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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
Antibodies that Immunospecifically Bind to TRAIL Receptors
Field of the Invention
[0001] The present* invention relates to antibodies and related molecules that
immunospecifically bind to TRAIL receptor, TR4. 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-TR4 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 TR4.
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, at least eighteen members of
the TNF
ligand superfamily have been identified and at least nineteen members of the
TNF-
receptor superfamily have been characterized (See, e.g., Locksley et el., Cell
(2001)
104:487-501).
[0004] Among the ligands there are included TNF-a, lymphotoxin-a (LT- a, also
known as TNF-f3), LT-f3 (found in complex heterotrimer LT-a2-P), FasL, CD4OL,
CD27L, CD3OL, 4-1BBL, OX4OL and nerve growth factor (NGF). The superfamily of
TNF receptors includes the p55TNF receptor, p75TNF receptor, TNF receptor-
related
protein, FAS antigen or APO-1, CD40, CD27, CD30, 4-1BB, 0X40, low affinity p75
and
NGF-receptor (Meager, A., Biologicals, 22:291-295 (1994)).
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WO 2004/016753 CA 02494372 2005-
02-04 PCT/US2003/025457
[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-a are involved in the pathogenesis of a wide range of diseases,
including
endotoxic shock, cerebral malaria, tumors, autoimmune disease, AIDS and graft-
host
rejection (Beutler, 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 TNFR1
(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)). 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., Cell 81, 479-
482 (1995);2
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
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., I Biol Chem 270, 7795-8 (1995);
F.C.
Kischkel, et al., EMBO 14, 5579-5588 (1995)), which in turn binds and
presumably
activates FLICE/MACH1, a member of the ICE/CED-3 family of pro-apoptotic
proteases
(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., Immunol 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 has been ascribed the name Apoptosis Inducing Molecule I (AIM-I)
(Intenation Application No. WO 97/33899) and TNF-related apoptosis-inducing
ligand or
(TRAIL) (Wiley, S.R. et al., Immunity 3:673-682 (1995)). Pifti, 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. The amino acid sequence of TRAIL is given in SEQ ID NO:66.
[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
3
CA 02494372 2010-11-17
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] As many as five TRAIL receptors have been identified, including TR4
(also
known as TRAM receptor 1 (TRAM-R1) and death receptor 4 (DR4), Pan et al.,
Science
276:111-3 (1997), International Patent Application Nos. W098/32856,
W000/67793,
W099/37684, W02000/34355, W099/02653, SEQ ID NO:1); TR7 (also referred to as
TRAM receptor 2 (TRAIL-R2), DR5, and KILLER, 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. W098/46643, W099/09165, W099/11791,
W098/41629, W000/66156, and W098/35986, SEQ ID NO:3); TR1 (also referred to as
Osteoprotegrin (OPG) osteoclastogenesis inhibitory factor (OCIF), TNFRSF11B,
and
FTHMA-090 (International Patent Application Nos. W098/12344, W02000/54651,
W02001/04137, W066/26217, W098/07840, W02000/21554, W099/53942, and
W02001/03719, SEQ ID NO:5); TR5 (also referred to as TRAIL receptor 3 (TRAIL-
R3),
decoy receptor 1 (DcR1) and TR1D) (Degli-Esposti et al., J. Exp. Med. 186:1165-
70
(1997), International Patent Application Nos. W098/30693, W000/71150,
W099/00423,
EP867509, W098/58062, SEQ 173 NO:2); and TRIO (also referred to as TRAM
Receptor
4 (TRAIL-R4), DcR2, and TRUNDD, Pan et al., FEBS Left. 424:41-5 (1998), Degli-
Eposti et al., Immunity 7:813-20 (1997), International Patent Application Nos.
-
W098/54202, W000/73321, W02000/08155, W099/03992, WO 2000/34355 and
W09910484, SEQ NO:4). TR4 and TR7 contain death domains in their cytoplasmic
tails and the triggering of these receptors results in apoptosis. On the other
hand, TR1,
TR5 and TRIO can inhibit apoptosis induced by the cytotoxic ligand TRAM in
part
because of their absent or truncated cytoplasmic death domains, respectively.
[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
4
CA 02494372 2005-08-19
of TNF receptors, both normally and in disease states. In particular, there is
a need to
isolate and characterize antibodies that modulate the biological activities of
TRAIL
receptors.
Summary of the Invention
An object of the present invention is to provide antibodies that
immunospecifically bind to TRAIL receptors. In accordance with an aspect of
the
present invention, there is provided use of an antibody or fragment thereof
for the
preparation of a pharmaceutical composition for the treatment or prevention of
a
hematological cancer wherein said pharmaceutical composition also comprises a
member
selected from the group consisting of:
(a) ibritamomab tinxetan;
(b) imatinib mesylate;
(c) bortezomib; and
(d) a smac peptide or polypeptide;
wherein said antibody or fragment thereof comprises a VII and a VL domain that
is at
least 80% identical to a VII and a VL domain of any one of SEQ ID NOS:43-56,
or a VII
and VL domain of an antibody expressed by any one of the cells lines contained
in ATCC
Deposit Nos. PTA-3571, PTA-3570, and PTA-3675 and wherein said antibody or
fragment thereof the specifically binds TR4.
The present invention encompasses antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind to a TR4 polypeptide or polypeptide fragment or
variant of TR4.
In particular, the invention encompasses antibodies (including molecules
comprising, or
alternatively consisting of, antibody fragments or variants thereof) that
immimospecifically bind to a polypeptide or polypeptide fragment or variant of
human
TR4 such as that of SEQ ID NO:1. In some embodiments, an antibody of the
invention
that immunospecifically bind to a 'TR4 polypeptide, also bind TR7 (e.g., SEQ
ID NO:3),
but not other proteins, including (TR1, TR5, and TRIO (SEQ ID NOS:5, 2 and 4.)
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
5
CA 02494372 2005-08-19
variants thereof, or related molecules, that immunospecifically bind to TR4 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
TR4 function or TR4 ligand function or aberrant 'TR4 or 'TR4 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 TR4 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),
5a
CA 02494372 2010-11-17
infectious diseases (e.g., AIDS, herpes viral infections, and other viral
infections) and
proliferative disorders.
[00161 In highly preferred embodiments, antibodies of the present
invention are used
in methods and compositions for preventing, diagnosing, prognosing, treating
or
ameliorating the following types of cancer: breast cancer, lung cancer,
(including non-
small cell lung cancer), colon cancer, cancer of the urinary tract, bladder
cancer, kidney =
cancer, pancreatic cancer, liver cancer, stomach cancer, prostate cancer,
leukemia, Non-
Hodgkin's lymphoma, esophageal cancer, brain cancer, leukemia, ovarian cancer,
testicular cancer, melanoma, uterine cancer, cervical cancer, cancer of the
larynx, rectal
cancer, and cancers of the oral cavity. In specific embodiments, antibodies of
the
invention are administered in combination with chemotherapeutics such as
paclitaxel
(Taxo TM), irinotecan (Camptosai,m CPT-11), iri notecan analogs,
and gemcitabine
(GEMZARTm)) or other therapeutic agents useful in the treatment of cancers.
[0017] The present invention also encompasses methods and compositions
for
detecting, diagnosing, or proposing 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 TR4 or
a fragment
or variant thereof. In specific embodiments, the present invention also
encompasses
methods and compositions for detecting, diagnosing, or proposing diseases or
disorders
associated with TR4 function or TR4 ligand function or aberrant TR4 or TR4
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 TR4 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, Hashim.oto's disease, and immunodeficiency
syndrome),
= inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid
arthritis),
infectious diseases (e.g., AI)S, herpes virus infections, and other viral
infections), and
proliferative disorders.
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
[0018] Another embodiment of the present invention includes the use of the
antibodies
of the invention as a diagnostic tool to monitor the expression of TR4
expression on cells.
[0019] The present inventors have generated single chain Fv's (scFvs) that
immunospecifically bind TR4 polypeptides (e.g., SEQ ID NOs:1). Thus, the
invention
encompases these scFvs, listed in Table 1. In addition, the invention
encomasses cell lines
engineered to express antibodies corresponding to these scFvs which are
deposited with
the American Type Culture Collection ("ATCC") as of 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.
[0020] Further, the present invention encompasses the polynucleotides encoding
the
scFvs, as well as the amino acid sequences encoding the scFvs. Molecules
comprising, or
alternatively consisting of, fragments or variants of these scFvs (e.g., VH
domains, VH
CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of the
scFvs
referred to in Table 1), that immunospecifically bind to TR4 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 TR4 or fragments and variants thereof.
[0021] The present invention also provides antibodies that bind TR4
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 TR4 polypeptides which are coupled to a therapeutic or
cytotoxic
agent. The present invention also provides antibodies that bind TR4
polypeptides which
are coupled to a radioactive material.
[0022] The present invention also provides antibodies that bind TR4
polypeptides that
act as either TR4 agonists or TR4 antagonists. In specific embodiments, the
antibodies of
the invention stimulate apoptosis of TR4 expressing cells. In other specific
embodiments,
the antibodies of the invention inhibit TRAIL binding to TR4. In other
specific
embodiments, the antibodies of the invention upregulate TR4 expression.
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
[0023] The present invention also provides antibodies that inhibit apoptosis
of TR4
expressing cells. In other specific embodiments, the antibodies of the
invention
downregulate TR4 expression.
[0024] 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 (KID) of 10-9 M or less.
[0025] The present invention further provides antibodies that stimulate
apoptosis of
TR4 expressing cells better than an equal concentration of TRAIL polypeptide
stimulates
apoptosis of TR4 expressing cells.
[0026] The present invention further provides antibodies that stimulate
apoptosis of
TR4 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.
[0027] In further embodiments, antibodies of the invention have an off rate
(koff) of
10-3/sec or less. In preferred embodiments, antibodies of the invention have
an off rate
(kat) of 10-4/sec or less. In other preferred embodiments, antibodies of the
invention have
an off rate (koff) of 10-5/sec or less.
[0028] The present invention also provides for antibodies that preferentially
bind TR4
and/or TR7 relative to their ability to bind other proteins (including TR1,
TR5 and TRIO).
[0029] 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 TR4 binding antibodies.
[0030] 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 farther 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,
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
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.
[0031] The present invention also provides for fusion proteins comprising an
antibody
(including molecules comprising, or alternatively consisting of, antibody
fragments or
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.
[0032] 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 description of the Figures
[0033] Figure 1 shows the effect of T1014A04 treatment on SW480 tumor growth
in
Swiss nu/nu mice with or without Topotecan treatment at 0.3 mg/kg.
[0034] Figure 2 shows the effect of T1014A04 treatment on SW480 tumor growth
in
Swiss nu/nu mice with or without Topotecan treatment at 0.6 mg/kg.
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WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
[0035] Figure 3 shows the effect of 14G03 treatment on the growth of SW480
tumors
in vivo after 28 days with and without Topotecan treatment.
Detailed Description of the Invention
Definitions
[0036] 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
multimers and antibody fragments as well as variants (including derivatives)
of antibodies,
antibody multimers 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 VII domain of an antibody. Antibodies that
immunospecifically bind
to TR4 may have cross-reactivity with other antigens, e.g., another TRAIL
Receptor.
Preferably, antibodies that immunospecifically bind to TR4 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 TR4 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.
[0037] 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 immunoglobulin 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 1, or a fragment or variant thereof. In a preferred embodiment, the
immunoglobulin
is an IgG1 isotype. In another preferred embodiment, the immunoglobulin is an
IgG4
10
CA 02494372 2010-11-17
isotype. Immunoglobulins may have both a heavy and light chain. An array of
IgG, IgE,
IgM, IgD, IgA, and IgY heavy chains may be paired with a light chain of the
kappa or
lambda forms.
[0038] Antibodies of the invention may also include multimeric forms of
antibodies.
For example, antibodies of the invention may take the form of antibody dimers,
timers, or
higher-order multimers of monomeric immunoglobulin molecules. Dimers of whole
immunoglobulin molecules or of F(ab')2 fragments are tetravalent, whereas
dimers of Fab
fragments or scFv molecules are bivalent. Individual monomers withon an
antibody
multimer may be identical or different, i.e., they may be heteromeric or
homomeric
antibody multimers. For example, individual antibodies within a multimer may
have the
same or different binding specificities.
[0039] Multimerization of antibodies may be accomplished through natural
aggregation of antibodies or through chemical or recombinant linking
techniques known
in the art. For example, some percentage of purified antibody preparations
(e.g., purified
IgG1 molecules) spontaneously form protein aggregates containing antibody
homodimers,
and other higher-order antibody multimers. Alternatively, antibody homodimers
may be
formed through chemical linkage techniques known in the art. For example,
heterobifunctional crosslinking agents including, but not limited to, SMCC
[succinimidyl
4-(maleimidomethyl)cyclohexane-1-carboxylate] and SATA [N-succinimidyl S-
acethylthio-acetate] (available, for example, from Pierce Biotechnology, Inc.
(Rockford,
IL)) can be used to form antibody multimers. An exemplary protocol for the
formation of
antibody homodimers is given in Ghetie et al., Proceedings of the National
Academy of
Sciences USA (1997) 94:7509-7514.
Antibody homodimers can be converted to Fab'2 homodimers through digestion
with pepsin. Another way to form antibody homodimers is through the use of the
autophilic T15 peptide described in Zhao and Kohler, The Journal of Immunology
(2002)
25:396-404.
[0040] Alternatively, antibodies can be made to multimerize through
recombinant
DNA techniques. IgM and IgA naturally form antibody multimers through the
interaction
with the mature J chain polypeptide (e.g., SEQ ID NO:67). Non-IgA or non-IgM
molecules, such as IgG molecules, can be engineered to contain the J chain
interaction
domain of IgA or IgM, thereby conferring the ability to form higher order
multimers on
the non-IgA or non-IgM molecules. (see, for example, Chintalacharuvu et al.,
(2001)
11
CA 02494372 2010-11-17
Clinical Immunology 101:21-31. and Frigerio et al., (2000) Plant Physiology
123:1483-
94.). IgA dimers are
naturally secreted into the lumen of mucosa-lined organs. This secretion is
mediated
through interaction of the J chain with the polymeric IgA receptor (pIgR) on
epithelial
cells. If secretion of an IgA form of an antibody (or of an antibody
engineered to to
contain a J chain interaction domain) is not desired, it can be greatly
reduced by
expressing the antibody molecule in association with a mutant J chain that
does not
interact well with pIgR (e.g., SEQ ID NOS:68-70; Johansen et al., The Journal
of
Immunology (2001) 167:5185-5192).
SEQ ID NO:68 is a mutant form of a human mature J chain with C134S
mutation compared to the mature form of human J chain (SEQ ID NO:67). SEQ ID
NO:69 is a mutant form of a human mature J chain with amino acids 113-137
deleted
compared to the mature form of human J chain (SEQ ID NO:67). SEQ ID NO:70
shows a
mutant form of human mature I chain with C109S and C134S mutation compared to
the
mature form of human J chain (SEQ ID NO:67). Expression of an antibody with
one of
these mutant J chains will reduce its ability to bind to the polymeric IgA
receptor on
epithelial cells, thereby reducing transport of the antibody across the
epithelial cell and its
resultant secretion into the lumen of mucosa lined organs. ScFv dimers can
also be
formed through recombinant techniques known in the art; an example of the
construction
of scFv dimers is given in Goel et al., (2000) Cancer Research 60:6964-6971.
Antibody multimers may be purified
using any suitable method known in the art, including, but not limited to,
size exclusion
chromatography.
[0041] Unless otherwise defined in the specification, specific binding or
immunospecifc binding by an anti-TR4 antibody means that the anti-TR4 antibody
binds
TR4 but does not significantly bind to (i.e., cross react with) proteins other
than TR4, such
as other proteins in the same family of proteins). An antibody that binds TR4
protein and
does not cross-react with other proteins is not necessarily an antibody that
does not bind
said other proteins in all conditions; rather, the TR4-specific antibody of
the invention
preferentially binds TR4 compared to its ability to bind said other proteins
such that it will
be suitable for use in at least one type of assay or treatment, i.e., give low
background
levels or result in no unreasonable adverse effects in treatment. It is well
known that the
portion of a protein bound by an antibody is known as the epitope. An epitope
may either
12
WO 2004/016753 CA 02494372 2005-02-04
PCT/US2003/025457
be linear (i.e., comprised of sequential amino acids residues in a protein
sequences) or
conformational (i.e., comprised of one or more amino acid residues that are
not contiguous
in the primary structure of the protein but that are brought together by the
secondary,
tertiary or quaternary structure of a protein). Given that TR4-specific
antibodies bind to
epitopes of TR4, an antibody that specifically binds TR4 may or may not bind
fragments
of TR4 and/or variants of TR4 (e.g., proteins that are at least 90% identical
to TR4)
depending on the presence or absence of the epitope bound by a given TR4-
specific
antibody in the TR4 fragment or variant. Likewise, TR4-specific antibodies of
the
invention may bind species orthologues of TR4 (including fragments thereof)
depending
on the presence or absence of the epitope recognized by the antibody in the
orthologue.
Additionally, TR4-specific antibodies of the invention may bind modified forms
of TR4,
for example, TR4 fusion proteins. In such a case when antibodies of the
invention bind
TR4 fusion proteins, the antibody must make binding contact with the TR4
moiety of the
fusion protein in order for the binding to be specific. Antibodies that
specifically bind to
TR4 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] In some embodiments the present invention encompasses antibodies
that
immunospecifcally or specifically bind both TR4 and TR7. Specific binding or
immunospecifc binding by an antibody that immunospecifically binds TR4 and TR7
means that the antibody binds TR4 and TR7 but does not significantly bind to
(i.e., cross
react with) proteins other than TR4 or TR7, such as other proteins in the same
family of
proteins). An antibody that binds TR4 and TR7 proteins and does not cross-
react with
other proteins is not necessarily an antibody that does not bind said other
proteins in all
conditions; rather, the antibody that immunospcifically or specifically binds
both TR4 and
TR7 preferentially binds TR4 and TR7 compared to its ability to bind said
other proteins
such that it will be suitable for use in at least one type of assay or
treatment, i.e., give low
background levels or result in no unreasonable adverse effects in treatment.
It is well
known that the portion of a protein bound by an antibody is known as the
epitope. An ,
epitope may either be linear (i.e., comprised of sequential amino acids
residues in a
protein sequences) or conformational (i.e., comprised of one or more amino
acid residues
that are not contiguous in the primary structure of the protein but that are
brought together
by the secondary, tertiary or quaternary structure of a protein). Given that
antibodies that
bind TR4 and TR7 bind to epitopes common to TR4 and TR7, an antibody that
13
WO 2004/016753 ./ CA 02494372 2005-02-04 PCT/US2003/025457
specifically binds TR4 and TR7 may or may not bind fragments of TR4, TR7
and/or
variants of TR4 or TR7 (e.g., proteins that are at least 90% identical to TR4
or TR7,
respectively) depending on the presence or absence of the epitope bound by a
given
antibody in the TR4 or TR7 fragment or variant. Likewise, antibodies of the
invention
that immunospecifically bind TR4 and TR7 may bind species orthologues of TR4
and/or
TR7 (including fragments thereof) depending on the presence or absence of the
epitope
recognized by the antibody in the orthologues. Additionally, antibodies of the
invention
that immunospecifically bind TR4 and TR7 may bind modified forms of TR4 or
TR7, for
example, TR4 or TR7 fusion proteins. In such a case when antibodies of the
invention
bind fusion proteins, the antibody must make binding contact with the TR4 or
TR7 moiety
of the fusion protein in order for the binding to be specific. Antibodies that
specifically
bind to TR4 or TR7 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.
[0043] The term "variant" as used herein refers to a polypeptide that
possesses a
similar or identical function as a TR4 polypeptide, a fragment of a TR4
polypeptide, an
anti-TR4 antibody or antibody fragment thereof, but does not necessarily
comprise a
similar or identical amino acid sequence of a TR4 polypeptide, a fragment of a
TR4
polypeptide, an anti-TR4 antibody or antibody fragment thereof, or possess a
similar or
identical structure of a TR4 polypeptide, a fragment of a TR4 polypeptide, an
anti-TR4
antibody or antibody fragment thereof, respectively. A variant having a
similar amino
acid sequence 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 TR4
polypeptide (SEQ
ID NO:1), a fragment of, an anti-TR4 antibody or antibody fragment thereof
(including a
VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one
or more scFvs 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 TR4 (SEQ ID NO:1), a fragment of
a TR4
polypeptide, an anti-TR4 antibody or antibody fragment thereof (including a VH
domain,
VHCDR, VL domain, or VLCDR having an amino acid sequence of any one of those
14
WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
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 TR4 polypeptide, a fragment of
a TR4
polypeptide, an anti-TR4 antibody or antibody fragment thereof (including a
VII domain,
VHCDR, VL domain, or VLCDR having an amino acid sequence of any one or more
scFvs referred to in Table 1), described herein. A polypeptide with similar
structure to a
TR4 polypeptide, a fragment of a TR4 polypeptide, an anti-TR4 antibody or
antibody
fragment thereof, described herein refers to a polypeptide that has a similar
secondary,
tertiary or quaternary structure of a TR4 polypeptide, a fragment of a TR4
polypeptide, an
anti-TR4 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.,
% identity ¨
number of identical overlapping positions/total number of positions x 100%).
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
15
CA 02494372 2010-11-17
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
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.
[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 Comput. App!.
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 TR4
polypeptide, a fragment of a TR4 polypeptide, or an antibody of the invention
that
immunospecifically binds to a TR4 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 TR4 polypeptide, a fragment of a
'TR4
polypeptide, an antibody that immunospecifically binds to a TR4 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 TR4 polypeptide, a
fragment of
a TR4 polypeptide, or an anti-TR4 antibody, may be modified, e.g., by
glycosylation,
16
WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
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 TR4 polyp eptide, a fragment of a TR4 polyp
eptide, or an
anti-TR4 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 TR4 polypeptide, a fragment of a TR4 polypeptide, or an anti-TR4 antibody,
may
contain one or more non-classical amino acids. A polypeptide derivative
possesses a
similar or identical function as a TR4 polypeptide, a fragment of a TR4
polypeptide, or an
anti-TR4 antibody, described herein.
[0048] The term "epitopes" as used herein refers to portions of TR4 having
antigenic
or immunogenic activity in an animal, preferably a mammal. An epitope having
immunogenic activity is a portion of TR4 that elicits an antibody response in
an animal.
An epitope having antigenic activity is a portion of TR4 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
TR4, or an
anti-TR4 antibody (including molecules such as scFv's, that comprise, or
alternatively
consist of, antibody fragments or variants thereof).
[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-TR4 antibody
of the
invention and an amino acid sequence of a heterologous polypeptide (i.e., a
polypeptide
unrelated to an antibody or antibody domain).
17
CA 02494372 2010-11-17
[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.
100521 Antibodies of the present invention are preferably provided in an
isolated form,
and preferably are substantially purified. By "isolated" is intended an
antibody removed
from its native environment. Thus, for eaxample, a polypeptide produced and/or
contained
within a recombinant host cell is considered isolated for purposes of the
present invention.
[0053] By "isolated antibody" is intended an antibody removed from its native
environment. Thus, an antibody produced and/or contained within a recombinant
host cell
is considered isolated for purposes of the present invention.
Antibody Structure
[0054] 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-
terminal
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
defmes 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, 1 gG,
IgA, and
IgE, respectively. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed.,
2nd ed.
Raven Press, N.Y. (1989)). The
variable regions of each light/heavy chain pair form the antibody binding
site.
[0055] Thus, an intact IgG antibody has two binding sites. Except in
bifunctional or
bispecific antibodies, the two binding sites are the same.
[0056] 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
18
WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
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).'
[0057] 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. ImnzunoL 79: 315-321
(1990),
Kostelny et al. J Iminunol. 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
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)).
[0058] 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-TR4 Antibodies
[0059] Using phage display technology, the present inventors have identified
single
chain antibody molecules ("scFvs") that immunospecifically bind to TR4 (or
fragments or
variants thereof). Molecules comprising, or alternatively consisting of,
fragments or
variants of these scFvs (e.g., including VII domains, VII CDRs, VL domains, or
VL
CDRs having an amino acid sequence of any one of those referred to in Table
1), that
immunospecifically bind to TR4 (or fragments or variants thereof) are also
encompassed
by the invention, as are nucleic acid molecules that encode these scFvs,
and/or molecules.
[0060] In particular, the invention relates to scFvs comprising, or
alternatively
consisting of, an amino acid sequence selected from the group consisting of
SEQ ID NOs:
42-53, preferably SEQ ID NOs:42 and 43 as referred to in Table 1 below.
Molecules
comprising, or alternatively consisting of, fragments or variants of these
scFvs (e.g.,
including VII domains, VII CDRs, VL domains, or VL CDRs having an amino acid
sequence of any one of those referred to in Table 1), that immunospecifically
bind to TR4
19
WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
are also encompassed by the invention, as are nucleic acid molecules that
encode these
scFvs, and/or molecules (e.g., SEQ ID NOs:54-65).
[0061] ScFvs corresponding to SEQ ID NOS:42-53 were selected for their ability
bind
TR4 polypeptide.
[0062] The present invention provides antibodies (including molecules
comprising, or
alternatively consisting of, antibody fragments or variants thereof) that
immunospecifically bind to a polypeptide or a polypeptide fragment of TR4. In
particular,
the invention provides antibodies corresponding to the scFvs referred to in
Table 1. Such
scFvs may routinely be "converted" to immunoglobulin molecules by inserting,
for
example, the nucleotide sequences encoding the VII and/or VL domains of the
scFv into
an expression vector containing the constant domain sequences and engineered
to direct
the expression of the immunoglobulin molecule, as described in more detail in
Example 5
below.
[0063] NSO cell lines that express IgG1 antibodies that comprise the VET and
VL
domains of scFvs of the invention have been 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. Accordingly, in one embodiment, the invention provides
antibodies that
comprise the VH and VL domains of scFvs of the invention.
[0064] In a preferred embodiment, an antibody of the invention is the antibody
expressed by cell line NSO aTRAIL 1985 BU #81 P:15 6/21/01 (See Table 1).
[0065] In a preferred embodiment, an antibody of the invention is the antibody
expressed by cell line TRAIL (NSO) 14G03 #39 P:14 7/2/01 (See Table 1).
[0066] In a preferred embodiment, an antibody of the invention is the antibody
expressed by cell line NSO anti-TRAIL 14F08 #28 P:11 (See Table 1).
20
Table I: scFvs that Immunospecifically bind to TRAIL Receptors
o
scFv scFv scFv AAs of AAs of AAs of AAs of AAs of AAs of AAs of AAs
of Cell Line ATCC ATCC
t..)
=
protein DNA VII VII VII VII VL VL
VL VL Expressing Deposit Deposit =
.6.
-a
SEQ ID SEQ ID Domain CDRI CDR2 CDR3 Domain CDRI CDR2 CDR3 antibody Number Date
c,
NO: NO:
-4
u,
(44
T1014A04 42 54 1-118 26-35 50-66 99-107 135-
245 157-170 186-192 225-234 NSO aTRAIL PTA-3571 July 30, 2001
1985 BU #81
P:15 6/21/01
T1014G03 43 55 1-118 26-35 50-66 99-107 135-
245 157-170 186-192 225-234 TRAIL (NSO) PTA-3570 July 30, 2001
14G03 #39
P:14 7/2/01
T1014A02 44 56 1-116 26-35 50-65 98-105 134-
244 156-168 - 184-190 223-233
T1014Al2 45 57 1-118 26-35 50-66 99-107 135-245 157-170 186-
192 225-234 n
T1014B01 46 58 1-118 26-35 50-66 99-107 135-245 157-170 186-
192 225-234 0
iv
a,
T1014B11 47 59 1-118 26-35 50-66 99-107 135-245 157-170 186-
192 225-234
a,
ui
I') T1014F08 48 60 1-118 26-35 50-66 99-107
135-245 157-170 186-192 225-234 NSO anti- PTA-3675 August 29,
¨1
iv
TRAIL 14F08 2001
iv
#28P:11 0
0
T1014G04 49 61 1-118 26-35 50-66 99-107 135-245 157-170 186-
192 225-234 in
1
0
T1015A02 50 62 1-123 26-37 52-67 100-112 140-250 162-174
190-196 229-239
iv
1
0
T1015A07 51 63 1-118 26-35 50-66 99-107 135-245 157-170 186-
192 225-234 a,
T1015E01 52 64 1-118 26-35 50-66 99-107 135-245 157-170 186-
192 225-234
11006F07 53 65 1-125 26-35 50-66 99-114 142-249 164-174 190-
196 229-238
00
n
,-i
cp
t..)
=
=
(44
N
CA
4=,
CA
--1
CA 02494372 2010-11-17
[0067] The present invention encompasses antibodies (including molecules
comprising, or
alternatively consisting of, antibody fragments or variants thereof) that
immunospecifically bind to a TR4 polypeptide or a fragment, variant, or fusion
protein
thereof. A TR4 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. In some embodiments, antibodies of the present
invention may
immunospecifically bind to both TR4 as described above and to TR7 (SEQ ID
NO:3) or
the polypeptide encoded by the cDNA in clone HLYBX88 contained in ATCC Deposit
97920 deposited Mar. 7, 1997. TRAIL receptors may be produced through
recombinant
expression of nucleic acids encoding the polypeptides of SEQ ID NOS:1-5, (TR4,
TR5,
TR7, TRIO, and TR1; e.g., the cDNAs in the ATCC Deposit Numbers 97853, (TR4)
97798 (TR5, deposited November 20, 1996), 97920 (TR7), or 209040 (TRIO,
deposited
May 15, 1997).
[0068] In one embodiment, the antibodies of the invention preferentially
bind TR4
(SEQ ID NO:1), or fragments, variants, or fusion proteins thereof (e.g., the
extracellular
region of TR4 fused to an Fe domain) relative to their ability to bind other
proteins
including TR1, TR5, TR7, or TR10 (SEQ ID NOS:5, 2, 3, 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 NOS:1 and 3), or fragments or variants
thereof relative to their ability to bind other proteins including TR1, TR5 or
TRIO (SEQ
ID NOS:5, 2 and 4) or fragments, variants, or fusion proteins thereof. In
other preferred
embodiments, the antibodies of the invention bind TR1 TR4, TR5, TR7 and TRIO
(SEQ
ID NOS:5, 1, 2, 3, and 4). An antibody's ability to preferentially bind one
antigen
compared to another antigen may be determined using any method known in the
art.
TR4 Polypeptides
[0069] 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
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 W000/67793.
22
WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
[0070] 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.
[0071] 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, 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 TR4 polypeptides.
[0072] Antibodies of the invention may bind TR4 homomers or heteromers. As
used
herein, the term homomer, 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 polyp eptide 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). In additional embodiments, antibodies of the
invention
bind at least a homodimer, at least a homotrimer, or at least a homotetramer
of TR4.
[0073] In specific embodiments antibodies of the presnt invention bind TR4
homotrimers (e.g., containing TR4 proteins having identical or different
polypeptide
sequences).
[0074] 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
antibodies of the invention bind at least a homodimer, at least a homotrimer,
or at least a
homotetramer containing one or more TR4 polypeptides.
23
CA 02494372 2010-11-17
[0075] In specific embodiments antibodies of the presnt invention bind a TR4
heterotrimer (e.g., containing 1 or 2 TR4 proteins and 2 or 1, respectively,
TR7 proteins).
[0076] MuRimers 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 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:1 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
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 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).
[0077] 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
24
CA 02494372 2010-11-17
using linker molecules and linker molecule length optimization techniques
known in the
art (see, e.g., US Patent Number 5,478,925).
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). 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).
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).
[0078] 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).
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).
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).
In another embodiment, two or more TR4
25
CA 02494372 2010-11-17
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. 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.
[0079] 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
occurring 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.
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 mulfimeric 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.
[0080] Certain members of the TNF family of proteins are believed to exist in
trimeric
form (Beutler and Huffel, Science 264:667, 1994; Banner etal., 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 No. 5,716,805. In specific embodiments,
antibodies of the invention bind TR4-leucine zipper fusion protein trimers.
[00811 Other peptides derived from naturally occurring trimeric proteins may
be
employed in preparing trimeric TR4. hi specific embodiments, antibodies of the
invention
bind TR4- fusion protein monomers and/or TR4 fusion protein trimers.
[0082] Antibodies of the invention that bind TR4 receptor polypeptides may
bind
them as isolated polypeptides or in their naturally occurring state. By
"isolated
26
WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
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 TR4
polypeptide is
substantially purified by the one-step method described in Smith and Johnson,
Gene
67:31-40 (1988). Thus, antibodies of the present invention may bind
recombinantly
produced TR4 receptor polypeptides. In a specific embodiment, antibodies of
the present
invention bind a TR4 receptor expressed on the surface of a cell, wherein said
TR4
polypeptide is encoded by a polynucleotide encoding amino acids 1 to 468 of
SEQ ID
NO:1 operably associated with a regulatory sequence that controls expression
of said
polyaucleotide.
[0083] Antibodies of the present invention may bind TR4 polypeptide fragments
comprising or alternatively, consisting of, an amino acid sequence contained
in SEQ ID
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.
[0084] Preferably, antibodies of the present invention bind polypeptide
fragments
selected from the group: a polypeptide comprising or alternatively, consisting
of, the TR4
27
CA 02494372 2005-02-04
WO 2004/016753
PCT/US2003/025457
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 NO: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 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:1); 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 15 amino
acid residues) depending on the criteria used to define each domain.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
[0085] 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 JD NO:1. In another highly preferred embodiment,
antibodies
28
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
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
NO: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 ligand 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. 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) induce cell death of the cell expressing
the TR4
receptor.
[0086] 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 NO:1), 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 parameters of these computer programs.
[0087] The data representing the structural or functional athibutes 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
Gamier-
29
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
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; Colutnn 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 IX 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;
Column XII represents the Jameson-Wolf antigenic index score; and Column XIII
represents the Emini surface probability plot.
[0088] 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.
[0089] 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:l. 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., 1, 2, 3 , or 4) of the same or different region features set
out above and in
Table 2.
30
CA 02494372 2005-02-04
WO 2004/016753
PCT/US2003/025457
Table 2
Res Position I
II
III IV V
VI VII VIII IX X
XI XII
XIII
Met 1
B
0.12 .
-0.10 0.90
.
.
.
.
.
.
.
.
Ala 2.
.
.
.
.
.
C
-0.08 *
* .
0.25 1.08
Pro
3.
.
.
.
.
.
c
0.42
*
*
.
0.10
0.86
Pro 4.
.
.
.
T
C
-0.04 *
* .
1.05 1.69
.
Pro 5 A. . . . T . 0.31 . * F 1.00 1.24
Ala 6 A. . . . T . 0.10 . * F 1.00 1.10
Arg 7 A. . . . T . 0.34 . * . 0.10 0.58
Val
8.
.
.
. B
B
.
-0.03 .
* .
-0.30 0.37
His
9.
.
.
.
. B
B
-0.52 .
* .
-0.30 0.37
Leu 10.
.
.
.
. B
B
-1.12 .
* .
-0.60 0.17
*
Gly 11
B
B
-1.12 .
-0.60 0.18
.
.
.
.
.
.
Ala 12.
.
.
. B
B
.
-2.09 .
* .
-0.60 0.14
*
Phe 13
B
B
-1.54 .
-0.60 0.12
.
.
.
.
.
.
Leu 14
B
B
-1.72 .
-0.60 0.18
.
.
.
.
.
.
.
Ala 15
B
B
-0.91 .
-0.60 0.27
.
.
.
.
.
.
.
Val
16
B
B
-0.78 .
-0.60 0.51
.
.
.
.
.
.
.
Thr 17.
.
.
. B
B
.
-0.53 .
. F
-0.45 0.95
Pro 18.
.
.
.
. B
c -0.13 .
. F 0.05 0.93
Asn 19. . . . . T C 0.09 . . F 0.60 1.69
Pro
20.
.
.
.
.
T
c
0.09
.
.
F
0.60
1.18
Gly
21.
.
.
.
T
T
.
0.64
.
.
F
0.65
0.77
Ser
22.
.
.
.
.
T
C
0.61
.
.
F
0.45
0.64
Ala
23.
.
.
.
.
.
C 0.51 . . F 0.25 0.41
Ala
24.
.
.
.
.
T
c
0.51
.
.
F
0.45
0.60
Ser
25.
.
B
.
.
T
.
0.13
.
.
F
0.85
0.78
Gly 26
A.
.
.
.
T
.
-0.11 .
. F 0.85 0.78
Thr 27
A.
.
.
.
. T
-0.40 .
. F 0.85 0.78
Glu 28
A A.
.
.
.
.
-0.40 .
. F 0.45 0.58
Ala 29
A A
-0.12 .
0.30 0.60
.
.
.
.
.
.
.
Ala 30
A A
-0.03 .
0.30 0.60
.
.
.
.
.
.
.
Ala
31
A
A.
.
.
.
.
.
.
.
. 001
0.30
0.53
Ala 32
A
A.
.
.
.
.
.
. . 0.37
-0.30 0.71
Thr 33
A.
.
.
.
. T
-0.49 *
. F
1.00 1.40
Pro 34
A.
.
.
.
. T
-0.19 .
. F 1.00 1.03
Ser
35.
.
.
.
. B
T
0.06
.
.
F
0.40
1.07
Lys
36.
.
.
.
. B
T
0.34
.
.
F
0.25
0.73
Val
37.
.
B
B
.
.
.
0.63 .
. F
-0.15 0.64
Trp
38.
.
.
.
. B
B
0.36 .
. F
-0.15 0.64
Gly 39.
.
.
.
. B
B
0.22 *
* F
-0.15 0.32
Ser 40.
.
.
.
.
.
C
0.63 *
* F
-0.05 0.43
Ser 41.
.
.
.
.
T
c
-0.30 *
* F
0.45 0.80
Ala 42. . . . . T c 0.56 * * F 1.05 0.57
Gly 43. . . . . T C 0.63 * * F 1.35 0.73
Arg 44. .
. .
. B
T
1.09 * * F 1.49 0.84
Ile
45.
.
.
.
.
. B
1.04
*
*
F
1.78
1.63
Glu 46. .
. . . . B
1.00 * * F 2.12 1.63
Pro 47. .
. .
. B
T
1.24 * * F 2.51 0.83
Arg 48. . . .
. T T
1.70 * * F 3.40 1.17
Gly 49. . . .
. T T
1.24 * * F 3.06 1.32
Gly 50= = . .
. T T
1.54 * * F 2.57 0.84
31
CA 02494372 2005-02-04
WO 2004/016753
PCT/US2003/025457
Table 2 (continued)
Res Position I
II
III IV V
VI VII VIII IX X
XI XII
XIII
Gly 51. . . . . T C 0.73 * * F 2.03 0.44
Arg 52. . . . . T C 0.73 * * F 1.39 0.36
Gly 53. .
. .
. B
T
0.31 * * F 0.85 0.57
Ala
54.
.
.
.
. B
T
0.36
.
*
F
0.85
0.83
Leu
55.
.
. B
.
.
.
0.10
.
*
F
0.65
0.57
Pro 56.
.
. B
.
.
.
0.10 .
* F -0.25 0.57
Thr 57.
.
. B
.
.
.
-0.01 .
* F
-0.25 0.55
Ser
58.
.
.
.
. B
T
0.30
.
.
F
0.10
1.16
Met 59. .
. .
. B
T
0.54 . . F 0.40 1.02
Gly
60.
.
.
.
. B
T
1.14
.
.
F
0.25
0.70
Gln
61.
.
.
.
T
T
.
1.06
.
.
F
0.65
0.81
His
62.
.
.
.
.
.
C
0.78
.
*
F
0.40
1.10
Gly 63. . . . . T C 1.19 . * F 0.60 1.12
Pro
64.
.
.
.
.
T C 1.20 . * F 1.20 1.27
Ser
65.
.
.
.
.
T
C
1.66
.
*
F
1.05
0.94
Ala 66. .
. B
.
. T
1.07 . * F 1.30 1.86
Arg
67.
.
.
.
. B
.
0.76
*
*
.
1.29
1.22
Ala
68.
.
.
.
. B
.
1.21
*
*
.
1.48
0.90
Arg
69.
.
.
. B
T
.
0.83
.
*
.
2.17
1.74
Ala 70. .
. .
. B
T
0.92 . * F 2.51 0.90
Gly 71. . . .
. T T
1.17 . * F 3.40 1.37
Arg 72. . . . . T C 0.84 . * F 2.71 0.69
Ala
73.
.
.
.
.
T
C
1.54
*
.
F
2.48
1.06
Pro 74. . . . . T C 1.22 * . F 2.70 2.10
Gly
75.
.
.
.
.
T C 1.22 * . F 2.62 1.66
Pro
76.
.
.
.
.
T C 1.68 * * F 2.24 1.66
Arg
77.
.
.
.
.
.
C
1.57
*
.
F
2.60
2.10
Pro 78.
. . . . A B
1.57 * . F 1.94 3.68
Ala 79.
. . . . A B
1.48 * . F 1.68 2.40
Arg 80.
. A B
. . . 1.61 * * F 1.42 1.64
Glu 81.
. A B
. . . 1.93 * * F 1.16 1.64
Ala 82 A A. . . . . 1.01 * * F 0.90 3.19
Ser 83 A. . . .
. T
1.33 * * F 1.30 1.34
Pro 84 A. . . .
. T
1.07 * * F 1.30 1.52
Arg 85 A. . . .
. T
0.92 * * F 1.00 1.12
Leu
86
A.
.
.
.
. T
0.97
.
*
.
0.85
1.13
Arg
87
A.
.
B
.
.
.
1.24
.
*
.
0.75
1.46
Val
88
A.
.
B
.
.
.
0.84
*
*
.
0.75
1.08
His 89
A.
.
B
.
.
.
1.10 .
* .
-0.15 1.13
Lys 90 A. . B . . . 0.29 * * F 0.90 1.16
Thr 91. .
. . . B B
0.24 * * F 0.00 1.35
Phe 92.
.
.
.
. B
B
-0.72 *
* .
-0.30 0.74
Lys 93.
.
.
.
. B
B
-0.72 *
* .
-0.30 0.27
Phe 94
B
B
-1.03 *
-0.60 0.14
.
.
.
.
.
.
.
Val 95
B
B
-1.93 *
-0.60 0.16
.
.
.
.
.
.
.
Val 96.
.
.
.
. B
B
-2.43 .
* .
-0.60 0.06
Val 97.
.
.
.
. B
B
-2.54 .
* .
-0.60 0.06
Gly 98.
.
.
.
. B
B
-2.59 .
* .
-0.60 0.06
Val 99
B
B
-2.74 .
-0.60 0.15
.
.
.
.
.
.
.
Leu 100.
.
.
.
.
.
. B
B
-274 *
-0.60 0.15
.
32
CA 02494372 2005-02-04
WO 2004/016753
PCT/US2003/025457
Table 2 (continued)
Res Position I
II
III IV V
VI VII VIII IX X
XI XII
XIII
Leu
101
B
B
-2.10 *
-0.60 0.11
.
.
.
.
.
.
.
Gin 102
B
B
-1.54 *
-0.60 0.23
.
.
.
.
.
.
.
Val
103
B
B
-1.50 .
-0.60 0.37
.
.
.
.
.
.
.
Val
104
B
T
-1.23 .
-0.20 0.61
.
.
.
.
.
.
.
Pro
105.
.
B
.
.
. T
-1.01 *
. F
0.25 0.35
Ser
106 A.
.
.
.
. T
-0.51 *
. F
-0.05 0.48
Ser 107 A.
.
.
.
. T
-1.40 *
* F
0.25 0.94
Ala
108
A.
.
.
.
.
.
-0.50 .
* F 0.05 0.43
Ala
109
A.
.
.
.
.
.
-0.46 .
* .
0.50 0.63
Thr
110
A.
.
.
.
.
.
-0.28 .
* .
-0.10 0.39
Ile
111 A.
.
.
.
.
.
0.02 .
* .
-0.10 0.53
Lys
112.
.
.
.
. B
.
0.32
.
*
.
0.50
0.87
Leu 113. .
. . . B
. 0.61 . * F 1.05 1.04
His 114. .
. . B
. . 0.31 . * F 1.30 1.99
Asp 115. . . . . T C 0.28 * * F 1.80 0.70
Gln 116. . . . T T . 0.86 . * F 1.65 0.84
Ser
117.
.
.
.
T
T
.
0.81
.
.
F
2.50
0.89
Ile
118.
.
.
.
T
T
.
1.62
.
.
F
2.25
0.92
Gly
119.
.
.
.
.
.
C
1.37
.
.
F
1.00
0.92
Thr
120.
.
.
.
.
.
C
1.37
.
.
F
0.45
0.72
Gin 121. . B . . . C 1.33 . . F 0.65 1.79
Gin 122. .
. . . B
. 1.33 .
. F 0.20 2.46
Trp 123
B
2.01 .
0.05 2.28
.
.
.
.
.
.
.
.
Glu 124
C 1.54 .
0.25 2.04
.
.
.
.
.
.
.
.
His 125
C 1.51 .
0.10 0.97
.
.
.
.
.
.
.
.
Ser
126.
.
.
.
.
T C 1.51 . . F 0.45 0.91
Pro 127. . . . T T . 0.70 . . F 1.55 0.91
Leu 128. . . . T T . 0.32 . . F 0.65 0.55
Gly 129. . . . T T . 0.11 . . F 0.65 0.22
Glu 130.
.
.
.
T .
.
-0.07 .
