0.38 -0.11 . F 2.801.05
.
Gly 270 A . . . . T . -0.220.00 . F 1.370.60
.
Val 271 A . . . . . . 0.07 0.69 . . 0.440.32
.
Asn 272 . . B . . . . -0.140.64 . . 0.160.30
.
Met 273 . . B . . . . -0.280.59 . . 0.180.46
.
Leu 274 . . . . . . C 0.03 0.59 . . 0.400.62
.
Ser 275 . . . . . T C 0.08 -0.06 . F 1.950.66
.
Pro 276 . . . . . T C 0.93 -0.07 . F 2.250.90
.
Gly 277 . . . . . T C 0.90 -0.69 . F 3.001.89
.
Glu 278 A . . . . T . 0.69 -0.87 . F 2.501.92
.
Ser 279 A A . . . . . 0.69 -0.57 . F 1.801.02
.
Glu 280 A A . . . . . 0.99 -0.31 . F 1.050.85
.
His 281 A A . . . . . 0.99 -0.74 . F 1.050.85
.
Leu 282 A A . . . . . 0.74 -0.31 . . 0.300.98
.
Leu 283 A A . . . . . 0.74 -0.20 . . 0.300.57
.
Glu 284 A A . . . . . 0.46 -0.20 . F 0.450.73
.
Pro 285 A A . . . . . 0.46 -0.20 . F 0.450.89
.
Ala 286 A A . . . . . 0.60 -0.89 . F 0.901.88
.
Glu 287 A A . . . . . 1.11 -1.57 . F 0.902.13
.
Ala 288 A A . . . . . 1.92 -1.19 . F 0.901.84
.
Glu 289 A A . . . . . 2.03 -1.21 . F 0.903.16
*
Arg 290 A A . . . . . 2.36 -1.71 . F 0.903.57
*
Ser 291 A . . . . T . 3.06 -1.71 . F 1.306.92
*
Gln 292 A . . . . T . 2.24 -2.21 . F 1.307.83
*
Arg 293 A . . . . T . 2.02 -1.53 . F 1.303.30
.
Arg 294 A . . . . T . 1.17 -0.84 . F 1.302.03
.
Arg 295 . . . B T . . 0.84 -0.59 * F 1.150.87
.
Leu 296 . . B B . . . 0.56 -0.56 * . 0.600.69
.
Leu 297 . . B B . . . 0.56 -0.06 * . 0.300.35
.
Val 298 . . . B . . C 0.44 0.34 * . 0.200.29
*
Pro 299 . . . . . T C -0.010.34 . . 0.900.61
*
Ala 300 . . . . . T C -0.120.09 * F 1.350.73
*
131
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Table 5 (continued)
ResPositionI II III IV V VI VII VIII IX XXI XII XIII XIV
Asn301 . . . . . T C 0.48 -0.60.. F 2.70 1.65
Glu302 . . . . . T C 0.98 -0.81.. F 3.00 1.65
Gly303 . . . . . . C 1.83 -0.76.. F 2.50 2.35
Asp304 . . . . . T C 1.73 -1.26.. F 2.40 2.54
Pro305 . . . . . T C 1.51 -1.17.* F 2.10 2.11
Thr306 A . . . . T . 1.62 -0.49.* F 1.30 1.76
Glu307 A . . . . T . 1.62 -0.91** F 1.30 2.07
Thr308 A . . B . . . 1.30 -0.51** F 0.90 2.31
Leu309 A . . B . . . 0.60 -0.37** F 0.45 0.86
Arg310 A . . B . . . 0.81 -0.07** . 0.30 0.43
Gln311 A . . B . . . 1.12 -0.07** . 0.30 0.50
Cys312 A . . . . T . 0.42 -0.56** . 2.15 1.01
Phe313 A . . . . T . 0.14 -0.46** . 0.70 0.45
Asp314 . . . . T T . 0.96 0.04 ** . 0.50 0.26
Asp315 A . . . . T . 0.03 -0.36** . 0.70 0.81
Phe316 A A . . . . . -0.82-0.24*. . 0.30 0.77
Ala317 A A . . . . . -0.37-0.39*. . 0.30 0.34
Asp318 A A . . . . . -0.370.04 ** . -0.300.32
Leu319 A A . . . . . -0.370.83 .. . -0.600.32
Val320 . A . . . . C -0.670.04 .. . -0.100.52
Pro321 . A . . . . C -0.26-0.07.. . 0.50 0.42
Phe322 . . . . T T . 0.33 0.84 .. . 0.20 0.54
Asp323 A . . . . T . 0.12 0.16 .. . 0.25 1.25
Ser324 A . . . . T . 0.12 -0.06.. F 1.00 1.25
Trp325 A . . . . T . 0.38 0.20 ** F 0.40 1.19
Glu326 A A . . . . . 0.70 0.03 *. F -0.150.71
Pro327 A A . . . . . 1.44 0.03 *. . -0.151.03
Leu328 A A . . . . . 0.63 -0.36*. . 0.45 1.96
Met329 A A . . . . . 0.59 -0.59*. . 0.60 0.93
Arg330 A A . . . . . 0.07 -0.16*. . 0.30 0.60
Lys331 A A . . . . . -0.530.10 *. . -0.300.60
Leu332 A A . . . . . -0.320.03 *. . -0.300.60
Gly333 A A . . . . . 0.49 -0.59*. . 0.60 0.51
Leu334 A A . . . . . 1.09 -0.19*. . 0.30 0.41
Met335 A A . . . . . 0.09 -0.19** . 0.30 0.86
Asp336 A A . . . . . 0.09 -0.19.* F 0.45 0.61
Asn337 A A . . . . . 0.04 -0.61** F 0.90 1.48
G1u338 A A . . . . . -0.20-0.66** F 0.90 1.11
Ile339 A A . . . . . 0.66 -0.77** F 0..750.67
Lys340 A A . . . . . 0.67 -0.77.* F 0.75 0.83
Val341 A A . . . . . 0.67 -0.67.* . 0.60 0.49
Ala342 A A . . . . . 0.08 -0.67.. . 0.75 1.20
Lys343 A A . . . . . -0.51-0.86.* . 0.60 0.61
Ala344 A A . . . . . 0.03 -0.36.* . 0.30 0.83
Glu345 A A . . . . . -0.04-0.57*. . 0.60 0.81
Ala346 A A . . . . . 0.92 -0.57*. . 0.60 0.55
Ala347 A A . . . . . 1.51 -0.57.* . 0.75 1.07
Gly348 A . . . . . . 1.16 -1.07.* . 0.95 1.03
His349 A . . . . T . 0.93 -0.59.. . 1.15 1.47
Arg350 A . . . . T . 0.69 -0.40.. F 1.00 1.20
132
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Table 5 (continued)
ResPositionI II III IV V VI VII VIII IX X XII XIII XIV
XI
Asp351 A . . . . T . 0.97 -0.14. F 1.00 1.90
.
Thr352 A . . . . T . 0.96 -0.09. F 1.00 2.02
.
Leu353 A . . B . . . 0.49 0.03 . . -0:151.02
.
Tyr354 A . . B . . . -0.37 0.71 . . -0.600.50
*
Thr355 A . . B . . . -0.43 1.40 . . -0.600.24
*
Met356 A . . B . . . -0.72 0.91 * . -0.600.59
.
Leu357 A . . B . . . -1.27 1.14 * . -0.600.40
.
Ile358 A . . B . . . -0.46 1.03 * . -0.600.20
*
Lys359 A . . B . . . -0.17 0.94 * . -0.600.33
*
Trp360 A . . B . . . -0.17 0.33 * . 0.00 0.81
*
Val361 A . . B . . . 0.09 0.13 * . 0.45 1.66
*
Asn362 . . . . . T C 1.01 -0.13* F 1.95 0.82
.
Lys363 . . . . . T C 1.90 -0.13* F 2.40 1.53
*
Thr364 . . . . . T C 1.27 -1.04* F 3.00 3.44
.
Gly365 . . . . . T C 1.26 -1.19* F 2.70 2.16
.
Arg366 . A . . T . . 1.26 -1.20* F 2.20 1.45
.
Asp367 . A . . . . C 1.22 -0.56* F 1.55 0.75
.
Ala368 A A . . . . . 0.87 -0.54. F 1.20 1.03
.
Ser369 A A . . . . . 0.37 -0.49. . 0.30 0.76
.
Val370 A A . . . . . -0.10 0.20 . . -0.300.37
*
His371 A A . . . . . -0.21 0.89 . . -0.600.30
*
Thr372 A A . . . . . -0.80 0.39 * . -0.300.38
*
Leu373 A A . . . . . -1.02 0.50 * . -0.600.52
*
Leu374 A A . . . . . -0.72 0.54 * . -0.600.31
.
Asp375 A A . . . . . -0.18 0.04 * . -0.300.38
.
Ala376 A A . . . . . -0.96 0.04 * . -0.300.66
.
Leu377 A A . . . . . -0.99 0.04 * . -0.300.66
.
Glu378 A A . . . . . -0.18 -0.21* . 0.30 0.39
.
Thr379 A A . . . . . 0.74 -0.21* F 0.45 0.67
*
Leu380 A A . . . . . -0.07 -0.71* F 0.90 1.59
.
Gly381 A A . . . . . -0.07 -0.71* F 0.75 0.76
.
Glu382 A A . . . . . 0.79 -0.21* F 0.45 0.53
.
Arg383 A A . . . . . 0.79 -0.70* F 0.90 1.28
.
Leu384 A A . . . . . 1.14 -0.99* F 0.90 2.24
*
Ala385 A A . . . . . 1.07 -1.41* F 0.90 2.59
*
Lys386 A A . . . . . 1.41 -0.73* F 0.75 0.93
.
Gln387 A A . . . . . 1.41 -0.73* F 0.90 1.95
*
Lys388 A A . . . . . 1.27 -1.41* F 0.90 3.22
*
Ile389 A A . . . . . 1.27 -1.41. F 0.90 2.19
*
Glu390 A A . . . . . 1.04 -0.73* F 0.90 1.04
*
Asp391 A A . . . . . 0.70 -0.44. F 0.45 0.43
*
His392 A A . . . . . 0.40 -0.06* . 0.30 0.82
*
Leu393 A A . . . . . 0.01 -0.36* . 0.30 0.64
*
Leu394 A A . . . . . 0.94 0.07 * F -0.150.38
*
Ser395 A . . . . T . 0.24 0.07 * F 0.25 0.55
*
Ser396 A . . . . T . -0.36 0.36 * F 0.25 0_58
*
Gly397 . . . T T . -0.57 0.29 . F 0.65 0.70
.
Lys398 A ~. . . . T . -0.57 0.36 . F 0.25 0.82
.
Phe399 A A . . . . . 0.24 0.66 . . -0.600.50
.
Met400 . A B . . . . 0.20 0.27 . . -0.300.88
*
133
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Table 5 (continued)
ResPositionI II III IV V VI VII VIII IX XI XII XIII XIV
X
Tyr401 . A B . . . . 0.50 0.27 * . -0.300.44
.
Leu402 A A . . . . . 0.26 0.67 * . -0.600.81
.
Glu403 A A . . . . . 0.21 0.39 * . -0.300.82
.
Gly404 A . . . . . . 0.61 -0.23 * F 0.65 0.88
.
Asn405 A . . . . T . 0.62 -0.60 * F 1.30 1.43
.
Ala406 A . . . . T . 0.27 -0.79 * F 1.15 0.83
.
Asp407 A . . . . T . 0.78 -0.17 * F 0.85 0.83
.
Ser408 A . . . . T . 0.39 -0.21 * F 0.85 0.69
.
Ala409 A . . . . . . 0.34 -0.19 * . 0.50 0.88
.
Met410 A . . . . . . -0.04 -0.26 . . 0.50 0.67
.
Ser411 A . . . . . . 0.16 0.17 . . -0.100.64
.
134
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[0203] In another aspect, the invention provides an antibody that binds a
peptide or
polypeptide comprising, or alternatively, consisting of, one, two, three,
four, five or more,
epitope-bearing portions of a TR7. 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).
[0204] 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," Sciezzce 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.
[0205] Antigenic epitope-bearing peptides and polypeptides are therefore
useful to
raise antibodies, including monoclonal antibodies, that bind to a TR7
polypeptide. See,
for instance, Wilson et al., Cell 37:767-778 (1984) at 777. Antigenic epitope-
bearing
peptides and polypeptides 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 SEQ ID N0:3.
[0206] Antibodies of the invention may bind one or more antigenic TR7
polypeptides
or peptides including, but not limited to: a polypeptide comprising, or
alternatively
consisting of, amino acid residues from about 62 to about 110 of SEQ ID N0:3,
about 119
to about 164 of 5EQ ID N0:3, about 224 to about 271 of SEQ ID N0:3, about 275
to
about 370 of SEQ ID N0:3, about 69 to about 80 of SEQ 1D N0:3, about 88 to
about 95
of SEQ ID N0:3, about 99 to about 103 of SEQ ID N0:3, about 119 to about 123
of SEQ
ID NO:3, about 130 to about 135 of SEQ ID N0:3, about 152 to about 163 of SEQ
ID
N0:3, about 226 to about 238 of SEQ ID NO:3, about 275 to about 279 of SEQ ID
N0:3,
135
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about 301 to about 305 of SEQ ID N0:3, and/or about 362 to about 367 of SEQ ID
N0:3.
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 TR7 receptor protein.
[0207] Epitope-bearing TR7 peptides and polypeptides 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).
[0208] As one of skill in the art will appreciate, TR7 receptor polypeptides
and the
epitope-bearing fragments thereof described herein (e.g., corresponding to a
portion of the
extracellular domain, such as, for example, amino acid residues 52 to 184 of
SEQ ID
N0:3 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 irc 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 TR7 protein or protein
fragment
alone (Fountoulakis et al., J. Biocl2em. 270:3958-3964 (1995)). TR7 fusion
proteins may
be used as an immunogen to elicit anti-TR7 antibodies. Thus, antibodies of the
invention
may bind fusion proteins that comprise all or a portion of a TRAIL receptor
polypeptide
such as TR7.
[0209] 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. Antibodies of the
present invention
may also bind such modified TR7 polypeptides or TR7 polypeptide fragments or
variants.
136
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[0210] For instance, 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 or loss of the ability to be
bound by a
specific antibody. 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 TR7 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 TR7 (preferably antibodies that bind
specifically to TR7)
will retained irrespective of the size or location of the deletion. In fact,
polypeptides
composed of as few as six TR7 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.
[0211] 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 TR7 ligand) may still be retained. For example, the ability of
shortened
TR7 polypeptides to induce and/or bind to antibodies which recognize the
complete or
mature forms of the polypeptides generally will be retained when less than the
majority of
the residues of the complete or mature polypeptide are removed from the N-
terminus.
Whether a particular polypeptide lacking N-terminal residues of a complete
polypeptide
retains such immunologic activities can readily be determined by routine
methods
described herein and otherwise known in the art. It is not unlikely that a TR7
polypeptide
with a large number of deleted N-terminal amino acid residues may retain some
biological
or immunogenic activities.
[0212] Accordingly, the present invention further provides antibodies that
bind
polypeptides having one or more residues deleted from the amino terminus of
the TR7
amino acid sequence shown in SEQ ID NO:3 up to the alanine residue at position
number
406 and polynucleotides encoding such polypeptides. In particular, the present
invention
provides antibodies that bind polypeptides comprising the amino acid sequence
of residues
n5-411 of SEQ ID N0:3 where n5 is an integer from 2 to 406 corresponding to
the position
of the amino acid residue in SEQ ID N0:3.
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[0213] More in particular, the invention provides antibodies that bind
polypeptides
comprising, or alternatively consisting of, the amino acid sequence of
residues: E-2 to 5-
411; Q-3 to S-411; R-4 to S-411; G-5 to S-411; Q-6 to S-411; N-7 to S-411; A-8
to S-411;
P-9 to S-411; A-10 to S-41I; A-11 to S-411; S-12 to S-411; G-13 to S-411; A-14
to S-411;
R-15 to S-411; K-16 to S-41I; R-17 to S-4I l; H-28 to S-411; G-19 to S-4I1; P-
20 to 5-
411; G-21 to S-411; P-22 to S-411; R-23 to S-411; E-24 to S-411; A-25 to S-
411; R-26 to
S-411; G-27 to S-4I1; A-28 to S-411; R-29 to S-411; P-30 to S-4I1; G-31 to S-
411; P-32
to S-411; R-33 to S-411; V-34 to S-411; P-35 to S-411; K-36 to S-411; T-37 to
S-411; L-
38 to S-411; V-39 to S-411; L-40 to S-411; V-41 to S-411; V-42 to S-4I1; A-43
to S-411;
A-44 to S-411; V-45 to S-4I I; L-46 to S-41I; L-47 to S-411; L-48 to S-411; V-
49 to 5-
411; S-50 to S-411; A-51 to S-411; E-52 to S-411; S-53 to S-411; A-54 to S-
411; L-55 to
S-4I1; I-56 to S-4I I; T-57 to S-411; Q-58 to S-411; Q-59 to S-411; D-60 to S-
411; L-61
to S-411; A-62 to S-411; P-63 to S-4I1; Q-64 to S-411; Q-65 to S-411; R-66 to
S-411; A-
67 to S-411; A-68 to S-411; P-69 to S-411; Q-70 to S-411; Q-71 to S-411; K-72
to S-411;
R-73 to S-4I l; S-74 to S-411; S-75 to S-411; P-76 to S-411; S-77 to S-411; E-
78 to S-411;
G-79 to S-4I I; L-80 to S-411; C-81 to S-411; P-82 to S-411; P-83 to S-411; G-
84 to 5-
411; H-85 to S-411; H-86 to S-411; I-87 to S-411; S-88 to S-411; E-89 to S-
411; D-90 to
S-411; G-91 to S-411; R-92 to S-411; D-93 to S-411; C-94 to S-411; I-95 to S-
411; S-96
to S-411; C-97 to S-41I; K-98 to S-411; Y-99 to S-4I1; G-100 to S-411; Q-101
to S-41I;
D-102 to S-411; Y-103 to S-411; S-104 to S-411; T-105 to S-411; H-106 to S-
411; W-107
to S-411; N-108 to S-411; D-109 to S-4I1; L-l I0 to S-411; L-111 to S-411; F-
112 to S-
411; C-113 to S-411; L-114 to S-411; R-115 to S-411; C-116 to S-411; T-117 to
S-411; 8-
118 to S-411; C-119 to S-411; D-120 to S-411; S-121 to S-411; G-122 to S-411;
E-123 to
S-411; V-124 to S-411; E-125 to S-411; L-126 to S-411; S-127 to S-411; P-128
to S-411;
C-129 to S-411; T-130 to S-411; T-131 to S-411; T-132 to S-41I; R-133 to S-
411; N-134
to S-411; T-I35 to S-411; V-136 to S-411; C-137 to S-411; Q-138 to S-411; C-
139 to 5-
411; E-140 to S-411; E-141 to S-411; G-142 to S-411; T-143 to S-411; F-144 to
S-411; 8-
145 to S-411; E-146 to S-411; E-147 to S-411; D-148 to S-411; S-149 to S-411;
P-150 to
S-411; E-151 to S-411; M-152 to S-411; C-153 to S-411; R-154 to S-411; K-155
to S-411;
C-156 to S-411; R-157 to S-411; T-158 to S-411; G-I59 to S-411; C-160 to S-
411; P-161
to S-411; R-162 to S-411; G-163 to S-411; M-164 to S-411; V-165 to S-411; K-
166 to 5-
411; V-167 to S-411; G-168 to S-411; D-169 to S-411; C-170 to S-411; T-171 to
S-411;
P-172 to S-411; W-173 to S-411; S-174 to S-411; D-175 to S-411; I-176 to S-
411; E-177
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to S-411; C-178 to S-411; V-179 to S-411; H-180 to S-411; K-181 to S-411; E-
182 to 5-
411; S-183 to S-411; G-184 to S-411; I-185 to S-411; I-186 to S-411; I-187 to
S-411; 6-
188 to S-411; V-189 to S-411; T-190 to S-411; V-191 to S-411; A-192 to S-411;
A-193 to
S-411; V-194 to S-411; V-195 to S-411; L-196 to S-411; I-197 to S-411; V-198
to S-411;
A-199 to S-411; V-200 to S-411; F-201 to S-411; V-202 to S-411; C-203 to S-
411; K-204
to S-411; S-205 to S-411; L-206 to S-411; L-207 to S-411; W-208 to S-411; K-
209 to S-
411; K-210 to S-41 l; V-211 to S-411; L-212 to S-41 l; P-213 to S-411; Y-214
to S-411;
L-215 to S-411; K-216 to S-411; G-217 to S-411; I-218 to S-411; C-219 to S-
411; S-220
to S-411; G-221 to S-411; G-222 to S-411; G-223 to S-411; G-224 to S-411; D-
225 to 5-
411; P-226 to S-411; E-227 to S-411; R-228 to S-411; V-229 to S-411; D-230 to
S-411;
R-231 to S-411; S-232 to S-411; S-233 to S-411; Q-234 to S-411; R-235 to S-
411; P-236
to S-411; G-237 to S-411; A-238 to S-411; E-239 to S-411; D-240 to S-411; N-
241 to 5-
411; V-242 to S-411; L-243 to S-411; N-244 to S-411; E-245 to S-411; I-246 to
S-411; V-
247 to S-411; S-248 to S-411; I-249 to S-411; L-250 to S-411; Q-251 to S-411;
P-252 to
S-411; T-253 to S-411; Q-254 to S-411; V-255 to S-411; P-256 to S-411; E-257
to S-411;
Q-258 to S-411; E-259 to S-411; M-260 to S-411; E-261 to S-411; V-262 to S-
411; Q-263
to S-411; E-264 to S-411; P-265 to S-411; A-266 to S-411; E-267 to S-411; P-
268 to 5-
411; T-269 to S-411; G-270 to S-411; V-271 to S-411; N-272 to S-411; M-273 to
S-411;
L-274 to S-411; S-275 to S-411; P-276 to S-411; G-277 to S-411; E-278 to S-
411; S-279
to S-411; E-280 to S-411; H-281 to S-411; L-282 to S-411; L-283 to S-411; E-
284 to 5-
411; P-285 to S-411; A-286 to S-411; E-287 to S-411; A-288 to S-411; E-289 to
S-411; 8-
290 to S-411; S-291 to S-411; Q-292 to S-411; R-293 to S-411; R-294 to S-411;
R-295 to
S-411; L-296 to S-411; L-297 to S-411; V-298 to S-411; P-299 to S-411; A-300
to S-411;
N-301 to S-411; E-302 to S-411; G-303 to S-411; D-304 to S-411; P-305 to S-
411; T-306
to S-411; E-307 to S-411; T-308 to S-411; L-309 to S-411; R-310 to S-411; Q-
311 to 5-
411; C-312 to S-411; F-313 to S-411; D-314 to S-411; D-315 to S-411; F-316 to
S-411;
A-317 to S-411; D-318 to S-411; L-319 to S-411; V-320 to S-411; P-321 to S-
411; F-322
to S-411; D-323 to S-411; S-324 to S-411; W-325 to S-411; E-326 to S-411; P-
327 to 5-
411; L-328 to S-411; M-329 to S-411; R-330 to S-411; K-331 to S-411; L-332 to
S-411;
G-333 to S-411; L-334 to S-411; M-335 to S-411; D-336 to S-411; N-337 to S-
411; E-338
to S-411; I-339 to S-411; K-340 to S-411; V-341 to S-411; A-342 to S-411; K-
343 to 5-
411; A-344 to S-411; E-345 to S-411; A-346 to S-411; A-347 to S-411; G-348 to
S-411;
H-349 to S-411; R-350 to S-411; D-351 to S-411; T-352 to S-411; L-353 to S-
411; Y-354
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to S-411; T-355 to S-411; M-356 to S-411; L-357 to S-411; I-358 to S-411; K-
359 to 5-
411; W-360 to S-411; V-361 to S-411; N-362 to S-411; K-363 to S-411; T-364 to
S-411;
G-365 to S-411; R-366 to S-411; D-367 to S-411; A-368 to S-411; S-369 to S-
411; V-370
to S-411; H-371 to S-411; T-372 to S-411; L-373 to S-411; L-374 to S-411; D-
375 to 5-
411; A-376 to S-411; L-377 to S-411; E-378 to S-411; T-379 to S-411; L-380 to
S-411; 6-
381 to S-411; E-382 to S-411; R-383 to S-411; L-384 to S-411; A-385 to S-411;
K-386 to
S-411; Q-387 to S-411; K-388 to S-411; I-389 to S-411; E-390 to S-411; D-391
to S-411;
H-392 to S-411; L-393 to S-411; L-394 to S-411; S-395 to S-411; S-396 to S-
411; G-397
to S-411; K-398 to S-411; F-399 to S-411; M-400 to S-411; Y-401 to S-411; L-
402 to 5-
411; E-403 to S-411; G-404 to S-411; N-405 to S-411; and/or A-406 to S-411 of
the TR7
sequence shown in SEQ ID N0:3.
[0214] In another embodiment, N-terminal deletions of the TR7 polypeptide can
be
described by the general formula n6 to 184 where n6 is a number from 1 to 179
corresponding to the amino acid sequence identified in SEQ ID N0:3. In
specific
embodiments, antibodies of the invention bind N terminal deletions of the TR7
comprising, or alternatively consisting of, the amino acid sequence of
residues: E-2 to 6-
184; Q-3 to G-184; R-4 to G-184; G-5 to G-184; Q-6 to G-184; N-7 to G-184; A-8
to 6-
184; P-9 to G-184; A-10 to G-184; A-11 to G-184; S-12 to G-184; G-13 to G-184;
A-14 to
G-184; R-15 to G-184; K-16 to G-184; R-17 to G-184; H-18 to G-184; G-19 to G-
184; P-
20 to G-184; G-21 to G-184; P-22 to G-184; R-23 to G-184; E-24 to G-184; A-25
to 6-
184; R-26 to G-184; G-27 to G-184; A-28 to G-184; R-29 to G-184; P-30 to G-
184; G-31
to G-184; P-32 to G-184; R-33 to G-184; V-34 to G-184; P-35 to G-184; K-36 to
G-184;
T-37 to G-184; L-38 to G-184; V-39 to G-184; L-40 to G-184; V-41 to G-184; V-
42 to 6-
184; A-43 to G-184; A-44 to G-184; V-45 to G-184; L-46 to G-184; L-47 to G-
184; L-48
to G-184; V-49 to G-184; S-50 to G-184; A-51 to G-184; E-52 to G-184; S-53 to
G-184;
A-54 to G-184; L-55 to G-184; I-56 to G-184; T-57 to G-184; Q-58 to G-184; Q-
59 to 6-
184; D-60 to G-184; L-61 to G-184; A-62 to G-184; P-63 to G-184; Q-64 to G-
184; Q-65
to G-184; R-66 to G-184; A-67 to G-184; A-68 to G-184; P-69 to G-184; Q-70 to
G-184;
Q-71 to G-184; K-72 to G-184; R-73 to G-184; S-74 to G-184; S-75 to G-184; P-
76 to 6-
184; S-77 to G-184; E-78 to G-184; G-79 to G-184; L-80 to G-184; C-81 to G-
184; P-82
to G-184; P-83 to G-184; G-84 to G-184; H-85 to G-184; H-86 to G-184; I-87 to
G-184;
S-88 to G-184; E-89 to G-184; D-90 to G-184; G-91 to G-184; R-92 to G-184; D-
93 to 6-
184; C-94 to G-184; I-95 to G-184; S-96 to G-184; C-97 to G-184; K-98 to G-
184; Y-99
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to G-184; G-100 to G-184; Q-101 to G-184; D-102 to G-184; Y-103 to G-184; S-
104 to
G-184; T-105 to G-184; H-106 to G-184; W-107 to G-184; N-108 to G-184; D-109
to 6-
184; L-110 to G-184; L-111 to G-184; F-112 to G-184; C-113 to G-184; L-114 to
G-184;
R-115 to G-184; C-116 to G-184; T-117 to G-184; R-118 to G-184; C-119 to G-
184; D-
120 to G-184; S-121 to G-184; G-122 to G-184; E-123 to G-184; V-124 to G-184;
E-125
to G-184; L-126 to G-184; S-127 to G-184; P-128 to G-184; C-129 to G-184; T-
130 to 6-
184; T-131 to G-184; T-132 to G-184; R-133 to G-184; N-134 to G-184; T-135 to
G-184;
V-136 to G-184; C-137 to G-184; Q-138 to G-184; C-139 to G-184; E-140 to G-
184; E-
141 to G-184; G-142 to G-184; T-143 to G-184; F-144 to G-184; R-145 to G-184;
E-146
to G-184; E-147 to G-184; D-148 to G-184; S-149 to G-184; P-150 to G-184; E-
151 to 6-
184; M-152 to G-184; C-153 to G-184; R-154 to G-184; K-155 to G-184; C-156 to
G-184;
R-157 to G-184; T-158 to G-184; G-159 to G-184; C-160 to G-184; P-161 to G-
184; 8-
162 to G-184; G-163 to G-184; M-164 to G-184; V-165 to G-184; K-166 to G-184;
V-167
to G-184; G-168 to G-184; D-169 to G-184; C-170 to G-184; T-171 to G-184; P-
172 to 6-
184; W-173 to G-184; S-174 to G-184; D-175 to G-184; I-176 to G-184; E-177 to
G-184;
C-178 to G-184; and/or V-179 to G-184; of the TR7 extracellular domain
sequence
shown in SEQ ID N0:3.
[0215] Also as mentioned above, even if deletion of one or more amino acids
from the
C-terminus of a protein results in modification of loss of one or more
biological functions
of the protein, other functional activities (e.g., biological activities,
ability to multimerize,
ability to bind TR7 ligand (e.g., TRAIL)) may still be retained. For example,
the ability of
the shortened TR7 polypeptide to induce andlor bind to antibodies which
recognize the
complete or mature forms of the polypeptide generally will be retained when
less than the
majority of the residues of the complete or mature polypeptide are removed
from the
C-terminus. Whether a particular polypeptide lacking C-terminal residues of a
complete
polypeptide retains such immunologic activities can readily be determined by
routine
methods described herein and otherwise known in the art. It is not unlikely
that a TR7
polypeptide with a large number of deleted C-terminal amino acid residues may
retain
some biological or immunogenic activities. In fact, peptides composed of as
few as six
TR7 amino acid residues may often evoke an immune response.
[0216] Accordingly, the present invention further provides antibodies that
bind
polypeptides having one or more residues deleted from the carboxy terminus of
the amino
acid sequence of the TR7 polypeptide shown in SEQ ID N0:3 up to the glutamic
acid
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residue at position number 52. In particular, the present invention provides
antibodies that
bind polypeptides comprising the amino acid sequence of residues 52-ms of SEQ
ID
N0:3, where ms is an integer from 57 to 410 corresponding to the position of
the amino
acid residue in SEQ ID N0:3.