. F 0.45 0.22
Leu
131
.
B
-0.11 *
0.18 0.42
.
.
.
.
.
.
.
Cys
132.
.
.
. B
.
.
-0.20 *
. F
1.21 0.42
Pro 133. .
. B
.
. T
0.58 * * F 1.69 0.32
Pro 134. . . . T T . 1.03 . * F 1.47 0.53
Gly 135. . . . T T . 0.73 . * F 2.80 1.94
Ser 136. . . . . T C 1.54 * . F 2.32 1.68
His
137.
.
.
.
.
.
C
2.32
*
.
F
2.48
1.88
Arg 138. .
. B
. . . 2.32 * . F 2.34 3.72
Ser
139.
.
. B
.
.
.
2.19 * . F 2.40 4.29
Glu 140. . . . T . . 1.94 * . F 2.86 3.12
Arg 141. . . . T T . 1.58 * . F 3.40 1.61
Pro 142. . . . T T . 1.61 . * F 2.91 0.64
Gly 143. . . . T T . 1.61 . * F 2.57 0.60
Ala 144. . . . T T . 1.24 . * . 2.08 0.60
Cys
145.
.
.
.
T
.
.
0.93
.
*
.
1.41
0.21
Asn
146.
.
. B
.
.
.
0.82
.
*
.
0.84
0.30
Arg 147
B
0.69 *
1.01 0.52
.
.
.
.
.
.
.
.
Cys 148. .
. B
. T . 0.18 * . F 1.83 0.96
Thr 149. .
. B
. T . 0.42 * . F 1.70 0.44
Glu 150. .
. B
. T . 0.84 * . F 1.53 0.22
33
CA 02494372 2005-02-04
WO 2004/016753
PCT/US2003/025457
Table 2 (continued)
Res Position I
II
III IV V
VI VII VIII IX X
XI XII
XIII
Gly 151. .
. . B
T . 0.53 * . F 0.76 0.65
Val 152. .
. . B B
. 0.42 . * F 0.19 0.65
Gly 153.
.
. B
B
.
.
. . 0.50 .
-0.13 0.61
Tyr
154=
=
=
.
=
.
. B
B
0.51 .
-0.60 0.62
Thr
155.
.
. B
B
.
.
0.51 .
. F
-0.30 1.12
Asn 156. . .
. B
. C 0.86 . . F 0.20 1.81
Ala 157. . . .
. T T
0.90 . . F 0.80 1.86
Ser
158.
.
.
.
. T
T
0.54
.
.
F
0.80
1.06
Asn 159. . . .
. T T
0.20 . . F 0.35 0.57
Asn 160.
.
.
.
T
T
.
-0.16 *
. F
0.35 0.57
Leu
161.
.
.
. A B
.
. -0.97 *
-0.60 0.23
.
Phe
162.
.
.
. A
B
.
-0
.
. .59 .
-0.60 0.12
Ala 163
A B
-0.96 .
-0.60 0.11
.
.
.
.
.
.
.
Cys
164
A
B
-1.27 *
-0.60 0.07
.
.
.
.
.
.
.
Leu
165.
.
.
.
.
. . B
T
-1.86 .
-0.20 0.12
Pro
166
B
T
-1.71 *
-0.20 0.12
.
.
.
.
.
.
.
Cys
167
T
T
-0.97 *
0.20 0.12
.
.
.
.
.
.
.
Thr 168 A
T
-0.68 .
0.10 0.30
.
.
.
.
.
.
.
Ala 169 A
-0.01 .
0.50 0.26
.
.
.
.
.
.
.
.
Cys 170 A
T
0.80 .
0.70 0.80
.
.
.
.
.
.
.
Lys 171 A. . . . T . 1.01 . . F 1.15 0.96
Ser 172 A. . . . T . 1.68 . * F 1.30 1.65
Asp 173 A. . . . T . 2.10 . * F 1.30 5.33
Glu 174 A A. . . . . 2.39 . * F 0.90 5.22
Glu 175 A A. . . . . 2.84 . * F 1.24 5.22
Glu 176 A A. . . . . 2.13 . * F 1.58 4.83
Arg 177.
. A
.
. T
. 2.12 . . F 2.32 1.50
Ser
178.
.
.
.
.
T
C
1.81
.
.
F
2.86
1.25
Pro 179. . . . T T . 1.50 * . F 3.40 1.04
Cys 180. . . . T T . 1.61 * . F 2.61 0.77
Thr 181. . . . T T . 1.61 * . F 2.67 1.12
Thr 182. . . . T . . 1.19 * * F 2.38 1.16
Thr 183. . . . T T . 0.90 . . F 2.49 3.13
Arg 184. . . . T T . 0.44 . . F 2.40 2.19
Asn
185.
.
.
.
T
T
.
1.11
.
.
F
2.50
0.81
Thr 186. . . . T T . 0.76 * . F 2.25 0.98
Ala 187
T
1.11 *
1.65 0.27
.
.
.
.
.
.
.
.
Cys 188
T
1.21 *
1.40 0.33
.
.
.
.
.
.
.
.
Gin 189
B
0.76 *
0.75 0.36
.
.
.
.
.
.
.
.
Cys 190
B
0.44 .
0.50 0.35
.
.
.
.
.
.
.
.
Lys 191. .
. . B
T . 0.06 . * F 0.85 0.94
Pro
192.
.
.
.
T
T
.
0.76
.
.
F
0.65
0.47
Gly 193. . . .
. T T
1.42 . * F 1.74 1.72
Thr 194. .
. .
. B
T
1.42 . * F 1.68 1.38
Phe 195. .
. . . . B
2.09 . * F 1.82 1.49
Arg 196. . . .
. . T
1.74 . * F 2.56 2.42
Asn 197. . = = T T . 1.37 . * F 3.40 2.25
Asp 198. . . .
. T T
1.71 . * F 3.06 2.63
Asn 199. = = = = T C 1.42 . * F 2.52 2.32
Ser 200 A. = . = T . 1.46 . * F 1.98 1.43
34
CA 02494372 2005-02-04
WO 2004/016753
PCT/US2003/025457
Table 2 (continued)
Res Position I
II III IV V
VI VII VIII IX X
XI XII
XIII
Ala
201
A.
=
=
=
=
=
1.46
.
*
.
1.14
0.46
Glu
202
A.
=
=
.
.
=
.
= 150
*
0.80
0.56
=
Met
203
A.
=
=
.
.
=
0.83
*
1.11
0.83
.
.
Cys 204 A
T
0.53 *
1.62 0.44
.
.
.
.
.
.
.
Arg 205
T T
0.52 *
2.33 0.34
.
.
.
.
.
.
.
Lys 206. . . . T T . 0.77 * . F 2.49 0.50
Cys 207. . . . T T . 0.10 * . F 3.10 0.92
Ser 208. . . . T . . 0.49 * * F 2.59 0.25
Thr 209. . . . T . . 1.27 * * F 1.98 0.19
Gly 210. . . .
. T
. 0.81 * . F 1.67 0.71
Cys 211. .
. B
. T . 0.17 * * F 1.16 0.53
Pro 212.
.
.
.
T
T
.
-0.02 *
* F
1.25 0.36
Arg 213. . . . T T . 0.32 * * F 0.65 0.27
Gly 214.
.
.
. B
T
.
-0.22 *
* .
0.85 1.01
Met 215. .
. . . B B
0.17 * * . 0.30 0.48
Val 216
B B
0.83 * *
0.79 0.49
.
.
.
.
.
.
Lys
217=
=
.
.
= B
B
0.38
*
*
.
0.98
0.83
Val
218.
.
.
.
. B
B
-0.04 *
* F
1.32 0.45
Lys 219. . B B . . . 0.09 . * F 1.51 0.88
Asp
220.
.
. B
.
.
.
0.40
.
*
F
1.90
0.68
Cys
221.
.
. B
.
.
.
0.96
.
*
F
0.81
0.96
Thr
222.
.
.
.
.
T
C
0.91
.
*
F
1.62
0.65
Pro
223.
.
.
.
T
T
.
0.88
.
*
F
1.63
0.65
Trp
224.
.
.
.
T
T
.
0.83
.
*
F
0.54
0.84
Ser
225
A.
.
.
.
. T
0.17
.
.
F
1.00
1.01
Asp 226 A A.
.
.
.
.
-0.02 .
. F 0.45 0.35
Ile
227
A
A.
.
.
.
.
.
026 *
. .
-0.30 0.25
Glu 228 A A. . . . . . 051 *
. 0.30 0.25
.
Cys 229
A B
0.80 *
0.60 0.30
.
.
.
.
.
.
.
Val 230 A A. . . . . 0.80 * * . 0.60 0.74
His
231
A
A.
.
.
.
.
0.46
*
*
.
0.60
0.58
Lys
232
A
A.
.
.
.
.
1.34 * . F 0.60 1.06
Glu 233. A . .
. T
. 1.00 * . F 1.30 2.30
Ser 234. . . .
. T T
1.63 * . F 1.70 1.68
Gly 235. . . .
. T T
2.49 * . F 1.70 1.14
Asn 236. . . .
. T T
1.63 * . F 1.40 1.06
Gly
237.
.
.
.
.
T
C
1.30
*
.
F
0.45
0.55
His 238
B
C
0.44 .
-0.40 0.59
.
.
.
.
.
.
.
Asn 239
B
C
-0.14 .
-0.40 0.27
.
.
.
.
.
.
.
Ile 240
B B
-0.61 .
-0.60 0.19
.
.
.
.
.
.
.
Trp 241
B B
-1.47 .
-0.60 0.12
.
.
.
.
.
.
.
Val
242
B B
-1.98 .
-0.60 0.05
.
=
=
.
.
.
.
Ile 243
B B
-2.26 .
-0.60 0.06
.
.
.
.
.
.
.
Leu 244
B B
-3.07 .
-0.60 0.08
.
.
.
.
.
.
.
Val
245
B B
-3.03 .
-0.60 0.09
-
=
=
.
.
.
.
Val
246
B B
-3.60 .
-0.60 0.09
=
.
=
.
.
.
.
Thr 247
B B
-2.96 .
-0.60 0.08
.
.
.
.
.
.
.
Leu 248
B B
-2.88 .
-0.60 0.17
.
.
=
.
.
.
.
Val
249=
.
=
.
. B B
-2.88 .
* .
-0.60 0.19
Val 250
B B
-2.83 .
-0.60 0.11
=
=
=
=
.
.
.
CA 02494372 2005-02-04
WO 2004/016753 PCT/US2003/025457
Table 2 (continued)
Res Position I II III IV V VI VII VIII IX X XI XII XIII
Pro 251 . . . . -2.83 . . . -0.60 0.11
Leu 252 . B B . -3.11 . . . -0.60 0.11
Leu 253 A . . . = -3.16 . . . -0.60 0.15
Leu 254 A = B . = = -3.11 . = = -0.60 0.07
Val 255 A . = . = -3.14 . = . -0.60 0.07
Ala 256 A = . B . . . -3.79 . = = -0.60 0.06
Val 257 . B . . . -3.64 . . . -0.60 0.05
Leu 258 . . . -3.50 . . . -0.60 0.04
Ile 259 . B B . . . -3.36 . . . -0.60 0.02
Val 260 . B B . . . -3.39 . . . -0.60 0.02
Cys 261 . B . . -3.14 . . . -0.60 0.01
Cys 262 . . B B . . . -2.59 . . . -0.60 0.02
Cys 263 . B . . . -2.12 . . . -0.60 0.03
Ile 264 . B . . . -1.90 . . . -0.60 0.06
Gly 265. . . . T T . -1.39 . . F 0.35 0.06
Ser 266. . . . T T . -1.07 . . F 0.35 0.11
Gly 267. . . . T T . -0.40 . . F 0.65 0.16
Cys 268. . . . T T . 0.06 . . F 1.25 0.27
Gly 269. . . . T . . 0.99 . * F 1.39 0.31
Gly 270. . . T . . 0.67 . . F 2.03 0.62
Asp 271. . . . . T C 0.37 . . F 2.37 0.62
Pro 272. . . . T T . 0.71 * * F 2.91 0.62
Lys 273. . . . T T . 1.49 * * F 3.40 1.05
Cys 274. . B . T . 0.98 * * . 2.51 1.23
Met 275. . B B . . 0.66 * * . 1.62 0.59
Asp 276. . B B . . . -0.04 * * . 1.28 0.16
Arg 277. . B B . . . -0.12 . * . 0.04 0.26
Val 278. . B B . . . -0.06 . * . -0.60 0.27
Cys 279 . B B . . . -0.20 . . . 0.30 0.32
Phe 280. . B B . . . 0.06 . * . -0.60 0.13
Trp 281 . B B . . . -0.60 0.18
Arg 282 . . . . . -0.60 0.28
Leu 283 B B . . . -0.71 . . . -0.60 0.26
Gly 284. . . B T . . -0.39 . * . -0.20 0.49
Leu 285. . . B . . C 0.10 . * . 0.50 0.25
Leu 286. . . B . . C 0.04 . * . 0.20 0.46
Arg 287. . B . . C -0.66 . . F 0.65 0.46
Gly 288. . . . . T C 0.16 . . F 1.35 0.57
Pro 289. . . . . T C 0.50 . * F 2.70 1.19
Gly 290. . . . . T C 1.31 * * F 3.00 1.01
Ala 291 A. . . . T . 1.53 . * F 2.50 1.65
Glu 292 A. . . . . . 1.39 . . F 2.00 1.08
Asp 293 A. . . . . . 1.73 . . F 1.70 1.48
Asn 294 A. . . . T . 1.94 . * . 1.45 2.36
Ala 295 A . . . . T . 1.40 . . . 1.15 2.36
His 296 A . = . . T . 1.18 * . . 1.00 0.99
Asa 297 A. . . . T . 0.88 . . . 0.10 0.51
Glu 298 A. = . = . . 0.88 * . . -0.10 0.67
Ile 299 A. = . = . . 0.29 * * . -0.10 0.80
Leu 300 A. . . = . . 0.88 * * . -0.10 0.50
36
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Table 2 (continued)
Res Position I
II
III IV V
VI VII VIII IX X
XI XII
XIII
Ser
301
A.
.
.
.
.
.
0.61
*
.
F
0.65
0.48
Asn 302 A.
.
.
.
T
.
-0.20 *
. F 0.25 0.92
Ala 303 A.
.
.
.
T
.
-0.50 *
. F 0.25 0.92
Asp 304 A. . . .
. T
0.08 * . F 0.85 0.92
Ser
305.
.
.
.
.
T
C
0.19
*
.
F
1.05
0.83
Leu 306.
.
.
.
. B
C
-0.37 *
. F 0.05 0.71
Ser 307.
.
. B
B
.
.
-0.67 *
. F
-0.15 0.31
Thr 308
B
B
-0.08 *
-0.60 0.31
.
.
.
=
.
.
.
Phe 309
B
B
-0.08 *
-0.30 0.66
.
.
.
=
=
.
.
Val 310 A.
.
.
.
. B
0.22 .
. F -0.15 0.85
Ser 311 A A. . . . . 0.43 . . F 0.00 1.03
Glu 312 A A. . . = = 0.73 . . F 0.00 1.17
Gin 313 A A. . . . . 0.74 . . F 0.90 2.73
Gin 314 A A. . . . . 1.44 . . F 0.90 2.73
Met 315 A A. . . . . 2.30 . . F 0.90 2.73
Glu 316 A A. . . . . 2.39 . . F 0.90 2.73
Ser 317 A A. . . . . 1.80 . * F 0.90 2.44
Gin 318 A A. . . . . 1.80 . * F 0.90 2.49
Glu 319 A A. . . . . 0.99 . * F 0.90 2.40
Pro 320 A A. . . . . 1.28 . * F 0.90 1.48
Ala 321 A A. . . . . 0.93 . . F 0.60 1.23
Asp 322 A A.
. . B
. 0.38 . . F 0.45 0.70
Leu 323 A A.
.
. B
.
0.07 .
. F -0.15 0.34
Thr 324.
A B
B
.
.
.
-0.79 .
. F -0.15 0.48
Gly 325
A B
B
-0.58 .
-0.30 0.21
.
.
.
.
.
.
Val 326
B
B
-0.29 .
-0.60 0.45
.
.
.
.
.
.
.
Thr 327
B
B
-0.50 .
-0.60 0.42
.
.
.
.
.
.
.
Val 328.
.
. B
B
.
.
-0.03 .
* F
-0.17 0.65
Gin 329. .
. . . B B
0.28 . * F 0.11 0.87
Ser
330.
.
.
.
.
T
C
0.03
.
*
F
2.04
1.05
Pro
331.
.
.
.
.
T
C
0.89
.
*
F
2.32
1.42
Gly
332.
.
.
.
. T
T
0.53
.
*
F
2.80
1.42
Glu 333 A. . . .
. T
0.58 . * F 1.97 0.57
Ala 334.
.
.
. .
. B
-0.23 .
* . 0.74 0.30
Gin 335
B
-0.28 .
0.46 0.25
.
.
.
.
.
.
.
.
Cys 336
B
-0.28 .
0.18 0.14
.
.
.
.
.
.
.
.
Leu 337.
.
.
.
.
. B
-0.52 .
* .
-0.40 0.22
Leu 338.
.
.
.
.
. B
-0.52 .
* .
-0.40 0.13
Gly 339. A .
.
. . C -0.52 .
* F 0.05 0.42
Pro 340 A A.
.
.
.
.
-0.52 .
* F -0.15 0.51
Ala 341 A A.
.
.
.
.
-0.20 .
* F 0.60 1.07
Glu 342 A A. . . . = 0.31 . * F 0.90 1.07
Ala 343 A A. . . . . 1.12 * * F 0.75 0.93
Glu 344 A A. . . . . 1.58 . * F 0.90 1.60
Gly 345 A A. . = . = 1.90 . * F 0.90 1.80
Ser 346 A. . . .
. T
2.60 . * F 1.30 3.50
Gin 347 A. . . .
. T
1.79 . * F 1.30 3.96
Arg 348 A. . . .
. T
1.57 . * F 1.30 3.30
Arg 349. .
. .
. B
T
0.71 . * F 1.30 2.03
Arg 350. .
. = = B B
0.84 . * F 0.75 0.87
37
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Table 2 (continued)
Res Position I
II III IV V
VI VII VIII IX X
XI XII
XIII
Leu 351.
B B .
. . 0.56 .
* . 0.60
0.69
Leu 352. .
B B .
. . 0.56 = * . 0.30 0.35
Val 353.
. B B .
. . 0.10 *
* . -0.30 0.29
Pro 354 .
. B . .
. . -0.20 0.35
Ala 355.
. . . T T
. -0.71 .
* . 0.50 0.43
Asn 356.
. . . .
T C -0.11 .
. F 1.65 0.96
Gly 357. . . . . T C 0.39 . . F 1.95 0.96
Ala 358. . . . . . C 1.24 . . F 2.20 1.37
Asp 359. . . . . T C 1.14 . . F 3.00 1.48
Pro 360 A. . . . T . 0.92 * . F 2.50 2.16
Thr 361 A. . . .
T . 0.32 . . F 1.90 1.76
Glu 362 A.
. . .
T . -0.14 .
. F 1.60 1.04
Thr 363 A.
. B .
. . -0.26 .
. F 0.15 0.56
Leu 364 A
B .
. -0.96 *
. . -0.60 0.33
Met 365 A
. B .
. -0.74 *
. . -0.60 0.17
Leu 366 A
.
.
. . -0.60 0.19
Phe 367 A
.
. .
. . -0.60 0.47
Phe 368 A
. . B .
. . -1.37 *
. . -0.60 0.41
Asp 369 A
. . B .
. .
. . -0.60 0.50
Lys 370 A A
. . .
. . -0.84 *
. . -0.30 0.93
Phe 371 A
A B .
. -0.89 *
. . -0.30 0.75
Ala 372 A A.
B .
. .
. -0.30 0.34
Asn 373 .
A B B .
. .
. . -0.60 0.26
Ile 374 .
B B .
. -0.40 *
. . -0.60 0.26
Val 375 .
A B B .
. -0.74 .
. . -0.60 0.43
Pro 376 .
A . B .
. C -0.33 ,.
. . -0.10 0.36
Phe 377 .
. . T T
0.26 .
. . 0.20 0.54
Asp 378. . . . T T . 0.26 . . F 0.80 1.21
Ser 379. . . . T T . 0.33 . . F 1.40 1.35
Trp 380 A. . . .
T . 0.59 * * F 0.40 1.29
Asp 381 A
A. . .
. . 0.91 *
. F -0.15 0.76
Gln 382 A
A. . .
. . 161 * .
. . -0.15 1.11
Leu 383 A
A. . .
. . 080 * .
. . -0.15 1.84
Met 384 A A. . . . . 1.10 * . . 0.30 0.91
Arg 385 A A. . . . . . 058 * . . 0.30 0.87
Gin 386 A A.
. .
. . 027 * .
. . -0.30 0.87
Leu 387 A A. . . . . 0
.31 *
. 0.45 1.27
Asp 388 A
A. . .
. . .
Leu 389 A A. . . . . 1.72 * . F 0.60 1.21
Thr 390 A. . . .
T . 0.72 * . F 1.30 2.54
Lys 391 A. . . .
T . 0.72 . * F 1.30 1.07
Asn 392 A. . . .
T . 0.68 * * F 1.30 2.16
Glu 393 A.
. .
T . -0.18 *
. F 1.30 1.11
Ile 394. . B B = . . 0.74 * . F 0.75 0.41
Asp 395 .
B B =
. 0.47 *
. 0.60 0.50
Val 396
B B .
. . 0.08 *
* . 0.60 0.29
Val 397 .
. B B .
. . -0.23 .
. . 0.51 0.41
Arg 398 .
. B .
T . -0.82 *
. . 1.12 0.36
Ala 399 .
. B .
T . -0.28 *
. . 0.73 0.49
Gly 400.
. . .
. -0.49 *
. F 2.09 0.65
38
CA 02494372 2005-02-04
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PCT/US2003/025457
Table 2 (continued)
Res Position I
II III IV V
VI VII VIII IX X
XI XII
XIII
Thr 401. . . . . T C 0.02 * * F 2.10 0.51
Ala 402. . . . = . C 0.88 * * F 1.09 0.50
Gly 403. . . . . T C 0.18 * * F 1.68 0.85
Pro 404.
.
.
.
. T C -0.04 .
. F 1.47 0.59
Gly 405. . . . . T C 0.06 . . F 1.26 0.48
Asp 406 A.
.
.
. T .
-0.22 .
. F 0.25 0.76
Ala 407 A A.
=
.
.
.
. . -0.23 .
-0.30 0.50
Leu 408 A A
-0.70 .
-0.60 0.50
.
.
.
.
=
=
.
Tyr 409 A A
-1.09 *
-0.60 0.25
.
.
.
.
=
.
.
Ala 410 A A
-0.70 *
-0.60 0.24
.
.
.
.
.
.
.
Met
411 A A.
.
.
.
.
. -0.99 *
-0.60 0.59
.
Leu 412 A A.
.
.
.
.
. -1.26 *
-0.60 0.39
.
Met
413 A A.
.
.
.
.
. -0.44 *
-0.60 0.29
.
Lys 414 A A.
.
.
.
.
. B
-016 *
-0.60 0.47
.
Trp 415 A A.
. . B
.
. . 0.12 *
0.15 1.14
Val 416 A A.
. . . B
0.38 * * . 0.45 1.66
Asn 417 A. . . .
. T
1.30 * . F 1.75 0.82
Lys 418 A. . . .
. T
1.90 * . F 2.20 1.53
Thr
419.
.
.
.
.
T C 1.27 * . F 3.00 3.32
Gly 420. . . . . T C 1.26 * . F 2.70 2.08
Arg 421. . . . T . . 1.22 * . F 2.40 1.40
Asn 422. . . . . T C 1.19 * . F 1.65 0.68
Ala 423
B
T
0.83 .
1.00 0.93
.
.
.
.
.
.
.
Ser 424
B
T
0.33 .
0.70 0.69
.
.
.
.
.
.
.
Ile 425.
.
. B
.
. T
-0.13 .
* .
-0.20 0.35
His
426.
A
B
.
.
.
.
-0.24 .
* .
-0.60 0.29
Thr 427.
.
. A B
.
.
-0.83 *
* .
-0.60 0.36
Leu 428 A A.
.
.
.
.
-1.06 *
* .
-0.60 0.52
Leu 429 A A.
.
.
.
.
-0.76 *
* .
-0.60 0.31
Asp 430 A A.
.
.
.
.
0.24 *
* .
-0.30 0.38
Ala 431 A A.
.
.
.
.
-0.32 *
* .
0.30 0.89
Leu
432
A
A.
.
.
.
.
-0.01 *
* .
0.75 1.07
Glu 433 A A. . . . . 0.80 * * . 0.75 1.11
Arg
434
A
A.
.
.
.
.
1.72 * * F 0.90 1.90
Met 435 A A. . . . . 1.69 * * F 0.90 4.52
Glu
436
A
A.
.
.
.
.
1.69 * * F 0.90 3.55
Glu 437 A A. . . . . 2.54 * . F 0.90 1.83
Arg 438 A A. . . . . 2.54 * * F 0.90 3.70
His 439 A A. . . . . 2.48 * * F 0.90 3.70
Ala 440 A A. . . . . 2.19 * * F 0.90 4.28
Lys 441 A A. = = = . 2.19 * * F 0.90 1.53
Glu 442 A A. - = . . 2.19 * . F 0.90 1.95
Lys 443 A A. . . . . 1.27 * * F 0.90 3.22
Ile 444 A A. .
. . 0.49 * * F 0.90 1.33
.
Gin 445 A A. = . . . 0.22 * * F 0.75 0.63
Asp 446 A A.
-
=
.
=
0.18 *
* F
-0.15 0.23
Leu 447 A A
-0.12 *
-0.30 0.56
=
.
=
.
=
.
.
Leu 448 A A
-0.51 *
0.55 0.43
=
=
=
.
.
.
.
Val 449 A A= = = = . 0.42 * . F 0.95 0.26
Asp 450 A.
.
.
.
T
.
-0.28 *
. F
1.60 0.62
39
CA 02494372 2005-02-04
WO 2004/016753 PCT/US2003/025457
Table 2 (continued)
Res Position I II III IV V VI VII VIII IX X XI XII XIII
Ser 451. . . . T T . -1.17 * . F 2.25 0.65
Gly 452. . . . T T . -0.60 * . F 2.50 0.62
Lys 453. . B . . T . -0.60 . . F 1.25 0.58
Phe 454 . A B . . . . 0.26 . . . 0.15 0.36
Ile 455 A B . . . 0.26 . . . 0.20 0.62
Tyr 456 . A B . . . . 0.21 . . . 0.55 0.52
Leu 457 . A B . . . . 0.24 . . . -0.03 0.59
Glu 458. A B . . . . -0.14 . . F 0.54 1.22
Asp 459. A . . T . . 0.26 . . F 1.66 0.77
Gly 460. . . . T T . 0.56 . . F 2.78 1.26
Thr 461. . . . . T C -0.06 * . F 2.70 0.73
Gly 462. . . . . T C 0.46 * . F 2.13 0.33
Ser 463. . . . . T C -0.36 . . F 1.26 0.44
Ala 464 A . . . . . . -0.36 . . . 0.14 0.25
Val 465 . . B . . . . -0.40 . . . 0.17 0.44
Ser 466 . . B . . . -0.48 . . . -0.10 0.42
Leu 467 . B . . . -0.52 . . . -0.10 0.53
Glu 468 A . . . . . -0.61 . . . 0.50 0.92
40
WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
[0090] 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).
[0091] 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
2/9:660-666. 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.
[0092] 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 ID NO: 1.
[0093] 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 ID NO:1; a polypeptide comprising amino acid
residues
from about 114 to about 160 of SEQ ID NO:1; a polypeptide comprising amino
acid
residues from about 169 to about 240 of SEQ ID NO:1; a polypeptide comprising
amino
acid residues from about 267 to about 298 of SEQ ID NO:1; a polypeptide
comprising
amino acid residues from about 330 to about 364 of SEQ ID NO:1; a polypeptide
comprising amino acid residues from about 391 to about 404 of SEQ ID NO:1;
and/or a
41
CA 02494372 2005-02-04
WO 2004/016753 PCT/US2003/025457
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. 4,631,211 to Houghten et al. (1986).
[0094] 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 Biochem 270:3958-3964 (1995)). Thus, antibodies of the invention may bind
fusion
proteins that comprise all or a portion of a TR4 polypeptide such as TR4.
[0095] 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 TR4 polypeptides or TR4 polypeptide fragments or
variants.
[0096] For instance, for many proteins, including the extracellular domain of
a
membrane associated protein or the mature form(s) 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
42
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
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. Chem., 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 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-
terminal deletions including the cysteine residue at position 109 (C-109) in
SEQ ID NO: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.
[0097] 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.
[0098] 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:1 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:1, where n1 is an integer from 2 to 463 corresponding to the
position of the
amino acid residue in SEQ ID NO: 1.
[0099] 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
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WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
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-12 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-19 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; 1-45 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-91 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-101 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-115 to E-468; Q-
116 to E-
468; S-117 to E-468; 1-118 to E-468; G-119 to E-468; T-120 to E-468; Q-121 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; P434 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-141 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-
148 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;
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WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
T-186 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; P492 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-216 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; 1-227 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; 1-240 to E-468; W-241 to E-468; V-242 to E-468; 1-243 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; 1-259 to E-
468; V-260
to E-468; C-261 to E-468; C-262 to E-468; C-263 to E-468; 1-264 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; R-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; 1-299 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-310 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
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
to E-468; 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; 1-374 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; 1-394 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; 0-403 to
E-468;
P-404 to E-468; 0-405 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; 1-425
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; 11-439 to E-468; A-440 to E-
468; K-441
to E-468; E-442 to E-468; K-443 to E-468; 1-444 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; 1-455 to E-468; Y-456 to E-
468; L-457
to E-468; E-458 to E-468; D-459 to E-468; 0-460 to E-468; T-461 to E-468; 0-
462 to E-
468; and/or S-463 to E-468 of the TR4 sequence of SEQ ID NO:l.
[0100] 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 NO: 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; 0-11 to 11-238; A-12 to 11-238; F-13 to 11-
238; L-14 to
11-238; A-15 to H-238; V-16 to II-238; T-17 to H-238; P-18 to II-238; N-19 to
H-238; P-
20 to H-238; 0-21 to II-238; S-22 to 11-238; A-23 to 11-238; A-24 to 11-238; S-
25 to H-
238; G-26 to II-238; T-27 to 14-238; E-28 to II-238; A-29 to H-238; A-30 to H-
238; A-31
to 11-238; A-32 to II-238; T-33 to H-238; P-34 to 11-238; S-35 to 11-238; K-36
to H-238;
V-37 to H-238; W-38 to 11-238; 0-39 to H-238; S-40 to 11-238; S-41 to 11-238;
A-42 to H-
238; G-43 to 11-238; R-44 to H-238; 1-45 to 11-238; E-46 to 14-238; P-47 to 11-
238; R-48
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WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
to H-238; G-49 to 11-238; G-50 to 11-238; G-51 to 11-238; R-52 to H-238; G-53
to 11-238;
A-54 to 11-238; L-55 to 11-238; P-56 to H-238; T-57 to H-238; S-58 to 11-238;
M-59 to H-
238; G-60 to 11-238; Q-61 to H-238; H-62 to H-238; G-63 to 11-238; P-64 to 11-
238; S-65
to H-238; A-66 to H-238; R-67 to H-238; A-68 to 11-238; R-69 to 11-238; A-70
to 11-238;
G-71 to 11-238; R-72 to 11-238; A-73 to 11-238; P-74 to H-238; G-75 to 11-238;
P-76 to H-
238; R-77 to 11-238; P-78 to H-238; A-79 to 11-238; R-80 to H-238; E-81 to H-
238; A-82
to 11-238; S-83 to 11-238; P-84 to 11-238; R-85 to H-238; L-86 to H-238; R-87
to H-238;
V-88 to 11-238; 11-89 to 11-238; K-90 to H-238; T-91 to 11-238; F-92 to 11-
238; K-93 to H-
238; F-94 to 11-238; V-95 to 11-238; V-96 to 11-238; V-97 to H-238; G-98 to 11-
238; V-99
to H-238; L-100 to 11-238; L-101 to 11-238; Q-102 to 11-238; V-103 to 11-238;
V-104 to
H-238; P405 to 11-238; S-106 to H-238; S-107 to H-238; A-108 to 11-238; A-109
to H-
238; T-110 to H-238; I-111 to H-238; K-112 to 11-238; L-113 to H-238; 11-114
to 11-238;
D-115 to H-238; Q-116 to H-238; S-117 to 11-238; 1-118 to H-238; G-119 to H-
238; T-
120 to 11-238; Q-121 to 11-238; Q-122 to 11-238; W-123 to 11-238; E-124 to 11-
238; 11-125
to H-238; S-126 to 11-238; P-127 to 11-238; L-128 to H-238; G-129 to 11-238; E-
130 to H-
238; L-131 to H-238; C-132 to H-238; P-133 to 11-238; P-134 to H-238; G-135 to
11-238;
S-136 to 11-238; 11-137 to 11-238; R-138 to H-238; S-139 to H-238; E-140 to 11-
238; R-
141 to 11-238; P-142 to 11-238; G-143 to H-238; A-144 to 11-238; C-145 to H-
238; N-146
to 11-238; R-147 to 11-238; C-148 to H-238; T-149 to H-238; E-150 to 11-238; G-
151 to H-
238; V-152 to H-238; G-153 to 11-238; Y-154 to 11-238; T-155 to 11-238; N-156
to 11-238;
A-157 to H-238; S-158 to H-238; N-159 to 11-238; N-160 to 11-238; L-161 to H-
238; F-
162 to H-238; A-163 to 11-238; C-164 to H-238; L-165 to H-238; P-166 to 11-
238; C-167
to H-238; T-168 to 11-238; A-169 to 11-238; C-170 to 11-238; K-171 to 11-238;
S-172 to H-
238; D-173 to H-238; E-174 to 1I-238; E-175 to 11-238; E-176 to H-238; R-177
to 11-238;
S-178 to 11-238; P-179 to 11-238; C-180 to 11-238; T-181 to 11-238; T-182 to
11-238; T-
183 to 11-238; R-184 to 11-238; N-185 to 11-238; T-186 to H-238; A-187 to 11-
238; C-188
to 11-238; Q-189 to 11-238; C-190 to H-238; K-191 to 11-238; P-192 to 11-238;
G-193 to
11-238; T-194 to 11-238; F-195 to 11-238; R-196 to 11-238; N-197 to 11-238; D-
198 to H-
238; N-199 toll-238; S-200 to 11-238; A-201 to 11-238; E-202 to 11-238; M-203
to 11-238;
C-204 to H-238; R-205 to H-238; K-206 to 11-238; C-207 to H-238; S-208 to 11-
238; T-
209 to 11-238; G-210 to H-238; C-211 to H-238; P-212 to 11-238; R-213 to 11-
238; G-214
to 11-238; M-215 to 11-238; V-216 to 11-238; K-217 to 11-238; V-218 to 11-238;
K-219 to
11-238; D-220 to 11-238; C-221 to 11-238; T-222 to 11-238; P-223 to 11-238; W-
224 to H-
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WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
238; S-225 to H-238; D-226 to H-238; 1-227 to II-238; E-228 to H-238; C-229 to
11-238;
V-230 to 11-238; H-231 to II-238; K-232 to II-238; and/or E-233 to 11-238; of
the TR4
extracellular domain sequence of SEQ ID NO: 1.
[0101] 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.
[0102] 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.
[0103] 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 1-455; 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 1-444; 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 II-426; A-24 to 1-425; 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 G-
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
1-394;
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 1-374; 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 1-299; 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 G-
269; A-24 to C-268; A-24 to G-267; A-24 to S-266; A-24 to G-265; A-24 to 1-
264; A-24
to C-263; A-24 to C-262; A-24 to C-261; A-24 to V-260; A-24 to 1-259; 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 1-243; A-24 to V-242; A-24 to
W-241;
A-24 to 1-240; 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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WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
235; A-24 to S-234; A-24 to E-233; A-24 to K-232; A-24 to 11-231; A-24 to V-
230; A-24
to C-229; A-24 to E-228; A-24 to 1-227; 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-201; 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 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-174; 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 11-137; A-24 to S-136; A-24 to G-135; A-24 to P-
134; A-24
to P433; 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 11-125; 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-119; A-24 to 1-118; A-24 to S-
117; 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
11-62; 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 1-45; A-24
to R-44;
50
WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
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:l.
[0104] 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: 1. 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-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-201; 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 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-174; 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-121; A-24 to T-
120; A-24
to G-119; A-24 to 1-118; A-24 to S-117; 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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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
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 1-45; 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 NO:1.
[0105] 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 NO: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.
[0106] 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- m1 and/or
n2- m2 of
SEQ ID NO:1, where n,n2, and m2 are integers as described above.
[0107] 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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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
[0108] 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.
[0109] 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.
[0110] 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.U. et al.,
Science
247:1306-1310 (1990).
[0111] 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 sub stituent 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 Pc
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.
[0112] 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.
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CA 02494372 2005-02-04
WO 2004/016753 PCT/US2003/025457
The prevention of aggregation is highly desirable. Aggregation of proteins not
only
results in a loss of activity but can also be problematic when preparing
pharmaceutical
formulations, because they can be immunogenic. (Pinckard et al., Clin Exp.
Immunol.
2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al.
Grit. Rev.
Therapeutic Drug Carrier Systems /0:307-377 (1993)).
[0113] The replacement of amino acids can also change the selectivity of
binding to
cell surface receptors. Ostade et al., Nature 367:266-268 (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 TR4
receptor
that contains one or more amino acid substitutions, deletions or additions,
either from
natural mutations or human manipulation.
[0114] 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
S erine
Threonine
Methionine
Glycine
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
[0115] In specific embodiments, the number of substitutions, additions or
deletions in
the amino acid sequence of SEQ ID NO: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, 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.
[0116] 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: M1 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; L10 replaced with A, G, I, S, T, M, or V; Gll replaced with A, I, L, S, T,
M, or V; Al2
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; T17 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 V; A42 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;
145
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 replaced with N; H62 replaced with K, or R; G63 replaced with A, I, L, S,
T, M, or
55
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
V; S65 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; V104 replaced with A, G, I, L, S, T, or M; S106 replaced with
A, G, I, L, T,
M, or V; S107 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; Il 1 1 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; S117 replaced with A, G, I, L, T, M, or V; 1118 replaced with
A, G, L, S,
T, M, or V; G119 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; S126 replaced with A, G, I, L, T,
M, or V;
L128 replaced with A, G, I, S, T, M, or V; G129 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; G135 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; R138 replaced with H, or K; S139 replaced with A, G, I, L, T, M, or V; E140
replaced
with D; R141 replaced with H, or K; G143 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; R147 replaced with
H, or K;
T149 replaced with A, G, I, L, S, M, or V; E150 replaced with D; G151 replaced
with A,
I, L, S, T, M, or V; V152 replaced with A, 0,1, L, S, T, or M; G153 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; S158 replaced
with A,
56
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
G, I, L, 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; S172
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; R177 replaced with H, or K; S178 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; R184 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; G193 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;
R196 replaced with H, or K; N197 replaced with Q; D198 replaced with E; N199
replaced
with Q; S200 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; R205
replaced with
H, or K; K206 replaced with H, or R; S208 replaced with A, G, I, L, T, M, or
V; T209
replaced with A, G, I, L, S, M, or V; G210 replaced with A, I, L, S, T, M, or
V; R213
replaced with H, or K; G214 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;
S225
replaced with A, G, I, L, T, M, or V; D226 replaced with E; 1227 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; G235 replaced with A, I, L, S, T, M, or V; N236
replaced with
Q; G237 replaced with A, I, L, S, T, M, or V; H238 replaced with K, or R; N239
replaced
with Q; 1240 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; 1243 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
57
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
replaced with A, G, I, L, S, T, or M; L258 replaced with A, G, I, S, T, M, or
V; 1259
replaced with A, G, L, S, T, M, or V; V260 replaced with A, G, I, L, S, T, or
M; 1264
replaced with A, G, L, S, T, M, or V; G265 replaced with A, I, L, S, T, M, or
V; S266
replaced with A, G, I, L, T, M, or V; G267 replaced with A, I, L, S, T, M, or
V; G269
replaced with A, I, L, S, T, M, or V; G270 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; R277 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; R282 replaced
with H, or
K; L283 replaced with A, G, I, S, T, M, or V; G284 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;
R287 replaced with H, or K; G288 replaced with A, I, L, S, T, M, or V; G290
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;
1299
replaced with A, G, L, S, T, M, or V; L300 replaced with A, G, I, S, T, M, or
V; S301
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; S305 replaced with A, G, I, L, T, M, or V;
L306
replaced with A, G, I, S, T, M, or V; S307 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; S311 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;
G325 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; S330 replaced with A, G, I, L, T, M, or V; G332 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, 1, S, T, M, or V; G339 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; G345 replaced with A, I, L, S, T, M, or V; S346 replaced with
A, G, I, L,
T, M, or V; Q347 replaced with N; R348 replaced with H, or K; R349 replaced
with H, or
58
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
K; R350 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; G357 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; 1374
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; S379 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; R385
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;
1394
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; R398 replaced with
H, or K;
A399 replaced with G, I, L, S, T, M, or V; G400 replaced with A, I, L, S, T,
M, or V;
T401 replaced with A, G, I, L, S, M, or V; A402 replaced with G, I, L, S, T,
M, or V;
G403 replaced with A, I, L, S, T, M, or V; G405 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; G420
replaced with A, I,
L, S, T, M, or V; R421 replaced with H, or K; N422 replaced with Q; A423
replaced with
G, I, L, S, T, M, or V; S424 replaced with A, G, I, L, T, M, or V; 1425
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; L429 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; L432 replaced
with A,
G, I, S, T, M, or V; E433 replaced with D; R434 replaced with H, or K; M435
replaced
59
WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
with A, G, I, L, S, T, or V; E436 replaced with D; E437 replaced with D; R438
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;
1444
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; S451 replaced with
A, G, I, L,
T, M, or V; G452 replaced with A, I, L, S, T, M, or V; K453 replaced with H,
or R; F454
replaced with W, or Y; 1455 replaced with A, G, L, S, T, M, or V; Y456
replaced with F,
or W; L457 replaced with A, G, I, 5, T, M, or V; E458 replaced with D; D459
replaced
with E; G460 replaced with A, I, L, S, T, M, or V; T461 replaced with A, G, I,
L, S, M, or
V; G462 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;
S466 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 NO: 1.
[0117] 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;
Gil replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Al2 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
60
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
with D, E, H, K, R, N, 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; 145 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,
61
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
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; S106 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; S107 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;
1111
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
62
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q116 replaced
with D, E, H,
K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S117 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; 1118 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G119
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; 11125 replaced with D,
E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; S126 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; G129 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; C132 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; 0135 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
S136 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 11137 replaced with
D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R138 replaced with D, E, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, P, or C; S139 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; R141
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; 0143 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; R147 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, 5, T, M, V, N, Q, F, W, Y, P, or C; G151 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; V152 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
G153 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; S158 replaced with D, E,
H, K, R, N,
63
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
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; S172 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; R177 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, P, or C; S178 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; R184 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;
G193
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; R196 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; D198 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; S200 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
64
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
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; R205 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
K206 replaced
with 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; S208 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; G210
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; R213 replaced with D, E, A, G, I, L, S, T, M, V. N, Q, F,
W, Y, P, or
C; G214 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; C221 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; S225 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;
1227 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; 1<232
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; S234 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; G235 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; G237 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;
1240 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;
1243 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L244 replaced with
D, E, H, K,
65
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
R, N, 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; 1259 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; C263
replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; 1264 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; G265 replaced with D, E, H, K, R, N, Q, F, W,
Y, P. or
C; S266 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G267 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; G269 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
G270
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; C274 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; R277 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;
C279 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; R282 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; G284 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;
R287 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G288
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,
66
WO 2004/016753 CA 02494372 2005-02-04 ..
PCT/US2003/025457
S, T, M, V, N, Q, F, W, Y, or C; G290 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; 1299 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; S301 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; S305
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; S307 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;
S311 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; G325 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; S330
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; G332 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;
67
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
A334 replaced with D, E, H, K, R, N, Q, 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; G339 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; G345 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S346
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; R348 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, P, or C; R349 replaced with D, E, A, G, I, L, S. T, M, V, N, Q, F, W, Y, P,
or C; R350
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; G357 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; 1374
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
68
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S379 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; R385 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; 1394 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; R398
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; G400 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; G403 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;
G405
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; W415 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; G420
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; R421 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,
69
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
or C; A423 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S424 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; 1425 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; L429 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; R434
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; R438
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; 1444 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;
S451 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G452 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; 1455
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; L457 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; G460 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; G462
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S463 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; S466 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
70
WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
or C; L467 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; and/or E468
replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C of SEQ ID NO:l.
[0118] 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 in vitro, or in vitro proliferative activity. Sites that
are critical for
ligand-receptor binding can also be determined by structural analysis such as
crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith
et al., J. MoL
Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)). 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.
[0119] 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.
[0120] 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. /3:4331 (1986); and Zoller et al., Nucl. Acids
Res. /0:6487
(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)).
[01211 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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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
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.
[0122] 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 polyp eptides 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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NO:1, and portions of such polypeptides with at least 30 amino acids and more
preferably
at least 50 amino acids.