[0217] More in particular, the invention provides antibodies that bind
polypeptides
comprising, or alternatively consisting of, the amino acid sequence of
residues: E-52 to M-
410; E-52 to A-409; E-52 to S-408; E-52 to D-407; E-52 to A-406; E-52 to N-
405; E-52 to
G-404; E-52 to E-403; E-52 to L-402; E-52 to Y-401; E-52 to M-400; E-52 to F-
399; E-52
to K-398; E-52 to G-397; E-52 to S-396; E-52 to S-395; E-52 to L-394; E-52 to
L-393; E-
52 to H-392; E-52 to D-391; E-52 to E-390; E-52 to I-389; E-52 to K-388; E-52
to Q-387;
E-52 to K-386; E-52 to A-385; E-52 to L-384; E-52 to R-383; E-52 to E-382; E-
52 to 6-
381; E-52 to L-380; E-52 to T-379; E-52 to E-378; E-52 to L-377; E-52 to A-
376; E-52 to
D-375; E-52 to L-374; E-52 to L-373; E-52 to T-372; E-52 to H-371; E-52 to V-
370; E-52
to S-369; E-52 to A-368; E-52 to D-367; E-52 to R-366; E-52 to G-365; E-52 to
T-364; E-
52 to K-363; E-52 to N-362; E-52 to V-361; E-52 to W-360; E-52 to K-359; E-52
to I-
358; E-52 to L-357; E-52 to M-356; E-52 to T-355; E-52 to Y-354; E-52 to L-
353; E-52 to
T-352; E-52 to D-351; E-52 to R-350; E-52 to H-349; E-52 to G-348; E-52 to A-
347; E-52
to A-346; E-52 to E-345; E-52 to A-344; E-52 to K-343; E-52 to A-342; E-52 to
V-341;
E-52 to K-340; E-52 to I-339; E-52 to E-338; E-52 to N-337; E-52 to D-336; E-
52 to M-
335; E-52 to L-334; E-52 to G-333; E-52 to L-332; E-52 to K-331; E-52 to R-
330; E-52 to
M-329; E-52 to L-328; E-52 to P-327; E-52 to E-326; E-52 to W-325; E-52 to S-
324; E-52
to D-323; E-52 to F-322; E-52 to P-321; E-52 to V-320; E-52 to L-319; E-52 to
D-318; E-
52 to A-317; E-52 to F-316; E-52 to D-315; E-52 to D-314; E-52 to F-313; E-52
to C-312;
E-52 to Q-311; E-52 to R-310; E-52 to L-309; E-52 to T-308; E-52 to E-307; E-
52 to T-
306; E-52 to P-305; E-52 to D-304; E-52 to G-303; E-52 to E-302; E-52 to N-
301; E-52 to
A-300; E-52 to P-299; E-52 to V-298; E-52 to L-297; E-52 to L-296; E-52 to R-
295; E-52
to R-294; E-52 to R-293; E-52 to Q-292; E-52 to S-291; E-52 to R-290; E-52 to
E-289; E-
52 to A-288; E-52 to E-287; E-52 to A-286; E-52 to P-285; E-52 to E-284; E-52
to L-283;
E-52 to L-282; E-52 to H-281; E-52 to E-280; E-52 to S-279; E-52 to E-278; E-
52 to 6-
277; E-52 to P-276; E-52 to S-275; E-52 to L-274; E-52 to M-273; E-52 to N-
272; E-52 to
V-271; E-52 to G-270; E-52 to T-269; E-52 to P-268; E-52 to E-267; E-52 to A-
266; E-52
to P-265; E-52 to E-264; E-52 to Q-263; E-52 to V-262; E-52 to E-261; E-52 to
M-260; E-
52 to E-259; E-52 to Q-258; E-52 to E-257; E-52 to P-256; E-52 to V-255; E-52
to Q-254;
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E-52 to T-253; E-52 to P-252; E-52 to Q-251; E-52 to L-250; E-52 to I-249; E-
52 to 5-
248; E-52 to V-247; E-52 to I-246; E-52 to E-245; E-52 to N-244; E-52 to L-
243; E-52 to
V-242; E-52 to N-241; E-52 to D-240; E-52 to E-239; E-52 to A-238; E-52~to G-
237; E-
52 to P-236; E-52 to R-235; E-52 to Q-234; E-52 to S-233; E-52 to S-232; E-52
to R-231;
E-52 to D-230; E-52 to V-229; E-52 to R-228; E-52 to E-227; E-52 to P-226; E-
52 to D-
225; E-52 to G-224; E-52 to G-223; E-52 to G-222; E-52 to G-221; E-52 to S-
220; E-52 to
C-219; E-52 to I-218; E-52 to G-217; E-52 to K-216; E-52 to L-215; E-52 to Y-
214; E-52
to P-213; E-52 to L-212; E-52 to V-211; E-52 to K-210; E-52 to K-209; E-52 to
W-208;
E-52 to L-207; E-52 to L-206; E-52 to S-205; E-52 to K-204; E-52 to C-203; E-
52 to V-
202; E-52 to F-201; E-52 to V-200; E-52 to A-199; E-52 to V-198; E-52 to I-
197; E-52 to
L-196; E-52 to V-195; E-52 to V-194; E-52 to A-193; E-52 to A-192; E-52 to V-
191; E-
52 to T-190; E-52 to V-189; E-52 to G-188; E-52 to I-187; E-52 to I-186; E-52
to I-185;
E-52 to G-184; E-52 to S-183; E-52 to E-182; E-52 to K-181; E-52 to H-180; E-
52 to V-
179; E-52 to C-178; E-52 to E-177; E-52 to I-176; E-52 to D-175; E-52 to S-
174; E-52 to
W-173; E-52 to P-172; E-52 to T-171; E-52 to C-170; E-52 to D-169; E-52 to G-
168; E-
52 to V-167; E-52 to K-166; E-52 to V-165; E-52 to M-164; E-52 to G-163; E-52
to 8-
162; E-52 to P-161; E-52 to C-160; E-52 to G-159; E-52 to T-158; E-52 to R-
157; E-52 to
C-156; E-52 to K-155; E-52 to R-154; E-52 to C-153; E-52 to M-152; E-52 to E-
151; E-
52 to P-150; E-52 to S-149; E-52 to D-148; E-52 to E-147; E-52 to E-146; E-52
to R-145;
E-52 to F-144; E-52 to T-143; E-52 to G-142; E-52 to E-141; E-52 to E-140; E-
52 to C-
139; E-52 to Q-138; E-52 to C-137; E-52 to V-136; E-52 to T-135; E-52 to N-
134; E-52 to
R-133; E-52 to T-132; E-52 to T-131; E-52 to T-130; E-52 to C-129; E-52 to P-
128; E-52
to S-127; E-52 to L-126; E-52 to E-125; E-52 to V-124; E-52 to E-123; E-52 to
G-122; E-
52 to S-121; E-52 to D-120; E-52 to C-119; E-52 to R-118; E-52 to T-117; E-52
to C-116;
E-52 to R-115; E-52 to L-114; E-52 to C-113; E-52 to F-112; E-52 to L-111; E-
52 to L-
110; E-52 to D-109; E-52 to N-108; E-52 to W-107; E-52 to H-106; E-52 to T-
105; E-52
to 5-104; E-52 to Y-103; E-52 to D-102; E-52 to Q-101; E-52 to G-100; E-52 to
Y-99; E-
52 to K-98; E-52 to C-97; E-52 to S-96; E-52 to I-95; E-52 to C-94; E-52 to D-
93; E-52 to
R-92; E-52 to G-91; E-52 to D-90; E-52 to E-89; E-52 to S-88; E-52 to I-87; E-
52 to H-
86; E-52 to H-85; E-52 to G-84; E-52 to P-83; E-52 to P-82; E-52 to C-81; E-52
to L-80;
E-52 to G-79; E-52 to E-78; E-52 to S-77; E-52 to P-76; E-52 to S-75; E-52 to
S-74; E-52
to R-73; E-52 to K-72; E-52 to Q-71; E-52 to Q-70; E-52 to P-69; E-52 to A-68;
E-52 to
A-67; E-52 to R-66; E-52 to Q-65; E-52 to Q-64; E-52 to P-63; E-52 to A-62; E-
52 to L-
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61; E-52 to D-60; E-52 to Q-59; E-52 to Q-58; and/or E-52 to T-57; of the TR7
sequence
shown in SEQ ID N0:3.
[0218] In another embodiment, antibodies of the invention bind C-terminal
deletions
of the TR7 polypeptide that can be described by the general formula 52-m~
where m~ is a
number from 57 to 183 corresponding to the amino acid sequence identified in
SEQ m
N0:3. In specific embodiments, antibodies of the invention bind C terminal
deletions of
the TR7 polypeptide comprising, or alternatively, consisting of, amino acid
residues: E-52
to S-183; E-52 to E-182; E-52 to K-181; E-52 to H-180; E-52 to V-179; E-52 to
C-178; E-
52 to E-177; E-52 to I-176; E-52 to D-175; E-52 to S-174; E-52 to W-173; E-52
to P-172;
E-52 to T-171; E-52 to C-170; E-52 to D-169; E-52 to G-168; E-52 to V-167; E-
52 to K-
166; E-52 to V-165; E-52 to M-164; E-52 to G-163; E-52 to R-162; E-52 to P-
161; E-52
to C-160; E-52 to G-159; E-52 to T-158; E-52 to R-157; E-52 to C-156; E-52 to
K-155; E-
52 to R-154; E-52 to C-153; E-52 to M-152; E-52 to E-151; E-52 to P-150; E-52
to S-149;
E-52 to D-148; E-52 to E-147; E-52 to E-146; E-52 to R-145; E-52 to F-144; E-
52 to T-
143; E-52 to G-142; E-52 to E-141; E-52 to E-140; E-52 to C-139; E-52 to Q-
138; E-52 to
C-137; E-52 to V-136; E-52 to T-135; E-52 to N-134; E-52 to R-133; E-52 to T-
132; E-52
to T-131; E-52 to T-130; E-52 to C-129; E-52 to P-128; E-52 to S-12?; E-52 to
L-126; E-
52 to E-125; E-52 to V-124; E-52 to E-123; E-52 to G-122; E-52 to S-121; E-52
to D-120;
E-52 to C-119; E-52 to R-118; E-52 to T-117; E-52 to C-116; E-52 to R-115; E-
52 to L-
114; E-52 to C-113; E-52 to F-112; E-52 to L-111; E-52 to L-110; E-52 to D-
109; E-52 to
N-108; E-52 to W-107; E-52 to H-106; E-52 to T-105; E-52 to S-104; E-52 to Y-
103; E-
52 to D-102; E-52 to Q-101; E-52 to G-100; E-52 to Y-99; E-52 to K-98; E-52 to
C-97; E-
52 to S-96; E-52 to I-95; E-52 to C-94; E-52 to D-93; E-52 to R-92; E-52 to G-
91; E-52 to
D-90; E-52 to E-89; E-52 to S-88; E-52 to I-87; E-52 to H-86; E-52 to H-85; E-
52 to G-
84; E-52 to P-83; E-52 to P-82; E-52 to C-81; E-52 to L-80; E-52 to G-79; E-52
to E-78;
E-52 to S-77; E-52 to P-76; E-52 to S-75; E-52 to S-74; E-52 to R-73; E=52 to
K-72; E-52
to Q-71; E-52 to Q-70; E-52 to P-69; E-52 to A-68; E-52 to A-67; E-52 to R-66;
E-52 to
Q-65; E-52 to Q-64; E-52 to P-63; E-52 to A-62; E-52 to L-61; E-52 to D-60; E-
52 to Q-
59; E-52 to Q-58; and/or E-52 to T-57; of the TR7 extracellular domain
sequence shown
in SEQ ~ N0:3.
[0219] The invention also provides antibodies that bind polypeptides having
one or
more amino acids deleted from both the amino and the carboxyl termini of a TR7
144
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
polypeptide, which may be described generally as having residues n5- m5 andlor
n~- m6 of
SEQ >D N0:3, where n5, n6, ms, and m~ are integers as described above.
[0220] Also included are antibodies that bind a polypeptide consisting of a
portion of
the complete TR7 amino acid sequence encoded by the cDNA clone contained in
ATCC
Deposit No. 97920, where this portion excludes from 1 to about 78 amino acids
from the
amino terminus of the complete amino acid sequence encoded by the cDNA clone
contained in ATCC Deposit No. 97920, or from 1 to about 233 amino acids from
the
carboxy terminus, or any combination of the above amino terminal and carboxy
terminal
deletions, of the complete amino acid sequence encoded by the cDNA clone
contained in
ATCC Deposit No. 97920.
[0221] Preferably, antibodies of the present invention bind the N- and C-
terminal
deletion mutants comprising only a portion of the extracellular domain; i.e.,
within
residues 52-184 of SEQ ID N0:3, since any portion therein is expected to ~be
soluble.
[0222] It will be recognized in the art that some amino acid sequence of TR7
can be
varied without significant effect of the structure or function of the protein.
If such
differences in sequence are contemplated, it should be remembered that there
will be
critical areas on the protein which determine activity. Such areas will
usually comprise
residues which make up the ligand binding site or the death domain, or which
form tertiary
structures which affect these domains.
[0223] Thus, the invention further includes antibodies that bind variations of
the TR7
protein which show substantial TR7 protein activity or which include regions
of TR7, such
as the protein portions discussed below. Such mutants include deletions,
insertions,
inversions, repeats, and type substitutions. Guidance concerning which amino
acid
changes are likely to be phenotypically silent can be found in Bowie, J.U. et
al., Sciefzce
247:1306-1310 (1990).
[0224] Thus, antibodies of the present invention may bind a fragment,
derivative, or
analog of the polypeptide of SEQ ID N0:3, or that encoded by the cDNA in ATCC
deposit 97920. Such fragments, variants or derivatives may be (i) one in which
at least 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
residue may or may not be one encoded by the genetic code, or (ii) one in
which one or
more of the amino acid residues includes a substituent group, or (iii) one in
which the
145
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
mature polypeptide is fused with another compound, such as a compound to
increase the
half-life of the polypeptide (for example, polyethylene glycol), or (iv) one
in which the
additional amino acids are fused to the mature polypeptide, such as an IgG Fc
fusion
region peptide or leader or secretory sequence or a sequence which is employed
for
purification of the mature polypeptide or a proprotein sequence. Such
fragments,
derivatives and analogs are deemed to be within the scope of those skilled in
the art from
the teachings herein.
[0225] Of particular interest are substitutions of charged amino acids with
another
charged amino acids and with neutral or negatively charged amino acids. The
latter results
in proteins with reduced positive charge to improve the characteristics of the
TR7 protein.
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.
Imm,unol.
2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al.
Crit. Rev.
Therapeutic Drug Carrier Systems 10:307-377 (1993)).
[0226] The replacement of amino acids can also change the selectivity of
binding to
cell surface receptors. Ostade et al., Nature 361:266-268 (1993) describes
certain
mutations resulting in selective binding of TNF-alpha to only one of the two
known types
of TNF receptors. Thus, the antibodies of the present invention may bind a TR7
receptor
that contains one or more amino acid substitutions, deletions or additions,
either from
natural mutations or human manipulation.
[0227] 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 3 above).
[0228] In specific embodiments, the number of substitutions, additions or
deletions in
the amino acid sequence of SEQ ID N0:3 and/or any of the polypeptide fragments
described herein (e.g., the extracellular 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, 10-1, 5-10, 1-5, 1-
3 or 1-2.
[0229] In specific embodiments, the antibodies of the invention bind TR7
polypeptides
or fragments or variants thereof (especially a fragment comprising or
alternatively
consisting of, the extracellular soluble domain of TR7), that contains any one
or more of
the following conservative mutations in TR7: Ml replaced with A, G, I, L, S,
T, or V; E2
replaced with D; Q3 replaced with N; R4 replaced with H, or K; G5 replaced
with A, I, L,
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CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
S, T, M, or V; Q6 replaced with N; N7 replaced with Q; A8 replaced with G, I,
L, S, T, M,
or V; A10 replaced with G, I, L, S, T, M, or V; A11 replaced with G, I, L, S,
T, M, or V;
S12 replaced with A, G, I, L, T, M, or V; G13 replaced with A, I, L, S, T, M,
or V; A14
replaced with G, I, L, S, T, M, or V; R15 replaced with H, or K; K16 replaced
with H, or
R; R17 replaced with H, or K; H18 replaced with K, or R; G19 replaced with A,
I, L, S, T,
M, or V; G21 replaced with A, I, L, S, T, M, or V; R23 replaced with H, or K;
E24
replaced with D; A25 replaced with G, I, L, S, T, M, or V; R26 replaced with
H, or K;
G27 replaced with A, I, L, S, T, M, or V; A28 replaced with G, I, L, S, T, M,
or V; R29
replaced with H, or K; G31 replaced with A, I, L, S, T, M, or V; R33 replaced
with H, or
K; V34 replaced with A, G, I, L, S, T, or M; K36 replaced with H, or R; T37
replaced with
A, G, I, L, S, M, or V; L38 replaced with A, G, I, S, T, M, or V; V39 replaced
with A, G,
I, L, S, T, or M; L40 replaced with A, G, I, S, T, M, or V; V41 replaced with
A, G, I, L, S,
T, or M; V42 replaced with A, G, I, L, S, T, or M; A43 replaced with G, I, L,
S, T, M, or
V; A44 replaced with G, I, L, S, T, M, or V; V45 replaced with A, G, I, L, S,
T, or M; L46
replaced with A, G, I, S, T, M, or V; L47 replaced with A, G, I, S, T; M, or
V; LAB
replaced with A, G, I, S, T, M, or V; V49 replaced with A, G, I, L, S, T, or
M; S50
replaced with A, G, I, L, T, M, or V; A51 replaced with G, I, L, S, T, M, or
V; E52
replaced with D; S53 replaced with A, G, I, L, T, M, or V; A54 replaced with
G, I, L, S, T,
M, or V; L55 replaced with A, G, I, S, T, M, or V; I56 replaced with A, G, L,
S, T, M, or
V; T57 replaced with A, G, I, L, S, M, or V; Q58 replaced with N; Q59 replaced
with N;
D60 replaced with E; L61 replaced with A, G, I, S, T, M, or V; A62 replaced
with G, I, L,
S, T, M, or V; Q64 replaced with N; Q65 replaced with N; R66 replaced with H,
or K;
A67 replaced with G, I, L, S, T, M, or V; A68 replaced with G, I, L, S, T, M,
or V; Q70
replaced with N; Q71 replaced with N; K72 replaced with H, or R; R73 replaced
with H,
or K; S74 replaced with A, G, I, L, T, M, or V; S75 replaced with A, G, I, L,
T, M, or V;
S77 replaced with A, G, I, L, T, M, or V; E78 replaced with D; G79 replaced
with A, I, L,
S, T, M, or V; L80 replaced with A, G, I, S, T, M, or V; G84 replaced with A,
I, L, S, T,
M, or V; H85 replaced with K, or R; H86 replaced with K, or R; I87 replaced
with A, G,
L, S, T, M, or V; S88 replaced with A, G, I, L, T, M, or V; E89 replaced with
D; D90
replaced with E; G91 replaced with A, I, L, S, T, M, or V; R92 replaced with
H, or K; D93
replaced with E; I95 replaced with A, G, L, S, T, M, or V; S96 replaced with
A, G, I, L, T,
M, or V; K98 replaced with H, or R; Y99 replaced with F, or W; 6100 replaced
with A, I,
L, S, T, M, or V; Q101 replaced with N; D102 replaced with E; Y103 replaced
with F, or
147
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
W; 5104 replaced with A, G, I, L, T, M, or V; T105 replaced with A, G, I, L,
S, M, or V;
H106 replaced with K, or R; W107 replaced with F, or Y; N108 replaced with Q;
D109
replaced with E; L110 replaced with A, G, I, S, T, M, or V; L111 replaced with
A, G, I, S,
T, M, or V; F112 replaced with W, or Y; L114 replaced with A, G, I, S, T, M,
or V; 8115
replaced with H, or K; T117 replaced with A, G, I, L, S, M, or V; 8118
replaced with H,
or K; D'120 replaced with E; 5121 replaced with A, G, I, L, T, M, or V; 6122
replaced
with A, I, L, S, T, M, or V; E123 replaced with D; V124 replaced with A, G, I,
L, S, T, or
M; E125 replaced with D; LI26 replaced with A, G, I, S, T, M, or V; S127
replaced with
A, G, I, L, T, M, or V; T130 replaced with A, G, I, L, S, M, or V; T131
replaced with A,
G, I, L, S, M, or V; T132 replaced with A, G, I, L, S, M, or V; 8133 replaced
with H, or
K; N134 replaced with Q; T135 replaced with A, G, I, L, S, M, or V; V136
replaced with
A, G, I, L, S, T, or M; Q138 replaced with N; E140 replaced with D; E141
replaced with
D; 6142 replaced with A, I, L, S, T, M, or V; T143 replaced with A, G, I, L,
S, M, or V;
F144 replaced with W, or Y; 8145 replaced with H, or K; E146 replaced with D;
E147
replaced with D; D148 replaced with E; 5149 replaced with A, G, I, L, T, M, or
V; E151
replaced with D; M152 replaced with A, G, I, L, S, T, or V; 8154 replaced with
H, or K;
K155 replaced with H, or R; 8157 replaced with H, or K; T158 replaced with A,
G, I, L,
S, M, or V; 6159 replaced with A, I, L, S, T, M, or V; 8162 replaced with H,
or K; 6163
replaced with A, I, L, S, T, M, or V; M164 replaced with A, G, I, L, S, T, or
V; V165
replaced with A, G, I, L, S, T, or M; K166 replaced with H, or R; V 167
replaced with A,
G, ~, L, S, T, or M; 6168 replaced with A, I, L, S, T, M, or V; D169 replaced
with E; T171
replaced with A, G, I, L, S, M, or V; W 173 replaced with F, or Y; S 174
replaced with A,
G, I, L, T, M, or V; D175 replaced with E; I176 replaced with A, G, L, S, T,
M, or V;
E177 replaced with D; V 179 replaced with A, G, I, L, S, T, or M; H180
replaced with K,
or R; K181 replaced with H, or R; E182 replaced with D; S 183 replaced with A,
G, I, L, T,
M, or V; 6184 replaced with A, I, L, S, T, M, or V; I185 replaced with A, G,
L, S, T, M,
or V; I186 replaced with A, G, L, S, T, M, or V; I187 replaced with A, G, L,
S, T, M, or
V; 6188 replaced with A, I, L, S, T, M, or V; V189 replaced with A, G, I, L,
S, T, or M;
T190 replaced with A, G, I, L, S, M, or V; V191 replaced with A, G, I, L, S,
T, or M;
A192 replaced with G, I, L, S, T, M, or V; A193 replaced with G, I, L, S, T,
M, or V;
V 194 replaced with A, G, I, L, S, T, or M; V 195 replaced with A, G, I, L, S,
T, or M;
L196 replaced with A, G, I, S, T, M, or V; I197 replaced with A, G, L, S, T,
M, or V;
V198 replaced with A, G, I, L, S, T, or M; A199 replaced with G, I, L, S, T,
M, or V;
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WO 02/079377 PCT/USO1/42996
V200 replaced with A, G, I, L, S, T, or M; F201 replaced with W, or Y; V202
replaced
with A, G, I, L, S, T, or M; K204 replaced with H, or R; 5205 replaced with A,
G, I, L, T,
M, or V; L206 replaced with A, G, I, S, T, M, or V; L207 replaced with A, G,
I, S, T, M,
or V; W208 replaced with F, or Y; K209 replaced with H, or R; K210 replaced
with H, or
R; V211 replaced with A, G, I, L, S, T, or M; L212 replaced with A, G, I, S,
T, M, or V;
Y214 replaced with F, or W; L215 replaced with A, G, I, S, T, M, or V; K216
replaced
with H, or R; 6217 replaced with A, I, L, S, T, M, or V; I218 replaced with A,
G, L, S, T,
M, or V; S220 replaced with A, G, I, L, T, M, or V; 6221 replaced with A, I,
L, S, T, M,
or V; 6222 replaced with A, I, L, S, T, M, or V; 6223 replaced with A, I, L,
S, T, M, or
V; 6224 replaced with A, I, L, S, T, M, or V; D225 replaced with E; E227
replaced with
D; 8228 replaced with H, or K; V229 replaced with A, G, I, L, S, T, or M; D230
replaced
with E; 8231 replaced with H, or K; S232 replaced with A, G, I, L, T, M, or V;
5233
replaced with A, G, I, L, T, M, or V; Q234 replaced with N; 8235 replaced with
H,~ or K;
6237 replaced with A, I, L, S, T, M, or V; A238 replaced with G, I, L, S, T,
M, or V;
E239 replaced with D; D240 replaced with E; N241 replaced with Q; V242
replaced with
A, G, I, L, S, T, or M; L243 replaced with A, G, I, S, T, M, or V; N244
replaced with Q;
E245 replaced with D; I246 replaced with A, G, L, S, T, M, or V; V247 replaced
with A,
G, I, L, S, T, or M; S248 replaced with A, G, I, L, T, M, or V; I249 replaced
with A, G, L,
S, T, M, or V; L250 replaced with A, G, I, S, T, M, or V; Q251 replaced with
N; T253
replaced with A, G, I, L, S, M, or V; Q254 replaced with N; V255 replaced with
A, G, I,
L, S, T, or M; E257 replaced with D; Q258 replaced with N; E259 replaced with
D; M260
replaced with A, G, T, L, S, T, or V; E261 replaced with D; V262 replaced with
A, G, I, L,
S, T, or M; Q263 replaced with N; E264 replaced with D; A266 replaced with G,
I, L, S,
T, M, or V; E267 replaced with D; T269 replaced with A, G, I, L, S, M, or V;
6270
replaced with A, I, L, S, T, M, or V; V271 replaced with A, G, I, L, S, T, or
M; N272
replaced with Q; M273 replaced with A, G, I, L, S, T, or V; L274 replaced with
A, G, I, S,
T, M, or V; S275 replaced with A, G, I, L, T, M, or V; 6277 replaced with A,
I, L, S, T,
M, or V; E278 replaced with D; 5279 replaced with A, G, I, L, T, M, or V; E280
replaced
with D; H281 replaced with K, or R; L282 replaced with A, G, I, S, T, M, or V;
L283
replaced with A, G, I, S, T, M, or V; E284 replaced with D; A286 replaced with
G, I, L, S,
T, M, or V; E287 replaced with D; A288 replaced with G, I, L, S, T, M, or V;
E289
replaced with D; 8290 replaced with H, or K; 5291 replaced with A, G, I, L, T,
M, or V;
Q292 replaced with N; 8293 replaced with H, or K; 8294 replaced with H, or K;
8295
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WO 02/079377 PCT/USO1/42996
replaced with H, or K; L296 replaced with A, G, I, S, T, M, or V; L297
replaced with A,
G, I, S, T, M, or V; V298 replaced with A, G, I, L, S, T, or M; A300 replaced
with G, I, L,
S, T, M, or V; N301 replaced with Q; E302 replaced with D; 6303 replaced with
A, I, L,
S, T, M, or V; D304 replaced with E; T306 replaced with A, G, I, L, S, M, or
V; E307
replaced with D; T308 replaced with A, G, I, L, S, M, or V; L309 replaced with
A, G, I, S,
T, M, or V; 8310 replaced with H, or K; Q311 replaced with N; F313 replaced
with W, or
Y; D314 replaced with E; D315 replaced with E; F316 replaced with W, or Y;
A317
replaced with G, I, L, S, T, M, or V; D318 replaced with E; L319 replaced with
A, G, I, S,
T, M, or V; V320 replaced with A, G, I, L, S, T, or M; F322 replaced with W,
or Y; D323
replaced with E; 5324 replaced with A, G, I, L, T, M, or V; W325 replaced with
F, or Y;
E326 replaced with D; L328 replaced with A, G, I, S, T, M, or V; M329 replaced
with A,
G, I, L, S, T, or V; 8330 replaced with H, or K; K331 replaced with H, or R;
L332
replaced with A, G, I, S, T, M, or V; 6333 replaced with A, I, L, S, T, M, or
V; L334
replaced with A, G, I, S, T, M, or V; M335 replaced with A, G, I, L, S, T, or
V; D336
replaced with E;.N337 replaced with Q; E338 replaced with D; I339 replaced
with A, G,
L, S, T, M, or V; K340 replaced with H, or R; V341 replaced with A, G, I, L,
S, T, or M;
A342 replaced with G, I, L, S, T, M, or V; K343 replaced with H, or R; A344
replaced
with G, I, L, S, T, M, or V; E345 replaced with D; A346 replaced with G, I, L,
S, T, M, or
V; A347 replaced with G, I, L, S, T, M, or V; 6348 replaced with A, I, L, S,
T, M, or V;
H349 replaced with K, or R; 8350 replaced with H, or K; D351 replaced with E;
T352
replaced with A, G, I, L, S, M, or V; L353 replaced with A, G, I, S, T, M, or
V; Y354
replaced with F, or W; T355 replaced with A, G, I, L, S, M, or V; M356
replaced with A,
G, I, L, S, T, or V; L357 replaced with A, G, I, S, T, M, or V; I358 replaced
with A, G, L,
S, T, M, or V; K359 replaced with H, or R; W360 replaced with F, or Y; V361
replaced
with A, G, I, L, S, T, or M; N362 replaced with Q; K363 replaced with H, or R;
T364
replaced with A, G, I, L, S, M, or V; 6365 replaced with A, I, L, S, T, M, or
V~, R366
replaced with H, or K; D367 replaced with E; A368 replaced with G, I, L, S, T,
M, or V;
S369 replaced with A, G, I, L, T, M, or V; V370 replaced with A, G, I, L, S,
T, or M;
H371 replaced with K, or R; T372 replaced with A, G, I, L, S, M, or V; L373
replaced
with A, G, I, S, T, M, or V; L374 replaced with A, G, I, S, T, M, or V; D375
replaced with
E; A376 replaced with G, I, L, S, T, M, or V; L377 replaced with A, G, I, S,
T, M, or V;
E378 replaced with D; T379 replaced with A, G, I, L, S, M, or V; L380 replaced
with A,
G, I, S, T, M, or V; 6381 replaced with A, I, L, S, T, M, or V; E382 replaced
with D;
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8383 replaced with H, or K; L384 replaced with A, G, I, S, T, M, or V; A385
replaced
with G, I, L, S, T, M, or V; K386 replaced with H, or R; Q387 replaced with N;
K388
replaced with H, or R; I389 replaced with A, G, L, S, T, M, or V; E390
replaced with D;
D391 replaced with E; H392 replaced with K, or R; L393 replaced with A, G, I,
S, T, M,
or V; L394 replaced with A, G, I, S, T, M, or V; 5395 replaced with A, G, I,
L, T, M, or
V; 5396 replaced with A, G, I, L, T, M, or V; 6397 replaced with A, I, L, S,
T, M, or V;
K398 replaced with H, or R; F399 replaced with W, or Y; M400 replaced with A,
G, I, L,
S, T, or V; Y401 replaced with F, or W; L402 replaced with A, G, I, S, T, M,
or V; E403
replaced with D; 6404 replaced with A, I, L, S, T, M, or V; N405 replaced with
Q; A406
replaced with G, I, L, S, T, M, or V; D407 replaced with E; 5408 replaced with
A, G, I, L,
T, M, or V; A409 replaced with G, I, L, S, T, M, or V; M410 replaced with A,
G, I, L, S,
T, or V; and/or S411 replaced with A, G, I, L, T, M, or V of SEQ lD N0:3.
[0230] In specific embodiments, the antibodies of the invention bind TR7
polypeptides
or fragments or variants thereof (especially a fragment comprising or
alternatively
consisting of, the extracellular soluble domain of TR7), that contains any one
or more of
the following non-conservative mutations in TR7: M1 replaced with D, E, H, K,
R, N, Q,
F, W, Y, P, or C; E2 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C;
Q3 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R4
replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G5 replaced with D, E,
H, K, R, N, Q,
F, W, Y, P, or C; Q6 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C;
N7 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A8
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; P9 replaced with D, E, H, K, R, A, G,
I, L, S, T, M,
V, N, Q, F, W, Y, or C; A10 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; A11
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S 12 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; G13 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A14
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R15 replaced with D, E,
A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; K16 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, P, or C; R17 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; H18
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Gl9
replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; P20 replaced with D, E, H, K, R, A, Q, I, L,
S, T, M, V,
N, Q, F, W, Y, or C; G21 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
P22
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R23
replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E24 replaced with
H, K, R, A, G,
151
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A25 replaced with D, E, H, K, R, N,
Q, F, W, Y,
P, or C; R26 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; G27
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A28 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; R29 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or
C; P30 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, ~or
C; G31
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P32 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R33 replaced with D, E, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, P, or C; V34 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; P35
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K36
replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T37 replaced with
D, E, H, K, R,
N, Q, F, W, Y, P, or C; L38 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; V39
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L40 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; V41 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V42
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A43 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; A44 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V45
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L46 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; IA.7 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L48
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V49 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; S50 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A51
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E52 replaced with H, K,
R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; S53 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; A54 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L55 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; I56 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; T57
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q58 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; Q59 replaced with D, E, H, K, R, A, G,
I, L, S, T, M,
V, F, W, Y, P, or C; D60 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; L61 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A62 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; P63 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, or C; Q64 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or
C; Q65 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;
R66 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A67 replaced with
D, E, H, K, R,
N, Q, F, W, Y, P, or C; A68 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; P69
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; Q70
replaced
152
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; Q71 replaced
with D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K72 replaced with D, E, A, G, I,
L, S, T, M, V,
N, Q, F, W, Y, P, or C; R73 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; S74 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S75 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; P76 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, or C; S77 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E78
replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G79 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; L80 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
C81 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
P82 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P83 replaced
with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G84 replaced with D, E, H,
K, R, N, Q,
F, W, Y, P, or C; H85 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C;
H86 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I87
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; S88 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; E89 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; D90
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G91
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; R92 replaced with D, E, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, P, or C; D93 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; C94 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; I95
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S96 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; C97 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, or P; K98 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; Y99
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; 6100
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; Q101 replaced with D, E, H, K, R, A, G, I,
L, S, T, M,
V, F, W, Y, P, or C; D102 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; Y103 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
5104
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T105 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; H106 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or
C; W107 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
N108 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; D109 replaced
with H, K, R, .
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L110 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; Ll l l replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
F112 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; C 113 replaced with
D, E, H, K,
153
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L114 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; Rl 15 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C;
C116 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
T117
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 8118 replaced with D, E,
A, G, I, L,
S, T, M, V; N, Q, F, W, Y, P, or C; C 119 replaced with D, E, H, K, R, A, G,
I, L, S, T, M,
V, N, Q, F, W, Y, or P; D 120 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y,
P, or C; S 121 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6122
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; E123 replaced with H, K, R, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, P, or C; V124 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
E125
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L126
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; S 127 replaced with D, E, H, K, R, N,
Q, F, W, Y, P,
or C; P128 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; C129
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; T130
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; T131 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; T132 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 8133
replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N134 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; T135 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; V136 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C137 replaced
with D, E,
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Q138 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; C 139 replaced with D, E, H, K, R, A,
G, I, L, S, T,
M, V, N, Q, F, W, Y, or P; E140 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F,
W, Y, P, or C; E141 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C;
6142 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T143 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; F144 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V,
P, or C; 8145 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; E146
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E147
replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D148 replaced with H,
K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S 149 replaced with D, E, H, K, R,
N, Q, F, W, Y,
P, or C; P150 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, or C;
E151 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
M152 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; C153 replaced with D, E, H, K, R,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or P; 8154 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, P, or C; K155 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
154
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
C156 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
8157
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T158
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; 6159 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; 0160 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or
P; P161
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; 8162
replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 6163 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; M164 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C;
V165 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K166 replaced with
D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V167 replaced with D, E, H, K, R, N,
Q, F, W, Y,
P, or C; 6168 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D169
replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C 170 replaced with D,
E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or P; T171 replaced with D, E, H, K, R, N,
Q, F, W, Y,
P, or C; P172 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, or C;
W 173 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S
174 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; D175 replaced with H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; I176 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
E177 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
C178 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V 179
replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; H180 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, P, or C; K181 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
E182 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S
183 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6184 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; I185 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I186
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; I187 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; 6188 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V189 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; T190 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
V191 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A192 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; A193 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C;
V 194 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V 195 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; L196 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; I197
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V 198 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; A199 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V200
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F201 replaced with D, E,
H, K, R, N,
155
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
Q, A, G, I, L, S, T, M, V, P, or C; V202 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; C203 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or
P; K204
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 5205
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; L206 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; L207 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W208 replaced
with D, E, H,
K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K209 replaced with D, E, A, G, I,
L, S, T, M,
V, N, Q, F, W, Y, P, or C; K210 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y,
P, or C; V211 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L212
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; P213 replaced with D, E, H, K, R, A, G, I,
L, S, T, M,
V, N, Q, F, W, Y, or C; Y214 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,
T, M, V, P, '
or C; L215 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K216 replaced
with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 6217 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; I218 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C219
replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; 5220 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; 6221 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or
C; 6222 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6223 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; 6224 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
D225 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
P226 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E227 replaced
with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 8228 xeplaced with D, E, A,
G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; V229 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or
C; D230 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
8231
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 5232
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; S233 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; Q234.replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;
8235
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P236
replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; 6237 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; A238 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; E239
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D240
replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N241 replaced with D,
E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; V242 replaced with D, E, H, K, R, N,
Q, F, W, Y,
P, or C; L243 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N244
replaced With D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E245 replaced with H, K,
R, A, G, I,
156
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
L, S, T, M, V, N, Q, F, W, Y, P, or C; I246 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; V247 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 5248 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; I249 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
L250 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q251 replaced with
D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; P252 replaced with D, E, H, K, R,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; T253 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or
C; Q254 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;
V255
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P256 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E257 replaced with H, K, R, A, G, I,
L, S, T, M,
V,~N, Q, F, W, Y, P, or C; Q258 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W,
Y, P, or C; E259 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
M260 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E261 replaced with
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V262 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; Q263 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C;
E264 replaced with H, K, R, A, G, I, L; S, T, M, V, N, Q, F, W, Y, P, or C;
P265 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A266 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; E267 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, P, or C; P268 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y,
or C; T269 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6270 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; V271 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or
C; N272 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;
M273
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L274 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; 5275 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
P276
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; 6277
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E278 replaced with H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; 5279 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
E280 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
H281 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L282 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; L283 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; E284
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P285
replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A286 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; E287 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
,: Y, P, or C; A288 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E289
replaced with
157
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 8290 replaced with D,
E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; 5291 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; Q292 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; 8293
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 8294
replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 8295 replaced with D, E, A,
G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; L296 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or
C; L297 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V298 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; P299 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, or C; A300 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
N301
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E302
replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 6303 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; D304 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y,
P, or C; P305 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, or C;
T306 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E307 replaced with
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T308 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; L309 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 8310
replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q311 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; C312 replaced with D, E, H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, or P; F313 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V,
P, or C; D314 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; D315
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F316
replaced with
D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A317 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; D318 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; L319 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V320 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; P321 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, or C; F322 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or
C; D323 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
5324
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W325 replaced with D, E,
H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; E326 replaced with H, K, R, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, P, or C; P327 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, or C; L328 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M329
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; 8330 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, P, or C; K331 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C;
158
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
L332 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6333 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; L334 replaced,with D, E, H, K, R, N, Q, F, W, Y, P,
or C;
M335 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D336 replaced with
H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N337 replaced with D, E, H, K,
R, A, G, I,
L, S, T, M, V, F, W, Y, P, or C; E338 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, P, or C; I339 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
K340 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V341 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; A342 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C;
K343 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A344
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E345 replaced with H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; A346 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
A347 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6348 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; H349 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y,
P, or C; 8350 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; D351
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T352
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; L353 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; Y354 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
T355
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M356 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; L357 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
I358
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K359 replaced with D, E,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; W360 replaced with D, E, H, K, R, N, Q, A,
G, I, L, S,
T, M, V, P, or C; V361 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
N362
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K363
replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T364 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; 6365 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
8366
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D367
replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A368 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; 5369 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V370
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H371 replaced with D, E,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; T372 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; L373 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L374 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; D375 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, P, or C; A376 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L377
replaced
159
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E378 replaced with H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; T379 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
L380 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6381 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; E382 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, P, or C; 8383 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; L384
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A385 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; K386 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or
C; Q387 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;
K388
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I389
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; E390 replaced with H, K, R, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, P, or C; D391 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; H392 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
L393
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L394 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; 5395 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
S396
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6397 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; K398 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or
C; F399 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
M400 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y401 replaced with D, E, H, K, R,
N, Q, A, G,
I, L, S, T, M, V, P, or C; L402 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; E403
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 6404
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; N405 replaced with D, E, H, K, R, A, G,
I, L, S, T,
M, V, F, W, Y, P, or C; A406 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; D407
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 5408
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; A409 replaced with D, E, H, K, R, N, Q,
F, W, Y,
P, or C; M410 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; and/or 5411
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C of SEQ ID N0:3.
[0231] Amino acids in the TR7 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, Scieface 244:1081-1085
(1989)). The latter procedure introduces single alanine mutations at every
residue in the
molecule. The resulting mutant molecules are then tested for biological
activity such as
receptor binding or in vitro, or in vitro proliferative activity. Sites that
are critical for
ligand-receptor binding can also be determined by structural analysis such as
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crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith
et al., T. Mol.
Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)). In
preferred
embodiments, antibodies of the present invention bind regions of TR7 that are
essential for
TR7 function. In other preferred embodiments, antibodies of the present
invention bind
regions of TR7 that are essential fox TR7 function and inhibit or abolish TR7
function. In
other preferred embodiments, antibodies of the present invention bind regions
of TR7 that
are essential for TR7 function and enhance TR7 function.
[0232] Additionally, protein engineering may be employed to improve or alter
the
characteristics of TR7 polypeptides. Recombinant DNA technology known to those
skilled in the art can be used to create novel mutant proteins or polypeptides
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. Antibodies
of the present invention may bind such modified TR7 polypeptides.
[0233] Non-naturally occurring TR7 variants that may be bound by the
antibodies of
the invention 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 et al.,
PIZilos. Traps. R. Soc. London SerA 317:415 (1986)).
[0234] Thus, the invention also encompasses antibodies that bind TR7
derivatives and
analogs that have one or more amino acid residues deleted, added, or
substituted to
generate TR7 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 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 acid positions on any one or more of the
glycosylation
recognitions sequences in the TR7 polypeptides, and/or an amino acid deletion
at the
second position of any one or more such recognition sequences will prevent
glycosylation
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of the TR7 at the modified tripeptide sequence (see, e.g., Miyajimo et al.,
EMBO J
5(6):1193-1197). Additionally, one or more of the amino acid residues of TR7
polypeptides (e.g., arginine and lysine residues) may be deleted or
substituted with another
residue to eliminate undesired processing by proteases such as, for example,
furins or
kexins.
[0235] The antibodies of the present invention also include antibodies that
bind a
polypeptide comprising, or alternatively, consisting of the polypeptide
encoded by the
deposited cDNA (the deposit having ATCC Accession Number 97920) including the
leader; the mature polypeptide encoded by the deposited the cDNA minus the
leader (i.e.,
the mature protein); a polypeptide comprising or alternatively, consisting of,
amino acids
about 1 to about 411 in SEQ ID NO:3; a polypeptide comprising or
alternatively,
consisting of, amino acids about 2 to about 411 in SEQ ID NO:3; a polypeptide
comprising or alternatively, consisting of, amino acids about 52 to about 411
in SEQ ID
N0;3; a polypeptide comprising or alternatively, consisting of, the TR7
extracellular
domain; a polypeptide comprising or alternatively, consisting of, the TR7
cysteine rich
domain; a polypeptide comprising or alternatively, consisting of, the TR7
transmembrane
domain; a polypeptide comprising or alternatively, consisting of, the TR7
intracellular
domain; a polypeptide comprising or alternatively, consisting of, the
extracellular and
intracellular domains with all or part of the transmembrane domain deleted;
and a
polypeptide comprising or alternatively, consisting of, the TR7 death domain;
as well as
polypeptides which are at least 80% identical, more preferably at least 90% or
95%
identical, still more preferably at least 96%, 97%, 98%, or 99% identical to
the
polypeptides described above, and also include portions of such polypeptides
with at least
30 amino acids and more preferably at least 50 amino acids.
[0236] By a polypeptide having an amino acid sequence at least, for example,
95%
"identical" to a reference amino acid sequence of a TR7 polypeptide is
intended that the
amino acid sequence of the polypeptide is identical to the reference sequence
except that
the polypeptide sequence may include up to five amino acid alterations per
each 100
amino acids of the reference amino acid of the TR7 polypeptide. 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
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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.
[0237] As a practical matter, whether any particular polypeptide is at least
90%, 95%,
96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown
in FIGS.
lA-B (SEQ ID N0:3), the amino acid sequence encoded by deposited cDNA clones,
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.
[0238] In a specific embodiment, the identity between a reference (query)
sequence (a
sequence of the present invention) and a subject sequence, also referred to as
a global
sequence alignment, is determined using the FASTDB computer program based on
the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred
parameters
used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch
Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1,
Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window
Size=500 or the length of the subject amino acid sequence, whichever is
shorter.
According to this embodiment, 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
the query sequence, the percent identity is corrected by calculating the
number of residues
of the query sequence that are N- and C-terminal of the subject sequence,
which are not
matched/aligned with a corresponding subject residue, as a percent of the
total bases of the
query sequence. A determination of whether a residue is matched/aligned is
determined
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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
matchedltotal 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 matchedlaligned with the query sequnce are
manually
corrected for. No other manual corrections are made for the purposes of this
embodiment.
[0239] The polypeptide of the present invention could be used as a molecular
weight
marker on SDS-PAGE gels or on molecular sieve gel filtration columns and as a
source
for generating antibodies that bind the TR7 polypeptides, using methods well
known to
those of skill in the art.
[0240] The present application is also directed to antibodies that bind
proteins
containing polypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to
the TR7
polypeptide sequence set forth herein as ns-ms, and/or n6-m6. In preferred
embodiments,
the application is directed to antibodies that bind proteins containing
polypeptides at least
90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid
sequence of the specific TR7 N- and C-terminal deletions recited herein.
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[0241] In certain preferred embodiments, antibodies of the invention bind TR7
proteins of the invention comprise fusion proteins as described above wherein
the TR7
polypeptides are those described as n5-ms, and n6-m6, herein.
TR10
[0242] In certain embodiments of the present invention, the antibodies of the
present
invention bind TR10 polypeptide, or fragments or variants thereof. The
following section
describes the TR10 polypeptides, fragments and variants that may be bound by
the
antibodies of the invention in more detail. The TR10 polypeptides, fragments
and variants
which may be bound by the antibodies of the invention are also described in,
for example,
International Publication Numbers W098/54202 and WO00/73321 which are herein
incorporated by reference in their entireties.
[0243] In certain embodiments, the antibodies of the present invention
immunospecifically bind TR10 polypeptide. An antibody that immunospecifically
binds
TR10 may, in some embodiments, bind fragments, variants (including species
orthologs
of TR10), multimers or modified forms of TRIO. For example, an antibody
immunospecific for TR10 may bind the TR10 moiety of a fusion protein
comprising all or
a portion of TR10.
[0244] TR10 proteins may be found as monomers or multimers (i.e., dimers,
trimers,
tetramers, and higher multimers). Accordingly, the present invention relates
to antibodies
that bind TR10 proteins found as monomers or as part of multimers. In specific
embodiments, antibodies of the invention bind TR10 monomers, dimers, trimers
or
tetramers. In additional embodiments, antibodies of the invention bind at
least dimers, at
least trimers, or at least tetramers containing one or more TR10 polypeptides.
[0245] Antibodies of the invention may bind TR10 homomers or heteromers. As
used
herein, the term homomer, refers to a multimer containing only TR10 proteins
of the
invention (including TR10 fragments, variants, and fusion proteins, as
described herein).
These homomers may contain TR10 proteins having identical or different
polypeptide
sequences. In a specific embodiment, a homomer of the invention is a multirner
containing only TR10 proteins having an identical polypeptide sequence. In
another
specific embodiment, antibodies of the invention bind TR10 homomers containing
TR10
proteins having different polypeptide sequences. In specific embodiments,
antibodies of
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the invention bind a TR10 homodimer (e.g., containing TR10 proteins having
identical or
different polypeptide sequences) or a homotrimer (e.g., containing TR10
proteins having
identical or different polypeptide sequences). In additional embodiments,
antibodies of
the invention bind at least a homodimer, at least a homotrimer, or at least a
homotetramer
of TR10.
[0246] As used herein, the term heteromer refers to a multimer containing
heterologous proteins (i.e., proteins containing polypeptide sequences that do
not
correspond to a polypeptide sequences encoded by the TR10 gene) in addition to
the TR10
proteins of the invention. In a specific embodiment, antibodies of the
invention bind a
heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments,
the
antibodies of the invention bind at least a homodimer, at least a homotrimer,
or at least a
homotetramer containing one or more TR10 polypeptides.
[0247] Multimers bound by one or more antibodies 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
bound
by one or more antibodies of the invention, such as, for example, homodimers
or
homotrimers, are formed when TR10 proteins contact one another in solution. In
another
embodiment, heteromultimers bound by one or more antibodies of the invention,
such as,
for example, heterotrimers or heterotetramers, are formed when proteins of the
invention
contact antibodies to the TR10 polypeptides (including antibodies to the
heterologous
polypeptide sequence in a fusion protein) in solution. In other embodiments,
multimers
bound by one or more antibodies of the invention are formed by covalent
associations with
and/or between the TR10 proteins. 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 N0:4 or the polypeptide encoded by the
deposited cDNA clone of ATCC Deposit 209040). 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 TR10 fusion protein. In one example, covalent associations are
between the
heterologous sequence contained in a fusion protein (see, e.g., US Patent
Number
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5,478,925). In a specific example, the covalent associations are between the
heterologous
sequence contained in a TR10-Fc fusion protein (as described herein). In
another specific
example, covalent associations of fusion proteins are between heterologous
polypeptide
sequences from another TNF family ligand/receptor member that is capable of
forming
covalently associated multimers, such as for example, oseteoprotegerin (see,
e.g.,
International Publication No. WO 98/49305, the contents of which are herein
incorporated
by reference in its entirety).
[0248] The multimers that may be bound by one or more antibodies 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, multirners that may be bound by one or more
antibodies 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 that may be bound by one or more antibodies 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 that may be
bound by one
or more antibodies of the invention (see, e.g., US Patent Number 5,478,925,
which is
herein incorporated by reference in its entirety).
[0249] Alternatively, multimers that may be bound by one or more antibodies of
the
invention may be generated using genetic engineering techniques known in the
art. In one
embodiment, proteins contained in multimers that may be bound by one or more
antibodies 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). Tn a specific
embodiment,
polynucleotides coding for a homodimer that may be bound by one or more
antibodies of
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the invention are generated by ligating a polynucleotide sequence encoding a
TR10
polypeptide 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 (lacl~ing 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 TR10 polypeptides 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). In another embodiment, two or more
TR10
polypeptides are joined through synthetic linkers (e.g., peptide, carbohydrate
or soluble
polymer linkers). Examples include those peptide linkers described in U.S.
Pat. No.
5,073,627 (hereby incorporated by reference). Proteins comprising multiple
TR10
polypeptides separated by peptide linkers may be produced using conventional
recombinant DNA technology. In specific embodiments, antibodies of the
invention bind
proteins comprising multiple TR10 polypeptides separated by peptide linkers.
[0250] Another method for preparing multimer TR10 polypeptides involves use of
TR10 polypeptides fused to a leucine zipper or isoleucine polypeptide
sequence. Leucine
zipper domains and isoleucine zipper domains are polypeptides that promote
multimerization of the proteins in which they are found. Leucine zippers were
originally
identified in several DNA-binding proteins (Landschulz et al., Science
240:1759, (1988)),
and have since been found in a variety of different proteins. Among the known
leucine
zippers are naturally occurring peptides and derivatives thereof that dimerize
or trimerize.
Examples of leucine zipper domains suitable for producing soluble multimeric
TR10
proteins are those described in PCT application WO 94/10308, hereby
incorporated by
reference. Recombinant fusion proteins comprising a soluble TR10 polypeptide
fused to a
peptide that dimerizes or trimerizes in solution are expressed in suitable
host cells, and the
resulting soluble multimeric TR10 is recovered from the culture supernatant
using
techniques known in the ant. In specific embodiments, antibodies of the
invention bind
TR10-leucine zipper fusion protein monomers and/or TR10-leucine zipper fusion
protein
multimers.
[0251] . Certain members of the TNF family of proteins are believed to exist
in trimeric
form (Beutler and Huffel, Science 264:667, 1994; Banner et al., Cell 73:431,
1993). Thus,
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trimeric TR10 may offer the advantage of enhanced biological activity.
Preferred leucine
zipper moieties are those that preferentially form trimers. One example is a
leucine zipper
derived from lung surfactant protein D (SPD), as described in Hoppe et al.
(FEBS Letters
344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby
incorporated
by reference. In specific embodiments, antibodies of the invention bind TR10-
leucine
zipper fusion protein trimers.
[0252] Other peptides derived from naturally occurring trimeric proteins may
be
employed in preparing trimeric TR10. In specific embodiments, antibodies of
the
invention bind TR10- fusion protein monomers and/or TR10 fusion protein
trimers.
[0253] The TR10 polypeptides are preferably provided in an isolated form, and
preferably are substantially purified. 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 TR10 polypeptide is substantially purified by the one-
step method
described in Smith and Johnson, Gene 67:31-40 (1988).
[0254] Antibodies of the present invention may bind TR10 polypeptides
comprising or
alternatively, consisting of, an amino acid sequence contained in SEQ ID N0:4,
encoded
by the cDNA contained in ATCC Deposit Number 209040, or encoded by nucleic
acids
which hybridize (e.g., under stringent hybridization conditions) to the
nucleotide
sequence contained in ATCC Deposit Number 209040, 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. Antibodies of the present invention may bind polypeptide
fragments,
including, for example, fragments that comprise or alternatively, consist of
from about
amino acid residues: 1 to 55, 56 to 105, 106 to 155, 156 to 212, 213 to 230,
231 to 281,
282 to 282, and/or 283 to 386 of SEQ ID N0:4. Moreover, polypeptide fragments
can be
at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175
or 200 amino
acids in length. 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.
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[0255] In, specific embodiments, polypeptide fragments of the invention
comprise, or
alternatively consist of, amino acid residues: 1-55, 56 to 212, 213 to 230,
231 to 286,
and/or 353 to 363 as depicted in SEQ ID N0:4.
[0256] Preferably, antibodies of the present invention bind polypeptide
fragments
selected from the group: (a) a polypeptide comprising or alternatively,
consisting of, the
TR10 extracellular domain (predicted to constitute amino acid residues from
about 56 to
about 212 in SEQ ID N0:4; (b) a polypeptide comprising or alternatively,
consisting of,
both TR10 cysteine rich domains (both of which rnay be found in the protein
fragment
consisting of amino acid residues from about 81 to about 182 in SEQ ID N0:4);
(c) a
polypeptide comprising or alternatively, consisting of, the TR10 cysteine rich
domain
consisting of amino acid residues from about 81 to about 135 in SEQ ID N0:4);
(d) a
polypeptide comprising or alternatively, consisting of, the TR10 cysteine rich
domain
consisting of amino acid residues from about 136 to about 182 in SEQ ID NO:l);
(e) a
polypeptide comprising or alternatively, consisting of, the TR10 transmembrane
domain
(predicted to constitute amino acid residues from about 213 to about 230 in
SEQ ID N0:4;
(f) a polypeptide comprising or alternatively, consisting of, the TR10
intracellular domain
(predicted to constitute amino acid residues from about 231 to about 386 in
SEQ ID
N0:4); (g) a polypeptide comprising or alternatively, consisting of, the TR10
partial death
domain (predicted to constitute amino acid residues from about 353 to about
363 in SEQ
ID N0:4); (h) a polypeptide comprising, or alternatively, consisting of, one,
two, three,
four or more, epitope bearing portions of the TRIO receptor protein (g) any
combination of
polypeptides (a)-(h).
[0257] It is believed that one or both of the extracellular cysteine rich
motifs of TR10
is important for interactions between TR10 and its ligands (e.g., TRAIL).
Accordingly, in
highly preferred embodiments, antibodies of the present invention bind TR10
polypeptide
fragments comprising, or alternatively consisting of amino acid residues 81 to
135, and/or
136 to 182 of SEQ ID N0:4. In another highly preferred embodiment, antibodies
of the
present invention bind TR10 polypeptides comprising, or alternatively
consisting of both
of the extracellular cysteine rich motifs (amino acid residues 81 to 182 of
SEQ ID N0:4.)
In another preferred embodiment, antibodies of the present invention bind TR10
polypeptides comprising, or alternatively consisting the extracellular soluble
domain of
TR10 (amino acid residues 56-212 of SEQ ID N0:4.) In highly preferred
embodiments,
the antibodies of the invention that bind all or a portion of the
extracellular soluble domain
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of TR10 (e.g., one or both cysteine rich domains) prevent TRAIL ligand from
binding to
TR10. In other highly preferred embodiments, the antibodies of the invention
that bind all
or a portion of the extracellular soluble domain of TR10 (e.g., one or both
cysteine rich
domains) agonize the TR10 receptor.
[0258] Antibodies of the invention may also bind fragments comprising, or
alternatively, consisting of structural or functional attributes of TR10. 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"),
hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic
regions, surface forming regions, and high antigenic index regions (i.e.,
containing four or
more contiguous amino acids having an antigenic index of greater than or equal
to 1.5, as
identified using the default parameters of the Jameson-Wolf program) of
complete (i.e.,
full-length) TR10. Certain preferred regions are those set out in Table 6 and
include, but
are not limited to, regions of the aforementioned types identified by analysis
of the amino
acid sequence depicted in (SEQ ID N0:4), such preferred regions include;
Garnier-
Robson predicted alpha-regions, beta-regions, turn-regions, and coil-regions;
Chou-
Fasman predicted alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle
predicted
hydrophilic regions; Eisenberg alpha and beta amphipathic regions; Emini
surface-forming
regions; and Jameson-Wolf high antigenic index regions, as predicted using the
default
A
parameters of these computer programs.
[0259] The data representing the structural or functional attributes of TR10
set forth in
Table 6, as described above, was generated using the various modules and
algorithms of
the DNA*STAR set on default parameters. Column I represents the results of a
Garnier-
Robson analysis of alpha helical regions; Column II represents the results of
a Chou-
Fasman analysis of alpha helical regions; Column III represents the results of
a Gamier
Robson analysis of beta sheet regions; Column IV represents the results of a
Chou-
Fasman analysis of beta sheet regions; Column V represents the results of a
Gamier
Robson analysis of turn regions; Column VI represents the results of a Chou-
Fasman
analysis of turn regions; Column VII represents the results of a Gamier Robson
analysis of
coil regions; Column VIII represents a Kyte-Doolittle hydrophilicity plot;
Column IX
represents a Hopp-Woods hydrophobicity plot; Column X represents the results
of an
Eisenberg analysis of alpha amphipathic regions; Column XI represents the
results of an
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Eisenberg analysis of beta amphipathic regions; Column XII represents the
results of a
Karplus-Schultz analysis of flexible regions; Column XIII represents the
Jameson-Wolf
antigenic index score; and Column XIV represents the Emini surface probability
plot.
[0260] In a preferred embodiment, the data presented in columns VIII, IX,
XIII, and
XIV of Table 6 can be used to determine regions of TR10 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 XIV 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.
[0261] The above-mentioned preferred regions set out in Table 6 include, but
are not
limited to, regions of the aforementioned types identified by analysis of the
amino acid
sequence set out in SEQ ID N0:4. As set out in Table 6, such preferred regions
include
Gamier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions,
Chou-Fasman
alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic
regions,
Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible
regions,
Jameson-Wolf regions of high antigenic index and Emini surface-forming
regions.
Preferably, antibodies of the present invention bind TR10 polypeptides or TR10
polypeptide fragments and variants comprising regions of TR10 that combine
several
structural features, such as several (e.g., l, 2, 3 , or 4) of the same or
different region
features set out above and in Table 6.
172
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Table 6
Res I II III IV V VI VII VIIT IX X XI XIIXIITXN
Position
Met1 . . B . . . . -0.440.93. . . -0.400.32
Gly2 . . B . . . . -0.060.93. . . -0.400.25
Leu3 . . . . T . . 0.03 0.90. . . 0.000.33
Trp4 . . . . T . . -0.430.86. . . 0.000.45
Gly5 . . . . . . C -0.260.89. . F -0.050.34
-
Gln6 . . B . . . . 0.03 0.89* . F -0.250.64
Ser7 . . B . . . . -0.210.69. . F -0.250.87
Val8 . . B . . . . 0.30 0.27* . F 0.050.89
Pro9 . . B . . . . 0.29 0.23* . F 0.050.69
Thr10 . . . . . T C 0.04 0.21. * F 0.450.69
Ala11 . . B . . T . 0.I6 0.33. * F 0.250.94
Ser12 . . B . . T . -0.13-0.31. * F 1.001.19
Ser' . . B . . T . 0.38 -0.24* . F 1.130.83
13
Ala14 . . B . . . . 0.70 -0.30* . F 1.210.81
Arg15 . . B . . T . 0.77 -0.80* . F 2.141.19
Ala16 . . B . . T . 1.14 -0.43* . F 2.121.39
Gly17 . . . . T T . 1.10 -0.39* . F 2.802.13
Arg18 . . B . . T . 0.81 -0.46* . F 2.121.08
Tyr19 . . B . . T . 1.51 0.04* . F 1.241.08
Pro20 . . B . . T . 1.09 -0.46* . F 1.562.13
Gly21 . . B . . T . 1.09 -0.40. . F 1.281.57
Ala22 . . B . . T . 1.13 0.10. . F 0.701.01
Arg23 . . B . . . . 0.68 -0.27. . F 1.250.88
Thr24 . . B . . . . 0.61 -0.27. * F 1.550.88
Ala25 . . B . . T . 0.93 -0.21. * F 2.201.25
Ser26 . . . . . T C 1.07 -0.71* * F 3.001.25
Gly27 . . . . T T . 1.37 -0.29* * F 2.601.34
Thr28 . . . . . T C 0.44 0.14* * F 1.501.40
Arg29 . . B . . T . -0.060.33. . F 0.850.86
Pro30 . . B . . T . 0.53 0.63. . F 0.250.72
Trp31 . . B . . T . 0.62 0.20* . . 0.100.83
Leu32 . . B . . T . 1.01 0.14* * . 0.100.65
Leu33 . . B . . . . 0.43 O. * * . -O.IO0.85
I4
Asp34 . . B . . T . -0.490.40* . F 0.250.56
Pro35 A . . . . T . -0.230.17. * F 0.250.56
Lys36 A . . . . T . -0.64-0.51* * F 1.301.37
Ile37 A . . . . T . -0.69-0.41* . . 0.700.71
Leu38 A . . B . . . -0.730.23* * . -0.300.34
Lys39 . . B B . . . -1.430.44* * . -0.600.13
Phe40 . . B B . . . -2.111.23* . . -0.600.16
V 41 . . B B . . . -3.011.23* . . -0.600.13
al
Val42 . . B B . . . -2.711.19* . . -0.600.05
Phe43 . . B B . . . -2.761.69* * . -0.600.06
Ile44 . . B B . . . -3.611.54* . . -0.600.06
Val45 . . B B . . . -3.721.59. . . -0.600.06
Ala46 . . B B . . . -3.081.63. . . -0.600.06
V 47 . . B B . . . -3.081.27. * . -0.600.13
al
Leu48 . . B B . . . -2.271.23* * . -0.600.13
Leu49 . . B B . . . -2.230.59. * . -0.600.26
Pro50 . . B B . . . -1.380.73. * . -0.600.26
Val51 . . B B . . . -1.090.09. * . -0.300.53
Arg52 . . B B . . . -0.82-0.21. * . 0,300.86
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Table 6 (continued)
Res I II III IV V VI VII VIII IX X XI XIIXIIIXIV
Position
Val53 . . B B . . . -032 -0.40. * . 0.300.56
Asp54 . . B B . . . -0.40-0.34. * F 0.601.09
Ser55 . . B B . . . -0.40-0.30* * F 0.450.39
Ala56 . . B B . . . 0.57 0.13* * F -0.150.81
Thr57 . . B B . . . 0.46 -0.51. * F 0.750.95
Ile58 . . B B . . . 1.31 -0.11. . F 0.601.23
Pro59 . . . . . . C 1.31 -0.50. . F 1.302.03
Arg60 . A . . T . . 0.76 -1.00. . F 1.302.44
Gln61 . A . . T . . 1.13 -0.84. . F 1.302.58
Asp62 . A . . T . . 1.44 -1.10* . F 1.302.58
Glu63 . A B . . . . 2.33 -1.13* . F 0.902.28
Val64 . A B . . . . 2.23 -0.73* . F 0.902.28
Pro65 . . B . . . . 1.27 -0.64* . F 1.101.97
Gln66 . . . B T . . 0.68 -0.00. . F 0.850.85
Gln67 . . B B . . . 0.47 0.50. . F -0.301.15
Thr68 . . B B . . . 0.47 0.29. . F 0.001.15
V 69 . . B B . . . 1.32 0.26. . F 0.221.15
al
Ala70 . . B B . . . 1.53 0.26. . F 0.441.15
Pro71 . . B B . . . 1.64 0.26. . F 0.661.38
Gln72 . . B . . . . 1.76 -0.23* . F 1.683.64
Gln73 . . B . . . . 1.77 -0.87* . F 2.207.06
Gln74 . . B . . T . 1.81 -0.99* . F 2.186.12
Arg75 . . B . . T . 2.44 -0.73* . F 1.962.91
Arg76 . . . . T T . 2.66 -1.13* . F 2.143.37
Ser77 . . . . . T C 2.66 -1.53* . F 1.723.37
Leu78 . A . . . . C 2.66 -1.93* . F 1.102.98
Lys79 . A . . . . C 1.99 -1.93* . F 1.102.63
Glu80 A A . . . . . 1.67 -1.36* . F 0.901.05
Glu81 A A . . . . . 0.97 -1.31* . F 0.901.97
Glu82 A A . . . . . 0.92 -1.50. . F 0.751.00
Cys83 A . . . . T . 1.43 -1.07. . F 1.150.57
Pro84 A . . . . T . 1.36 -0.69. . F 1.150.44
Ala85 A . . . . T . 1.47 -0.19. . F 0.850.35
.