[0123] 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 polyp eptide 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.
[0124] 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.
[0125] 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
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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 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.
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=
[0126] 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 n'-m1, 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.
[0127] 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 n1-ml, and/or n2-m2 herein.
TR7
[0128] 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, W000/66156, and W098/35986.
[0129] In certain embodiments, the antibodies of the present invention
immunospecifically bind TR7 polyp eptide. 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.
[0130] 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, timers or tetramers.
In
additional embodiments, the multimers of the invention are at least dimers, at
least
trimers, or at least tetramers.
[0131] 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).
These homomers may contain TR7 proteins having identical or different
polypeptide
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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.
[0132] 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.
[0133] 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 polyp eptide sequence of the protein
(e.g., the
polypeptide sequence recited in SEQ ID NO: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)
polypeptide. In another instance, the covalent associations are the
consequence of
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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 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).
[0134] 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).
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). 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).
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).
[0135] 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
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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).
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).
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).
In another embodiment, two or more TR7
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. 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.
[0136] 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.
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
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CA 02494372 2010-11-17
in the art. In specific embodiments, antibodies of the invention bind TR7-
leucine zipper
fusion protein monomers and/or TR7-leucine zipper fusion protein multimers.
[0137] 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 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 No. 5,716,805. In specific embodiments,
antibodies of the invention bind TR7-leucine zipper fusion protein trimers.
[0138] 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.
[0139] Antibodies that bind TR7 receptor polypeptides may bind them as
isolated
polypeptides or in their naturally occurring state. 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, Gene 67:31-40 (1988). Thus,
antibodies
of the present invention may bind recombinantly produced TR7 receptor
polypeptides. In
a specific embodiment, antibodies of the present invention bind a TR7 receptor
expressed
on the surface of a cell comprising a polynucleotide encoding amino acids 1 to
411 of
SEQ ID NO:3 operably associated with a regulatory sequence that controls gene
expression.
[0140] 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 ID NO: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
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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 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
411 of SEQ ID NO: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.
[0141] 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 NO: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 NO: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 NO: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
polyp eptide 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 NO: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 ID NO: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
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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
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.
[01421 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 NO: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 NO: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 NO: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.
[0143] 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.
[0144] 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
tarn-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
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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 4 and include,
but are not
limited to, regions of the aforementioned types identified by analysis of the
amino acid
sequence of SEQ ID NO:3, 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 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.
[0145] The data representing the structural or functional antibutes of TR7 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
Gamier-
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 rdsults
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.
[0146] In a preferred embodiment, the data presented in columns VIII, IX,
XIII, and
XIV of Table 4 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 xrv 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 4 present the result of different
analysees of
the TR7 protein sequence.
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[0147] 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 NO:3. As set out in Table 4, such preferred regions
include
Garnier-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 TR7 polypeptides or TR7
polypeptide
fragments and variants comprising regions of TR7 that combine several
structural features,
such as several (e.g., 1, 2, 3 , or 4) of the same or different region
features set out above
and in Table 4.
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Table 4
Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV
Met 1 A .. . . . . 1.11 -0.70 . * . 1.29 2.18
Glu 2 A .. . . . . 1.50 -0.70 . * . 1.63 1.69
Gin 3 A .. . . T . 1.89 -0.73 . * . 2.17 2.28
Arg 4. . . . T T . 1.69 -0.76 . * . 2.91 3.71
Gly 5. . . . T T . 1.87 -0.87 . * F 3.40 2.17
Gin 6. . . . T T . 1.88 -0.44 . * F 2.76 1.93
Asn 7. . . . . . C 1.29 -0.34 . * F 1.87 1.00
Ala 8. . . . . . C 0.99 0.16 . . F 1.08 1.02
Pro 9. . . . . . C 0.53 0.11 . * . 0.44 0.79
Ala 10 A .. . . . . 0.29 0.14 . * . -0.10 0.48
Ala 11 A .. . T . 0.40 0.24 . . . 0.10 0.48
Ser 12 A .. . . T . 0.44 -0.26 . * F 0.85 0.61
Gly 13 A .. . . T . 1.14 -0.69 . * F 1.30 1.22
Ala 14 A .. . . T . 1.32 -1.19 . * F 1.30 2.36
Arg 15 A .. . T . . 1.57 -1.19 . * F 1.50 2.39
Lys 16. . . . T . . 1.94 -1.14 . . F 1.50 2.39
Arg 17. . . . T . . 1.90 -1.14 . * F 1.80 3.66
His 18. . . . . . C 2.03 -1.21 * * F 1.90 1.85
Gly 19. . . . . T C 2.73 -0.79 * * F 2.40 1.43
Pro 20. . . . . T C 2.62 -0.79 * * F 2.70 1.43
Gly 21. . . . . T C 1.99 -0.79 * . F 3.00 1.82
Pro 22. . . . . T C 1.99 -0.79 . * F 2.70 1.86
Arg 23 . A. . . . C 1.68 -1.21 * . F 2.30 2.35
Glu 24 . A B. . . . 1.43 -1.21 * . F 2.10 2.35
Ala 25 . A. . T . . 1.76 -1.14 * . F 2.50 1.54
Arg 26 . A. . T . . 1.89 -1.57 * . F 2.50 1.54
Gly 27. . . . . . 1.76 -1.14 * . F 3.00 1.37
Ala 28. . . . . C 1.43 -0.71 * * F 2.70 1.35
Arg 29. . . . . T C 1.54 -0.79 * * F 2.66 1.06
Pro 30. . . . . T C 1.28 -0.79 * * F 2.62 2.10
Gly 31. . . . . T C 0.96 -0.57 * * F 2.58 1.54
Pro 32. . . . . T C 1.34 -0.64 * * F 2.54 1.22
Arg 33. . . . . . C 1.62 -0.64 * * F 2.60 1.58
Val 34. . . . . . C 0.70 -0.59 * * F 2.34 2.30
Pro 35. . B . . . . 0.06 -0.33 * * F 1.58 1.23
Lys 36. . . . . -0.41 -0.11 * . F 0.97 0.46
Thr 37. . B B . . . -1.06 0.57 * * F -0.19 0.52
Leu 38. B B . . . -2.02 0.57 * * . -0.60 0.25
Val 39. . . . . -1.76 0.79 . . . -0.60 0.09
Leu 40 A .. B . . . -2.13 1.29 . . . -0.60 0.06
Val 41 A .. B . . . -3.03 1.30 . . . -0.60 0.08
Val 42 A .. B . . . -3.53 1.26 . . . -0.60 0.08
Ala 43 A .. B . . . -3.53 1.30 . . . -0.60 0.08
Ala 44 A .. B . . . -3.49 1.30 . . . -0.60 0.09
Val 45 A .. B . . . -3.53 1.34 . . . -0.60 0.10
Leu 46 A .. B . . . -2.98 1.34 . . . -0.60 0.07
Leu 47 A .. B . . . -2.71 1.23 . . . -0.60 0.09
Leu 48 A .. B . . . -2.12 1.23 . . . -0.60 0.13
Val 49 A .. B . . . -1.83 0.59 . . . -0.60 0.27
Ser 50 A .. B . . . -1.57 0.29 . * . -0.30 0.44
84
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Table 4 (continued)
Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV
Ala 51 A A. . . . . -1.57 0.10 . . . -0.30 0.54
Glu 52 A A. . . . . -1.64 0.10 . . . -0.30 0.60
Ser 53 A A. B . . . -1.14 0.14 . . . -0.30 0.31
Ala 54 A A. B . . . -0.29 0.24 . . . -0.30 0.45
Leu 55 A A. B . . . 0.01 0.14 . . . -0.30 0.45
Ile 56 A A. B . . . 0.60 0.54 . . . -0.60 0.58
Thr 57 A A. B . . . -0.21 0.16 . . F -0.15 0.96
Gin 58 A A. B . . . -0.50 0.34 . . F -0.15 0.96
Gin 59 A A. B . . . -0.12 0.16 . . F 0.00 1.38
Asp 60 . A. B T . . 0.69 -0.10 . . F 1.00 1.48
Leu 61 . A. . . . C 1.58 -0.19 . * F 0.80 1.48
Ala 62 . A. . . . C 2.00 -0.19 . * F 0.80 1.48
Pro 63 . A. . . . C 1.41 -0.59 . * F 1.10 1.73
Gin 64 . A. . T . . 0.82 -0.09 . * F 1.00 2.13
Gin 65 A A. . . . . 0.61 -0.27 . * F 0.60 2.13
Arg 66 A A. . . . . 1.42 -0.34 . * F 0.60 2.13
Ala 67 A A. . . . . 2.01 -0.37 . * F 0.94 2.13
Ala 68 A A. . . . . 2.27 -0.37 * * F 1.28 2.13
Pro 69 A A. . . . . 2.38 -0.77 * * F 1.92 2.17
Gin 70 . A. . T . . 2.08 -0.77 * . F 2.66 4.21
Gin 71. . . . T T . 1.67 -0.89 * * F 3.40 5.58
Lys 72. . . T T . 2.04 -1.00 . . F 3.06 4.84
Arg 73. . . . T T . 2.33 -1.00 . . F 2.97 4.32
Ser 74. . . . . T C 2.54 -1.01 . . F 2.68 3.34
Ser 75. . . . . T C 2.20 -1.41 . . F 2.59 2.89
Pro 76. . . . T T . 1.39 -0.99 . . F 2.70 1.46
Ser 77. . . . T T . 0.68 -0.30 . . F 2.50 0.90
Glu 78. . . . T T . 0.36 -0.11 . * F 2.25 0.36
Gly 79. . . . T . . 0.44 -0.07 . . F 1.80 0.36
Leu 80. . . . T . . 0.40 -0.07 . . F 1.55 0.42
Cys 81. . . . . . C 0.58 -0.03 . . . 0.95 0.24
Pro 82. . . . . T C 0.84 0.47 * . F 0.15 0.33
Pro 83. . . T T . -0.04, 0.54 * . F 0.35 0.54
Gly 84. . . . T T . 0.00 0.54 * . . 0.20 0.70
His 85. . . . . T C 0.81 0.36 * . . 0.30 0.61
His 86. . . . . . C 1.48 -0.07 * . . 0.70 0.68
Ile 87. . . . . . C 1.34 -0.50 * * . 1.19 1.15
Ser 88. . . . . . C 1.67 -0.50 * * F 1.53 0.84
Glu 89. . . . T . . 2.01 -1.00 * * F 2.52 1.21
Asp 90. . . . T . . 1.38 -1.50 * * F 2.86 2.88
Gly 91. . . . T T . 0.52 -1.61 * * F 3.40 1.15
Arg 92. . . . T T . 1.11 -1.31 * * F 2.91 0.47
Asp 93. . . . T T . 0.74 -0.93 . * F 2.57 0.37
Cys 94. . . . T T . 0.79 -0.36 . * . 1.78 0.20
Ile 95. . . . T . . 0.54 -0.79 . * . 1.54 0.21
Ser 96. . . . T . . 0.54 -0.03 . * . 1.18 0.19
Cys 97. . . . T T . 0.43 0.40 . * . 0.76 0.36
Lys 98. . . T T . 0.43 0.23 . . . 1.34 0.88
Tyr 99. . . . T T . 0.86 -0.46 . * F 2.52 1.10
Gly 100. . . = T T . 1.44 -0.09 . * F 2.80 3.22
85
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Table 4
Res Position I II III IV V VI VII VIII IX X XI XII
XIII XIV
Gin 101. . . T T . 1.43 -0.27 * . F
2.52 2.16
Asp 102. . . . T T . 2.07 0.21 * * F
1.64 1.99
Tyr 103. . . . T T . 1.73 -0.04 * * F
1.96 2.73
Ser 104. . . . T T . 1.98 0.44 * . F 0.78 1.66
Thr 105. . . . T . . 2.32 0.44 * . F 0.30 1.60
His 106. . . . . . 1.51 0.44 * . .
0.15 1.70
Trp 107. . . . . 0.70 0.37 * . .
0.65 1.05
Asn 108. . . . T T . 0.24 0.67 . . .
0.20 0.60
Asp 109. . . . T . -0.12 0.97 * . .
0.20 0.38
Leu 110 A .. . . T . -0.62 1.04 * * .
-0.20 0.19
Leu 111. . . B T . . -0.48 0.81 * * .
-0.20 0.10
Phe 112. . . B T . . -0.86 0.41 * * .
-0.20 0.12
Cys 113. . . . . -1.17 0.99 * * .
-0.20 0.08
Leu 114. . B T . . -1.06 0.79 . * .
-0.20 0.13
Arg 115. . B T . . -0.91 0.10 . * .
0.10 0.30
Cys 116. . . B T . . -0.10 -0.11 . . .
0.70 0.30
Thr 117. . . B T . . 0.30 -0.69 . * .
1.00 0.61
Arg 118. . . B T . . 0.62 -0.99 . . F
1.49 0.42
Cys 119. . . . T T . 1.43 -0.56 * . F
2.23 0.77
Asp 120. . . . T T . 0.47 -1.13 * . F
2.57 0.92
Ser 121. . . T T . 1.13 -0.97 . * F
2.91 0.35
Gly 122. . . . T T . 0.63 -0.97 . * F
3.40 1.13
Glu 123 . A. T . . 0.22 -0.86 . * F
2.51 0.56
Val 124 A A. . . . . 0.68 -0.47 . * F
1.47 0.56
Glu 125 . A. . T . . 0.01 -0.43 . * .
1.38 0.87
Leu 126 . A. . T . . 0.00 -0.29 . * .
1.04 0.27
Ser 127. . . . . T C 0.03 0.20 . * F
0.45 0.52
Pro 128. . . . T T . -0.28 0.04 . * F
0.93 0.44
Cys 129. . . T T . 0.69 0.53 . * F
0.91 0.77
Thr 130. . . . T T . 0.69 -0.16 . * F
2.24 1.12
Thr 131. . . . T . . 1.19 -0.14 . * F
2.32 1.16
Thr 132. . . . T T . 0.63 -0.09 . * F
2.80 3.13
Arg 133. . . . T T . 0.18 -0.01 . . F
2.52 1.61
Asn 134. . . . T T . 0.84 0.07 . . F 1.49 0.60
Thr 135. . . . T T . 0.49 -0.01 . . F
1.81 0.72
Val 136. . . . T . C 0.80 0.07 * . .
0.58 0.20
Cys 137 . A. . T . . 1.11 0.07 * . .
0.10 0.21
Gin 138 . A B. . . . 0.66 -0.33 * . .
0.30 0.25
Cys 139 . A. . T . . 0.34 -0.39 . . .
0.70 0.34
Glu 140 A A. . . . . -0.04 -0.54 * * F
0.75 0.91
Glu 141 A A. . . . . 0.92 -0.33 * * F
0.45 0.46
Gly 142 . A. . T . . 1.59 -0.73 . * F
1.30 1.67
Thr 143 A A. . . . . 1.59 -1.30 . * F
0.90 1.67
Phe 144 A A. . . . . 2.26 -1.30 . * F
0.90 1.67
Arg 145 A A. . . . . 1.96 -1.30 . * F
0.90 2.81
Glu 146 A A. . . . . 1.74 -1.34 . * F
0.90 2.61
Glu 147 A A. . . . . 2.09 -1.40 . * F
0.90 4.66
Asp 148 A A. . . . . 1.80 -2.19 . * F
0.90 4.12
Ser 149 A .. . . . 1.83 -1.57 . * F
1.30 2.35
Pro 150 A .. . . . 1.83 -1.00 . . F
1.15 0.73
86
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Table 4 (continued)
Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV
Glu 151 A .. . . . 1.88 _i.00* . F 1.15 0.85
Met 152 A .. . . T . 1.21 -1.00 * * . 1.49 1.28
Cys 153 A .. . . T . 1.32 -0.81 * * . 1.68 0.44
Arg 154 A .. . . T . 1.31 -1.24 * . . 2.02 0.50
Lys 155. . . T T . 1.18 -0.76 * * F 2.91 0.73
Cys 156. . . . T T . 0.51 -0.94 * . F 3.40 1.35
Arg 157 . .. . T . . 0.90 -0.94 * . F 2.71 0.37
Thr 158 . .. . T . . 1.68 -0.51 * . F 2.37 0.28
Gly 159. . . T . . 1.22 -0.51 * . F 2.43 1.04
Cys 160. . . . . T C 0.58 -0.66 . * F 2.19 0.53
Pro 161 . .. T T . 0.39 -0.04 . * F 2.00 0.36
Arg 162. . . . T T . 0.32 0.11 . * F 1.65 0.27
Gly 163. . . . . -0.22 -0.31 * * . 2.50 1.01
Met 164. . B B . . . -0.22 -0.24 * * . 1.30 0.48
Val 165 . . B B. . . 0.44 -0.24 * * . 1.30 0.24
Lys 166. . B B . . . -0.01 -0.24 * * . 1.30 0.41
Val 167 . . B. . T . -0.43 -0.10 * * F 1.85 0.22
Gly 168. . . . T T . -0.30 -0.23 . . F 2.25 0.44
Asp 169 . .. T T . 0.01 -0.44 . . F 2.50 0.34
Cys 170. . . . T T . 0.57 0.47 . * F 1.35 0.48
Thr 171 . .. . . T C 0.52 0.21 . * F 1.20 0.65
Pro 172. . . . T T . 0.49 -0.21 . * F 1.75 0.65
Trp 173. . . . T T . 0.83 0.47 . * F 0.60 0.84
Ser 174 A .. , . T . 0.17 -0.10 . * F 1.00 1.01
Asp 175 A A. . . . . -0.02 -0.01 . . F 0.45 0.35
Ile 176 A A. . . . . 0.26 0.20 * * . -0.30 0.25
Glu 177 A A. . . . . 0.51 -0.21 * . . 0.30 0.25
Cys 178 A A. . . . . 0.80 -0.60 * . . 0.60 0.30
Val 179 A A. . . . . 0.80 -0.60 * * . 0.60 0.74
His 180 A A. . . . . 0.46 -0.90 . * . 0.60 0.58
Lys 181 A A. . . . . 0.46 -0.47 * . F 0.60 1.06
Glu 182 A .. . . T . -0.43 -0.36 * . F 1.00 1.00
Ser 183 A .. . . T . -0.66 -0.31 . . F 0.85 0.52
Gly 184 A .. . T T . -0.14 -0.13 . . F 1.25 0.18
Ile 185 A .. . . T . -0.97 0.30 . . . 0.10 0.10
Ile 186 . . B B. . . -1.32 0.94 . * . -0.60 0.06
Ile 187 . . B B. . . -2.18 1.04 . . . -0.60 0.08
Gly 188 . . B B. . . -2.47 1.26 . * . -0.60 0.09
Val 189 . . B B. . . -2.71 1.07 . . . -0.60 0.13
Thr 190 A .. B . . . -2.68 0.89 . * . -0.60 0.18
Val 191 A .. B . . . -2.64 0.84 . . . -0.60 0.14
Ala 192 A .. B . . . -2.57 1.06 . * . -0.60 0.14
Ala 193 A .. B . . . -3.11 1.10 . . . -0.60 0.08
Val 194 A .. B . . . -3.11 1.30 . . . -0.60 0.07
Val 195 A .. B . . . -3.39 1.30 . . . -0.60 0.05
Leu 196 A .. B . . . -3.39 1.30 . . . -0.60 0.05
Ile 197 A .. B . . . -3.50 1.44 . . . -0.60 0.05
Val 198 A .. B . . = -3.77 1.59 . = . -0.60 0.06
Ala 199 A .. B . . = -3.58 1.59 . . . -0.60 0,06
Val 200 A .= B = . . -2.68 1.47 . = = -0.60 0.04
87
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Table 4 (continued)
Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV
Phe 201 A .. B . . . -2.17 0.79 . . . -0.60
0.12
Val 202 A .. B . . . -2.09 0.53 . . . -0.60
0.16
Cys 203 A. . . . T . -2.04 0.71 . . . -0.20
0.17
Lys 204 A .. . . T . -1.74 0.76 . . . -0.20
0.17
Ser 205 A .. . . T . -0.84 0.89 . . . -0.20
0.24
Leu 206 A .. . . T . -0.10 0.24 . . . 0.10
0.88
Leu 207 A A. . . . . -0.10 -0.33 . . . 0.30
0.88
Trp 208 A A. . . . . -0.24 0.31 . . . -0.30
0.49
Lys 209 A A. . . . . -0.50 0.61 . . . -0.60
0.49
Lys 210 A A. . . . . -0.44 0.36 * . . -0.30
0.91
Val 211 A A. . . . . -0.44 0.43 * * . -0.45
1.36
Leu 212 . A B. . . . 0.41 0.20 * * . -0.30
0.56
Pro 213 . A B. . . - 0.36 0.20 * . . -0.30
0.56
Tyr 214. . . . . -0.58 0.63 * . . -0.20
0.75
Leu 215. . B T . . -1.29 0.67 * * . -0.20
0.64
Lys 216. . . B T . . -0.73 0.56 * . . -0.20
0.22
Gly 217. . B B . . . -0.27 0.51 * . . -0.60
0.19
Ile 218. . B B . . . -0.40 0.19 * . . -0.30
0.23
Cys 219. . B . . T . -0.50 -0.07 * . . 0.70
0.11
Ser 220. . . . T T . -0.03 0.36 . * F 0.65
0.11
Gly 221. . . . T . -0.08 0.36 . . F 0.65
0.16
Gly 222. . . . T . 0.06 -0.33 . . F 1.25
0.49
Gly 223. . . . . . C 0.94 -0.47 . . F 0.85
0.57
Gly 224. . . . . . C 1.72 -0.86 * . F 1.15
0.99
Asp 225. . . . . T C 1.17 -1.29 . * F 1.50
1.97
Pro 226. . . . . T C 1.51 -1.07 * . F 1.84
1.47
Glu 227. . B . . T . 1.97 _1.50* . F 1.98 2.49
Arg 228. . B . T . 2.01 -1.93 * . F 2.32
2.92
Val 229. . . . T . . 2.06 -1.54 * . F 2.86
2.53
Asp 230. . . . T T . 2.06 -1.59 * . F 3.40
1.96
Arg 231. . . T T . 2.38 -1.19 * * F 3.06
1.73
Ser 232. . . . T T . 2.17 -1.19 * . F 2.72
4.57
Ser 233. . . . T T . 1.71 -1.40 * * F 2.72
4.23
Gin 234. . . . . . C 1.98 -0.97 * * F 2.32
2.14
Arg 235. . . . . T C 1.98 -0.47 * * F 2.22
1.61
Pro 236. . . . . T C 1.87 -0.86 * * F 2.86
2.08
Gly 237. . . . T T . 2.17 -1.24 . * F 3.40
2.01
Ala 238. . . . . T C 1.61 -1.24 . * F 2.86
1.65
Glu 239 A .. . . . . 0.80 -0.60 . * F 1.97
0.79
Asp 240 A .. . . . . 0.69 -0.34 . * F 1.33
0.66
Asn 241 A .. . . . . 0.90 -0.37 * . . 0.99
1.05
Val 242 A .. . . . . 0.36 -0.87 * . - 0.95
1.05
Leu 243 A .. . . . . 0.09 -0.19 * . . 0.50
0.44
Asn 244 A .. B . . . -0.21 0.46 * . . -0.60
0.20
Glu 245 A .. B . . . -1.10 0.44 * . . -0.60
0.37
Ile 246 A .. B . . . -1.91 0.49 * . . -0.60
0.31
Val 247 A .. B . . . -1.06 0.49 * . . -0.60
0.16
Ser 248. . B B . . . -0.46 0.49 * . . -0.60
0.16
Ile 249. B B . . . -0.77 0.91 * . - -0.60
0.35
Leu 250. . . B . . C -0.77 0.71 . . - -0.40
0.69
88
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Table 4 (continued)
Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV
Gin 251. . . . . T C -0.73 0.47 . . F 0.15 0.89
Pro 252. . . . . T C -0.09 0.73 . . F 0.15 0.94
Thr 253. . . . . T C 0.21 0.47 . . F 0.30 1.76
Gin 254. . . . . T C 1.10 -0.21 . . F 1.20 1.76
Val 255 . A. . . . C 1.91 -0.21 . . F 0.80 1.97
Pro 256 . A. . . . C 1.31 -0.64 . . F 1.10 2.37
Glu 257 A A. . . . . 1.52 -0.51 . * F 0.90 1.35
Gin 258 A A. . . . . 0.98 -0.91 . * F 0.90 3.16
Glu 259 A A. . . . . 0.98 -0.91 . * F 0.90 1.51
Met 260 A A. . . . . 1.83 -0.94 . * F 0.90 1.51
Glu 261 A A. . . . . 1.83 -0.94 . * . 0.75 1.51
Val 262 A A. . . . . 1.24 -0.91 . * F 0.90 1.35
Gin 263 A A. . . . . 1.24 -0.41 . * F 0.60 1.38
Glu 264 A A. . . . . 1.03 -1.03 . * F 0.90 1.38
Pro 265 A A. . . . . 1.32 -0.60 . * F 1.18 2.88
Ala 266 A A. . . . . 0.98 -0.76 . * F 1.46 2.40
Glu 267 A .. . . T . 0.98 -0.73 . * F 2.14 1.37
Pro 268 A .. . . T . 0.98 -0.09 . . F 1.97 0.66
Thr 269. . . . T T . 0.38 -0.11 . . F 2.80 1.05
Gly 270 A .. . . T . -0.22 0.00 . . F 1.37 0.60
Val 271 A .. . . . . 0.07 0.69 . . . 0.44 0.32
Asn 272. . . . . . -0.14 0.64 . . . 0.16 0.30
Met 273. B . . . . -0.28 0.59 . . . 0.18 0.46
Leu 274. . . . . . C 0.03 0.59 . . . 0.40 0.62
Ser 275. . . . . T C 0.08 -0.06 . . F 1.95 0.66
Pro 276. . . . . T C 0.93 -0.07 . . F 2.25 0.90
Gly 277. . . . . T C 0.90 -0.69 . . F 3.00 1.89
Glu 278 A .. . . T . 0.69 -0.87 . . F 2.50 1.92
Ser 279 A A. . . . . 0.69 -0.57 . . F 1.80 1.02
Glu 280 A A. . . . . 0.99 -0.31 . . F 1.05 0.85
His 281 A A. . . . . 0.99 -0.74 . . F 1.05 0.85
Leu 282 A A. . . . . 0.74 -0.31 . . . 0.30 0.98
Leu 283 A A. . . . . 0.74 -0.20 . . . 0.30 0.57
Glu 284 A A. . . . . 0.46 -0.20 . . F 0.45 0.73
Pro 285 A A. . . . . 0.46 -0.20 . . F 0.45 0.89
Ala 286 A A. . . . . 0.60 -0.89 . . F 0.90 1.88
Glu 287 A A. . . . . 1.11 -1.57 . . F 0.90 2.13
Ala 288 A A. . . . . 1.92 -1.19 . . F 0.90 1.84
Glu 289 A A. . . . . 2.03 -1.21 * . F 0.90 3.16
Arg 290 A A. . . - . 2.36 -1.71 * . F 0.90 3.57
Ser 291 A .. . T . 3.06 -1.71 * . F 1.30 6.92
Gin 292 A .= = . . 2.24 -2.21 * . F 1.30 7.83
Arg 293 A .. . . . 2.02 -1.53 . . F 1.30 3.30
Arg 294 A .= . . T . 1.17 -0.84 . . F 1.30 2.03
Arg 295. . . B T . . 0.84 -0.59 . * F 1.15 0.87
Leu 296. . B B . . . 0.56 -0.56 . * . 0.60 0.69
Leu 297. . . . . 0.56 -0.06 . * . 0.30 0.35
Val 298. . B . . C 0.44 0.34 * * . 0.20 0.29
Pro 299. . . . . T C -0.01 0.34 * . . 0.90 0.61
Ala 300. . . . . T C -0.12 0.09 * * F 1.35 0.73
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Table 4 (continued)
Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV
Asn 301. . . . . T C 0.48 -0.60 . . F 2.70 1.65
Glu 302. . . . . T C 0.98 -0.81 . . F 3.00 1.65
Gly 303. . . . . C 1.83 -0.76 . . F 2.50 2.35
Asp 304. . . . . T C 1.73 -1.26 . . F 2.40 2.54
Pro 305. . . T C 1.51 -1.17 . * F 2.10 2.11
Thr 306 A .. . . T 1.62 -0.49 . * F 1.30 1.76
Glu 307 A .. . T . 1.62 -0.91 * * F 1.30 2.07
Thr 308 A .. B . . 1.30 -0.51 * * F 0.90 2.31
Leu 309 A .. D . . . 0.60 -0.37 * * F 0.45 0.86
Arg 310 A .. B . . . 0.81 -0.07 * * . 0.30 0.43
Gin 311 A .. B . . 1.12 -0.07 * * . 0.30 0.50
Cys 312 A .. . T . 0.42 -0.56 * * . 1.15 1.01
Phe 313 A .. . . 0.14 -0.46 * * . 0.70 0.45
Asp 314. . . 0.96 0.04 * * . 0.50 0.26
Asp 315 A .. T . 0.03 -0.36 * * . 0.70 0.81
Phe 316 A A. . . . -0.82 -0.24 * . . 0.30 0.77
Ala 317 A A. . . . . -0.37 -0.39 * . . 0.30 0.34
Asp 318 A A. . . . . -0.37 0.04 * * . -0.30 0.32
Leu 319 A A. . . . -0.37 0.83 . . . -0.60 0.32
Val 320 . A. . . . C -0.67 0.04 . . . -0.10 0.52
Pro 321 . A. . C -0.26 -0.07 . . . 0.50 0.42
Phe 322. . T T 0.33 0.84 . . . 0.20 0.54
Asp 323 A .. T . 0.12 0.16 . . . 0.25 1.25
Ser 324 A .. . . . 0.12 -0.06 . . F 1.00 1.25
Trp 325 A .. . . T . 0.38 0.20 * * F 0.40 1.19
Glu 326 A A. . . . 0.70 0.03 * . F -0.15 0.71
Pro 327 A A. . . . . 1.44 0.03 * . . -0.15 1.03
Leu 328 A A. . . . . 0.63 -0.36 * . . 0.45 1.96
Met 329 A A. . . . . 0.59 -0.59 * . . 0.60 0.93
Arg 330 A A. . . . . 0.07 -0.16 * . . 0.30 0.60
Lys 331 A A. . . . . -0.53 0.10 * . . -0.30 0.60
Leu 332 A A. . . . . -0.32 0.03 * . . -0.30 0.60
Gly 333 A A. . . . . 0.49 -0.59 * . . 0.60 0.51
Leu 334 A A. . . . . 1.09 -0.19 * . . 0.30 0.41
Met 335 A A. . . . . 0.09 -0.19 * * . 0.30 0.86
Asp 336 A A. . . . . 0.09 -0.19 . * F 0.45 0.61
Asn 337 A A. . . . . 0.04 -0.61 * * F 0.90 1.48
Glu 338 A A. . . . . -0.20 -0.66 * * F 0.90 1.11
Ile 339 A A. . . . . 0.66 -0.77 * * F 0.75 0.67
Lys 340 A A. . . . . 0.67 -0.77 . * F 0.75 0.83
Val 341 A A. . . . . 0.67 -0.67 . * . 0.60 0.49
Ala 342 A A. . . . . 0.08 -0.67 . . . 0.75 1.20
Lys 343 A A. . . . . -0.51 -0.86 . * . 0.60 0.61
Ala 344 A A. . ' . . 0.03 -0.36 . * . 0.30 0.83
Glu 345 A A. . . . . -0.04 -0.57 * . . 0.60 0.81
Ala 346 A A. . . . . 0.92 -0.57 * . . 0.60 0.55
Ala 347 A A. . . . . 1.51 -0.57 . * . 0.75 1.07
Gly 348 A .. . . . 1.16 -1.07 . * . 0.95 1.03
His 349 A .. . . . 0.93 -0.59 . . . 1.15 1.47
Arg 350 A .. . . . 0.69 -0.40 . . F 1.00 1.20
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Table 4 (continued)
Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV
Asp 351 A .. . . T . 0.97 -0.14 . . F 1.00 1.90
Thr 352 A. . T . 0.96 -0.09 . . F 1.00 2.02
Leu 353 A .. B . . . 0.49 0.03 . . . -0.15 1.02
Tyr 354 A .. B . . . -0.37 0.71 . * . -0.60 0.50
Thr 355 A .. B . . . -0.43 1.40 . * . -0.60 0.24
Met 356 A .. B . . . -0.72 0.91 * . . -0.60 0.59
Leu 357 A .. B . . . -1.27 1.14 * . . -0.60 0.40
Ile 358 A .. B . . . -0.46 1.03 * * . -0.60 0.20
Lys 359 A .. B . . . -0.17 0.94 * * . -0.60 0.33
Trp 360 A .. B . . . -0.17 0.33 * * . 0.00 0.81
Val 361 A .. B . . . 0.09 0.13 * * . 0.45 1.66
Asn 362. . . . . T C 1.01 -0.13 * . F 1.95 0.82
Lys 363. . . . . T C 1.90 -0.13 * * F 2.40 1.53
Thr 364. . . . . T C 1.27 -1.04 * . F 3.00 3.44
Gly 365. . . . . T C 1.26 -1.19 * . F 2.70 2.16
Arg 366 . A. . T . . 1.26 -1.20 * . F 2.20 1.45
Asp 367 . A. . . . C 1.22 -0.56 * . F 1.55 0.75
Ala 368 A A. . . . . 0.87 -0.54 . . F 1.20 1.03
Ser 369 A A. . . . . 0.37 -0.49 . . . 0.30 0.76
Val 370 A A. . . . . -0.10 0.20 . * . -0.30 0.37
His 371 A A. . . . . -0.21 0.89 . * . -0.60 0.30
Thr 372 A A. . . . . -0.80 0.39 * * . -0.30 0.38
Leu 373 A A. . . . . -1.02 0.50 * * . -0.60 0.52
Leu 374 A A. . . . . -0.72 0.54 * . . -0.60 0.31
Asp 375 A A. . . . . -0.18 0.04 * . . -0.30 0.38
Ala 376 A A. . . . . -0.96 0.04 * . . -0.30 0.66
Leu 377 A A. . . . . -0.99 0.04 * . . -0.30 0.66
Glu 378 A A. . . . . -0.18 -0.21 * . . 0.30 0.39
Thr 379 A A. . . . . 0.74 -0.21 * * F 0.45 0.67
Leu 380 A A. . . . . -0.07 -0.71 * . F 0.90 1.59
Gly 381 A A. . . . . -0.07 -0.71 * . F 0.75 0.76
Glu 382 A A. . . . . 0.79 -0.21 * . F 0.45 0.53
Arg 383 A A. . . . . 0.79 -0.70 * . F 0.90 1.28
Leu 384 A A. . . . . 1.14 -0.99 * * F 0.90 2.24
Ala 385 A A. . . . . 1.07 -1.41 * * F 0.90 2.59
Lys 386 A A. . . . . 1.41 -0.73 * . F 0.75 0.93
Gin 387 A A. . . . . 1.41 -0.73 * * F 0.90 1.95
Lys 388 A A. . . . . 1.27 -1.41 * * F 0.90 3.22
Ile 389 A A. . . . . 1.27 -1.41 . * F 0.90 2.19
Glu 390 A A. . . . . 1.04 -0.73 * * F 0.90 1.04
Asp 391 A A. . . . . 0.70 -0.44 . * F 0.45 0.43
His 392 A A. . . . . 0.40 -0.06 * * . 0.30 0.82
Leu 393 A A. = . . . 0.01 -0.36 * * . 0.30 0.64
Leu 394 A A. = . . . 0.94 0.07 * * F -0.15 0.38
Ser 395 A .= . . . 0.24 0.07 * * F 0.25 0.55
Ser 396 A .. . . . -0.36 0.36 * * F 0.25 0.58
Gly 397. . . . T . -0.57 0.29 . . F 0.65 0.70
Lys 398 A .. . . T . -0.57 0.36 . . F 0.25 0.82
Phe 399 A A. . . . . 0.24 0.66 . . . -0.60 0.50
Met 400 . A B. . . . 0.20 0.27 . * . -0.30 0.88
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Table 4 (continued)
Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV
Tyr 401 . A B. . . . 0.50 0.27 . * . -0.30
0.44
Leu 402 A A. . . . 0.26 0.67 . * . -0.60
0.81
Glu 403 A A. . . . . 0.21 0.39 . * . -0.30
0.82
Gly 404 A .. . . . 0.61 -0.23 . * F 0.65
0.88
Asn 405 A .. . . . 0.62 -0.60 . * F 1.30
1.43
Ala 406 A .. . . . 0.27 -0.79 . * F 1.15
0.83
Asp 407 A .. . . T . 0.78 -0.17 . * F 0.85
0.83
Ser 408 A .. . . T . 0.39 -0.21 . * F 0.85
0.69
Ala 409 A .. . . . 0.34 -0.19 . * . 0.50
0.88
Met 410 A .. . . . . -0.04 -0.26 . . . 0.50
0.67
Ser 411 A .. . . . . 0.16 0.17 . . . -0.10
0.64
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[0148] 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).
[0149] 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," 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.
[0150] 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 NO:3.
[0151] 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 NO:3,
about 119
to about 164 of SEQ ID NO:3, about 224 to about 271 of SEQ ID NO:3, about 275
to
about 370 of SEQ ID NO:3, about 69 to about 80 of SEQ ID NO:3, about 88 to
about 95
of SEQ ID NO:3, about 99 to about 103 of SEQ ID NO:3, about 119 to about 123
of SEQ
ID NO:3, about 130 to about 135 of SEQ ID NO:3, about 152 to about 163 of SEQ
ID
NO:3, about 226 to about 238 of SEQ ID NO:3, about 275 to about 279 of SEQ ID
NO:3,
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about 301 to about 305 of SEQ ID NO:3, and/or about 362 to about 367 of SEQ lD
NO: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.
[0152] 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).
[0153] 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
NO: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 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 TR7 protein or protein
fragment
alone (Fountoulakis et al., J. Biochem. 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 TR4 polypeptide
such as TR7.
[0154] 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.
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[0155] For instance, for many proteins, including the extracellular domain of
a
membrane associated protein or the mature form(s) 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.
[0156] 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.
[0157] 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 NO:3 where n5 is an integer from 2 to 406 corresponding to
the position
of the amino acid residue in SEQ ID NO:3.
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[0158] More in particular, the invention provides antibodies that bind
polypeptides
comprising, or alternatively consisting of, the amino acid sequence of
residues: E-2 to S-
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-411; 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-411; R-17 to S-411; H-18 to S-411; G-19 to S-
411; P-20 to
S-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-411; A-28 to S-411; R-29 to S-411; P-30 to S-411; 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-411; A-
43 to S-
411; A-44 to S-411; V-45 to S-411; L-46 to S-411; L-47 to S-411; L-48 to S-
411; V-49 to
S-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-411; 1-56 to S-411; 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-411; 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-411; 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-411; L-80 to S-411; C-81 to S-411; P-82 to S-411; P-83 to S-
411; G-84
to S-411; II-85 to S-411; H-86 to S-411; 1-87 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; 1-95
to S-411;
S-96 to S-411; C-97 to S-411; K-98 to S-411; Y-99 to S-411; G-100 to S-411; Q-
101 to S-
411; 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-411; L-110 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; R-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-411; R-
133 to S-
411; N-134 to S-411; T-135 to S-411; V-136 to S-411; C-137 to S-411; Q-138 to
S-411;
C-139 to S-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; R-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-159 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 S-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; 1-176
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to S-411; E-177 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 S-411; S-183 to S-411; G-184 to S-411; 1-185 to S-411; 1-186 to
S-411; I-
187 to S-411; G-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; 1-197
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-411; V-211 to S-411; L-212 to S-411; 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; 1-218 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 S-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 S-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; 1-249 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 S-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 S-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; R-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 S-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 S-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; 1-339 to S-411; K-340 to S-411; V-341 to S-411; A-
342 to S-
411; K-343 to S-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
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to S-411; Y-354 to S-411; T-355 to S-411; M-356 to S-411; L-357 to S-411; 1-
358 to S-
411; K-359 to S-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 S-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; G-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; 1-389 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 S-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 lD NO:3.
[0159] 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 NO: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 G-
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 G-
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 G-
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 G-
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; 1-56 to G-184; T-57 to G-184; Q-58 to G-184; Q-
59 to G-
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 0-184; R-66 to G-184; A-67 to 0-184; A-68 to G-184; P-69 to 0-184; Q-70 to
G-184;
Q-71 to G-184; K-72 to G-184; R-73 to 0-184; S-74 to G-184; S-75 to G-184; P-
76 to G-
184; S-77 to G-184; E-78 to G-184; G-79 to 0-184; L-80 to G-184; C-81 to G-
184; P-82
to 0-184; P-83 to 0-184; G-84 to 0-184; H-85 to G-184; H-86 to G-184; 1-87 to
G-184;
S-88 to G-184; E-89 to G-184; D-90 to G-184; 0-91 to G-184; R-92 to 0-184; D-
93 to G-
184; C-94 to G-184; 1-95 to G-184; S-96 to 0-184; C-97 to G-184; K-98 to 0-
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 G-
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 G-
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 G-
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; R-
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 G-
184; W-173 to G-184; S-174 to G-184; D-175 to G-184; 1-176 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 NO:3.
[0160] 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 and/or bind to antibodies which
recognize the
complete or mature forms of the polypeptide generally will be retained when
less than the
majority of the residues of the complete or mature polypeptide are removed
from the
C-terminus. Whether a particular polypeptide lacking C-terminal residues of a
complete
polypeptide retains such immunologic activities can readily be determined by
routine
methods described herein and otherwise known in the art. It is not unlikely
that a 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.
[0161] 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 NO: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-m5 of SEQ
ID
NO:3, where m5 is an integer from 57 to 410 corresponding to the position of
the amino
acid residue in SEQ ID NO:3.
[0162] 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 AL409; 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 1-389; 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 G-
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 1-339; 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 5-
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 11-281; E-52 to E-280; E-52 to S-279; E-52 to
E-278; E-
52 to G-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
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to Q-254; E-52 to T-253; E-52 to P-252; E-52 to Q-251; E-52 to L-250; E-52 to
1-249; E-
52 to S-248; E-52 to V-247; E-52 to 1-246; 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 1-218; 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
1-197; 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 1-187; E-52 to 1-
186; E-52 to
1-185; 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 1-176; 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-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 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 1-95; 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
1-87; 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
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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
sequence shown in SEQ ID NO:3.
[0163] In another embodiment, antibodies of the invention bind C-terminal
deletions
of the TR7 polypeptide that can be described by the general formula 52-m6
where m6 is a
number from 57 to 183 corresponding to the amino acid sequence identified in
SEQ ID
NO: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 11-180; E-52 to V-179; E-52 to
C-178; E-
52 to E-177; E-52 to 1-176; 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 P428; 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 11-106; 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 1-95; 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 1-87; E-52 to 11-86; E-52 to 11-85;
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 ID NO:3.
[0164] 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
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
polypeptide, which may be described generally as having residues n5- m5 and/or
n6- m6 of
SEQ ID NO:3, where n5, n6, m5, and m6 are integers as described above.
[0165] 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.
[0166] 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 NO:3, since any portion therein is expected to be
soluble.
[0167] 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.
[0168] 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., Science
247:1306-1310 (1990).
[0169] Thus, antibodies of the present invention may bind a fragment,
derivative, or
analog of the polypeptide of SEQ ID NO: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
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
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.
[0170] 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. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987);
Cleland et
al. Grit. Rev. Therapeutic Drug Carrier Systems /0:307-377 (1993)).
[0171] 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.
[0172] 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).
[0173] In specific embodiments, the number of substitutions, additions or
deletions in
the amino acid sequence of SEQ ID NO: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.
[0174] 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: M1 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
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WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
with A, I, L, 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; All 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; L48
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; 156 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; 187 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; 195 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; G100 replaced
with A, I,
L, S, T, M, or V; Q101 replaced with N; D102 replaced with E; Y103 replaced
with F, or
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WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
W; S104 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; R115
replaced with H, or K; T117 replaced with A, G, I, L, S, M, or V; R118
replaced with H,
or K; D120 replaced with E; S121 replaced with A, G, I, L, T, M, or V; G122
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; L126 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; R133 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; G142 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; R145 replaced with H, or K; E146 replaced with D;
E147
replaced with D; D148 replaced with E; S149 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; R154 replaced with
H, or K;
K155 replaced with H, or R; R157 replaced with H, or K; T158 replaced with A,
G, I, L,
S, M, or V; G159 replaced with A, I, L, S, T, M, or V; R162 replaced with H,
or K; G163
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; V167
replaced with A,
G, I, L, S, T, or M; G168 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; W173 replaced with F, or Y; S174
replaced
with A, G, I, L, T, M, or V; D175 replaced with E; 1176 replaced with A, G, L,
S, T, M, or
V; E177 replaced with D; V179 replaced with A, G, I, L, S, T, or M; 11180
replaced with
K, or R; K181 replaced with H, or R; E182 replaced with D; S183 replaced with
A, G, I,
L, T, M, or V; G184 replaced with A, I, L, S, T, M, or V; 1185 replaced with
A, G, L, S, T,
M, or V; 1186 replaced with A, G, L, S, T, M, or V; 1187 replaced with A, G,
L, S, T, M,
or V; G188 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;
V194 replaced with A, G, I, L, S, T, or M; V195 replaced with A, G, I, L, 5,
T, or M;
L196 replaced with A, G, I, S, T, M, or V; 1197 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 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
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; S205 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; G217 replaced with A, I, L, S, T, M, or V; 1218 replaced with A,
G, L, S, T,
M, or V; S220 replaced with A, G, I, L, T, M, or V; G221 replaced with A, I,
L, S, T, M,
or V; G222 replaced with A, I, L, S, T, M, or V; G223 replaced with A, I, L,
S, T, M, or
V; G224 replaced with A, I, L, S, T, M, or V; D225 replaced with E; E227
replaced with
D; R228 replaced with H, or K; V229 replaced with A, G, I, L, S, T, or M; D230
replaced
with E; R231 replaced with H, or K; S232 replaced with A, G, I, L, T, M, or V;
S233
replaced with A, G, I, L, T, M, or V; Q234 replaced with N; R235 replaced with
H, or K;
G237 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; 1246 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; 1249 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, I, 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;
G270
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; G277 replaced with A,
I, L, S, T,
M, or V; E278 replaced with D; S279 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; R290 replaced with H, or K; S291 replaced with A, G, I, L, T,
M, or V;
Q292 replaced with N; R293 replaced with H, or K; R294 replaced with H, or K;
R295
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WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
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; G303 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; R310 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; S324 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; R330 replaced with H, or K; K331 replaced with H, or R;
L332
replaced with A, G, I, S, T, M, or V; G333 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; 1339 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; G348 replaced with A, I, L, S,
T, M, or V;
H349 replaced with K, or R; R350 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; 1358 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; G365 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; G381 replaced with A, I, L, S, T, M, or V; E382 replaced
with D;
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
R383 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; 1(386 replaced with H, or R; Q387 replaced with
N; 1(388
replaced with H, or R; 1389 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; S395 replaced with A, G, I,
L, T, M, or
V; S396 replaced with A, G, I, L, T, M, or V; G397 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; G404 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; S408 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 ID NO:3.
[0175] 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; All replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S12
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;
G19 replaced
with D, E, H, K, R, N, Q, 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;
P22 replaced with D, E, H, K, R, A, G, 1, 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,
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WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
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; L47 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; 156 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
110
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
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; 187
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; 195
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; G100
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;
S104
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; L111 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; C113 replaced with
D, E, H, K,
111
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
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; R115 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
C116 replaced with 1-30 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; R118 replaced with D, E,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; C119 replaced with D, E, H, K, R, A, G, I,
L, S, T, M,
V, N, Q, F, W, Y, or P; D120 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W,
Y, P, or C; S121 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G122
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; S127 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; R133
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; C139 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;
G142 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; R145 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,
L, S, T, M, V, N, Q, F, W, Y, P, or C; S149 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; R154 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;
112
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
C156 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
R157
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; G159 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; C160 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; R162
replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G163 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; G168 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; C170 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;
W173 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S174
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; 1176 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; V179 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;
S183 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G184 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; 1185 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 1186
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; 1187 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; G188 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;
V194 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V195 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; 1197
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V198 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,
113
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
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, 0, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S205
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; 0217 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; 1218 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; S220 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; G221 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or
C; 0222 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G223 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; G224 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; R228 replaced 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;
R231
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S232
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;
R235
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; G237 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,
114
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
L, S, T, M, V, N, Q, F, W, Y, P, or C; 1246 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; S248 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; 1249 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; G270 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; S275 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; G277
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; S279 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
115
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R290 replaced with D,
E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; S291 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; R293
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R294
replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R295 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; G303 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; R310
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;
S324
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; R330 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,
116
WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
P, or C; L332 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G333
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; 1339 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; G348 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; R350 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;
1358
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; G365 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
R366
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; S369 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
117
CA 02494372 2005-02-04
WO 2004/016753
PCT/US2003/025457
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; G381 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; R383 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; 1389
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; S395 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; G397 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; G404
replaced withr
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; S408
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 S411
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C of SEQ ID NO:3.
[0176] 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, Science 244:1081-1085
(1989)). The latter procedure introduces single alanine mutations at every
residue in the
molecule. The resulting mutant molecules are then tested for biological
activity such as
receptor binding or in vitro, or in vitro proliferative activity. Sites that
are critical for
ligand-receptor binding can also be determined by structural analysis such as
118
WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
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 TR7 that are
essential
for TR7 function. In other preferred embodiments, antibodies of the present
invention bind
regions of TR7 that are essential for 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.
[0177] 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.
[0178] 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. /3:4331
(1986); and Zoller et al., NucL Acids Res. /0:6487 (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 3/7:415 (1986)).
[0179] 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
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
second position of any one or more such recognition sequences will prevent
glycosylation
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.
[0180] 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 polyp eptide 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
NO: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 polyp eptide 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.
[0181] 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
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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.
[0182] 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.
1A-B (SEQ ID NO: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.
[0183] 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
matched/aligned with a corresponding subject residue, as a percent of the
total bases of the
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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 sequnce are
manually
corrected for. No other manual corrections are made for the purposes of this
embodiment.
[0184] 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.
[0185] 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 n5-m5, 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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[0186] In certain preferred embodiments, antibodies of the invention bind TR7
proteins of the invention comprise fusion proteins as described above wherein
the TR7
polyp eptides are those described as n5-m5, and n6-1116, herein.
Antibodies of the invention may bind Modified TRAIL Receptor Polyp eptides
[0187] It is specifically contemplated that antibodies of the present
invention may bind
modified forms of TR4 proteins SEQ ID NO:1). In those embodimjents where an
antibody of the present invention specifically binds both TR4 and TR7 (SEQ ID
NO:3), it
is also specifically contemplated that those antibodies may bind modified
forms of TR4
and/or TR7. Modified forms of TR7 would include, for example, modified forms
of TR7
that correspond to the modified forms of TR4 described below.
[0188] In specific embodiments, antibodies of the present invention bind TR4
polypeptides (such as those decribed above) including, but not limited to
naturally purified
TR4 polypeptides, TR4 polypeptides produced by chemical synthetic procedures,
and TR4
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, TR4 polypeptides may also
include an
initial modified methionine residue, in some cases as a result of host-
mediated processes.
[0189] In addition, TR4 proteins that are bound by antibodies of the present
invention
can be chemically synthesized using techniques known in the art (e.g., 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 TR4 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 TR4 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, sarco sine,
citrulline,
homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,
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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).
[0190] The invention additionally, encompasses antibodies that bind TR4
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.
[0191] Additional post-translational modifications to TR4 polypeptides for
example,
e.g., N-linked or 0-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 0-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.
[0192] Also provided by the invention are antibodies that bind chemically
modified
derivatives of TR4 polypeptides 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
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.
[0193] 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
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biological activity, the ease in handling, the degree or lack of antigenicity
and other known
effects of the polyethylene glycol to a therapeutic protein or analog). For
example, the
polyethylene glycol may have an average molecular weight of about 200, 500,
1000, 1500,
2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,
8500,
9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500,
14,000,
14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500,
19,000, 19,500,
20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000,
70,000, 75,000,
80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
[0194] As noted above, the polyethylene glycol may have a branched structure.
Branched polyethylene glycols are described, for example, in U.S. Patent No.
5,643,575;
Morpurgo et al., AppL Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al.,
Nucleosides Nucleotides /8:2745-2750 (1999); and Caliceti et al., Bioconjug.
Chem.
10:638-646 (1999).
[0195] 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, (coupling PEG to G-CSF), see
also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of
GM-CSF
using tresyl chloride). For example, polyethylene glycol may be covalently
bound
through amino acid residues via a reactive group, such as, a free amino or
carboxyl group.
Reactive groups are those to which an activated polyethylene glycol molecule
may be
bound. The amino acid residues having a free amino group may include lysine
residues
and the N-terminal amino acid residues; those having a free carboxyl group may
include
aspartic acid residues, glutarnic 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.
[0196] 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
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(e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and
combinations thereof) of
the protein.
[01971 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 chemical modification at
the N-
terminus 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.
[01981 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.,
Grit. Rev. Thera.
Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol.
68:1-18 (1998);
U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO
98/32466.
[01991 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
(C1S02CH2CF3). 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.
[02001 Polyethylene glycol can also be attached to proteins using a number of
different intervening linkers. For example, U.S. Patent No. 5,612,460
discloses
urethane linkers for
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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.
Pegylated protein products produced using the reaction chemistries
set out herein are included within the scope of the invention.
[0201] The number of polyethylene glycol moieties attached to each TR4
polypeptide
(i.e., the degree of substitution) may also vary. For example, the pegylated
proteins of the
invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,
17, 20, or more
polyethylene glycol molecules. Similarly, the average degree of substitution
within ranges
such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-
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).
[0202] As mentioned the antibodies of the present invention may bind TR4
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 in the
same or
varying degrees at several sites in a given TR4 polypeptide. TR4 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 TR4 polyp eptides 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,
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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 Enzymol /82:626-646 (1990); Rattan et al., Ann NY Acad
Sci 663:48-
62 (1992)).
Anti-TR4 Antibodies
[0203] In one embodiment, the invention provides antibodies (e.g., antibodies
comprising two heavy chains and two light chains linked together by disulfide
bridges)
that immunospecifically bind TR4 (SEQ ID NO:1) 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 scFvs or 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 bind TR4 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 scFvs
or cell lines referred to in Table 1. Immunospecific binding to TR4
polypeptides may be
determined by immunoassays known in the art or described herein for assaying
specific
antibody-antigen binding. Molecules comprising, or alternatively consisting
of, fragments
or variants of these antibodies that immunospecifically bind to TR4 are also
encompassed
by the invention, as are nucleic acid molecules encoding these antibodies
molecules,
fragments and/or variants (e.g., SEQ ID NOs:54-65).
[0204] In one embodiment of the present invention, antibodies that
immunospecifically bind to a TR4 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 scFvs or cell lines referred to in Table 1 and/or any one of the light
chains expressed
by at least one of the scFvs or cell lines referred to in Table 1.
[0205] In another embodiment of the present invention, antibodies that
immunospecifically bind to TR4 or a fragment or variant thereof, comprise a
polypeptide
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having the amino acid sequence of any one of the VII domains of at least one
of the scFvs
referred to in Table 1 and/or any one of the VL domains of at least one of the
scFvs
referred to in Table 1. In preferred embodiments, antibodies of the present
invention
comprise the amino acid sequence of a VII domain and VL domain from a single
scFv
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
scFvs
referred to in Table 1. Molecules comprising, or alternatively consisting of,
antibody
fragments or variants of the VII and/or VL domains of at least one of the
scFvs referred to
in Table 1 that immunospecifically bind to a TR4 are also encompassed by the
invention,
as are nucleic acid molecules encoding these VII and VL domains, molecules,
fragments
and/or variants.
[0206] The present invention also provides antibodies that
immunospecificially bind
to a polypeptide, or polypeptide fragment or variant of TR4, 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 VII domain of one or
more scFvs
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 VII
domain of
one or more scFvs referred to in Table 1. In another embodiment, antibodies
that
immunospecifically bind TR4, comprise, or alternatively consist of, a
polypeptide having
the amino acid sequence of a VII CDR2 contained in a VII domain of one or more
scFvs
referred to in Table 1. In a preferred embodiment, antibodies that
immunospecifically
bind TR4, comprise, or alternatively consist of a polypeptide having the amino
acid
sequence of a VII CDR3 contained in a VII domain of one or more scFvs referred
to in
Table 1. Molecules comprising, or alternatively consisting of, these
antibodies, or
antibody fragments or variants thereof, that immunospecifically bind to TR4 or
a TR4
fragment or variant thereof are also encompassed by the invention, as are
nucleic acid
molecules encoding these antibodies, molecules, fragments and/or variants
(e.g., SEQ ID
NOs:54-65).
[0207] The present invention also provides antibodies that immunospecificially
bind
to a polypeptide, or polypeptide fragment or variant of TR4, 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 VL domain of one or
more scFvs
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referred to in Table 1. In particular, the invention provides antibodies that
immunospecifically bind TR4, comprising, or alternatively consisting of, a
polypeptide
having the amino acid sequence of a VL CDR1 contained in a VL domain of one or
more
scFvs referred to in Table 1. In another embodiment, antibodies that
immunospecifically
bind TR4, comprise, or alternatively consist of, a polypeptide having the
amino acid
sequence of a VL CDR2 contained in a VL domain of one or more scFvs referred
to in
Table 1. In a preferred embodiment, antibodies that immunospecifically bind
TR4,
comprise, or alternatively consist of a polypeptide having the amino acid
sequence of a
VL CDR3 contained in a VL domain of one or more scFvs referred to in Table 1.
Molecules comprising, or alternatively consisting of, these antibodies, or
antibody
fragments or variants thereof, that immunospecifically bind to TR4 or a TR4
fragment or
variant thereof are also encompassed by the invention, as are nucleic acid
molecules
encoding these antibodies, molecules, fragments and/or variants (e.g., SEQ ID
NOs:54-65).
[0208] The present invention also provides antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants)
that
immunospecifically bind to TR4 polypeptide or a fragment or variant of a TR4,
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 a VII domain or VL domain
of one
or more scFvs referred to in Table 1. In particular, the invention provides
for antibodies
that immunospecifically bind to a polypeptide or polypeptide fragment or
variant of TR4,
wherein said antibodies comprise, or alternatively consist of, a VII CDR1 and
a VL
CDR1, a VII CDR1 and a VL CDR2, a VH CDR1 and a VL CDR3, a VII CDR2 and a
VL CDR1, VH CDR2 and VL CDR2, a VH CDR2 and a VL CDR3, a VH CDR3 and a
VII CDR1, a VII CDR3 and a VL CDR2, a VII CDR3 and a VL CDR3, or any
combination thereof, of the VII CDRs and VL CDRs contained in a VII domain or
VL
domain of one or more scFvs referred to in Table 1. In a preferred embodiment,
one or
more of these combinations are from the same scFv as disclosed in Table 1.
Molecules
comprising, or alternatively consisting of, fragments or variants of these
antibodies, that
immunospecifically bind to TR4 are also encompassed by the invention, as are
nucleic
acid molecules encoding these antibodies, molecules, fragments or variants
(e.g., SEQ ID
NOs:54-65).
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Nucleic Acid Molecules Encoding anti-TR4 Antibodies
[0209] 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
specific
embodiments, the nucleic acid molecules encoding an antibody of the invention
comprise,
or alternatively consist of SEQ ID NOs:54-65 or fragments or variants thereof.
[0210] 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
VII domain
having an amino acid sequence of any one of the VH domains of at least one of
the scFvs
referred to in Table 1 and a VL domain having an amino acid sequence of VL
domain of
at least one of the scFvs 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 VII domain having an amino acid sequence of any
one of the
VII domains of at least one of the scFvs referred to in Table 1 or a VL domain
having an
amino acid sequence of a VL domain of at least one of the scFvs referred to in
Table 1.
[0211] 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 TR4 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 VII 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
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
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, asp aragine, 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, tr-
yptophan, 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 TR4).
[0212] 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 on 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
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 may routinely be expressed and the
functional and/or biological activity of the encoded protein, (e.g., ability
to
immunospecifically bind TR4) can be determined using techniques described
herein or by
routinely modifying techniques known in the art.
[0213] 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 TR4 or a fragment or variant 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 VII or
VL domains
of one or more scFvs 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
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
followed by one or more washes in 0.1xSSC/0.2% SDS 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 in Molecular
Biology, Vol. I,
Green 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.
[02141 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
immunospecifically binds to TR4 or fragments or variants of TR4, 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 VH domain of at least one of the
scFvs referred
to in Table 1.
[0215] In another embodiment, an antibody (including a molecule comprising, or
alternatively consisting of, an antibody fragment or variant thereof), that
immunospecifically binds to TR4 or a fragment or variant of TR4, 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 at least one of the
scFvs referred
to in Table 1.
Methods of Producing Antibodies
[02161 Antibodies in accordance with the invention are preferably prepared the
utilization of a phage scFv display library. Technologies utilized for
achieving the same
are disclosed in the patents, applications, and references disclosed herein.
[0217] In phage display methods, functional antibody domains are displayed on
the
surface of phage particles which carry the polynucleotide sequences encoding
them. In
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
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CA 02494372 2010-11-17
. .
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. coil and the E. coli is infected with
helper phage.
Phage used in these methods are typically filamentous phage including fd and
M13 and
the VII 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 (Le., 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
Brinlanan et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J.
Immunol.
Methods 184:177-186 (1995); Kettleborough et al., But J. limnunol. 24:952-958
(1994);
Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology
57:191-
280(1994); PCT publications WO 90/02809;
WO 91/10737; WO 92/01047; WO 92/18719; WO 93/1 1236; WO 95/15982; WO
95/20401; W097/13844; 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.
[0218] 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 one or more
scFvs
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 of the scFvs
referred
to in Table 1 may be expressed in all possible combinations using a phage
display library,
allowing for the selection of VH/VL combinations that bind TR4 polypeptides
with
preferred binding characteristics such as improved affinity or improved off
rates.
Additionally, VII and VL segments - the CDR regions of the VH and VL domains
of the
scFvs referred to in Table 1, in particular, may be mutated in vitro.
Expression of VII and
VL domains with "mutant" CDRs in a phage display library allows for the
selection of
VH/VL combinations that bind TR4 polypeptides with preferred binding
characteristics
such as improved affinity or improved off rates.
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Additional Methods of Producing Antibodies
[0219] 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
including but not limited to myeloma cell lines and 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.
[0220] One way to produce the antibodies of the invention would be to clone
the VH
and/or VL domains of the scFvs referred to in Table 1. In order to isolate the
VH and VL
domains from bacteria transfected with a vector containing the scFv, PCR
primers
complementary to VH or VL nucleotide sequences (See Example 5), may be used to
amplify the VH and VL sequences. 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. The VH and VL domains can then be sequenced using conventional
methods
known in the art. Alternatively, the VH and VL domains may be amplified using
vector
specific primers designed to amplify the entire scFv, (i.e. the VH doamin,
linker and VL
domain.)
[0221] 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
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CA 02494372 2010-11-17
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)).
[0222] 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.
[0223] 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 VII 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 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.
[0224] 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 immunoglobilin 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 Barr virus
transformed B
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CA 02494372 2010-11-17
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.
[0225) 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), to generate
antibodies having a different amino acid sequence, for example to create amino
acid
substitutions, deletions, and/or insertions.
[0226] In a specific embodiment, VII and VL domains of one or more scFvs
referred
to in Table 1, 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 VII and/or VL domains of one or
more scFvs
referred to in Table 1, or fragments or variants thereof, is inserted within
framework
regions using recombinant DNA techniques known in the art. The framework
regions
may be naturally occurring or consensus framework regions, and preferably
human
framework regions (see, e.g., Chothia et Mol. Biol. 278: 457-479 (1998) for a
listing
of human framework regions).
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
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
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.
[0227] 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.
[0228] An important practical application of such a strategy is the
"humanization" of
the mouse humoral immune system. Introduction of human immunoglobulin (Ig)
loci into
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
milestone towards fulfilling the promise of antibody therapy in human disease.
[0229] 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.
[0230] 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
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CA 02494372 2010-11-17
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.
[0231] This general strategy was demonstrated in connection with the
generation of
the first XenoMouseTmstrains as published in 1994. See Green et al. Nature
Genetics
7:13-21 (1994). The XenoMouselm strains were engineered with yeast artificial
chromosomes (YACS) containing 245 kb and10 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, and human Ig constant regions might recapitulate substantially the
full
repertoire that is characteristic of the human humoral response to infection
and
immunization. The work 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 XenoMouseTm mice. See
Mendez et al.
Nature Genetics 15:146-156 (1997), Green and Jakobovits J Exp. Med. 188:483-
495
(1998), Green, Journal of Immunological Methods 231:11-23 (1999) and PCT
Publication WO 98/24893.
[0232] Such approach is further discussed and delineated in U.S. Patent Nos.
5,258,492, 5,939,598, 6,114,598, 6,075,181, 6,162,963 and 6,150,584, and PCT
Publications WO 94/02602, WO 96/33735 and WO 98/24893.
139
CA 02494372 2010-11-17
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 Bl, 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.
[0233] 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
multi-dose utilizations of the antibody. Thus, it would be desirable to
provide fully human
antibodies against TR4 polypeptides in order to vitiate concerns and/or
effects of HAMA
or HACA responses.
[0234] Monoclonal antibodies specific for TR4 polypeptides may be prepared
using
hybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler et al.,
Eur. J.
Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976);
Hammerling et al.,
in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 571-681
(1981)).
Briefly, XenoMouseTm mice may be immunized with TR4 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 TR4 polyp etides.
[0235] For some uses, including in 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. In a specific
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CA 02494372 2010-11-17
embodiment, antibodies of the present invention comprise one or more VII 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 VII 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
CDRs corresponding to one or more of the VII or VL domains of one or more
scFvs
referred to in Table 1, or fragments or variants thereof, and framework
regions (and,
optionally one or more CDRs not present in the antibodies expressed by scFvs
referred to
in Table 1) from a human immunoglobulin molecule. In a preferred embodiment,
an
antibody of the present invention comprises a VII CDR3, VL CDR3, or both,
corresponding to the same scFv, or different scFvs selected from the scFvs
referred to in
Table 1, or fragments or variants thereof, and framework regions from a human
immunoglobulin.
[0236] A chimeric antibody is a molecule in which different portions of the
antibody
are derived from different immunoglobulin molecules such as antibodies having
a human
variable region 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);
Gullies et al.,
J. Immunol. Methods 125:191-202 (1989); U.S. Patent Nos. 5,807,715; 4,816,567;
and
4,816,397. 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 VII CDR3s or VL CDR3s of a VII
or
VL domain of one or more of the scFvs referred to in Table 1, or a variant
thereof, and
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CA 02494372 2010-11-17
non-human framework regions or human framework regions different from those of
the
frameworks in the corresponding scFv 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
residues at
particular positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089;
Rieclunann et al.,
Nature 352:323 (1988)).
[0237] Intrabodies 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., Gene Ther. 4:11-15
(1997);
Rondon and Marasco, Annu. Rev. MicrobioL 51:257-283 (1997); Proba etal., J.
MoL Biol.
275:245-253 (1998); Cohen et al., Oncogene 17:2445-2456 (1998); Ohage and
Steipe, J.
MoL Biol. 291:1119-1128 (1999); Ohage etal., J. MoL Biol. 291:1129-1134
(1999); Wirtz
and Steipe, Protein ScL 8:2245-2250 (1999); Zhu et al., J. ImmunoL Methods
231:207-
222 (1999); and references cited therein.
[0238] 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 vector(s) containing a polyriucleotide
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 vector(s) 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
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CA 02494372 2010-11-17
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
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) 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.
[0239] The expression vector(s) 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, vectors encoding both the heavy and
light chains
may be co-expressed in the host cell for expression of the entire
immunoglobulin
molecule, as detailed below.
[0240] 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, bactetiophage 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., Saccharornyces, Pichia) transformed with recombinant yeast expression
vectors
containing antibody coding sequences; insect cell systems infected with
recombinant virus
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CA 02494372 2010-11-17
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, BIM, 293, 3T3, NSO cells) harboring
recombinant expression constructs containing promoters derived from the genome
of
mammalian cells (e.g., metallothionein promoter) or from mammalian viruses
(e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably,
bacterial cells
such as Escherichia coli, and more preferably, eukaryotic cells, especially
for the
expression of whole recombinant antibody molecule, are used for the expression
of a
recombinant antibody molecule. For example, mammalian cells such as Chinese
hamster
ovary cells (CHO), in conjunction with a vector such as the major intermediate
early gene
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)).
[0241] 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.
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
[0242] 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).
[0243] In mammalian host cells, a number of viral-based expression systems may
be
utilized, hi cases where an adenovirus is used as an expression vector, the
antibody
coding sequence of interest may be ligated to an adenovirus
transcription/translation
control complex, e.g., the late promoter and tripartite leader sequence. This
chimeric gene
may then be inserted in the adenovirus genome by in vitro or in vivo
recombination.
Insertion in a non-essential region of the viral genome (e.g., region El or
E3) will result in
a recombinant virus that is viable and capable of expressing the antibody
molecule in
infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 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)).
[0244] 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, MDCK, 293, 3T3, W138, and in particular, breast
cancer
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and
normal
mammary gland cell line such as, for example, CRL7030 and HsS78Bst.
[0245] 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
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.
[0246] 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,
Aim. 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 &
146
CA 02494372 2010-11-17
Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual,
Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds),
Current
Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et
al., J.
Mol. Biol. 150:1 (1981).
[0247] 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, VoL3. (Academic Press, New York, 1987)). When a marker in the vector
system
expressing antibody is amplifiable, increase in the level of inhibitor present
in culture of
host cell will increase the number of copies of the marker gene. Since the
amplified
region is associated with the coding sequence of the antibody, production of
the antibody
will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
[0248] Vectors which use glutamine synthase (GS) or DBFR as the selectable
markers
can be amplified in the presence of the drugs methionine sulphoximine 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; W086/05807; W089/01036; W089/10404; and W091/06657.
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 aL, Bio/technology
10:169(1992) and
in Biblia and Robinson BiotechnoL Prog. 11:1 (1995).
[0249] 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,
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WO 2004/016753 CA 02494372 2005-02-04PCT/US2003/025457
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 77:2 197 (1980)). The
coding
sequences for the heavy and light chains may comprise cDNA or genomic DNA.
[0250] 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,
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.
[0251] Antibodies of the present invention include naturally purified
products,
products of chemical synthetic procedures, and products produced by
recombinant
techniques from a prokaryotic or eukaryotic host, including, for example,
bacterial, yeast,
higher plant, insect and mammalian cells. Depending upon the host employed in
a
recombinant production procedure, the antibodies of the present invention may
be
glycosylated or may be non-glycosylated. In addition, antibodies of the
invention may also
include an initial modified methionine residue, in some cases as a result of
host-mediated
processes.
[0252] Antibodies of the invention can be chemically synthesized using
techniques
known in the art (e.g., see Creighton, 1983, Proteins: Structures and
Molecular Principles,
W.H. Freeman & Co., N.Y., and Hunkapiller, M., et al., 1984, Nature 310:105-
111). For
example, a peptide corresponding to a fragment of an antibody of the invention
can be
synthesized by use of a peptide synthesizer. Furthermore, if desired,
nonclassical amino
acids or chemical amino acid analogs can be introduced as a substitution or
addition into
the antibody 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, omithine,
norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,
cysteic acid, t-
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PCT/US2003/025457
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).
[0253] The invention encompasses antibodies 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.
[0254] Additional post-translational modifications encompassed by the
invention
include, for example, e.g., N-linked or 0-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 0-linked carbohydrate chains,
and
addition or deletion of an N-terminal methionine residue as a result of
procaryotic host cell
expression. The antibodies may also be modified with a detectable label, such
as an
enzymatic, fluorescent, radioisotopic or affinity label to allow for detection
and isolation
of the antibody.
[0255] Examples of suitable enzymes include horseradish peroxidase,
alkaline
phosphatase, beta-galactosidase, glucose oxidase or acetylcholinesterase;
examples of
suitable prosthetic group complexes include streptavidin/biotin and
avidin/biotin;
examples of suitable fluorescent materials include biotin, umbelliferone,
fluorescein,
fluorescein isothiocyanate, rho damine, dichlorotriazinylamine fluorescein,
dansyl chloride
or phycoerythrin; an example of a luminescent material includes luminol;
examples of
bioluminescent materials include luciferase, luciferin, and aequorin; and
examples of
suitable radioactive material include a radioactive metal ion, e.g., alpha-
emitters such as,
for example, 213Ii or other radioisotopes such as, for example, iodine (1311,
125L 1231, 121-rx
carbon (14C), sulfur (35S), tritium (3H), indium (iismin, 113min, 112in, "In),
and technetium
("Tc, 99"'Tc), thallium (2mu), gallium (68Ga, 67Ga), palladium (1 3Pd),
molybdenum
(99Mo), xenon (133Xe), fluorine (18F), 153sm, 177Ln, 159Gd, 149pm, 140La,
175yb, 166}{0, 90y,
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CA 02494372 2010-11-17
47sc, 186Re, 188Re, 142pr, 105¨ ,"Ru, 68Ge, "Co, 65Zn, "Sr, 3213, 153Gd, 169¨
,Yb 51Cr, 54Mn,
75Se, 113Sn, and "7Tin.
[0256] In specific embodiments, antibodies of the invention may be
labeled with
Europium. For example, antibodies of the invention may be labelled with
Europium using
the DELFIATM Eu-labeling kit (catalog# 1244-302, Perkin Elmer Life Sciences,
Boston,
MA) following manufacturer's instructions.
[0257] In specific embodiments, antibodies of the invention are attached
to
macrocyclic chelators useful for conjugating radiometal ions, including but
not limited to,
iiih 177u4 90y, 166llo, 153sm, 215Bi and 225Ac to polypeptides. In a preferred
embodiment, the radiometal ion associated with the macrocyclic chelators
attached to
antibodies of the invention is 111In. In another preferred embodiment, the
radiometal ion
associated with the macrocyclic chelator attached to antibodies polypeptides
of the
invention is 90Y. In specific embodiments, the macrocyclic chelator is
1,4,7,10-
tetraazacyclododecane-N,N',N",Nm-tetraacetic acid (DOTA). In specific
embodiments,
the macrocyclic chelator is a-(5-isothiocyanato-2-methoxypheny1)-1,4,7,10-
tetraaza-
cyclododecane-1,4,7,10-tetraacetic acid. In other specific embodiments, the
DOTA is
attached to the antibody of the invention via a linker molecule. Examples of
linker
molecules useful for conjugatinga macrocyclic chelator such as 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.
In addition, U.S. Patents 5,652,361 and 5,756,065,
disclose chelating agents that may be conjugated to antibodies, and methods
for making
and using them.
[0258] In one embodiment, antibodies of the invention are labeled with
biotin. In
other related embodiments, biotinylated antibodies of the invention may be
used, for
example, as an imaging agent or as a means of identifying one or more TRAIL
receptor
coreceptor or ligand molecules.
[0259) Also provided by the invention are chemically modified derivatives
of
antibodies of the invention which may provide additional advantages such as
increased
solubility, stability and in vivo or in vitro circulating time of the
polypeptide, or decreased
immunogenicity (see U. S. Patent No. 4,179,337). The chemical moieties for
derivitization may be selected from water soluble polymers such as
polyethylene glycol,
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CA 02494372 2010-11-17
ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran,
polyvinyl
alcohol and the like. The antibodies 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.
[02601 The polymer may be of any molecular weight, and may be branched or
unbranched. For polyethylene glycol, the preferred molecular weight is between
about 1
kDa and about 100 kDa (the term "about" indicating that in preparations of
polyethylene
glycol, some molecules will weigh more, some less, than the stated molecular
weight) for
ease in handling and manufacturing. Other sizes may be used, depending on the
desired
therapeutic profile (e.g., the duration of sustained release desired, the
effects, if any on
biological activity, the ease in handling, the degree or lack of antigenicity
and other known
effects of the polyethylene glycol to a therapeutic protein or analog). For
example, the
polyethylene glycol may have an average molecular weight of about 200, 500,
1000, 1500,
2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,
8500,
9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500,
14,000,
14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500,
19,000, 19,500,
20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000,
70,000, 75,000,
80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
[02611 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., App!. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al.,
Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug.
Chem.
10:638-646(1999).
[0262] The polyethylene glycol molecules (or other chemical moieties) should
be
attached to the antibody with consideration of effects on functional or
antigenic domains
of the antibody. There are a number of attachment methods available to those
skilled in
the art, e.g., EP 0 401 384, (coupling PEG to G-CSF), see
also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of
GM-CSF
using tresyl chloride). For example, polyethylene glycol may be covalently
bound
through amino acid residues via a reactive group, such as, a free amino or
carboxyl group.
Reactive groups are those to which an activated polyethylene glycol molecule
may be
bound. The amino acid residues having a free amino group may include, for
example,
lysine residues and the N-terminal amino acid residues; those having a free
carboxyl group
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CA 02494372 2010-11-17
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.
[0263] As suggested above, polyethylene glycol may be attached to proteins,
e.g.,
antibodies, 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.
[0264] One may specifically desire antibodies chemically modified at the N-
terminus
of either the heavy chain or the light chain or both. Using polyethylene
glycol as an
illustration, 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
chemical
modification at the N-terminus 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.
[0265] As indicated above, pegylation of the antibodies of the invention may
be
accomplished by any number of means. For example, polyethylene glycol may be
attached to the antibody either directly or by an intervening linker.
Linkerless systems for
attaching polyethylene glycol to proteins are described in Delgado et al.,
Crit. Rev. Thera.
Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern: J. of Hematol.
68:1-18 (1998);
U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO
98/32466.
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CA 02494372 2010-11-17
[0266] One system for attaching polyethylene glycol directly to amino acid
residues of
antibodies without an intervening linker employs tresylated MPEG, which is
produced by
the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride
(C1S02CH2CF3). Upon reaction of protein with tresylated MPEG, polyethylene
glycol is
directly attached to amine groups of the protein. Thus, the invention includes
antibody-
polyethylene glycol conjugates produced by reacting antibodies of the
invention with a
polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
[0267] Polyethylene glycol can also be attached to antibodies using a number
of
different intervening linkers. For example, U.S. Patent No. 5,612,460,
discloses
urethane linkers for connecting polyethylene glycol to proteins.
Antibody-polyethylene glycol conjugates
wherein the polyethylene glycol is attached to the antibody by a linker can
also be
produced by reaction of antibodies 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.
Pegylated antibody
products produced using the reaction chemistries set out herein are included
within the
scope of the invention.
[0268] The number of polyethylene glycol moieties attached to each antibody of
the
invention (i.e., the degree of substitution) may also vary. For example, the
pegylated
antibodies 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
antibody molecule. Methods for detennining the degree of substitution are
discussed, for
example, in Delgado et al., Cut. Rev. Thera. Drug Carrier Sys. 9:249-
304(1992).
Characterization of anti-TR4 Antibodies
[0269] Antibodies of the present invention (including molecules comprising, or
alternatively consisting of, antibody fragments or variants thereof) may also
be described
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PCT/US2003/025457
or specified in terms of their binding to TR4 polypeptides or fragments or
variants of TR4
polypeptides. In specific embodiments, antibodies of the invention bind TR4
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 le M, le M, 5 X 10-4M, le m, 5 X 10-5
M, or
10-5 M. More preferably, antibodies of the invention bind TR4 polypeptides or
fragments
or variants thereof with a dissociation constant or KD less than or equal to 5
X 1 0-6 M, 10-6
M, 5 X le A4, 10-7 M, 5 X le M, or 10-8 M. Even more preferably, antibodies of
the
invention bind TR4 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 1040 M, 10-i0M, 5
X 10-11 M,
1041 5 10-12 A4, 1012 A4, 5 -13-M, 10-13 M, 5 X 1044 M, 10-14 m¨,5 X 10-
15 M, or 10-15
M. The invention encompasses antibodies that bind TR4 polypeptides with a
dissociation
constant or KD that is within any one of the ranges that are between each of
the individual
recited values.
[0270] In specific embodiments, antibodies of the invention bind TR4
polypeptides or
fragments or variants thereof with an off rate (kat) of less than or equal to
5 X 10-2 sec-1,
10-2 sec-1, 5 X 10-3 sec1 or le sec-1. More preferably, antibodies of the
invention bind
TR4 polypeptides or fragments or variants thereof with an off rate (koff) less
than or equal
to 5 X 10-4 sec-1, 10-4 sec-1, 5 X 10-5 sec-1, or 10-5 sec4 5 X 10-6 sec4, 10-
6 sec-1, 5 X 10-7
sec 4 or le sec-1. The invention encompasses antibodies that bind TR4
polypeptides with
an off rate (koff) that is within any one of the ranges that are between each
of the individual
recited values.
[0271] In other embodiments, antibodies of the invention bind TR4
polypeptides or
fragments or variants thereof with an on rate (lcon) of greater than or equal
to 103 AT1 sec-1,
X 103 M-1 sec-1, 104 M-1 sec4 or 5 X 104 M-1 sec-1. More preferably,
antibodies of the
invention bind TR4 polypeptides or fragments or variants thereof with an on
rate (Icon)
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-1 sec-1. The invention encompasses antibodies that bind TR4
polypeptides with
on rate (kon) that is within any one of the ranges that are between each of
the individual
recited values.
[02721 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 TR4
polypeptides
(SEQ ID NOS:1). In another embodiment, the antibodies of the
invention
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immunospecifically bind to a polypeptide or polypeptide fragment or variant of
simian
TR4 polypeptides. In yet another embodiment, the antibodies of the invention
immunospecifically bind to a polypeptide or polypeptide fragment or variant of
murine
TR4 polypeptides. In one embodiment, the antibodies of the invention bind
immunospecifically to human and simian TR4 polypeptides. In another
embodiment, the
antibodies of the invention bind immunospecifically to human TR4 polypeptides
and
murine TR4 polypeptides. More preferably, antibodies of the invention,
preferentially
bind to human TR4 polypeptides compared to murine TR4 polypeptides.
[0273] In preferred embodiments, the antibodies of the present invention
(including
molecules comprising, or alternatively consisting of, antibody fragments or
variants
thereof), immunospecifically bind to TR4 polypeptides and do not cross-react
with any
other antigens. In preferred embodiments, the antibodies of the invention
immunospecifically bind to TR4 polypeptides (e.g., SEQ ID NOS:1 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., TR1,
TR5, TRIO BCMA, TACI, CD30, CD27, 0X40, 4-1BB, CD40, NGFR, TNFR1, TNFR2,
Fas, and NGFR).
[0274] In another embodiment, the antibodies of the present invention
(including
molecules comprising, or alternatively consisting of, antibody fragments or
variants
thereof), immunospecifically bind to TR4 polypeptides and cross-react with
other
antigens. In other embodiments, the antibodies of the invention
immunospecifically bind
to TR4 polypeptides (e.g., SEQ ID NOS:1 or fragments or variants thereof) and
cross-
react with one or more additional members of the Tumor Necrosis Factor
Receptor Family
polypeptides (e.g., TR1, TR5, TRIO BCMA, TACT, CD30, CD27, 0X40, 4-1BB, CD40,
NGFR, TNFR1, TNFR2, Fas, and NGFR).
[0275] In a preferred embodiment, antibodies of the invention preferentially
bind TR4
(SEQ ID NO:1), or fragments and variants thereof relative to their ability to
bind TR1,
TR5, TR7, or TRIO (SEQ ID NOS:2-5) 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 TR1, TR5 or TRIO (SEQ ID NOS:5, 2 and 4) or fragments or variants
thereof. In
other preferred embodiments, the antibodies of the invention bind TR1, TR4,
TR5, TR7
and TRIO (SEQ ID NOS:5, 1, 2, 3 and 4). An antibody's ability to
preferentially bind one
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antigen compared to another antigen may be determined using any method known
in the
art.
[0276] 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 (i.e., 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
affinity (i.e., KD) that is at least two orders of magnitude less than the
antibody's KD for
the second antigen.
[0277] 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
(koff) that is less
than the antibody's koff 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 koff that is at least one order of magnitude less than the
antibody's koff 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
koff that is at least
two orders of magnitude less than the antibody's koff for the second antigen.
[0278] 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 TR4
polypeptides
(e.g., membrane-embedded TRAIL receptors), the ability to stimulate TR4
mediated
biological activity (e.g., to stimulate apoptosis of TR4 expressing cells, see
Example 4);
the ability to substantially block TR4 ligand (e.g. TRAIL (SEQ ID NO:66), 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 TR4 expression on the surface of
cells. Other
biological activities that antibodies against TR4 polypeptides may have,
include, but are
not limited to, the ability to inhibit TR4 mediated biological activity (e.g.,
to inhibit
apoptosis of TR4 expressing cells) or the ability to downregulate '1R4
expression on the
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surface of cells. Optionally, the antibodies of the invention will bind to the
same or
closely associated (e.g., overlapping) 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.
[0279] The present invention also provides for antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof), that
stimulate TR4 mediated biological activities. In one embodiment, an antibody
that
stimulates TR4 mediated biological activities comprises, or alternatively
consists of a VII
and/or a VL domain of at least one of the scFvs referred to in Table 1, or
fragment or
variant thereof. In a specific embodiment, an antibody that stimulates TR4
mediated
biological activities comprises, or alternatively consists of a VII and a VL
domain of any
one of the scFvs referred to in Table 1, or fragment or variant thereof.
Nucleic acid
molecules encoding these antibodies are also encompassed by the invention.
[0280] The present invention also provides for antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof), that
stimulate apoptosis of TR4 expressing cells (see Example 4). In one
embodiment, an
antibody that stimulates apoptosis of TR4 expressing cells comprises, or
alternatively
consists of a VII and/or a VL domain of at least one of the scFvs referred to
in Table 1, or
fragment or variant thereof. In a specific embodiment, an antibody that
stimulates
apoptosis of TR4 expressing cells comprises, or alternatively consists of a
VII and a VL
domain of any one of the scFvs referred to in Table 1, or fragment or variant
thereof.
Nucleic acid molecules encoding these antibodies are also encompassed by the
invention.
[0281] 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 TR4 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). 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
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an anti Ig Fe 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.
[0282] 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 TR4 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 TR4 expressing cells (See, for example, Example 4). In
a specific
embodiment, antibodies of the invention stimulate apoptosis of TR4 expressing
cells
better than an equal concentration (in terms of, for example,
nanograms/milliliter) of
TRAIL polypeptide (including TRAIL polyp eptide fragments, variants or fusion
proteins)
stimulates apoptosis of TR4 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, nanograms/milliliter) of TRAIL polypeptide (including TRAIL
polypeptide
fragments, variants or fusion proteins) stimulates apoptosis of TR4 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,
nanograms/milliliter) of TRAIL polypeptide (including TRAIL polypeptide
fragments,
variants or fusion proteins) stimulates apoptosis of TR4 expressing cells in
the presence of
2 micrograms/milliliter of cycloheximide.
[0283] 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 TR4
expressing cells when
administered in combination with a chemotherapeutic drug (or other therapeutic
agents
useful in the treatment of cancers), than either the chemotherapeutic drug (or
other
therapeutic agents useful in the treatment of cancers) or the antibodies alone
stimulate
apoptosis of receptor expressing cells. In specific embodiments, antibodies of
the present
invention, stimulate more apoptosis of TR4 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 TR4 expressing cells when administered
in
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combination with cycloheximide, than either cycloheximide or the antibodies
alone
stimulate apoptosis of receptor expressing cells.
[0284] The present invention also provides for antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof), that
block or inhibit the binding of TRAIL to a TR4 polypeptide (see Example 3). In
one
embodiment, an antibody that blocks or inhibits the binding of TRAIL to TR4
comprises,
or alternatively consists of a VH and/or a VL domain of at least one of the
scFvs 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 TR4 comprises, or alternatively
consists of a
VH and a VL domain of any one of the scFvs referred to in Table 1, or fragment
or variant
thereof. Nucleic acid molecules encoding these antibodies are also encompassed
by the
invention.
[0285] The present invention also provides for fusion proteins comprising, or
alternatively consisting of, an antibody (including molecules comprising, or
alternatively
consisting of, antibody fragments or variants thereof), that
immunospecifically binds to
TR4, and a heterologous polypeptide. Preferably, the heterologous polypeptide
to which
the antibody is fused to is useful for function or is useful to target the TR4
expressing
cells. In specific embodiments the invention encompasses bispecific antibodies
that in
which one antibody binding site is specific for TR4 and the second antibody
binding site is
specific for a heterologous polypeptide such as TR7 or a tumor specific
antigen. In an
alternative preferred embodiment, the heterologous polypeptide to which the
antibody is
fused to is useful to target the antibody to a tumor cell. In one embodiment,
a fusion
protein of the invention comprises, or alternatively consists of, a
polypeptide having the
amino acid sequence of any one or more of the VH domains of an antibody of the
invention or the amino acid sequence of any one or more of the VL domains of
an
antibody of the invention or fragments or variants thereof, and a heterologous
polypeptide
sequence. In another embodiment, a fusion protein of the present invention
comprises, or
alternatively consists of, a polypeptide having the amino acid sequence of any
one, two,
three, or more of the VH CDRs of an antibody of the invention, or the amino
acid
sequence of any one, two, three, or more of the VL CDRs of an antibody of the
invention,
or fragments or variants thereof, and a heterologous polypeptide sequence. In
a preferred
embodiment, the fusion protein comprises, or alternatively consists of, a
polypeptide
having the amino acid sequence of, a VH CDR3 of an antibody of the invention,
or
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CA 02494372 2010-11-17
fragment or variant thereof, and a heterologous polypeptide sequence, which
fusion
protein immunospecifically binds to TR4. In another embodiment, a fusion
protein
comprises, or alternatively consists of a polypeptide having the amino acid
sequence of at
least one VH domain of an antibody of the invention and the amino acid
sequence of at
least one VL domain of an antibody of the invention or fragments or variants
thereof, and
a heterologous polypeptide sequence. Preferably, the VH and VL domains of the
fusion
protein correspond to a single antibody (or scFv or Fab fragment) of the
invention. In yet
another embodiment, a fusion protein of the invention comprises, or
alternatively consists
of a polypeptide having the amino acid sequence of any one, two, three or more
of the VII
CDRs of an antibody of the invention and the amino acid sequence of any one,
two, three
or more of the VL CDRs of an antibody of the invention, or fragments or
variants thereof,
and a heterologous polypeptide sequence. Preferably, two, three, four, five,
six, or more of
the VHCDR(s) or VLCDR(s) correspond to single antibody (or scFv or Fab
fragment) of
the invention. Nucleic acid molecules encoding these fusion proteins are also
encompassed by the invention.