Gly86 A . . . . T . 1.17 -0.19. * F 1.001.27
Ser87 A . . . . . . 1.17 -0.37. * F 0.801.10
His88 . . . . . . C 1.59 -0.80. * F 1.581.88
Arg89 . . B . . . . 1.49 -0.54. * F 1.662.98
Ser90 . . B . . . . 1.73 -0.49. * F 1.643.21
Glu91 . . . . T . . 1.49 -0.44. * F 2.322.33
Tyr92 . . . . T T . 1.12 -0.44. * F 2.801.20
Thr93 . . . . T T . 1.16 0.13. * F 1.770.48
Gly94 . . . . T T . 0.83 0.14* * . 1.340.45
'
Ala95 . . . . T T . 0.47 0.57. . . 1.010.44
Cys96 . . . . T . . 0.16 0.39. . . 1.080.16
Asn97 . . . . . T C 0.40 0.39. . . 1.050.24
Pro98 . . . . T T . 0.37 -0.04* . F 2.250.41
Cys99 . . . . T T . -0.14-0.11* . F 2.500.76
Thr100 . . B . . T . 0.44 -0.04* . F 1.850.35
Glu101 . . B . . . . 0.87 -0.44* . F 1.400.38
Gly102 . . B . . T . 0.56 -0.11* * F 1.501.10
Val103 . . B . . T . -0.12-0.20. . . 1.101.10
Asp104 . . B . . T . -0.04-0.00. . . 0.700.45
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Table 6 (continued)
Res III IV V VI VII VIII IX X XI XIIXIIIXIV
Position
I
II
Tyr105 . . B . . T . -0.030.50. * . -0.200.46
Thr106 . . B . . . . -0.030.46. . . -0.400.82
Ile107 . . B . . . . 0.31 0.21. * . -0.100.79
Ala108 . . B . . T . 0.36 0.61. * . -0.200.81
Ser109 . . . . T T . 0.14 0.54. . F 0.350.46
Asn110 . . . . T T . 0.09 0.49. . F 0.501.02
Asn111 . . . . T T . -0.270.19. . F 0.801.36
Leu112 . . . . . T C -0.190.26. . F 0.450.54
Pro113 . . . . T T . -0.410.56. . F 0.350.28
Sex114 . . . . T T . -0.780.84. . . 0.200.14
Cys115 . . B . . T . -1.091.01* . . -0.200.09
Leu116 . . B B . . . -1.940.81* . . -0.600.09
Leu117 . . B B . . . -1.801.03* . . -0.600.05
Cys118 . . B B . . . -1.541.21* . . -0.600.05
Thr119 . . B B . . . -1.540.64* . . -0.600.12
Val120 . . B B . . . -1.220.34* . . -0.300.19
Cys121 . . B . . T . -0.410.09* . . 0.100.35
Lys122 . . B . . T . 0.09 -0.09* . F 0.850.42
Ser123 . . . . T T . 0.76 -0.09. . F 1.590.81
Gly124 . . . . T T . 1.11 -0.33. . F 2.082.44
Gln125 . . . . T . . 1.67 -0.90. . F 2.522.44
Thr126 . . . . T . . 2.03 -0.51. . F 2.862.44
Asn127 . . . . T T . 1.32 -0.51. . F 3.403.31
Lys128 . . . . T T . 1.31 -0.37. . F 2.761.02
Ser129 . . . . T T . 1.34 -0.29. . F 2.421.02
Ser130 . . . . T T . 1.03 -0.29* . F 1.930.92
Cys131 . . B B . . . 1.46 -0.20* . F 0.790.66
Thr132 . . B B . . . 1.46 -0.20* . F 0.450.97
Thr133 . . B B . . . 1.10 -0.59* * F 0.901.21
Thr134 . . . . T T . 0.54 -0.49. . F 1.403.25
Arg135 . . . . T T . 0.18 -0.41. . F 1.401.67
Asp136 . . . . T T . 0.84 -0.33* . F 1.250.62
Thr137 . . B . . T . 0.49 -0.41* . . 0.700.75
Val138 . . B B . . . 0.80 -0.33* . . 0.610.20
Cys139 . . B B . . . 1.16 -0.33* . . 0.920.21
Gln140 . . B B . . . 0.70 -0.33* . . 1.230.29
Cys141 . . B B . . . 0.40 -0.39* . . 1.540.39
Glu142 . . . . T T . 0.01 -0.64* * F 3.100.98
Lys143 . . . . T T . 0.87 -0.43* . F 2.490.49
Gly144 . . . . T T . 1.53 -0.43. * F 2.671.58
Ser145 . . . . T T . 1.58 -1.00. * F 3.001.52
Phe146 A . . . . . . 2.24 -1.00. * F 2.431.52
Gln147 . . . . T . . 1.94 -0.60. * F 2.862.47
Asp148 . . . . T T . 1.69 -0.64. * F 3.402.47
Lys149 . . . . T T . 2.03 -0.60. * F 3.064.42
Asn150 . . . . . T C 1.73 -1.39. * F 2.524.42
Ser151 . . . . . T C 1.77 -1.17* * F 2.182.62
Pro152 . . . . T . . 1.88 -0.60* . F 1.690.70
Glu153 . . . . T . . 1.57 -0.60* . F 1.350.85
Met154 . . B B . . . 0.86 -0.51* . . 0.600.92
Cys155 . . B B . . . 0.97 -0.33* . . 0.300.32
Arg156 . . B B . . . 0.96 -0.76* . . 0.600.36
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Table 6 (continued)
Res I II III IV ' VI VII VIII IX X XI XIIXIIIXN
Position V
Thr 157 . . B B . . . 0.82 -0.27* . . 0.640.53
Cys 158 . . . . T T . 0.16 -0.46* . F 1.930.97
Arg 159 . . . . T T . 0.54 -0.46* . F 2.270.27
Thr 160 . . . . T T . 1.32 -0.03* . F 2.610.28
Gly 161 . . . . T T . 0.87 -0.51* . F 3.401.04
Cys 162 . . . . . T C 0.58 -0.66. * F 2.710.53
Pro 163 . . . . T T . 0.39 -0.04. * F 2.270.36
Arg 164 . . . . T T . 0.32 0.11* * F 1.330.27
Gly 165 . . B . . T . -0.22-0.31* * . 1.191.01
Met 166 . . B . . . . -0.18-0.24* * . 0.500.48
Val 167 . . B . . . . 0.49 -0.29* * . 0.500.33
Lys 168 . . B . . . . 0.03 0.11* * . -0.100.54
Val 169 . . B . . T . -0.390.26* * . 0.100.29
Ser 170 . . B . . T . -0.260.13* * F 0.590.57
Asn 171 . . B . . T . 0.46 -0.09* * F 1.530.44
Cys 172 . . B . . T . 1.01 -0.09* * F 2.021.16
Thr 173 . . B . . T . 0.97 -0.34* * F 2.361.16
Pro 174 . . . . T T . 0.93 -0.73* * F 3.401.20
Arg 175 . . . . T T . 1.28 -0.44* * F 2.761.57
Ser 176 . . B . T T . 0.61 -1.01. * F 2.722.18
Asp 177 . . . . T . . 1.32 -0.93. * F 2.030.76
Ile 178 . . B . . . . 1.63 -1.36. * F 1.290.77
Lys 179 . . B . . . . 1.84 -0.96. * F 0.950.93
Cys 180 . . B . . T . 1.43 -1.34. * F 1.150.96
Lys 181 . . B . . T . 1.14 -0.96. * F 1.301.83
Asn 182 A . . . . T . 0.56 -1.14. * F 1.150.93
Glu 183 A . . . . T . 1.14 -0.64. * F 1.301.75
Ser 184 A A . . . . . 0.80 -0.83. * F 1.181.17
Ala 185 A A . . . . . 1.16 -0.44. . F 1.010.98
Ala 186 A A . . . . . 0.77 -0.36. . F 1.290.81
Ser 187 A A . . . . . 0.81 0.07. . F 0.970.60
Ser 188 . . . . T T . 0.50 -0.31* . F 2.801.19
Thr 189 . . . . T T . 0.59 -0.33* * F 2.521.70
Gly 190 . . . . T T . 0.59 -0.40. . F 2.241.96
Lys 191 . . . . . T C 0.59 -0.29. . F 1.761.48
Thr 192 . A . . . . C 0.89 -0.17. . F 1.081.03
Pro 193 . A . . . . C 1.19 -0.66. . F 1.101.81
Ala 194 A A . . . . . 1.19 -1.09. . F 0.901.57
Ala 195 A A . . . . . 0.68 -0.60. . F 0.901.57
Glu 196 A A . . . . . 0.32 -0.44. . F 0.450.75
Glu 197 A A . B . . . 0.32 -0.39* . F 0.601.07
Thr 198 A A . B . . . -0.36-0.40* . F 0.601.53
Val 199 A . . B . . . -0.58-0.21* . F 0.450.62
Thr 200 A . . B . . . -0.330.47* . F -0.450.30
Thr 201 A . . B . . . -0.930.90* . . -0.600.20
Ile 202 A A . B . . . -1.741.03* . . -0.600.27
Leu 203 . A B B . . . -2.021.07* . . -0.600.15
Gly 204 . A B . . . . -1.471.09* . . -0.600.11
Met 205 . A B . . . . -1.370.99. . . -0.600.21
Leu 206 . A B . . . . -1.300.73* . . -0.600.39
Ala 207 . A B . . . . -0.440.80. . . -0.600.61
Ser 208 . . . . . T C 0.12 0.87. . . 0.000.84
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Table 6 (continued)
Res I II III IV V VI VIIVIII IX X XI XII XIIIXIV
Position
Pro 209 A . . . . T . -0.341.01 . . . -0.051.60
Tyr 210 A . . . . T . -0.631.01 . . . -0.051.31
His 211 . . B . . T . -0.711.20 . . . -0.200.68
Tyr 212 . . B B . . . -1.011.50 . . . -0.600.31
Leu 213 . . B B . . . -1.571.76 . . . -0.600.14
Ile 214 . . B B . . . -2.211.64 . . . -0.600.08
Ile 215 . . B B . . . -2.781.79 . . . -0.600.04
Ile 216 . . B B . . . -3.601.71 . . -0.600.04
V 217 . . B B . . . -4.241.67 . . . -0.600.04
al
Val 218 . . B B . . . -4.321.67 . . . -0.600.04
Leu 219 . B B . . . -4.241.67 . . . -0.600.04
Val 220 . . B B . . . -3.941.67 . . . -0.600.04
Ile 221 . . B B . . . -3.911.53 . . . -0.600.06
Ile 222 . . B B . . . -3.911.53 . . . -0.600.05
Leu 223 . . B B . . . -3.911.49 . . . -0.600.05
Ala 224 . . B B . . . -3.961.49 . . . -0.600.05
Val 225 . . B B . . . -3.441.44 . . . -0.600.06
Val 226 . . B B . . . -3.261.19 . . . -0.600.07
Val 227 . . B B . . . -2.671.29 . . . -0.600.06
V 228 . . B B . . . -2.521.17 . * . -0.600.11
al
Gly 229 . . B B . . . -1.821.10 . * . -0.600.08
Phe 230 A . . . . T . -0.920.46 . * . -0.200.21
Ser 231 A . . . . T . -0.02-0.19. * . 0.700.55
Cys 232 A . . , . T . 0.13 -0.83. * . 1.151.12
Arg 233 A . . , . T . 0.10 -0.47. * F 1.001.12
Lys 234 . A . B T . . 0.14 -0.57. * F 1.150.58
Lys 235 A A . B . . . 0.60 -0.57* * F 0.901.46
Phe 236 . A B B . . . 0.09 -0.39* * . 0.451.17
Ile 237 . A B B . . . 0.80 0.30 * * -0.300.48
Ser 238 . . B B . . . 0.34 0.30 * * . -0.300.48
Tyr 239 . . B B . . . -0.590.73 * . . -0.600.55
Leu 240 . . B B . . . -1.300.63 * * . -0.600.55
Lys 241 . . B B . . . -0.900.51 * . . -0.600.22
Gly 242 . . B B . . . -0.360.51 * . . -0.600.19
Ile 243 . . B B . . . -0.400.19 * . . -0.300.23
Cys 244 . . B . . T . -0.50-0.07* . . 0.700.11
Ser 245 . . B . . T . -0.030.36 * . F 0.250.11
Gly 246 . . . . T T . -0.420.36 * . F 0.650.16
Gly 247 . . . . T T . -0.290.10 . . F 0.920.29
Gly 248 . . . . . . C 0.60 -0.04* . F 1.390.34
Gly 249 . . . . . . C 1.38 -0.43* * F 1.660.59
Gly 250 . . . . . T C 0.82 -0.86* * F 2.581.17
Pro 251 . . . . . T C 1.13 -0.64* . F 2.700.87
Glu 252 . . B . . T . 1.59 -0.57* . F 2.381.20
Arg 253 . . B . . T . 1.08 -1.00* . F 2.112.38
Val 254 . . B B . . . 0.61 -0.79* . . 1.291.14
His 255 . . B B . . . 0.26 -0.53* * . 0.870.54
Arg 256 . . B B . . . 0.58 0.26 * . . -0.300.24
Val 257 . . B B . . . 0.69 0.26 * * . -0.300.63
Leu 258 . . B B . . . 0.69 -0.39* * . 0.640.91
Phe 259 . . B B . . . 1.24 -0.89* * . 1.280.91
Arg 260 . . B . : T . 0.61 -0.50. * . 1.871.65
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Table 6 (continued)
Res I II III IV V VI VIIVIII IX X XI XII XIIIXTV
Position
Arg 261 . . . . T T . 0.29 -0.57. * F 3.061.07
Arg 262 . . . . T T . 0.84 -0.83. * F 3.401.91
Ser 263 . . . . T T . 1.77 -1.23* * F 3.061.31
Cys 264 . . . . . T C 1.61 -1.23. * F 2.521.31
Pro 265 . . . . T T . 1.29 -0.59. * ' 2.230.50
F
Ser 266 . . . . T T . 0.83 -0.16. * F 1.860.57
Arg 267 . . B . . T . 0.13 -0.11* . F 1.541.06
Val 268 . . B . . T . 0.43 -0.19. * F 1.660.69
Pro 269 . . B . . T . 1.10 -0.61. * F 2.230.89
Gly 270 . . . . . T C 1.31 -1.00* * F 2.700.76
Ala 271 A . B . . T . 1.02 -0.60. * F 2.381.65
Glu 272 A . . . . . . 1.02 -0.74* * F 1.911.08
Asp 273 A . . . . . . 1.88 -1.17* * F 1.642.13
Asn 274 A . . . . T . 2.09 -1.20* * F 1.573.39
Ala 275 A . . . . T . 2.12 -1.70. * F 1.303.39
Arg 276 A . . . . T . 1.90 -1.21. * F 1.642.93
Asn 277 A . . . . T . 1.60 -0.53. * F 1.981.50
Glu 278 A . . . . . . 1.60 -0.54. * F 2.121.99
Thr 279 A . . . . . . 1.71 -0.64. * F 2.461.64
Leu 280 . . . . T T . 2.06 -0.64* . F 3.401.99
Ser 281 . . . . T T . 1.13 -0.29* . F 2.761.80
Asn 282 . . . . T T . 1.13 0.40 * . F 1.521.03
Arg 283 . . . . T T . 0.92 0.31 * . F 1.482.17
Tyr 284 . . . . T . . 0.92 0.06 * . F 0.942.50
Leu 285 . . B . . . . 1.73 0.16 * . F 0.202.24
Gln 286 . . B . . T . 1.18 0.16 . . F 0.401.98
Pro 287 . . . . . T C 0.88 0.80 . . F 0.150.94
Thr 288 . . . . . T C 0.77 0.43 . . F 0.301.53
Gln 289 . . B . . T . 1.01 -0.26. . F 1.001.53
Val 290 . A B . . . . 1.82 -0.26. . F 0.601.71
Ser 291 . A B . . . . 0.93 -0.69* . F 0.902.05
Glu 292 . A B . . . . 1.14 -0.49* * F 0.450.83
Gln 293 . A B . . . . 1.11 -0.49. * F 0.601.94
Glu 294 A A . . . . . 1.11 -0.70. * F 0.901.43
Ile 295 A A . . . . . 1.97 -0.69. . F 0.901.43
Gln 296 A A . . . . . 1.46 -0.69. * F 0.901.43
Gly 297 A A . . . . . 0.87 -0.40. * F 0.450.68
Gln 298 A A . . . . . 0.87 0.10 . * F -0.150.98
Glu 299 A A . . . . . 0.06 -0.59. * F 0.750.98
Leu 300 A A . . . . . 0.63 -0.30. * F 0.450.82
Ala 301 A A . . . . . 0.29 -0.24. . . 0.300.68
Glu 302 A A . . . . . -0.22-0.21. . . 0.300.39
,
Leu 303 A A . B . . . -0.530.43 . . . -0.600.35
Thr 304 A A . B . . . -1.390.23 . . . -0.300.50
Gly 305 A A . B . . . -0.580.37 . . . -0.300.21
Val 306 . . B B . . . -0.290.37 . . . -0.300.45
Thr 307 . . B B . . . -0.500.07 . . F 0.150.42
Val 308 . . . B . . C 0.31 0.01 . . F 0.650.65
Glu 309 . . . B . . C 0.62 -0.41. . F 1.701.53
Ser 310 . . . . . T C 0.76 -1.06. * F 2.701.83
Pro 311 . . . . . T C 1.61 -1.11* * F 3.003.82
Glu 312 A . . . . T . 2.03 -1.36* * F 2.503.82
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Table 6 (continued)
Res I II IIIIV V VI VII VIII IX X XI XII XIIIXN
Position
Glu 313 A . . . . T . 2.08 -1.36* . F 2.205.58
Pro 314 A . . . . . . 1.27 -1.06* . F 1.702.97
Gln 315 A A . . . . . 1.57 -0.80* . F 1.201.42
Arg 316 A A . . . . . 1.78 -0.80* . F 0.901.42
Leu 317 A A . . . . . 1.19 -0.40* * F 0.601.59
Leu 318 A A . . . . . 1.19 -0.33* * F 0.450.93
Glu 319 A A . . . . . 0.81 -0.73* * F 0.750.82
Gln 320 A A . . . . . 0.81 -0.23* * F 0.601.00
Ala 321 A A . . . . . 0.36 -0.91* * F 0.902.10
Glu 322 A A . . . . . 0.50 -1.17* . F 0.901.20
Ala 323 A A . . . . . 1.31 -0.60* * F 0.750.37
Glu 324 A A . . . . . 1.42 -0.60. * F 0.750.64
Gly 325 A A . . . . . 1.53 -1.10. * F 0.750.72
Cys 326 A A . . . . . 223 -1.10. * F 0.901.40
Gln 327 A A . . . . . 1.42 -1.60. * F 0.901.58
Arg 328 A A . . . . . 1.20 -0.91. * F 0.901.32
Arg 329 . A B B . . . 0.34 -0.66. * F 0.902.03
Arg 330 . A B B . . . 0.48 -0.59. * . 0.600.87
Leu 331 . A B B . . . 0.29 -0.56. * . 0.600.69
Leu 332 . A B B . . . 0.29 0.09 * * . -0.300.26
Val 333 . A B B . . . 0.18 0.49 * * . -0.600.21
Pro 334 . A B . . . . -0.52 0.49 * . . -0.600.43
Val 335 . . B . . . . -0.63 0.30 * * . -0.100.53
Asn 336 . . B . . . . -0.12 -0.39. . F 0.801.19
Asp 337 A . . . . T . 0.10 -0.64. . F 1.301.03
Ala 338 A . . . . T . 0.96 -0.57. . F 1.301.41
Asp 339 A . . . T . 0.28 -1.21. . F 1.301.46
Ser 340 A . . . . T . 0.83 -0.93. . F 1.150.61
Ala 341 A . . o . . . . 0.52 -0.54. . F 0.950.81
Asp 342 A . . B . . . -0.29 -0.56. . F 0.750.70
Ile 343 A . . B . . . -0.51 0.13 . * F -0.150.43
Ser 344 . A B B . . . -0.51 0.43 . * F -0.450.35
Thr 345 . A B B . . . -0.80 -0.07. * F 0.450.35
Leu 346 A A . B . . . -0.51 0.43 * * . -0.600.51
Leu 347 A A . B . . . -1.10 0.13 * * . -0.300.51
Asp 348 A A . B . . . -0.52 0.24 * * . -0.300.36
Ala 349 A A . . . . . -1.03 0.24 . * . -0.300.62
Ser 350 A A . . . . . -0.72 0.24 * * . -0.300.62
Ala 351 A A . . . . . 0.09 -0.44* * . 0.300.65
Thr 352 A A . . . . . 0.56 -0.44. * . 0.451.11
Leu 353 A A . . . . . 0.52 -0.51. * F 0.750.82
Glu 354 A A . . . . . 0.52 -0.40. * F 0.601.10
Glu 355 A A . . . . . 0.87 -0.40. . F 0.450.77
Gly 356 A A . . . . . 1.46 -0.89. . F 0.901.87
.
His 357 A A . . . . . 1.46 -1.57. * F 0.901.87
Ala 358 A A . . . . . 1.38 -1.09. * F 0.901.56
Lys 359 A A . . . . . 1.38 -0.40. * F 0.601.10
Glu 360 A A . . . . . 1.38 -0.43. * F 0.601.41
Thr 361 A A . . . . . 1.72 -0.93* * F 0.902.32
Ile 362 A A . . . . . 0.94 -1.03. * F 0.902.01
Gln 363 A A . . . . . 0.68 -0.34. * F 0.450.96
Asp 364 A A . . . . . 0.29 0.30 . * F -0.150.49
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Table 6 (continued)
Res I II III IV V VI VII VIIIIX X XI XII XIIIXIV
Position
Gln 365 A A . . . . . -0.01 0.24 . . F 0.060.70
Leu 366 . A B . . . . 0.30 -0.06* * F 0.870.54
Val 367 . A B . . . . 1.23 -0.46* * F 1.080.56
Gly 368 . . . . . T C 0.42 -0.46* . F 1.890.64
Ser 369 . . . . . T C -0.28 -0.17* . F 2.100.64
Glu 370 A . . . . T . -0.52 -0.07. * F 1.690.75
Lys 371 A . . . . T . 0.29 0.04 . . F 1.031.19
Leu 372 A A . . . . . 1.14 -0.39. . F 1.021.54
Phe 373 A A . . . . . 1.49 -0.77* . . 0.961.54
Tyr 374 A A . . . . . 1.79 -0.77* . . 0.751.28
Glu 375 A A . . . . . 1.20 -0.77* . F 0.902.70
Glu 376 A A . . . . . 0.81 -0.96* . F 0.903.15
Asp 377 A A . . . . . 1.32 -1.31. . F 0.901.99
Glu 378 A . . . . T . 1.43 -1.69. . F 1.301.54
Ala 379 A . . . . T . 1.37 -1.19. . F 1.150.90
Gly 380 A . . . . T . 1.07 -0.70. . F 1.150.78
Ser 381 A . . . . T . 0.40 -0.31* . F 0.850.60
Ala 382 A . . . . T . -0.41 0.26 * . F 0.250.32
Thr 383 A . . . . T . -0.80 0.44 . . F -0.050.27
Ser 384 A . . . . T . -0.60 0.44 . . F -0.050.25
Cys 385 . . B . . T . -0.64 0.49 . . . -0.200.32
Leu 386 . . B . . . . -0.73 0.41 . . . -0.400.28
180
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[0262] In another aspect, the invention provides an antibody that binds a
peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide described
herein. The
epitope of this polypeptide portion is an immunogenic or antigenic epitope of
a
polypeptide of the invention. 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 moxlecule 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 51:3998- 4002 (1983).
[0263] As to the selection of peptides or polypeptides bearing an antigenic
epitope
(i.e., that contain a region of a protein molecule to which an antibody can
bind), it is well
known in that art that relatively short synthetic peptides that mimic part of
a protein
sequence are routinely capable of eliciting an antiserum that reacts with the
partially
mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green,
N. and
Learner, R.A. (1983) Antibodies that react with predetermined sites on
proteins. Science
219: 660-666. Peptides capable of eliciting protein-reactive sera are
frequently represented
in the primary sequence of a protein, can be characterized by a set of simple
chemical
rules, and are confined neither to immunodominant regions of intact proteins
{i.e.,
immunogenic epitopes) nor to the amino or carboxyl terminals.
[0264] Antigenic epitope-bearing peptides and polypeptides are therefore
useful to
raise antibodies, including monoclonal antibodies, that bind to a TR10
polypeptide of the
invention. See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777.
Antigenic
epitope-bearing peptides and polypeptides 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 SEQ ID N0:4.
[0265] Antibodies of the invention may bind one or more antigenic TR10
polypeptides
or peptides including, but not limited to: a polypeptide comprising amino acid
residues
from about 57 to about 113 of SEQ ID N0:4; a polypeptide comprising amino acid
residues from about 130 to about 197 of SEQ ID N0:4; a polypeptide comprising
amino
acid residues from about 75 to about 142 of SEQ )D N0:4; a polypeptide
comprising
amino acid residues from about 280 to about 283 of SEQ ID N0:4; and/or a
polypeptide
comprising amino acid residues from about 195 to about 228 of SEQ ID N0:4. In
this
context "about" includes the particularly recited range, larger or smaller by
several (5, 4,
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3, 2, or 1) amino acids, at either terminus or at both termini. As indicated
above, the
inventors have determined that the above polypeptide fragments are antigenic
regions of
the TR10 protein.
[0266] Epitope-bearing TR10 peptides and polypeptides may be produced by any
conventional means. Houghten, R.A., "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 X2:5131-5135 (1985). This
"Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described
in U.S.
Patent No. 4,631,211 to Houghten et al. (1986).
[0267] As one of skill in the art will appreciate, TR10 polypeptides and the
epitope-bearing fragments thereof described herein (e.g., corresponding to a
portion of the
extracellular domain such as, for example, amino acid residues 56 to 212 of
SEQ ID N0:4
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 TR10 protein or protein fragment alone
(Fountoulakis et
al., J Biochem 270:3958-3964 (1995)). Thus, antibodies of the invention may
bind fusion
proteins that comprise all or a portion of a TRAIL receptor polypeptide such
as TR10.
[0268] 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. Antibodies of the
present invention
may also bind such modified TR10 polypeptides or TR10 polypeptide fragments or
variants.
[0269] For instance, 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
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without substantial loss of biological function, or loss of the ability to be
bound by a
specific antibody. For instance, Ron et al., J. Biol. Cherrz., 268:2984-2988
(1993) reported
modified KGF proteins that had heparin binding activity even if 3, 8, or 27
amino-terminal
amino acid residues were missing.
[0270] As mentioned above, even if deletion of one or more amino acids from
the
N-terminus of a protein results in modification of loss of one or more
biological functions
of the protein, other functional activities (e.g., biological activities,
ability to multimerize,
ability to bind TR10 ligand) may still be retained. For example, the ability
of shortened
TR10 polypeptides to induce and/or bind to antibodies which recognize the
complete or
mature forms of the polypeptides generally will be retained when less than the
majority of
the residues of the complete or mature polypeptide are removed from the N-
terminus.
Whether a particular polypeptide lacking N-terminal residues of a complete
polypeptide
retains such immunologic activities can readily be determined by routine
methods
described herein and otherwise known in the art. It is not unlikely that a
TR10
polypeptide 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
TR10 amino acid residues may often evoke an immune response.
[0271] Accordingly, the present invention further provides antibodies that
bind
polypeptides having one or more residues deleted from the amino terminus of
the TR10
amino acid sequence SEQ ID NO:4 up to the alanine residue at position number
382 and
polynucleotides encoding such polypeptides. In particular, the present
invention provides
polypeptides comprising the amino acid sequence of residues n7-386 of SEQ ID
N0:4,
where n7 is an integer from 2 to 381 corresponding to the position of the
amino acid
residue in SEQ ~ N0:4.
[0272] More in particular, the invention provides antibodies that bind
polypeptides
comprising, or alternatively consisting of, the amino acid sequence of
residues of G-2 to
L-386; L-3 to L-386; W-4 to L-386; G-5 to L-386; Q-6 to L-386; S-7 to L-386; V-
8 to L-
386; P-9 to L-386; T-10 to L-386; A-11 to L-386; S-12 to L-386; S-13 to L-386;
A-14 to
L-386; R-15 to L-386; A-16 to L-386; G-17 to L-386; R-18 to L-386; Y-19 to L-
386; P-20
to L-386; G-21 to L-386; A-22 to L-386; R-23 to L-386; T-24 to L-386; A-25 to
L-386; S-
26 to L-386; G-27 to L-386; T-28 to L-386; R-29 to L-386; P-30 to L-386; W-31
to L-386;
L-32 to L-386; L-33 to L-386; D-34 to L-386; P-35 to L-386; K-36 to L-386; I-
37 to L-
386; L-38 to L-386; K-39 to L-386; F-40 to L-386; V-41 to L-386; V-42 to L-
386; F-43 to
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L-386; I-44 to L-386; V-45 to L-386; A-46 to L-386; V-47 to L-386; L-48 to L-
386; L-49
to L-386; P-50 to L-386; V-51 to L-386; R-52 to L-386; V-53 to L-386; D-54 to
L-386; S-
55 to L-386; A-56 to L-386; T-57 to L-386; I-58 to L-386; P-59 to L-386; R-60
to L-386;
Q-61 to L-386; D-62 to L-386; E-63 to L-386; V-64 to L-386; P-65 to L-386; Q-
66 to L-
386; Q-67 to L-386; T-68 to L-386; V-69 to L-386; A-70 to L-386; P-71 to L-
386; Q-72 to
L-386; Q-73 to L-386; Q-74 to L-386; R-75 to L-386; R-76 to L-386; S-77 to L-
386; L-78
to L-386; K-79 to L-386; E-80 to L-386; E-81 to L-386; E-82 to L-386; C-83 to
L-386; P-
84 to L-386; A-85 to L-386; G-86 to L-386; S-87 to L-386; H-88 to L-386; R-89
to L-386;
S-90 to L-386; E-91 to L-386; Y-92 to L-386; T-93 to L-386; G-94 to L-386; A-
95 to L-
386; C-96 to L-386; N-97 to L-386; P-98 to L-386; C-99 to L-386; T-100 to L-
386; E-101
to L-386; G-102 to L-386; V-103 to L-386; D-104 to L-386; Y-105 to L-386; T-
106 to L-
386; I-107 to L-386; A-108 to L-386; S-109 to L-386; N-110 to L-386; N-111 to
L-386;
L-112 to L-386; P-113 to L-386; S-114 to L-386; C-115 to L-386; L-116 to L-
386; L-117
to L-386; C-118 to L-386; T-119 to L-386; V-120 to L-386; C-121 to L-386; K-
122 to L-
386; S-123 to L-386; G-124 to L-386; Q-125 to L-386; T-126 to L-386; N-127 to
L-386;
K-128 to L-386; S-129 to L-386; S-130 to L-386; C-131 to L-386; T-132 to L-
386; T-133
to L-386; T-134 to L-386; R-135 to L-386; D-136 to L-386; T-137 to L-386; V-
138 to L-
386; C-139 to L-386; Q-140 to L-386; C-141 to L-386; E-142 to L-386; K-143 to
L-386;
G-144 to L-386; S-145 to L-386; F-146 to L-386; Q-147 to L-386; D-148 to L-
386; K-149
to L-386; N-150 to L-386; S-151 to L-386; P-152 to L-386; E-153 to L-386; M-
154 to L-
386; C-155 to L-386; R-156 to L-386; T-157 to L-386; C-158 to L-386; R-159 to
L-386;
T-160 to L-386; G-161 to L-386; C-162 to L-386; P-163 to L-386; R-164 to L-
386; G-165
to L-386; M-166 to L-386; V-167 to L-386; K-168 to L-386; V-169 to L-386; S-
170 to L-
386; N-171 to L-386; C-172 to L-386; T-173 to L-386; P-174 to L-386; R-175 to
L-386;
S-176 to L-386; D-177 to L-386; I-178 to L-386; K-179 to L-386; C-180 to L-
386; K-181
to L-386; N-182 to L-386; E-183 to L-386; S-184 to L-386; A-185 to L-386; A-
186 to L-
386; S-187 to L-386; S-188 to L-386; T-189 to L-386; G-190 to L-386; K-191 to
L-386;
T-192 to L-386; P-193 to L-386; A-194 to L-386; A-195 to L-386; E-196 to L-
386; E-197
to L-386; T-198 to L-386; V-199 to L-386; T-200 to L-386; T-201 to L-386; I-
202 to L-
386; L-203 to L-386; G-204 to L-386; M-205 to L-386; L-206 to L-386; A-207 to
L-386;
S-208 to L-386; P-209 to L-386; Y-210 to L-386; H-211 to L-386; Y-212 to L-
386; L-213
to L-386; I-214 to L-386; I-215 to L-386; I-216 to L-386; V-217 to L-386; V-
218 to L-
386; L-219 to L-386; V-220 to L-386; I-221 to L-386; I-222 to L-386; L-223 to
L-386; A-
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WO 02/079377 PCT/USO1/42996
224 to L-386; V-225 to L-386; V-226 to L-386; V-227 to L-386; V-228 to L-386;
G-229
to L-386; F-230 to L-386; S-231 to L-386; C-232 to L-386; R-233 to L-386; K-
234 to L-
386; K-235 to L-386; F-236 to L-386; I-237 to L-386; S-238 to L-386; Y-239 to
L-386; L-
240 to L-386; K-241 to L-386; G-242 to L-386; I-243 to L-386; C-244 to L-386;
S-245 to
L-386; G-246 to L-386; G-247 to L-386; G-248 to L-386; G-249 to L-386; G-250
to L-
386; P-251 to L-386; E-252 to L-386; R-253 to L-386; V-254 to L-386; H-255 to
L-386;
R-256 to L-386; V-257 to L-386; L-258 to L-386; F-259 to L-386; R-260 to L-
386; R-261
to L-386; R-262 to L-386; S-263 to L-386; C-264 to L-386; P-265 to L-386; S-
266 to L-
386; R-267 to L-386; V-268 to L-386; P-269 to L-386; G-270 to L-386; A-271 to
L-386;
E-272 to L-386; D-273 to L-386; N-274 to L-386; A-275 to L-386; R-276 to L-
386; N-277
to L-386; E-278 to L-386; T-279 to L-386; L-280 to L-386; S-281 to L-386; N-
282 to L-
386; R-283 to L-386; Y-284 to L-386; L-285 to L-386; Q-286 to L-386; P-287 to
L-386;
T-288 to L-386; Q-289 to L-386; V-290 to L-386; S-291 to L-386; E-292 to L-
386; Q-293
to L-386; E-294 to L-386; I-295 to L-386; Q-296 to L-386; G-297 to L-386; Q-
298 to L-
386; E-299 to L-386; L-300 to L-386; A-301 to L-386; E-302 to L-386; L-303 to
L-386;
T-304 to L-386; G-305 to L-386; V-306 to L-386; T-307 to L-386; V-308 to L-
386; E-309
to L-386; S-310 to L-386; P-311 to L-386; E-312 to L-386; E-313 to L-386; P-
314 to L-
386; Q-315 to L-386; R-316 to L-386; L-317 to L-386; L-318 to L-386; E-319 to
L-386;
Q-320 to L-386; A-321 to L-386; E-322 to L-386; A-323 to L-386; E-324 to L-
386; G-325
to L-386; C-326 to L-386; Q-327 to L-386; R-328 to L-386; R-329 to L-386; R-
330 to L-
386; L-331 to L-386; L-332 to L-386; V-333 to L-386; P-334 to L-386; V-335 to
L-386;
N-336 to L-386; D-337 to L-386; A-338 to L-386; D-339 to L-386; S-340 to L-
386; A-341
to L-386; D-342 to L-386; I-343 to L-386; S-344 to L-386; T-345 to L-386; L-
346 to L-
386; L-347 to L-386; D-348 to L-386; A-349 to L-386; S-350 to L-386; A-351 to
L-386;
T-352 to L-386; L-353 to L-386; E-354 to L-386; E-355 to L-386; G-356 to L-
386; H-357
to L-386; A-358 to L-386; K-359 to L-386; E-360 to L-386; T-361 to L-386; I-
362 to L-
386; Q-363 to L-386; D-364 to L-386; Q-365 to L-386; L-366 to L-386; V-367 to
L-386;
G-368 to L-386; S-369 to L-386; E-370 to L-386; K-371 to L-386; L-372 to L-
386; F-373
to L-386; Y-374 to L-386; E-375 to L-386; E-376 to L-386; D-377 to L-386; E-
378 to L-
386; A-379 to L-386; G-380 to L-386; and/or S-381 to L-386 of the TR10
sequence shown
in SEQ ID N0:4.