[0286] 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
TR4 or a fragment or variant of TR4, 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 TR4 or a fragment or variant of TR4, 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)).
Antibodies that have been
identified to immunospecifically bind to TR4 or a fragment or variant of TR4
can then be
assayed for their specificity and affinity for TR4 or a fragment or variant of
1R4, using or
routinely modifying techniques described herein or otherwise known in the art.
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CA 02494372 2010-11-17
[0287] The antibodies of the invention may be assayed for immunospecific
binding to
TR4 polypeptides and cross-reactivity with other antigens by any method known
in the
art. Immunoassays which can be used to analyze immtmospecific binding and
cross-
reactivity include, but are not limited to, competitive and non-competitive
assay systems
using techniques such as BIAcoremanalysis (See, e.g., Example 2), FACS
(fluorescence
activated cell sorter) analysis, immunofluorescence, inununocytochemistry,
radioimmunoassays, 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.
1, John Wiley
& Sons, Inc., New York).
Exemplary immunoassays are described briefly below (but are not intended by
way of
limitation).
[0288] 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 Fe antibody, and the antigen in the form or a TRAIL receptor-Pc 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 TR4 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
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signal detected as well as other variations of ELISAs known in the art. For
further
discussion regarding ELISAs see, e.g., Ausubel et al., eds, 1994, Current
Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
[0289] 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 1251), 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 TR4
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, a
TR4 polypeptide is incubated with an antibody of the present invention
conjugated to a
labeled compound (e.g., compound labeled with 3H or 1251) in the presence of
increasing
amounts of an unlabeled second anti-TR4 antibody. This kind of competitive
assay
between two antibodies, may also be used to determine if two antibodies bind
the same,
closely associated (e.g., overlapping) or different epitopes.
[0290] In a preferred embodiment, BlAcoreTmkinetic 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. BIAcoremkinetic 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.
[0291] 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 NaC1, 0.01 M sodium phosphate at pH 7.2, 1%
Trasylol) TM
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
sepharos1; 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 immunoprecipitate a particular antigen
can be
assessed by, e.g., western blot analysis. One of skill in the art would be
knowledgeable as
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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 TM
further discussion regarding immunoprecipitation protocols see, e.g., Ausubel
et al., eds,
1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,
New
York at 10.16.1.
[0292] 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-Tweeir 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
[0293] 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 polyp eptides to antibodies of the invention
that are
specific for particular cell surface antigens or which bind antigens that bind
particular cell
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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 16, 1998), 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),
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.
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 Fe
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. Immunol.
146:2446-2452 (1991),
[0294] 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; A.sbkenazi et
al.,
Proc. Natl. Acad. Sci. USA 88: 10535-10539 (1991); Zheng et al., J. Immunol.
154:5590-
5600 (1995); and Vii et al., Proc. Natl. Acad. Sci. USA 89:11357- 11341
(1992).
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CA 02494372 2010-11-17
[0295] Additional fusion proteins of the invention may be generated
through the
techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-
shuffling
(collectively referred to as "DNA shuffling"). DNA shuffling may be employed
to
modulate the activities of 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).
embodiment, polynucleotides encoding antibodies of the invention may be
altered by In one
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 pol3mucleotide encoding an antibody which portions immuno specifically bind
to TR4
may be recombined with one or more components, motifs, sections, parts,
domains,
fragments, etc. of one or more heterologous molecules.
[0296] 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 etal., 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
heraagglutinin "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).
[0297] 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
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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
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, luminol; examples of
bioluminescent
materials include, but are not lmited to, luciferase, luciferin, and aequorin;
and examples
of suitable radioactive material include, but are not limited to, iodine
(1211, 1231, 1251, 1311),
carbon (14C), sulfur (35S), tritium (3H), indium (111in, 1121n, 113mhi, 115mi,
), n technetium
(99Tc,991TITc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (1 3Pd),
molybdenum
(99Mo), xenon (135Xe), fluorine (18F), 153sm, 177u, 159Gd, 149pm, 140La,
175y1, 166H0, 90y,
47se, 186Re, 188Re, 142pr, 105- , Rh and 97Ru.
[0298] 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, 213b.-.=l, or other radioisotopes such as, for example,
103pd, 135xe, 1311, 68Ge,
57Co, 65Zn, 85Sr, 32P, 35S, 90y, 153sm, 153Gd, 169yb, 51cr, 54mn, 75se, 113sn,
90y, i7Tin,
186Re, 188Re and 166 Ho. In specific embodiments, an antibody or
fragment thereof is
attached to macrocyclic chelators that chelate radiometal ions, including but
not limited to,
177Lu, 90Y, 166,-A0.-, and 153Sm, to polypeptides. In specific
embodiments, the macrocyclic
chelator is 1,4,7,10-tetraazacyclododecane-N,N,N",Nm-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,
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CA 02494372 2010-11-17
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.
[0299] Additional chelating agents are known in the art. Chelating agents may
be
attached to antibodies of the invention to facilitate labeling said antibodies
with metal ions
including, but not limited to, radionuclides or fluorescent labels. For
example, see
Subramanian, R. and Meares, C.F., "Bifunctional Chelating Agents for
Radiometal-
labeled monoclonal Antibodies," in Cancer Imaging with Radiolabeled Antibodies
(D. M.
Goldenberg, Ed.) Kluwer Academic Publications, Boston; Saji, H., "Targeted
delivery of
radiolabeled imaging and therapeutic agents: bifunctional
radiopharmaceuticals." Grit.
Rev. Ther. Drug Carrier Syst. /6:209-244 (1999); Srivastava S.C. and Mease
R.C.,
"Progress in research on ligands, nuclides and techniques for labeling
monoclonal
antibodies." Int. J. Rad. Appl. Instrum. B /8:589-603 (1991); and Liu, S. and
Edwards,
D.S., "Bifunctional chelators for therapeutic lanthanide
radiophannaceuticals." Bioconjug.
Chem. /2:7-34 (2001). Any chelator which can be covalently bound to an
antibody may
be used according to the present invention. The chelator may further comprise
a linker
moiety that connects the chelating moiety to the antibody.
[0300] In one embodiment, antibodies of the invention are attached to an
acyclic
chelator such as diethylene triamine-N,N,N,N",N"-pentaacetic acid (DPTA),
analogues of
DPTA, and/or derivatives of DPTA. As non-limiting examples, the chelator may
be 2-(p-
isothiocyanatobenzy1)-6- methyldiethylenetriaminepentaacetic acid (1B4M-DPTA,
also
known as MX-DTPA), 2-methyl-6-(rho-nitrobenzy1)-1,4,7- triazaheptane-
N,N,M,N",N"-
pentaacetic acid (nitro-1B4M-DTPA or nitro-MX-DTPA); 2-(p-
isothiocyanatobenzy1)-
cyclohexyldiethylenetriaxninepentaacetic acid (CHX-DTPA), or N42-amino-3-(rho-
nitrophenyl)propyli-trans-cyclohexane-1,2-diamine-N,N,N"-pentaacetic acid
(nitro-CHX-
A-DTPA).
[0301] In another embodiment, antibodies of the invention are attached to an
acyclic
terpyridine chelator such as 6,6"-bisUN,N,N",N"-
tetra(carboxymethyl)amino]methy1]-4'-
(3-amino-4-methoxypheny1)-2,2%6',2 "- texpyridine (TMT-amine).
[0302] In specific embodiments, the macrocyclic chelator which is attached to
the
antibody of the invention is 1,4,7,10-tetraazacyclododecane-N,N,Y,W-
tetraacetic acid
(DOTA). In other specific embodiments, the DOTA is attached to an antibody of
the
invention via a linker molecule. Examples of linker molecules useful for
conjugating
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CA 02494372 2010-11-17
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.
In addition, U.S. Patents 5,652,361 and
5,756,065, disclose chelating agents that may be conjugated to antibodies,
and
methods for making and using them.
Though U.S. Patents 5,652,361 and 5,756,065 focus on conjugating chelating
agents to antibodies, one skilled in the art could readily adapt the method
disclosed therein
in order to conjugate chelating agents to other polypeptides.
[0303] Bifunctional chelators based on macrocyclic ligands in which
conjugation is
via an activated arm, or functional group, attached to the carbon backbone of
the ligand
can be employed using techniques described in the art, such as those described
by M. Moi
et al., J Amer. Chem. Soc. 49:2639 (1989) (2-p-nitrobenzy1-1,4,7,10-
tetraazacyclododecane-NX,N",Nm-tetraacetic acid); S. V. Deshpande et al., J.
Nud Med.
31:473 (1990); G. Ruser et al., Bioconj. Chem. 1:345 (1990); C. J. Broan et
al., J. C. S.
Chem. Comm. 23:1739 (1990); and C. J. Anderson et al., J. Nucl. Med. 36:850
(1995).
[0304] In one embodiment, a macrocyclic chelator, such as polyazamacrocyclic
chelators, optionally containing one or more carboxy, amino, hydroxamate,
phosphonate,
or phosphate groups, are attached to antibodies of the invention. In another
embodiment,
the chelator is a chelator selected from the group consisting of DOTA,
analogues of
= DOTA, and derivatives of DOTA.
[0305] In one embodiment, a suitable chelator molecule that may be attached
to the
antibodies of the invention include a chelator selected from the group: DOXA
(1-oxa-
4,7,10-triazacyclododecanetriacetic acid), NOTA (1,4,7-
triazacyclononanetriacetic acid),
TETA (1,4,8,11-tetraa7acyclotetradecanetetraacetic acid), and THT (4'-(3-amino-
4-
methoxy-pheny1)-6,6"-bis(M,N-dicarboxymethyl-N-methylhydra zino)-2,2':6',2"-
terpyridine), and analogs and derivatives thereof. See, e.g., Ohmono et al.,
J. Med. Chem.
35: 157-162 (1992); Kung et aL, J. NucL Med. 25: 326-332 (1984); Jurisson et
al., Chem.
Rev. 93:1137-1156 (1993); and U.S. Patent No. 5,367,080. Other suitable
chelators
include chelating agents disclosed in U.S. Patent Nos. 4,647,447; 4,687,659;
4,885,363;
EP-A-71564; W089/00557; and EP-A-232751.
[0306] In another embodiment, suitable macrocyclic carboxylic acid chelators
which
can be used in the present invention include a chelator selected from the
group: 1,4,7,10-
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PCT/US2003/025457
tetraazacyclododecane-N,N,N",N"-tetraacetic
acid
(DOTA);
1,4,8,12-
tetraazacyc1opentadecane-N,1V1,NW"-tetraacetic acid (1 5N4); 1,4,7-
triazacyclononane-
N,N',N"-triacetic acid (9N3); 1,5,9-triazacyclododecane-N,N,N"-triacetic acid
(1 2N3);
and 6-bromoacetamido-benzyl- 1 ,4,8, 1 1 -tetraazacyclotetradecane- N,AP,N",Nm-
tetraacetic
acid (BAT).
[0307]
A preferred chelator that can be attached to the antibodies of the invention
is
a-(5-isothiocyanato-
2-methoxypheny1)- 1,4,7,1 0-tetraazacyclododecane- 1 ,4,7, 1 0-
tetraacetic acid, which is also known as Me0-DOTA-NCS. A salt or ester of a-(5-
isothiocyanato- 2-methoxypheny1)- 1,4,7,1 0-tetraazacyclododecane- 1 ,4,7,1 0-
tetraacetic
acid may also be used.
[0308]
Antibodies of the invention to which chelators such as those described are
covalently attached may be labeled (via the coordination site of the chelator)
with
radionuclides that are suitable for therapeutic, diagnostic, or both
therapeutic and
diagnostic purposes. Examples of appropriate metals include, but are not
limited to, Ag,
At, Au, Bi, Cu, Ga, Ho, In, Lu, Pb, Pd, Pm, Pr, Rb, Re, Rh, Sc, Sr, Tc, Tl, Y,
and Yb.
Examples of the radionuclide used for diagnostic purposes include, but are not
limited to,
Fe, Gd, 111in, 67-m, or -- 6g
k.)-
Ga. In another embodiment, the radionuclide used for diagnostic
purposes is mIn or 67Ga. Examples of the radionuclide used for therapeutic
purposes
66
1H0, 165Dy, 90y, 115mbi, 52Fe, or 72
include, but are not limited to,
Ga. In one embodiment,
the radionuclide used for diagnostic purposes is 166110 or 90Y. Examples of
the
radionuclides used for both therapeutic and diagnostic purposes include, but
are not
limited to, 1535m, 177Lu, 159Gd, 175Yb, or 475c. In one embodiment, the
radionuclide is
1535m, 177Lu, 175yb, or 159Gd.
[0309]
Preferred metal radionuclides that may be used according to the present
invention include a radionuclide selected from 90y, 99m-1,c, 1111n, 47se,
67Ga, 51cr, 177msn,
67cii, 167-m,
T
97Ru, 188Re, 177Lu, 199Au, 475c, 67Ga, 51Cr, 1771n511, 67CU, 167TM, 95RU,
188Re,
177LU, 199Au, 203pb and 141ce.
[0310]
In a particular embodiment, antibodies of the invention to which chelators are
covalently attached may be labeled with a metal ion selected from the group
consisting of
90y, 111in, 177Lu, 166H0, 215Bi, and 225Ac.
[0311]
Moreover, y-emitting radionuclides, such as 99mTc 11 ,1In 67Ga, and 169yb have
may be used for diagnostic imaging, while 13-emitters, such as 67 cu, 111Ag,
186Re, and 90Y
are useful for the applications in tumor therapy. Also other useful
radionuclides include y-
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WO 2004/016753 CA 02494372 2005-02-04
PCT/US2003/025457
emitters, such as 99mTc, 67Ga, and 169Yb, and [3-emitters, such as
67Cu, inAg, 186Re,
188Re and 90Y, as well as other radionuclides of interest such as 21 im,
212Bi, rrin, 86Rb
105Rh, '53 Sm, 198Au, 149pm, 85sr, 142pr, 214pb, 109pd, 166H0, 208T1, an 44Sc.
Antibodies of
the invention to which chelators are covalently attached may be labeled with
the
radionuclides described above or others known in the art.
[0312] In another embodiment, antibodies of the invention to which
chelators are
covalently attached may be labeled with paramagnetic metal ions including ions
of
transition and lanthanide metal, such as metals having atomic numbers of 21-
29, 42, 43,
44, or 57-71, in particular ions of metals selected from Cr, V, Mn, Fe, Co,
Ni, Cu, La, Ce,
Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu. The paramagnetic
metals used
in compositions for magnetic resonance imaging include the elements having
atomic
numbers of 22 to 29, 42, 44 and 58-70.
[0313] In another embodiment, antibodies of the invention to which
chelators are
covalently attached may be labeled with fluorescent metal ions including
lanthanides, in
particular a member selected from La, Ce, Pr, Nd, Pm, Sm, Eu (e.g., 152¨n),
Gd, Tb, Dy,
Ho, Er, Tm, Yb, and Lu.
[0314] In another embodiment, antibodies of the invention to which
chelators are
covalently attached may be labeled with heavy metal-containing reporters
including atoms
of a metal selected from Mo, Bi, Si, and W.
[0315] Radiolabeled antibodies of the invention may be used not only to
kill cells to
which they bind, but also may be useful to kill neighboring cells. For
example, expression
of TR4 may not be universal on all the cells of the tumor. However, because
the energy
from certain radioactive decay events can span more than a single cell
diameter,
radiolabeled antibodies of the invention may be used to kill cells that do not
express TR4,
e.g., cancerous cells, but which are in close proximity to cells that do
express TR4.
[0316] 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, etopo side, tenopo side, vincristine,
vinblastine,
colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, 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 limited
to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,
cytarabine, 5-
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CA 02494372 2010-11-17
fiuorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil,
melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiarnine platinum
(II)
(DDP) cisplatin), anthracyclines (e.g., daunombicin (formerly daunomycin) and
doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),
bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and
vinblastine).
[0317] 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; and direct coupling reactions (e.g., Bolton-Hunter and Chloramine-T
reaction).
[0318] 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 97/35899), AIM II (see,
International
Publication No. WO 97/34911), Fas Ligand (Takahashi et al., ha. Immunol.,
6:1567-1574
(1994)), VEGI (see, International Publication No. WO 99/23105), a thrombotic
agent or
an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological
response modifiers
such as, for example, lymphokines, interleukin-1 (IL- 1), interleukin-2 (IL-
2), interletikin-
6 (LL-6), granulocyte macrophage colony stimulating factor (GM-CSF),
granulocyte
colony stimulating factor (G-CSF), or other growth factors.
[0319] In specific embodiments antibodies of the invention are conjugated with
a a
polypeptide cytotoxin. An example of a suitable polypeptide cytotoxin is a
ribosome-
inactivating protein. Type I ribosome-inactivating proteins are single-chain
proteins, while
type II ribosome-inactivating proteins consist of two nonidentical subunits (A
and B
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
chains) joined by a disulfide bond (for a review, see Soria et al., Targeted
Diagn. Ther.
7:193 (1992)). Useful type I ribosome-inactivating proteins include
polypeptides from
Saponaria officinalis (e.g., saporin-1, saporin-2, saporin-3, saporin-6),
Momordica
charantia (e.g, momordin), Byronia dioica (e.g., bryodin, bryodin-2),
Trichosanthes
kirilowii (e.g., trichosanthin, trichokirin), Gelonium multiflorum (e.g.,
gelonin),
Phytolacca americana (e.g., pokeweed antiviral protein, pokeweed antiviral
protein-II,
pokeweed antiviral protein-S), Phytolacca dodecandra (e.g., dodecandrin,
Mirabilis
antiviral protein), and the like. Ribosome-inactivating proteins are
described, for example,
by Walsh et al., U.S. Pat. No. 5,635,384.
[0320] Suitable type II ribosome-inactivating proteins include polypeptides
from
Ricinus communis (e.g., ricin), Abrus precatorius (e.g., abrin), Adenia
digitata (e.g.,
modeccin), and the like. Since type II ribosome-inactiving proteins include a
B chain that
binds galactosides and a toxic A chain that depurinates adensoine, type II
ribosome-
inactivating protein conjugates should include the A chain. Additional useful
ribosome-
inactivating proteins include bouganin, clavin, maize ribosome-inactivating
proteins,
Vaccaria pyramidata ribosome-inactivating proteins, nigrine b, basic nigrine
1, ebuline,
racemo sine b, luffin-a, luffin-b, luffin-S, and other ribosome-inactivating
proteins known
to those of skill in the art. See, for example, Bologuesi and Stirpe,
International
Publication No. W098/55623, Colnaghi et al., International Publication No.
W097/49726, Hey et al., U.S. Pat. No. 5,635,384, Bolognesi and Stirpe,
International
Publication No. W095/07297, Arias et al., International Publication No.
W094/20540,
Watanabe et al., J. Biochem. 106:6 977 (1989); Islam et al., Agric. Biol.
Chem. 55:229
(1991), and Gao et al., FEBS Lett. 347:257 (1994).
[0321] 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.
[0322] Techniques for conjugating a therapeutic moiety to antibodies are well
known,
see, e.g., Amon 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
172
CA 02494372 2010-11-17
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).
[0323] 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.
[0324] 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
factor(s) and/or cytolcine(s) can be used as a therapeutic.
Uses of Antibodies of the Invention
[0325] Antibodies of the present invention may be used, for example, but not
limited
to, top, 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 TR4
polypeptides in biological samples. See, e.g., Harlow et al., Antibodies: A
Laboratory
Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).
Immunophenotyping
[0326] 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-
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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)).
[0327] 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
[0328] The present invention provides antibodies (including antibody fragments
or
variants thereof), that can be used to identify epitopes of a TR4 polypeptide.
In particular,
the antibodies of the present invention can be used to identify epitopes of a
human TR4
polypeptide (e.g., SEQ ID NOS:1) or a TR4 polypeptide expressed on human
cells; a
murine TR4 or a TR4 polypeptide expressed on murine cells; a rat TR4
polypeptide
receptor or a TR4 polypeptide expressed on rat cells; or a monkey TR4
polypeptide or a
TR4 polypeptide expressed on monkey cells, using techniques described herein
or
otherwise known in the art. Fragments which function as epitopes 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 TR4
polyp eptides.
Diagnostic Uses of Antibodies
[0329] Labeled antibodies of the invention (including molecules comprising, or
alternatively consisting of, antibody fragments or variants thereof) which
specifically bind
to a TR4 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 TR4 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 TR4.
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[0330] The invention provides for the detection of expression of a TR4
polypeptide
comprising: (a) assaying the expression of a TR4 polypeptide in a biological
sample from
an individual using one or more antibodies of the invention that
immunospecifically binds
to TR4; and (b) comparing the level of TR4 polypeptide in the biological
sample with a
standard level of TR4 polypeptide, (e.g., the level in normal biological
samples).
[0331] The invention provides for the detection of aberrant expression of a
TR4
polypeptide comprising: (a) assaying the expression of a TR4 polypeptide in a
biological
sample from an individual using one or more antibodies of the invention that
immunospecifically binds to TR4; and (b) comparing the level of a TR4
polypeptide in the
biological sample with a standard level of a TR4 polypeptide, e.g., in normal
biological
samples, whereby an increase or decrease in the assayed level of a TR4
polypeptide
compared to the standard level of a TR4 polypeptide is indicative of aberrant
expression.
[0332] By "biological sample" is intended any fluids and/or cells obtained
from an
individual, body fluid, body tissue, body cell, cell line, tissue culture, or
other source
which may contain a TR4 polypeptide protein or mRNA. Body fluids include, but
are not
limited to, sera, plasma, urine, synovial fluid, spinal fluid, saliva, and
mucous. Tissues
samples may be taken from virtually any tissue in the body. Tissue samples may
also be
obtained from autopsy material. Methods for obtaining tissue biopsies and body
fluids
from mammals are well known in the art. Where the biological sample is to
include
mRNA, a tissue biopsy is the preferred source.
[0333] Antibodies of the invention (including molecules comprising, or
alternatively
consisting of, antibody fragments or variants thereof) which specifically bind
to a TR4
polypeptide can be used for diagnostic purposes to detect, diagnose, prognose,
or monitor
cancers and other hyperproliferative disorders, and/or diseases or conditions
associated
therewith. The invention provides for the detection of aberrant expression of
TR4
polypeptide comprising: (a) assaying the expression of TR4 polypeptide in a
biological
sample from an individual using one or more antibodies of the invention that
immunospecifically binds to a TR4 polypeptide; and (b) comparing the level of
a TR4
polypeptide with a standard level of TR4 polypeptide, e.g., in normal
biological samples,
whereby an increase or decrease in the assayed level of TR4 polypeptide
compared to the
standard level of TR4 polypeptide is indicative of a cancer and/or a
hyperproliferative
disorder.
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[0334] TRAIL has been shown in some instances to selectively kill tumor cells
(See,
for example, Oncogene 19:3363-71 (2000)). This may be a result of differential
expression of TRAIL receptors on normal and cancerous cells. Thus, in specific
embodiments, an increase in the assayed level of a TR4 polypeptide is
indicative of a
cancer and/or a hyperproliferative disorder.
[0335] Other reports suggest that decreased TR4 expression by tumor cells may
be a
mechanism by which tumor cells evade the immune system (See, for example, Int.
J.
Oncol. 16:917-25 (2000)) Thus, in other specific embodiments, a decrease in
the assayed
level of TR4 polypeptide is indicative of a cancer and/or a hyperproliferative
disorder.
[0336] One aspect of the invention is the detection and diagnosis of a disease
or
disorder associated with aberrant expression of TR4 in an animal, preferably a
mammal
and most preferably a human. In one embodiment, diagnosis comprises: a)
administering
(for example, parenterally, subcutaneously, or intraperitoneally) to a subject
an effective
amount of a labeled antibody of the invention (including molecules comprising,
or
alternatively consisting of, antibody fragments or variants thereof) that
immunospecificaLly binds to a TR4 polypeptide; b) waiting for a time interval
following
the administering for permitting the labeled antibody to preferentially
concentrate at sites
in the subject where TR4 polypeptide is expressed (and for unbound labeled
molecule to
be cleared to background level); c) determining background level; and d)
detecting the
labeled antibody in the subject, such that detection of labeled antibody or
fragment thereof
above the background level and above or below the level observed in a person
without the
disease or disorder indicates that the subject has a particular disease or
disorder associated
with aberrant expression of TR4 polypeptide. Background level can be
determined by
various methods including, comparing the amount of labeled molecule detected
to a
standard value previously determined for a particular system.
[0337] It will be understood in the art that the size of the subject and the
imaging
system used will determine the quantity of imaging moiety needed to produce
diagnostic
images. In the case of a radioisotope moiety, for a human subject, the
quantity of
radioactivity injected will normally range from about 5 to 20 millicuries of
99Tc. The
labeled antibody will then preferentially accumulate at the location of cells
which contain
the specific protein. In vivo tumor imaging is described in S.W. Burchiel et
al.,
"Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments."
(Chapter
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13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and
B. A.
Rhodes, eds., Masson Publishing Inc. (1982).
[0338] Depending on several variables, including the type of label used and
the mode
of administration, the time interval following the administration for
permitting the labeled
molecule to preferentially concentrate at sites in the subject and for unbound
labeled
molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours
or 6 to 12
hours. In another embodiment, the time interval following administration is 5
to 20 days
or 5 to 10 days.
[0339] In one embodiment, monitoring of the disease or disorder is carried out
by
repeating the method for diagnosing the disease or disorder, for example, one
month after
initial diagnosis, six months after initial diagnosis, one year after initial
diagnosis, etc.
[0340] Presence of the labeled molecule can be detected in the patient using
methods
known in the art for in vivo scanning. These methods depend upon the type of
label used.
Skilled artisans will be able to determine the appropriate method for
detecting a particular
label. Methods and devices that may be used in the diagnostic methods of the
invention
include, but are not limited to, computed tomography (CT), whole body scan
such as
position emission tomography (PET), magnetic resonance imaging (MRI), and
sonography.
[0341] In a specific embodiment, the molecule is labeled with a radioisotope
and is
detected in the patient using a radiation responsive surgical instrument
(Thurston et al.,
U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled
with a
fluorescent compound and is detected in the patient using a fluorescence
responsive
scanning instrument. In another embodiment, the molecule is labeled with a
positron
emitting metal and is detected in the patient using positron emission-
tomography. In yet
another embodiment, the molecule is labeled with a paramagnetic label and is
detected in
a patient using magnetic resonance imaging (MRI).
Therapeutic Uses of Antibodies
[0342] One or more antibodies of the present invention (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind to TR4 may be used locally or systemically in the body
as a
therapeutic. The present invention is further directed to antibody-based
therapies which
involve administering antibodies of the invention (including molecules
comprising, or
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alternatively consisting of, antibody fragments or variants thereof) to an
animal, preferably
a mammal, and most preferably a human, for preventing or treating one or more
of the
disclosed diseases, disorders, or conditions. Therapeutic compounds of the
invention
include, but are not limited to, antibodies of the invention and nucleic acids
encoding
antibodies (and anti-idiotypic antibodies) of the invention as described
herein. In one
embodiment, the antibodies of the invention can be used to treat, ameliorate
or prevent
diseases, disorders or conditions, including, but not limited to, any one or
more of the
diseases, disorders, or conditions described herein. The treatment and/or
prevention of
diseases, disorders, or conditions includes, but is not limited to,
alleviating symptoms
associated with those diseases, disorders or conditions. Antibodies of the
invention may be
provided in pharmaceutically acceptable compositions as known in the art or as
described
herein. 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 TR4 binding antibodies.
Therapeutic Uses of Antibodies for Treating Cancers
[0343] In highly preferred embodiments, antibodies of the invention that bind
TR4
and stimulate apoptosis of TR4 expressing cells are used to treat, prevent or
ameliorate
cancer. In other highly preferred embodiments, antibodies of the invention
that bind a
TR4 polypeptide are used to treat, prevent or ameliorate cancer. In specific
embodiments,
antibodies of the invention are used to inhibit the progression or metastasis
of cancers and
other related disorders. Cancers and related disorders, include, but are not
limited to,
colon cancer, cervical cancer, leukemia (including acute leukemias (e.g.,
acute
lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic,
promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic
leukemias
(e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic
leukemia)),
polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's
disease),
multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and
solid
tumors including, but not limited to, sarcomas and carcinomas such as
fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous
cell
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carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous
gland carcinoma, papillary carcinoma, papillary adenocarcinomas,
cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,
testicular
tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,
epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma,
pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,
melanoma, neuroblastoma, and retinoblastoma.
[0344] In highly preferred embodiments, antibodies of the invention that bind
TR4
and stimulate apoptosis of TR4 expressing cells are used to treat, prevent or
ameliorate
renal cancer.
[0345] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat, prevent or ameliorate renal cancer.
[0346] In highly preferred embodiments, antibodies of the invention that bind
TR4
and stimulate apoptosis of TR4 expressing cells are used to treat, prevent or
ameliorate
melanoma.
[0347] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat, prevent or ameliorate melanoma.
[0348] In highly preferred embodiments, antibodies of the invention that bind
TR4
and stimulate apoptosis of TR4 expressing cells are used to treat, prevent or
ameliorate
cancers of the liver such as hepatomas.
[0349] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat, prevent or ameliorate cancers of the liver such as hepatomas.
[0350] In highly preferred embodiments, antibodies of the invention that bind
TR4
and stimulate apoptosis of TR4 expressing cells are used to treat, prevent or
ameliorate
cancers of the central nervous system such as medulloblastoma, neuroblastoma,
and
glioblastoma.
[0351] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat, prevent or ameliorate cancers of the central nervous system
such as
medulloblastoma, neuroblastoma, and glioblastoma.
[0352] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat, prevent or hematological cancers such as multiple myeloma, non-
Hodgkin's
lymphoma, chronic lymphocytic leukemia and chronic myelgenous leukemia.
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[0353] In highly preferred embodiments, antibodies of the invention that bind
TR4
and stimulate apoptosis of TR4 expressing cells are used to treat, prevent or
ameliorate
multiple myeloma.
[0354] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat, prevent or ameliorate multiple myleoma.
[0355] In other highly preferred embodiments, antibodies of the invention that
bind
TR4 and stimulate apoptosis of TR4 expressing cells are used to treat, prevent
or
ameliorate non-Hodgkin's lymphoma.
[0356] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat, prevent or ameliorate non-Hodgkin's lymphoma. Non hodgkin's
lymphomas,
include but are not limited to, B cell lymphomas such as precursor B
lymphpblastic
lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic lymphoma,
lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, extranodal
marginal
zone - MALT lymphoma, nodal marginal zone lymphoma, follicular lymphoma,
mantle
cell lymphoma, diffuse large B-cell lymphoma, primary mediastinal large B-cell
lymphoma, primary effusion lymphoma and Burkitt's lymphoma) and T-cell
lymphomas
such as precursor (peripheral) T-cell lymphoblastic lymphoma, adult T-cell
lymphoma,
extranodal Natural Killer/T-cell, nasal type lymphoma, enteropathy type T-cell
lymphoma,
hepatosplenic T-cell lymphoma, subcutaneous panniculitis like T-cell lymphoma,
skin
(cutaneous) lymphomas (including mycosis fungoides and Sezary syndrome),
anaplastic
large cell lymphoma, peripheral T-cell, not otherwise specified lymphoma, and
angioimmunoblastic T-cell lymphoma.
[0357] In other highly preferred embodiments, antibodies of the invention that
bind
TR4 and stimulate apoptosis of TR4 expressing cells are used to treat, prevent
or
ameliorate chronic lymphocytic leukemia (CLL).
[0358] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat, prevent or ameliorate chronic lymphocytic leukemia (CLL).
[0359] In other highly preferred embodiments, antibodies of the invention that
bind
TR4 and stimulate apoptosis of TR4 expressing cells are used to treat, prevent
or
ameliorate chronic myelogenous leukemia (CML).
[0360] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat, prevent or ameliorate chronic myelogenous leukemia (CML).
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[0361] In highly preferred embodiments, antibodies of the invention that bind
TR4
and stimulate apoptosis of TR4 expressing cells are used to treat, prevent or
meliorate
prostate cancer and/or metastatic prostate cancer.
[0362] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat, prevent or ameliorate prostate cancer and/or metastatic
prostate cancer.
[0363] It has been demonstrated, in accordance with the present invention that
the
expression of TRAIL receptor TR4 on lung carcinoma tissue, bladder carcinoma
tissue
and Ovarian carcinoma tissue. Additionally, it has been demonstrated, in
accordance with
the present invention that TRAIL receptor TR4 is expressed on primary breast,
colon,
lung, and stomach tumor tissue. (See Example 9).
[0364] Thus, in highly preferred embodiments, antibodies of the invention that
bind
TR4 and stimulate apoptosis of TR4 expressing cells are used to treat lung
cancer,
including but not limited to non-small cell lung cancer.
[0365] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat lung cancer, including but not limited to non-small cell lung
cancer.
[0366] In other highly preferred embodiments, antibodies of the invention that
bind
TR4 and stimulate apoptosis of TR4 expressing cells are used to treat bladder
cancer.
[0367] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat bladder cancer.
[0368] In other highly preferred embodiments, antibodies of the invention that
bind
TR4 and stimulate apoptosis of TR4 expressing cells are used to treat ovarian
cancer.
[0369] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat ovarian cancer.
[0370] In other highly preferred embodiments, antibodies of the invention that
bind
TR4 and stimulate apoptosis of TR4 expressing cells are used to treat breast
cancer and/or
breast cancers that have metastasized.
[0371] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat breast cancer and/or breast cancers that have metastasized.
[0372] In other highly preferred embodiments, antibodies of the invention that
bind
TR4 and stimulate apoptosis of TR4 expressing cells are used to treat colon
cancer and/or
colorectal cancer.
[0373] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat colon cancer and/or colorectal cancer.
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[0374] In other highly preferred embodiments, antibodies of the invention that
bind
TR4 and stimulate apoptosis of TR4 expressing cells are used to treat stomach
cancer.
[0375] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat stomach cancer.
[0376] In other highly preferred embodiments, antibodies of the invention that
bind
TR4 and stimulate apoptosis of TR4 expressing cells are used to treat, prevent
or
ameliorate renal cancer, melanoma, pancreatic cancer and cancers of the liver
such as
hepatomas. In other preferred embodiments, antibodies of the invention that
bind TR4 are
used to treat, prevent or ameliorate renal cancer, melanoma, pancreatic cancer
and cancers
of the liver such as hepatomas.
[0377] In other highly preferred embodiments, antibodies of the invention that
bind
TR4 and stimulate apoptosis of TR4 expressing cells are used to treat, prevent
or
ameliorate leukemia.
[0378] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat, prevent or ameliorate leukemia.
[0379] In other highly preferred embodiments, antibodies of the invention that
bind
TR4 and stimulate apoptosis of TR4 expressing cells are used to treat, prevent
or
ameliorate myelodysplastic syndrome.
[0380] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat, prevent or ameliorate myelodysplastic syndrome.
[0381] In other highly preferred embodiments, antibodies of the invention that
bind
TR4 and stimulate apoptosis of TR4 expressing cells are used to treat, prevent
or
ameliorate bone cancers including but not limited to Ewing's sarcoma and
osteosarcoma.
[0382] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat, prevent or ameliorate bone cancers including but not limited to
Ewing's
sarcoma and osteosarcoma.
[0383] In other highly preferred embodiments, antibodies of the invention that
bind
TR4 and stimulate apoptosis of TR4 expressing cells are used to treat, prevent
or
ameliorate bone cancers including but not limited to Ewing's sarcoma and
rhabdomyo sarcoma.
[0384] In other preferred embodiments, antibodies of the invention that bind
TR4 are
used to treat, prevent or ameliorate bone cancers including but not limited to
Ewing's
sarcoma and rhabdomyo sarcoma.
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[0385] In another embodiment, antibodies of the invention that bind TR4 and,
optionally, stimulate apoptosis of TR4 expressing cells, are used to treat
diseases and/or
disorders associated with increased cell survival, or the inhibition of
apoptosis, including
cancers (such as follicular lymphomas, carcinomas with p53 mutations, and
hormone-
dependent tumors, including, but not limited to colon cancer, cardiac tumors,
pancreatic
cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal
cancer, testicular
cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,
osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast
cancer,
prostrate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders
(such as
multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary
cirrhosis, Behcet's
disease, Crohn's disease, polymyositis, systemic lupus erythematosus and
immune-related
glomerulonephritis rheumatoid arthritis) and viral infections (such as herpes
viruses, pox
viruses and adenoviruses), information graft v. host disease, acute graft
rejection, and
chronic graft rejection. In preferred embodiments, the antibodies and antibody
compositions of the invention are used to inhibit growth, progression, and/or
metastasis of
cancers, in particular those listed above. In preferred embodiments the
antibodies and
antibody compositions of the invention are not hepatotoxic, in vitro or in
vivo.
[0386] In preferred embodiments, the antibodies of the invention that are used
to treat,
prevent or ameliorate the cancers described above specifically and/or
preferentially bind
TR4. In other preferred embodiments, the antibodies of the invention that are
used to
treat, prevent or ameliorate the cancers described above specifically and/or
preferentially
bind TR4 and TR7.
[0387] In preferred embodiments, the antibodies of the invention are used to
treat,
prevent or ameliorate radiation resistant cancers and/or cancers that are
resistant to one or
more chemotherapeutic agents or other therapeutic agents useful in the
treatment of
cancers.
Additional Therapeutic Uses of Antibodies
[0388] In another embodiment, the invention provides methods and compositions
for
inhibiting the growth of or killing TR4 expressing cells, comprising, or
alternatively
consisting of, administering to an animal in which such inhibition of growth
or killing of
TR4 expressing cells is desired, antibody or antibody compositions of the
invention (e.g.,
antibody fragments and variants, antibody mixtures, antibody multimers, fusion
proteins
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of the invention, and antibodies in combination with other therapeutic
compounds such as
chemotherapeutic agents) in an amount effective to inhibit the growth of or
kill TR4
expressing cells.
[0389] In one aspect, the present invention is directed to a method for
enhancing
apoptosis induced by a TNF-family ligand (especially TRAIL (SEQ ID NO:66)),
which
involves contacting a cell which expresses a TR4 polypeptide with an effective
amount of
an antibody or antibody composition of the invention, preferably an agonistic
anti-TR4
antibody, capable of inducing or increasing TR4 mediated signaling. In another
aspect,
the present invention is directed to a method for enhancing apoptosis induced
by a TNF-
family ligand (especially TRAIL (SEQ ID NO:66)), which involves contacting a
cell
which expresses a TR4 and/or TR7 polypeptide with an effective amount of an
antibody or
antibody composition of the invention, preferably an agonistic antibody that
specifically
binds both TR4 and TR7, capable of inducing or increasing TR4 and/or TR7
mediated
signaling. Preferably, TR4 and/or TR7 mediated signaling is increased or
induced by an
antibody of the invention to treat a disease wherein decreased apoptosis or
decreased
cytokine and adhesion molecule expression is exhibited.
[0390] In one aspect, the present invention is directed to a method for
inducing
apoptosis of TR4 and/or TR7 expressing cells, which involves contacting a cell
which
expresses TR4 and/or TR7, with an effective amount of an antibody or antibody
composition of the invention, preferably an agonistic anti-TR4, and/or an anti-
TR4 and
TR7 antibody (i.e., an antibody that immunospecifically binds both TR4 and
TR7),
capable of inducing or increasing TRAIL receptor mediated signaling,
especially TR4 and
TR7 mediated signalling.
[0391] In a further aspect, the present invention is directed to a method for
inhibiting
apoptosis induced by a TNF-family ligand (especially TRAIL (SEQ ID NO:66)),
which
involves contacting a cell which expresses a TR4 polypeptide, with an
effective amount of
an antibody or antibody composition of the invention, preferably an
antagonistic anti-TR4
antibody, capable of decreasing TR4 mediated signaling. In another aspect, the
present
invention is directed to a method for inhibiting apoptosis induced by a TNF-
family ligand
(especially TRAIL (SEQ ID NO:66)), which involves contacting a cell which
expresses a
TR4 and/or TR7 polypeptide, with an effective amount of an antibody or
antibody
composition of the invention, preferably an antagonistic antibody that
specifically binds
both TR4 and TR7, capable of decreasing TR4 and/or TR7 mediated signaling.
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Preferably, TR4 and/or TR7 mediated signaling is decreased to treat a disease
wherein
increased apoptosis or NFKB expression is exhibited.
[0392] In one aspect, the present invention is directed to a method for
inhibiting
apoptosis of TR4 and/or TR7 expressing cells, which involves contacting a cell
which
expresses TR4 and/or TR7, with an effective amount of an antibody or antibody
composition of the invention, preferably an antagonistic anti-TR4, and/or an
anti-TR4 and
TR7 antibody (i.e., an antibody that immunospecifically binds both TR4 and
TR7),
capable of decreasing TRAIL receptor mediated signaling, especially TR4 and
TR7
mediated signalling.
[03931 By TR4 "agonist" is intended naturally occurring and synthetic
compounds
capable of enhancing or potentiating apoptosis mediated by TRAIL receptor. By
TR4
"antagonist" is intended naturally occurring and synthetic compounds capable
of inhibiting
apoptosis mediated by TRAIL receptor. Whether any candidate "agonist" or
"antagonist"
of the present invention can enhance or inhibit, respectively, apoptosis can
be determined
using art-known TNF-family ligand/receptor cellular response assays, including
those
described in more detail below.
[0394] The antibodies of the invention can be used to treat, ameliorate or
prevent
diseases, disorders or conditions associated with aberrant expression and/or
activity of
TR4 or TR4 ligand, including, but not limited to, any one or more of the
diseases,
disorders, or conditions described herein. The treatment and/or prevention of
diseases,
disorders, or conditions associated with aberrant TR4 expression and/or
activity or
aberrant TR4 ligand expression and/or activity includes, but is not limited
to, alleviating
symptoms associated with those diseases, disorders or conditions. Antibodies
of the
invention may be provided in pharmaceutically acceptable compositions as known
in the
art or as described herein.
[0395] Further, antibodies of the present invention (including molecules
comprising,
or alternatively consisting of, antibody fragments or variants thereof) which
activate
TRAIL receptor-mediated biological activities (e.g., the induction of
apoptosis in TRAIL
receptor expressing cells) can be administered to an animal to treat, prevent
or ameliorate
a disease or disorder described herein, particularly cancers and other
hyperproliferative
disorders. These antibodies may potentiate or activate either all or a subset
of the
biological activities of TRAIL receptor, for example, by inducing a
conformational change
in TRAIL receptor. In a specific embodiment, an antibody of the present
invention that
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increases TR4 activity by at least 5%, at least 10%, at least 15%, at least
20%, at least
25%, 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%, at least 99%, at least two-fold, at least three-fold, at
least four fold, at
least five fold, at least ten-fold, at least twenty-fold, at least fifty-fold,
or at least one
hundred-fold relative to TR4 activity in absence of the antibody is
administered to an
animal to treat, prevent or ameliorate a disease or disorder. In another
embodiment, a
combination of antibodies, a combination of antibody fragments, a combination
of
antibody variants, or a combination of antibodies, antibody fragments and/or
antibody
variants that increase TR4 activity by at least 5%, at least 10%, at least
15%, at least 20%,
at least 25%, 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%, at least 99%, at least two-fold, at least three-fold,
at least four
fold, at least five fold, at least ten-fold, at least twenty-fold, at least
fifty-fold, or at least
one hundred-fold relative to TR4 activity in absence of the said antibodies or
antibody
fragments and/or antibody variants, is administered to an animal to treat,
prevent or
ameliorate a disease or disorder.
[0396] Further, antibodies of the present invention (including molecules
comprising,
or alternatively consisting of, antibody fragments or variants thereof) which
activate TR4-
mediated biological activities (e.g., the induction of apoptosis in TR4
expressing cells) can
be administered to an animal to treat, prevent or ameliorate a disease or
disorder
associated with aberrant TR4 expression, lack of TR4 function, aberrant TR4
ligand
expression, or lack of TR4 ligand function. These antibodies may potentiate or
activate
either all or a subset of the biological activities of TRAIL receptor, for
example, by
inducing a conformational change in TRAIL receptor. In a specific embodiment,
an
antibody of the present invention that increases TR4 activity by at least 5%,
at least 10%,
at least 15%, at least 20%, at least 25%, 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%, at least 99%, at least
two-fold, at least
three-fold, at least four fold, at least five fold, at least ten-fold, at
least twenty-fold, at least
fifty-fold, or at least one hundred-fold relative to TR4 activity in absence
of the antibody
is administered to an animal to treat, prevent or ameliorate a disease or
disorder associated
with aberrant TR4 expression, lack of TR4 function, aberrant TR4 ligand
expression, or
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lack of TR4 ligand function. In another embodiment, a combination of
antibodies, a
combination of antibody fragments, a combination of antibody variants, or a
combination
of antibodies, antibody fragments and/or antibody variants that increase TR4
activity by at
least 5%, at least 10%, at least 15%, at least 20%, at least 25%, 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%, at
least 99%, at
least two-fold, at least three-fold, at least four fold, at least five fold,
at least ten-fold, at
least twenty-fold, at least fifty-fold, or at least one hundred-fold relative
to TR4 activity in
absence of the said antibodies or antibody fragments and/or antibody variants,
is
administered to an animal to treat, prevent or ameliorate a disease or
disorder associated
with aberrant TR4 expression or lack of TR4 function or aberrant TR4 ligand
expression
or lack of TR4 ligand function.
[0397] Antibodies of the present invention (including molecules comprising, or
alternatively consisting of, antibody fragments or variants thereof) that
function as
agonists or antagonists of a TRAIL receptor, preferably of TR4 signal
transduction, can be
administered to an animal to treat, prevent or ameliorate a disease or
disorder associated
with aberrant TR4 expression, lack of TR4 function, aberrant TR4 ligand
expression, or
lack of TR4 ligand function. For example, antibodies of the invention which
mimic the
action of TRAIL binding to TR4, in full or in part, TR4 agonists, may be
administered to
an animal to treat, prevent or ameliorate a disease or disorder associated
with aberrant
TR4 expression, lack of TR4 function, aberrant TR4 ligand expression, or lack
of TR4
ligand function. As an alternative example, antibodies of the invention which
disrupt or
prevent the interaction between TR4 and its ligand or inhibit, reduce, or
prevent signal
transduction through TR4, may be administered to an animal to treat, prevent
or
ameliorate a disease or disorder associated with aberrant TR4 expression, lack
of TR4
function, aberrant TR4 ligand expression, or lack of TR4 ligand function.
Antibodies of
the invention which do not prevent TR4 from binding its ligand but inhibit or
downregulate TR4 signal transduction can be administered to an animal to
treat, prevent or
ameliorate a disease or disorder associated with aberrant TR4 expression, lack
of TR4
function, aberrant TR4 ligand expression, or lack of TR4 ligand function. The
ability of an
antibody of the invention to enhance, inhibit, upregulate or downregulate TR4
signal
transduction may be determined by techniques described herein or otherwise
known in the
art. For example, TRAIL-induced receptor activation and the activation of
signaling
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molecules can be determined by detecting the association of adaptor proteins
such as
FADD and TRADD with TR4, by immunoprecipitation followed by western blot
analysis
(for example, as described herein).
[0398] Further, antibodies of the present invention (including molecules
comprising,
or alternatively consisting of, antibody fragments or variants thereof) which
activate TR4-
mediated biological activities (e.g., the induction of apoptosis in TR4
expressing cells) can
be administered to an animal to treat, prevent or ameliorate a disease or
disorder
associated with aberrant TR4 expression, lack of TR4 function, aberrant TR4
ligand
expression, or lack of TR4 ligand function. These antibodies may potentiate or
activate
either all or a subset of the biological activities of TRAIL receptor, for
example, by
inducing a conformational change in TRAIL receptor. In a specific embodiment,
an
antibody of the present invention that increases TR4 activity by at least 5%,
at least 10%,
at least 15%, at least 20%, at least 25%, 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%, at least 99%, at least
two-fold, at least
three-fold, at least four fold, at least five fold, at least ten-fold, at
least twenty-fold, at least
fifty-fold, or at least one hundred-fold relative to TR4 activity in absence
of the antibody
is administered to an animal to treat, prevent or ameliorate a disease or
disorder associated
with aberrant TR4 expression, lack of TR4 function, aberrant TR4 ligand
expression, or
lack of TR4 ligand function. In another embodiment, a combination of
antibodies, a
combination of antibody fragments, a combination of antibody variants, or a
combination
of antibodies, antibody fragments and/or antibody variants that increase TR4
activity by at
least 5%, at least 10%, at least 15%, at least 20%, at least 25%, 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%, at
least 99%, at
least two-fold, at least three-fold, at least four fold, at least five fold,
at least ten-fold, at
least twenty-fold, at least fifty-fold, or at least one hundred-fold relative
to TR4 activity in
absence of the said antibodies or antibody fragments and/or antibody variants
is
administered to an animal to treat, prevent or ameliorate a disease or
disorder associated
with aberrant TR4 expression or lack of TR4 function or aberrant TR4 ligand
expression
or lack of TR4 ligand function.
[0399] In a specific embodiment, an antibody of the present invention
(including
molecules comprising, or alternatively consisting of, antibody fragments or
variants
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thereof) that inhibits or downregulates, in full or in part, TR4 activity
(e.g., stimulation of
apoptosis) by at least 95%, at least 90%, at least 85%, at least 80%, at least
75%, at least
70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at
least 35%, at
least 30%, at least 25%, at least 20%, or at least 10% relative to TR4
activity in absence of
the antibody is administered to an animal to treat, prevent or ameliorate a
disease or
disorder associated with aberrant TR4 expression, excessive TR4 function,
aberrant TR4
ligand expression, or excessive TR4 ligand function. In another embodiment, a
combination of antibodies, a combination of antibody fragments, a combination
of
antibody variants, or a combination of antibodies, antibody fragments, and/or
variants that
inhibit or downregulate TR4 activity by at least 95%, at least 90%, at least
85%, at least
80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at
least 50%, at
least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least
25%, at least
20%, or at least 10% relative to TR4 activity in absence of said antibodies,
antibody
fragments, and/or antibody variants, are administered to an animal to treat,
prevent or
ameliorate a disease or disorder associated with aberrant TR4 expression,
excessive TR4
function, aberrant TR4 ligand expression, or excessive TR4 ligand function.
[0400] In one embodiment, therapeutic or pharmaceutical compositions of the
invention are administered to an animal to treat, prevent or ameliorate a
disease or
disorder diseases associated with increased apoptosis including, but not
limited to, AIDS,
neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease,
Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration),
myelodysplastic syndromes (such as aplastic anemia), ischemic injury (such as
that caused
by myocardial infarction, stroke and rep erfusion injury), toxin-induced liver
disease (such
as that caused by alcohol), septic shock, cachexia and anorexia. In another
embodiment,
therapeutic or pharmaceutical compositions of the invention are administered
to an animal
to treat, prevent or ameliorate bone marrow failure, for example, aplastic
anemia and
myelodysplastic syndrome.
[0401] Therapeutic or pharmaceutical compositions of the invention, may also
be
administered to treat, prevent, or ameliorate organ rejection or graft-versus-
host disease
(GVHD) and/or conditions associated therewith. Organ rejection occurs by host
immune
cell destruction of the transplanted tissue through an immune response.
Similarly, an
immune response is also involved in GVHD, but, in this case, the foreign
transplanted
immune cells destroy the host tissues. Cellular death induced by immune cell
effector
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functions is apoptotic death. Thus, the administration of antibodies of the
invention, (e.g.,
those that inhibit apoptosis), may be an effective therapy in preventing organ
rejection or
GVHD.
[0402] In another embodiment, therapeutic or pharmaceutical compositions of
the
invention are administered to an animal to treat, prevent or ameliorate
infectious diseases.
Infectious diseases include diseases associated with yeast, fungal, viral and
bacterial
infections. Viruses associated with viral infections which can be treated or
prevented in
accordance with this invention include, but are not limited to, retroviruses
(e.g., human
T-cell lymphotrophic virus (HTLV) types I and II and human immunodeficiency
virus
(HIV)), herpes viruses (e.g., herpes simplex virus (HSV) types I and II,
Epstein-Barr virus,
HHV6-HHV8, and cytomegalovirus), arenavirues (e.g., lassa fever virus),
paramyxoviru.ses (e.g., morbillivirus virus, human respiratory syncytial
virus, mumps, and
pneumovirus), adenoviruses, bunyaviruses (e.g., hantavirus), cornaviruses,
filoviruses
(e.g., Ebola virus), fiaviviru.ses (e.g., hepatitis C virus (HCV), yellow
fever virus, and
Japanese encephalitis virus), hepadnaviruses (e.g., hepatitis B viruses
(HBV)),
orthomyoviruses (e.g., influenza viruses A, B and C), papovaviruses (e.g.,
papillomavirues), picornaviruses (e.g., rhinoviruses, enteroviruses and
hepatitis A viruses),
poxviruses, reoviruses (e.g., rotavirues), togaviruses (e.g., rubella virus),
rhabdoviruses
(e.g., rabies virus). Microbial pathogens associated with bacterial infections
include, but
are not limited to, Streptococcus pyogenes, Streptococcus pneumoniae,
Neisseria
gonorrhoea, Neisseria meningitidis, Corynebacterium dzphtheriae , Clostridium
botulinum, Clostridium perfringens, Clostridium tetani, Haemophilus
influenzae,
Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromotis,
Staphylococcus
aureus, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa,
Campylobacter
(Vibrio) fetus, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus,
Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia
pseudotuberculosis,
Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella
typhimurium,
Treponema pallidum, Trepoizema pertenue, Treponenza carateneum, Borrelia
vincentii,
Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium
tuberculosis,
Toxoplasma gondii, Pneumocystis carinii, Francisella tularensis, Brucella
abortus,
Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki,
Rickettsia
tsutsugumushi, Clzlamydia spp., and Helicobacter pylori.
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[0403] In another embodiments, antibodies and antibody compositions of the
present
invention are used to treat, prevent, or ameliorate diseases associated with
increased
apoptosis including, but not limited to, AIDS, neurodegenerative disorders
(such as
Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,
Retinitis
pigmentosa, Cerebellar degeneration), brain tumor or prion associated
disease);
autoimmune disorders (such as, multiple sclerosis, Rheumatoid Arthritis,
Sjogren's
syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease,
Crohn's disease,
polymyositis, systemic lupus erythematosus and immune-related
glomerulonephritis and
rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia),
graft v. host
disease, ischemic injury (such as that caused by myocardial infarction, stroke
and
reperfusion injury), liver injury (e.g., hepatitis related liver injury,
ischemia/reperfusion
injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver
disease (such as
that caused by alcohol), septic shock, cachexia and anorexia. In preferred
embodiments,
anti-TR4 antagonistic antibodies, prevent TRAIL from binding to the TRAIL
receptors to
which the antibodies are bound, but do not transduce the biological signal
that results in
apoptosis) are used to treat the diseases and disorders listed above.
[0404] Many of the pathologies associated with HIV are mediated by apoptosis,
including HIV-induced nephropathy and HIV encephalitis. Thus, in additional
preferred
embodiments, antibodies, preferably antagonistic anti-TR4 antibodies, of the
invention are
used to treat AIDS and pathologies associated with AIDS. Another embodiment of
the
present invention is directed to the use of antibodies of the invention to
reduce
TRAIL-mediated death of T cells in HIV-infected patients.
[0405] In additional embodiments, antibodies of the present invention,
particularly
antagonistic anti-TR4 antibodies, are administered in combination with other
inhibitors of
T cell apoptosis. For example, Fas-mediated apoptosis has been implicated in
loss of T
cells in HIV individuals (Katsikis et al., J. Exp. Med. 181:2029-2036, 1995).
Thus, a
patient susceptible to both Fas ligand mediated and TRAIL mediated T cell
death may be
treated with both an agent that blocks TRAIL/TR4 interactions and an agent
that blocks
Fas-ligand/Fas interactions. Suitable agents for blocking binding of Fas-
ligand to Fas
include, but are not limited to, soluble Fas polypeptides; mulitmeric forms of
soluble Fas
polypeptides (e.g., dimers of sFas/Fc); anti-Fas antibodies that bind Fas
without
transducing the biological signal that results in apoptosis; anti-Fas-ligand
antibodies that
block binding of Fas-ligand to Fas; and muteins of Fas-ligand that bind Fas
but do not
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transduce the biological signal that results in apoptosis. Preferably, the
antibodies
employed according to this method are monoclonal antibodies. Examples of
suitable
agents for blocking Fas-ligand/Fas interactions, including blocking anti-Fas
monoclonal
antibodies, are described in International application publication number WO
95/10540.
[0406] Suitable agents, which also block binding of TRAIL to a TR4 that may be
administered with the antibodies of the present invention include, but are not
limited to,
soluble TR4 polypeptides (e.g., a soluble form of OPG, TR5 (International
application
publication number WO 98/30693); a soluble form of TR4 (International
publication
number WO 98/32856); TR7/DR5 (International application publication number WO
98/41629); and TRIO (International application publication number WO
98/54202));
multimeric forms of soluble TR4 polypeptides; and TR4 antibodies that bind the
TR4
without transducing the biological signal that results in apoptosis, anti-
TRAIL antibodies
that block binding of TRAIL to one or more TRAIL receptors, and muteins of
TRAIL that
bind TRAIL receptors but do not transduce the biological signal that results
in apoptosis.
[0407] In rejection of an allograft, the immune system of the recipient animal
has not
previously been primed to respond because the immune system for the most part
is only
primed by environmental antigens. Tissues from other members of the same
species have
not been presented in the same way that, for example, viruses and bacteria
have been
presented. In the case of allograft rejection, inununosuppressive regimens are
designed to
prevent the immune system from reaching the effector stage. However, the
immune
profile of xenograft rejection may resemble disease recurrence more that
allograft
rejection. In the case of disease recurrence, the immune system has already
been
activated, as evidenced by destruction of the native islet cells. Therefore,
in disease
recurrence the immune system is already at the effector stage. Antibodies of
the present
invention (e.g., agonistic antibodies of the invention) are able to suppress
the immune
response to both allografts and xenografts because lymphocytes activated and
differentiated into effector cells will express the TR4 polypeptides, and
thereby are
susceptible to compounds which enhance apoptosis. Thus, the present invention
further
provides a method for creating immune privileged tissues. Antagonist of the
invention
can further be used in the treatment of Inflammatory Bowel-Disease.
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[0408] Antibodies and antibody compositions of the invention may be useful for
treating inflammatory diseases, such as rheumatoid arthritis, osteoarthritis,
psoriasis,
septicemia, and inflammatory bowel disease.
[0409] In addition, due to lymphoblast expression of TR4 polypeptides,
antibodies and
antibody compositions of the invention may be used to treat this form of
cancer. Further,
antibodies and antibody compositions of the invention may be used to treat
various
chronic and acute forms of inflammation such as rheumatoid arthritis,
osteoarthritis,
psoriasis, septicemia, and inflammatory bowel disease.
[0410] In one embodiment, antibodies and antibody compositions of the
invention
may be used to treat cardiovascular disorders, including peripheral artery
disease, such as
limb ischemia.
[0411] Cardiovascular disorders include cardiovascular abnormalities, such as
arterio-
arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations,
congenital
heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart
defects
include aortic coarctation, cor triatriatum, coronary vessel anomalies,
crisscross heart,
dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger
complex,
hypoplastic left heart syndrome, levocardia, tetralogy of fallot,
transposition of great
vessels, double outlet right ventricle, tricuspid atresia, persistent truncus
arteriosus, and
heart septal defects, such as aortopulmonary septal defect, endocardial
cushion defects,
Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.
[0412] Cardiovascular disorders also include heart disease, such as
arrhythmias,
carcinoid heart disease, high cardiac output, low cardiac output, cardiac
tamponade,
endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive
heart failure,
congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart
hypertrophy,
congestive cardiomyopathy, left ventricular hypertrophy, right ventricular
hypertrophy,
post-infarction heart rupture, ventricular septal rupture, heart valve
diseases, myocardial
diseases, myocardial ischemia, pericardial effusion, pericarditis (including
constrictive and
tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart
disease, rheumatic heart disease, ventricular dysfunction, hyperemia,
cardiovascular
pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and
cardiovascular
tuberculosis.
[0413] Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial
flutter,
bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block,
sinoatrial block,
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long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-
excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome,
tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal
tachycardia,
supraventricular tachycardia, accelerated idioventricular rhythm,
atrioventricular nodal
reentry tachycardia, ectopic atrial tachycardia, ectopic junctional
tachycardia, sinoatrial
nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and
ventricular
tachycardia.
[0414] Heart valve disease include aortic valve insufficiency, aortic valve
stenosis,
hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve
prolapse, mitral
valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve
insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve
insufficiency,
and tricuspid valve stenosis.
[0415] Myocardial diseases include alcoholic cardiomyopathy, congestive
cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis,
pulmonary
subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy,
endocardial
fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial
reperfusion injury,
and myocarditis.
[0416] Myocardial ischemias include coronary disease, such as angina pectoris,
coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary
vasospasm,
myocardial infarction and myocardial stunning.
[0417] Cardiovascular diseases also include vascular diseases such as
aneurysms,
angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease,
Klippel-
Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic
diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial
occlusive diseases,
arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders,
diabetic angiopathies,
diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids,
hepatic veno-
occlusive disease, hypertension, hypotension, ischemia, peripheral vascular
diseases,
phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST
syndrome, retinal
vein occlusion, Scimitar syndrome, superior vena cava syndrome,
telangiectasia, atacia
telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose
veins, varicose
ulcer, vasculitis, and venous insufficiency.
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[0418] Aneurysms include dissecting aneurysms, false aneurysms, infected
aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary
aneurysms, heart aneurysms, and iliac aneurysms.
[0419] Arterial occlusive diseases include arteriosclerosis, intermittent
claudication,
carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion,
Moyamoya
disease, renal artery obstruction, retinal artery occlusion, and
thromboangiitis obliterans.
[0420] Cerebrovascular disorders include carotid artery diseases, cerebral
amyloid
angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis,
cerebral
arteriovenous malformation, cerebral artery diseases, cerebral embolism and
thrombosis,
carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral
hemorrhage,
epidural hematoma, sub dural hematoma, subaraxhnoid hemorrhage, cerebral
infarction,
cerebral ischemia (including transient), sub clavian steal syndrome,
periventricular
leukomalacia, vascular headache, cluster headache, migraine, and
vertebrobasilar
insufficiency.
[0421] Embolisms include air embolisms, amniotic fluid embolisms, cholesterol
embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and
thromoboembolisms. Thrombosis include coronary thrombosis, hepatic vein
thrombosis,
retinal vein occlusion, carotid artery thrombosis, sinus thrombosis,
Wallenberg's
syndrome, and thrombophlebitis.
[0422] Ischemia includes cerebral ischemia, ischemic colitis, compartment
syndromes,
anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and
peripheral
limb ischemia. Vasculitis includes aortitis, arteritis, Behcet's Syndrome,
Churg-Strauss
Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans,
hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous
vasculitis, and
Wegener's granulomatosis.
[0423] In one embodiment, antibodies and antibody compositions of the
invention is
used to treat thrombotic microangiopathies. One such disorder is thrombotic
thrombocytopenic purpura (TTP) (Kwaan, H.C., Semin. Hematol. 24:71 (1987);
Thompson et al., Blood 80:1890 (1992)). Increasing TTP-associated mortality
rates have
been reported by the U.S. Centers for Disease Control (Torok et al., Am. J.
Hematol. 50:84
(1995)). Plasma from patients afflicted with TTP (including HIV+ and HIV-
patients)
induces apoptosis of human endothelial cells of dermal microvascular origin,
but not large
vessel origin (Laurence et al., Blood 87:3245 (1996)). Plasma of TTP patients
thus is
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thought to contain one or more factors that directly or indirectly induce
apoptosis. As
described in International patent application number WO 97/01715, TRAIL is
present in
the serum of up patients, and is likely to play a role in inducing apoptosis
of
microvascular endothelial cells. Another thrombotic
microangiopathy is hemolytic-uremic syndrome (HUS) (Moake, J.L., Lancet,
343:393
(1994); Melnyk et al., (Arch. Intern. Med., 155:2077 (1995); Thompson et al.,
supra).
Thus, in one embodiment, the invention is directed to use of antibodies and
antibody
compositions of the invention to treat the condition that is often referred to
as "adult HUS"
(even though it can strike children as well). A disorder known as
childhood/diarrhea-associated HUS differs in etiology from adult HUS. In
another
embodiment, conditions characterized by clotting of small blood vessels may be
treated
using of antibodies and antibody compositions of the invention. Such
conditions include,
but are not limited to, those described herein. For example, cardiac problems
seen in
about 5-10% of pediatric AIDS patients are believed to involve clotting of
small blood
vessels. Breakdown of the microvasculature in the heart has been reported in
multiple
sclerosis patients. As a further example, treatment of systemic lupus
erythematosus (SLE)
is contemplated. In one embodiment, antibodies and antibody compositions of
the
invention, preferably antagonistic anti-TR4 antibodies of the invention, may
be
administered in vivo to a patient afflicted with a thrombotic microangiopathy.
Thus, the
present invention provides a method for treating a thrombotic microangiopathy,
involving
use of an effective amount of an antibody or antibody composition of the
invention.
[0424] Antibodies and antibody compositions of the invention may be employed
in
combination with other agents useful in treating a particular disorder. For
example, in an
in vitro study reported by Laurence et al. (Blood 87:3245 (1996)), some
reduction of TTP
plasma-mediated apoptosis of microvascular endothelial cells was achieved by
using an
anti-Fas blocking antibody, aurintricarboxylic acid, or normal plasma depleted
of
cryoprecipitate. Thus, a patient may be treated with an antibody or antibody
composition
of the invention in combination with an agent that inhibits Fas-ligand-
mediated apoptosis
of endothelial cells, such as, for example, an agent described above. In one
embodiment,
antibodies of the invention and an anti-FAS blocking antibody are both
administered to a
patient afflicted with a disorder characterized by thrombotic microanglopathy,
such as
TTP or HUS. Examples of blocking monoclonal antibodies directed against Fas
antigen
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CA 02494372 2010-11-17
(CD95) are described in International patent application publication number WO
95/10540.
[0425] The naturally occurring balance between endogenous stimulators and
inhibitors
of angiogenesis is one in which inhibitory influences predominate (Rastinejad
et al., Cell
56:345-355 (1989)). In those rare instances in which neovascularization occurs
under
normal physiological conditions, such as wound healing, organ regeneration,
embryonic
development, and female reproductive processes, angiogenesis is stringently
regulated and
spatially and temporally delimited. Under conditions of pathological
angiogenesis such as
that characterizing solid tumor growth, these regulatory controls fail.
Unregulated
angiogenesis becomes pathologic and sustains progression of many neoplastdc
and non-
neoplastic diseases. A number of serious diseases are dominated by abnormal
neovascularization including solid tumor growth and metastases, arthritis,
some types of
eye disorders, and psoriasis. See, e.g., reviews by Moses et al., Biotech.
9:710-714
(1991); Folkman et al., N. Engl. J Med., 353:1757-1771 (1995); Auerbach et
at., J.
Microvasc. Res. 29:401-411(1985); Folkman, Advances in Cancer Research, eds.
Klein
and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J.
Opthalmol.
94:715-743 (1982); and Folkman et at., Science 221:719-725 (1983). In a number
of
pathological conditions, the process of angiogenesis contributes to the
disease state. For
example, significant data have accumulated which suggest that the growth of
solid tumors
is dependent on angiogenesis. Folkman and Klagsbrun, Science 235:442-447
(1987).
[0426] The present invention provides for treatment of diseases or disorders
associated
with neovascularization by administration of an antibody or antibody
compositions of the
invention. Malignant and metastatic conditions which can be treated with the
polynucleotides and polypeptides of the invention include, but are not limited
to those
malignancies, solid tumors, and cancers described herein and otherwise known
in the art
(for a review of such disorders, see Fishman et at., Medicine, 2d Ed., J. B.
Lippincott Co.,
Philadelphia (1985)).
[0427) Additionally, ocular disorders associated with neovascularization
which can be
treated with an antibody or antibody composition of the invention include, but
are not
limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma,
retrolental
flbroplasia, uveitis, retinopathy of prematurity macular degeneration, comeal
graft
neovascularization, as well as other eye inflammatory diseases, ocular tumors
and diseases
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
associated with choroidal or iris neovascularization. See, e.g., reviews by
Waltman et al.,
Am. J. Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-
312 (1978).
[0428] Additionally, disorders which can be treated with an antibody or
antibody
composition of the invention include, but are not limited to, hemangioma,
arthritis,
psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing,
granulations,
hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber
syndrome,
pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
[0429] Antibodies and antibody compositions of the invention are useful in
the
diagnosis and treatment or prevention of a wide range of diseases and/or
conditions. Such
diseases and conditions include, but are not limited to, cancer (e.g., immune
cell related
cancers, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma,
cancer
associated with mutation or alteration of p53, brain tumor, bladder cancer,
uterocervical
cancer, colon cancer, colorectal cancer, non-small cell carcinoma of the lung,
small cell
carcinoma of the lung, stomach cancer, etc.), lymphoproliferative disorders
(e.g.,
lymphadenopathy), microbial (e.g., viral, bacterial, etc.) infection (e.g.,
HIV-1 infection,
HIV-2 infection, herpesvirus infection (including, but not limited to, HSV-1,
HSV-2,
CMV, VZV, 1{HV-6, HHV-7, EBV), adenovirus infection, poxvirus infection, human
papilloma virus infection, hepatitis infection (e.g., HAY, HBV, HCV, etc.),
Helicobacter
pylori infection, invasive Staphylococcia, etc.), parasitic infection,
nephritis, bone disease
(e.g., osteoporosis), atherosclerosis, pain, cardiovascular disorders (e.g.,
neovascularization, hypovascularization or reduced circulation (e.g., ischemic
disease
(e.g., myocardial infarction, stroke, etc.))), AIDS, allergy, inflammation,
neurodegenerative disease (e.g., Alzheimer's disease, Parkinson's disease,
amyotrophic
lateral sclerosis, pigmentary retinitis, cerebellar degeneration, etc.), graft
rejection (acute
and chronic), graft vs. host disease, diseases due to osteomyelodysplasia
(e.g., aplastic
anemia, etc.), joint tissue destruction in rheumatism, liver disease (e.g.,
acute and chronic
hepatitis, liver injury, and cirrhosis), autoimmune disease (e.g., multiple
sclerosis,
rheumatoid arthritis, systemic lupus erythematosus, autoimmune
lymphoproliferative
syndrome (ALPS), immune complex glomerulonephritis, autoimmune diabetes,
autoimmune thrombocytopenic purpura, Grave's disease, Hashimoto's thyroiditis,
etc.),
cardiomyopathy (e.g., dilated cardiomyopathy), diabetes, diabetic
complications (e.g.,
diabetic nephropathy, diabetic neuropathy, diabetic retinopathy), influenza,
asthma,
psoriasis, glomerulonephritis, septic shock, and ulcerative colitis.
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[0430] Antibodies and antibody compositions of the invention are useful in
promoting
angiogenesis, wound healing (e.g., wounds, burns, and bone fractures).
[0431] Antibodies and antibody compositions of the invention are also useful
as an
adjuvant to enhance immune responsiveness to specific antigen, such as in anti-
viral
immune responses.
[0432] More generally, antibodies and antibody compositions of the invention
are
useful in regulating (i.e., elevating or reducing) immune response. For
example,
antibodies and antibody compositions of the invention may be useful in
preparation or
recovery from surgery, trauma, radiation therapy, chemotherapy, and
transplantation, or
may be used to boost immune response and/or recovery in the elderly and
immunocompromised individuals. Alternatively, antibodies and antibody
compositions of
the invention are useful as immunosuppressive agents, for example in the
treatment or
prevention of autoimmtme disorders. In specific embodiments, antibodies and
antibody
compositions of the invention are used to treat or prevent chronic
inflammatory, allergic or
autoimmune conditions, such as those described herein or are otherwise known
in the art.
Therapeutic/Prophylactic Compositions and Administration
[0433] The invention provides methods of treatment, inhibition and prophylaxis
by
administration to a subject of an effective amount of antibody (or fragment or
variant
thereof) or pharmaceutical composition of the invention, preferably an
antibody of the
invention. In a preferred aspect, an antibody or fragment or variant thereof
is substantially
purified (i.e., substantially free from substances that limit its effect or
produce undesired
side-effects). The subject is preferably an animal, including but not limited
to, animals
such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a
mammal, and
most preferably a human.
[0434] Formulations and methods of administration that can be employed when
the
compound comprises a nucleic acid or an immunoglobulin are described above;
additional
appropriate formulations and routes of administration can be selected from
among those
described herein below.
[0435] Various delivery systems are known and can be used to administer
antibody or
fragment or variant thereof of the invention, e.g., encapsulation in
liposomes,
microparticles, microcapsules, recombinant cells capable of expressing the
antibody or
antibody fragment, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.
Biol. Chem.
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral
or other
vector, etc. Methods of introduction include, but are not limited to,
intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,
intracerebral,
epidural, and oral routes. The compositions may be administered by any
convenient route,
for example by infusion or bolus injection, by absorption through epithelial
or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.)
and may be
administered together with other biologically active agents. Administration
can be
systemic or local. In addition, it may be desirable to introduce the
pharmaceutical
compositions of the invention into the central nervous system by any suitable
route,
including intraventricular and intrathecal injection; intraventricular
injection may be
facilitated by an intraventricular catheter, for example, attached to a
reservoir, such as an
Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use
of an
inhaler or nebulizer, and formulation with an aerosolizing agent.
[0436] In a specific embodiment, it may be desirable to administer the
pharmaceutical
compositions of the invention locally to the area in need of treatment; this
may be
achieved by, for example, and not by way of limitation, local infusion during
surgery,
topical application, e.g., in conjunction with a wound dressing after surgery,
by injection,
by means of a catheter, by means of a suppository, or by means of an implant,
said
implant being of a porous, non-porous, or gelatinous material, including
membranes, such
as sialastic membranes, or fibers. Preferably, when administering a protein,
including an
antibody, of the invention, care must be taken to use materials to which the
protein does
not absorb.
[0437] In another embodiment, the composition can be delivered in a vesicle,
in
particular a liposome (see Langer, Science 249:1527-1535 (1990); Treat et al.,
in
Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler
(eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 3 17-
327; see
generally ibid.).
[0438] In yet another embodiment, the composition can be delivered in a
controlled
release system. In one embodiment, a pump may be used (see Langer, supra;
Sefton, CRC
Crit. Ref. Biomed. Eng. 14:20 1 (1987); Buchwald et al., Surgery 88:507
(1980); Saudek
et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric
materials can
be used (see Medical Applications of Controlled Release, Langer and Wise
(eds.), CRC
Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug
Product Design
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and
Peppas,
J., Macromol. Sci. Rev. Macromol. Chem. 23:71 (1983); see also Levy et al.,
Science
228:190 (1985); During et al., Ann. Neurol. 25:35 1 (1989); Howard et al.,
J.Neurosurg. 7
1:105 (1989)). In yet another embodiment, a controlled release system can be
placed in
proximity of the therapeutic target, i.e., the brain, thus requiring only a
fraction of the
systemic dose (see, e.g., Goodson, in Medical Applications of Controlled
Release, supra,
vol. 2, pp. 115-138 (1984)).
[0439] Other controlled release systems are discussed in the review by Langer
(Science 249:1527-1535 (1990)).
[0440] In a specific embodiment where the composition of the invention is a
nucleic
acid encoding a protein, the nucleic acid can be administered in vivo to
promote
expression of its encoded protein, by constructing it as part of an
appropriate nucleic acid
expression vector and administering it so that it becomes intracellular, e.g.,
by use of a
retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or
by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating
with lipids or
cell-surface receptors or transfecting agents, or by administering it in
linkage to a
homeobox- like peptide which is known to enter the nucleus (see e.g., Joliot
et al., Proc.
Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid
can be
introduced intracellularly and incorporated within host cell DNA for
expression, by
homologous recombination.
[0441] The present invention also provides pharmaceutical compositions. Such
compositions comprise a therapeutically effective amount of an antibody or a
fragment
thereof, and a pharmaceutically acceptable carrier. In a specific embodiment,
the term
"pharmaceutically acceptable" means approved by a regulatory agency of the
Federal or a
state government or listed in the U.S. Pharmacopeia or other generally
recognized
pharmacopeia for use in animals, and more particularly in humans. The term
"carrier"
refers to a diluent, adjuvant, excipient, or vehicle with which the
therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids, such as
water and oils,
including those of petroleum, animal, vegetable or synthetic origin, such as
peanut oil,
soybean oil, mineral oil, sesame oil and the like. Water is a preferred
carrier when the
pharmaceutical composition is administered intravenously. Saline solutions and
aqueous
dextrose and glycerol solutions can also be employed as liquid carriers,
particularly for
injectable solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose,
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sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,
glycerol monostearate,
talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the
like. The composition, if desired, can also contain minor amounts of wetting
or
emulsifying agents, or pH buffering agents. These compositions can take the
form of
solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-
release
formulations and the like. The composition can be formulated as a suppository,
with
traditional binders and carriers such as triglycerides. Oral formulation can
include
standard carriers such as pharmaceutical grades of mannitol, lactose, starch,
magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of
suitable
pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences"
by E.W.
Martin. Such compositions will contain a therapeutically effective amount of
the antibody
or fragment thereof, preferably in purified form, together with a suitable
amount of carrier
so as to provide the form for proper administration to the patient. The
formulation should
suit the mode of administration.
[0442] In a preferred embodiment, the composition is formulated in accordance
with
routine procedures as a pharmaceutical composition adapted for intravenous
administration to human beings. Typically, compositions for intravenous
administration
are solutions in sterile isotonic aqueous buffer. Where necessary, the
composition may
also include a solubilizing agent and a local anesthetic such as lignocamne to
ease pain at
the site of the injection. Generally, the ingredients are supplied either
separately or mixed
together in unit dosage form, for example, as a dry lyophilized powder or
water free
concentrate in a hermetically sealed container such as an ampoule or sachette
indicating
the quantity of active agent. Where the composition is to be administered by
infusion, it
can be dispensed with an infusion bottle containing sterile pharmaceutical
grade water or
saline. Where the composition is administered by injection, an ampoule of
sterile water for
injection or saline can be provided so that the ingredients may be mixed prior
to
administration.
[0443] The compositions of the invention can be formulated as neutral or salt
forms.
Pharmaceutically acceptable salts include those formed with anions such as
those derived
from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those
formed with
cations such as those derived from sodium, potassium, ammonium, calcium,
ferric
hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
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[0444] The amount of the composition of the invention which will be effective
in the
treatment, inhibition and prevention of a disease or disorder associated with
aberrant
expression and/or activity of a polyp eptide of the invention can be
determined by standard
clinical techniques. In addition, in vitro assays may optionally be employed
to help
identify optimal dosage ranges. The precise dose to be employed in the
formulation will
also depend on the route of administration, and the seriousness of the disease
or disorder,
and should be decided according to the judgment of the practitioner and each
patient's
circumstances. Effective doses may be extrapolated from dose-response curves
derived
from in vitro or animal model test systems.
[0445] For antibodies, the dosage administered to a patient is typically 0.1
mg/kg to
100 mg/kg of the patient's body weight. Preferably, the dosage administered to
a patient
is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more
preferably 1 mg/kg
to 10 mg/kg (e.g., 3 mg/kg or 5 mg/kg) of the patient's body weight.
Generally, human
antibodies have a longer half-life within the human body than antibodies from
other
species due to the immune response to the foreign polypeptides. Thus, lower
dosages of
human antibodies and less frequent administration is often possible. Further,
the dosage
and frequency of administration of therapeutic or pharmaceutical compositions
of the
invention may be reduced by enhancing uptake and tissue penetration (e.g.,
into the brain)
of the antibodies by modifications such as, for example, lipidation.
[0446] Antibodies of the invention may be formulated in pharmaceutically
acceptable
carriers. A formulation of an antibody of the invention may comprise a buffer.
Buffers
are well-known in the art and may be routinely applied to maintain the desired
pH of the
solution compositions of the invention. Suitable buffers for use in the
preparation of a
pharmaceutical composition of the invention include, for example, those
described below.
[0447] Suitable buffers for use in the preparation of a antibody composition
of the
invention may include, but are not limited to, citrate, acetate, phosphate,
carbonate,
diphosphate, glycyl-glycine-piperazine-2HC1-NaOH; MES-Na0H-NaC1; TRIS-malic
acid-NaOH; MES-NaOH; ACES-NaOH-NaCl; BES-NaOH-NaCl; MOPS-NaOH-NaCl;
TES-NaOH-NaCl; MOPS-KOH; HEPES-Na0H-NaC1; TRIS-HC1; HEPPSO-NaOH;
TAPS-NaOH-NaCl; HEPPS (EPPS)-NaOH; citric acid-disodiumhydrogenphosphate;
boric acid-citric acid-potassium dihydrogen phosphate-Diethyl- barbituric acid-
NaOH;
citric acid-sodium citrate; sodium acetate-acetic acid; histidine; phosphate;
potassium
hydrogenphthalate-NaOH; cacodylic acid sodium salt-HC1; potassium dihydrogen
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phosphate-di sodium hydro genphosphate; potassium dihydro gen-phosphate-Na0H;
sodium dihydrogen phosphate- disodium hydrogen phosphate; imidazole-HC1;
sodium
tetraborate-boric acid; 2-amino-2-methyl-1,3-propanediol-HC1; diethanolamine-
HC1;
potassium chloride-boric acid-NaOH; boric acid-NaOH-KC1; glycine-NaOH; and
sodium
carbonate-sodium hydrogen carbonate.
[0448] In one embodiment, the buffer is a citrate buffer or an acetate buffer.
In
another embodiment, the buffer includes an acetate buffer having a
concentration of about
1 to about 50 mM and having a NaC1 concentration of about 1 to about 500 mM.
In
another embodiment, the buffer includes an acetate buffer having a
concentration of about
mM and having a NaC1 concentration of about 140 mM. Suitable acetate buffers
include acetate buffers having a concentration of about 1, 20, 25, 50, 75,
100, 200, 250,
300, 400, or 500 mM. Suitable buffers and solutions include those having a
NaC1
concentration of about 1, 50, 75, 100, 125, 140, 150, 175, 200, 225, 250, 275,
300, 350,
400, 450, or 500 mM. An additional suitable buffer is a HEPES buffer, in
particular a
HEPES buffer having a concentration of about 10, 20, 30, 40, 50, 60, 70, 80,
90, or 100
mM. In an additional embodiment, the solution comprises a HEPES buffer having
a
concentration of about 50 mM.
[0449] In other embodiments, antibodies of the invention are formulated in a
citrate
buffered solution that has a pH in the range of 5.5 to 6.5. In further
embodiments,
antibodies of the invention are formulated in a citrate buffered solution that
has a pH of
approximately or exactly 6Ø In other embodiments, antibodies of the
invention are
formulated in a citrate buffered solution that has a pH in the range of 5.5 to
6.5 and which
also contains between 0 and 2.0%, preferably between 0 and 0.1% and more
preferably
less than 0.05%, of a surfactant such as Tweeir80 or polysorbate 80.
[0450] In one embodiment, antibodies of the invention are formulated in 10 mM
sodium citrate, 1.9% glycine, 0.5% sucrose, 0.02% polysorbate 80, pH 6.5.
[0451] In other embodiments, antibodies of the invention are formulated in a
histidine
buffered solution that has a pH in the range of 6.5 to 7.5. In other
embodiments,
antibodies of the invention are formulated in a histidine buffered solution
that has a pH in
the range of 6.5 to 7.5 and which also contains between 0 and 2.0%, preferably
between 0
and 0.1% and more preferably less than 0.05%, of a surfactant such as Tv.veeir
80 or
polysorbate 80.
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CA 02494372 2010-11-17
[04521 In other embodiments, antibodies of the invention are formulated
in a
phosphate buffered solution that has a pH in the range of 7.0 to 8Ø In other
embodiments, antibodies of the invention are formulated in a phosphate
buffered solution
that has a pH in the range of 7.0 to 8.0 and which also contains between 0 and
2.0%,
preferably between 0 and 0.1% and more preferably less than 0.05%, of a
surfactant such
as Tweeirim80 or polysorbate 80.
[0453] Generally, administration of products of a species origin or
species reactivity
(in the case of antibodies) that is the same species as that of the patient is
preferred. Thus,
in a preferred embodiment, human antibodies, fragments, or variants, (e.g.,
derivatives), or
nucleic acids, are administered to a human patient for therapy or prophylaxis.
[0454] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or
neutralizing antibodies of the invention (including molecules comprising, or
alternatively
consisting of, antibody fragments or variants thereof) that immunospecifically
bind to
TR4, or polynucleotides encoding antibodies that immunospecifically bind to
TR4, for
both immunoassays and therapy of disorders related to TR4 polynucleotides or
polypeptides, including fragments thereof. Such antibodies will preferably
have an
affinity for TR4 and/or TR4 polyp eptide fragments. Preferred binding
affinities include
those 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, 5 X 104 M, 10-4 M, 5 X 105 M, or 10-5 M. More preferably,
antibodies of
the invention bind TR4 polypeptides or fragments or variants thereof with a
dissociation
constant or KD less than or equal to 5 X 11316 M, 1016 M, 5 X 10-7 M, 1(117M,
5 X 10-8M, or
10-8 M. Even more preferably, antibodies of the invention bind TR4
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 1040 M, 1(110M, 5 X 1041 M, 1C111 iv/, 5 1(112 M 10112 M,
5 X 43M,
10-13M, 5 10-14 m, 10-14 M, 5 X 105 M, or 10-15 M. In a preferred
embodiment,
antibodies of the invention induce apoptosis of TR4 expressing cells.
[0455] As discussed in more detail below, the antibodies of the present
invention may
be used either alone or in combination with other compositions. The antibodies
may
further be recombinantly fused to a heterologous polypeptide at the N- or C-
terminus or
chemically conjugated (including covalent and non-covalent
conjugations) to
polypeptides or other compositions. For example, antibodies of the present
invention may
be recombinantly fused or conjugated to molecules useful as labels in
detection assays and
effector molecules such as heterologous polypeptides, drugs, radionuclides, or
toxins. See,
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e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No.
5,314,995; and EP 396,387.
[0456] The antibody and antibody compositions of the invention may be
administered
alone or in combination with other therapeutic agents, including but not
limited to
chemotherapeutic agents, antibiotics, antivirals, anti-retroviral agents,
steroidal and non-
steroidal anti-inflammatories, conventional immunotherapeutic agents and
cytokines.
Combinations may be administered either concomitantly, e.g., as an admixture,
separately
but simultaneously or concurrently; or sequentially. This includes
presentations in which
the combined agents are administered together as a therapeutic mixture, and
also
procedures in which the combined agents are administered separately but
simultaneously,
e.g., as through separate intravenous lines into the same individual.
Administration "in
combination" further includes the separate administration of one of the
compounds or
agents given first, followed by the second.
[0457] In preferred embodiments, antibodies of the invention that are
administered to
an animal, preferably a human, for therapeutic uses are multimeric antibodies.
In specific
embodiments, antibodies of the invention are homodimeric IgG molecules. In
other
specific embodiments, antibodies of the invention are homodimeric IgG1
molecules. In
specific embodiments, antibodies of the invention are homotrimeric IgG
molecules. In
other specific embodiments, antibodies of the invention are trimeric IgG1
molecules. In
other specific embodiments, antibodies of the invention are higher-order
multimers of IgG
molecules (e.g., tetramers, penatmers and hexamers]. In still further specific
embodiments, antibodies of the IgG molecules comprising the higher order
multimers of
IgG molecules are IgG1 molecules.
[0458] Alternatively, antibodies of the invention for therapeutic uses may
be
administered in combination with crosslinking agents known in the art,
including but not
limited to, anti-IgG antibodies.
Combination Therapies with anti-TR4 antibodies, TRAIL, Apoptosis
Inducing Peptides and/or Chemotherapeutic agents
[0459] Anti-TR4 antibodies may be administered in combination with other
anti-TR4
antibodies, TRAIL, chemotherapeutics and/or other therapeutic agents useful in
the
treatment of cancers.
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[0460] In specific embodiments, an antibody of the invention that specifically
binds
TR4 is used or administered in combination with a second antibody that
specifically binds
TR7. In another embodiment, the antibodies specific for TR4 and TR7 are
agonistic
antibodies that induce apoptosis of TR4 expressing cells (e.g., cells that
express TR4 and
TR7). In a specific embodiment, the combination of anti-TR4 treatment and anti-
TR7
treatment induces more apoptosis of TR4 and TR7 expressing cells than either
anti-TR4
antibody treatment or anti-TR7 antibody treatment alone. The anti-TR4 and anti-
TR7
antibodies can be administered either simultaneously, sequentially, or a
combination of
simultaneous or sequential administration throughout the dosage regimen. In
another
specific embodiment anti-TR4 and anti-TR7 antibodies are used or administered
in
combination with a chemotherapeutic drug, such as those described herein (see,
for
example, below and Example 4). In a particular embodiment, the synergistic
induction of
apoptosis resulting from anti-TR4 and anti-TR7 antibody treatment, is more
evident or
more pronounced when the anti-TR4 and anti-TR7 antibodies are used or
administered in
combination with a chemotherapeutic agent and/or a cross-linking reagent.
104611 In a preferred embodiment, compositions of the invention are
administered in
combination with a chemotherapeutic agent. Chemotherapeutic agents that may be
administered with the compositions of the invention include, but are not
limited to,
antibiotic derivatives (e.g., doxombicin (adriamycin), bleomycin,
daunorubicin, and
dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g.,
fluorouracil, 5-FU,
methotrexate, floxtuidine, interferon alpha-2b, glutamic acid, plicamycin,
mercaptopurine,
and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU,
cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea,
procarbazine,
mitomycin, busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,
medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,
estradiol,
megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate,
chlorotrianisene, and
testolactone); nitrogen mustard derivatives (e.g., mephalen, chorarnbucil,
mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations
(e.g.,
bethamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase,
mitotane,
vincristine sulfate, vinblastine sulfate, etoposide, Topotecan, 5-
Fluorouracil, paclitaxel
(Tax), Cisplatin, Cytarabine, and IFN-gamma, irinotecan (Camptosair,m CPT-11),
irinotecan analogs, gemcitabine ((GEMZARTm), and oxaliplatin, ifosamide,
nitosourea
compounds).