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[0273] In another embodiment, N-terminal deletions of the TR10 polypeptide can
be
described by the general formula n$-212, where n$ is a number from 2 to 207,
corresponding to the position of amino acid identified in SEQ ID N0:4.
[0274] In specific embodiments, antibodies of the invention bind N terminal
deletions of the TR10 comprising, or alternatively consisting of, the amino
acid sequence
of residues: G-2 to Y-212; L-3 to Y-212; W-4 to Y-212; G-5 to Y-212; Q-6 to Y-
212; S-7
to Y-212; V-8 to Y-212; P-9 to Y-212; T-10 to Y-212; A-11 to Y-212; S-12 to Y-
212; S-
13 to Y-212; A-14 to Y-212; R-15 to Y-212; A-16 to Y-212; G-17 to Y-212; R-18
to Y-
212; Y-19 to Y-212; P-20 to Y-212; G-21 to Y-212; A-22 to Y-212; R-23 to Y-
212; T-24
to Y-212; A-25 to Y-212; 5-26 to Y-212; G-27 to Y-212; T-28 to Y-212; R-29 to
Y-212;
P-30 to Y-212; W-31 to Y-212; L-32 to Y-212; L-33 to Y-212; D-34 to Y-212; P-
35 to Y-
212; K-36 to Y-212; I-37 to Y-212; L-38 to Y-212; K-39 to Y-212; F-40 to Y-
212; V-41
to Y-212; V-42 to Y-212; F-43 to Y-212; I-44 to Y-212; V-45 to Y-212; A-46 to
Y-212;
V-47 to Y-212; L-48 to Y-212; L-49 to Y-212; P-50 to Y-212; V-51 to Y-212; R-
52 to Y-
212; V-53 to Y-212; D-54 to Y-212; S-55 to Y-212; A-56 to Y-212; T-57 to Y-
212; I-58
to Y-212; P-59 to Y-212; R-60 to Y-212; Q-61 to Y-212; D-62 to Y-212; E-63 to
Y-212;
V-64 to Y-212; P-65 to Y-212; Q-66 to Y-212; Q-67 to Y-212; T-68 to Y-212; V-
69 to Y-
212; A-70 to Y-212; P-71 to Y-212; Q-72 to Y-212; Q-73 to Y-212; Q-74 to Y-
212; R-75
to Y-212; R-76 to Y-212; S-77 to Y-212; L-78 to Y-212; K-79 to Y-212; E-80 to
Y-212;
E-81 to Y-212; E-82 to Y-212; C-83 to Y-212; P-84 to Y-212; A-85 to Y-212; G-
86 to Y-
212; S-87 to Y-212; H-88 to Y-212; R-89 to Y-212; S-90 to Y-212; E-91 to Y-
212; Y-92
to Y-212; T-93 to Y-212; G-94 to Y-212; A-95 to Y-212; C-96 to Y-212; N-97 to
Y-212;
P-98 to Y-212; C-99 to Y-212; T-100 to Y-212; E-101 to Y-212; G-102 to Y-212;
V-103
to Y-212; D-104 to Y-212; Y-105 to Y-212; T-106 to Y-212; I-107 to Y-212; A-
108 to Y-
212; S-109 to Y-212; N-110 to Y-212; N-111 to Y-212; L-112 to Y-212; P-113 to
Y-212;
S-114 to Y-212; C-115 to Y-212; L-116 to Y-212; L-117 to Y-212; C-118 to Y-
212; T-
119 to Y-212; V-120 to Y-212; C-121 to Y-212; K-122 to Y-212; S-123 to Y-212;
G-124
to Y-212; Q-125 to Y-212; T-126 to Y-212; N-127 to Y-212; K-128 to Y-212; S-
129 to Y-
212; S-130 to Y-212; C-131 to Y-212; T-132 to Y-212; T-133 to Y-212; T-134 to
Y-212;
R-135 to Y-212; D-136 to Y-212; T-137 to Y-212; V-138 to Y-212; C-139 to Y-
212; Q-
140 to Y-212; C-141 to Y-212; E-142 to Y-212; K-143 to Y-212; G-144 to Y-212;
S-145
to Y-212; F-146 to Y-212; Q-147 to Y-212; D-148 to Y-212; K-149 to Y-212; N-
150 to
Y-212; S-151 to Y-212; P-152 to Y-212; E-153 to Y-212; M-154 to Y-212; C-155
to Y-
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212; R-156 to Y-212; T-157 to Y-212; C-158 to Y-212; R-159 to Y-212; T-160 to
Y-212;
G-161 to Y-212; C-162 to Y-212; P-163 to Y-212; R-164 to Y-212; G-165 to Y-
212; M-
166 to Y-212; V-167 to Y-212; K-168 to Y-212; V-169 to Y-212; S-170 to Y-212;
N-171
to Y-212; C-172 to Y-212; T-173 to Y-212; P-174 to Y-212; R-175 to Y-212; S-
176 to Y-
212; D-177 to Y-212; I-178 to Y-212; K-179 to Y-212; C-180 to Y-212; K-181 to
Y-212;
N-182 to Y-212; E-183 to Y-212; S-184 to Y-212; A-185 to Y-212; A-186 to Y-
212; 5-
187 to Y-212; S-188 to Y-212; T-189 to Y-212; G-190 to Y-212; K-191 to Y-212;
T-192
to Y-212; P-193 to Y-212; A-194 to Y-212; A-195 to Y-212; E-196 to Y-212; E-
197 to Y-
212; T-198 to Y-212; V-199 to Y-212; T-200 to Y-212; T-201 to Y-212; I-202 to
Y-212;
L-203 to Y-212; G-204 to Y-212; M-205 to Y-212; L-206 to Y-212; and/or A-207
to Y-
212 of the TR10 extracellular domain sequence shown in SEQ ID N0:4.
[0275] Also as mentioned above, even if deletion of one or more amino acids
from the
C-terminus of a protein results in modification of loss of one or more
biological functions
of the protein, other functional activities (e.g., biological activities
(e.g., ability to inhibit
TRAIL induced cell death ifZ vivo or in vitro, and/or regulate (e.g., inhibit)
B cell
proliferation, and/or regulate hematopoiesis), ability to multimerize, ability
to bind TR10
ligand (e.g., TRAIL, and/or ligands on the surface of NK cells and/or
endothelial cells)
may still be retained. For example the ability of the shortened TR10
polypeptide 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 TR10 polypeptide with a large number of
deleted
C-terminal amino acid residues may retain some biological or immunogenic
activities. In
fact, peptides composed of as few as six TR10 amino acid residues may often
evoke an
immune response.
[0276] Accordingly, the present invention further provides bantibodies that
bind
polypeptides having one or more residues deleted from the carboxy terminus of
the amino
acid sequence of the TR10 polypeptide shown in SEQ ll~ N0:4, up to the
glutamine
residue at position number 61, and polynucleotides encoding such polypeptides.
In
particular, the present invention provides polypeptides comprising the amino
acid
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CA 02426710 2003-04-23
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sequence of residues 56-m7 of SEQ ID N0:4, where m' is an integer from 61 to
385
corresponding to the position of the amino acid residue in SEQ ID N0:4.
[0277] More in particular, the invention provides antibodies that bind
polypeptides
comprising, or alternatively consisting of, the amino acid sequence of
residues A-56 to C-
385; A-56 to S-384; A-56 to T-383; A-56 to A-382; A-56 to S-381; A-56 to G-
380; A-56
to A-379; A-56 to E-378; A-56 to D-377; A-56 to E-376; A-56 to E-375; A-56 to
Y-374;
A-56 to F-373; A-56 to L-372; A-56 to K-371; A-56 to E-370; A-56 to S-369; A-
56 to 6-
368; A-56 to V-367; A-56 to L-366; A-56 to Q-365; A-56 to D-364; A-56 to Q-
363; A-56
to I-362; A-56 to T-361; A-56 to E-360; A-56 to K-359; A-56 to A-358; A-56 to
H-357;
A-56 to G-356; A-56 to E-355; A-56 to E-354; A-56 to L-353; A-56 to T-352; A-
56 to A-
351; A-56 to S-350; A-56 to A-349; A-56 to D-348; A-56 to L-347; A-56 to L-
346; A-56
to T-345; A-56 to S-344; A-56 to I-343; A-56 to D-342; A-56 to A-341; A-56 to
S-340; A-
56 to D-339; A-56 to A-338; A-56 to D-337; A-56 to N-336; A-56 to V-335; A-56
to P-
334; A-56 to V-333; A-56 to L-332; A-56 to L-331; A-56 to R-330; A-56 to R-
329; A-56
to R-328; A-56 to Q-327; A-56 to C-326; A-56 to G-325; A-56 to E-324; A-56 to
A-323;
A-56 to E-322; A-56 to A-321; A-56 to Q-320; A-56 to E-319; A-56 to L-318; A-
56 to L-
317; A-56 to R-316; A-56 to Q-315; A-56 to P-314; A-56 to E-313; A-56 to E-
312; A-56
to P-311; A-56 to S-310; A-56 to E-309; A-56 to V-308; A-56 to T-307; A-56 to
V-306;
A-56 to G-305; A-56 to T-304; A-56 to L-303; A-56 to E-302; A-56 to A-301; A-
56 to L-
300; A-56 to E-299; A-56 to Q-298; A-56 to G-297; A-56 to Q-296; A-56 to I-
295; A-56
to E-294; A-56 to Q-293; A-56 to E-292; A-56 to S-291; A-56 to V-290; A-56 to
Q-289;
A-56 to T-288; A-56 to P-287; A-56 to Q-286; A-56 to L-285; A-56 to Y-284; A-
56 to 8-
283; A-56 to N-282; A-56 to S-281; A-56 to L-280; A-56 to T-279; A-56 to E-
278; A-56
to N-277; A-56 to R-276; A-56 to A-275; A-56 to N-274; A-56 to D-273; A-56 to
E-272;
A-56 to A-271; A-56 to G-270; A-56 to P-269; A-56 to V-268; A-56 to R-267; A-
56 to S-
266; A-56 to P-265; A-56 to C-264; A-56 to S-263; A-56 to R-262; A-56 to R-
261; A-56
to R-260; A-56 to F-259; A-56 to L-258; A-56 to V-257; A-56 to R-256; A-56 to
H-255;
A-56 to V-254; A-56 to R-253; A-56 to E-252; A-56 to P-251; A-56 to G-250; A-
56 to 6-
249; A-56 to G-248; A-56 to G-247; A-56 to G-246; A-56 to S-245; A-56 to C-
244; A-56
to I-243; A-56 to G-242; A-56 to K-241; A-56 to L-240; A-56 to Y-239; A-56 to
S-238;
A-56 to I-237; A-56 to F-236; A-56 to K-235; A-56 to K-234; A-56 to R-233; A-
56 to C-
232; A-56 to S-231; A-56 to F-230; A-56 to G-229; A-56 to V-228; A-56 to V-
227; A-56
to V-226; A-56 to V-225; A-56 to A-224; A-56 to L-223; A-56 to I-222; A-56 to
I-221; A-
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56 to V-220; A-56 to L-219; A-56 to V-218; A-56 to V-217; A-56 to I-216; A-56
to I-215;
A-56 to I-214; A-56 to L-213; A-56 to Y-212; A-56 to H-211; A-56 to Y-210; A-
56 to P-
209; A-56 to S-208; A-56 to A-207; A-56 to L-206; A-56 to M-205; A-56 to G-
204; A-56
to L-203; A-56 to I-202; A-56 to T-201; A-56 to T-200; A-56 to V-199; A-56 to
T-198; A-
56 to E-197; A-56 to E-196; A-56 to A-195; A-56 to A-194; A-56 to P-193; A-56
to T-
192; A-56 to K-191; A-56 to G-190; A-56 to T-189; A-56 to S-188; A-56 to S-
187; A-56
to A-186; A-56 to A-185; A-56 to S-184; A-56 to E-183; A-56 to N-182; A-56 to
K-181;
A-56 to C-180; A-56 to K-179; A-56 to I-178; A-56 to D-177; A-56 to S-176; A-
56 to 8-
175; A-56 to P-174; A-56 to T-173; A-56 to C-172; A-56 to N-171; A-56 to S-
170; A-56
to V-169; A-56 to K-168; A-56 to V-167; A-56 to M-166; A-56 to G-165; A-56 to
R-164;
A-56 to P-163; A-56 to C-162; A-56 to G-161; A-56 to T-160; A-56 to R-159; A-
56 to C-
158; A-56 to T-157; A-56 to R-156; A-56 to C-155; A-56 to M-154; A-56 to E-
153; A-56
to P-152; A-56 to S-151; A-56 to N-150; A-56 to K-149; A-56 to D-148; A-56 to
Q-147;
A-56 to F-146; A-56 to S-145; A-56 to G-144; A-56 to K-143; A-56 to E-142; A-
56 to C-
141; A-56 to Q-140; A-56 to C-139; A-56 to V-138; A-56 to T-137; A-56 to D-
136; A-56
to R-135; A-56 to T-134; A-56 to T-133; A-56 to T-132; A-56 to C-131; A-56 to
S-130;
A-56 to S-129; A-56 to K-128; A-56 to N-127; A-56 to T-126; A-56 to Q-125; A-
56 to 6-
124; A-56 to S-123; A-56 to K-122; A-56 to C-121; A-56 to V-120; A-56 to T-
119; A-56
to C-118; A-56 to L-117; A-56 to L-116; A-56 to C-115; A-56 to S-114; A-56 to
P-113;
A-56 to L-112; A-56 to N-111; A-56 to N-110; A-56 to S-109; A-56 to A-108; A-
56 to I-
107; A-56 to T-106; A-56 to Y-105; A-56 to D-104; A-56 to V-103; A-56 to G-
102; A-56
to E-101; A-56 to T-100; A-56 to C-99; A-56 to P-98; A-56 to N-97; A-56 to C-
96; A-56
to A-95; A-56 to G-94; A-56 to T-93; A-56 to Y-92; A-56 to E-91; A-56 to S-90;
A-56 to
R-89; A-56 to H-88; A-56 to S-87; A-56 to G-86; A-56 to A-85; A-56 to P-84; A-
56 to C-
83; A-56 to E-82; A-56 to E-81; A-56 to E-80; A-56 to K-79; A-56 to L-78; A-56
to S-77;
A-56 to R-76; A-56 to R-75; A-56 to Q-74; A-56 to Q-73; A-56 to Q-72; A-56 to
P-71; A-
56 to A-70; A-56 to V-69; A-56 to T-68; A-56 to Q-67; A-56 to Q-66; A-56 to P-
65; A-56
to V-64; A-56 to E-63; A-56 to D-62; and/or A-56 to Q-61 of the TR10 sequence
shown in
SEQ ID N0:4.
[0278] In another embodiment, antibodies of the invention bind polypeptides
having
one or more residues deleted from the carboxy terminus of the amino acid
sequence of the
TR10 polypeptide shown in SEQ ID N0:4, up to the glutamine residue at position
number
61, and polynucleotides encoding such polypeptides. In particular, the present
invention
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provides polypeptides comprising the amino acid sequence of residues 56-m$ of
SEQ ID
N0:4, where m8 is an integer from 61 to 212 corresponding to the position of
the amino
acid residue in SEQ ID N0:4.
[0279] More in particular, the invention provides antibodies that bind
polypeptides
comprising, or alternatively consisting of, the amino acid sequence of
residues A-56 to C-
385; A-56 to S-384; A-56 to T-383; A-56 to A-382; A-56 to S-381; A-56 to G-
380; A-56
to A-379; A-56 to E-378; A-56 to D-377; A-56 to E-376; A-56 to E-375; A-56 to
Y-374;
A-56 to F-373; A-56 to L-372; A-56 to K-371; A-56 to E-370; A-56 to S-369; A-
56 to 6-
368; A-56 to V-367; A-56 to L-366; A-56 to Q-365; A-56 to D-364; A-56 to Q-
363; A-56
to I-362; A-56 to T-361; A-56 to E-360; A-56 to K-359; A-56 to A-358; A-56 to
H-357;
A-56 to G-356; A-56 to E-355; A-56 to E-354; A-56 to L-353; A-56 to T-352; A-
56 to A-
351; A-56 to S-350; A-56 to A-349; A-56~to D-348; A-56 to L-347; A-56 to L-
346; A-56
to T-345; A-56 to S-344; A-56 to I-343; A-56 to D-342; A-56 to A-341; A-56 to
S-340; A-
56 to D-339; A-56 to A-338; A-56 to D-337; A-56 to N-336; A-56 to V-335; A-56
to P-
334; A-56 to V-333; A-56 to L-332; A-56 to L-331; A-56 to R-330; A-56 to R-
329; A-56
to R-328; A-56 to Q-327; A-56 to C-326; A-56 to G-325; A-56 to E-324; A-56 to
A-323;
A-56 to E-322; A-56 to A-321; A-56 to Q-320; A-56 to E-319; A-56 to L-318; A-
56 to L-
317; A-56 to R-316; A-56 to Q-315; A-56 to P-314; A-56 to E-313; A-56 to E-
312; A-56
to P-311; A-56 to S-310; A-56 to E-309; A-56 to V-308; A-56 to T-307; A-56 to
V-306;
A-56 to G-305; A-56 to T-304; A-56 to L-303; A-56 to E-302; A-56 to A-301; A-
56 to L-
300; A-56 to E-299; A-56 to Q-298; A-56 to G-297; A-56 to Q-296; A-56 to I-
295; A-56
to E-294; A-56 to Q-293; A-56 to E-292; A-56 to S-291; A-56 to V-290; A-56 to
Q-289;
A-56 to T-288; A-56 to P-287; A-56 to Q-286; A-56 to L-285; A-56 to Y-284; A-
56 to 8-
283; A-56 to N-282; A-56 to S-281; A-56 to L-280; A-56 to T-279; A-56 to E-
278; A-56
to N-277; A-56 to R-276; A-56 to A-275; A-56 to N-274; A-56 to D-273; A-56 to
E-272;
A-56 to A-271; A-56 to G-270; A-56 to P-269; A-56 to V-268; A-56 to R-267; A-
56 to 5-
266; A-56 to P-265; A-56 to C-264; A-56 to S-263; A-56 to R-262; A-56 to R-
261; A-56
to R-260; A-56 to F-259; A-56 to L-258; A-56 to V-257; A-56 to R-256; A-56 to
H-255;
A-56 to V-254; A-56 to R-253; A-56 to E-252; A-56 to P-251; A-56 to G-250; A-
56 to 6-
249; A-56 to G-248; A-56 to G-247; A-56 to G-246; A-56 to S-245; A-56 to C-
244; A-56
to I-243; A-56 to G-242; A-56 to K-241; A-56 to L-240; A-56 to Y-239; A-56 to
S-238;
A-56 to I-237; A-56 to F-236; A-56 to K-235; A-56 to K-234; A-56 to R-233; A-
56 to C-
232; A-56 to S-231; A-56 to F-230; A-56 to G-229; A-56 to V-228; A-56 to V-
227; A-56
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to V-226; A-56 to V-225; A-56 to A-224; A-56 to L-223; A-56 to I-222; A-56 to
I-221; A-
56 to V-220; A-56 to L-219; A-56 to V=218; A-56 to V-217; A-56 to I-216; A-56
to I-215;
A-56 to I-214; A-56 to L-213; and/or A-56 to Y-212; of the TR10 sequence shown
in SEQ
ID N0:4.
[0280] The invention also provides polypeptides having one or more amino acids
deleted from both the amino and the carboxyl termini, which may be described
generally
as having residues n7- m7 and/or n8- m$ of SEQ >I7 N0:4 (i.e., SEQ 1D N0:4),
where n7,
n8, m7 and m$ are integers as described above. Thus, any of the above listed N-
or C-
terminal deletions can be combined to produce a N- and C-terminal deleted TR10
polypeptide.
[0281] Also included are antibodies that bind a polypeptide consisting of a
portion of
the complete TR10 amino acid sequence encoded by the cDNA clone contained in
ATCC
Deposit No. 209040, where this portion excludes from 1 to about 80 amino acids
from the
amino terminus of the complete amino acid sequence encoded by the cDNA clone
contained in ATCC Deposit No. 209040, or from 1 to about 204 amino acids from
the
carboxy terminus, or any combination of the above amino terminal and carboxy
terminal
deletions, of the complete amino acid sequence encoded by the cDNA clone
contained in
ATCC Deposit No. 209040.
[0282] Preferably, antibodies of the present invention bind fragments of TR10
comprising a portion of the extracellular domain; i.e., within residues 56 to
212 of SEQ ID
N0:4, since any portion therein is expected to be soluble.
[0283] It will be recognized in the art that some amino acid sequences of TR10
can be
varied without significant effect on the structure or function of the protein.
If such
differences in sequence are contemplated, it should be remembered that there
will be
critical areas on the protein which determine activity. Thus, the invention
further includes
antibodies that bind variations of the TR10 receptor, which show substantial
TR10
receptor activity or which include regions of TR10 proteins, 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 J.U. Bowie et al., SciefZCe
247:1306-
1310 (1990).
[0284] The antibodies of the present invention may bind a fragment,
derivative, or
analog of the polypeptide of SEQ ID N0:4, or that encoded by the deposited
cDNA in
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WO 02/079377 PCT/USO1/42996
ATCC deposit 209040. Such fragments, variants or derivatives may be (i) one in
which at
least 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 residue may or may not be one encoded by the genetic
code, or (ii)
one in which one or more of the amino acid residues includes a substituent
group, or (iii)
one in which the mature polypeptide is fused with another compound, such as a
compound
to increase the half-life of the polypeptide (for example, polyethylene
glycol), or (iv) one
in which the additional amino acids are fused to the mature polypeptide, such
as an IgG Fc
fusion region peptide or leader or secretory sequence or a sequence which is
employed for
purification of the mature polypeptide or a proprotein sequence. Such
fragments,
derivatives and analogs are deemed to be within the scope of those skilled in
the art from
the teachings herein.
[0285] 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
TR10
receptor protein. 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., Clifa
Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987);
Cleland et
al. Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993)).
[0286] The replacement of amino acids can also change the selectivity of
binding to
cell surface receptors. Ostade et al., Nature 361:266-268 (1993), describes
certain
mutations resulting in selective binding of TNF-oc to only one of the two
known types of
TNF receptors. Thus, the antibodies of the present invention may bind a TR10
receptor
that contains one or more amino acid substitutions, deletions, or additions,
either from
natural mutations or human manipulation.
[0287] 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 3 above). In specific embodiments, the number of
substitutions,
additions or deletions in the amino acid sequence of SEQ ID N0:4 and/or any of
the
polypeptide fragments described herein (e.g., the extracellular domain or
intracellular
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WO 02/079377 PCT/USO1/42996
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, 10-1, 5-10, 1-5, 1-3 or 1-2.
[0288] In specific embodiments, the antibodies of the invention bind TR10
polypeptides or fragments or variants thereof (especially a fragment
comprising or
alternatively consisting of, the extracellular soluble domain of TR10), that
contains any
one or more of the following conservative mutations in TR10: M1 replaced with
A, G, I,
L, S, T, or V; G2 replaced with A, I, L, S, T, M, or V; L3 replaced with A, G,
I, S, T, M,
or V; W4 replaced with F, or Y; G5 replaced with A, I, L, S, T, M, or V; Q6
replaced with
N; S7 replaced with A, G, I, L, T, M, or V; V8 replaced with A, G, I, L, S, T,
or M; T10
replaced with A, G, I, L, S, M, or V; A11 replaced with G, I, L, S, T, M, or
V; S12
replaced with A, G, I, L, T, M, or V; S13 replaced with A, G, I, L, T, M, or
V; A14
replaced with G, I, L, S, T, M, or V; R15 replaced with H, or K; A16 replaced
with G, I, L,
S, T, M, or V; G17 replaced with A, I, L, S, T, M, or V; R18 replaced with H,
or K; Y19
replaced with F, or W; G21 replaced with A, I, L, S, T, M, or V; A22 replaced
with G, I,
L, S, T, M, or V; R23 replaced with H, or K; T24 replaced with A, G, I, L, S,
M, or V;
A25 replaced with G, I, L, S, T, M, or V; S26 replaced with A, G, I, L, T, M,
or V; G27
replaced with A, I, L, S, T, M, or V; T28 replaced with A, G, I, L, S, M, or
V; R29
replaced with H, or K; W31 replaced with F, or Y; L32 replaced with A, G, I,
S, T, M, or
V; L33 replaced with A, G, I, S, T, M, or V; D34 replaced with E; K36 replaced
with H, or
R; I37 replaced with A, G, L, S, T, M, or V; L38 replaced with A, G, I, S, T,
M, or V; K39
replaced with H, or R; F40 replaced with W, or Y; V41 replaced with A, G, I,
L, S, T, or
M; V42 replaced with A, G, I, L, S, T, or M; F43 replaced with W, or Y; I44
replaced with
A, G, L, S, T, M, or V; V45 replaced with A, G, I, L, S, T, or M; A46 replaced
with G, I,
L, S, T, M, or V; V47 replaced with A, G, I, L, S, T, or M; L48 replaced with
A, G, I, S, T,
M, or V; L49 replaced with A, G, I, S, T, M, or V; V51 replaced with A, G, I,
L, S, T, or
M; R52 replaced with H, or K; V53 replaced with A, G, I, L, S, T, or M; D54
replaced
with E; S55 replaced with A, G, I, L, T, M, or V; A56 replaced with G, I, L,
S, T, M, or V;
T57 replaced with A, G, I, L, S, M, or V; I58 replaced with A, G, L, S, T, M,
or V; R60
replaced with H, or K; Q61 replaced with N; D62 replaced with E; E63 replaced
with D;
V64 replaced with A, G, I, L, S, T, or M; Q66 replaced with N; Q67 replaced
with N; T68
replaced with A, G, I, L, S, M, or V; V69 replaced with A, G, I, L, S, T, or
M; A70
replaced with G, I, L, S, T, M, or V; Q72 replaced with N; Q73 replaced with
N; Q74
replaced with N; R75 replaced with H, or K; R76 replaced with H, or K; S77
replaced with
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CA 02426710 2003-04-23
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A, G, I, L, T, M, or V; L78 replaced with A, G, I, S, T, M, or V; K79 replaced
with H, or
R; E80 replaced with D; E81 replaced with D; E82 replaced with D; A85 replaced
with G,
I, L, S, T, M, or V; G86 replaced with A, I, L, S, T, M, or V; S87 replaced
with A, G, I, L,
T, M, or V; H88 replaced with K, or R; R89 replaced with H, or K; S90 replaced
with A,
G, I, L, T, M, or V; E91 replaced with D; Y92 replaced with F, or W; T93
replaced with
A, G, I, L, S, M, or V; G94 replaced with A, I, L, S, T, M, or V; A95 replaced
with G, I, L,
S, T, M, or V; N97 replaced with Q; T100 replaced with A, G, I, L, S, M, or V;
E101
replaced with D; 6102 replaced with A, I, L, S, T, M, or V; V103 replaced with
A, G, I, L,
S, T, or M; D104 replaced with E; Y105 replaced with F, or W; T106 replaced
with A, G,
I, L, S, M, or V;1107 replaced with A, G, L, S, T, M, or V; A108 replaced with
G, I, L, S,
T, M, or V; S 109 replaced with A, G, I, L, T, M, or V; N110 replaced with Q;
N111
replaced with Q; L112 replaced with A, G, I, S, T, M, or V; 5114 replaced with
A, G, I, L,
T, M, or V; L116 replaced with A, G, I, S, T, M, or V; L117 replaced with A,
G, I, S, T,
M, or V; T119 replaced with A, G, I, L, S, M, or V; V120 replaced with A, G,
I, L, S, T,
or M; K122 replaced with H, or R; S123 replaced with A, G, I, L, T, M, or V;
6124
replaced with A, I, L, S, T, M, or V; Q125 replaced with N; T126 replaced with
A, G, I, L,
S, M, or V; N127 replaced with Q; K128 replaced with H, or R; S 129 replaced
with A, G,
I, L, T, M, or V; 5130 replaced with A, G, I, L, T, M, or V; T132 replaced
with A, G, I, L,
S, M, or V; T133 replaced with A, G, I, L, S, M, or V; T134 replaced with A,
G, I, L, S,
M, or V; 8135 replaced with H, or K; D136 replaced with E; T137 replaced with
A, G, I,
L, S, M, or V; V138 replaced with A, G, I, L, S, T, or M; Q140 replaced with
N; E142
replaced with D; K143'replaced with H, or R; 6144 replaced with A, I, L, S, T,
M, or V;
5145 replaced with A, G, I, L, T, M, or V; F146 replaced with W, or Y; Q147
replaced
with N; D148 replaced with E; K149 replaced with H, or R; N150 replaced with
Q; 5151
replaced with A, G, I, L, T, M, or V; E153 replaced with D; M154 replaced with
A, G, I,
L, S, T, or V; 8156 replaced with H, or K; T157 replaced with A, G, I, L, S,
M, or V;
8159 replaced with H, or K; T160 replaced with A, G, I, L, S, M, or V; 6161
replaced
with A, I, L, S, T, M, or V; 8164 replaced with H, or K; 6165 replaced with A,
I, L, S, T,
M, or V; M166 replaced with A, G, I, L, S, T, or V; V167 replaced with A, G,
I, L, S, T,
or M; K168 replaced with H, or R; V169 replaced with A, G, I, L, S, T, or M;
5170
replaced with A, G, I, L, T, M, or V; N171 replaced with Q; T173 replaced with
A, G, I,
L, S, M, or V; 8175 replaced with H, or K; S 176 replaced with A, G, I, L, T,
M, or V;
D177 replaced with E; I178 replaced with A, G, L, S, T, M, or V; K179 replaced
with H,
194
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or R; K181 replaced with H, or R; N182 replaced with Q; E183 replaced with D;
5184
replaced with A, G, I, L, T, M, or V; A185 replaced with G, I, L, S, T, M, or
V; A186
replaced with G, I, L, S, T, M, or V; S 187 replaced with A, G, I, L, T, M, or
V; S 188
replaced with A, G, I, L, T, M, or V; T189 replaced with A, G, I, L, S, M, or
V; 6190
replaced with A, I, L, S, T, M, or V; K191 replaced with H, or R; T192
replaced with A,
G, I, L, S, M, or V; A194 replaced with G, I, L, S, T, M, or V; A195 replaced
with G, I, L,
S, T, M, or V; E196 replaced with D; E197 replaced with D; T198 replaced with
A, G, I,
L, S, M, or V; V 199 replaced with A, G, I, L, S, T, or M; T200 replaced with
A, G, I, L, S,
M, or V; T201 replaced with A, G, I, L, S, M, or V; I202 replaced with A, G,
L, S, T, M,
or V; L203 replaced with A, G, I, S, T, M, or V; 6204 replaced with A, I, L,
S, T, M, or
V; M205 replaced with A, G, I, L, S, T, or V; L206 replaced with A, G, I, S,
T, M, or V;
A207 replaced with G, I, L, S, T, M, or V; 5208 replaced with A, G, I, L, T,
M, or V;
Y210 replaced with F, or W; H211 replaced with K, or R; Y212 replaced with F,
or W;
L213 replaced with A, G, I, S, T, M, or V; I214 replaced with A, G, L, S, T,
M, or V; I215
replaced with A, G, L, S, T, M, or V; I216 replaced with A, G, L, S, T, M, or
V; V217
replaced with A, G, I, L, S, T, or M; V218 replaced with A, G, I, L, S, T, or
M; L219
replaced with A, G, I, S, T, M, or V; V220 replaced with A, G, I, L, S, T, or
M; I221
replaced with A, G, L, S, T, M, or V; I222 replaced with A, G, L, S, T, M, or
V; L223
replaced with A, G, I, S, T, M, or V; A224 replaced with G, I, L, S, T, M, or
V; V225
replaced with A, G, I, L, S, T, or M; V226 replaced with A, G, I, L, S, T, or
M; V227
replaced with A, G, I, L, S, T, or M; V228 replaced with A, G, I, L, S, T, or
M; 6229
replaced with A, I, L, S, T, M, or V; F230 replaced with W, or Y; 5231
replaced with A,
G, I, L, T, M, or V; 8233 replaced with H, or K; K234 replaced with H, or R;
K235
replaced with H, or R; F236 replaced with W, or Y; I237 replaced with A, G, L,
S, T, M,
or V; 5238 replaced with A, G, I, L, T, M, or V; Y239 replaced with F, or W;
L240
replaced with A, G, I, S, T, M, or V; K241 replaced with H, or R; 6242
replaced with A, I,
L, S, T, M, or V; I243 replaced with A, G, L, S, T, M, or V; S245 replaced
with A, G, I, L,
T, M, or V; 6246 replaced with A, I, L, S, T, M, or V; 6247 replaced with A,
I, L, S, T,
M, or V; 6248 replaced with A, I, L, S, T, M, or V; 6249 replaced with A, I,
L, S, T, M,
or V; 6250 replaced with A, I, L, S, T, M, or V; E252 replaced with D; 8253
replaced
with H, or K; V254 replaced with A, G, I, L, S, T, or M; H255 replaced with K,
or R;
8256 replaced with H, or K; V257 replaced with A, G, I, L, S, T, or M; L258
replaced
with A, G, I, S, T, M, or V; F259 replaced with W, or Y; 8260 replaced with H,
or K;
195
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8261 replaced with H, or K; 8262 replaced with H, or K; 5263 replaced with A,
G, I, L,
T, M, or V; 5266 replaced with A, G, I, L, T, M, or V; 8267 replaced with H,
or K; V268
replaced with A, G, I, L, S, T, or M; 6270 replaced with A, I, L, S, T, M, or
V; A271
replaced with G, I, L, S, T, M, or V; E272 replaced with D; D273 replaced with
E; N274
replaced with Q; A275 replaced with G, I, L, S, T, M, or V; 8276 replaced with
H, or K;
N277 replaced with Q; E278 replaced with D; T279 replaced with A, G, I, L, S,
M, or V;
L280 replaced with A, G, I, S, T, M, or V; 5281 replaced with A, G, I, L, T,
M, or V;
N282 replaced with Q; 8283 replaced with H, or K; Y284 replaced with F, or W;
L285
replaced with A, G, I, S, T, M, or V; Q286 replaced with N; T288 replaced with
A, G, I, L,
S, M, or V; Q289 replaced with N; V290 replaced with A, G, I, L, S, T, or M;
5291
replaced with A, G, I, L, T, M, or V; E292 replaced with D; Q293 replaced with
N; E294
replaced with D; I295 replaced with A, G, L, S, T, M, or V; Q296 replaced with
N; 6297
replaced with A, I, L, S, T, M, or V; Q298 replaced with N; E299 replaced with
D; L300
replaced with A, G, I, S, T, M, or V; A301 replaced with G, I, L, S, T, M, or
V; E302
replaced with D; L303 replaced with A, G, I, S, T, M, or V; T304 replaced with
A, G, I, L,
S, M, or V; 6305 replaced with A, I, L, S, T, M, or V; V306 replaced with A,
G, I, L, S, T,
or M; T307 replaced with A, G, I, L, S, M, or V; V308 replaced with A, G, I,
L, S, T, or
M; E309 replaced with D; S310 replaced with A, G, I, L, T, M, or V; E312
replaced with
D; E313 replaced with D; Q315 replaced with N; 8316 replaced with H, or K;
L317
replaced with A, G, I, S, T, M, or V; L318 replaced with A, G, I, S, T, M, or
V; E319
replaced with D; Q320 replaced with N; A321 replaced with G, I, L, S, T, M, or
V; E322
replaced with D; A323 replaced with G, I, L, S, T, M, or V; E324 replaced with
D; 6325
replaced with A, I, L, S, T, M, or V; Q327 replaced with N; 8328 replaced with
H, or K;
8329 replaced with H, or K; 8330 replaced with H, or K; L331 replaced with A,
G, I, S,
T, M, or V; L332 replaced with A, G, I, S, T, M, or V; V333 replaced with A,
G, I, L, S,
T, or M; V335 replaced with A, G, I, L, S, T, or M; N336 replaced with Q; D337
replaced
with E; A338 replaced with G, I, L, S, T, M, or V; D339 replaced with E; 5340
replaced
with A, G, I, L, T, M, or V; A341 replaced with G, I, L, S, T, M, or V; D342
replaced with
E; I343 ,replaced with A, G, L, S, T, M, or V; 5344 replaced with A, G, I, L,
T, M, or V;
T345 replaced with A, G, I, L, S, M, or V; L346 replaced with A, G, I, S, T,
M, or V;
L347 replaced with A, G, I, S, T, M, or V; D348 replaced with E; A349 replaced
with G, I,
L, S, T, M, or V; 5350 replaced with A, G, I, L, T, M, or V; A351 replaced
with G, I, L, S,
T, M, or V; T352 replaced with A, G, I, L, S, M, or V; L353 replaced with A,
G, I, S, T,
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M, or V; E354 replaced with D; E355 replaced with D; 6356 replaced with A, I,
L, S, T,
M, or V; H357 replaced with K, or R; A358 replaced with G, I, L, S, T, M, or
V; K359
replaced with H, or R; E360 replaced with D; T361 replaced with A, G, I, L, S,
M, or V;
I362 replaced with A, G, L, S, T, M, or V; Q363 replaced with N; D364 replaced
with E;
Q365 replaced with N; L366 replaced with A, G, I, S, T, M, or V; V367 replaced
with A,
G, I, L, S, T, or M; 6368 replaced with A, I, L, S, T, M, or V; 5369 replaced
with A, G, I,
L, T, M, or V; E370 replaced with D; K371 replaced with H, or R; L372 replaced
with A,
G, I, S, T, M, or V; F373 replaced with W, or Y; Y374 replaced with F, ox W;
E375
replaced with D; E376 replaced with D; D377 replaced with E; E378 replaced
with D;
A379 replaced with G, I, L, S, T, M, or V; 6380 replaced with A, I, L, S, T,
M, or V;
5381 replaced with A, G, I, L, T, M, or V; A382 replaced with G, I, L, S, T,
M, or V;
T383 replaced with A, G, I, L, S, M, or V; 5384 replaced with A, G, I, L, T,
M, or V;
and/or L386 replaced with A, G, I, S, T, M, or V of SEQ ID N0:4.