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[0462] Therapeutic agents, useful in the treatment, prevention, amelioration
and/or
cure of cancers, with which antibodies of the present invention may be
administered,
include, for example, biological agents (e.g., inhibitors of signaling
pathways, inhibitors of
gene transcription, inhibitors of multi-drug resistance (MDR) mechanisms,
inhibitors of
angiogenesis, inhibitors of matrix metalloproteinases, proteasome inhibitors,
hormones
and hormone antagonists, and compounds of unknown mechanism), chemotherapeutic
agents (e.g., alkylating agents, antimetabolites, farnesyl transferase
inhibitors, mitotic
spindle inhibitors (plant-derived alkaloids), nucleotide analogs, platinum
analogs, and
topoisomerase inhibitors), corticosteroids, gene therapies, immunotherapeutic
agents (e.g.,
monoclonal antibodies, cytokines and vaccines), phototherapy, radiosensitizing
agents,
treatment support agents (e.g., anti-emetic agents, analgesic agents and
hematopoietic
agents), and other miscellaneous drug types. Therapeutic procedures, useful in
the
treatment, prevention, amelioration and/or cure of cancers, with which
agonistic
antibodies of the present invention may be administered, include, for example,
but are not
limited to, surgical procedures and radiation therapies.
[0463] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, prevention, amelioration and/or cure of cancers.
[0464] In specific embodiments, antibodies of the present invention may be
administered in combination with one or more chemotherapeutic or other
therapeutic
agents useful in the treatment, prevention, amelioration and/or cure of
cancers including,
but not limited to, 8106 (Anti-tenascin monoclonal antibody), 2-
chlorodeoxyadenosine,
A007 (4-4'-dihydroxybenzophenone-2, 4-dinitrophenylhydrazone), Abarelix
(Abarelix-
Depot-M , PPI-149, R-3827); Abiraterone acetate (CB-7598, CB-7630), ABT-627
(ET-
1 inhibitor), ABX-EGF (anti-EGFr MAb), Acetyldinaline (CI-994, GOE-5549, GOR-
5549, PD-130636), AG-2034 (AG-2024, AG-2032, GARFT [glycinamide ribonucleoside
transformylase] inhibitor), Alanosine, Aldesleukin (IL-2, Proleukin0),
Alemtuzumab
(CampathO), Alitretinoin (Panretin , LGN-1057), Allopurinol (Aloprim ,
ZyloprimS),
Altretamine (Hexalen , hexamethylmelamine, Hexastat0), Amifostine (Ethyol ),
Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC 603071), Aminoglutethimide
(CytadrenO), Aminolevulinic acid (Levulan , Kerastick8), Aminopterin,
Amsacrine,
Anastrozole (Arimidex ), Angiostatin, Annamycin (AR-522, annamycin LF, Aronex
),
Anti-idiotype therapy (BsAb), Anti-CD19/CD3 MAb (anti-CD19/CD3 scFv, anti-NHL
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MAb), APC-8015 (Provenge , Dendritic cell therapy), Aplidine (AplidinC,
AplidinaC),
Arabinosylguanine (Ara-G, GW506U78, Nelzarabine , Compound 506U78), Arsenic
trioxide (Trisenox , ATO, AtrivexC), Avorelin (Metereline, MF-6001, EP-
23904),
B43-Genistein (anti-CD19 Ab/genistein conjugate), B43-PAP (anti-CD19
Ab/pokeweed
antiviral protein conjugate), B7 antibody conjugates, BAY 43-9006 (Raf kinase
inhibitor),
BBR 3464, Betathine (Beta-LT), Bevacizumab (Anti-VEGF monoclonal antibody,
rhuMAb-VEGF), Bexarotene (TargretinC, LGD1069), BIBH-1 (Anti-FAP MAb), BIBX-
1382, Biclutamide (CasodexC), Biricodar dicitrate (Incel , Incel MDR
Inhibitor),
Bleomycin (BlenoxaneC), BLP-25 (MUC-1 peptide), BLyS antagonists, BMS-214662
(BMS-192331, BMS-193269, BMS-206635), BNP-1350 (BNPI-1100, Karenitecins),
Boronated Protoporphyrin Compound (PDIT, Photodynamic Immunotherapy),
Bryostatin-
1 (BryostatinC, BMY-45618, NSC-339555), Budesonide (Rhinocort0), Busulfan
(Busulfex , MyleranS), C225 (IMC-225, EGFR inhibitor, Anti-EGFr MAb,
CetuximabC), C242-DM1 (huC242-DM1), Cabergoline (Dostinex8), Capecitabine
(Xeloda , Doxifluridine , oral 5-FU), Carbendazin (FB-642), Carboplatin
(Paraplatin , CBDCA), Carboxyamidotriazole (NSC 609974, CAI, L-651582),
Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer ), CC49-zeta gene therapy, CEA-
cide (Labetuzumab0, Anti-CEA monoclonal antibody, hMN-14), CeaVacC (MAb
3H1), Celecoxib (CelebrexC), CEP-701 (KT-5555), Cereport (Lobradimil , RMP-
7),
Chlorambucil (LeukeranS), CHML (Cytotropic Heterogeneous Molecular Lipids),
Cholecaliferol, CI-1033 (Pan-erbB RTK inhibitor), Cilengitide (EMD-121974,
integrin
alphavbeta3 antagonist), Cisplatin (Platinol , CDDP), Cisplatin-epinephrine
gel
(IntraDose , FocaCist8), Cisplatin-liposomal (SPI-077), 9-cis retinoic acid (9-
cRA),
Cladribine (2-CdA, LeustatinS), Clofarabine (chloro-fluoro-araA), Clonadine
hydrochloride (Duraclon0), CMB-401 (Anti-PEM MAb/calicheamycin), CMT-3 (COL-3,
MetastatS), Cordycepin, Cotara0 (chTNT-1/B, [131I]-chTNT-1/B), CN-706, CP-
358774
(Tarceva , OSI-774, EGFR inhibitor), CP-609754, CP IL-4-toxin (IL-4 fusion
toxin), CS-
682, CT-2584 (Apra , CT-2583, CT-2586, CT-3536), CTP-37 (Avicine C, hCG
blocking
vaccine), Cyclophosphamide (CytoxanC, NeosarC, CTX), Cytarabine (Cytosar-U ,
ara-
C, cytosine arabinoside, DepoCytC), D-limonene, DAB389-EGF (EGF fusion toxin),
Dacarbazine (DTIC), Daclizumab0 (Zenapax0), Dactinomycin (Cosmegene),
Daunomycin (DaunorubicinC, CerubidineC), Daunorubicin (DaunoXome ,
DaunorubicinC, Cerubidine ), DeaVac (CEA anti-idiotype vaccine), Decitabine
(5-aza-
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2'-deoxyytidine), Declopramide (Oxi-104), Denileukin diftitox (OntakC),
Depsipeptide
(FR901228, FK228), Dexamethasone (DecadronC), Dexrazoxane (ZinecardC),
Diethylnorspermine (DENSPM), Diethylstilbestrol (DES), Dihydro-5-azacytidine,
Docetaxel (Taxotere , TaxaneC), Dolasetron mesylate (AnzemetC), Dolastatin-10
(DOLA-10, NSC-376128), Doxorubicin (Adriamycin , Rubex8), DPPE, DX-
8951f (DX-8951), Edatrexate, EGF-P64k Vaccine, Elliott's B Solution , EMD-
121974,
Endostatin, Eniluracil (776c85), E09 (E01, E04, E068, E070, E072), Epirubicin
(Ellence , EPI, 4' epi-doxorubicin), Epratuzumab (LymphocideC, humanized anti-
CD22, HAT), Erythropoietin (EPOC, Epogen , ProcritC), Estramustine (Emcyt6),
Etanidazole (Radinyle), Etoposide phosphate (Etopophos ), Etoposide (VP-16,
VepesidC), Exemestane (Aromasin , Nikidess0), Exetecan mesylate (DX-8951, DX-
8951f), Exisulind (SAAND, Aptosyn , cGMP-PDE2 and 5 inhibitor), F19 (Anti-FAP
monoclonal antibody, iodinated anti-FAP MAb), Fadrozole (Afema , Fadrozole
hydrochloride, Arensing), Fenretinide (4HPR), Fentanyl citrate (Actiq0),
Filgrastim
(Neupogen , G-CSF), FK-317 (FR-157471, FR-70496), Flavopiridol (HMR-1275),
F1y3/f1k2 ligand (Mobista8), Fluasterone, Fludarabine (Fludara , FAMP),
Fludeoxyglucose (F-18 ), Fluorouracil (5-FU, Adrucil , Fluoroplex , Efudex8),
Flutamide (Eulexin0), FMdC (KW-2331, MDL-101731), Formestane (LentaronC),
Fotemustine (Muphoran , MustophoranC), FUDR (Floxuridine0), Fulvestrant
(Faslodex8), G3139 (Genasense , GentaAnticode , Bc1-2 antisense), Gadolinium
texaphyrin (Motexafin gadolinium, Gd-Tex , XcytrinC), Galarubicin
hydrochloride (DA-
125), GBC-590, Gastrimmune (Anti-gastrin-17 immunogen, anti-gl 7),
Gemcitabine
(Gemto , Gemzar0), Gentuzumab-ozogamicin (Mylotarg ), GL331, Globo H
hexasaccharide (Globo H-KLH0), Glufosfamide (13-D-g1ucosyl-isofosfamide
mustard,
D19575, INN), Goserelin acetate (Zoladexe), Granisetron (Kytri1C), GVAX (GM-
CSF
gene therapy), Her-2/Neu vaccine, Herceptin (Trastuzumab , Anti-HER-2
monoclonal
antibody, Anti-EGFR-2 MAb), HSPPC-96 (HSP cancer vaccine, gp96 heat shock
protein-
peptide complex), Hu1D10 (anti-HLA-DR MAb, SMART 1D10), HumaLYM (anti-CD20
MAb), Hydrocortisone, Hydroxyurea (Hydrea ), Hypericin (VIMRxyne), 1-131
Lipidiol , Ibritumomab tiuxetan (ZevalinC), Idarubicin (IdamycinC, DMDR,
IDA),
Ifosfamide (IFEXC), Imatinib mesylate (STI-571, Imatinib , Glivec , Gleevec ,
Abl
tyrosine kinase inhibitor), INGN-101 (p53 gene therapy/retrovirus), INGN-201
(p53 gene
therapy/adenovirus), Interferon alpha (Alfaferone , Alpha-IF ), Interferon
alpha 2a
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(Intron MD), Interferon gamma (Gamma-interferon, Gamma 1000, Gamma-IF),
Interleukin-2 (ProleiukinR0), Intoplicine (RP 60475), Irinotecan (Camptosar ,
CPT-11,
Topotecin , CaptoCPT-1), Irofulven (MGI-114, Ivofulvan, Acylfulvene analogue),
ISIS-
2053 (PKC-alpha antisense), ISIS-2503 (Ras antisense), ISIS-3521 (PKC-alpha
antisense),
ISIS-5132 (K-ras/raf antisense), Isotretinoin (13-CRA, 13-cis retinoic acid,
AccutaneS),
Ketoconazole (NizoralC), KRN-8602 (MX, MY-5, NSC-619003, MX-2), L-778123 (Ras
inhibitors), L-asparaginase (Elspar , Crastining, Asparaginase medacC,
Kidrolasee),
Leflunomide (SU-101, SU-0200), Letrozole (Femara0), Leucovorin (Leucovorin ,
WellcovorinC), Leuprolide acetate (Viadur , Lupron , Leuprogel , Eligard8),
Leuvectin (cytofectin + IL-2 gene, IL-2 gene therapy), Levamisole
(Ergamisole),
Liarozole (Liazal, Liazol, R-75251, R-85246, Ro-85264), Lmb-2 immunotoxin
(anti-
CD25 recombinant immuno toxin, anti-Tac(Fv)-PE38), Lometrexol (T-64, T-
904064),
Lomustine (CCNUS, CeeNUO), LY-335979, Lym-1 (131-I LYM-1), Lymphoma vaccine
(Genitope), Mannan-MUC1 vaccine, Marimastat (BB-2516, TA-2516, MMP
inhibitor),
MDX-447 (MDX-220, BAB-447, EMD-82633, H-447, anti-EGFr/FcGammaR1r),
Mechlorethamine (Nitrogen Mustard, HN2, Mustargen0), Megestrol acetate
(Megace0,
Panacea)), Melphalan (L-PAM, Alkeran , Phenylalanine mustard), Mercaptopurine
(6-
mercaptopurine, 6-MP), Mesna (MesnexO), Methotexate0 (MTX, Mexate , FolexC),
Methoxsalen (Uvadex0), 2-Methoxyestradiol (2-ME, 2-ME2), Methylprednisolone
(Solumedro10), Methyltestosterone (Android-10C, Tested , VirilonC), MGV,
Mitomycin C (MitomycinC, Mutamycin , Mito Extra ), Mitoxantrone (Novantrone0,
DHAD), Mitumomab0 (BEC-2, EMD-60205), Mivobulin isethionate (CI-980), MN-14
(Anti-CEA immunoradiotherapy, 131I-MN-14, 188Re-MN-14), Motexafin Lutetium
(Lutrin , OptrinC, Lu-Tex , lutetium texaphyrin, LucynC, AntrinS), MPV-2213ad
(FinrozoleS), MS-209, Muc-1 vaccine, NaPro Paclitaxel, Nelarabine (Compound
506,
U78), Neovastat (AE-941, MMP inhibitor), Neugene compounds (Oncomyc-NG,
Resten-NG, myc antisense), Nilutamide (Nilandron8), NovoMAb-G2 scFv (NovoMAb-
G2 IgM), 06-benzylguanine (BG, Procept6), Octreotide acetate (Sandostatin LARC
Depot), Odansetron (Zofrane), Onconase (RanpirnaseC), OncoVAX-CL, OncoVAX-CL
Jenner (GA-733-2 vaccine), OncoVAX-P (OncoVAX-PrPSA), Onyx-015 053 gene
therapy), Oprelvekin (Neumage6), Orzel (Tegafur + Uracil + Leucovorin),
Oxaliplatin
(Eloxatine , EloxatinC), Pacis (BCG, live), Paclitaxel (Paxene , TaxolO),
Paclitaxel-
DHA (TaxoprexinC), Pamidronate (Aredia8), PC SPES, Pegademase (Adagen ,
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Pegademase bovine), Pegaspargase (OncosparS), Peldesine (BCX-34, PNP
inhibitor),
Pemetrexed disodium (Alimta , MTA, multitargeted antifo late, LY 231514),
Pentostatin
(Nipent , 2-deoxycoformycin), Perfosfamide (4-hydroperoxycyclophosphamide, 4-
HC),
Perillyl alcohol (perilla alcohol, perillic alcohol, perillol, NSC-641066),
Phenylbutyrate,
Pirarubicin (THP), Pivaloyloxymethyl butyrate (AN-9, PivanexC), Porfimer
sodium
(Photofrine), Prednisone, Prinomastat (AG-3340, MMP inhibitor), Procarbazine
(MatulaneC), PROSTVAC, Providence Portland Medical Center Breast Cancer
Vaccine,
PS-341 (LDP-341, 26S proteasome inhibitor), PSMA MAb (Prostate Specific
Membrane
Antigen monoclonal antibody), Pyrazoloacridine (NSC-366140, PD-115934),
Quinine,
R115777 (Zarnestrag), Raloxifene hydrochloride (Evista , Keoxifene
hydrochloride),
Raltitrexed (Tomudex , ZD-1694), Rebeccamycin, Retinoic acid, R-flurbiprofen
(FlurizanO, E-7869, MPC-7869), RFS-2000 (9-nitrocamptothecan, 9-NC,
rubitecang),
Rituximab (RituxanC, anti-CD20 MAb), RSR-13 (GSJ-61), Satraplatin (BMS-
182751,
JM-216), SCH 6636, SCH-66336, Sizofilan (SPG, SizofiranC, Schizophyllan ,
SonifilanC), SKI-2053R (NSC-D644591), Sobuzoxane (MST-16, Perazoling),
Squalamine (MSI-1256F), SR-49059 (vasopressin receptor inhibitor, Via),
Streptozocin
(ZanosarS), SU5416 (Semaxanib , VEGF inhibitor), SU6668 (PDGF-TK inhibitor), T-
67 (T-138067, T-607), Talc (Sclerosol0), Tamoxifen (Nolvadex8), Taurolidine
(TaurolinC), Temozolamide (Temodar , NSC 362856), Teniposide (VM-26, Vumon0),
TER-286, Testosterone (Andro , Androderm , Testoderm TTS , Testoderm , Depo-
Testosterone , Andro gel , depoAndro 8) , Tf-CRM107 (Transferrin-CRM-107),
Thalidomide, Theratope, Thioguanine (6-thioguanine, 6-TG), Thiotep a
(triethylenethiophosphaoramide, Thioplex8), Thymo sin alpha I (Zadaxin ,
Thymalfasine), Tiazofurin (Tiazole ), Tirapazamine (SR-259075, SR-4233,
Tirazone ,
Win-59075), TNP-470 (AGM-1470, Fumagillin), Tocladesine (8-C1-cAMP), Topotecan
(Hycamtin , SK&F-104864, NSC-609699, EvotopinS), Toremifene (Estrimex ,
FarestonC), Tositumomab (Bexxar8), Tretinoin (Retin-A , Atragen , ATRA,
Vesanoid0), TriAb (anti-idiotype antibody immune stimulator), Trilostane
(Modrefen0), Triptorelin pamoate (Trelstar Depot , Decapepty18), Trimetrexate
(NeutrexinC), Troxacitabine (BCH-204, BCH-4556, Troxaty16), TS-1, UCN-01 (7-
hydroxystaurosporine), Valrubicin (Valstar8), Valspodar (PSC 833), Vapreotide
(BMY-
41606), Vaxid (B-cell lymphoma DNA vaccine), Vinblastine (Velban , VLB),
Vincristine (Oncovin , Onco TCSC, VCR, Leurocristine0), Vindesine (Eldisine ,
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Fildesin0), Vinorelbine (Navelbine6), Vitaxin (LM-609, integrin alphavbeta3
antagonistic MAb), WF10 (macrophage regulator), WHI-P131, WT1 Vaccine, )CR-
5000
(DACA), XR-9576 ()CR-9351, P-glycoprotein/MDR inhibitor), ZD-9331, ZD-1839
(IRESSAC), and Zoledronate (Zometae).
[0465] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, prevention, amelioration and/or cure of cancers. .
[0466] In further specific embodiments, antibodies of the present
invention may be
administered in combination with one or more combinations of therapeutic
agents useful
in the treatment, prevention, amelioration and/or cure of cancers including,
but not limited
to, 9-aminocamptothecin + G-CSF, Adriamycin + Blenoxane + Vinblastine +
Dacarbazine (ABVD), BCNU (Carmustine) + Etoposide + Ara-C (Cytarabine) +
Melphalen (BEAM), Bevacizumab + Leucovorin, Bleomycin + Etoposide + Platinol
(Cisplatin) (BEP), Bleomycin + Etoposide + Adriamycin + Cyclophosphamide +
Vincristine + Procarbazine + Prednisone (BEACOPP), Bryostatin + Vincristine,
Busulfan
+ Melphalan, Carboplatin + Cereport , Carboplatin + Cyclophosphamide,
Carboplatin +
Paclitaxel, Carboplatin + Etoposide + Bleomycin (CEB), Carboplatin + Etoposide
+
Thiotepa, Cisplatin + Cyclophosphamide, Cisplatin + Docetaxel, Cisplatin +
Doxorubicin,
Cisplatin + Etoposide, Cisplatin + Gemcitabine, Cisplatin + Interferon alpha,
Cisplatin +
Irinotecan, Cisplatin + Paclitaxel, Cisplatin + Teniposide, Cisplatin +
Vinblastine,
Cisplatin + Vindesine, Cisplatin + Vinorelbine, Cisplatin + Cytarabine +
Ifosfamide,
Cisplatin + Ifosfamide + Vinblastine, Cisplatin + Vinblastine + Mitomycin C,
Cisplatin +
Vincristine + Fluorouracil, Cisplatin + Vincristine + Lomustine, Cisplatin +
Vinorelbine +
Gemcitabine, Cisplatin + Carmustine + Dacarbazine + Tamoxifen, Cisplatin +
Cyclophosphamide + Etoposide + Vincristine, Cisplatin (Platino10) + Oncovin +
Doxorubicin (Adriamycin ) + Etoposide (CODE), Cisplatin + Cytarabine +
Ifosfamide +
Etoposide + Methotrexate, Cyclophosphamide + Adriamycin (Doxorubicin),
Cyclophosphamide + Melphalan, Cyclophosphamide + SCH 6636, Cyclophosphamide +
Adriamycin + Cisplatin (Platinolg) (CAP), Cyclophosphamide + Adriamycin +
Vincristine (CAV), Cyclophosphamide + Doxorubicin + Teniposide + Prednisone,
Cyclophosphamide + Doxorubicin + Teniposide + Prednisone + Interferon alpha,
Cyclophosphamide + Epirubicin + Cisplatin (PlatinolO) (CEP), Cyclophosphamide
+
Epirubicin + Fluorouracil, Cyclophosphamide + Methotrexate + Fluoruracil
(CMF),
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Cyclophosphamide + Methotrexate + Vincristine (CMV), Cyclophosphamide +
Adriamycin + Methotrexate + Fluorouracil (CAMF), Cyclophosphamide +
Adriamycin + Methotrexate + Procarbazine (CAMP), Cyclophosphamide +
Adriamycin + Vincristine + Etoposide (CAV-E), Cyclophosphamide + Adriamycin
+
Vincristine + Prednisone (CHOP), Cyclophosphamide + Novantrone (Mitoxantrone)
+
Vincristine (Oncovorin) + Prednisone (CNOP), Cyclophosphamide + Adriamycin +
Vincristine + Prednisone + Rit-uximab (CHOP + Rituximab), Cyclophosphamide +
Adriamycin + Vincristine + Teniposide (CAV-T), Cyclophosphamide + Adriamycin
+
Vincristine alternating with Platinol + Etoposide (CAV/PE), Cyclophosphamide
+
BCNU (Carmustine) + VP-16 (Etoposide) (CBV), Cyclophosphamide+ Vincristine +
Prednisone (CVP), Cyclophosphamide + Oncovin + Methotrexate + Fluorouracil
(COMF), Cytarabine + Methotrexate, Cytarabine + Bleomycin + Vincristine +
Methotrexate (CytaBOM), Dactinomycin + Vincristine, Dexamethasone + Cytarabine
+
Cisplatin (DHAP), Dexamethasone + Ifosfamide + Cisplatin + Etoposide (DICE),
Docetaxel + Gemcitabine, Docetaxel + Vinorelbine, Doxorubicin + Vinblastine +
Mechlorethamine + Vincristine + Bleomycin + Etoposide + Prednisone (Stanford
V),
Epirubicin + Gemcitabine, Estramustine + Docetaxel, Estramustine + Navelbine,
Estramustine + Paclitaxel, Estramustine + Vinblastine, Etoposide (VepesidO) +
Ifosfamide + Cisplatin (PlatinolO) (VIP), Etoposide + Vinblastine + Adriamycin
(EVA),
Etoposide (VepesidO) + Ifosfamide + Cisplatin + Epirubicin (VIC-E), Etoposide
+
Methylprednisone + Cytarabine + Cisplatin (ESHAP), Etoposide + Prednisone +
Ifosfamide + Cisplatin (EPIC), Fludarabine + Mitoxantrone + Dexamethasone
(FMD),
Fludarabine + Dexamethasone + Cytarabine (ara-C) + Cisplatin (Platino10)
(FluDAP),
Fluorouracil + Bevacizumab , Fluorouracil + CeaVac , Fluorouracil +
Leucovorin,
Fluorouracil + Levamisole, Fluorouracil + Oxaliplatin, Fluorouracil +
Raltitrexed,
Fluorouracil + SCH 6636, Fluorouracil + Trimetrexate, Fluorouracil +
Leucovorin +
Bevacizumab , Fluorouracil + Leucovorin + Oxaliplatin, Fluorouracil +
Leucovorin +
Trimetrexate, Fluorouracil + Oncovin + Mitomycin C (F0Mi), Hydrazine +
Adriamycin + Methotrexate (HAM), Ifosfamide + Docetaxel, Ifosfamide +
Etoposide,
Ifosfamide + Gemcitabine, Ifosfamide + Paclitaxel, Ifosfamide + Vinorelbine,
Ifosfamide
+ Carboplatin + Etoposide (ICE), Ifosfamide + Cisplatin + Doxorubicin,
Irinotecan +
C225 (Cetuximab8), Irinotecan + Docetaxel, Irinotecan + Etoposide, Irinotecan
+
Fluorouracil, Irinotecan + Gemcitabine, Mechlorethamine + Oncovin
(Vincristine) +
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Procarbazine (MOP), Mechlorethamine + Oncovin (Vincristine) + Procarbazine +
Prednisone (MOPP), Mesna + Ifosfamide + Idarubicin + Etoposide (MIZE),
Methotrexate
+ Interferon alpha, Methotrexate + Vinblastine, Methotrexate + Cisplatin,
Methotrexate
with leucovorin rescue + Bleomycin + Adriamycin + Cyclophosphamide + Oncovorin
+
Dexamethasone (m-BACOD), Mitomycin C + Ifosfamide + Cisplatin (Platinole)
(MIP),
Mitomycin C + Vinblastine + Paraplatin (MVP), Mitoxantrone + Hydrocortisone,
Mitoxantrone + Prednisone, Oncovin + SCH 6636, Oxaliplatin + Leucovorin,
Paclitaxel
+ Doxorubicin, Paclitaxel + SCH 6636, Paraplatin + Docetaxel, Paraplatin +
Etoposide, Paraplatin + Gemcitabine, Paraplatin + Interferon alpha,
Paraplatin +
Irinotecan, Paraplatin + Paclitaxel, Paraplatin + Vinblastine, Carboplatin
(Paraplatin )
+ Vincristine, Paraplatin@ + Vindesine, Paraplatin + Vinorelbine, Pemetrexed
disodium
+ Gemcitabine, Platinol (Cisplatin) + Vinblastine + Bleomycin (PVB),
Prednisone +
Methotrexate + Adriamycin + Cyclophosphamide + Etoposide (ProMACE),
Procarbazine
+ Lomustine, Procarbazine + Lomustine + Vincristine, Procarbazine + Lomustine
+
Vincristine + Thioguanine, Procarbazine + Oncovin + CCNU + Cyclophosphamide
(POCC), Quinine + Doxorubicin, Quinine + Mitoxantrone + Cytarabine, Thiotepa +
Etoposide, Thiotepa + Busulfan + Cyclophosphamide, Thiotepa + Busulfan +
Melphalan,
Thiotepa + Etoposide + Carmustine, Thiotepa + Etoposide + Carboplatin,
Topotecan +
Paclitaxel, Trimetrexate + Leucovorin, Vinblastine + Doxorubicin + Thiotepa,
Vinblastine
+ Bleomycin + Etoposide + Carboplatin, Vincristine + Lomustine + Prednisone,
Vincristine (Oncovin ) + Adriamycin + Dexamethasone (VAD), Vincristine
(Oncovin ) + Adriamycin + Procarbazine (YAP), Vincristine + Dactinomycin +
Cyclophosphamide, and Vinorelbine + Gemcitabine.
[0467] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
combinations of
therapeutic agents in the treatment, prevention, amelioration and/or cure of
cancers.
[0468] In a specific embodiment, antibody and antibody compositions of the
invention
are administered in combination with CHOP (cyclophosphamide, doxorubicin,
vincristine,
and prednisone) or any combination of the components of CHOP. In another
embodiment, antibody and antibody compositions of the invention are
administered in
combination with Rituximab. In a further embodiment, antibody and antibody
compositions of the invention are administered with Rituximab and CHOP, or
lymphomaRituximab and any combination of the components of CHOP.
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[0469] In additional preferred embodiments, antibody compositions of the
invention
are administered in combination with Rituximab (RituxanTM) and/or Ibritumomab
Tiuxetan (ZevalinTM, e.g., either (In-111) Ibritumomab Tiuxetan or (Y-90)
Ibritumomab
Tiuxetan). In a specific embodiment, antibody compositions of the invention
are
administered in combination with Rituximab and/or Ibritumomab Tiuxetan for the
treatment of non-Hodgkin's lymphoma.
[0470] In additional preferred embodiments, antibody compositions of the
invention
are administered in combination with imatinib mesylate (GleevecO: 44(4-Methyl-
I-
piperazinyl)methyll -N-P-methy1-3 -[ [4-(3 -pyridiny1)-2-pyrimidinyl] amino]-
phenylibenzamide methanesulfonate). In a specific embodiment, antibody
compositions
of the invention are administered in combination with imatinib mesylate for
the treatment
of chronic myelogenous leukemia.
[0471] In additional preferred embodiments, antibody compositions of the
invention
are administered in combination with bortezomib (VelcadeTM [(1R)-3-methy1-1-
[[(2S)-1-
oxo-3-pheny1-2-[(pyrazinylcarbonyl) amino]propyl]aminoThutyl] boronic acid).
In a
specific embodiment, antibody compositions of the invention are administered
in
combination with bortezomib for the treatment of multiple myeloma.
[0472] In additional preferred embodiments, antibody compositions of the
invention
are administered in combination with Alemtuzumab (Campath8). In a specific
embodiment, antibody compositions of the invention are administered in
combination with
Alemtuzumab for the treatment of chronic lymphocytic leukemia.
[0473] In additional preferred embodiments, antibody compositions of the
invention
are administered in combination with fludarabine phosphate (FludaraC: 9H-Purin-
6-
amine, 2-fluoro-9-(5-0-phosphono-P-D-arabinofuranosyl) (2-fluoro-ara-AMP)). In
a
specific embodiment, antibody compositions of the invention are administered
in
combination with fludarabine phosphate for the treatment of chronic
lymphocytic
leukemia.
[0474] In additional preferred embodiments, the compositions of the invention
are
administered in combination with TRAIL polypeptides or fragments or variants
thereof,
particularly of the extracellular soluble domain of TRAIL.
[0475] .In one embodiment, the compositions of the invention are administered
in
combination with other members of the TNF family or antibodies specific for
TNF
receptor family members. TNF, TNF'-related or TNF-like molecules that may be
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CA 02494372 2010-11-17
administered with the compositions of the invention include, but are not
limited to, soluble
forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-
beta
(found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD3OL,
CD4OL,
4-1BBL, DcR3, OX4OL, TNF-gamma (International Publication No. WO 96/14328),
TRAIL, AIM-II (International Publication No. WO 97/34911), APRIL (J. Exp. Med.
188(6):1185-1190), endokine-alpha (International Publication No. WO 98/07880),
TR6
(International Publication No. WO 98/30694), OPG, and neutrokine-alpha
(International
Publication No. WO 98/18921, 0X40, and nerve growth factor (NGF), and soluble
forms
of Fas, CD30, CD27, CD40 and 4-D3B, TR2 (International Publication No. WO
96/34095), DR3 (International Publication No. WO 97/35904), TR5 (International
Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694),
TR7
(International Publication No. WO 98/41629), TRANK, TR9 (International
Publication
No. WO 98/56892),TR10 (International Publication No. WO 98/54202),312C2
(International Publication No. WO 98/06842), and TR12, and soluble forms
CD154,
CD70, and CD153.
[0476] In one embodiment, the antibody compositions of the invention are
administered in combination with apoptosis inducing polypeptides. In a
specific
embodiment, antibodies of the invention are administered in combination with
Smac
(second mitochondria-derived activator of caspases) proteins, also known as
DIABLO
(direct IAP (inhibitor of apoptosis) binding protein with low pl) (GenBank
Accession No.
:NP_063940 ). Smac is a 239
amino acid protein. The N-terminal 55 amino acids serve as a mitochondrial
targeting
sequence which is cleaved after import to the mitochondria. Apoptosis inducing
polypeptides may be delivered using techniques known in the art. For example,
one way
to deliver Smac protein would be through the delivery of a nucleic acid
encoding either
the full length or mature form of Smac (amino acids 56-239 of GenBank
Accession No.
:NP_063940, a cytosolic form that bypasses mitochondrial processing).
Alternatively,
antibody compositions of the invention may be administered in combination with
cell
permeable, synthetic Smac peptides which are capable of inhibiting LAP
proteins (e.g.,
those containing amino acid residues 56-62 of GenBank Accession No.
:NP_063940;
AVPIAQK as described in Chai et al., (2000) Nature 406:855-862 and Fulda et
al., (2002)
Nature Medicine 8:808-815.
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[0477] In one embodiment, an antibody composition of the invention is
administered
in combination with a histone deacetylase inhibitor (e.g., depsipeptide (e.g.,
FK-288 and
FR901228), MS-275, and the triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-
oic acid
(CDDO) or other molecules related to CDDO, valproic acid, suberoylanilide
hydroxamic
acid (SAHA), pyroxamide, trapoxin, (depsipeptide), and N-acetyl dinaline (CI-
994).
[0478] In another embodiment, an antibody compositions of the invention is
administered in combination with inhibitors of ERK1/2.
[0479] In another embodiment, an antibody compositions of the invention is
administered in combination with proteasome inhibitors such as PS-341 (LDP-
341, 26S
proteasome inhibitor).
[0480] In another embodiment, an antibody compositions of the invention is
administered in combination with a COX-2 inhibitor.
[0481] In specific embodiments antibodies of the present invention may be
administered in combination with one or more therapeutic agents, as described
above, in
the treatment, prevention, amelioration and/or cure of hematological cancer
(e.g.,
leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, non-
Hodgkin's
lymphoma, multiple myeloma), colorectal cancer, lung cancer, brain cancer,
skin cancer,
breast cancer, prostate cancer, pancreatic cancer, hepatic cancer, ovarian
cancer, as well as
endothelioma, osteoblastoma, osteoclastoma, Ewing's sarcoma, and Kaposi's
sarcoma.
[0482] In further particular embodiments, antibodies of the present invention
are used
to treat, ameliorate and/or prevent hematological cancers. Antibodies of the
present
invention may be used in combination with one or more surgical and/or
radiological
procedures and /or therapeutic agents to treat, ameliorate and/or prevent
hematological
cancers. Hematological cancers which may be treated using antibodies of the
present
invention include, but are not limited to, non-Hodgkin's lymphoma (e.g., small
lymphocytic lymphoma, follicular center cell lymphoma, lymphoplasmacytoid
lymphoma,
marginal zone lymphoma, mantle cell lymphoma, immunoblastic lymphoma,
burkitt's
lymphoma, lymphoblastic lymphoma, peripheral T-cell lymphoma, anaplastic large
cell
lymphoma and intestinal T-cell lymphoma), leukemia, acute lymphocytic
leukemia,
chronic lymphocytic leukemia and plasma cell neoplasms including multiple
myeloma.
[0483] In preferred embodiments, agonistic antibodies of the present invention
are
used to treat, ameliorate and/or prevent hematological cancers. Agonistic
antibodies of the
present invention may be used in combination with one or more surgical and/or
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radiological procedures and /or therapeutic agents to treat, ameliorate and/or
prevent
hematological cancers. Hematological cancers which may be treated using
agonistic
antibodies of the present invention include, but are not limited to, non-
Hodgkin's
lymphoma (e.g., small lymphocytic lymphoma, follicular center cell lymphoma,
lymphoplasmacytoid lymphoma, marginal zone lymphoma, mantle cell lymphoma,
immunoblastic lymphoma, burkitt's lymphoma, lymphoblastic lymphoma, peripheral
T-
cell lymphoma, anaplastic large cell lymphoma and intestinal T-cell lymphoma),
leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and plasma
cell
neoplasms including multiple myeloma.
[0484] In one preferred embodiment, agonistic antibodies of the invention are
used to
treat plasma cell neoplasms. In a specific embodiment, that plasma cell
neoplasm is
multiple myeloma.
[0485] In another preferred embodiment, agonistic antibodies of the invention
are used
to treat non-Hodgkin's lymphoma.
[0486] In another preferred embodiment, agonistic antibodies of the invention
are used
to treat leukemia. In a specific embodiment, that leukemia is acute
lymphocytic leukemia.
In another specific embodiment, that leukemia is chronic lymphocytic leukemia.
[0487] Antibodies of the present invention may be administered in combination
with
one or more surgical and/or radiological procedures useful in the treatment of
hematological cancer including, but not limited to, bone marrow
transplantation, external
beam radiation and total body irradiation.
[0488] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more surgical and/or radiological
procedures
useful in the treatment of hematological cancer including, but not limited to,
bone marrow
transplantation, external beam radiation and total body irradiation.
[0489] In one preferred embodiment, agonistic antibodies of the present
invention may
be administered in combination with one or more surgical and/or radiological
procedures
useful in the treatment of multiple myeloma including, but not limited to,
allogeneic bone
marrow transplantation and peripheral stem cell support.
[0490] In another preferred embodiment, agonistic antibodies of the present
invention
may be administered in combination with one or more surgical and/or
radiological
procedures useful in the treatment of non-Hodgkin's lymphoma including, but
not limited
to, allogeneic bone marrow transplantation and peripheral stem cell support.
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[0491] In further specific embodiments, agonistic antibodies of the present
invention
may be administered in combination with one or more surgical and/or
radiological
procedures useful in the treatment of leukemia including, but not limited to,
allogeneic
bone marrow transplantation and peripheral stem cell support. In one specific
preferred
embodiment, agonistic antibodies of the invention are used to treat acute
lymphocytic
leukemia (ALL). In another specific preferred embodiment, agonistic antibodies
of the
invention are used to treat chronic lymphocytic leukemia (CLL).
[0492] Antibodies of the present invention may be administered in combination
with
one or more therapeutic agents useful in the treatment of multiple myeloma
including, but
not limited to, Alkylating agents, Anthracyclines, Carmustine (DTI-015, BCNU,
BiCNU,
Gliadel Wafer ), Cyclophosphamide (Cytoxan , Neosar , CTX), Dexamethasone
(Decadron0), Doxorubicin (Adriamycin , Doxi1C, Rubex8), Melphalan (L-PAM,
Alkeran , Phenylalanine mustard), Prednisone, Thalidomide and Vincristine
(Oncovorin , Onco TCS , VCR, Leurocristine6).
[0493] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, amelioration and/or prevention of multiple myeloma.
[0494] Preferred combinations of therapeutic agents useful in the treatment of
multiple
myeloma which may be administered in combination with antibodies of the
present
invention include, but are not limited to, Cyclophosphamide + Prednisone,
Melphalan +
Prednisone (MP), Vincristine + Adriamycin + Dexamethasone (VAD), Vincristine
+
Carmustine + Melphalan + Cyclophosphamide + Prednisone (VBMCP; the M2
protocol),
and Vincristine + Melphalan + Cyclophosphamide + Prednisone alternating with
Vincristine + Carmustine + Doxorubicin + Prednisone (VMCP/VBAP).
[0495] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agent
combinations in the treatment, amelioration and/or prevention of multiple
myeloma.
[0496] Antibodies of the present invention may be administered in combination
with
one or more therapeutic agents useful in the treatment of non-Hodgkin's
lymphoma
including, but not limited to, 2-chlorodeoxyadenosine, Amifostine (Ethyol ,
Ethiofos ,
WR-272), Bexarotene (Targretin , Targretin gel , Targretin oral , LGD1069),
Bleomycin (Blenoxane0), Busulfan (Busulfex , Myleran ), Carboplatin
(ParaplatinC,
CBDCA), Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer ), Chlorambucil
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(LeukeranC), Cisplatin (Platinol , CDDP), Clathibine (2-CdA, LeustatinC),
Cyclophosphamide (CytoxanC, NeosarC, CTX), Cytarabine (Cytosar-U , ara-C,
cytosine
arabinoside, DepoCytC), Dacarbazine (DTIC), Daunorubicin (Daunomycin,
DaunoXome , DaunorubicinC, Cerubidine Denileukin diftitox (Ontake),
Dexamethasone (Decadron ), Dolasetron mesylate (AnzemetC), Doxorubicin
(Adriamycin , Doxil , RubexC), Erythropoietin (EPOS, Epogen , Procrit8),
Etoposide
phosphate (EtopophosC), Etoposide (VP-16, VepesidC), Fludarabine (Fludara ,
FAMP),
Granisetron (Kytri18), Hydrocortisone, Idarubicin (IdamycinC, DMDR, IDA),
Ifosfamide
(IFEX8), Interferon alpha (Alfaferone , Alpha-IF ), Interferon alpha 2a
(Intron AC),
Mechlorethamine (Nitrogen Mustard, HN2, Mustargen8), Melphalan (L-PAM,
AlkeranC,
Phenylalanine mustard), Methotrexate (MTX, Mexate , Folex0),
Methylprednisolone
(SolumedrolO), Mitoxantrone (Novantrone , DHAD), Ondansetron (Zofran0),
Pentostatin (Nipent , 2-deoxycoformycin), Perfosfamide (4-
hydroperoxycyclophosphamide, 4-HC), Prednisone, Procarbazine (Matulane 8),
Rituximab (RituxanC, anti-CD20 MAb), Thiotepa (triethylenethiophosphaoramide,
ThioplexC), Topotecan (Hycamtin , SK&F-104864, NSC-609699, EvotopinC),
Vinblastine (VelbanC, VLB), Vincristine (OncovinC, Onco TCS , VCR,
Leurocristine9) and Vindesine (Eldisine , Fildesing).
[0497] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, amelioration and/or prevention of non-Hodgkin's lymphoma.
[0498] Preferred combinations of therapeutic agents useful in the treatment of
non-
Hodgkin's lymphoma which may be administered in combination with antibodies of
the
present invention include, but are not limited to, Adriamycin + Blenoxane +
Vinblastine
+ Dacarbazine (ABVD), Anti-idiotype therapy (BsAb) + Interferon alpha, Anti-
idiotype
therapy (BsAb) + Chlorambucil, Anti-idiotype therapy (BsAb) + Interleukin-2,
BCNU
(Carmustine) + Etoposide + Ara-C (Cytarabine) + Melphalen (BEAM), Bleomycin +
Etoposide + Adriamycin + Cyclophosphamide + Vincristine + Procarbazine +
Prednisone
(BEACOPP), Bryostatin + Vincristine, Cyclophosphamide + BCNU (Carmustine) + VP-
16 (Etoposide) (CBV), Cyclophosphamide + Vincristine + Prednisone (CVP),
Cyclophosphamide + Adriamycin (Hydroxyldaunomycin) + Vincristine (Oncovorin)
+
Prednisone (CHOP), Cyclophosphamide + Novantrone (Mitoxantrone) + Vincristine
(Oncovorin) + Prednisone (CNOP), Cyclophosphamide + Doxorubicin + Teniposide +
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Prednisone, Cyclophosphamide + Adriamycin (Hydroxyldaunomycin) + Vincristine
(Oncovorin) + Prednisone + Rituximab (CHOP + Rituximab), Cyclophosphamide +
Doxorubicin + Teniposide + Prednisone + Interferon alpha, Cytarabine +
Bleomycin +
Vincristine + Methotrexate (CytaBOM), Dexamethasone + Cytarabine + Cisplatin
(DHAP), Dexamethasone + Ifosfamide + Cisplatin + Etoposide (DICE), Doxorubicin
+
Vinblastine + Mechlorethamine + Vincristine + Bleomycin + Etoposide +
Prednisone
(Stanford V), Etoposide + Vinblastine + Adriamycin (EVA), Etoposide +
Methylprednisone + Cytarabine + Cisplatin (ESHAP), Etoposide + Prednisone +
Ifosfamide + Cisplatin (EPIC), Fludarabine, Mitoxantrone + Dexamethasone
(F'MD),
Fludarabine, Dexamethasone, Cytarabine (ara-C), + Cisplatin (Platino10)
(FluDAP),
Ifosfamide + Cisplatin + Etoposide (ICE), Mechlorethamine + Oncovin
(Vincristine) +
Procarbazine + Prednisone (MOPP), Mesna + Ifosfamide + Idarubicin + Etoposide
(MIZE), Methotrexate with leucovorin rescue + Bleomycin + Adriamycin +
Cyclophosphamide + Oncovorin + Dexamethasone (m-BACOD), Prednisone +
Methotrexate + Adriamycin + Cyclophosphamide + Etoposide (ProMACE), Thiotepa +
Busulfan + Cyclophosphamide, Thiotepa + Busulfan + Melphalan, Topotecan +
Paclitaxel, and Vincristine (OncovinC) + Adriamycin + Dexamethasone (VAD).
[0499] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agent
combinations in the treatment, amelioration and/or prevention of non-Hodgkin's
lymphoma.
[0500] Further examples of therapeutic agents useful in the treatment of non-
Hodgkin's lymphoma which may be administered in combination with antibodies of
the
present invention include, but are not limited to, A007 (4-4'-
dihydroxybenzophenone-2, 4-
dinitrophenylhydrazone), AG-2034 (AG-2024, AG-2032, GARFT [glycinamide
ribonucleoside transformylase] inhibitor), Aldesleukin (IL-2, Proleukine),
Alemtuzumab
(Campathe), Alitretinoin (Panretin , LGN-1057), Altretamine (Hexalen ,
hexamethylmelamine, HexastatO), Aminocamptothecin (9-AC, 9-Aminocamptothecin,
NSC 603071), Anti-CD19/CD3 MAb (anti-CD19/CD3 scFv, anti-NHL MAb), Anti-
idiotype therapy (BsAb), Arabinosylguanine (Ara-G, GW506U78), Arsenic trioxide
(Trisenox , ATO), B43-Genistein (anti-CD19 Ab/genistein conjugate), B7
antibody
conjugates, Betathine (Beta-LT), BLyS antagonists, Bryostatin-1 (Bryostatin ,
BMY-
45618, NSC-339555), CHML (Cytotropic Heterogeneous Molecular Lipids),
Clofarabine
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(chloro-fluoro-araA), Daclizumab (Zenapax ), Depsipeptide (FR901228, FK228),
Dolastatin-10 (DOLA-10, NSC-376128), Epirubicin (Ellence , EPI, 4' epi-
doxorubicin),
Epratuzumab (Lymphocide , humanized anti-CD22, HAT), F1y3/f1k2 ligand
(Mobistae),
G3139 (Genasense , GentaAnticodeC, Bc1-2 antisense), Hu1D10 (anti-HLA-DR MAb,
SMART 1D10), HumaLYM (anti-CD20 MAb), Ibritumomab tiuxetan (ZevalinC),
Interferon gamma (Gamma-interferon, Gamma 100 , Gamma-IF), Irinotecan
(Camptosar , CPT-11, Topotecin , CaptoCPT-1), ISIS-2053, ISIS-3521 (PKC-alpha
antisense), Lmb-2 immunotoxin (anti-CD25 recombinant immuno toxin, anti-
Tac(Fv)-
PE38), Leuvectine (cytofectin + IL-2 gene, IL-2 gene therapy), Lym-1 (131-1
LYM-1),
Lymphoma vaccine (Genitope), Nelarabine (Compound 506, U78), Neugene compounds
(Oncomyc-NG , Resten-NG , myc antisense), NovoMAb-G2 scFv (NovoMAb-G2
IgM), 06-benzylguanine (BG, ProceptC), Oxaliplatin (EloxatineC, EloxatinO),
Paclitaxel
(Paxene , Taxo18), Paclitaxel-DHA (Taxoprexing), Peldesine (BCX-34, PNP
inhibitor),
Rebeccamycin and Rebeccamycin analogues, SCH-66336, Sobuzoxane (MST-16,
PerazolinC), SU5416 (SemaxanibC, VEGF inhibitor), TER-286, Thalidomide, TNP-
470
(AGM-1470), Tositumomab (BexxarC), Valspodar (PSC 833), Vaxid (B-cell lymphoma
DNA vaccine), Vinorelbine (Navelbine0), WF10 (macrophage regulator) and XR-
9576
(XR-9351, P-glycoprotein/MDR inhibitor).
[0501] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, amelioration and/or prevention of non-Hodgkin's lymphoma.
[0502] Antibodies of the present invention may be administered in combination
with
one or more therapeutic agents useful in the treatment of acute lymphocytic
leukemia
including, but not limited to, Amsacrine, Carboplatin (Paraplatin , CBDCA),
Carmustine
(DTI-015, BCNU, BiCNU, Gliadel Wafer ), Cholecaliferol, Cyclophosphamide
(Cytoxan , NeosarC, CTX), Cytarabine (Cytosar-U , ara-C, cytosine arabinoside,
DepoCytO), Daunorubicin (Daunomycin, DaunoXome , DaunorubicinC, Cerubidine0),
Dexamethasone (Decadron0), Doxorubicin (Adriamycin , Doxi1C, RubexC),
Etoposide
(VP-16, VepesidC), Filgrastam (Neupogen , G-CSF, LeukineC), Fludarabine
(Fludarag, FAMP), Idarubicin (Idamycin0, DMDR, IDA), Ifosfamide (IFEXC),
Imatinib
mesylate (STI-571, Imatinib , Glivec , Gleevec , Abl tyrosine kinase
inhibitor),
Interferon gamma (Gamma-interferon, Gamma 100 , Gamma-IF), L-asparaginase
(Elspar , Crastinine, Asparaginase medac , Kidrolasee), Mercaptopurine (6-
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mercaptopurine, 6-MP), Methotrexate (MTX, Mexate , Folex ), Mitoxantrone
(Novantrone , DHAD), Pegaspargase (Oncospare), Prednisone, Retinoic acid,
Teniposide (VM-26, Vumon0), Thioguanine (6-thioguanine, 6-TG), Topotecan
(Hycamtin , SK&F-104864, NSC-609699, Evotopine), Tretinoin (Retin-A , Atragen
,
ATRA, VesanoidO) and Vincristine (Oncovorin , Onco TCS , VCR, Leurocristine0).
[0503] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, amelioration and/or prevention of acute lymphocytic
leukemia.
[0504] Further examples of therapeutic agents useful in the treatment of
acute
lymphocytic leukemia which may be administered in combination with antibodies
of the
present invention include, but are not limited to, Aminocamptothecin (9-AC, 9-
Aminocamptothecin, NSC 603071), Aminopterin, Annamycin (AR-522, annamycin LF,
AronexS), Arabinosylguanine (Ara-G, GW506U78, Nelzarabine6), Arsenic trioxide
(Trisenox , ATO, Atrivexe), B43-Genistein (anti-CD19 Ab/genistein conjugate),
B43-
PAP (anti-CD19 Ab/pokeweed antiviral protein conjugate), Cordycepin, CS-682,
Decitabine (5-aza-2' -deoxyytidine), Dolastatin-10 (DOLA-10, NS C-376128),
03139
(Genasense , GentaAnticode , Bc1-2 antisense), Irofulven (MGI-114, Ivofulvan,
Acylfulvene analogue), MS-209, Phenylbutyrate, Quinine, TNP-470 (AGM-1470,
Fumagillin), Trimetrexate (Neutrexin0), Troxacitabine (BCH-204, BCH-4556,
Troxaty18), UCN-01 (7-hydroxystaurosporine), WHI-P131 and WT1 Vaccine.
[0505] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, amelioration and/or prevention of acute lymphocytic
leukemia.
[0506] Preferred combinations of therapeutic agents useful in the treatment
of acute
lymphocytic leukemia which may be administered in combination with antibodies
of the
present invention include, but are not limited to, Carboplatin + Mitoxantrone,
Carmustine
+ Cyclophosphamide + Etoposide, Cytarabine + Daunorubicin, Cytarabine +
Doxorubicin,
Cytarabine + Idarubicin, Cytarabine + Interferon gamma, Cytarabine + L-
asparaginase,
Cytarabine + Mitoxantrone, Cytarabine + Fludarabine and Mitoxantrone,
Etoposide +
Cytarabine, Etoposide + Ifosfamide, Etoposide + Mitoxantrone, Ifosfamide +
Etoposide +
Mitoxantrone, Ifosfamide + Teniposide, Methotrexate + Mercaptopurine,
Methotrexate +
Mercaptopurine + Vincristine + Prednisone, Phenylbutyrate + Cytarabine,
Phenylbutyrate
+ Etoposide, Phenylbutyrate + Topotecan, Phenylbutyrate + Tretinoin, Quinine +
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Doxorubicin, Quinine + Mitoxantrone + Cytarabine, Thioguanine + Cytarabine +
Amsacrine, Thioguanine + Etoposide + Idarubicin, Thioguanine + Retinoic acid +
Cholecaliferol, Vincristine + Prednisone, Vincristine + Prednisone and L-
asparaginase,
Vincristine + Dexamethasone/Prednisone + Asparaginase + Daunorubicin/
Doxorubicin,
Vincristine + Dexamethasone/Preclnisone + Asparaginase + Daunorubicin/
Doxorubicin +
Filgrastim, Vincristine + Dexamethasone/Pralnisone + Asparaginase +
Daunorubicin/Doxorubicin + Cyclophosphamide + Methotrexate, and Vincristine +
Dexamethasone/Predni sone + Asparaginase + Daunorubicin/Doxorubicin +
Cyclophosphamide + Methotrexate + Filgrastim.
[0507] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agent
combinations in the treatment, amelioration and/or prevention of acute
lymphocytic
leukemia.
[0508] Antibodies of the present invention may be administered in combination
with
one or more therapeutic agents useful in the treatment of chronic lymphocytic
leukemia
including, but not limited to, Chlorambucil (LeukeranC), Cladribine (2-CdA,
LeustatinC),
Cyclophosphamide (CytoxanC, Neosar , CTX), Cytarabine (Cytosar-U , ara-C,
cytosine
arabinoside, DepoCyt , cytarabine ocfosfate, ara-CMP), Doxorubicin
(AdriamycinC,
Doxil , RubexC), Fludambine (Fludara , FAMP), Pentostatin (Nipent , 2-
deoxycoformycin), Prednisone and Vincristine (Oncovorin , Onco TCS , VCR,
Leurocristine8).
[0509] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, amelioration and/or prevention of chronic lymphocytic
leukemia.
[0510] Further examples of therapeutic agents useful in the treatment of
chronic
lymphocytic leukemia which may be administered in combination with antibodies
of the
present invention include, but are not limited to, Alemtuzumab (CampathC),
Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC 603071), Aminopterin,
Annamycin (AR-522, annamycin LF, Aronex0), Arabinosylguanine (Ara-G, GW506U78,
NelzarabineC, Compound 506U78), Arsenic trioxide (Trisenox , ATO, AtrivexC),
Bryostatin-1 (Bryo statinC, BMY-45618, NS C-339555), CS-682, Dolastatin-10 (D
OLA-
10, NSC-376128), Filgrastim (Neupogen , G-CSF, Leukine), Flavopkidol (NSC-
649890,
HMER-1275), G3139 (Genasense , GentaAnticodeC, Bc1-2 antisense), Irofulven
(MGI-
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114, Ivofulvan, Acylfulvene analogue), MS-209, Phenylbutyrate, Rituximab
(Rituxan ,
anti-CD20 MAb), Thalidomide, Theophylline, TNP-470 (AGM-1470, Fumagillin), UCN-
01 (7-hydroxystaurosporine) and WHI-P131.
[0511] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, amelioration and/or prevention of chronic lymphocytic
leukemia.
[0512] Preferred combinations of therapeutic agents useful in the treatment of
chronic
lymphocytic leukemia which may be administered in combination with antibodies
of the
present invention include, but are not limited to, Fludarabine + Prednisone,
and
Cyclophosphamide + Doxorubicin + Vincristine + Prednisone (CHOP).
[0513] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agent
combinations in the treatment, amelioration and/or prevention of chronic
lymphocytic
leukemia.
[0514] In further particular embodiments, antibodies of the present invention
are used
to treat, ameliorate and/or prevent colorectal cancer. Antibodies of the
present invention
may be used in combination with one or more surgical and/or radiological
procedures and
/or therapeutic agents to treat, ameliorate and/or prevent colorectal cancer.
Colorectal
cancers which may be treated using antibodies of the present invention
include, but are not
limited to, colon cancer (e.g., early stage colon cancer (stage I and II),
lymph node
positive colon cancer (stage III), metastatic colon cancer (stage IV)) and
rectal cancer.
[0515] In preferred embodiments, agonistic antibodies of the present invention
are
used to treat, ameliorate and/or prevent colorectal cancer. Agonistic
antibodies of the
present invention may be used in combination with one or more surgical and/or
radiological procedures and /or therapeutic agents to treat, ameliorate and/or
prevent
colorectal cancer. Colorectal cancers which may be treated using agonistic
antibodies of
the present invention include, but are not limited to, colon cancer (e.g.,
early stage colon
cancer (stage I and II), lymph node positive colon cancer (stage III),
metastatic colon
cancer (stage IV)) and rectal cancer.
[0516] In one preferred embodiment, agonistic antibodies of the invention are
used to
treat colon cancer.
[0517] Antibodies of the present invention may be administered in combination
with
one or more therapeutic agents useful in the treatment of colorectal cancer
including, but
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not limited to, Capecitabine (Xeloda , Doxifluridine , oral 5-FU),
Fluorouracil (5-FU,
Adrucil , Fluoroplex , EfudexO), Irinotecan (Camptosar , CPT-11, Topotecin ,
CaptoCPT-1), Leucovorin (Leucovorin , Wellcovorin0), and Levamisole
(Ergamisol0).
[0518] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, amelioration and/or prevention of colorectal cancers.
[0519] Preferred combinations of therapeutic agents useful in the treatment of
colorectal cancer which may be administered in combination with antibodies of
the
present invention include, but are not limited to, Fluorouracil + Leucovorin,
and
Fluorouracil + Levamisole.
[0520] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agent
combinations in the treatment, amelioration and/or prevention of colorectal
cancers.
[0521] Further examples of therapeutic agents useful in the treatment of
colorectal
cancer which may be administered in combination with antibodies of the present
invention
include, but are not limited to, Aminocamptothecin (9-AC, 9-Aminocamptothecin,
NSC
603071), Aplidine (Aplidin , Aplidinag), Bevacizumab (Anti-VEGF monoclonal
antibody, rhuMAb-VEGF), C225 (IMC-225, EGFR inhibitor, Anti-EGFr MAb,
Cetuximab8), C242-DM1 (huC242-DM1), CC49-zeta gene therapy, CEA-cide
(Labetuzumab , Anti-CEA monoclonal antibody, hIVIN-14), CeaVac (MAb 3H1), CP-
609754, CTP-37 (Avicine , hCG blocking vaccine), Declopramide (Oxi-104),
Eniluracil
(776c85), F19 (Anti-FAP monoclonal antibody, iodinated anti-FAP MAb), FMdC (KW-
2331, MDL-101731), FUDR (Floxuridine8), Gemcitabine (Gemto , Gemzar0),
Herceptine (Trastuzumab , Anti-HER-2 monoclonal antibody, Anti-EGFR-2 MAb),
Intoplicine (RP 60475), L-778123 (Ras inhibitors), Leuvectin (cytofectin + IL-
2 gene,
IL-2 gene therapy), MN-14 (Anti-CEA immunoradiotherapy, 131LmN_ 14, 188Re-4N-
14),
OncoVAX-CL, OncoVAX-CL-Jenner (GA-733-2 vaccine).Orzel (Tegafur + Uracil +
Leucovorin), Oxaliplatin (Eloxatine , Eloxating), Paclitaxel-DHA
(Taxoprexin0),
Pemetrexed disodium (Alimta , MTA, multitargeted antifolate, LY 231514),
R115777
(Zarnestra0), Raltitrexed (Tomudex , ZD-1694), SCH 66336, SU5416 (Semaxanib ,
VEGF inhibitor), Tocladesine (8-C1-cAMP), Trimetrexate (NeutrexinS), TS-1, and
ZD-
9331 .
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[0522] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, amelioration and/or prevention of colorectal cancers.
[0523] Further exemplary combinations of therapeutic agents useful in the
treatment
of colorectal cancer which may be administered in combination with antibodies
of the
present invention include, but are not limited to, Aminocamptothecin + G-CSF,
Bevacizumab + Fluorouracil, Bevacizumab + Leucovorin, Bevacizumab +
Fluorouracil + Leucovorin, Cyclophosphamide + SCH 6636, Fluorouracil + CeaVac
,
Fluorouracil + Oxaliplatin, Fluorouracil + Raltitrexed, Fluorouracil + SCH
6636,
Fluorouracil + Trimetrexate, Fluorouracil + Leucovorin + Oxaliplatin,
Fluorouracil +
Leucovorin + Trimetrexate, Irinotecan + C225 (Cetuximab8), Oncovin + SCH
6636,
Oxaliplatin + Leucovorin,. Paclitaxel + SCH 6636, Pemetrexed disodium +
Gemcitabine,
and Trimetrexate + Leucovorin.
[0524] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agent
combinations in the treatment, amelioration and/or prevention of colorectal
cancers.
[0525] In further particular embodiments, antibodies of the present invention
are used
to treat, ameliorate and/or prevent lung cancer. Antibodies of the present
invention may be
used in combination with one or more surgical and/or radiological procedures
and /or
therapeutic agents to treat, ameliorate and/or prevent lung cancer. Lung
cancer which may
be treated using antibodies of the present invention includes, but is not
limited to, non-
small cell lung cancer (NSCLC) including early stage NSCLC (i.e., Stage IA/D3
and Stage
IIA/IIB), Stage IRA NSCLC, Stage IIA(unresectable)/MB NSCLC and Stage IV
NSCLC,
small cell lung cancer (SCLC) including limited stage SCLC and extensive stage
SCLC as
well as Malignant Pleural Mesothelioma.
[0526] In preferred embodiments, agonistic antibodies of the present invention
are
used to treat, ameliorate and/or prevent lung cancer. Agonistic antibodies of
the present
invention may be used in combination with one or more surgical and/or
radiological
procedures and /or therapeutic agents to treat, ameliorate and/or prevent lung
cancer. Lung
cancer which may be treated using agonistic antibodies of the present
invention includes,
but is not limited to, non-small cell lung cancer (NSCLC) including early
stage NSCLC
(i.e., Stage IA/II3 and Stage IIAJII13), Stage IIIA NSCLC, Stage
IIA(unresectable)/IIIB
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NSCLC and Stage IV NSCLC, small cell lung cancer (SCLC) including limited
stage
SCLC and extensive stage SCLC as well as Malignant Pleural Mesothelioma.
[0527] In one preferred embodiment, agonistic antibodies of the invention are
used to
treat non-small cell lung cancers.
[0528] Antibodies of the present invention may be administered in combination
with
one or more therapeutic agents useful in the treatment of lung cancer
including, but not
limited to, BAY 43-9006 (Raf kinase inhibitor), Carboplatin (Paraplatin ,
CBDCA),
Chlorambucil (Leukeran8), Cisplatin (Platinol , CDDP), Cisplatin-epinephrine
gel
(IntraDose , FocaCistO), Cyclophosphamide (Cytoxan , Neosar , CTX), Docetaxel
(Taxotere , Taxane8), Doxorubicin (Mriamycin , Doxil , RubexO), Edatrexate,
Epirubicin (Ellence , EPI, 4' epi-doxorubicin), Etoposide phosphate (Etopophos
),
Etoposide (VP-16, Vepesid8), Gemcitabine (Gemto , Gemzar8), Herceptin
(Trastuzumab , Anti-HER-2 monoclonal antibody, Anti-EGFR-2 MAb), Ifosfamide
(IFEXO), Irinotecan (Camptosar , CPT-11, Topotecin , CaptoCPT-1), Lomustine
(CCNU , CeeNUO), Mechlorethamine (Nitrogen Mustard, HN2, Mustargeng),
Melphalan (L-PAM, Alkeran , Phenylalanine mustard), Methotrexate (MTX, Mexate
,
Folex ), Mitomycin C (Mitomycin , Mutamycine, Mito Extra ), Paclitaxel (Paxene
,
TaxolO), Paclitaxel-DHA (Taxoprexin8), Porfimer sodium (Photofrin0),
Procarbazine
(Matulane ), SKI-2053R (NSC-D644591), Teniposide (VM-26, Vumone), Topotecan
(Hycamtin , SK&F-104864, NSC-609699, EvotopinC), Vinblastine (Velban , VLB),
Vincristine (Oncovin , Onco TCS , VCR, Leurocristine8), Vindesine (Eldisine ,
Fildesin8), and Vinorelbine (Navelbine6).
[0529] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, amelioration and/or prevention of lung cancers.
[0530] Further examples of therapeutic agents useful in the treatment of lung
cancer
which may be administered in combination with antibodies of the present
invention
include, but are not limited to, ABX-EGF (anti-EGFr MAb), Acetyldinaline (CI-
994),
AG-2034 (AG-2024, AG-2032, GARFT [glycinamide ribonucleoside transformylase]
inhibitor), Alanosine, Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC
603071),
Angiostatin, Aplidine (Aplidin , Aplidina6), BBR 3464, Bexarotene (Targretin ,
LGD1069), BTBH-1 (Anti-FAP MAb), BIBX-1382, BLP-25 (MUC-1 peptide),
Bryostatin-1 (Bryostatin , BMY-45618, NS C-339555), Budesonide (Rhinocort8),
C225
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(IMC-225, EGFR inhibitor, Anti-EGFr MAb, Cetuximab0), Capecitabine (XelodaC,
Doxifluridinee, oral 5-FU), Carboxyamidotriazole (NSC 609974, CAI, L-651582),
CEA-
cideC (Labetuzumab , Anti-CEA monoclonal antibody, hMN-14), Cereport
(Lobradimil0, RMP-7), CI-1033 (Pan-erbB RTK inhibitor), CilengitideC (EMD-
121974,
integrin alphavbeta3 antagonist), 9-cis retinoic acid (9-cRA), Cisplatin-
liposomal (SPI-
077), CMB-401 (Anti-PEM MAb/calicheamycin), CMT-3 (Metastate), CP-358774
(TarcevaC, OSI-774, EGFR inhibitor), CT-2584 (Apra ), DAB389-EGF (EGF fusion
toxin), DeaVacC (CEA anti-idiotype vaccine), Decitabine (5-aza-2'-
deoxyytidine),
Diethylnorsp ermine (DENSPM), Dihydro-5-azacytidine, EGF-P64k Vaccine,
Endostatin,
Etanidazole (Radiny1C), Exetecan mesylate (DX-8951, DX-8951f), Exisulind
(SAAND,
Aptosyn , cGMP-PDE2 and 5 inhibitor), FK-317 (FR-157471, FR-70496),
Flavopiridol
(HMR-1275), Fotemustine (MuphoranC, MustophoranC), G3139 (GenasenseC,
GentaAnticodeC, Bc1-2 antisense), Gadolinium texaphyrin (Motexafm gadolinium,
Gd-
Tex , Xcytrin8), GBC-590, GL331, Galarubicin hydrochloride (DA-125),
GlufosfamideC (P-D-glucosyl-isofosfamide mustard, D19575, INN), GVAX (GM-CSF
gene therapy), INGN-101 (p53 gene therapy/retrovirus), INGN-201 (p53 gene
therapy/adenovirus), Irofulven (MGI-114), ISIS-2053, ISIS-3521 (PKC-alpha
antisense),
ISIS-5132 (K-ras/raf antisense), Isotretinoin (13-CRA, 13-cis retinoic acid,
AccutaneC),
Lometrexol (T-64, T-904064), Marimastat (BB-2516, TA-2516, MMP inhibitor),
MDX-
447 (BAB-447, EMD-82633, H-447, anti-EGFr/FcGammaR1r), MGV, Mitumomab0
(BEC-2, EMD-60205), Mivobulin isethionate (CI-980), Neovastate (AE-941, MMP
inhibitor), Onconase (RanpirnaseO), Onyx-015 (p53 gene therapy), Pemetrexed
disodium
(AlimtaC, MTA, multitargeted antifolate, LY 231514), Pivaloyloxymethyl
butyrate (AN-
9, PivanexC), PrinomastatC (AG-3340, MMP inhibitor), PS-341 (LDP-341, 26S
proteasome inhibitor), Pyrazoloacridine (NSC-366140, PD-115934), R115777
(ZarnestraC), Raltitrexed (TomudexC, ZD-1694), R-flurbiprofen (FlurizanC, E-
7869,
MPC-7869), RFS-2000 (9-nitrocamptothecan, 9-NC, rubitecanC), RSR-13 (GSJ-61),
Satraplatin (BMS-182751, JM-216), SCH-66336, Sizofilane (SPG, Sizofiran ,
SchizophyllanC, Sonifilane), Squalamine (MSI-1256F), SR-49059 (vasopressin
receptor
inhibitor, Via), SU5416 (Semaxanib , VEGF inhibitor), Taurolidine (TaurolinC),
Temozolamide (Temodare, NSC 362856), Thalidomide, Thymosin alpha I (ZadaxinC,
ThymalfasinC), Tirapazamine (SR-259075, SR-4233, TirazoneC, Win-59075), TNP-
470
(AGM-1470), TriAb (anti-idiotype antibody immune stimulator), Tretinoin
(Retin-AC,
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Atragen(D, ATRA, VesanoidC), Troxacitabine (BCH-204, BCH-4556, Troxaty1C),
Vitaxin (LM-609, integrin alphavbeta3 antagonistic MAb), XR-9576 (P-
glycoprotein/MDR inhibitor), and ZD-1839 (TRES SAC).
[0531] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, amelioration and/or prevention of lung cancers.
[0532] Preferred combinations of therapeutic agents useful in the treatment of
lung
cancer which may be administered in combination with antibodies of the present
invention
include, but are not limited to, Cisplatin + Docetaxel, Cisplatin + Etoposide,
Cisplatin +
Gemcitabine, Cisplatin + Interferon alpha, Cisplatin + Irinotecan, Cisplatin +
Paclitaxel,
Cisplatin + Teniposide, Cisplatin + Vinblastine, Cisplatin + Vindesine,
Cisplatin +
Vinorelbine, Cisplatin + Vinblastine + Mitomycin C, Cisplatin + Vinorelbine +
Gemcitabine, Cisplatin (P1atinols1)) + Oncovin + Doxorubicin (Adriamycin ) +
Etoposide (CODE), Cyclophosphamide + Adriamycin + Cisplatin (PlatinolC)
(CAP),
Cyclophosphamide + Adriamycin + Vincristine (CAV), Cyclophosphamide +
Epirubicin
+ Cisplatin (PlatinolC) (CEP), Cyclophosphamide + Methotrexate + Vincristine
(CMV),
Cyclophosphamide + Adriamycin , Methotrexate + Fluorouracil (CAMP),
Cyclophosphamide + Adriamycin , Methotrexate + Procarbazine (CAMP),
Cyclophosphamide + Adriamycin , Vincristine + Etoposide (CAV-
E),Cyclophosphamide
+ Adriamycin , Vincristine + Teniposide (CAV-T), Cyclophosphamide + Oncovin ,
Methotrexate + Fluorouracil (COMF), Cyclophosphamide + Adriamycin +
Vincristine,
alternating with Cisplatin + Etoposide (CAV/PE), Docetaxel + Gemcitabine,
Docetaxel +
Vinorelbine, Etoposide (Vepeside) + Ifosfamide + Cisplatin (Platinole) (VIP),
Etoposide
(VepesidO) + Ifosfamide, Cisplatin + Epirubicin (VIC-E), Fluorouracil +
Oncovin +
Mitomycin C (F0Mi), Hydrazine + Adriamycin + Methotrexate (HAM), Ifosfamide +
Docetaxel, Ifosfamide + Etoposide, Ifosfamide + Gemcitabine, Ifosfamide +
Paclitaxel,
Ifosfamide + Vinorelbine, Ifosfamide + Carboplatin + Etoposide (ICE),
Irinotecan +
Docetaxel, Irinotecan + Etoposide, Irinotecan + Gemcitabine, Methotrexate +
Cisplatin,
Methotrexate + Interferon alpha, Methotrexate + Vinblastine, Mitomycin C +
Ifosfamide
+ Cisplatin (Platino10) (MIP), Mitomycin C + Vinblastine + Paraplatin (MVP),
Paraplatin + Docetaxel, Paraplatin + Etoposide, Paraplatin + Gemcitabine,
Paraplatin + Interferon alpha, Paraplatin + Irinotecan, Paraplatin +
Paclitaxel,
Paraplatin + Vinblastine, Paraplatin + Vindesine, Paraplatin + Vinorelbine,
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Procarbazine + Oncovin + CCNU (Lomustine) + Cyclophosphamide (POCC),
Vincristine (OncovinO) + Adriamycin + Procarbazine (VAP), and Vinorelbine +
Gemcitabine.
[0533] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agent
combinations in the treatment, amelioration and/or prevention of lung cancers.
[0534] In further particular embodiments, antibodies of the present invention
are used
to treat, ameliorate and/or prevent head and neck cancers including brain
cancers.
Antibodies of the present invention may be used in combination with one or
more surgical
and/or radiological procedures and /or therapeutic agents to treat, ameliorate
and/or
prevent head and neck cancers including brain cancers. Brain cancers which may
be
treated using antibodies of the present invention include, but are not limited
to, gliomas
such as astrocytomas and oligodendromas, non-glial tumors such as neuronal,
meningeal,
ependymal and choroid plexus cell tumors, and metastatic brain tumors such as
those
originating as breast, lung, prostate and skin cancers.
[0535] In further preferred embodiments, agonistic antibodies of the present
invention
are used to treat, ameliorate and/or prevent head and neck cancers including
brain cancers.
Agonistic antibodies of the present invention may be used in combination with
one or
more surgical and/or radiological procedures and /or therapeutic agents to
treat, ameliorate
and/or prevent head and neck cancers including brain cancers. Brain cancers
which may
be treated using agonistic antibodies of the present invention include, but
are not limited
to, gliomas such as astrocytomas and oligodendromas, non-glial tumors such as
neuronal,
meningeal, ependymal and choroid plexus cell tumors, and metastatic brain
tumors such
as those originating as breast, lung, prostate and skin cancers.
[05361 In one preferred embodiment, agonistic antibodies of the invention are
used to
treat brain tumors. In a further preferred embodiment, agonistic antibodies of
the invention
are used to treat glioblastoma multiforrne.
[0537] Antibodies of the present invention may be administered in combination
with
one or more radiological procedures useful in the treatment of brain cancers
including, but
not limited to, external beam radiation therapy, stereotactic radiation
therapy, conformal
radiation therapy, intensity-modulated radiation therapy (IMRT), and radio
surgery.
[0538] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more radiological procedures useful in
the
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treatment of brain cancers including, but not limited to, external beam
radiation therapy,
stereotactic radiation therapy, conformal radiation therapy, intensity-
modulated radiation
therapy (DMRT), and radio surgery.
[0539] Antibodies of the present invention may be administered in combination
with
one or more therapeutic agents useful in the treatment of brain cancers
including, but not
limited to, Bleomycin (Blenoxane ), Busulfan (Busulfex , Mylerane),
Carboplatin
(Paraplatin , CBDCA), Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer ),
Cisplatin (Platinol , CDDP), Cisplatin-epinephrine gel (IntraDose ,
FocaCist8),
Cyclophosphamide (Cytoxan , CTX), Cytarabine (Cytosar-U , ara-C, cytosine
arabinoside, DepoCyt ), Dacarbazine (DTICO), Dactinomycin (CosmegenS),
Daunorubicin (Daunomycin, DaunoXorne , Daunorubicine, CerubidineS), Docetaxel
(Taxotere 0, TaxaneS), Dexamethasone (DecadronO), Etoposide phosphate
(Etopophose), Etoposide (VP-16, Vepesid ), Fluorouracil (5-FU, Adrucil ),
Hydroxyurea (HydreaC), Ifosfamide (IFEX8), Lomustine (CCNUS, CeeNUO),
Melphalan (L-PAM, Alkeran , Phenylalanine mustard), Mercaptopurine (6-
mercaptopurine, 6-MP), Methchlorethamine (Nitrogen Mustard, HN2, MustargenS),
Methotrexate0 (MTX, Mexate , Folex6), Paclitaxel (Paxene0, Taxo16), Paclitaxel-
DHA (TaxoprexinS), Procarbazine (Matulane8), Temozolamide (Temodar , NSC
362856), Teniposide (VM-26, Vumon8), Thioguanine (6-thioguanine, 6-TG),
Thiotepa
(triethylenethiophosphaoramide), Topotecan (Hycamtin , SK&F-104864, NSC-
609699,
Evotopin0), and Vincristine (Oncovin0, Onco TCS , VCR, Leurocristine6).
[0540] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, amelioration and/or prevention of brain cancers.
[0541] Further examples of therapeutic agents useful in the treatment of brain
cancers
which may be administered in combination with antibodies of the present
invention
include, but are not limited to, 8106 (Anti-tenascin monoclonal antibody), BMX-
1382,
Cereport (Lobradimil , RMP-7), Cilengitide (EMD-121974, integrin alphavbeta3
antagonist), CMT-3 (Metastat8), Cotara (chTNT-1/B, [131I]-chTNT-1/B), CP IL-4-
toxin
(IL-4 fusion toxin), Fenretinide (4HPR), Fotemustine (Muphorani14,
Mustophoran0),
Gemcitabine (Gemto , Gemzar0), Hypericin0 (VIMRxynS), Imatinib mesylate (STI-
571, Imatinib , Glivec , Gleevec0, Abl tyrosine kinase inhibitor), Irinotecan
(Camptosar0, CPT-11, Topotecin , CaptoCPT-1), Leflunomide (SU-101, SU-0200),
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Mivobulin isethionate (CI-980), 06-benzylguanine (BG, Procept8), Prinomastat
(AG-
3340, MMP inhibitor), R115777 (Zarnestrag), SU6668 (PDGF-TK inhibitor), T-67
(T-
138067, T-607), Tamoxifen (NolvadexO), Tf-CRM107 (Transferrin-CRM-107),
Thalidomide, Tiazofurin (TiazoleS), Vapreotide (BMY-41606), Vinorelbine
(Navelbine ), and XR-5000 (DACA).
[05421 In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, amelioration and/or prevention of brain cancers.
[0543] Preferred combinations of therapeutic agents useful in the treatment of
brain
cancers which may be administered in combination with antibodies of the
present
invention include, but are not limited to, Busulfan + Melphalan, Carboplatin +
Cereport ,
Carboplatin + Etoposide, Carboplatin + Etoposide + Thiotepa, Cisplatin +
Etoposide,
Cisplatin + Cytarabine + Ifosfamide, Cisplatin + Vincristine + Lomustine,
Cisplatin +
Cyclophosphamide + Etoposide + Vincristine, Cisplatin + Cytarabine +
Ifosfamide +
Etoposide + Methotrexate, Cyclophosphamide + Melphalan, Cytarabine +
Methotrexate,
Dactinomycin + Vincristine, Mechlorethamine + Oncovin (Vincristine) +
Procarbazine
(MOP), Mechlorethamine + Oncovin (Vincristine) + Procarbazine + Prednisone
(MOPP), Carboplatin (Paraplating) + Etoposide, Carboplatin (Paraplating) +
Vincristine,
Procarbazine + Lomustine, Procarbazine + Lomustine + Vincristine, Procarbazine
+
Lomustine + Vincristine + Thioguanine, Thiotepa + Etoposide, Thiotepa +
Etoposide +
Carmustine, Thiotepa + Etoposide + Carboplatin, Vinblastine + Bleomycin +
Etoposide +
Carboplatin, and Vincristine + Lomustine + Prednisone.
[0544] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
combinations of
therapeutic agents in the treatment, amelioration and/or prevention of brain
cancers.
[0545] In specific embodiments antibodies of the present invention are used to
treat,
ameliorate and/or prevent skin cancers including basal cell carcinoma,
squamous cell
carcinoma and malignant melanoma. Antibodies of the present invention may be
used in
combination with one or more surgical and/or radiological procedures and /or
therapeutic
agents to treat, ameliorate and/or prevent skin cancers.
[0546] In preferred embodiments agonistic antibodies of the present invention
are used
to treat, ameliorate and/or prevent skin cancers including basal cell
carcinoma, squamous
cell carcinoma and malignant melanoma. Agonistic antibodies of the present
invention
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may be used in combination with one or more surgical and/or radiological
procedures and
/or therapeutic agents to treat, ameliorate and/or prevent skin cancers.
[0547] Antibodies of the present invention may be administered in combination
with
one or more therapeutic agents useful in the treatment of skin cancers
including, but not
limited to, Bleomycin (Blenoxane ), Carmustine (DTI-015, BCNU, BiCNU, Gliadel
Wafer ), Cisplatin (Platinol , CDDP), Dacarbazine (DTIC), Interferon alpha 2b
(Intron
AO), Interleukin-2 (ProleiukinRO), Tamoxifen (Nolvadex0), Temozolamide
(Temodar ,
NSC 362856), Vinblastine (Velban , VLB), Vincristine (Oncovin , Onco TCS ,
VCR,
Leurocristine8), and Vindesine (Eldisine , Fildesin0). Combinations of
therapeutic
agents useful in the treatment of skin cancers include, but are not limited
to, Cisplatin +
Carmustine + Dacarbazine + Tamoxifen.
[0548] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, amelioration and/or prevention of skin cancers.
[0549] In further particular embodiments, antibodies of the present invention
are used
to treat, ameliorate and/or prevent breast cancer. Antibodies of the present
invention may
be used in combination with one or more surgical and/or radiological
procedures and /or
therapeutic agents to treat, ameliorate and/or prevent breast cancer. Breast
cancers which
may be treated using antibodies of the present invention include, but are not
limited to,
ductal carcinoma, stage I, stage II, stage III and stage IV breast cancers as
well as invasive
breast cancer and metastatic breast cancer.
[0550] In preferred embodiments, agonistic antibodies of the present invention
are
used to treat, ameliorate and/or prevent breast cancer. Agonistic antibodies
of the present
invention may be used in combination with one or more surgical and/or
radiological
procedures and /or therapeutic agents to treat, ameliorate and/or prevent
breast cancer.
Breast cancers which may be treated using agonistic antibodies of the present
invention
include, but are not limited to, ductal carcinoma, stage I, stage II, stage
III and stage IV
breast cancers as well as invasive breast cancer and metastatic breast cancer.
[0551] In one preferred embodiment, agonistic antibodies of the invention are
used to
treat metastatic breast cancer.
[0552] Antibodies of the present invention may be administered in combination
with
one or more surgical and/or radiological procedures useful in the treatment of
breast
cancer.
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[0553] In preferred embodiments, agonistic antibodies of the present invention
may be
administered in combination with one or more surgical and/or radiological
procedures
useful in the treatment of breast cancer.
[0554] Antibodies of the present invention may be administered in combination
with
one or more therapeutic agents useful in the treatment of breast cancer
including, but not
limited to, Amifostine (EthyolC), Aminoglutethimide (CytadrenC), Anastrozole
(ArimidexO), Bleomycin (BlenoxaneC), Capecitabine (Xeloda , Doxifluridine ,
oral 5-
FU), Cisplatin (Platinol , CDDP), Cisplatin-epinephrine gel (IntraDose ,
FocaCistC),
Cyclophosphamide (Cytoxan , NeosarC, CTX), Docetaxel (Taxotere , TaxaneC),
Doxorubicin (AdriamycinC, Doxil , Rubex414), Epirubicin (Ellence , EPI, 4' epi-
doxorubicin), Exemestane (AromasinC, NikidessC), Fadrozole (Afema , Fadrozole
hydrochloride, Arensing), Fluorouracil (5-FU, Adrucil , Fluoroplex , EfudexC),
Herceptin (TrastuzumabC, Anti-HER-2 monoclonal antibody, Anti-EGFR-2 MAb),
Ifosfamide (IFEXC), Letrozole (Femara0), Leucovorin (Leucovorin ,
WellcovorinS),
Mechlorethamine (Nitrogen Mustard, HN2, MustargenS), Megestrol acetate (Megace
,
PallaceC), Melphalan (L-PAM, AlkeranC, Phenylalanine mustard), Methotrexate
(MTX, Mexate , Folex8), Methyltestosterone (Android-10C, Testred , VirilonC),
Mitomycin C (MitomycinC, Mutamycin , Mito Extra ), Orzel (Tegafur + Uracil +
Leucovorin), Paclitaxel (Paxene , TaxolC), Sobuzoxane (MST-16, Perazoline),
Tamoxifen (NolvadexC), Testosterone (Andro , Androderm , Testoderm TTSO,
Testoderm , Depo-Testosterone , Androgel , depoAndroC), Vinblastine (Velban ,
VLB), Vincristine (OncovinC, Onco TCSC, VCR, Leurocristine8), and Vinorelbine
(NavelbineO).
[0555] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, amelioration and/or prevention of breast cancers.
[0556] Further examples of therapeutic agents useful in the treatment of
breast cancer
which may be administered in combination with antibodies of the present
invention
include, but are not limited to, Aldesleukin (IL-2, Proleuking), Altretamine
(HexalenC,
hexamethylmelamine, HexastatC), Angiostatin, Annamycin (AR-522, annamycin LF,
Aronex0), Biricodar dicitrate (Incel , Incel MDR Inhibitor), Boronated
Protoporphyrin
Compound (PDIT, Photodynamic Immunotherapy), Bryostatin-1 (Bryostatin, BMY-
45618, NSC-339555), Busulfan (Busulfex , MyleranC), Carmustine (DTI-015, BCNU,
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WO 2004/016753 CA 02494372 2005-02-04 PCT/US2003/025457
BiCNU, Gliadel Wafer ), D-limonene, Dacarbazine (DTIC), Daunorubicin
(Daunomycin,
DaunoXome , DaunorubicinO, CerubidineC), Dolastatin-10 (DOLA-10, NSC-376128),
DPPE, DX-8951f (DX-8951), EMD-121974, Endostatin, E09 (E01, E04, E068, E070,
E072), Etoposide phosphate (EtopophosC), Etoposide (VP-16, VepesidC),
Fluasterone,
Fludarabine (Fludarae, FAMP), Flutamide (EulexinO), Formestane (LentaronC),
Fulvestrant (Faslodex8), Galarubicin hydrochloride (DA-125), Gemcitabine
(GemtoO,
Gemzare), Her-2/Neu vaccine, Hydroxyurea (HydreaC), Idarubicin (IdamycinO,
DMDR,
IDA), Interferon alpha 2a (Intron AO), Interferon gamma (Gamma-interferon,
Gamma
100C, Gamma-IF), Irinotecan (Camptosar0, CPT-11, Topotecin0, CaptoCPT-1),
Ketoconazole (Nizoral0), KRN-8602 (MX, MY-5, NSC-619003, MX-2), L-asparaginase
(ElsparC), Leuprolide acetate (ViadurO, LupronS), Lomustine (CCNUC, CeeNUC),
LY-
335979, Mamian-MUC1 vaccine, 2-Methoxyestradiol (2-ME, 2-ME2), Mitoxantrone
(NovantroneC, DHAD), Motexafin Lutetium (LutrinC, Opting, Lu-Tex , lutetium
texaphyrin, LucynC, AntrinO), MPV-2213ad (Finrozole8), MS-209, Muc-1 vaccine,
NaPro Paclitaxel, Perillyl alcohol (perilla alcohol, perillic alcohol,
perillol, NSC-641066),
Pirarubicin (THP), Procarbazine (MatulaneC), Providence Portland Medical
Center Breast
Cancer Vaccine, Pyrazoloacridine (NSC-366140, PD-115934), Raloxifene
hydrochloride
(EvistaC, Keoxifene hydrochloride), Raltitrexed (TomudexC, ZD-1694),
Rebeccamycin,
Streptozocin (ZanosarC), Temozolamide (TemodarC, NSC 362856), Theratope,
Thiotepa
(triethylenethiophosphaoramide, ThioplexC), Topotecan (HycamtinC, SK&F-104864,
NSC-609699, EvotopinC), Toremifene (Estrimexe, FarestonS), Trilostane
(ModrefenC),
and XR-9576 (XR-9351, P-glycoprotein/MDR inhibitor).
[0557] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agents
in the treatment, amelioration and/or prevention of breast cancers.
[0558] Preferred combinations of therapeutic agents useful in the treatment of
breast
cancer which may be administered in combination with antibodies of the present
invention
include, but are not limited to, Cyclophosphamide + Adriamycine (Doxorubicin),
Cyclophosphamide + Epirubicin + Fluorouracil, Cyclophosphamide + Methotrexate
+
Fluorouracil (CMF), Paclitaxel + Doxorubicin, and Vinblastine + Doxorubicin +
Thiotepa.
[0559] In preferred embodiments, agonistic antibodies of the invention are
administered in combination with one or more of the above-described
therapeutic agent
combinations in the treatment, amelioration and/or prevention of breast
cancers.
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[0560] In further particular embodiments, antibodies of the present invention
are used
to treat, ameliorate and/or prevent prostate cancer. Antibodies of the present
invention
may be used in combination with one or more surgical and/or radiological
procedures and
/or therapeutic agents to treat, ameliorate and/or prevent prostate cancer.
Prostate cancer
which may be treated using antibodies of the present invention includes, but
is not limited
to, benign prostatic hyperplasia, malignant prostate cancer (e.g., stage I,
stage II, stage III
or stage IV) and metastatic prostate cancer.
[0561] In preferred embodiments, agonistic antibodies of the present invention
are
used to treat, ameliorate and/or prevent prostate cancer. Agonistic antibodies
of the present
invention may be used in combination with one or more surgical and/or
radiological
procedures and /or therapeutic agents to treat, ameliorate and/or prevent
prostate cancer.
Prostate cancer which may be treated using agonistic antibodies of the present
invention
includes, but is not limited to, benign prostatic hyperplasia, malignant
prostate cancer
(e.g., stage I, stage II, stage III or stage IV) and metastatic prostate
cancer.
[0562] In one preferred embodiment, agonistic antibodies of the invention are
used to
treat malignant prostate cancer. In a further preferred embodiment, agonistic
antibodies of
the invention are used to treat metastatic prostate cancer.
[0563] Antibodies of the present invention may be administered in combination
with
one or more surgical, radiological and/or hormonal procedures useful in the
treatment of
prostate cancer including, but not limited to, prostatectomy (e.g., radical
retropubic
prostatectomy), external beam radiation therapy, brachytherapy, orchiectomy
and
hormone treatment (e.g., LHRH agonists, androgen receptor inhibitors).
[0564] In preferred embodiments, agonistic antibodies of the present invention
may be
administered in combination with one or more surgical, radiological and/or
hormonal
procedures useful in the treatment of prostate cancer including, but not
limited to,
prostatectomy (e.g., radical retropubic prostatectomy), external beam
radiation therapy,
brachytherapy, orchiectomy and hormone treatment (e.g., LHRH agonists,
androgen
receptor inhibitors).
[0565] Antibodies of the present invention may be administered in combination
with
one or more therapeutic agents useful in the treatment of prostate cancer
including, but not
limited to, Aminoglutethimide (CytadrenC), Biclutamide (Casodexe),
Cyclophosphamide
(CytoxanC, NeosarC, CTX), Diethylstilbestrol (DES), Doxorubicin (AdriamycinC,
Doxi1C, Rubex6), Flutamide (EulexinC), Hydrocortisone, Ketoconazole
(Nizorale),
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