[0289] In specific embodiments, the antibodies of the invention bind TR10
polypeptides or fragments or variants thereof (especially a fragment
comprising or
alternatively consisting of, the extracellular soluble domain of TR10), that
contains any
one or more of the following non-conservative mutations in TR10: Ml replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; G2 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
L3 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W4 replaced with D,
E"H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; G5 replaced with D, E, H, K, R, N, .Q,
F, W, Y, P, or
C; Q6 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;
S7 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; V8 replaced with D, E, H, K, R, N,
Q, F, W,
Y, P, or C; P9 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C;
T10 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or. C; A11 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; S 12 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; S 13
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A14 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; R15 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or
C; A16 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G17 replaced with
D, E, H,
K, R, N, Q, F, W, Y, P, or C; R18 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, P, or C; Y19 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; P20
xeplaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G21
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A22 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; R23 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; T24
197
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replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A25 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; S26 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
G27
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T28 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; R29 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or
C; P30 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or
C; W31
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L32
replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L33 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
D34 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P35
replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K36 replaced
with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I37 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; L38 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K39
replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F40 replaced with D, E,
H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; V41 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; V42. replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F43 replaced with
D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; I44 replaced with D, E, H, K, R, N,
Q, F, W, Y, P,
or C; V45 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A46 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; V47 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
L48 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; LA.9 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; P50 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, or C; V51 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R52
replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V53 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; D54 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; S55 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A56 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; T57 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; I58
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P59 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R60 replaced with D, E, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, P, or C; Q61 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y,
P, or C; D62 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; E63
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V64
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; P65 replaced with D, E, H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, or C; Q66 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W,
Y, P, or C; Q67 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; T68
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V69 replaced with D, E,
H, K, R, N,
198
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
Q, F, W, Y, P, or C; A70 replaced with D, E, H, I~, R, N, Q, F, W, Y, P, or C;
P71
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; Q72
replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; Q73 replaced
with D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; Q74 replaced with D, E, H, K, R,
A, G, I, L, S,
T, M, V, F, W, Y, P, or C; R75 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y,
P, or C; R76 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; S77
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L78 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; K79 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or
C; E80 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
E81 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E82 replaced
with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C83 replaced with D, E, H, K, R,
A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or P; P84 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, or C; A85 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G86
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S87 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; H88 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; R89
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S90
replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; E91 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, P, or C; Y92 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,
V, P, or C;
T93 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G94 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; A95 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; C96
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; N97
replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; P98 replaced
with D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; C99 replaced with D, E, H, K,
R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or P; T100 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; E101 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
6102
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V 103 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; D104 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; Y105 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
T106
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I107 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; A108 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
5109
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N110 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; N111 replaced with D, E, H, K, R, A, G,
I, L, S, T,
M, V, F, W, Y, P, or C; L112 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; P113
199
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S
114 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; C115 replaced with D, E, H, K, R,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or P; L116 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
L117 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C118 replaced with
D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; T119 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; V 120 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C
121 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; K122 replaced
with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S 123 replaced with D, E, H,
K, R, N, Q, F,
W, Y, P, or C; 6124 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q125
replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T126 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; N127 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F,
W, Y, P, or C; K128 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
S 129 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S 130 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; C131 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, or P; T132 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T133
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; T134 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; 8135 xeplaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; D136
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T137
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; V138 replaced with D, E, H, K, R, N, Q,
F, W, Y,
P, or C; C139 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, or P;
Q140 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;
C141 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; E142 replaced
with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K143 replaced with D, E, A,
G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; 6144 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or
C; S145 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F146 replaced
with D, E, H,
K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q147 replaced with D, E, H, K, R,
A, G, I, L,
S, T, M, V, F, W, Y, P, or C; D148 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, P, or C; K149 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
N150 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;
S151 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; P152 replaced with D, E, H, K, R,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; E153 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, P, or C; M154 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
C155
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; 8156
replaced
200
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T157 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; C158 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, or P; 8159 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
T160 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6161 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; C162 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, or P; P163 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or
C; 8164 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
6165 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; M166 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; V167 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K168
replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V 169 replaced with D,
E, H, K, R, N,
Q, F, W, Y, P, or C; S170 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
N171
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; C 172
replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; T173 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; P174 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, or C; 8175 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
5176 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D177 replaced with
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I178 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; K179 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; C180
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; K181
replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N182 replaced with
D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E183 replaced with H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; S 184 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
A185 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A186 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; S 187 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; S 188
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T189 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; 6190 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
K191
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T192
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; P193 replaced with D, E, H, K, R, A, G, I,
L, S, T, M,
V, N, Q, F, W, Y, or C; A194 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; A195
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E196 replaced with H, K,
R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; E197 replaced with H, K, R, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, P, or C; T198 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; V199
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T200 replaced with D, E,
H, K, R, N,
201
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
Q, F, W, Y, P, or C; T201 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
I202
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L243 replaced with D, E,
H, K, R, N,
Q, F; W, Y, P, or C; 6204 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
M205
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L206 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; A207 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
5208
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P209 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; Y210 replaced with D, E, H, K, R, N,
Q, A, G, I,
L, S, T, M, V, P, or C; H211 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; Y212 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
L213
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I214 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; I215 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
I216
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V217 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; V218 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L219
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V220 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; I221 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
I222
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L223 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; A224 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V225
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V226 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; V227 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V228
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6229 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; F230 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or
C; S231 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C232 replaced
with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; 8233 replaced with D, E, A,
G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; K234 replaced With D, E, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, P, or C; K235 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
F236 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; I237
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; 5238 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; Y239 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
L240
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K241 replaced with D, E,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; 6242 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; I243 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C244 replaced
with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; 5245 replaced with D, E, H,
K, R, N, Q,
F, W, Y, P, or C; 6246 replaced with D, E, H, K, R, N, Q,~F, W, Y, P, or C;
6247
202
CA 02426710 2003-04-23
WO 02/079377 PCT/USO1/42996
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6248 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; 6249 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
6250
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P251 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, ar C; E252 replaced with H, K, R, A, G, I,
L, S, T, M,
V, N, Q, F, W, Y, P, or C; 8253 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y,
P, or C; V254 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H255
replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 8256 replaced with D, E, A,
G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; V257 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or
C; L258 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F259 replaced
with D, E, H,
K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; 8260 replaced with D, E, A, G, I,
L, S, T, M,
V, N, Q, F, W, Y, P, or C; 8261 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y,
P, or C; 8262 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; S263
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C264 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or P; P265 replaced with D, E, H, K, R, A,
G, I, L, S,
T, M, V, N, Q, F, W, Y, or C; 5266 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
8267 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V268
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; P269 replaced with D, E, H, K, R,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; 6270 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or
C; A271 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E272 replaced
with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D273 replaced with H, K, R, A,
G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; N274 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V,
F, W, Y, P, or C; A275 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
8276
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N277
replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E278 replaced with H, K,
R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; T279 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; L280 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 5281 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; N282 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V,
F, W, Y, P, or C; 8283 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C;
Y284 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L285
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q286 replaced with D, E, H, K, R,
A, G, I, L,
S, T, M, V, F, W, Y, P, or C; P287 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, or C; T288 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
Q289
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V290
replaced with
203
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D, E, H, K, R, N, Q, F, W, Y, P, or C; S291 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; E292 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; Q293
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E294
replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I295 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; Q296 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y,
P, or C; 6297 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q298
replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E299 replaced with H, K,
R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; L300 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; A301 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E302 replaced
with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L303 replaced with D, E, H,
K, R, N, Q,
F, W, Y, P, or C; T304 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
6305 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; V306 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; T307 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V308
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; E309 replaced with H, K, R, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, P, or C; 5310 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; P311
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E312
replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E313 replaced
with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P314 replaced with D, E, H, K,
R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or C; Q315 replaced with D, E, H, K, R, A, G, I,
L, S, T, M,
V, F, W, Y, P, or C; 8316 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or
C; L317 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L318 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; E319 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F,
W, y, P, or C; Q320 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,
Y, P, or C;
A321 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E322 replaced with
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A323 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; E324 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
6325 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 0326 replaced with
D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Q327 replaced with D, E, H, K,
R, A, G, I,
L, S, T, M, V, F, W, Y, P, or C; 8328 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F,
W, y, P, or C; 8329 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, y,
P, or C;
8330 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L331
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L332 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; V333 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P334
replaced with
204
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D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; V335 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; N336 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W,
Y, P, or C; D337 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
A338 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D339 replaced with
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S340 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; A341 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D342
replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I343 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; S344 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; T345
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L346 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; L347 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
D348
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A349
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; 5350 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; A351 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T352 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L353 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
E354 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
E355 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 6356 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; H357 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, P, or C; A358 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K359
replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E360 replaced with H, K,
R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; T361 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; I362 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q363 replaced
with D, E,
H, K, R, A, G, I, L, 5, T, M, V, F, W, Y, P, or C; D364 replaced with H, K, R,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; Q365 replaced with D, E, H, K, R, A, G, I,
L, S, T, M,
V, F, W, Y, P, or C; L366 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V367
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6368 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; 5369 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
E370
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K371
replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L372 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; F373 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or
C; Y374 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
E375 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E376 replaced
with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D377 replaced with H, K, R, A,
G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; E378 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q,
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F, W, Y, P, or C; A379 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
6380
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 5381 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; A382 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
T383
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 5384 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; C385 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, or P; and/or L386 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C of
SEQ ID N0:4.
[0290] Amino acids in the TR10 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
molecule. The resulting mutant molecules are then tested for biological
activity such as
receptor binding or in vitro proliferative activity. Sites that are critical
for ligand-receptor
binding can also be determined by structural analysis such as crystallization,
nuclear
magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol.
224:899-904
(1992) and de Vos et al. Science 255:306-312 (1992)). In preferred
embodiments,
antibodies of the present invention bind regions of TR10 that are essential
for TR10
function. In other preferred embodiments, antibodies of the present invention
bind regions
of TR10 that are essential for TR10 function and inhibit or abolish TR10
function. In other
preferred embodiments, antibodies of the present invention bind regions of
TR10 that are
essential for TR10 function and enhance TR10 function.
[0291] To improve or alter the characteristics of TR10 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.
[0292] Non-naturally occurring TR10 variants that may be bound by the
antibodies of
the invention 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.,
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Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (see
e.g., Wells et al.,
Philos. Trans. R. Soc. London SerA 317:415 (1986)).
[0293] Thus, the invention also encompasses antibodies that bind TR10
derivatives
and analogs that have one or more amino acid residues deleted, added, or
substituted to
generate TR10 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 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 acid positions on any one or more of the
glycosylation
recognitions sequences in the TR10 polypeptides, andlor an amino acid deletion
at the
second position of any one or more such recognition sequences will prevent
glycosylation
of the TR10 at the modified tripeptide sequence (see, e.g., Miyajimo et al.,
EMBO J
5(6):1193-1197). Additionally, one or more of the amino acid residues of TR10
polypeptides (e.g., arginine and lysine residues) may be deleted or
substituted with another
residue to eliminate undesired processing by proteases such as, for example,
furins or
kexins.
[0294] The antibodies of the present invention also include antibodies that
bind a
polypeptide comprising, or alternatively, consisting of the polypeptide
encoded by the ,
deposited cDNA (the deposit having ATCC Accession Number 209040) 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 from about 1 to about
386 in SEQ
m N0:4; a polypeptide comprising, or alternatively, consisting of amino acids
from about
2 to about 386 in SEQ ID N0:4; a polypeptide comprising, or alternatively,
consisting of
amino acids from about 56 to about 386~in SEQ >D NO:4; a polypeptide
comprising, or
alternatively, consisting of the extracellular domain; a polypeptide
comprising, or
alternatively, consisting of the cysteine rich domain; 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; and a polypeptide comprising, or
alternatively,
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WO 02/079377 PCT/USO1/42996
consisting of the partial death domain; as well as polypeptides which are at
least 80%
identical, more preferably at least 80%, 85%, 90%, or 95% identical, still
more preferably
at least 96%, 97%, 98%, or 99% identical to the polypeptides described above
(e.g., the
polypeptide encoded by the deposited cDNA clone (the deposit having ATCC
Accession
Number 209040), the polypeptide of SEQ ID N0:4 (SEQ ID N0:4)), and also
include
portions of such polypeptides with at least 30 amino acids and more preferably
at least 50
amino acids.
[0295] By a polypeptide having an amino acid sequence at least, for example,
95%
"identical" to a reference amino acid sequence of a TR10 polypeptide is
intended that the
amino acid sequence of the polypeptide is identical to the reference sequence
except that
the polypeptide sequence may include up to five amino acid alterations per
each 100
amino acids of the reference amino acid of the TR10 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.
[0296] 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 SEQ ID N0:4, or to the amino acid sequence encoded by the deposited
cDNA
clone, 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.
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[0297] 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 a1. (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=1,
Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window
Size=500 or the length of the subject amino acid sequence, whichever is
shorter.
According to this embodiment, 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
matchedlaligned 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°lo 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
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deletions are internal deletions so there are no residues at the N- or C-
termini of the
subject sequence which are not matchedlaligned 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.
[0298] The present application is also directed to antibodies that bind
polypeptides at
least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the TR10
polypeptide
sequence set forth as n7-m7, and/or n$- m8 herein. In preferred embodiments,
the
application is directed to proteins containing polypeptides at least 80%, 85%,
90%, 95%,
96°70, 97%, 98% or 99% identical to polypeptides having the amino acid
sequence of the
specific TR10 N- and C-terminal deletions recited herein. Polynucleotides
encoding these
polypeptides are also encompassed by the invention.
[0299] In certain preferred embodiments, antibodies of the invention bind TR10
fusion
proteins as described above wherein the TR10 portion of the fusion protein are
those
described as n7-m7, and/or n$- m8 herein.
Antibodies of the invention may bind Modified TRAIL Receptor Polypeptides
[0300] It is specifically contmeplated that antibodies of the present
invention may bind
modified forms of TRAIL Receptor proteins (e.g., TR4, TRS, TR7, and/or TR10
(SEQ ID
NOS:1-4, respectively)
[0301] In specific embodiments, antibodies of the present invention bind TRAIL
receptor polypeptides (such as those decribed above) including, but not
limited to naturally
purified TRAIL receptor polypeptides, TRAIL receptor polypeptides produced by
chemical synthetic procedures, and TRAIL receptor polypeptides produced by
recombinant techniques from a prokaryotic or eukaryotic host, including, for
example,
bacterial, yeast, higher plant, insect and mammalian cells using, for example,
the
recombinant compositions and methods described above. Depending upon the host
employed in a recombinant production procedure, the polypeptides may be
glycosylated or
non-glycosylated. In addition, TRAIL Receptor polypeptides may also include an
initial
modified methionine residue, in some cases as a result of host-mediated
processes.
[0302]. In addition, TRAIL Receptor proteins that are bound by antibodies of
the
present invention can be chemically synthesized using techniques known in the
art (e.g.,
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see Creighton, Proteins: Structures and Molecular Principles, W.H. Freeman &
Co., N.Y.
(1983), and Hunkapiller, et al., Nature 310:105-111 (1984)). For example, a
peptide
corresponding to a fragment of a TRAIL Receptor polypeptide 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
TRAIL
Receptor polypeptide sequence. Non-classical amino acids include, but are not
limited to,
to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino
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).
[0303] The invention additionally, encompasses antibodies that bind TRAIL
Receptor
polypeptides which are differentially modified during or after translation,
e.g., by
glycosylation, acetylation, phosphorylation, amidation, derivatization by
known
protecting/blocking groups, proteolytic cleavage, linkage to an antibody
molecule or other
cellular ligand, etc. Any of numerous chemical modifications may be carried
out by
known techniques, including but not limited to, specific chemical cleavage by
cyanogen
bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation,
formylation,
oxidation, reduction, metabolic synthesis in the presence of tunicamycin; etc.
[0304] Additional post-translational modifications to TRAIL Receptor
polypeptides
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.
[0305] Also provided by the invention are antibodies that bind chemically
modified
derivatives of TRAIL Receptor polypeptide which may provide additional
advantages
such as increased solubility, stability and circulating time of the
polypeptide, or decreased
immunogenicity (see U. S. Patent No. 4,179,337). The chemical moieties for
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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.
[0306] 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, 5504, 6000, 6500, 7000, 7500, 8000,
8500,
9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500,
14,000,
14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500,
19,000, 19,500,
20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000,
70,000, 75,000,
80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
[0307] 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. Bioclae»z. Bioteclzzzol. 56:59-72 (1996); Vorobjev et
al.,
Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bzocozzjug.
Chem.
10:638-646 (1999), the disclosures of each of which are incorporated herein by
reference.
[0308] 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. HernatoZ. 20:1028-1035 (1992) (reporting pegylation of
GM-CSF
using taresyl 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
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and the N-terminal amino acid residues; those having a free carboxyl group may
include
aspartic acid residues, glutamic acid residues and the C-terminal amino acid
residue.
Sulfhydryl groups may also be used as a reactive group for attaching the
polyethylene
glycol molecules. Preferred for therapeutic purposes is attachment at an amino
group,
such as attachment at the N-terminus or lysine group.
[0309] As suggested above, polyethylene glycol may be attached to proteins via
linkage to any of a number of amino acid residues. For example, polyethylene
glycol can
be linked to a proteins via covalent bonds to lysine, histidine, aspartic
acid, glutamic acid,
or cysteine residues. One or more reaction chemistries may be employed to
attach
polyethylene glycol to specific amino acid residues (e.g., lysine, histidine,
aspartic acid,
glutamic acid, or cysteine) of the protein or to more than one type of amino
acid residue
(e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and
combinations thereof) of
the protein.
[0310] One may specifically desire proteins chemically modified at the N-
terminus.
Using polyethylene glycol as an illustration of the present composition, one
may select
from a variety of polyethylene glycol molecules (by molecular weight,
branching, etc.),
the proportion of polyethylene glycol molecules to protein (or peptide)
molecules in the
reaction mix, the type of pegylation reaction to be performed, and the method
of obtaining
the selected N-terminally pegylated protein. The method of obtaining the N-
terminally
pegylated preparation (i.e., separating this moiety from other monopegylated
moieties if
necessary) may be by purification of the N-terminally pegylated material from
a
population of pegylated protein molecules. Selective proteins chemically
modified at the
N-terminus modification may be accomplished by reductive alkylation which
exploits
differential reactivity of different types of primary amino groups (lysine
versus the N-
terminal) available for derivatization in a particular protein. Under the
appropriate
reaction conditions, substantially selective derivatization of the protein at
the N-terminus
with a carbonyl group containing polymer is achieved.
[0311] 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. Tl2era.
Drug Carrier Sys. 9:249-304 (1992); Francis et al., Interyc. J. of Hematol.
68:1-18 (1998);
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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.
[0312] 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
(C1SO2CH2CF3). 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.
[0313] 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 1,1'-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.
[0314] The number of polyethylene glycol moieties attached to each TRAIL
Receptor
polypeptide (z.e., the degree of substitution) may also vary. For example, the
pegylated
proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 12, 15,
17, 20, or more polyethylene glycol molecules. Similarly, the average degree
of
substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-
11,10-12, 11-
13, 12-14, 13-15, 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 al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304
(1992).
[0315] As mentioned the antibodies of the present invention may bind TRAIL
Receptor polypeptides that are modified by either natural processes, such as
posttranslational processing, or by chemical modification techniques which are
well
known in the art. It will be appreciated that the same type of modification
may be present
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in the same or varying degrees at several sites in a given TRAIL Receptor
polypeptide.
TRAIL Receptor polypeptides may be branched, for example, as a result of
ubiquitination,
and they may be cyclic, with or without branching. Cyclic, branched, and
branched cyclic
TRAIL Receptor polypeptides may result from posttranslation natural processes
or may be
made by synthetic methods. Modifications include acetylation, acylation, ADP-
ribosylation, amidation, covalent attachment of flavin, covalent attachment of
a heme
moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent
attachment
of a lipid or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking,
cyclization, disulfide bond formation, demethylation, formation of covalent
cross-links,
formation of cysteine, formation of pyroglutamate, formylation, gamma-
carboxylation,
glycosylation, GPI anchor formation, hydroxylation, iodination, methylation,
myristoylation, oxidation, pegylation, proteolytic processing,
phosphorylation,
prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated
addition of
amino acids to proteins such as arginylation, and ubiquitination. (See, for
instance,
PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL
COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,
New York, pgs. 1-12 (1983); Seifter et al., Meth Eyizymol 182:626-646 (1990);
Rattan et
al., Ann NYAcad Sci 663:48-62 (1992)).
[0316] In one embodiment, the invention provides antibodies (each consisting
of two
heavy chains and two light chains linked together by disulfide bridges to form
an
antibody) that immunospecifically binds one or more TRAIL receptor
polypeptides (e.g.,
SEQ ID NOS:1-4) or fragments or variants thereof, wherein the amino acid
sequence of
the heavy chain and the amino acid sequence of the light chain are the same as
the amino
acid sequence of a heavy chain and a light chain expressed by one or more cell
lines
referred to in Table 1. In another embodiment, the invention provides
antibodies (each
consisting of two heavy chains and two light chains linked together by
disulfide bridges to
form an antibody) that immunospecifically binds one or more TRAIL receptor
polypeptides (e.g., SEQ ID NOS:1-4) or fragments or variants thereof, wherein
the amino
acid sequence of the heavy chain or the amino acid sequence of the light chain
are the
same as the amino acid sequence of a heavy chain or a light chain expressed by
one or
more cell lines referred to in Table 1. Immunospecific binding to TRAIL
receptor
polypeptides may be determined by immunoassays known in the art or described
herein
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for assaying specific antibody-antigen binding. Molecules comprising, or
alternatively
consisting of, fragments or variants of these antibodies that
immunospecifically bind to
one or more TRAIL, receptor are also encompassed by the invention, as are
nucleic acid
molecules encoding these antibodies molecules, fragments andlor variants.
[0317] In one embodiment of the present invention, antibodies that
immunospecifically bind to a TRAIL receptor or a fragment or variant thereof,
comprise a
polypeptide having the amino acid sequence of any one of the heavy chains
expressed by
at least one of the cell lines referred to in Table 1 and/or any one of the
light chains
expressed by at least one of the cell lines referred to in Table 1. In another
embodiment of
the present invention, antibodies that immunospecifically bind to a TRAIL
receptor or a
fragment or variant thereof, comprise a polypeptide having the amino acid
sequence of
any one of the VH domains of a heavy chain expressed by at least one of the
cell lines
referred to in Table 1 and/or any one of the VL domains of a light chain
expressed by at
least one of the cell lines referred to in Table 1. In preferred embodiments,
antibodies of
the present invention comprise the amino acid sequence of a VH domain and VL
domain
expressed by the same cell line selected from the group consisting of cell
lines referred to
in Table 1 In alternative embodiments, antibodies of the present invention
comprise the
amino acid sequence of a VH domain and a VL domain from different cell lines
referred
to in Table 1. Molecules comprising, or alternatively consisting of, antibody
fragments or
variants of the VH and/or VL domains expressed by at least one of the cell
lines referred
to in Table 1 that immunospecifically bind to a TRAIL receptor are also
encompassed by
the invention, as are nucleic acid molecules encoding these VH and VL domains,
molecules, fragments and/or variants.
[0318] The present invention also provides antibodies that immunospeeificially
bind to
a polypeptide, or polypeptide fragment or variant of a TRAIL receptor, wherein
said
antibodies comprise, or alternatively consist of, a polypeptide having an
amino acid
sequence of any one, two, three, or more of the VH CDRs contained in a heavy
chain
expressed by one or more cell lines referred to in Table 1. In particular, the
invention
provides antibodies that immunospecifically bind a TRAIL receptor, comprising,
or
alternatively consisting of, a polypeptide having the amino acid sequence of a
VH CDR1
contained in a heavy chain expressed by one or more cell lines referred to in
Table 1. In
another embodiment, antibodies that immunospecifically bind a TRAIL receptor,
comprise, or alternatively consist of, a polypeptide having the amino acid
sequence of a
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VH CDR2 contained in a heavy chain expressed by one or more cell lines
referred to in
Table 1. In a preferred embodiment, antibodies that immunospecifically bind a
TRAIL
receptor, comprise, or alternatively consist of a polypeptide having the amino
acid
sequence of a VH CDR3 contained in a heavy chain expressed by one or more cell
lines
referred to in Table 1. Molecules comprising, or alternatively consisting of,
these
antibodies, or antibody fragments or variants thereof, that immunospecifically
bind to
TRAIL receptor or a TRAIL receptor fragment or variant thereof are also
encompassed by
the invention, as are nucleic acid molecules encoding these antibodies,
molecules,
fragments andlor variants.
[0319] The present invention also provides antibodies that immunospecificially
bind to
a polypeptide, or polypeptide fragment or variant of a TRAIL receptor, wherein
said
antibodies comprise, or alternatively consist of, a polypeptide having an
amino acid
sequence of any one, two, three, or more of the VL CDRs contained in a light
chain
expressed by one or more cell lines referred to in Table 1. In particular, the
invention
provides antibodies that immunospecifically bind a TRAIL receptor, comprising,
or
alternatively consisting of, a polypeptide having the amino acid sequence of a
VL CDR1
contained in a light chain expressed by one or more cell lines referred to in
Table 1. In
another embodiment, antibodies that immunospecifically bind a TRAIL receptor,
comprise, or alternatively consist of, a polypeptide having the amino acid
sequence of a
VL CDR2 contained in a light chain expressed by one or more cell lines
referred to in
Table 1. In a preferred embodiment, antibodies that immunospecifically bind a
TRAIL
receptor, comprise, or alternatively consist of a polypeptide having the amino
acid
sequence of a VL CDR3 contained in a light chain expressed by one or more cell
lines
referred to in Table 1. Molecules comprising, or alternatively consisting of,
these
antibodies, or antibody fragments or variants thereof, that immunospecifically
bind to
TRAIL receptor or a TRAIL receptor fragment or variant thereof are also
encompassed by
the invention, as are nucleic acid molecules encoding these antibodies,
molecules,
fragments and/or variants.
[0320] The present invention also provides antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants)
that
immunospecifically bind to a TRAIL receptor polypeptide or polypeptide
fragment or
variant of a TRAIL receptor, wherein said antibodies comprise, or
alternatively consist of,
one, two, three, or more VH CDRs and one, two, three or more VL CDRs, as
contained in
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a heavy chain or light chain expressed by one or more cell lines referred to
in Table 1. In
particular, the invention provides for antibodies that immunospecifically bind
to a
polypeptide or polypeptide fragment or variant of a TRAIL receptor, wherein
said
antibodies comprise, or alternatively consist of, a VH CDR1 and a VL CDR1, a
VH CDR1
and a VL CDR2, a VH CDR1 and a VL CDR3, a VH CDR2 and a VL CDRl, VH CDR2
and VL CDR2, a VH CDR2 and a VL CDR3, a VH CDR3 and a VH CDR1, a VH CDR3
and a VL CDR2, a VH CDR3 and a VL CDR3, or any combination thereof, of the VH
CDRs and VL CDRs contained in a heavy chain or light chain expressed by one or
more
cell lines referred to in Table 1. In a preferred embodiment, one or more of
these
combinations are from the same antibody as disclosed in Table 1. Molecules
comprising,
or alternatively consisting of, fragments or variants of these antibodies,
that
immunospecifically bind to TRAIL receptor are also encompassed by the
invention, as are
nucleic acid molecules encoding these antibodies, molecules, fragments or
variants.
Nucleic Acid Molecules Encoding anti-TRAIL Receptor Antibodies
[0321] The present invention also provides for nucleic acid molecules,
generally
isolated, encoding an antibody of the invention (including molecules
comprising, or
alternatively consisting of, antibody fragments or variants thereof). In a
specific
embodiment, a nucleic acid molecule of the invention encodes an antibody
(including
molecules comprising, or alternatively consisting of, antibody fragments or
variants
thereof), comprising, or alternatively consisting of, a VH domain having an
amino acid
sequence of any one of the VH domains expressed by at least one of the cell
lines referred
to in Table 1 and a VL domain having an amino acid sequence of any one of the
VL
domains expressed by at least one of the cell lines referred to in Table 1. In
another
embodiment, a nucleic acid molecule of the invention encodes an antibody
(including
molecules comprising, or alternatively consisting of, antibody fragments or
variants
thereof), comprising, or alternatively consisting of, a VH domain having an
amino acid
sequence of any one of the VH domains expressed by at least one of the cell
lines referred
to in Table 1 or a VL domain having an amino acid sequence of any one of the
VL
domains expressed by at least one of the cell lines referred to in Table 1.
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[0322] The present invention also provides antibodies that comprise, or
alternatively
consist of, variants (including derivatives) of the antibody molecules (e.g.,
the VH
domains and/or VL domains) described herein, which antibodies
immunospecifically bind
to a TRAIL receptor or fragment or variant thereof. Standard techniques known
to those
of skill in the art can be used to introduce mutations in the nucleotide
sequence encoding a
molecule of the invention, including, for example, site-directed mutagenesis
and
PCR-mediated mutagenesis which result in amino acid substitutions. Preferably,
the
variants (including derivatives) encode less than 50 amino acid substitutions,
less than 40
amino acid subsitutions, less than 30 amino acid substitutions, less than 25
amino acid
substitutions, less than 20 amino acid substitutions, less than 15 amino acid
substitutions,
less than 10 amino acid substitutions, less than 5 amino acid substitutions,
less than 4
amino acid substitutions, less than 3 amino acid substitutions, or less than 2
amino acid
substitutions relative to the reference VH domain, VHCDR1, VHCDR2, VHCDR3, VL
domain, VLCDR1, VLCDR2, or VLCDR3. A "conservative amino acid substitution" is
one in which the amino acid residue is replaced with an amino acid residue
having a side
chain with a similar charge. Families of amino acid residues having side
chains with
similar charges have been defined in the art. These families include amino
acids with
basic side chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,
leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched
side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine, phenylalanine,
tryptophan, histidine). Alternatively, mutations can be introduced randomly
along all or
part of the coding sequence, such as by saturation mutagenesis, and the
resultant mutants
can be screened for biological activity to identify mutants that retain
activity (e.g., the
ability to bind a TRAIL receptor).
[0323] For example, it is possible to introduce mutations only in framework
regions or
only in CDR regions of an antibody molecule. Introduced mutations may be
silent or .
neutral missense mutations, i.e., have no, or little, effect an an antibody's
ability to bind
antigen. These types of mutations may be useful to optimize codon usage, or
improve a
hybriodma's antibody production. Alternatively, non-neutral missense mutations
may alter
an antibody's ability to bind antigen. The location of most silent and neutral
missense
mutations is likely to be in the framework regions, while the location of most
non-neutral
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missense mutations is likely to be in CDR, though this is not an absolute
requirement.
One of skill in the art would be able to design and test mutant molecules with
desired
properties such as no alteration in antigen binding activity or alteration in
binding activity
(e.g, improvements in antigen binding activity or change in antibody
specificity).
Following mutagenesis, the encoded protein rnay routinely be expressed and the
functional
and/or biological activity of the encoded protein, (e.g., ability to
immunospecifically bind
a TRATL receptor) can be determined using techniques described herein or by
routinely
modifying techniques known in the art.
[0324] In a specific embodiment, an antibody of the invention (including a
molecule
comprising, or alternatively consisting of, an antibody fragment or variant
thereof), that
immunospecifically binds TRAIL receptor polypeptides or fragments or variants
thereof,
comprises, or alternatively consists of, an amino acid sequence encoded by a
nucleotide
sequence that hybridizes to a nucleotide sequence that is complementary to
that encoding
one of the VH or VL domains expressed by one or more cell lines referred to in
Table 1.
under stringent conditions, e.g., hybridization to filter-bound DNA in 6X
sodium
chloride/sodium citrate (SSC) at about 45° C followed by one or more
washes in
0.2xSSC/0.1% SDS at about 50-65° C, under highly stringent conditions,
e.g.,
hybridization to filter-bound nucleic acid in 6xSSC at about 45° C
followed by one or
more washes in 0.lxSSC/0.2% SD5 at about 68° C, or under other
stringent hybridization
conditions which are known to those of skill in the art (see, for example,
Ausubel, F.M. et
al., eds. , 1989, Current Protocols ih Molecular Biology, Vol. I, Grreen
Publishing
Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6
and 2.10.3).
Nucleic acid molecules encoding these antibodies are also encompassed by the
invention.
[0325] It is well known within the art that polypeptides, or fragments or
variants
thereof, with similar amino acid sequences often have similar structure and
many of the
same biological activities. Thus, in one embodiment, an antibody (including a
molecule
comprising, or alternatively consisting of, an antibody fragment or variant
thereof), that
imrnunospecifically binds to a TRAIL, receptor polypeptide or fragments or
variants of a
TRAIL receptor polypeptide, comprises, or alternatively consists of, a VH
domain having
an amino acid sequence that is at least 35%, at least 40%, at least 45%, at
least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least
85%, at least 90%, at least 95%, or at least 99% identical, to the amino acid
sequence of a
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VH domain of a heavy chain expressed by at least one of the cell lines
referred to in Table
1.
[0326] In another embodiment, an antibody (including a molecule comprising, or
alternatively consisting of, an antibody fragment or variant thereof), that
immunospecifically binds to a TRAIL receptor polypeptide or fragments or
variants of a
TRAIL receptor polypeptide, comprises, or alternatively consists of, a VL
domain having
an amino acid sequence that is at least 35%, at least 40%, at least 45%, at
least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least
85%, at least 90%, at least 95%, or at least 99% identical, to the amino acid
sequence of a
VL domain of a light chain expressed by at least one of the cell lines
referred to in Table
1.
Methods of Producing Antibodies
[0327] Antibodies in accordance with the invention are preferably prepared the
utilization of a transgenic mouse that has a substantial portion of the human
antibody
producing genome inserted but that is rendered deficient in the production of
endogenous,
murine, antibodies (e.g., XenoMouse strains available from Abgenix Inc.,
Fremont, CA).
Such mice, then, are capable of producing human immunoglobulin molecules and
antibodies and are deficient in the production of murine immunoglobulin
molecules and
antibodies. Technologies utilized for achieving the same are disclosed in the
patents,
applications, and references disclosed herein.
XenoMouse Technology
[0328] The ability to clone and reconstruct megabase-sized human loci in YACs
and
to introduce them into the mouse germline provides a powerful approach to
elucidating the
functional components of very large or crudely mapped loci as well as
generating useful
models of human disease. Furthermore, the utilization of such technology for
substitution
of mouse loci with their human equivalents could provide unique insights into
the
expression and regulation of human gene products during development, their
communication with other systems, and their involvement in disease induction
and
progression.
[0329] An important practical application of such a strategy is the
"humanization" of
the mouse humoral immune system. Introduction of human immunoglobulin (Ig)
loci into
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mice in which the endogenous Ig genes have been inactivated offers the
opportunity to
study the mechanisms underlying programmed expression and assembly of
antibodies as
well as their role in B cell development. Furthermore, such a strategy could
provide an
ideal source for production of fully human monoclonal antibodies (Mabs) an
important
riiilestone towards fulfilling the promise of antibody therapy in human
disease.
[0330] Fully human antibodies are expected to minimize the immunogenic and
allergic responses intrinsic to mouse or mouse-derivatized Monoclonal
antibodies and thus
to increase the efficacy and safety of the administered antibodies. The use of
fully human
antibodies can be expected to provide a substantial advantage in the treatment
of chronic
and recurring human diseases, such as cancer, which require repeated antibody
administrations.
[0331] One approach towards this goal was to engineer mouse strains deficient
in
mouse antibody production with large fragments of the human Ig loci in
anticipation that
such mice would produce a large repertoire of human antibodies in the absence
of mouse
antibodies. Large human Ig fragments would preserve the large variable gene
diversity as
well as the proper regulation of antibody production and expression. By
exploiting the
mouse machinery for antibody diversification and selection and the lack of
immunological
tolerance to human proteins, the reproduced human antibody repertoire in these
mouse
strains should yield high affinity antibodies against any antigen of interest,
including
human antigens. Using the hybridoma technology, antigen-specific human
Monoclonal
antibodies with the desired specificity could be readily produced and
selected.
[0332] This general strategy was demonstrated in connection with the
generation of
the first XenoMouseT""strains as published in 1994. See Green et al. Nature
Gehetics
7:13-21 (1994). The XenoMouseT"~ strains were engineered with yeast artificial
chromosomes (YACS) containing 245 kb andl0 190 kb-sized germline configuration
fragments of the human heavy chain locus and kappa light chain locus,
respectively, which
contained core variable and constant region sequences. Id. The human Ig
containing YACs
proved to be compatible with the mouse system for both rearrangement and
expression of
antibodies and were capable of substituting for the inactivated mouse Ig
genes. This was
demonstrated by their ability to induce B-cell development, to produce an
adult-like
human repertoire of fully human antibodies, and to generate antigen-specific
human
monoclonal antibodies. These results also suggested that introduction of
larger portions of
the human Ig loci containing greater numbers of V genes, additional regulatory
elements,
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and human Ig constant regions might recapitulate substantially the full
repertoire that is
characteristic of the human humoral response to infection and immunization.
The worlc of
Green et al. was recently extended to the introduction of greater than
approximately 80%
of the human antibody repertoire through introduction of megabase sized,
germline
configuration YAC fragments of the human heavy chain loci and kappa light
chain loci,
respectively, to produce XenoMouseT"~ mice. See Mendez et al. Nature Genetics
15:146-156 (1997), Green and Jakobovits J Exp. Med. 188:483-495 (1998), Green,
Journal of Irnfraunological Methods 231:11-23 (1999) and U.S. Patent
Application Serial
No. 08/759,620, filed December 3, 1996, the disclosures of which are hereby
incorporated
by reference.
[0333] Such approach is further discussed and delineated in U.S. Patent
Application
Serial Nos. 07/466,008, filed January 12, 1990, 07/710,515, filed November 8,
1990,
07/919,297, filed July 24, 1992, 07/922,649, filed July 30, 1992, filed
08/031,801, filed
March 15,1993, 08/112,848, filed August 27, 1993, 08/234,145, filed April 28,
1994,
08/376,279, filed January 20, 1995, 081430, 938, April 27, 1995, 0-81464,584,
filed June 5,
1995, 08/464,582, filed June 5, 1995, 08/471,191, filed June 5, 1995,
08/462,837, filed
June 5, 1995, 08/486,853, filed June 5, 1995, 08/486,857, filed June 5, 1995,
08/486,859,
filed June 5, 1995, 081462,513, filed June 5, 1995, 08/724,752, filed October
2, 1996, and
081759,620, filed December 3,1996. See also Mendez et al. Nature Genetics
15:146-156
(1997) and Green and Jakobovits J Exp. Med. 188:483 495 (1998). See also
European
Patent No., EP 0 471 151 B 1, grant published June 12, 1996, International
Patent
Application No., WO 94/02602, published February 3, 1994, International Patent
Application No., WO 96/34096, published October 31, 1996, and WO 98/24893,
published June 11, 1998. The disclosures of each of the above-cited patents,
applications,
and references are hereby incorporated by reference in their entirety.
[0334] Human anti-mouse antibody (HAMA) responses have led the industry to
prepare chimeric or otherwise humanized antibodies. While chimeric antibodies
have a
human constant region and a murine variable region, it is expected that
certain human
anti-chimeric antibody (HACA) responses will be observed, particularly in
chronic or
mufti-dose utilizations of the antibody. Thus, it would be desirable to
provide fully human
antibodies against TRAIL receptor polypeptides in order to vitiate concerns
and/or effects
of HAMA or HACA responses.
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[0335] Monoclonal antibodies specific for TRAIL receptor polypeptides were
prepared using hybridoma technology. (Kohler et al., Nature 256:495 (1975);
Kohler et
al., Eur. 3. 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.~.,
pp. 571-681 (1981)). Briefly, XenoMouseT"~ mice were immunized with TRAIL
receptor
polypeptides. After immunization, the splenocytes of such mice were 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 such a selection are then assayed to identify
clones
which secrete antibodies capable of binding the TRAIL receptor polypetides.
[0336] In one embodiment, the present invention provides hybridoma cell lines
expressing an antibody of the invention. In specific embodiments, the
hybridoma cell line
of the invention is 1.2. In another specific embodiment, the hybridoma cell
line of the
invention is 1.2.1. In another specific embodiment, the hybridoma cell line of
the
invention is 1.2.2. In another specific embodiment, the hybridoma cell line of
the
invention is 1.2.3. In another specific embodiment, the hybridoma cell line of
the
invention is 1.3. In another specific embodiment, the hybridoma cell line of
the invention
is 1.3.1. In another specific embodiment, the hybridoma cell line of the
invention is 1.3.2.
In another specific embodiment, the hybridoma cell line of the invention is
1.3.3. In
another specific embodiment, the hybridoma cell line of the invention is 7.1.
In another
specific embodiment, the hybridoma cell line of the invention is 7.1.1. In
another specific
embodiment, the hybridoma cell line of the invention is 7.1.2. In another
specific
embodiment, the hybridoma cell line of the invention is 7.1.3. In another
specific
embodiment, the hybridoma cell line of the invention is 7.3. In another
specific
embodiment, the hybridoma cell line of the invention is 7.3.1. In another
specific
embodiment, the hybridoma cell line of the invention is 7.3.2. In another
specific
embodiment, the hybridoma cell line of the invention is 7.3.3. In another
specific
embodiment, the hybridoma cell line of the invention is 7.8. In another
specific
embodiment, the hybridoma cell line of the invention is 7.8.1. In another
specific
embodiment, the hybridoma cell line of the invention is 7.8.2. In another
specific
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embodiment, the hybridoma cell line of the invention is 7.8.3. In another
specific
embodiment, the hybridoma cell line of the invention is 7.10. In another
specific
embodiment, the hybridoma cell line of the invention is 7.10.1. In another
specific
embodiment, the hybridoma cell line of the invention is 7.10.2. In another
specific
embodiment, the hybridoma cell line of the invention is 7.10.3. In another
specific
embodiment, the hybridoma cell line of the invention is 7.12. In another
specific
embodiment, the hybridoma cell line of the invention is 7.12.1. In another
specific
embodiment, the hybridoma cell line of the invention is 7.12.2. In another
specific
embodiment, the hybridoma cell line of the invention is 7.12.3. In another
specific
embodiment, the hybridoma cell line of the invention is 8.3. In another
specific
embodiment, the hybridoma cell line of the invention is 8.3.1. In another
specific
embodiment, the hybridoma cell line of the invention is 8.3.2.
Additional Methods of Producing Antibodies
[0337] Antibodies of the invention (including antibody fragments or variants)
can be
produced by any method known in the art. For example, it will be appreciated
that
antibodies in accordance with the present invention can be expressed in cell
lines other
than hybridoma cell lines. Sequences encoding the cDNAs or genomic clones for
the
particular antibodies can be used for transformation of a suitable mammalian
or
nonmammalian host cells or to generate phage display libraries, for example.
Additionally,
polypeptide antibodies of the invention may be chemically synthesized or
produced
through the use of recombinant expression systems.
[0338] One way to produce the antibodies of the invention would be to clone
the VH
and/or VL domains expressed by any one or more of the hybridoma cell lines
referred to
in Table 1. In order to isolate the VH and VL domains from the hybridoma cell
lines,
PCR primers including VH or VL nucleotide sequences (See Example 5), may be
used to
amplify the expressed VH and VL sequences contained in total RNA isolated from
hybridoma cell lines. The PCR products may then be cloned using vectors, for
example,
which have a PCR product cloning site consisting of a 5' and 3' single T
nucleotide
overhang, that is complementary to the overhanging single adenine nucleotide
added onto
the 5' and 3' end of PCR products by many DNA polymerases used for PCR
reactions.
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The VH and VL domains can then be sequenced using conventional methods known
in the
art.
[0339] The cloned VH and VL genes may be placed into one or more suitable
expression vectors. By way of non-limiting example, PCR primers including VH
or VL
nucleotide sequences, a restriction site, and a flanking sequence to protect
the restriction
site may be used to amplify the VH or VL sequences. Utilizing cloning
techniques known
to those of skill in the art, the PCR amplified VH domains may be cloned into
vectors
expressing the appropriate immunoglobulin constant region, e.g., the human
IgG1 or IgG4
constant region for VH domains, and the human kappa or lambda constant regions
for
kappa and lambda VL domains, respectively. Preferably, the vectors for
expressing the
VH or VL domains comprise a promoter suitable to direct expression of the
heavy and
light chains in the chosen expression system, a secretion signal, a cloning
site for the
immunoglobulin variable domain, immunoglobulin constant domains, and a
selection
marker such as neomycin. The VH and VL domains may also be cloned into a
single
vector expressing the necessary constant regions. The heavy chain conversion
vectors and
light chain conversion vectors are then co-transfected into cell lines to
generate stable or
transient cell lines that express full-length antibodies, e.g., IgG, using
techniques known to
those of skill in the art (See, for example, Guo et al., J. Clin. Endocrinol.
Metab. 82:925-
31 (1997), and Ames et al., J. Immunol. Methods 184:177-86 (1995) which are
herein
incorporated in their entireties by reference).
[0340] The invention provides polynucleotides comprising, or alternatively
consisting
of, a nucleotide sequence encoding an antibody of the invention (including
molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof). The
invention also encompasses polynucleotides that hybridize under high
stringency, or
alternatively, under intermediate or lower stringency hybridization
conditions, e.g., as
defined supra, to polynucleotides complementary to nucleic acids having a
polynucleotide
sequence that encodes an antibody of the invention or a fragment or variant
thereof.
[0341] The polynucleotides may be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. If the amino acid
sequences
of the VH domains, VL domains and CDRs thereof, are known, nucleotide
sequences
encoding these antibodies can be determined using methods well known in the
art, i.e., the
nucleotide codons known to encode the particular amino acids are assembled in
such a
way to generate a nucleic acid that encodes the antibody, of the invention.
Such a
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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.
[0342] Alternatively, a polynucleotide encoding an antibody (including
molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) 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 or Epstein Barn virus
transformed B
cell lines that 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.
[0343] Once the nucleotide sequence of the antibody (including molecules
comprising,
or alternatively consisting of, antibody fragments or variants thereof) is
determined, the
nucleotide sequence of the antibody may be manipulated using methods well
known in the
art for the manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site
directed mutagenesis, PCR, etc. (see, for example, the techniques described in
Sambrook
et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring
Harbor
Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998, Current
Protocols in
Molecular Biology, John Wiley & Sons, NY, which are both incorporated by
reference
herein in their entireties), to generate antibodies having a different amino
acid sequence,
for example to create amino acid substitutions, deletions, and/or insertions.
[0344] In a specific embodiment, VH and VL domains of heavy and light chains
expressed by one or more cell lines referred to in Table l, or fragments or
variants thereof,
are inserted within framework regions using recombinant DNA techniques known
in the
art. In a specific embodiment, one, two, three, four, five, six, or more of
the CDRs of
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heavy and light chains expressed by one or more cell lines referred to in
Table 1, or
fragments or variants thereof, is inserted within framework regions using
recombinant
DNA techniques known in the art. The frameworlc 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
frameworlc
regions, the contents of which are hereby incorporated by reference in its
entirety).
Preferably, the polynucleotides generated by the combination of the framework
regions
and CDRs encode an antibody (including molecules comprising, or alternatively
consisting of, antibody fragments or variants thereof) that specifically binds
to a TRAIL
receptor. Preferably, as discussed supra, polynucleotides encoding variants of
antibodies
or antibody fragments having one or more amino acid substitutions may be made
within
the framework regions, and, preferably, the amino acid substitutions do not
significantly
alter 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, or
antibody fragments or variants, lacking one or more intrachain disulfide
bonds. Other
alterations to the polynucleotide are encompassed by the present invention and
fall within
the ordinary skill of the art.
[0345] For some uses, such as for in vitro affinity maturation of an antibody
of the
invention, it may be useful to express the VH and VL domains of the heavy and
light
chains expressed by one or more cell lines referred to in Table 1 as single
schain
antibodies or Fab fragments in a phage display library. For example, the cDNAs
encoding
the VH and VL domains expressed by the cell lines referred to in Table 1 rnay
be
expressed in all possible combinations using a phage display library, allowing
for the
selection of VH/VL combinations that bind a TRAIL receptor polypeptides with
preferred
binding characteristics such as improved affinity or improved off rates.
Additionally, VH
and VL segments - the CDR regions of the VH and VL domains expressed by the
cell
lines referred to in Table 1, in particular, may be mutated in vitro.
Expression of VH and
VL domains with "mutant" CDRs in a phage display library allows for the
selection of
VH/VL combinations that bind a TRAIL receptor polypeptides with preferred
binding
characteristics such as improved affinity or improved off rates.
[0346] In phage display methods, functional antibody domains are displayed on
the
surface of phage particles which carry the polynucleotide sequences encoding
them. In
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particular, DNA sequences encoding VH and VL domains are amplified from animal
cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues) or
synthetic
cDNA libraries. The DNA encoding the VH and VL domains are joined together by
an
scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or
pComb 3
HSS). The vector is electroporated in E. coli and the E. coli is infected with
helper phage.
Phage used in these methods are typically filamentous phage including fd and
M13 and
the VH and VL domains are usually recombinantly fused to either the phage gene
III or
gene VIII. Phage expressing an antigen binding domain that binds to an antigen
of interest
(i.e., a TRAIL receotor polypeptide or a fragment thereof) can be selected or
identified
with antigen, e.g., using labeled antigen or antigen bound or captured to a
solid surface or
bead. Examples of phage display methods that can be used to make the
antibodies of the
present invention include, but are not limited to, 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. 24:952-958 (1994); Persic et
al., Gene 187
9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT
application
No. PCT/GB91/01 134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;
WO 92/18719; WO 93/1 1236; WO 95/15982; WO 95/20401; WO97/13844; 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; 5,516,717; 5,780,225; 5,658,727; 5,735,743 and
5,969,108; each of
which is incorporated herein by reference in its entirety.
[0347] For some uses, including i~z vivo use of antibodies in humans and in
vitro
detection assays, it may be preferable to use human or chimeric antibodies.
Completely
human antibodies are particularly desirable for therapeutic treatment of human
patients.
See also, U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT publications WO
98/46645,
WO 98/50435, WO 98/24893, W098/16654, WO 96/34096, WO 96/35735, and WO
91/10741; each of which is incorporated herein by reference in its entirety.
In a specific
embodiment, antibodies of the present invention comprise one or more VH and VL
domains of the invention and constant regions from another immunoglobulin
molecule,
preferably a human immunoglobulin molecule. In a specific embodiment,
antibodies of
the present invention comprise one or more CDRs corresponding to the VH and VL
domains of the invention and framework regions from another immunoglobulin
molecule,
preferably a human immunoglobulin molecule. In other embodiments, an antibody
of the
present invention comprises one, two, three, four, five, six or more VL CDRs
or VH
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CDRs corresponding to one or more of the VH or VL domains expressed by one or
more
cell lines referred to in Table 1, or fragments or variants thereof, and
frameworle regions
(and, optionally one or more CDRs not derived from the antibodies expressed by
cell lines
referred to in Table 1) from a human immunoglobulin molecule. In a preferred
embodiment, an antibody of the present invention comprises a VH CDR3, VL CDR3,
or
both, corresponding to the same cell line, or different cell lines selected
from the cell lines
referred to in Table 1, or fragments or variants thereof, and framework
regions from a
human immunoglobulin.
[0348] A chimeric antibody is a molecule in which different portions of the
antibody
are derived from different immunoglobulin molecules such as antibodies having
a variable
region derived from a human antibody and a non-human (e.g., murine)
immunoglobulin
constant region or vice versa. Methods for producing chimeric antibodies are
known in
the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques
4:214
(1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); U.S. Patent
Nos.
5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by
reference in their
entirety. Chimeric antibodies comprising one or more CDRs from human species
and
framework regions from a non-human immunoglobulin molecule (e.g., framework
regions
from a murine, canine or feline immunoglobulin molecule) (or vice versa) can
be
produced 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 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. 5,565,352). In a preferred embodiment, chimeric antibodies comprise
a human
CDR3 having an amino acid sequence of any one of the VH CDR3s or VL CDR3s of a
heavy or light chain expressed by one or more of the cell lines referred to in
Table 1, or a
variant thereof, and non-human framework regions or human framework regions
different
from those of the frameworks in the corresponding cell line disclosed in Table
1. Often,
framework residues in the 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 sequence comparison to identify unusual
framework
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residues at particular positions. (See, e.g., Queen et al., U.S. Patent No.
5,585,089;
Riechmann et al., Nature 352:323 (1988), which are incorporated herein by
reference in
their entireties.)
[0349] Tntrabodies are antibodies, often scFvs, that are expressed from a
recombinant
nucleic aicd molecule and engineered to be retained intracellularly (e.g.,
retained in the
cytoplasm, endoplasmic reticulum, or periplasm). Intrabodies may be used, for
example,
to ablate the function of a protein to which the intrabody binds. The
expression of
intrabodies may also be regulated through the use of inducible promoters in
the nucleic
acid expression vector comprising the intrabody. Intrabodies of the invention
can be
produced using methods known in the art, such as those disclosed and reviewed
in Chen et
al., Hum. Gene Ther. 5:595-601 (1994); Marasco, W.A., Ge>ze Ther. 4:11-15
(1997);
Rondon and Marasco, Annu. Rev. Microbiol. 51:257-283 (1997); Proba et al., J.
Mol. Biol.
275:245-253 (1998); Cohen et al., Ozzcogezze 17:2445-2456 (1998); Ohage and
Steipe, J.
Mol. Biol. 291:1119-1128 (1999); Ohage et al., J. Mol. Biol. 291:1129-1134
(1999); Wirtz
and Steipe, Protein Sci. 5:2245-2250 (1999); Zhu et al., J. Inzmurzol.
Metlzods 231:207-222
(1999); and references cited therein.
[0350] Recombinant expression of an antibody of the invention (including
antibody
fragments or variants thereof (e.g., a heavy or light chain of an antibody of
the invention),
requires construction of an expression vectors) containing a polynucleotide
that encodes
the antibody. Once a polynucleotide encoding an antibody molecule (e.g., a
whole
antibody, a heavy or light chain of an antibody, or portion thereof
(preferably, but not
necessarily, containing the heavy or light chain variable domain)), of the
invention has
been obtained, the vectors) 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 (e.g., a
whole
antibody, a heavy or light chain of an antibody, a heavy or light chain
variable domain of
an antibody, or a portion thereof, or a heavy or light chain CDR, a single
chain Fv, or
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fragments or variants thereof), 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, the contents of each of which are hereby incorporated by reference
in its
entirety) and the variable domain of the antibody may be cloned into such a
vector for
expression of the entire heavy chain, the entire light chain, or both the
entire heavy and
light chains.
[0352] The expression vectors) is(are) 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
polynucleotide(s) encoding an antibody of the invention (e.g., whole antibody,
a heavy or
light chain thereof, or portion thereof, or a single chain antibody, or a
fragment or variant
thereof), operably linked to a heterologous promoter. In preferred
embodiments, for the
expression of entire antibody molecules, vectoxs 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.
[0352] A variety of host-expression vector systems may be utilized to express
the
antibody molecules of the invention. Such host-expression systems represent
vehicles by
which the coding sequences of interest may be produced and subsequently
purified, but
also represent cells which may, when transformed or transfected with the
appropriate
nucleotide coding sequences, express an antibody molecule of the invention in
situ. These
include, but are not limited to, bacteriophage particles engineered to express
antibody
fragments or variants teherof (single chain antibodies), 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, Picl2ia) transformed with recombinant yeast expression
vectors
containing antibody coding sequences; insect cell systems infected with
recombinant virus
expression vectors (e.g., baculovirus) containing antibody coding sequences;
plant cell
systems infected with recombinant virus expression vectors (e.g., cauliflower
mosaic
virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant
plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or
mammalian
cell systems (e.g., COS, CHO, BHK, 293, 3T3, NSO cells) harboring recombinant
expression constructs containing promoters derived from the genome of
mammalian cells
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(e.g., metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late
promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such
as
Escherichia cola, and more preferably, eukaryotic cells, especially for the
expression of
whole recombinant antibody molecule, are used for the expression of a
recombinant
antibody molecule. For example, mammalian cells such as Chinese hamster ovary
cells
(CHO), in conjunction with a vector such as the major intermediate early gene
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
(1990);
Bebbington et al., Bio/Techniques 10:169 (1992); Keen and Hale, Cytotechnology
18:207
(1996)). These references are incorporated in their entirities by refernce
herein.
[0353 In bacterial systems, a number of expression vectors may be
advantageously
selected depending upon the use intended for the antibody molecule being
expressed. For'
example, when a large quantity of such a protein is to be produced, for the
generation of
pharmaceutical compositions of an antibody molecule, vectors which direct the
expression
of high levels of fusion protein products that are readily purified may be
desirable. Such
vectors include, but are not limited to, the E. coli expression vector pUR278
(Ruther et al.,
EMBO 1. 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
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 5-
transferase (GST). In general, such fusion proteins are soluble and can easily
be purified
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.
[0354] In an insect system, Autographa californica nuclear polyhedrosis virus
(AcNPV) may be used as a vector to express foreign genes. The virus grows in
Spodoptera frugiperda cells. Antibody coding sequences 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).
[0355) 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
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coding sequence of interest may be ligated to an adenovirus
transcriptionltranslation
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 8 1:355-
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, e.g.,
Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
[0356] 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, NSO, MDCI~, z93, 3T3, W138, and in particular, breast
cancer
cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and
normal
mammary gland cell line such as, for example, CRL7O30 and HsS78Bst.
[0357] For long-term, high-yield production of recombinant proteins, stable
expression is preferred. For example, cell lines which stably express the
antibody may be
engineered. Rather than using expression vectors which contain viral origins
of
replication, host cells can be transformed with DNA controlled by appropriate
expression
control elements (e.g., promoter, enhancer, sequences, transcription
terminators,
polyadenylation sites, etc.), and a selectable marker. Following the
introduction of the
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CA 02426710 2003-04-23
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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 compositions that interact directly or indirectly
with the
antibody molecule.
[035$] 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 22:8
17 (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); TIB TECH 11(5):155-2 15 (May, 1993));
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); I~riegler, 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-Garapin et
al., J.
Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their
entireties.
[0359] 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
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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 coding sequence of the antibody, production of
the antibody
will also increase (Grouse et al., Mol. Cell. Biol. 3:257 (1983)).
[0360] Vectors which use glutamine synthase (GS) or DHFR as the selectable
markers
can be amplified in the presence of the drugs methionine sulphoxixnine or
methotrexate,
respectively. An advantage of glutamine synthase based vectors are the
availabilty of cell
lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase
negative.
Glutamine synthase expression systems can also function in glutamine synthase
expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) by providing
additional
inhibitor to prevent the functioning of the endogenous gene. A glutamine
synthase
expression system and components thereof are detailed in PCT publications:
W087/04462; WO86/05807; W089/01036; W089/10404; and W091/06657 which are
incorporated in their entireties by reference herein. Additionally, glutamine
synthase
expression vectors that may be used according to the present invention are
commercially
available from suplliers, including, for example Lonza Biologics, Inc.
(Portsmouth, NH).
Expression and production of monoclonal antibodies using a GS expression
system in
murine myeloma cells is described in Bebbington et ad., Bioltechnology
10:169(1992) and
in Biblia and Robinson Biotechreol. Prog. 11:1 (1995) which are incorporated
in their
entirities by reference herein.
[0361] 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
is preferably
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.
[0362] Once an antibody molecule of the invention (including molecules
comprising,
or alternatively consisting of, antibody fragments or variants thereof) has
been chemically
synthesized or recombinantly expressed, it may be purified by any method known
in the
art for purification of an immunoglobulin molecule, or more generally, a
protein molecule,
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such as, 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. Further, the antibodies of the present invention may
be fused to
heterologous polypeptide sequences described herein or otherwise known in the
art, to
facilitate purification.
Characterization of anti-TRAIL Receptor Antibodies
[0363] Antibodies of the present invention (including molecules comprising, or
alternatively consisting of, antibody fragments or variants thereof) may also
be described
or specified in terms of their binding to TRAIL receptor polypeptides or
fragments or
variants of TRAIL receptor polypeptides. In specific embodiments, antibodies
of the
invention bind TRAIL receptor polypeptides, or fragments or variants thereof,
with a
dissociation constant or KD of less than or equal to 5 X 10-2 M, 10-2 M, 5 X
10-3 M,10-3 M,
X 10-4 M, 10-4 M, 5 X 10-5 M, or 10-$ M. More preferably, antibodies of the
invention
bind TRAIL receptor polypeptides or fragments or variants thereof with a
dissociation
constant or KD less than or equal to 5 X 10-6 M, 10-6 M, 5 X 10-7 M, 10-7 M, 5
X 10-8 M, or
10-8 M. Even more preferably, antibodies of the invention bind TRAIL receptor
polypeptides or fragments or variants thereof with a dissociation constant or
KD less than
or equal to 5 X 10-9 M,10-9 M, 5 X 10-1° M, 101° M, 5 X 10-11 M,
10-11 M, 5 X 10-12 M,
10-12 M, 5 X -13 M,10-13 M, 5 X 10-14 M, 10-14 M, 5 X 10-15 M, or 10-15 M. The
invention
encompasses antibodies that bind TRAIL Receptor polypeptides with a
dissociation
constant or Kn that is within one of the ranges that are between each of the
individual
recited values.
[0364] In specific embodiments, antibodies of the invention bind TRAIL
receptor
polypeptides or fragments or variants thereof with an off rate (ko~) of less
than or equal to
5 X 10-2 sec'1, 10-2 sec 1, 5 X 10y3 sec 1 or 10-3 sec 1. More preferably,
antibodies of the
invention bind TRAIL receptor polypeptides or fragments or variants thereof
with an off
rate (ko~) less than or equal to 5 X 10-4 sec 1, 10-4 sec 1, 5 X 10-5 sec 1,
or 10-5 sec 15 X 10-6
sec 1, 10-6 sec 1, 5 X 10-7 sec 1 or 10-7 sec 1. The invention encompasses
antibodies that bind
TRAIL Receptor polypeptides with an off rate (ko~) that is within one of the
ranges that
are between each of the individual recited values.
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[0365] In other embodiments, antibodies of the invention bind TRAIL receptor
polypeptides or fragments or variants thereof with an on rate (kon) of greater
than or equal
to 103 M-1 sec-1, 5 X 103 M-1 sec 1, 104 M-1 sec 1 or 5 X 104 M-1 sec-1. More
preferably,
antibodies of the invention bind TRAIL receptor polypeptides or fragments or
variants
thereof with an on rate (kon) greater than or equal to 105 M-1 sec'1, 5 X 105
M-1 sec-1, 106
M-1 sec-1, or 5 X 106 M-1 sec 1 or 107 M-I sec-1. The invention encompasses
antibodies that
bind TRAIL Receptor polypeptides with on rate (kon) that is within one of the
ranges that
are between each of the individual recited values.
[0366] The antibodies of the invention (including molecules comprising, or
alternatively consisting of, antibody fragments or variants thereof)
immunospecifically
bind to a polypeptide or polypeptide fragment or variant of human TRAIL
receptor
polypeptides (SEQ ID NOS:1-4). In another embodiment, the antibodies of the
invention
immunospecifically bind to a polypeptide or polypeptide fragment or variant of
simian
TRAIL receptor polypeptides. In yet another embodiment, the antibodies of the
invention
immunospecifically bind to a polypeptide or polypeptide fragment or variant of
murine
TRAIL receptor polypeptides. In one embodiment, the antibodies of the
invention bind
immunospecifically to human and simian TRAIL receptor polypeptides. In another
embodiment, the antibodies of the invention bind immunospecifically to human
TRAIL
receptor polypeptides and murine TRAIL receptor polypeptides. More preferably,
antibodies of the invention, preferentially bind to human TRAIL receptor
polypeptides
compared to murine TRAIL receptor polypeptides.
[0367] In preferred embodiments, the antibodies of the present invention
(including
molecules comprising, or alternatively consisting of, antibody fragments or
variants
thereof), immunospecifically bind to TRAIL receptor polypeptides and do not
cross-react
with any other antigens. In preferred embodiments, the antibodies of the
invention
immunospecifically bind to TRAIL receptor polypeptides (e.g., SEQ ID NOS:1-4
or
fragments or variants thereof) and do not cross-react with one or more
additional members
of the Tumor Necrosis Factor Tumor Necrosis Factor Receptor Family
polypeptides (e.g.,
BCMA, TACI, CD30, CD2.7, OX40, 4-1BB, CD40, NGFR, TNFR1, TNFR2, Fas, and
NGFR).
[0368] In another embodiment, the antibodies of the present invention
(including
molecules comprising, or alternatively consisting of, antibody fragments or
variants
thereof), immunospecifically bind to TRAIL receptor polypeptides and cross-
react with
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other antigens. In other embodiments, the antibodies of the invention
immunospecifically
bind to TRAIL receptor polypeptides (e.g., SEQ ID NOS:1-4 or fragments or
variants
thereof) and cross-react with one or more additional members of the Tumor
Necrosis
Factor Receptor Family polypeptides (e.g., BCMA, TACI, CD30, CD27, OX40, 4-
1BB,
CD40, NGFR, TNFR1, TNFR2, Fas, and NGFR).
[0369] In a preferred embodiment, antibodies of the invention preferentially
bind TR4
(SEQ ID N0:1), or fragments and variants thereof relative to their ability to
bind TR5,
TR7, or TR10 (SEQ m NOS:2-4) or fragments or variants thereof. In another
preferred
embodiment, antibodies of the invention preferentially bind TR7, or fragments
and
variants thereof relative to their ability to bind TR4, TRS, or TR10 (SEQ ID
NOS:1, 2, and
4) or fragments or variants thereof. In other preferred embodiments, the
antibodies of the
invention preferentially bind to TR4 and TR7 (SEQ ID NOS:1 and 3), or
fragments and
variants thereof relative to their ability to bind TR5 or TR10 (SEQ ID NOS:2
and 4) or
fragments or variants thereof. In other preferred embodiments, the antibodies
of the
invention preferentially bind to TR5 and TR10 (SEQ ~ NOS:2 and 4), or
fragments and
variants thereof relative to their ability to bind TR4 or TR7 (SEQ ID NOS:1
and 3) or
fragments or variants thereof. In other preferred embodiments, the antibodies
of the
invention bind TR4, TRS, TR7 and TR10 (SEQ ID NOS:1-4). In another embodiment,
antibodies of the invention preferentially bind TR5 (SEQ ID N0:2), or
fragments and
variants thereof relative to their ability to bind TR4, TR7 or TR10 (SEQ lD
NOS:1, 4 and
5) or fragments or variants thereof. In another embodiment, antibodies of the
invention
preferentially bind TR10 (SEQ ID N0:4), or fragments and variants thereof
relative to
their ability to bind TR4, TRS, or TR7 (SEQ ID NOS: l-3) or fragments or
variants
thereof. An antibody's ability to preferentially bind one antigen compared to
another
antigen may be determined using any method known in the art.
[0370] By way of non-limiting example, an antibody may be considered to bind a
first
antigen preferentially if it binds said first antigen with a dissociation
constant (KD) that is
less than the antibody's KD for the second antigen. In another non-limiting
embodiment,
an antibody may be considered to bind a first antigen preferentially if it
binds said first
antigen with an affinity (KD) that is at least one order of magnitude less
than the
antibody's KD for the second antigen. In another non-limiting embodiment, an
antibody
may be considered to bind a first antigen preferentially if it binds said
first antigen with an
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affinity (KD) that is at least two orders of magnitude less than the
antibody's KD for the
second antigen.
[0371] In another non-limiting embodiment, an antibody may be considered to
bind a
first antigen preferentially if it binds said first antigen with an off rate
(ko~) that is less than
the antibody's ko~ for the second antigen. In another non-limiting embodiment,
an
antibody may be considered to bind a first antigen preferentially if it binds
said first
antigen with a ko~ that is at least one order of magnitude less than the
antibody's ko~ for
the second antigen. In another non-limiting embodiment, an antibody may be
considered
to bind a first antigen preferentially if it binds said first antigen with a
ko~ that is at least
two orders of magnitude less than the antibody's ko~ for the second antigen.
[0372] The invention also encompasses antibodies (including molecules
comprising,
or alternatively consisting of, antibody fragments or variants thereof) that
have one or
more of the same biological characteristics as one or more of the antibodies
described
herein. By "biological characteristics" is meant, the in vitro or in vivo
activities or
properties of the antibodies, such as, for example, the ability to bind to
TRAIL receptor
polypeptides (e.g., membrane-embedded TRAIL receptors), the ability to
stimulate TRAIL
receptor mediated biological activity (e.g., to stimulate apoptosis of TRAIL
receptor
expressing cells, see Example 4); the ability to substantially block TRAIL
receptor ligand
(e.g. TRAIL (SEQ ID N0:5), also known as AIM-I, International Application No.
WO
97/35899 and U.S. Patent Application 5,771,223), or a fragment, variant or
fusion protein
thereof, binding to TRAIL receptor, see Example 3; or the ability to
upregulate TRAIL
receptor expression on the surface of cells. Other biological activities that
antibodies
against TRAIL, receptor polypeptides may have, include, but are not limited
to, the ability
to inhibit TRAIL receptor mediated biological activity (e.g., to inhibit
apoptosis of TRAIL
receptor expressing cells) or the ability to downregulate TRAIL receptor
expression on
the surface of cells. Optionally, the antibodies of the invention will bind to
the same
epitope as at least one of the antibodies specifically referred to herein.
Such epitope
binding can be routinely determined using assays known in the art.
[0373] The present invention also provides for antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof), that
stimulate one or more TRAIL receptor polypeptide mediated biological
activities. In one
embodiment, an antibody that stimulates one or more TRAIL receptor polypeptide
mediated biological activities comprises, or alternatively consists of a VH
and/or a VL
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domain of a heavy chain and/or a light chain, respectively, expressed by at
least one of the
cell lines referred to in Table 1, or fragment or variant thereof. In a
specific embodiment,
an antibody that stimulates one or more TRAIL receptor polypeptide mediated
biological
activities comprises, or alternatively consists of a VH and a VL domain of a
heavy chain
arid a light chain, respectively, expressed by any one of the cell lines
referred to in Table
1, or fragment or variant thereof. Nucleic acid molecules encoding these
antibodies are
also encompassed by the invention.
[0374] The present invention also provides for antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof), that
stimulate apoptosis of TRAIL receptor expressing cells (see Example 4). In one
embodiment, an antibody that stimulates apoptosis of TRAIL receptor expressing
cells
comprises, or alternatively consists of a VH and/or a VL domain of a heavy
chain and/or a
light chain, respectively, expressed by at least one of the cell lines
referred to in Table 1,
or fragment or variant thereof. In a specific embodiment, an antibody that
stimulates
apoptosis of TRAIL receptor expressing cells comprises, or alternatively
consists of a VH
and a VL domain of a heavy chain and a light chain, respectively, expressed by
any one of
the cell lines referred to in Table 1, or fragment or variant thereof. Nucleic
acid molecules
encoding these antibodies are also encompassed by the invention.
[0375] In preferred embodiments, the present invention also provides for
antibodies
(including molecules comprising, or alternatively consisting of, antibody
fragments or
variants thereof), that stimulate apoptosis of TRAIL receptor expressing cells
equally well
in the presence or absence of antibody cross-linking reagents, such as for
example anti-Ig
Fc reagents cells (See, for example, Example 4 and Figures 2, 3, and 4). In a
specific
embodiment, antibodies of the present invention stimulate apoptosis of HeLa
cells, equally
well in the presence or absence of an anti-Ig Fc antibody cross-linking
reagent. In another
specific embodiment, antibodies of the present invention stimulate apoptosis
of HeLa
cells, equally well in the presence or absence of an anti-Ig Fc antibody cross-
linking
reagent in the presence of 2 micrograms/milliliter of cycloheximide. In
another
embodiment, antibodies of the present invention stimulate apoptosis of SW480
cells,
equally well in the presence or absence of an anti Ig Fc antibody cross-
linking reagent. In
another specific embodiment, antibodies of the present invention stimulate
apoptosis of
SW480 cells, equally well in the presence or absence of an anti-Ig Fc antibody
cross-
linking reagent in the presence of 2 micrograms/milliliter of cycloheximide.
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[0376] In other preferred embodiments, the present invention also provides for
antibodies (including molecules comprising, or alternatively consisting of,
antibody
fragments or variants thereof), that stimulate apoptosis of TRAIL receptor
expressing cells
at least as well as an equal concentration (in terms of, for example,
nanograms/milliliter)
of TRAIL polypeptide (including TRAIL polypeptide fragments, variants or
fusion
proteins) stimulates apoptosis of TRAIL receptor expressing cells (See, for
example,
Example 4 and Figures 2, 3, and 4). In a specific embodiment, antibodies of
the invention
stimulate apoptosis of TRAIL receptor expressing cells better than an equal
concentration
(in terms of, for example, nanograms/milliliter) of TRAIL polypeptide
(including TRAIL
polypeptide fragments, variants or fusion proteins) stimulates apoptosis of
TRAIL receptor
expressing cells. In a specific embodiment, antibodies of the invention
stimulate apoptosis
of HeLa cells better than an equal concentration (in terms of, for example,
nanogramslmilliliter) of TRAIL polypeptide (including TRAIL polypeptide
fragments,
variants or fusion proteins) stimulates apoptosis of TRAIL receptor expressing
cells. In
another specific embodiment, antibodies of the present invention stimulate
apoptosis of
HeLa cells better than an equal concentration (in terms of, for example,
nanogramslmilliliter) of TRAIL polypeptide (including TRAIL polypeptide
fragments,
variants or fusion proteins) stimulates apoptosis of TRAIL receptor expressing
cells in the
presence of 2 micrograms/milliliter of cycloheximide.
[0377] In other preferred embodiments, the present invention also provides for
antibodies (including molecules comprising, or alternatively consisting of,
antibody
fragments or variants thereof), that stimulate more apoptosis of TRAIL
receptor
expressing cells when administered in combination with a chemotherapeutic
drug, than
either the chemotherapeutic drug or the antibodies alone stimulate apoptosis
of receptor
expressing cells (see, for example, Figure 6). In specific embodiments,
antibodies of the
present invention, stimulate more apoptosis of TRAIL receptor expressing cells
when
administered in combination with Topotecan, than either Topotecan or the
antibodies
alone stimulate apoptosis of receptor expressing cells. In specific
embodiments, antibodies
of the present invention, stimulate more apoptosis of TRAIL, receptor
expressing cells
when administered in combination with cycloheximide, than either cycloheximide
or the
antibodies alone stimulate apoptosis of receptor expressing cells.
[0378] The present invention also provides for antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof), that
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blocks or inhibits the binding of TRAIL to a TRAIL receptor polypeptide (see
Example 3).
In one embodiment, an antibody that blocks or inhibits the binding of TRAIL to
a TRAIL
receptor polypeptide comprises, or alternatively consists of a VH and/or a VL
domain of a
heavy chain and/or a light chain, respectively, expressed by at least one of
the cell lines
referred to in Table 1, or fragment or variant thereof. In a specific
embodiment, an
antibody that blocks or inhibits the binding of TRAIL to a TRAIL receptor
polypeptide
comprises, or alternatively consists of a VH and a VL domain of a heavy chain
and a light
chain, respectively, expressed by any one of the cell lines referred to in
Table 1, or
fragment or variant thereof. Nucleic acid molecules encoding these antibodies
are also
encompassed by the invention.
[0379] Antibodies of the present invention (including antibody fragments or
variants
thereof) may be characterized in a variety of ways. In particular, antibodies
and related
molecules of the invention may be assayed for the ability to
immunospecifically bind to
TRAIL receptor polypeptides or a fragment or variant of TRAIL receptor
polypeptides
using techniques described herein or routinely modifying techniques known in
the art.
Assays for the ability of the antibodies of the invention to
immunospecifically bind
TRAIL receptor polypeptides or a fragment of TRAIL receptor polypeptides may
be
performed in solution (e.g., Houghten, Bio/Techniques 13:412-421(1992)), on
beads (e.g.,
Lam, Nature 354:82-84 (1991)), on chips (e.g., Fodor, Nature 364:555-556
(1993)), on
bacteria (e.g., U.S. Patent No. 5,223,409), on spores (e.g., Patent Nos.
5,571,698;
5,403,484; and 5,223,409), on plasmids (e.g., Cull et al., Proc. Natl. Acad.
Sci. USA
89:1865-1869 (1992)) or on phage (e.g., Scott and Smith, Science 249:386-390
(1990);
Devlin, Science 249:404-406 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA
87:7178-7182 (1990); and Felici, J. Mol. Biol. 222:301-310 (1991)) (each of
these
references is incorporated herein in its entirety by reference). Antibodies
that have been
identified to immunospecifically bind to TRAIL receptor polypeptides or a
fragment or
variant of TRAIL receptor polypeptides can then be assayed for their
specificity and
affinity for TRAIL receptor polypeptides or a fragment of TRAIL receptor
polypeptides
using or routinely modifying techniques described herein or otherwise known in
the art.
[0380] The antibodies of the invention may be assayed for immunospecific
binding to
TRAIL receptor polypeptides and cross-reactivity with other antigens by any
method
known in the art. Immunoassays which can be used to analyze immunospecific
binding
and cross-reactivity include, but are not limited to, competitive and non-
competitive assay
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systems using techniques such as BIAcore analysis (See, e.g., Example 2), FAGS
(fluorescence activated cell sorter) analysis (see, e.g., Example 4 and Figure
1),
immunofluorescence, immunocytochemistry, radioimrnunoassays, ELISA (enzyme
linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays,
western
blots, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays,
agglutination assays, complement-fixation assays, immunoradiometric assays,
fluorescent
immunoassays, and 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. l, 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).
[0381] ELISAs comprise preparing antigen, coating the well of a 96-well
microtiter
plate with the antigen, washing away antigen that did not bind the wells,
adding the
antibody of interest conjugated to a detectable compound such as an enzymatic
substrate
(e.g., horseradish peroxidase or alkaline phosphatase) to the wells and
incubating for a
period of time, washing away unbound antibodies or non-specifically bound
antibodies,
and detecting the presence of the antibodies specifically bound to the antigen
coating the
well. 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. Alternatively,
the antigen
need not be directly coated to the well; instead the ELISA plates may be
coated with an
anti-Ig Fc antibody, and the antigen in the form or a TRAIL receptor-Fc fusion
protein,
may be bound to the anti-Ig Fc coated to the plate. This may be desirable so
as to
maintain the antigen protein (e.g., the TRAIL receptor polypeptides) in a more
native
conformation than it may have when it is directly coated to a plate. In
another alternative,
instead of coating the well with the antigen, the antibody may be coated to
the well. In
this case, the detectable molecule could be the antigen conjugated to a
detectable
compound such as an enzymatic substrate (e.g., horseradish peroxidase or
alkaline
phosphatase). 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. l, John Wiley & Sons, Inc., New
York at
11.2.1.
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[0382] The binding affinity of an antibody (including an scFv or other
molecule
comprising, or alternatively consisting of, antibody fragments or variants
thereof) 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., antigen
labeled with
3H or 1~'SI), or fragment or variant thereof 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 the present invention for
TRAIL receptor
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, TRAIL receptor polypeptide is incubated with an antibody of the
present
invention conjugated to a labeled compound (e.g., compound labeled with 3H or
lasl) in
the presence of increasing amounts of an unlabeled second anti-TRAIL receptor
antibody.
This kind of competitive assay between two antibodies, may also be used to
determine if
two antibodies bind the same or different epitopes.
[0383] In a preferred embodiment, BIAcore kinetic analysis is used to
determine the
binding on and off rates of antibodies (including antibody fragments or
variants thereof) to
a TRAIL receptor, or fragments of a TRAIL receptor. BIAcore kinetic analysis
comprises
analyzing the binding and dissociation of antibodies from chips with
immobilized TRAIL
receptors on their surface as described in detail in Example 2.
[0384] Immunoprecipitation protocols generally comprise lysing a population of
cells
in a lysis buffer such as RIPA buffer (1% 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., 1 to 4 hours) at 40 degrees C, adding protein A
and/or protein G
sepharose beads to the cell lysate, incubating for about an hour or more at 40
degrees C,
washing the beads in lysis buffer and resuspending the beads in SDS/sample
buffer. The
ability.of the antibody of interest to irnmunoprecipitate 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,
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1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,
New
York at 10.16.1.
[0385] Western blot analysis generally comprises preparing protein samples,
electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-PAGE
depending on the molecular weight of the antigen), transferring the protein
sample from
the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon,
blocking the
membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing
the
membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with
primary
antibody (the antibody of interest) diluted in blocking buffer, washing the
membrane in
washing buffer, blocking the membrane with a secondary antibody (which
recognizes the
primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic
substrate
(e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule
(e.g., 32P or
1251) 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.
Antibody Conjugates
[0386] The present invention encompasses antibodies (including antibody
fragments
or variants thereof), recombinantly fused or chemically conjugated (including
both
covalent and non-covalent conjugations) to a heterologous polypeptide (or
portion thereof,
preferably at least 10, at least 20, at least 30, at least 40, at least 50, at
least 60, at least 70,
at least 80, at least 90 or at least 100 amino acids of the polypeptide) to
generate fusion
proteins. The fusion does not necessarily need to be direct, but may occur
through linker
sequences. For example, antibodies of the invention may be used to target
heterologous
polypeptides to particular cell types (e.g., cancer cells), either in vitro or
in vivo, by fusing
or conjugating the heterologous polypeptides to antibodies of the invention
that are
specific for particular cell surface antigens or which bind antigens that bind
particular cell
surface receptors. Antibodies of the invention may also be fused to albumin
(including but
not limited to recombinant human serum albumin (see, e.g., U.S. Patent No.
5,876,969,
issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No. 5,766,883,
issued June
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16, 1998, herein incorporated by reference in their entirety)), resulting in
chimeric
polypeptides. In a preferred embodiment, polypeptides and/or antibodies of the
present
invention (including fragments or variants thereof) are fused with the mature
form of
human serum albumin (i.e., amino acids 1- 585 of human serum albumin as shown
in
Figures 1 and 2 of EP Patent 0 322 094) which is herein incorporated by
reference in its
entirety. In another preferred embodiment, polypeptides and/or antibodies of
the present
invention (including fragments or variants thereof) are fused with polypeptide
fragments
comprising, or alternatively consisting of, amino acid residues 1-z of human
serum
albumin, where z is an integer from 369 to 419, as described in U.S. Patent
5,766,883
herein incorporated by reference in its entirety. Polypeptides and/or
antibodies of the
present invention (including fragments or variants thereof) may be fused to
either the N-
or C-terminal end of the heterologous protein (e.g., immunoglobulin Fc
polypeptide or
human serum albumin polypeptide). Polynucleotides encoding fusion proteins of
the
invention are also encompassed by the invention. Such fusion proteins may, for
example,
facilitate purification and may increase half-life in vivo. Antibodies fused
or conjugated
to heterologous polypeptides 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 93/2 1232; 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. Irmnunol.
146:2446-2452 (1991), which are incorporated by reference in their entireties.
[0387] The present invention further includes compositions comprising, or
alternatively consisting of, heterologous polypeptides fused or conjugated to
antibody
fragments. For example, the heterologous polypeptides may be fused or
conjugated to a
Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, or a portion thereof.
Methods
for fusing or conjugating polypeptides to antibody portions are known in the
art. See, e.g.,
U.S. Patent Nos. 5,356,603; 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 9 1/06570; Ashkenazi et
al.,
Proc. Natl. Acad. Sci. USA 88: 10535-10539 (1991); Zheng et al., J. Irmnunol.
154:5590-
5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11357- 11341 (1992)
(said
references incorporated by reference in their entireties).
[0388] 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
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modulate the activities of antibodies (including molecules comprising, or
alternatively
consisting of, antibody fragments or variants thereof), such methods can be
used to
generate antibodies with altered activity (e.g., antibodies with higher
affinities and lower
dissociation rates). 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-
35 (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, polynucleotides encoding antibodies of the invention 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
portions of
a polynucleotide encoding an antibody which portions immunospecifically bind
to TRAIL
receptor may be recombined with one or more components, motifs, sections,
parts,
domains, fragments, etc. of one or more heterologous molecules.
[0389] Moreover, the antibodies of the present invention (including antibody
fragments or variants thereof), can be fused to marker sequences, such as a
polypeptides to
facilitate purification. In preferred embodiments, the marker amino acid
sequence is a
hexa-histidine polypeptide, 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
hemagglutinin "HA"
tag, which corresponds to an epitope derived from the influenza hemagglutinin
protein
(Wilson et al., Cell 37:767 (1984)) and the FLAG~ tag (Stratagene, La Jolla,
CA).
[0390] The present invention further encompasses antibodies (including
antibody
fragments or variants thereof), conjugated to a diagnostic or therapeutic
agent. The
antibodies can be used diagnostically to, for example, monitor or prognose 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, but are not limited to, various enzymes, prosthetic groups,
fluorescent materials,
luminescent materials, bioluminescent materials, radioactive materials,
positron emitting
metals using various positron emission tomographies, and nonradioactive
paramagnetic
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metal ions. The detectable substance may be coupled or conjugated either
directly to the
antibody 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, but are not
limited to,
horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase;
examples of suitable prosthetic group complexes include, but are not limited
to,
streptavidin/biotin and avidin/biotin; examples of suitable fluorescent
materials include,
but are not limited to, umbelliferone, fluorescein, fluorescein
isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an
example of a
luminescent material includes, but is not limited to, lunninol; examples of
bioluminescent
materials include, but are not lmited to, luciferase, luciferin, and aequorin;
and examples
121 123 125 131
of suitable radioactive material include, but are not limited to, iodine ( I,
I, I, I),
carbon (14C), sulfur (3sS), tritium (3IT), indium (111In, 112In, 113mIn,
llsmIn), technetium
(99TC~99mTC), thallium (2olTi), gallium (68Ga, 67Ga), palladium (lo3Pd),
molybdenum
(9~Mo), xenon (l3sXe), fluorine (1sF), ls3Sm~ 177Lu, 1s90d~ 149Pm~ l4oLa~
17s~~ 166Ho~ 9oy~
a.7Sc~ ls6Re~ lssRe~ 142Pr~ losRh, and 97Ru.
[0391] Further, an antibody of the invention (including an scFv or other
molecule
comprising, or alternatively consisting of, antibody fragments or variants
thereof), may be
coupled or 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, 21381, or other radioisotopes such as, for example, lo3Pd, l3s~e,
1311, 6sGe,
57C~~ 65zn~ 85Sr, 32p~ 355 90Ya 153Sm~ 153~d~ 169~~ 5lCr' S4Mn, 75Se~ 113Sna
90Y~ 117T1n,
ls6Re, lssRe and 166Ho. In specific embodiments, an antibody or fragment
thereof is
attached to macrocyclic chelators that chelate radiometal ions, including but
not limited to,
177Lu~ 9oY~ 166Ho, and ls3Sm, to polypeptides. In specific embodiments, the
macrocyclic
chelator is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid
(DOTA). In other
specific embodiments, the DOTA is attached to the an antibody of the invention
or
fragment thereof via a linker molecule. Examples of linker molecules useful
for
conjugating DOTA to a polypeptide are commonly known in the art - see, for
example,
DeNardo et al., Clin Cancer Res. 4(10):2483-90, 1998; Peterson et al.,
Bioconjug. Chem.
10(4):553-7, 1999; and Zimmerman et al., Nucl. Med. Biol. 26(8):943-50, 1999
which are
hereby incorporated by reference in their entirety.
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[0392] A cytotoxin or cytotoxic agent includes any agent that is detrimental
to cells.
Examples include, but are not limited to, paclitaxol, cytochalasin B,
gramicidin D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine,
colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
W ithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine,
lidocaine, propranolol, thymidine kinase, endonuclease, RNAse, and puromycin
and
frragments, variants or homologs thereof. Therapeutic agents include, but are
not limuted
to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,
cytarabine, 5-
fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil,
melphalan, carmustine (BSN~ and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine 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).
[0393] 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; 5,714,711;
5,696,239; 5,652,371;
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) and direct coupling reactions (e.g., Bolton-Hunter and Chloramine-T
reaction).
[0394] The antibodies of the invention which are conjugates 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, but are not limited to, for example, a toxin such as
abrin, ricin A,
alpha toxin, pseudomonas exotoxin, or diphtheria toxin, saporin, momordin,
gelonin,
pokeweed antiviral protein, alpha-sarcin and cholera 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 97135899), AIM II (see,
International
Publication No. WO 97134911), Fas Ligand (Takahashi et al., Int. Irn~raufaol.,
6:1567-1574
(1994)), VEGI (see, International Publication No. WO 99/23105), a thrombotic
agent or
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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), interleulun-
6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF),
granulocyte
colony stimulating factor (G-CSF), or other growth factors.
[0395] Antibodies of the invention (including antibody fragments or variants
thereof),
may also be attached to solid supports, which are particularly useful for
immunoassays or
purification of the target antigen. Such solid supports include, but are not
limited to, glass,
cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or
polypropylene.
[0396] Techniques for conjugating a 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).
[0397] Alternatively, an antibody of the invention 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.
[039$] An antibody of the invention (including an other molecules comprising,
or
alternatively consisting of, an antibody fragment or variant thereof), 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.
Uses of Antibodies of the Invention
[0399] 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 TRAIL
receptor
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polypeptides in biological samples. See, e.g., Harlow et al., Antibodies: A
Laboratory
Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by
reference
herein in its entirety).
Immunophenotyping
[0400] The antibodies of the invention may be utilized for immunophenotyping
of cell
lines and biological samples (See, for example, Example 4). 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, particularly of tumors and cancer
cells.
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)).
[0401] 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 hematopoietic stem and progenitor cells capable of undergoing
proliferation
and/or differentiation, as might be found in human umbilical cord blood.
Epitope Mapping
[0402] The present invention provides antibodies (including antibody fragments
or
variants thereof), that can be used to identify epitopes of a TRAIL receptor
polypeptide.
In particular, the antibodies of the present invention can be used to identify
epitopes of a
human TRAIL receptor polypeptide (e.g., SEQ ID NOS:1-4) or a TRAIL receptor
polypeptide expressed on human cells; a murine TRAIL receptor or a TRAIL
receptor
polypeptide expressed on murine cells; a rat TRAIL receptor polypeptide
receptor or a
TRAIL receptor polypeptide expressed on rat cells; or a monkey TRAIL receptor
polypeptide or a TRAIL receptor polypeptide expressed on monkey cells, using
techniques
described herein or otherwise known in the art. Fragments which function as
epitopes
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may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl.
Acad. Sci.
USA 82:5131-5135 (1985), further described in U.S. Patent No. 4,711,211.)
Identified
epitopes of antibodies of the present invention may, for example, be used as
vaccine
candidates, i.e., to immunize an individual to elicit antibodies against the
naturally
occuring forms of TRAIL receptor polypeptides.
Diagnostic Uses of Antibodies
[0403] Labeled antibodies of the invention (including molecules comprising, or
alternatively consisting of, antibody fragments or variants thereof) which
specifically bind
to a TRAIL receptor polypeptide can be used for diagnostic purposes to detect,
diagnose,
prognose, or monitor diseases and/or disorders. In specific embodiments,
labeled
antibodies of the invention (including molecules comprising, or alternatively
consisting of,
antibody fragments or variants thereof) which specifically bind to a TRAIL
receptor
polypeptide can be used for diagnostic purposes to detect, diagnose, prognose,
or monitor
diseases and/or disorders associated with the aberrant expression and/or
activity of a
TRAIL receptor polypeptide or a TRAIL receptor polypeptide receptor.
[0404] The invention provides for the detection of expression of a TRAIL
receptor
polypeptide comprising: (a) assaying the expression of a TRAIL receptor
polypeptide in a
biological sample from an individual using one or more antibodies of the
invention that
immunospecifically binds to a TRAIL receptor polypeptide; and (b) comparing
the level
of a TRAIL receptor polypeptide with a standard level of a TRAIL receptor
polypeptide,
(e.g., the level in normal biological samples).
[0405] The invention provides for the detection of aberrant expression of a
TRAIL
receptor polypeptide comprising: (a) assaying the expression of a TRAIL
receptor
polypeptide in a biological sample from an individual using one or more
antibodies of the
invention that immunospecifically binds to a TRAIL receptor polypeptide; and
(b)
comparing the level of a TRAIL receptor polypeptide with a standard level of a
TRAIL
receptor polypeptide, e.g., in normal biological samples, whereby an increase
or decrease
in the assayed level of a TRAIL receptor polypeptide compared to the standard
level of a
TRAIL receptor polypeptide is indicative of aberrant expression.
[0406] By "biological sample" is intended any fluids andlor cells obtained
from an
individual, body fluid, body tissue, body cell, cell line, tissue culture, or
other source
which may contain a TRAIL receptor polypeptide protein or mRNA. Body fluids
include,
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